<%BANNER%>

Larval Competition and Adult Susceptibility to Arbovirus Infection in Container Mosquitoes


PAGE 1

LARVAL COMPETITION AND ADULT SU SCEPTIBILITY TO ARBOVIRUS INFECTION IN CONTAINER MOSQUTIOES By BARRY W. ALTO A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2006

PAGE 2

Copyright 2006 by Barry W. Alto

PAGE 3

This dissertation is dedicated to my moth er, Barbara A. Larson, for her undying support

PAGE 4

iv ACKNOWLEDGMENTS I am thankful for valuable advice and re views from my dissertation committee, L. P. Lounibos, S. Juliano, C. Lord, J. Mar uniak, C. Mores, C. Osenberg, and W. Tabachnick. I am especially grateful to my advisor L. P. Lounibos for years of guidance, support, useful discussions, and reviews. I am grateful to J. Butler, B. Coon, and K. McKenzie for assistance with methods and e quipment necessary to construct the silicon membrane system; C. Jennings for supplying me with citrated blood; G. OMeara for providing me with cages for bloodfeeding trials and data on mosquito larval densities from field collections; P. Grimstad and R. Nasci for providing me with Aedes eggs; N. Nishimura, R. Escher, I. Tobar, and B. Wa gner for daily maintenance of the competition studies and for measuring wing lengths; J. Ma runiak, S. Higgs, and D. Bowers for advice with cell culture and Sindbis virus assays; S. Fernandez at Walter Reed Army Institute of Research for generously providing me with the plaque assay protocol and the dengue-2 virus (16803) used in the mosquito infecti on study; M. Reiskind for dengue virus RNA extraction and quantitative RT-P CR research, as well as valuab le discussions and reviews that improved the dissertation; D. Baptiste for providing Vero cells and aiding in dengue virus RNA extraction; D. Chisenhall, J. Dyer K. Pesko, and S. Richards for assistance in bloodfeeding mosquitoes.

PAGE 5

v TABLE OF CONTENTS page ACKNOWLEDGMENTS.................................................................................................iv LIST OF TABLES............................................................................................................vii LIST OF FIGURES...........................................................................................................ix ABSTRACT....................................................................................................................... xi CHAPTER 1 INTRODUCTION AND REVE W OF THE LITERATURE.......................................1 Introductory Statement.................................................................................................1 Water-filled Containers................................................................................................1 Mosquitoes....................................................................................................................3 Aedes albopictus ....................................................................................................3 Aedes aegypti .........................................................................................................5 Dengue Virus................................................................................................................7 Introduction...........................................................................................................7 Human Infection....................................................................................................8 Infection Cycle in the Mosquito Vector................................................................9 Sylvatic Dengue Cycles.......................................................................................11 Viral Isolates and Experiment al Infection/Transmission....................................12 Sindbis Virus..............................................................................................................14 Introduction.........................................................................................................14 Human and Reservoir Infection...........................................................................15 Viral Isolates and Experiment al Infection/Transmission....................................16 Competition and Vect or Competence.........................................................................21 2 AGE-DEPENDENT BLOODFEEDING OF Aedes aegypti AND Aedes albopictus ON ARTIFICIAL AND LIVING HOSTS................................................27 Introduction.................................................................................................................27 Materials and Methods...............................................................................................29 Experimental Protocol.........................................................................................29 Data Analyses......................................................................................................31 Results........................................................................................................................ .33 Discussion...................................................................................................................34

PAGE 6

vi 3 LARVAL COMPETITION DIFFERENTIALLY AFFECTS ARBOVIRUS INFECTION IN Aedes MOSQUITOES.....................................................................39 Introduction.................................................................................................................39 Materials and Methods...............................................................................................43 Competition Study...............................................................................................43 Infection Study....................................................................................................45 Results........................................................................................................................ .49 Competition Study...............................................................................................49 Infection Study....................................................................................................50 Discussion...................................................................................................................52 4 LARVAL COMPETITION A ND SUSCEPTIBILITY OF Aedes aegypti AND Aedes albopictus TO INFECTION BY DENGUE VIRUS........................................65 Introduction.................................................................................................................65 Materials and Methods...............................................................................................69 Competition Study...............................................................................................69 Infection Study....................................................................................................72 Viral propagation..........................................................................................72 Oral infection of mosquitoes........................................................................73 Blood meal plaque assay..............................................................................76 Mosquito homogenization, plaque assay, and RNA extraction...................77 Quantitative RT-PCR...................................................................................78 Species by Compe tition Comparison..................................................................80 Results........................................................................................................................ .81 Competition Study...............................................................................................81 Infection Study....................................................................................................82 Species by Compe tition Comparison..................................................................85 Discussion...................................................................................................................86 5 COMPETITION, ARBOVIRUS INFECTI ON, AND FUTURE EXPERIMENTS111 Competition and Enhanced Infection.......................................................................111 Future Studies...........................................................................................................116 Field-collected Mosquitoes for Infection Experiments.....................................116 Mechanisms Responsible for Competition-enhanced Infection........................117 Other Epidemiologically Signifi cant Factors: Adult Survival..........................119 Other Ecological Interacti ons in the Larval Stages...........................................121 Conclusions...............................................................................................................122 LIST OF REFERENCES.................................................................................................123 BIOGRAPHICAL SKETCH...........................................................................................153

PAGE 7

vii LIST OF TABLES Table page 2-1 Test for equal slopes among re gressions of proportion bloodfed of Aedes aegypti and A. albopictus versus age....................................................................................38 2-2 Intercept and slope es timates for simple linear regressions of proportion bloodfed of Aedes aegypti and A. albopictus versus age.........................................38 3-1 Multivariate ANOVA for main effects a nd multivariate pairwise contrasts of competitive treatment effects on female Aedes albopictus and A. aegypti ..............62 3-2 ANCOVA for the effects of compe titive treatment and size covariate on body titer for Aedes albopictus and A. aegypti females with disseminated infections.....63 3-3 Product moment correlation coefficients (r1,2) for the relationship between population growth measurements (time to emergence, survivorship, size, and ) and infection parameters..........................................................................................64 4-1 MANOVA and multivariate pairwise contrasts of competitive treatment effects on female Aedes albopictus and A. aegypti ............................................................104 4-2 Multivariate ANOVA for main effects a nd multivariate pairwise contrasts of competitive treatment effects on female Aedes albopictus and A. aegypti proportion infected and proportion with disseminated infection...........................105 4-3 ANCOVA (after testing for equality of slopes) for the effects of competitive treatment and size covariate on body titer proportion infected, and proportion with disseminated infection for Aedes albopictus and A. aegypti females............106 4-4 Product-moment correlation coefficients (r1,2) for the relationship between population growth measurements (time to emergence, survivorship, size, and ) and infection parameters for A. albopictus (df=25) and A. aegypti (df=29)..........108 4-5 Two-way MANOVA of species ( A. albopictus and A. aegypti ) and competitive treatment ( A. albopictus : A. aegypti 160:0, 320:0, 0:160, a nd 0:320) effects on female population growth measurements...............................................................109 4-6 Two-way MANOVA of species ( A. albopictus and A. aegypti ) and competitive treatment ( A. albopictus : A. aegypti 160:0, 320:0, 0:160, a nd 0:320) effects on proportion infected and proportion w ith disseminated infections..........................110

PAGE 8

viii 4-7 Two-way ANOVA for species ( A. albopictus and A. aegypti ) and competitive treatment ( A. albopictus : A. aegypti 160:0, 320:0, 0:160, a nd 0:320) effects on body titer................................................................................................................110

PAGE 9

ix LIST OF FIGURES Figure page 2-1 Least squares means ( SE) for proportion bloodfed females on the siliconmembrane system for 3-15 day old Aedes albopictus and A. aegypti ......................37 2-2 Least squares means ( SE) for pr oportion bloodfed females on restrained chickens for 3-15 day old Aedes albopictus and A. aegypti. ....................................37 3-1 Aedes albopictus least squares means (SE) for female survivorship and size at emergence.................................................................................................................58 3-2 Aedes aegypti least squares means (SE) for female survivorship and time to emergence.................................................................................................................58 3-3 Least squares means ( SE) for estimated finite rate of increase, for Aedes albopictus and A. aegypti .........................................................................................59 3-4 Bivariate plots of least squares means ( SE) for three dependent variables for Aedes albopictus females fed on a Sindbis virus blood meal..................................60 3-5 Least squares means for body titer and size of adult Aedes albopictus females with disseminated and isolated Sindbis virus infections..........................................61 4-1 Aedes albopictus least squares means ( SE) for female size and time to emergence.................................................................................................................97 4-2 Aedes aegypti least squares means ( SE) for female size and time to emergence.97 4-3 Least squares means ( SE) for estimated finite rate of increase, for A. albopictus and A. aegypti .........................................................................................98 4-4 Least squares ( SE) for proportion of A. albopictus and A. aegypti infected and disseminated infections............................................................................................99 4-5 Bivariate plots of least squa res means ( SE) for proportion of A. albopictus infected and disseminated infections........................................................................99 4-6 Bivariate plots of least squa res means ( SE) for proportion of A. aegypti infected and disseminated infections......................................................................100

PAGE 10

x 4-7 Least squares means for body titer and size of adult A. albopictu s females with disseminated (i.e., infection spread beyond the midgut, infecting secondary target organs) dengue-2 virus infections................................................................100 4-8 Least squares means for propor tion infected and size of adult A. albopictus females...................................................................................................................101 4-9 Least-squares means for proportion disse minated infections and size of adult A. albopictus females..................................................................................................101 4-10 Least-squares means for body titer and size of adult A. aegypti females with disseminated (i.e., infection spread beyond the midgut, infecting secondary target organs) dengue-2 virus infections................................................................102 4-11 Least-squares means for propor tion infected and size of adult A. aegypti females. Numbers in the figure ke y represent the ratio of larval A. albopictus to A. aegypti. ...............................................................................................................102 4-12 Least-squares means for proportion disse minated infections and size of adult A. aegypti females. Numbers in the figure key represent the ratio of larval A. albopictus to A. aegypti. .........................................................................................103 4-13 Two-way ANOVA for species ( A. albopictus and A. aegypti ) and competitive treatment ( A. albopictus : A. aegypti 160:0, 320:0, 0:160, a nd 0:320) effects on Least-squares means for body titer.........................................................................103

PAGE 11

xi Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy LARVAL COMPETITION AND ADULT SU SCEPTIBILITY TO ARBOVIRUS INFECTION IN CONTAINER MOSQUITOES By Barry W. Alto August 2006 Chair: L. Philip Lounibos Major Department: Entomology and Nematology Larval competition is well-documented am ong container mosquitoes and influences life history traits such as survivorship, deve lopment, and adult size. Few studies have attempted to address how biol ogical interactions experienced by larvae may impact adult susceptibility to arboviral inf ection, subsequent viral spread to secondary tissues (i.e., disseminated infection), and viral body titer. With Sindbis, an arbovirus frequently used in vector research, Aedes albopictus mosquitoes had higher infection rates but lower body titer and dissemination rates than A. aegypti For both A. albopictus and A. aegypti competition affected population growth measurements, with uncrowded larval conditions consistently resulting in shorter time to adult emergence, increased survivorship, a dult size, and better population performance than crowded conditions. For A. albopictus but not for A. aegypti more intense intraand interspecific competition resulted in highe r Sindbis virus infection rates, body titers, and dissemination rates compared to low competition conditions. Whole body titers of

PAGE 12

xii virus increased with mosquito size irrespective of competition. However, between competitive treatments, mosquitoes from lo w competition conditions had greater mean size, with lower infection a nd lower whole body titers than smaller mosquitoes from high competition conditions. The results of expe riments on this model system indicate the importance of the larval environment, esp ecially competitive conditions, on adult vector competence. With dengue virus, the most impor tant arbovirus afflicting humans, A. aegypti had lower dengue virus infection rates and body titers but highe r dissemination rates than A. albopictus. Higher levels of intraand interspecific competition enhanced A. albopictu s infection and dissemination rates with dengue virus Similar effects of competition on mosquito infection parameters with unrel ated Sindbis and dengue viruses suggest a generalizable mechanism of environmental influences on infection parameters. The experimental results indicate th at larval conditions are an important aspect of vector competence and should be included in futu re epidemiological considerations and modeling of arbovirus transmission.

PAGE 13

1 CHAPTER 1 INTRODUCTION AND REVE W OF THE LITERATURE Introductory Statement Larval competition is well-documented am ong container mosquitoes but its effects on adult susceptibility to arboviral infection remain unclear This research addresses the question whether larval competition among and between mosquitoes Aedes aegypti and A. albopictus influences adult susceptibility to Sindbis and dengue virus infection. A silicon membrane bloodfeeding system was evaluated as a method to administer infectious bloodmeals for subsequent competition and infection studies. The purpose of the following literature review is to provide a working know ledge of container habitats and basic biology of the two mosquito species used in the current experiments. Also, I summarize the vector biology research of dengue and Sindbis viruses as well as place the current question concerning competition and susceptibility to ar boviral infection in context to studies of similar nature. Water-filled Containers Phytotelmata are parts of te rrestrial plants such as l eaf axils of tank bromeliads, bamboo internodes, pitchers of carnivorous plants, Heliconia bracts, fallen leaves or fruit husks, and treeholes which hold bodies of wa ter (Frank and Lounibos 1983). Artificial containers serve as analogs of phytotelmata and come in a variety of forms such as discarded tires, cans, vases, jars, cisterns, a nd plastic debris. Both natural and artificial containers are habitats for a variety of arth ropods having aquatic life stages. Containers may be favorable model systems for inve stigating entomological and ecological

PAGE 14

2 processes because they harbor small, discrete aquatic commun ities. Diptera are the most taxonomically diverse group among insect orders inhabiting phytotelma ta (Fish 1983). In particular, mosquitoes are the most extensivel y studied dipterous insects within container communities, in part, because they often ar e the most abundant macroinvertebrates (Fish 1983) and may be vectors of arthropod-borne (arbo) viruses and other vertebrate pathogens. Nutrient resources in these systems come in the form of inputs of allochthonous plant detritus (fallen leaves, flower parts) (Lounibos et al. 1993, 1992, Kitching 1971), macroinvertebrate carcasses (Daugherty et al. 2000, Sota et al. 1998, Heard 1994, Naeem 1988, Bradshaw and Holzapfel 1986), throughfall, and stem flow (Kaufman et al. 1999, Walker et al. 1991, Walker and Merritt 1988, Carpenter 1982a). Th e latter two resource inputs refer to precipitation that has fallen through the canopy or down the branches and trunk of trees, respectively. Decomposing plan t detritus is recognized as the predominant nutrient base for treeholes, and, perhaps, ar tificial container co mmunities (Maci and Bradshaw 2000, Lounibos et al. 1992, Carpenter 1983, Fish and Carpenter 1982, Kitching 1971). Leaf litter is likely a lower quality nutrient resource compared to macroinvertebrate carcasses due to intrinsic differences in carbon:nitrogen ratios among these resources (Cloe and Garman 1996). Some macroinvertebrates may directly consume plant detritus (e.g., Helodes and Prionocyphon beetles, Paradise and Kuhn 1999, Barrera 1996a, Carpenter 1982b), however most container mosquitoes consume microorganisms associated with the detritus and water column (e.g., Walker and Merritt 1988, Fish and Carpenter 1982). Spatial and te mporal differences in the quality and quantity of nutrient resources have important consequences for container communities.

PAGE 15

3 For example, mosquito larval stages are confined to containers, and so habitat characteristics (e.g., nutrients, competition, predation, temperature) largely determine mosquito population growth measurements. Rate of leaf decomposition, and associated bacteria and algae, is often positively corre lated with nutritional value and mosquito productivity (e.g., Dieng et al. 2002, Yanoviak 1999, Fish and Carpenter 1982, Swift et al. 1979), and differences in degradation among leaf types are due to the environmental conditions and properties of the leaf species (Yanoviak 1999, Lonard and Juliano 1995, Fish and Carpenter 1982). Mosquitoes Aedes albopictus The Asian tiger mosquito Aedes albopictus (Skuse), native to Southeast (SE) Asia and the Pacific and Indian Ocean regions, inva ded container habitats in the U.S., Europe, West Africa and South America during the last 30 years (reviewed in Eritja et al. 2005, Juliano and Lounibos 2005, Lounibos 2002). Aedes albopictus is second only to A. aegypti in terms of importance as a vector of dengue virus (DENV). Although small introductions of A. albopictus in the continental U.S. were found previously in used tires shipped to a port in Oakland, CA (Eads 1972) and a cemetery in Memphis, TN (Reiter and Darsie 1984), none became established. It is believed that A. albopictus first became established in the continental U.S. in Houston, Texas in 1985 (Sprenger and Wuithiranyagool 1986). Populations of A. albopictus in the U.S. are believed to be derived from temperate Japan (Hawle y et al. 1987), whereas Brazilian A. albopictus are of tropical origin (Birungi and Muns termann 2002). Successful spread of A. albopictus in the eastern U.S. was facilitated by immatu re stages using artificial container habitats, in particular used or discarde d tires (Moore 1999, Reiter 1998). Aedes albopictus is

PAGE 16

4 adapted to both tropical and temperate clima tic regions and capable of using a wide range of suitable container habitats such as man-made c ontainers (e.g., discarded tires, cemetery vases, cans), natural tree holes, bamboo internodes, and other phytotelmata (Hawley 1988). Adult females deposit desiccation resistan t eggs on walls of containers, and these eggs hatch when flooded by water (Hawley 1988). Embryonated eggs are able to survive several months at 20-25C and at moderate to high humidity (44-90%) (Sota and Mogi 1992, Hawley 1988). The embryonation period is temperature-dependent but usually can be completed within two days to just over a week. Aedes albopictus from temperate Japan have photoperiodically induc ible egg diapause, whereas A. albopictus of tropical origins ordinarily do not (Lounibos et al 2003a, Pumpuni et al 1992, Hawley et al. 1987). In continental Asia and the U.S., 0C and -5C are the approximate northern maximum isotherms for overwintering range and northward expansion, respectively (Nawrocki and Hawley 1987). Larvae in water-filled containers filte r-feed and browse on decomposing plant detritus and microorganisms. Studies quantifying development time of larval stages have usually been performed at 25 C and under optimal nutrition. These conditions allow for complete larval development in 510 days (Hawley 1988). Low temperature, crowded larval conditions, and nutrient depr ivation greatly increase development time (e.g., Lounibos et al. 2002, Alto and Ju liano 2001ab, Briegel and Timmermann 2001, Teng and Apperson 2000). The non-feeding pupal stage lasts 1-3 days. Pupal size is determined to a large extent by larval de nsity and food supply, how ever other factors (e.g., temperature) may also be important (Hawley 1988).

PAGE 17

5 Studies quantifying adult long evity have largely been c onducted in the laboratory and are likely to overestimate longevity in the field. Under laboratory conditions, females can live for several weeks and pe rhaps as long as a few months. High temperature coupled with low humidity de creases adult longevity (Alto and Juliano 2001b, Mogi et al. 1996). Female A. albopictus blood feed diurnally and are able to blood feed within 2-3 days of emergence (Hawley 1988). Aedes albopictus is an opportunistic biter taking blood meals from a variety of hosts, including humans (Ponlawat and Harrington 2005, Nie bylski et al. 1994, Savage et al. 1993). It is capable of dispersing > 800 m in suburban settings (H onrio et al. 2003). Female fecundity is positively correlated with size but other f actors may also influence fecundity (e.g., temperature, size of blood meal) (e.g., Armbruster and Hutchinson 2002, Lounibos et al 2002, Briegel and Timmermann 2001, Blackmo re and Lord 2000, Briegel 1990, 1985). Aedes aegypti The yellow fever mosquito Aedes aegypti (L.) has its origins in Africa. Water vessels aboard slave ships are thought to have transpor ted immature stages of A. aegypti from West Africa to the West ern Hemisphere during the 15th to 17th centuries (Christophers 1960). However, A. aegypti may have established in Portugal and Spain prior to its arrival in the Western Hemisphere (Tabachnick 1991). It is likely that A. aegypti spread subsequently to the Mediterran ean, tropical Asian, and Pacific Islands during the 18th, 19th, and 20th centuries, respectively (Tabachnick 1991). In Sub-Saharan Africa, at least 2 forms of A. aegypti exist, differing genetica lly, morphologically, and behaviorally (Tabachnick et al. 1979 Mattingly 1957). The sylvan form, A. aegypti formosus (Walker), is darkly colored and found in natural phytotelmata (treeholes) and confined to East Af rica (Christophers 1960). Aedes aegypti formosus feed on a variety of

PAGE 18

6 vertebrates, including primates, but prim arily feed on reptiles and small mammals (McClelland and Weitz 1963) The domestic form, A. aegypti aegypti (L.), is lighter in color and highly anthropophili c, blood feeding predominantly on humans and occupying artificial containers in its immature stages. The domestic form is referred to as A. aegypti and the sylvan form as A. aegypti formosus Adults of A. aegypti commonly oviposit and blood feed in human dwellings. The adaptation to artificial containe r habitats and blood feeding on humans has made A. aegypti highly successfully in spreading throughout much of tropical to mild temperate regions (Lounibos 2002, Christophers 1960). Aedes aegypti is considered the primary vect or of DENV. Female A. aegypti adults deposit desiccation resistant eggs within a wide range of artificial containers, both outdoors and indoors, in urban environments. Eggs of A. aegypti are more resistant to mortality induced by high temperatures and desiccation as compared to A. albopictus (Juliano et al. 2002, Sota and Mogi 1992). The embryonation period is similar to that of A. albopictus and there is no evidence that eggs, or any developmental stage of A. aegypti are capable of diapause. A. aegypti larvae develop more rapidly than A. albopictus on artificial nutrient resources (e.g., yeast, albumin) but develop more slowly compared to A. albopictus on leaf litter (B.W. Alto, personal observation). This is consiste nt with the observation that larval resistance to starvation was maximized with leaf litter for A. albopictus and with liver powder (non-natural) food for A. aegypti (Barrera 1996b). Pupal developmental period is similar to A. albopictus and highly dependent on temperature ( 2 3 d at 2327C) (Christophers 1960).

PAGE 19

7 In extreme cases, maximum survival of adu lt females in laboratory settings may be >100 days, however, longevity is highly depe ndent on abiotic condi tions (temperature, humidity) (Mogi et al. 1996) as well as adult size and nutrient availability (water, carbohydrates, blood) (Christophers 1960). Re gular access to carbohydrates and blood, coupled with high humidity and 28C is optimal for A. aegypti adult longevity (Christophers 1960). A mark-release-recap ture field study in Kenya determined longevity of adult A. aegypti during the rainy season (April-May 1972, environmental conditions unspecified) (Trpis and Hausermann 1986). Mean-maximum adult female and male longevity was 10.7-42 and 5.8-8 d, respectively. Aedes aegypti often prefer human hosts for blood meals and may imbibe multip le blood meals in a single gonotrophic cycle (Scott et al. 2000ab, 1993ab). Large A. aegypti consume more than twice as much blood as small individuals, and subsequent efficien cy of yolk synthesis derived from the blood meal is positively related to female size (B riegel 1990). Adult dispersal may be hundreds of meters (e.g., 100 to >800 m) in rural and urban dengue endemic regions (Harrington et al. 2005, Honrio et al. 2003). Dengue Virus Introduction Dengue virus (DENV) consists of the dengue serotypes 1-4. These are the etiological agents of human disease that ra nge in severity from undifferentiated dengue infection (asymptomatic or m ildly symptomatic), classical dengue fever (DF), dengue hemorrhagic fever (DHF), to dengue shock syndrome (DSS) (Gubler 1997, Gubler et al. 1981). DF is characterized by an abrupt febril e illness with associat ed malaise, headache, retro-orbital pain, rash, and extreme muscle and joint pain. DF is not known to be associated with mortality. Initial sympto ms of DHF resemble those of DF followed by

PAGE 20

8 thrombocytopenia, hemorrhagic manifestations, and plasma leakage due to increased vascular permeability from the release of circulating factors in infected white blood cells (e.g., monocytes, T cells ). DSS is char acterized by severe DHF followed by shock where patients experience restlessness, rapi d and weak pulse, subnor mal temperature, and low blood pressure. DENV is considered am ong the most important vector-transmitted arboviruses and its geographic range places 2.5 b illion humans at risk (review in Gubler 2002). Annually 50-100 million cases of DF occur in tropical cities with hundreds of thousands of cases of DHF (<1-15% DHF mortality) (Gubler 2002). An A. aegypti eradication program initiated by the Pan American Health Organization in the 1940s and 1950s was successful at limiting Aedes aegypti distribution in the Americas (Gubler 1997). However, the program was disbanded in the 1970s followed by A. aegypti reinfestation in most areas wh ich the program had targeted. Dengue activity has increased in recent decad es and poses a major global public health problem (Gubler 1997). Although the reasons are complex and not fully understood, factors that may contribute to increased dengue activity in clude reinvasion of tropical America by the primary vector of DENV, A. aegypti ineffective mosquito control in areas associated with dengue, unplanned ur banization, abundant man-made larval habitats, and increased and rapid human travel. Human Infection A study on naturally acquired DENV of se rotypes 1, 3, and 4 in Central Java, Indonesia, showed patients w ith viremia ranging from 103.8 to > 108.0 MID50/ml (Mosquito Intrathoracic Inocula tion Dose for 50% infection) th at lasted for 5-6 days in some cases (Gubler et al. 1979). Similarly in Jakarta, Indonesian patients with dengue fever showed a viremia range of 2-12 days w ith an average of 4-5 days (Gubler et al.

PAGE 21

9 1981). Viral titer in thes e patients ranged from 103.8 to 107.2 MID50/ml, with DENV serotype 4 infected patients showing 102 times lower titer (Gubler et al. 1981). Gubler et al. (1981) did not find that disease severity was signifi cantly affected by duration or magnitude of viremia. However, fatal DHF cases had large amounts of circulating DENV (Gubler et al. 1981). DENV pathogenesi s is difficult to study because there are no in vivo or in vitro models manifesting pathology simila r to humans (Leitmeyer et al. 1999). Despite unique human pathology, nonhu man primates and mice have served traditionally as human surrogates in dengue laboratory models (Gubler 1997). Infection Cycle in the Mosquito Vector The DENV transmission cycle includes a human reservoir and a mosquito vector, although sylvatic cycles occur between monke ys and mosquitoes in tropical Africa and Asia. After imbibing an infectious bloodm eal, arboviruses (e.g., DENV) are deposited in the mosquito midgut and an infection may initiate. Biological transmission of arboviruses includes acquisiti on from an infectious bloodmeal, replication in the mosquito, dissemination of virus throughout the body of a mosquito resulting in a generalized infection, movement of virus into salivary glan ds via hemolymph or neural pathways, and transmission to a host by subs equent bloodfeeding (H ardy et al. 1983). Additional biological routes include transovarial and ve nereal transmission. Successful biological transmission of an arbovirus requires that several internal physical barriers must be overcome in the mosquito. The ingested infectious blood is deposited into the posterior midgut. Within a matter of hours viruses migrate toward the microvillar margins of the mesenteronal epit helial cells (midgut cells) (Hardy et al. 1983). Mechanisms by which viruses enter the midgut cells are not well known, but

PAGE 22

10 attachment with receptor-mediated-entry is t hought to be a common mechanism. Once in a midgut cell, the virus releases its nuclei c acid and replicates. The midgut infection barrier is the first barrier that the arbovi rus must overcome in the infection process (Gomez-Machorro et al. 2004, Bosio et al 2000, Woodring et al 1996, Hardy et al. 1983). Crossing of this barrier is thought to be dose-dependent such that the likelihood of infection increases with increas ed viral titer in the blood me al (Lord et al. 2006, Hardy et al. 1983). At this stage, arbovi ral infection is limited to th e mesenteronal epithelial cells. The next barrier to overcome in the arboviral infection cycl e is the midgut escape barrier (Bennett et al. 2005ab, Bennett et al. 2002, My les et al. 2004, Woodring et al. 1996). Crossing of this barrier, which consists of multilayer basal laminae, is also thought to be dose-dependent (DeFoliart et al. 1987). If arboviruses fail to overcome the midgut escape barrier, then infections are limited to the mesenterona l epithelial cells. If the midgut escape barrier is overcome, the arboviru s enters the hemocoel from where it can disseminate, via hemolymph, to other tissues and organs (e.g., fat body, foregut, hindgut, ovarioles, salivary glands). These midgut barriers have important epidemiological significance because they, in part, determin e whether mosquitoes become potential arboviral transmitters. Intrathoracic inoc ulation of arboviruses (ie., bypass midgut barriers) is highly efficient at infecting mosquitoes, and may effectively eliminate interspecific differences in vector competen ce, even species that may otherwise show refractoriness to arboviral inf ection or transmission, perhaps due to barriers, (i.e., genetic refractoriness) (Woodring et al. 1996, Hardy et al. 1978). The final two barriers to transmission by mosquitoes are the salivary gland infection barrier and the saliv ary gland escape barrier (Grims tad 1985 et al., Hardy et al.

PAGE 23

11 1983). Crossing of the salivary gland inf ection barrier is dose-dependent and timedependent. The reason for the time-dependency is that, in many instances, the longevity of the female adult mosquito and the extrin sic incubation period are similar. The time from initial ingestion of the infectious blood meal until the time the mosquito can transmit the arbovirus is the extrinsic incubation period. Thus, for successful transmission of the arbovirus to another host, it must pass all the barriers and infect the salivary glands before the mosquito dies. The paired salivary glands consist of a single layer of cuboidal epithelial cells surrounded by a basal lamina. If the salivary gland infection barrier is ove rcome, the arbovirus may replicate in the cuboidal cells. Finally, if the salivary gland escape barrier is overcome, vi rus is incorporated into the saliva and is potentially transmitted to a vertebrate host dur ing the next blood feeding. These barriers determine the intrinsic ability of a mosquito to become infected and subsequently transmit a pathogen (i.e., vector competen ce). Mosquitoes with disseminated DENV infection are capable of transmitting virus fo r the remainder of their life (Rodhain and Rosen 1997). Sylvatic Dengue Cycles Sylvatic dengue cycles are known to occu r in West Africa and Malaysia involving Aedes species mosquitoes and monkeys (Diallo et al. 2003, Wang et al. 2000, De Silva et al. 1999, Gubler 1997, Rodhain 1991, Rudnick 1978, 1965). Sylvatic dengue cycles are not known in the Western Hemisphere (Rodha in and Rosen 1997). However, antibodies to DENV have been recovered from bats a nd other mammals in Costa Rica, Ecuador, and French Guiana (de Thoisy et al. 2004, Platt et al. 2000). Further research on sylvatic cycles in the Western Hemisphere is needed (e.g., viral isolati on) since neutralizing antibodies may cross-react with other relate d viruses in those regions (Scott 2001, Innis

PAGE 24

12 1997). Monkeys infected with DENV are not known to exhibit human-like DF or DHF symptoms. In West Africa, DENV-2 has been isolated from A. africanus (Theobald), A. luteocephalus (Newstead), A. opok (Corbet and Van Someren), A. furcifer (Edwards), and A. taylori (Edwards) (Diallo 2003, Wang et al. 2000). In Malaysia, it is believed that the canopy dwelling mosquito A. niveus (Ludlow) serves as the vector for all the DENV serotypes to monkeys (Wang et al. 2000). DE NV isolates from nonprimate reservoirs are not known to exist, although ne utralizing antibodies have be en found from mammals in the Eastern Hemisphere (Rudnick 1965). Viral Isolates and Experimental Infection/Transmission DENV is one of > 70 arboviruses within the genus Flavivirus (family Flaviviridae) (White and Fenner 1994). Flaviviruses are e nveloped, spherical virions with a diameter of 40-50 nm. They have a linear plus sense singl e stranded RNA genome of 10.5-11 kb and are capped at the 5 terminus but not polyadenylated at the 3 terminus. Endemic/epidemic dengue cycles between humans and the primary and secondary vectors, A. aegypti and A. albopictus, as well as A. polynesiensis (Marks). Field collections of naturally infected mosquito es suggest that other vectors may include A. mediovittatus (Coquillett), A. scutellaris (Walker), A. cooki (Belkin), and A. hebrideus (Edwards) (Rodhain and Rose n 1997, Freier and Rosen 1988). It is thought that the DENV serotypes that now cause epidemics independently evolved from sylvatic progenitors 100 to 1,500 years ago, presumably when DENV adapted to peridomestic A. albopictus and later to A. aegypti (Moncayo et al. 2004, Wang et al. 2000). Thus, sylvatic DENV serotypes, typically cycling between sylvatic mosquitoes and monkeys, differ from endemic/epidemic DENV serotypes transmitted by A. aegypti, A. albopictus

PAGE 25

13 and other anthropophilic Aedes mosquitoes (Moncayo et al. 2004). All sylvatic DENV serotypes are found in Malaysia whereas on ly sylvatic DENV-2 occurs in Africa, suggesting that DENV may have originated in the Asian-Oceanic region (Wang et al. 2000). If this hypothesis is true, then the peridomestic transmission of DENV was most likely initially vectored by A. albopictus since A. aegypti did not establis h in Asia until the later half of the 19th century (Tabachnick 1991, Smith 1956). Both A. albopictus and A. aegypti are more susceptible to infection wi th endemic/epidemic DENV-2 than to sylvatic DENV-2, supporting the hypothesis of emergence of endemic/epidemic dengue by viral adaptation to peridomestic Aedes spp. (Moncayo et al. 2004). Endemic/epidemic DENV serotypes di ffer in their ability to infect A. albopictus and A. aegypti (e.g., Moncayo et al. 2004, Armstrong and Rico-Hesse 2003, 2001, Rosen et al. 1985). Previous literature sugges ted that all DENV sero types infected and disseminated in A. aegypti more poorly than in other Aedes species, including A. albopictus (Rodhain and Rosen 1997, Rosen et al. 1985, Gubler et al. 1979). However, these studies used highly adapted laboratory colonies of these Aedes species, which may have altered vector competence due to founder effects, genetic drift, and unintentional artificial selection impose d on laboratory colonies (Arm strong and Rico-Hesse 2001, Lorenz et al. 1984). A laboratory study on the F1 F2 progeny of field collected mosquitoes in Vietnam and Thailand showed that A. aegypti were more readily orally infected than A. albopictus (mosquito head assays) with SE Asian DENV-2 (Vazeille et al. 2003). Similarly, laboratory colonies of Aedes collected in Taiwan ( F5) showed A. aegypti had significantly higher sa livary gland infection a nd transmission rates for DENV-1 compared to A. albopictus (Chen et al. 1993). The conflicting observations in

PAGE 26

14 the cited studies demonstrate that it is unclear whether A. albopictus or A. aegypti is the more competent DENV vector. It appears th at vector competence of these two species critically depends on underlying genetic differe nces in the strains of mosquitoes, the strains of DENV, and the environmental cond itions under which the laboratory analyses are conducted. The complexity of investig ating vector competence mechanisms and variation has been discussed elsewhere (Tabachnick 1994). Vertical transmission of of arboviruse s (e.g., transovarial) may serve as a mechanism to survive inhospitable environm ental conditions (e.g., cold temperatures, drought). For arboviruses that cycle between mosquito and humans (e.g., DENV in the Western Hemisphere), verti cal transmission may facilitate endemic maintenance, especially when human cases are not occurri ng. Experimental studies have shown that both A. albopictus and A. aegypti are capable of vertical transmission of DENV as determined by detection of DENV antigen or viral isolation among immature stages (e.g., Joshi et al. 2006, 2002 1996, Rodhain and Ro sen 1997, Bosio et al. 1992, Rosen et al. 1983). For example, field collections of immature stages of A. aegypti in India and Burma showed definitive evidence of DENV-2 and DENV-3 vertical transmission because infected field mosquito s upernatant fluid was inoculated in Toxorhynchites splendens (Weidemann) mosquitoes, allowed to repl icate, and DENV antigen was positively recovered from head tissues (Thenm ozhi et al. 2000, Khin and Than 1983). Sindbis Virus Introduction Sindbis virus (SINV) was first isolated from mosquitoes Culex univittatus (Theobald), Culex pipiens (L.), and a juvenile hooded crow Corvus corone sardonius in 1952 in Sindbis Egypt, 30 km north of Cair o (Taylor et al. 1955). SINV has a wide

PAGE 27

15 geographic distribution in Aust ralia, Scandinavia, South Af rica, Middle East, and Asia (Laine et al. 2004, Dohm et al. 1995, Niklasson 1989, Tesh 1982). SINV has been given distinct names based on the geographic regi on of isolation i.e., Ockelbo (Sweden), Pogosta (Finland), Karelian (Russia), and SINV (other regions) (Laine et al. 2004). In all instances the viruses are similar and represen t geographically distinct genotypes (Kurkela et al. 2004, Laine et al. 2004, Sammels et al. 1999, Lundstr m 1999, Norder et al. 1996, Shirako et al. 1991, Lundstrm et al. 1993a, Olson and Trent 1985). SINV is not known to occur in the Americas. The wide distributi on is partially attributab le to migratory birds which serve as reservoirs and transport SI NV over large distances (e.g., Buckley et al. 2003, Brummer-Korvenkontio et al. 2002, Lundstr m et al. 2001, Lundstrm et al. 1993b). Human and Reservoir Infection Clinical symptoms of SINV infection were described from Uganda in 1961 (Woodall et al. 1962). The most complete record of human cases include SINV epidemics in Sweden, Finland, and Russia during late summer and fall (August October) (Laine et al. 2004, Lundstrm et al. 1991). Human SINV infections produce a self-limited febrile disease characterized by ar thralgia, rash, headache, fatigue, and fever (Laine et al. 2004, 2000, Tesh 1982). It is not unc ommon for chronic arth ralgia to last for several years after full recove ry from other symptoms (e.g., Kurkela et al. 2005, Laine et al. 2000, Turunen et al. 1998, Ni klasson et al. 1988, Niklasson and Espmark 1986). SINV is not known to cause human mortality, although morbidity is common. Although SINV has a wide geographic distri bution, human disease with clinical symptoms has been limited to Northern Europe and South Africa (Lundstrm 1994, Niklasson et al. 1988). Nucleotide sequences of genes encoding capsid (C) and envelope

PAGE 28

16 protein (E2) showed th at SINV strains from Northern Eu rope were most closely related to those strains from South Africa compared to strains from other geographic regions (Norder et al. 1996, Shirako et al. 1991). In tercontinental exchange of SINV strains may be facilitated by migratory birds because 25% of the 252 bird species that breed in Scandinavia overwinter in Africa (Norder et al. 1996, Lundstrm et al. 1993a, Shirako et al. 1991). Initially SINV infection was regarded as a minor human disease because there were few human cases. However, several out breaks occurred in South Africa in 1974 involving C. univittatus and Culex theileri (Theobald) (Jupp et al. 1986a, McIntosh et al. 1976, 1967, 1964), and Northern Europe in the 1980s including Sweden (Lundstrm et al. 1991, Espmark and Niklasson 1984, Niklas son et al. 1984, Skogh and Espark 1982), Finland (Kurkela et al 2004, Brummer-Korvenkontio et al. 2002, BrummerKorvenkontio and Kuusisto 1981), and Russia (Lvov et al. 1984, 1982). Mosquito vectors of SINV in Northern Europe and Russia include Culex spp., Culiseta spp., A. cinereus (Meigen), and Aedes communis (DeGeer) (Lundstrm 1999, 1994). SINV is the most common virus isolated from mosquitoes in Australia where the principal vectors include Culex annulirostris (Skuse) and Aedes normanensis (Taylor) (Niklasson 1989). However, clinical symptoms in Australia have only been reported on a few occasions (Sammels et al. 1999). Viral Isolates and Experimental Infection/Transmission SINV is a prototype Alphavirus in the family Togaviridae. Alphaviruses are enveloped, spherical virions with icosahedral cap sids and a diameter of 70 nm (White and Fenner 1994). They have a linear plus se nse single stranded R NA genome 11-12 kb, are capped at the 5 terminus, and are polyadenylated at the 3 terminus. The most complete

PAGE 29

17 record of SINV isolates from field-collected mosquitoes and experimental infection / transmission studies are from South Africa a nd Sweden. Typically, zoonotic circulation of SINV occurs between ornithophilic Culex and Culiseta spp. and passerine birds, although other vector species and avian orders may also be involved in transmission cycles (Lundstrm 1999, 1994, Lundstrm et al 1993b, Francy et al. 1989). The single SINV isolate from an arthropod, not a mosquito, was from a Hyalomma marginatum tick (Koch) in Italy (Sicily) in 1975 (Gresikova et al. 1978). Viral is olates from fieldcollected birds during SINV epidemics in South Africa (1960s1970s) showed high SINV immune rates for several bird species. Vira l isolates from fieldcollected ornithophilic mosquitoes C. univittatus and C. theileri showed infection rates of 0.65% and 0.083%, respectively. Culex univittatus was observed to readily feed on humans and was regarded as the main epidemic vector for SINV in the region (McIntosh et al. 1978, 1976). However, C. neavei (Theobald) may be an important vector in costal South Africa, although it may not acquire a dissemi nated infection as readily as C. univittatus (Jupp et al. 1986b). Laboratory experiments were used to determine the vector potential of Culex spp. derived from South Africa (Jupp et al. 1972, Jupp and McIntosh 1970ab). Culex univittatus (F6-15) was readily infected (50-83%) after SINV infectious bloodmeals (separate feeding trials with a range of viral titers 10 3.6 5.6) and 57-66% transmitted SINV to avian hosts after taking a subsequent bloodmeal (Jupp and McIntosh 1970a). A similar experiment showed higher (74-100%) C. theileri (F3-11) infection with similar SINV titers, but transmission was much lower (9%) (Jupp et al. 1972). Both infection (016%) and transmission (0-50%) in C. pipiens (F4-10) were lower than for the other Culex spp. (Jupp and McIntosh 1970b).

PAGE 30

18 Sweden is dominated by mammalophilic Aedes species (Francy et al. 1989, Jaenson and Niklasson 1986). Field collections made in central Sweden showed that 60% of all Aedes spp. collected were A. cinereus whereas only 3.4 and 3.7% (of total mosquitoes collected) were Culex and Culiseta spp., respectively. Despite their infrequency in collections, C. pipiens C. torrentium (Martini), and C. morsitans (Theobald) accounted for 80% of all SINV isolates (Francy et al. 1989). Minimum in fection rates were 7-14% for C. pipiens and C. torrentium and 2-5% for C. morsitans Minimum infection rates for A. cinereus were 0.5%. Experimental inoculati ons in indigenous bird species showed that Passeriforms (105.8 to 107.5 Plaque forming units/ml) (PFU/ml) had significantly higher and longer viremia compared to Anseriforms (103.7 to 104.5 PFU/ml ) (Lundstrm et al. 1993b). Passeriform thrushes ( Turdus spp.) and finches ( Fringilla spp.) are the major SINV reservoirs in Sweden (Lunds trm et al. 2001, Lundstrm 1994). The presence of neutralizing antibodies to SINV in passerine reservoirs was detected in summer but not spring bird migrants (Fra ncy et al. 1989). It is likely that Culex and Culiseta spp. are important vectors in the enzo otic cycle involving passerine birds, whereas A. cinereus and A. communis are probable bridge vect ors to humans (Francy et al. 1989, Jaenson and Niklasson 1986, Lundstr m 1999, 1994). This epidemiological hypothesis is supported by SI NV isolates from a susp ected bridge vector A. communis of SINV human infections in Russia (Lvov et al. 1984). Experimental infection and transmission studi es were used to determine the vector potential of Culex spp. from central Sweden (Lundstrm et al. 1990ab). C. torrentium (F3-6) infected and transmitted SINV to avian hosts more efficiently than C. pipiens (F410). Even at low infectious bloodm eal titers (<2.0 PFU/ml), 50% of C. torrentium were

PAGE 31

19 infected with SINV. Blood meal titers of >3.0 PFU/ml resulted in 90-100% infection and 100% transmission. For C. pipiens 3.0-3.0 PFU/ml resulted in 4% infection, whereas higher titers (6.0-8.9 PFU/ml) resu lted in 42 to 55% infectio n and 14 to 37% transmission (Lundstrm et al. 1990a). A laboratory experiment determined the e ffect of natural temperature regimes (10, 17, 24, cyclic 10-24C) during the transmission season in Sweden on Culex spp. vector competence (Lundstrm et al. 1990b). Low temperature significantly reduced transmission potential of C. pipiens as measured by SINV dissemination, compared to higher temperatures. In c ontrast, dissemination in C. torrentium was rapid and unaffected by temperature regimes. This result was unexpected and contrary to the established thought that extrin sic incubation period is invers ely related to temperature (e.g., Reisen et al. 2006). Thus, although both Culex spp. may serve as enzootic vectors in Sweden, C. pipiens transmission potential may be broken under cooler conditions, whereas C. torrentium is likely to persist as an efficien t SINV vector in co ol weather. An identical experiment using Aedes spp. showed similar SINV infection between temperature regimes, however, transmissi on was lower and occurred later at low temperature compared to high temperat ure (Turell and Lundstrm 1990). Low temperatures were associated with longer extrinsic incubation periods in A. taeniorhynchus and A. aegypti, which are not known as natural vectors of SINV (Lundstrm et al. 1990b). Adult A. communis A. cinereus and A. excrucians were collected from Sweden and allowed to blood feed on SINV infected chickens (104.2 PFU/ml) (Turell et al. 1990). All three Aedes spp. were highly susceptible to SINV infection (96-100%) and had

PAGE 32

20 dissemination rates rangi ng from 51-100%. Although A. communis failed to refeed, the other Aedes spp. had a 50% transmission rate. These Aedes spp. are competent SINV vectors and should be regarded as potential links between the enzootic SINV cycle and human infections in Scandinavia. SINV has been repeatedly isolated from both A. communis and A. cinereus during episodes of human infection. Further, these Aedes spp. are active day biters on mammals, including humans but will also bite birds (Turell et al. 1990). Laboratory experiments with SINV using easily colonized mosquito species (e.g., A. aegypti A. albopictus ) have proven useful to addre ss questions about experimental infection and transmission, genetically modi fied arboviruses (e.g., SINV gene expression vectors), and the dynamics of arboviral tissue tropism and pa thology in mosquito vectors (e.g., Bowers et al. 2003, Bowers et al. 1995, Jackson et al. 1993, Xiong et al. 1989). High (108.4 PFU/ml) SINV titers resulted in greater infection compared to moderate (105.3 PFU/ml) SINV titers (Percent infected for high-moderate titers; A. albopictus 90-49%; C. pipiens 48-0%, A. aegypti untested-18%). Aedes albopictus had greater dissemination (66%) and transmission rates (53%) compared to A. aegypti (9 and 7%) at the lower titer (Dohm et al. 1995). A study on SINV replication and tissue trop ism following intrathoracic inoculation in A. albopictus showed temporal and organ-specific distribution of the virus during the extrinsic incubation period (B owers et al. 1995). Many or gans had maximal infection within 3-4 days after infection because th e gut barriers were bypassed by intrathoracic inoculation. Some organs were refractory to infection (e.g., ovarioles, malpighian tubules), whereas others had tr ansient or persistent infect ions, perhaps indicating viral

PAGE 33

21 modulation by the mosquito vector or SI NV (e.g., Bowers et al. 1995, Luo and Brown 1993, Murphy et al. 1975). SINV-associated pa thology of the salivary glands and midgut muscle tissue of A. albopictus has been observed (Bower s et al. 2003). Typically, arboviruses have few cytopathic affects on mosquito cells ( in vitro and in vivo ) (Hardy et al. 1983). Similarly, a study on A. aegypti following oral infection showed rapid infection of many organs within several days after feeding with the salivary glands being infected by day 5 (Jackson et al. 1993). As was the case for A. albopictus some organs of A. aegypti were refractory to infection (e.g., ovar ioles, malpighian tubules). Unlike A. albopictus there was no indication that the distribution of SINV in organs changed from days 6-14 (Jackson et al. 1993). Competition and Vector Competence Classic laboratory and field research es tablished evidence for the importance of interspecific competition for a variety of systems (e.g., Connell 1961, Birch 1953, Hairston 1951, Crombie 1947, Park 1948). Desp ite numerous studies, establishing the existence and importance of competition in nature may be difficult and has been historically a topic of deba te (e.g., Hairston et al. 1960). Reviews on this topic have provided concise evidence that interspecific co mpetition is widespread in natural systems for a variety of organisms (e.g., Reit z and Trumble 2002, Connell 1983, Schoener 1983, Crombie 1947). More recently, interspecific competition has been invoked as a mechanism by which competitively superior inva sive plant and animal species alter the distribution and abundance of establishe d species (e.g., Juliano and Lounibos 2005, Levine et al. 2002, Reitz a nd Trumble 2002, Byers and Goldwasser 2001, Mack et al. 2000, Holaway 1999, Petren and Case 1996, D Antonio and Vitousek 1992).

PAGE 34

22 Intraand interspecitic competition between larval mosquitoes is common and plays an important role in dermining populat ion growth measurements. Competition has been demonstrated in laborat ory and field experiments fo r several mosquito vector species that occupy a variety of aquatic habitats (e.g., Costanzo et al. 2005ab, Peck and Walton 2005, Juliano and Lounibos 2005, Brak s et al. 2004, Juliano et al. 2004, YeEbiyo et al. 2003, Gimnig et al. 2002, Gleise r et al. 2000ab, Schneider et al. 2000, Juliano 1998, Barrera 1996b, Lonard and Juliano 1995, Broadie and Bradshaw 1991). Mechanisms involved in mosquito competition have largely been attributable to limiting resources (e.g., food) (e.g., Juliano 1 998, Barrera 1996b), although interference competition, mediated by direct physical cont act or chemical excretions, may also be important. However, studies with mosquitoes have yielded mixed results and additional studies are needed to evaluate the role of interference competition in natural systems (Broberg and Bradshaw 1995, Broadie and Bradshaw 1991, Dye 1984, 1982, Moore and Whitacre 1972, Moore and Fisher 1969). Res ource type (e.g., leaves) and abundance and larval density affect mosquito fitness such th at high intraand interspecific larval density and low resources result in increased larval development time and mortality and decreased adult size, fecundity, longevity, and per capita rate of growth (e.g., Alto et al. 2005, Costanzo et al. 2005ab, Peck and Walt on 2005, Juliano et al. 2004, Lounibos et al. 2003b, 1993, Gimnig et al. 2002, Daugherty et al 2000, Schneider et al. 2000, Teng and Apperson 2000, Yanoviak 1999, Juliano 1998, Lonard and Juliano 1995, Hawley 1985). Competition is well documented among container mosquitoes and may be important to some mosquitoes in other aquatic habitats. Arbovirus-mosquito research has mainly focused on intrinsic (e.g., genetic) a nd extrinsic (e.g., temperature, blood meal

PAGE 35

23 viral titer) factors of adult biology that determine vector competence (Tabachnick 1994). Few studies have attempted to address how biological conditions experienced by larvae may determine subsequent adult vector competence. It is likely that effects of larval compet ition have an impact on the adult stage and influence adult vector competen ce of arboviruses. The most extensive research has been conducted on the effect of nutrient depriva tion on mosquito vector competence. Low food availability among Ochlerotatus triseriatus (Say) produced smaller adults. These small adults transmitted La Crosse virus (LAC V) at higher rates than did larger adults that resulted from well-fed larvae. However, the infection rates in these mosquitoes were independent of adult body size (Grimsta d and Haramis 1984, Grimstad and Walker 1991). Enhanced transmission efficiency of small O. triseriatus adults was associated with higher virus titers and dissemination rate s compared to larger adults. Additional support for size-dependent transmission come s from field-collected pupae that were orally infected as adults w ith LACV and had disseminati on and transmission rates that were inversely correlated with a dult size (Paulson and Hawley 1991). The effect of larval nutrition and adu lt size on infection parameters has been investigated in other mosquito species. Large A. aegypti adults produced under varying conditions of larval crowding and food availa bility had a greater proportion of DENV-2 disseminated infection (New Guinea C strain) than did smaller females (Sumanochitrapon et al. 1998). Similar results were found for A. aegypti susceptibility to infection with Ross River virus (RRV) over a range of blood meal titers. Differences between infection of small and large adults became less distinct at greater blood meal titers (Nasci and Mitchell 1994) perhaps suggesting that high titers simply overwhelm

PAGE 36

24 the differences seen at lower titers. C onversely, titer (midgut and head) as well as DENV-2 (Puerto Rico and Ibo strains) dissemina tion were independent of A. aegypti body size (Bosio et al. 1998). Lo w food availability among larvae of C. tritaeniorhynchus (Giles) produced smaller adults that had shorter periods between initial infection of Japanese encephal itis virus (JEV) and subsequent virus secretion in the saliva than did larger adults from we ll-fed larvae. Additionally, small C. tritaeniorhynchus adults had greater JEV transmission than did la rge adults (Takahashi 1976). Baqar et al. (1980) showed a trend, although not significant, that increased larval densities and decreased larval nutrition resulted in small adults with increased infection susceptibility of C. tritaeniorhynchus to West Nile virus (WNV). In fection and transm ission rates of Murray Valley encephalitis virus (MVEV) were unaltered between two larval nutritional regimes producing different sized adult C. annulirostris Additionally, neither body nor salivary gland viral titers were altered by larval nutrition (Kay et al. 1989). Similar nonsignificant effects of the larval en vironment and adult size were found for C. tarsalis (Coquillett) infection and transmission of St. Louis encephalitis virus (SLEV) and Western Equine encephalomyelitis viru s (WEEV) (Reisen et al. 1997). Size-dependent differences in mesenteronal tissues may, in part, explain differences in dissemination and transmissi on of arboviruses by adults of different sizes (Grimstad and Walker 1991). Fewer basal lamina layers were present in the mesenteron of small adult O. triseriatus (4-6 layers) as compared to large adults (1016 layers) which weakened the midgut escape barrier (MEB) and thus enhanced dissemination and transmission rates (Grimstad and Walker 1991, Paulson and Hawley 1991). An alternative hypothesis explaining observed negative rela tionships between size (wing

PAGE 37

25 length) and vector competence may be relate d to the number of vi ral particles imbibed relative to mosquito body size. Large mosquitoes imbibe greater volume of blood, and thus virus, than smaller mosquitoes. Add itional support comes from greater viral titers found in freshly bloodfed large adults than in small adults (Nasci and Mitchell 1994). However, when the amount of virus imbibe d was corrected for mosquito body weight, small adults imbibe proportionally more viru s than large adults in proportion to their body weight (Nasci and Mitchell 1994, Grimst ad and Haramis 1984). This explanation may hold true for a number of mosquito sp ecies since blood meal titer is positively related to infection, dissemination, and transm ission (e.g., Turell et al. 2001, Dohm et al. 1995, Grimstad and Haramis 1984, Kramer et al. 1981). The previous examples illustrate that larval nutrition affects adult mosquito infection and transmission of mosquito arboviruses. However, the mechanism and details are dependent on the particular mosquito-virus system. Some of the studies support the hypothesis that larval resource competition enhances vector competence. Resource competition alters numerous mosquito life history traits, however, these studies have limited the focus to a single life history trait, adult size. Further, they did not address whether the effect of resource competition on vector competence was causally related to adult size, or alternatively (additionally) related to other physiological conditions correlated with adult size (Grimstad a nd Walker 1991, Paulson and Hawley 1991). Further, drawing conclusions about common themes from a limited number of studies would be premature and perhaps misleading. Thus, controlled experi ments are required to determine quantitatively the effects of larval competition on vector competence for multiple mosquito-virus systems, as well as to disentangle which mosquito life history

PAGE 38

26 traits (e.g., size, development time) are most important in determining vector competence parameters (infection, body viral titer, disse minated infection). Results from such experiments may support with greater quant itative detail th e hypothesis that resource competition affects mosquito vectoring ability, or may offer alternative explanations for the larval competition-adult vector competen ce relationship. The chapters that follow describe the development of an artifici al bloodfeeding system used in delivering arbovirus infectious bl ood meals as well as experiments that evaluate the effects of competition on A. aegypti and A. albopictus population growth measurements and SINV and DENV infection parameters The use of these Aedes species to investigate the relationship between competition and arbovi ral infection is important because; competition is well-documented between these Aedes species, competition has important ecological effects on their distri bution and abundance, and these Aedes are the most important vectors of human arboviruses.

PAGE 39

27 27 CHAPTER 2 AGE-DEPENDENT BLOODFEEDING OF Aedes aegypti AND Aedes albopictus ON ARTIFICIAL AND LIVING HOSTS Introduction Since its introduction to the Americas in the mid 1980s (Hawley et al. 1987, Sprenger and Wuithiranyagool 1986), Aedes albopictus has spread rapidly and colonized much of the southeastern U.S. and Brazil. In parts of the eastern U.S., the invasion of A. albopictus is associated with declines in th e abundance, and in some instances displacement, of Aedes aegypti in rural and suburban area s (Mekuria and Hyatt 1995, O'Meara et al. 1995, Hornby et al. 1994, H obbs et al. 1991). However, these Aedes coexist in urban areas of south Florida. R ecent comparative studies attempting to explain the observed distributions of these Aedes have investigated egg de siccation (Juliano et al. 2002, Sota and Mogi 1992), larval compe tition (Lounibos et al. 2002, Daugherty et al. 2000, Juliano 1998, Barrera 1996a), adult de siccation (Mogi et al. 1996), and reproductive and metabolic differences (Klowden and Chambers 1992). One major concern about the A. albopictus invasion in the Americas has been its potential as an arboviral disease vector (e .g., DENV). In recent decades, the range of A. aegypti the primary vector of DENV in the Amer icas, has increased, and dengue activity has surged (Gubler 1997). The range of A. albopictus in the U.S. is more extensive than that of A. aegypti and its range in the U.S. is likely to continue to expand (e.g., Madon et al. 2002). Aedes albopictus is a competent laboratory ve ctor of numerous arboviruses

PAGE 40

28 (Mitchell 1991, Shroyer 1986) including DENV in Asia and Hawaii; however, the degree to which A. albopictus is involved in arbovirus transmissi on in the Americas is unclear. With exceptions of transovarial and vene real transmission, successful biological transmission of arboviruses requires acquisition of an infectious bloodmeal, or at least probing behavior. For A. aegypti research investigating factor s that influence the normal sequence of events in succe ssful acquisition of a bloodmeal (e.g., host-seeking, probing, bloodfeeding) have mainly focused on m easurements of host-seeking behavior (Klowden and Fernandez 1996, Klowden and Briegel 1994, Bowen 1991, Klowden et al. 1988, Klowden and Lea 1984, 1979ab, 1978). Davis (1984) showed a linear increase in hostseeking behavior of A. aegypti from 1 to 5 days post-emergence followed by a constant high response until the end of observations at 15 days. A st udy measuring probing behavior in A. aegypti over a 21-day period showed a rhyt hmic pattern in probing activity in response to a convection current of consta nt heat and moisture, but no probing pattern was observed in response to a human host (Burgess 1959). However, the design of this latter experiment was weak (e.g., experimental units were not replicated), and there was little statistical suppor t for the conclusion of rhythmic behavior. Few studies have measured age-related acquisition of the in itial bloodmeal, an important factor in determining vector potential. Those that have done so have focused on bloodfeeding over a short interval. Seat on and Lumsden (1941) showed a general increase in bloodfeeding associated with age for 1-5-day-old starved virgin A. aegypti followed by decreased bloodfeeding on day 6. They suggest ed that the decrea sed response on day 6 was attributable to female exhaustion. A si milar increase in bloodfeeding with increasing age was found for 3 strains of 1-4-day-old starved A. aegypti fed on chickens and

PAGE 41

29 membrane systems (Bishop and Gilchrist 1946). In order to quantify age-dependent acquisition of a bloodmeal, th e present study compares bl oodfeeding patterns of A. albopictus and A. aegypti starting from the time of firs t responsiveness to a bloodmeal (Hawley 1988, Christophers 1960) up to 15 days post-emergence. Materials and Methods Experimental Protocol Aedes eggs used to initiate the experiment s were derived from laboratory colonies at the Florida Medical Entomology La boratory in Vero Beach, FL. Both Aedes spp. originated from Fall, 2000, field collections of larvae from water-fille d cemetery vases in Hillsborough County, FL, near Tampa. Colonies were housed in 0.03 m3 cages at (mean SD) 24.6 0.4 C, 76.6 6.7% RH, and a 14:10 (L:D) h photoperiod regime including a 1 h dawn and dusk. Colonies had access to 20% sucrose solution ad libitum and weekly bloodmeals from domestic chickens (handled in accordance with the National Institutes of Health guidelines for the use of laboratory animals). Females were provided with water-containing cups lined with paper to wel as oviposition substrates. Eggs were hatched, by species, in metal pans with 1.0 li ter tap water and 0.30 g of a 1:1 lactalbumin and brewers yeast mixture. Following hatching, approximately 300-500 larvae were reared in pans, with water and f ood substrate changed every 2 days. As soon as pupation occurred, inspections of the rearing pans were made daily, and pupae were transferred into 40-ml vials with water until emergence. Vials were checked daily between 1600 and 1800 h for newly emerged adults. These adults were transferred, by species, to cylindrical cages ( 11 x 9.5 cm, ht x diam) with nylon mesh tops and maintained under similar conditions as the parental generation except for

PAGE 42

30 bloodfeeding. Female density per cage ranged from 5 to 47 with means SE of 14.8 9.9 and 14.2 8.2 for A. aegypti and A. albopictus respectively. At least one male was present in each cage for every 3-4 females, although many cages had equal numbers of males and females. Examination of scatter plots of residuals versus predicted values (Draper and Smith 1966) showed no evidence that the number of males per cage was in any way related to proportions of females that bloodfed. In Experiment 1, cages with Aedes females were haphazardly assigned to an age treatment (e.g., 3, 4, 15 days old). Each cage containing same-age adults ranging from 3 through 15 days old was offered a bloodmeal from a silicon membrane feeding system (Butler et al. 1984). Thus, same-age females were tested on many different days. Females were deprived of sucrose, but not water, 24 h prior to bloodfeeding trials. Before the start of a feeding trial, c itrated bovine blood was heated to (mean SD) 37.8 1.1 C in 1.5 ml circular wells and covered with a silicon membrane. Next, the membrane feeding system was positioned over the mesh top of the cage for two 15-min periods separated by a 15-min interval. Feed ing trials were performed at (mean SD) 23.2 0.5 C and 48.1 4.2% RH. After a feeding trial, the number of females that successfully acquired a bloodmeal was recorded. If blood wa s visually detected in the female gut, it was scored as having a bloodmeal. Thus no attempt was made to distinguish between meals of different volumes. All feeding tr ials were performed in the late afternoon, within 2-3 h of each other. For Experiment 2, 3-15 day old Aedes were allowed to bloodfeed from a restrained domestic chicken. The methods for mosqu ito husbandry and a dult exposure during feeding trials were the same as those used in Experiment 1. For all feeding trials,

PAGE 43

31 uniformly sized and aged (6-8 wks old) chickens were restrained inside 0.03 m3 cages into which adult Aedes were released and allowed to f eed for 30 min. Female density per cage ranged from 5 51 with means SE of 28.1 9.9 and 24.1 9.7, for A. aegypti and A. albopictus respectively. Larger cages were used for bloodfeeding in Experiment 2 to provide greater space for the normal seque nce of events involved in bloodmeal acquisition (Clements 1999). Immediately fo llowing feeding trials, chickens were removed from the cages, and then Aedes were removed from the cage using an electric aspirator and killed by placing them at -20 C for < 1h. The number of female Aedes that had successfully bloodfed was recorded as in Experiment 1. Data Analyses For Experiments 1 and 2, proportions bloodfed for each species were calculated as the numbers of females that acquired a bl oodmeal during a trial divided by the total numbers of females offered the bloodmeal. E xperimental units were defined as the cage of adult Aedes offered blood. Difficulties in predicti ng the number of females that would emerge and survive to the day of feeding pr ecluded equal sample sizes for each unique species-by-age treatment. For Experiment 1, the numbers of replicates for each A. aegypti and A. albopictus by age treatment were (mean SD) 5 1 and 5 2, respectively (127 total cages). In Experi ment 2, each unique species-by-age treatment was restricted to 3 replic ates, except for 15-day old A. aegypti, which had 4 replicates (79 total cages). For both experiments, effects of female de nsity per cage was test ed as a continuous variable (PROC GLM, SAS Institute 1989, Sokal and Rohlf 1995). Raw data adequately met assumptions of normality and homoge neous variance except for membrane-fed A.

PAGE 44

32 albopictus where the proportion bloodfed was transformed by log10(x + 1) to meet the assumption of normality. Because effects of female density on proportion bloodfed were all non-significant ( P >> 0.10 in all cases), analysiss of effects of female age were performed on proportion bloodfed, which wa s assessed by treating age as a continuous independent variable and comparing regression lines for each species by feeding protocol treatment. This tests for equal slopes among species-feeding protocol groups to determine whether the regression relationshi ps were similar (SAS Institute 1989, Sokal and Rohlf 1995). Graphical presentation of the data appeared to show age-dependent periodicity in feeding incidence. Therefore, separate Runs Up and Down Tests were performed for the proportion bloodfed for each species-feeding protocol combination (Sokal and Rohlf 1995, Zar 1996). A run was defined as a temporal sequence of increases or decreases in the proportion bloodfed. Difference between mean proportion bloodfed for consecutive age groups was determined and resulted in a sequence of positive and negative changes in proportion bloodfed across female ages (e.g., + + + = 2 runs). These tests determined whether the number of runs for proportion blood-fed among females of different ages was significantly different from random expectation. As an additional test to a ddress the apparent age-depe ndent periodic pattern of feeding, four regressions were run (one for each species-feeding protocol combination) of proportion blood-fed versus age, each with a sine function of age according to the model: y = a + bA + c sin (dA),

PAGE 45

33 where y is the proportion bloodfed, a is the intercept, b is the slope, A is female age, c is a parameter affecting the am plitude of the sine function, and d is a parameter affecting the frequency of the sine function. Several different initial parameter estimates were used to determine whether the addition of a sine wave function improved the fit of the regression (SAS Institute 1989, PROC NLIN). If either c or d parameters were not significant, the slope ( b ) was removed and the reduced model tested. Subsequently, if either c or d were not significant, both c and d were removed from the model and a linear regression was performed, includ ing the slope, to determine whether or not there was a trend in age-dependent blood-feeding. Results Regardless of age, a higher proportion of both A. albopictus and A. aegypti bloodfed on the restrained chicken (mean SE; 59.8 2.4 and 81.3 2.3%, respectively) compared to the membrane system (mean SE; 30.8 2.7 and 55.6 2.6%, respectively). Treating age as a continuous independent variable, there were significant age, species, feeding protocol, and age x feeding protocol effects (Table 2-1). All other effects were not significant Slopes of proportion bloodfed vs. age were significantly positive for both Aedes species feeding on the membrane system and were not significantly different from zero for both Aedes species feeding on the restrained chicken (Table 2-2). Although these slope s were significant, the low r2 values suggest that the linear relationships were weak (Fig. 2-1, Ta ble 2-2). In addition, the sine function contributed significantly to the regression for A. aegypti (P < 0.0001, r2 = 0.391, proportion fed = 0.188 + 0.042*(age) + 0.11*sine (9.91*age)). For both Aedes species

PAGE 46

34 fed on the restrained chicken, the sine function did not cont ribute significantly to the regression (Table 2-2). Averaged over both species, slopes for proportion bloodfed on the membrane system were significantly great er, as shown by the age x feeding protocol interaction, than those for Aedes fed on the restrained chicke n (Tables 2-1, 2-2). Runs Up and Down Tests for A. albopictus and A. aegypti fed on restrained chickens showed that the numbe r of runs was significantly different from random (both P < 0.0275) with the number of runs being greater than expected compared to random (Fig. 2-2). Thus, proportion bloodfed on chickens showed a significant pattern of alternate increases and decreases on altern ate days of female mosquito age. Runs Up and Down Tests were not significant (both P > 0.05) for either Aedes species fed on the membrane system (Fig. 2-1). Discussion In Experiment 1, using the membrane feed ers, there was a significant increase in proportion bloodfed as age increased (Table 2-2, Fig. 2-1). In Experiment 2, with restrained chickens, there was no significan t increase in bloodfeeding associated with increased age (Table 2-2, Fig. 2-2). Fu rther, the membrane-fed and chicken-fed mosquitoes showed significantl y different trends (Table 2-2) Thus, the temporal pattern of bloodfeeding is strongly affected by th e blood source used in experiments. Hostrelated cues (e.g., CO2, surface area) may be partially responsible for the observed differences in pattern of bloodfeeding and should be taken into consideration in bloodfeeding research using Aedes mosquitoes of different ag es, especially for silicon membrane systems The lack of significantly positive slopes for chicken-fed mosquitoes is likely due to a higher proportion of bloodfed younger Aedes as compared to the membrane-fed mosquitoes.

PAGE 47

35 Results showed significant age effects on bloodfeeding for both A. aegypti and A. albopictus. Davis (1984), in a study with nave A. aegypti females of uniform ages ranging from 1 through 15 day old, showed a linear increase in hos t-seeking behavior for 1-5-day-old females, whereas females > 5 days old showed a consistently high (e.g., 94%) response to a human hand. Results from the current study suggest that bloodfeeding for these Aedes over a similar period of time, shows some similarities to host-seeking response observed by Davis. However, proportions bloodfeeding appear additionally to exhibit distinct periodic patterns on alternate days of female mosquito age. Significant but weak positive re lationships were found for A. albopictus and A. aegypti feeding versus age on the membrane syst em (Fig. 2-1, Table 2-2), and no positive relationships for feeding versus age on the restrained chicken (Fig. 2-2, Table 2-2). Additionally, slopes for the two Aedes species, as a single group, fed on the membrane system were significantly different from those fed on the restrained chicken (Table 2-2). Also, there appears to be an age-dependent periodic patter n in bloodfeeding incidences. The periodic pattern is most obvious among 3-13 -day-old adults of each species fed on the restrained chicken, where the number of runs was significantly greater than that expected for random daily variation. Li kewise, for the proportion of bloodfed A. aegypti on the membrane system versus age, a sine function made a significant contribution to the fit, providing further eviden ce for periodicity This resu lt is surprising, because this was a short time series and typically, time seri es analyses have the potential to provide good fits when there are > 50 observations (Chatfield 1989). Unlike some previous research, Aedes in the current study were experime ntally nave (i.e., never given a previous bloodmeal), thus any periodicity in the time series is likely attributable to

PAGE 48

36 endogenous factors. Periodicity could be an artifact of unknown exogenous factors, although most obvious factors were controle d (e.g., temperature, humidity, feeding times). These results lend support to previous reports of a possible periodic pattern in probing behavior of non-bloodfed A. aegypti (Burgess 1959) and hos t-seeking behavior in non-bloodfed Anopheles gambiae sensu stricto (Takken et al. 1998). Hormone levels (e.g., juvenile hormone, ecdysteroids) vary at different times throughout the duration of adult female life. Juvenile hormone has been shown to be involved in initiating bloodfeeding for Culex pipiens (L.) and C. quinquefasciatus (Say) (Meola and Petralia 1980), and C. nigripalpus (Theobald) (Hancock and Fost er 2000). The processes by which synergistic and antagonistic effects of juvenile hormone and ecdysteriods, from day to day, influence consumption of th e initial bloodmeal, es pecially long after emergence (e.g., 15 days), is unknown. Given the lack of data on endogenous hormone fluctuation during the life span of unfed fema les, it is speculative to suggest that these hormones may contribute to the apparent age-dependent differences observed in proportion bloodfed of A. aegyp ti and A. albopictus

PAGE 49

37 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 102468101214Age (days)Proportion bloodfed3456789101112131415 a Aedes aegypti (runs = 9) Aedes albopictus (runs = 7) Figure 2-1. Least squares means ( SE) fo r proportion bloodfed females on the siliconmembrane system for 3-15 day old Aedes albopictus and A. aegypti Line drawn through means shows the best-fit linear regression for A. aegypti (solid) and A. albopictus (broken). 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 102468101214Age (days)Proportion bloodfed3456789101112131415 A edes aegypti (runs = 12) A edes albopictus (runs = 11) Figure 2-2. Least squares means ( SE) fo r proportion bloodfed females on restrained chickens for 3-15 day old Aedes albopictus and A. aegypti.

PAGE 50

38Table 2-1. Test for equal slopes am ong regressions of proportion bloodfed of Aedes aegypti and A. albopictus versus age Source df Type III SS MS F P Age 1 1.3739 1.3739 30.69 < 0.0001 Feeding Protocol 1 1.4164 1.4164 31.64 < 0.0001 Species 1 0.1824 0.1824 4.07 0.0449 Feeding Protocol X Species 1 0.0697 0.0697 1.56 0.2136 Age X Feeding Protocol 1 0.2671 0.2671 5.97 0.0155 Age X Species 1 0.0412 0.0412 0.92 0.3384 Age X Feeding Protocol X Species 1 0.1094 0.1094 2.44 0.1197 Error df 198 Table 2-2. Intercept and slope estimates for si mple linear regression s of proportion bloodfed of Aedes aegypti and A. albopictus versus age. Slopes for groups followed by di fferent letters are significantly different. Source Intercept SE Slope SE r2 df F P Membrane System A. aegypti 0.1703 0.0779 0.0434 0.0082 0.3171 1, 61 28.33 < 0.0001 A. albopictus 0.1084 0.0778 0.0226 0.0081 0.1110 1, 62 7.74 0.0071 Chicken Host A. aegypti 0.7225 0.0593 0.0103 0.0059 0.0723 1, 38 2.96 0.0934 A. albopictus 0.4600 0.0879 0.0153 0.0090 0.0720 1, 37 2.87 0.0987 a b

PAGE 51

39 CHAPTER 3 LARVAL COMPETITION DIFFERENTIALLY AFFECTS ARBOVIRUS INFECTION IN Aedes MOSQUITOES Introduction Biotic interactions among organisms pl ay an important role in regulating population growth and in shaping communities. Among biotic interactions, competition has received a great deal of attention especially in the field of invasion biology where competitively superior invasive species disp lace or otherwise alte r the distribution of established species (e.g., Julia no et al. 2004, Holway 1999, Petren et al. 1993). Although the most obvious effects of competition are re duced growth and survivorship, there are less obvious indirect effects mediated by comp etitively induced differences in life history traits (e.g., morphological or beha vioral trait-mediated indire ct effects; Abrams 1995). Indirect effects describe interactions between two species mediated by a third species (e.g., exploitative competition, appare nt competition, trophic cascades, indirect mutualism, interaction modifications) (Woo tton 1994, Osenberg et al. 1992). Although different authors have applied multiple terms to similar types of indirect interactions (e.g., Morrison 1999), there is a consensus on cl assifying indirect effects as densitymediated or trait-mediated (Altwegg 2002). Density-mediated indirect effects occur when abundance of one species indirectly al ter the abundance of another species through effects produced by altering th e abundance of an intermediate species. Trait-mediated indirect effects occur when one species alters traits (e.g., be havioral, morphological) in a second species in ways that change the intera ction between the second and third species.

PAGE 52

40 The most frequently studied trait-mediated i ndirect effects involve predatory species that induce prey behavioral modifi cation (e.g., reduced activity, incr ease use of refuges) that indirectly alter competitive interactions among those prey (Relyea 2000, Werner and Anholt 1996, Werner 1992, 1991). Less attention has been given to indir ect effects of competition among organisms with complex life cycles, where the impact of competition in one life stage has consequences for species interactions in subsequent stages (Altwegg 2002). Adult lifehistory traits of organisms w ith complex life cycles are, to a large extent, products of their larval environment. For example, e ffects of competition include reduced growth, development, and survivorship. Competitive-induced differences in adult life-history traits such as size may alter species intera ctions with enemies, including predators, pathogens, and parasites. Although nutrient limited conditions and physiological stress indirectly result in greater su sceptibility to infection with pathogens or parasites in a single life stage (Kiesecker and Skelly 2001, Murray et al. 1998, Oppliger et al. 1998, Matson and Waring 1984), little is known about effects of competition in juvenile life history stages on susceptibility to inf ection in subsequent adult stages. Water-filled containers are well suited to investigations of competitively induced indirect effects because they harbor simple communities subject to variable resource availability. Among the organisms o ccupying aquatic container communities, mosquitoes are the best studied because of the role of adu lts as vectors of pathogens. Resource availability and larval density in containers both influence mosquito survivorship, growth, and a dult size (e.g., Juliano et al. 2004, Lounibos et al. 2002). Effects of competitive interactions among larval stages may carry over to the adult stage

PAGE 53

41 and affect vector competence, which descri bes the ability to become infected and subsequently to transmit a pathogen afte r imbibing an infectious bloodmeal (Hardy 1988). Biological transmission of arboviruses in cludes acquisition of the virus by the vector from an infectious bloodmeal, repli cation, dissemination of virus to the salivary glands, and transmission to a host by b ite (Higgs 2004, Hardy 1988). Successful completion of this process requires that inf ection and dissemination barriers within the mosquito be overcome (Hardy 1988, Hardy et al. 1983). For example, if arboviruses fail to pass through the midgut, then infection is limited to th e midgut cells and, although the mosquito is infected, it cannot transmit vi rus (Hardy et al. 1983). Larval competition may have important consequences for adult arbovirus infection parameters. Typically, pupal and adult sizes of container breeding mosquitoes are positively related to the feeding rate experienced by larvae (e.g., Ch ristophers 1960). Resource competition, and associated low food availability, am ong larvae of the treehole mosquito Ochlerotatus triseriatus produced smaller adults that transmitted La Crosse encephalitis virus (LACV) at higher rates than did larg er adults from well-fed la rvae. Infection rates were independent of adult body size (Grimstad and Walker 1991, Grimstad and Haramis 1984), although when O. triseriatus reared from field-colle cted pupae were orally infected with LACV, disseminated infecti on and transmission rates were negatively correlated with adult size (Paulson an d Hawley 1991). In contrast, large Aedes aegypti adults produced under varying conditions of larval crowding and food availability disseminated dengue virus serotype 2 (DENV2) more efficiently than did smaller females (Sumanochitrapon et al. 1998). T hus, it appears that ecological conditions

PAGE 54

42 encountered by larvae can have variable eff ects on the interaction of mosquitoes with arboviruses. Investigations of competitive effects on pat hogen transmission, other than size-related effects, remain rare. The goal of our study was to determine the effects of larval competition on growth and survivorship of two well known container mosquito species, A. albopictus and A. aegypti as well as their subsequent competen ce for arboviral infection and dissemination using Sindbis virus (SINV). SINV is a model Alphavirus that cycles between reservoir bird hosts and Aedes and Culex vector species (Seabaugh et al. 1998 and therein), and is widely used in experimental vector biology research (Olson et al. 1996, Dohm et al. 1995). Aedes albopictus is an invasive container breedi ng mosquito native to Asia which became established in large areas of the U. S., Europe, Africa, and South America during the last two decades (Lounibos 2002). In the southern U.S., the spread of A. albopictus coincided with reductions in range an d abundance of the resident exotic A. aegypti in artificial containers (reviewed by Juliano et al. 2004). Aedes albopictus is an important vector of several arboviruses aff ecting humans and second only to A. aegypti in global importance as a vector of DENV (Louni bos 2002, Gubler and Kuno 1997). These species frequently encounter each other in ar tificial containers, in which interspecific competition has been well documented (Braks et al. 2004, Juliano et al. 2004, Barrera 1996b, Black et al. 1989, Ho et al. 1989), wh ich probably explains displacements of A. aegypti by A. albopictus (Juliano 1998). This study test s whether variation in population growth parameters known to arise from intraand interspecific competition (Juliano et al. 2004, Lounibos et al. 2002) have carryover effect s in the adult stage, and are associated with variation in su sceptibility to SINV infection dynamics.

PAGE 55

43 Materials and Methods Competition Study Aedes albopictus Lake Charles strain (Nasci et al. 1989) and A. aegypti Rockefeller strain were used in the experiments. Thes e mosquitoes were the progeny of genetically well-characterized strains. Aedes albopictus was obtained from a collection made at Lake Charles, Louisiana in 1987 and has been propagated under laboratory conditions since 1987. The A. aegypti Rockefeller strain was obtai ned from a long-standing colony at the University of Notre Dame. The competition experiment between A. albopictus and A. aegypti used 5-liter plastic containers filled with 4000 ml of tap water, 500 ml oak leaf infusion water (OMeara et al. 1989), and 0.2 g of larval food (1:1, by weight, albumin: yeast). Three days after addi ng the initial contents to cont ainers, a supplemental 500 ml oak infusion and 0.2 g larval food was added. Initial food resources were incubated for 5 d before the addition to each container of first instar (< 24 h old) mosquitoes. Ten days later, I removed 50% of the liquid contents except larvae, and added 0.1 g larval food, 250 ml oak infusion water, and 2,250 ml tap wate r. Previous studies showed that this protocol provided sufficient resources for mo squitoes to complete development without negating the effects of larv al competition (B.W. Alto, unpublished data ). Competition treatments consisted of species/densities of A. albopictus : A. aegypti -160:0, 320:0, 160:160, 0:320, and 0:160. Ten replicates were used per treatment, for a total of 50 containers kept at 28 1C and 14:10 L:D regime. Containers were checked daily, and pupae transferred to sealed 20 ml vials with tap water until adult emergence. Emerged adults were kept, by species, in cylindri cal cages (11 x 9.5 cm, ht. x diameter) and provided with 10% sucrose and an oviposition cup. The experiment was maintained until the last adult had emerged.

PAGE 56

44 Measurements of population growth correlate s were used to estimate the effect of competition on female A. albopictus and A. aegypti population growth. Mean female size (wing length) and mean time to emergence we re calculated for each replicate. Female survivorship per replicate was calculated as ( number of adult females) / (total number of original larvae) of a given species. An estimated finite rate of increase ( ) was also calculated for each replicate container. ln [(1/ No) x Ax f(wx) ] = exp(r ) = exp D + [ x x Ax f(wx) / x Ax f(wx) ] is a transformation of r a composite index of population performance (Juliano 1998). r is an estimate of r = dN / Ndt, wh ich describes the per capita growth rate. No is the initial number of females in a cohort (assumed to be 50 %), Ax is the number of females emerging on day x wx is mean female size on day x f(wx) is a function relating the number of eggs produced by a female to her size, and D is the time (in days) from emergence to oviposition. For A. albopictus and A. aegypti, D is assumed to be 14 and 12 d, respectively (Juliano 1998, Livdahl a nd Willey 1991). We used the following fecundity-size relationships ( f(wx) ) to calculate : A. aegypti (Briegel 1990) : f(wx) = 2.50( wx 3) 8.616 r2 = 0.875, N = 206, and P <0.001

PAGE 57

45 A. albopictus (Lounibos et al. 2002) : f(wx) = 78.02 ( wx) 121.24 r2 = 0.713, N = 91, and P <0.001 In both cases wx = wing length in mm. Effects of A. albopictus and A. aegypti competition were analyzed by individual Mu ltivariate Analyses of Variance (MANOVA) to determine competitive treatment effects on the population growth correlates time to emergence, survivorship to emergence, and adult size. Raw data adequately met assumptions of univariate normality and homogeneous variances for all correlates used in the MANOVAs. For all analyses, significant e ffects were further analyzed by contrasts of pairs of main effect multivariate means with a sequential Bonferroni adjustment for experimentwise =0.05. Standardized canonical coefficients (SCC) were used to determine the relative contribution of each of the response variab les to significant multivariate effects as well as their relations hip to each other (e.g., positive or negative) (Scheiner 2001). Competitive effects on A. albopictus and A. aegypti were analyzed using one-way ANOVAs with treatment as a cat egorical variable (SAS Institute 1989). Significant effects were further analyzed by pa irwise comparisons of main effect means (Ryan-Einot-Gabriel-Welsch te st, SAS Institute 1989). Infection Study For each replicate from the competition study, newly emerged females and males were housed, by species, in cages (11 x 9.5 cm ht. x diameter) and provided 10% sucrose and an oviposition cup. This arrangement facilitated mating and oviposition and enabled

PAGE 58

46 the delivery of infectious bloodmeals to multiple females of approximately the same age. Because larval competition increases developmental time, adults from the competition containers emerged over several weeks. Th erefore, multiple cages were used to house adults for each replicate to ensure that the fe males given an infectious blood meal were of similar ages (4-10 d old). SINV infection rates do not diffe r over the age range of 4-10 d for these Aedes species (Dohm et al. 1995). Thus, tests for the effects of larval competition on subsequent adult infection were performed on the same individual mosquitoes. Adults were housed in cages within an incubator at 26 1C and 14:10 L:D photoperiod. Adult females of each species were deprived of sucrose but not water for 24 h, then allowed to bloodfeed for 30 min. on a citrated bovine blood-SINV mixture maintained at 37 1C in a silicon membrane system (Butler et al. 1984). SINV (MRE16 strain) titers used in bloodfeeding trials were 105.3 tissue culture dose re quired to infect 50% of wells (TCID50) (Reed and Muench 1938). TCID50 is the quantity of virus that is required to infect 50% of the tissue cultures, so that viral titers (= number of virus particles / ml) can be determined. Viral titer refers to the amount of virus in solution. Virus titers were similar to those produced in wild bird reservoirs in nature (Ockelbo virus, a closely related strain of Sindbis vi rus (Lundstrom et al. 1993)). Titers were determined by 10-fold serial dilutions in 96-well plates seeded with 6.0 x 105 Vero cells / ml (10 wells per dilution). TCID50 was determined by cytopathic effects after a 7 d incubation (Reed and Muench 1938). Vero cells infected with SINV virus exhibit stereotypical cytopathic effects, so infection was unambiguous. To avoid the possibility of reductions in titer with repeated th awing and freezing, all blood meals had virus derived from single stock placed in 1.5ml ali quots that were frozen (-80C) and thawed

PAGE 59

47 only once. The infection study was conducted in a biosafety level-2 facility appropriate for SINV at the Florida Medical Entomol ogy Laboratory in Vero Beach, Florida. Females that failed to take a blood meal during the first trial were given a second trial 18 h later. After the second feeding attempt, unfed females were removed from the cages, and bloodfed females were held for a 16 d extrinsic incubation period (EIP). The time from initial ingestion of the infectious blood meal until the time the mosquito can transmit the arbovirus is the EIP. Females surviving the EIP we re killed and individually stored at -80 C and, subsequently, their wings were removed (to be measured as an indicator of female size). B odies and legs were ground into a powder separately in 1 ml diluent (Leibovitz L-15 media, 5% fetal bovine serum, and gentamicin), centrifuged at 21000 m/s2 for 12 min. at 4 C, and filtered (0.22m). Proportion females infected, body titer (log10 TCID50), and proportion of infected female s with disseminated infection (i.e., with positive infected legs) were determined using 10-fold serial dilutions in triplicate wells of 96-well plates seeded with Vero cells by TCID50. Infection was determined using a 1/10 dilution of the body stock solution, and body titer was determined using a full range of dilutions. When describing infection of mosquitoes, negative describes the abse nce of a viral infection, and positive describes a mosquito with a viral infection in the midgut, and perhaps other organs. An infection limited to the midgut is called an isolated infection, whereas an infection spread beyond the midgut, infecting secondary target organs (e.g., salivary glands, head, legs), is called a dissemi nated infection. Disseminate d infection is a recognized indicator of a mosquitos ab ility to transmit virus via bi ting (Gubler and Kuno 1997). So, dissemination of infection in positive female s was determined by assaying undiluted leg

PAGE 60

48 stock solution (Turell et al. 1984). In this st udy, isolated infections refer to mosquitoes with positively infected bodies but absence of infection in legs, whereas disseminated infections refer to positively infected bodies and legs. Assa ying salivary glands may be a more direct indicator of a mosquitos ability to transmit virus. However, extraction of the salivary glands may result in contamination with surrounding tissue. Thus, assays of mosquito legs were used in order to avoid this contamination probl em and still obtain a good indication of ability to tr ansmit (Turell et al. 1984). Prior to analyzing effects of competitiv e treatment on arboviral infection, interspecific differences in susceptibility were analyzed using MANOVA and SCC on the response variables proporti on infected, body titer, and pr oportion with disseminated infection. Next, individual MANOVAs for A. albopictus and A. aegypti were used to determine the effect of larval competiti on on response variables: proportion infected, body titer, and proportion with disseminated inf ection as described above. Multivariate contrasts with sequential Bonferroni adjustment for experimentwise =0.05 (Rice 1989, Scheiner 2001) were used to compare high density treatments ( 320:0, 160:160) vs. the low density treatment (160:0), and then to compare the two high density treatments. For A. albopictus and A. aegypti effects of mean fema le size on body titer were tested by treating size as a covariate in an analysis of covariance (ANCOVA) with competitive treatment and competition x size interactions. Significant effects were further analyzed by all possible pairwise co mparisons of treatmen t means (sequential Bonferroni adjustment; Rice 1989). Effects of mean female si ze on body titer were expected to be most pronounced in female s with disseminated infections, and this analysis was the primary interest.

PAGE 61

49 Product-moment correlation coefficients (r1,2) were used to describe the relationship between population growth measur ements (time to emergence, survivorship, size, ) and infection parameters: proportion infected, body titer of females with isolated infection, body titer of females with di sseminated infection, and proportion with disseminated infection among co mpetitive treatments of A. albopictus and A. aegypti. These analyses allowed for a test of the strength of positive or negative relationships among population growth measurements and infection. Results Competition Study For both A. albopictus and A. aegypti, competitive treatments significantly affected population growth measurements (Table 3-1), with uncrowded larval conditions consistently resulting in shorter time to emer gence, greater survivor ship, and greater adult size compared to crowded conditions (Figs. 3-1, 3-2). For A. albopictus SCC showed that differences in adult size followed by surv ivorship to emergence contributed the most to the significant competition effect as well as to subsequent treatment differences (Table 3-1). Although time to emergence was s horter at uncrowded larval conditions, it contributed less than the other population growth measurements ( A. albopictus time to emergence SE d; 160:0, 13.58 0.23, 320:0, 15.62 0.23, 160:160, 15.93 0.24) (Table 3-1). For A. aegypti SCC showed that differences in survivorship to emergence followed by time to emergence contributed the mo st to the significant competition effect as well as pairwise-differences (Table 3-1). Size contributed far le ss to the significant competition effect ( A. aegypti mean wing length SE mm; 0:160, 2.65 0.05, 0:320, 2.39 0.05, 160:160, 2.47 0.07) (Table 3-1). For both species, competitive treatments significantly affected ( F2, 26 = 191.84, P < 0.0001; F2, 19 = 51.94, P < 0.0001; A.

PAGE 62

50 albopictus and A. aegypti respectively) and was significantly grea ter in the pattern: 160 larvae > 320 larvae > 160+160 larvae (Fig. 3-3). Thus, interand intraspecific competition had major population-level effects. Infection Study Prior to analyzing effects of competiti on on arboviral infection, interspecific differences in susceptibility were first ex amined. Proportions infected, whole body titer, and proportions with disseminated infec tion were significantly different between A. albopictus and A. aegypti Pillais trace (3, 37) = 0.76, P < 0.0001 Proportion infected (SCC = 1.23) was the most important variable in the overall interspecific difference, followed by proportion with disseminated in fection (SCC = -0.66) and whole body titer (SCC = -0.57). The opposite signs of the SCC showed that there was a negative relationship between the variables across the species, so that A. albopictus had a greater proportion of infected individuals, a lower body titer, and a lower proportion disseminated infection compared to A. aegypti (LS means SE for A. albopictus and A. aegypti proportion infected, 0.94 0.03 and 0.58 0.04; body titer, 4.08 0.14 and 5.58 0.22 TCID50; and proportion with di sseminated infection, 0.67 0.03 and 1.00 0, respectively). Interspecific competition had significant effects on proportion infected, whole body titer, and proportion of A. albopictus with disseminated infection Pillais trace (6,24) = 0.52, P = 0.025 Proportion infected (SCC = 1.12) made the greatest co ntribution to the multivariate differences among treatments, and body titer (SCC = 0.23) and proportion with disseminated infection (SCC = 0.06) contributed less. Aedes albopictus at low density alone (160/containe r) had a significantly lower proportion infected, lower

PAGE 63

51 proportion with disseminated infection, and lower body titer compared to high density treatments Pillais trace (3,25) = 0.38, P = 0.011 (Fig. 3-4, A and B). Proportion infected was the major contri butor to this effect (SCC = 1.14), whereas titer (SCC = 0.27) and proportion with disseminated inf ection (SCC = -0.08) contributed little. The two high density treatments di d not differ significantly Pillais trace (3,24) = 0.14, P = 0.319 (Fig. 3-4, A and B). For the infection st udy, mortality during the extrinsic incubation period resulted in few A. aegypti females from the 160:160 treatment. Therefore, only means for intraspecific density treatments are reported for A. aegypti There were no significant effects of competition on infection parameters for A. aegypti Pillais trace (2,11) = 0.23, P = 0.301 Mean SE females assayed per treatment replicate were; 160:0 (10.0 1.84), 320:0 (11.89 1.15), 160:160 (albo) (6.44 0.63), 0:320 (6.11 1.12), and 0:160 (5.20 0.92). Females with disseminated infections are capable of transmitting virus and therefore are of epidemiologic significance. Fo r these females, an analysis of covariance with mean female size as a covariate showed significant effects of size and competition on whole body viral titer for A. albopictus with disseminated infections, but no significant size x competition interaction (Tab le 3-2). Thus, effects of mean body size and of competition are independent. Estimat ed slopes were positive, indicating that within a competitive treatment body titer in creased with size for mosquitoes with disseminated infection (Fig. 3-5). Pairwi se comparisons of adjusted means among treatments showed that significant differen ces in body titer followed the pattern 160:160 > 320:0 > 160:0 (mean SE: 5.80 0.23, 5.13 0.22, 3.23 0.32 TCID50, respectively).

PAGE 64

52 For A. aegypti with disseminated infections, there were no significant competitive treatment or covariate effects (Table 3-2). Product-moment correlations showed signi ficant relationships between infection and all correlates of population growth for A. albopictus (Table 3-3). In particular, increased time to emergence, a result of intraand interspecific competition, was positively correlated with infection rate for A. albopictus but survivorship, size, and were negatively correlated with infection ra te (Table 3-3). Also, survivorship and were significantly negatively correlated with mean A. albopictus body titer for females with disseminated infections. All correlati ons between population growth parameters of A. aegypti and infection parameters we re non-significant (Table 3-3). Discussion The two experiments in this study were desi gned to quantify the effects of intraand interspecific larval comp etition, and then to determine whether competitive effects carried over into the adult st age and influenced competence for arbovirus infection. For both Aedes species in the competition experiment, all population growth measurements clearly showed that higher larval densities resulted in poorer performance (Figs. 3-1, 3-2, 3-3). Analyses of survivorship, time to emer gence, and size at emergence suggested that the effects of intraand interspecific co mpetition were similar. However, for both Aedes species, a synthesis of multiple growth measurements ( ) showed that interspecific competition was more intense than in traspecific competition (Fig. 3-3). A variety of model systems have show n that the outcome of interspecific competition depends on resource type (e .g., Sanders and Gordon 2003, Tilman 1982). Contrasting outcomes have b een obtained with these two Aedes species, A. aegypti

PAGE 65

53 having the competitive advantage over A. albopictus with nutritious larval food (e.g., liver powder, yeast), but not with low-nutrient, more natural resource s (e.g., leaf litter) (Braks et al. 2004, Juliano 1998, Barrera 1996b, Black et al. 1989). The current experiment used a combination of natural (leaf infusion) and s upplemental (albumin, yeast) resources and, for both Aedes species, interspecific competition was greater than intraspecific competition as measured by The intention in designing the competition experiment was not to mimic natural resource s, rather to use a resource base known to maximize the production of Aedes females for the infecti on study, without negating the effects of competition. These objectives were met since competitive interactions were detected and sufficient numbers of adults were obtained for the infection study. Although the experimental design was cons trained to maximize adult production without negating competition, mosquito dens ities and sizes confor med to observations from field conditions. In the current experiment, densities were 0.032 and 0.064 larvae/ml for the 160 and 320 larvae treatments respectively. Sampling of the entire contents of water-holding golf cart tires in Broward, Indian River, and Monroe counties in Florida (Dec. 1996 or Jan. 1997 April 1998) s howed that larval densities were within the range observed in tires occupied by A. albopictus A. aegypti or both species (N=790, mean SE, 0.17 0.02, range 0.00083 3.08 larvae/ml) (G. F. OMeara, unpublished). Also, A. albopictus adult female wing lengths (Fig. 3-1) were within the range of A. albopictus collected at tire sites in East St. Louis, USA (N = 180, mean SE, 2.43 0.02 mm, range 1.84 2.95) (B.W. Alto and S.A. Juliano, unpublished). Similarly, both A. albopictus and A. aegypti female wing lengths in the cu rrent study were within the range of field-collected females of these species from tire sites in so uthwestern Louisiana

PAGE 66

54 (N=150, mean SE 2.68 0.02 mm, range 2.04 3.12; N=115, 2.64 0.03, range 1.92 3.12, respectively) (Nasci 1990). Wing length, as a surrogate of adult size, is a good indicator of larval e nvironmental conditions (e .g., food resources, larval density) (Juliano 1998, references therein). Thus, the experime ntal set-up produced adult females that parallel those sizes found in nature. The infection component revealed that larval competition altered adult mosquito susceptibility to arboviral inf ection and potential for virus transmission. In particular, competitively stressed A. albopictus females were more likely to become infected and have higher SINV titers and dissemination than females reared with less competition. Results are consistent with other model systems where competition, in the form of nutrient-limitation or stressors, enhanced suscep tibility to infection with pathogens or parasites (Kiesecker and Skelly 2001, Murra y et al. 1998, Oppliger et al. 1998, Matson and Waring 1984). In the current study, infectio n rate was the variable most sensitive to the impact of larval competition. Intraa nd interspecific competi tion altered subsequent A. albopictus interactions with SINV, suggesting that biotic interactions in early developmental stages may be important in determining adult arboviral infection parameters among mosquitoes. This type of indirect effect may be viewed as an interaction modification since a change in de nsity of one species a lters the nature of a direct interaction between tw o other species (Wootton 1993). On the other hand, effects of competition on A. aegypti infection parameters were not observed, and reasons for differences between the two Aedes species in responses to competitive treatments are unknown. Although there was less statistical power in the A. aegypti tests due to lower sample sizes, biological expl anations could include sp ecies-specific qualitative

PAGE 67

55 differences in the availability of midgut re ceptor sites used by SI NV or escape barriers (e.g., midgut escape barrier) that may be differentially affected by competition. These results suggest species-specific differences in how larval competition affects adult competence for arboviral infection parameters. Similarly, in plant communities, studies have demonstrated species-specific res ponses to indirect e ffects (e.g., indirect facilitation), most likely attri butable to differences among sp ecies in life history traits (e.g., Pages et al. 2003, Levine 1999). The correlation coefficients demonstrating that infection rates were significantly associated with all correlate s of population growth (Table 3-3) represent the first evidence that life history traits, in addition to adult size, change parameters associated with vector competence. Furthermore, corr elations between life-history traits and infection parameters in A. albopictus showed that negative e ffects of competition on population growth are associated with enhanced vector potential (Table 3-3). The observation that larger A. albopictus females with disseminated infections had significantly greater bod y titer can be explained as simply a size phenomenon, i.e., more tissue is available for virus propagation. For body titer, there were i ndependent effects of both mean adult size and competition. The lack of a significant size x competition interaction showed that the effect of si ze on body titer was similar among competitive treatments (Fig. 3-5). More importantly, de nsity-dependent differenc es in body titer were found, with greater mean body titer among A. albopictus reared under competitive conditions. Significant density-d ependent differences in bod y titer were identical to significant density-dependent differences in except in the opposite direction (Figs. 33, 3-5). Specifically, more intense competition as measured by a lower resulted in

PAGE 68

56 greater body titer. These results demonstr ate both size-dependent and size-independent effects of competition on infection dyna mics that have opposite effects across competition treatments. Within a competitive treatment, larger mosquitoes have greater body titer, but between competitive treatments larger mosquitoes from low-competition larval rearing conditions have lower body titer and a lower proportion infected, compared to smaller mosquitoes emerging from high-co mpetition conditions. Overall, the results demonstrate that competitive stress experienced by A. albopictus larval stages carried over to the adult stage and si gnificantly influenced suscep tibility to infection and dissemination. Over and above the effect s of competition, the two Aedes species differed in susceptibility to infection a nd dissemination. Other studies have shown interspecific differences for quantitative aspects of infec tion (Turell et al. 2001, Gubler et al. 1979). However, previous research using these Aedes species, as well as other mosquito species, did not quantify the variables most important for interspecific differences (e.g., infection, body titer, dissemination) or the positive and negative interrela tionships among these variables across species (i.e., see SCC). A. albopictus was significantly more susceptible to infection than was A. aegypti as seen in other studies (Tur ell et al. 2001, Gubler et al. 1979). Conversely, although A. aegypti had lower infection rates, those females that were infected had significantly higher body titer a nd dissemination rates compared to infected A. albopictus Infection contributed approximate ly twice as much as body titer and dissemination to interspecific differences. Factors limiting body tit er and dissemination (e.g., midgut escape barrier) were less efficient (or not expressed) in A. aegypti compared to A. albopictus under these conditions. These results suggest fundamental differences in

PAGE 69

57 physiology between these Aedes species that alter their susceptibility to arboviral infection and dissemination, and these diffe rences are likely to have important epidemiological consequences. If effects of competition on vector infec tion with SINV apply to arboviruses such as DENV and West Nile virus, these results may have important implications for human health. The current study suggests th at competition experienced by larval A. albopictus may enhance the threat posed by this speci es in pathogen transmission. Uncrowded larval rearing at low densities, used in most laboratory studies of vector competence, do not accurately reflect conditions in natu re where competition is often strong and widespread (Juliano et al. 2004) The current study suggests that indirect effects are important in determining mosquito vector ab ility, and that the effect may be species specific. Failure to consider larval stresse s may result in misleading estimates of relative susceptibility to infection for A. albopictus and A. aegypti and by extension, other arboviral vectors. This report is the first to quantify how larval competition may affect arbovirus infection in adult mo squitoes, and demonstrates th e species-specificity of the process from infection to dissemination. Futu re assessments of v ector potential should consider the species-specific effects of larval conditions that reflect competitive conditions observed in nature.

PAGE 70

58 0.15 0.2 0.25 0.3 0.35 0.4 2.12.22.32.42.52.6Size at emergence (mm)Proportion surviving 160:0 320:0 160:160a a b Figure 3-1. Aedes albopictus least squares means (SE) for female survivorship and size at emergence. Different lowercase le tters indicate significant differences between bivariate means. Competition treatments consisted of species density ratios of A. albopictus : A. aegypti :0, 320:0, and 160:160. 0.1 0.15 0.2 0.25 0.3 0.35 0.4 12.51313.51414.5Time to Emer g ence ( da y s ) Proportion surviving 0:160 0:320 160:160a b b Figure 3-2. Aedes aegypti least squares means (SE) for female survivorship and time to emergence. Different lowercase lette rs indicate significant differences between bivariate means. Competition treatments consisted of species density ratios of A. albopictus : A. aegypti :160, 0:320, and 160:160.

PAGE 71

59 1.02 1.06 1.1 1.14 1.180123456Competitive Treatment ( A. albopictus: A. aegypti )Estimated finite rate of increase A. albopictus A. aegyptia b c A B C160:0320:0160:1600:3200:160 Figure 3-3. Least squares means ( SE) fo r estimated finite rate of increase, for Aedes albopictus and A. aegypti Points without bars have standard errors too small to appear on the graph. Different lo wercase and uppercase letters indicate significant differences between means for A. albopictus and A. aegypti respectively.

PAGE 72

60 A0.80 0.85 0.90 0.95 1.00 0.40.50.60.70.80.9 Proportion with disseminated infectionProportion infected 160:0 320:0 160:160 B0.80 0.85 0.90 0.95 1.00 3.03.54.04.55.0 log10 (body titer)Proportion Infected 160:0 320:0 160:160 Figure 3-4. Bivariate plots of least squares means ( SE) for three dependent variables for Aedes albopictus females fed on a Sindbis virus blood meal. (A) Proportion of infected females vs. propor tion with disseminated infection. (B) Proportion of infected females vs. body titer. In both graphs, the dashed ellipse indicates multivariate means that are not significantly different. Numbers in the figure key represent the ratio of A. albopictus to A. aegypti.

PAGE 73

61 0 1 2 3 4 5 6 7 2.02.22.42.6 Size (wing length, mm)log10 (body titer) 160:0 (disseminated) 320:0 (disseminated) 160:160 (disseminated) 160:0 (isolated) 320:0 (isolated) 160:160 (isolated) Figure 3-5. Least squares means for body titer and size of adult Aedes albopictus females with disseminated (i.e., infection spread beyond the midgut, infecting secondary target organs such as body, le gs) and isolated (i.e ., infection limited to the midgut) Sindbis virus infections The size effect on females with disseminated infections gives a slop e of 5.48 (SE = 1.28). Solid and dased lines drawn through bivariate means show the best fit for A. albopictus with disseminated infections in three comp etitive treatment conditions. Numbers in the figure key represent the ratio of A. albopictus to A. aegypti

PAGE 74

62Table 3-1. Multivariate ANOVA for main effects and multivariate pa irwise contrasts of competitive treatment effects on female Aedes albopictus and A. aegypti population growth measurements: time to emergence, survivorship to emergence, and adult size. Comparison df Pillais trace P Standardized Canonical Coefficients Time Surv. Size A. albopictus Competitive treatment 6 1.02 < 0.0001 0.79 1.19 1.97 160,0 vs. 320,0 3 0.90 < 0.0001 0.88 1.18 1.89 160,0 vs. 160,160 3 0.91 < 0.0001 0.73 1.19 2.02 320,0 vs. 160,160 3 0.13 0.3274 Error df 26 A. aegypti Competitive treatment 6 0.93 0.0006 1.11 2.01 0.27 0,160 vs. 0,320 3 0.88 < 0.0001 1.12 1.99 0.28 0,160 vs. 160,160 3 0.76 < 0.0001 1.05 2.09 0.20 0,320 vs. 160,160 3 0.14 0.4472 Error df 19

PAGE 75

63Table 3-2. ANCOVA for the effects of competitiv e treatment and size c ovariate on body titer for Aedes albopictus and A. aegypti females with disseminated infections. Source df F P A. albopictus, disseminated Size 1 18.38 0.0002 Competitive treatment 2 15.20 < 0.0001 Size x competition 2 0.26 0.7750 Error df 25 A. aegypti disseminated Size 1 1.74 0.2170 Competitive treatment 2 0.74 0.5036 Size x competition 2 2.62 0.1336 Error df 11

PAGE 76

64Table 3-3. Product moment correlation coefficients (r1,2) for the relationship between populat ion growth measurements (time to emergence, survivorship, size, and ) and infection parameters. Infection pa rameters include infection, body titer of females with isolated infection, and dissemination for Aedes albopictus (df=25) and A. aegypti (df=12). An infection limited to the midgut is called an isolate d infection, whereas an in fection spread beyond the midgut, infecting secondary target organs (e.g., salivary glands, head, legs), is called a dissemi nated infection. Asterisks denote significant correlati on coefficients (* P < 0.05; ** P < 0.001). No r1,2 values are reported for A. aegypti dissemination and body titer (isolated) since all infected individuals had disseminated infections. A. albopictus A. aegypti Infection parameters and growth parameters Infection Time to Emergence 0.50 ** 0.08 Survivorship 0.50 ** 0.07 Size 0.64 ** 0.17 0.55 ** 0.10 Body Titer (disseminated) Time to Emergence 0.26 0.24 Survivorship 0.38 0.38 Size 0.00098 0.13 0.40 0.19 Dissemination Time to Emergence 0.04 Survivorship 0.22 Size 0.20 0.13

PAGE 77

65 CHAPTER 4 LARVAL COMPETITION AND SUSCEPTIBILITY OF Aedes aegypti AND Aedes albopictus TO INFECTION BY DENGUE VIRUS Introduction Dengue virus (DENV) is an arthropod borne (arbo) virus. Ther e are four different serotypes of DENV that are the cause of morb idity and mortality throughout much of the tropical world. Approximately 50-100 million ca ses of dengue fever (DF) occur annually with hundreds of thousand of cases of dengue hemorrhagic fever (DHF), a lifethreatening form of dengue. In Southeast Asia, range expansion of the primary vector Aedes aegypti (L.) and human migration have co ntributed to hyperendemicity (cocirculation of multiple serotypes in a single location) and associated epidemic DF and DHF (Gubler 2002). Regions with hype rendemic DENV are expanding, and recent introductions of Southeast Asian genotypes of DENV to the Western Hemisphere pose an increased risk of transmission in the tropica l Americas resulting in greater number of cases of severe DHF (Cologna et al. 2005, Co logna and Rico-Hesse 2003, Rico-Hesse et al. 1997, Lewis et al. 1993). The yellow fever mosquito A. aegypti and Asian tiger mosquito A. albopictus (Skuse) are considered the primary and secondary vectors of DENV, respectively (Rodhain and Rosen 1997). However, the relative importance of these Aedes species in DENV transmission in nature is difficult to de termine, especially in regions where they coexist. Aedes aegypti and A. albopictus have sympatric and allopa tric breeding sites in Southeast Asia as well as in many locations in the Western Hemisphere where dengue is

PAGE 78

66 endemic and a serious health risk to hu mans. Geographic strains of both these Aedes species vary in their susceptibility to DENV infection (e.g., Be nnett et al. 2002, Failloux et al. 2002, Vazeille-Falcoz et al. 1999, Boromisa et al. 1987 Gubler et al. 1979, Gubler and Rosen 1976). Additionally, mosquito infection parameters may be altered by different serotypes and strains of DENV (e.g., Moncayo et al. 2004, Armstrong and RicoHesse 2003, 2001, Rosen et al. 1985, Whitehead et al. 1971). For example, Southeast Asian DENV-2 strains, which are more virule nt than American genotypes, consistently show significantly greater disseminated infection in A. aegypti compared to American DENV-2 strains (Cologna et al. 2005, Ar mstrong and Rico-Hesse 2003, 2001), but no comparable data exist for A. albopictus Contrasting outcomes of dengue infection have been obtained for these two Aedes species in laboratory and fi eld studies. Studies using well-established laboratory mosquito strains, suggested that DENV (serotypes 1, 2, 3, 4) were less efficient at infecting and causing disseminated infections in A. aegypti compared to other Aedes species, including A. albopictus (Rodhain and Rosen 1997, Rosen et al. 1985, Gubler et al. 1979). Similarly, a greater proportion of A. albopictus had disseminated DENV-2 virus infection compared to sylvatic A. aegypti formosus (Vazeille et al. 2001). In contrast laborat ory experiments showed that a greater proportion of A. aegypti (F1-F2 generation) had disseminated DENV-2 infections compared to A. albopictus although the proportion of A. albopictus with disseminated infections increased with subsequent labor atory generations (Vazei lle et al. 2003). In summary, research to date is equivocal whether A. albopictus or A. aegypti is the superior vector. Rather, it is likely that the gene tic background of both th e virus and mosquito

PAGE 79

67 species play important roles in determini ng vector competence for dengue (Tabachnick 1994). The establishment of A. albopictus in new regions, especially where A. aegypti is absent, also spreads the risk for DENV transmission since human movement and transport may place the reservoir, virus, and vector in close proximity to one another. In the southern U.S., the introduction and spread of A. albopic tus was associated with declines in resident A. aegypti (Juliano et al. 2004, OMeara et al. 1995, Mekuria and Hyatt 1995, Hornby et al. 1994, Hobbs et al. 1991). The two Aedes species occupy similar container habitats, and interspecific competition among the la rval stages is welldocumented and a likely contributor to the observed decline of A. aegypti (e.g., Costanza et al. 2005a, Braks et al. 2004, Juliano et al. 2004, Lounibos et al. 2002, Barrera 1996). Competition, due to resource limitation or high larval density, has been demonstrated for many mosquito species and is usually reflected by an increase in larval development time and mortality, and decrease in adult size (e .g., Alto et al. 2005ab, Juliano and Lounibos 2005, Peck and Walton 2005, Braks et al. 2004, Juliano et al. 2004, Ye-Ebiyo et al. 2003, Gimnig et al. 2002, Lounibos et al. 2002, Schneider et al. 2000, Teng and Apperson 2000, Lonard and Juliano 1995, Broadie and Bradshaw 1991). The effects of larval competition likely im pact the adult stage, and therefore may also influence adult suscep tibility to pathogens (e.g., arboviruses), including DENV (Vazeille et al. 2003, Black et al. 2002, Sumanochitrapon et al. 1998). However, this field of investigation has not been well explored. Small O. triseriatus adults derived from competitive larval environments unde r laboratory conditions or from field collections had similar La Crosse virus (LACV) infection rates compared to large adults,

PAGE 80

68 but greater dissemination and transmissi on (Grimstad and Walker 1991, Paulson and Hawley 1991, Grimstad and Haramis 1984). Large A. aegypti adults, reared with abundant resources and low larval density, had greater incidence of disseminated DENV2 viral infection compared to smaller adults (Sumanochitr apon et al. 1998). Explicit examination of the effects of intraand interspecific competition between A. albopictus and A. aegypti on vector competence has only been examined in a model using SINV (Alto et al. 2005a). These experiments de monstrated that competition resulted in increased development time to emergence and decreased survivorship, size, and a performance index ( ) for both species, and enhanced A. albopictus SINV infection parameters, but not A. aegypti parameters. Specifically, competitively stressed A. albopictus had greater infection, disseminated in fection, and body titer than unstressed individuals, and proportion infect ed contributed the most to th ese significant effects (Alto et al. 2005a). While the results of this previous work were compelling, A. aegypti and A. albopictus are not vectors of SINV in nature. Ther efore, the artificial nature of the virusvector association limits applying these re sults more generally. To examine the generality of patterns observed in this previous work, and to use a virus of primary importance to human health, I conducted an experiment to test whether competition among larvae affects DENV infection parameters, and whether population growth measurements are associated with DENV infection parameters, for A. aegypti and A. albopictus For this study, infection parameters refer to proportion of females infected with DENV, proportion of females with disseminated infection, and DENV body titer.

PAGE 81

69 Materials and Methods Competition Study Mosquitoes used the experiment were the same continuously propagated colony strains described elsewhere (Alto et al. 2005a). Aedes albopictus Lake Charles strain and A. aegypti Rockefeller strain represent long-standi ng laboratory colonies (Nasci et al. 1989, Craig and Vandehey 1962). The current e xperimental setup was similar to that used to investigate the affects of larval competition on adult Aedes infection with SINV (Alto et al. 2005a). A similar experimental design was desirable because it facilitated comparison between experiments to evaluate whether competition has similar affects on Aedes infection parameters for arboviruses in two different families (i.e., SINV in Togaviridae, DENV in Flaviviridae). Larval food resources used in the compe tition experiment were identical to those described by Alto et al. (2005a), except th at supplemental resources were added on day 13 instead of day 10. Briefly, la rval rearing vessels consiste d of 5-L plastic containers filled with 4000 ml tap water, 500 ml oak leaf infusion water (OMeara et al. 1989), and 0.2 g larval food (1:1 albumin: yeast). Food resources were allowed to incubate for 5 d before newly hatched (<24 h old) mosquitoes were added to experime ntal containers. Three days after adding the larvae, a suppl emental 500 ml oak infusion and 0.2 g larval food were added. Thirteen days later, 50% of the liquid was removed, except larvae, and 0.1 g larval food, 250 ml oak leaf infusion wa ter, and 2250 ml tap water were added. Competition treatments consisted of species density ratios of A. albopictus : A. aegypti (i.e.., 160:0, 320:0, 160:160, 0:320, and 0:160). Ten replicates were used for each treatment, except for 320:0 and 0:320, whic h had 11 replicates. Containers were maintained under constant environmen tal conditions (28C 1C, 14:10 L:D

PAGE 82

70 photoperiod). Pupae were removed from cont ainers daily and stored in 20-ml waterfilled vials until adult emergence. Larval r earing in the competition experiment lasted until all pupae emerged as adults or larvae had died. Measurements on individuals and cohorts were used to evaluate competitive treatment effects on A. albopictus and A. aegypti population growth. Mean female size (wing length in mm) and mean time to adult emergence (days) were determined for each treatment replicate. Wing length was measured as the distance from the axillary incision to the distal point on the lateral margin of the wing, excluding the wing fringe. Female survivorship per replicate was calculated as ( number of adult females) / (total number of original larvae) of a given species. Estimated finite rate of increase ( ) was calculated for each replicate: ln [(1/ No) x Ax f(wx) ] = exp(r ) = exp D + [ x x Ax f(wx) / x Ax f(wx) ] where is a composite index of performa nce based on a transformation of r (Juliano 1998, Livdahl 1984, 1982, Li vdahl and Sugihara 1984). No is the initial number of females in a cohort (assumed to be 50 %), Ax is the number of females emerging on day x and wx is mean female size on day x D is the time (in days) from emergence to oviposition. For A. albopictus and A. aegypti, D is assumed to be 14 and 12 d, respectively (Livdahl and Willey 1991, Juliano 1998) Mean female size per day, used to calculate was obtained from all females assayed fo r viral infection as well as all unfed

PAGE 83

71 females obtained from the entire duration of the experiment. Number of eggs produced by a female was estimated from female size based on a regression function f(wx) The following fecundity-size relationships ( f(wx) ) were used to calculate : A. aegypti (Briegel 1990) : f(wx) = 2.50( wx 3) 8.616 r2 = 0.875, N = 206, and P <0.001 A. albopictus (Lounibos et al. 2002) : f(wx) = 78.02 ( wx) 121.24 r2 = 0.713, N = 91, and P <0.001 In both cases wx is wing length in millimeters. Population growth measurements (time to emergence, size of females assayed for infection, survivor ship) were analyzed, separately for A. albopictus and A. aegypti by Multivariate Analyses of Variance (MANOVA) to quantify the effect of comp etition. Thus, MANOVA used sizes of females assayed for DENV infection and was calculated based on sizes of females assayed for infection as well as all unfed females obtained over the duration of the experiment. Significant effects were further analyzed by all po ssible contrasts of pairs of main effect multivariate means using the se quential Bonferroni method (experimentwise = 0.05). Standardized canonica l coefficients (SCC) were used to describe the relative contribution of each population growth measurem ent to significant multivariate effects as well as their relationship to each other (e .g., positive or negative: SAS Institute 2002, Scheiner 2001). Competitive treatment effects on A. albopictus and A. aegypti were analyzed by separate one-way ANOVA, and si gnificant effects were further analyzed by

PAGE 84

72 pairwise comparisons of main effect m eans (Ryan-Einot-Gabriel-Welsch test, SAS Institute 2002). Raw data adequately met assumptions of univariate normality and homogeneous variances for all population growth measurements in MANOVA and ANOVA, except A. albopictus development time, which showed departure from normality. No common transformations improved normality, however, MANOVA, using Pillais trace, is robust to departures from normality (Scheiner 2001). Also, the highly significant treatment effects and similar direct ion of competitive effects suggest that the departure from normality had little effect in determining the results. Infection Study Viral propagation A Southeast Asian genotype of DENV-2 was originally isolated from a patient in Thailand in 1974. This virus isolate ha d been passed once in the mosquito Toxorhynchites amboinensis (Doleschall), 3 times in Vero cells, twice in A. albopictus C6/36 cells, and 3 additional passages in Ve ro cells (S. Fernandez, pers. comm.). Subsequently, the DENV stock was pa ssed twice in Vero cells. T-75 cm2 flasks with confluent monolayers of Vero cells were i ndividually inoculated with 3 ml media (Leibovitz L-15 media, 10% fetal bovine serum (FBS), 50 g/ml gentamicin) containing 200 l DENV 2 stock. T-75 cm2 flasks were rocked for 1 h at 37C, to allow for adsorption, after which media were added to bring the total volume to 10 ml and incubated at 35C. Media in T-75 cm2 flasks was renewed on days 4 and 8 and harvested for infectious bloodmeals on day 11. Freshly recovered media-virus suspension (i.e., unfrozen) from day 11 was used as the sour ce virus for use in infectious bloodmeals offered to mosquitoes (Miller et al. 1982). In fectious bloodmeals using previously frozen

PAGE 85

73 (-80C) DENV-2 stock showed significantly lower infection and dissemination in both Aedes species compared to fresh grown virus, even at similar titers ( unpublished data ), in agreement with observations for other ar boviruses, including DENV-2 (Turell 1988, Miller 1987, Miller et al. 1982). All proce dures involving DENV-2 were performed in a biosafety level-3 facility. Oral infection of mosquitoes Adult mosquitoes from the larval competition experiment were housed by treatment replicate and species in wax-co ated cardboard containers (14 cm high x 11 cm diameter) and provided with an oviposition c up, 20% sucrose, and water. Sucrose and water were renewed every 48 h. Sucrose, but not water, was removed from mosquitoes 24 h prior to blood feeding. Infectious blood meals were offered to 4-7 d old females using a silicon membrane feed er system (Alto et al. 2005a 2003, Butler et al. 1984). Citrated bovine blood was combined with DENV-2 stock in a 4 to 1 ratio, respectively to provide a blood meal titer of 6.2 log10 PFU/0.2 ml. Membrane f eeders with infectious blood were heated at 37C in an in cubator for 20 min. and offered to Aedes females for 30 min. An aliquot of infecti ous blood was immediately frozen at -80C and later tested by plaque assay to determine th e titer of blood meals offered to Aedes females. Next, mosquitoes were cold anesthetized and fully bloodfed females we re identifie d, using a stereo microscope within a glove box, isolat ed, and held for a 12 d extrinsic incubation period at 28C 1C and provided with sucrose, water and a 14:10 h light:dark photoperiod regime. Sucrose and water were renewed every 48 h. Mosquitoes that survived the extrinsic incubation period were individually stored in vials at -80C and, subsequently, their wings were removed and me asured as an indicator of female size (see above).

PAGE 86

74 Interspecific ( A. aegypti versus A. albopictus ) differences in DENV-2 susceptibility to infection were evaluated by MANOVA and SCC on the response variables proportion infected, and proportion with disseminated infection. A one-way ANOVA tested for interspecific differen ces in body titer of Aedes females with disseminated DENV-2 infections. Next, individual one -way MANOVAs and SCC, for each Aedes species, were used to determine competitive treatment e ffects on proportion in fected, and proportion with disseminated infection. Significant e ffects were further analyzed by all possible pairwise contrasts of pairs of bivariate means using the sequential Bonferroni method (experimentwise = 0.05). Raw data adequately met assumptions of univariate normality and homogeneous variances for analyses, except for proportion A. albopictus infected, which showed de parture from normality. No common transformations, including arcsine square root, improved norma lity. The sensitivity of departure from normality was assessed by analyzing the proportion A. albopictus infection using a Kruskal-Wallis nonparametric test which is a weaker test but does not assume normality. Results of the nonparametric test gave the same conclusions as parametric analyses, thus I am confident that effects on proportion A. albopictus infected are not artifacts produced through departure in normality Further, MANOVA, using Pillais trace, is robust to departures in normality (Scheiner 2001). The effects of mean female size on proportion infected, proportion with disseminated infection, and body titer of fema les with disseminated infection were assessed by treating size as a covariate in an analysis of covariance (ANCOVA), with competition treatment and competition x size inte ractions as categorical variables (SAS Institute 2002). ANCOVAs involving body titer used size based on wing length

PAGE 87

75 measurements of females with disseminated DENV-2 infections. ANCOVAs involving proportion infected and proporti on with disseminated infection used size based on wing length measurements of all females assa yed for DENV-2 infection. Initially all ANCOVAs tested for equality of slopes for each size by competitive treatment (i.e., each competitive treatment has its own slope estimate). ANCOVAs determined to have common slopes (i.e., no significant size x competitive treatment interaction) were retested for the equality of the intercepts. ANCOVAs determined to have similar intercepts were re-tested as ANOVAs with competitive treatment and no size covariate. Significant effects were further analyzed by all possibl e pairwise comparisons of treatment means (Tukey-Kramer adjustment of experimentwise = 0.05, SAS Institute 2002). Raw data adequately met assumptions of univari ate normality, homogeneous variances, and linearity, however, the proportion A. albopictus infected with DENV showed some departure from normality. No common transf ormations, including arcsine square root, improved normality. Product-moment correlation coefficients (r1,2) were used to describe the relationship between population growth measur ements (time to emergence, survivorship, size, ) and infection parameters: proportion infected, proportion with disseminated infection, and body titer of females with disseminated infection among competitive treatments of the two Aedes species. Thus, these anal yses pool all competitive treatments. The correlation analyses of body tite r and size were based on sizes of females with disseminated DENV-2 infections. Correla tion coefficients quantify the strength of the relationship (positive or negative) be tween population growth measurements and infection parameters.

PAGE 88

76 Blood meal plaque assay Titrations of DENV-2 infectious blood m eals were performed by plaque assays in duplicate 6-well plates of confluent monolayers of Vero cells maintained with Leibovitz L-15 media, 10% FBS, and 50 g/ml gentamicin. 10-fold seri al dilutions of infectious blood meal samples were made by combining a 0.2 ml DENV-2 blood meal sample with 1.8 ml media (2X Eagles Minimum Essential Medium (EMEM) cont aining Earles Basic Salt Solution, 10% FBS, 100 U/ml penicillin, 100 g/ml streptomycin), thus creating a 10-1 dilution. This process was repeated to yield a full range of dilutions from 10-1 to 10-9. At the time of viral inoculation, medi a covering cell monolayers in the wells was removed and wells were individually inoculated with 0.2 ml of the se rial dilutions. Sixwell plates were gently rocked for 1 h incubation at 35C and a 5% CO2 atmosphere. Following incubation, the first overlay of agarose was applied to the cell monolayer. The first and second overlays of agarose described here provided sufficient reagents to complete ten 6-well plate plaque assays. Briefly, 1.8 g Seaplaque low melting agarose (FMC Biotechnology) was added to 100 ml of double distilled water. The solution was heated until completely melted and then cooled to 40C. In a separate flask, 10 ml FBS was combined with 2 ml nonessential amino acid solution, 100U/ml penicillin and 100 g/ml streptomycin. In another se parate flask, 1 ml of L-glutamine and 250 g/ml of Amphotericin B were added to 100 ml of 2X EMEM. Next, the EMEM mixture was added to the agarose followed by the FBS mixture. Each well received 3 ml of the first overlay of reagents Six-well plates remained motionless for 5 min. to allow for the agarose to gel and then well-plate covers were removed for 15 min.

PAGE 89

77 to facilitate drying. Finally, well plate covers were replac ed and the 6-well plates were incubated for 6 d at 35C and a 5% CO2 atmosphere. The second overlay of agar ose was applied on the 6th day of incubation. Briefly, 1.8 g Seaplaque low melting agarose was comb ined with 2.0 g sodium chloride and 200 ml double distilled water. The solution was heated until completely melted and then cooled to 40C. Next, 9 ml neutral red solution (Sigma Cat: N2889) was added to the solution and each well received 3 ml of the seco nd overlay reagents. Six-well plates were treated similar as the first overlay except that plates were incubated for 24 h at 35C and 5% CO2 atmosphere. Plaques were counted and e xpressed in plaque forming units (PFU) per 0.2 ml of test inoculum. Mosquito homogenization, plaque assay, and RNA extraction For each mosquito, wings and legs were separated from bodies using forceps sterilized with 70% ethanol followed by intens e flaming (Turell et al. 1984). Wings were measured and used as an i ndicator of female size for calculations. Bodies were assayed to determine infection and whole body viral titer, whereas legs were assayed as an indicator of disseminated infection (Tur ell et al. 1984). Bodies and legs were homogenized separately in 2 ml flat botto m vials containing 1 ml media (Leibovitz L-15 media, 10% FBS, 100 U/ml of penicillin, 100 g/ml streptomycin, and 250 g/ml Amphotericin B) and 2 zinc plated steel BBs (Daisy). Homogenization was performed by placing vials into a TissueLyser (Qia gen) for 6 min. at 25 Hz followed by centrifugation at 3148 x g for 4 min. and 4C. Body infection and disseminated infection were determined by plaque assays using und iluted body and leg stock solutions. Plaque assays were performed similarly to assays of blood meal titer, except that 12-well plates

PAGE 90

78 were used instead of 6-well plates and addi tional antibiotics were added. A single well was inoculated for each body and leg stock solu tion of each tested female. Only females determined to have disseminated DENV-2 infections (i.e., positive infection in legs) were subsequently assayed for body titer. Plaque assays for female bodies and legs were scored as positive or negative with no attemp t to count number of plaques. Subsequently, body homogenates of females with dissemina ted infections were thawed and DENV-2 RNA was extracted from 140 l of the sample using QIAa mp viral RNA Mini Kits (Qiagen) and then assayed by quantitative real-time (RT)-PCR (Armstrong and RicoHesse 2003, 2001). Quantitative RT-PCR Quantitative RT-PCR allowed for determinati on of the relative amounts of virus in the body (viral titer), as meas ured by cDNA amplification, standardized with a plaque assay (Richardson et al. 2006, Bustin 2000). A commercially available quantitative RTPCR kit, SuperScript III Platinum one-step qua ntitative RT-PCR system (Invitrogen), and fluorogenic probe hydrol ysis (TaqMan) technology was used to detect DENV-2 RNA. DENV-2 vi rus specific primers targeted the capsid gene (Forward 237-251 bp, Reverse 305-284 bp) (Callahan et al. 2001). Primer sequences were: Forward (5-CAT GGC CCT KGT GGC G3) and Reverse (5-CCC CAT CTY TTC AGT ATC CCT G-3) (Callahan et al. 2001). The DENV-2 specific dual-labeled fluorogenic oligonucleotide probe included a 5-reporter dye and a 3-quencher dye (250 nm DLB 5 6-FAM / 3 BHQ-2 (5-TCC TTC GTT TCC TAA CAA TCC-3) (Callahan et al. 2001).

PAGE 91

79 Reactions used a thermostable enzyme, Taq DNA polymerase, derived from a bacterium Thermus aquaticus (Holland et al. 1991). The oligonucleotide probe anneals to the target RNA sequence downstream from a primer site. Under these conditions, the reporter and quencher dyes are in close proxi mity so the quencher inhibits fluorescence emission. The 5 nuclease activity of Taq DNA polymerase cleaves the probe during extension so the reporter and quencher dye s separate and results in a detectable fluorescence signal which is recorded by the li ghtcycler. PCR products are detected by the generation of a fluorescent si gnal, and the intensity of the signal is directly related to product accumulation. Regular cycling of temperature allows for denaturation, annealing, and extension steps that are repe ated resulting in expone ntial growth of the target amplicon (DENV-2 cDNA). Each reaction included: 0.4 l SuperScript III RT/Platimum Taq mix, 10 l 2X reaction mix (a buffer system, MgSO4, dNTPs and stabilizers), 1 l forward primer (10 M), 1 l reverse primer (10 M), 0.5 l fluorogenic probe (10 M), 4.2 l DEPC treated H2O, and 2 l test sample (positive or negative for DENV-2 RNA). Reactions were performed in glass capil lary tubes in a thermocycler LightCycler 2.0 Instrument equipped with LightCycler software versi on 3.5 (Roche Molecular Biochemicals). The thermal cycle included: RT, 30 min. at 48C; Denaturing, 2 min. at 95C; followed by 45 cycles of PCR, 15 s at 95C, 1 min. at 60C. Each lightcyler set of reactions included a negative control (water) a nd positive control standard (DENV-2 stock RNA, 10-5 dilution). The positive control was an indi cator of cDNA synthesis and served as a known standard of DENV-2 RNA used to produce cDNA. Plaque forming units (PFU) were calculated by a standa rd curve method that compared cDNA synthesis to in vitro

PAGE 92

80 PFU for the same full range of positive DENV2 RNA stock virus titr ated in parallel by quantitative RT-PCR and plaque assay (R ichardson et al. 2006, Bustin 2000). Quantitative RT-PCR estimates crossing points which are cycle numbers that correspond to the point at which exponential growth of the target amplicon occurs. Positive and dilute test samples have high crossing points, since there wa s little initial RNA, whereas concentrated test samples have low crossi ng points since there were greater amounts of initial RNA. A standard curve was generate d by assaying a full range of 10-fold serial dilutions of DENV-2 stock (7.2 log10 PFU/0.2 ml) by plaque assay (see blood meal plaque assay) to quantify PFU, as we ll as by quantitative RT-PCR testing DENV-2 cDNA synthesis. Three replicates were us ed for each dilution assayed by quantitative RT-PCR (slope = -3.007, intercept = 34.54, r2 = 0.9604). Plaque assays determined that 2.2 log10 PFU/0.2 ml corresponded to the 10-5 dilution. These estimates were used to transform crossing points to PFU in determining body titer for Aedes females with disseminated DENV-2 infections. Species by Competition Comparison An additional set of analyses were used as alternative methods to address mosquito species by competitive treatment effects. Th e intention of the design of the competition experiment should be primarily thought of as two experiments; One experiment for intraand interspecific competition for A. albopictus (160:0, 320:0, and 160:160) and another experiment for intraand interspecific competition for A. aegypti (0:160, 0:320, and 160:160). However, it is possible to analyze subsets of the experimental treatments to further isolate species and competition effects on population growth measurements and infection parameters. Population growth m easurements (time to emergence, size of females assayed for infection, survivorship ) were analyzed by a two-way MANOVA and

PAGE 93

81 SCC with mosquito species ( A. albopictus and A. aegypti ) and competitive treatment (160:0, 320:0, 0:160, and 0:320) as factors. Treatments involving both species present (e.g., 160:160) were intentionly omitted, thus isolating treatment effects. Similarly, infection parameters (proporti on infected, proportion with di sseminated infection) were analyzed by a two-way MANOVA and SCC w ith mosquito species and competitive treatment as factors. Species and comp etitive treatment effects on body titer were analyzed using a two-way ANOVA and significa nt effects were further analyzed by all possible pairwise comparisons of treatm ent means (Tukey-Kramer adjustment of experimentwise = 0.05, SAS Institute 2002). Additiona lly, separate one-paired t-tests were used to address species effects on pr oportion infected, proportion with disseminated infection, and body titer in th e interspecific competitive treatment (160:160). Results Competition Study For both A. albopictus and A. aegypti competitive treatments significantly affected population growth measurements (Table 4-1) in the pattern 160 larvae < 320 larvae = 160:160 larvae (Figs. 4-1, 4-2). Greater competi tion consistently resulted in significantly smaller adult size, longer time to em ergence, and lower survivorship ( A. albopictus mean SE proportion surviving; 160:0, 0.4 2 0.03, 320:0, 0.27 0.03, 160:160, 0.32 0.03; A. aegypti mean SE proportion surviving; 0:160, 0.36 0.02, 0:320, 0.31 0.01, 160:160, 0.33 0.02) than all less intense competitive treatments (Figs. 4-1, 4-2). For both Aedes SCC showed that differences in adult size and time to emergence contributed the most to the significant competition effect as well as to subsequent treatment differences (Table 4-1). For both species, competitive treatments significantly affected ( A. albopictus F2, 28 = 90.44, P < 0.0001; A. aegypti F2, 28 = 150.84, P < 0.0001) and

PAGE 94

82 was significantly greater in the pattern: 160 larvae > 320 larvae > 160+160 larvae (Fig. 4-3). Infection Study The infection study produced 2508 mosquito es that successfully completed the extrinsic incubation period. Six infectious blood meals were given over the course of the experiment and plaque assays showed some variation between the blood meal viral titer used in the experiment (mean SE; 6.47 0.098 log10 PFU/ 0.2ml, 6.2-6.8 log10 PFU/0.2 ml range). It was desirable to compare competitive treatments for females exposed to identical blood meal titers. Logistic cons traints precluded determining whether different bloodmeal viral titers produced differences in mosquito infection parameters. Further, different numbers of females were avai lable for each bloodfeeding trial with many treatments having few females available to bloodfeed. This presented a problem since it prevented comparisons of all competitive trea tments within a give n bloodfeeding trial. Thus, females were compared using those th at completed the extrinsic incubation period from the first bloodfeeding (6.2 log10 PFU/0.2ml) which represented ca. 50 % of the total number of females available to assay. Assaying less than all available females for DENV-2 infection assumes that females from th e first bloodfeeding ar e representative of all females from the competition experiment Similar outcomes of competition-induced changes in infection parameters from a previo us study support this a ssumption (Alto et al. 2005a). Mean SE females assayed per tr eatment replicate were; 160:0 (16.56 1.41), 320:0 (12.5 1.60), 160:160 ( A. albopictus ) (8.29 2.07), 160:160 ( A. aegypti ) (17.90 0.98), 0:320 (19.54 0.45), and 0:160 (20.00 0).

PAGE 95

83 Interspecific differences (i.e., A. albopictus versus A. aegypti ) in susceptibility to infection with DENV-2 were compared and pr oportions infected a nd proportions with disseminated infection were signifi cantly different between the two Aedes species (Pillais trace 2, 56 = 0.58, P < 0.0001). Proportion with disseminated infection (SCC = 1.19) contributed more to the overall inters pecific difference than proportion infected (SCC = -0.88). The opposite signs of the SCCs showed a negative relationship between infection and dissemination, from the fact that A. albopictus had a greater proportion of infected females but a lower proportion of disseminated infections compared to A. aegypti (Fig. 4-4). Body titer was signifi cantly different between species ( F1, 47 = 85.26, P < 0.0001) and A. albopictus (mean SE, 4.6 0.06 log10 PFU/0.2 ml) had greater body titer compared to A. aegypti (mean SE, 3.9 0.04 log10 PFU/0.2 ml). Competition had significant effects on th e proportion infected and proportion with disseminated infections for A. albopictus (Table 4-2). Proportion infected provided the largest contribution to bivariate differen ces among treatments and proportion with disseminated infection contri buted less (Table 4-2). Aedes albopictus at low density had significantly lower proportion infected and disseminated infection compared to high density intraand interspecific competition (Table 4-2, Fig. 4-5). The two high density treatments did not differ significantly (Table 4-2, Fig. 4-5). Competitive treatments resulted in similar trends of infection and dissemination for A. aegypti however, there were no significant effect s (Table 4-2, Fig. 4-6). Separate analyses of covariance with m ean female size as a covariate and mean body titer, proportion infected, and proportion with disseminated infection showed no significant competitive treatment or covariate effects on body titer for A. albopictus

PAGE 96

84 (Table 4-3, Fig. 4-7). There were marginally significant effects of size x competition interaction, as shown by regression lines for the three competitive treatments, and significant competition e ffects for proportion of A. albopictus infected (Table 4-3, Fig. 48). Pairwise comparisons of LS means among competitive treatments showed a significantly greater proportion of A. albopictus infected at both high density intraand interspecific competitive treatments than low density treatment (LS means shown in Fig. 4-5). Proportions infected were not significantly different between high density intraand interspecific competitive treatments (LS means shown in Fig. 4-5). There was no significant size effect for proportion of A. albopictus infected (Table 4-3, Fig. 4-8). There were marginally significant effects of competition on A. albopictus proportion with disseminated infection (Table 4-3). Pa irwise comparisons of LS means among competitive treatments showed marg inally significant differences ( P = 0.0501) in proportion of A. albopictus with disseminated infection between low density versus high density interspecific competition with no ot her significant effects (LS means shown in Fig. 4-5). There was no significan t size effect for proportion of A. albopictus with disseminated infection (Table 4-3, Fig. 4-9). For A. aegypti there were no significant competitive treatment or covariate eff ects (Table 4-3, Figs. 4-10, 4-11, 4-12). Product-moment correlations showed si gnificant relationships between the proportions of A. albopictus infected and with disseminated DENV-2 infections and time to emergence and size (Table 4-4). Time to emergence, positively a ssociated with intraand interspecific competition, was positively co rrelated with the proportion infected and disseminated infection whereas size was negatively correlated with the proportion infected and disseminated in fection (Table 4-4).

PAGE 97

85 Species by Competition Comparison A two-way MANOVA showed significant species ( A. albopictus and A. aegypti ) and competitive treatment (160:0, 320:0, 0:160, 0:320) effects on population growth measurements, but no species x competitive treatment interaction (Table 4-5). SCC showed that differences in adult size and tim e to emergence contributed the most to the significant species and competitive treatment effects (Table 4-5). Aedes aegypti had significantly shorter time to emergence (mean SE d; 12.81 0.29 and 13.74 0.29 for A. aegypti and A. albopictus respectively) and larger body size (mean SE mm; 2.83 0.02 and 2.74 0.02 for A. aegypti and A. albopictus respectively) than A. albopictus Crowded larval conditions (320 larvae) result ed in smaller size (mean SE mm; 2.65 0.02 and 2.92 0.02 for 320 and 160 larvae, respec tively) and longer time to emergence (mean SE d; 15.49 0.29 and 11.07 0.30 fo r 320 and 160 larvae, respectively). A two-way MANOVA showed significant species ( A. albopictus and A. aegypti ) and competitive treatment (160:0, 320:0, 0:160, 0:320) effects on proportion infected and proportion with disseminated infection, but no species x competitive treatment interaction (Table 4-6). SCC showed that differences in the proportion of females with disseminated infections contributed the most to the signi ficant species effect, whereas differences in the proportion infected contributed the most the significant competitive treatment effect (Table 4-6). Aedes aegypti had lower infection rates (mean SE proportion infected; 0.75 0.02 and 0.88 0.02 for A. aegypti and A. albopictus respectively) but higher dissemination rates (mean SE proportion wi th disseminated infection; 0.60 0.03 and 0.32 0.03 for A. aegypti and A. albopictus respectively) than A. albopictus. Crowded larval conditions (320 larvae) resulted in higher infection rates (mean SE proportion infection; 0.88 0.02 and 0.76 0.03 for 320 and 160 larvae, respectively) and

PAGE 98

86 dissemination rates (mean SE proportion wi th disseminated infection; 0.50 0.03 and 0.42 0.03 for 320 and 160 larvae, respectively) than uncrowded conditions (160 larvae). A two-way ANOVA showed significant species and species x competitive treatment interaction effect s on body titer (Table 4-7). Aedes albopictus had significantly greater body titer than A. aegypti but this interspecific effect depended on competitive treatment (Fig. 4-13). The species x competitiv e treatment interaction was attributable to less interspecific difference ( A. albopictus versus A. aegypti ) in body titer from crowded larval conditions (320 larvae) compared to uncrowded conditions (160 larvae) (Fig. 413). Separate one paired t-tests on infection parameters in the interspecific competitive treatment (i.e., 160:160) showed significan t differences in the proportion infected, proportion with disseminated in fections, and body titer (All P < 0.05 ). Aedes albopictus had significantly greater proportion infected (mean SE; A. albopictus A. aegypti ; 0.93 0.04, 0.82 0.03), lower proportion with di sseminated infection (mean SE; A. albopictus A. aegypti ; 0.47 0.09, 0.71 0.05), and grea ter body titer (mean SE; A. albopictus A. aegypti 4.50 0.08, 3.99 0.05) than A. aegypti Discussion All population growth measurements showed consistently poorer performance for mosquitoes reared at high larval density (Fig s. 4-1, 4-2, 4-3). These results are consistent with those of a similar experiment, using Aedes species in tests of competitive treatment effects on adult SINV infection (Alto et al. 2005a). The goal of the SINV and DENV experiments was to maximize adult mosquito production, to assess vector competence, without negating the effects of larval comp etition. To achieve this, a combination of natural (oak leaf infusion) a nd artificial (yeast, albumin) la rval food resources was used.

PAGE 99

87 Previous laboratory and field research shows contrasting outcomes of competition between these Aedes species dependent upon larval resource type, with A. aegypti an equal or superior competitor with protein-rich resources (e.g., liver powder, yeast) and A. albopictus a superior competitor with plant detr itus (e.g., leaves) (Juliano and Lounibos 2005, Braks et al. 2004, Juliano 1998, Barrera 1996, Black et al. 1989). The present study confirms the ability to replicate envir onmetal variables that provide for larval competition between A. aegypti and A. albopictus Higher levels of intraand interspecific competition significantly enhanced DENV2 infection and dissemination for A. albopictus. Phenotypic expres sion of vector competence in adult mosquitoes was signifi cantly altered by competitive conditions of the aquatic larval environment. These results may be the pr oduct of norms of reaction of the genes controlling vector competence in these mosquitoes. Other studies on competition and phenotypic expression of a trait have shown that the phenotypic expression of virulence is altered when hos ts were exposed to different competitive conditions or nutrient gradients (e.g., Bedho mme et al. 2004, Scheiner 1993). Vector competences studies have shown that there is evidence that a great amount of phenotypic variance in DENV-2 infection in A. aegypti is associated with environmental and random experimental effects (e.g., Bosio et al. 2000). The proportion of infected A. albopictus females contributed more to the competitiv e treatment effects than the proportion of females with disseminated infections (Table 42). This observation s uggests that initial infection in the adult mosquito midgut is most influenced by larval competition.than escaping the midgut and infecting ot her organs (i.e., dissemination).

PAGE 100

88 The lack of significant covariate (size) effects in the analyses of covariance suggests that size had no direct effect on vector competence within competitive treatments for both species (160, 320, 160:160) (Table 4-3). A marginally significant interaction between the covariate an d competitive treatment for proportion A. albopictus infected (Table 4-3) suggests that the eff ect of mosquito size on infection may depend on competition experienced. Among replicates of the low density treatments, which produced females of the largest average si zes, proportion infect ed declined with increasing mean body size. This decline is not evident in smaller females from high density treatments (Fig. 4-8). Significant or marginally significant effects of competition were observed for A. albopictus because more intense competition resulted in greater DENV-2 infection and disseminated infection (Figs. 4-8, 4-9). Thus, differences in Aedes size, within competitive treatments make little or no contributi on to the differences observed in infection parameters. Although mean size may contribute little to differences in infection parameters within competitive treatments, mean size wa s significantly correlated with infection parameters across competitive treatments, perh aps due to competitive effects (Table 4-4, Figs. 4-8, 4-9). Specifically, small adult female s, associated with intraand interspecific competition, had enhanced infection and dissemination rates for A. albopictus (Table 44). Under the conditions of the current study small A. albopictus produced by competitive treatments may pose a greater health risk for DENV transmission compared to larger conspecifics. This effect may be enhanced because small adults may bloodfeed more frequently than larger adults (Scott et al. 2000b). However, competitive treatment differences in size may be correlated with overall competitive stress, making it difficult to

PAGE 101

89 determine whether size alone was correlated with variation in infection parameters between treatment groups. Female longevity, host attack rates, and bloodfeeding success, important contributors to vector potential, are positively related to size (Xue et al. 1995, Willis and Nasci 1994, Nasci 1991, Nasci 1987, Nasci 1986ab, Hawley 1985, Haramis 1985, 1983), so determining the epidemiologi cal importance for DENV transmission of different sized individuals coming from differing competitive environments requires interpretation of multiple life history parameters. Contrary to infection and dissemination results, no significant correlations were observed between size and body titer for either Aedes species (Table 4-4) consistent with results using two A. aegypti strains and DENV-2 (Bosio et al. 1998). Thus, size of A. albopictus mosquitoes is correlated with DENV-2 infection and dissemination, but not body titer. This suggests that different mechanisms are responsible for associati ons between size and different vector competence traits. A midgut barrier has been proposed for mosqu itoes that become infected but fail to spread arboviruses beyond the midgut to sec ondary tissues (i.e., disseminate) (Thomas et al. 1993, Houk et al. 1986, Weaver et al 1984, Hardy et al. 1983). Enhanced dissemination and transmission of LACV by small O. triseriatus adults was suggested to be a result of fewer basal lamina layers present in smaller adults, thus weakening the midgut escape barrier (Grimstad and Wa lker 1991, Paulson and Hawley 1991). Midgut basal laminae thickness was signi ficantly different among three A. albopictus strains, suggesting genetic control, and thickness was inversely related to dissemination of DENV-1. However, within an A. albopictus strain, basal laminae thickness did not differ between disseminated and nondisseminated female infections (Thomas et al. 1993).

PAGE 102

90 Thus, although basal laminae th ickness may vary with adult size and, in part, determine dissemination, other mechanical and physiological factors are also like ly involved such as stress-induced midgut perforation or viral modulation. A description of the mechanism(s) responsible for the observed results was beyond the scope of these studies. Similar results were reported for A. albopictus vector competence for SINV (Alto et al. 2005a), and may suggest a common physiolo gical (e.g., repressed innate immune function) (Sanders et al. 2005) or mechanical (e.g., leaky midgut hypot hesis) (Chandler et al. 1998, Weaver et al. 1991, Weaver 1986, Hardy et al. 1983, Miles et al. 1973, Boorman 1960) mechanism(s) responsible for the observed results. Correlation analyses suggested that life hi story traits, in addition to size, were associated with DENV-2 infection parameters (Table 4-4). Similar to size correlations, longer time to emergence for A. albopictus associated with greater intraand interspecific competition, was associated with enhanced infection and dissemination (Table 4-4). Taken together, results show consistent asso ciations of reduced fitness measurements with higher A. albopictus DENV-2 infection parameters, but not A. aegypti suggesting species differences in infecti on responses. These results agree with other reports investigating competitive stress a nd infectibility for a variety of systems, where competition in the form of nutrien t limitation (Koella a nd Srensen 2002, Beck and Levander 2000, Oppliger et al. 1998, Su wanchaichinda and Paskewitz 1998, Morris and Potter 1997, Lively et al. 1995, Matson and Waring 1984, Steinhaus 1958) and other stressors (Lafferty and Holt 2003, Kieseck er and Skelly 2001) increases host susceptibility to infection by pathogens and parasites.

PAGE 103

91 The current experimental results reflect only females assayed from the first infectious bloodfeeding. These females had shorter time to emergence than females given infectious bloodmeals at later dates. Th is experimental approach assumes that the females assayed for DENV infection are represen tative, in terms of infection parameters, of all females from the entire duration of the competition experiment. It is not clear how longer time to emergence, and associated di fferences in subsequent adult physiology, may alter infection parameters. Intuition sugg ests there may be a positive relationship so that longer time to emergen ce is associated with enhan ced infection parameters, as suggested by the correlation analyses (Table 4-4). However, this reasoning may be simplistic and may not capture temporal diffe rences in competitiv e interactions among larvae. For example, adult females associat ed with longer time to emergence may be initially exposed as larvae to intense competition, followed by release from competition when competitors are removed from th e environment (mortality, emergence to adulthood). Thus, differences in infection pa rameters may be associated with temporal differences in intensity of competition. Additio nal studies in the future will specifically focus on addressing the relations hip between time to emergence and infection parameters. Results from these studies will address whether females emerging early in a competitive experiment are similar, in terms of infecti on parameters, to females with longer time to emergence. Beyond the effects of competition, an inte rspecific comparison showed significant differences between A. aegypti and A. albopictus infection parameters. Aedes albopictus had a significantly greater proportion of DENV infected fe males, but a lower proportion of disseminated females, compared to A. aegypti The mosquitoes used in the current

PAGE 104

92 research were derived from well-establishe d laboratory colonies. Although it is well accepted that laboratory colonization may alter A. albopictus and A. aegypti susceptibility for DENV and yellow fever virus (YFV) respec tively (Vazeille et al 2003, Lorenz et al. 1984), the current research results do agree wi th results for multiple mosquito strains (F1-2 generation) and different DENV serotypes (DENV-1, mosquitoes F5) (Chen et al. 1993). A. aegypti had significantly greater DENV-2 disseminated infection compared to A. albopictus using three Southeast Asian DENV-2 stra ins, including an almost identical strain used in the current experiment (V azeille et al. 2003). The current experiment represents a detailed comparison of DENV-2 vector competence between A. aegypti and A. albopictus including information on species-sp ecific infection, dissemination, and whole body titer. Accurate description of relative viral susceptibility of A. aegypti and A. albopictus requires a comparison of multiple infection parameters, especially since some pairs of infection parameters yield interspe cific differences of opposite direction (e.g., infection: A. albopictus > A. aegypti dissemination; A. albopictus < A. aegypti ) (Fig. 44). However, it is important to recognize th at vector competence is only one compenent in determining DENV transmission in nature (i .e., vectorial capacity) and the roles of these two vector species in DENV transm ission may be dynamically changing depending on both genetic and environmental parameters. Species-specific variation in DENV-2 inf ection parameters may be viewed as fundamental differences in physiology between these Aedes species and the outcome of infection. Genetic studies on DENV-2 have mapped several quantitative trait loci controlling DENV-2 midgut inf ection (Gomez-Machorro et al. 2004, Bosio et al. 2000) and dissemination (Bennett et al. 2005a) in A. aegypti Similar studies have not been

PAGE 105

93 done on A. albopictus However, quantitative trait loci controlling infection parameters may depend on mosquito and virus strains. In addition to differences in infection and dissemination, A. albopictus had on average significantly greater viral body titer compared to A. aegypti yet it disseminated DENV-2 poorl y. Bosio et al. (1998) showed that DENV-2 dissemination rates in A. aegypti were independent of midgut virus titer. Interspecific differe nces in body titer of Aedes females with disseminated infections suggest that factors limiting viral replication in A. aegypti were more efficient compared to A. albopictus These results were unexpected a nd may represent species-specific differences in viral selection pressure within the vector host. This study represents the most extensive DENV body titer comparison betw een these two Aedes species so it is not clear whether other mosquito and DENV strains show Aedes -specific differences in viral load. A more efficient midgut escape barrier in A. albopictus than A. aegypti may select for greater viral replication, and associated body titer, since failure to escape the midgut results in failure to infect the next host. Alternatively, A. albopictus may merely be a better physiological environment for DENV-2 replication than A. aegypti regardless of barriers The observed Aedes -specific difference in body tit er represents a favorable situation to test hypotheses th at integrate host-pathogen theo ry and vectorial capacity. Vectorial capacity, the daily rate at which future pathogen inoculations arise from a current infective case, are sensitive to changes in daily survivorship of the vector. Thus, small changes in DENV-induced adult survivor ship (Fernandez et al. 2003) may result in relatively large differences in vectorial capacity (Dye 1986). Although arboviruses are assumed to have little negative effects on their arthropod vectors, many exceptions are

PAGE 106

94 known (Platt et al. 1997, Fara n et al. 1987, Turell et al 1985, Beaty et al. 1980, Tesh 1980, Grimstad et al. 1980, Mims et al. 1966). Host-parasite theory suggests parasite/pathogen load is inversely relate d to host fitness (e .g., Anderson and May 1978, May and Anderson 1978). Future experiments should address DENV-induced differences in adult Aedes longevity and reproductive succ ess and its contribution to the relative importance of A. aegypti and A. albopictus in DENV transmission (e.g., longevity and vectorial cap acity), especially in symp atric locations for these Aedes species where dengue is endemic. The observation that A. aegypti had a greater proportion of females with disseminated infections suggest th at the midgut escape barrier in A. aegypti (Bennett et al. 2005ab, Bosio et al. 2000, 1998) wa s less efficient at limiting spread of viral infection to secondary tissues compared to A. albopictus These species-specific differences in dissemination are consistent with the observation that A. aegypti is the more important vector in DENV transmission to humans. However, in the current study, the actual number of DENV transmitting mosquitoes (a ssumes dissemination is an indicator of potential to transmit) may be more similar between A. aegypti and A. albopictus when simultaneously accounting for both proportion infection ( A. albopictus > A. aegypti ) and proportion with disseminated infections ( A. albopictus < A. aegypti ) (Fig. 4-4). The proportion of potential transmitters are equiva lent to the product of the proportion of females infected and the proportion of females with disseminated infection ( A. albopictus 0.33; A. aegypti 0.49). The species by competition comparison an alyses (i.e., two-way MANOVAs and two-way ANOVA) showed si gnificant effects of Aedes species and competitive

PAGE 107

95 treatments on population growth measurements a nd infection parameters (Table 4-5, 4-6). Fundamental physiological differences in Aedes size and time to emergence were responsible for the species eff ect (Table 4-5, SCC), so that A. aegypti performed better than A. albopictus under the experimental conditions. Other authors have demonstrated that A. aegypti develop more quickly than A. albopictus with nutrient rich resources (e.g, albumin, yeast) (Klowden and chambers 1992) Also, interspecific differences in infection parameters (Table 4-6) show A. aegypti was less resistant to limiting the spread of DENV infection, as shown by higher dissem ination rates. Similar to the other analyses, competitive treatment effects were realized by longer time to emergence and smaller adult size (Table 4-5). Also, competitive treatment effects enhanced infection parameters (Table 4-6), especially proportion females infected. There were no species x competitive treatment interactions for populat ion growth measurements or infection parameters. These later results we re surprising since it suggests that A. albopictus and A. aegypti infection parameters respond similarly to competition. These results differ compared to results from the analyses done separately for each species (i.e., one-way MANOVAs). However, in these later analyses A. aegypti did show a trend, although not significant, for competition-induced differences in infection parameters (Fig. 4-6). Discrepancy between results of different anal yses on infection parameters may be due to greater power in the current analyses compared to analyses of each species separately. Species x competitive treatment interactions were found for DENV body titer (Table 4-7) so that differences in Aedes species body titers were more similar under crowded larval conditions (Fig. 4-13), perhaps due to enhanced stress. Although these analyses were

PAGE 108

96 interesting, they are limited to capturing only the results from intraspecific competitive treatments. Competitive interactions among larval stages alter adult A. albopictus infection parameters for DENV with the general association of increased larval competition increasing adult susceptibility to DENV infection. This phenomenon suggests the importance of an under explored connection between mosquito larval ecology and the epidemiology of arboviruses. Mathematical models of arthropod-borne infectious diseases have generally ignored the effects of larval environment on vector competence, assuming a black box approach to all infec tion dynamics within the vector (Dye 1986). The results of this study sugge st that larval conditions are an important aspect of DENV biology, and should be included in future cons iderations of dengue epidemiology. Recent studies have begun to relate landscape m easures (e.g., vegetation cover, rainfall, temperature, and container de nsity) and vector ecology, spec ifically in the context of competition at the larval stag e (Barrera et al. 2006, Schneid er et al. 2004, Morrison et al. 2004). The current research bridges the gap between these studies associating landscape and DENV vector potential (Kolivras 2006, Barrera et al 2006, Morrison et al. 2004, Schneider et al. 2004, Kuno 1997). In this study, I have shown that environmentalinduced differences in adult size, as well as other life history traits, may play an important role in determining vector co mpetence and vectorial capacity (e.g., altered infection parameters, longevity, bloodfeeding frequency) and should be considered in vector intervention methods aimed at reducing dengue and in building re alistic, predictive models of dengue epidemiology (e.g., Schneider et al. 2004, Luz et al 2003, Smith et al. 2004, Focks et al. 2000, 1995).

PAGE 109

97 9 10 11 12 13 14 15 16 17 18 2.52.62.72.82.93Size at emergence (mm)Time to emergence (days) 160:0 320:0 160:160 a a b Figure 4-1. Aedes albopictus least squares means ( SE) for female size and time to emergence. Different letters indicate significant differences between bivariate means. Competition treatments consisted of species density ratios of A. albopictus : A. aegypti :0, 320:0, 160:160. 9 10 11 12 13 14 15 16 17 18 2.52.62.72.82.93Size at emergence (mm)Time to emergence (days) 0:160 0:320 160:160 a a b Figure 4-2. Aedes aegypti least squares means ( SE) for female size and time to emergence. Different letters indicate significant differences between bivariate means. Competition treatments consisted of species density ratios of A. albopictus : A. aegypti :160, 0:320, 160:160.

PAGE 110

98 1.08 1.1 1.12 1.14 1.16 1.18 1.2 0123456Competitive treatment ( A. albopictus:A. aegypti )Estimated finite rate of increase A. albopictus A. aegypti 160:0320:0160:1600:3200:160a b c A B C Figure 4-3. Least squares means ( SE) fo r estimated finite rate of increase, for A. albopictus and A. aegypti Points without bars have standard errors too small to be visible. Different lowercase an d uppercase letters i ndicate significant differences between means for A. albopictus and A. aegypti respectively.

PAGE 111

99 0.6 0.7 0.8 0.9 1 0.20.30.40.50.60.7Proportion with disseminated infectionProportion infected A. albopictus A. aegypti A B Figure 4-4. Least squares ( SE) for proportion of A. albopictus and A. aegypti infected and disseminated infections. Uppercase letters indicate significant differences between bivariate means. 0.5 0.6 0.7 0.8 0.9 1 0.150.250.350.450.550.65Proportion with disseminated infectionProportion infected 160:0 320:0 160:160 A B B Figure 4-5. Bivariate plots of least squares means ( SE) for proportion of A. albopictus infected and disseminated infections. Competition treatments consisted of species density ratios of A. albopictus : A. aegypti 160:0, 320:0, 160:160. Different letters indicate significant differences between bivariate means.

PAGE 112

100 0.6 0.65 0.7 0.75 0.8 0.85 0.9 0.40.50.60.70.8Proportion with disseminated infectionProportion infected 0:160 0:320 160:160 A A A Figure 4-6. Bivariate plots of least squares means ( SE) for proportion of A. aegypti infected and disseminated infections. Competition treatments consisted of species density ratios of A. albopictus : A. aegypti 0:160, 0:320, 160:160. Absences of different letters indica te no significant di fferences between bivariate means. 3.6 3.8 4 4.2 4.4 4.6 4.8 5 2.42.52.62.72.82.933.1Size (wing length mm)log10 PFU/0.2 ml 160:0 320:0 160:160 Figure 4-7. Least squares means for body titer and size of adult A. albopictu s females with disseminated (i.e., infection spread beyond the midgut, infecting secondary target organs) dengue-2 virus infections. Numbers in the figure key represent the ratio of larval A. albopictus to A. aegypti

PAGE 113

101 y = 0.0287x + 0.8975 r2 = 0.0027 y = -0.4423x + 2.0716 r2 = 0.1154 y = -2.2168x + 7.2284 r2 = 0.37630 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 2.42.52.62.72.82.93Size (wing length mm)Proportion Infected 160:0 320:0 160:160 Figure 4-8. Least squares means for proportion infected and size of adult A. albopictus females. Solid and dashed lines draw n through bivariate means show the best fit for A. albopictus in three competitive treatme nt conditions. Regression equations, with associated r2 values, are shown for competitive treatments. Numbers in the figure key repr esent the ratio of larval A. albopictus to A. aegypti. 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 2.42.52.62.72.82.93Size (wing length mm)Proportion dissemination 160:0 320:0 160:160 Figure 4-9. Least-squares means for proportion disseminated infections and size of adult A. albopictus females. Numbers in the figure key represent the ratio of larval A. albopictus to A. aegypti.

PAGE 114

102 3.4 3.6 3.8 4 4.2 4.4 4.6 4.8 5 2.42.52.62.72.82.933.1Size (wing length mm)log10 PFU/0.2 ml 0:160 0:320 160:160 Figure 4-10. Least-squares mean s for body titer and size of adult A. aegypti females with disseminated (i.e., infection spread beyond the midgut, infecting secondary target organs) dengue-2 virus infections. Numbers in the figure key represent the ratio of larval A. albopictus to A. aegypti. 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 2.52.62.72.82.933.1Size (wing length mm)Proportion Infected 0:160 0:320 160:160 Figure 4-11. Least-squares means for proportion infected and size of adult A. aegypti females. Numbers in the figure ke y represent the ratio of larval A. albopictus to A. aegypti.

PAGE 115

103 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 12.52.62.72.82.933.1Size (wing length mm)Proportion dissemination 0:160 0:320 160:160 Figure 4-12. Least-squares means for propor tion disseminated infections and size of adult A. aegypti females. Numbers in the fi gure key represent the ratio of larval A. albopictus to A. aegypti. 3.5 3.7 3.9 4.1 4.3 4.5 4.7 4.9100140180220260Competitive treatment (number of larvae)log10PFU/0.2 ml A. albopictus (160) A. albopictus (320) A. aegypti (160) A. aegypti (320)160320 a a b b Figure 4-13. Two-wa y ANOVA for species ( A. albopictus and A. aegypti ) and competitive treatment ( A. albopictus : A. aegypti 160:0, 320:0, 0:160, and 0:320) effects on Least-squares means for body titer.

PAGE 116

104Table 4-1. MANOVA and multivariate pairwise contrasts of competitive treatment effects on female Aedes albopictus and A. aegypti population growth measurements: time to emergence, survivorship to emergence, and adult size. Standardized Canonical Coefficients Comparison df Pillais trace P Time Survivorship Size A. albopictus Competitive treatment 6 0.86 < 0.0001 0.84 0.31 1.13 160:0 vs. 320:0 3 0.71 < 0.0001 0.76 0.43 1.13 160:0 vs. 160:160 3 0.74 < 0.0001 0.90 0.22 1.12 320:0 vs. 160:160 3 0.11 0.3826 Error df 28 A. aegypti Competitive treatment 6 0.88 < 0.0001 0.96 0.27 1.62 0:160 vs. 0:320 3 0.85 < 0.0001 0.99 0.28 1.59 0:160 vs. 160:160 3 0.78 < 0.0001 0.90 0.23 1.69 0:320 vs. 160:160 3 0.17 < 0.1844 Error df 28

PAGE 117

105Table 4-2. Multivariate ANOVA for main effects and multivariate pa irwise contrasts of competitive treatment effects on female Aedes albopictus and A. aegypti proportion infected and proportion with di sseminated infection. Standardized Canonical Coefficients Comparison df Pillais trace P Infection Dissemination A. albopictus Competitive treatment 4 0.51 0.0057 1.04 0.61 160:0 vs. 320:0 2 0.41 0.0022 1.11 0.49 160:0 vs. 160:160 2 0.36 0.0060 0.89 0.77 320:0 vs. 160:160 2 0.06 0.4897 Error df 24 A. aegypti Competitive treatment 4 0.23 0.1394 Error df 28

PAGE 118

106Table 4-3. ANCOVA (after testi ng for equality of slopes) for the effects of competitive trea tment and size covariate on body t iter, proportion infected, and proportion with disseminated infection for Aedes albopictus and A. aegypti females. Body titer refers only to females with disseminated dengue-2 infections. Size x competition interactions with P > 0.05 are not shown except for marginally significant inte ractions. Error dfs reflect final reduced analyses (ANCOVA or ANOVA). Source df F P A. albopictus Titer (infection disseminated) Size 1 0.99 0.3335 Competitive treatment 2 1.99 0.1672 Error df 17 Infection Size 1 1.90 0.1812 Competitive treatment 2 7.10 0.0038 Size x competition 2 3.39 0.0529 Error df 21 Dissemination Size 1 1.87 0.1843 Competitive treatment 2 3.24 0.0568 Error df 24 A. aegypti Titer (infection disseminated) Size 1 0.01 0.9285 Competitive treatment 2 2.38 0.1123 Error df 26

PAGE 119

107Table 4-3 (continued) Infection Size 1 1.71 0.2022 Competitive treatment 2 2.87 0.0737 Error df 28 Dissemination Size 1 0.27 0.6064 Competitive treatment 2 2.32 0.1167 Error df 28

PAGE 120

108Table 4-4. Product-moment correlation coefficients (r1,2) for the relationship between populatio n growth measurements (time to emergence, survivorship, size, and ) and infection parameters for A. albopictus (df=25) and A. aegypti (df=29). Replicates in which only a single female body titer was measured were excluded from correlations, resulting in 18 and 27 dfs for A. albopictus and A. aegypti respectively. ** P < 0.01, *** P < 0.001, and **** P < 0.0001 show significant correlation coefficients. A sequential Bonfer onni adjustment corrected experimentwise =0.05 for 12 comparisons for A. albopictus and A. aegypti respectively. A. albopictus (r1,2) A. aegypti (r1,2) Infection Parameter Growth Correlates Infection Time to Emergence 0.50 ** 0.38 Survivorship 0.15 0.22 Size 0.62 **** 0.41 0.43 0.45 Dissemination Time to Emergence 0.55 *** 0.15 Survivorship 0.04 0.39 Size 0.50 ** 0.22 0.39 0.41 Body Titer (disseminated) Time to Emergence 0.43 0.26 Survivorship 0.05 0.22 Size 0.28 0.29 0.31 0.37

PAGE 121

109Table 4-5. Two-way MANOVA of species ( A. albopictus and A. aegypti ) and competitive treatment ( A. albopictus : A. aegypti 160:0, 320:0, 0:160, and 0:320) effects on female population growth measurements: time to emergence, survivorship to emergence, and adult size. Standardized Canonical Coefficients Comparison df Pillais trace P Time Survivorship Size Species 3 0.25 0.0133 0.82 0.04 1.69 Competitive treatment (trt.) 3 0.86 < 0.0001 1.23 0.53 1.24 Species x Competitive trt. 3 0.13 0.1523 Error df 38

PAGE 122

110Table 4-6. Two-way MANOVA of species ( A. albopictus and A. aegypti ) and competitive treatment ( A. albopictus : A. aegypti 160:0, 320:0, 0:160, and 0:320) effects on pr oportion infected and proportion w ith disseminated infections. Standardized Canonical Coefficients Comparison df Pillais trace P Infection Dissemination Species 2 0.64 < 0.0001 0.86 1.24 Competitive treatment (trt.) 2 0.27 < 0.0035 1.17 0.45 Species x Competitive trt. 2 0.08 0.2136 Error df 37 Table 4-7. Two-way ANOVA for species ( A. albopictus and A. aegypti ) and competitive treatment ( A. albopictus : A. aegypti 160:0, 320:0, 0:160, and 0:320) effects on body titer. Comparison df F P Species 1 72.65 < 0.0001 Competitive treatment (trt.) 1 0.83 0.3706 Species x Competitive trt. 1 5.18 0.0299 Error df 31

PAGE 123

111 CHAPTER 5 COMPETITION, ARBOVIRUS INFECTI ON, AND FUTURE EXPERIMENTS Competition and Enhanced Infection Typically, vector competence studies are performed on adult mosquitoes derived from larvae that developed with a surplus of food and space. Rearing larvae in this manner, particularly container species such as A. aegypti and A. albopictus fails to recreate their stressful larval environment. Our lack of knowledge of how larval conditions, particularly competition, inter act with vector competence is a gap in understanding the transmission of arboviruses. In the current rese arch, the effects of larval competition, as determined by populat ion growth measurements, were preserved while allowing sufficient adult production to examine quantitatively how larval competition impacted arbovirus infection. For both larval competition experiments, intense competition was associated with reductions in mosquito fitness measurements (Chapter 3, Figs. 3-1, 3-2, 3-3; Chapter 4, Figs. 4-1, 4-2, 4-3). Previous studies usi ng highly nutritious re sources (yeast, liver powder) showed that A. aegypti was the superior larval co mpetitor (Barrera 1996, Black et al. 1989). However, the outcome of in terspecific competition experiments between A. albopictus and A. aegypti using natural resources (leaves) showed that A. albopictus was the superior larval compet itor (Juliano and Lounibos 2005, Braks et al. 2004, Juliano 1998). Supplementing natural reso urces (leaves) with inverteb rate carcasses, a nutrient rich resource, reduces resource limitation and the competitive advantage of A. albopictus, and may promote coexistence (Daugherty et al. 2000). A comparative study of leaves

PAGE 124

112 and enriched resources, confirmed these variable outcomes and concluded that the outcome of competition between these species is likely to depend on resource type (Barrera 1996b), as in othe r systems (e.g., Tilman 1982). In the current studies, interspecific competition between these Aedes species as measured by was more intense than intraspecific competition (Chapt er 3, Fig. 3-3; Chapter 4, Fig. 4-3). These results suggest an unstable competitive equilibrium, where the outcome of competition depends not only on resources (see above) but also on the initial abundance of the two species, so that the more abundant species co mpetitively excludes the other species (i.e., Grover 1997). Similar designs in the two competiti on experiments allowed for robust crossexperiment comparisons of infection para meters in adult mosquitoes. The two competition experiments yielded similar competitive treatment effects on adult infection parameters for the two unrelated viruses. Specifically, reduced mosquito fitness, associated with intense competition, enhan ced infection parameters for SINV and DENV (Chapter 3, Table 3-3, Figs. 3-4, 3-5; Chapter 4, Table 4-4, Fig. 4-5). Aedes albopictus infection parameters showed strong effect s of larval competition, with proportion infected with SINV and DENV contributing th e most to competitive treatment effects relative to the other infection parameters meas ured (Chapter 3, SCC in text; Chapter 4, Table 4-2). Aedes aegypti showed no competition-induced changes in infection parameters for the viruses examined suggesting differences in competition-induced responses of A. aegypti and A. albopictus infection parameters (Tables 3-3, 4-4). Although general effects of competition were similar for both experiments, population growth measurements showed that the intensity of competition was slightly

PAGE 125

113 less in the DENV experiment (Chapter 3, Figs 3-1, 3-2, 3-3; cf. Ch apter 4, Figs. 4-1, 4-2, 4-3). However, time to emergence in the high density treatments (320 and 160:160) were longer in the DENV experiment than in th e SINV experiment (Chapter 3, Fig. 3-2, LS means in text; cf. Chapter 4, Figs. 4-1, 4-2). Adult size and competitive treatments had independent and opposite effects on SINV body titer for A. albopictus so that larger size and more intense competitive conditions were both associated w ith greater body titer (Chapter 3, Fig. 3-5). The lack of size e ffects within competitive treatments on DENV infection parameters (Chapter 4, Table 43) may suggest virus-specific effects on infection parameters associated with size. Alternatively, lack of size effects within competitive treatments may be related to larger sizes of mosquitoes in the DENV experiment, compared to the SINV experiment. Sizes of A. albopictus and A. aegypti from uncrowded larval conditions (160 larvae ) in the SINV experiment were similar to sizes of the two Aedes species from crowed larval c onditions (320 and 160:160 larvae) in the DENV experiment (Chapter 3, Fig. 3-1, in te xt; cf. Chapter 4, Figs. 4-1, 4-2). Larger sized mosquitoes in the DENV experiment may be, in part, a result of sampling females for size and infection only from the first DE NV infectious blood feeding (~ 50% of the females). These mosquitoes emerged to adulthood early in the competition experiment when larval food resources may have been more abundant relative to later in the experiment. In addition to size differences, fe males assayed for the DENV experiment had shorter time to emergence and it is not clear how these factor s may alter infection parameters. Differences between experiments in the expression of infection parameters associated with adult size may depend on the range of adult sizes. Size effects on DENV infection parameters were observed between competitive treatments (Chapter 4, Table 4-

PAGE 126

114 4) suggesting that size was related to DENV infection parameters, although over a greater range of sizes than observed in the SINV experiment. However, competitive treatment differences in size may be correlated with ot her life history traits, as well as overall competitive stress, making it difficult to determine whether size alone was correlated with variation in in fection parameters between treatment groups. Laboratory colonies of these Aedes species have traditionally been accepted as representative of their natu ral populations. However, laboratory colonization may alter infection parameters due to founder effects, genetic drift, and unintentional selection imposed on laboratory colonies (Wallis et al 1985, Lorenz et al. 1984). For example, laboratory colonies may be established from a small number of indivi duals and thus there is a higher probability of missing rare alleles compared to a colony derived from a larger number of founders (Munstermann 1994). La boratory colonization with associated selection and drift, even af ter only a few generations, may reduce heterozygosity and the number of alleles (Munstermann 1994, 1980). Despite these shortcomings the current experiments on competition and infection used well-established laboratory colonies of Aedes mosquitoes for two major reasons. First, it was unclear whether larval competition would have strong, subtle, or any effect on ad ult infection paramete rs. Minimizing intrapopulation variation in response to viral in fection, associated with greater genetic variability, should increase the likelihood of dete cting competition-induced effects on adult infection parameters. Secondly, the use of identical laborator y mosquito strains, and similar experimental design between e xperiments, allowed for comparisons of infection parameters for SINV and DENV. The use of F1 generation Aedes whose parents were collected from natural field populations from different years (i.e., SINV

PAGE 127

115 versus DENV experiments), would have c onfounded cross experiment comparisons of mosquito infection parameters for SINV and DENV due to undefined genetic variation in the vector populations. In addition to competitive effects on these Aedes species, there were also interspecific differences in infection parameters. A comp arison of mosquito species showed that A. albopictus had greater infection but lo wer dissemination of SINV and DENV than A. aegypti (Chapter 3, in text; cf. Chapter 4, Fig. 4-4). In contrast, viral body titer was significantly greater in A. aegypti for SINV and greater in A. albopictus for DENV (Chapter 3, in text; cf. Chapter 4, in te xt). Similar responses of the two vector species to SINV and DENV infection and disse mination may suggest similar mechanisms controlling these processes. Specifically, midgut infection barriers are more efficient in A. aegypti than A. albopictus and midgut escape barriers are more efficient in A. albopictus than A. aegypti There is a growing literatur e on the genetic controls of mosquito vector competence, particularly of A. aegypti vector competence for DENV. Genetic studies with DENV-2 have identifie d candidate genes (e.g., early trypsin gene coding for the proteolytic enzyme trypsin) asso ciated with susceptibility to infection (Gorrochotegui-Escalante et al. 2005) and mapped several quanti tative trait loci controlling midgut infection (Gomez-Machor ro et al. 2004, Bosio et al. 2000) and dissemination in A. aegypti (Bennett et al. 2005a), although si milar studies are lacking for A. albopictus These studies demonstrate that there is extensive geneti c variation in the types of loci and the genes at individual loci that control vector competence of A. aegypti for DENV. Species-specific differences in body titer suggest differen ces in the abilities of these closely related species to limit SINV and DENV replication.

PAGE 128

116 Future Studies Field-collected Mosquitoes for Infection Experiments The current experiments showed similar di rectional effects of competition-induced changes in SINV and DENV infection parameters for A. albopictus It is unclear whether genetically more heterogeneous mosquitoes would have similar in fection responses. Further insight may be gained from experiment s using mosquitoes more representative of natural populations. Future studies should consider experiments on larval competition and infection parameters using F1 generation Aedes whose parents were collected from natural field populations. The outcome of such additional experiments would answer whether conclusions based on Aedes laboratory colonies apply to field populations, and whether trends in infection parameters induced by competitive interactions are similar among more genetically heterogeneous populations. Laboratory competition experiments using F1 generation Aedes might be coupled with infection experiments using ad ults obtained from field-collected A. albopictus and A. aegypti pupae (e.g., Paulson and Hawley 1991). The size of Aedes adults emerged from field-collected pupae is an accurate indi cator of larval conditions (e.g., competition, temperature) because body size is fixed afte r emergence to adulthood. Moreover, female size is positively related to fitness. Sizes of A. aegypti and A. albopictus females in the SINV and DENV experiments were within th e range of sizes of field-collected individuals. However, there is a broader range of sizes of both these Aedes from field collections (Schneider et al. 2004, Scott et al. 2000b, Willis and Nasci 1994, Nasci 1991, 1990, 1986ab). The proposed experiment will determine whether a broader range of adult Aedes sizes are associated with a broader range of infection parameters.

PAGE 129

117 Mechanisms Responsible for Competition-enhanced Infection The focus of this research was to determine whether larval competition alters adult infection parameters for different arboviruse s. An investigation of the mechanisms responsible for the observed results was beyond the scope of these studies. However, it may be useful to consider mechanisms that may be responsible fo r competition-induced changes in adult infection parameters Similar effects of competition on A. albopictus infection parameters for unrelated arboviruse s highlight the importance of previously unappreciated ecological factor s in determining mosquito vector potential and may suggest a common physiological mechanism(s) responsible for the observed results (e.g., repressed innate immune system) (Sanders et al. 2005, Dimopoulos 2003). Mounting an immune response (melanization response, anti microbial peptides) comes at a cost of reduced fecundity in A. aegypti and Anopheles gambiae (Giles) (Schwartz and Koella 2004, Ahmed et al. 2002), demonstrating a trad e-off between host immunity and fitness components (Sheldon and Verhulst 1996). If similar trade-offs occur in the current system, competitively stressed individuals may be less able to mount an effective immune response, or perhaps pay a highe r fitness price comp ared to unstressed individuals. A simple mechanical mechanism may acc ount for the effects of competition on virus infection and dissemination. It is lik ely that arboviruses, found within ingested blood meals, enter mosquito midgut cells by membrane fusion or receptor-mediated endocytosis (e.g., Barth and Schatzmayr 1992, Hase et al. 1989, Hardy et al. 1983), followed by several days of midgut replication, and finally dissemination via hemolymph to secondary tissues. Several studies have invoked a leaky midgut hypothesis to account for the appearance of arboviruses in the hemolymph and secondary tissues

PAGE 130

118 shortly after (minutes to hours) imbibing an infectious blood meal (Chandler et al. 1998, Weaver et al. 1991, Weaver 1986, Hardy et al 1983, Miles et al. 1973, Boorman 1960). The leaky midgut hypothesis contends that rapid spread of inf ectious viruses to secondary tissues is facilitated by physical disrupti ons in the midgut epithelium and associated tissues (e.g., Weaver et al. 1991) or thru in tercellular junctional spaces (Houk and Hardy 1979). Dual infectious bloodmeals, with bot h microfilarial parasi tes and arboviruses, showed that physical disruption of the midgut by microfilarial penetration facilitated greater arboviral infection (V aughan et al. 1999, Zytoon et al. 1993, Turell et al. 1984). Similar results were obtained when perfor ations were experimentally induced in mosquito midguts (Zytoon et al. 1993). Midgut stress and disruptions have been shown to occur during bloodfeeding, presumably due to midgut distension or over-distension, and to facilitate direct arboviral access to the midgut (Weaver et al. 1991, Houk and Hardy 1979). Thus, midgut stress and disruptions facilitate greater ar boviral infection, as well as dissemination, since arboviruses bypa ss the midgut barriers and arrive in the interior of midgut cells or the mosquito body cavity (hemocoel) withou t replication in the midgut epithelium. This phenomenon is lik ely to have important epidemiological significance because it allows for arboviral infection in mosquito species that may otherwise show refractoriness to arboviral infection or transmission, perhaps due to barriers, (i.e., genetic refractoriness) (H ardy et al. 1978), and it reduces the extrinsic incubation period which enhances vectorial capacity. A testable hypothesis is that competition-induced enhancement of mosquito arboviral infection and dissemination may be attributable to midgut stress that allo ws arboviruses to enter (infect) and leave (disseminate), as well as bypass, the midgut tissues of competitively stressed mosquitoes

PAGE 131

119 with greater ease than less stressed individuals. Support fo r this hypothesis would come from an experiment showing that radiolabel ed virus in bloodmeals can be detected at greater levels in the midgut and hemocoel of competitively stressed mosquitoes than in less stressed individuals shortly after e ngorgement (Weaver et al. 1991). Additional support would come from morphological dete ction, via electron microscopy (Grimstad and Walker 1991), of differences in midgut tissues such as pe rforations or basal laminae thickness, between competitively stre ssed and unstressed individuals. Other Epidemiologically Significant Factors: Adult Survival Aedes albopictus had much greater viral body titer compared to A. aegypti yet it was a poorer disseminator of DENV (Chapt er 4, Fig. 4-4). These results were unexpected and may represent species-specific differences in viral selection pressure within the vector host. Host-parasite theory suggests parasite/pathogen load is inversely related to host fitness (Anderson and May 1978, May and Anderson 1978). Although arboviruses are assumed to have little nega tive effects on their arthropod vectors, many exceptions are known, such as retarded la rval development (Beaty et al. 1980, Tesh 1980), reduced adult longevity, including DENV-3 infected A. aegypti (e.g., Joshi et al. 2002, Faran et al. 1987), fecund ity (e.g., Turell et al. 1985, Tesh 1980), and damage to the salivary glands with redu ced ability to re-feed (e.g., Pl att et al. 1997, Turell et al. 1985, Grimstad et al. 1980, Mims et al. 1966). No comparative studies exist for the effect of DENV infection on adult A. aegypti and A. albopictus longevity. Aedes speciesspecific differences in DENV body titer pose new questions and opportunities for understanding the relative roles of A. aegypti and A. albopictus in DENV transmission. The widespread expansion of A. albopictus in the last three decades has increased the number of locations where these Aedes species are sympatric and dengue is endemic. A

PAGE 132

120 clearer understanding of the re lative importance of these sp ecies in DENV maintenance and transmission will improve dengue prediction and control. It is assumed that the more anthropophilic A. aegypti is the more important dengue vector. However, it is likely that other factors are involved in determining vector potential, and the relative importance of A. aegypti and A. albopictus may depend on ecological conditions, including host preference and availability, larv al nutrition, adult temperature and relative humidity. One readily testable hypot hesis involves DENVinduced differences in vector longevity. Measurements of vectorial capacity, the daily rate at which future pathogen inoculations arise from a current infective case, are especially sensitive to changes in the: (1) probability a vect or feeds on a host in a given day, (2) duration of the extrinsi c incubation period, and (3) daily survivorship rate of the vector. Even small changes in DENV-induced adult survivorship (Fernandez et al. 2003) may result in relatively large differences in vectorial capacity estimates for A. albopictus and A. aegypti (Dye 1986). A testable hypothesis is that A. aegypti is, in part, a more efficient DENV transmitter because Aedes species-specific differences in viral load lead to species-specific differences in adult survi vorship. The first step in addressing this hypothesis would be to establish that a nega tive relationship exists between dengue viral load and adult survivorship. Th is hypothesis predicts that th e greater viral load carried by A. albopictus results in a greater reduction in adult survivorship compared to A. aegypti An experimental test of this hypot hesis would involve offering adult Aedes bloodmeals containing DENV or lacking DE NV (i.e., control). Measurements would be made on adult DENV body titer and daily survivorshi p. Support for the hypothesis would come from detecting an interacti on between species treatment ( A. albopictus and A. aegypti )

PAGE 133

121 and bloodmeal (DENV infected and uninfected) showing that A. albopictus was more detrimentally affected by DENV infection than A. aegypti Other Ecological Interactions in the Larval Stages These competition experiments attempted to describe the importanc e of a biological interaction i.e., competition, as a contributor to vector competence. Other potentially important biological in teractions that may affect a dult mosquito vector competence include larval parasitism (e.g., Bedhomme et al. 2005, Agnew et al. 1999, Washburn et al. 1991), apparent competition (e.g., Julia no and Lounibos 2005), and predation (e.g., Griswold and Lounibos 2005, Kesavaraju and Juliano 2004). Take for example predation, like competition, is widespread and plays an important role in determining mosquito performance and represents an untested ecological factor that may alter mosquito susceptibility to arboviral infection. Reduction in survival is perhaps the most obvious, and readily measurable, outcome of predation. However, other population growth measurements (development time, size at emergence) may be influenced by predation and interact with each other in determining survivorship. For example, predation typically reduces prey development time due to preferential consumption of slowly developing prey as well as release fr om competition. Future studies on predation should initially establish whethe r larval predation alters arbovi ral infection parameters for surviving adult mosquitoes compared to pr edator-free conditions. These studies would also address whether there were similaritie s in the associations between population growth measurements between predatorand competition-induced differences in infection parameters. Relationships between population growth measurements and predation treatments in determining infection parameters are likely to differ for mosquito prey species that

PAGE 134

122 show facultative changes in behavi oral responses to predation (e.g., O. triseriatus ) compared to prey species that show little change in behavior (e.g., A. albopictus ). A behavioral study on water-borne cues from a predator induced more frequent low-risk behaviors in native mosquito O. triseriatus but not invasive A. albopictus (Kesavaraju and Juliano 2004). Aedes albopictus outcompetes O. triseriatus however, predatormediated coexistence, attributable to diffe rences in prey behavioral response, may promote coexistence between these two co mpeting species (Griswold and Lounibos 2005, Juliano and Lounibos 2005). A more deta iled manipulation of predation, or in combination with competition, treatments might include actual versus perceived predation (e.g., caged predator) which would hold survivorship constant and allow for a more detailed description of the relations hip of other population growth measurements (size, development time) and infection para meters. Thus, this later treatment would identify predator-induced indirect effects (e.g., behavioral, morphological: Relyea 2000) that alter mosquito vector infection parameters. Conclusions Competition in the larval stages enhanced arboviral infection parameters of adult A. albopictus using a model (SINV) and natural (DE NV) arbovirus-vector system. These effects may apply generally to mosquito-virus systems suggesting th at larval conditions are an important aspect of vector comp etence and should be included in future considerations of arbovirus transmission. Thes e results, coupled with future experiments, may lead to a clearer unders tanding of the rela tionship between larv al ecology and adult vector competence and vectorial capacity.

PAGE 135

123 LIST OF REFERENCES Abrams, P.A. 1995. Implications of dynamica lly variable traits for identifying, classifying and measuring di rect and indirect effects in ecological communities. American Naturalist 146 : 112-134. Agnew, P., S. Bedhomme, C. Haussy, and Y. Michalakis. 1999. Age and size at maturity of the mosquito Culex pipiens infected by microsporidian parasite Vavraia culicis Proceedings of the Royal Society of London Series B 266 : 947-952. Ahmed, A.M., S.L. Baggott, R. Maingon, and H. Hurd. 2002. The costs of mounting an immune response are reflected in the reproductive fitness of the mosquito Anopheles gambiae Oikos 97 : 371-377. Alto, B.W., L.P. Lounibos, S. Higgs, a nd S.A. Juliano. 2005a. Larval competition differentially affects arbovirus infection in Aedes mosquitoes. Ecology 86 : 32793288. Alto, B.W., S.P. Yanoviak, L.P. Lounibos, a nd B.G. Drake. 2005b. Effects of elevated atmospheric C02 on water chemistry and mosquito growth under competitive conditions in container hab itats. Florida Entomologist 88 : 372-382. Alto, B.W., L.P. Lounibos, and S.A. Ju liano. 2003. Age-dependent bloodfeeding of Aedes aegypti and Aedes albopictus on artificial and living hosts. Journal of the American Mosquito Control Association. 19 : 347-352. Alto B.W., and S.A. Juliano. 2001a. Temperature effects on the dynamics of Aedes albopictus (Diptera: Culicidae) populations in the laboratory. J ournal of Medical Entomology 38 : 548-556. Alto B.W., and S.A. Juliano. 2001b. Precipitatio n and temperature effects on populations of Aedes albopictus (Diptera: Culicidae): implications for range expansion. Journal of Medical Entomology 38 : 646-656. Altwegg, R. 2002. Trait-mediated indirect eff ects and complex life-cyles in two European frogs. Evolutionary and Ecology Research 4 : 519-536. Anderson, R.M. and R.M. May. 1978. Regulat ion and stability of host-parasite population interactions. I. Regulatory Processes. Journal of Animal Ecology. 47 : 219-247.

PAGE 136

124 Armstrong, P.M., and R. Rico-Hesse. 2003. Effici ency of dengue serotype 2 virus strains to infect and disseminate in Aedes aegypti American Journal of Tropical Medicine and Hygiene 68 : 539-544. Armbruster, P., and R.A. Hutchinson. 2002. Pupa l mass and wing length as indicators of fecundity in Aedes albopictus and Aedes geniculatus (Diptera: Culicidae). Journal of Medical Entomology 39 : 699-704. Armstrong, P.M., and R. Rico-Hesse. 2001. Differential susceptibility of Aedes aegypti to infection by the American and Sout heast Asian genotypes of dengue type 2 virus. Vector Borne and Zoonotic Diseases 1 : 159-168. Baqar, S., C.G. Hayes, and T. Ahmed. 1980. Th e effect of larval rearing conditions and adult age on the susceptibility of Culex tritaeniorhynchus to infection with West Nile virus. Mosquito News 40 : 165-171. Barrera, R. 1996a. Species concurrence and th e structure of a community of aquatic insects in tree holes. Journal of Vector Ecology 21 : 66-80. Barrera, R. 1996b. Competition a nd resistance to starvation in larvae of containerinhabiting Aedes mosquitoes. Ecological Entomology 21 : 117-127. Barrera, R., M. Amador, and G.C. Cl ark. 2006. Ecological fa ctors influencing Aedes aegypti (Diptera: Culicidae) productivity in artifi cial containers in Salinas, Puerto Rico. Journal of Medical Entomology 43 : 484-492. Barth, O.M., and H.G. Schatzmayr. 1992. Brazi lian dengue virus type 1 replicating in mosquito cell cultures. Memri as do Instituto Oswaldo Cruz 87 : 1-7. Beaty, B.J., R.B. Tesh, and T.H.G. Aitke n. 1980. Transovarial transmission of yellow fever virus in Stegomyia mosquitoes. American Journal of Tropical Medicine and Hygiene 29 : 125-132. Beck, M.A., and O.A. Levander. 2000. Host nutritional status and its effect on a viral pathogen. The Journal of Infectious Diseases 182 (Suppl 1): S93-S96. Bedhomme, S., P. Agnew, C. Sidobre, and Y. Michalakis. 2005. Prevalence-dependent costs of parasite virulence. P ublic Library of Science Biology 3 : 1403-1408. Bedhomme, S., P. Agnew, C. Sidobre, a nd Y. Michalakis. 20 04. Virulence reaction norms across a food gradient. Proceedings of the Royal Society of London, Series B 271 : 739-744. Bennett, K.E., D. Flick, K.H. Fleming, R. Jochim, B.J. Beaty, and W.C. Black, IV. 2005a. Quantitative trait loci that control dengue-2 virus dissemination in the mosquito Aedes aegypti Genetics 170 : 185-194.

PAGE 137

125 Bennett, K.E., B.J. Beaty, and W.C. Black, IV. 2005b. Selection of D2S3, an Aedes aegypti (Diptera: Culicid ae) strain with high oral su sceptibility to dengue 2 virus and D2MEB, a strain with a midgut barrier to dengue 2 escape. Journal of Medical Entomology 42 : 110-119. Bennett, K.E., K.E. Olson, M. de L. Munoz, I. Fernandez-Sala, J. A. Farfan-Ale, S. Higgs, W.C. Black IV, and B.J. Beaty. 2002. Variation in vector competence for dengue 2 virus among 24 collections of Aedes aegypti from Mexico and the United States. American Journal of Tropical Medicine and Hygiene 67 : 85-92. Birch, L.C. 1953. Experimental background to the study of the distribution and abundance of insects. III. The relation be tween innate capacity for increase and survival of different species of beetles living together on the same food. Evolution 7 : 136-144. Birungi, J., and L.E. Munsterm ann. 2002. Genetic structure of Aedes albopictus (Diptera: Culicidae) populations based on mitochondr ial ND5 sequences: Evidence for an independent invasion into Brazil and United States. Annals of the Entomological Society of America 95 : 125-132. Bishop A, and B.M. Gilchrist. 1946. Experiments upon the feeding of Aedes aegypti through nimal membranes with a view to applying this method to the chemotherapy of malaria. Parasitology 37 : 85-100. Black, W.C. IV, K.E. Bennett, N. Gorrochot egui-Escalante, C.V. Barillas-Mury, I. Fernandez-Salas, M. de L. Munoz, J.A. Farfan-Ale, K.E. Olson, and B.J. Beaty. 2002. Flavivirus susceptibility in Aedes aegypti Archives of Medical Research 33 : 379-388. Black, W.C., IV, K.S. Rai, B.J. Turc o, and D.C. Arroyo. 1989. Laboratory study of competition between United States strains of Aedes albopictus and Aedes aegypti (Diptera: Culicidae). Journal of Medical Entomology 32 : 847-852. Blackmore, M.S. and C.C. Lord. 2000. The relationship between size and fecundity in Aedes albopictus Journal of Vector Ecology 25 : 212-217. Boromisa, R.D., K.S. Rai, and P.R. Grimst ad. 1987. Variation in the vector competence of geographic strains of Aedes albopictus for dengue 1 virus. Journal of the American Mosquito Control Association 3 : 378-386. Boorman, J. 1960. Observations on the amount of virus present in the hemolymph of A. aegypti infected with Uganda S, yellow fever and Semlikie Forest virus. Transactions of the Royal Society of Tropical Medicine and Hygiene 54 : 362-365. Bosio, C.F., R.E. Fulton, M.L. Salasek, B. J. Beaty, and W.C. Black. 2000. Quantitative trait loci that control vector compet ence for dengue-2 virus in the mosquito Aedes aegypti Genetics 156 : 687-698.

PAGE 138

126 Bosio, C.F., B.J. Beaty, and W.C. Black. 1998. Quantitative genetics of vector competence for dengue-2 virus in Aedes aegypti American Journal of Tropical Medicine and Hygiene 59 : 965-970. Bosio, C.F., R.E. Thomas, P.R. Grimstad, and K.S. Rai. 1992. Variation in the efficiency of vertical transmission of dengue-1 virus by strains of Aedes albopictus (Diptera: Culicidae). Journal of Medical Entomology 29 : 985-989. Bowen M.F. 1991. The sensory physiology of host-seek ing behavior in mosquitoes. Annual Review of Entomology 36 : 139-158. Bowers, D.F., C.G. Coleman, and D.T. Brow n. 2003. Sindbis virus-associated pathology in Aedes albopictus (Diptera: Culicidae). Journal of Medical Entomology 40 : 698705. Bowers, D.F., B.A. Abell, and D.T. Brow n. 1995. Replication and ti ssue tropism of the Alphavirus Sindbis in the mosquito Aedes albopictus. Virology 212 : 1-12. Bradshaw, W.E., and C.M. Holzapfel. 1986. Ge ography of density-depen dent selection in pitcher-plant mosquitoes. Pages 48-65 in F. Taylor and R. Karban, editors. The evolution of insect life cycles Springer, New York, NY. Braks, M.A. H., N.A. Honrio, L.P. Louni bos, R. Lourenco-de-Oliveira, and S.A. Juliano. 2004. Interspecific competition betw een two invasive species of container mosquitoes, Aedes aegypti and Aedes albopictus (Diptera: Culicidae), in Brazil. Annals of the Entomological Society of America 97 : 130-139. Briegel, H. 1990. Metabolic relationship between female body size, reserves, and fecundity of Aedes aegypti Journal of Insect Physiology 36 : 165-172. Briegel, H. 1985. Mosquito reproduction: In complete utilizati on of the blood meal protein for ogenesis. Journal of Insect Physiology 31 : 15-21. Briegel, H., and S.E. Timmermann. 2001. Aedes albopictus (Diptera: Culicidae): Physiological aspects of development and reproduction. Journal of Medical Entomology 38 : 566-571. Broadie, K.S., and W.E. Bradshaw. 1991. Mech anisms of interference competition in the western tree-hole mosquito, Aedes sierrensis Ecological Entomology 16 : 145-154. Broberg, L., and W.E. Bradshaw. 19 95. Density-dependent development in Wyeomyia smithii (Diptera, Culicidae) Intraspecifi c competiton is not the result of interference. Annals of the En tomological Society of America 88 : 465-470. Brummer-Korvenkontio, M., O. Vapalahti, P. K uusisto, P. Saikku, T. Manni, P. Koskela, T. Nygren, H. Brummer-Korvenkontio, and A. Vaheri. 2002. Epidemiology of Sindbis virus infection in Fi nland 1981-96: possible fact ors explaining a peculiar disease pattern. Epidemiology and Infection 129 : 335-345.

PAGE 139

127 Brummer-Korvenkontio, M., P. and Kuusisto. 1981. Onko Suomen Lnsiosa sstynyt Pogostalta (Has western Finland been spared the Pogosta?). Suom Lkril 32 : 2606-2607. Buckley, A., A. Dawson, S.R. Moss, S.A. Hinsley, P.E. Bellamy, and E.A. Gould. 2003. Serological evidence of West Nile virus Usutu virus and Sindbis virus infection of birds in the UK. Journal of General Virology 84 : 2807-2817. Burgess, L. 1959. Probing behaviour of Aedes aegypti (L.) in response to heat and moisture. Nature 184 : 1968-1969. Bustin, S.A. 2000. Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. Journal of Molecular Endocrinology 25 : 169193. Butler, J.F., W.R. Hess, R.G. Endris, and K.H. Holscher. 1984. In vitro feeding of Ornithodoros ticks for rearing and assessment of disease transmission. Acarology VI : 1075-1081. Byers, J.E., and L. Goldwasser. 2001. Exposing the mechanism and timing of impact of nonindigenous species on native species. Ecology 82 : 1330-1343. Callahan, J.D., S-J. L. Wu, A. Dion-Schultz, B.E. Mangold, L.F. Peruski, D.M. Watts, K.R. Porter, G.R. Murphy, W. Suhar yono, C-C King, C.G. Hayes, and J.J. Temenak. 2001. Development and evaluati on of serotypeand group-specific flourogenic reverse transcriptase PCR (Taq Man) assays for dengue virus. Journal of Clinical Microbiology 39 : 4119-4124. Carpenter, S.R. 1983. Resource limitation of larval treehole mosquitoes subsisting on beech detritus. Ecology 64 : 219-223. Carpenter, S.R. 1982a. Stemflow chemistry: Effects on population dynamics of detritivorous mosquitoes in tree-hole ecosystems. Oecologia 53 : 1-6. Carpenter, S.R. 1982b. Comparisons of equations for decay of leaf litter in tree-hole ecosystems. Oikos 39 : 17-22 Chandler, L.J., C.D. Blair, and B.J. B eaty. 1998. La Crosse virus infection of Aedes triseriatus (Diptera: Culicidae) ovaries before dissemination of virus from the midgut. Journal of Medical Entomology 35 : 567-572. Chatfield, C. 1989. The analysis of time series. An introduction. Chapman and Hall, New York, NY. Chen, W-J, H-L. Wei, E-L. Hsu, and E-R. Chen. 1993. Vector competence of Aedes albopictus and Ae. aegypti (Diptera: Culicidae) to Dengue 1 virus on Taiwan: Development of the virus in orally and pare nterally infected mosquitoes. Journal of Medical Entomology 30 : 524-530.

PAGE 140

128 Christophers, R.C. 1960. Aedes aegypti The yellow fever mos quito: Its life history, bionomics, and structure. Cambridge University Press, New York, NY. Clements, A.N. 1999. The biology of mosquitoes, volum e 2, sensory reception and behaviour. CABI Publishing, New York, NY. Cloe, W.W., III, and G.C. Garman. 1996. The energetic importance of terrestrial arthropod inputs to three warm-water streams. Freshwater Biology 36 : 105-114. Cologna, R., P.M. Armstrong, and R. Rico-H esse. 2005. Selection for virulent dengue viruses occurs in humans and mosquitoes. Journal of Virology 79 : 853-859. Cologna, R., and R. Rico-Hesse. 2003. Ameri can genotype structures decrease dengue virus output from human monocytes and dendritic cells. Journal of Virology 77 : 3929-3938. Connell, J.H. 1983. On the prevalence and relative importance of interspecific competition: Evidence from field experiments. American Naturalist 122 : 661-696. Connell, J.H. 1961. The influence of interspe cific competition and other factors on the distribution of the barnacle Chthamalus stellatus Ecology 42 : 710-723. Costanzo, K.S., B. Kesavaraju, and S.A. Ju liano. 2005a. Condition-specific competition in container mosquitoes: The role of noncompeting life-history stages. Ecology 86 : 3289-3295. Costanzo, K.S., K. Mormann, and S.A. Juliano. 2005b. Asymmetrical competition and patterns of abundance of Aedes albopictus and Culex pipiens (Diptera: Culicidae). Journal of Medical Entomology 42 : 559-570. Craig, G.B., Jr., and R.C. Vande hey. 1962. Genetic variability in Aedes aegypti (Diptera: Culicidae) I. Mutations affecting color pattern. Annals of the Entomological Society of America 55 : 47-58. Crombie, A.C. 1947. Interspecific competition. Journal of Animal Ecology 16 : 44-73. DAntonio, C.M., and P.M. Vitousek. 1992. Biol ogical invasions by exotic grasses, the grass/fire cycle, and global change. Annual Review of Ecology and Systematics 23 : 63-87. Daugherty, M.P., B.W. Alto, and S.A. Juliano. 2 000. Invertebrate carcasses as a resource for competing Aedes albopictus and Aedes aegypti (Diptera: Culicidae). Journal of Medical Entomology 37 : 364-372. Davis, E.E. 1984. Development of lactic acid-recept or sensitivity and host-seeking behaviour in newly emerged female Aedes aegypti mosquitoes. Journal of Insect Physiology 30 : 211-215.

PAGE 141

129 DeFoliart, G.R., P.R. Grimstad, and D.M. Watts. 1987. Advances in mosquito-borne arbovirus/vector research. A nnual Review of Entomology 32 : 479-505. De Silva, A.M., W.P.J. Dittus, P.H. Amer asinghe, and F.P. Amerasinghe. 1999. Serologic evidence for an epizootic dengue virus infecting Toque Macaques ( Macaca sinica ) at Polonnaruwa, Sri Lanka. American Jour nal of Tropical Medicine and Hygiene 60 : 300-306. de Thoisy, B., P. Dussrt, and M. Kazanji. 2004. Wild terrestrial rainforest mammals as potential reservoirs for flaviviruses (yellow fever, dengue 2 and St. Louis encephalitis viruses) in French Guiana. Transactions of the Royal Society of Tropical Medicine and Hygiene 98 : 409-412. Diallo M., Y. Ba, A.A. Sall, O.M. Diop, J.A. Ndione, M. Mondo, L. Girault, and C. Mathiot. 2003. Amplification of the sylvatic cycle of dengue virus type 2, Senegal, 1999-2000: Entomologic findings and epid emiologic considerations. Emerging Infectious Diseases 9 : 362-367. Dieng, H, C. Mwandawiro, M. Boots, R. Mo rales, T. Satho, N. Tuno, Y. Tsuda, and M. Takagi. 2002. Leaf litter decay pro cess and the growth performance of Aedes albopictus larvae (Diptera: Culicidae). Journal of Vector Ecology 27 : 31-38. Dimopoulos, D. 2003. Insect immunity and it s implication in mosquito malaria interactions. Cellular Microbiology 5 : 3-14. Dohm, D.J., T.M. Logan, J.F. Barth, and M. J. Turell. 1995. Laboratory transmission of Sindbis virus by Aedes albopictus Ae. aegypti and Culex pipiens (Diptera: Culicidae). Journal of Medical Entomology 32 : 818-821. Draper, N.R., and H. Smith. 1966. Applied regression analysis. Wiley, New York, NY. Dye, C. 1986. Vectorial Capacity: must we measure all its components? Parisitology Today 2 : 203-208. Dye, C. 1984. Competition amongst larval Aedes aegypti : the role of interference. Ecological Entomology 9 : 355-357. Dye, C. 1982. Intraspecific competition amongst larval Aedes aegypti : food exploitation or chemical interference ? Ecological Entomology 7 : 39-46. Eads, R.B. 1972. Recovery of Aedes albopictus from used tires shiped to United States ports. Mosquito News 32 :113-114. Eritja, R., R. Escosa, J. Lucientes, E. Mar qus, R. Molina, D. Roiz, and S. Ruiz. 2005. Worldwide invasion of vector mosquito es: present European distribution and challenges for Spain. Biological Invasions 7 : 87-97.

PAGE 142

130 Espmark, A., and B. Niklasson. 1984. Ockel bo disease in Sweden: epidemiological, clinical, and virological data from the 1982 outbreak. American Journal of Tropical Medicine and Hygiene 33 : 1203-1211. Failloux, A-B, M. Vazeille, and F. Rodhain. 2002. Geographic genetic variation in populations of the dengue virus vector Aedes aegypti Journal of Molecular Evolution 55 : 653-663. Faran, M.F., M.J. Turell, W. S. Romoser, R.G. Routier, P.H. Gibbs, T.L. Cannon, and C.L. Bailey. 1987. Reduced survival of adult Culex pipiens infected with Rift Valley Fever virus. American Journa l of Tropical Medicine and Hygiene 37 : 403409. Fernndez, Z., A.C. Moncayo, A.S. Carrara, O.P. Forattini, and S.C. Weaver. 2003. Vector competence of rural and urban strains of Aedes (Stegomyia) albopictus (Diptera: Culicidae) from So Paulo State, Brazil for IC, ID, and IF subtypes of Venezuelan equine encephalitis viru s. Journal of Medical Entomology 40 : 522-527. Fish, D. 1983. Phytotelmata: flora and fauna. Pages 1-27 in J.H. Frank and L.P. Lounibos, editors. Phytotelmata: terrestria l plants as hosts for aquatic insect communities. Plexus, Medford, New Jersey. Fish, D., and S.R. Carpenter. 1982. Leaf litter and larval mosquito dynamics in tree-hole ecosystems. Ecology 63 : 283-288. Focks, D.A., R.J. Brenner, J. Hayes, and E. Daniels. 2000. Transmission thresholds for dengue in terms of Aedes aegypti pupae per person with di scussion of their utility in source reduction efforts. American J ournal of Tropical Me dicine and Hygiene 62 : 11-18. Focks, D.A, E. Daniels, D.G. Haile, and J. E. Keesling. 1995. A simulation model of the epidemiology of urban dengue fever: Literature analysis, model development, preliminary validation, and samples of si mulation results. American Journal of Tropical Medicine and Hygiene 53 : 489-506. Francy, D.B., T.G.T. Jaenson, J.O. Lundstrom, E.B. Schildt, A. Espmark, B. Henriksson, and B. Niklasson. 1989. Ecological studies of mosquitoes and birds as hosts of Ockelbo virus in Sweden and isolations of Inkoo and Batai viruses from mosquitoes. American Journal of Tropical Medicine and Hygiene 41 : 355-363. Frank, J.H., and L.P. Lounibos. 1983. Phytotelmata : Terrestrial plants as hosts for aquatic insect communities. Plexus, Medford, New Jersey. Freier, J.E., and L. Rosen. 1988. Vertic al transmission of dengue viruses by Aedes mediovittatus American Journal of Tr opical Medicine and Hygiene 39 : 218-222.

PAGE 143

131 Gimnig, J.E., M. Ombok, S. Otieno, M.G. Kaufman, J.M. Vulule, and E.D. Walker. 2002. Density-dependent development of Anopheles gambiae (Diptera: Culicidae) larvae in artificial habitats Journal of Medical Entomology 39 : 162-172. Gleiser, R.M., J. Urrutia, and D.E. Gorla. 2000a. Effects of crow ding on populations of Aedes albifasciatus larvae under laboratory conditions. Entomologia Experimentalis et Applicata 95 : 135-140. Gleiser, R.M., J. Urrutia, and D.E. Gorla. 2000b. Body size variation of the floodwater mosquito Aedes albifasciatus in central Argentina. Medical and Veterinary Entomology 14 : 38-43. Gomez-Machorro, C., K.E. Bennett, M. del L. Munoz, and W.C. Black, IV. 2004. Quantitative trait loci affecting dengue mi dgut infection barriers in an advanced intercross line of Aedes aegypti Insect Molecular Biology 13 : 637-648. Gorrochotegui-Escalante, N. S. Lozano-Fuen tes, K.E. Bennett, A. Molina-Cruz, B.J. Beaty, and W. Black IV. 2005. Association mapping of segregating sites in the early trypsin gene and su sceptibility to dengue-2 virus in the mosquito Aedes aegypti Insect Biochemistry and Molecular Biology 35 : 771-788. Gresikova, M., M. Sekeyova, G. Tempera, S. Guglielmino, and A. Castro. 1978. Identification of a Sindbis vi rus strain isolated from Hyaloma marginatum ticks in Sicily. Acta Virol. (Praha) 22 : 231-232. Grimstad, P.R., and E.D. Walker. 1991. Aedes triseriatus (Dipera: Culicidae) and La Crosse virus. IV. Nutritional deprivation of larvae affects the adult barriers to infection and transmission. J ournal of Medical Entomology 28 : 378-386. Grimstad, P.R., S.L. Paulson, and G.B. Craig, Jr. 1985. Vector competence of Aedes hendersoni (Diptera: Culicidae) for La Crosse virus and evidence of a salivarygland escape barrier. Jo urnal of Medical Entomology 22 : 447-453. Grimstad, P.R. and L.D. Haramis. 1984. Aedes triseriatus (Diptera: Culicidae) and La Crosse virus. III. Enhanced oral transmission by nutrition-deprived mosquitoes. Journal of Medical Entomology 21 : 249-256. Grimstad, P.R., Q.E. Ross, and G.B. Craig, Jr. 1980. Aedes triseriatus (Diptera: Culicidae) and La Crosse virus II. Modifi cation of mosquito feeding behavior by virus infection. Journal of Medical Entomology. 17 : 1-7. Griswold, M.W., and L.P. Lounibos. 2005. Does differential predation permit invasive and native mosquito larvae to coexist in Florida? Ecological Entomology 30 : 122127. Grover, J.P. 1997. Resource competiti on. Chapman and Hall, New York, NY.

PAGE 144

132 Gubler, D.J. 2002. Epidemic dengue/dengue hemorrhagic fever as a public health, social and economic problem in the 21st century. Trends in Microbiology 10 : 100-103. Gubler, D.J. 1997. Dengue and dengue hemorrhagic fever: its history and resurgence as a global public health problem. Pages 1-22 in D.J. Gubler and G. Kuno, editors. Dengue and dengue hemorrhagic fever. CABI Publishing, New York, NY. Gubler, D.J., and G. Kuno. 1997. Dengue a nd dengue hemorrhagic fever. CABI Publishing, New York, NY. Gubler, D.J., W. Suharyono, R. Tan, M. Abidi n, and A. Sie. 1981. Viraemia in patients with naturally acquired dengue infect ion. Bulletin of th e World Health Organization 59 : 623-630. Gubler, D.J., S. Nalim, R. Tan, H. Saipa n, and J. Sulianti Saroso. 1979. Variation in susceptibility to oral infection with dengue viruses among geographic strains of Aedes aegypti American Journal of Tropi cal Medicine and Hygiene. 28 : 10451052. Gubler, D.J., and L. Rosen. 1976. Variation among geographic strains of Aedes albopictus in susceptibility to infection with dengue viruses. Am erican Journal of Tropical Medicine and Hygiene 25 : 318-325. Hancock R.G., and Foster W.A. 2000. Exoge nous juvenile hormone and methoprene, but not male accessory gland substances or ova riectomy, affect the blood/nectar choice of female Culex nigripalpus mosquitoes. Medical Veterinary Entomology 14 : 376382 Hairston, N.G. 1951. Interspecific competiti on and its probable influence upon the vertical distribution of Appal achian salamanders of the genus Plethodon Ecology 32 : 266-274. Hairston, N.G., F.E. Smith, and L.B. Sl obodkin. 1960. Community structure, population control, and competition. American Naturalist 94 : 421-425. Haramis, L.D. 1985. Larval nutrition adult body size, and the biology of Aedes triseriatus Pages 431-437 in L.P. Lounibos J.R. Rey, and J.H Frank, editors. Ecology of mosquitoes: Proceedings of a workshop. Florida Medical Entomology Laboratory, Vero Beach, FL. Haramis, L.D. 1983. Increased adult size correlated with parity in Aedes triseriatus Mosquito News 43 : 77-79. Hardy, J.L. 1988. Susceptibility and resistan ce of vector mosquitoes. Pages 87-126 in T.P. Monath, editor. The arboviruses: Epidemiology and ecology. CRC Press, Boca Raton, FL.

PAGE 145

133 Hardy, J.L., E.J. Houk, L.D. Kramer, and W.C. Reeves. 1983. Intrinsi c factors affecting vector competence of mosquitoes for ar boviruses. Annual Review of Entomology 28 : 229-262. Hardy, J.L., G. Apperson, S. M. Asman, and W.C. Reeves. 1978. Selection of a strain of Culex tarsalis highly resistant to infection follo wing ingestion of Western equine encephalomyelitis virus. American J ournal of Tropical Medicine and Hygiene 27 : 313-321. Harrington, L.A., T.W. Scott, K. Lerdthusnee, R.C. Coleman, A. Costero, G.G. Clark, J.J. Jones, S. Kitthawee, P. Kittayapong, R. Sithiprasasna, and J.D. Edman. 2005. Dispersal of the dengue vector Aedes aegypti within and between rural communities. American Journal of Tropical Medicine and Hygiene 72 : 209-220. Hase, T., P.L. Summers, and K.H. Eckels. 1989. Flavivirus entry into cultured mosquito cells and human peripheral blood monocytes. Archives of Virology 104 : 129-143. Hawley, W.A. 1988. The biology of Aedes albopictus Journal of the American Mosquito Control Association 4 (Supplement): 1-40. Hawley, W.A. 1985. The effect of larval de nsity on adult longevity of a mosquito, Aedes sierrensis : epidemiological consequences. Journal of Animal Ecology 54 : 955-964. Hawley, W.A., P. Reiter, R.S. Copela nd, C.B. Pumpuni, and G.B. Craig, Jr. 1987. Aedes albopictus in North America: Probable introduc tion in used tires from Northern Asia. Science 236 : 1114-1116. Heard, S.B. 1994. Pitcher-plant midges and mosquitoes: a processing chain commensalism. Ecology 75 : 1647-1660. Higgs, S. 2004. How do mosquito vectors liv e with their viruses? Pages 103-137 in S.H. Gillespie, G.L. Smith, and A. Osbourn, editors. .Microbe-Vector Interactions in Vector-Borne Diseases. Cambridge University Press, Cambridge, UK. Ho, B.C., A. Ewert, and L. Chew 1989. Interspecific competition among Aedes aegypti Ae. albopictus and Ae. triseriatus (Diptera: Culicidae): larval development in mixed cultures. Journal of Medical Entomology 26 : 615-623. Hobbs J.H., Hughes E.A., and B.H. Eichold, II. 1991. Replacement of Aedes aegypti by Aedes albopictus in Mobile Alabama. Journal of the American Mosquito Control Association 7 : 488-489. Holland, P.M., R.D. Abramson, R. Watson, and D.H. Gelfand. 1991. Detection of specific polymerase chain reaction produc t by utilizing the 5----3 exonuclease activity of Thermus aquaticus DNA polymerase. Proceedings of the National Academy of Sciences 88 : 7276-7280.

PAGE 146

134 Holway, D.A. 1999. Competitive mechanisms unde rlying the displacement of native ants by the invasive Argentine ant. Ecology 80 : 238-251. Honrio, N.A., W.C. Silva, P.J. Leite, J.M. Gonalves, L.P. Lounibos, and R. Lourencode-Oliveira. 2003. Dispersal of Aedes aegypti and Aedes albopictus (Diptera: Culicidae) in an Urban endemic dengue area in the State of Rio de Janeiro, Brazil. Memorias do Instituto Oswaldo Cruz 98 : 191-198. Hornby, J.A., Moore D.E., and Miller T.W., Jr. 1994. Aedes albopictus distribution, abundance, and colonization in Lee County, Florida, and its effect on Aedes aegypti Journal of the American Mosquito Control Association 10 : 397-402. Houk, E.J., L.D. Kramer, J.S. Hardy, and S. B. Presser. 1986. An interspecific mosquito model for the mesenteronal infection barri er to Western equine encephalomyelitis virus ( Culex tarsalis and Culex pipiens ). American Journal of Tropical Medicine and Hygiene 35 : 632-641. Houk, E.J., and J.L. Hardy. 1979. In vivo nega tive staining of the midgut continuous junction in the mosquito, Culex tarsalis (Diptera: Culicidae). Acta Tropica 36 : 267275. Innis, B.L. 1997. Antibody responses to dengue virus infection. Pages 245-271 in D.J. Gubler and G. Kuno, editors. Dengue a nd dengue hemorrhagic fever. CABI publishing, New York, NY. Jackson, A.C., J.C. Bowen, and A.E.R. Downe. 1993. Experimental infection of Aedes aegypti (Diptera: Culic idae) by the oral ro ute with Sindbis virus. Journal of Medical Entomology 30 : 332-337. Jaenson, T.G.T., and B. Niklasson. 1986. Feed ing patterns of mos quitoes (Diptera: Culicidae) in relation to the transmission of Ockelbo disease in Sweden. Bulletin of Entomological Research 76 : 375-383. Joshi, V., R.C. Sharma, Y. Sharma, S. Adha, K. Sharma, H. Singh, A. Purohit, and M. Singhi. 2006. Importance of socioeconomic st atus and tree holes in distribution of Aedes mosquitoes (Diptera: Culicidae) in J odhpur, Rajasthan, India. Journal of Medical Entomology 43 : 330-336. Joshi, V., D.T. Mourya, and R.C. Shar ma. 2002. Persistence of dengue-3 through transovarial transmission passage in successive generations of Aedes aegypti mosquitoes. American Journal of Tropical Medicine and Hygiene. 67 : 158-161. Joshi, V., M. Singhi, R.C. Chaudhary. 1996. Transovarial transmission of dengue 3 by Aedes aegypti Transactions of the Royal So ciety of Tropical Medicine and Hygiene 90 : 643-644. Juliano, S.A. 1998. Species introduction and replacement among mosquitoes: interspecific resource competition or apparent competition? Ecology 79 : 255-268.

PAGE 147

135 Juliano, S.A., and L.P. Lounibos. 2005. Ecol ogy of invasive mosquitoes: effects on resident species and on hu man health. Ecology Letters 8 : 558-574. Juliano, S.A., L.P. Lounibos, and G.F. OMeara. 2004. A field test for competitive effects of Aedes albopictus on Aedes aegypti in south Florida: differences between sites of coexistence and exclusion? Oecologia 139 : 583-593. Juliano, S.A., G.F. OMeara, J.R. Morrill, and M.M. Cutwa. 2002. Desiccation and thermal tolerance of eggs and the coexis tence of competing mosquitoes. Oecologia 130 : 458-469. Jupp, P.G., N.K. Blackburn, D.L. Thompson, and G.M. Meenehan. 1986a. Sindbis and West Nile virus infections in the Witw atersrand-Pretoria region. South African Journal of Medical Science 70 : 218-220. Jupp, P.G., B.M. McIntosh, and N.K. Blackburn. 1986b. Experimental assessment of the vector competence of Culex ( Culex ) neavei Theobald with West Nile and Sindbis viruses in South Africa. Transactions of the Royal Society of Tropical Medicine and Hygiene 80 : 226-230. Jupp, P.G., B.M. McIntosh, and D.B. Dickinson. 1972. Quantitative experiments on the vector capability of Culex ( Culex ) theileri Theobald with West Nile and Sindbis viruses. Journal of Medical Entomology 9 : 393-395. Jupp, P.G., and B.M. McIntosh. 1970a. Quantitat ive experiments on the vector capability of Culex ( Culex ) univittatus Theobald with West Nile and Sindbis viruses. Journal of Medical Entomology 7 : 371-373. Jupp, P.G., and B.M. McIntosh. 1970b. Quantitative experiments on the vector capability of Culex ( Culex ) pipiens fatigans Wiedemann with West Nile and Sindbis viruses. Journal of Medical Entomology 7 : 353-356. Kaufman, M.G., E.D. Walker, T.W. Smith, R. W. Merritt, and M.J. Klug. 1999. Effects of larval mosquitoes ( Aedes triseriatus ) and stemflow on microbial community dynamics in container habitats. Appl ied and Environmental Microbiology 65 : 2661-2673. Kay, B.H., J.D. Edman, I.D. Fanning, and P. Mo ttram. 1989. Larval diet and the vector competence of Culex annulirostris (Diptera: Culicidae) for Murray Valley encephalitis virus. Journal of Medical Entomology 26 : 487-488. Kesavaraju, B., and S.A. Julia no. 2004. Differential behavioral responses to water-borne cues to predation in two container dwelling mosquitoes. Annals of the Entomological Society of America 97 :194-201

PAGE 148

136 Khin, M.M. and K.A. Than. 1983. Transova rial transmission of dengue 2 virus by Aedes aegypti in nature. American Journal of Tropical Medicine and Hygiene 32 : 590594. Kiesecker, J.M., and D.K. Skelly. 2001. Eff ects of disease and pond drying on gray tree frog growth, development, and survival. Ecology 82 : 1956-1963. Kitching, R.L. 1971. An ecological study of wa ter-filled treeholes and their position in the woodland ecosystem. Journal of Animal Ecology 40 : 281-302. Klowden, M.J., and N.M. Fernandez. 1996. Effects of age and mating on the host seeking behavior of Aedes aegypti mosquitoes. Journal of Vector Ecology 21 : 156158. Klowden, M.J., and H. Briegel. 1994. Mosquito gonotrophic cycle and multiple feeding potential: Contrasts between Anopheles and Aedes Journal of Medical Entomology 31 : 618-622. Klowden, M.J., and G.M. Chambers. 1992. Reproductive and metabolic differences between Aedes aegypti and Ae. albopictus (Diptera: Culicidae). Journal of Medical Entomology 29 :467-471. Klowden M.J., J.L. Blackmer, and G.M. Chambers. 1988. Effects of larval nutrition on the host-seeking behavior of adult Aedes aegypti mosquitoes. Journal of the American Mosquito Control Association 4 : 73-75. Klowden M.J., and A.O. Lea. 1984. Blood feed ing affects age-related changes in the host-seeking behavior of Aedes aegypti (Diptera: Culicidae) during oocyte maturation. Journal of Medical Entomology 21 : 274-277. Klowden M.J., and A.O. Lea. 1979a. Humoral inhibition of host-seeking in Aedes aegypti during oocyte maturation. J ournal of Insect Physiology 25 : 231-235. Klowden M.J., and A.O. Lea. 1979b. Abdominal distention term inates subsequent hostseeking behaviour of Aedes aegypti following a blood meal. Journal of Insect Physiology 25 : 583-585. Klowden M.J., and A.O. Lea. 1978. Blood meal size as a factor affecting continued hostseeking by Aedes aegypti (L.). American Journal of Tropical Medicine and Hygiene 27 : 827-831. Koella, J.C., and F.L. Srensen. 2002. Eff ect of adult nutriti on on the melanization immune response of the malaria vector Anopheles stephensi Medical and Veterinary Entomology 16 : 316-320. Kolivras, K.N. 2006. Mosquito habitat a nd dengue risk potential in Hawaii: A conceptual framework and GIS appl ication. The Professional Geographer 58 : 139154.

PAGE 149

137 Kramer, L.D., J.L. Hardy, S.B. Presser, and E.J. Houk. 1981. Dissemination barriers for western equine encephalomyelitis virus in Culex tarsalis infected after ingestion of low viral doses. American Journal of Tropical Medicine and Hygiene 30 : 190-197. Kuno, G. 1997. Factors influencing the tran smission of dengue viruses. Pages 61-88 in D.J. Gubler and G. Kuno, editors. Denuge and dengue hemorrhagic fever. CABI Publising, New York, NY. Kurkela, S., T. Manni, J. Myllynen, A. Va heri, and O. Vapalahti. 2005. Clinical and laboratory manifestations of Sindbis viru s infection: Prospective study, Finland, 2002-2003. Journal of Infectious Diseases 191 : 1820-1828. Kurkela, S., T. Manni, A. Vaheri, and O. Vapalahti. 2004. Causative agent of Pogosta disease isolated from blood and skin lesions. Emerging Infectious Diseases 10 : 889-894. Lafferty, K.D., and R.D. Holt. 2003. How should environmental stress affect the population dynamics of di sease? Ecology Letters 6 : 654-664. Laine, M., R. Luukkainen, J. and A. To ivanen. 2004. Sindbis viruses and other alphaviruses as cause of human arthritic di sease. Journal of Internal Medicine 256 : 457-471. Laine, M., R. Luukkainen, J. Jalava, J. Il onen, P. Kuusisto, and A. Toivanen. 2000. Prolonged arthritis associated with Si ndbis-related (Pogosta) virus infection. Rheumatology 39 : 1272-1274. Leitmeyer, K.C., D.W. Vaughn, D.M. Watts, R. Salas, I. Villalobos de Chacon, C. Ramos, and R. Rico-Hesse. 1999. Dengue viru s structural differences that correlate with pathogenesis. Journal of Virology 73 : 4738-4747. Lonard, P.M., and S.A. Juliano. 1995. Effect of leaf litter and de nsity on fitness and population performance of the hole mosquito Aedes triseriatus Ecological Entomology 20 : 125-136. Levine, J.M. 1999. Indirect facilitation: Ev idence and predictions from a riparian community. Ecology 80 : 1762-1769. Levine, J.M., M. Vila, C.M. DAntonio, J.S. Dukes, K. Grigulis, and S. Lavorel. 2002. Mechanisms underlying the impacts of exotic plant invasions. Proceedings of the Royal Society of London B. 270 : 775-781. Lewis, J.A., G-J Chang, R.S. Lanciotti, R. M. Kinney, L.W. Mayer, and D.W. Trent. 1993. Phylogenetic relationships of Dengue-2 viruses. Virology 197 : 216-224. Livdahl, T.P. 1984. Interspecific interac tions and the r-K continuum: laboratory comparisons of geographic strains of Aedes triseriatus Oikos 42 : 193-202.

PAGE 150

138 Livdahl, T.P. 1982. Competition within and between hatching cohorts of a treehole mosquito. Ecology 63 : 1751-1760. Livdahl, T.P., and M.S. Willey. 1991. Prospect s for an invasion: Competition between Aedes albopictus and native Aedes triseriatus Science 253 : 189-191. Livdahl, T.P., and G. Sugihara. 1984. Non-lin ear interactions of populations and the importance of estimating per capita rates of change. Journal of Animal Ecology 53 : 573-580. Lively, C.M., S.G. Johnson, L.F. Delph, and K. Clay. 1995. Thinning reduces the effect of rust infection on jewelweed ( Impatiens capensis ). Ecology 76 : 1859-1862. Lord, CC., C.R. Rutledge, and W.J. Tab achnick. 2006. Relationships between host viremia and vector susceptibility for arbovi ruses. Journal of Medical Entomology 43 : 623-630. Lorenz, L., B.J. Beaty, T.H.G. Aitken, G. P. Wallis, and W.J. Tabachnick. 1984. The effect of colonization upon Aedes aegypti susceptibility to oral infection with yellow fever virus. American Journa l of Tropical Medicine and Hygiene 33 : 690694. Lounibos, L.P. 2002. Invasions by insect vect ors of human disease. Annual Review of Entomology 47 : 233-266. Lounibos, L.P., R.L. Escher, and R. Lourenco -de-Oliveira. 2003a. As ymmetric evolution of photoperiodic diapause in temperature and tropical invasi ve populations of Aedes albopictus (Diptera: Culicidae). Annals of the Entomological Society of America 96 : 512-518. Lounibos, L.P., G.F. OMeara, N. Nishimura, and R.L. Escher. 2003b. Interactions with native mosquito larvae regulate the production of Aedes albopictus from bromeliads in Florida. Ecological Entomology 28 : 551-558. Lounibos, L.P., S. Suarez, Z. Menendez, N. Nishimura, R.L. Escher, S.M. OConnell, and J.R. Rey. 2002. Does temperature aff ect the outcome of larval competition between Aedes aegypti and Aedes albopictus ? Journal of Vector Ecology 27 : 8695. Lounibos, L.P., N. Nishimura, and R.L. Es cher. 1993. Fitness of a treehole mosquito: influences of food type and predation. Oikos 66 : 114-118. Lounibos, L.P., N. Nishimura, and R.L. Esch er. 1992. Seasonality of oak litterfall in Southeastern Florida. Florida Scientist 55 : 92-98.

PAGE 151

139 Lundstrm, J.O. 1999. Mosquito-b orne viruses in Western Eu rope: A review. Journal of Vector Ecology 24 : 1-39. Lundstrm, J.O., K.M. Lindstrm, B. Olse n, R. Dufva, and D.S. Krakower. 2001. Prevalence of Sindbis virus neutralizi ng antibodies among Swedish passerines indicates that thrushes are the main amplifying hosts. Journal of Medical Entomology 38 : 289-297. Lundstrm, J.O. 1994. Vector competence of Western European mosquitoes for arboviruses: A review of field and experime ntal studies. Bulletin for the Society of Vector Ecology 19 : 23-36. Lundstrm, J.O., S. Vene, J-F. Saluzzo, a nd B. Niklasson. 1993a. Antigenic comparison of Ockelbo virus isolates from Sweden a nd Russia with Sindbis virus isolates from Europe, Africa, and Australia: Further evidence for variati on among Alphaviruses. American Journal of Tropi cal Medicine and Hygiene 49 : 531-537. Lundstrm, J.O., M.J. Turell, and B. Nikla sson. 1993b. Viremia in three orders of birds (Anseriformes, Galliformes and Passerifor mes) inoculated with Ockelbo virus. Journal of Wildlife Disease 29 : 189-195. Lundstrm, J.O., S. Vene, A. Espmark, M. Engvall, and B. Niklasson. 1991. Geographic and temporal distribution of Ockelbo disease in Sweden. Epidemiology and Infection 106 : 567-574. Lundstrm, J.O., B. Niklasson, and D.B. Francy. 1990a. Swedish Culex torrentium and Cx. pipiens (Diptera: Culicidae) as experimental vectors of Ockelbo virus. Journal of Medical Entomology 27 : 561-563. Lundstrm, J.O., M.J. Turell, and B. Ni klasson. 1990b. Effect of environmental temperature on the vector competence of Culex pipiens and Cx. torrentium for Ockelbo virus. American Journal of Tropical Medicine and Hygiene 43 : 534-542. Luo, T., and D. Brown. 1993. Purification and ch aracterization of a Sindbis virus-induced peptide which stimulates its own producti on and blocks RNA synthesis. Virology 194 : 44-49. Luz, P.M., C.T. Codeo, E. Massad, and C.J. Struchiner. 2003. Uncertainties regarding dengue modeling in Rio de Janeiro, Brazil. Memrias do Instituto Oswaldo Cruz 98 : 871-878. Lvov, D.K., T.M. Skvortsova, L.K. Berezina et al. 1984. Isolation of Karelian fever agent from Aedes communis mosquitoes. Lancet 2 : 399-400. Lvov, D.K., T.M. Skvortsova, N.G. Kondashina, et al. 1982. Etiology of Karelian fever, a new arbovirus infection. Voprosy Virus 27 : 690-692.

PAGE 152

140 Maci, A., and W.E. Bradshaw. 2000. Seasonal availability of resources and habitat degradation for the west ern tree-hole mosquito, Aedes sierrensis Oecologia 125 : 55-65. Mack, R.N., D. Simberloff, W.M. Lonsdale, H. Evans, M. Clout, and F. A. Bazzaz. 2000. Biotic invasions: Causes, epidemiol ogy, global consequences, and control. Ecological Applications 10 : 689-710. Madon M.B., M.S. Mulla, M.W. Shaw, S. Kluh, and J.E. Hazelrigg. 2002. Introduction of Aedes albopictus (Skuse) into Southern Ca lifornia and potential for its establishment. Jour nal of Vector Ecology 27 : 149-154. Matson P.A., and R.H. Waring. 1984. Effects of nutrient and light limitation on mountain hemlock: susceptibility to laminated root rot. Ecology 65 : 1517-1524. Mattingly, P.F. 1957. Genetical aspects of the Aedes aegypti problem. I. Taxonomy and bionomics. Annals of Tropical Medicine and Parasitology. 51 : 392-408. May, R.M., and R.M. Anderson. 1978. Regulat ion and stability of host-parasite population interactions. II. Destabilizi ng processing. Journal of Animal Ecology 47 : 249-267. McClelland, G.A.H., and B. Weitz. 1963. Serologi cal identification of the natural hosts of Aedes aegypti (L.) and some other mosquitoes (Diptera, Culicidae) caught resting in vegetation in Kenya and Uganda. Anna ls of Tropical Medicine and Parasitology 57 : 214-224. McIntosh B.M., P.G. Jupp, and I. Dos Sant os. 1978. Infection by Sindbis and West Nile viruses in wild populations of Culex (C ulex ) univittatus Theobald (Diptera: Culicidae) in South Africa. Journal of th e Entomological Society of South Africa 41 : 57-61. McIntosh B.M., P.G. Jupp, I. Dos Santos, and G.M. Meenehan. 1976. Epidemics of West Nile and Sindbis viruse s in South Africa with Culex (C ulex ) univittatus Theobald as vector. South African Journal of Medical Science 72 : 295-300. McIntosh B.M., P.G. Jupp, G.M. McGillivray, and J. Sweetnam. 1967. Ecological studies on Sindbis and West Nile viruses in South Africa. I. Viral activ ity as revealed by infection of mosquitoes and sentinel fo wls. South African Journal of Medical Science 32 : 1-14. McIntosh B.M., G.M. McGillivray, D.B. Di ckinson, H. Malherbe. 1964. Illness caused by Sindbis and West Nile viruses in S outh Africa. South African Journal of Medical Science 38:291-294. Mekuria Y., and M.G. Hyatt. 1995. Aedes albopictus in South Carolina. Journal of the American Mosquito Control Association 11 : 468-470.

PAGE 153

141 Meola RW., and R.S. Petralia. 1980. Juvenile hormone induction of biting behavior in Culex mosquitoes. Science 209 :1548-1550. Miles, J.A., J.S. Pillai, and T. Maguire. 1973. Multiplicati on of Whataroa virus in mosquitoes. Journal of Medical Entomology 10 : 176-185. Miller, B.R. 1987. Increased yellow fever vi rus infection and di ssemination rates in Aedes aegypti mosquitoes orally exposed to fres hly grown virus. Transactions of the Royal Society of Tropi cal Medicine and Hygiene 81 : 1011-1012. Miller, B.R., B.J. Beaty, T.H. G. Aitken, K. H. Eckels, and P.K. Russell. 1982. Dengue-2 vaccine: Oral infection, transmission, and lack of evidence for reversion in the mosquito, Aedes aegypti American Journal of Tropi cal Medicine and Hygiene 31 : 1232-1237. Mims, C.A., M.F. Day, and I.D. Marshall. 1966. Cy topathic effect of Semliki Forest virus in the mosquito Aedes aegypti American Journal of Tropical Medicine and Hygiene 22 : 332-337. Mitchell, C.J. 1991. Vector competence of North and South American strains of Aedes albopictus for certain arboviruses: A review. Journal of the American Mosquito Control Association 7 : 446-451. Mogi, M., I. Miyagi, K. Abadi, and Syafruddi n. 1996. Interand intraspecific variation in resistance to desiccation by adult Aedes ( Stegomyia ) spp. (Diptera: Culicidae) from Indonesia. Journal of Medical Entomology 33 : 53-57. Moncayo, A.C., Z. Fernandex, D. Ortiz, F. Dial lo, A. Sall, S. Hartman, C.T. Davis, L. Coffey, C.C. Mathiot, R.B. Tesh, and S.C. Weaver. 2004. Dengue emergence and adaptation to peridomestic mosquitoes. Emerging Infectious Diseases 10 : 17901796. Moore, C.G., 1999. Aedes albopictus in the United States: current status and prospects for further spread. Journal of the Amer ican Mosquito Control Association 15 : 221227. Moore, C.G., and D.M. Whitacre. 1972. Competition in mosquitoes. 2. Production of Aedes aegypti larval growth retardants at vari ous densities and nutrition levels. Annals of the Entomological Society of America 65 : 915-918. Moore, C.G., and B.R. Fisher. 1969. Competitio n in mosquitoes. Density and species ratio effects on growth, mortality, f ecundity, and the production of growth retardant. Annals of the En tomological Society of America 62 : 1325-1331. Morris, J.G., Jr., and M. Potter. 1997. Emer gence of new pathogens as a function of changes in host susceptibility. Emerging Infectious Diseases 3 : 435-441.

PAGE 154

142 Morrison, L.W. 1999. Indirect effects of phor id fly parasitoids on the mechanisms of interspecific competition among ants. Oecologia 121 : 113-122. Morrison, A.C., K. Gray, A. Getis, H. Astete M. Sihuincha, D. Focks, D. Watts, J.D. Stancil, J.G. Olson, P. Blair, and T.W. Scott. 2004. Temporal and geographic patterns of Aedes aegypti (Diptera: Culicidae) production in Iquitos, Peru. Journal of Medical Entomology 41 : 1123-1142. Munstermann, L.E. 1994. Unexpected geneti c consequences of colonization and inbreeding Allozyme tracking in Cu licidae (Diptera). Annals of the Entomological Society of America 87 : 157-164. Munstermann, L.E. 1980. Distinguis hing geographic stains of the Aedes altropalpus grwoup (Diptera: Culicidae) by analysis of enzyme variation. Annals of the Entomological Society of America 73 : 699-704. Murphy, F.A., S.G. Whitfield, W.D. Sudia, a nd R.W. Chamberlain. 197 5. Interactions of vector with vertebrate pat hogenic viruses. Pages 25-48 in K. Maramorosch and Shope, editors. Invertebra te immunity. Academic Press, New York, NY. Murray, D.L., L.B. Keith, and J.R. Cary. 1998. Do parasitism and nutritional status interact to affect production in snowshoe hares? Ecology 79 : 1209-1222. Myles, K.M., D.J. Pierro, and K.E. Ols on. 2004. Comparison of the transmission of two genetically distinct Sindbis vi ruses after oral infection of Aedes aegypti (Diptera: Culicidae). Journal of Medical Entomology 41 : 95-106. Nasci, R.S. 1991. Influence of larval and adult nutrition on biting persistence in Aedes aegypti (Diptera: Culicidae). Jour nal of Medical Entomology 28 : 522-526. Nasci, R.S. 1990. Relationship of wing length to adult dry weight in several mosquito species (Diptera: Culicidae). J ournal of Medical Entomology 27 : 716-719. Nasci, R.S. 1987. Adult body size and parity in field populations of the mosquitoes Anopheles crucians Aedes taeniorhynchus and Aedes sollicitans Journal of the American Mosquito Control Association 3 : 636-637. Nasci, R.S. 1986a. The size of emerging and host-seeking Aedes aegypti and the relation of size to blood-feeding success in the fi eld. Journal of the American Mosquito Control Association 2 : 61-62. Nasci, R.S. 1986b. Relationship between adult mosquito (Diptera: Culicidae) body size and parity in field populations Environmental Entomology 15 : 874-876. Nasci, R.S., and C.J. Mitchell. 1994. Larval diet, adult size, and susceptibility of Aedes aegypti (Diptera: Culicidae) to infection with Ross River virus. Journal of Medical Entomology 31 : 123-126.

PAGE 155

143 Nasci, R.S., S.G. Hare, and F.S. Willis. 1989. Interspecific mating between Louisiana strains of Aedes albopictus and Aedes aegypti in the field and laboratory. Journal of the American Mosquito Control Association 5 : 416-421. Naeem, S. 1988. Resource heterogeneity foster s coexistence of a mite and a midge in pitcher plants. Ecological Monographs 58 : 215-227. Nawrocki, S.J., and W.A. Hawley. 1987. Estima tion of the northern lim its of distribution of Aedes albopictus in North America. Journal of the American Mosquito Control Association 3 : 314-317. Niebylski, M.L., H.M. Savage, R.S. Nasc i, and G.B. Craig, Jr. 1994. Blood hosts of Aedes albopictus in the United States. Journal of the American Mosquito Control Association. 10 : 447-450. Niklasson, B. 1989. Sindbis and Sindbi s-like viruses. Pages 167-176 in T.P. Monath, editor. The arboviruses: epidemiology a nd ecology, volume 4. CRC, Boca Raton, FL. Niklasson, B., A. Espmark, and J.O. Lunds trom. 1988. Occurrence of arthralgia and specific IgM antibodies three to four y ears after Ockelbo di sease. Journal of Infectious Diseases 157 : 832-835. Niklasson, B., and A. Espmark. 1986. Ockel bo disease: arthralg ia 3-4 years after infection with a Sindbis virus related agent. Lancet i : 1039-1040. Niklasson, B., A. Espmark, J.W. LeDuc, T.P. Gargan, W. Ennis, R.B. Tesh, and A.J. Main, Jr. 1984. Association of a sindbis-like virus with Ockelbo disease in Sweden. American Journal of Tropi cal Medicine and Hygiene 33 : 1212-1217. Norder H., J.O. Lundstrm, O. Kozuch, and L.O. Magnius. 1996. Gene tic relatedness of Sindbis virus strains from Europe, Middle East, and Africa. Virology 222 : 440-445. Olson, K.E., S. Higgs, P.J. Gaines, A.M. Powers, B.S. Davis, K.I. Kamrud, J.O. Olson, C.D. Blair, and B.J. Beaty. 1996. Genetica lly engineered resi stance to dengue-2 virus transmission in mosquitoes. Science 272 : 884-886. Olson, K., and D.W. Trent. 1985. Genetic a nd antigenic variations among geographical isolates of Sindbis virus. Journal of General Virology 66 : 797-810. OMeara, G.F., L. F. Evans, Jr., A.D. Gettman, and J.P. Cuda. 1995. Spread of Aedes albopictus and decline of Ae. aegypti (Diptera: Culicidae) in Florida. Journal of Medical Entomology 32 : 554-562. OMeara, G.F., F.E. Vose, and D.B. Carl son. 1989. Environmental factors influencing oviposition by Culex ( Culex ) (Diptera: Culicidae) in two types of traps. Journal of Medical Entomology 26 : 528-534.

PAGE 156

144 Oppliger, A., J. Clobert, J. Lecomte, P. Lo renzon, K. Boudjemadi, and H.B. John-Alder. 1998. Environmental stress increases the prev alence and intensity of blood parasite infection in the common lizard Lacerta vivipara Ecology Letters 1 : 129-138. Osenberg, C.W., G.C. Mittelb ach, and P.C. Wainwright. 1992. Two-stage life histories in fish: The interaction between juvenile competition and adult performance. Ecology 73 : 255-267. Pages, J-P., G. Pache, D. Joud, N. Magnan, and R. Michalet. 2003. Di rect and indirect effects of shade on four forest tree se edlings in the French Alps. Ecology 84 : 27412750. Paradise C.J., and K. Kuhn. 1999. Interactive e ffects of pH and leaf litter on a shredder, the scirtid beetle, Helodes pulchella inhabiting tree-holes. Freshwater Biology 41 : 43-49. Park, T. 1948. Experimental studies of inters pecies competition. I. Competition between populations of the flour beetles, Tribolium confusum Duval and Tribolium castaneum Herbst. Ecological Monographs 18 : 265-308. Paulson, S.L., and W.A. Hawley. 1991. Effect of body size on the vector competence of field and laboratory populations of Aedes triseriatus for La Crosse virus. Journal of the American Mosquito Control Association 7 : 170-175. Peck, G.W., and W.E. Walton. 2005. Effect of different assemblages of larval foods on Culex quinquefasciatus and Culex tarsalis (Diptera: Culicidae) growth and whole body stoichiometry. Envir onmental Entomology 34 : 767-774. Petren, K. and T.J. Case. 1996. An experi mental demonstration of exploitation competition in an ongoing invasion. Ecology 77 : 118-132. Petren, K., D.T. Bolger, and T.J. Case. 1993. Mechanisms in the competitive success of an invading sexual gecko over an asexual native. Science 259 : 354-358. Platt, K.B., J.A. Mangiafico, O.J. Rocha, M.E. Zaldivar, J. Mora, G. Trueba, and W.A. Rowley. 2000. Detection of dengue virus neut ralizing antibodies in bats from Costa Rica and Ecuador. Journal of Medical Entomology 37 : 965-967. Platt, K.B., K.J. Linthicum, K.S.A. Myint, B.L. Innis, B. Lerdthusnee, and D.W. Vaughn. 1997. Impact of dengue virus infection on feeding behavior of Aedes aegypti American Journal of Tropi cal Medicine and Hygiene 57 : 119-125. Ponlawat, A., and L.C. Harringt on. 2005. Blood feeding patterns of Aedes aegypti and Aedes albopictus in Thailand. Journal of Medical Entomology 42 : 844-849. Pumpuni, C.B., J. Knepler, and G.B. Craig, Jr 1992. Influence of te mperature and larval nutrition on diapause in ducing photoperiod of Aedes albopictus Journal of the American Mosquito Control Association 8 : 223-227.

PAGE 157

145 Reed, L.J., and H. Muench. 1938. A simple method of estimating fifty per cent endpoints. American Journal of Hygiene 27 : 493-497. Reisen, W.K., Y. Fang, and V.M. Martinez. 2006. Effects of temperature on the transmission of West Nile virus by Culex tarsalis (Diptera: Culicidae). Journal of Medical Entomology 43 : 309-317. Reisen, W.K., J.L. Hardy, and S.B. Presser. 1997. Effects of water quality on the vector competence of Culex tarsalis (Diptera: Culicidae) for Western Equine encephalomyelitis (Togaviridae) and St. Louis encephalitis (Flaviviridae) viruses. Journal of Medical Entomology 34 : 631-643. Reiter, P. 1998. Aedes albopictus and world trade in used tires, 1988-1995: the shape of things to come? Journal of the Amer ican Mosquito Control Association 14 : 83-94. Reiter, P., and R.F. Darsie. 1984. Aedes albopictus in Memphis, Tennesse (USA): An achievement of modern tran sportation? Mosquito News 44 : 396-399. Reitz, S.R., and J.T. Trumble. 2002. Competitive displacement among insects and arachnids. Annual Review of Entomology 47 : 435-465. Relyea, R.A. 2000. Trait-mediated indirect effects in larval anurans: reversing competition with the thre at of predation. Ecology 81 : 2278-2289. Rice, W.R. 1989. Analyzing tables of statistical tests. Evolution 43 : 223-225. Richardson, J., A. Molina-Cruz, M.I. Salazar, and W.Black IV. Quntitative analysis of dengue-2 virus RNA during the extrinsi c incubation period in individual Aedes aegypti American Journal of Tropical Medicine and Hygiene 74 : 132-141. Rico-Hesse, R., L.M. Harrison, R.A. Salas, D. Tovar, A. Nisalak, C. Romas, J. Boshell, M.T.R. de Mesa, R.M.R. Nogueira, and A.T. da Rosa. 1997. Origins of dengue type 2 viruses associated with increased pathogenicity in the Americas. Virology 230 : 244-251. Rodhain, F. 1991. The role of monkeys in the biology of dengue and yellow fever. Comparative Immunology, Microbio logy and Infectious Disease 14 : 9-19. Rodhain, F, and L. Rosen. 1997. Mosquito vector s and dengue virus-vector relationships. Pages 45-60 in D.J. Gubler and G. Kuno, editors. Dengue and dengue hemorrhagic fever. CABI Publishing, New York, NY. Rosen, L., L.E. Roseboom, D.J. Gubler, J.C. Lien, and B.N. Chaniotis. 1985. Comparative susceptibility of mosquito sp ecies and strains to oral and parenteral infection with Dengue and Japanese Encepha litis viruses. American Journal of Tropical Medicine and Hygiene 34 : 603-615.

PAGE 158

146 Rosen, L., D.A. Shroyer, R.B. Tesh, J.E. Freier, and J.C. Lien. 1983. Transovarial transmission of dengue viruses by mosquitoes: Aedes albopictus and Aedes aegypti American Journal of Tropi cal Medicine and Hygiene 32 : 1108-1119. Rudnick, A. 1978. Ecology of dengue virus. Asian Journal of Infectious Disease 2 : 156160. Rudnick, A. 1965. Studies of the ecology of de ngue in Malaysia: a preliminary report. Journal of Medical Entomology 2 : 203-208. Sammels, L.M., M.D. Lindsay, M. Poidinger, R.J. Coelen, and J.S. Mackenzie. 1999. Geographic distribution and evolution of Sindbis virus in Australia. Journal of General Virology 80 : 739-748. Sanders, H.R., B.D. Foy, A.M. Evans, L.S. Ross, B.J. Beaty, K.E. Olson, and S.S. Gill. 2005. Sindbis virus induces transport proce sses and alters expression of innate immunity pathway genes in the midgut of the disease vector, Aedes aegypti Insect Biochemistry and Molecular Biology 35 : 1293-1307. Sanders, N.J., and D.M. Gordon. 2003. Res ource-dependent interactions and the organization of desert ant communities. Ecology 84 : 1024-1031. SAS Institute. 2002. SAS/STAT users guide, ve rsion 9. SAS Institute, Inc., Cary, NC. SAS Institute Inc. 1989. SAS/STAT users guide, version 6 SAS Institute, Inc., Cary, NC. Savage H.M., M.L. Niebylski, G.C. Smith, C.J. Mitchell, and G.B. Craig, Jr. 1993. Hostfeeding patterns of Aedes albopictus (Diptera: Culicidae) at a temperate North American site. Journal of Medical Entomology 30 : 27-34. Scheiner, S.M. 2001. MANOVA: multiple response variables and multispecies interactions. Pages 99-115 in S.M. Scheiner and J. Gurevitch, editors. Design and analysis of ecological experiments. S econd edition. Oxford University Press, Oxford, UK. Scheiner, S.M. 1993. Genetics and evolution of phenotypic plasticit y. Annual Review of Ecology and Systematics 24 : 35-68. Schneider, J.R., A.C. Morrison, H. Astete, T. W. Scott, and M.L. Wilson. 2004. Adult size and distribution of Aedes aegypti (Diptera: Culicidae) associated with larval habitats in Iquitos, Peru. Journal of Medical Entomology 41 : 634-642. Schneider, P., W. Takken, and P. McCall. 2000. Interspecific competition between sibling species larvae of Anopheles arabiensis and An. gambiae Medical and Veterinary Entomology 14 : 165-170.

PAGE 159

147 Schoener, T.W. 1983. Field experiments on interspecific competition. American Naturalist. 122 : 240-285. Schwartz, A., and J.C. Koella. 2004. The cost of immunity in the yellow fever mosquito, Aedes aegypti depends on immune activation. Jo urnal of Evolutionary Biology 17 : 834-840. Scott, T.W. 2001. Are bats really involved in dengue virus transmission? Journal of Medical Entomology 38 : 771-772. Scott, T.W., A.C. Morrison, L.H. Lorenz, G.G. Clark, D. Strickman, P. Kittayapong, H. Zhou, and J.D. Edman. 2000a. Longitudinal studies of Aedes aegypti (Diptera: Culicidae) in Thailand and Puerto Rico: population dynamics. Journal of Medical Entomology 37 : 77-88. Scott, T.W., P.H. Amerasinghe, A.C. Morris on, L.H. Lorenz, G.G. Clark, D. Strickman, P. Kittayapong, and J.D. Edman. 2000b. Longitudinal studies of Aedes aegypti (Diptera: Culicidae) in Thailand and Pu erto Rico: Blood feeding frequency. Journal of Medical Entomology 37 : 89-101. Scott, T.W., G.G. Clark, P.H. Amerasinghe, L.H. Lorenz, P. Reiter, and J.D. Edman. 1993a. Detection of multiple blood feeding patterns in Aedes aegypti (Diptera: Culicidae) during a single gonotrophic cycle using histological technique. Journal of Medical Entomology 30 : 94-99. Scott, T.W., E. Chow, D. Strickman, P. Kittayapong, and J.D. Edman. 1993b. Blood feeding patterns of Aedes aegypti (Diptera: Culicidae) coll ected in a rural Thai village. Journal of Medical Entomology 30 : 922-927. Seabaugh, R.C., K.E. Olson, S. Higgs, J.O. Carlson, B.J. Beaty. 1998. Development of a chimeric Sindbis vi rus with enhanced per os infection of Aedes aegypti Virology 243 : 99-112. Seaton D.R., Lumsden W.H.R. 1941. Observations on the effects of age, fertilization and light on biting by Aedes aegypti (L.) in a controlled microclimate. Annals of Tropical Medicine and Parasitology 35 : 23-36. Sheldon, B.C. and S. Verhulst. 1996. Ecologi cal immunology: costly parasite defences and trade-offs in evolutionary eco logy. Trends in Ecology and Evolution 11 : 317321. Shirako, Y. B. Niklasson, J.M. Dalrymple, E. G. Strauss, and J.H. Strauss. 1991. Structure of the Ockelbo virus genome and its relati onship to other sindbi s viruses. Virology 182 : 753-764. Shroyer, D.A. 1986. Aedes albopictus and arboviruses: A concise re view of the literature. Journal of the American Mo squito Control Association 2 : 424-428.

PAGE 160

148 Skogh, M., and A. Espmark. 1982. Ockelbo dis ease: epidemic arthritis-exanthema syndrome in Sweden caused by Sindbis-virus like agent. Lancet 3 : 795-796. Smith, C.E.G. 1956. The history of dengue in tr opical Asia and its probable relationship to the mosquito Aedes aegypti Journal of Tropical Medicine and Hygiene 59 : 243251. Smith, D.L., J. Dushoff, and F.E. McKenz ie. 2004. The risk of a mosquito-borne infection in a heterogeneous environm ent. Public Library of Science 2 : 1957-1964. Sokal R.R., and F.J. Rohlf. 1995. Biometry. W.H. Freeman and Company, New York, NY. Sota, T., M., Mogi, and K. Kato. 1998. Local and regional-scale f ood web structure in Nepenthes alata pitchers. Biotropica 30 : 82-91. Sota T, and Mogi M. 1992. Interspecific variation in desiccation survival time of Aedes ( Stegomyia ) mosquito eggs is correlated with habitat and egg size. Oecologia 90 : 353-358. Sprenger, D., and T. Wuithiranyagool. 1986. The discovery and distribution of Aedes albopictus in Harris County, Texas. Journal of the American Mosquito Control Association 2 : 217-219. Steinhaus, E.A. 1958. Crowding as a possible st ress factor in insect disease. Ecology 39 : 503-514. Sumanochitrapon, W., D. Strickman, R. Sithipra sasna, P. Kittayapong, and B.L. Innis. 1998. Effect of size and ge ographic origin of Aedes aegypti on oral infection with Dengue-2 virus. American Journal of Tropical Medicine and Hygiene 58 : 283-286. Suwanchaichinda, C., and S.M. Paskewitz. 1998. Effects of larval nutrition, adult body size, and adult temperature on the ability of Anopheles gambiae (Diptera: Culicidae) to melanize Sephadex beads. Journal of Medical Entomology 35 : 157161. Swift, M.J., O.W. Heal, and J.M. A nderson. 1979. Decomposition in terrestrial ecosystems. University of California Press, Berkeley, CA. Tabachnick, W.J. 1994. Genetics of insect ve ctor competence for arboviruses. Pages 93108 in K.F. Harris, editor. Advances in disease vector re search, Volume 10. Springer-verlag, New York, NY. Tabachnick, W.J. 1991. Evolutionary genetics and arthropod-borne disease. The yellow fever mosquito. American Entomologist 37 : 14-24. Tabachnick, W.J., L.E. Munstermann, and J. R. Powell. 1979. Genetic distinctness of sympatric forms of Aedes aegypti in East Africa. Evolution 33 : 287-295.

PAGE 161

149 Takahashi, M. 1976. The effects of envir onmental and physiological conditions of Culex tritaeniorhynchus on the pattern of transmission of Japanese encephalitis virus. Journal of Medical Entomology 13 : 275-284. Takken W, M.J. Klowden, and G.M. Chambers. 1998. Effect of body size on host seeking and blood meal utilization in Anopheles gambiae sensu stricto (Diptera: Culicidae): the disadvantage of being sm all. Journal of Medical Entomology 35 : 639-645. Taylor, R.M., H.S. Hurlbut, T.H. Work, J.R. Kingston, and T.E. Frothingham. 1955. Sindbis virus: a newly recognized arthropodtransmitted virus. American Journal of Tropical Medicine and Hygiene 4 : 844-862. Teng H-J, and C. Apperson. 2000. Development and survival of immature Aedes albopictus and Aedes triseriatus (Diptera: Culicidae) in the laboratory: Effects of density, food, and competition on response to temperature. Journal of Medical Entomology 37 : 40-52. Tesh, R.B. 1982. Arthritides caused by mos quito-borne viruses. Annual Review of Medicine 33 :31-40. Tesh, R.B. 1980. Experimental studies on the tr ansovarial transmission of Kunjin and San Angelo viruses in mosquitoes. Ameri can Journal of Tropical Medicine and Hygiene 29 : 657-666. Thenmozhi, V., S.C. Tewari, R. Manavaia n, A. Balasubramanian, and A. Gajanans. 2000. Natural vertical transmission of dengue viruses in Aedes aegypti in southern India. Transactions of the Royal Society of Tropical Medicine and Hygiene 94 : 507-. Thomas, R.E., W.K. Wu, D. Verleye, and K.S. Rai. 1993. Midgut basal lamina thickness and dengue-1 virus dissemination rates in laboratory strains of Aedes albopictus (Diptera: Culicidae). Journa l of Medical Entomology 30 : 326-331. Tilman, D. 1982. Resource competition and community structure. Princeton University Press, Princeton, NJ. Trpis, M., and W. Hausermann. 1986. Disper sal and other population parameters of Aedes aegypti in an African village and their possible significance in epidemiology of vector-borne diseases. American J ournal of Tropical Medicine and Hygiene 35 : 1263-1279. Turell, M.J. 1988. Reduced Rift Valley Feve r virus infection rates in mosquitoes associated with pledget feedings. Amer ican Journal of Tr opical Medicine and Hygiene 39 : 597-602. Turell, M.J., M.L. OGuinn, D.J. Dohm, a nd J.W. Jones. 2001. Vector competence of North American mosquitoes (Diptera: Culicidae) for West Nile virus. Journal of Medical Entomology 38 : 130-134.

PAGE 162

150 Turell, M.J., and J.O. Lundstrm. 1990. Eff ect of environmental temperature on the vector competence of Aedes aegypti and Ae. taeniorhynchus for Ockelbo virus. American Journal of Tropi cal Medicine and Hygiene 43 : 543-550. Turell, M.J., J.O. Lundstrm, and B. Nikl asson. 1990. Transmission of Ockelbo virus by Aedes cinereus Ae. communis and Ae. excrucians (Diptera: Culicidae) collected in an enzootic area in central Swed en. Journal of Medical Entomology 27 : 266-268. Turell, M.J., T.P. Gargan, II, and C.L. Bailey. 1985. Culex pipiens (Diptera: Culicidae) morbidity and mortality associated with Ri ft Valley Fever virus infection. Journal of Medical Entomology 22 : 332-337. Turell, M.J. P.A. Rossignol, A. Spielman, C.A. Rossi, and C.L. Bailey. 1984. Enhanced arbovirus transmission by mosquitoes that concurrently ingested microfilariae. Science 225 : 1039-1041. Turell, M.J., T.P. Gargan II, and C.L. Baile y. 1984. Replication and dissemination of rift valley fever virus in Culex pipiens American Journal of Tropical Medicine and Hygiene 33 : 176-181. Turunen, M., P. Kuusisto, P.-E. Uggeldahl, and A. Toivanen. 1998. Pogosta disease: Clinical observations during an outbreak in the province of north Karelia, Finland. British Journal of Rheumatology 37 : 1177-1180. Vaughan. J.A., M. Trpis, and M.J. Turell. 1999. Brugia malayi microfilariae (Nematoda: Filaridae) enhance the inf ectivity of Venezuelan equine encephalitis virus to Aedes mosquitoes (Diptera: Culicidae). Journal of Medical Entomology 36 : 758-763. Vazeille, M., L. Rosen, L. Mousson, and A-B. Failloux. 2003. Low oral receptivity for dengue type 2 viruses of Aedes albopictus from Southeast Asia compared with that of Aedes aegypti American Journal of Tr opical Medicine and Hygiene 68 : 203208. Vazeille, M., L. Mousson, I. Rakatoarivony, R. Villeret, F. Rodhain, J-B Duchemin, and A-B. Failloux. 2001. Population genetic struct ure and competence as a vector for dengue type 2 virus of Aedes aegypti and Aedes albopictus from Madagascar. American Journal of Tropi cal Medicine and Hygiene 65 : 491-497. Vazeille-Falcoz, M., L. Mousson, R. R odhain, E. Chungue, and A-B. Failloux. 1999. Variation in oral susceptibility to de ngue type 2 virus of populations of Aedes aegypti from the islands of Tahiti and Moorea, French Polynesia. American Journal of Tropical Medicine and Hygiene 60 : 292-299. Walker, E.D., D.L. Lawson, R.W. Merritt, W.T. Morgan, and M.J. Klug. 1991. Nutrient dynamics, bacterial populations, and mos quito productivity in treehole ecosystems and microorganisms. Ecology 72 : 1529-1546.

PAGE 163

151 Walker, E.D., and R.W. Merritt. 1988. The significance of leaf detritus to mosquito (Diptera: Culicidae) productivity from treeholes. Environmental Entomology 17 : 199-206. Wallis, G.P., T.H.G. Aitken, B.J. Beaty, L. Lorenz, G.D. Amato, and W.J. Tabachnick. 1985. Selection for susceptibil ity and refractoriness of Aedes aegypti to oral infection with yellow fever virus. Amer ican Journal of Tropical Medicine and Hygiene. 34 : 1225-1231. Wang, E., H. Ni, R. Xu, A.D. T., Barrett, S.J. Watowich, D.J. Gubler, and S.C. Weaver. 2000. Evolutionary relationships of endemic/ epidemic and sylvatic dengue viruses. Journal of Virology 74 : 3227-3234. Washburn, J.O., D.R. Mercer, and J.R. A nderson. 1991. Regulatory role of parasites: Impact on host population shifts with resource availability. Science 253 : 185-188. Weaver, S.C. 1986. Electron microscopic analys is of infection patterns for Venezuelan equine encephalomyelitis virus in the vector mosquito Culex ( Melanoconion ) taeniopus American Journal of Tropical Medicine and Hygiene. 35 : 624-631. Weaver, S.C., T.W. Scott, L.H. Lorenz, and P.M. Repik. 1991. Detection of Eastern Equine Encephalomyelitis virus deposition in Culiseta melanura following ingestion of radiolabeled virus in blo od meals. American Journal of Tropical Medicine and Hygiene. 44 : 250-259. Weaver, S.C., W.F. Scherer, E.W. Cu pp, and D.A. Castello. 1984. Barriers to dissemination of Venezuelan encephalitis vi ruses in the Middle American enzootic vector mosquito, Culex ( Melanoconion ) taeniopus American Journal of Tropical Medicine and Hygiene 33 : 953-960. Werner, E.E. 1992. Individual behavior and higher-order species interactions. The American Naturalist 140 : S5-S32. Werner, E.E. 1991. Nonlethal effects of a pr edator on competitive interactions between two anuran larvae. Ecology 72 : 1709-1720. Werner, E.E., and B.R. Anholt. 1996. Predator -induced behavioral indirect effects: Consequences to competitive interactions in anuran larvae. Ecology 77 : 157-169 White, D.O, and F.J. Fenner. 1994. Medical vi rology. Academic Press, New Yorik, NY. Whitehead, R.H. T.M. Yuill, D.J. Goul d, and P. Simasathien. 1971. Experimental infection of Aedes aegypti and Aedes albopictus with dengue viruses. Transactions of the Royal Society of Tr opical Medicine and Hygiene 65 : 661-667. Willis, F.S. and R.S. Nasci. 1994. Aedes albopictus (Diptera: Culicidae) population density and structure in Southwest Loui siana. Journal of Medical Entomology 31 : 594-599.

PAGE 164

152 Woodring, J.L., S. Higgs, and B.J. Beat y. 1996. Natural cycles of vector-borne pathogens. Pages 51-72. i n B.J. Beaty and W. C. Mar quardt, editors. The biology of disease vectors. University Press of Colorado, Niwot, CO. Wootton, J.T. 1994. The nature and consequenc es of indirect effects in ecological communities. Annual Review of Ecology and Systematics 25 : 443-466. Wootton, J.T. 1993. Indirect effects and ha bitat use in an intertidal community: Interaction chains and interaction mo difications. The American Naturalist 141 : 7189. Woodall J.P., M.C. Williams, and J.M. Elli ce. 1962. Sindbis infection in man. East Afrrican Virus Research 12 : 17. Xue, R-D. D.R. Barnard, and C.E. Schreck. 1995. Influence of body size and age of Aedes albopictus on human host attack rates and the repellency of Deet. Journal of the American Mosquito Control Association 11 : 50-53. Xiong, C., R. Levis, P. Shen, S. Schlesi nger, C.M. Rice, and H.W. Huang. 1989. Sindbis virus: An efficient, broad host range vect or for gene expression in animal cells. Science 243 : 1188-1191. Yanoviak, S. P. 1999. Effects of leaf litte r species on macroinvertebrate community properties and mosquito yield in Neotr opical tree hole microcosms. Oecologia 120 : 147-155. Ye-Ebiyo, Y., R.J. Pollack, A. Kiszewski, and A. Spielman. 2003. Enhancement of development of larval Anopheles arabiensis by proximity to flowering maize ( Zea mays ) in turbid water and when crowded. Am erican Journal of Tropical Medicine and Hygiene 68 : 748-752. Zar, J.H. 1996. Biostatistical analys is. Prentice Hall, Englewood Cliffs, NJ. Zytoon, E.M. H.I. El-Belbasi, and T. Ma tsumura. 1993. Mechanisms of increased dissemination of Chikungunya virus in Aedes albopictus mosquitoes concurrently ingesting microfilariae of Dirofilaria immitis American Journal of Tropical Medicine and Hygiene. 49 : 201-207.

PAGE 165

153 BIOGRAPHICAL SKETCH Barry W. Alto was born in Virginia, MN, in the heart of the Great North Woods. He spent a great deal of his childhood en joying the outdoors. After graduating from Virginia High School, he moved to St. Paul MN, and received his Bachelor of Arts degree in biology from University of St. Th omas. His schooling continued in Normal, IL, where he received his Master of Scie nce degree in biology from Illinois State University.


xml version 1.0 encoding UTF-8
REPORT xmlns http:www.fcla.edudlsmddaitss xmlns:xsi http:www.w3.org2001XMLSchema-instance xsi:schemaLocation http:www.fcla.edudlsmddaitssdaitssReport.xsd
INGEST IEID E20110217_AAAABY INGEST_TIME 2011-02-17T18:20:34Z PACKAGE UFE0014961_00001
AGREEMENT_INFO ACCOUNT UF PROJECT UFDC
FILES
FILE SIZE 8423998 DFID F20110217_AABQQD ORIGIN DEPOSITOR PATH alto_b_Page_113.tif GLOBAL false PRESERVATION BIT MESSAGE_DIGEST ALGORITHM MD5
698262457c02d0ff8ea23ecdc627fdef
SHA-1
40538cac8370c2400b4ada424b3b4aebee6abf24
25917 F20110217_AABQPO alto_b_Page_026.QC.jpg
4bf97216585cd433c0e9c517490b5902
e64a3a00c9de19761c5ddc646d2534d5fb7ffd32
17719 F20110217_AABQQE alto_b_Page_071.pro
1063e47fd7bf264c694e31a45f56a5d9
d7a56781617cbb5fb95c347d848924fda9c22615
1051981 F20110217_AABQPP alto_b_Page_097.jp2
bac72ae305772abe9ebab80219b12bf5
341bd14deac252d1c9e73db16cfa6df472babd90
1051975 F20110217_AABQQF alto_b_Page_067.jp2
92ce9c008cf2a3727f340a3a6cf0a32d
2e41d84618e84e47db4db00857ce91c9abc299d1
1833 F20110217_AABQPQ alto_b_Page_123.txt
69c2cca2f7bd52f101b0ec855fa9e7d8
211d759f2b785ece574649d8fa35e24c5797a931
26564 F20110217_AABQQG alto_b_Page_036.QC.jpg
119b1a3da6bb0df38013664b11b72d2b
ea5a19c27df4aca622ae712e97606d44370dc139
24717 F20110217_AABQPR alto_b_Page_073.pro
6314ff35345adc41fb13a8fd1fdd7f43
25fa31f438f85b7cf52fcf70949c0ca4664be86c
2115 F20110217_AABQQH alto_b_Page_120.txt
c754eb3c23fab708196d9b3e9ca56a9b
1ed8cde2d94af37b2e99cd5670db80d0e2168791
2488 F20110217_AABQPS alto_b_Page_140.txt
101053df3b4d6f93a6ca7e93572f5855
4b5e6e1c3d42c490fab3ddceff617e17b3b9af66
41550 F20110217_AABQPT alto_b_Page_039.pro
e72a7b3a8a783960de851b51ac6edcfb
800668ff86d834f17dbd6bba50bbbed3481d641a
49191 F20110217_AABQQI alto_b_Page_019.pro
f0585af09abebfd837750587b71314df
da8f85ff7490c2d68684ddaf150bfbd7e39ed8f6
24527 F20110217_AABQPU alto_b_Page_090.QC.jpg
15d2d8f09788c6f75be9f81a8bcea59e
9b946e7a704ad2ebadacb2a3bac40ad35bb0adbd
1051965 F20110217_AABQQJ alto_b_Page_032.jp2
81ebe21424330f18d0edc9dbb48621e8
2f1d1289987f9faf1cc81fe7272dffafccbd5c0c
52793 F20110217_AABQPV alto_b_Page_132.pro
545201c2a4f59c930197bc263fc10419
b328586800d40fe56e530036a5e85022ea2a124d
1051955 F20110217_AABQQK alto_b_Page_142.jp2
755805f770774660cb43ddcb175d0337
e3b2b907209ea6695977ddef21dd15110cbbede1
F20110217_AABQPW alto_b_Page_126.tif
36a3c883d5aa2c02f9200e049b910f5a
f4f08fa97c0d96f264c1edb7895227731771d547
F20110217_AABQQL alto_b_Page_125.tif
a08dc6d377e8f1a37d0158e01b79c7c7
4358c81c39ae179a57d1ffba80d0dceb66b8993b
6193 F20110217_AABQPX alto_b_Page_043thm.jpg
93f32600b7495b775af3d4090b0d3147
6fdc9bb72b71eb5ca2fb2957213a2cb982a251c8
F20110217_AABQRA alto_b_Page_079.tif
d27bb35de02e063f9b72e7f320cb3563
16c37d09d4a7e5138aaf515f1196967c07e8b359
F20110217_AABQQM alto_b_Page_015.tif
171852fc04ab1f2007ca5e5e50b64567
bf6e521e2d3f5090c99fe8b16d2e60a87057e332
F20110217_AABQPY alto_b_Page_103.tif
09673328eb9a0fc6606f9debb425d7bc
bef2884e57fff955b3ff6ec20c07d62608906d0c
F20110217_AABQRB alto_b_Page_145.tif
608b2e162f6295d7ed5e56c03d7349b5
ac6eb99c2bf4e4f2fdee4f5809efac369318dd32
567059 F20110217_AABQQN alto_b_Page_118.jp2
cdef67b3e5ec15ed65ba801b2352b839
8f5b01482c262917b191e5e8dfe412db794a64a1
F20110217_AABQPZ alto_b_Page_107.tif
af61ca588e595a9c1f34ab8d41996c8c
f5738d5a42c41657cd6aed1d81af04b6492c379f
8301 F20110217_AABQRC alto_b_Page_001.pro
77db1b4f0ab1bf17d28bb1dab1dcc308
52f8312cf0d2914e0fcf90021a287bd0ad7fd8db
11866 F20110217_AABQRD alto_b_Page_165.pro
e7ab8968b8ffb3f40bc321988327b631
345ef6778aa232d8de4ec7c988f8d5d9fe1e1283
49522 F20110217_AABQQO alto_b_Page_015.pro
093b114116bfc796b5eb20bffa786b7a
2f6665132f1779b338b4c92ee847a6d6909ef053
F20110217_AABQRE alto_b_Page_038.tif
527dfb5e75b73b2224bbe84ce62fc014
0c325ac25234c564e04aaab2b772b9b87ff92b5e
1051925 F20110217_AABQQP alto_b_Page_058.jp2
b44daae92940dab167879c1cb1d14634
c5d7164da8d8ac95e35c9772963a3fbaf2564fc7
28223 F20110217_AABQRF alto_b_Page_155.QC.jpg
1bf4babe680ee44754ee7421ebb228af
6fdb557dcc17d850e70b30193dae4179b2f6ad72
F20110217_AABQQQ alto_b_Page_010.tif
e95f8dac02875538e3b0b815ad2b1e04
1765539c718cb224bce5c1e19efd48d4d53c8c4e
8425398 F20110217_AABQRG alto_b_Page_074.tif
0eea7edecaae906bbea8dd320ee8dbcb
abaf4e4e7d22185d31fdd219c563e09ceeacb211
F20110217_AABQQR alto_b_Page_136.tif
adf2f40458f54704a3125fd6e93fa938
071aad70cf7238fee03e56a29c0e0defe79dc94d
44759 F20110217_AABQRH alto_b_Page_051.pro
c4d8793e00fad9cbcca14576a7f0dba7
59b3c3846d4806d7cf82c72b6504b61724fc3f49
50413 F20110217_AABQQS alto_b_Page_014.pro
36ac48d6eeedea7fb2e6e027ebc63976
62f9d814c48010dbb188a67b5849e9635d5a9309
529454 F20110217_AABQRI alto_b_Page_072.jp2
049ff4fcbc8d69517c515957513d96b9
ca83d8de86e097ce372b24914b048417e3ef7ecf
F20110217_AABQQT alto_b_Page_036.tif
8f8c13f144e164fc7bc0d06682048506
d7243412999cebf8ebb79a55fd89508231df3040
79737 F20110217_AABQRJ alto_b_Page_016.jpg
135392e229921c43edc227fd7a043ddf
1421b40e9d663d1335e9c8744a07884ed4c40ee4
16879 F20110217_AABQQU alto_b_Page_112.QC.jpg
6fd3d8aeb831f5a044a900c4cc2992eb
b5d4e97bbe3d721beae913bb015e86cd3d3f7297
24894 F20110217_AABQRK alto_b_Page_031.QC.jpg
8e1be58558c639c5fffe3b7c7d400038
23c5fb4395a314535691e309384216000c48ba2d
951077 F20110217_AABQQV alto_b_Page_039.jp2
6b3e70da88b15ab73dbbc39b68dd0b3c
0c76e77cf0ccf9fc55217a10aa729d2f75cbf428
26493 F20110217_AABQRL alto_b_Page_126.QC.jpg
0a74ab9fee3064724ff7d64cce38a1fc
dd4725cde95adaa51dcfd2abd489a582b35d4997
7828 F20110217_AABQQW alto_b_Page_165.QC.jpg
063f199d40fea500e034d886e87b930a
243c2eccfafab643351a648768ef09560898fbf3
15125 F20110217_AABQSA alto_b_Page_038.QC.jpg
6bd720d004c03e8766e3d51d5ea2daa5
8f42308db135fe46b2ead20dc053b05bc0f0d0ce
47472 F20110217_AABQRM alto_b_Page_041.pro
537638cfec8c5900d0a6acfd47c4d19c
2f3c8d404c9c8863cb060e2c7c36ae8878bc3f79
25390 F20110217_AABQQX alto_b_Page_032.QC.jpg
10e5e49750099b5426cfc36c0c82beb0
1a38c7d830cd103bfd12ea3d56b751e4e580763a
26960 F20110217_AABQSB alto_b_Page_093.QC.jpg
921edfb827f91b2af14f89499a8f2226
32c7bc5fb24690f56d16bd660f3e7ac87c1075e6
47795 F20110217_AABQRN alto_b_Page_115.jpg
3e61baa9593768013406b2c8a93aca1f
28e7bfce40bee791138d7d301500203cb74bc7e3
1051948 F20110217_AABQQY alto_b_Page_023.jp2
ff7fd5c4be5841e0e312cdcef4598c4b
3c4288da3e3bcafb6af3d1ae2d9149f1b0b221b3
1051970 F20110217_AABQSC alto_b_Page_018.jp2
5d9d5123ddd8e3c3696dd6a4eef74e65
46330853e02c904c8a2f874005853e9fc8e86260
1051950 F20110217_AABQRO alto_b_Page_151.jp2
130d1dc6ee9381845197a087fc9cd1eb
124e603e921eb86bc10c7986d1b26577e5860886
63818 F20110217_AABQQZ alto_b_Page_153.pro
91341dc34c87cb5a4e4477c24e4d040a
b1bf1f6e0be7f43a34e1e6b03de7d70ff3b62d42
2572 F20110217_AABQSD alto_b_Page_149.txt
dd64e8b33a170cbbe1193e2a6f05eabd
b4648798bbf4993791fac9263f091bd1cb64d7f0
633268 F20110217_AABQSE alto_b_Page_120.jp2
ab8b634d5ad1db22e79bc60b761dee8e
5ff49054f8d2f56726a4a70238e9236d8d3b4a5d
2582 F20110217_AABQRP alto_b_Page_153.txt
416b7f262469b6ec8b29887b123fef39
c8b57eb6e2c876066bb3ad5c9d657dc52437e5aa
F20110217_AABQSF alto_b_Page_034.tif
4243308c4e9285b294e0375cddd8025d
97717c5c2a8288d5da6edd3444574232df6048b2
1875 F20110217_AABQRQ alto_b_Page_016.txt
9f2de921682bd9dbc0b6b84aed1fca96
9cf268461ad93ff731f392cbc9a51c9a4f963719
68324 F20110217_AABQSG alto_b_Page_083.jpg
0ef94cde4aaea1c0c5b950c11d626114
2a9a1d323b161c4d1d72a8e9916dc3f470d29ccc
6452 F20110217_AABQRR alto_b_Page_121.QC.jpg
8a4664c1ac26e741d3e120b3a540944e
fe0bb9f5cad75ff9f82e93d85b149c453ca16e36
53695 F20110217_AABQSH alto_b_Page_097.pro
cff0f0204cd13db654bad9c61c9d5e2f
9716b2c127463f7b1177c784a8a1048549080a4e
84048 F20110217_AABQRS alto_b_Page_107.jpg
608df0e340bbf3d02fd2c679eb6b5297
87e389aa829dcd601ddd5e5b360939570a89fca7
F20110217_AABQSI alto_b_Page_048.tif
5ca1323e3670fe41949d49b3c43d9e70
4406b6a20a5a7b4ddd4bac09020c70d11ddf67dd
86617 F20110217_AABQRT alto_b_Page_034.jpg
acefbadb1cf88337011466547b82676d
e91b3cd01a2bf642c5cea7a60580d8b72d3bcb95
6031 F20110217_AABQSJ alto_b_Page_106thm.jpg
2148563fa1e40a2b0e8630668b5ff0cc
fbd5c67598034f1e29522e742498de24499dd0aa
1051976 F20110217_AABQRU alto_b_Page_125.jp2
900c770177b17d93690a77855c36790c
ae65d1daba1437849d0be593c553abc5216c4da4
51933 F20110217_AABQSK alto_b_Page_012.jpg
5c7853a2b04f4bbd4dfe788ad397b658
c82761fcd55c0993f6961ff1f299ee1b1ff226b4
82726 F20110217_AABQRV alto_b_Page_053.jpg
1a6572a6e8b8ab856e6047dbec5ebdea
c94154bc7a1c9fdd37ae9714e7a4b665db71f92f
6195 F20110217_AABQSL alto_b_Page_078thm.jpg
0ef155acb42ef08095b7e51c93897557
434d525b4c058bb8bae152c56e2c4391238c4fb9
1051963 F20110217_AABQRW alto_b_Page_147.jp2
81adc5b951c28a026e21aaa36b340190
641284fe1ae56e787bcb9eec83b92ded2d5fcf0e
27825 F20110217_AABQSM alto_b_Page_149.QC.jpg
7f517e546407734de005e626ab3afcda
ac5b10bea339c688a0e97946ee541d9189f5fa7a
6427 F20110217_AABQRX alto_b_Page_055thm.jpg
4a0d6ea77d80cd17c24ef3b46860aec7
a3d8598a2accf938bf5ff76c7245fe86bdfb4a22
891094 F20110217_AABQTA alto_b_Page_011.jp2
d6fb0300a65228e5f74a7484184ea5e4
730eafac342a7cc4a9dbdee08525c05e2681691a
F20110217_AABQSN alto_b_Page_089.jp2
44c852495b58f7b0ba1e97c42652e323
0034702bfda81f5d2465bac6b9acca472daf4f59
1912 F20110217_AABQRY alto_b_Page_106.txt
059fc14f6f0f42ae5fb20f62f6b904c2
c8d6f76d2f28b88596c8b00cf045c20895af2dc0
2715 F20110217_AABQTB alto_b_Page_154.txt
696512fb450943264f6f305f477533fe
30061e56421673c3998f6ebc57bfc2d491bc8c74
1051967 F20110217_AABQSO alto_b_Page_127.jp2
da11753c02b0e1a63add01102deb7fa2
3805e08531517b99dafb93c6d605a6651c5261e7
76700 F20110217_AABQRZ alto_b_Page_005.pro
ff31a76a383ec54f5a99297eb0ba2361
27b55a2b56043eb4dd35e8ebdaa76229ad22ffd9
6221 F20110217_AABQTC alto_b_Page_096thm.jpg
025625711dc16ba0730bcd4a86a292ea
8b8bc2180011d57604f40134f9fb7ddfdd714219
695175 F20110217_AABQSP alto_b_Page_113.jp2
aa76a75529c08bd0b92be2c26bc40a42
2a0052e99b320b0a51e17cdc25eafde2b5ae82b6
2065 F20110217_AABQTD alto_b_Page_047.txt
5c2d3dfe35b2d7759b1f78ab3916445c
b80f801c19158071cb4d34243f48441f0113ee4f
6291 F20110217_AABQTE alto_b_Page_021thm.jpg
f2261921ecd88de0f13b1d3f2c1c605e
bafcbdd7a8984c96205df327da4044b9943483f3
103192 F20110217_AABQSQ alto_b_Page_145.jpg
701d640fa0abff691e219051eaa9feb5
c6be8e0fc046e7d5962c46507267f8962fb871d3
2072 F20110217_AABQTF alto_b_Page_022.txt
07f3702459fee8efb7a47b5d413f04fe
862489446ea4cb927c04c27091412cfa59de4854
1112 F20110217_AABQSR alto_b_Page_111.txt
69e2de343aa7f5a9f89b27db9e412749
a224a455681196ecce6fb6489083afd9ac73ff45
5884 F20110217_AABQTG alto_b_Page_099thm.jpg
be473f6f4ab7e6621614c7ac5e920077
c18a83a2964057b14e78c4c3fd6090533e1217c1
82910 F20110217_AABQSS alto_b_Page_098.jpg
df0a050e730d0ec23c8860be4c8479e4
ac8917b800b40f0e346c34c85a1b9661ced4dda3
965005 F20110217_AABQTH alto_b_Page_013.jp2
0fdac93f715946520c1894321b0bab08
8a62c9cae25c2aa8ae2dea6c8a4f072b043a5f0b
2015 F20110217_AABQST alto_b_Page_100.txt
014130627b958212a9f054f6a90f30de
b57fea082b28e4d4082ee4de065d61aff9c1d134
49418 F20110217_AABQTI alto_b_Page_028.pro
e5e0a95f078c58a90269083bd887f4c4
c941ef804267d74f8b3754d1a014ac2ca32ff32f
50280 F20110217_AABQSU alto_b_Page_061.pro
0be860ab1c5a1fddea1663030d9ba97c
af94ad8af08e5f1d53af151f01dade16d3bc461d
25318 F20110217_AABQTJ alto_b_Page_106.QC.jpg
9af90e90052066c512969a9ee37870c6
7800a60ac972052e0a30bfa95f54442d716bd139
F20110217_AABQSV alto_b_Page_031.tif
febc17264cbf2dccb35522b8b579ee2e
abd1d9642568654988cf3dd675218fae5c4b56cd
83603 F20110217_AABQTK alto_b_Page_028.jpg
30514683e859169e16609a4c69304016
53a487c1caaf7ccfa9afe629f81658df0ce47385
27127 F20110217_AABQSW alto_b_Page_065.QC.jpg
f666a86eb3dc149455de61d36ed46671
8bf2573b115fcfa404cb0d0ae428c3893c61bff5
84179 F20110217_AABQTL alto_b_Page_086.jpg
ab3d613c634c2c61a2af226870b8e270
466b2b2f86a59e5eb7b0c647aeb00b1999e9f07b
30447 F20110217_AABQSX alto_b_Page_012.pro
1a084050053bfe51474f32f0bd66a28d
42f44ecb05d401faa4283537283e30db48804606
19458 F20110217_AABQUA alto_b_Page_009.QC.jpg
8a7276b05d79d856cf66f4d237b5873a
59fd37f13a178e93eb6779561d50b0a845402fa9
1936 F20110217_AABQTM alto_b_Page_032.txt
b858c8a7ef78df7db49dbbecb0b1239d
7ee1dfcf93759fe25d77b996639c06d745bf3315
108190 F20110217_AABQSY alto_b_Page_142.jpg
2e097cb31a096468da166af2043fc942
f3eac45bde6c9dfedd37906c7caad053435de252
F20110217_AABQUB alto_b_Page_080.tif
35773ba338a72f3bde357e44aa02e409
1b9bb1e155cb19907024dcbf44c22fb5f0134765
F20110217_AABQTN alto_b_Page_020.tif
de30e0808c9545ba0831c1fbe853f99c
feb9f124fb79e0a3319a7104b21401711f008cef
2064 F20110217_AABQSZ alto_b_Page_059.txt
88616389309ccece8c79339e684282e3
e9b84772036ca8443f436ef4e3469bf4df98a7c7
6418 F20110217_AABQUC alto_b_Page_132thm.jpg
c6e275633214104dd87ca3b94aab3831
77456342eb5c85795d3cdc2bee597c9cb6ed5208
F20110217_AABQTO alto_b_Page_135.tif
f82b1f9e8206985159679a9e50989018
bc89b0fb241f9e9fee2478118469119e940d2aea
36528 F20110217_AABQUD alto_b_Page_118.pro
fb84fc7b624b4395259cf5574e1487b1
e94ed4aa6afd043c48e615ada5bba52817a256e1
925586 F20110217_AABQTP alto_b_Page_057.jp2
66fca109a3e6b624c5a4e6bff71bddbf
19b36dd29149052e99c4b54d4c89c32997e7c2e4
1051956 F20110217_AABQUE alto_b_Page_164.jp2
eb317bb4263c4516fad564f48081c1bc
eefff7fb14fa6eb2dad238c2887f99876c9a4015
1217 F20110217_AABQTQ alto_b_Page_012.txt
d0bab39b11a3f4161f103ebb5cb2ae4a
13c1ead454a6a54c6a497c5b74ec3e2daaa03333
F20110217_AABQUF alto_b_Page_001.tif
8115aecd7ee58c3b6192842b37c0a5c2
a571a0b7a247b247514ca8b810f10ea4c88f3154
6753 F20110217_AABQUG alto_b_Page_149thm.jpg
4a2ad36c08f96f5c08496a49458ff28b
c682413e28a723c8f9674e1230fc320399aa86bf
F20110217_AABQTR alto_b_Page_153.tif
cc26c37f96d804889289e4bcdb4e1dcd
c1f302ed177066c9c7b93c81742cc3d8094e6fe7
1988 F20110217_AABRAA alto_b_Page_133.txt
bfff6df1d98dd521e50dfc1d0382ef3d
49c9805f406f853904b8732443fb21b216f66ea1
52678 F20110217_AABQUH alto_b_Page_047.pro
88d6f94838121897edc6b5c73d077437
7d566576a4d879da14e8673b9fb354af53e721cf
84359 F20110217_AABQTS alto_b_Page_035.jpg
7130e950cbda0120f7acc4260c8d9c8c
66255aee6e4fa2c433f924e3e6d6f62ed76aa38d
6593 F20110217_AABRAB alto_b_Page_001.QC.jpg
dc48c01b1f98e087e924843d4de44ad8
cd9bb81431713e06982f2a093d874325ae582378
49143 F20110217_AABQUI alto_b_Page_032.pro
19820322eabf3816a844837b2ae7783e
480127e90da99d586666a58137363cfc5a69ebc2
17566 F20110217_AABQTT alto_b_Page_048.QC.jpg
b10d5be1862ced29d9c15e6c062fae9a
df1041e4a2acd627308021439ac5a14e8403d37b
86493 F20110217_AABRAC alto_b_Page_059.jpg
70ca5e85ea708ca3a64b2f3ef0049c84
269950f85d56c4180bd48d00ff5eda5572edd4f0
1998 F20110217_AABQUJ alto_b_Page_164.txt
917f5891a4dd3b5a200bc35bab6b3510
42ec8a836984c8095fc2a9a9662687f00f226388
49665 F20110217_AABQTU alto_b_Page_042.pro
308e72d3bd1ee2e0c82a68e4ae4b58cb
364a00ee2fdd228ce3a1bbe6553c0c7790bdc4d3
6847 F20110217_AABRAD alto_b_Page_003.jpg
6c8e024dde28e8bab571f687b54cfa36
b903ecb2a65bc77fe2a3cedbaf1ebd1afa5d46d5
F20110217_AABQUK alto_b_Page_129.tif
005a0a71eb1177e85e34c67f1dca1858
d9c8df442141b7211d0a5fd3b62aca1c2e281cae
1776 F20110217_AABQTV alto_b_Page_112.txt
3010ec8342794ccc55c702931abbdc0b
2cce601a9cfa3bbd44f76cd4d912b56808f94dc0
18013 F20110217_AABRAE alto_b_Page_049.QC.jpg
55db552c9af2f50da812359d11669328
a6e7481e6f73f7ee9d5292af4620def313648e05
F20110217_AABQUL alto_b_Page_028.tif
428c070b1b3ac564298505c87ea47db8
016cb81ac4174fa2f2dfc7e68802fe85e5866bd9
37538 F20110217_AABQTW alto_b_Page_004.pro
089793930e6d0f5f0cb3e415541a7a6d
d44c8df14a14b6c74ad815c21fcae1b8e2a23cf2
1932 F20110217_AABRAF alto_b_Page_089.txt
c7257997e0cadfd2f01ef57674200bb7
e73f9fe8ee738633f2857e6acee8730420e4c30e
2023 F20110217_AABQVA alto_b_Page_037.txt
3d8e3ea50555ec6f8af1e5aef47d8509
afbe2baec91fa06609c51ab18831168fd460aa1f
37201 F20110217_AABQUM alto_b_Page_076.jpg
7a8aed07376a51e8c17e5ea01f5c0525
0b1ef2d8b9af7e6b7a3454213c6442fb336974ae
26400 F20110217_AABQTX alto_b_Page_029.QC.jpg
5726ca03b5ddc92440a3e594e74e4da6
31ce47d586d482cfdc4f63fba3d4b0149d68e454
F20110217_AABRAG alto_b_Page_164.tif
d5dea1b7c5efeb48134b5a71d9ab75d3
b689ed2fe115afdae50d9618d10919b964f570a6
27164 F20110217_AABQVB alto_b_Page_025.QC.jpg
8089ab01c99156d402cfc349f0379403
3bde669d1a0a2b375d0e4fe25d72b30139578b0e
61105 F20110217_AABQUN alto_b_Page_148.pro
493526eaf20f8e3ccadbaf7b7c32720e
c5cd0a56c3c738a549d4586c94c8209faef931c9
87158 F20110217_AABQTY alto_b_Page_130.jpg
cb000cbae6498996fa526982d3da93fd
23b780f87bbfd7322247dfe293ea398488532b0b
1104 F20110217_AABRAH alto_b_Page_117.txt
72cec22390c2c47ca0790e5adae066fe
fe47fd00ff9e0f5f9a1b25fd6aa4b99e5684ece3
2612 F20110217_AABQVC alto_b_Page_161.txt
ad23b672c26a151a47f3ca89bff34422
747c0784fc7db0e220de2a10a3608ba29a54580b
1836 F20110217_AABQUO alto_b_Page_084.txt
ae61722c6b6cc6a002d83218e7a811c0
4719f449cf735894e23ee407a27090f2088ffa4b
1365 F20110217_AABQTZ alto_b_Page_002.QC.jpg
d511d6d2a865a347f695daeaaf2a13cc
0136ce02d50f5cccdae5f952eca85a6b0589fa6f
6636 F20110217_AABRAI alto_b_Page_161thm.jpg
34c56fb160e3cfbebb80abbe4ec8fc18
7976b920846c1e899a970e989e35b5cb4dc46934
1041267 F20110217_AABQVD alto_b_Page_045.jp2
2db5c1789ba58329a62112b25de462ff
b505d0a32ab84862147add60d19c7b21e236b9af
26352 F20110217_AABQUP alto_b_Page_116.pro
8db270834889bca03c9332ee4381deed
fc1ac1809c272f1a8846118ac4124ca21e65c7f5
1592 F20110217_AABRAJ alto_b_Page_115.txt
a8b6562da4c9a84208e13d3da0fe32d5
739488802b4176a6dd0e21c2033b5067ccf56121
27425 F20110217_AABQVE alto_b_Page_125.QC.jpg
6c5dd1cd097dc0d1e431f4adbdddb553
59e44c87742d7ad37775392ffdab1c96093f8312
87298 F20110217_AABQUQ alto_b_Page_058.jpg
0afcde0d40d990f2f8298b746b73587c
42cee9e64c5e16cb478450e31fa1176df3147092
1051942 F20110217_AABRAK alto_b_Page_034.jp2
2395e4c9c422dfad4f74d39bf53da5d6
da1027fdd8387fdf9f122a62bd944a95d11530bc
1051954 F20110217_AABQUR alto_b_Page_061.jp2
5d1dbaed2dfe55d75a6e2ac948a0aa37
756f1498e1a73edb2a60abfcef75e88a27d47c01
1974 F20110217_AABRAL alto_b_Page_042.txt
9e04cd8dc98e5014b724d224fee7f6ae
0a8f5381036be1ee64ad46237ed46e0ab27d484c
1971 F20110217_AABQVF alto_b_Page_021.txt
28187aae6298f93c81d9e332aa21beb9
0f643cc05866c7a4b107826d61af8b7ae0cb2931
26331 F20110217_AABRBA alto_b_Page_028.QC.jpg
b018703d56bced5769d170b4d03f1331
068a11ce38317c3e1c2085eb17dea1a2d8148932
1109 F20110217_AABRAM alto_b_Page_038.txt
29d6aea35ea4cffba85183aac23b7261
67842769d31f4256370bef3df7d8f6759bdd4554
5433 F20110217_AABQVG alto_b_Page_044thm.jpg
d95f663526b3f35d1428de6d418691f3
0b71446310bae10959bb9b48158e04bac9348e1a
6515 F20110217_AABQUS alto_b_Page_125thm.jpg
702bb54f5ee0b03ca7068325a0ee8ac9
f508ff1d121a27e578541ff1b00dc84628b3e2d6
3115 F20110217_AABRBB alto_b_Page_005.txt
d18838204554be0bf1224f67c7e2fd2b
a0ffb25c9a437247ae4bf2f56d697c6694b15c8a
2594 F20110217_AABRAN alto_b_Page_152.txt
772f1f56d170b3a9ad3c3b22ee4e8654
174ae75a61d1fef1d30721ad8e02ddd74af5b649
1226 F20110217_AABQVH alto_b_Page_119thm.jpg
47270d54a11a671e8396677fab12efb9
351e97575231d8ed2ca0bf9ca9c62c1ee81b8fc1
26188 F20110217_AABQUT alto_b_Page_066.QC.jpg
17e347fcbd07c26eef53d7f3482bbf34
1b5f39b3149047f14ecf8698cdd3c2a941357953
246632 F20110217_AABRBC alto_b_Page_001.jp2
1bd5c28e01209b88340f6b0278945cb3
77dcfe9ad00392f016a182f7b19efa1eb7ead550
1051944 F20110217_AABRAO alto_b_Page_017.jp2
92793fd5f3217fe03b71e56e3d4883ab
96ee5c8d259a53ec6dac8feb1c8cf3389b1bf80c
2545 F20110217_AABQVI alto_b_Page_118thm.jpg
58f82d628131a8fd0404def058815e44
cb5f9b1d9a1601db2e2a4a43547dea202dee7448
22552 F20110217_AABQUU alto_b_Page_039.QC.jpg
51ab41f0ddff0847cf4242b7750a6efe
df2459def17ebbb165cfc45e27e5ab3e85837a99
6522 F20110217_AABRBD alto_b_Page_153thm.jpg
2bbf343abe5088a61e4215fab6ee7b21
8bb928c05d5fef064b40a32b5171e1f3fddae1f0
1033607 F20110217_AABRAP alto_b_Page_134.jp2
d4cb32ee9a95d9c4d46a003b7ce21212
7389d642d4226b91ab0e634d54d54463e14ba2ce
25779 F20110217_AABQVJ alto_b_Page_107.QC.jpg
4f38e88b225cd4cfbe29c8f4f9ed5d58
9c2350343a51bc4d3496ab2f3b6072acc36f4046
85196 F20110217_AABQUV alto_b_Page_093.jpg
62666d96267823d454b7bbf1e800e365
1a4ccc80e7f5dff63870091013e68d66856a276d
6392 F20110217_AABRBE alto_b_Page_130thm.jpg
e52b48ee9761b87ce018d3b82216973e
a16dee2e2dba4ee4694e0946a3dd872c27790dd7
1051935 F20110217_AABRAQ alto_b_Page_031.jp2
cde4a60f628769fbfed30e375541b53e
f221277b06461194ca0b144353ecbf5be7166978
49814 F20110217_AABQVK alto_b_Page_081.pro
c53a5139654daf1c52372781f63efd0f
281a9bc3a53ab6ac7eb38a3fa0aa2113275f9020
47657 F20110217_AABQUW alto_b_Page_109.jpg
900b786b5b948c355557854666ea8615
89be4ba4f0e805656c0f4ceba73ecae871bb510e
49991 F20110217_AABRBF alto_b_Page_079.pro
41b3ececb1a013c8255d2479183cdd9c
3580f594e2da904868220df113743a996918edf3
42870 F20110217_AABRAR alto_b_Page_013.pro
5898f2719c3ff13bf2b1d173e0f556e9
10954d8d200f1d701d292d58fa26b71e921cc8f6
1051985 F20110217_AABQVL alto_b_Page_096.jp2
fd88b33c9deb079b9d7464ea8adc8cf5
2ae3937e9f5299065f558833aa096552584aa427
81871 F20110217_AABQUX alto_b_Page_108.jpg
82b4ab81e2526ece165fbd5fd6d13af4
1ecd6638ebe5fe0eaf49b5f5d4d187f63fffe77d
26819 F20110217_AABRBG alto_b_Page_059.QC.jpg
73b2291e52979985cc2871f99e8254c1
f9dbad08257f81d09e27d8ea31965dd48df7074b
2016 F20110217_AABQWA alto_b_Page_105.txt
414bee33baeff6fc99d4186acc4e7c22
0ab6e67c4979ff2c307a44b940016034a1dcd4d0
859 F20110217_AABRAS alto_b_Page_008thm.jpg
c2b708e6edae63c18b8fbcaadbca07c1
5aa7e87a5c51b18f144a2758b58e8799001d9035
1051966 F20110217_AABQVM alto_b_Page_084.jp2
e14f89d71576c36bbc7b14cf6b37a830
78bbd8842d9c712fe38b755d8848b948beb557da
50053 F20110217_AABQUY alto_b_Page_021.pro
22e3bc159b012ce20562cd877680b843
6b5971a964c1580105b086d8faf4e84f64a16791
28269 F20110217_AABRBH alto_b_Page_142.QC.jpg
63c85c409a309d26215be5956b7ea67e
a556cfe5b80ae4e617b1ddd38ec1eebd8a65e03e
44884 F20110217_AABQWB alto_b_Page_072.jpg
f2aa1016ed2ac06a1a720c2291eb12e6
95490ab994d4c1c310e66590b1e3b1c32a27c4da
628 F20110217_AABRAT alto_b_Page_119.txt
ddc1e99861ec0a68552a556725a413e0
1973c64dde81fcc6d49a521c532100dfafa1a885
1944 F20110217_AABQVN alto_b_Page_028.txt
2eac164d6a4d8444ba4c79983c66cc90
6a63cbaccea437f1a06f261ad4d1c53ca22123b5
107063 F20110217_AABQUZ alto_b_Page_152.jpg
77942b3608101a4b072d1cacb4c29d26
f2ed1c70958c74928b4d21f05203ab34c7aa9fdb
101739 F20110217_AABRBI alto_b_Page_144.jpg
eed1c57006badc3710c18588109ab7e3
6ada62ee5d47002f568da89e7d5694c82a5e416f
1051934 F20110217_AABQWC alto_b_Page_162.jp2
8b82ce1cf17961b27baf7be1873bca82
eb3b6b73cdff29598d4ed06afceb4615a3a3fe95
6007 F20110217_AABRAU alto_b_Page_128thm.jpg
269ca1e1ed36a9308b2d101a5772be1f
5c075d2483a20f5049aa35b707ea3117ddfcdf85
F20110217_AABQVO alto_b_Page_039.tif
70fe6f0f4961ea1f47b5310480862f6b
61264d2dde5b0c14074144e2d4351fe699f3e841
85655 F20110217_AABRBJ alto_b_Page_027.jpg
7d71f3eded419fb3895710e779517a78
e4fd943169ad03a1a8c758f7a414b766769f4a03
75377 F20110217_AABQWD alto_b_Page_134.jpg
bd89837c42de2c7494b6244fd751f5db
c1c4ecb54024c5308356bb5eee0d7d7a7276d653
51675 F20110217_AABRAV alto_b_Page_092.pro
ce870859c566f4481c554ac78bef1141
e157b9ed464d95f22b01cc2a069ed0af5add5d47
67663 F20110217_AABQVP alto_b_Page_146.pro
228a90706ab4527e0cc1aa449a523ddc
f905283ebf29317f2fab98844f8b39a70d537753
F20110217_AABRBK alto_b_Page_143.tif
b411fbdb7651d3882e3a792c416c77fa
76d2019b025e326dc53f66ba59703f65ffd9fec5
1947 F20110217_AABQWE alto_b_Page_121thm.jpg
0865fa3c75648a2ec113373ae2336a4f
2f321f9ce5078ae62cde05332eda9b32189c63b7
61745 F20110217_AABRAW alto_b_Page_141.pro
716eac79692eaf7b083dc3cb2f36031f
c8060f499bd776dbd7631d58381c01e902389fec
49300 F20110217_AABQVQ alto_b_Page_085.pro
5487befd6d288cef538564087e50db40
546e0b39386f07c26bb605aab66023c125cc88a6
26853 F20110217_AABRBL alto_b_Page_098.QC.jpg
0f8f7f200e803169757e6cf20b1b3edc
fbdef8397a2e30e81c003eadd120012fbe4dd82e
21410 F20110217_AABQWF alto_b_Page_117.jpg
faaa9940e98b737ffe53ccf44781be8e
6abc096f6ad8a116743f9a0e2004b95d92b249f0
64510 F20110217_AABRAX alto_b_Page_155.pro
17f4ced386759591a134accf221649a0
5a74358478430d8ba29e49e65d84a67b3efae025
29844 F20110217_AABQVR alto_b_Page_156.QC.jpg
d221808cbd32ab98f2d4afcda4df0d16
d34a4fde3c4a7395e5d65309203012fd1b39db1c
37798 F20110217_AABRCA alto_b_Page_069.pro
18e00396f2860d30a30dfaaef2ed2101
2569e92e05acad538a88bf101abed4b54c8ce328
36019 F20110217_AABRBM alto_b_Page_050.pro
22d408668985eb6f7157a3f26a0d0664
45b5e1d18b6682419bf4bdbca3dc9aff07c3efbf
F20110217_AABQWG alto_b_Page_009.tif
95b78135124c540dd40444f6bc06b9ef
82ae60d5824c7fbefa9879a7db63d38e906199db
81983 F20110217_AABRAY alto_b_Page_079.jpg
90c766fba68f8cae6d36d5e536fca753
d5fcac5cabeea3056b42cdd0a0565d5e25d062e6
87676 F20110217_AABQVS alto_b_Page_017.jpg
c5a81b4d887f381890141cc3c76569df
522ee52d45e7ab01b21ad7024a7a1d8eab4def1c
44777 F20110217_AABRCB alto_b_Page_123.pro
01e4b486a80ffa58369e453a1222302f
2b97cd48902a53166b34410c315d598980fd27ed
25495 F20110217_AABRBN alto_b_Page_014.QC.jpg
25e832d45bcfecf5f3ae5c02e7c7fda5
158cb1f15a464b80fd50f93d6b202ccee6c4d6ed
52041 F20110217_AABQWH alto_b_Page_034.pro
d99ed8737a69400388cbe67129a56f39
85e2312041aa6a52689584c3a09536e1a189c24e
5607 F20110217_AABRAZ alto_b_Page_134thm.jpg
3ea41c6e27128c632df7631a9082b2be
9607af3f8cd5bfc5e5df0049939173f287b589b3
83941 F20110217_AABRBO alto_b_Page_055.jpg
13f54c05882dfb68d49b4810d5d3425f
d622f8a172e9ef8dbaf4175a0c2d78a0134e8627
7062 F20110217_AABQWI alto_b_Page_008.pro
3702cbc28993ad2ae9559b89d164c5cb
4efa86f232422fc938ed892904c06079b10bc8bb
53841 F20110217_AABQVT alto_b_Page_131.pro
9b9aa6d13d7ceaaa8d09fa69c49bd55c
d348cd59185ee66267b7c277a4d84cd740723e42
48473 F20110217_AABRCC alto_b_Page_038.jpg
ca38fe6ac9ce43cd0f9ba495334b122b
a39e516188234be41136cc2dbe710a6ff05cec18
3918 F20110217_AABRBP alto_b_Page_012thm.jpg
5ba10e46d2d825b16315b957b0284528
8049cf31f3b611708f8e32ccaa1f9d022eff5894
1946 F20110217_AABQWJ alto_b_Page_128.txt
42688fd4e5b8a30e9573fb0bc4ab11ff
f7bc3738e5934a139eb55042a90db8cc8304a0c8
64057 F20110217_AABQVU alto_b_Page_157.pro
1ae3cd7d0fd6a991a35e70c32ac47c30
d95764ab8dc1c098d1699ed1fff612bed0509dea
24307 F20110217_AABRCD alto_b_Page_064.QC.jpg
750c49132f8f65cb23645b70223c4ea9
86eadbdecd73a3a72420b49cbfad5afcded0c34e
F20110217_AABRBQ alto_b_Page_027.tif
017625d229d9d23b0142a29c56ba732d
0b7c5e6cf4d996d89fb74093a0862b22d3ec397c
F20110217_AABQWK alto_b_Page_006.tif
ad88700d08a0e7820712baf1c324d669
3dd34f7a257fc9858df81a89d8f718454880c759
73298 F20110217_AABQVV alto_b_Page_044.jpg
52625bc368474970e83add9e842fa635
fa4685dd0154fe22aad4c672b80776e51321c767
1051927 F20110217_AABRCE alto_b_Page_088.jp2
b5ca35bbf37a836b25a5c04244213aa3
1280a203f5aa682c1b548308a037c0ec1808c7ae
F20110217_AABRBR alto_b_Page_065.tif
7bdc10b667cc78f0ad9934c08b4809f1
a0e8776c2cedbc2c28f5b1db26b89291f5e943d8
F20110217_AABQWL alto_b_Page_158.tif
6eac33463dc3598b552210948536458a
ef10f54652853286f70c5d4dbaa57f2cf52dbb65
F20110217_AABQVW alto_b_Page_013.tif
64d2e8dd94792b26d47df69b96f7d220
667d4f1fe97897cbceb92b9ed425d07f88ed5ee8
F20110217_AABRCF alto_b_Page_041.tif
4c13c81fcb30115efe3e4db976d2f304
c9e6e2832b2a9295e948def1ff030319691b7210
F20110217_AABQXA alto_b_Page_137.jp2
ff7258a410308e26f78bebcc4fb75311
29826291c718fa82920f69352305bd661dfb2241
348173 F20110217_AABRBS alto_b_Page_121.jp2
1b525105a1f39b40499b9925788e28a6
ac3bd43cc3ec40923e6382c0b9a0e844d3ee81be
75646 F20110217_AABQWM alto_b_Page_051.jpg
db192544b238ff0a98644a25a5b37971
e47ab8035ffdda878d75018571568b17ba7c5386
80936 F20110217_AABQVX alto_b_Page_041.jpg
94369a7b5aabfe5fb454a7a90671eed0
9155939df5a62ba06def6e15c9ec24969558927b
83951 F20110217_AABRCG alto_b_Page_021.jpg
306e6921c2e42f4bdc21ecb555e033d7
e1924e7c41460b99f5b9fe7a77b9f27512fc91a8
1978 F20110217_AABQXB alto_b_Page_014.txt
6e3a163e934545a5dec468aaac293343
7aa7618dc848dcb9dd1088e82887141b05cd93a4
F20110217_AABRBT alto_b_Page_131.tif
f09b916825bb7482db4781fa3345514c
c530b2088873626bdfdbcfb8a8450aae6bc36ff9
6163 F20110217_AABQWN alto_b_Page_014thm.jpg
7a04c9997bb2b6553260300399bdb915
df860583640f1406f0e5d7106d877c0689616218
45364 F20110217_AABQVY alto_b_Page_111.jpg
5f8e389b19c20555b417b9fc6a8056c1
110f6567030d3668d89d7f09989e7fb5919b81f6
25933 F20110217_AABRCH alto_b_Page_095.QC.jpg
a394204ffabf55e61c5ce5ca8aa5d9c7
fecd6b63a7abd52882cd4a5f43137816db40ff50
152 F20110217_AABQXC alto_b_Page_003.txt
550a2d28a54fdcf966a904a589ffe62c
e26a2bccd77bf967a809760620b9e5634a75c6fe
2671 F20110217_AABRBU alto_b_Page_163.txt
4157f70f2832e925ec681bcd282f893c
3ddbff237f20f66237247427206d50c578864526
12648 F20110217_AABQWO alto_b_Page_075.pro
060c551e6d1ee42f9aacef26eb4eced3
65f6cf5cb3260c7e6c78ab881b2de9050a1c2703
F20110217_AABQVZ alto_b_Page_010.txt
036bc38de22fb6d408f7cd260f70d68a
1704e0e18a92c212072f3e2e4ae89972a987e1d2
85199 F20110217_AABRCI alto_b_Page_129.jpg
12966bb91d44a6096589e46c885f2d1f
d519b7688c5f86fcfb362a33c94fa2bfa10295d5
3183 F20110217_AABQXD alto_b_Page_119.QC.jpg
a1fef96756594e5732e281a09928d30b
e9daec905b6e106de21239ed163a900dc5cfc715
F20110217_AABRBV alto_b_Page_040thm.jpg
3f8f5f7ee4e504c3069da37f084f4d6e
20da14d260be05cd4690826cf71b35e6a5c5e684
6305 F20110217_AABQWP alto_b_Page_086thm.jpg
e4beb975560330d38e0c37b85092e397
2402a7acebca3f78bc1cf4d43c5d9b4f1b33f300
26755 F20110217_AABRCJ alto_b_Page_023.QC.jpg
40e62c05630df9749d1ef830fcd29980
cf2cdb73c0e457dccf77aaa2fcbfdcc81eec40fd
1938 F20110217_AABQXE alto_b_Page_063.txt
708cdc0e28177c813cdf793931ffb1bb
74957c1e9bdb9e47531c9d9be261be0bd36d0846
53184 F20110217_AABRBW alto_b_Page_049.jpg
4cc18e9c3c338783ed91205df2d341bf
d6277434338af0c87a2d1e72efbfec5a2f127031
26587 F20110217_AABQWQ alto_b_Page_132.QC.jpg
2e22328db9709adeb83268fb5d35eb80
8f223d8fafaf7c2607adde23d68c5b41eb73d239
471451 F20110217_AABRCK alto_b_Page_074.jp2
bef47bf0671726a0d04fa84ba2ce3502
c9ea410d2c9f76a289b0e2bf3137935b02b87161
42325 F20110217_AABQXF alto_b_Page_077.pro
bec5b28a4f209f08c1c9190923fd82f4
5f86dd3756d4797bbb09af9b290f6824b7265318
1482 F20110217_AABRBX alto_b_Page_122.txt
b929c42a7dcda192e31d0d32b7ad3722
2700ae970634d429d176b57549cc9ee0f6dfd014
7035 F20110217_AABQWR alto_b_Page_156thm.jpg
a19c624f0e93ecd498caedfa6a181bc0
c55701ae152b970dbb661e5684969fbfcb30c382
1051932 F20110217_AABRCL alto_b_Page_007.jp2
c1b33851e8b896c3cd23dfc8d8ea029f
fd3f69214be44c2d7391e33673cd92f188522d06
83892 F20110217_AABQXG alto_b_Page_061.jpg
2da74f97ad3308eb1136551260a5384f
21933dbb8f8563a859e9e64179456db3a60bbed7
22315 F20110217_AABRBY alto_b_Page_007.QC.jpg
2ac0628c75c154ac21575085713fba8d
b1c47c3631d6f31331e62b6dcc68907f20cf5dc6
F20110217_AABQWS alto_b_Page_154.jp2
e86ea09243e2d40c462143b435711b78
a148c2968283241ac623e6cce1eb8129815aa081
48686 F20110217_AABRDA alto_b_Page_164.pro
80d22bbaa54e0d9c51de0dac12871768
c2c68840bebd77af614f6faab6cf2e28e01a150d
1051978 F20110217_AABRCM alto_b_Page_027.jp2
3b54eb2eca76615286a4b6cf2251babe
72310d02220764307764a6b677a055ecd3b1b2bb
1958 F20110217_AABQXH alto_b_Page_033.txt
b198d190a3ffa0400a62e174df77f9fe
3781224d0075d17f50838daa1ed73b642c13efd1
4820 F20110217_AABRBZ alto_b_Page_056thm.jpg
2185370254d6d8a23559b4784717aa74
11edc044b8ec49ad8dcb66b3654d242d6b9447af
618487 F20110217_AABQWT alto_b_Page_112.jp2
6632c6276976e96351c70d75d9e94416
a7e8e45967996e967df5225f6aa059e12b9e9d41
1051983 F20110217_AABRDB alto_b_Page_163.jp2
fd471a0a63ccd96bac7baa7eb4941978
96cf77acc45bcb82c6a9eed2d00e93bc90109a58
378389 F20110217_AABRCN alto_b_Page_117.jp2
89ce5ef19b6569f29eb9afdbc7c023b5
62326f21b8a29e0ee4306f0be2145e8369dcca93
F20110217_AABQXI alto_b_Page_115.tif
003f493af7611ad8959f8d4241c6ecb4
12652e4406dc8e7facbfca13ec6cd802e0400a97
51821 F20110217_AABQAA alto_b_Page_023.pro
3c7c17b33308ec1935d97cca972f34c2
7dc08fcaa74130f2b7ffa43f6280af2186a31028
1822 F20110217_AABRDC alto_b_Page_094.txt
36c5b77805004bedeae8732bc759d1df
0173e51814602ae8215d79dde1dc87fdee81e2e6
63876 F20110217_AABRCO alto_b_Page_152.pro
ab3d609b3ad742009f12eb006f7ec4aa
3440bedb7f829c2954cccf28c7016962d7ce98c8
F20110217_AABQXJ alto_b_Page_093.tif
ad89a57af9bf189907dd3d28cce1cc93
6e9268d421b558769ea980a41651ff5376dccf85
28393 F20110217_AABQWU alto_b_Page_145.QC.jpg
a9ae2b9d16b8d9eae3df3e9b0e2cc70f
1c8b8b8ad00a994e6fa67df6c36dc955d133c848
1982 F20110217_AABQAB alto_b_Page_101.txt
ac5140b63a67547a5300e624f2a26097
7aeef7e73b5d29cba002e85f19b31f6522b09e78
6819 F20110217_AABRDD alto_b_Page_152thm.jpg
704b0e3887b610ae128f72f3c76d48ff
6b7ef295b4b92b76fcfe129a9d12d62459c938b2
83643 F20110217_AABRCP alto_b_Page_042.jpg
efd5df0239a0f28f78502613c9d17a4a
32ee6104e73c50e8ab753b03ebaeff51258f974e
2058 F20110217_AABQXK alto_b_Page_017.txt
09116cac531a924111c82333211bef76
97d01a0d9277aef71b19b4fd27c5b5a65497af40
5831 F20110217_AABQWV alto_b_Page_080thm.jpg
9ea8ff5f8daf6fe9f49b3b1077e30ed7
7c4f6ea7d3d9f07ec5ec37a9880d5afe77ed0e1d
79338 F20110217_AABQAC alto_b_Page_031.jpg
a01fd3e557ad0758cb1047662d1aa7f7
84050a077d024588b3a074c8fd21bd2b26b4057d
1725 F20110217_AABRDE alto_b_Page_011.txt
f6b67f2034f46103e1e4bd9e553a6316
8cc99de879f1a52f6fee6522ba12ddeb2ec5b85c
84291 F20110217_AABRCQ alto_b_Page_105.jpg
e440ef82e7ea5e340e35af0b54ed6e50
9e556abc1baf5c366b4da4650cace7d33712115c
6321 F20110217_AABQXL alto_b_Page_037thm.jpg
e71d169bf38a9602fd3de6acbb7dd644
dc9011bb8d16cb4cd067869f6cb61b1643a42fc4
6373 F20110217_AABQWW alto_b_Page_144thm.jpg
e0a3480543990083c965999c26ef5239
48c4a92a613c5eab4cb7117e5f9c15dd2fc5727a
6263 F20110217_AABQAD alto_b_Page_126thm.jpg
87f988307f5ef3b60fdef001e4173a60
eb7472ec9aaaf20b456a9c73bb18af856606c73c
51350 F20110217_AABRDF alto_b_Page_066.pro
c6ff5280143cce374ff2c6482b3dabae
d454c81a35d097d01a888cfc813c1a6cd6318d59
82258 F20110217_AABRCR alto_b_Page_046.jpg
b9bcf030d81cccec18b158a25824f2bc
abcbed7e7e61b0cb36a91d94aa4bc07327e8596f
F20110217_AABQXM alto_b_Page_024.jp2
72b5918102efa8b3bbf37fe63ca055d8
e487e3238cfe0099ee40e426d23519e96acfab1a
25850 F20110217_AABQWX alto_b_Page_067.QC.jpg
d0c3f153a7889bbdfa9f55cd840b5c2f
c998a09da13caeee083b13badb11e2f0583ab32d
72231 F20110217_AABQAE alto_b_Page_009.jpg
9d0e343ed5a9cc1825d69452a41f64b4
2097c6cbb7932e230f9038b5466e405ef053d361
84920 F20110217_AABRDG alto_b_Page_054.jpg
fa9822d7ee6d2769abbb9063a3195f18
d2541c91467ac1b6b5421c5fe2dc96d8c54eff3f
1051982 F20110217_AABQYA alto_b_Page_036.jp2
64b84746c787a6a187a4247d1737f105
3ba5adabfac1a83fde611f29f27e616d9ab79232
F20110217_AABRCS alto_b_Page_150.jp2
06d2063fe41853833bd1b6f7f72c78ba
7d9c2d40c0abc8ec92b794ba2b10d3c9bff30a5d
27030 F20110217_AABQXN alto_b_Page_027.QC.jpg
50ac6234fe2018a761dd274bbb06af04
808ec735101f90049bea7268c9ada443166e69a4
68741 F20110217_AABQWY alto_b_Page_156.pro
000e3f72438a03db2e9d2de6a006aa0a
71a91827f5e78888555ee77400332acfef00b469
3259 F20110217_AABQAF alto_b_Page_008.QC.jpg
dd95675468e4c84216448811b9b8677e
34ddd8045d41ff8a160f5d8eaece460f52c88127
2002 F20110217_AABRDH alto_b_Page_035.txt
e692244e7821be6b02b5aadcde34a425
1f0c5f836732f687eee814a8123ee0d035f5f6fd
53229 F20110217_AABQYB alto_b_Page_112.jpg
1249a9da85ad0205379dc66cacdc3e45
987588085d1af890e2b751acec7c2b097b9bd3b7
F20110217_AABRCT alto_b_Page_144.tif
a9fc41d4080011467785d6cc5f85b44b
eebd636b693195ffdb6d9f60de6c9b5b7b2ab6a8
1051969 F20110217_AABQXO alto_b_Page_130.jp2
82b9549ce5a86dfc6d7166aa1de87e40
cbee1ec3e2e5ae238121f5d3a55d864ce8b9c1e3
1741 F20110217_AABQWZ alto_b_Page_039.txt
269484d3c2336bb7c1c248f7fbe4a2c0
243329a074f130a9bf8095c5a90ae7d64ded222c
5966 F20110217_AABQAG alto_b_Page_063thm.jpg
7f8e3cd9bb8c50847d9b6aca5e5a3a9f
4fbadb235cc033c091d76ea9e56a5080afb6ef5f
50368 F20110217_AABRDI alto_b_Page_107.pro
05684bc2a4de4c3058676cd4347dabf1
f7a15bc0ff32df4ebbcbd56a38a39aad3b06a0c9
26189 F20110217_AABQYC alto_b_Page_101.QC.jpg
88af75b1ef6a8928df26b7db8d571bd7
613fc00d19053b57bf636e9d9372fc4df46e1590
30089 F20110217_AABRCU alto_b_Page_113.pro
1f4ae2a7239c1e114180545a03f88411
029577d79939d0eaf2c19ee5b3fb382545f1b0e2
60887 F20110217_AABQXP alto_b_Page_160.pro
a894bd1b2a6a1f400ca62cb6a83327e9
cc46a54a6efe44997ee1acde7c348decfc335417
F20110217_AABQAH alto_b_Page_040.jp2
86a68f8410241e3051ef47c67ae2dbee
21d6a94e28677b6f1107e98be05ab6a7ea39afaf
6081 F20110217_AABRDJ alto_b_Page_045thm.jpg
e1fd17eed186f90d0ee260976df0eb16
677a8774c7d129658c557addd58b9206466a6e1f
1051977 F20110217_AABQYD alto_b_Page_158.jp2
a481c3caa477a9f3e981574d835cf642
f68cf7b66de58866ed0d9cfb6c8e8df2d7269b82
25577 F20110217_AABRCV alto_b_Page_070.pro
c9798c275b5d87ccb41521f738f032f9
7ffe2b7bae633284946b651e50e6b4166160290f
61784 F20110217_AABQXQ alto_b_Page_147.pro
a426cf7cb670c4cdc0ceaa5b0de6d50e
ea5a334a861d470d5c502c5e43dbeea0d512e76d
F20110217_AABQAI alto_b_Page_042.tif
639eabcc6a71cb420478c0ce970ca28c
297a5284c3e10e4ec0d32206d6ab35c0807d41d0
23836 F20110217_AABRDK alto_b_Page_165.jpg
545f9aa742793b9945d21330e12bef7f
948bece45ce9fee0767a4100e4ac6a673f9c3ee3
F20110217_AABQYE alto_b_Page_095.tif
7429f95aec58478f609d5771f8f08a14
c158dfffde78704870aadf94c78c9bf0fc85c421
851800 F20110217_AABRCW alto_b_Page_004.jp2
b342c712c7630c0f28afe7b926c701d6
e71f3eb3b681abad0235c821a493c318b9546e36
64734 F20110217_AABREA alto_b_Page_005.jpg
ac1284ef6de4dd25dbe5ba273615e9cc
07a537b18ec54ee59b367c8accf9013685958e28
971375 F20110217_AABQXR alto_b.pdf
e3c060483be40ac37e627b79ed7141f1
7f0f45acb52e41929baba322f1064d3bf81d21cb
5201 F20110217_AABQAJ alto_b_Page_164thm.jpg
debdb22a3a71b63b9e3732124239a8d9
a4979fe0d709c36cb71a4fb189b28eedf9c5e524
49311 F20110217_AABRDL alto_b_Page_024.pro
0a3d350c413d753b04f3114b8bff566b
54c5733f06c1da89842e8eb649f29b809ea5f19f
29664 F20110217_AABQYF alto_b_Page_162.QC.jpg
ec99a2a12cf3f1e70d0e4e25362f4854
7ef72ca90211e33c64ada4dc77012c366ed1ee5e
2050 F20110217_AABRCX alto_b_Page_093.txt
cfa3525354bcf0b85900e1e908165482
b36258378ccbfac45ea0a843e63866c711209b49
F20110217_AABQXS alto_b_Page_094.tif
b32d7eb83f8ddec71f38e73af695b8d9
4e8d165619ef305cc12720c474c632840664520c
5978 F20110217_AABQAK alto_b_Page_102thm.jpg
c44e99a9968eec59928fd281f94dce45
a807bda9a8297ae3e7665de6846c98fcaab1e824
4824 F20110217_AABRDM alto_b_Page_009thm.jpg
176b60a20f7f1af40c56c97712afa2b0
7810563eb0a79c9d441ea0aa3e9adbbe624c582d
48452 F20110217_AABQYG alto_b_Page_128.pro
da4f1f7546faefce5cf54e9ce7f001ed
50f89c066ca44b4f181b7031760c2369f265defd
2799 F20110217_AABRCY alto_b_Page_076thm.jpg
28b7dd5f990cdf511ad4b99635e520da
03b1fb3764fabcce9d46b90d8ddd44c404eb0907
F20110217_AABREB alto_b_Page_078.jp2
46eb8e2cad00230a967c41a7b1b1201a
2abec676b3940f3d0946cf145b80439446ce523a
82416 F20110217_AABQXT alto_b_Page_089.jpg
845c551e12ce9786d059573dc70ece9c
7f503b1047aabd0c52065969f9aaa8ab812081d8
1051974 F20110217_AABQAL alto_b_Page_060.jp2
a64b6ab64dec434f53762f4cde4ddfbb
129cfc7662bd73e8e78c0a5164407f0502fded16
2005 F20110217_AABRDN alto_b_Page_096.txt
5a062f61deab1dcd1fc362695153c5c1
cc7b0470289915eece66bf18bd2c0ed3acec9b16
99921 F20110217_AABQYH alto_b_Page_147.jpg
119a099822bea04681f7f06c8c455600
4afb3c4f8a22e936cdfa770e65c70e7480651da9
3036 F20110217_AABRCZ alto_b_Page_050thm.jpg
5f21e52f39ecbc7c7a516df079cee901
2f4901c850465f32fec1426603f873c8b3844f26
2500 F20110217_AABREC alto_b_Page_141.txt
bcfb28643445e875729bfd5d3c38728b
beda1307a57d291e73e373688014f15460300597
4890 F20110217_AABQXU alto_b_Page_011thm.jpg
ce6c61766ce136976902ed9da0a9f35a
244f661d8be80117703b24bc26b6b3fb117734a9
F20110217_AABQBA alto_b_Page_054.jp2
d604093c75fd131978919cd6ab5beb97
838fc4e2a6d60b8a6b7a8eac2cd37b2389156c6b
1448 F20110217_AABQAM alto_b_Page_056.txt
895207a9479f4bc3ebfc1dbc42486be8
1857ba19594e324bab91cf35ec12414aa507dce8
89555 F20110217_AABRDO alto_b_Page_097.jpg
699943b33c9ef15b2a7347fd0390dbda
e1bbc80516a73364a591523b93fd00a0787e123b
F20110217_AABQYI alto_b_Page_043.tif
6b74c389e0db174a018d71a281a104ea
48d5ec901f1af30a22ab5167faad039952a9bde9
48971 F20110217_AABRED alto_b_Page_063.pro
733a403c35e6e5f984a4b0d30f02a72d
3da7e58513387dce946227c1afb67b0ee07062c6
85165 F20110217_AABQBB alto_b_Page_067.jpg
3de4693ada0a8746a35a4da83b22bd2f
dd80699513d6f512e95f059115865cbf52415ef8
6004 F20110217_AABQAN alto_b_Page_026thm.jpg
3ee163807d0311ee3ce36bc82df88187
cb3e59af1096c3324e68ded2fde678861ae27d66
F20110217_AABRDP alto_b_Page_099.tif
6d6860b3f8fd69aa0ac32fcb33718350
0d78939a43b2c0b4b92091a6d6166400faff7ab2
1698 F20110217_AABQYJ alto_b_Page_090.txt
1c3fc23b3fac7301667868ba5b218804
c989478fc283e534456eab30ce05050ab53dec7e
F20110217_AABREE alto_b_Page_085.txt
76d64a15e5f79fc1098c79c3408b953b
1f1f596ee40ee798b8a5b7041909b70d55c194ba
1120 F20110217_AABRDQ alto_b_Page_002.pro
9324feaf586d28f48ac1db42755164c0
3d42481f3a4554a1a632ee83b0e909f741a39c1a
1975 F20110217_AABQXV alto_b_Page_107.txt
d35fbe928f8ca7cc586b1efc8f022ed7
746eff56016d9d2cfe599814cb8084a453ef6609
51410 F20110217_AABQBC alto_b_Page_105.pro
9f2d51af22a205e7d34f58cd392106c5
a6e0f9230ee5138abc81e979ee50bf6b550cb29a
2244 F20110217_AABQAO alto_b_Page_076.txt
d47a96c6b976645d88fdeee140dcfcf2
e1af2a0b53c48609039e2d075b5ee4ed61e1b973
4239 F20110217_AABQYK alto_b_Page_048thm.jpg
70879ab64512bda4a979b9e2c5840c44
a402a076aed98510e11934a295abbc56cff456c1
84865 F20110217_AABREF alto_b_Page_024.jpg
dba8157bab89ece49794a42a20176c83
bf263d194e06e147960c9d35090aa851c5aa4b0d
62211 F20110217_AABRDR alto_b_Page_139.pro
2516c67e32c1c9879328f37f0908d063
af439ce84b59fc17d1fa4522a7da7682abc46b65
F20110217_AABQXW alto_b_Page_042.jp2
b176859d59442dce4737717a7f7d276b
118dcd588fdbae50fd7afdc482ec4181f229eb5f
1051986 F20110217_AABQBD alto_b_Page_098.jp2
2f1183850ce7023db8f779220a70d28d
5893908d4a662d00c3dea77bf57e49136fb508cc
5480 F20110217_AABQAP alto_b_Page_082thm.jpg
a4a133386618cdcae7d0dc2450c26ee2
d89c1285267dd3de6e68dab4e8ef13e9fdb75d20
24974 F20110217_AABQYL alto_b_Page_102.QC.jpg
1f0c8db69f7221983da31eed1a55c8c3
bf7192e8ac69d659edfa58e7f4cd7d6fc26d3e16
102367 F20110217_AABREG alto_b_Page_155.jpg
4595d709749774ba344396e50a8a3db0
15bb6d7a36a44bbd45438d047816bb49c4e15f5b
2524 F20110217_AABRDS alto_b_Page_139.txt
e09c28d3069e54b0ec1ff4284234fc8f
944d8f0517d6cc5067b111c4682f0267565f6621
F20110217_AABQXX alto_b_Page_059.tif
644bda72bfa31d03b211a51108d1cb80
8d1d8bf8fb058461536faa71708a9b3c6cafc1ab
F20110217_AABQBE alto_b_Page_026.tif
561e686da78e9e7b19160ea47c64d321
0412e045e6b87a528a37accd480e91b089c8672b
26292 F20110217_AABQAQ alto_b_Page_068.QC.jpg
e00e7be15edfeb8290dac397add7ef31
b7884393e920963cb0c00df823e0515d187c8cf9
23914 F20110217_AABQZA alto_b_Page_045.QC.jpg
01aa647a44baed2d204e9eb9b76ef7e0
3c7bf4aee399305006178b64ee6be2306d704a95
88335 F20110217_AABQYM alto_b_Page_125.jpg
ca36f3d074645db892ce530bc33ad796
a6840a99ac0561044102b31fe06155a5bd22eb7e
4283 F20110217_AABREH alto_b_Page_111thm.jpg
6b43181332d399df2771ab9f453e31c5
50c8bdc9db20d1869e108de15f82ba4985863f12
1996 F20110217_AABRDT alto_b_Page_027.txt
b9a83d8d3c98dd6adbade7ee0bd88cb4
0398ce33b06efbc4f42bce646cfe606c78168853
49154 F20110217_AABQXY alto_b_Page_124.pro
4b2e37ac9cb2d0a48c08a101c1ed72c1
c45dfd64ee98c63074068f8c2b6394e1eb37f217
2754 F20110217_AABQBF alto_b_Page_138.txt
f4eea5af3aca28957d031310bda86cc1
5363b3889e78369440ecc5b70179bf584d6ec9bc
6036 F20110217_AABQAR alto_b_Page_061thm.jpg
a5952d0ce36d1452ba0cebc04c2412b5
4def375b242d9259ac369b7fc6f663fb6e86c64d
6393 F20110217_AABQZB alto_b_Page_104thm.jpg
86630abd590e000c5fd915b51978c847
5cd56d5265ee30319ded416d1a590888527ad786
F20110217_AABQYN alto_b_Page_160.tif
6e4fd7a7d6090d98a9d8e6418af785eb
c51528f250c2d1dbd4ec051ce05025effb638691
1013651 F20110217_AABREI alto_b_Page_123.jp2
128e7a57c98587efdb92f0239d15dbf3
c7fbb84d2ca90a00ba5e598505d4e660cb9f4c57
6250 F20110217_AABRDU alto_b_Page_098thm.jpg
e1a26597c93f098728c428a8ca2a96cd
ccb72e730a2fba7bcfd43608477852a000601c93
51226 F20110217_AABQXZ alto_b_Page_100.pro
51a01dd7c0caf3f90feb3c2c0e1a9cad
922530db492464cbde45d0a89171acfb6b37304c
81427 F20110217_AABQBG alto_b_Page_087.jpg
6106e33de6c9d95e1007529bfa14e627
250dcc0e89a10aaa0abe1c2bb4020c5382274c29
84922 F20110217_AABQAS alto_b_Page_037.jpg
d5ae8f188b8b0f31f08beb459c64be14
e501eb1162e21b3f3988ffc7eadf1d4ae275cea3
112398 F20110217_AABQZC alto_b_Page_156.jpg
6628fe53340dfc06999902f376b1049a
9589c133672031a74cc0385fb584d58a148398fa
6875 F20110217_AABQYO alto_b_Page_138thm.jpg
ac35670900d8f61c0a79b54105804fb2
f6a30eb6b2712efa17276fdbb0e94dedf33efecd
2020 F20110217_AABREJ alto_b_Page_066.txt
250cece06a3df17ed7216644fbda257e
b06d501f52b1ca273ff1a4aeb0eaa503f0dc56d6
33536 F20110217_AABRDV alto_b_Page_114.pro
5dac4bf00c72f11df4fb766340ed8e98
cd51df5ebd9f1d3fb450f2a94ac02793d33872b5
87206 F20110217_AABQBH alto_b_Page_040.jpg
1887a5b17a58c64cbc13f95a02c0e735
a471765c605bb57cb74550996cacce61438e56a6
1995 F20110217_AABQAT alto_b_Page_081.txt
ea28c3c38839fc0abe4eabff2e3fd6d5
433e056c3cb40c27b3eb8cd6b6acd783d773b7f4
25317 F20110217_AABQZD alto_b_Page_087.QC.jpg
01e3c3b87b347cd4420c1969250de585
829fa5e0c2a7d200269e57b0af04e4186e68faeb
19217 F20110217_AABQYP alto_b_Page_056.QC.jpg
35e7812eec4895fb515fcf25b1bdd72b
8d65ac4b8ebb13c2246d0370ff7ad02559a98c52
6505 F20110217_AABREK alto_b_Page_047thm.jpg
9ba1fa43b1270ed194e604bf41a60b3b
f65b1aa8fbae095ddc5c412496218ee3e6cb4cc5
4197 F20110217_AABRDW alto_b_Page_109thm.jpg
26fbed7be0939c5786ff81866c44584d
c5a4a27b35bf7c4b91850e7d84252c79367ed44e
5934 F20110217_AABQBI alto_b_Page_018thm.jpg
3f7bb29fb697159ba78cef55b7876601
74f94e168f55b67b17b19be258844b562ae1e64b
F20110217_AABQAU alto_b_Page_092.tif
bf1d7240773f69e8a6c4837563677972
5d6fdab0f8decd1f1ea63af7bb2d4a63b686e8d2
6256 F20110217_AABQZE alto_b_Page_052thm.jpg
569dd248698bae6662f0abbc1d566151
cb6d09b5d41728eab8822b9481765fc44830bae0
64536 F20110217_AABQYQ alto_b_Page_145.pro
325b1ed5c5cea0efa85af9d8f9534ad8
fef488e6d96268fa97af5bf57b442e28fb2ac896
1051940 F20110217_AABRFA alto_b_Page_139.jp2
459551b4382a16b6fa8eb1347e629cf2
9f242dad538a2e70647632c89ac7c9e0553048a2
14340 F20110217_AABREL alto_b_Page_073.QC.jpg
02ae1378a32f629bbc752473d0b45d23
907e126be7484e229e40650c27ca0ad442548f35
803760 F20110217_AABRDX alto_b_Page_005.jp2
e3f9d9fe6198e3425aa69976da95bf2d
a3a36be7748d318b51ca54955faf2c1c373bc658
990556 F20110217_AABQBJ alto_b_Page_009.jp2
5e2f1adcc5f4a57339372eb166195d8a
411939a074627426476a021a21a2541c6e4a45af
521 F20110217_AABQAV alto_b_Page_165.txt
d9cdda89db18fa32a486f3a2df1afa9a
b9c3f8a4825c18bfded18a7d3e79faa6ccc9fe12
F20110217_AABQZF alto_b_Page_064.jp2
255ec9bfd814b0565560daa497bec502
4ee60cc83643ec9e093752ccb0870914e4ff3c2f
1005090 F20110217_AABQYR alto_b_Page_090.jp2
e61a0701b8bc5006ac494987c26b05ad
78a20cbf6c9634c224dd01ed192f801633a2c446
F20110217_AABRFB alto_b_Page_108.txt
a472f6e3e6dbd8f96f7fdf67506daa17
6852269569d8dc4119f99a99491020a877859fcc
24516 F20110217_AABREM alto_b_Page_041.QC.jpg
676b8eead743900e95da0070f62ac455
0041e20851894744f5ce827d7817b84b6178df40
701727 F20110217_AABRDY alto_b_Page_076.jp2
f436cfec3c7835c1bdbfafdcb3f0b2ee
13904d2570f258e465accd144f4ae82b441516f2
80526 F20110217_AABQBK alto_b_Page_099.jpg
f19b4f4b047dd6ae85c204127d2ab668
2841d2defb03db40d2fca0c3cb3d83e6d040c371
27318 F20110217_AABQAW alto_b_Page_140.QC.jpg
4dc5207c65481c6d1328432a028c6ce0
6ed2299164b6c8a252f7a875a951acbe1f4b36e4
2549 F20110217_AABQZG alto_b_Page_136.txt
45e0fea5340dcab833ab46f18a10a306
39a01962a99bc82c8b2f06016bb68e68b4cbde14
1896 F20110217_AABQYS alto_b_Page_102.txt
ab285ba9963569e1d808edb533bc80a6
67aeea84ef5b8132d39105df1b2532ca61a61499
11077 F20110217_AABREN alto_b_Page_050.QC.jpg
b14f48acec7667bd873ab29eb849e28b
e9c14cd8186c41ca0cfe37a67fbdfae35f694238
26360 F20110217_AABRDZ alto_b_Page_061.QC.jpg
e11d3024d67ec0cbe7ffe0e38ebd5a22
e8555f445f003f12a067967da03ff64f33c4bac2
F20110217_AABQBL alto_b_Page_151.tif
af9b14ff72b6f5a06e8726f07e294b9c
cc9d00a0706572cca2f24c60b7716b163f0332af
18361 F20110217_AABQAX alto_b_Page_110.pro
27e7d3815742c8da688476403f641328
8c445a0ea983a0a6a4a92cec2838c0160961e33f
28398 F20110217_AABQZH alto_b_Page_152.QC.jpg
60b96e2ebb4ccec5f3e7e6ee9aa246c8
4b0cbf60c1e0837f3010cd576d137e6b924bab2a
68810 F20110217_AABQYT alto_b_Page_137.pro
1e6fb6c01365d689604e9dbfe5f2562d
1c627e2e28a9aa8e6fe66eb4a5aedd0af7e2cd17
78626 F20110217_AABRFC alto_b_Page_084.jpg
45b446af3425b6858e853f2da805fe88
ef71bb1ad57eccd1cb872c01eb3b4a024d22a1dc
23686 F20110217_AABREO alto_b_Page_094.QC.jpg
b10ff7605d0de929ff00b7225a189059
a787707fd928d7ff16766b657737e0e91064f3a0
6685 F20110217_AABQBM alto_b_Page_155thm.jpg
5e07abb1d1f7a514598483e5bcd8f510
34726f9ade27cfae74ccaca141d9099f284e0245
6536 F20110217_AABQAY alto_b_Page_145thm.jpg
807509601a34b30d7c5bbb38e781c5bf
e19d0713561185ae6af74db1a892c03fe5dace5e
32527 F20110217_AABQZI alto_b_Page_048.pro
4b47943c5f056a561e19297b5af9c1df
3aa9f04ef0ca0f8e6903b2fa80a430c1ef44c38b
1051958 F20110217_AABQYU alto_b_Page_152.jp2
95bdd86d8ea48f81c205970e8838c5ea
a2d8a57aa508019fc72481d22505021d3f61bd14
11614 F20110217_AABQCA alto_b_Page_008.jpg
d83f4f33f02c9099ad31a5dc5030178f
ce6b6c164d3fcdb2340f42ab175863760e7bf801
F20110217_AABRFD alto_b_Page_104.tif
15edc6168ce5c7ec4bbd0c830b4887b0
d2888d33c9071b05b05a4bdef984e21d033ee926
6294 F20110217_AABREP alto_b_Page_150thm.jpg
08bfc26a6453addd73e0ece3ef74c3e2
323729983b9b0209ced2995763796cc1a64d8c50
F20110217_AABQBN alto_b_Page_153.jp2
4954c189b6481cf91d17d97251bf0f2a
e84ebf708b009441f8801a2f9d75352caef3e689
63279 F20110217_AABQAZ alto_b_Page_069.jpg
62215361e88e2d06572494b1057be0ae
2ce1e22a6f89616b4445e02177883195f66fd461
2625 F20110217_AABQZJ alto_b_Page_142.txt
8f2c61fb1435c66dbd81801d86de05f2
2463a9346d845a572d53eff4b59a4c979f539731
1726 F20110217_AABQYV alto_b_Page_044.txt
6a8960293aeb3a3ac326a062e0b13fb5
0e946261d5ff66e67ddbab4dddc5d1c3e3133c6c
6174 F20110217_AABQCB alto_b_Page_046thm.jpg
724af1f65709c0dcf1d80a9b22f1c442
6ebb35cff93e2e05cd3114911a445bdc562f0007
27778 F20110217_AABRFE alto_b_Page_097.QC.jpg
d77874607956730339f84b5f838bc134
c8b0dd855aab1ef0526bfc222aa7779400e787d5
70283 F20110217_AABREQ alto_b_Page_057.jpg
96ac7fb9de70386a11820f457c671b34
e726db49196d1d6e8dcf84445732f8f2a00b2699
F20110217_AABQBO alto_b_Page_133.tif
2f92430a61daebb6d001c5328be471d6
9eb0b7d1445d077cd4505e18192dce8d4deb54fc
49059 F20110217_AABQZK alto_b_Page_114.jpg
09cc2f6996a97eae783803b2ebe4d40f
115828fb46b322df2cf0e82c1fa3fec802dbb2f2
51860 F20110217_AABQCC alto_b_Page_104.pro
907a82bc342b9b6abe2540cd2083a4cc
0bf8a92e67b77d9d3fb06ab3d5e711aa605f0f26
76161 F20110217_AABRFF alto_b_Page_090.jpg
1a9289ad65017eae3975b91feb0bccb7
3eecc94f012791a0a1eab2fc433598345fb6e06e
2765 F20110217_AABRER alto_b_Page_162.txt
39b7c4e5ba999890c70831914b8ab2c2
ca8e53de0903cef6481b0f3fef7ae63fccb91fd1
50119 F20110217_AABQBP alto_b_Page_091.pro
13c0e21e1d58baa88a36d03af2914b2a
e569bacf5d4a8eba71fe62672f0909f04925f3ad
2131 F20110217_AABQZL alto_b_Page_135.txt
e44997f9fd22dcfe19abeb347a304bef
fa668488753bc5d9eb34b64fffae103c530c9118
F20110217_AABQYW alto_b_Page_051.tif
72cafdd6f347fe14710dcdf68352902b
463a9477f3c0bc5bb01eb4e4f20e630445b62c36
1904 F20110217_AABQCD alto_b_Page_062.txt
78b59867e332949fc5b6f6ddb44d9f6c
154b48c858d9ef9a9d6723907ede2bd50b4a4427
5498 F20110217_AABRFG alto_b_Page_135thm.jpg
2487c720b2816bf5aa0a065efe2ab470
0717edb8634c746eb595a9a1e0c8a064a2c57b1c
25915 F20110217_AABRES alto_b_Page_150.QC.jpg
ddc57721e347134d51000888487b29be
e5e9759d7c55b43df4be1c118871caff498ca054
5870 F20110217_AABQZM alto_b_Page_090thm.jpg
ed66d7675bb385d21672cf56fd1c75d2
cb068f3e4a265eacf132f37850cabd93e2f1596c
26207 F20110217_AABQYX alto_b_Page_019.QC.jpg
f24188e38d43898171683a63c6e49f51
a306ee7ad304472d9c211316426389be7d991b93
F20110217_AABQCE alto_b_Page_141.jp2
0df04b7c15f998e3d7eeb47be728726f
78ae238e84814914ce28d488edf7b853667f1a67
2009 F20110217_AABQBQ alto_b_Page_054.txt
b9e4fcfac907604617c7814a837540b4
0daea360afb9b2ca71009b1648c44b137bd304bd
F20110217_AABRFH alto_b_Page_080.jp2
a4019b71f9f6745ddc4a54f554066855
67a2f590cb956093cd11684c26bd736b46046706
19976 F20110217_AABRET alto_b_Page_117.pro
6fbbf8bf47c138807d88e67c17757dfd
fb26aae49130cf3f080800dd9c48817b0f39222c
F20110217_AABQZN alto_b_Page_139.tif
999e179b1b57217053715702fccb014c
07542c6d4a7e5d9b8e14b416d32e08009f522f96
14752 F20110217_AABQYY alto_b_Page_109.QC.jpg
728cbb955fef362a5b3a07d3c2688348
f397be71151375c43a5743ef6d78ac53d4112974
4534 F20110217_AABQCF alto_b_Page_115thm.jpg
2fb17043c75f86468a67cae0d0198c54
192b983c7922dbdb573bfbe15fbcda5cde3cae2b
22845 F20110217_AABQBR alto_b_Page_077.QC.jpg
7a9f5f2d506b5daa05a46bc9406f1ffe
aa83cda78bf3581e6f464bf220570c9de1fc0a53
53095 F20110217_AABRFI alto_b_Page_058.pro
1c4e68bc11dd9ea6bc48a69ab3286348
8b0ad206a58c270132c69519b3efdfbd7f961d45
65802 F20110217_AABREU alto_b_Page_143.pro
37dac49a6f8218338bc3572d7c5f940a
4d9f44a07e7a3b389a180bcaa2c44080549fd12a
52336 F20110217_AABQZO alto_b_Page_040.pro
34f70289cfbcf954d089a85a762d375f
9deb87338cd77b9ad770efdc089dd6770de450d5
48986 F20110217_AABQYZ alto_b_Page_089.pro
d102136c02e77358cb3c5e02f97fb602
097aaaef407925ec6b8eac6bd8d9f9a325b6ae65
27556 F20110217_AABQCG alto_b_Page_148.QC.jpg
1bd6db79e6cd16b2ef0cc30631b60825
2438106ea09061b6a229d988957efc0f13e67e05
47045 F20110217_AABQBS alto_b_Page_018.pro
bea452d67505d2259c1ebd6d80e4f00e
41e0f605711d1c1909346635963da01ed35f0951
F20110217_AABRFJ alto_b_Page_018.tif
f33880364e7f9e903e0ff81a40e4317d
ad313757a6946e0156c734625ea9bc3d9df48145
6325 F20110217_AABREV alto_b_Page_035thm.jpg
edd4966c6308959c6a93d99c499b6382
0b8624dbbdf746a361f29aaa655a365d4cea85bd
47878 F20110217_AABQZP alto_b_Page_087.pro
caa833da61bb1511e32889f750060632
46e35543fc4c0f17664bbac1d868b90f86054392
525746 F20110217_AABQCH alto_b_Page_115.jp2
95d0860a995de95f6fa0d9ca8876867c
66eef06390802ed4c5219a56204f66db6c0ac42d
6178 F20110217_AABQBT alto_b_Page_093thm.jpg
b6abfa6351fa941f8bce6b5ff80f6c59
94b8e95f82cb68134532feca28679a02a385690a
15106 F20110217_AABRFK alto_b_Page_111.QC.jpg
86e77af8e81dcc2bf953f1cf844cc097
9caa96d52edfa98d75582a50075502a1ff06aae9
F20110217_AABREW alto_b_Page_124.tif
c5c0dd3346187425afb1b0adec95dbf2
fb3f33cd46b822adb859645dc35953a35eb5713b
F20110217_AABQZQ alto_b_Page_142.tif
ba603156dcbb7a4ff67eb9f780d6346f
276d188745cea6d143736036d417bea139edb0ac
81742 F20110217_AABQCI alto_b_Page_128.jpg
4139b07ec13e998a13a9d2329515c03a
6dd29015873577e72c79a6b83548a2327e704e8c
2660 F20110217_AABQBU alto_b_Page_007.txt
6503dfe864d3d6bd33b992b43ceadee1
4236478384e0ff1185505e117d4bf82a615a5c56
109 F20110217_AABRGA alto_b_Page_002.txt
c95380a47f3a3d02aa4442bb65cc2c40
25c05d75fb5291c23a748c2b61fc229d353bdc1f
26442 F20110217_AABRFL alto_b_Page_020.QC.jpg
878b7cb5b5211bb7081787e621ba9473
955882efb125ca41ad0a037ded592ef3da3b5c19
F20110217_AABREX alto_b_Page_119.tif
fb4f6a8c9a67e08b8e3d7b0323f83a7f
98d45427361bea48a078966f8696d743afe9cad7
2909 F20110217_AABQZR alto_b_Page_071thm.jpg
f68554b14d809785cf4f2217fe163c3c
d3fc1399983b02bf3dad5a6780dd77d4b813132f
6341 F20110217_AABQCJ alto_b_Page_023thm.jpg
cd08b66c4337ab6d59d8e7ec0a2fc65e
3dccab60bb90e88e3db561d78cf621e2447f07d8
80406 F20110217_AABQBV alto_b_Page_088.jpg
c4124eb2641a9731fedd9d8bcd0acfc7
8424e407acb0dce14f978ee9387ae7fdf573f289
1957 F20110217_AABRGB alto_b_Page_078.txt
4457b0d36e10773b975b96880f5a3c07
4fe935db2e69ce1e8d50d0f68becc876749d3a65
50472 F20110217_AABRFM alto_b_Page_127.pro
bbd0576511fdbb0157c663ab9ae04575
ec94688f9423ac63daac9bc50319807c616dfeb6
287428 F20110217_AABREY alto_b_Page_075.jp2
221cad37abf29701bc3da1f4c196831b
b1dda5d439311e398827a94589b0328f36c82277
83767 F20110217_AABQZS alto_b_Page_033.jpg
1697c9d6cd04425dbf7098febbe8c987
0c8b6a52c874ba76845544cf8b67eda0744f67c0
51028 F20110217_AABQCK alto_b_Page_096.pro
5ddfb869026365a5250e2292c68012b3
1284b7b1865dbac1544905463d6c39e4abda9703
23457 F20110217_AABQBW alto_b_Page_123.QC.jpg
76bcbf62946ebe37b1d264c1a72ea4a9
f6f21e1ea98c46fe17fe8d0305b2172fca12f54a
6112 F20110217_AABRGC alto_b_Page_067thm.jpg
6f856cdba0f4880cb5b0c807ef3c726b
187d3716e41b02a70ed7843a7f58c0762af8e520
85076 F20110217_AABRFN alto_b_Page_135.jpg
bbd26fa97225507f785d76ecb4269862
055b648ae91ad8ca86c3ea818409b6df220ddce7
2106 F20110217_AABREZ alto_b_Page_131.txt
aefc8756a56c24c4c0a872b8410102bc
405680818f12ecd1ca11efea4da4d72b8e995198
F20110217_AABQZT alto_b_Page_152.tif
94e4aec4946653869cacbc1457de3aff
ac01c4cd6d38148f4ebbabbdcfa984a18316b270
F20110217_AABQDA alto_b_Page_161.tif
1ce60842bc83ce3e2a9e5c848aa73b43
bfcf48dfaa83627d2635d5a09a158a6496cd0518
6062 F20110217_AABQCL alto_b_Page_031thm.jpg
3f200ddabdb4bb22e4acf74c0c27b997
3e8721786e6f975d6d2cee7c80f091b03f4c4756
F20110217_AABQBX alto_b_Page_089.tif
aa273d19f03e60419510e7dae8c85873
23521a1bc9cc67d43abc6da2cb071d8672ff3c3f
F20110217_AABRFO alto_b_Page_037.tif
d31ccb3cc80a188717436cefbe6ad9f3
08a555686124c808cf5f1865de599124b6344186
108529 F20110217_AABQZU alto_b_Page_163.jpg
63a522f105f555e2df27a27514504287
f0c31893b2f2d3d0472f79e6f79bf26f5174a6be
F20110217_AABQCM alto_b_Page_111.tif
785b51932eb41981185f6878b8e2fa61
61cffe68bc16deff0a1720397b886cd79001b5e5
695572 F20110217_AABQBY alto_b_Page_012.jp2
399e9bb7edb8557d29d882779cd03bc4
2bd4620c43e21ed87520eba1bab06438ee2eea8f
6532 F20110217_AABRGD alto_b_Page_025thm.jpg
5340f6632f8e336c99c9a7269528841d
bc9a0b2a81b63eb2f31524c37c214886aa47fef0
68335 F20110217_AABRFP alto_b_Page_162.pro
9fad4f88729c683773f6350286fc894d
92cb236392462c9348ac7fdd8d4571ad7d10fc7d
36048 F20110217_AABQZV alto_b_Page_120.pro
369f980d5367dfaf8c5e412dc7e09539
eb27245bfe3d1ef629a8c84c4af004641d70c486
6234 F20110217_AABQDB alto_b_Page_133thm.jpg
1502ed1dbe4c12de5c0d4f19d23a66e7
3617cee293c4749f14152c0ffcd97647a2c22cfa
82785 F20110217_AABQCN alto_b_Page_052.jpg
715cdcff1cbe0d6a886b2a6fc8d2e63f
c1f6a96b20cac8df17cc64d0ef9c6e46a16ebc08
F20110217_AABQBZ alto_b_Page_056.tif
1921865b2b64ea23fb5ff93ef04bd93d
b68314321c773beb9cabb3033cc6d0ba30aeeb64
27795 F20110217_AABRGE alto_b_Page_141.QC.jpg
d318a515f4c9478c58c41c9280cc7076
5ebf3cebce629b8d66001502a518492a592c2e89
F20110217_AABRFQ alto_b_Page_032.tif
a364e9fca0aff6ca2384dd0ee346a657
bd47e160cc6dda134e1440dc0dd63fb5c1ab7bc3
22325 F20110217_AABQZW alto_b_Page_083.QC.jpg
def2c521b296efa7b2b3d4ebf3fd6b0d
d7f2b755b1cfc84dba6e1be32ff970fe50ff6a33
F20110217_AABQDC alto_b_Page_100.jp2
50324ac8f319358d59f210363d2bb58d
cdb7a73bd3ab855b3fbdfafc8e659a9d723f5fbb
F20110217_AABQCO alto_b_Page_033.tif
d38ced47ef5cd6ac172a15456e126048
25a381d0f179348a095b5b6f7962d3c3601b98b1
2082 F20110217_AABRGF alto_b_Page_165thm.jpg
f85246e49ef400a794a9db91158481d8
aa41695dd215863b0bd70f555d834658bc0035c9
1964 F20110217_AABRFR alto_b_Page_046.txt
1ce48291b4b0ba2f3de0cedb8325c876
09f645a6b1ea884e3ae886e7e6684a2d557fd3de
6337 F20110217_AABQDD alto_b_Page_024thm.jpg
d4cd671d877dca5c50222e6e2c108908
a23fb89f4f9b52daa77704feba1107bb9f00dd0b
50600 F20110217_AABQCP alto_b_Page_103.pro
63a8e4ee053f8034c39241d38d95772d
0b7c89a7f5532a970282b82edb774f2ed7f69dd0
724784 F20110217_AABRGG alto_b_Page_010.jp2
13217b5ee54452b8911e15bdda64d0d9
dbd5a5bec47cb9646c548e8d4e29b22c968046d2
F20110217_AABRFS alto_b_Page_140.tif
60fc2c23b0f7c30374e0d99939f21304
9c9cccecd91ab900633bc5b90ad02716cbb7c805
F20110217_AABQZX alto_b_Page_091.jp2
bb320a7fca8d66ace7757898df5eb2e1
b0fad43942d91892771f4ae08e55ea80808b60bc
2600 F20110217_AABQDE alto_b_Page_009.txt
f71fbe88166de2aaae5369869df6f765
e50949ece0e08eb1530c99f2cd8d108187db8975
80385 F20110217_AABQCQ alto_b_Page_102.jpg
94b9ca91848e3e11a5e06a67c1b3767e
a7ccf32abb48459008fc7a4fffa43348a3001fd4
60728 F20110217_AABRGH alto_b_Page_158.pro
b74e0b907baac8ab38d5866b62dbc8f1
b4dc833a5ab9ab5edaf7778382da3aa441b78719
26403 F20110217_AABRFT alto_b_Page_096.QC.jpg
20744dbe8b48147cafea507cda6b2d4e
ff80ce6c7e0c305c08e1e7e7207e7b413d3e27f1
24980 F20110217_AABQZY alto_b_Page_099.QC.jpg
f55c2e1ca261717bf791f614685e80e5
660fba421810657a9b4e39f5a5f307c138c664db
26215 F20110217_AABQDF alto_b_Page_127.QC.jpg
c1f13c6de769d3694f859e02c218fabd
95cbea07aea1b9b77b4d66438efdaff6fb072e7b
28236 F20110217_AABQCR alto_b_Page_131.QC.jpg
10742d32e9416bb4bdeddd4eeb3c6c50
91685c77c5bbc990264bd47a946bd1c7d31df748
475772 F20110217_AABRGI alto_b_Page_116.jp2
4bdfdaeb84b9e95e9706d0472230d85c
a154ba6d9ff8392718014680fb05d928ee6dc1e3
1676 F20110217_AABRFU alto_b_Page_082.txt
8c3f2e45f061cb22b11053321c9257a3
e1cf8e37c083e0dfc763cf97f1a1b86be9b9a25d
5019 F20110217_AABQZZ alto_b_Page_004thm.jpg
7332bfed7b125a9580f63686ab38c6cb
3b46cf9974ebaa76fae2a79d8e8b6f9008446acb
538277 F20110217_AABQDG alto_b_Page_114.jp2
10bce0438a85ec568f9fb0270be6165a
d74e3b26a02664db1a5ef3b09324b325863b8bf7
6399 F20110217_AABQCS alto_b_Page_054thm.jpg
606995f654607a015495ad27c46a7ecf
d8fb521ad26e02ccfece8fb77c7ee00a705f6e8f
6330 F20110217_AABRGJ alto_b_Page_127thm.jpg
cbc02b49e1efb7c18c0874631524c770
1671e4102a314813c2c9ce5bfc47c9417152871f
6139 F20110217_AABRFV alto_b_Page_016thm.jpg
8388fd8b2a81035e64fed53e255b2f98
3efae927236b51b495a56797a73e23eb902e3eff
50380 F20110217_AABQDH alto_b_Page_070.jpg
1aea21095a26cb93ad756333e851f3fd
21a5da75fa23aa1a1621dbca579a45ce5e6f782d
F20110217_AABQCT alto_b_Page_130.tif
0f65d5d09913af6b1a918af3dcce2e19
d2f35bcd3c2e36a3a6f229601453bcc1c3b4ae33
83924 F20110217_AABRGK alto_b_Page_164.jpg
eedd1a717b499a22368f90c1b17f1c32
6d302e365da1548e7747ce7b67633ad5e89efec1
F20110217_AABRFW alto_b_Page_090.tif
2c69c2348b133228d20a8e327548f621
0c2a9e1cad646ceb0b271d45d00067ce4df62c1a
1051923 F20110217_AABQDI alto_b_Page_148.jp2
63db6565d5240a971642a35298b20265
a5b4abecfdb5e77175fbd33ebb0285ef51f70117
6635 F20110217_AABQCU alto_b_Page_148thm.jpg
61534c6faf30309660b393e3b498adfb
262d3868c2c924c8ed55f7a9113b647f237d4972
21100 F20110217_AABRHA alto_b_Page_057.QC.jpg
23b3a7654fa347ef3db7d66000755866
b15e3116e0d064e0e115531a78f5a7af792e7a53
81694 F20110217_AABRGL alto_b_Page_043.jpg
2bc3994d6b5e349fe207a21faa751466
3cbdb609cb06bce583061cbd70dd429098de3af1
25507 F20110217_AABRFX alto_b_Page_002.jp2
6544f85bad0899d81c2abbc5ad57b1dd
9918af13ec7290dfdfea5c94639f6319fea0fab2
83338 F20110217_AABQDJ alto_b_Page_060.jpg
0602d14b41746a68651ac671fb107b69
625bd34aa3b88c116dbf4fbbd161fe6f5d5c5cbb
6441 F20110217_AABQCV alto_b_Page_089thm.jpg
d5b3d30ca9e6f56652218431c60dc632
a9fc2e1b7dbe4ca8f7726bfeae9fa50fe4e4df15
84811 F20110217_AABRHB alto_b_Page_091.jpg
7811bbd662cbda2f2c34edf4c3b4146d
7d0363b81f65157061ed42b54efaa408ef97ed26
26644 F20110217_AABRGM alto_b_Page_024.QC.jpg
604f8e218e5bed16965908e20eb2851a
d36a1da10b8f61438aa1b3545a73c651b363f6d9
49718 F20110217_AABRFY alto_b_Page_053.pro
ad009bd8d99f1c0492c8387644a03cea
7795b9e266065c89527e80d8179bfbe28857f850
6318 F20110217_AABQDK alto_b_Page_017thm.jpg
3451b9dc7e5844b96dbeacb563f567f1
6aaded4104484308f8cc873ea4281d2757a06f5c
109313 F20110217_AABQCW alto_b_Page_146.jpg
eb810135e4d9f1ca7a77448d2f34f7ed
427d62ef732bc6f4c3ff47743c2a7c7738d58c4e
6115 F20110217_AABRHC alto_b_Page_062thm.jpg
e6854023e0db126a1f990166aa606e3d
210b55e1b6c309344f4cab0beeb137bbeaf26b68
51563 F20110217_AABRGN alto_b_Page_037.pro
1b4a02f574f04e474bb2aabe28df9dcf
ab9ef0f0b9ed72ed1b91b7baeb0115ba24f9eb63
78427 F20110217_AABRFZ alto_b_Page_080.jpg
0a3a18abe32761f920cab414b79b620b
cdfecb646fcf348ad1ec23aa7e02d229324ed7fc
2071 F20110217_AABQDL alto_b_Page_132.txt
5a572350dc616ec4f25b865b08214acb
38dfa486b69eb6f4476b8fb4c30f28e9e3e11267
25963 F20110217_AABQCX alto_b_Page_105.QC.jpg
bfeab832c947565e4d7cda9789dc10af
10e51a2ed6fe75c81d17a2850f4deb94c000a4b3
6421 F20110217_AABQEA alto_b_Page_042thm.jpg
442bb6bb4a6c5daa948b3c04e54c9d34
df3547950ab040303acddc94bc1d1dcf64cf3555
2078 F20110217_AABRHD alto_b_Page_130.txt
00608e590f297a5320de9dd292709b4c
50327961f00eff73381b9a00a88c262cd58efd10
59097 F20110217_AABRGO alto_b_Page_113.jpg
316b3c5552b5a7ed80ec7c88d614325e
92779c6950c36cec31fdfd544afb71b6c50f6733
26031 F20110217_AABQDM alto_b_Page_091.QC.jpg
2f887fc20dfc125f5cfa79c864046ffb
fbab02b8d12621550e53de8e03da084e01d3cdc0
95010 F20110217_AABQCY alto_b_Page_140.jpg
3f7e60de1e4f48d62712a814bfb89e0c
3e32ef166c15014b2bb6ec46e26b40c344e534c1
26129 F20110217_AABQEB alto_b_Page_086.QC.jpg
4b61d3224d921febcf7166a25d9022f6
2b1fc3039991230d2eaecfa578b4656a2d8b5054
83946 F20110217_AABRGP alto_b_Page_100.jpg
d17ab1f46e913d4061b2ee9b8d611d39
43b8a544bb617bcc6c5a16cc71d566ae42c5666f
F20110217_AABQDN alto_b_Page_007.tif
cc2a2d07fa6b18a55c1fb29710d45c7e
f98c06cc730a5e36eb11a730f1a71fe7c749cec6
25044 F20110217_AABQCZ alto_b_Page_108.QC.jpg
e877e635fc2c9f419a741702785e5634
f6b03e20cabbce1b5149c45c75609e04ac383846
F20110217_AABRHE alto_b_Page_077.tif
789fa21d8a256185c05f1d915f85f0a6
da5315189e16038e80fca13886727a34bdd94130
1290 F20110217_AABRGQ alto_b_Page_073.txt
3b9782b4055ff310b38b0735975a8554
6f31b061b482f4c3e0f7644e7f40a0684583b99f
6096 F20110217_AABQDO alto_b_Page_060thm.jpg
594702f634a3a1c94fa161ff4e4bcc76
ad069a2d23ea993f8d5707c8a2cd19b39375e8b1
9738 F20110217_AABQEC alto_b_Page_076.QC.jpg
6eaab8577abe147ea447c8ada47a2cb9
c97b814b9f3a30743008609713e04a977be4ca9a
48086 F20110217_AABRHF alto_b_Page_062.pro
677f7bf02aa2f9d966605b713fad082f
d06dc626fd399d00d263f33aa27ad906d1fe88f4
5643 F20110217_AABRGR alto_b_Page_051thm.jpg
39c2fd0254e87a7cef8147af35cab5ca
fd460662c9f5572f33baaeeaf194461a3874ee69
1689 F20110217_AABQDP alto_b_Page_003.QC.jpg
311c561c69f95c7cb104c09045746172
4397044f8cc7783cf1a9190636d7f03ea32d07ed
F20110217_AABQED alto_b_Page_146.jp2
afdb2f681b88fc905bd5a5ba97895362
79e4ed99d87eeecd0cc76f6c1af2d61a91081232
9725 F20110217_AABRHG alto_b_Page_110.QC.jpg
31b4246790414728aff85ff4843c5ce3
1a835b32ad39c5e156b30f33c41eec2a5a157e4a
3544 F20110217_AABRGS alto_b_Page_038thm.jpg
9ff800cb792a62a0ddb6ab104e6281f9
af76d7c5fb5e4b75ffb902b9792d82154d2b9b9e
33685 F20110217_AABQDQ alto_b_Page_120.jpg
fec3dedee305ece4ecd780a7d7b19fb1
91bbeef121f269d12a4b83a2377949a8519dd270
26707 F20110217_AABQEE alto_b_Page_022.QC.jpg
75804f0ba9baae096cf166fe1aff2867
2a9cd8cf3bbf564587c30f684e6acfb8f232433f
83066 F20110217_AABRHH alto_b_Page_103.jpg
e4ac04f6263acafbd5b7a81a7c271d67
0e7069dd110a7c1161127420b6f9cf96bfbce9b9
F20110217_AABRGT alto_b_Page_011.tif
5f719588e104b0f920319570b17de8b6
97a2debbeecfcaf61e8c889ad167ea7478114b7c
572 F20110217_AABQDR alto_b_Page_075.txt
a138e653747dfa6286039c16cf37c56b
acd35f4655f906f4413bd66350ae1e7facaa8422
569289 F20110217_AABQEF alto_b_Page_109.jp2
08449444f92460a2b2b136fd68c0450d
b576671f3ddeabcb689e672194b654341a5272d2
50494 F20110217_AABRHI alto_b_Page_020.pro
d4cdc35e62ca1a23731faedcbc7398fb
935326fb5a22065f9f1de44507b8f73dff288c65
987 F20110217_AABRGU alto_b_Page_072.txt
7074aaac0af243fe8eac0c4754cb1a30
6debaba2edea7934c6af50f9b7681a2d382743bd
F20110217_AABQDS alto_b_Page_134.txt
4ee6160670ca9399d39c4ba733982746
0ef6ab4407d4f4185981b8a4aa69cfdf2ce67789
F20110217_AABQEG alto_b_Page_148.tif
1bb790e08c012cfc3667680e2fb92889
7fec9ec1294f173b7ac7921ef132568ddf5afdee
1051964 F20110217_AABRHJ alto_b_Page_108.jp2
b438168c95d93cb1dea8391f36512b69
efd44f571a779d93d9995ec52ce2c0ec777d21d4
2640 F20110217_AABRGV alto_b_Page_120thm.jpg
b1b4a34a5432deea1478c2d0ab07c6fe
c227d7d7019eaeb10f4b3e27150d5494ceab339e
2667 F20110217_AABQDT alto_b_Page_143.txt
caa9e91714f4d0201f498dc6642998dd
21df61ae73ffcb6503276457b8bc2fd1a661107d
F20110217_AABQEH alto_b_Page_052.jp2
0c68c8307572a64d2c9837a25fd7ece2
af49bc459dc52089fda38fb28f05729cd11f4635
F20110217_AABRHK alto_b_Page_055.tif
173c0efa742d4307c650a3acd4e9cb74
58fa8e459a8a31ffb110820b102b6d5c53a82bac
63415 F20110217_AABRGW alto_b_Page_149.pro
a0678713bd2a49e7c5394860509ef603
947996a02b49d06dd42bb19b3bd93d27cb1b5f38
62214 F20110217_AABQDU alto_b_Page_159.pro
18f1b8dafe8e2dee5b8817bf976854f3
144457ac029952f851de159fa365490002e0cc97
16021 F20110217_AABQEI alto_b_Page_012.QC.jpg
0475c8cd52023fd6ce53ecf6418a532d
6c014775d11e79c52347fe9367a6cc186aad9d40
31257 F20110217_AABRIA alto_b_Page_115.pro
3fa94152634985911db92c61073c2d70
8a4acfcebc6e6b1b9750b446cb80d7f4027e9a90
6783 F20110217_AABRHL alto_b_Page_162thm.jpg
8115c2c08d48124a49b1064b6717858d
94c557219f834530ff44d98ad58efda0d6e1100c
F20110217_AABRGX alto_b_Page_064.tif
148ef4507aca0cfb07c4c93d0db161c6
306254e6c72360dfcf41ec61bc6a210a0a0f53e1
5340 F20110217_AABQDV alto_b_Page_083thm.jpg
3d1fe5553bbe30c76c4cfe6573aa0997
67bce47dc5b32f5580ea50fe13d9740352baa43c
F20110217_AABQEJ alto_b_Page_046.tif
080712fcd04d82f1ec7d2ee0626cc04e
4600c6f2fdd7b0ace5596f2d0a22c8e3fbe6b168
86865 F20110217_AABRIB alto_b_Page_092.jpg
ea11500a0647b093779f616bac4bf623
ae611643e914a0a70bf8a165886ca012d6b5ff98
14531 F20110217_AABRHM alto_b_Page_010.QC.jpg
f5683a73e18144cbe4fc20632b7e4fa7
5f7e3a05f7c859b1b214700dfabaaff2c0c7c4f7
548481 F20110217_AABRGY alto_b_Page_111.jp2
85d5140e435020c1bf95242a5de9ba0d
cd5249f54946b7576de09cc5e46ad01c67bf399b
83815 F20110217_AABQDW alto_b_Page_101.jpg
a9d9e007642eafe52b862229e9e1b112
5051405a3eda53de98cb0ad708cab4fc9dad459d
8516 F20110217_AABQEK alto_b_Page_118.QC.jpg
b8a9b6b7b9852b811ca40cdc553a74eb
ab6c327bab89c4c5de67e1683f8f55990cf1a672
F20110217_AABRIC alto_b_Page_084.tif
f9275a78657da2b151f9e0b07dd56814
9b23f7b6eba8925276a52338e20e6f84d15d0985
F20110217_AABRHN alto_b_Page_014.jp2
2278ddbcceb7e53e34beb670d88f1fbe
06854b773508096c5bbb4eaa56fd8572b02a56c2
2053 F20110217_AABRGZ alto_b_Page_040.txt
d889b18421384b1e2f5ccbe8c1d4f316
68668fddd123f83bb53342d9001d0794030e120f
21924 F20110217_AABQDX alto_b_Page_013.QC.jpg
10001337e7cb08f821a2f008db951c1b
f80680ce46fd29ab29c3b9912c8796c7de0b4f88
50432 F20110217_AABQFA alto_b_Page_055.pro
ab022a3d973bd8fa5d42c9de197236d0
1788b2e7b1b757ad24d93a40faa3206c4b098b81
F20110217_AABQEL alto_b_Page_052.tif
f8a65ffda29929cacaae4307a454b7e0
123fcb5646a3589cbf7fd9f6b2ff6c02aa0aeea2
25416 F20110217_AABRID alto_b_Page_079.QC.jpg
219730654cadec232c7f77a0b31972f1
053e8570bf4f0b889f11873fb3273f3dee940bb1
15551 F20110217_AABRHO alto_b_Page_070.QC.jpg
455c7b94a3e4ab26025441ebf5fcf628
0b888729091007b32eaf7c54d8eca0db8d6bcc0f
F20110217_AABQDY alto_b_Page_070.tif
b8c8c5d1ab10eb3564e049f9c27cac8c
44ce9f5fdb12da4cfba628588df28b0d398ac638
F20110217_AABQFB alto_b_Page_004.tif
700ed684852b903c07f73f2394f0c4ed
c0f72d97e1b5f7cbb57a65994d09ce3c69790ca2
5024 F20110217_AABQEM alto_b_Page_075.QC.jpg
e6d56a9f93f3acf604cf4a828aaedf3f
919513995fe02960ef014215ca5dcafd270136fb
5893 F20110217_AABRIE alto_b_Page_087thm.jpg
533b860460dade1aa6daba684147d909
eb6b2e52d82be306de1d15e56a4df6df48f2da3b
2035 F20110217_AABRHP alto_b_Page_068.txt
8b7677ccb15dd04710f4a435cb6d9c9f
e96b22ca81b391bcf87cd26becbc097349d2a830
F20110217_AABQDZ alto_b_Page_104.txt
db945c9d69cae61420a0b4943c3f3984
eda54c1f7ef074e458eac4ffde6dfce5a660749f
F20110217_AABQFC alto_b_Page_055.jp2
0fe11a31c697493f16894f267448376d
4f6961e233de5a84ad5b441b3946d82dff095f2d
F20110217_AABQEN alto_b_Page_016.jp2
306e5211280f7c2cf529a5e59a7c57d3
497cd3a40c71f7dd401c3b9422603ba84282377d
F20110217_AABRHQ alto_b_Page_086.tif
09c8156aaeadfb86dc526d590ecc65de
8b71825e50c86abae1cf9d4ddb7901e9e9f80838
86730 F20110217_AABQEO alto_b_Page_065.jpg
64f2d3cb1a11596a33775baa810ed1f8
c9ef6d8dc7e818cf77bc9e45e5078a5864afe791
F20110217_AABRIF alto_b_Page_019.tif
53c0cc6d6912386120c0359a02bf1e3e
635cda61438e138d260b103ad6f8a230ec6dbe5e
1051921 F20110217_AABRHR alto_b_Page_132.jp2
cd3b166cd43c87a17158e2db72f62a77
441236a93c27883dc3f302c93ea59642107bec77
2017 F20110217_AABQFD alto_b_Page_030.txt
5800f294b72b115b3d37b3e342e5b02f
a7857ee09ee90895d12a9cad6a3e92ec53fca71f
F20110217_AABQEP alto_b_Page_035.tif
ce309e68cf8d01f67118983e5fa4e538
c455c2364fa414205291356af1a42677e065935d
1647 F20110217_AABRIG alto_b_Page_083.txt
870f42dc8a19ccedf3b077d654ec12da
3d6883922d56ec0d1eabf58296d8a0e2a6e2c134
F20110217_AABRHS alto_b_Page_087.tif
af87601ac4750b33a0b619ed029ce717
107545411fa10da280dedf8b33c07f95b38be62d
6147 F20110217_AABPZK alto_b_Page_032thm.jpg
d7c8da009bb5336014ff12c6ba6b1ed6
965a07760279ba4ebfb5fd66ccc567fb907f64ce
50769 F20110217_AABQFE alto_b_Page_027.pro
85ade93e16287b434a9897fc3fec0ca1
c2fc71d3dbcab65f855cfe3ee2645d32a21483e0
F20110217_AABQEQ alto_b_Page_045.tif
a926799fde0eaede8d66908a13256ca3
6ff40773b38eba0a3833ad7238bbb2227883a616
1808 F20110217_AABRIH alto_b_Page_013.txt
a3225973150561ddd1db6cbca6139fd3
146b2054b23a706b24a200eb2331698cdf50d370
6447 F20110217_AABRHT alto_b_Page_091thm.jpg
633316bb12d67d94e7c4bff06c963ee9
3c868e8d2b669be15f9a4b2771476afcdf35050d
6314 F20110217_AABPZL alto_b_Page_029thm.jpg
5b964efc84fa8a3b5a24a1cb0d32e931
fb4b9717f5e5d9295d7a7bb64c26039a05bced2c
67572 F20110217_AABQFF alto_b_Page_011.jpg
8ae3fcc9241504f3a1a675e2d8582f4d
e3939e67009011a5f98727d6aa87360543dd9061
51260 F20110217_AABQER alto_b_Page_095.pro
e3f8fd357b76f109e12002bce281d25b
8550575de9a2f1207465851136b3ce0baa2553f5
25645 F20110217_AABRII alto_b_Page_103.QC.jpg
55b804a2b2e51d4a2e05609de21cba65
02c9d172c94620cc818ff9e18829ec6c3b24f65f
119553 F20110217_AABRHU alto_b_Page_008.jp2
d5db27f104d9684b21ddbe35eabc6729
4904fdfcc6fefd68056055afe86f47dd342f35a6
F20110217_AABPZM alto_b_Page_012.tif
32fe6936f26d4964e0f655790e3ebbe6
87fa2cb3b4f460188e6809cb64ee09fb58b054c3
48065 F20110217_AABQFG alto_b_Page_102.pro
6a35de67dc83b64178986d39ca541670
974f824275d68c88fcc7e5c7d2b2172591b543e8
6101 F20110217_AABQES alto_b_Page_041thm.jpg
3a8e20cce824eeb489b7c26d0e7b873b
33b6e7ac28eec81382fdc2708daff5527c7c7fef
6864 F20110217_AABRIJ alto_b_Page_146thm.jpg
7f9c8ffdd74ca4845ff4fea716a66ccf
4473709fe6a591f9124825c5ce028d2f800c7b5e
67139 F20110217_AABRHV alto_b_Page_154.pro
52a441f7f76951b12f4aae072980c4fe
0b22f80afdb0d8293ce47be998a35ee189b348aa
F20110217_AABPZN alto_b_Page_022.jp2
4f49c8782a9352b1186df5f15afa8c0a
c20488867263372438aa95c61e5ed28d1ff8644b
6189 F20110217_AABQFH alto_b_Page_015thm.jpg
c7032204df6076d87be08d1f604dcf0d
9c729e4c44a2830a3d51022ba30dd22bf5483719
F20110217_AABQET alto_b_Page_044.tif
7994ba9e297820fc9443b0ea8458086d
f5007958f8311281debc2b62397b2fa2b852579e
1051924 F20110217_AABRIK alto_b_Page_103.jp2
cdb80744fe8122523d9837b1bbdd7861
26c38e2b14774f497cc5db59b9ba3bdc7a5d5ae9
1981 F20110217_AABRHW alto_b_Page_127.txt
7f057e3c358ce8c574b7c9f1474acad2
ba58a7b94c31225502e7dea5c3bfea3cb064172a
F20110217_AABPZO alto_b_Page_155.tif
f3699f592bdb0e112a8bf3344312fbd3
ab2d9dd3b6c42fa9e5f17ca1c715a0202bcbd7a5
F20110217_AABQFI alto_b_Page_157.jp2
02feb1d783df8232c13db3d1d27b809d
537b5ee71799597f6b4ce968456afd1bbda01f64
F20110217_AABQEU alto_b_Page_047.jp2
a8aab0bf2833997b5b53d1b276ae1382
2f5d003a2df855941ede0b504828e01417255345
1051973 F20110217_AABRJA alto_b_Page_043.jp2
515abb34771b80c426355bccb863960d
8b3371e078a0859bcb87436f892683c4083d0abc
29374 F20110217_AABRIL alto_b_Page_146.QC.jpg
3b22504f77269ef7e738a2ed1553609a
2d34c2c6ae010bb9bd02360b7c6ab3d6bbd038f5
111757 F20110217_AABRHX alto_b_Page_138.jpg
c4bcbcd21994935d71dfc32c27ed641a
6100128bdb4e321b426eb575767f7e27e1efe9f7
5005 F20110217_AABPZP alto_b_Page_049thm.jpg
764919549a57884cefad5e6462e94631
21cb9e8522b565a218e9363a0ded6044f93b3d2d
1051949 F20110217_AABQFJ alto_b_Page_041.jp2
5b470f0d7459f6b1ca9c0278df48f04a
bfbffc91fe8fca83ec7e3fdba9f8d43f11b1cac9
6172 F20110217_AABQEV alto_b_Page_020thm.jpg
5b885946b1fabc933944f9b59bf90c53
87e8955a7fb65664f7074bc2de82c20786b61dfa
22479 F20110217_AABRJB alto_b_Page_082.QC.jpg
f694f7236139b135bc115c333d7b7d3a
99525eb4e883cb32ea0ede6b9093184cc9aed2bc
6141 F20110217_AABRIM alto_b_Page_084thm.jpg
b1e9fc6762be86b9bc21b8f7e355fcfd
b540887aa172245c8820e16cbd8f861874a70d7e
2450 F20110217_AABRHY alto_b_Page_144.txt
27a16c16ab095a9c5e987ca49eb8f55d
cb06c0ef531d3787c169e4c05acaeb3509f22e1d
6577 F20110217_AABQFK alto_b_Page_143thm.jpg
a607956484c4f2239a6e7cfe7a5a41dd
7673130a003e945a21be77610568b864ef71beb2
82608 F20110217_AABQEW alto_b_Page_014.jpg
4bef91eeae9fabc9a627dab7726c5a79
55695e5da955ba59d143f78397cf008814f78f2b
F20110217_AABPZQ alto_b_Page_076.tif
99ceddf6acb13d19209adec50391e870
e00dbd112460461cd1e04e9a5cd478ad85007682
F20110217_AABRJC alto_b_Page_025.tif
9fe7f8f65ebf6a73b2bc71a787e06fe9
4b9604131f199e6100ec9c2c1d4f269aca245795
4408 F20110217_AABRIN alto_b_Page_070thm.jpg
709c61f7bee5841ccbf2336e0998ba0c
549c4ecdea7b46857ae0129eb16db098453626d5
29585 F20110217_AABRHZ alto_b_Page_118.jpg
7cdd354935b134c94c7d81392961b1c3
eed91031ebd4adbef66b13be9026b28442d27466
1051941 F20110217_AABQGA alto_b_Page_133.jp2
29523c55ce68fe39dafcd85e0c0f04af
0f4ff7a6d12ce690d56eeca77368e4fc432aead7
18904 F20110217_AABQFL alto_b_Page_006.QC.jpg
848fc1856988a91c91aaafc67d8c1b99
8159a25496581f25d4c3a7627de5d3dec78714ad
F20110217_AABQEX alto_b_Page_137.tif
219036d394998e45c5a6e7780db5ad88
327088426a0b33d0c541565427617120fdba4dde
F20110217_AABPZR alto_b_Page_017.tif
1d69ccc4bb87d43e2345a7bdc42123db
c467ceb903749bcf5f14dca80184981b9b0cb2cc
1051937 F20110217_AABRJD alto_b_Page_028.jp2
97f1a7c515d3540a22b967daffab5e41
0b8c5275f571f1ac488cb7aa63ada846e10b7903
24755 F20110217_AABRIO alto_b_Page_062.QC.jpg
48d06db21bf0944cfacd8679d25f0bcd
60b84c400a605b986623f84035393a8f8fccdda5
25565 F20110217_AABQGB alto_b_Page_060.QC.jpg
1d9bf86784e89d2724aad75ad2f61b01
3db7623c1fa3b54e07c3df1bb095252d3046a74e
987357 F20110217_AABQFM alto_b_Page_077.jp2
448bd4422d7111c3d6f7886b00913891
bd1a07d944001f06509652904465d607199071ab
50970 F20110217_AABQEY alto_b_Page_098.pro
f41d8c505669a124fed1f4c0236a4075
a35249099ac1a91e1bbfc3ea78f9080df784e18f
F20110217_AABPZS alto_b_Page_116.tif
10cf2cc2b7800665ceb88792954bb633
52de7d29624e901ed4f5841525d281bdc911fc85
82082 F20110217_AABRJE alto_b_Page_026.jpg
892539743ed239892dc472f282968452
423a426f0495a53584c8c9a593c9eddd2cee2387
F20110217_AABRIP alto_b_Page_066.jp2
87f3ea0b53cb8d7fd8ccc215e72534fb
6984d353793b494f6fdfeb0ec0caaab70343a7c5
83996 F20110217_AABQGC alto_b_Page_127.jpg
04291d8fe4bd9b58c09bc865ab8f48fb
3b1ae304407e33002077f3d5c0f9117aefbdaa14
6498 F20110217_AABQFN alto_b_Page_058thm.jpg
f3a201de058ab46c38596abdac8cea11
4551dcfeb01385b365092084eaf13db47ac1c6a0
28150 F20110217_AABQEZ alto_b_Page_158.QC.jpg
e2ba80fb872a116d8be72978182b03b0
9cb715a07c3b9cd5a33a412c46d12cdfb75cdfcd
14071 F20110217_AABPZT alto_b_Page_072.QC.jpg
166a30fde8d5836a74b5627521b03836
86f5e200524b3634ab6c8c3524b9948b48cb49a8
2614 F20110217_AABRJF alto_b_Page_145.txt
4ae4747bfa4f6bea2da420960ff64a03
011f42e808b701d3a9b8d3f1436df538cccc07c2
47959 F20110217_AABRIQ alto_b_Page_073.jpg
ed2eb2bd18d8c86f551cd6c106b6f309
adf3cf6ad547e5fe1c850968652b507ccbfe28e7
76873 F20110217_AABQGD alto_b_Page_094.jpg
1905c01cf030339cbf34ace63a255aff
bec8730fa94d60274be6d8c59e4a909fcd85a82e
1428 F20110217_AABQFO alto_b_Page_113.txt
e230340f19a83ad4f76c179cc3d16e68
04618aee7ec7faff4ed00bd58a451e6a4dc96b9f
2464 F20110217_AABPZU alto_b_Page_160.txt
c45a486bcfae4ff2d95220289655aaac
431cc21d6d88defd81a427f07d07cc6ad90719a3
F20110217_AABRIR alto_b_Page_072.tif
4af04a9a111bde8850c10fdb18a55775
b71e42c885ffa921006c0098c110be7a706e6f53
1889 F20110217_AABQFP alto_b_Page_043.txt
32cb5dfbcdaf2f0a804270947b5d01ae
a0517f9590681c8734412dac22d24db841f058fa
343215 F20110217_AABPZV alto_b_Page_110.jp2
15d9d084a4001e5f57324bbd41602627
42645688c710b4afeeb1763bb33ab53f90849453
5763 F20110217_AABRJG alto_b_Page_094thm.jpg
89df3e146fa69c72252d9eb3eb7ae1a2
3a2ebf6b655d6ba73c6c14cfb0181171d9cc944d
F20110217_AABRIS alto_b_Page_022thm.jpg
0b24add5ecced2d40e169e28ff0c5970
45167b5db4ac6799468ed17fb163492ea811f74a
2507 F20110217_AABQGE alto_b_Page_147.txt
240def6ef7384497702bb6cc4d84edc9
4a5cc799f53bab8adc8e5d86d5ef4184cac798ac
F20110217_AABQFQ alto_b_Page_079.jp2
abb83da86a1a4828ba712acdfe8bfa8f
6bfd24aed659114bc2a08abdef38e24858c0eedd
80752 F20110217_AABPZW alto_b_Page_063.jpg
c80aa91aac74fa817a812536925991fa
ebdef9f8b2b584a76f042eed35cb212373eab152
26109 F20110217_AABRJH alto_b_Page_035.QC.jpg
c4e4f70f5f1b160d825892b65172f75d
2f89a34c3e94ca132032aec73d8ee0b7cf7a2c64
26294 F20110217_AABRIT alto_b_Page_074.jpg
6fadd1caddd06fe80b570f6f0cdf3b23
d97a07e1d32519e6e00d769667342f783d9cff0e
2034 F20110217_AABQGF alto_b_Page_095.txt
d66807bcd5ab6b91becbaf5808537e32
4114f624ef98d89b69e7b0c3b56e2b9926782a9a
55953 F20110217_AABQFR alto_b_Page_048.jpg
355269e878bd47d055919a81256e673f
53713b848fab17f50ed43a90af85da8f71a61839
17551 F20110217_AABPZX alto_b_Page_075.jpg
4b027f4ecf2d9033eab5c3888a6c1405
33985d6d6538c15a10e49e62a88636a36ceaf087
5612 F20110217_AABRJI alto_b_Page_123thm.jpg
a8998e13145b381c7c486460fc621033
14d0ad9c5edc99ba44198a1661641ff97d388946
F20110217_AABRIU alto_b_Page_030.jp2
e80911525eb8f38c0736951004a0d9c6
3661a3830bed359bdc6abce42cf625d808058fae
25924 F20110217_AABQGG alto_b_Page_078.QC.jpg
9f06768a7cb5faf3ab9ca9d7378ec58f
47683ed08f5c052ccee94265c73f51191b85bb53
1051968 F20110217_AABQFS alto_b_Page_131.jp2
a08e98f60aa19b6f7760011fd66902ec
c84000201b8b348dd4a77081c49a268719b3c631
1457 F20110217_AABRJJ alto_b_Page_074.txt
8802072a0f5b223b72154df018f8a3bd
1a05f67279e77bf976e4dc248632157b0e1fdead
2024 F20110217_AABRIV alto_b_Page_092.txt
d3b4042f1ce1e02d4988bed79d248700
079dba4b49f9e4c223a759822dd6b26cc37bf037
84407 F20110217_AABQGH alto_b_Page_068.jpg
a98d2ab6c3e956a63204fdd45f4f823e
5e40dadc88e51a8f996016deb46bca9f0c8ec811
F20110217_AABQFT alto_b_Page_053.tif
f98d1ecd2e30ef39cd6fc6ed00c4018d
5230f6951123c395542e93f356445808a8662f9d
25253 F20110217_AABPZY alto_b_Page_124.QC.jpg
521bcb630214f6acc9d0b87733dd4b89
110dbf77d62477056568a017a8a1f92f79b6dda5
588360 F20110217_AABRJK alto_b_Page_049.jp2
85e0b77550be7ef14213dbbacd49f838
4e4bab2de69d20e09be6059f16d641471807e9a1
F20110217_AABRIW alto_b_Page_123.tif
5f23fd11fad5df0c1de9e10760aa6ffb
37dae7aab5200a2623a2a087378070ea28479cea
F20110217_AABQGI alto_b_Page_082.tif
cd2d77a5ee86333c6ba737ade0308b75
46514c02bdee88fca090b1d755a7dca87a0db46f
25834 F20110217_AABQFU alto_b_Page_055.QC.jpg
faca5bac4c50f6478e6cc4eb8a7f48b5
47dd23d706911bd63592081f6c5f59e2f66280f4
83277 F20110217_AABPZZ alto_b_Page_019.jpg
585ffb2d583e9d74e03902b6d7eefd01
6fc24f117de767fc39f12514feb4308433a0d69d
F20110217_AABRKA alto_b_Page_066.tif
c252e8e819ccfe92d499e41ed973722b
15b61bdc0e672d2ad27e4d39a765fbdd8f39eba2
2047 F20110217_AABRJL alto_b_Page_126.txt
700455ea814e27e0dc6920606f205dc4
31531c678e8b192e7e23276d927ae5946161fe1d
51269 F20110217_AABRIX alto_b_Page_030.pro
b0a736ffd46a8a810347dce46c268e2d
9b9bce1e6060b7229daca0f9438fc5e0b033b770
2482 F20110217_AABQGJ alto_b_Page_148.txt
1df3c4e7c5a3cc587af848e4977d5b1f
4bf41b04dbed694e8c1d0e91c2483d5ffbdebbab
1051904 F20110217_AABQFV alto_b_Page_102.jp2
e1c72e0faf99ca1777fac166bf956b69
df4215e4fc7864a50481b8c6c2cb7912b79e09d2
6090 F20110217_AABRKB alto_b_Page_103thm.jpg
c6ef7efad4cf2d58a05aff7e85595e69
810ed4911c1430b6e3ef0c467705a6c046a524bd
1051972 F20110217_AABRJM alto_b_Page_026.jp2
3e3b7475bcca00551cce5dd06d744f0a
d791a579e2084fbd0c545798318c42733f98f740
4041 F20110217_AABRIY alto_b_Page_073thm.jpg
59127544125402f2d2ab3a38e898a39d
318a797fd7ca90f67be8ab63853c9391fd424b84
2068 F20110217_AABQGK alto_b_Page_117thm.jpg
0be599cc0c7b2ed0551293f7f81a2149
5def9340cafe09ff19a8fc413087eef662f4f68c
F20110217_AABQFW alto_b_Page_061.tif
9f2c3eb6cd352cf6b73132cc99aad71e
289868da2090725205db2b5596154f1c87a41ebf
2025 F20110217_AABRKC alto_b_Page_061.txt
148a50cfaccde2cc99202f960a6a68cc
e55158764823b2ab87150dee5a9ddbd2e6c6066e
2455 F20110217_AABRJN alto_b_Page_158.txt
7c593c72ae7119bcc567fba7fdbb5202
d2357f3d9435e53a8eb3ad4e2086f27cb5d7a3a6
1051962 F20110217_AABRIZ alto_b_Page_104.jp2
4f70e323df0ecebc9581d3e7b91263bf
ed6b5dc2acc6b1fb55443665afa25a929f717054
1051847 F20110217_AABQGL alto_b_Page_136.jp2
d8d1b0589d005db9e84c730ca7674b3c
dd00e5b7c93bb1c907d70d437a244c6e837ef323
25163 F20110217_AABQFX alto_b_Page_016.QC.jpg
513accd58f53649f2bdf29a70bb0550e
900de68471247690e5895d7f11c1d60fc2d8d8a9
1952 F20110217_AABQHA alto_b_Page_064.txt
873506c7fafd7803da538faedd211cd0
e985c314b0d772353be03dd38394912d73881479
2529 F20110217_AABRKD alto_b_Page_159.txt
19b3af471dd5b0fd15343cc656c5cab4
eb2864a792259cc6acd3eab16393a959f6a299e2
6663 F20110217_AABRJO alto_b_Page_157thm.jpg
e9c830b4ee6a2f2d9359d8308a6489c6
cb648b2b1fab823d1448724f5e3e29435c041fe1
1141 F20110217_AABQGM alto_b_Page_110.txt
28817e0587295296183395f567dad15b
d702e81582b6a712e7d07f6a3c1ffd2a7d2db807
1953 F20110217_AABQFY alto_b_Page_053.txt
be462ad3ced7ba08aaa7de9882c88813
ade7a42d5625691b2ef65ac410150c7299b31355
80283 F20110217_AABQHB alto_b_Page_064.jpg
1f116e5bef91e784cd484e9bd3588ada
b159b2602abf8bbc13556a200b9697d6af45ab94
F20110217_AABRKE alto_b_Page_097.tif
676b80eae973b57336ad67d798542221
6ffbe5dd7474aceeaab5b6af0846a65ff399db7b
30068 F20110217_AABRJP alto_b_Page_137.QC.jpg
5664261ff88415fd6ded81edf338ef95
2f71926fe61fd20ecfc9ce9955179cf83cc6eff2
33422 F20110217_AABQFZ alto_b_Page_112.pro
39a948062130aed3a29b46a4733fc88d
2aec1cc4971048eec7549366c42758181567d767
52206 F20110217_AABQHC alto_b_Page_135.pro
ff7ee3cc97eb12c4ef1056e6e954fd78
3a4070a9d9791c03ee42c7e1785e91128df1d200
7894 F20110217_AABQGN alto_b_Page_074.QC.jpg
40d8776e9b292ad8ebc98d0e93164dfc
6122d322fcd5a688bc305b0a058274a5cc8e1585
6687 F20110217_AABRKF alto_b_Page_163thm.jpg
9e0722c940018bd641c08a1913bdbcbc
cfb74d457ddaed1fc5ccc0d7305ca277dfaff8cb
1908 F20110217_AABRJQ alto_b_Page_041.txt
88a4f5e5b86b25a4a137d895a39f245d
9a7913f6d9447a9bb128892c811e645d8d3ea8d2
1914 F20110217_AABQHD alto_b_Page_075thm.jpg
40d89a6165edca7eda3e4b029c59a9c8
a1b166ff7451696e22aeaf0418e49b302d0ea110
42620 F20110217_AABQGO alto_b_Page_076.pro
a7c79b99b55a5d2e23401f7c1d8f411c
6fc2c87a738961f480c75c44403780c4f8cf23c0
50491 F20110217_AABRKG alto_b_Page_101.pro
127bb6e1b954f197720cd26158da09a3
dbbde08cfd02a1517db97049aa72f48c0f9d44d7
26789 F20110217_AABRJR alto_b_Page_030.QC.jpg
52056c9a5638e95d61f1947dcd05747c
84a8c4a1bf237b2925091ee6ef5b83f16ebb3e3c
82998 F20110217_AABQHE alto_b_Page_085.jpg
a3e6e125a0e68f671e157142cdd812a0
5faed16cce9776e46ee006451abb9636c92a6a47
F20110217_AABQGP alto_b_Page_098.tif
33da4966fee75fe03628268adea96448
dc62939d05b194922c312d432e5e15c9931fa404
F20110217_AABRJS alto_b_Page_128.tif
44a704bf1d3c3200ab31bc8d38f4c9bf
d13d727630082cc4be72732b17118644d6820af9
1992 F20110217_AABQGQ alto_b_Page_103.txt
fc8cd2b50d87d5e840de43c6b8ca8810
dbea985943e44df3ad6ad2f48b37329a0a56c013
6019 F20110217_AABRKH alto_b_Page_108thm.jpg
d5e7e767d2ef3827019eef7ee946ba80
4582c526856f080f408005879c8973d8c26bc3a6
6328 F20110217_AABRJT alto_b_Page_033thm.jpg
862eea656e4091033a978156a03b0449
610198350bf63853b68a5c5823315c653fc1acaf
24651 F20110217_AABQHF alto_b_Page_043.QC.jpg
aec2bc47c946effcd227c4d701fafbb4
165ba56d9c324c93c6f29f0e763b8f9956ce37a8
28145 F20110217_AABQGR alto_b_Page_151.QC.jpg
22515b08188ed2c28fa6dbc7184ec61a
6adb5d2f17c1de810508f0dd4be02cf966f29162
F20110217_AABRKI alto_b_Page_114.tif
7245909010ff85ba1a27c14454c75fdc
c675b095890a8ad7135285f50a8c2f7232d2dc73
F20110217_AABRJU alto_b_Page_098.txt
b29a0c398ec07638b01b58c4936dd24e
bb927be9a2754d469b632c669a2b890667102918
F20110217_AABQHG alto_b_Page_135.jp2
ced1cbe0ab2dcf2cfe14914ebf4d8ee8
f0ccd082f60c582a01247fb44e7775c4736f9e8b
F20110217_AABQGS alto_b_Page_144.jp2
52e9c5126a0a434695a8267569d95f2a
94dfbc8ab0cb888e748d6ee1712da63e75f7dd56
1976 F20110217_AABRKJ alto_b_Page_091.txt
d97a4a7e7269c49c7dbf4fdb4d019056
b2cd44c902928568e606a1d85e7bb8f46e82aa35
5183 F20110217_AABRJV alto_b_Page_057thm.jpg
7e20dca3ee4d5347996731f9b203afbd
cc535bc94a0d2cbfaa69f1ee1ca37428f124414d
F20110217_AABQHH alto_b_Page_108.tif
7d52b84cffd3b666fe0f286eab50b3f0
de66de77b9e2261383733d3e5fb829ccdd4264d0
F20110217_AABQGT alto_b_Page_062.tif
386b72ac374bfc135d4fd56acfeb8d71
1cc244c19adf83971bfaa8f533ffd63c2482f709
6199 F20110217_AABRKK alto_b_Page_019thm.jpg
96258b0fe368f6980b7cfd31b1e41272
3bec7c5d5bed86cfbed508c85e410bd3b2d333c1
F20110217_AABRJW alto_b_Page_035.jp2
fba178679f452d3ac46db30bf9b8396b
f46d8d3d9a73313932e3d10081987f0ca597c932
F20110217_AABQHI alto_b_Page_105.jp2
383a3fd94529cb25022a8f9fd0c0ded1
269f4d0ccac17acc3456e7003e966723cbf95b33
82776 F20110217_AABQGU alto_b_Page_006.jpg
2fde32dbdc317aba0cb3fcb9b11faae8
4d76a558330ce18d1baf13b2eeb3f6664f84a494
F20110217_AABRLA alto_b_Page_023.tif
47f6b51ff3f47265ab54ca3ce4fd7a71
f05cdc8f7293463adca8a2c21100076e1ec39312
22072 F20110217_AABRKL alto_b_Page_001.jpg
bd4f2fe6c0aa4c9d78a40a4073aa50ea
15f55b6f3fd6bb307556c5ba5e34a8d1e4c9354a
27020 F20110217_AABRJX alto_b_Page_092.QC.jpg
86e806fadf84f7cd648d61581807fbb1
d8dd90920e24082b42bac6142ee057d048f17ba9
F20110217_AABQHJ alto_b_Page_100.tif
b245bb03a497fe59e9439e4037eb792d
1da34c56dac443422ff13d2105a4607cec926025
752625 F20110217_AABQGV alto_b_Page_048.jp2
9403030cc62e85031f47b8a96842b66e
a2bb5b1fd288d0212779a3f491676b52b665c888
F20110217_AABRLB alto_b_Page_030.tif
52738caadc607138a35a045948b72360
482269f55b7203db7479ee824c80f3c56aee0f93
81685 F20110217_AABRKM alto_b_Page_124.jpg
bb782e5376de61d7d0d42a7e1a6a81d7
a882f8be1fa748c8d40a2c19e301fb9ba5e61bb3
48878 F20110217_AABRJY alto_b_Page_046.pro
c0c95653ad83f7a4f0a7e1b389fd407e
787bf53eba98d62ea883fbde96b8ed09bd80d3ba
16564 F20110217_AABQHK alto_b_Page_121.pro
c5fb55d91a46d4975a4afeeee7f8ba86
0eac33d37b34d43d0959b18116986782b51bd615
1868 F20110217_AABQGW alto_b_Page_051.txt
bd1b4ccfc20eb4c9068cc732a528fb4e
12942dfb9e1dfd629341bc41eb8d18737d608319
F20110217_AABRLC alto_b_Page_040.tif
10561f0e54bdd354f1fe426645fd53a6
0c5d2464f476a883734646a394a10211712af1d0
52103 F20110217_AABRKN alto_b_Page_017.pro
a255c56afdec406f7395cca6bc527351
7296c24b43e580c0ad4a4e9f04249083c25f84f7
45503 F20110217_AABRJZ alto_b_Page_134.pro
6cea7b7f5bdad13632fed2165e5f33ce
400f6bf5b9f96ee19eda437c3783cdf9146dd539
1051957 F20110217_AABQIA alto_b_Page_161.jp2
15738df9c3edfe4e4ea50f7281b0bdb3
a87b656a5f3fb3de9836c78595b93be27631b1d8
113689 F20110217_AABQHL alto_b_Page_137.jpg
ed158de1094ce3db2e850f9043a3326e
c210fccdd8a17a5ea2f8c292391cb72c544ebc8f
F20110217_AABQGX alto_b_Page_138.tif
9371c12caa92bd03fb31a637dc19f8e9
58481c29b63b7d2bb0b164774386bc51a0f02084
F20110217_AABRLD alto_b_Page_047.tif
b41ebac498a227120002e1e9493cfc2f
98d05a3a7cc4c3bce7babfcbfa7d99971a7e5b17
F20110217_AABRKO alto_b_Page_021.tif
6437639542ef9261051ffa63748267e0
d976bb7110ee5c0127b18f8c380bac694d08b16c
26857 F20110217_AABQIB alto_b_Page_133.QC.jpg
a8861612ec69ce7e473bcd69f63c4e7b
3113a650557620e495cf0a13f149ed1ec2c7028e
25832 F20110217_AABQHM alto_b_Page_046.QC.jpg
c8487dd61a581922d47349c9d55036ef
db915e121801033110112a7ec03dfd17e169e7bf
27727 F20110217_AABQGY alto_b_Page_159.QC.jpg
ee373c9b6b34a7ef36eeaf5568c6aaff
3c727f46cc7dc91c3c8a38ec265923198f056c17
F20110217_AABRLE alto_b_Page_049.tif
b033a4a6ae91889e3d5b6237a4acbe69
4beb07193d48dd9bd835d36dfa7c03858e2c0b93
2589 F20110217_AABRKP alto_b_Page_157.txt
b9c084025471d03507e1f7b33ba45f28
f64d2ef93da128dbea2c19f1bda7ef42122f8ed9
910531 F20110217_AABQIC alto_b_Page_082.jp2
4d8b9619eb282e847ae2aa0e6c9500f9
1966307b1dff0b2e466332ac1192bf0da267d53b
47535 F20110217_AABQHN alto_b_Page_031.pro
9d63657d512b50b4b02befd019c52bc4
6ea6391793c212c32d40e2bb68e5dc9287063b4a
1764 F20110217_AABQGZ alto_b_Page_114.txt
8311953bfa11a9af3fa33898ebb1f0bb
8c9a8f468e3065b48237f722e637eb8e228adb17
F20110217_AABRLF alto_b_Page_050.tif
7d7942a5fc0cc6536ce4bfca7a23a92a
7c0345547f7ae1489a6de9c01d4d3b9055717de8
101464 F20110217_AABRKQ alto_b_Page_141.jpg
ba843949afdb14dc577872b013294774
10c61843868070cf3fecd20587c90e83452b0a43
1830 F20110217_AABQID alto_b_Page_050.txt
60f25e0933a3172e9cf3b7612a0ada26
3cdfdc5222af6b546d847d024885df3af6205f5c
26681 F20110217_AABQHO alto_b_Page_042.QC.jpg
99a8f5d76f47d81558249371c602e4d1
737992a40839cd818edf4417f25d506bd61133fd
F20110217_AABRLG alto_b_Page_054.tif
6aa7c7101b6ef25cfbd3aa9a5c07fd81
b1fc3d851e1ce1d3d424c617946304d237f7b168
6433 F20110217_AABRKR alto_b_Page_059thm.jpg
5b00044b20b75888f0cb53961b4d4be9
8c4771c7e7609824f9b769243c515ef840c75c80
F20110217_AABQIE alto_b_Page_117.tif
dd61e4dc56a8b8e4a149c6e25707edf8
618582e851b671148931b82bcd7502f2ef6cc00d
102906 F20110217_AABQHP alto_b_Page_158.jpg
c2c76a1209cd2c18293beb7582a42747
ba012daebfe29f80c275e856986382ded3f4265a
F20110217_AABRLH alto_b_Page_063.tif
a965fb2c5f621bdefd87a41c495c42b5
2864be8a7ea0283b6fbf2138ab74557b6aa8c95c
4997 F20110217_AABRKS alto_b_Page_114thm.jpg
74bbb6d1d4bb762d2091e441d6c75cda
631216b6adf665c1e60d2f857ebdb631dc6bb1b0
F20110217_AABQIF alto_b_Page_129.txt
2ae6a6f64c8c8eb35da6e92235db4b25
af965cdb8b87db907424084c1ce3e3c3156bf87a
77802 F20110217_AABQHQ alto_b_Page_045.jpg
1099fbc27a93dd4bf4172aadfe18a181
e3076225e008239a383821eedc8cd17d5a472beb
F20110217_AABRKT alto_b_Page_083.tif
dacd656344fa11c2886d22f89bd7d1a6
33847939789f36e926d25e4e9537f0db75e59aa8
1051939 F20110217_AABQHR alto_b_Page_092.jp2
808951aff13af06e968399d693d95b63
6cb0d13e9492217490f92fe3b15b386475feae44
F20110217_AABRLI alto_b_Page_068.tif
fa4b50ddd8854ba7b2d0940c3726b2f9
08fff599fc928301c2705ea18cd061f4fc48fbe4
192259 F20110217_AABRKU UFE0014961_00001.mets FULL
b83e26d0fd5d63cfffed83a17274680f
3bf7dca52c360790c3bb3f2ede37aaa41ad3a9e9
16059 F20110217_AABQIG alto_b_Page_115.QC.jpg
e15bfea9005f6104a21be7db43d41b52
c7e88d52967797a2b17a7d95474fa1380a5d5b79
1950 F20110217_AABQHS alto_b_Page_026.txt
4ead620773c98c00b388340b59b11db5
3d50a07b002a1e7e1b4fc503f70a3280a3a48321
F20110217_AABRLJ alto_b_Page_071.tif
55c53bdc715a85ff0d53928aa7ae2dc2
9f3ac752e99d54124d086606441f38114e53c46a
1980 F20110217_AABQIH alto_b_Page_060.txt
c1d56bd5fc9251cc6e8c9b15a16df307
b2cf49553ffd43db23834543dc0467abba2ecf91
F20110217_AABQHT alto_b_Page_159.jp2
a75498a0865c488cfb2323e40b779363
0854ee8328ac7a2ea79c405fb13389473dade76b
F20110217_AABRLK alto_b_Page_085.tif
931471ee587cbcb7143929bdc386482c
af73ea8e8f7244a4cd82ac79bcd844532c80d754
F20110217_AABQII alto_b_Page_105.tif
a8bdad82e6cb5fd9d1c5ca3c3c1b0922
f9f9c4bc13b3935496d700675236105167541a1a
40702 F20110217_AABQHU alto_b_Page_057.pro
707c2d15b476a58c63020642c5b2e3c3
8ea90d8f809486f9391d0a0e2ea4949f34a38ada
3919 F20110217_AABRMA alto_b_Page_006.txt
b1f2104c5b82d301d10a00df73c54ccc
ab92e12757519bf63c052a14039c3557f05007e2
F20110217_AABRLL alto_b_Page_088.tif
3fc21bc9436e0ce1ed7f2f21e2ef5e34
743f913fec552e2fb8114ec11d2fa786c738abce
F20110217_AABRKX alto_b_Page_002.tif
beea3179b8180352c5c5df9410f24b8b
fc9a7defb584e2331f0eba7e0de05101b956afe3
2493 F20110217_AABQIJ alto_b_Page_116thm.jpg
55e5102e9bf8ba18d3a8d4914f486a1c
eb9dbca22d903170ee0d924a3d133c6ebee4da9a
6395 F20110217_AABQHV alto_b_Page_036thm.jpg
5a0eb16e5d115c0f607f00f0d2383d53
cfd6177b868d213b7f999d025448944b7b1f71d5
1968 F20110217_AABRMB alto_b_Page_019.txt
b0de3340ef99672f36e65465af48e764
6b6b0618016b8becf622f5c7779e2d6e92be1991
F20110217_AABRLM alto_b_Page_106.tif
dca7ebd9be3a4ffe96a639360a9a04b0
39b2c23c44475bc737dafe483a49c9ce3df816fc
F20110217_AABRKY alto_b_Page_005.tif
d25298372c706314a28d8b7ca3d70da4
1d9360611fd6b9ae35fcb5ba5745796c2eb26f44
F20110217_AABQIK alto_b_Page_029.tif
099018b78a9d80a2d66e27d256b5382c
cf638ed610b23c7d18a37d0a4281a6ec9246b0c0
16830 F20110217_AABQHW alto_b_Page_114.QC.jpg
aa58e463268015e7ba7917b42b90b137
a116ae9ca422d7422f35ee82cb237ed0c8bfeb64
1999 F20110217_AABRMC alto_b_Page_029.txt
53d08fd65421b801832a22ddd4a25f54
535e8a9e73cb8c11eb929dfaae79c5e2d58adec3
F20110217_AABRLN alto_b_Page_109.tif
c95aceb7416eeae52f97e9dbd7663473
9430e62431dac0e48372cd7de4a4ebb9863e1c6b
F20110217_AABRKZ alto_b_Page_008.tif
5f41b2aa777e67c5484a12469becfd9b
d0ab7f0fe91b01311d4468ad038e11744f58411c
112018 F20110217_AABQIL alto_b_Page_162.jpg
e69e562493e56d89c91479cf1fdf4b12
42a8e7a3e3fc60a84cf9ea2e36cff7bd6ae6be0c
83638 F20110217_AABQHX alto_b_Page_081.jpg
d837f7d862aa52dd92009092ae284f02
5404732b7529ab18b2dfbab6f5f1c632ac716f84
1051909 F20110217_AABQJA alto_b_Page_068.jp2
4cc5a384b7f05ec772695eb001331861
d69e6e6bdf0d751e51d8c9c5167c953db78b3a46
2030 F20110217_AABRMD alto_b_Page_036.txt
b0d95261a6973ac62d4c74950a20023d
8c3baf2a5b19705b38e90c20621962d3022a1bb0
F20110217_AABRLO alto_b_Page_121.tif
33ef4f16372b78cf072396cf240a5043
2f89dee9d34a869fa297cafe72c95add16ced520
F20110217_AABQIM alto_b_Page_154.tif
1ee513decb0145ec9176b886df116264
ca3f38f2ff7609f6d140e394bd3fc96fc0c67660
1969 F20110217_AABQHY alto_b_Page_079.txt
dc5245677c807ff0208029453e21cd6a
12797f441c6c5a45f60f6569b7e567e75c52155b
7998 F20110217_AABQJB alto_b_Page_116.QC.jpg
a959f5b9884c5027db0e568f666fc4bf
1abd14f918434e2ad763f6d680e417c6d2c02fb3
992 F20110217_AABRME alto_b_Page_049.txt
0ac3fcd73f6b1077af83d0d0d9e5f02f
610d1cd2e81089781ebb22e7be33827d4313f361
F20110217_AABRLP alto_b_Page_127.tif
5db6cc95b4d6a80f6030815faccd8873
d9abea9d4a13910ab5a9b3fcf951c6b68f725197
76610 F20110217_AABQIN alto_b_Page_123.jpg
f40e0bf70db1708b2475afa29bd4c7f6
331e839d16d2b7f9a5744ec6c7cf87cae10ce6e1
1865 F20110217_AABQHZ alto_b_Page_018.txt
333a947f92cf51a5c61b34c34ece56ac
089340bc55f8f555edcbde05dce8a62df44b08e6
101805 F20110217_AABQJC alto_b_Page_139.jpg
296e23ae90d7c1477366050dee21bee4
4ad2fb63af1a3beab21e0d939f9ab2f146dfd628
2001 F20110217_AABRMF alto_b_Page_052.txt
18a5cebbabb6be2057f97cc2c488938e
7d178c7a4d0806c5ebd27cb75aaf607f6fd0badb
F20110217_AABRLQ alto_b_Page_134.tif
23f80bf414d0682d031780d0b8045084
06a250dbbac0a547d14a260c1440d31a514517e5
26747 F20110217_AABQIO alto_b_Page_116.jpg
b02f20c63e44187313f58e9aeccdcdd6
f50204e8389bc806494642f5dd19aa5e56fd8171
6425 F20110217_AABQJD alto_b_Page_105thm.jpg
b31f39fde60fcfd99ceba6d32f029011
54efb330f5d746fb93d30f6add9afc5a55281662
2012 F20110217_AABRMG alto_b_Page_055.txt
c6a529422ed92e96a3bed7c1ec7b722f
5cc450ddbe843db1aec75870f0067a7ca9845bf0
F20110217_AABRLR alto_b_Page_141.tif
6d03974fd281811f566a002f712694e2
646d9c2a05e09ca358a8f747039a01c2da844754
25949 F20110217_AABQIP alto_b_Page_100.QC.jpg
5705302a8a7b9664232bf08f9ba2f492
9dac688f7d4c87a0a053cd5ae459f2bd67b296d6
56823 F20110217_AABQJE alto_b_Page_003.jp2
527c6fef46df131627f399b0447f994c
4a1d170daf028b2ee4e5c484304de575d9860372
2083 F20110217_AABRMH alto_b_Page_058.txt
f7ef0e711eded315a7eb4be7e54ee618
d938105be4351139cf8e62cd3befe5a39f0f8874
F20110217_AABRLS alto_b_Page_146.tif
f480b5eee58ffa8621bc843d87136919
f036f237e15576fe41e924dee221fa41454db392
F20110217_AABQIQ alto_b_Page_006.jp2
bb767758be8b7c40e4400bfabf9f7f23
b0b0bcc7c1a338b030f7cb55689c7cba7a5d99c7
6120 F20110217_AABQJF alto_b_Page_088thm.jpg
84043cf0f51edf4fa1517e70b417c64e
380c1e8c83de512b2e7e713c99f80f4da8e6e48f
F20110217_AABRMI alto_b_Page_065.txt
9bbb87a5980515dbefabf62164b078a1
8f1a3f562d6c2696c0571302c16961872d073220
F20110217_AABRLT alto_b_Page_149.tif
f01ea0adbb452094bc94db711066d33d
4bc9cd4ee3a027edd9db7b70e776b8b9f90d8876
47484 F20110217_AABQIR alto_b_Page_043.pro
91274b635bcbfd3f9fdd0274e3dcfe89
2d66a039229ed09e4b7b8d8103c995e2c3a6cc60
6946 F20110217_AABQJG alto_b_Page_137thm.jpg
83fd541a8b319895e878bde5ad8f0b64
7a8c295be49067a9425c7d806ff026ad87619dee
F20110217_AABRLU alto_b_Page_157.tif
0d740b386068e1eecfea3555e3623cb6
a1bdaac80cbf3a65868e3609e1823c0881372094
24087 F20110217_AABQIS alto_b_Page_109.pro
635a8395c5f237cf0991b62c201dace3
f4924673c443809cfd4c2a52d6e8a3f08a8e3418
1507 F20110217_AABRMJ alto_b_Page_069.txt
821e5e99bf471f31e98813b3c41d2a16
7fdfd78aa8e28cd4b7552fcf9a6f10e6b2767625
F20110217_AABRLV alto_b_Page_159.tif
a1f7613d064e52887ecf7132d46281d6
4393a9a08e667b7663bad4cc807148364c0fe23c
6411 F20110217_AABQIT alto_b_Page_027thm.jpg
d59d889a6f00b51a988368a4b7ed8ab0
b5d16a8ecd2ad2df564d968e39958ef89f0dd1fa
F20110217_AABQJH alto_b_Page_101thm.jpg
6b653f6559fb21437d1d0a98cc18a494
9bd089603eb8b956702f10610210c348a8944c83
1240 F20110217_AABRMK alto_b_Page_070.txt
aa7a2d8e96c4572101bfc61752e7e2c0
3893331e27e76612b8f0de543fd4725a2c8aa8ad
F20110217_AABRLW alto_b_Page_163.tif
c7ce9e5aabf7a1d7969d40dfa60f4a87
6459140bdce9a66723e1179ef79a72d89e303a8d
F20110217_AABQIU alto_b_Page_015.txt
0f94bdfe9688490d14f107bc338e3386
45edcf0d0031ad25e34ae7398951a6ac3929bac3
6464 F20110217_AABQJI alto_b_Page_158thm.jpg
ee21185c4e583ba779f4a5e2c2957685
cce0c4d49a712f6ec0a9599c44af52536b21d48a
27921 F20110217_AABRNA alto_b_Page_038.pro
5f71cd98cf7b87f23141e621d9cac0bd
700224f396fca14139b5ac8554fa45eecfb0738c
997 F20110217_AABRML alto_b_Page_071.txt
f63d2b9c1a97338bfc2155908f135d41
695b1656e28c35cfc3fd562d1ede922805e8eaad
F20110217_AABRLX alto_b_Page_165.tif
4658097888202f3d6cd6b965f7e792a5
4a99519ed07b57b427e5dbc4906365b967dde72f
6417 F20110217_AABQIV alto_b_Page_065thm.jpg
1f691db509bf2d67d4a65ccc2e692099
2cf5b9544d32cbc234fb9b0b922021a923e76693
587391 F20110217_AABQJJ alto_b_Page_070.jp2
cc2e5e1fa2fd75665a72a3a93dc70794
226d32daba088ad1832347b1b1396b60986699d3
19660 F20110217_AABRNB alto_b_Page_049.pro
96329b90f848e56ed7986bcecb63527c
7b21e53cd2a30d1e1f21fd378cadca83a0b9eea4
1973 F20110217_AABRMM alto_b_Page_086.txt
e5142d51d495aa56476ea1e78c1f8062
bc9a3e4f3c9b3e35ee43eb324b1b7bd864e0a4ee
464 F20110217_AABRLY alto_b_Page_001.txt
266bcba170ca713d095ba19fba129637
ed651d256f844823fb9a43419bdadf2bb541e7d2
49208 F20110217_AABQIW alto_b_Page_108.pro
88a61f69f33e779ad98e41096fb743a1
585c9735e40759bb19ae8c6b9547acbc83658600
27379 F20110217_AABQJK alto_b_Page_144.QC.jpg
3e059c34707f63f5c3613d944260b568
1c59f9a2a5ce4811b0a77b498a936accfae052fd
50841 F20110217_AABRNC alto_b_Page_052.pro
98795ca8e83ef5dcc2ff9b06f751ae98
b37cc48e959584186b8e8cc75b77edb5078e04ae
1881 F20110217_AABRMN alto_b_Page_088.txt
6f695395f3548ce26134844c23e1f2a3
3b06b0a84efff794a46da48d1f22c2ae830af28d
1531 F20110217_AABRLZ alto_b_Page_004.txt
c6c36e5cd60a448020ca7cc1194a0ec4
a64aa545410f0f8ee58732909c590dbb2f6c30bf
6614 F20110217_AABQIX alto_b_Page_140thm.jpg
350c474d09a7e768da7cf06b149e68bf
559926a607aec9897a88df77547b2afea52e4cb5
F20110217_AABQKA alto_b_Page_147.tif
3444c55198461edc54c484dd713915c2
80153842f8284ec6e200bf9e5d0a84b6a781bcf8
27982 F20110217_AABQJL alto_b_Page_122.pro
674f3d3a762fe31478883e7445a6eb4d
36f04cef3569d15bdac3caa3f4cabcb9ee395a58
51173 F20110217_AABRND alto_b_Page_054.pro
8f1597d94a1e9bec4e28c71389233bf6
a6e8fbfdd9250aa992f41c89aa68520162f172f2
1211 F20110217_AABRMO alto_b_Page_109.txt
25c88724f2677436c2f6f325b9466eb5
c8ed14e16b744f6cffff3f8e9c246b8154890c92
23652 F20110217_AABQIY alto_b_Page_111.pro
ccd1492581ee7980c2211361a4541077
9620f658e13da23f61dddaeafc9ae0a8d8e0239f
9420 F20110217_AABQKB alto_b_Page_119.jpg
88b7ec0947360d9c96dc4526f14aa166
63aab426d866a5ec4c8c9ebbe9e404f109e304ac
50469 F20110217_AABQJM alto_b_Page_133.pro
c65f808597a4f88d21543a279370515d
fbf15319a7a2cb7fa108458d455bf87abca61f6a
33746 F20110217_AABRNE alto_b_Page_056.pro
72e244237711ef750f07443cbd90e395
146f04bc2e5efe6f18fec1ffba800d1cb259552c
2787 F20110217_AABRMP alto_b_Page_137.txt
7926e3ef96b6d2814ed4f0dafcd24b8c
2cc50db90f249ed5372d7e646b8eb324f148a2c6
6149 F20110217_AABQIZ alto_b_Page_081thm.jpg
f648eca15689b7e1b2762fbfea6c26dc
d7dc361484aa0c34611a1f692a142ecbde8a0ef3
51126 F20110217_AABQKC alto_b_Page_129.pro
dd6f5aab99fbdb46713226c336569ab8
c61cb5c37df185ca79f08b41bbfebfd7803307dd
59596 F20110217_AABQJN alto_b_Page_056.jpg
8fcd7b922beb701260afd0580f009f3d
e9019fbde5f6513955d0fb67cab1eea026a5563e
50172 F20110217_AABRNF alto_b_Page_060.pro
8352261c43df5813afdd10e388fe207e
7c55c52d1a24f94b91a710d5bfc278553576e269
2610 F20110217_AABRMQ alto_b_Page_155.txt
e81881bd3bb655bc043cad2fe88f8aae
0d07e7159a2c616431b433e3edb874484c086b39
2029 F20110217_AABQKD alto_b_Page_023.txt
e765bf5ffdcf2e3a114bc19176d5bb66
c47a94f455809378856eb026617d2038028ee469
F20110217_AABQJO alto_b_Page_014.tif
43f7e544ab35d33614f67d4610096739
19008ea69ef76dc5283985aa714641e24328da38
48396 F20110217_AABRNG alto_b_Page_064.pro
821658ca87d8592ceb5f404d077f6dea
c8a1be4780f7ee46db4d96bdcba5f9dcf9a6ad84
2777 F20110217_AABRMR alto_b_Page_156.txt
81bc6957fa0532a8e1ea57cc582a9fa9
957b5a5c40213bccd6daf01db13f8e9946c24b6e
24459 F20110217_AABQKE alto_b_Page_080.QC.jpg
87883f0b94084d39287465b501663208
9eb99b7b8bade2d4bdbb3d3bceea180a5196c5ac
F20110217_AABQJP alto_b_Page_120.tif
a4ef700afa2d97f6e32508307e0f342d
7a5c05943cacc4c512d626082b08d383cc5758c7
51599 F20110217_AABRNH alto_b_Page_067.pro
5292ed836ae7857987ec4dca06f7fca3
bb0222eacd88d5e04c1961ec3558e881e6c04a4d
2546 F20110217_AABRMS alto_b_Page_003.pro
1b2b2a65b0cb1125e4c37f45c6adb348
1ab9b6838ed686da358622ec59c189b8377e30b7
28842 F20110217_AABQKF alto_b_Page_154.QC.jpg
de9fa08eaf3477b60509cf7b56501d56
a4395c3a9d5d303de4f996d818cf962f38b5ff5f
86361 F20110217_AABQJQ alto_b_Page_036.jpg
33e46d532ec0c6cd7fdf091e60c48229
1bdafc57001e91e2ee3b33b6c9f4cfbf07c4cf78
22038 F20110217_AABRNI alto_b_Page_072.pro
c4c2a929feecb782aeaf488104fa5fa9
e3470d677fd38d9c1869c03ff7fdcc376422c4bf
63491 F20110217_AABRMT alto_b_Page_009.pro
9dd0e4fdb3b0f3acc4be342d0651b8e2
5b541c1720a9e04c495dc5bc4a5edbecbad91b99
24960 F20110217_AABQKG alto_b_Page_063.QC.jpg
ffb5523c4a4e1aa62293186161b6d879
35fbfd445f60b4312e3bdaa4320e5e6c3fe041a2
F20110217_AABQJR alto_b_Page_025.txt
a58694e29a768e32e5b2636c2001fb9d
76dbb8978ae71166ac9a7a38864b4c5b6c8a9180
49910 F20110217_AABRNJ alto_b_Page_078.pro
a7d0e00ae37a1df3191c9ed2dbbec6de
a3386b571e5e1d79e07684182022d9b0cf2715a2
47470 F20110217_AABRMU alto_b_Page_016.pro
c69506a5293bec48ea9ac3a7ec586f8a
3bee87f72b27f93a5fc85fc9eea07c56502f6c55
26123 F20110217_AABQKH alto_b_Page_081.QC.jpg
61693b6dcd183e9df4d587f3ea4a850b
d22d9627f385ddd3c630ce44341cf15947511c54
2581 F20110217_AABQJS alto_b_Page_110thm.jpg
171d2e7cd00b2c11911a2efd30dd3ab4
656670c010bc69751df1ea2da704d92df0b74721
52800 F20110217_AABRMV alto_b_Page_022.pro
3481feb232c8c931bfef3199fb0e386d
3738b909209b5f9db4ca43be22eaae47b94228e0
26903 F20110217_AABQJT alto_b_Page_130.QC.jpg
bf87e01b91cdf4a4ef22a582e3bebaa2
9b018c0d6d40a3793cc80131f9737c05c6ff223c
47002 F20110217_AABRNK alto_b_Page_080.pro
8ff10f6201ae41286fb9a84c5821c740
6ffd7046098b6bf7192f7d85966bd80beb58664b
51369 F20110217_AABRMW alto_b_Page_025.pro
8a0638c7b10a30b6bea1ab65d634bcd6
e53e16e557b169b09e5d1f48d41c32da8741af7a
F20110217_AABQKI alto_b_Page_067.tif
585886c691f6aacb4ab5a38bb25d6aa2
30604f744b76dcc4bd1364bbc1f5f2e0187e01ad
20735 F20110217_AABQJU alto_b_Page_121.jpg
1d7650154361bdd2ea9b10e9c6158630
51c626b2a899bae116b664dfb43a95f3bfaccf42
4124 F20110217_AABROA alto_b_Page_002.jpg
a19087c15a528d7feb4525e1c3254875
3573ed26a8fc10c3a302863bab4167759d3e26a9
40528 F20110217_AABRNL alto_b_Page_082.pro
c7ffc6c98a92427df66f14658c4339d5
f3d3b75f118b2e8e9714dc75bd688ebc55f8ca02
50985 F20110217_AABRMX alto_b_Page_029.pro
862b034b71d78569f9828ddce8714f10
a808d40e93bdfb7df94959dec666e2bc5f903f61
61669 F20110217_AABQKJ alto_b_Page_140.pro
47597d09e5cd900a9863d346a7d4c2b2
1204adfe7a3807b054bf449acd0fa5cb254bdf4e
5522 F20110217_AABQJV alto_b_Page_113thm.jpg
a3dc9430c39096048440daf9b313db3e
4e3e59abdd682884d49ca3a2d831de10446a21fd
65551 F20110217_AABROB alto_b_Page_004.jpg
28a2c80c21ca63ac69c02746ab3faeb9
2d5493ae7c9b82069d0af1a81f34ca97403249c1
41031 F20110217_AABRNM alto_b_Page_083.pro
97c0dc2acdee4f66d670ae5ce444e171
a485617eab986007748eadd380d52676d50721f7
50871 F20110217_AABRMY alto_b_Page_035.pro
f89f5c21818c2edb9b2b621de57c7d0d
3b45d8d507a2d102818fd8ce034bede955492110
65883 F20110217_AABQKK alto_b_Page_163.pro
34a8a637cec90097f1e795e5b74214da
543bf99744a5507b9f06e50320480affcb44b6bb
6124 F20110217_AABQJW alto_b_Page_100thm.jpg
6d4809f8e77cf6a51a110ab240758345
54a51d6e430bc6bfc9fc01a26850ffadc9908fab
57556 F20110217_AABROC alto_b_Page_010.jpg
6936c82d9127963714f4cbbe2c983682
54e7ab7e5505a6273c6f2056e12bbd772bf74a9c
46694 F20110217_AABRNN alto_b_Page_084.pro
64e81641fe88e87f1557be5c6af1ffd2
7cb00d3ae46288aecca33d574caf645ad11fca4f
51693 F20110217_AABRMZ alto_b_Page_036.pro
f9a979ea0a634da8ad3b84542ee68378
58c48ec91e6eb72e8eb98392ea8a91a644e8cbef
1986 F20110217_AABQLA alto_b_Page_020.txt
129d2aa82ea9699ec01378922c335dfd
76e6a34f0160453140e2654bfcd0b0a2df5fc217
F20110217_AABQKL alto_b_Page_162.tif
b8c1dd24dbcba6640f5fd75266773eff
d8f731524b9393a4ad997babf98137088140b44b
45029 F20110217_AABQJX alto_b_Page_010.pro
dd8f1f1b5c77c090519fa873f74c7c61
7a0ec0dfca79d51336a5389d3c4a78dc10eb15a9
72805 F20110217_AABROD alto_b_Page_013.jpg
c56c004f0c7198346700a128e5c63cdb
1b7cc4d62e89dc21c61e0f78fae26ec3e22e0b0c
47678 F20110217_AABRNO alto_b_Page_088.pro
78bffb196eaf43a7ce9d4a75b60e3812
7bd23ea7216eccddad51bfc82e1be571de601d9f
F20110217_AABQLB alto_b_Page_063.jp2
2fd060fe27d8cefc9662a751366c66c5
6f1eea5c5406f1ca0466cfd4869223a4cf5f8e5b
F20110217_AABQKM alto_b_Page_024.txt
0fbd09e739f9f4a51cbe449451a72458
20a7ff49c3190cb5f49b8f8af66278da50cb4eee
84500 F20110217_AABQJY alto_b_Page_020.jpg
9d8026daae6e39a14d04a92ae332b598
89de9f433fa1dae73241cba052349a92dd1cc402
82545 F20110217_AABROE alto_b_Page_015.jpg
57322b6662522b49b3cf39f5603e344d
996c7bcbe147d4cf041de0052cc8feceb495b939
42789 F20110217_AABRNP alto_b_Page_090.pro
06e88e8ccbd3de3abb3fd54e4c6cd701
ed2a3a89c2f68b38762a2278e831dbf1387c4f73
84200 F20110217_AABQLC alto_b_Page_066.jpg
90bf9c8d90ace56e2cda5cf85ef119c0
4b550db6cb8af478aa6b0e26da9b72032894b7bb
84994 F20110217_AABQKN alto_b_Page_133.jpg
73b82c0aaa5ea19bea88e26b991c3799
d0beb35e15b12c965d670c5224951872a2166b22
877 F20110217_AABQJZ alto_b_Page_121.txt
98a78d254c92c51bbacd661943f2f81f
9cad2626556c6a627e69de93d1c52663bb6b6861
78439 F20110217_AABROF alto_b_Page_018.jpg
516cf6d580e5790c36a196131c3915d3
f1e0ad8209f48615f8bf053e9c741c3eaae1b790
F20110217_AABRNQ alto_b_Page_093.pro
16fe59e26bd5d3190fbfc9adca2059d1
7fbf454cf05b9bd23b5161782a12918a9678f633
638036 F20110217_AABQLD alto_b_Page_038.jp2
9a5bef1652a45800ed460678e023b100
01ff1a97804c5fdc7b0d302092f7fbc925f4a781
24593 F20110217_AABQKO alto_b_Page_084.QC.jpg
4d24eae8b3bd509dc76fe1c49cf5b4a6
0899022acdb68ffdfd370bbb448fe65b8943e635
85669 F20110217_AABROG alto_b_Page_022.jpg
79f12e454971555f4516655cf5bb1587
fdee1b1d94b3ca67eea45142e3ece713ea682c98
45813 F20110217_AABRNR alto_b_Page_094.pro
4e9687b495c669ab51ba872960f53b1c
628d4096bf180830a5ff401dd923667cab5b8ee1
2514 F20110217_AABQLE alto_b_Page_151.txt
0dc6b0c2a533892e7868e7446d3001ff
a586f08845f9b5d69cc5deda7781a4497def5748
2763 F20110217_AABQKP alto_b_Page_122thm.jpg
367ad9f3cbbfc9e523a559669b886e41
e96c08e03497a299138c3a4eb6a4552daacc3dcd
86665 F20110217_AABROH alto_b_Page_025.jpg
f89e6fdb27a636f15004c0b77c05e367
2d9f4fa7a1d9ab0af29df92f58682686fb830bb0
48561 F20110217_AABRNS alto_b_Page_099.pro
5046d78fb6a07bd25475653ced017612
c438c40f4bcf20e9aea318881420bcbc9354c125
F20110217_AABQLF alto_b_Page_057.tif
019a2a40e3289815f1fbaa82c074b48a
0bb00d1be1f984d001418115f902da0695445e95
F20110217_AABQKQ alto_b_Page_073.tif
47076dc382f639c654e46fd21ffe1b44
3b15ab5895e12fbfb58f846c140835ba0b82765d
86669 F20110217_AABROI alto_b_Page_029.jpg
6de08954fbff7fefe40fc89c8ce5716f
873a329907f42b6ef44066b39b65e939a6f54edf
52725 F20110217_AABRNT alto_b_Page_125.pro
f2eeb5ed957a24e23257848f78de7afa
7a26094d98953ed3e895b803be1c2ef5f67ca074
F20110217_AABQLG alto_b_Page_058.tif
7411bab9f9ab9cc3a643218a54ff99be
8fb876a9b428d1a62cebe53264dc61094d49ba53
30265 F20110217_AABQKR alto_b_Page_110.jpg
b8ede285d754aaa3b69ba98969aa0a9e
76d54eb5a5303c6df498bb74d285c24062ec250a
85986 F20110217_AABROJ alto_b_Page_030.jpg
2a2a26ff4a0a80041c6901aeafe43863
80f91c44c362ebc8a77e3bf3faef6de43f1e0df5
52170 F20110217_AABRNU alto_b_Page_126.pro
bd740743a05150c5d7748e1883e01d57
b086b4d82b0de6797abb81a03a7afc948d126492
6109 F20110217_AABQLH alto_b_Page_107thm.jpg
ab36d56f6ec0094a0c5c3753610b757d
1cc3b2215f05ce430f03d2df5a55e6f98f89576d
6549 F20110217_AABQKS alto_b_Page_136thm.jpg
343dd36bb84932bf43b80b3482bca351
e8200a9ae2852fcd66c366bd92962ef47ac95486
81930 F20110217_AABROK alto_b_Page_032.jpg
48e38fd5cd6955d830bc7be8d993c11a
95354d0807d58b4f036967a09d10bca9feff9622
53150 F20110217_AABRNV alto_b_Page_130.pro
f7b52fc71e241510487d6bf5db259a2f
527ea68bcbd4332e6e97e323cf8af135f582f957
F20110217_AABQLI alto_b_Page_039thm.jpg
f506e15fb161e17732f3d777dfa3824b
5c5531435774ccb065249a4b8f52cf3d4f4f04de
64294 F20110217_AABQKT alto_b_Page_161.pro
31bcc8b35b237b736ad56913d531fe01
751a1411add90199ae762d2725f5edd52c65e12b
63113 F20110217_AABRNW alto_b_Page_136.pro
f051fd3edbdd5f126b113682aebe67a0
2cfe9526b30b3f5f342c568955c2a8a54b653c1c
1913 F20110217_AABQKU alto_b_Page_099.txt
a005e4a86f488b8f1387fc0dbfb0bd8d
1195bf7a297b403ea918ae94729eed58a3e3d5e1
109739 F20110217_AABRPA alto_b_Page_157.jpg
acab194470a465dc9791699cb8171092
afc57b4edc916a0087d9c0dd94de6efa493fdc7b
71060 F20110217_AABROL alto_b_Page_039.jpg
99998d959165914f76b052d9b47f59c8
94479026c8a4f5b887be85b1a2820a4c20921f69
68513 F20110217_AABRNX alto_b_Page_138.pro
4a6596cd9ee39054aa7f8caf5b34f8d4
1f59bb54f7fb352a913e39fd90e6680b03d1fce1
108784 F20110217_AABQLJ alto_b_Page_143.jpg
5c143ea534cd1ba4a1e2abcbf7890b9b
8933d977a17c406bd15ff4d0933a81a9d5e2a00a
6750 F20110217_AABQKV alto_b_Page_159thm.jpg
7eaa5433f432bb3946d8fa16d714a4e5
ffbe26c1ead696023ca681551d0d810a937e9db4
101648 F20110217_AABRPB alto_b_Page_160.jpg
62e6d29a942eaa4d6136083aebcf2fae
bf11adc9678411c8d393791433ebabad8b83b747
37334 F20110217_AABROM alto_b_Page_050.jpg
c8003954355a4683a802a922e13c1d99
c4476c85e67c27b12cd4d3f509ee4f3f61897538
56847 F20110217_AABRNY alto_b_Page_150.pro
2c3e3c13c4fe3e412538601196e2882c
8faa0249578098df85ab5dae2a6eaed63c7afe47
39074 F20110217_AABQKW alto_b_Page_011.pro
3ee225c75101b7117c0b0aa2fb7da85a
32d9d34aff77e1b7db57bd09c6a04ffc5e91da21
6275 F20110217_AABQLK alto_b_Page_066thm.jpg
e17fa78193dea5a854c19b61e039301b
fcc36d1b7fe0b1df6cebcc96c4435f4c37a1a956
106334 F20110217_AABRPC alto_b_Page_161.jpg
3fbea63e133fcbbb7754ed607bb549ae
47c6f27000bb6429b73b0c13afa431f393f62063
31213 F20110217_AABRON alto_b_Page_071.jpg
d545acfc88eff75a9e3aa70d4abb0ab0
9616af0b0a3c25ed1eaeff5b4bb53aec5a79da5d
61774 F20110217_AABRNZ alto_b_Page_151.pro
04a15928819d0616702cdd9eba3092a8
02fca321052434f49d783fdc70af2493f9b08e6e
F20110217_AABQKX alto_b_Page_132.tif
0ac1967d0c57552c4a3151f73cc4be88
b08bb9eebf132ce60d23c1492c6a63abf56079a1
46967 F20110217_AABQMA alto_b_Page_045.pro
74e905458c91c1d860c4723132a6b923
5b2c9c889df63c8cc5f0115aad2755fab9661013
F20110217_AABQLL alto_b_Page_003.tif
053ca86e757fa6defb8c2d0d870dfe10
7303c40cca9cb52d43004f4ae9f20a4fda6f2259
1051910 F20110217_AABRPD alto_b_Page_015.jp2
941f42f4a9915eff5ace78eabdd35f85
105741288a64a46271da69d061921a2ed78d1b19
73992 F20110217_AABROO alto_b_Page_077.jpg
e86a46f25ab984c31b68b7a80a844109
6a5d9330137f2315b544e69159fbb8b29f9c828a
25943 F20110217_AABQKY alto_b_Page_015.QC.jpg
89ad97766e83390b2cc3d219df861fa6
50899ca93e1a491cdf213f0c4e2a80df846673c0
26235 F20110217_AABQMB alto_b_Page_074.pro
81d80e62c0b640a0ef9bf91f4cf2323e
3a1fc2adde0a77e88552d71306e9c45d7923afad
F20110217_AABQLM alto_b_Page_091.tif
c320f1978aa91272f564521fe920fd5f
7b975fe32dcd5cc0fcc142180d00f4d1eba366df
1051980 F20110217_AABRPE alto_b_Page_020.jp2
0792d732e93d40fb9528f8d413c5ea52
2aadb88f978996e1be6d5e6c5ff6e18f6de82ebd
84251 F20110217_AABROP alto_b_Page_078.jpg
db32299ec1b4806175870d414e14df2a
cb5bfd0f480278137d7ac6c6a1d0cf59189efa4d
F20110217_AABQKZ alto_b_Page_037.jp2
99a352520f2a79a12751c1538ee0484a
ae8f6549b24066b6da07f7aa0dc5b36156e80b23
25440 F20110217_AABQMC alto_b_Page_128.QC.jpg
be21839eb64a980c9f4a20e93b9190e1
a4828b4e2d0c9bab2f85a92ebaac1a4e17420790
F20110217_AABQLN alto_b_Page_024.tif
7cd466ef0d06590fa48b8187956ee767
fd5492eba28caf9d3d1332f1b0f9e22210c4148d
F20110217_AABRPF alto_b_Page_025.jp2
f9c0eeec7a839379e7f21898f2024c99
ae7601d70e4933b80d1618cf71acb04c38d7860a
F20110217_AABROQ alto_b_Page_095.jpg
6af8eb80576b777a16a0c93dc667a3e8
eb3452a6c9635d8bb558d0965d354c20a6d8719f
94882 F20110217_AABQMD alto_b_Page_150.jpg
e69653ea1541740a38d3391661868b4a
8d0308590a6057f9097fe8fce5c195c2588b3b61
6617 F20110217_AABQLO alto_b_Page_151thm.jpg
39e3b3f20b883ce1f051d1de317321bd
4fae79f094033f897a62e5a8e787cbc1a20a08b6
F20110217_AABRPG alto_b_Page_046.jp2
7c2d620f8c94300edd583d7a8b25af90
5d9540c0e9527a72c7e842931b893566ca7caa40
85265 F20110217_AABROR alto_b_Page_096.jpg
f7dd45c042217d95b8c783a17214dff2
bd9d60776f817152511bc31866e2ef4ec3ea3880
6309 F20110217_AABQME alto_b_Page_068thm.jpg
2ea651e3292e15a6c8b7032fbbccabac
32151a451444ef27c84ef66f4a73ec21ddf9b856
50110 F20110217_AABQLP alto_b_Page_086.pro
442fb78fd10adf05053988e5c85c96d8
c3cf3e0a1b95029161f1d4cd2e9cbec9bc9b2075
689136 F20110217_AABRPH alto_b_Page_050.jp2
9a2505546de66238fae2113bffd70100
35a9404f5545cd9af406f674eb50eaa9269a293e
80330 F20110217_AABROS alto_b_Page_106.jpg
6f3a2375e3721b73904d56c3d957cf36
31c604c09040ac6d60984a876cad640fcabdaad4
43329 F20110217_AABQMF alto_b_Page_044.pro
a88c91dc8dc7aecc08e7a869c66a1617
ed0d786400ca4ea0943d982be9c9263f3c9a2c9b
F20110217_AABQLQ alto_b_Page_060.tif
78f1ced96d2cdbcc6bde6ce58fa18d2b
1a9a252a0674922867febaf39e548e82cf6e929b
1017788 F20110217_AABRPI alto_b_Page_051.jp2
fa5d910b759e8f4ab2254cc35413fc48
6084132bc192900796bc1aacf67fb39f0ba0ccb6
31065 F20110217_AABROT alto_b_Page_122.jpg
33bcc0fe6ae52ec93ec4851dfb605670
8fa38cc386c76ed6e9ece6060814a1f5efc507d5
51902 F20110217_AABQMG alto_b_Page_068.pro
91e42be5573ef7234b1514076b505f09
f962ab6633731bb878aed982b5e75e1ea9f8cf72
4757 F20110217_AABQLR alto_b_Page_112thm.jpg
63b5d28fa3c54d13b6525d8ca754c38b
a38203eb203b59650e2a372edc9a8b6582262025
775096 F20110217_AABRPJ alto_b_Page_056.jp2
0054630ac66a48469fb651c77dc9f3d0
4f115f31fb51db3c7e2d861c6ca457047fda2e85
85722 F20110217_AABROU alto_b_Page_126.jpg
a7361d6192298b51321d31ad2cb7f795
3005c6d99b3d6045e2626a529115192cd40f223f
F20110217_AABQMH alto_b_Page_019.jp2
d83deae6e3355c27b1fac95533c6a427
0d08cb249d5d3f9eae978645d0caabfc665e705a
2503 F20110217_AABQLS alto_b_Page_074thm.jpg
39658442525352f57fefa10fe8bab370
e694560f175d1679da4865833c6b12db08e5288a
1051953 F20110217_AABRPK alto_b_Page_059.jp2
5f10895e595698e4b7ffa7b13cb4cbab
d01ef8518e5e922478089469fff7bea47e84e3d6
89143 F20110217_AABROV alto_b_Page_131.jpg
73338de3da9382eeb88c79fd8bfa68ba
4be7dce17e3c0fe8166a3a891e38cc2bfb54523c
6586 F20110217_AABQMI alto_b_Page_141thm.jpg
183a4d751ad22c75ecf97d898802ed88
478de32e41926c5d2698742db86ce696332245e3
1291 F20110217_AABQLT alto_b_Page_048.txt
399246ed3b4cdfbeee16666d489d16c9
c9dcaa300320c03784f1c43fe57fbebfbc99e26e
F20110217_AABRPL alto_b_Page_062.jp2
7c72d675d4af495284c1e76c9be80b69
90e69ea2fa2b2c97aab3c1bf16eeb0cb0a307c0a
101067 F20110217_AABROW alto_b_Page_148.jpg
8b35ab77ac3b90249ac8685587a189cb
3d072711734227d30c0ec24d1507324a511e3b7f
77908 F20110217_AABQMJ alto_b_Page_062.jpg
431fb072518c42b0c12e819bdea3ebd0
37a4d8d70f890e4dcb9870610fe29fb66cc484c0
F20110217_AABQLU alto_b_Page_069.tif
f74d72dec6af5c93d19e4d03045a32e0
713f0356d9c4c0b96a6357aa54c5741d62957fe0
541856 F20110217_AABRQA alto_b_Page_122.jp2
876852ed47e8d429f55a9d95ce60725a
fcfbb3ecb0110d4948ebf351c2a403138e93c843
104280 F20110217_AABROX alto_b_Page_151.jpg
63ca5ff578bc94bacce10bc9917b3b59
b7a2fd30a5606da446dfaae7dbfc6236263bb368
52832 F20110217_AABQLV alto_b_Page_065.pro
cccea84272afb27c5efd65097dfa855f
1b820bda68db6b5739f23138fbba61b4965ee6e0
F20110217_AABRQB alto_b_Page_124.jp2
4fef3299327b3c393a73b5384daa4276
e58c069f238bab6d7a9564c29b03cf9436e2e5a2
F20110217_AABRPM alto_b_Page_065.jp2
cf6c2329f0bb4dc371f99cf1c21979ac
5db26ead78fca160db8449264ac483bb27577506
104607 F20110217_AABROY alto_b_Page_153.jpg
0c1e9a91ff03d19af63f0c1d651da47e
09cc64f1850c6ebd5c73a5c06355bdefcfad8265
27745 F20110217_AABQMK alto_b_Page_139.QC.jpg
f901094ddcf766b4e8847c99a90e5fa4
7331550ae2eb1a38ea0d098f6ddb8f2d05211b06
2315 F20110217_AABQLW alto_b_Page_150.txt
31546ba10d8ed742119783d8c9bb990c
a7488dc0537ee9dc1e9edd31b4a9ee0fa4d86b07
F20110217_AABRQC alto_b_Page_126.jp2
aeef10965be7737a4deeb0dc22d45e84
3a79f5fe49a5cfccb63dc54b64950edfd0d9f418
354484 F20110217_AABRPN alto_b_Page_071.jp2
095387f30f43d6bba2c30da2c396c50c
9a2ebebbbcba191a5737f672fb2df29927dbae47
103688 F20110217_AABROZ alto_b_Page_154.jpg
c5b54b6a1e5627dc5dc4ca2bad23a8f8
cb694eace27e6aa6d23358f5f5914e29fda8fde4
F20110217_AABQNA alto_b_Page_112.tif
bf16a047a5cc06c882481cc9afbd2720
8174ced858c725f19ec657aed2e197f1e63feeb1
F20110217_AABQML alto_b_Page_143.jp2
c7ed8e30f7b0d8c68a36d00f656bfb49
12ab137800b06443497ad0c388ea576dc7abbf16
85034 F20110217_AABQLX alto_b_Page_023.jpg
8a6a6747b3f85644c5724f5e9f0bc64d
5b363dae46ec14a7acc380e9542592c527ab34f5
1051901 F20110217_AABRQD alto_b_Page_129.jp2
766dad40550b21c27924f97f5bb93179
a86303ecff0b4e7965f6f419e5a526df25863c6e
571327 F20110217_AABRPO alto_b_Page_073.jp2
aac1f3568920f2da965b767d0813f5de
6d3acd9784f173662c2d19000826a65e2291fa1a
2061 F20110217_AABQNB alto_b_Page_125.txt
5f9f974d66c5a98584eb8e11e50878ec
fb8233c494d59432705dbd83a920960a12092d7e
9699 F20110217_AABQMM alto_b_Page_122.QC.jpg
d6a901002f4a1c2056433ffd0a32f85a
9369b0c914798ccabddd6b598960929b5e320c4c
842711 F20110217_AABQLY alto_b_Page_069.jp2
87f0d56f3a5039d6ea01ad0be48dc14b
38e3d24f5f14f46dfa7ff4d894d3aa1e5504d8c7
F20110217_AABRQE alto_b_Page_138.jp2
36bdaabf062fd1b5a52a5b3bfd16d127
b9f19b28d416830322177d714cec295fcc9f12bb
1051931 F20110217_AABRPP alto_b_Page_081.jp2
d077a7226f5408d57fbf77c9dc27bc60
db59516d74b9c02903df1a677a28a20d6a9746a4
F20110217_AABQNC alto_b_Page_156.tif
d62b2cb2909e67dc8b355950690efa21
df783593db24f7101033890fe6f6a935c286af35
F20110217_AABQMN alto_b_Page_081.tif
1520621c05e539209b2a0f52ac107cf9
8a4e653a3205c688da0961aa73a39fd286c8122b
25708 F20110217_AABQLZ alto_b_Page_053.QC.jpg
1a874a7c2d1db220d04d702796f23b89
9825deaeff7ce0131fe8e5e457db6915fa56bb87
F20110217_AABRQF alto_b_Page_155.jp2
82e18f215d4c7efb67bd000006010b13
17df416c9d67d8bc2f3262b544ea16be8b792de8
927039 F20110217_AABRPQ alto_b_Page_083.jp2
5bb7a6987d9048a5807c4989d0d69759
e454395c885b18937b94aac11cc6b65f91056d0d
1051943 F20110217_AABQND alto_b_Page_145.jp2
6c0c1c402d0a39d9d80f062d444ee26b
df0eecf5f5d25115a206e2a336bca8c7aa6dbc39
288 F20110217_AABQMO alto_b_Page_008.txt
875fe09aaf1f235278ebad7e1e95bf88
b214c009df7c33803a963d1c038f13185b56aeae
F20110217_AABRQG alto_b_Page_160.jp2
7f33203b8378193f1500c645a7663cc6
4a812a7a130e3e8fb5ebcc7745d2a8e16bb724da
1051878 F20110217_AABRPR alto_b_Page_086.jp2
3d38167a625daab6fbdbce6855e7ecb9
b097c0779ff96ab16cac7c928d3b7c287d6aa3cd
29115 F20110217_AABQNE alto_b_Page_163.QC.jpg
49811707117658a7252cdfd446a8f14b
1647b80e71b9239bcbd4ff39969127d177de31da
1885 F20110217_AABQMP alto_b_Page_087.txt
70c881286cde30827925af9dc8999580
d4abac8734598d1aeff235a81e0c07c9664f5de2
279709 F20110217_AABRQH alto_b_Page_165.jp2
000ff3fd42b9b584654ae06a8698a861
496ea291fb255b47aa2910f909eec0f03c2dabac
1051951 F20110217_AABRPS alto_b_Page_093.jp2
57061e11f6a1798008e5ae7293830917
6042c1196930de257303d99abef2222bc948ae23
6333 F20110217_AABQNF alto_b_Page_079thm.jpg
db80a0db6941e08915ebf912d222aec6
3b9a913795954fe5804948ae8a5032b4e209a1bc
83212 F20110217_AABQMQ alto_b_Page_007.jpg
fca60f35eab966b0869c088033efb20f
070d4de57a0d8c2f7f6be55c9999f0e45786c207
22960 F20110217_AABRQI alto_b_Page_134.QC.jpg
ebc5fe0a08b1e0c05d4d0f422999dcb1
ca375295f963de9bf4dc6c157dab55d86d6d7773
1027059 F20110217_AABRPT alto_b_Page_094.jp2
8e3ab4842173d2ffac767be533f396b3
c8539836606160d98cb3bb99daa29f4a8877b808
95757 F20110217_AABQNG alto_b_Page_006.pro
cc009336a23e0f8d1103df36675b5c3c
2fca44a01bf62a6ccef744cbd9df819a22d484fb
1399 F20110217_AABQMR alto_b_Page_116.txt
4712f75f17ba1030de6a668b31d5b49f
d761b2826c062578ca8897d72015aefa6e0f9b2f
6001 F20110217_AABRQJ alto_b_Page_064thm.jpg
64c5712e20909272a7ab514b0c166cef
83fa178ec4cd522f4b746dc73aa45e38ead9d85f
1051912 F20110217_AABRPU alto_b_Page_095.jp2
73c440a46896500df961b9d1b4c1dddd
39710f6b093c27f3f6d067ed1e95170adf3ecd3d
29846 F20110217_AABQNH alto_b_Page_138.QC.jpg
93425c5e52a3720a642bbcc916b7239e
501e2b8ae15ac7709a1b9fe16cd5c7c91cd24a8b
5126 F20110217_AABQMS alto_b_Page_007thm.jpg
82f5d7639f8ad2ff5101f6f03294a998
d912f794c140f90b18042aa1c801e81ffdc79e0a
6304 F20110217_AABRQK alto_b_Page_092thm.jpg
02a56e96d82c4516b3f21f58579ab554
79e8bbe062a53380950122a44e741f7fc0726bc5
1051947 F20110217_AABRPV alto_b_Page_099.jp2
ee4c41727cdc0a624e96723077ac7e62
269b8dc3bd3b0f690e45e34d0dcb746496cf07a6
10556 F20110217_AABQNI alto_b_Page_071.QC.jpg
d3978d1a9763fd57383596561dfb6236
f281d2e7d2e3bfed2f1cb43e6e07434e5ebe65ea
F20110217_AABQMT alto_b_Page_016.tif
635839493018ccb969f769601dbc1b63
04cc59736f5e22d090d5c1d7c354b85d7a2a4c2a
6546 F20110217_AABRQL alto_b_Page_142thm.jpg
ee7b6f64bae18100046d30b9c67487c9
4a1e2dd6ad1d3f2d8d512e6e26eb6513da466692
F20110217_AABRPW alto_b_Page_101.jp2
77728fed791db012fe7fed1bef339fec
1451a61a4151576c4a4585da2e0ef8bf35bcb2f2
23561 F20110217_AABQNJ alto_b_Page_051.QC.jpg
64c490607404c97aac645af062b8dcd9
57c881a9f1936df9726b7be9f172a2eb84525f71
F20110217_AABQMU alto_b_Page_110.tif
dbf53e4d9f433b0d1fc9f993a8ed3d96
ee8c422bcf9d958013782aa28c3dce691fed3109
25641 F20110217_AABRRA alto_b_Page_085.QC.jpg
cf01cbf260cf979f83358575fdb83040
946e48d199396007703c44d15543a7dcd6e4e16c
24549 F20110217_AABRQM alto_b_Page_018.QC.jpg
de61acd9ed9e3b1275392b47b4f7fda2
92adf32051450765606dc3c7a414f25705d774c9
F20110217_AABRPX alto_b_Page_106.jp2
aed84e663274a3d356fa6d62c2156664
60f387ff19cb5d314bd7d916dbf395d4bb182462
87436 F20110217_AABQNK alto_b_Page_047.jpg
ea1fe2781f2d0ef7f143ba6e27056d54
845a22b9d7819d8d4627101a04ab5e9aa953918a
86686 F20110217_AABQMV alto_b_Page_104.jpg
5a70c6b79f7f3dfb19f0130467103faf
23e12be37b71c1446faf666b0468875614a4c303
28224 F20110217_AABRRB alto_b_Page_160.QC.jpg
b5a797c8836acb107eea1d96db198351
4a47ad5978c60cf6056961726bf5412ccb62cd80
1051961 F20110217_AABRPY alto_b_Page_107.jp2
e4e8c7b19657756cc8baf9107b50da8c
ad8dd736a3569c3056d46e13eb744805b890a36b
25938 F20110217_AABQMW alto_b_Page_033.QC.jpg
d8e9227d298c8af3189e756146ed1cff
62b2519acdc6c2d8be1ce38b4658aebd210d9d9d
23393 F20110217_AABRRC alto_b_Page_044.QC.jpg
54dc76e594d2ead6c55deb6b4b7594ae
808ad11d7232a9971f1a8f0305b1744252bf6e59
28265 F20110217_AABRQN alto_b_Page_161.QC.jpg
8464f81d2b6edcb1e22b3e7bef8f3c1b
c64c9860c5d00cac557fa51e1e9d619d396053df
133577 F20110217_AABRPZ alto_b_Page_119.jp2
618aab3c5a4ba6e396904e2f14c945ee
1b018afb393bda8448cb05dafaf3b52634bb644e
49234 F20110217_AABQNL alto_b_Page_033.pro
9ed3e22fcd27b194619f2897d7a116f5
72c8184ab129f84c298530491d0a48ffceb5b136
F20110217_AABQMX alto_b_Page_078.tif
83937fc8f1149181534f7391d5532f42
b7ac9f0b9ff6b37b2350a7b80d9d60c133a42d26
F20110217_AABQOA alto_b_Page_156.jp2
0e5111b5bca61ed3be9d174d994f9585
96ebf3f7a7caa00d7a4eb9aa1606bd910ab28156
19820 F20110217_AABRRD alto_b_Page_069.QC.jpg
ffac2dbe6ccad17cb27e2a82d56983d6
09c40677f008e8e66d8429268c14aaaaaebafe92
6462 F20110217_AABRQO alto_b_Page_117.QC.jpg
cd25b7d44b12ec42027cdb9e8fe58f38
d5f96a77066714fbccf813cc4487b731528e8405
1879 F20110217_AABQNM alto_b_Page_031.txt
9a07c55ad0261d71c6f73c00937b9307
73ad3529228d91ce44102273f832f914c30ae96a
9473 F20110217_AABQMY alto_b_Page_120.QC.jpg
9a3aa1dca16a6438305ef3c8a60d7547
3f0586049d88af168b870b50f0329f7feebcb7e8
1849 F20110217_AABQOB alto_b_Page_080.txt
6eaf92c1f9806efa1daec501eaddac0c
37fee703ffc272de8569469859722b6ce541fae3
6491 F20110217_AABRRE alto_b_Page_131thm.jpg
306fcc8cbf4bc6b26e734d2ac00a4af5
a2517cf0624718670713f09b48dc9a21fb348856
26126 F20110217_AABRQP alto_b_Page_021.QC.jpg
db0a851e56558c6af4ccee01b083093a
534f6bbf740788c3d2756e88a1d17a184b5bba8e
6748 F20110217_AABQNN alto_b_Page_160thm.jpg
700aa27e940bda242b4479e5ef70a223
913e62c512b1d070a6d36c0d9c84c052dcb790ac
F20110217_AABQMZ alto_b_Page_122.tif
bc609eaea2391a1a6966ad25b7cf26c2
c2bf778f55a1c6a3ed403afff60b806f58977528
29096 F20110217_AABQOC alto_b_Page_157.QC.jpg
7069b639d12dc4592c7a4df8f5ac29d2
488eac62c79f8e8f9555b18a3d995b977629448b
25811 F20110217_AABRRF alto_b_Page_052.QC.jpg
2ee695607eca46c4bf813819fe26a6d2
57ae3af66d6e54226d7434f16fd728ea3faee2f9
6648 F20110217_AABRQQ alto_b_Page_154thm.jpg
168406ae9df1452bec8e96ed348b104f
5808eb73325fbb8e4fcd9eb21fd98b61bd997689
1937 F20110217_AABQNO alto_b_Page_124.txt
82dacffc6015ef9131b8fa6f968d41d3
077e6317b7df8295aa8c4dcc207349d6287734a9
103290 F20110217_AABQOD alto_b_Page_149.jpg
eb7d4b90aaeff51838ade207d0fa2b44
b67e9e1f6ed786e4cb51b94dd2ccec93cded8733
23477 F20110217_AABRRG alto_b_Page_135.QC.jpg
ecf97fe789f638419f5bb59733fe89bf
674d0119005b7c673783bfea852efb32590e2ecc
22317 F20110217_AABRQR alto_b_Page_164.QC.jpg
693c5b82045af9e84a4d3b6e245dfde1
001bb2bf69d96c67a7a7da3bd865cbffffbca7ed
1051979 F20110217_AABQNP alto_b_Page_149.jp2
57cc1b481714cf702c302c8b087691e3
901a841c2e80253a66e4d973b854affa8cc8913f
1051915 F20110217_AABQOE alto_b_Page_087.jp2
043c4fb1cf3682ec8b0bcacd192b4c53
9e4f6d258bbd1041bff85a2112d9116822827873
25994 F20110217_AABRRH alto_b_Page_037.QC.jpg
804e71d91e191e0c35ad94125e78e349
b968466dad0d05b7a12b50167b8073c594deb001
27713 F20110217_AABRQS alto_b_Page_017.QC.jpg
c63a3a29d74af9196579cccb42e4efe6
7ea2884ec2d848d95a0c453079543bf38f6e71f3
F20110217_AABQNQ alto_b_Page_096.tif
8f06da83d7d74cf81dc4aad5f802c29b
bf9722416d1b613b9d2487d5721f6576c8665117
27002 F20110217_AABQOF alto_b_Page_034.QC.jpg
d66d9a846d1333198b08693d5c96a6c4
63a473f70d4169a3fa31b73c950106f13a6ae3ee
26798 F20110217_AABRRI alto_b_Page_104.QC.jpg
277a542e567c6aa2d8157c72fdece646
c8bd3f42509bb47a07d3db435f320d382cdd1b60
26323 F20110217_AABRQT alto_b_Page_054.QC.jpg
1034e08cb47acc3feea5078d791c8a1f
9877c0f42cb812e77bb040e2c927c0d4b8be9251
48361 F20110217_AABQNR alto_b_Page_106.pro
099f5ae1da38393d2170dcd73893b4ef
0beacdded6329a49a5485d0f9973c0c42095416d
1051959 F20110217_AABQOG alto_b_Page_033.jp2
6220a69d40cf6282bd38fdc047a5754d
396c44325c0c521572c7b73b5fba2f2b9f860ff9
28065 F20110217_AABRRJ alto_b_Page_136.QC.jpg
ce2d413bb76f8e3cfe1d05f7e940e9d3
7ee5bfb4296e7dde536c129c157e990a2eb18d20
6486 F20110217_AABRQU alto_b_Page_034thm.jpg
0419d610cfb710874e59c423ce92948d
c0f57759a73e0c4ab977db404e31160e01583126
515 F20110217_AABQNS alto_b_Page_002thm.jpg
4d2911e7f782509967c035a3834d832d
b57fe9e70040dc7a1b4b8f8a78cd22cf9192c9a1
F20110217_AABQOH alto_b_Page_101.tif
ebe79fa5f9fd0fe4f2f7f374f7313fc1
0d44c2066a5253dc40f0b7437683f455ab24b00b
3968 F20110217_AABRRK alto_b_Page_072thm.jpg
cdf52b1d7d3816600179afa7a6392084
83a2f3e0eeef9f6d512424a0e9ae2b773b205d78
3283 F20110217_AABRQV alto_b_Page_005thm.jpg
27cc6c7eec23f7529f0e9f7e5ffc6eb7
6c4f765e371665862c3df85ac9a3e226f1865298
65372 F20110217_AABQNT alto_b_Page_007.pro
f11cab7a07908f81c9095306b4a68641
e8c086902819802bd6f20a033331fdfc7814c5f9
6286 F20110217_AABQOI alto_b_Page_053thm.jpg
d49060d489a0b2ca01e0459c2dbe84fb
2ec1b4d9c1be86bdc57a06166d6b4c2f37bf8278
25347 F20110217_AABRRL alto_b_Page_089.QC.jpg
6a462e01d706efb35353499091d04aee
256db6b6e83b359f750580469f964f23804860c1
20198 F20110217_AABRQW alto_b_Page_004.QC.jpg
000b6d34eb6b08221a5f9cf72e08e826
346a1e510fb8056c052f62946573be2a1f89a256
52466 F20110217_AABQNU alto_b_Page_059.pro
0c648bc2a77cdf9fa2a52295a459a879
72db177c0fe274605d8d7ec6c88bbb4a1f096eac
F20110217_AABQOJ alto_b_Page_075.tif
f2c01378d46cf98b937dcdfc91bfade7
e3d01b63892a473c102a37551a7e88b04df4ed37
6053 F20110217_AABRRM alto_b_Page_124thm.jpg
032d0354de4f0ddf9bfa9ca9b63c416e
3fabed7eed7749c52703c8e228b2f52dcaf7a847
672 F20110217_AABRQX alto_b_Page_003thm.jpg
d338e37e41b22ecaf3886fc81bc4d64a
449d3e3db2f90dd752d8cced9195964470dc6d1f
F20110217_AABQNV alto_b_Page_128.jp2
a1e7692341d46e3dd79cd68578e3b75d
9283dda636ee6f7b2be40696594d5f9a1d85415b
6384 F20110217_AABQOK alto_b_Page_147thm.jpg
ef43688eacb52cc760496f9cf666a87f
9df9ce8036aebbc8731bd464318f0060d788edd7
263721 F20110217_AABRRN UFE0014961_00001.xml
80b514de05080d4a859c03903a3dd5ec
38c9c7a1bf4df14222424011a52ee77d719a4374
26725 F20110217_AABRQY alto_b_Page_047.QC.jpg
9be01fa7253b757c28d545ff3650d8e6
b4ada59143b67e79e12e3ebac8813ebd2a3e309e
2049 F20110217_AABQNW alto_b_Page_034.txt
92f367fc5d7bbaadd4fd6a404b149f08
2ba60ff6ac9e3fdbcdde8f028d6fd2bffc209ca4
1761 F20110217_AABQOL alto_b_Page_077.txt
de01181feccf6de1c4226a810151e621
eb1aa17c8df2c192f53f1ff6f42a51596696c3b4
4846 F20110217_AABRQZ alto_b_Page_069thm.jpg
4c818fdb0c197dc54824d91fb7f3fe3c
bcc20c44f1d4728ce9a4b6c811050662d99ccabf
F20110217_AABQNX alto_b_Page_067.txt
4f0258df3f96540a4345abe322959be0
0772372f96d0be777e5fc403faae01292aacf9a5
6661 F20110217_AABQPA alto_b_Page_139thm.jpg
cee984bf738114dabb488815cd831c57
209ea5ded741a79568954c4939ce89064a8bb18b
19992 F20110217_AABRRO alto_b_Page_011.QC.jpg
0330797795a6ef2233618875814fdc8d
ccb81029bd1e8233b03c511eeac16e87cdcf0aa1
6211 F20110217_AABQNY alto_b_Page_129thm.jpg
c88c97ad0413d73ace59f138766c36c5
f8704777ef832d0a080eba61d13042a2195fed05
F20110217_AABQPB alto_b_Page_118.tif
ca5cd7e5a832d1dde9b3abf1ffa3c15b
b9cceaecc3148863b6853b6f4ef74b80ec7cc525
103385 F20110217_AABQOM alto_b_Page_136.jpg
901904fe885ffb85ead037fec8c01ad4
6b40d196d70720cd8372108b3475596b0abc29a9
25265 F20110217_AABRRP alto_b_Page_088.QC.jpg
06af735acdb6c8632f9caea9aa6aa8db
89478b8b8802b74748b88f9683ae70bb5281cbdc
18673 F20110217_AABQNZ alto_b_Page_113.QC.jpg
6d4ca42c5aa0d7c14f569a170759245b
42a6395ae015e8a994c6baee1e6143ed89c8eab0
F20110217_AABQPC alto_b_Page_140.jp2
97f41819c7ced2f03261ab106a07a4f3
94a1e0418d2c3b31a93aff1e34072b1ad12be1ea
86743 F20110217_AABQON alto_b_Page_132.jpg
efcd12f6af629fdf6f92224fbf609e03
c56616ff37460aa04cdc1aff64f637d3d191f6f6
26895 F20110217_AABRRQ alto_b_Page_129.QC.jpg
415987a041e3b63aaf300fd2efd8e08c
4af309a7676c46a3dc8941af6fa174302926d668
1633 F20110217_AABQPD alto_b_Page_057.txt
3905cd3a7874e8c4d757e7a2b1b7c18d
2b5bc441680911d58c5bc04080783aefefd4a5dd
27007 F20110217_AABQOO alto_b_Page_040.QC.jpg
66d8eb331e4f203499afc28be074687a
f6534b28ea25096ff6420736be25cfb3f80cd2fa
29289 F20110217_AABRRR alto_b_Page_143.QC.jpg
764492a315defb263b834127261b3a12
26359fe3a4c0bfc35c29b42d29549d29e505bec9
14215 F20110217_AABQPE alto_b_Page_005.QC.jpg
aeb19532bd41f9ee7c9fb015f86dd3df
e6e99343c1d37b4f9c4fa59d1565cf4871ff3613
F20110217_AABQOP alto_b_Page_022.tif
4f2da9139233427f9967522e913da816
8f021dcf89706372f2236369eff67526c66c3128
28343 F20110217_AABRRS alto_b_Page_153.QC.jpg
85ea561fdd50fa86260f4368c692787f
2dfe61baff132b3e19bd3f4f2f395e5f87759a91
5362 F20110217_AABQPF alto_b_Page_013thm.jpg
61b7db4c83bc0d0269fff3a0dc34cc21
3a9beffe07a263b11b8c286ddbbdd5e50077f5da
986810 F20110217_AABQOQ alto_b_Page_044.jp2
54c23bd4a33f0501bef9a3e629fbb998
d0fff3c809cff8182b2742d76cc2d96501033720
1829 F20110217_AABRRT alto_b_Page_001thm.jpg
f2b7b0a12941a159347a5bd2d5df5e57
dde5f975b4b5510d075b43b3697e63caf7574fc6
2739 F20110217_AABQPG alto_b_Page_146.txt
cc115a338daf1a9fe6f4d9530b26a036
b74d76ce80488f8ce0387a065da224285901b31b
48454 F20110217_AABQOR alto_b_Page_026.pro
a5b2b5b6e2a8bf99edf13f61e0f23dfb
6796229e9ec620b1fdcd4049d275aa1d4d24726e
4134 F20110217_AABRRU alto_b_Page_006thm.jpg
4ed6517ee84f0d8c0600a791e8c930ed
ffbd00b50ddf8abba40db7d5645df5457c28a369
60080 F20110217_AABQPH alto_b_Page_144.pro
9858004267be4b6068f276eaa75f089f
5ed4258a9a7ff70009c0b2f41a20d4e5bc055ae3
F20110217_AABQOS alto_b_Page_053.jp2
e5d3e39d6c7d3621bdb030774eec821b
bb83c994ba528c54f72a806b28d3cab552621f94
3301 F20110217_AABRRV alto_b_Page_010thm.jpg
46a76dbb7e6b8cafb4269b1748965d42
61e6bbf94f795bde726027ddbb6dd1360a774094
1907 F20110217_AABQPI alto_b_Page_045.txt
de5f9efdf1838530028b9b7bb138021e
e2f482b457ab411b8ce4e258a437d73441343361
2146 F20110217_AABQOT alto_b_Page_097.txt
2bbd430d8c3ad19e9389bfd8d8234308
5301e581600eb3121c95074990a41d2aca170aad
6098 F20110217_AABRRW alto_b_Page_028thm.jpg
d00c4ed4015a76959c55dd4da699c5b2
fb63ec976db559b516566c0a0da665248a55992c
2027 F20110217_AABQPJ alto_b_Page_118.txt
4274f3f95562aa29b50c35e1c36bff5a
381ebb265c248cb40635c11500ec440454dda0d7
6252 F20110217_AABQOU alto_b_Page_095thm.jpg
df6a8217a3ebe2128b86022c41738d1a
214a2f359eae91f0aa751d83259a0eacffae4e97
6423 F20110217_AABRRX alto_b_Page_030thm.jpg
20f9ddf8ed6612f467b5ba576281f4cf
46e9cccabc2dd39de5b860aaca6e1470f278113a
64554 F20110217_AABQPK alto_b_Page_142.pro
9f5be0394915dab38398740337256e0f
cd123364d8bab2d5782dba4f784dd4ae0fa9d7c7
F20110217_AABQOV alto_b_Page_029.jp2
2b21dac37d6dc27a8dc26feb10cda6ef
286ab164f0fda1675f8a80c1c6a0523f92b8dcb6
5747 F20110217_AABRRY alto_b_Page_077thm.jpg
35443f6ba89e217629aca0f93d0ce43b
cf93dd17e2adc543b0b98345504e4066fa02bb58
F20110217_AABQPL alto_b_Page_150.tif
f9abf938d9da680881d4c5a3505d79fc
f98db7e242bb49d68821ba7c50d948b470ca21b3
27328 F20110217_AABQOW alto_b_Page_058.QC.jpg
07e8dd3fc25f45f7875f4e2660ba3ea4
2d15c865573f09af6bcee617f41c09968db7482a
6214 F20110217_AABRRZ alto_b_Page_085thm.jpg
dcf5f7029782c83bd8470d8a9485a2a3
7fd01f956acb2f7166747406c6d9ba6d3124c6ca
9703 F20110217_AABQQA alto_b_Page_119.pro
c5836d405353c1e8683879e4f7810b74
3ba1678c449d6434dfb846541546069dd241b1aa
69271 F20110217_AABQPM alto_b_Page_082.jpg
32402b0923e307145cb3988bb606f756
f7c7d6953f5c663fa4da243a75d85daabbc40e8f
F20110217_AABQOX alto_b_Page_102.tif
5416acd43611161ce5249e1fa6845ce2
a73df78fd7b279154d7a94857f5a8693b94d7a51
102262 F20110217_AABQQB alto_b_Page_159.jpg
d6d2aa5112b1b874c7ff0536408366f1
61b1ed86d2b7223e2351bfc409491320f6fa79a1
F20110217_AABQOY alto_b_Page_021.jp2
54f7f9f10cb0aaf9b72968a2fd136473
eb948052fe8855a66bbf193a16d7b3f7cc2293b5
27109 F20110217_AABQQC alto_b_Page_147.QC.jpg
3980aed9c83fabb033fe1aa8daafe9a0
a44c9bdf430ce59d30cce3f6fbb0e83d8dc82121
1051946 F20110217_AABQPN alto_b_Page_085.jp2
6021e3cb789b0d11a170be48ce6a6636
3ac61b8a5d228648136f243bb0418d37b4d27d17
6340 F20110217_AABQOZ alto_b_Page_097thm.jpg
6ee13212dea7ff69a2454ed5bcd4ea4a
781febd2a10928450d2ca93b35e9c19ada55dffc


Permanent Link: http://ufdc.ufl.edu/UFE0014961/00001

Material Information

Title: Larval Competition and Adult Susceptibility to Arbovirus Infection in Container Mosquitoes
Physical Description: Mixed Material
Copyright Date: 2008

Record Information

Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
System ID: UFE0014961:00001

Permanent Link: http://ufdc.ufl.edu/UFE0014961/00001

Material Information

Title: Larval Competition and Adult Susceptibility to Arbovirus Infection in Container Mosquitoes
Physical Description: Mixed Material
Copyright Date: 2008

Record Information

Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
System ID: UFE0014961:00001


This item has the following downloads:


Full Text












LARVAL COMPETITION AND ADULT SUSCEPTIBILITY TO ARBOVIRUS
INFECTION IN CONTAINER MOSQUTIOES















By

BARRY W. ALTO


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA


2006

































Copyright 2006

by

Barry W. Alto

































This dissertation is dedicated to my mother, Barbara A. Larson, for her undying support















ACKNOWLEDGMENTS

I am thankful for valuable advice and reviews from my dissertation committee, L.

P. Lounibos, S. Juliano, C. Lord, J. Maruniak, C. Mores, C. Osenberg, and W.

Tabachnick. I am especially grateful to my advisor L. P. Lounibos for years of guidance,

support, useful discussions, and reviews. I am grateful to J. Butler, B. Coon, and K.

McKenzie for assistance with methods and equipment necessary to construct the silicon

membrane system; C. Jennings for supplying me with citrated blood; G. O'Meara for

providing me with cages for bloodfeeding trials and data on mosquito larval densities

from field collections; P. Grimstad and R. Nasci for providing me with Aedes eggs; N.

Nishimura, R. Escher, I. Tobar, and B. Wagner for daily maintenance of the competition

studies and for measuring wing lengths; J. Maruniak, S. Higgs, and D. Bowers for advice

with cell culture and Sindbis virus assays; S. Fernandez at Walter Reed Army Institute of

Research for generously providing me with the plaque assay protocol and the dengue-2

virus (16803) used in the mosquito infection study; M. Reiskind for dengue virus RNA

extraction and quantitative RT-PCR research, as well as valuable discussions and reviews

that improved the dissertation; D. Baptiste for providing Vero cells and aiding in dengue

virus RNA extraction; D. Chisenhall, J. Dyer, K. Pesko, and S. Richards for assistance in

bloodfeeding mosquitoes.
















TABLE OF CONTENTS



A C K N O W L E D G M E N T S ................................................................................................. iv

LIST OF TA BLE S ....................................................... .. ........... ............ .. vii

LIST OF FIGURES ......... ......................... ...... ........ ............ ix

ABSTRACT .............. ................. .......... .............. xi

CHAPTER

1 INTRODUCTION AND REVIEW OF THE LITERATURE............. .............

Intro du ctory Statem ent ........................................... .......................... ..................... 1
W ater-filled C containers ............................ .. ...................... ........ .. ... ............... 1
M mosquitoes ................................................ 3
Aedes albopictus ..... ........... ......... .......... ........ 3
Aedes aegypti ................. .... ......... ...... ........ 5
Dengue Virus .................. ............... ........7.......
Introduction ................................................................. 7
Human Infection ........... ......... ... ................. 8
Infection Cycle in the Mosquito Vector .............. .................... ...........9
Sylvatic D engue C ycles .............................. ... ....... .......... ................ 11
Viral Isolates and Experimental Infection/Transmission ....................................12
Sindbis Virus ...................... ....... ... .. ... .. .. ................... 14
Intro du action ....................................................................... 14
Human and Reservoir Infection .................................................... ........ ................15
Viral Isolates and Experimental Infection/Transmission ..................................16
Com petition and V ector Com petence..................................... ........................ 21

2 AGE-DEPENDENT BLOODFEEDING OF Aedes aegypti AND Aedes
albopictus ON ARTIFICIAL AND LIVING HOSTS ......... .. ............. 27

Introdu action ...................................... ................................................. 2 7
M materials and M methods ....................................................................... ..................29
E x p erim ental P rotocol .............................................................. .....................2 9
D ata A n aly se s ................................................................................ 3 1
R e su lts ...........................................................................................3 3
D isc u ssio n ............................................................................................................. 3 4


v









3 LARVAL COMPETITION DIFFERENTIALLY AFFECTS ARBOVIRUS
INFECTION IN Aedes M OSQUITOES.......................................... ..................... 39

In tro d u ctio n ........................................................................................................... 3 9
M materials and M methods ....................................................................... ..................43
C om p petition Stu dy .................................................................... .....................4 3
Infection Study .......................... .............. ................. .... ....... 45
R e su lts ...........................................................................................4 9
C om p petition Stu dy .................................................................... .....................4 9
Infection Study .......................... .............. ................. .... ....... 50
D discussion ..................................... .................. ............... ........... 52

4 LARVAL COMPETITION AND SUSCEPTIBILITY OF Aedes aegypti AND
Aedes albopictus TO INFECTION BY DENGUE VIRUS ............... .................... 65

Introduction .............. ..... ... ................................................ 65
M materials and M methods ....................................................................... ..................69
Com petition Study .................................................................. ............... 69
Infection Study .......................... .............. ................. .... ....... 72
V iral propagation ........ ................................................. ........ .............. 72
O ral infection of m osquitoes ........................................ .......................... 73
Blood meal plaque assay .............. ................... ...............76
Mosquito homogenization, plaque assay, and RNA extraction .................77
Quantitative RT-PCR ............... ............................ .. ................. 78
Species by Com petition Com prison ...................................... ............... 80
R results ........... ............. ........................... 81
Com petition Study ........... .... ................ ........ ... ........ .. ............
Infection Stu dy ..............................................................82
Species by Com petition Com prison ...................................... ............... 85
D iscu ssion ................... ......... .......... ..... ..... ............... .. ............. 86

5 COMPETITION, ARBOVIRUS INFECTION, AND FUTURE EXPERIMENTS 111

Com petition and Enhanced Infection ......................................................................111
F future Stu dies ........................................... ..... ..................... ... ......... 116
Field-collected Mosquitoes for Infection Experiments................................... 116
Mechanisms Responsible for Competition-enhanced Infection ........................117
Other Epidemiologically Significant Factors: Adult Survival ..........................119
Other Ecological Interactions in the Larval Stages .................. .... .......... 121
C o n clu sio n s.................................................... ................ 12 2

L IST O F R E FE R E N C E S ........................................................................ ................... 123

BIOGRAPHICAL SKETCH ............................................................. ............... 153















LIST OF TABLES


Table p

2-1 Test for equal slopes among regressions of proportion bloodfed of Aedes aegypti
and A. albopictus versus age ............................................................................. 38

2-2 Intercept and slope estimates for simple linear regressions of proportion
bloodfed ofAedes aegypti and A. albopictus versus age ......................................38

3-1 Multivariate ANOVA for main effects and multivariate pairwise contrasts of
competitive treatment effects on female Aedes albopictus and A. aegypti..............62

3-2 ANCOVA for the effects of competitive treatment and size covariate on body
titer for Aedes albopictus and A. aegypti females with disseminated infections. ....63

3-3 Product moment correlation coefficients (rl,2) for the relationship between
population growth measurements (time to emergence, survivorship, size, and V')
and infection param eters ................................................ .............................. 64

4-1 MANOVA and multivariate pairwise contrasts of competitive treatment effects
on female Aedes albopictus and A. aegypti................. ..................104

4-2 Multivariate ANOVA for main effects and multivariate pairwise contrasts of
competitive treatment effects on female Aedes albopictus and A. aegypti
proportion infected and proportion with disseminated infection .........................105

4-3 ANCOVA (after testing for equality of slopes) for the effects of competitive
treatment and size covariate on body titer, proportion infected, and proportion
with disseminated infection for Aedes albopictus and A. aegypti females ............106

4-4 Product-moment correlation coefficients (rl,2) for the relationship between
population growth measurements (time to emergence, survivorship, size, and V')
and infection parameters for A. albopictus (df=25) and A. aegypti (df=29) ..........108

4-5 Two-way MANOVA of species (A. albopictus and A. aegypti) and competitive
treatment (A. albopictus: A. aegypti, 160:0, 320:0, 0:160, and 0:320) effects on
female population growth measurements...................... ...................... 109

4-6 Two-way MANOVA of species (A. albopictus and A. aegypti) and competitive
treatment (A. albopictus: A. aegypti, 160:0, 320:0, 0:160, and 0:320) effects on
proportion infected and proportion with disseminated infections..........................110









4-7 Two-way ANOVA for species (A. albopictus and A. aegypti) and competitive
treatment (A. albopictus: A. aegypti, 160:0, 320:0, 0:160, and 0:320) effects on
body titer. ................................................................ .. ..... ......... 110
















LIST OF FIGURES


Figure page

2-1 Least squares means ( SE) for proportion bloodfed females on the silicon-
membrane system for 3-15 day old Aedes albopictus and A. aegypti..................37

2-2 Least squares means ( SE) for proportion bloodfed females on restrained
chickens for 3-15 day old Aedes albopictus and A. aegypti...................................37

3-1 Aedes albopictus least squares means (+SE) for female survivorship and size at
em erg en ce ...................... .. .. ......... .. .. ..............................................5 8

3-2 Aedes aegypti least squares means (+SE) for female survivorship and time to
em erg en ce ...................... .. .. ......... .. .. ..............................................5 8

3-3 Least squares means ( SE) for estimated finite rate of increase, k', for Aedes
albop ictus and A aegyp ti .............................................................. .....................59

3-4 Bivariate plots of least squares means ( SE) for three dependent variables for
Aedes albopictus females fed on a Sindbis virus blood meal ................................60

3-5 Least squares means for body titer and size of adult Aedes albopictus females
with disseminated and isolated Sindbis virus infections............... ..................61

4-1 Aedes albopictus least squares means ( SE) for female size and time to
em erg en ce ...................... .. .. ......... .. .. ..............................................9 7

4-2 Aedes aegypti least squares means ( SE) for female size and time to emergence .97

4-3 Least squares means ( SE) for estimated finite rate of increase, k', for A.
albop ictus and A aegyp ti ........................................... ........................................ 98

4-4 Least squares ( SE) for proportion ofA. albopictus and A. aegypti infected and
dissem inated infections ...................... .. .... ................................ ...........99

4-5 Bivariate plots of least squares means ( SE) for proportion ofA. albopictus
infected and dissem inated infections................................... ......................... 99

4-6 Bivariate plots of least squares means ( SE) for proportion ofA. aegypti
infected and dissem inated infections.................................. ........................ 100









4-7 Least squares means for body titer and size of adult A. albopictus females with
disseminated (i.e., infection spread beyond the midgut, infecting secondary
target organs) dengue-2 virus infections ....................................... .............100

4-8 Least squares means for proportion infected and size of adult A. albopictus
fem ales ............................................................................ 10 1

4-9 Least-squares means for proportion disseminated infections and size of adult A.
a lbop ictu s fem ales ................................................................ .... .... .................... 10 1

4-10 Least-squares means for body titer and size of adult A. aegypti females with
disseminated (i.e., infection spread beyond the midgut, infecting secondary
target organs) dengue-2 virus infections .......... ............................. .............102

4-11 Least-squares means for proportion infected and size of adult A. aegypti
females. Numbers in the figure key represent the ratio of larval A. albopictus to
A a egyp ti. ...................................................................................... 102

4-12 Least-squares means for proportion disseminated infections and size of adult A.
aegypti females. Numbers in the figure key represent the ratio of larval A.
albopictus to A aegypti. ............................................... .............................. 103

4-13 Two-way ANOVA for species (A. albopictus and A. aegypti) and competitive
treatment (A. albopictus: A. aegypti, 160:0, 320:0, 0:160, and 0:320) effects on
Least-squares m eans for body titer .......................... ........... ............. .................. 103















Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

LARVAL COMPETITION AND ADULT SUSCEPTIBILITY TO ARBOVIRUS
INFECTION IN CONTAINER MOSQUITOES

By

Barry W. Alto

August 2006

Chair: L. Philip Lounibos
Major Department: Entomology and Nematology

Larval competition is well-documented among container mosquitoes and influences

life history traits such as survivorship, development, and adult size. Few studies have

attempted to address how biological interactions experienced by larvae may impact adult

susceptibility to arboviral infection, subsequent viral spread to secondary tissues (i.e.,

disseminated infection), and viral body titer.

With Sindbis, an arbovirus frequently used in vector research, Aedes albopictus

mosquitoes had higher infection rates but lower body titer and dissemination rates than A.

aegypti. For both A. albopictus and A. aegypti, competition affected population growth

measurements, with uncrowded larval conditions consistently resulting in shorter time to

adult emergence, increased survivorship, adult size, and better population performance

than crowded conditions. For A. albopictus, but not for A. aegypti, more intense intra-

and interspecific competition resulted in higher Sindbis virus infection rates, body titers,

and dissemination rates compared to low competition conditions. Whole body titers of









virus increased with mosquito size irrespective of competition. However, between

competitive treatments, mosquitoes from low competition conditions had greater mean

size, with lower infection and lower whole body titers than smaller mosquitoes from high

competition conditions. The results of experiments on this model system indicate the

importance of the larval environment, especially competitive conditions, on adult vector

competence.

With dengue virus, the most important arbovirus afflicting humans, A. aegypti had

lower dengue virus infection rates and body titers but higher dissemination rates than A.

albopictus. Higher levels of intra- and interspecific competition enhanced A. albopictus

infection and dissemination rates with dengue virus. Similar effects of competition on

mosquito infection parameters with unrelated Sindbis and dengue viruses suggest a

generalizable mechanism of environmental influences on infection parameters. The

experimental results indicate that larval conditions are an important aspect of vector

competence and should be included in future epidemiological considerations and

modeling of arbovirus transmission.














CHAPTER 1
INTRODUCTION AND REVIEW OF THE LITERATURE

Introductory Statement

Larval competition is well-documented among container mosquitoes but its effects

on adult susceptibility to arboviral infection remain unclear. This research addresses the

question whether larval competition among and between mosquitoes Aedes aegypti and

A. albopictus influences adult susceptibility to Sindbis and dengue virus infection. A

silicon membrane bloodfeeding system was evaluated as a method to administer

infectious bloodmeals for subsequent competition and infection studies. The purpose of

the following literature review is to provide a working knowledge of container habitats

and basic biology of the two mosquito species used in the current experiments. Also, I

summarize the vector biology research of dengue and Sindbis viruses as well as place the

current question concerning competition and susceptibility to arboviral infection in

context to studies of similar nature.

Water-filled Containers

Phytotelmata are parts of terrestrial plants such as leaf axils of tank bromeliads,

bamboo internodes, pitchers of carnivorous plants, Heliconia bracts, fallen leaves or fruit

husks, and treeholes which hold bodies of water (Frank and Lounibos 1983). Artificial

containers serve as analogs of phytotelmata and come in a variety of forms such as

discarded tires, cans, vases, jars, cisterns, and plastic debris. Both natural and artificial

containers are habitats for a variety of arthropods having aquatic life stages. Containers

may be favorable model systems for investigating entomological and ecological









processes because they harbor small, discrete aquatic communities. Diptera are the most

taxonomically diverse group among insect orders inhabiting phytotelmata (Fish 1983). In

particular, mosquitoes are the most extensively studied dipterous insects within container

communities, in part, because they often are the most abundant macroinvertebrates (Fish

1983) and may be vectors of arthropod-borne (arbo) viruses and other vertebrate

pathogens.

Nutrient resources in these systems come in the form of inputs of allochthonous

plant detritus (fallen leaves, flower parts) (Lounibos et al. 1993, 1992, Kitching 1971),

macroinvertebrate carcasses (Daugherty et al. 2000, Sota et al. 1998, Heard 1994, Naeem

1988, Bradshaw and Holzapfel 1986), throughfall, and stem flow (Kaufman et al. 1999,

Walker et al. 1991, Walker and Merritt 1988, Carpenter 1982a). The latter two resource

inputs refer to precipitation that has fallen through the canopy or down the branches and

trunk of trees, respectively. Decomposing plant detritus is recognized as the predominant

nutrient base for treeholes, and, perhaps, artificial container communities (Macia and

Bradshaw 2000, Lounibos et al. 1992, Carpenter 1983, Fish and Carpenter 1982,

Kitching 1971). Leaf litter is likely a lower quality nutrient resource compared to

macroinvertebrate carcasses due to intrinsic differences in carbon:nitrogen ratios among

these resources (Cloe and Garman 1996). Some macroinvertebrates may directly

consume plant detritus (e.g., Helodes and Prionocyphon beetles, Paradise and Kuhn

1999, Barrera 1996a, Carpenter 1982b), however most container mosquitoes consume

microorganisms associated with the detritus and water column (e.g., Walker and Merritt

1988, Fish and Carpenter 1982). Spatial and temporal differences in the quality and

quantity of nutrient resources have important consequences for container communities.









For example, mosquito larval stages are confined to containers, and so habitat

characteristics (e.g., nutrients, competition, predation, temperature) largely determine

mosquito population growth measurements. Rate of leaf decomposition, and associated

bacteria and algae, is often positively correlated with nutritional value and mosquito

productivity (e.g., Dieng et al. 2002, Yanoviak 1999, Fish and Carpenter 1982, Swift et

al. 1979), and differences in degradation among leaf types are due to the environmental

conditions and properties of the leaf species (Yanoviak 1999, Leonard and Juliano 1995,

Fish and Carpenter 1982).

Mosquitoes

Aedes albopictus

The Asian tiger mosquito Aedes albopictus (Skuse), native to Southeast (SE) Asia

and the Pacific and Indian Ocean regions, invaded container habitats in the U.S., Europe,

West Africa and South America during the last 30 years (reviewed in Eritja et al. 2005,

Juliano and Lounibos 2005, Lounibos 2002). Aedes albopictus is second only to A.

aegypti in terms of importance as a vector of dengue virus (DENV). Although small

introductions ofA. albopictus in the continental U.S. were found previously in used tires

shipped to a port in Oakland, CA (Eads 1972) and a cemetery in Memphis, TN (Reiter

and Darsie 1984), none became established. It is believed that A. albopictus first became

established in the continental U.S. in Houston, Texas in 1985 (Sprenger and

Wuithiranyagool 1986). Populations of A. albopictus in the U.S. are believed to be

derived from temperate Japan (Hawley et al. 1987), whereas Brazilian A. albopictus are

of tropical origin (Birungi and Munstermann 2002). Successful spread of A. albopictus

in the eastern U.S. was facilitated by immature stages using artificial container habitats,

in particular used or discarded tires (Moore 1999, Reiter 1998). Aedes albopictus is









adapted to both tropical and temperate climatic regions and capable of using a wide range

of suitable container habitats such as man-made containers (e.g., discarded tires,

cemetery vases, cans), natural tree holes, bamboo internodes, and other phytotelmata

(Hawley 1988).

Adult females deposit desiccation resistant eggs on walls of containers, and these

eggs hatch when flooded by water (Hawley 1988). Embryonated eggs are able to survive

several months at 20-250C and at moderate to high humidity (44-90%) (Sota and Mogi

1992, Hawley 1988). The embryonation period is temperature-dependent but usually can

be completed within two days to just over a week. Aedes albopictus from temperate

Japan have photoperiodically inducible egg diapause, whereas A. albopictus of tropical

origins ordinarily do not (Lounibos et al. 2003a, Pumpuni et al. 1992, Hawley et al.

1987). In continental Asia and the U.S., 0C and -5C are the approximate northern

maximum isotherms for overwintering range and northward expansion, respectively

(Nawrocki and Hawley 1987).

Larvae in water-filled containers filter-feed and browse on decomposing plant

detritus and microorganisms. Studies quantifying development time of larval stages have

usually been performed at 250C and under optimal nutrition. These conditions allow

for complete larval development in 5-10 days (Hawley 1988). Low temperature,

crowded larval conditions, and nutrient deprivation greatly increase development time

(e.g., Lounibos et al. 2002, Alto and Juliano 2001ab, Briegel and Timmermann 2001,

Teng and Apperson 2000). The non-feeding pupal stage lasts 1-3 days. Pupal size is

determined to a large extent by larval density and food supply, however other factors

(e.g., temperature) may also be important (Hawley 1988).









Studies quantifying adult longevity have largely been conducted in the laboratory

and are likely to overestimate longevity in the field. Under laboratory conditions,

females can live for several weeks and perhaps as long as a few months. High

temperature coupled with low humidity decreases adult longevity (Alto and Juliano

2001b, Mogi et al. 1996). Female A. albopictus blood feed diurnally and are able to

blood feed within 2-3 days of emergence (Hawley 1988). Aedes albopictus is an

opportunistic biter taking blood meals from a variety of hosts, including humans

(Ponlawat and Harrington 2005, Niebylski et al. 1994, Savage et al. 1993). It is capable

of dispersing > 800 m in suburban settings (Honorio et al. 2003). Female fecundity is

positively correlated with size but other factors may also influence fecundity (e.g.,

temperature, size of blood meal) (e.g., Armbruster and Hutchinson 2002, Lounibos et al

2002, Briegel and Timmermann 2001, Blackmore and Lord 2000, Briegel 1990, 1985).

Aedes aegypti

The yellow fever mosquito Aedes aegypti (L.) has its origins in Africa. Water

vessels aboard slave ships are thought to have transported immature stages of A. aegypti

from West Africa to the Western Hemisphere during the 15th to 17th centuries

(Christophers 1960). However, A. aegypti may have established in Portugal and Spain

prior to its arrival in the Western Hemisphere (Tabachnick 1991). It is likely that A.

aegypti spread subsequently to the Mediterranean, tropical Asian, and Pacific Islands

during the 18th, 19th, and 20th centuries, respectively (Tabachnick 1991). In Sub-Saharan

Africa, at least 2 forms ofA. aegypti exist, differing genetically, morphologically, and

behaviorally (Tabachnick et al. 1979, Mattingly 1957). The sylvan form, A. aegypti

formosus (Walker), is darkly colored and found in natural phytotelmata (treeholes) and

confined to East Africa (Christophers 1960). Aedes aegyptiformosus feed on a variety of









vertebrates, including primates, but primarily feed on reptiles and small mammals

(McClelland and Weitz 1963). The domestic form, A. aegypti aegypti (L.), is lighter in

color and highly anthropophilic, blood feeding predominantly on humans and occupying

artificial containers in its immature stages. The domestic form is referred to as A. aegypti

and the sylvan form as A. aegyptiformosus. Adults of A. aegypti commonly oviposit and

blood feed in human dwellings. The adaptation to artificial container habitats and blood

feeding on humans has made A. aegypti highly successfully in spreading throughout

much of tropical to mild temperate regions (Lounibos 2002, Christophers 1960). Aedes

aegypti is considered the primary vector of DENV.

Female A. aegypti adults deposit desiccation resistant eggs within a wide range of

artificial containers, both outdoors and indoors, in urban environments. Eggs of A.

aegypti are more resistant to mortality induced by high temperatures and desiccation as

compared to A. albopictus (Juliano et al. 2002, Sota and Mogi 1992). The embryonation

period is similar to that of A. albopictus and there is no evidence that eggs, or any

developmental stage ofA. aegypti, are capable of diapause.

A. aegypti larvae develop more rapidly than A. albopictus on artificial nutrient

resources (e.g., yeast, albumin) but develop more slowly compared to A. albopictus on

leaf litter (B.W. Alto, personal observation). This is consistent with the observation that

larval resistance to starvation was maximized with leaf litter for A. albopictus and with

liver powder (non-natural) food for A. aegypti (Barrera 1996b). Pupal developmental

period is similar to A. albopictus and highly dependent on temperature (-2 3 d at 23-

27C) (Christophers 1960).









In extreme cases, maximum survival of adult females in laboratory settings may be

>100 days, however, longevity is highly dependent on abiotic conditions (temperature,

humidity) (Mogi et al. 1996) as well as adult size and nutrient availability (water,

carbohydrates, blood) (Christophers 1960). Regular access to carbohydrates and blood,

coupled with high humidity and -280C is optimal for A. aegypti adult longevity

(Christophers 1960). A mark-release-recapture field study in Kenya determined

longevity of adult A. aegypti during the rainy season (April-May 1972, environmental

conditions unspecified) (Trpis and Hausermann 1986). Mean-maximum adult female and

male longevity was 10.7-42 and 5.8-8 d, respectively. Aedes aegypti often prefer human

hosts for blood meals and may imbibe multiple blood meals in a single gonotrophic cycle

(Scott et al. 2000ab, 1993ab). Large A. aegypti consume more than twice as much blood

as small individuals, and subsequent efficiency of yolk synthesis derived from the blood

meal is positively related to female size (Briegel 1990). Adult dispersal may be hundreds

of meters (e.g., 100 to >800 m) in rural and urban dengue endemic regions (Harrington et

al. 2005, Honorio et al. 2003).

Dengue Virus

Introduction

Dengue virus (DENV) consists of the dengue serotypes 1-4. These are the

etiological agents of human disease that range in severity from undifferentiated dengue

infection (asymptomatic or mildly symptomatic), classical dengue fever (DF), dengue

hemorrhagic fever (DHF), to dengue shock syndrome (DSS) (Gubler 1997, Gubler et al.

1981). DF is characterized by an abrupt febrile illness with associated malaise, headache,

retro-orbital pain, rash, and extreme muscle and joint pain. DF is not known to be

associated with mortality. Initial symptoms of DHF resemble those of DF followed by









thrombocytopenia, hemorrhagic manifestations, and plasma leakage due to increased

vascular permeability from the release of circulating factors in infected white blood cells

(e.g., monocytes, T cells ). DSS is characterized by severe DHF followed by shock

where patients experience restlessness, rapid and weak pulse, subnormal temperature, and

low blood pressure. DENV is considered among the most important vector-transmitted

arboviruses and its geographic range places 2.5 billion humans at risk (review in Gubler

2002). Annually 50-100 million cases of DF occur in tropical cities with hundreds of

thousands of cases of DHF (<1-15% DHF mortality) (Gubler 2002).

An A. aegypti eradication program initiated by the Pan American Health

Organization in the 1940s and 1950s was successful at limiting Aedes aegypti distribution

in the Americas (Gubler 1997). However, the program was disbanded in the 1970s

followed by A. aegypti reinfestation in most areas which the program had targeted.

Dengue activity has increased in recent decades and poses a major global public health

problem (Gubler 1997). Although the reasons are complex and not fully understood,

factors that may contribute to increased dengue activity include reinvasion of tropical

America by the primary vector of DENV, A. aegypti, ineffective mosquito control in

areas associated with dengue, unplanned urbanization, abundant man-made larval

habitats, and increased and rapid human travel.

Human Infection

A study on naturally acquired DENV of serotypes 1, 3, and 4 in Central Java,

Indonesia, showed patients with viremia ranging from 103.8 to > 108.0 MIDso/ml

(Mosquito Intrathoracic Inoculation Dose for 50% infection) that lasted for 5-6 days in

some cases (Gubler et al. 1979). Similarly in Jakarta, Indonesian patients with dengue

fever showed a viremia range of 2-12 days with an average of 4-5 days (Gubler et al.









1981). Viral titer in these patients ranged from 103.8 to 107.2 MIDo0/ml, with DENV

serotype 4 infected patients showing 102 times lower titer (Gubler et al. 1981). Gubler

et al. (1981) did not find that disease severity was significantly affected by duration or

magnitude of viremia. However, fatal DHF cases had large amounts of circulating

DENV (Gubler et al. 1981). DENV pathogenesis is difficult to study because there are

no in vivo or in vitro models manifesting pathology similar to humans (Leitmeyer et al.

1999). Despite unique human pathology, nonhuman primates and mice have served

traditionally as human surrogates in dengue laboratory models (Gubler 1997).

Infection Cycle in the Mosquito Vector

The DENV transmission cycle includes a human reservoir and a mosquito vector,

although sylvatic cycles occur between monkeys and mosquitoes in tropical Africa and

Asia. After imbibing an infectious bloodmeal, arboviruses (e.g., DENV) are deposited in

the mosquito midgut and an infection may initiate. Biological transmission of

arboviruses includes acquisition from an infectious bloodmeal, replication in the

mosquito, dissemination of virus throughout the body of a mosquito resulting in a

generalized infection, movement of virus into salivary glands via hemolymph or neural

pathways, and transmission to a host by subsequent bloodfeeding (Hardy et al. 1983).

Additional biological routes include transovarial and venereal transmission.

Successful biological transmission of an arbovirus requires that several internal

physical barriers must be overcome in the mosquito. The ingested infectious blood is

deposited into the posterior midgut. Within a matter of hours viruses migrate toward the

microvillar margins of the mesenteronal epithelial cells midgutt cells) (Hardy et al.

1983). Mechanisms by which viruses enter the midgut cells are not well known, but









attachment with receptor-mediated-entry is thought to be a common mechanism. Once in

a midgut cell, the virus releases its nucleic acid and replicates. The midgut infection

barrier is the first barrier that the arbovirus must overcome in the infection process

(Gomez-Machorro et al. 2004, Bosio et al. 2000, Woodring et al. 1996, Hardy et al.

1983). Crossing of this barrier is thought to be dose-dependent such that the likelihood of

infection increases with increased viral titer in the blood meal (Lord et al. 2006, Hardy et

al. 1983). At this stage, arboviral infection is limited to the mesenteronal epithelial cells.

The next barrier to overcome in the arboviral infection cycle is the midgut escape barrier

(Bennett et al. 2005ab, Bennett et al. 2002, Myles et al. 2004, Woodring et al. 1996).

Crossing of this barrier, which consists of multilayer basal laminae, is also thought to be

dose-dependent (DeFoliart et al. 1987). If arboviruses fail to overcome the midgut

escape barrier, then infections are limited to the mesenteronal epithelial cells. If the

midgut escape barrier is overcome, the arbovirus enters the hemocoel from where it can

disseminate, via hemolymph, to other tissues and organs (e.g., fat body, foregut, hindgut,

ovarioles, salivary glands). These midgut barriers have important epidemiological

significance because they, in part, determine whether mosquitoes become potential

arboviral transmitters. Intrathoracic inoculation of arboviruses (ie., bypass midgut

barriers) is highly efficient at infecting mosquitoes, and may effectively eliminate

interspecific differences in vector competence, even species that may otherwise show

refractoriness to arboviral infection or transmission, perhaps due to barriers, (i.e., genetic

refractoriness) (Woodring et al. 1996, Hardy et al. 1978).

The final two barriers to transmission by mosquitoes are the salivary gland

infection barrier and the salivary gland escape barrier (Grimstad 1985 et al., Hardy et al.









1983). Crossing of the salivary gland infection barrier is dose-dependent and time-

dependent. The reason for the time-dependency is that, in many instances, the longevity

of the female adult mosquito and the extrinsic incubation period are similar. The time

from initial ingestion of the infectious blood meal until the time the mosquito can

transmit the arbovirus is the extrinsic incubation period. Thus, for successful

transmission of the arbovirus to another host, it must pass all the barriers and infect the

salivary glands before the mosquito dies. The paired salivary glands consist of a single

layer of cuboidal epithelial cells surrounded by a basal lamina. If the salivary gland

infection barrier is overcome, the arbovirus may replicate in the cuboidal cells. Finally, if

the salivary gland escape barrier is overcome, virus is incorporated into the saliva and is

potentially transmitted to a vertebrate host during the next blood feeding. These barriers

determine the intrinsic ability of a mosquito to become infected and subsequently

transmit a pathogen (i.e., vector competence). Mosquitoes with disseminated DENV

infection are capable of transmitting virus for the remainder of their life (Rodhain and

Rosen 1997).

Sylvatic Dengue Cycles

Sylvatic dengue cycles are known to occur in West Africa and Malaysia involving

Aedes species mosquitoes and monkeys (Diallo et al. 2003, Wang et al. 2000, De Silva et

al. 1999, Gubler 1997, Rodhain 1991, Rudnick 1978, 1965). Sylvatic dengue cycles are

not known in the Western Hemisphere (Rodhain and Rosen 1997). However, antibodies

to DENV have been recovered from bats and other mammals in Costa Rica, Ecuador, and

French Guiana (de Thoisy et al. 2004, Platt et al. 2000). Further research on sylvatic

cycles in the Western Hemisphere is needed (e.g., viral isolation) since neutralizing

antibodies may cross-react with other related viruses in those regions (Scott 2001, Innis









1997). Monkeys infected with DENV are not known to exhibit human-like DF or DHF

symptoms. In West Africa, DENV-2 has been isolated from A. africanus (Theobald), A.

luteocephalus (Newstead), A. opok (Corbet and Van Someren), A. furcifer (Edwards), and

A. taylori (Edwards) (Diallo 2003, Wang et al. 2000). In Malaysia, it is believed that the

canopy dwelling mosquito A. niveus (Ludlow) serves as the vector for all the DENV

serotypes to monkeys (Wang et al. 2000). DENV isolates from nonprimate reservoirs are

not known to exist, although neutralizing antibodies have been found from mammals in

the Eastern Hemisphere (Rudnick 1965).

Viral Isolates and Experimental Infection/Transmission

DENV is one of > 70 arboviruses within the genus Flavivirus (family Flaviviridae)

(White and Fenner 1994). Flaviviruses are enveloped, spherical virions with a diameter

of 40-50 nm. They have a linear plus sense single stranded RNA genome of 10.5-11 kb

and are capped at the 5' terminus but not polyadenylated at the 3' terminus.

Endemic/epidemic dengue cycles between humans and the primary and secondary

vectors, A. aegypti and A. albopictus, as well as A. polynesiensis (Marks). Field

collections of naturally infected mosquitoes suggest that other vectors may include A.

mediovittatus (Coquillett), A. scutellaris (Walker), A. cooki (Belkin), and A. hebrideus

(Edwards) (Rodhain and Rosen 1997, Freier and Rosen 1988). It is thought that the

DENV serotypes that now cause epidemics independently evolved from sylvatic

progenitors 100 to 1,500 years ago, presumably when DENV adapted to peridomestic

A. albopictus and later to A. aegypti (Moncayo et al. 2004, Wang et al. 2000). Thus,

sylvatic DENV serotypes, typically cycling between sylvatic mosquitoes and monkeys,

differ from endemic/epidemic DENV serotypes transmitted by A. aegypti, A. albopictus,









and other anthropophilic Aedes mosquitoes (Moncayo et al. 2004). All sylvatic DENV

serotypes are found in Malaysia whereas only sylvatic DENV-2 occurs in Africa,

suggesting that DENV may have originated in the Asian-Oceanic region (Wang et al.

2000). If this hypothesis is true, then the peridomestic transmission of DENV was most

likely initially vectored by A. albopictus since A. aegypti did not establish in Asia until

the later half of the 19th century (Tabachnick 1991, Smith 1956). Both A. albopictus and

A. aegypti are more susceptible to infection with endemic/epidemic DENV-2 than to

sylvatic DENV-2, supporting the hypothesis of emergence of endemic/epidemic dengue

by viral adaptation to peridomestic Aedes spp. (Moncayo et al. 2004).

Endemic/epidemic DENV serotypes differ in their ability to infect A. albopictus

and A. aegypti (e.g., Moncayo et al. 2004, Armstrong and Rico-Hesse 2003, 2001, Rosen

et al. 1985). Previous literature suggested that all DENV serotypes infected and

disseminated in A. aegypti more poorly than in other Aedes species, including A.

albopictus (Rodhain and Rosen 1997, Rosen et al. 1985, Gubler et al. 1979). However,

these studies used highly adapted laboratory colonies of these Aedes species, which may

have altered vector competence due to founder effects, genetic drift, and unintentional

artificial selection imposed on laboratory colonies (Armstrong and Rico-Hesse 2001,

Lorenz et al. 1984). A laboratory study on the F1 F2 progeny of field collected

mosquitoes in Vietnam and Thailand showed that A. aegypti were more readily orally

infected than A. albopictus (mosquito head assays) with SE Asian DENV-2 (Vazeille et

al. 2003). Similarly, laboratory colonies of Aedes collected in Taiwan (> F5) showed A.

aegypti had significantly higher salivary gland infection and transmission rates for

DENV-1 compared to A. albopictus (Chen et al. 1993). The conflicting observations in









the cited studies demonstrate that it is unclear whether A. albopictus or A. aegypti is the

more competent DENV vector. It appears that vector competence of these two species

critically depends on underlying genetic differences in the strains of mosquitoes, the

strains of DENV, and the environmental conditions under which the laboratory analyses

are conducted. The complexity of investigating vector competence mechanisms and

variation has been discussed elsewhere (Tabachnick 1994).

Vertical transmission of of arboviruses (e.g., transovarial) may serve as a

mechanism to survive inhospitable environmental conditions (e.g., cold temperatures,

drought). For arboviruses that cycle between mosquito and humans (e.g., DENV in the

Western Hemisphere), vertical transmission may facilitate endemic maintenance,

especially when human cases are not occurring. Experimental studies have shown that

both A. albopictus and A. aegypti are capable of vertical transmission of DENV as

determined by detection of DENV antigen or viral isolation among immature stages (e.g.,

Joshi et al. 2006, 2002 1996, Rodhain and Rosen 1997, Bosio et al. 1992, Rosen et al.

1983). For example, field collections of immature stages ofA. aegypti in India and

Burma showed definitive evidence ofDENV-2 and DENV-3 vertical transmission

because infected field mosquito supernatant fluid was inoculated in Toxorhynchites

splendens (Weidemann) mosquitoes, allowed to replicate, and DENV antigen was

positively recovered from head tissues (Thenmozhi et al. 2000, Khin and Than 1983).

Sindbis Virus

Introduction

Sindbis virus (SINV) was first isolated from mosquitoes Culex univittatus

(Theobald), Culexpipiens (L.), and a juvenile hooded crow Corvus corone sardonius in

1952 in Sindbis Egypt, 30 km north of Cairo (Taylor et al. 1955). SINV has a wide









geographic distribution in Australia, Scandinavia, South Africa, Middle East, and Asia

(Laine et al. 2004, Dohm et al. 1995, Niklasson 1989, Tesh 1982). SINV has been given

distinct names based on the geographic region of isolation i.e., Ockelbo (Sweden),

Pogosta (Finland), Karelian (Russia), and SINV (other regions) (Laine et al. 2004). In all

instances the viruses are similar and represent geographically distinct genotypes (Kurkela

et al. 2004, Laine et al. 2004, Sammels et al. 1999, Lundstr6m 1999, Norder et al. 1996,

Shirako et al. 1991, Lundstr6m et al. 1993a, Olson and Trent 1985). SINV is not known

to occur in the Americas. The wide distribution is partially attributable to migratory birds

which serve as reservoirs and transport SINV over large distances (e.g., Buckley et al.

2003, Brummer-Korvenkontio et al. 2002, Lundstr6m et al. 2001, Lundstr6m et al.

1993b).

Human and Reservoir Infection

Clinical symptoms of SINV infection were described from Uganda in 1961

(Woodall et al. 1962). The most complete record of human cases include SINV

epidemics in Sweden, Finland, and Russia during late summer and fall (August -

October) (Laine et al. 2004, Lundstr6m et al. 1991). Human SINV infections produce a

self-limited febrile disease characterized by arthralgia, rash, headache, fatigue, and fever

(Laine et al. 2004, 2000, Tesh 1982). It is not uncommon for chronic arthralgia to last for

several years after full recovery from other symptoms (e.g., Kurkela et al. 2005, Laine et

al. 2000, Turunen et al. 1998, Niklasson et al. 1988, Niklasson and Espmark 1986).

SINV is not known to cause human mortality, although morbidity is common.

Although SINV has a wide geographic distribution, human disease with clinical

symptoms has been limited to Northern Europe and South Africa (Lundstr6m 1994,

Niklasson et al. 1988). Nucleotide sequences of genes encoding capsid (C) and envelope









protein (E2) showed that SINV strains from Northern Europe were most closely related

to those strains from South Africa compared to strains from other geographic regions

(Norder et al. 1996, Shirako et al. 1991). Intercontinental exchange of SINV strains may

be facilitated by migratory birds because 25% of the 252 bird species that breed in

Scandinavia overwinter in Africa (Norder et al. 1996, Lundstr6m et al. 1993a, Shirako et

al. 1991).

Initially SINV infection was regarded as a minor human disease because there were

few human cases. However, several outbreaks occurred in South Africa in 1974

involving C. univittatus and Culex theileri (Theobald) (Jupp et al. 1986a, McIntosh et al.

1976, 1967, 1964), and Northern Europe in the 1980's including Sweden (Lundstr6m et

al. 1991, Espmark and Niklasson 1984, Niklasson et al. 1984, Skogh and Espark 1982),

Finland (Kurkela et al. 2004, Brummer-Korvenkontio et al. 2002, Brummer-

Korvenkontio and Kuusisto 1981), and Russia (Lvov et al. 1984, 1982). Mosquito

vectors of SINV in Northern Europe and Russia include Culex spp., Culiseta spp., A.

cinereus (Meigen), and Aedes communis (DeGeer) (Lundstr6m 1999, 1994). SINV is the

most common virus isolated from mosquitoes in Australia where the principal vectors

include Culex annulirostris (Skuse) and Aedes normanensis (Taylor) (Niklasson 1989).

However, clinical symptoms in Australia have only been reported on a few occasions

(Sammels et al. 1999).

Viral Isolates and Experimental Infection/Transmission

SINV is a prototype Alphavirus in the family Togaviridae. Alphaviruses are

enveloped, spherical virions with icosahedral capsids and a diameter of 70 nm (White and

Fenner 1994). They have a linear plus sense single stranded RNA genome 11-12 kb, are

capped at the 5' terminus, and are polyadenylated at the 3' terminus. The most complete









record of SINV isolates from field-collected mosquitoes and experimental infection /

transmission studies are from South Africa and Sweden. Typically, zoonotic circulation

of SINV occurs between ornithophilic Culex and Culiseta spp. and passerine birds,

although other vector species and avian orders may also be involved in transmission

cycles (Lundstrom 1999, 1994, Lundstrom et al. 1993b, Francy et al. 1989). The single

SINV isolate from an arthropod, not a mosquito, was from a Hyalomma marginatum tick

(Koch) in Italy (Sicily) in 1975 (Gresikova et al. 1978). Viral isolates from field-

collected birds during SINV epidemics in South Africa (1960s-1970s) showed high SINV

immune rates for several bird species. Viral isolates from field-collected ornithophilic

mosquitoes C. univittatus and C. theileri showed infection rates of 0.65% and 0.083%,

respectively. Culex univittatus was observed to readily feed on humans and was regarded

as the main epidemic vector for SINV in the region (McIntosh et al. 1978, 1976).

However, C. neavei (Theobald) may be an important vector in costal South Africa,

although it may not acquire a disseminated infection as readily as C. univittatus (Jupp et

al. 1986b). Laboratory experiments were used to determine the vector potential of Culex

spp. derived from South Africa (Jupp et al. 1972, Jupp and McIntosh 1970ab). Culex

univittatus (F6-15) was readily infected (50-83%) after SINV infectious bloodmeals

(separate feeding trials with a range of viral titers 10 3.6 5.6) and 57-66% transmitted

SINV to avian hosts after taking a subsequent bloodmeal (Jupp and McIntosh 1970a). A

similar experiment showed higher (74-100%) C. theileri (F3-11) infection with similar

SINV titers, but transmission was much lower (9%) (Jupp et al. 1972). Both infection (0-

16%) and transmission (0-50%) in C. pipiens (F4-10) were lower than for the other Culex

spp. (Jupp and McIntosh 1970b).









Sweden is dominated by mammalophilic Aedes species (Francy et al. 1989, Jaenson

and Niklasson 1986). Field collections made in central Sweden showed that 60% of all

Aedes spp. collected were A. cinereus, whereas only 3.4 and 3.7% (of total mosquitoes

collected) were Culex and Culiseta spp., respectively. Despite their infrequency in

collections, C. pipiens, C. torrentium (Martini), and C. morsitans (Theobald) accounted

for 80% of all SINV isolates (Francy et al. 1989). Minimum infection rates were -7-14%

for C. pipiens and C. torrentium, and -2-5% for C. morsitans. Minimum infection rates

for A. cinereus were 0.5%. Experimental inoculations in indigenous bird species showed

that Passeriforms (105.8 to 107.5 Plaque forming units/ml) (PFU/ml) had significantly

higher and longer viremia compared to Anseriforms (103.7 to 104.5 PFU/ml ) (Lundstrom

et al. 1993b). Passeriform thrushes (Turdus spp.) and finches (Fringilla spp.) are the

major SINV reservoirs in Sweden (Lundstr6m et al. 2001, Lundstr6m 1994). The

presence of neutralizing antibodies to SINV in passerine reservoirs was detected in

summer but not spring bird migrants (Francy et al. 1989). It is likely that Culex and

Culiseta spp. are important vectors in the enzootic cycle involving passerine birds,

whereas A. cinereus and A. communis are probable bridge vectors to humans (Francy et

al. 1989, Jaenson and Niklasson 1986, Lundstr6m 1999, 1994). This epidemiological

hypothesis is supported by SINV isolates from a suspected bridge vector A. communis of

SINV human infections in Russia (Lvov et al. 1984).

Experimental infection and transmission studies were used to determine the vector

potential of Culex spp. from central Sweden (Lundstr6m et al. 1990ab). C. torrentium

(F3-6) infected and transmitted SINV to avian hosts more efficiently than C. pipiens (F4-

10). Even at low infectious bloodmeal titers (<2.0 PFU/ml), 50% of C. torrentium were









infected with SINV. Blood meal titers of >3.0 PFU/ml resulted in 90-100% infection and

100% transmission. For C. pipiens, 3.0-3.0 PFU/ml resulted in 4% infection, whereas

higher titers (6.0-8.9 PFU/ml) resulted in 42 to 55% infection and 14 to 37% transmission

(Lundstr6m et al. 1990a).

A laboratory experiment determined the effect of natural temperature regimes (10,

17, 24, cyclic 10-240C) during the transmission season in Sweden on Culex spp. vector

competence (Lundstr6m et al. 1990b). Low temperature significantly reduced

transmission potential of C. pipiens, as measured by SINV dissemination, compared to

higher temperatures. In contrast, dissemination in C. torrentium was rapid and

unaffected by temperature regimes. This result was unexpected and contrary to the

established thought that extrinsic incubation period is inversely related to temperature

(e.g., Reisen et al. 2006). Thus, although both Culex spp. may serve as enzootic vectors

in Sweden, C. pipiens transmission potential may be broken under cooler conditions,

whereas C. torrentium is likely to persist as an efficient SINV vector in cool weather. An

identical experiment using Aedes spp. showed similar SINV infection between

temperature regimes, however, transmission was lower and occurred later at low

temperature compared to high temperature (Turell and Lundstr6m 1990). Low

temperatures were associated with longer extrinsic incubation periods in A.

taeniorhynchus and A. aegypti, which are not known as natural vectors of SINV

(Lundstr6m et al. 1990b).

Adult A. communis, A. cinereus, and A. excrucians were collected from Sweden

and allowed to blood feed on SINV infected chickens (104.2 PFU/ml) (Turell et al. 1990).

All three Aedes spp. were highly susceptible to SINV infection (96-100%) and had









dissemination rates ranging from 51-100%. Although A. communis failed to refeed, the

other Aedes spp. had a 50% transmission rate. These Aedes spp. are competent SINV

vectors and should be regarded as potential links between the enzootic SINV cycle and

human infections in Scandinavia. SINV has been repeatedly isolated from both A.

communis and A. cinereus during episodes of human infection. Further, these Aedes spp.

are active day biters on mammals, including humans, but will also bite birds (Turell et al.

1990).

Laboratory experiments with SINV using easily colonized mosquito species (e.g.,

A. aegypti, A. albopictus) have proven useful to address questions about experimental

infection and transmission, genetically modified arboviruses (e.g., SINV gene expression

vectors), and the dynamics of arboviral tissue tropism and pathology in mosquito vectors

(e.g., Bowers et al. 2003, Bowers et al. 1995, Jackson et al. 1993, Xiong et al. 1989).

High (108.4 PFU/ml) SINV titers resulted in greater infection compared to moderate (105.3

PFU/ml) SINV titers (Percent infected for high-moderate titers; A. albopictus, 90-49%;

C. pipiens, 48-0%, A. aegypti, untested-18%). Aedes albopictus had greater

dissemination (66%) and transmission rates (53%) compared to A. aegypti (9 and 7%) at

the lower titer (Dohm et al. 1995).

A study on SINV replication and tissue tropism following intrathoracic inoculation

in A. albopictus showed temporal and organ-specific distribution of the virus during the

extrinsic incubation period (Bowers et al. 1995). Many organs had maximal infection

within 3-4 days after infection because the gut barriers were bypassed by intrathoracic

inoculation. Some organs were refractory to infection (e.g., ovarioles, malpighian

tubules), whereas others had transient or persistent infections, perhaps indicating viral









modulation by the mosquito vector or SINV (e.g., Bowers et al. 1995, Luo and Brown

1993, Murphy et al. 1975). SINV-associated pathology of the salivary glands and midgut

muscle tissue of A. albopictus has been observed (Bowers et al. 2003). Typically,

arboviruses have few cytopathic affects on mosquito cells (in vitro and in vivo) (Hardy et

al. 1983). Similarly, a study on A. aegypti following oral infection showed rapid

infection of many organs within several days after feeding with the salivary glands being

infected by day 5 (Jackson et al. 1993). As was the case for A. albopictus, some organs

ofA. aegypti were refractory to infection (e.g., ovarioles, malpighian tubules). Unlike A.

albopictus, there was no indication that the distribution of SINV in organs changed from

days 6-14 (Jackson et al. 1993).

Competition and Vector Competence

Classic laboratory and field research established evidence for the importance of

interspecific competition for a variety of systems (e.g., Connell 1961, Birch 1953,

Hairston 1951, Crombie 1947, Park 1948). Despite numerous studies, establishing the

existence and importance of competition in nature may be difficult and has been

historically a topic of debate (e.g., Hairston et al. 1960). Reviews on this topic have

provided concise evidence that interspecific competition is widespread in natural systems

for a variety of organisms (e.g., Reitz and Trumble 2002, Connell 1983, Schoener 1983,

Crombie 1947). More recently, interspecific competition has been invoked as a

mechanism by which competitively superior invasive plant and animal species alter the

distribution and abundance of established species (e.g., Juliano and Lounibos 2005,

Levine et al. 2002, Reitz and Trumble 2002, Byers and Goldwasser 2001, Mack et al.

2000, Holaway 1999, Petren and Case 1996, D'Antonio and Vitousek 1992).









Intra- and interspecitic competition between larval mosquitoes is common and

plays an important role in dermining population growth measurements. Competition has

been demonstrated in laboratory and field experiments for several mosquito vector

species that occupy a variety of aquatic habitats (e.g., Costanzo et al. 2005ab, Peck and

Walton 2005, Juliano and Lounibos 2005, Braks et al. 2004, Juliano et al. 2004, Ye-

Ebiyo et al. 2003, Gimnig et al. 2002, Gleiser et al. 2000ab, Schneider et al. 2000, Juliano

1998, Barrera 1996b, Leonard and Juliano 1995, Broadie and Bradshaw 1991).

Mechanisms involved in mosquito competition have largely been attributable to limiting

resources (e.g., food) (e.g., Juliano 1998, Barrera 1996b), although interference

competition, mediated by direct physical contact or chemical excretions, may also be

important. However, studies with mosquitoes have yielded mixed results and additional

studies are needed to evaluate the role of interference competition in natural systems

(Broberg and Bradshaw 1995, Broadie and Bradshaw 1991, Dye 1984, 1982, Moore and

Whitacre 1972, Moore and Fisher 1969). Resource type (e.g., leaves) and abundance and

larval density affect mosquito fitness such that high intra- and interspecific larval density

and low resources result in increased larval development time and mortality and

decreased adult size, fecundity, longevity, and per capital rate of growth (e.g., Alto et al.

2005, Costanzo et al. 2005ab, Peck and Walton 2005, Juliano et al. 2004, Lounibos et al.

2003b, 1993, Gimnig et al. 2002, Daugherty et al. 2000, Schneider et al. 2000, Teng and

Apperson 2000, Yanoviak 1999, Juliano 1998, Leonard and Juliano 1995, Hawley 1985).

Competition is well documented among container mosquitoes and may be

important to some mosquitoes in other aquatic habitats. Arbovirus-mosquito research has

mainly focused on intrinsic (e.g., genetic) and extrinsic (e.g., temperature, blood meal









viral titer) factors of adult biology that determine vector competence (Tabachnick 1994).

Few studies have attempted to address how biological conditions experienced by larvae

may determine subsequent adult vector competence.

It is likely that effects of larval competition have an impact on the adult stage and

influence adult vector competence of arboviruses. The most extensive research has been

conducted on the effect of nutrient deprivation on mosquito vector competence. Low

food availability among Ochlerotatus triseriatus (Say) produced smaller adults. These

small adults transmitted La Crosse virus (LACV) at higher rates than did larger adults

that resulted from well-fed larvae. However, the infection rates in these mosquitoes were

independent of adult body size (Grimstad and Haramis 1984, Grimstad and Walker

1991). Enhanced transmission efficiency of small 0. triseriatus adults was associated

with higher virus titers and dissemination rates compared to larger adults. Additional

support for size-dependent transmission comes from field-collected pupae that were

orally infected as adults with LACV and had dissemination and transmission rates that

were inversely correlated with adult size (Paulson and Hawley 1991).

The effect of larval nutrition and adult size on infection parameters has been

investigated in other mosquito species. Large A. aegypti adults produced under varying

conditions of larval crowding and food availability had a greater proportion of DENV-2

disseminated infection (New Guinea C strain) than did smaller females

(Sumanochitrapon et al. 1998). Similar results were found for A. aegypti susceptibility to

infection with Ross River virus (RRV) over a range of blood meal titers. Differences

between infection of small and large adults became less distinct at greater blood meal

titers (Nasci and Mitchell 1994), perhaps suggesting that high titers simply overwhelm









the differences seen at lower titers. Conversely, titer midgutt and head) as well as

DENV-2 (Puerto Rico and Ibo strains) dissemination were independent of A. aegypti

body size (Bosio et al. 1998). Low food availability among larvae of C.

tritaeniorhynchus (Giles) produced smaller adults that had shorter periods between initial

infection of Japanese encephalitis virus (JEV) and subsequent virus secretion in the saliva

than did larger adults from well-fed larvae. Additionally, small C. tritaeniorhynchus

adults had greater JEV transmission than did large adults (Takahashi 1976). Baqar et al.

(1980) showed a trend, although not significant, that increased larval densities and

decreased larval nutrition resulted in small adults with increased infection susceptibility

of C. tritaeniorhynchus to West Nile virus (WNV). Infection and transmission rates of

Murray Valley encephalitis virus (MVEV) were unaltered between two larval nutritional

regimes producing different sized adult C. annulirostris. Additionally, neither body nor

salivary gland viral titers were altered by larval nutrition (Kay et al. 1989). Similar

nonsignificant effects of the larval environment and adult size were found for C. tarsalis

(Coquillett) infection and transmission of St. Louis encephalitis virus (SLEV) and

Western Equine encephalomyelitis virus (WEEV) (Reisen et al. 1997).

Size-dependent differences in mesenteronal tissues may, in part, explain differences

in dissemination and transmission of arboviruses by adults of different sizes (Grimstad

and Walker 1991). Fewer basal lamina layers were present in the mesenteron of small

adult 0. triseriatus (4-6 layers) as compared to large adults (10-16 layers) which

weakened the midgut escape barrier (MEB) and thus enhanced dissemination and

transmission rates (Grimstad and Walker 1991, Paulson and Hawley 1991). An

alternative hypothesis explaining observed negative relationships between size (wing









length) and vector competence may be related to the number of viral particles imbibed

relative to mosquito body size. Large mosquitoes imbibe greater volume of blood, and

thus virus, than smaller mosquitoes. Additional support comes from greater viral titers

found in freshly bloodfed large adults than in small adults (Nasci and Mitchell 1994).

However, when the amount of virus imbibed was corrected for mosquito body weight,

small adults imbibe proportionally more virus than large adults in proportion to their

body weight (Nasci and Mitchell 1994, Grimstad and Haramis 1984). This explanation

may hold true for a number of mosquito species since blood meal titer is positively

related to infection, dissemination, and transmission (e.g., Turell et al. 2001, Dohm et al.

1995, Grimstad and Haramis 1984, Kramer et al. 1981).

The previous examples illustrate that larval nutrition affects adult mosquito

infection and transmission of mosquito arboviruses. However, the mechanism and details

are dependent on the particular mosquito-virus system. Some of the studies support the

hypothesis that larval resource competition enhances vector competence. Resource

competition alters numerous mosquito life history traits, however, these studies have

limited the focus to a single life history trait, adult size. Further, they did not address

whether the effect of resource competition on vector competence was causally related to

adult size, or alternatively (additionally) related to other physiological conditions

correlated with adult size (Grimstad and Walker 1991, Paulson and Hawley 1991).

Further, drawing conclusions about common themes from a limited number of studies

would be premature and perhaps misleading. Thus, controlled experiments are required

to determine quantitatively the effects of larval competition on vector competence for

multiple mosquito-virus systems, as well as to disentangle which mosquito life history









traits (e.g., size, development time) are most important in determining vector competence

parameters (infection, body viral titer, disseminated infection). Results from such

experiments may support with greater quantitative detail the hypothesis that resource

competition affects mosquito vectoring ability, or may offer alternative explanations for

the larval competition-adult vector competence relationship. The chapters that follow

describe the development of an artificial bloodfeeding system used in delivering

arbovirus infectious blood meals as well as experiments that evaluate the effects of

competition on A. aegypti and A. albopictus population growth measurements and SINV

and DENV infection parameters. The use of these Aedes species to investigate the

relationship between competition and arboviral infection is important because;

competition is well-documented between these Aedes species, competition has important

ecological effects on their distribution and abundance, and these Aedes are the most

important vectors of human arboviruses.














CHAPTER 2
AGE-DEPENDENT BLOODFEEDING OF Aedes aegypti AND Aedes albopictus ON
ARTIFICIAL AND LIVING HOSTS

Introduction

Since its introduction to the Americas in the mid 1980s (Hawley et al. 1987,

Sprenger and Wuithiranyagool 1986), Aedes albopictus has spread rapidly and colonized

much of the southeastern U.S. and Brazil. In parts of the eastern U.S., the invasion of A.

albopictus is associated with declines in the abundance, and in some instances

displacement, ofAedes aegypti in rural and suburban areas (Mekuria and Hyatt 1995,

O'Meara et al. 1995, Hornby et al. 1994, Hobbs et al. 1991). However, these Aedes

coexist in urban areas of south Florida. Recent comparative studies attempting to explain

the observed distributions of these Aedes have investigated egg desiccation (Juliano et al.

2002, Sota and Mogi 1992), larval competition (Lounibos et al. 2002, Daugherty et al.

2000, Juliano 1998, Barrera 1996a), adult desiccation (Mogi et al. 1996), and

reproductive and metabolic differences (Klowden and Chambers 1992).

One major concern about the A. albopictus invasion in the Americas has been its

potential as an arboviral disease vector (e.g., DENV). In recent decades, the range of A.

aegypti, the primary vector of DENV in the Americas, has increased, and dengue activity

has surged (Gubler 1997). The range of A. albopictus in the U.S. is more extensive than

that of A. aegypti, and its range in the U.S. is likely to continue to expand (e.g., Madon et

al. 2002). Aedes albopictus is a competent laboratory vector of numerous arboviruses









(Mitchell 1991, Shroyer 1986) including DENV in Asia and Hawaii; however, the degree

to which A. albopictus is involved in arbovirus transmission in the Americas is unclear.

With exceptions of transovarial and venereal transmission, successful biological

transmission of arboviruses requires acquisition of an infectious bloodmeal, or at least

probing behavior. For A. aegypti, research investigating factors that influence the normal

sequence of events in successful acquisition of a bloodmeal (e.g., host-seeking, probing,

bloodfeeding) have mainly focused on measurements of host-seeking behavior (Klowden

and Fernandez 1996, Klowden and Briegel 1994, Bowen 1991, Klowden et al. 1988,

Klowden and Lea 1984, 1979ab, 1978). Davis (1984) showed a linear increase in host-

seeking behavior ofA. aegypti from 1 to 5 days post-emergence followed by a constant

high response until the end of observations at 15 days. A study measuring probing

behavior in A. aegypti over a 21-day period showed a rhythmic pattern in probing activity

in response to a convection current of constant heat and moisture, but no probing pattern

was observed in response to a human host (Burgess 1959). However, the design of this

latter experiment was weak (e.g., experimental units were not replicated), and there was

little statistical support for the conclusion of rhythmic behavior. Few studies have

measured age-related acquisition of the initial bloodmeal, an important factor in

determining vector potential. Those that have done so have focused on bloodfeeding

over a short interval. Seaton and Lumsden (1941) showed a general increase in

bloodfeeding associated with age for 1-5-day-old starved virgin A. aegypti followed by

decreased bloodfeeding on day 6. They suggested that the decreased response on day 6

was attributable to female exhaustion. A similar increase in bloodfeeding with increasing

age was found for 3 strains of 1-4-day-old starved A. aegypti fed on chickens and









membrane systems (Bishop and Gilchrist 1946). In order to quantify age-dependent

acquisition of a bloodmeal, the present study compares bloodfeeding patterns of A.

albopictus and A. aegypti starting from the time of first responsiveness to a bloodmeal

(Hawley 1988, Christophers 1960) up to 15 days post-emergence.

Materials and Methods

Experimental Protocol

Aedes eggs used to initiate the experiments were derived from laboratory colonies

at the Florida Medical Entomology Laboratory in Vero Beach, FL. Both Aedes spp.

originated from Fall, 2000, field collections of larvae from water-filled cemetery vases in

Hillsborough County, FL, near Tampa. Colonies were housed in 0.03 m3 cages at (mean

SD) 24.6 0.40C, 76.6 6.7% RH, and a 14:10 (L:D) h photoperiod regime including

a 1 h dawn and dusk. Colonies had access to 20% sucrose solution ad libitum and

weekly bloodmeals from domestic chickens (handled in accordance with the National

Institutes of Health guidelines for the use of laboratory animals). Females were provided

with water-containing cups lined with paper towel as oviposition substrates. Eggs were

hatched, by species, in metal pans with 1.0 liter tap water and 0.30 g of a 1:1 lactalbumin

and brewers yeast mixture. Following hatching, approximately 300-500 larvae were

reared in pans, with water and food substrate changed every 2 days.

As soon as pupation occurred, inspections of the rearing pans were made daily,

and pupae were transferred into 40-ml vials with water until emergence. Vials were

checked daily between 1600 and 1800 h for newly emerged adults. These adults were

transferred, by species, to cylindrical cages (11 x 9.5 cm, ht x diam) with nylon mesh tops

and maintained under similar conditions as the parental generation except for









bloodfeeding. Female density per cage ranged from 5 to 47 with means SE of 14.8

9.9 and 14.2 8.2 for A. aegypti and A. albopictus, respectively. At least one male was

present in each cage for every 3-4 females, although many cages had equal numbers of

males and females. Examination of scatter plots of residuals versus predicted values

(Draper and Smith 1966) showed no evidence that the number of males per cage was in

any way related to proportions of females that bloodfed.

In Experiment 1, cages with Aedes females were haphazardly assigned to an age

treatment (e.g., 3, 4, ..., 15 days old). Each cage containing same-age adults ranging

from 3 through 15 days old was offered a bloodmeal from a silicon membrane feeding

system (Butler et al. 1984). Thus, same-age females were tested on many different days.

Females were deprived of sucrose, but not water, 24 h prior to bloodfeeding trials.

Before the start of a feeding trial, citrated bovine blood was heated to (mean SD) 37.8 +

1.1C in 1.5 ml circular wells and covered with a silicon membrane. Next, the membrane

feeding system was positioned over the mesh top of the cage for two 15-min periods

separated by a 15-min interval. Feeding trials were performed at (mean SD) 23.2

0.50C and 48.1 4.2% RH. After a feeding trial, the number of females that successfully

acquired a bloodmeal was recorded. If blood was visually detected in the female gut, it

was scored as having a bloodmeal. Thus no attempt was made to distinguish between

meals of different volumes. All feeding trials were performed in the late afternoon,

within 2-3 h of each other.

For Experiment 2, 3-15 day old Aedes were allowed to bloodfeed from a restrained

domestic chicken. The methods for mosquito husbandry and adult exposure during

feeding trials were the same as those used in Experiment 1. For all feeding trials,









uniformly sized and aged (6-8 wks old) chickens were restrained inside 0.03 m3 cages

into which adult Aedes were released and allowed to feed for 30 min. Female density per

cage ranged from 5 51 with means SE of 28.1 9.9 and 24.1 9.7, for A. aegypti and

A. albopictus, respectively. Larger cages were used for bloodfeeding in Experiment 2 to

provide greater space for the normal sequence of events involved in bloodmeal

acquisition (Clements 1999). Immediately following feeding trials, chickens were

removed from the cages, and then Aedes were removed from the cage using an electric

aspirator and killed by placing them at -200C for < Ih. The number of female Aedes that

had successfully bloodfed was recorded as in Experiment 1.

Data Analyses

For Experiments 1 and 2, proportions bloodfed for each species were calculated as

the numbers of females that acquired a bloodmeal during a trial divided by the total

numbers of females offered the bloodmeal. Experimental units were defined as the cage

of adult Aedes offered blood. Difficulties in predicting the number of females that would

emerge and survive to the day of feeding precluded equal sample sizes for each unique

species-by-age treatment. For Experiment 1, the numbers of replicates for each A.

aegypti and A. albopictus by age treatment were (mean SD) 5 1 and 5 2,

respectively (127 total cages). In Experiment 2, each unique species-by-age treatment

was restricted to 3 replicates, except for 15-day old A. aegypti, which had 4 replicates (79

total cages).

For both experiments, effects of female density per cage was tested as a continuous

variable (PROC GLM, SAS Institute 1989, Sokal and Rohlf 1995). Raw data adequately

met assumptions of normality and homogeneous variance except for membrane-fed A.









albopictus where the proportion bloodfed was transformed by logio(x + 1) to meet the

assumption of normality. Because effects of female density on proportion bloodfed were

all non-significant (P >> 0.10 in all cases), analysis of effects of female age were

performed on proportion bloodfed, which was assessed by treating age as a continuous

independent variable and comparing regression lines for each species by feeding protocol

treatment. This tests for equal slopes among species-feeding protocol groups to

determine whether the regression relationships were similar (SAS Institute 1989, Sokal

and Rohlf 1995).

Graphical presentation of the data appeared to show age-dependent periodicity in

feeding incidence. Therefore, separate Runs Up and Down Tests were performed for the

proportion bloodfed for each species-feeding protocol combination (Sokal and Rohlf

1995, Zar 1996). A run was defined as a temporal sequence of increases or decreases in

the proportion bloodfed. Difference between mean proportion bloodfed for consecutive

age groups was determined and resulted in a sequence of positive and negative changes in

proportion bloodfed across female ages (e.g., + + + = 2 runs). These tests determined

whether the number of runs for proportion blood-fed among females of different ages

was significantly different from random expectation.

As an additional test to address the apparent age-dependent periodic pattern of

feeding, four regressions were run (one for each species-feeding protocol combination) of

proportion blood-fed versus age, each with a sine function of age according to the model:


y = a bA + c sin(dA),









where y is the proportion bloodfed, a is the intercept, b is the slope, A is female age,

c is a parameter affecting the amplitude of the sine function, and d is a parameter

affecting the frequency of the sine function. Several different initial parameter estimates

were used to determine whether the addition of a sine wave function improved the fit of

the regression (SAS Institute 1989, PROC NLIN). If either c or d parameters were not

significant, the slope (b) was removed and the reduced model tested. Subsequently, if

either c or dwere not significant, both c and dwere removed from the model and a linear

regression was performed, including the slope, to determine whether or not there was a

trend in age-dependent blood-feeding.

Results

Regardless of age, a higher proportion of both A. albopictus and A. aegypti

bloodfed on the restrained chicken (mean SE; 59.8 2.4 and 81.3 2.3%, respectively)

compared to the membrane system (mean SE; 30.8 2.7 and 55.6 2.6%,

respectively).

Treating age as a continuous independent variable, there were significant age,

species, feeding protocol, and age x feeding protocol effects (Table 2-1). All other

effects were not significant. Slopes of proportion bloodfed vs. age were significantly

positive for both Aedes species feeding on the membrane system and were not

significantly different from zero for both Aedes species feeding on the restrained chicken

(Table 2-2). Although these slopes were significant, the low r2 values suggest that the

linear relationships were weak (Fig. 2-1, Table 2-2). In addition, the sine function

contributed significantly to the regression for A. aegypti (P < 0.0001, r2 = 0.391,

proportion fed = 0.188 + 0.042*(age) + 0.11*sine (9.91*age)). For both Aedes species









fed on the restrained chicken, the sine function did not contribute significantly to the

regression (Table 2-2). Averaged over both species, slopes for proportion bloodfed on

the membrane system were significantly greater, as shown by the age x feeding protocol

interaction, than those for Aedes fed on the restrained chicken (Tables 2-1, 2-2).

Runs Up and Down Tests for A. albopictus and A. aegypti fed on restrained

chickens showed that the number of runs was significantly different from random (both P

< 0.0275) with the number of runs being greater than expected compared to random (Fig.

2-2). Thus, proportion bloodfed on chickens showed a significant pattern of alternate

increases and decreases on alternate days of female mosquito age. Runs Up and Down

Tests were not significant (both P > 0.05) for either Aedes species fed on the membrane

system (Fig. 2-1).

Discussion

In Experiment 1, using the membrane feeders, there was a significant increase in

proportion bloodfed as age increased (Table 2-2, Fig. 2-1). In Experiment 2, with

restrained chickens, there was no significant increase in bloodfeeding associated with

increased age (Table 2-2, Fig. 2-2). Further, the membrane-fed and chicken-fed

mosquitoes showed significantly different trends (Table 2-2). Thus, the temporal pattern

of bloodfeeding is strongly affected by the blood source used in experiments. Host-

related cues (e.g., C02, surface area) may be partially responsible for the observed

differences in pattern of bloodfeeding and should be taken into consideration in

bloodfeeding research using Aedes mosquitoes of different ages, especially for silicon

membrane systems. The lack of significantly positive slopes for chicken-fed mosquitoes

is likely due to a higher proportion of bloodfed younger Aedes as compared to the

membrane-fed mosquitoes.









Results showed significant age effects on bloodfeeding for both A. aegypti and A.

albopictus. Davis (1984), in a study with naive A. aegypti females of uniform ages

ranging from 1 through 15 day old, showed a linear increase in host-seeking behavior for

1-5-day-old females, whereas females > 5 days old showed a consistently high (e.g.,

94%) response to a human hand. Results from the current study suggest that

bloodfeeding for these Aedes, over a similar period of time, shows some similarities to

host-seeking response observed by Davis. However, proportions bloodfeeding appear

additionally to exhibit distinct periodic patterns on alternate days of female mosquito age.

Significant but weak positive relationships were found for A. albopictus and A. aegypti

feeding versus age on the membrane system (Fig. 2-1, Table 2-2), and no positive

relationships for feeding versus age on the restrained chicken (Fig. 2-2, Table 2-2).

Additionally, slopes for the two Aedes species, as a single group, fed on the membrane

system were significantly different from those fed on the restrained chicken (Table 2-2).

Also, there appears to be an age-dependent periodic pattern in bloodfeeding incidences.

The periodic pattern is most obvious among 3-13-day-old adults of each species fed on

the restrained chicken, where the number of runs was significantly greater than that

expected for random daily variation. Likewise, for the proportion of bloodfed A. aegypti

on the membrane system versus age, a sine function made a significant contribution to

the fit, providing further evidence for periodicity This result is surprising, because this

was a short time series and typically, time series analyses have the potential to provide

good fits when there are > 50 observations (Chatfield 1989). Unlike some previous

research, Aedes in the current study were experimentally naive (i.e., never given a

previous bloodmeal), thus any periodicity in the time series is likely attributable to









endogenous factors. Periodicity could be an artifact of unknown exogenous factors,

although most obvious factors were controled (e.g., temperature, humidity, feeding

times). These results lend support to previous reports of a possible periodic pattern in

probing behavior of non-bloodfed A. aegypti (Burgess 1959) and host-seeking behavior

in non-bloodfed Anopheles gambiae sensu strict (Takken et al. 1998). Hormone levels

(e.g., juvenile hormone, ecdysteroids) vary at different times throughout the duration of

adult female life. Juvenile hormone has been shown to be involved in initiating

bloodfeeding for Culexpipiens (L.) and C. quinquefasciatus (Say) (Meola and Petralia

1980), and C. nigripalpus (Theobald) (Hancock and Foster 2000). The processes by

which synergistic and antagonistic effects of juvenile hormone and ecdysteriods, from

day to day, influence consumption of the initial bloodmeal, especially long after

emergence (e.g., 15 days), is unknown. Given the lack of data on endogenous hormone

fluctuation during the life span of unfed females, it is speculative to suggest that these

hormones may contribute to the apparent age-dependent differences observed in

proportion bloodfed ofA. aegypti and A. albopictus.







* Aedes aegypti (runs = 9)
0 Aedes albopictus (runs = 7)


{I{


3 4 5 6 7 8 9 10 11 12 13 14 15
Age (days)
Figure 2-1. Least squares means ( SE) for proportion bloodfed females on the silicon-
membrane system for 3-15 day old Aedes albopictus and A. aegypti. Line
drawn through means shows the best-fit linear regression for A. aegypti (solid)
and A. albopictus (broken).


T oI


S0.3 Aedes aegypti (runs =12)
0.2 0 Aedes albopictus (runs = 11)
0.2
3 4 5 6 7 8 9 10 11 12 13 14 15
Age (days)
Figure 2-2. Least squares means ( SE) for proportion bloodfed females on restrained
chickens for 3-15 day old Aedes albopictus and A. aegypti.


li i j

i i









Table 2-1. Test for equal slopes among regressions of proportion bloodfed ofAedes aegypti and A. albopictus versus age

Source df Type III SS MS F P

Age 1 1.3739 1.3739 30.69 < 0.0001
Feeding Protocol 1 1.4164 1.4164 31.64 < 0.0001
Species 1 0.1824 0.1824 4.07 0.0449
Feeding Protocol X Species 1 0.0697 0.0697 1.56 0.2136
Age X Feeding Protocol 1 0.2671 0.2671 5.97 0.0155
Age X Species 1 0.0412 0.0412 0.92 0.3384
Age X Feeding Protocol X Species 1 0.1094 0.1094 2.44 0.1197
Error df 198



Table 2-2. Intercept and slope estimates for simple linear regressions of proportion bloodfed ofAedes aegypti and A. albopictus
versus age. Slopes for groups followed by different letters are significantly different.

Source Intercept SE Slope e SE r2 df F P

Membrane System A. aegypti 0.1703 0.0779 0.0434 + 0.0082 a 0.3171 1,61 28.33 < 0.0001
A. albopictus 0.1084 0.0778 0.0226 0.0081 a 3.1110 1,62 7.74 0.0071
Chicken Host A. aegypti 0.7225 0.0593 0.0103 + 0.0059 b 0.0723 1, 38 2.96 0.0934
A. albopictus 0.4600 0.0879 0.0153 + 0.0090 J 0.0720 1, 37 2.87 0.0987














CHAPTER 3
LARVAL COMPETITION DIFFERENTIALLY AFFECTS ARBOVIRUS INFECTION
IN Aedes MOSQUITOES

Introduction

Biotic interactions among organisms play an important role in regulating

population growth and in shaping communities. Among biotic interactions, competition

has received a great deal of attention especially in the field of invasion biology where

competitively superior invasive species displace or otherwise alter the distribution of

established species (e.g., Juliano et al. 2004, Holway 1999, Petren et al. 1993). Although

the most obvious effects of competition are reduced growth and survivorship, there are

less obvious indirect effects mediated by competitively induced differences in life history

traits (e.g., morphological or behavioral trait-mediated indirect effects; Abrams 1995).

Indirect effects describe interactions between two species mediated by a third

species (e.g., exploitative competition, apparent competition, trophic cascades, indirect

mutualism, interaction modifications) (Wootton 1994, Osenberg et al. 1992). Although

different authors have applied multiple terms to similar types of indirect interactions

(e.g., Morrison 1999), there is a consensus on classifying indirect effects as "density-

mediated" or "trait-mediated" (Altwegg 2002). Density-mediated indirect effects occur

when abundance of one species indirectly alter the abundance of another species through

effects produced by altering the abundance of an intermediate species. Trait-mediated

indirect effects occur when one species alters traits (e.g., behavioral, morphological) in a

second species in ways that change the interaction between the second and third species.









The most frequently studied trait-mediated indirect effects involve predatory species that

induce prey behavioral modification (e.g., reduced activity, increase use of refuges) that

indirectly alter competitive interactions among those prey (Relyea 2000, Werner and

Anholt 1996, Werner 1992, 1991).

Less attention has been given to indirect effects of competition among organisms

with complex life cycles, where the impact of competition in one life stage has

consequences for species interactions in subsequent stages (Altwegg 2002). Adult life-

history traits of organisms with complex life cycles are, to a large extent, products of

their larval environment. For example, effects of competition include reduced growth,

development, and survivorship. Competitive-induced differences in adult life-history

traits such as size may alter species interactions with enemies, including predators,

pathogens, and parasites. Although nutrient limited conditions and physiological stress

indirectly result in greater susceptibility to infection with pathogens or parasites in a

single life stage (Kiesecker and Skelly 2001, Murray et al. 1998, Oppliger et al. 1998,

Matson and Waring 1984), little is known about effects of competition in juvenile life

history stages on susceptibility to infection in subsequent adult stages.

Water-filled containers are well suited to investigations of competitively induced

indirect effects because they harbor simple communities subject to variable resource

availability. Among the organisms occupying aquatic container communities,

mosquitoes are the best studied because of the role of adults as vectors of pathogens.

Resource availability and larval density in containers both influence mosquito

survivorship, growth, and adult size (e.g., Juliano et al. 2004, Lounibos et al. 2002).

Effects of competitive interactions among larval stages may carry over to the adult stage









and affect vector competence, which describes the ability to become infected and

subsequently to transmit a pathogen after imbibing an infectious bloodmeal (Hardy

1988).

Biological transmission of arboviruses includes acquisition of the virus by the

vector from an infectious bloodmeal, replication, dissemination of virus to the salivary

glands, and transmission to a host by bite (Higgs 2004, Hardy 1988). Successful

completion of this process requires that infection and dissemination barriers within the

mosquito be overcome (Hardy 1988, Hardy et al. 1983). For example, if arboviruses fail

to pass through the midgut, then infection is limited to the midgut cells and, although the

mosquito is 'infected,' it cannot transmit virus (Hardy et al. 1983). Larval competition

may have important consequences for adult arbovirus infection parameters. Typically,

pupal and adult sizes of container breeding mosquitoes are positively related to the

feeding rate experienced by larvae (e.g., Christophers 1960). Resource competition, and

associated low food availability, among larvae of the treehole mosquito Ochlerotatus

triseriatus produced smaller adults that transmitted La Crosse encephalitis virus (LACV)

at higher rates than did larger adults from well-fed larvae. Infection rates were

independent of adult body size (Grimstad and Walker 1991, Grimstad and Haramis

1984), although when 0. triseriatus reared from field-collected pupae were orally

infected with LACV, disseminated infection and transmission rates were negatively

correlated with adult size (Paulson and Hawley 1991). In contrast, large Aedes aegypti

adults produced under varying conditions of larval crowding and food availability

disseminated dengue virus serotype 2 (DENV-2) more efficiently than did smaller

females (Sumanochitrapon et al. 1998). Thus, it appears that ecological conditions









encountered by larvae can have variable effects on the interaction of mosquitoes with

arboviruses. Investigations of competitive effects on pathogen transmission, other than

size-related effects, remain rare.

The goal of our study was to determine the effects of larval competition on growth

and survivorship of two well known container mosquito species, A. albopictus and A.

aegypti, as well as their subsequent competence for arboviral infection and dissemination

using Sindbis virus (SINV). SINV is a model Alphavirus that cycles between reservoir

bird hosts and Aedes and Culex vector species (Seabaugh et al. 1998 and therein), and is

widely used in experimental vector biology research (Olson et al. 1996, Dohm et al.

1995). Aedes albopictus is an invasive container breeding mosquito native to Asia which

became established in large areas of the U.S., Europe, Africa, and South America during

the last two decades (Lounibos 2002). In the southern U.S., the spread of A. albopictus

coincided with reductions in range and abundance of the resident exotic A. aegypti in

artificial containers (reviewed by Juliano et al. 2004). Aedes albopictus is an important

vector of several arboviruses affecting humans and second only to A. aegypti in global

importance as a vector of DENV (Lounibos 2002, Gubler and Kuno 1997). These

species frequently encounter each other in artificial containers, in which interspecific

competition has been well documented (Braks et al. 2004, Juliano et al. 2004, Barrera

1996b, Black et al. 1989, Ho et al. 1989), which probably explains displacements of A.

aegypti by A. albopictus (Juliano 1998). This study tests whether variation in population

growth parameters known to arise from intra- and interspecific competition (Juliano et al.

2004, Lounibos et al. 2002) have carryover effects in the adult stage, and are associated

with variation in susceptibility to SINV infection dynamics.









Materials and Methods

Competition Study

Aedes albopictus Lake Charles strain (Nasci et al. 1989) and A. aegypti Rockefeller

strain were used in the experiments. These mosquitoes were the progeny of genetically

well-characterized strains. Aedes albopictus was obtained from a collection made at

Lake Charles, Louisiana in 1987 and has been propagated under laboratory conditions

since 1987. The A. aegypti Rockefeller strain was obtained from a long-standing colony

at the University of Notre Dame. The competition experiment between A. albopictus and

A. aegypti used 5-liter plastic containers filled with 4000 ml of tap water, 500 ml oak leaf

infusion water (O'Meara et al. 1989), and 0.2 g of larval food (1:1, by weight, albumin:

yeast). Three days after adding the initial contents to containers, a supplemental 500 ml

oak infusion and 0.2 g larval food was added. Initial food resources were incubated for 5

d before the addition to each container of first instar (< 24 h old) mosquitoes. Ten days

later, I removed 50% of the liquid contents, except larvae, and added 0.1 g larval food,

250 ml oak infusion water, and 2,250 ml tap water. Previous studies showed that this

protocol provided sufficient resources for mosquitoes to complete development without

negating the effects of larval competition (B.W. Alto, unpublished data). Competition

treatments consisted of species/densities ofA. albopictus: A. aegypti -- 160:0, 320:0,

160:160, 0:320, and 0:160. Ten replicates were used per treatment, for a total of 50

containers kept at 28 + 1IC and 14:10 L:D regime. Containers were checked daily, and

pupae transferred to sealed 20 ml vials with tap water until adult emergence. Emerged

adults were kept, by species, in cylindrical cages (11 x 9.5 cm, ht. x diameter) and

provided with 10% sucrose and an oviposition cup. The experiment was maintained until

the last adult had emerged.









Measurements of population growth correlates were used to estimate the effect of

competition on female A. albopictus and A. aegypti population growth. Mean female size

(wing length) and mean time to emergence were calculated for each replicate. Female

survivorship per replicate was calculated as (number of adult females) / (total number of

original larvae) of a given species. An estimated finite rate of increase (V') was also

calculated for each replicate container.



In [(1/No,) YE, Af(w

= exp(r') = exp

D + [ xAf(w) / EAf(w)]



X' is a transformation of r', a composite index of population performance (Juliano

1998). r' is an estimate of r = dN /Ndt, which describes the per capital growth rate. No is

the initial number of females in a cohort (assumed to be 50 %), Ax is the number of

females emerging on day x, wis mean female size on day x,f(wx) is a function relating

the number of eggs produced by a female to her size, and D is the time (in days) from

emergence to oviposition. For A. albopictus and A. aegypti, D is assumed to be 14 and

12 d, respectively (Juliano 1998, Livdahl and Willey 1991). We used the following

fecundity-size relationships (f(wx)) to calculate V':



A. aegypti (Briegel 1990).

f(w) = 2.50(w,3) 8.616

r2 = 0.875, N= 206, and P<0.001











A. albopictus (Lounibos et al. 2002).

f(wx) = 78.02 (wx)- 121.24

r2= 0.713, N= 91, and P<0.001



In both cases w = wing length in mm. Effects of A. albopictus and A. aegypti

competition were analyzed by individual Multivariate Analyses of Variance (MANOVA)

to determine competitive treatment effects on the population growth correlates time to

emergence, survivorship to emergence, and adult size. Raw data adequately met

assumptions of univariate normality and homogeneous variances for all correlates used in

the MANOVAs. For all analyses, significant effects were further analyzed by contrasts

of pairs of main effect multivariate means with a sequential Bonferroni adjustment for

experimentwise ca=0.05. Standardized canonical coefficients (SCC) were used to

determine the relative contribution of each of the response variables to significant

multivariate effects as well as their relationship to each other (e.g., positive or negative)

(Scheiner 2001). Competitive effects on A. albopictus and A. aegypti V' were analyzed

using one-way ANOVAs with treatment as a categorical variable (SAS Institute 1989).

Significant effects were further analyzed by pairwise comparisons of main effect means

(Ryan-Einot-Gabriel-Welsch test, SAS Institute 1989).

Infection Study

For each replicate from the competition study, newly emerged females and males

were housed, by species, in cages (11 x 9.5 cm, ht. x diameter) and provided 10% sucrose

and an oviposition cup. This arrangement facilitated mating and oviposition and enabled









the delivery of infectious bloodmeals to multiple females of approximately the same age.

Because larval competition increases developmental time, adults from the competition

containers emerged over several weeks. Therefore, multiple cages were used to house

adults for each replicate to ensure that the females given an infectious blood meal were of

similar ages (4-10 d old). SINV infection rates do not differ over the age range of 4-10 d

for these Aedes species (Dohm et al. 1995). Thus, tests for the effects of larval

competition on subsequent adult infection were performed on the same individual

mosquitoes. Adults were housed in cages within an incubator at 26 1C and 14:10 L:D

photoperiod. Adult females of each species were deprived of sucrose but not water for 24

h, then allowed to bloodfeed for 30 min. on a citrated bovine blood-SINV mixture

maintained at 37 1C in a silicon membrane system (Butler et al. 1984). SINV (MRE-

16 strain) titers used in bloodfeeding trials were 105.3 tissue culture dose required to infect

50% of wells (TCID5o) (Reed and Muench 1938). TCID50 is the quantity of virus that is

required to infect 50% of the tissue cultures, so that viral titers (= number of virus

particles / ml) can be determined. Viral titer refers to the amount of virus in solution.

Virus titers were similar to those produced in wild bird reservoirs in nature (Ockelbo

virus, a closely related strain of Sindbis virus (Lundstrom et al. 1993)). Titers were

determined by 10-fold serial dilutions in 96-well plates seeded with 6.0 x 105 Vero cells /

ml (10 wells per dilution). TCID50 was determined by cytopathic effects after a 7 d

incubation (Reed and Muench 1938). Vero cells infected with SINV virus exhibit

stereotypical cytopathic effects, so infection was unambiguous. To avoid the possibility

of reductions in titer with repeated thawing and freezing, all blood meals had virus

derived from single stock placed in 1.5ml aliquots that were frozen (-800C) and thawed









only once. The infection study was conducted in a biosafety level-2 facility appropriate

for SINV at the Florida Medical Entomology Laboratory in Vero Beach, Florida.

Females that failed to take a blood meal during the first trial were given a second

trial 18 h later. After the second feeding attempt, unfed females were removed from the

cages, and bloodfed females were held for a 16 d extrinsic incubation period (EIP). The

time from initial ingestion of the infectious blood meal until the time the mosquito can

transmit the arbovirus is the EIP. Females surviving the EIP were killed and individually

stored at -800C and, subsequently, their wings were removed (to be measured as an

indicator of female size). Bodies and legs were ground into a powder separately in 1 ml

diluent (Leibovitz L-15 media, 5% fetal bovine serum, and gentamicin), centrifuged at

21000 m/s2 for 12 min. at 40C, and filtered (0.22im). Proportion females infected, body

titer logoo TCID5o), and proportion of infected females with disseminated infection (i.e.,

with positive infected legs) were determined using 10-fold serial dilutions in triplicate

wells of 96-well plates seeded with Vero cells by TCID5o.

Infection was determined using a 1/10 dilution of the body stock solution, and

body titer was determined using a full range of dilutions. When describing infection of

mosquitoes, "negative" describes the absence of a viral infection, and "positive"

describes a mosquito with a viral infection in the midgut, and perhaps other organs. An

infection limited to the midgut is called an "isolated infection," whereas an infection

spread beyond the midgut, infecting secondary target organs (e.g., salivary glands, head,

legs), is called a "disseminated infection." Disseminated infection is a recognized

indicator of a mosquito's ability to transmit virus via biting (Gubler and Kuno 1997). So,

dissemination of infection in positive females was determined by assaying undiluted leg









stock solution (Turell et al. 1984). In this study, isolated infections refer to mosquitoes

with positively infected bodies, but absence of infection in legs, whereas disseminated

infections refer to positively infected bodies and legs. Assaying salivary glands may be a

more direct indicator of a mosquito's ability to transmit virus. However, extraction of the

salivary glands may result in contamination with surrounding tissue. Thus, assays of

mosquito legs were used in order to avoid this contamination problem and still obtain a

good indication of ability to transmit (Turell et al. 1984).

Prior to analyzing effects of competitive treatment on arboviral infection,

interspecific differences in susceptibility were analyzed using MANOVA and SCC on the

response variables proportion infected, body titer, and proportion with disseminated

infection. Next, individual MANOVAs for A. albopictus and A. aegypti were used to

determine the effect of larval competition on response variables: proportion infected,

body titer, and proportion with disseminated infection as described above. Multivariate

contrasts with sequential Bonferroni adjustment for experimentwise a=0.05 (Rice 1989,

Scheiner 2001) were used to compare high density treatments (320:0, 160:160) vs. the

low density treatment (160:0), and then to compare the two high density treatments.

For A. albopictus and A. aegypti, effects of mean female size on body titer were

tested by treating size as a covariate in an analysis of covariance (ANCOVA) with

competitive treatment and competition x size interactions. Significant effects were

further analyzed by all possible pairwise comparisons of treatment means (sequential

Bonferroni adjustment; Rice 1989). Effects of mean female size on body titer were

expected to be most pronounced in females with disseminated infections, and this

analysis was the primary interest.









Product-moment correlation coefficients (rl,2) were used to describe the

relationship between population growth measurements (time to emergence, survivorship,

size, V') and infection parameters: proportion infected, body titer of females with isolated

infection, body titer of females with disseminated infection, and proportion with

disseminated infection among competitive treatments ofA. albopictus and A. aegypti.

These analyses allowed for a test of the strength of positive or negative relationships

among population growth measurements and infection.

Results

Competition Study

For both A. albopictus and A. aegypti, competitive treatments significantly affected

population growth measurements (Table 3-1), with uncrowded larval conditions

consistently resulting in shorter time to emergence, greater survivorship, and greater adult

size compared to crowded conditions (Figs. 3-1, 3-2). For A. albopictus, SCC showed

that differences in adult size followed by survivorship to emergence contributed the most

to the significant competition effect as well as to subsequent treatment differences (Table

3-1). Although time to emergence was shorter at uncrowded larval conditions, it

contributed less than the other population growth measurements (A. albopictus time to

emergence SE d; 160:0, 13.58 0.23, 320:0, 15.62 + 0.23, 160:160, 15.93 + 0.24)

(Table 3-1). For A. aegypti, SCC showed that differences in survivorship to emergence

followed by time to emergence contributed the most to the significant competition effect

as well as pairwise-differences (Table 3-1). Size contributed far less to the significant

competition effect (A. aegypti mean wing length SE mm; 0:160, 2.65 0.05, 0:320,

2.39 0.05, 160:160, 2.47 0.07) (Table 3-1). For both species, competitive treatments

significantly affected V' (F2,26 = 191.84, P < 0.0001; F2, 19 = 51.94, P < 0.0001; A.









albopictus and A. aegypti, respectively) and V' was significantly greater in the pattern:

160 larvae > 320 larvae > 160+160 larvae (Fig. 3-3). Thus, inter- and intraspecific

competition had major population-level effects.

Infection Study

Prior to analyzing effects of competition on arboviral infection, interspecific

differences in susceptibility were first examined. Proportions infected, whole body titer,

and proportions with disseminated infection were significantly different between A.

albopictus and A. aegypti [Pillai's trace (3, 37) = 0.76, P < 0.0001]. Proportion infected

(SCC = 1.23) was the most important variable in the overall interspecific difference,

followed by proportion with disseminated infection (SCC = -0.66) and whole body titer

(SCC = -0.57). The opposite signs of the SCC showed that there was a negative

relationship between the variables across the species, so that A. albopictus had a greater

proportion of infected individuals, a lower body titer, and a lower proportion

disseminated infection compared to A. aegypti (LS means SE for A. albopictus and A.

aegypti proportion infected, 0.94 0.03 and 0.58 0.04; body titer, 4.08 0.14 and 5.58

+ 0.22 TCID5o; and proportion with disseminated infection, 0.67 0.03 and 1.00 + 0,

respectively).

Interspecific competition had significant effects on proportion infected, whole body

titer, and proportion ofA. albopictus with disseminated infection [Pillai's trace (6,24) =

0.52, P = 0.025]. Proportion infected (SCC = 1.12) made the greatest contribution to the

multivariate differences among treatments, and body titer (SCC = 0.23) and proportion

with disseminated infection (SCC = 0.06) contributed less. Aedes albopictus at low

density alone (160/container) had a significantly lower proportion infected, lower









proportion with disseminated infection, and lower body titer compared to high density

treatments [Pillai's trace (3,25) = 0.38, P = 0.011] (Fig. 3-4, A and B). Proportion

infected was the major contributor to this effect (SCC = 1.14), whereas titer (SCC = 0.27)

and proportion with disseminated infection (SCC = -0.08) contributed little. The two

high density treatments did not differ significantly [Pillai's trace (3,24) = 0.14, P = 0.319]

(Fig. 3-4, A and B). For the infection study, mortality during the extrinsic incubation

period resulted in few A. aegypti females from the 160:160 treatment. Therefore, only

means for intraspecific density treatments are reported for A. aegypti. There were no

significant effects of competition on infection parameters for A. aegypti [Pillai's trace

(2,11) = 0.23, P = 0.301]. Mean SE females assayed per treatment replicate were;

160:0 (10.0 + 1.84), 320:0 (11.89 + 1.15), 160:160 (albo) (6.44 0.63), 0:320 (6.11 +

1.12), and 0:160 (5.20 + 0.92).

Females with disseminated infections are capable of transmitting virus and

therefore are of epidemiologic significance. For these females, an analysis of covariance

with mean female size as a covariate showed significant effects of size and competition

on whole body viral titer for A. albopictus with disseminated infections, but no

significant size x competition interaction (Table 3-2). Thus, effects of mean body size

and of competition are independent. Estimated slopes were positive, indicating that

within a competitive treatment body titer increased with size for mosquitoes with

disseminated infection (Fig. 3-5). Pairwise comparisons of adjusted means among

treatments showed that significant differences in body titer followed the pattern 160:160

> 320:0 > 160:0 (mean SE: 5.80 0.23, 5.13 0.22, 3.23 0.32 TCID50, respectively).









For A. aegypti with disseminated infections, there were no significant competitive

treatment or covariate effects (Table 3-2).

Product-moment correlations showed significant relationships between infection

and all correlates of population growth for A. albopictus (Table 3-3). In particular,

increased time to emergence, a result of intra- and interspecific competition, was

positively correlated with infection rate for A. albopictus, but survivorship, size, and V'

were negatively correlated with infection rate (Table 3-3). Also, survivorship and V'

were significantly negatively correlated with mean A. albopictus body titer for females

with disseminated infections. All correlations between population growth parameters of

A. aegypti and infection parameters were non-significant (Table 3-3).

Discussion

The two experiments in this study were designed to quantify the effects of intra-

and interspecific larval competition, and then to determine whether competitive effects

carried over into the adult stage and influenced competence for arbovirus infection. For

both Aedes species in the competition experiment, all population growth measurements

clearly showed that higher larval densities resulted in poorer performance (Figs. 3-1, 3-2,

3-3). Analyses of survivorship, time to emergence, and size at emergence suggested that

the effects of intra- and interspecific competition were similar. However, for both Aedes

species, a synthesis of multiple growth measurements (V') showed that interspecific

competition was more intense than intraspecific competition (Fig. 3-3).

A variety of model systems have shown that the outcome of interspecific

competition depends on resource type (e.g., Sanders and Gordon 2003, Tilman 1982).

Contrasting outcomes have been obtained with these two Aedes species, A. aegypti









having the competitive advantage over A. albopictus with nutritious larval food (e.g.,

liver powder, yeast), but not with low-nutrient, more natural resources (e.g., leaf litter)

(Braks et al. 2004, Juliano 1998, Barrera 1996b, Black et al. 1989). The current

experiment used a combination of natural (leaf infusion) and supplemental (albumin,

yeast) resources and, for both Aedes species, interspecific competition was greater than

intraspecific competition as measured by V'. The intention in designing the competition

experiment was not to mimic natural resources, rather to use a resource base known to

maximize the production of Aedes females for the infection study, without negating the

effects of competition. These objectives were met since competitive interactions were

detected and sufficient numbers of adults were obtained for the infection study.

Although the experimental design was constrained to maximize adult production

without negating competition, mosquito densities and sizes conformed to observations

from field conditions. In the current experiment, densities were 0.032 and 0.064

larvae/ml for the 160 and 320 larvae treatments, respectively. Sampling of the entire

contents of water-holding golf cart tires in Broward, Indian River, and Monroe counties

in Florida (Dec. 1996 or Jan. 1997 April 1998) showed that larval densities were within

the range observed in tires occupied by A. albopictus, A. aegypti, or both species (N=790,

mean SE, 0.17 0.02, range 0.00083 3.08 larvae/ml) (G. F. O'Meara, unpublished).

Also, A. albopictus adult female wing lengths (Fig. 3-1) were within the range of A.

albopictus collected at tire sites in East St. Louis, USA (N = 180, mean SE, 2.43 0.02

mm, range 1.84 2.95) (B.W. Alto and S.A. Juliano, unpublished). Similarly, both A.

albopictus and A. aegypti female wing lengths in the current study were within the range

of field-collected females of these species from tire sites in southwestern Louisiana









(N=150, mean SE 2.68 0.02 mm, range 2.04 3.12; N=115, 2.64 0.03, range 1.92 -

3.12, respectively) (Nasci 1990). Wing length, as a surrogate of adult size, is a good

indicator of larval environmental conditions (e.g., food resources, larval density) (Juliano

1998, references therein). Thus, the experimental set-up produced adult females that

parallel those sizes found in nature.

The infection component revealed that larval competition altered adult mosquito

susceptibility to arboviral infection and potential for virus transmission. In particular,

competitively stressed A. albopictus females were more likely to become infected and

have higher SINV titers and dissemination than females reared with less competition.

Results are consistent with other model systems where competition, in the form of

nutrient-limitation or stressors, enhanced susceptibility to infection with pathogens or

parasites (Kiesecker and Skelly 2001, Murray et al. 1998, Oppliger et al. 1998, Matson

and Waring 1984). In the current study, infection rate was the variable most sensitive to

the impact of larval competition. Intra- and interspecific competition altered subsequent

A. albopictus interactions with SINV, suggesting that biotic interactions in early

developmental stages may be important in determining adult arboviral infection

parameters among mosquitoes. This type of indirect effect may be viewed as an

interaction modification since "a change in density of one species alters the nature of a

direct interaction between two other species" (Wootton 1993). On the other hand, effects

of competition on A. aegypti infection parameters were not observed, and reasons for

differences between the two Aedes species in responses to competitive treatments are

unknown. Although there was less statistical power in the A. aegypti tests due to lower

sample sizes, biological explanations could include species-specific qualitative









differences in the availability of midgut receptor sites used by SINV or escape barriers

(e.g., midgut escape barrier) that may be differentially affected by competition. These

results suggest species-specific differences in how larval competition affects adult

competence for arboviral infection parameters. Similarly, in plant communities, studies

have demonstrated species-specific responses to indirect effects (e.g., indirect

facilitation), most likely attributable to differences among species in life history traits

(e.g., Pages et al. 2003, Levine 1999).

The correlation coefficients demonstrating that infection rates were significantly

associated with all correlates of population growth (Table 3-3) represent the first

evidence that life history traits, in addition to adult size, change parameters associated

with vector competence. Furthermore, correlations between life-history traits and

infection parameters in A. albopictus showed that negative effects of competition on

population growth are associated with enhanced vector potential (Table 3-3). The

observation that larger A. albopictus females with disseminated infections had

significantly greater body titer can be explained as simply a size phenomenon, i.e., more

tissue is available for virus propagation. For body titer, there were independent effects of

both mean adult size and competition. The lack of a significant size x competition

interaction showed that the effect of size on body titer was similar among competitive

treatments (Fig. 3-5). More importantly, density-dependent differences in body titer were

found, with greater mean body titer among A. albopictus reared under competitive

conditions. Significant density-dependent differences in body titer were identical to

significant density-dependent differences in V', except in the opposite direction (Figs. 3-

3, 3-5). Specifically, more intense competition as measured by a lower V' resulted in









greater body titer. These results demonstrate both size-dependent and size-independent

effects of competition on infection dynamics that have opposite effects across

competition treatments. Within a competitive treatment, larger mosquitoes have greater

body titer, but between competitive treatments larger mosquitoes from low-competition

larval rearing conditions have lower body titer and a lower proportion infected, compared

to smaller mosquitoes emerging from high-competition conditions. Overall, the results

demonstrate that competitive stress experienced by A. albopictus larval stages carried

over to the adult stage and significantly influenced susceptibility to infection and

dissemination.

Over and above the effects of competition, the two Aedes species differed in

susceptibility to infection and dissemination. Other studies have shown interspecific

differences for quantitative aspects of infection (Turell et al. 2001, Gubler et al. 1979).

However, previous research using these Aedes species, as well as other mosquito species,

did not quantify the variables most important for interspecific differences (e.g., infection,

body titer, dissemination) or the positive and negative interrelationships among these

variables across species (i.e., see SCC). A. albopictus was significantly more susceptible

to infection than was A. aegypti, as seen in other studies (Turell et al. 2001, Gubler et al.

1979). Conversely, although A. aegypti had lower infection rates, those females that were

infected had significantly higher body titer and dissemination rates compared to infected

A. albopictus. Infection contributed approximately twice as much as body titer and

dissemination to interspecific differences. Factors limiting body titer and dissemination

(e.g., midgut escape barrier) were less efficient (or not expressed) in A. aegypti compared

to A. albopictus under these conditions. These results suggest fundamental differences in









physiology between these Aedes species that alter their susceptibility to arboviral

infection and dissemination, and these differences are likely to have important

epidemiological consequences.

If effects of competition on vector infection with SINV apply to arboviruses such

as DENV and West Nile virus, these results may have important implications for human

health. The current study suggests that competition experienced by larval A. albopictus

may enhance the threat posed by this species in pathogen transmission. Uncrowded

larval rearing at low densities, used in most laboratory studies of vector competence, do

not accurately reflect conditions in nature where competition is often strong and

widespread (Juliano et al. 2004). The current study suggests that indirect effects are

important in determining mosquito vector ability, and that the effect may be species

specific. Failure to consider larval stresses may result in misleading estimates of relative

susceptibility to infection for A. albopictus and A. aegypti, and by extension, other

arboviral vectors. This report is the first to quantify how larval competition may affect

arbovirus infection in adult mosquitoes, and demonstrates the species-specificity of the

process from infection to dissemination. Future assessments of vector potential should

consider the species-specific effects of larval conditions that reflect competitive

conditions observed in nature.











b

+


0.4


0.35


0.3


0.25


0.2


I I I I I I
2.1 2.2 2.3 2.4 2.5 2.
Size at emergence (mm)


Figure 3-1. Aedes albopictus least squares means (+SE) for female survivorship and size
at emergence. Different lowercase letters indicate significant differences
between bivariate means. Competition treatments consisted of species density
ratios ofA. albopictus: A. aegypti-160:0, 320:0, and 160:160.

0.4 a
a


O 0:160
S0:320
0160:160


13.5


14.5


Time to Emergence (days)
Figure 3-2. Aedes aegypti least squares means (+SE) for female survivorship and time to
emergence. Different lowercase letters indicate significant differences
between bivariate means. Competition treatments consisted of species density
ratios of A. albopictus: A. aegypti-0:160, 0:320, and 160:160.


0160:0
*320:0
0160:160


0.15


0.35-

0.3-


0.25-

0.2


0.15-


n 1


I


12.5
12.5


-0-


a
a






59




m 1.18
a a

CC
S1.14 0
O b







C A. albopictus
O A. aegypti
1.1u I B





W 1.02

l 1.02 --- ^ ------ ^ ------ ^ ---
160:0 320:0 160:160 0:320 0:160

Competitive Treatment (A. albopictus: A. aegypti)



Figure 3-3. Least squares means ( SE) for estimated finite rate of increase, X', for Aedes
albopictus and A. aegypti. Points without bars have standard errors too small
to appear on the graph. Different lowercase and uppercase letters indicate
significant differences between means for A. albopictus and A. aegypti,
respectively.














0160:0
S320:0
0160:160


.4 .
0.4 0.5


0.6


Proportion with disseminated infection


1.00-


0.95-


0.90-


0.85-


0.80 1-
3.0


0160:0
S320:0
0160:160


I I I 5
3.5 4.0 4.5 5.


log10 (body titer)
Figure 3-4. Bivariate plots of least squares means ( SE) for three dependent variables
for Aedes albopictus females fed on a Sindbis virus blood meal. (A)
Proportion of infected females vs. proportion with disseminated infection. (B)
Proportion of infected females vs. body titer. In both graphs, the dashed
ellipse indicates multivariate means that are not significantly different.
Numbers in the figure key represent the ratio ofA. albopictus to A. aegypti.


1.00


0.95


0.90


0.85


0.80


i--..._._.. r___~_~~.~.----


I


.... .











O 160:0 (disseminated) 320:0 (disseminated)
O 160:160 (disseminated) 0 160:0 (isolated)
U 320:0 (isolated) O 160:160 (isolated)


0 0
S0 0



mm n D
D 0
DO
- O-

I I I


2.0


2.2


2.4


2.6


Size (wing length, mm)
Figure 3-5. Least squares means for body titer and size of adult Aedes albopictus females
with disseminated (i.e., infection spread beyond the midgut, infecting
secondary target organs such as body, legs) and isolated (i.e., infection limited
to the midgut) Sindbis virus infections. The size effect on females with
disseminated infections gives a slope of 5.48 (SE = 1.28). Solid and dased
lines drawn through bivariate means show the best fit for A. albopictus with
disseminated infections in three competitive treatment conditions. Numbers
in the figure key represent the ratio ofA. albopictus to A. aegypti.









Table 3-1. Multivariate ANOVA for main effects and multivariate pairwise contrasts of competitive treatment effects on female
Aedes albopictus and A. aegypti population growth measurements: time to emergence, survivorship to emergence, and
adult size.

Comparison df Pillai's trace P Standardized Canonical Coefficients

Time Surv. Size

A. albopictus
Competitive treatment 6 1.02 < 0.0001 -0.79 1.19 1.97
160,0 vs. 320,0 3 0.90 < 0.0001 0.88 1.18 1.89
160,0 vs. 160,160 3 0.91 < 0.0001 0.73 1.19 2.02
320,0 vs. 160,160 3 0.13 0.3274
Error df 26

A. aegypti
Competitive treatment 6 0.93 0.0006 1.11 2.01 0.27
0,160 vs. 0,320 3 0.88 < 0.0001 1.12 1.99 0.28
0,160 vs. 160,160 3 0.76 < 0.0001 -1.05 2.09 0.20
0,320 vs. 160,160 3 0.14 0.4472
Error df 19









Table 3-2. ANCOVA for the effects of competitive treatment and size
females with disseminated infections.


covariate on body titer for Aedes albopictus and A. aegypti


Source df F P


A. albopictus, disseminated
Size
Competitive treatment
Size x competition
Error df

A. aegypti, disseminated
Size
Competitive treatment
Size x competition
Error df


18.38
15.20
0.26


1.74
0.74
2.62


0.0002
< 0.0001
0.7750


0.2170
0.5036
0.1336









Table 3-3. Product moment correlation coefficients (rl,2) for the relationship between population growth measurements (time to
emergence, survivorship, size, and V') and infection parameters. Infection parameters include infection, body titer of
females with isolated infection, and dissemination for Aedes albopictus (df=25) and A. aegypti (df=12). An infection
limited to the midgut is called an "isolated infection," whereas an infection spread beyond the midgut, infecting secondary
target organs (e.g., salivary glands, head, legs), is called a "disseminated infection." Asterisks denote significant correlation
coefficients (* P < 0.05; ** P < 0.001). No rl,2 values are reported for A. aegypti dissemination and body titer (isolated)
since all infected individuals had disseminated infections.


A. albopictus A. aegypti
Infection parameters
and growth parameters

Infection
Time to Emergence 0.50 ** 0.08
Survivorship 0.50 ** -0.07
Size -0.64 ** 0.17
V -0.55 ** 0.10

Body Titer (disseminated)
Time to Emergence 0.26 0.24
Survivorship -0.38 0.38
Size -0.00098 0.13
V -0.40 0.19

Dissemination
Time to Emergence 0.04
Survivorship 0.22
Size -0.20
h' -0.13














CHAPTER 4
LARVAL COMPETITION AND SUSCEPTIBILITY OF Aedes aegypti AND Aedes
albopictus TO INFECTION BY DENGUE VIRUS

Introduction

Dengue virus (DENV) is an arthropod borne (arbo) virus. There are four different

serotypes of DENV that are the cause of morbidity and mortality throughout much of the

tropical world. Approximately 50-100 million cases of dengue fever (DF) occur annually

with hundreds of thousand of cases of dengue hemorrhagic fever (DHF), a life-

threatening form of dengue. In Southeast Asia, range expansion of the primary vector

Aedes aegypti (L.) and human migration have contributed to hyperendemicity (co-

circulation of multiple serotypes in a single location) and associated epidemic DF and

DHF (Gubler 2002). Regions with hyperendemic DENV are expanding, and recent

introductions of Southeast Asian genotypes of DENV to the Western Hemisphere pose an

increased risk of transmission in the tropical Americas resulting in greater number of

cases of severe DHF (Cologna et al. 2005, Cologna and Rico-Hesse 2003, Rico-Hesse et

al. 1997, Lewis et al. 1993).

The yellow fever mosquito A. aegypti and Asian tiger mosquito A. albopictus

(Skuse) are considered the primary and secondary vectors of DENV, respectively

(Rodhain and Rosen 1997). However, the relative importance of these Aedes species in

DENV transmission in nature is difficult to determine, especially in regions where they

coexist. Aedes aegypti and A. albopictus have sympatric and allopatric breeding sites in

Southeast Asia as well as in many locations in the Western Hemisphere where dengue is









endemic and a serious health risk to humans. Geographic strains of both these Aedes

species vary in their susceptibility to DENV infection (e.g., Bennett et al. 2002, Failloux

et al. 2002, Vazeille-Falcoz et al. 1999, Boromisa et al. 1987, Gubler et al. 1979, Gubler

and Rosen 1976). Additionally, mosquito infection parameters may be altered by

different serotypes and strains of DENV (e.g., Moncayo et al. 2004, Armstrong and Rico-

Hesse 2003, 2001, Rosen et al. 1985, Whitehead et al. 1971). For example, Southeast

Asian DENV-2 strains, which are more virulent than American genotypes, consistently

show significantly greater disseminated infection in A. aegypti compared to American

DENV-2 strains (Cologna et al. 2005, Armstrong and Rico-Hesse 2003, 2001), but no

comparable data exist for A. albopictus. Contrasting outcomes of dengue infection have

been obtained for these two Aedes species in laboratory and field studies. Studies using

well-established laboratory mosquito strains, suggested that DENV (serotypes 1, 2, 3, 4)

were less efficient at infecting and causing disseminated infections in A. aegypti

compared to other Aedes species, including A. albopictus (Rodhain and Rosen 1997,

Rosen et al. 1985, Gubler et al. 1979). Similarly, a greater proportion ofA. albopictus

had disseminated DENV-2 virus infection compared to sylvatic A. aegyptiformosus

(Vazeille et al. 2001). In contrast laboratory experiments showed that a greater

proportion ofA. aegypti (Fi-F2 generation) had disseminated DENV-2 infections

compared to A. albopictus, although the proportion of A. albopictus with disseminated

infections increased with subsequent laboratory generations (Vazeille et al. 2003). In

summary, research to date is equivocal whether A. albopictus or A. aegypti is the superior

vector. Rather, it is likely that the genetic background of both the virus and mosquito









species play important roles in determining vector competence for dengue (Tabachnick

1994).

The establishment ofA. albopictus in new regions, especially where A. aegypti is

absent, also spreads the risk for DENV transmission since human movement and

transport may place the reservoir, virus, and vector in close proximity to one another. In

the southern U.S., the introduction and spread of A. albopictus was associated with

declines in resident A. aegypti (Juliano et al. 2004, O'Meara et al. 1995, Mekuria and

Hyatt 1995, Horby et al. 1994, Hobbs et al. 1991). The two Aedes species occupy

similar container habitats, and interspecific competition among the larval stages is well-

documented and a likely contributor to the observed decline of A. aegypti (e.g., Costanza

et al. 2005a, Braks et al. 2004, Juliano et al. 2004, Lounibos et al. 2002, Barrera 1996).

Competition, due to resource limitation or high larval density, has been demonstrated for

many mosquito species and is usually reflected by an increase in larval development time

and mortality, and decrease in adult size (e.g., Alto et al. 2005ab, Juliano and Lounibos

2005, Peck and Walton 2005, Braks et al. 2004, Juliano et al. 2004, Ye-Ebiyo et al. 2003,

Gimnig et al. 2002, Lounibos et al. 2002, Schneider et al. 2000, Teng and Apperson

2000, Leonard and Juliano 1995, Broadie and Bradshaw 1991).

The effects of larval competition likely impact the adult stage, and therefore may

also influence adult susceptibility to pathogens (e.g., arboviruses), including DENV

(Vazeille et al. 2003, Black et al. 2002, Sumanochitrapon et al. 1998). However, this

field of investigation has not been well explored. Small 0. triseriatus adults derived

from competitive larval environments under laboratory conditions or from field

collections had similar La Crosse virus (LACV) infection rates compared to large adults,









but greater dissemination and transmission (Grimstad and Walker 1991, Paulson and

Hawley 1991, Grimstad and Haramis 1984). Large A. aegypti adults, reared with

abundant resources and low larval density, had greater incidence of disseminated DENV-

2 viral infection compared to smaller adults (Sumanochitrapon et al. 1998). Explicit

examination of the effects of intra- and interspecific competition between A. albopictus

and A. aegypti on vector competence has only been examined in a model using SINV

(Alto et al. 2005a). These experiments demonstrated that competition resulted in

increased development time to emergence and decreased survivorship, size, and a

performance index (V') for both species, and enhanced A. albopictus SINV infection

parameters, but not A. aegypti parameters. Specifically, competitively stressed A.

albopictus had greater infection, disseminated infection, and body titer than unstressed

individuals, and proportion infected contributed the most to these significant effects (Alto

et al. 2005a). While the results of this previous work were compelling, A. aegypti and A.

albopictus are not vectors of SINV in nature. Therefore, the artificial nature of the virus-

vector association limits applying these results more generally. To examine the

generality of patterns observed in this previous work, and to use a virus of primary

importance to human health, I conducted an experiment to test whether competition

among larvae affects DENV infection parameters, and whether population growth

measurements are associated with DENV infection parameters, for A. aegypti and A.

albopictus. For this study, infection parameters refer to proportion of females infected

with DENV, proportion of females with disseminated infection, and DENV body titer.









Materials and Methods

Competition Study

Mosquitoes used the experiment were the same continuously propagated colony

strains described elsewhere (Alto et al. 2005a). Aedes albopictus Lake Charles strain and

A. aegypti Rockefeller strain represent long-standing laboratory colonies (Nasci et al.

1989, Craig and Vandehey 1962). The current experimental setup was similar to that

used to investigate the affects of larval competition on adult Aedes infection with SINV

(Alto et al. 2005a). A similar experimental design was desirable because it facilitated

comparison between experiments to evaluate whether competition has similar affects on

Aedes infection parameters for arboviruses in two different families (i.e., SINV in

Togaviridae, DENV in Flaviviridae).

Larval food resources used in the competition experiment were identical to those

described by Alto et al. (2005a), except that supplemental resources were added on day

13 instead of day 10. Briefly, larval rearing vessels consisted of 5-L plastic containers

filled with 4000 ml tap water, 500 ml oak leaf infusion water (O'Meara et al. 1989), and

0.2 g larval food (1:1 albumin: yeast). Food resources were allowed to incubate for 5 d

before newly hatched (<24 h old) mosquitoes were added to experimental containers.

Three days after adding the larvae, a supplemental 500 ml oak infusion and 0.2 g larval

food were added. Thirteen days later, 50% of the liquid was removed, except larvae, and

0.1 g larval food, 250 ml oak leaf infusion water, and 2250 ml tap water were added.

Competition treatments consisted of species density ratios ofA. albopictus: A. aegypti

(i.e.., 160:0, 320:0, 160:160, 0:320, and 0:160). Ten replicates were used for each

treatment, except for 320:0 and 0:320, which had 11 replicates. Containers were

maintained under constant environmental conditions (280C 1C, 14:10 L:D









photoperiod). Pupae were removed from containers daily and stored in 20-ml water-

filled vials until adult emergence. Larval rearing in the competition experiment lasted

until all pupae emerged as adults or larvae had died.

Measurements on individuals and cohorts were used to evaluate competitive

treatment effects on A. albopictus and A. aegypti population growth. Mean female size

(wing length in mm) and mean time to adult emergence (days) were determined for each

treatment replicate. Wing length was measured as the distance from the axillary incision

to the distal point on the lateral margin of the wing, excluding the wing fringe. Female

survivorship per replicate was calculated as (number of adult females) / (total number of

original larvae) of a given species. Estimated finite rate of increase ()') was calculated

for each replicate:



In [(1/No) YE, Axf(w

= exp(r') = exp

D+[ [xxAf(w) / A/E f(w)]



where V' is a composite index of performance based on a transformation of r'

(Juliano 1998, Livdahl 1984, 1982, Livdahl and Sugihara 1984). No is the initial number

of females in a cohort (assumed to be 50 %), A, is the number of females emerging on

day x, and w, is mean female size on day x. D is the time (in days) from emergence to

oviposition. For A. albopictus and A. aegypti, D is assumed to be 14 and 12 d,

respectively (Livdahl and Willey 1991, Juliano 1998). Mean female size per day, used to

calculate V', was obtained from all females assayed for viral infection as well as all unfed









females obtained from the entire duration of the experiment. Number of eggs produced

by a female was estimated from female size based on a regression functionf(wx). The

following fecundity-size relationships (f(wx)) were used to calculate V':

A. aegypti (Briegel 1990).

f(w) = 2.50(w,3) 8.616

r2 = 0.875, N= 206, andP<0.001



A. albopictus (Lounibos et al. 2002):

f(w) = 78.02 (w,)- 121.24

r2= 0.713, N= 91, and P<0.001

In both cases w, is wing length in millimeters. Population growth measurements

(time to emergence, size of females assayed for infection, survivorship) were analyzed,

separately for A. albopictus and A. aegypti, by Multivariate Analyses of Variance

(MANOVA) to quantify the effect of competition. Thus, MANOVA used sizes of

females assayed for DENV infection and V' was calculated based on sizes of females

assayed for infection as well as all unfed females obtained over the duration of the

experiment. Significant effects were further analyzed by all possible contrasts of pairs of

main effect multivariate means using the sequential Bonferroni method (experimentwise

a = 0.05). Standardized canonical coefficients (SCC) were used to describe the relative

contribution of each population growth measurement to significant multivariate effects as

well as their relationship to each other (e.g., positive or negative: SAS Institute 2002,

Scheiner 2001). Competitive treatment effects on A. albopictus and A. aegypti V' were

analyzed by separate one-way ANOVA, and significant effects were further analyzed by









pairwise comparisons of main effect means (Ryan-Einot-Gabriel-Welsch test, SAS

Institute 2002). Raw data adequately met assumptions of univariate normality and

homogeneous variances for all population growth measurements in MANOVA and

ANOVA, except A. albopictus development time, which showed departure from

normality. No common transformations improved normality, however, MANOVA, using

Pillai's trace, is robust to departures from normality (Scheiner 2001). Also, the highly

significant treatment effects and similar direction of competitive effects suggest that the

departure from normality had little effect in determining the results.

Infection Study

Viral propagation

A Southeast Asian genotype of DENV-2 was originally isolated from a patient in

Thailand in 1974. This virus isolate had been passed once in the mosquito

Toxorhynchites amboinensis (Doleschall), 3 times in Vero cells, twice in A. albopictus

C6/36 cells, and 3 additional passages in Vero cells (S. Fernandez, pers. comm.).

Subsequently, the DENV stock was passed twice in Vero cells. T-75 cm2 flasks with

confluent monolayers of Vero cells were individually inoculated with 3 ml media

(Leibovitz L-15 media, 10% fetal bovine serum (FBS), 50 [tg/ml gentamicin) containing

200tl DENV-2 stock. T-75 cm2 flasks were rocked for 1 h at 370C, to allow for

adsorption, after which media were added to bring the total volume to 10 ml and

incubated at 350C. Media in T-75 cm2 flasks was renewed on days 4 and 8 and harvested

for infectious bloodmeals on day 11. Freshly recovered media-virus suspension (i.e.,

unfrozen) from day 11 was used as the source virus for use in infectious bloodmeals

offered to mosquitoes (Miller et al. 1982). Infectious bloodmeals using previously frozen









(-800C) DENV-2 stock showed significantly lower infection and dissemination in both

Aedes species compared to fresh grown virus, even at similar titers (unpublished data), in

agreement with observations for other arboviruses, including DENV-2 (Turell 1988,

Miller 1987, Miller et al. 1982). All procedures involving DENV-2 were performed in a

biosafety level-3 facility.

Oral infection of mosquitoes

Adult mosquitoes from the larval competition experiment were housed by

treatment replicate and species in wax-coated cardboard containers (14 cm high x 11 cm

diameter) and provided with an oviposition cup, 20% sucrose, and water. Sucrose and

water were renewed every 48 h. Sucrose, but not water, was removed from mosquitoes

24 h prior to blood feeding. Infectious blood meals were offered to 4-7 d old females

using a silicon membrane feeder system (Alto et al. 2005a, 2003, Butler et al. 1984).

Citrated bovine blood was combined with DENV-2 stock in a 4 to 1 ratio, respectively to

provide a blood meal titer of 6.2 logo PFU/0.2 ml. Membrane feeders with infectious

blood were heated at 37C in an incubator for 20 min. and offered to Aedes females for

30 min. An aliquot of infectious blood was immediately frozen at -80C and later tested

by plaque assay to determine the titer of blood meals offered to Aedes females. Next,

mosquitoes were cold anesthetized and fully bloodfed females were identified, using a

stereo microscope within a glove box, isolated, and held for a 12 d extrinsic incubation

period at 280C + 1C and provided with sucrose, water and a 14:10 h light:dark

photoperiod regime. Sucrose and water were renewed every 48 h. Mosquitoes that

survived the extrinsic incubation period were individually stored in vials at -80C and,

subsequently, their wings were removed and measured as an indicator of female size (see

above).









Interspecific (A. aegypti versus A. albopictus) differences in DENV-2 susceptibility

to infection were evaluated by MANOVA and SCC on the response variables proportion

infected, and proportion with disseminated infection. A one-way ANOVA tested for

interspecific differences in body titer ofAedes females with disseminated DENV-2

infections. Next, individual one-way MANOVAs and SCC, for each Aedes species, were

used to determine competitive treatment effects on proportion infected, and proportion

with disseminated infection. Significant effects were further analyzed by all possible

pairwise contrasts of pairs of bivariate means using the sequential Bonferroni method

(experimentwise c = 0.05). Raw data adequately met assumptions of univariate

normality and homogeneous variances for analyses, except for proportion A. albopictus

infected, which showed departure from normality. No common transformations,

including arcsine square root, improved normality. The sensitivity of departure from

normality was assessed by analyzing the proportion A. albopictus infection using a

Kruskal-Wallis nonparametric test which is a weaker test but does not assume normality.

Results of the nonparametric test gave the same conclusions as parametric analyses, thus

I am confident that effects on proportion A. albopictus infected are not artifacts produced

through departure in normality. Further, MANOVA, using Pillai's trace, is robust to

departures in normality (Scheiner 2001).

The effects of mean female size on proportion infected, proportion with

disseminated infection, and body titer of females with disseminated infection were

assessed by treating size as a covariate in an analysis of covariance (ANCOVA), with

competition treatment and competition x size interactions as categorical variables (SAS

Institute 2002). ANCOVAs involving body titer used size based on wing length









measurements of females with disseminated DENV-2 infections. ANCOVAs involving

proportion infected and proportion with disseminated infection used size based on wing

length measurements of all females assayed for DENV-2 infection. Initially all

ANCOVAs tested for equality of slopes for each size by competitive treatment (i.e., each

competitive treatment has its own slope estimate). ANCOVAs determined to have

common slopes (i.e., no significant size x competitive treatment interaction) were re-

tested for the equality of the intercepts. ANCOVAs determined to have similar intercepts

were re-tested as ANOVAs with competitive treatment and no size covariate. Significant

effects were further analyzed by all possible pairwise comparisons of treatment means

(Tukey-Kramer adjustment of experimentwise c = 0.05, SAS Institute 2002). Raw data

adequately met assumptions of univariate normality, homogeneous variances, and

linearity, however, the proportion A. albopictus infected with DENV showed some

departure from normality. No common transformations, including arcsine square root,

improved normality.

Product-moment correlation coefficients (rl,2) were used to describe the

relationship between population growth measurements (time to emergence, survivorship,

size, k') and infection parameters: proportion infected, proportion with disseminated

infection, and body titer of females with disseminated infection among competitive

treatments of the two Aedes species. Thus, these analyses pool all competitive

treatments. The correlation analyses of body titer and size were based on sizes of females

with disseminated DENV-2 infections. Correlation coefficients quantify the strength of

the relationship (positive or negative) between population growth measurements and

infection parameters.









Blood meal plaque assay

Titrations of DENV-2 infectious blood meals were performed by plaque assays in

duplicate 6-well plates of confluent monolayers of Vero cells maintained with Leibovitz

L-15 media, 10% FBS, and 50 [tg/ml gentamicin. 10-fold serial dilutions of infectious

blood meal samples were made by combining a 0.2 ml DENV-2 blood meal sample with

1.8 ml media (2X Eagle's Minimum Essential Medium (EMEM) containing Earle's Basic

Salt Solution, 10% FBS, 100 U/ml penicillin, 100 [tg/ml streptomycin), thus creating a

10-1 dilution. This process was repeated to yield a full range of dilutions from 10-1 to

10-9. At the time of viral inoculation, media covering cell monolayers in the wells was

removed and wells were individually inoculated with 0.2 ml of the serial dilutions. Six-

well plates were gently rocked for 1 h incubation at 350C and a 5% CO2 atmosphere.

Following incubation, the first overlay of agarose was applied to the cell

monolayer. The first and second overlays of agarose described here provided sufficient

reagents to complete ten 6-well plate plaque assays. Briefly, 1.8 g Seaplaque low melting

agarose (FMC Biotechnology) was added to 100 ml of double distilled water. The

solution was heated until completely melted and then cooled to 400C. In a separate flask,

10 ml FBS was combined with 2 ml non-essential amino acid solution, 100U/ml

penicillin and 100 [tg/ml streptomycin. In another separate flask, 1 ml of L-glutamine

and 250 [tg/ml of Amphotericin B were added to 100 ml of 2X EMEM. Next, the

EMEM mixture was added to the agarose followed by the FBS mixture. Each well

received 3 ml of the first overlay of reagents. Six-well plates remained motionless for 5

min. to allow for the agarose to gel and then well-plate covers were removed for 15 min.









to facilitate drying. Finally, well plate covers were replaced and the 6-well plates were

incubated for 6 d at 350C and a 5% CO2 atmosphere.

The second overlay of agarose was applied on the 6th day of incubation. Briefly,

1.8 g Seaplaque low melting agarose was combined with 2.0 g sodium chloride and 200

ml double distilled water. The solution was heated until completely melted and then

cooled to 400C. Next, 9 ml neutral red solution (Sigma Cat: N2889) was added to the

solution and each well received 3 ml of the second overlay reagents. Six-well plates were

treated similar as the first overlay except that plates were incubated for 24 h at 350C and

5% CO2 atmosphere. Plaques were counted and expressed in plaque forming units (PFU)

per 0.2 ml of test inoculum.

Mosquito homogenization, plaque assay, and RNA extraction

For each mosquito, wings and legs were separated from bodies using forceps

sterilized with 70% ethanol followed by intense flaming (Turell et al. 1984). Wings were

measured and used as an indicator of female size for V' calculations. Bodies were

assayed to determine infection and whole body viral titer, whereas legs were assayed as

an indicator of disseminated infection (Turell et al. 1984). Bodies and legs were

homogenized separately in 2 ml flat bottom vials containing 1 ml media (Leibovitz L-15

media, 10% FBS, 100 U/ml of penicillin, 100 [tg/ml streptomycin, and 250 [tg/ml

Amphotericin B) and 2 zinc plated steel BBs (Daisy). Homogenization was performed

by placing vials into a TissueLyser (Qiagen) for 6 min. at 25 Hz followed by

centrifugation at 3148 x g for 4 min. and 4C. Body infection and disseminated infection

were determined by plaque assays using undiluted body and leg stock solutions. Plaque

assays were performed similarly to assays of blood meal titer, except that 12-well plates









were used instead of 6-well plates and additional antibiotics were added. A single well

was inoculated for each body and leg stock solution of each tested female. Only females

determined to have disseminated DENV-2 infections (i.e., positive infection in legs) were

subsequently assayed for body titer. Plaque assays for female bodies and legs were

scored as positive or negative with no attempt to count number of plaques. Subsequently,

body homogenates of females with disseminated infections were thawed and DENV-2

RNA was extracted from 140 [tl of the sample using QIAamp viral RNA Mini Kits

(Qiagen) and then assayed by quantitative real-time (RT)-PCR (Armstrong and Rico-

Hesse 2003, 2001).

Quantitative RT-PCR

Quantitative RT-PCR allowed for determination of the relative amounts of virus in

the body (viral titer), as measured by cDNA amplification, standardized with a plaque

assay (Richardson et al. 2006, Bustin 2000). A commercially available quantitative RT-

PCR kit, SuperScriptTM III Platinum one-step quantitative RT-PCR system

(InvitrogenTM), and fluorogenic probe hydrolysis (TaqMan) technology was used to

detect DENV-2 RNA. DENV-2 virus specific primers targeted the capsid gene (Forward

237-251 bp, Reverse 305-284 bp) (Callahan et al. 2001). Primer sequences were:

Forward (5'-CAT GGC CCT KGT GGC G- 3') and Reverse (5'-CCC CAT CTY TTC

AGT ATC CCT G-3') (Callahan et al. 2001). The DENV-2 specific dual-labeled

fluorogenic oligonucleotide probe included a 5'-reporter dye and a 3'-quencher dye (250

nm DLB 5' 6-FAM / 3' BHQ-2 (5'-TCC TTC GTT TCC TAA CAA TCC-3') (Callahan

et al. 2001).









Reactions used a thermostable enzyme, Taq DNA polymerase, derived from a

bacterium Thermus aquaticus (Holland et al. 1991). The oligonucleotide probe anneals

to the target RNA sequence downstream from a primer site. Under these conditions, the

reporter and quencher dyes are in close proximity so the quencher inhibits fluorescence

emission. The 5' nuclease activity of Taq DNA polymerase cleaves the probe during

extension so the reporter and quencher dyes separate and results in a detectable

fluorescence signal which is recorded by the lightcycler. PCR products are detected by

the generation of a fluorescent signal, and the intensity of the signal is directly related to

product accumulation. Regular cycling of temperature allows for denaturation,

annealing, and extension steps that are repeated resulting in exponential growth of the

target amplicon (DENV-2 cDNA).

Each reaction included: 0.4 [tl SuperScriptTM III RT/Platimum Taq mix, 10 [tl 2X

reaction mix (a buffer system, MgSO4, dNTPs and stabilizers), 1 [tl forward primer

(10[tM), 1 [tl reverse primer (10[tM), 0.5 [tl fluorogenic probe (10[tM), 4.2 [tl DEPC

treated H20, and 2 [tl test sample (positive or negative for DENV-2 RNA). Reactions

were performed in glass capillary tubes in a thermocycler, LightCycler 2.0 Instrument

equipped with LightCycler software version 3.5 (Roche Molecular Biochemicals). The

thermal cycle included: RT, 30 min. at 480C; Denaturing, 2 min. at 950C; followed by 45

cycles of PCR, 15 s at 950C, 1 min. at 600C. Each lightcyler set of reactions included a

negative control (water) and positive control standard (DENV-2 stock RNA, 10-5

dilution). The positive control was an indicator of cDNA synthesis and served as a

known standard of DENV-2 RNA used to produce cDNA. Plaque forming units (PFU)

were calculated by a standard curve method that compared cDNA synthesis to in vitro









PFU for the same full range of positive DENV-2 RNA stock virus titrated in parallel by

quantitative RT-PCR and plaque assay (Richardson et al. 2006, Bustin 2000).

Quantitative RT-PCR estimates crossing points which are cycle numbers that correspond

to the point at which exponential growth of the target amplicon occurs. Positive and

dilute test samples have high crossing points, since there was little initial RNA, whereas

concentrated test samples have low crossing points since there were greater amounts of

initial RNA. A standard curve was generated by assaying a full range of 10-fold serial

dilutions of DENV-2 stock (7.2 logo PFU/0.2 ml) by plaque assay (see blood meal

plaque assay) to quantify PFU, as well as by quantitative RT-PCR testing DENV-2

cDNA synthesis. Three replicates were used for each dilution assayed by quantitative

RT-PCR (slope = -3.007, intercept = 34.54, r2= 0.9604). Plaque assays determined that

2.2 logo PFU/0.2 ml corresponded to the 10-5 dilution. These estimates were used to

transform crossing points to PFU in determining body titer for Aedes females with

disseminated DENV-2 infections.

Species by Competition Comparison

An additional set of analyses were used as alternative methods to address mosquito

species by competitive treatment effects. The intention of the design of the competition

experiment should be primarily thought of as two experiments; One experiment for intra-

and interspecific competition for A. albopictus (160:0, 320:0, and 160:160) and another

experiment for intra- and interspecific competition for A. aegypti (0:160, 0:320, and

160:160). However, it is possible to analyze subsets of the experimental treatments to

further isolate species and competition effects on population growth measurements and

infection parameters. Population growth measurements (time to emergence, size of

females assayed for infection, survivorship) were analyzed by a two-way MANOVA and









SCC with mosquito species (A. albopictus and A. aegypti) and competitive treatment

(160:0, 320:0, 0:160, and 0:320) as factors. Treatments involving both species present

(e.g., 160:160) were intentionly omitted, thus isolating treatment effects. Similarly,

infection parameters (proportion infected, proportion with disseminated infection) were

analyzed by a two-way MANOVA and SCC with mosquito species and competitive

treatment as factors. Species and competitive treatment effects on body titer were

analyzed using a two-way ANOVA and significant effects were further analyzed by all

possible pairwise comparisons of treatment means (Tukey-Kramer adjustment of

experimentwise c = 0.05, SAS Institute 2002). Additionally, separate one-paired t-tests

were used to address species effects on proportion infected, proportion with disseminated

infection, and body titer in the interspecific competitive treatment (160:160).

Results

Competition Study

For both A. albopictus and A. aegypti, competitive treatments significantly affected

population growth measurements (Table 4-1) in the pattern 160 larvae < 320 larvae =

160:160 larvae (Figs. 4-1, 4-2). Greater competition consistently resulted in significantly

smaller adult size, longer time to emergence, and lower survivorship (A. albopictus mean

+ SE proportion surviving; 160:0, 0.42 0.03, 320:0, 0.27 0.03, 160:160, 0.32 0.03;

A. aegypti mean SE proportion surviving; 0:160, 0.36 0.02, 0:320, 0.31 0.01,

160:160, 0.33 0.02) than all less intense competitive treatments (Figs. 4-1, 4-2). For

both Aedes, SCC showed that differences in adult size and time to emergence contributed

the most to the significant competition effect as well as to subsequent treatment

differences (Table 4-1). For both species, competitive treatments significantly affected

' (A. albopictus, F2,28 = 90.44, P < 0.0001; A. aegypti, F2,28 = 150.84, P < 0.0001) and









k' was significantly greater in the pattern: 160 larvae > 320 larvae > 160+160 larvae (Fig.

4-3).

Infection Study

The infection study produced 2508 mosquitoes that successfully completed the

extrinsic incubation period. Six infectious blood meals were given over the course of the

experiment and plaque assays showed some variation between the blood meal viral titer

used in the experiment (mean SE; 6.47 0.098 logo PFU/ 0.2ml, 6.2-6.8 logo PFU/0.2

ml range). It was desirable to compare competitive treatments for females exposed to

identical blood meal titers. Logistic constraints precluded determining whether different

bloodmeal viral titers produced differences in mosquito infection parameters. Further,

different numbers of females were available for each bloodfeeding trial with many

treatments having few females available to bloodfeed. This presented a problem since it

prevented comparisons of all competitive treatments within a given bloodfeeding trial.

Thus, females were compared using those that completed the extrinsic incubation period

from the first bloodfeeding (6.2 logo PFU/0.2ml) which represented ca. 50 % of the total

number of females available to assay. Assaying less than all available females for

DENV-2 infection assumes that females from the first bloodfeeding are representative of

all females from the competition experiment. Similar outcomes of competition-induced

changes in infection parameters from a previous study support this assumption (Alto et al.

2005a). Mean SE females assayed per treatment replicate were; 160:0 (16.56 1.41),

320:0 (12.5 1.60), 160:160 (A. albopictus) (8.29 2.07), 160:160 (A. aegypti) (17.90 +

0.98), 0:320 (19.54 0.45), and 0:160 (20.00 + 0).









Interspecific differences (i.e., A. albopictus versus A. aegypti) in susceptibility to

infection with DENV-2 were compared and proportions infected and proportions with

disseminated infection were significantly different between the two Aedes species

(Pillai's trace 2,56 = 0.58, P < 0.0001). Proportion with disseminated infection (SCC =

1.19) contributed more to the overall interspecific difference than proportion infected

(SCC = -0.88). The opposite signs of the SCCs showed a negative relationship between

infection and dissemination, from the fact that A. albopictus had a greater proportion of

infected females but a lower proportion of disseminated infections compared to A.

aegypti (Fig. 4-4). Body titer was significantly different between species (Fi, 47 = 85.26,

P < 0.0001) and A. albopictus (mean SE, 4.6 0.06 logo PFU/0.2 ml) had greater body

titer compared to A. aegypti (mean SE, 3.9 0.04 logo PFU/0.2 ml).

Competition had significant effects on the proportion infected and proportion with

disseminated infections for A. albopictus (Table 4-2). Proportion infected provided the

largest contribution to bivariate differences among treatments and proportion with

disseminated infection contributed less (Table 4-2). Aedes albopictus at low density had

significantly lower proportion infected and disseminated infection compared to high

density intra- and interspecific competition (Table 4-2, Fig. 4-5). The two high density

treatments did not differ significantly (Table 4-2, Fig. 4-5). Competitive treatments

resulted in similar trends of infection and dissemination for A. aegypti, however, there

were no significant effects (Table 4-2, Fig. 4-6).

Separate analyses of covariance with mean female size as a covariate and mean

body titer, proportion infected, and proportion with disseminated infection showed no

significant competitive treatment or covariate effects on body titer for A. albopictus









(Table 4-3, Fig. 4-7). There were marginally significant effects of size x competition

interaction, as shown by regression lines for the three competitive treatments, and

significant competition effects for proportion ofA. albopictus infected (Table 4-3, Fig. 4-

8). Pairwise comparisons of LS means among competitive treatments showed a

significantly greater proportion ofA. albopictus infected at both high density intra- and

interspecific competitive treatments than low density treatment (LS means shown in Fig.

4-5). Proportions infected were not significantly different between high density intra-

and interspecific competitive treatments (LS means shown in Fig. 4-5). There was no

significant size effect for proportion ofA. albopictus infected (Table 4-3, Fig. 4-8).

There were marginally significant effects of competition on A. albopictus proportion with

disseminated infection (Table 4-3). Pairwise comparisons of LS means among

competitive treatments showed marginally significant differences (P = 0.0501) in

proportion of A. albopictus with disseminated infection between low density versus high

density interspecific competition with no other significant effects (LS means shown in

Fig. 4-5). There was no significant size effect for proportion of A. albopictus with

disseminated infection (Table 4-3, Fig. 4-9). For A. aegypti, there were no significant

competitive treatment or covariate effects (Table 4-3, Figs. 4-10, 4-11, 4-12).

Product-moment correlations showed significant relationships between the

proportions ofA. albopictus infected and with disseminated DENV-2 infections and time

to emergence and size (Table 4-4). Time to emergence, positively associated with intra-

and interspecific competition, was positively correlated with the proportion infected and

disseminated infection whereas size was negatively correlated with the proportion

infected and disseminated infection (Table 4-4).









Species by Competition Comparison

A two-way MANOVA showed significant species (A. albopictus and A. aegypti)

and competitive treatment (160:0, 320:0, 0:160, 0:320) effects on population growth

measurements, but no species x competitive treatment interaction (Table 4-5). SCC

showed that differences in adult size and time to emergence contributed the most to the

significant species and competitive treatment effects (Table 4-5). Aedes aegypti had

significantly shorter time to emergence (mean SE d; 12.81 0.29 and 13.74 0.29 for

A. aegypti and A. albopictus, respectively) and larger body size (mean SE mm; 2.83

0.02 and 2.74 0.02 for A. aegypti and A. albopictus, respectively) than A. albopictus.

Crowded larval conditions (320 larvae) resulted in smaller size (mean SE mm; 2.65

0.02 and 2.92 0.02 for 320 and 160 larvae, respectively) and longer time to emergence

(mean SE d; 15.49 0.29 and 11.07 0.30 for 320 and 160 larvae, respectively).

A two-way MANOVA showed significant species (A. albopictus and A. aegypti)

and competitive treatment (160:0, 320:0, 0:160, 0:320) effects on proportion infected and

proportion with disseminated infection, but no species x competitive treatment interaction

(Table 4-6). SCC showed that differences in the proportion of females with disseminated

infections contributed the most to the significant species effect, whereas differences in

the proportion infected contributed the most the significant competitive treatment effect

(Table 4-6). Aedes aegypti had lower infection rates (mean SE proportion infected;

0.75 0.02 and 0.88 0.02 for A. aegypti and A. albopictus, respectively) but higher

dissemination rates (mean SE proportion with disseminated infection; 0.60 + 0.03 and

0.32 0.03 for A. aegypti and A. albopictus, respectively) than A. albopictus. Crowded

larval conditions (320 larvae) resulted in higher infection rates (mean SE proportion

infection; 0.88 0.02 and 0.76 0.03 for 320 and 160 larvae, respectively) and









dissemination rates (mean SE proportion with disseminated infection; 0.50 + 0.03 and

0.42 0.03 for 320 and 160 larvae, respectively) than uncrowded conditions (160 larvae).

A two-way ANOVA showed significant species and species x competitive

treatment interaction effects on body titer (Table 4-7). Aedes albopictus had significantly

greater body titer than A. aegypti but this interspecific effect depended on competitive

treatment (Fig. 4-13). The species x competitive treatment interaction was attributable to

less interspecific difference (A. albopictus versus A. aegypti) in body titer from crowded

larval conditions (320 larvae) compared to uncrowded conditions (160 larvae) (Fig. 4-

13).

Separate one paired t-tests on infection parameters in the interspecific competitive

treatment (i.e., 160:160) showed significant differences in the proportion infected,

proportion with disseminated infections, and body titer (All P < 0.05). Aedes albopictus

had significantly greater proportion infected (mean SE; A. albopictus, A. aegypti; 0.93

0.04, 0.82 0.03), lower proportion with disseminated infection (mean SE; A.

albopictus, A. aegypti; 0.47 0.09, 0.71 0.05), and greater body titer (mean SE; A.

albopictus, A. aegypti, 4.50 + 0.08, 3.99 0.05) than A. aegypti.

Discussion

All population growth measurements showed consistently poorer performance for

mosquitoes reared at high larval density (Figs. 4-1, 4-2, 4-3). These results are consistent

with those of a similar experiment, using Aedes species in tests of competitive treatment

effects on adult SINV infection (Alto et al. 2005a). The goal of the SINV and DENV

experiments was to maximize adult mosquito production, to assess vector competence,

without negating the effects of larval competition. To achieve this, a combination of

natural (oak leaf infusion) and artificial (yeast, albumin) larval food resources was used.









Previous laboratory and field research shows contrasting outcomes of competition

between these Aedes species dependent upon larval resource type, with A. aegypti an

equal or superior competitor with protein-rich resources (e.g., liver powder, yeast) and A.

albopictus a superior competitor with plant detritus (e.g., leaves) (Juliano and Lounibos

2005, Braks et al. 2004, Juliano 1998, Barrera 1996, Black et al. 1989). The present

study confirms the ability to replicate environmental variables that provide for larval

competition between A. aegypti and A. albopictus.

Higher levels of intra- and interspecific competition significantly enhanced DENV-

2 infection and dissemination for A. albopictus. Phenotypic expression of vector

competence in adult mosquitoes was significantly altered by competitive conditions of

the aquatic larval environment. These results may be the product of norms of reaction of

the genes controlling vector competence in these mosquitoes. Other studies on

competition and phenotypic expression of a trait have shown that the phenotypic

expression of virulence is altered when hosts were exposed to different competitive

conditions or nutrient gradients (e.g., Bedhomme et al. 2004, Scheiner 1993). Vector

competence studies have shown that there is evidence that a great amount of phenotypic

variance in DENV-2 infection in A. aegypti is associated with environmental and random

experimental effects (e.g., Bosio et al. 2000). The proportion of infected A. albopictus

females contributed more to the competitive treatment effects than the proportion of

females with disseminated infections (Table 4-2). This observation suggests that initial

infection in the adult mosquito midgut is most influenced by larval competition.than

escaping the midgut and infecting other organs (i.e., dissemination).









The lack of significant covariate (size) effects in the analyses of covariance

suggests that size had no direct effect on vector competence within competitive

treatments for both species (160, 320, 160:160) (Table 4-3). A marginally significant

interaction between the covariate and competitive treatment for proportion A. albopictus

infected (Table 4-3) suggests that the effect of mosquito size on infection may depend on

competition experienced. Among replicates of the low density treatments, which

produced females of the largest average sizes, proportion infected declined with

increasing mean body size. This decline is not evident in smaller females from high

density treatments (Fig. 4-8). Significant or marginally significant effects of competition

were observed for A. albopictus because more intense competition resulted in greater

DENV-2 infection and disseminated infection (Figs. 4-8, 4-9). Thus, differences in

Aedes size, within competitive treatments make little or no contribution to the differences

observed in infection parameters.

Although mean size may contribute little to differences in infection parameters

within competitive treatments, mean size was significantly correlated with infection

parameters across competitive treatments, perhaps due to competitive effects (Table 4-4,

Figs. 4-8, 4-9). Specifically, small adult females, associated with intra- and interspecific

competition, had enhanced infection and dissemination rates for A. albopictus (Table 4-

4). Under the conditions of the current study small A. albopictus produced by

competitive treatments may pose a greater health risk for DENV transmission compared

to larger conspecifics. This effect may be enhanced because small adults may bloodfeed

more frequently than larger adults (Scott et al. 2000b). However, competitive treatment

differences in size may be correlated with overall competitive stress, making it difficult to