<%BANNER%>

Interactions of Invasive Species in Mosquito Container Communities in Virginia


PAGE 1

INTERACTIONS OF INVASIVE SPECIES IN MOSQUITO CONTAINER COMMUNITIES IN VIRGINIA By JENNIFER S. ARMISTEAD A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2007 1

PAGE 2

2007 Jennifer S. Armistead 2

PAGE 3

To my husband Paul, whose love, friendship, pati ence and encouragement are heaven-sent, and to my father Gary, for showing me that in a ll things nature there is something marvelous. 3

PAGE 4

ACKNOWLEDGMENTS I would like to thank my advisor, L. P. L ounibos, for sharing his expertise and providing direction for my research, as well as for his conf idence, patience, and flexibility in working with me from a distance. I am grateful to my comm ittee members, G. F. OMeara for his insight in working with Ochlerotatus atropalpus and J.R. Arias for sharing his knowledge and ingenuity and for graciously allowing me to use his equipmen t and laboratory space. I would like to thank R. Escher, N. Nishimura, and M. Reiskind for their technical assistan ce during my time at the Florida Medical Entomology Labor atory, and at the Fairfax Count y Department of Health in Virginia I am appreciative of J. Frescholtz and J. van der Voort for thei r assistance in the field, and A. Joye for his help with GIS. I am grateful to L. McCuiston for providing the O. atropalpus and O. japonicus eggs used in this research. I would al so like to thank B. Harrison and J. Scott for engaging in discussions with me regarding O. japonicus 4

PAGE 5

TABLE OF CONTENTS page ACKNOWLEDGMENTS ...............................................................................................................4 ABSTRACT ...................................................................................................................................10 CHAPTER 1 INTRODUCTION AND LITERATURE REVIEW..............................................................12 Invasion Biology .....................................................................................................................12 Ochlerotatus japonicus ...........................................................................................................15 2 FIELD ASSESSMENT OF INTERSPECIF IC INTERACTIONS AMONG INVASIVE AND NATIVE CONTAINER-INHABITING MOSQUITOES............................................19 Introduction .............................................................................................................................19 Materials and Methods ...........................................................................................................21 Oviposition Traps ............................................................................................................21 Natural and Artificial Containers ....................................................................................23 Adult Surveillance ...........................................................................................................24 Data Analysis ..........................................................................................................................24 Oviposition Traps ............................................................................................................24 Natural and Artificial Containers ....................................................................................25 Adult Surveillance ...........................................................................................................27 Results .....................................................................................................................................28 Oviposition Traps ............................................................................................................28 Natural and Artificial Containers ....................................................................................29 Interspecific Associations ........................................................................................30 Habitat Comparisons ................................................................................................31 Aedes albopictus and Ochlerotatus japonicus .........................................................31 Ochlerotatus japonicus and Ochlerotatus triseriatus ..............................................34 Ochlerotatus atropalpus and Ochlerotatus japonicus .............................................34 Adult Surveillance ...........................................................................................................35 Discussion ...............................................................................................................................36 3 INTERSPECIFIC COMPETITION BETWEEN AEDES ALBOPICTUS AND OCHLOEROTATUS JAPONICUS .........................................................................................66 Introduction .............................................................................................................................66 Materials and Methods ...........................................................................................................69 Data Analysis ..........................................................................................................................71 Population Growth Correlates .........................................................................................71 Composite Index of Population Performance ..................................................................72 Results .....................................................................................................................................74 5

PAGE 6

Survivorship to Adulthood ..............................................................................................74 Developmental Time .......................................................................................................74 Female Wing Length .......................................................................................................75 Estimated Finite Rate of Increase ( ) .............................................................................75 Discussion ...............................................................................................................................75 4 INTERSPECIFIC COMPETITION BETWEEN OCHLEROTATUS ATROPALPUS AND OCHLEROTATUS JAPONICUS .................................................................................85 Introduction .............................................................................................................................85 Materials and Methods ...........................................................................................................87 Data Analysis ..........................................................................................................................89 Population Growth Correlates .........................................................................................89 Composite Index of Population Performance ..................................................................89 Results .....................................................................................................................................92 Survivorship to Adulthood ..............................................................................................92 Developmental Time .......................................................................................................92 Female Wing Length .......................................................................................................93 Estimated Finite Rate of Increase ( ) .............................................................................93 Discussion ...............................................................................................................................93 5 CONCLUSIONS ..................................................................................................................105 APPENDIX A DESCRIPTIVE STATISTICS AN D INFORMATION: CHAPTER 2................................109 B HABITAT COMPARISONS: RANK OR DERS OF MOSQUITO SPECIES....................110 LIST OF REFERENCES .............................................................................................................111 BIOGRAPHICAL SKETCH .......................................................................................................124 6

PAGE 7

LIST OF TABLES Table page 2-1 Frequency of occurrence of A. albopictus O. hendersoni and O. triseriatus in ovitraps set at three sites and two hei ghts in Fairfax, Virginia in 2006. ..........................................44 2-2 Frequency of occurrence, by month, of A. albopictus O. hendersoni and O. triseriatus in ovitraps in Fairfax County, Virginia in 2006. ................................................................45 2-3 Monthly abundance (mean numb er of eggs per trap) of A. albopictus, O. hendersoni and O. triseriatus collected in ovitraps. ....................................................................................46 2-4 Coefficients of association (C8) for the most abundant speci es in 191 artificial and natural container samples from Fairfax County, Virginia in 2006 ....................................47 2-5 Intraand interspecific mean crowding of the most abundant mosquito species collected from artificial and natural containers in Fairfax County, Virginia in 2006 .......................48 2-6 Species rank abundances compared for differe nt container habitats sampled in Fairfax County, Virginia from May September 2006. ................................................................49 2-7 Number of larvae and average instar number for A. albopictus and O. japonicus collected in Fairfax County, Virg inia by month, May September 2006. ........................50 2-8 Tests for significant heterogeneity of monthly instar distributions of A. albopictus and O. japonicus from June August 2006, ba sed on log-rank statistics.....................................51 2-9 Least square means ( SE) for intraspecific crowding (transformed by log10 ( x + 1)) among larvae of A. albopictus and O. japonicus June August 2006..............................52 2-10 Frequencies of collection of A. albopictus O. japonicus and O. triseriatus adult females in CO2-baited CDC light traps and gravid traps, 2004 2006.............................53 3-1 Means ( SE) of population growth correlates for A. albopictus and O. japonicus .................79 4-1 Means ( SE) of population growth correlates for O. japonicus and O. atropalpus ................99 A-1 Descriptive statistics and information for mosquito species collected in a survey of natural and artificial container habita ts in Fairfax County, Virginia in 2006 ..................109 B-1 Rank orders of immature mosquito abundan ces used for habitat comparisons of rock pools, tires, small and la rge artificial containers..............................................................110 7

PAGE 8

LIST OF FIGURES Figure page 2-1 Map of Fairfax County, Virginia showing loca tions of study areas that were sampled or censused repeatedly. ..........................................................................................................54 2-2 Proportion of mosquito-pos itive containers containing A. albopictus, O. japonicus or both A. albopictus and O. japonicus May September 2006. ...........................................55 2-3 Monthly abundance (mean number of mosquitoes co llected per container) of A. albopictus and O. japonicus (SE) from 91 mosquito-posit ive artificial containers........56 2-4 Seasonal occurrences (proportion of sp ecies-positive containe rs per month) of A. albopictus and O. japonicus collected from artificial containers in 2006 .........................57 2-5 Monthly instar distributions of A. albopictus from May September 2006 in Fairfax, Virginia. .............................................................................................................................58 2-6 Monthly instar distributions of O. japonicus from May September 2006 in Fairfax, Virginia. .............................................................................................................................60 2-7 Interspecific mean crowding of A. albopictus by O. japonicus and O. japonicus by A. albopictus ...........................................................................................................................60 2-8 Intraspecific mean crowding (density of conpspecifics encountered per unit resource, a) of A. albopictus and O. japonicus by month. .....................................................................61 2-9 Metamorphic success of A. albopictus and O. japonicus collected from containers in which the two species co-occurred. ...................................................................................62 2-10 Mean weekly abundance of A. albopictus collected in (A) CO2-baited light traps and (B) gravid traps over time, from 2004 through 2006, in Fairfax County, Virginia...........63 2-11 Mean weekly abundance of O. japonicus collected in (A) CO2-baited light traps and (B) gravid traps over time, from 2004 through 2006, in Fairfax County, Virginia...........64 2-12 Mean weekly abundance of O. triseriatus collected in (A) CO2-baited light traps and (B) gravid traps over time, from 2004 through 2006, in Fairfax County, Virginia...........65 3-1 Mean survivorship (proportion of the origin al number of larvae surviving to adulthood) of A. albopictus and O. japonicus (SE) ...........................................................................80 3-2 Means of median time to adulthood for female A. albopictus and O. japonicus (SE) ..........81 3-3 Means of median time to adulthood for male A. albopictus and O. japonicus (SE) .............82 3-4 Means of median wing lengths of A. albopictus and O. japonicus adult females (SE) ........83 8

PAGE 9

3-5 Mean estimates of population performance ( , an estimate of the finite rate of increase for the cohort) for female A. albopictus and O. japonicus adults (SE) ...........................84 4-1 Mean survivorship (proportion of the origin al number of larvae surviving to adulthood) of O. japonicus and O. atropalpus (SE) ........................................................................100 4-2 Means of median time to adulthood for female O. japonicus and O. atropalpus (SE) .......101 4-3 Means of median time to adulthood for male O. japonicus and O. atropalpus (SE) ..........102 4-4 Means of median wing lengths of O. japonicus and O. atropalpus adult females (SE) .....103 4-5 Mean estimates of population performance ( , an estimate of the finite rate of increase for the cohort) for female O. japonicus and O. atropalpus adults (SE) ........................104 9

PAGE 10

ABSTRACT OF THESIS PRESEN TED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE INTERACTIONS OF INVASIVE SPECIES IN MOSQUITO CONTAINER COMMUNITIES IN VIRGINIA By Jennifer S. Armistead May 2007 Chair: L. Philip Lounibos Major: Entomology and Nematology The success of an invasive species to become established in a new region often depends on its interactions with ecologically similar resi dent species. Introductions of disease vectors, particularly mosquitoes, are of significant importa nce as their invasions may have ecological and epidemiological consequences. Interactions of a recent invasive mosquito with resident species in containers in Virginia were evaluated th rough field surveys and controlled experiments. In my study, sampling of larvae from natural and artificial containe rs, trapping of adults, and ovitraps were used to confirm and quantif y co-occurrences and potential interactions of Ochlerotatus japonicus with resident mosquitoes in thes e habitats. Frequent and abundant occurrences of O. japonicus in rock pools were associated with the possible decline and displacement of native O. atropalpus Laboratory evaluation of the effects of larval resource competition on the population perfor mance of these two species suggest that interspecific competition is probable and likely to favor the success of O. japonicus over O. atropalpus Autogenous reproduction of O. atropalpus which requires a lengthe ned period of larval 10

PAGE 11

development to obtain nutrient reserves for e gg development, may disadvantage it in larval competition in conditions of limited resources. In my study, field collections of O. japonicus from artificial contai ners inhabited by larvae of resident mosquitoes demonstrated their coexis tence in these habitats. A field experiment that measured interand intraspecifi c effects of larval density on the population performance of A. albopictus and O. japonicus indicated the former species to be a superior competitor. However, the ability of O. japonicus to perform equally well in the presence of absence of A. albopictus suggests these two species will be able to coexist in artificial container ha bitats in nature. High larval densities, intraspecific mean crow ding, and superior population performance of A. albopictus under interrather than intraspecific conditions suggest that intraspecific competition may be most important in regul ating population growth of this species in cont ainer habitats. Interactions of O. japonicus with resident container-inhabiting mosquitoes appear to be influenced by species-specific differences in seasonality. The ability of O. japonicus to overwinter as larvae allows it to resume development earlier in the spring than both A. albopictus and O. atropalpus Consequent co-occurrence of older O. japonicus with early hatchlings of A. albopictus and O. atropalpus may favor the more recent invader during competition. Infrequent collections of O. japonicus in ovitraps suggest that use of this technique may be improved in alternative macrohab itats where this species is mo st abundant. While trapping of adults over a three-year pe riod indicated no changes in th e frequency or abundance of A. albopictus, significant population declines of both O. japonicus and O. triseriatus were observed over this period. Continued monitoring of all life st ages of these species over several years will be necessary to observe any significant populatio n changes or identify ecological processes at work since the invasion of O. japonicus 11

PAGE 12

CHAPTER 1 INTRODUCTION AND LITERATURE REVIEW Invasion Biology Biological invasions are occurring at an alar ming frequency worldwide, the impacts of which threaten biodiversity, eco system functioning, resource avai lability, national economies, and human health (Ruesink et al. 1995, Simberloff 1996). Nonindigenous organisms, including invertebrates, vertebra tes, plants, bacteria, and fungi, are spreading into new regions at unprecedented rates, becoming established in all but the most remote areas of the planet. Historically, some of the most important biologi cal invasions have involve d dispersal of disease vectors, particularly mosquitoes (Diptera: Culicid ae), a trend that has continued at an increasing rate over the past century (reviewed by Lounibos 2002, Juliano and Lounibos 2005). The most recent successful invasions of mosquitoes have resulted from human transport of immature stages. The most notable invasi ons and range expansions in th e United States have been by mosquitoes that occupy container habitats (Lounibos 2002), which is the topic of my study. The patterns and processes related to the intr oduction, establishment, spread, and impact of non-native species is the focus of invasion bi ology (Williamson 1996). The term invasive refers to an introduced species that has increased and spread (D aehler 2001, Richardson et al. 2000), with the potential to impact native species a nd ecosystems, or human activities (Juliano and Lounibos 2005). Common characteristics of i nvasive species are thought to include an r -selected life history strategy, range expans ion, high rate of population incr ease, ability to compete for resources and habitat with native species, repeated introductions, and associations with humans (reviewed by Sakai et al. 2001). The three most successful species of invasive mosquitoes, Aedes aegypti, A. albopictus and the Culex pipiens Complex, seem to have been favored by propagule (or invasion) frequency and previous invasion success (Lounibos 2002). A retrospective review 12

PAGE 13

of life history characteristics of successful invasive mosqu itoes by Juliano and Lounibos (2005) revealed that desiccation-resistant eggs and occupying human-dominated habitats are significantly associated with invasi ve status, while larval habitat, autogeny, and diapause are not; however data are absent for many species. Invasive organisms may impact native speci es through biotic interactions, such as predation, parasitism and competition, as well as ecosystems by affecting ecological processes, such as water and nutrient cy cling (Williamson 1996). Invasions may also have genetic and evolutionary consequences, re sulting in short-term change s, such as hybridization or introgression (Rhymer and Simberloff 1996), or longterm changes from genetic drift or natural selection (Williamson 1996, Sakai et al. 2001). Multip le processes will likely act in a single invasion system, and they may either facilitate invasion success and spread, which is often indicated by the decline or elimination of ecologically simila r species (Juliano 1998), or act as barriers to invasion (R osen et al. 1976, O Meara et al. 1989). Many ecological processes associated with i nvasive species have been shown to play important roles in structuring the mosquito communities of container habitats, including interspecific competition, predation, and parasitism Interspecific competition may be defined as any negative negative effect between two species or a negative zero effect where one species affects another but is not reciprocally affect ed. Interspecific competition is unavoidable unless the invader is filling a vacant ni che by exploiting a previously unoccupied habitat or unused resource (Williamson 1996). Interspecific compe tition among mosquitoes encompasses resource competition (Ho et al. 1989, Livdahl and Willey 1991, Daugherty et al. 2000, Juliano et al. 2004, Costanzo et al. 2005), chemical interference (Sunahara and Mogi 2002, Bdhomme et al. 2005), mating interference (Ribiero and Spielman 1986, Nasci et al. 1989), and hatching inhibition 13

PAGE 14

(Edgerly et al. 1993). Predation is a prominent feature of many c ontainer habitats utilized by mosquitoes. Differences in behavioral responses to water-borne cues from predation (Kesavaraju and Juliano 2004) are thought to re sult in selective predation on invasive (Grill and Juliano 1996, Griswold and Lounibos 2005a,b) or native (Lounibos et al. 2001) species. Intraguild predation, in which older immature stages prey upon newly hatched larvae of conspecifics or heterospecifics, is also known to occur in mosquito communities (Edgerly et al. 1999, Koenraadt and Takken 2003, Koenraadt et al. 2004). This ty pe of predation is often facilitated by differences in seasonality, in which one species ha tches earlier in the year than the other (Teng and Apperson 2000). Apparent competition cause d by shared gregarine parasites has been investigated in interactions of invasive mosqu itoes with multiple resident species with varying results (Juliano 1998, Alia badi and Juliano 2002). In addition to effects on resident species or ecosystems, invasive mosquitoes are of particular concern because they may also imp act human or vertebrate animal health. While interactions with resident spec ies usually occur among aquatic larv ae, it is the terrestrial adult phase of mosquitoes that is most likely to impact human health. Invasive mosquitoes in the New World have been associated with human dis ease outbreaks of yellow fever (Tabachnick 1991), dengue (Gubler 1997), and malaria (Soper a nd Wilson 1943, Lounibos 2002), and may be associated with the transmission of Eastern equi ne, LaCrosse, and West Nile encephalitis viruses (reviewed by Lounibos 2002, Juliano and Lounibos 2005). Human health may be affected by invasive mosquitoes in three ways (Juliano a nd Lounibos 2005): simultaneous introduction of a novel vector and novel pathogen (Tabachnick 1991, Gubler 1997), acquisition of a native pathogen by a novel vector (Soper and Wilson 1943, Gubler 1997, Lounibos 2002), or independent introductions of a novel vector and a novel pathogen (Ross 1911, Kramer and 14

PAGE 15

Bernard 2001). Simultaneous or independent in troductions of novel vectors and novel pathogens may create new public health threats due to th e high susceptibility of the host population, whereas if a novel vector becomes involved in an existing disease transmission cycle, it alters the nature of an existing public hea lth threat by changing the transmi ssion rate due to differences in vector efficiency (Juliano and Lounibos 2005). Ochlerotatus japonicus In 1998, Ochlerotatus japonicus, a container-inhabiting mosqu ito native to eastern Asia (Tanaka et al. 1979), was detected independently in light trap collections in New York and New Jersey (Peyton et al. 1999) and human biting co llections in Connecticut (Andreadis et al. 2001). The introduction of O. japonicus is presumed to have occurred vi a tire shipments (Peyton et al. 1999), a mechanism which accounted for multiple inte rceptions of larvae of this species in New Zealand (Heardon et al. 1999, Laird et al. 1994). Si nce its arrival in 1998, O. japonicus has spread throughout the eastern United States with reports from Alabama (Qualls and Mullen 2006), Georgia and South Carolina (Reeves a nd Korecki 2004), Indiana (Young et al. 2004), Maine (Foss and Dearborn 2001), Maryland (Sardelis and Turell 2001), Miss ouri (Gallitano et al. 2006), Ohio and Pennsylvania (Fonseca et al. 2001), Tennessee (Caldwell et al. 2005), Virginia (Harrison et al. 2002), and Vermont (Graham and Tu rmel 2001). On the West coast of the United States, O. japonicus is only known to occur in Wash ington state (Roppo et al. 2004). Examination of patterns of genetic divers ity using random amplified polymorphic DNA and sequences of mtDNA from populat ions spanning the range of O. japonicus in Japan and the United States revealed distinct genetic signa tures in U.S. populations, suggesting multiple introductions from Japan (Fonseca et al. 2001). In addition to the United States, O. japonicus has become established in Canada (Savignac et al. 2002, Thielman and Hunter 2006) Belgium (Widdel et al. 2005), and France (Schaffner et al. 2003). 15

PAGE 16

Ochlerotatus japonicus was previously known as Aedes japonicus until revisions of that genus by Reinert (2000), based on differences in th e primary characteristics of male and female genitalia, elevated the subgenus Ochlerotatus to generic level. Ochlerotatus japonicus is a member of the Chrysolineatus subgroup of the subgenus Finlaya Ochlerotatus japonicus sensu lato includes four morphologically similar subs pecies that occur throughout most of Japan, Taiwan, Korea, eastern China, and Russia (Tanaka et al. 1979): O. japonicus amamiensis (Tanaka, Mizusawa and Saugstad 1979), O. japonicus japonicus (Theobald 1901), O. japonicus shintienensis (Tsai and Lien 1950), and O. japonicus yaeyamensis (Tanaka, Mizusawa and Saugstad 1979). Only the subspecies O. japonicus japonicus which is common in Palearctic Japan and Korea, has been detect ed outside its native range (Fons eca et al. 2001); henceforth in this thesis, O. japonicus japonicus will be referred to simply as O. japonicus. In both the native range of this species and the United States, O. japonicus larvae inhabit a wide variety of natural (treeholes, rock pools) and artificial contai ners (buckets, tires, birdbaths), with rock pools being the pref erred habitat (Tanaka et al. 1979, Andreadis et al. 2001, Scott 2003). Surveys have revealed that the species may occur in tem porary ground water sources as well, although this is uncommon (LaCasse and Yamaguti 1950, Andreadis et al. 2001). Ochlerotatus japonicus larvae have been detected in containers with varying levels of sun exposure and water temperature (Oliver et al. 2003) Adults of this specie s have been shown to feed on avian and mammalian hosts in th e laboratory (Miyagi 1971), although bloodmeal analyses of wild-caught female s have all been mammalian in or igin (Scott 2003, Apperson et al. 2002). Ochlerotatus japonicus will bite humans that encroach on its habitat (Knight 1968), but are often protracted in their appro ach (B. Harrison personal communication). 16

PAGE 17

The introduction of O. japonicus into the United States is of considerable interest because of the vector potential of this species as well as the ecological consequences that may result from its invasion. Ochlerotatus japonicus has demonstrated the ability to transmit Japanese encephalitis (Sucharit et al. 1989, Takashima and Rosen 1989) and Getah (Takashima and Hashimoto 1985) viruses in the labo ratory, although it is not consid ered an important vector of either virus in its native range, having only b een implicated in the transmission of Japanese encephalitis virus in Far East Asia (Chagin and Kondratiev 1943). Ochlerotatus japonicus is also a competent laboratory vector of eastern equine encephalitis (Sardelis et al 2002a), LaCrosse (Sardelis et al. 2002b), St. Louis encephalitis (Sardelis et al. 2003) and West Nile (Sardelis and Turrell 2001, Turrell et al. 2001) vi ruses. However, in the United States only the latter has been recovered from wild-caught females of this species (Werner 2001, White et al. 2001, Scott 2003). This combination of a separately in troduced novel vector and novel pathogen could become epidemiologically significant in North America. Ochlerotatus japonicus is known to co-occur with numerous resident species in natural and artificial containers in North America (Andreadis et al. 2001, Sc ott et al. 2001a, Oliver et al. 2003, Thielman and Hunter 2006). This species ha s most frequently been recovered from containers inhabited by A. albopictus C. pipiens (Andreadis et al. 2001, Gallitano et al. 2006), Culex restuans (Scott et al. 2001a), O. atropalpus and O. triseriatus (Andreadis et al. 2001, Oliver et al. 2003), although it has also been known to occur with Anopheles punctipennis (Scott et al. 2001a) Anopheles quadrimaculatis (Thielman and Hunter 2006) and Culex territans (Oliver et al. 2003). However, ther e is currently no information concerning the nature of the interactions of O. japonicus with these species or the ecol ogical processes that have been 17

PAGE 18

operating during its invasion. Therefore, a study of the interactions of O. japonicus with resident species in natural and ar tificial container communities was proposed. The primary objective of my study was to c onfirm and assess the interactions of O. japonicus in mosquito container communities and ma ke predictions concerning the ecological processes that may be operating as a result of th e invasion of this species. This was accomplished through a field survey of all life stages conducted areas in which this species is known to occur in various types of mosquito container commun ities. As rock pools are the species preferred habitat, it is predicted that O. japonicus will most likely interact with and impact the native rock pool mosquito, O. atropalpus Furthermore, the tendency of O. japonicus to inhabit artificial containers suggests that this species will also likely encounter and interact with the Eastern treehole mosquito, O. triseriatu s, and its closely related sibling species, O. hendersoni as well the now resident invader A. albopictus. Because severe crowding and limiting resources are frequent in container habitats (Kitching 2000), it is predicted that interspecific competition via depletion of shared resources will be important in the in teractions of the larvae of O. japonicus and A. albopictus in artificial containers, and O. atropalpus in rock pools. Therefore, the effects of interspecific resource comp etition on the growth, survivorsh ip, and reproductive success of these species were measured in a series of field and laboratory experiments, in order to determine the impact of these larval conditions on the overall population growth of these species. 18

PAGE 19

CHAPTER 2 FIELD ASSESMENT OF INTERSPECIFIC INTERACTIONS AMONG INVASIVE AND NATIVE CONTAINER-INHABITING MOSQUITOES Introduction Naturally occurring throughout Japan, Korea, Taiwan, and China (Tanaka et al. 1979), Ochlerotatus japonicus is the most recently recognized mosquito species to invade the United States, which is thought to have occurred via the interna tional transport of used tires (Peyton et al. 1999, Lounibos 2002). Initially collected in the summer of 1998 in Connecticut (as reported by Andreadis et al. 2001), New York a nd New Jersey (Peyton et al. 1999), O. japonicus has since become established thr oughout the northeastern United States (reviewed by Scott 2003), and spread south to Georgia (Reeves and Kor ecki 2004), north into Canada (Savignac et al. 2002, Thielman and Hunter 2006), a nd west to Missouri (Gallitano et al. 2006). The spread of O. japonicus on the west coast has so far been lim ited to Washington State (Roppo et al. 2004). Distinct genetic profiles of O. japonicus collected from various site s in Japan and throughout its range in the United States suggest multiple introduc tions of the species in the U.S. (Fonseca et al. 2001). Although common and widely distri buted within its na tive range in eastern Asia (LaCasse and Yamaguti 1950, Tanaka et al 1979), O. japonicus attracted little scientific attention until its discovery in North America, wher e its potential involvement in the transmission of West Nile virus (Sardelis and Turell 2001) and other endemic arboviruses (S ardelis et al. 2002ab, 2003) has stimulated significant scientific interest. Knowledge of the bi ology and surveillance methods for the collection of O. japonicus has since improved remarkably. Collection records from both Japan (Sasa et al. 1947) and the United States (Andreadis et al. 2001) in dicate that adults are rarely taken in light traps, carbon dioxide (CO 2 ) baited traps, or biti ng collections. The use of 19

PAGE 20

infusion-baited gravid traps, typically in tended to attract mosquitoes of the genus Culex (Reiter 1983), has provided the most consis tent and greatest numbers of O. japonicus adults (Scott et al. 2001b). As a container-inhabiting mosquito, ovipos ition traps are a logical sampling method with many potential applications, includ ing detection in new areas, r outine population surveillance, and species distribution studies. However this technique has been employed with varying success in collecting O. japonicus (Andreadis et al. 2001). Modificatio n of the traditional oviposition trap using blocks of expanded polystyrene as an ovipos ition substrate in a variet y of aquatic habitats has been indicated as a suitable al ternative (Scott and Crans 2003). Ochlerotatus japonicus appears to be most easily and consistently collected in large numbers in the larval stage. Within its native range in Asia, O. japonicus larvae are found in a wide variety of natural and artifi cial containers, with rock pool s being the preferred oviposition substrate (LaCasse and Yamaguti 1950, Tanaka et al. 1979). In the United States, O. japonicus larvae are commonly found in artif icial (e.g. automobile tires, bird baths, gutters, flower pots) and natural (tree holes and rock pools) containe rs where they feed on organic detritus and microorganisms, often in close association with human dwellings (Andreadis et al. 2001). Larvae may be found in shaded or sunny locations (Sco tt 2003) in clean, clear wa ter containing decaying leaf litter and algae (Scott et al. 2001a). The likelihood of an invasive species coloni zing a new region depends on its ability to adapt to new environmental conditions and compet e with resident species that occupy a similar ecological niche. The indigenous mosquito species most likely to be affected by O. japonicus in the United States include the rock hole mosquito, O. atropalpus the Eastern treehole mosquito, O. triseriatu s and its closely rela ted sibling species, O. hendersoni as well as the now resident invader Aedes albopictus Frequent and abunda nt collections of O. japonicus larvae co-occurring 20

PAGE 21

with both O. atropalpus in rock pools and A. albopictus in artificial contai ners, provide a natural setting for interspecific larval resource compe tition (Andreadis et al. 2001) which some speculate may limit its invasion success (Juliano and Lounibos 2005). However at present, no field research has been published to confirm this conjecture following the establishment of O. japonicus in the United States. To better understand the ecological niche of O. japonicus with respect to the aforementioned resident containerinhabiting species, a broad-based survey of all life stages was conducted in Fairfax Count y, Virginia (latitude 38 50 N, longitude 77 7 W), a suburb of Washington, D.C., where all of these species ar e known to co-occur. Sampling of natural and artificial larval habi tats was used to confirm and qua ntify co-occurrences and potential interactions of O. japonicus with resident mosquitoes in th ese habitats. Routine trapping of adults over a three-year period was used to assess changes in frequency or abundance of the species of interest. Ovitraps were employed to better understand the seasonality of egg-laying behavior of these species as well as their tende ncies to oviposit at gr ound level versus in the canopy, or in residential areas vers us disturbed or undisturbed forest s. In particular, the current chapter focuses on the potential interactions of O. japonicus with A. albopictus in artificial containers and O. atropalpus in rock pools in an effort to make predictions regarding the competitive outcomes resulting from the co-occurrence of these species. Materials and Methods Oviposition Traps To monitor the distribution, abundance, and oviposition of O. japonicus and resident container-inhabiting mosquitoes in Fairfax C ounty, VA, standardized collections of eggs employed the use of ovitraps. These dark, wa ter-filled receptacles containing a removable oviposition substrate have been commonly us ed for these purposes (e.g., Kitron et. al. 1989, 21

PAGE 22

Lounibos et al. 2001). Ovitraps were construc ted from black polypropylene cups (400 ml capacity) filled with 200 ml of di stilled water and approximately 1 g of dried pin oak leaves ( Quercus palustris ), one of the most common trees in the study area. An oviposition stick, constructed from a single tongue depressor covere d with 76 lb. seed germination paper (Anchor Paper, St. Paul, MN), served as an oviposition substrate (Steinly et al. 1991). A hole, 2.5 cm in diameter, was drilled 2.5 cm from the top of each cup to allow for suspension and drainage of the ovitrap. Ovitraps were placed within or on the edge of three separa te sites in Fairfax County, VA (Figure 2-1) from May through September 2006. The study sites included a high-density residential development, an undisturbed forested streambed, and a rural forest disturbed from frequent dumping of trash. Ovitraps were secure d to trees using bundling twine at two heights, five at ground level and five at approximately 3m, that were pre-selected randomly at each site using a numbered 10 m by 10 m grid overlay of aerial photographs of each site. Vertical stratification was employed to increase th e likelihood of encountering native treehole mosquitoes, namely O. hendersoni and O. triseriatus, and to assess how frequently O. japonicus mosquitoes seek oviposition sites above ground-level. Ovitraps were set at each site simultaneously for a period of five days each mo nth, after which they were retrieved and the oviposition stick of each was stored separately in an individual plastic bag for transport to the laboratory. In the laboratory, each oviposition stick was examined using a dissecting microscope, and the number of eggs present on each was counted. Seed germination papers were carefully removed from the oviposition sticks and allowed to dry for seven days at room temperature to ensure embryonation, after which they were placed in the bottom portion of a plastic tray (21 cm 22

PAGE 23

high x 12 cm diameter) and flooded with 400 ml of tap water. Non-natural food, comprised of ground rabbit chow and brewers yeast in a 3:1 ratio, was added to each tray upon flooding every other day to ensure larval development. All mosqu itoes were identified in the fourth larval instar using keys (Slaff and Apperson 1989, Darsie and Ward 2005), and mortality at other immature stages was noted. Because intermittent hatchi ng is a common occurrence among the eggs of Aedes and Ochlerotatus mosquitoes, those eggs that did no t hatch after being submerged for a period of seven days were dried on the oviposition substrate for an additional seven days before reflooding. Those eggs that did not hatch after th ree cycles of drying and flooding were bleached and examined microscopically to assess their viability (Trpis 1970). Unhatched eggs were considered viable if the embryo was segmented, possessed ocelli and an eggbreaker (Christophers 1960). Natural and Artificial Containers To assess the potential for interactions in nature among the larvae of container-inhabiting mosquitoes in Fairfax, Virginia, a field survey of such habitats was conducted from May through September 2006 throughout the area (Figure 2-1) Although a few tree holes and ornamental bromeliad plants were sampled, rock pools were the only natural containers sampled in large numbers during the study. Only a single rive r system, the Potomac River and associated tributaries, were sampled for rock pool mosquito es. Containers were only sampled once, and the entire aquatic contents of each were removed using a turkey baster or siphon, measured, and concentrated by filtering through a fine mesh (small enough to retain first instars) sieve. Samples were transported to the laboratory in 18ounce (532.32 ml) plastic Whirl-pak bags. Those containers containing water but no mosquitoes were noted. The ge ocoordinates of each site were obtained with a global positioning system, and the degree of sun exposure (none, partial, or full) and container type were recorded. 23

PAGE 24

In the laboratory, the mosquitoes from each c ontainer were examined separately in a 34.3 x 24.4 cm plastic tray. Fourth instars were identif ied to species and counted. All other mosquito immatures were counted, sorted by stage and placed in separate 21 x 12 cm cylindrical growth chambers (BioQuip Products, Inc., Rancho Domingu ez, CA ) containing 400 ml of tap water and food, consisting of three parts gr ound rabbit chow and one part brewers yeast. Any predatory species present, such as Toxorhynchites rutilus were removed and stored separately. Mosquitoes collected from the field as pupae were identified upon emergence as adults while all others were identified as fourth instars. Mortality at all immature stages was noted. Adult Surveillance Adult mosquitoes were colle cted with carbon dioxide (CO 2 )-baited CDC light traps and gravid traps set at 70 pre-select ed trapping sites located throu ghout Fairfax County (Figure 2-1) from May through October over a three-year period from 2004 to 2006. One of each type of trap was set weekly at each site. Traps were set in the early afternoon and retrieved the following morning. All mosquitoes collected were sorted, c ounted and identified to the species level using keys and a dissecting microscope. Data Analysis Oviposition Traps G -tests (Sokal and Rohlf 1981) were used to test for heterogeneity among the frequencies of mosquito species collected at each site. Pairwise comparisons between species were conducted as necessary with additional G -tests using the Bonferroni adjustment to correct for experimentwise error. G -tests were also used to compare the frequency of occurrence of each individual species by height. Ag ain, pairwise comparisons between sites were conducted for each species with G -tests using the Bonferroni adjustment to correct for experimentwise error. Due to non-normal distributions not remediable by stan dard transformations, Friedmans two-way 24

PAGE 25

analysis of variance and Cochrans Q test were used to compare mean abundance per trap and frequency of occurrence, respectively, for each individual species among months. Natural and Artificial Containers G -tests (Sokal and Rohlf 1981) we re used to detect significan t differences in frequency of occurrence of each individual mosquito species, including A. albopictus, O. japonicus, Culex pipiens, C. restuans O. triseriatus, and O. hendersoni, by container type, fluid volume, and exposure to the sun. The coefficient of association (C 8 ), which ranges from +1 to -1, as described by Hurlbert (1969) was used to quantify co-o ccurrences of the six most abundant species collected during the container habitat survey. This metric quantifies frequencies of cooccurrence based on species presence-absence data Positive associations between species may reflect a common habitat preferen ce or interspecific attraction, while negative associations may result from different habitat pr eferences or interspecific repuls ion. Statistical significances of C 8 values were assessed with a corrected 2 formula (Pielou 1977) for approximating a discrete distribution. Fishers exact test wa s applied in those cases with ce ll totals less than or equal to five. Only samples from which one or more larvae or pupae were identif ied were included in calculations. To quantify frequencies of encounter among mosquitoes in containers as a function of habitat size, Lloyds (1967) indices of mean intraand interspecific crowding were used per unit resource (Rathcke 1976), in this case container fluid volume. Th e mean crowding of one species upon itself and each of the other species per unit resource was calculated. Interspecific mean crowding of species x by y per unit resource a was quantified as ( x i y i / a i )/ x i and intraspecific mean crowding by the same formula but substituting ( x i 1) for y i Mean interand intraspecific crowding were calculated per month to assess the potential for larval competition between and 25

PAGE 26

among O. japonicus and A. albopictus A two-way repeated measures -analysis of variance, with species, month, and interactions as effects was used to det ect significant differences in interspecific mean crowding betwee n the two species over time. Only those months in which the two species co-occurred more than five times were considered. One-way repeated measuresANOVAs were used to detect di fferences in intraspecific cr owding among months for each species. To compare differences in intraspecific crowding among O. japonicus larvae in artificial containers and rock pools, a two-way repeated measures ANOVA was used with container type, month, and interaction as effects. In terspecific crowding data were log 10 transformed and intraspecific crowdi ng data were log 10 (x + 1) transformed to meet assumptions of normality and homoscedacity. All analyses were performed with PROC MIXED in SAS (SAS 1989). The large number of samples from both artificial and natural containers permitted a comparison of the mosquito community structure of these habitats. Kendalls coefficient of rank correlation, tau ( ), was used to estimate the degree of similarity of rank order abundance of mosquito species in containers in Fairfax C ounty, Virginia (Ghent 1963). The ranked abundances of species in small vs. large artificial containe rs, and in rock pools ve rsus tires and artificial containers were compared. Only those species occurring at least once in both habitats were considered in the comparisons. Other analyses focused on poten tial interactions between A. albopictus and O. japonicus Instar distributions were co mpared using a Kolmogorov-Smir nov two-sample test, and the average instar number for each species was calculated per m onth. Heterogeneity of instar distributions of O. japonicus and A. albopictus for the months of June, July, and August was assessed with the life table procedure (PROC LIFETEST) in SAS (SAS 1989). The significance 26

PAGE 27

of heterogeneities was assessed with Wilcoxon and Log-rank tests, which were then used to construct z -statistics for post-hoc multiple comparisons among groups (Fox 1993). Metamorphic success of O. japonicus and A. albopictus was calculated per month as Williams means (Williams 1937, Haddow 1960), denoted by M W where log 10 ( M W +1) = [ log 10 ( n i +1)]/ N where n i is the number of pupae per total number of immatures in a series of N container samples. M W is obtained by subtracting one from the antilog quantity [ log( n+1)]/ N Williams mean is frequently used as a measure of central tendency in entomological collections with many zero values (Haddow 1960, Lounibos 1981, Lounibos et al. 2001). Metamorphic success of the two species was compared usi ng a two-way repeated measures ANOVA with species, month, and interaction as effects. Only those months in which the two species cooccurred more than five times were consid ered. One-way repeated measures-ANOVAs were used to detect differences in metamorphic success among months for each species. Metamorphic success of O. japonicus in rock pool and artifici al containers was also compared using a two-way repeated measures-ANOVA, with container t ype, month, and interaction as effects. All metamorphic success data were arcsine square ro ot transformed, to best meet assumptions of normality and homogeneity of variance. Analyses were performed with PROC MIXED in SAS (SAS 1989) Adult Surveillance The frequency of collection for A. albopictus, O. japonicus and O. triseriatus adult females was calculated per week for 2004 through 2006. Frequencies of collection were analyzed for each species using two-way repeated m easures ANOVAs, with year, trap type, and interactions as effects. Week ly abundances of adult female A. albopictus O. japonicus, and O. triseriatus collected in CO 2 -baited CDC light traps and gravid tr aps were calculated as arithmetic 27

PAGE 28

means (average number collected per trap night) as well as Williams means, denoted as log 10 ( M W +1) = [ log 10 ( n i +1)]/ N where n i is the number of individuals collected in each of N trap nights. To meet assumptions of normality and homoscedacity, a log 10 (x + 1) transformation was applied to Williams means before analyzing tr ends in abundance over time, from 2004 through 2006, for each species by linear regression. A ll analyses were performed with SPSS (SPSS 2002). Results Oviposition Traps Ochlerotatus japonicus was detected only once in a single ovitrap set at ground level at the disturbed forest site in June, and was not included in any subsequent analyses. Aedes albopictus O. hendersoni and O. triseriatus were the only other species collected. The frequencies of collection were homogeneous among all three species at the undistur bed forest site (G = 2.282, p = 0.319, df = 2), but were heterogene ous at both the residential ( G = 21.06, p < 0.001, df = 2) and disturbed forest sites ( G = 25.575, p < 0.001, df = 2), however A. albopictus was the only species detected at the former site (Table 2-1). Only A. albopictus and O. triseriatus were collected at the disturbed forest site, but pairwise comparisons did not indicate a significant difference in the frequency of occurrence be tween these two species (Table 2-1). The frequencies of occurrence among all three sp ecies in ovitraps placed above ground were homogeneous ( G = 0.59, p = 0.744, df = 2), but were heterogeneous at ground level ( G = 30.11, p < 0.001, df = 2). Pairwise comparisons indicated that A. albopictus was collected more frequently in ovitraps at ground level than either O. hendersoni or O. triseriatus (Table 2-1). G -tests for heterogeneity for the presence of A. albopictus indicated no significant difference in the frequency of collection of this species by site ( G = 3.206, p = 0.201, df = 2). The frequency of occurrence of O. triseriatus ( G = 12.468, p = 0.002, df = 2) and O. hendersoni 28

PAGE 29

( G = 7.284, p = 0.026, df = 2) was heterogeneous among s ites, however upon ex clusion of those sites where these species were never detected (r esidential and disturbed forest, respectively), these tests were not significant. The frequency of occurrence of A. albopictus was significantly greater in ovitraps at gro und level than above ground (G = 22.55, p < 0.001 df = 1). There was no significant difference in th e frequency of occurrence of O. triseriatus ( G = 0.445, p = 0.505, df = 1) or O. hendersoni ( G = 0.002, p = 0.966) by ovitrap height. The frequency of collection of A. albopictus was significantly diffe rent among months ( Q = 25.217, p < 0.001, df = 4), with the highest frequenc ies of collection occurring in July and August (Table 2-2). There was a significant difference among m onths for the frequency of collection of O. hendersoni ( Q = 24.8, p < 0.001, df = 4); a comparison of only the two months in which this species was collected was also significant ( Q = 6.0, p = 0.008, df = 1). The frequency of collection of O. triseriatus was significantly different among months ( Q = 26.4, p < 0.001, df =4), with the highest frequency of collection occurring in July (Table 2-2). Mean abundances of these three species followed trends similar to their monthly egg frequencies (Table 2-3). Natural and Artificial Containers Overall, 191 containers (131 ar tificial and 61 natural) were sampled for the presence of larvae and pupae during the study period, of which 134 were positive for mosquitoes. These included rock pools, tires, flower pot saucers, tarpaulins, drainpip es, French drains, birdbaths, cemetery vases, trashcans, drums, and other sm all miscellaneous artificial containers. The following 10 species were collected, in order of frequency of occurrence: A. albopictus O. japonicus, Culex pipiens C. restuans O. triseriatus, O. hendersoni, Anopheles punctipennis T. rutilus O. atropalpus, and Orthopodomyia signifera Descriptive statistics and collection information for each of these species are provided in Appendix A. Ochlerotatus atropalpus was 29

PAGE 30

collected from only four containers all of which were rock pools. Anopheles punctipennis was collected only from rock pools, while T. rutilus and O. signifera were collected only from artificial containers. These three rare species ha ve not been included in any subsequent analyses because they were collecte d in very few samples. Ochlerotatus japonicus was the only species to occur more frequently in rock pools than artificial containers ( G = 5.98, p = 0.015, df = 1) while A. albopictus ( G = 139.15, p < 0.001, df = 1), C. pipiens ( G = 25.6, p < 0.001, df = 1), C. restuans ( G = 14.73, p < 0.001, df = 1), O. hendersoni ( G = 24.95, p < 0.001, df = 1), and O. triseriatus ( G = 29.4, p < 0.001, df = 1) were collected significantly more freque ntly from artificial containers. Aedes albopictus ( G = 27.63, p < 0.001, df = 1), C. restuans ( G = 8.16, p = 0.004, df = 1), O. japonicus ( G = 83.65, p < 0.001, df = 1), and O. triseriatus ( G = 10.03, p < 0.001, df = 1) were collected more frequently from containers that were at least partially shaded, while there was no significant difference for C. pipiens ( G = 0.39, p = 0.84, df = 1), O. atropalpus ( G = 2.09, p = 0.15, df = 1), or O. hendersoni ( G = 3.29, p = 0.070, df = 1). The mean fluid volume (SE) of containers sa mpled was 2.42 l, with a median of 0.3 l, and range of 0.01 120 l. Aedes albopictus ( G = 35.37, p < 0.001, df = 1), O. japonicus ( G = 7.99, p = 0.005, df = 1), and O. triseriatus ( G = 4.61, p = 0.032, df = 1) were sampled more frequently from containers containing less than 1 l of wa ter, while there was no significant difference for O. hendersoni ( G = 0.22, p = 0.637, df = 1), C. pipiens ( G = 3.19, p = 0.07, df = 1), or C. restuans ( G = 2.01, p = 0.156, df = 1). Interspecific Associations Using the C 8 index of association, the 15 pairings from containers sampled in Fairfax County, Virginia revealed seven positive, six nega tive, and two zero associations (Table 2-4). Four of the positive associations were significant and those included pairings between C. pipiens 30

PAGE 31

and C. restuans A. albopictus and O. triseriatus A. albopictus and O. hendersoni and O. hendersoni and O. triseriatus. There were only two significant negative associations, that of O. japonicus and O. triseriatus and O. japonicus and A. albopictus. C 8 indices were recalculated excluding rock pools, to determine if this contai ner type contributed disp roportionately to those associations involving species co mmonly found in rock pools. However, this exclusion did not have any affect on the magnitude or direction of the associations. Mean intraand interspecifi c crowding was calculated for the six most abundant mosquito species collected from 191 c ontainers censused from May th rough September (Table 2-5). Aedes albopictus and C. restuans both encountered a higher density of conspecifics than any other mosquito species. O. japonicus, O. triseriatus, and C. pipiens encountered a higher density of A. albopictus than any other species or themselves, while O. hendersoni encountered a higher density of O. triseriatus than any other species or themselves. Habitat Comparisons Kendalls coefficient of rank correlation tau ( ) was positive in all three comparisons of rank abundance (Table 2-6), but only the comparison of small and large artificial containers was significant. These results indicate that while the rank order of abundances of mosquito species occurring in rock pools and artifi cial containers are similar, th ey are not significantly similar. Ochlerotatus japonicus was the most abundant species found in rock pools, while A. albopictus was the most abundant species found in all four s ubclasses of artificial co ntainers considered for this analysis (see Appendix B). A lthough only collected from rock pools, O. atropalpus ranked fifth in abundance in this habitat (see Appendix B). Aedes albopictus and Ochlerotatus japonicus Aedes albopictus and O. japonicus occurred either alone or together in 97.75% of those containers sampled from May through September that were positive for mosquito immatures. 31

PAGE 32

Aedes albopictus was collected from approximately half of these containers (50.75%) in the absence of O. japonicus which was collected from 26% of th ese containers in the absence of A. albopictus. The two species occurred together in 21% of these containers. Co-occurrenes of these two species occurred more freque ntly in medium-to-large sized ( G = 3.67, p = 0.05, df =1), shaded ( G = 29.56, p < 0.001, df = 1) artificial containers ( G = 17.65, p < 0.001, df = 1) containing less than 1 l of water ( G = 9.23, p = 0.002, df = 1). Aedes albopictus was most frequently sampled in the absence of O. japonicus in September, while the highest proportion of containers positive for only O. japonicus were sampled in July (Figure 2-2). The two species were found together most freque ntly in June (Figure 2-2). Aedes albopictus was consistently present in greater abundance than O. japonicus in artificial containers in all months except May (Figure 2-3). Ochlerotatus japonicus was most frequently collected from artificial containers in July while A. albopictus was most frequent in August (Figure 2-4). The average instar number, or age, of A. albopictus larvae was 2.77, ranging among months from 2.67 to 3.65, while that of O. japonicus was 3.86, ranging from 3.17 to 4.19 (Table 2-7). A comparison of the overa ll instar distributions of the two species from May through September by Kolmogorov-Smirnov two-sample test indicated the two were significantly different ( D = 18.130, p < 0.001). Significant heterogeneity am ong monthly instar distributions was detected from a log-rank test for both A. albopictus ( 2 = 13.46, p < 0.001, df = 2) and O. japonicus ( 2 = 24.22, p < 0.001, df = 2). Paired comparisons showed that the significant heterogeneity of instar distributions of A. albopictus was largely attributable to that of July, which was significantly different from June and August (Table 2-8). Paired comparisons for instar distributions of O. japonicus indicated highly significant differences among all months (Table 2-8), however graphical comparisons revealed no obvious differences (Figure 2-6. It 32

PAGE 33

should be noted that pupae compri sed the greatest proportion of O. japonicus sampled in each of the months considered (Figure 2-6). Investigation of the temporal dynamics of co-occurrences of A. albopictus and O. japonicus indicated that interspecific mean crow ding was not significant affected by species (F 1,24 = 0.46, p = 0.561) or month (F 2,22 = 0.39, p = 0.8839). Mean crowding of O. japonicus by A. albopictus was always more than that of A. albopictus by O. japonicus and was highest for both species in August (Figure 27). Intraspecific mean crowdi ng was significantly different among months for both A. albopictus (F 2,30 = 76.51, p < 0.001) and O. japonicus (F 2,29 = 9.36, p < 0.001). While intraspecific mean crowding of O. japonicus was greatest in July, A. albopictus encountered the greatest density of conspecifics per unit resour ce the following month in August (Figure 2-8), pairwise comparisons of least squa re means indicated that intraspecific competition was lower for both species in June than July or August (Table 2-9). Because O. japonicus was collected frequently in high dens ities from both artificial containe rs and rock pools, intraspecific mean crowding for this species was calculat ed for each habitat as 33.36 and 109.48, respectively. However, analysis by two-way repeated measur es-ANOVA did not indicate that intraspecific crowding of this species was effected by container type (F 1,23 = 1.77, p = 0.1962) or month (F 1,18 = 0.15, p = 0.6989). In containers where the two species co -occurred, the metamorphic successes of A. albopictus and O. japonicus, measured as Williams Mean (M w ) number of pupae collected per total number of immatures, were found to be 0.1234 and 0.1133, respectively. However, metamorphic success of was not effected by species (F 1,24 = 0.33, p = 0.5721) or month (F 2,22 = 0.24, p = 0.7871). Metamorphic success was greatest for O. japonicus in June, while there appeared to be no differen ce among months for that of A. albopictus (Figure 2-9). Repeated 33

PAGE 34

measures-ANOVAs indicated that there were no signi ficantly differences in the metamorphic success of A. albopictus (F 2,30 = 0.33, p = 0.5620) or O. japonicus (F 2,29 = 1.63, p = 0.2135) among months. While the me tamorphic success of O. japonicus was higher in rock pools (0.1479) than artificial containers (0.1047), this difference was not found to be significant among container type (F 1,26 = 2.09, p = 0.1599) or month (F 1,25 = 1.15, p = 0.2930). Ochlerotatus japonicus and Ochlerotatus triseriatus Ochlerotatus triseriatus was only collected from 15 contai ners during the course of the census, most of which (73.7%) were sampled in August. Because O. triseriatus occurred with O. japonicus in only five of these containers, analysis of the potential for interactions between these species was rather limited. In addition to the sign ificant negative association of these species as indicated by the C 8 index (Table 2-4), it was found that O. triseriatus encountered more conspecifics (34.47) than O. japonicus (19.21) than conspecifics per unit resource, however this was much less than the interspecific mean crowding of O. triseriatus by A. albopictus (136.7). Ochlerotatus japonicus encountered only 12.85 O. triseriatus compared to 139.3 A. albopictus, and 127.56 conspecifics per unit re source (Table 2-5). Finally, the metamorphic success of O. triseriatus was found to be 0.59, while that of O. japonicus was only 0.01 in containers in which the two species co-occurred. Ochlerotatus atropalpus and Ochlerotatus japonicus Because O. atropalpus was collected on only four occasions, and only twice with O. japonicus, an assessment of the potential for interactions of these species was limited. While O. atropalpus was recovered exclusively from ro ck pools exposed fully to the sun, O. japonicus was only collected from those that were at least partially shaded. A non-si gnificant coefficient of association (C 8 ) of zero was calculated for the two sp ecies when all container types were considered. When only rock pools were included, the coefficient decreased slightly to -0.04, but 34

PAGE 35

was still non-significant. These findings are not surprising considering th e limited collections of O. atropalpus from any container habitat. No effort was made to determine the metamorphic success or interspecific mean crowding of thes e two species because they only co-occurred twice. Adult Surveillance The frequency of collection of A. albopictus was affected by trap type (F 1,19 = 69.439, p < 0.001) but not by year (F 2,38 = 1.984, p =0.151), with a higher freque ncy of collection of this species in CO 2 -baited CDC light traps than gravid traps in all years (Table 2-10). The frequency of collection of O. japonicus was affected by both trap type (F 1,22 = 75.034, p < 0.001) and year (F 2,44 = 71.504, p < 0.001), with a higher frequency of colle ction of this species in gravid traps (Table 2-10). Pairwise comparisons indicated significantly different frequencies of collection of O. japonicus among all years, with a decline in frequency of collection in both CO 2 -baited CDC light traps and gravid traps with each subse quent year from 2004 to 2006 (Table 2-10). The frequency of collection of O. triseriatus was also affected by both trap type (F 1,22 = 9.245, p < 0.001) and year (F 2,44 = 67.027, p < 0.001), with a higher frequency of collection of this species in CO 2 -baited CDC light traps in al l years (Table 2-10). Pairwise comparisons indicated that O. triseriatus was collected more frequently in 2004 than 2006 in CO 2 -baited CDC light traps, and more frequently in 2004 than in 2005 or 2006 in gravid traps (Table 2-10). Linear regression analysis of the weekly abundance, given as the log 10 Williams mean (M w ) number of adult females collected per trap + 1, of A. albopictus as collected in both CO 2 baited CDC light traps and gravid traps, over time di d not indicate any significant change in abundance of this species from 2004 through 2006 (Figure 2-10). However, this time series analysis indicated that the mean abundances of both O. japonicus (Figure 2-11) and O. triseriatus (Figure 2-12) declined significan tly over this period of time. 35

PAGE 36

Discussion The results of this preliminary asse ssment demonstrate the coexistence of O. japonicus with resident container-inhabi ting mosquitoes in Fairfax Count y, Virginia, facilitated by what appears to be species-specific differences in habitat preference and seasonality. The negative associations of O. japonicus immatures with A. albopictus and O. triseriatus seem largely due to an apparent divergence in container preference, with O. japonicus occurring predominately in rock pools rather than artificial containers Furthermore, the limited collection of O. triseriatus from artificial containers suggests that perhaps this species prefers an alternative habitat, such as treeholes. Findings from the oviposition survey were similar. A wide distribution and high abundance of A. albopictus across macrohabitats was observed throughout the study period, with this species occurring more frequently at residential and disturbed habitats than other resident container-inhabiting mosquitoes, a trend similar to that which has been observed in Florida (Lounibos et al. 2001). Spatial a nd temporal distributions of O. hendersoni and O. triseriatus were not consistent with those observed in othe r areas, but this may be due to the collection effort of this study. Ochlerotatus triseriatus is known to seek out ovi position sites at ground level (Scholl and DeFoliart 1977) while O. hendersoni prefers to oviposit a bove ground level (Sinsko and Grimstad 1977, Clark and Crai g 1985), even up to 9 m (Beier et al. 1982). However, these results suggest that there is no significant diffe rence in the frequency of oviposition of either species at ground level or above ground. Wh ilee the period of ovi position activity of O. triseriatus typically extends beyond that of O. hendersoni into late summer (Scholl and DeFoliart 1977), the results of this st udy suggest a similar seasonal ity for these two species. While O. japonicus was only detected once throughout the oviposition study, possibly implying a univoltine population of this specie s in Fairfax County, Virginia, oviposition by and hatching of viable eggs from wild-caught females of this species collected from May through 36

PAGE 37

October in previous years (personal observation, unpublished data) suggests this is an artifact. While these findings are likely a reflection of in appropriate macrohabitat placement of ovitraps, it should be noted that others have had limite d success with this collection technique for O. japonicus in the past (Andreadis et al 2001). Ovitraps would perhaps have been more useful in collecting O. japonicus in different macrohabitats, particular ly near rock pools where this species is most abundant. Findings from the survey of artificial containe rs suggest that interspecific competition may be occurring in these habitats where O. japonicus coexists with resident mosquito species in the presence of limited resources; however this study suggests that the invasion of O. japonicus may not result in competition in artificial containers th at leads to displacement. This is supported by the abundances and co-occurences of O. japonicus with A. albopictus and O. triseriatus the two aedine mosquitoes this species is most likely to encounter in artificial containers in Fairfax County, Virginia. Metamorphic success of O. japonicus was not significantly different from that of A. albopictus, nor were there any significant differences in interspecific crowding of the two species among months. However, A. albopictus encountered a higher density of conspecifics than O. japonicus per unit resource, suggesting that intrarather than interspecific interactions may be more important in regulating the population gr owth of this species in artificial containers. Similarly, there appeared to be greater crowding of O. japonicus by conspecifics than by A. albopictus (Figures 2-7, 2-8). Furthermore, intras pecific crowding and metamorphic success of O. japonicus in artificial containers and rock pools, wh ere it is often the on ly species present, were similar. While these findings suggests th at intraspecific competition may be just as important as interspecific competition for this species, it is possible that there has been selection among O. japonicus since arriving in this area to avoid competition in ar tificial containers with 37

PAGE 38

A. albopictus ; however the plausibility of such selecti on is currently unknown due to the paucity of data regarding seasonal patterns and abundance of O. japonicus since its arrival in northern Virginia. Such speculation may be supported furt her by differences in the seasonal abundances and frequencies of occurrence of the two species in artificial containers. The success of O. japonicus in artificial containers in relation to A. albopictus seems to be facilitated by differences in seas onality and instar distributions. Ochlerotatus japonicus was collected most frequently and in greater a bundance early in the season in contrast to A. albopictus, which was most active in later months. Activity of O. japonicus during this time also happened to coincide with the greatest metamor phic success of this species, and was the only time when the interspecific mean crowding of O. japonicus on A. albopictus was higher than that of A. albopictus on O. japonicus. The different instar distributions of A. albopictus (Figure 2-5) and O. japonicus (Figure 2-6) promoted the early s eason metamorphic success of the latter species in artificial containers, with the presence of older instars of O. japonicus in May, June, and July perhaps giving this species a head start over A. albopictus. This suggests the capacity of this species to overwinter in such habitats in the larval stage, an observation that has been made by others in the species native range (LaCasse and Yamaguti 1950), and the United States in New Jersey (Scott 2003) and North Carolina (B Harrison personal communication). However, no data regarding the induction or termination of diapause in O. japonicus are currently available. Furthermore, the disparity in the age of O. japonicus and A. albopictus larvae, with the average instar of O. japonicus consistently higher than that of A. albopictus throughout all months of the study, may provide a competitive advantage to O. japonicus in containers with A. albopictus, allowing the former species to persist later into the season despite the high abundance of A. albopictus The importance of cohort structure in density-dependent intraspecific 38

PAGE 39

competition has been documented for O. triseriatus whose early hatching larvae experienced higher survivorship, faster development time, and higher per capita growth ra te, than cohorts that hatched later (Livdahl 1982, Edge rly and Livdahl 1992). Such an advantage may be due to the size-efficiency of the early cohort (Brooks a nd Dodson 1965), who as large filter feeders are more efficient and can exploit a wider range of food particles than smaller competitors, or even be cannibalistic (Koenekoop and Livdahl 1986), however evidence of the latter may have been an artifact of simple experimental conditions (Edgerly and Livdahl 1992). Furthermore, egg larva interactions betwee n larvae of early hatching cohorts and eggs of later cohorts are a known form of interference competition among containerinhabiting mosquitoes, in which the presence of feeding older larvae delayed hatching (Edgerly et al. 1993). The impact of interspecific larval resource competition, particularly among different instars, on the populati on growth of these two species needs to be explored experimentally to reveal the complexitie s of community dynamics. This study revealed somewhat different findings for interactions between O. japonicus and O. triseriatus In contrast to A. albopictus, O. triseriatus had five times the metamorphic success of O. japonicus in containers in which these species co -occurred, suggesting that this species is superior in larval resource utiliz ation. However, the abundance and of O. triseriatus and cooccurrences with O. japonicus were low in these collections. Although outside the scope of this study, interspecific interactions with O. triseriatus may be important for O. japonicus in treeholes, as these species have been collected fr om such habitats in Connecticut (Andreadis et al. 2001), and should be considered in future rese arch. Experimental studies should be viewed in the context of field observations of the freque ncy of interspecific interactions, seasonal distributions, and overwintering behaviors, as th ese life history traits may ultimately influence the community structure of treeholes in which these species may coexist. Aedes albopictus is 39

PAGE 40

known to be superior to O. triseriatus larvae in competition (Livdahl and Willey 1991), which coupled with the high inters pecific mean crowding of O. triseriatus by A. albopictus demonstrated in this study, suggests that intera ctions between these two species may be most important in determining the success of O. triseriatus populations in artifi cial containers in Fairfax County, Virginia. It would be of great value to compare th ese findings with that from similar studies conducted in areas where A. albopictus does not occur, as we ll as in eastern Asia where the native ranges of these species overlap; however such data are currently unavailable. The only comparable survey, conducted by Andreadis et al. (2001) in Conn ecticut shortly after the invasion of O. japonicus indicated that this species ra nked fourth in overall abundance (9.4%) in a tire survey, in which it was severely outnumbered by O. triseriatus The potential for larval resource competition between these two species in areas where A. albopictus does not occur seems likely, and results of this study suggest that O. triseriatus will likely be the superior competitor under such conditions. Broad-based surveillance of adult females of A. albopictus O. japonicus and O. triseriatus with CO 2 -baited CDC light traps and gravid trap s provided a limited perspective of the population trends of these species over time in Fairfax, Virginia. The adult population of A. albopictus did not alter in frequency or abundance during the three year surveillance period, which leads one to speculate that perhaps this species has been unaffected by the introduction of O. japonicus in the area. On the contrar y, the adult populations of both O. japonicus and O. triseriatus declined during the surveill ance period, although this trend seemed to be somewhat more severe for the former species. While thes e observations are likely attributable to annual variations in environmental cond itions (i.e. temperature and rainfall), they reflect the local decline, or possibly the eventual extinction, of O. japonicus populations following the 40

PAGE 41

introduction and initial expansion of this spec ies throughout the area. This phenomenon in which an invading species reaches a peak of density and then declines is often referred to as boom-andbust (Williamson 1996). Selective pressures from pred ation, competition, or lack of availability of accessible resources may promote this type of invasion trend (Williamson 1996). Additional years of consistent surveillance wi ll be required to fully appreciate these trends with respect to the invasion success of O. japonicus The successful establishment of O. japonicus in Fairfax County, Virg inia appears to be associated with a population declin e and potential displacement of O. atropalpus in local rock pools. The limited collection of this native rock pool mosquito while surveying these habitats is cause for concern as this species was once abundant throughout the area; in fact the type-form given by Coquillett (1902) was from nearby Plummers Island in Montgomery County, Maryland. A similar pattern of decline for O. atropalpus has been observed in rock pool communities of Connecticut (Andr eadis et al. 2001) and North Carolina (B. Harrison personal communication), and the absence of this species in rock pools has been noted in New Jersey (Scott et al. 2001a), however O. atropalpus is more common in tires than in rock pools in this area. It is important to note two major flooding ev ents, heavy rains and a hurricane, that occurred during this study limited rock pool collections in late June and early July, and again in early September. Although no conclusions could be based solely on the limited co-occurrence of these species during this study, these findings suggest that interspecific larv al resource competition with O. japonicus may have had profound affects on populations of O. atropalpus in areas where these two species co-occur. Differences in the overwintering strategies of these species have pr obably facilitated the decline of O. atropalpus which is known to diapause in th e egg stage rather than as larvae, 41

PAGE 42

whereas O. japonicus may diapause in either stage (LaCasse and Yamaguti 1950, Kamimura 1976, Scott 2003, B. Harrison personal communi cation). Similar to that observed for A. albopictus and O. japonicus in artificial containers, differences in the cohort structures of these two species in rock pools may exacerbate la rval competitive outcomes or promote hatching inhibition, intraguild predation, or cannibalism. The tendency of O. atropalpus to inhabit rock pools fully exposed to the sun, c ontrary to the preferences of O. japonicus may allow this species to persist in th is habitat, albeit probably in small numbers as highly flood prone rock pools tend to be in the more sunny locations (OMeara et al. 1997). Survival of O. atropalpus in an environment frequently subjected to floodi ng may be facilitated by seasonal variation in ovipositional behavior, deposition of different types of eggs, or variable delays before hatching. It is possible that this find ing is an artifact of sampling effort; therefore the rock pool microhabitat preferences of these species with respect to sun exposure should be researched further. Furthermore, as noted in Lounibos (2002), O. atropalpus has expanded its distribution by occupying artificial containers, primarily tires, and has itself been considered an invasive species both in the United States and abroad. Su ch tendencies may allow this species to persist through dispersal to new areas where O. japonicus does not occur, or may allow it to avoid competition to some extent in areas where the two species co-occur. However, it is interesting to note that O. atropalpus was not recovered from a ny tires during this study. In conclusion, the invasion of O. japonicus seems to be associated with the possible displacement O. atropalpus possibly through interspecific resour ce competition, in part of this species rock pool habitat. In artificial containers, O. japonicus larvae are most likely to interact with A. albopictus, particularly late in the season, duri ng the months of August and September, in small-sized shaded containers. The success of O. japonicus in artificial containers is most 42

PAGE 43

43 likely attributed to the earlier seasonal appearan ce, older age, and capacit y of this species to complete development in the presence of resident species. While surveillance cannot reveal any detrimental effects of O. japonicus on resident artificial cont ainer inhabiting mosquitoes, monitoring over subsequent years should continue to observe popul ation trends. While this study focused predominantly on the potential interspecific interactions of O. japonicus with resident mosquito species in artificial cont ainers and rock pools, other factor s, particularly predators, may be influencing the structure of these comm unities and should be investigated. Selective preference of predators (Grisw old and Lounibos 2005, 2006) and diffe rential responses of prey species to predators (Holt and Lawton 1994) ha ve been shown to influence interspecific interactions of container-inhab iting mosquitoes. Furthermore, the tolerance of these species to varying environmental conditions, intraguild pred ation, or differences in foraging behavior may be important in their interspecific interactions.

PAGE 44

Table 2-1. Frequency of occurrence of A. albopictus O. hendersoni and O. triseriatus in ovitraps set at three sites and two heights in Fairfax, Virginia in 2006. Frequency (no. pos. traps a /total) Site A. albopictus O. hendersoni O. triseriatus Residential 0.1957a 0b 0b Undisturbed forest 0.2128 0.1087 0.1304 Disturbed forest 0.3478a 0b 0.1739a Height Ground 0.4265a 0.0588b 0.1471b Above ground 0.0857 0.0571 0.0571 a Frequencies were calculated as the number of positive traps per total number of traps set at each site from May September 200 6. Lower case letters indicate signifi cant differences among sites resulti ng from pairwise comparisons with G -tests (df = 1, p = 0.05), using the Bonferroni method to adjust for experimentwise error. 44

PAGE 45

Table 2-2. Frequency of occurrence, by month, of A. albopictus, O. hendersoni and O. triseriatus in ovitraps in Fairfax County, Virginia in 2006. Frequency (no. pos. traps a /total) Species May June July August September A. albopictus 0.1a 0.2222ab 0.4231b 0.3929b 0.1481a O. hendersoni b 0 0.0370a 0.2692b 0 0 O. triseriatus 0a 0.1ab 0.3b 0.0333a 0.0333a a Frequencies were determined as the number of positive traps per total number of traps set each month. b Frequencies of collection of O. hendersoni were compared only for June and July; differences were significant at = 0.01 with Cochrans Q -test, df = 1. Lower case letters indica te significant differences among sites resulting from pairwise comparisons with Cochrans Q test (df = 1, p = 0.05), using the Bonferroni method to adjust for experimentwise error. 45

PAGE 46

Table 2-3. Monthly abundance (mean number of eggs per trap) of A. albopictus, O. hendersoni and O. triseriatus collected in ovitraps. Abundance (mean no. ( SE) /trap) a Species May June July August September A. albopictus 1.0 (0.93)a 1.19 (0.66)ab 11.92 (5.85)b 6.39 (2.73)b 0.67 (0.49)a O. hendersoni b 0 0.15 (0.15) 4.08 (2.17) 0 0 O. triseriatus 0a 0.22 (0.13)ab 8.69 (4.24)b 0.79 (0.79)ab 0.15 (0.15)a Total rainfall (cm) 5.61 35.61 9.04 2.62 16.03 a Abundance was determined as the mean number of eggs co llected per trap, based on all ovitraps set each month. b Mean abundances of O. hendersoni were compared only for June and Ju ly; differences were significant at = 0.05 with Friedmans two-way analysis of variance, df = 1. Lo wer case letters indicate si gnificant differences among s ites resulting from pairwise comparisons with Friedmans two-way analysis of variance test (df = 1, p = 0.05), using the Bonferroni method to adjust for experimentwise error. All other pair wise comparisons we re non-significant. 46

PAGE 47

Table 2-4. Coefficients of association (C 8 ) for the most abundant species in 191 artificial and natural container samples from Fairfax County, Virginia in 2006. Parentheses enclose C 8 values that exclude rock pools. Coefficient of association (C 8 ) Species C. restuans C. pipiens O. triseriatus O. japonicus O. hendersoni A. albopictus -0.056 -0.015 0.067*** -0.238*** (-0.207***) 0.037** O. hendersoni 0 -0.272 0.232** 0 (0) O. japonicus 0.075 (0.105) 0.042 (0.133) -0.048** (0) O. triseriatus -0.122 -0.140 C. pipiens 0.638*** ** p < 0.01, *** < 0.001 by 2 ; all other interspecific asso ciations are non-significant. 47

PAGE 48

Table 2-5. Intraand inte rspecific mean crowding of the most abundant mos quito species collected fr om artificial and natural containers in Fairfax County, Virgin ia in 2006. Mean crowding is defined as the mean density of species y encountered by species x per liter volume. Values in bold indicate intraspecific mean crowding. Mean crowding a (density of species y encountered by species x per liter volume) Species y Species x A. albopictus O. hendersoni O. japonicus O. triseriatus C. pipiens C. restuans A. albopictus 395.80 3.013 53.01 31.59 351.88 57.37 O. hendersoni 40.73 32.49 18.06 56.27 1.77 5.74 O. japonicus 139.3 9.03 127.56 12.85 51.56 83.06 O. triseriatus 136.7 11.90 19.21 34.47 25.93 39.26 C. pipiens 1098.43 2.48 37.56 12.96 126.98 52.33 C. restuans 20.16 2.51 57.41 4.33 34.05 116.60 a Interspecific mean crowding was determined from only those containers in whic h both species co-occurred. 48

PAGE 49

Table 2-6. Species rank abundances compared for different container habitats sampled in Fairfax County, Virginia from May September 2006. Kendalls was used as an index of similarity. Container habitat No. of samples No. of species t s a P Small artificial containers 97 7 Large artificial containers 33 9 0.714 8.101 < 0.001 Tires 29 7 Rock pools 59 7 0.200 0.537 0.709 All artificial containers 130 9 Rock pools 59 7 0.467 1.917 0.055 a Significance of Kendalls was tested by calculati ng the test statistic, t s which makes use of a normal approximation to test the null hypothesis that the true value of = 0: t s = / sqrt [( 2(2 n + 5)) /( 9n(n 1))], where n is the number of data pairs. 49

PAGE 50

Table 2-7. Number of larvae and average instar number for A. albopictus and O. japonicus collected in Fairfax County, Virginia by month, May September 2006. A. albopictus O. japonicus Month No. larvae Average instar number ( SE) No. larvae Average instar number ( SE) May 5 3.0 (0.71) 16 3.13 (0.49) June 204 2.67 (0.08) 98 4.18 (0.13) July 574 2.94 (0.05) 1836 3.67 (0.04) August 3427 2.69 (0.02) 998 4.19 (0.04) September 227 3.64 (0.05) 2 4.0 (1.0) Total 4437 2.77 (0.02) 2950 3.86 (0.03) 50

PAGE 51

Table 2-8. Tests for significant heterogene ity of monthly instar distributions of A. albopictus and O. japonicus from June August 2006, based on log-rank statistics. Paired comparisons with z -test A. albopictus O. japonicus July August July August Month z P z P z P z P June 3.66 < 0.001 0.203 0.416 4.86 < 0.001 3.06 0.001 July 2.34 0.01 4.61 < 0.001 Test 2 df P 2 df P Log-rank 13.46 2 < 0.001 24.22 2 < 0.001 Wilcoxon 16.46 2 < 0.001 45.94 2 < 0.001 51

PAGE 52

Table 2-9. Least square means ( SE) for intraspecific crowding (transformed by log 10 ( x + 1)) among larvae of A. albopictus and O. japonicus June August 2006. Means followed by lower case letter s that are not commonly sh ared are significantly different by pairwise comparisons (p < 0.05) with the Bonferroni adjust ment for experimentwise error. Least square means ( SE) Species June July August A .albopictus 0.6489 (0.1928)a 1.6176 (0.2164)b 1.8092 (0.1163)b O. japonicus 1.2286 (0.3570)a 2.9186 (0.2558)b 2.3577 (0.2408)ab 52

PAGE 53

Table 2-10. Frequencies of collection of A. albopictus O. japonicus, and O. triseriatus adult females in CO 2 -baited CDC light traps and gravid traps, 2004 2006. Means followe d by letters that are not commonly sh ared are significantly different by pairwise comparisons ( p < 0.05) with the Bonferroni adjust ment for experimentwise error. Mean ( SE) frequency of collection (no. pos. traps/total a ) CO 2 -baited CDC light traps Gravid traps Species 2004 2005 2006 2004 2005 2006 A. albopictus 0.423 (0.40) 0.433 (0.066) 0.327 (0.054) 0.064 (0.013) 0.046 (0.012) 0.061 (0.012) O. japonicus 0.213 (0.20)a 0.137 (0.016)b 0.046 (0.008)c 0.454 (0.034)a 0.281 (0.367)b 0.186 (0.023)c O. triseriatus 0.241 (0.30)a 0.176 (0.27)ab 0.135 (0.23)b 0.094 (0.015)a 0.046 (0.009)b 0.046 (0.12)b a Mean frequency of collection was determined from 23 weekly fre quencies (the number of positive tr aps per total traps set each w eek) for each species. 53

PAGE 54

Figure 2-1. Map of Fairfax County, Virginia showi ng locations of study areas that were sampled or censused repeatedly. Tr ap sites include both a CO 2 -baited light trap and a gravid trap. 54

PAGE 55

0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 May June July AugustSeptember Month A. albopicuts O. japonicus Both N = 6 N = 30 N = 64 N = 2 N = 20 Proportion of mosquito-positive containers 55 Figure 2-2. Proportion of mosquito-positive containers containing A. albopictus, O. japonicus, or both A. albopictus and O. japonicus May September 2006. Total numbers of mos quito-positive containers sampled are indicated at the top of each histogram bar. 55

PAGE 56

0 10 20 30 40 50 60 70 80 90 May June July AugustSeptember Month A. albopictus O. japonicus Mean no. mosquitoes per container 56 Figure 2-3. Monthly abundance (mean number of mosquitoes collected per container) of A. albopictus and O. japonicus (SE) from 91 mosquito-positive artificial containers. 56

PAGE 57

0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 May June July AugustSeptember Month A. albopictus O. japonicus N = 81 N = 22 Proportion of species-positive containers 57 Figure 2-4. Seasonal occurrences (proportion of species-positive containers per month) of A. albopictus and O. japonicus collected from artificial containers in 2006. The to tal numbers of species-pos itive containers sampled from May September are indicated in the figure legend. 57

PAGE 58

0.00 0.20 0.40 0.60 0.80 1.00 IIIIIIIVP Stage July n1= 14, n2= 574 IIIIIIIVP Stage 0.00 0.20 0.40 0.60 0.80 1.00 0.00 0.20 0.40 0.60 0.80 1.00May n1= 3, n2= 5 June n1= 18, n2= 204 August n1 = 53, n2= 3427 September n1= 5, n2= 227 Proportion of immatures collected Stage Stage I IIIIIIV P I IIIIIIV P 0.60 0.40 0.20 0.00 0.80 0.60 0.40 0.20 0.00 1.00 0.80 0.60 0.40 0.20 0.00 0.80 0.00 0.20 0.40 0.60 0.80 1.00 IIIIIIIVP Stage July n1= 14, n2= 574 IIIIIIIVP Stage 0.00 0.20 0.40 0.60 0.80 1.00 0.00 0.20 0.40 0.60 0.80 1.00May n1= 3, n2= 5 June n1= 18, n2= 204 August n1 = 53, n2= 3427 September n1= 5, n2= 227 Proportion of immatures collected Stage Stage I IIIIIIV P I IIIIIIV P 0.60 0.40 0.20 0.00 0.80 0.60 0.40 0.20 0.00 1.00 0.80 0.60 0.40 0.20 0.00 0.80 58 Figure 2-5. Monthly inst ar distributions of A. albopictus from May September 2006 in Fairfax, Virginia. Total numbers of positive containers (n 1 ) sampled and larvae collected (n 2 ) are indicated for each month. 58

PAGE 59

July n1 = 21, n2= 1836 IIIIIIIVPStage May n1= 1, n2= 1 June n1 = 10, n2= 98 August n1= 25, n2= 998 September n1= 1, n2= 2 Proportion of immatures collected 0.00 0.20 0.40 0.60 0.80 1.00 IIIIIIIVP Stage 0.00 0.20 0.40 0.60 0.80 1.00 0.00 0.20 0.40 0.60 0.80 1.00 Stage Stage I II IIIIV P I IIII IIV P 1.00 0.80 0.60 0.40 0.20 0.00 0.80 0.60 0.40 0.20 0.00 0.80 0.60 0.40 0.20 0.00 July n1 = 21, n2= 1836 IIIIIIIVPStage May n1= 1, n2= 1 June n1 = 10, n2= 98 August n1= 25, n2= 998 September n1= 1, n2= 2 Proportion of immatures collected 0.00 0.20 0.40 0.60 0.80 1.00 IIIIIIIVP Stage 0.00 0.20 0.40 0.60 0.80 1.00 0.00 0.20 0.40 0.60 0.80 1.00 Stage Stage I II IIIIV P I IIII IIV P 1.00 0.80 0.60 0.40 0.20 0.00 0.80 0.60 0.40 0.20 0.00 0.80 0.60 0.40 0.20 0.00 8 59 59

PAGE 60

Figure 2-6. Monthly inst ar distributions of O. japonicus from May September 2006 in Fairfax, Virginia. Total numbers of positive containers (n 1 ) sampled and larvae collected (n 2 ) are indicated for each month. 0 1 2 3 4 5 6 7 May June July AugustSeptember Month A. albopictus by O. japonicus O. japonicus by A. albopictus Interspecific mean crowding 60 Figure 2-7. Interspecific mean crowding of A. albopictus by O. japonicus and O. japonicus by A. albopictus. Interspecific mean crowding was calculated by month as the density of species y encountered by species x per unit resource a, in this case, container volume. Interspecific mean crowding was quantified as ( x i y i / a i )/ x i 60

PAGE 61

0 50 100 150 200 250 300 May June July AugustSeptember Month A. albopictus O. japonicus Intraspecific mean crowding 61 Figure 2-8. Intraspecific mean crowding (density of conpspecifics encountered per unit resource, a) of A. albopictus and O. japonicus by month. Intraspecific mean crowding was quantified as [x i (x i 1)/ a i ]/ x i where a is container volume. 61

PAGE 62

0 0.05 0.1 0.15 0.2 0.25 0.3 May June July AugustSeptember Month A. albopictus O. japonicus n = 8 n = 5 n = 13 Mean ( M w ) no. pupae per total no. immatures 62 Figure 2-9. Metamorphic success of A. albopictus and O. japonicus collected from containers in which the two species co-occurred. Numbers of samples for each month ar e indicated above each histogram bar. 62

PAGE 63

0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.00Log (Mean (Mw) number of adult females per trap + 1)2004 2005 2006 Year A Y = 6E-05x + 0.2372 r2 = 0.0002 p = 0.904 B Y = 0.001x + 0.0647 r2 = 0.0043 p = 0.6 0.60 0.70 0.40 0.50 0.20 0.30 0.00 0.10 0.20 0.25 0.15 0.05 0.10 0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.00Log (Mean (Mw) number of adult females per trap + 1)2004 2005 2006 Year A Y = 6E-05x + 0.2372 r2 = 0.0002 p = 0.904 B Y = 0.001x + 0.0647 r2 = 0.0043 p = 0.6 0.60 0.70 0.40 0.50 0.20 0.30 0.00 0.10 0.20 0.25 0.15 0.05 0.10 63 Figure 2-10. Mean weekly abundance of A. albopictus collected in (A) CO 2 -baited light traps and (B) gr avid traps over time, from 2004 through 2006, in Fairfax County, Virginia. Williams means (W M ) were transformed by log 10 (x + 1) to meet assumptions of normality and homogeneity of variance. 63

PAGE 64

0.00 0.04 0.08 0.12 0.16 0.20 0.00 0.10 0.20 0.30 0.40 0.50 0.00Log (Mean (Mw) number of adult females per trap + 1)2004 2005 2006 Year A Y = -0.008x + 0.1156 r2 = 0.4960 p < 0.001 B Y = -0.0015x + 0.2611 r2 = 0.3241 p < 0.001 0.16 0.20 0.12 0.04 0.08 0.00 0.30 0.40 0.20 0.10 0.00 0.04 0.08 0.12 0.16 0.20 0.00 0.10 0.20 0.30 0.40 0.50 0.00Log (Mean (Mw) number of adult females per trap + 1)2004 2005 2006 Year A Y = -0.008x + 0.1156 r2 = 0.4960 p < 0.001 B Y = -0.0015x + 0.2611 r2 = 0.3241 p < 0.001 0.16 0.20 0.12 0.04 0.08 0.00 0.30 0.40 0.20 0.10 64 Figure 2-11. Mean weekly abundance of O. japonicus collected in (A) CO 2 -baited light traps and (B) gr avid traps over time, from 2004 through 2006, in Fairfax County, Virginia. Williams means (W M ) were transformed by log 10 (x + 1) to meet assumptions of normality and homogeneity of variance. 64

PAGE 65

0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.00 0.02 0.04 0.06 0.08 0.10204 0.00Log (Mean (Mw) number of adult females per trap + 1)2004 2005 2006 Year A Y = -0.006x + 0.1345 r2 = 0.1386 p = 0.002 B Y = -0.002x + 0.0368 r2 = 0.1166 p = 0.004 0.25 0.30 0.15 0.20 0.05 0.10 0.00 0.06 0.08 0.04 0.02 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.00 0.02 0.04 0.06 0.08 0.10204 0.00Log (Mean (Mw) number of adult females per trap + 1)2004 2005 2006 Year A Y = -0.006x + 0.1345 r2 = 0.1386 p = 0.002 B Y = -0.002x + 0.0368 r2 = 0.1166 p = 0.004 0.25 0.30 0.15 0.20 0.05 0.10 0.00 0.06 0.08 0.04 0.02 Figure 2-12. Mean weekly abundance of O. triseriatus collected in (A) CO 2 -baited light traps and (B) gr avid traps over time, from 2004 through 2006, in Fairfax County, Virginia. Williams means (W M ) were transformed by log 10 (x + 1) to meet assumptions of normality and homogeneity of variance. 65 65

PAGE 66

CHAPTER 3 INTERSPECIFIC COMPETITION BETWEEN AEDES ALBOPICTUS AND OCHERLOTATUS JAPONICUS Introduction Aedes albopictus and Ochlerotatus japonicus are two of the most recently recognized exotic mosquito species to become established in the United States. Aedes albopictus was introduced into the United States from Japa n by way of tire shipments (Hawley et al. 1987, Reiter and Sprenger 1987), which led to its es tablishment in Texas in 1985 (Sprenger and Wuithiranyagool 1986). State and local mosquito su rveillance records indicate that it has since spread rapidly, becoming established across much of the eastern United States from southern Florida to New Jersey, Illinois, Indiana, and Ohio (Moore 1999). The westward spread of this invader has been much slower, presumably due to the drier summers in this region (Nawrocki and Hawley 1987). Aedes albopictus has been intercepted and destroyed on the west coast in California (Linthicum et al. 2003 ) and Washington (Craven et al 1988).The invasion success and rapid spread of A. albopictus in the United States has been attr ibuted to its generalized habitat and food requirements, ability to live in human -dominated habitats (H awley 1988), desiccation resistant eggs (Focks et al. 1994, Juliano et al. 2002), and superior larval competitive ability (Juliano 1998, Juliano et al. 2004). The introduction of O. japonicus into the United States was initially reported by Peyton et al. (1999) from light trap collections from New York and New Jersey in August and September 1998. However, an archival search by Andreadis et al. (2001) revealed that this species was actually first detected one month earlier in Connecticut. Ochlerotatus japonicus has since been detected along the East coast with reports as far south as Georgia (Reeves and Korecki 2004), north as Maine (Foss and Dearborn 2001), and west as Missouri (Gallitano et al. 2006), from 66

PAGE 67

what appear to be multiple introductions from Japan (Fonseca et al. 2001). Ochlerotatus japonicus appears to have become established on the west coast in Washington (Roppo et al. 2004), and has recently been detected in Mississippi and Nevada (Moore 2005). Like A. albopictus, the used tire trade is the susp ected mechanism of introduction of O. japonicus into the United States (Peyton et al. 1999, Lounibos 2002). The current US distributions of these two species overlap considerably, although O. japonicus appears to be more cold tolerant (Tanaka et al. 1997) than A. albopictus as evidenced by the former species more north erly native range. It has been predicted that the range of A. albopictus may eventually expand northward as far as the -5 C isotherm, as it does in Asia, however at such latitudes populations would likely not overwinter (Nawrocki and Hawley 1987). Furthermore, both species are container-inhabi ting mosquitoes commonly found in water-filled artificial container habitats such as automobile tires, bird baths, and flow er pot saucers; however rock pools are the preferred habitat of O. japonicus in its native range (Tanaka et al. 1979). The aquatic larvae of both species f eed on microorganisms and particul ate matter in the water column as well as on leaves and other orga nic detritus (Merritt et al. 1992). Severe crowding and limiting reso urces are frequent in these habitats, thus it is likely that larval resource competition, interor intraspecific, may have im portant affects on the growth, survivorship, and reproductive success of these species (Julia no and Lounibos 2005); therefore larval conditions may have a significant impact on overall population growth. Those species that can maintain positive population growth under inte rspecific conditions of greater density or lower resource availability than a competitor are considered to have a competitive advantage. Such a competitive advantage is even greater if one species can maintain positive population growth under conditions that result in nega tive population growth for a competitor. 67

PAGE 68

The role of interspecific competition in st ructuring communities of container-dwelling mosquitoes has been well documented, perhaps best so for A. albopictus and A. aegypti in the southeastern United States, where interspecifi c larval competition (Barrera 1996, Juliano 1998, Braks et al. 2004) was the probabl y cause of the declin e in range and abundance of the latter species throughout most of this area (OMear a et al. 1995, Juliano et al. 2004, Juliano and Lounibos 2005). Aedes albopictus has also been indicated experime ntally to be a superior larval competitor to O. triseriatus (Livdahl and Willey 1991) and Culex pipiens (Costanzo et al. 2005). While the invasion of an introduced species ma y negatively impact native or other introduced species as a result of interspeci fic larval competition, the effects of other interactions such as predation, habitat alteration, or apparent competit ion mediated by shared enemies should also be considered when assessing interspecific interactions. Understanding the invasion dynamics of A. albopictus and O. japonicus is important not only because of the ecological consequences re sulting from their interactions with native container-inhabiting mosquitoes, but also beca use these species may be of epidemiological significance. The invasion and establishment of A. albopictus and O. japonicus in the United States is cause for concern because of potential involvement of these species in the transmission cycle of human arboviruses. In its native range A. albopictus is a known vector of dengue virus, which has been isolated from wild-caught individuals of this species in Me xico (Ibaez-Bernal et al. 1997). This species was also implicated as the vector in the 2001 outbreak of dengue in Hawaii (Effler et al. 2005). Wild-caught A. albopictus in the United States have also been recovered infected with easte rn equine encephalitis (Mitch ell et al. 1992) and LaCrosse encephalitis viruses (Ger hardt et al. 2001). 68

PAGE 69

Although O. japonicus is not considered an important di sease vector in its native range in Asia, it may transmit Japanese encephalitis viru s (Takashima and Rosen 1989) and has also been indicated as a competent experimental vector of eastern equine encephalitis (Sardelis et al. 2002a), LaCrosse encephalitis virus (Sardelis et al. 2002b), and St. Loui s encephalitis viruses (Sardelis et al. 2003). Both A. albopictus and O. japonicus have shown to be competent vectors of West Nile virus in the laboratory (Turell et al. 2001), and wild-caught adults of both species have been recovered infected with this virus (Holick et al. 2002, Scott 2003, Godsey et al. 2005). The demonstrated ability of these species to be infected by and transmit numerous arboviruses indicates that their introductions and competitive interactions in the United States may have important public health c onsequences (Lounibos 2002). While considerable data exist regarding th e interspecific interactions and competitive outcomes of A. albopictus with numerous other c ontainer-inhabiting mosquito species, there are no comparable reports on O. japonicus. As A. albopictus and O. japonicus are known to frequently co-occur in container habitats, an investigation of larval competitive interactions between these two species was proposed. An e xperiment designed similar to those of Juliano (1998) and Braks et al. (2004), with the excep tion that only a single resource level was implemented, was conducted to measure the perfor mance of larvae of Virginia populations of these species competing for resources under fiel d conditions in Fairfax, Virginia. Comparisons were made between species for intraand interspe cific effects of larval density on survivorship, development time, body size, and population growth. Materials and Methods The experiment was conducted in a heavily fore sted streambed located directly behind the Fairfax County Department of Health in Fairfax, Virginia (latitude 38 50 N, longitude 69

PAGE 70

77 19W) from August to October 2006. Routine surveillance data collected by the Department of Health indicated that both A. albopictus and O. japonicus were commonly detected on this property as both larvae in artificial containers and as adults in CO 2 -baited CDC light traps and gravid traps. The A. albopictus and O. japonicus used in this experiment were the first generation progeny of gravid individuals co llected from Fairfax, Virginia. Interand intraspecific larval compet ition was investigated by monitoring the development of larvae at different densities in 400-ml black polypropylene cups (10.5 cm in height, 6.5 cm base diameter). Field surveys of cont ainer habitats in the ar ea indicated that these species co-occur in containers of similar shape and size in nature Three density combinations of A. albopictus : O. japonicus (10:0, 50:0, 0:10, 0:50, and 25:25) we re evaluated using a completely randomized block design. One replicate of each combination was placed at each of five experimental sites, spaced approximately 30m apar t, for a total of five replicates per treatment and 25 cups. Although both species have been f ound in containers with varying sun exposure (i.e., none, partial, and full exposure), all five s ites used were completely shaded to maintain experimental consistency. On 10 August, each cup was randomly labele d with a unique number and letter corresponding to one of the five treatments and sites, where they were secured to plastic stakes to prevent toppling. Food consisted of fallen pin oak leaves ( Quercus palustris ) that had been collected, washed, and dried at room temperature for one week prior to quartering, weighing, and sorting. To allow for the leaves to soak and be colonized by microorganisms, four days prior to the start o the experiment 1 g was added to each of the 25 cups containi ng 200 ml of distilled water. Each container was covered with fiberg lass screen (0.5 mm) and secured with a rubber band to prevent entry of other macrofauna and detritus. In the laboratory, eggs of A. albopictus 70

PAGE 71

and O. japonicus were synchronously hatched (Novak a nd Shroyer 1978), and 24 hours later larvae were counted into ali quots of 10, 25, and 50. Within on e hour after counting, the larvae were distributed into appropriate cups. Each container was monitored daily for the presence of pupae, which were collected and housed singly in sealed 50 ml vials containing wa ter from their respective field cup. Each vial was labeled with the appropriate site and treatm ent identifier before being secured to a plastic stake at the field site with a rubber band. Upon emergence, adults were killed by freezing before scoring by container, species, sex, and day of emergence. The experiment ended on 11 October when the final adult emerged. Ambient temperatur e was monitored hourly for the duration of the experiment with three Onset HOBO data loggers located in the middle and at either end of the experimental area. The average (SE) hourly ambient temperature recorded was 18.77.06 C, with a range of 7.76 C to 29.73 C (n = 4197). For two days of the experiment (31 August and 1 September), the area was subjected to intense wind and rain due to Hurricane Ernesto. To prevent damage to experimental apparatus and lo ss of data, the cups at each experimental site were successfully covered and secured with a tarpaulin during this time. Data Analysis Population Growth Correlates To quantify the effects of interand intraspecific competition on A. albopictus and O. japonicus, the mean survivorship, median developmen t time of males and females, and median body size at adulthood of females were analyzed by one-way ANOVA, followed by Tukeys honestly significantly different (HSD) post-hoc tests for pairwise comparisons of means in SPSS (SPSS 2002). Survivorship was calculated as the proportion of adults that emerged from the initial cohort of first instar la rvae. Development time was calculated as the number of days from 71

PAGE 72

hatching to adult emergence. Adult body size was estimated from the length of one wing, which was removed from each female and measured under a dissecting microscope with an ocular micrometer (Packer and Corbet 1989). Median rather than mean development time and wing length were calculated for each species and treatm ent because of the non-normal distributions of these variables within cohorts. Composite Index of Population Performance Survivorship, female development time a nd wing length were used to calculate a composite index of mosqu ito population performance ( ), which is an analog of the finite rate of increase as defined by Juliano (1998): where N 0 is the initial number of females (assumed to be 50% of the cohort), A X is the number of females eclosing on day x w X is the mean wing length of females eclosing on day x and f(w X ) is a function relating egg pr oduction to wing length. D is the time from adult eclosion to reproduction, taken as 14 days for A. albopictus (Livdahl and Willey 1991) and 12 days for O. japonicus (see below). Values of greater than one indicate th at the populati on is increasing, approximately equal to one that the population is stable, and less than one that the population is decreasing. D for O. japonicus was determined experimentally under controlled conditions of 26 C and 12h:12h light:dark in an insectary at the Fl orida Medical Entomology Laboratory in Vero Beach, Florida. Ochlerotatus japonicus eggs used for this experiment were obtained from the 72

PAGE 73

colony maintained at the Headlee Research Labo ratory Mosquito Research and Control Unit at Rutgers University in New Brunswick, New Je rsey. This colony orig inated from larval collections from a horse farm in New Egypt, Ocean County, New Jersey in 2001 (L. McCuiston personal communication). In the laboratory, eggs were hatched by fl ooding with water, and cohorts of larvae hatching within the same 24-hour period were groupe d together and placed in plastic trays within separate 0.028 m 3 (30.5 cm x 30.5 cm x 30.5 cm) cages. Each cohort was fed 100 mg of an artificial diet consisti ng of one part Brewers yeast and one part lactalbumen every other day. Beginning two days after emergence, adult females of each cohort were offered a bloodmeal from a restrained chicken placed within the ca ge daily for one hour. Cotton soaked in a 20% sucrose solution was provided as a source of car bohydrates for adults at all times. Upon visual inspection immediately following the bloodfeeding oppor tunity, those females that appeared to have fed to repletion were removed from the cage using a mouth aspirato r and placed singly in 12-dram plastic vials containing a 2.54 cm by 7. 62 cm strip of wet seed germination paper (Anchor Paper, St. Paul, MN) to serve as an oviposition substrate (Ste inly et al. 1991). The germination paper was checked daily for the presen ce of eggs, and was repl enished with water if necessary. The date of oviposition for each fema le was recorded, from which the average time from adult eclosion to oviposition for O. japonicus was calculated to be 12 days, with a range of 4 to 17 days (N = 144). A regression relating female wing length to fecundity for A. albopictus was obtained from Lounibos et al. (2002): f(w X ) = 78.02 w X 121.240 (r 2 = 0.713, N = 91, p < 0.001) 73

PAGE 74

where w X is the wing length in millimeters on day x, while that for O. japonicus was obtained from Lounibos et al. ( unpublished data), who used indi viduals originating from the same colony from Rutgers University mentioned previously, as follows: f(w X ) = 53.078 w X 113.91 (r 2 = 0.319, N = 79, p < 0.001), where w X is the wing length in millimeters on day x. For analyses of for A. albopictus and O. japonicus a one-way ANOVA was used with Tukeys honestly significantly different (HSD ) tests performed post-hoc for pairwise comparisons of means (SPSS 2002). Results Survivorship to Adulthood Mean survivorship to adulthood of A. albopictus was affected by trea tment but that of O. japonicus was not (Table 3-1, Figure 3-1). Mean survivorship of A. albopictus varied significantly among treatments (F 2,12 = 7.442, p = 0.008), and was significantly higher in the 10:0 treatment than the 50:0 or 25:25 treatments, whic h were not different from one another. With respect to individual density treatments, mean survivorship of A. albopictus was consistently higher than that of O. japonicus Developmental Time Median time from hatch to adulthood of both sexes was significantly affected by density treatment for both males (F 2,12 = 7.560, p = 0.008) and females (F 2,12 = 19.114, p < 0.001) of A. albopictus. Comparisons of means for A. albopictus showed that development time was significantly shorter for both males and females in the 10:0 and 25:25 density treatments (Table 3-1). Median development time of both male (F 2,12 = 7.09, p = 0.009) and female (F 2,12 = 10.194, p = 0.003) O. japonicus was significantly affected by treatm ents; however significant differences in pairwise comparisons differed between the sexes in this species (Table 3-1). With respect to 74

PAGE 75

individual density treatments, median de velopment times of both male and female A. albopictus were consistently faster than those of O. japonicus (Figures 3-2, 3-3). Female Wing Length Median wing lengths of both A. albopictus and O. japonicus females were significantly affected by density treatments (F 2,12 = 4.837, p = 0.029; F 2,12 = 9.584, p = 0.003). For both species, median wing length was significantly greater for females from 10:0 treatments; however for A. albopictus this difference was only significant in comparison to females from the 25:25 density treatment (Table 3-1, Figure 3-4). Estimated Finite Rate of Increase ( ) The mean estimated finite rate of increase of both species was si gnificantly affected by density treatments (Table 3-1, Figur e 3-5), although more pronounced for A. albopictus (F 2,12 = 23.585, p < 0.001) than O. japonicus (F 2,12 = 16.366, p < 0.001). Comparisons of means showed that of A. albopictus was significantly higher for the 10:0 treatment than the 25:25 treatment, which was significantly higher than that of th e 50:0 density treatment (Table 3-1). For O. japonicus, of the low-density treatment was significan tly higher than either of the high-density treatments. With respect to individual density trea tments, mean estimated finite rates of increase of A. albopictus were consistently higher than those of O. japonicus Discussion It is common opinion that the invasion su ccess and spread of non-native species is enhanced by superiority in interspecific comp etition, particularly when similar species and limited resources are encountered (Williamson 1996). It has been demonstrated that interspecific larval resource competition plays an important role in structuring the mosquito communities of artificial container habitats (J uliano and Lounibos 2005). Given th e results of this experiment, A. albopictus does appear to be a superior competitor to O. japonicus; however the non-significant 75

PAGE 76

impact of interspecific larval resource co mpetition on population performance suggests that O. japonicus will be able to coexist with A. albopictus in artificial container habitats in nature. Continued coexistence of A. albopictus and O. japonicus in artificial containers is supported by the relatively high survivorship of both species, which were not significantly different under interspecific (25: 25 treatment) versus intraspeci fic conditions (50:0 treatment) of the same mosquito density. Furthermore, the estimated finite rate of increase, , remained greater than one in al l species/density treatments, indi cating a population increase for both species under all experime ntal conditions (Table 3-1). However, the mean for A. albopictus was actually significantly higher under interspecifi c conditions than intraspecific conditions of the same mosquito density (Figure 3-5). This app ears to be due to the median development time of females from interspecific density treatments being significantly shorter than those from intraspecific treatments of the same mosquito density (Figure 3-2). Thes e findings suggest that intraspecific competition may be more important for regulating A. albopictus population growth in container habitats than interspecific competition with O. japonicus While neither species appeared to be detrimentally affected under interspecific conditions with respect to population performance, A. albopictus may have a slight competitive advantage over O. japonicus. This is supported by the consistently higher survivorship, shorter development time, and higher finite rate of increase of A. albopictus compared to O. japonicus across density treatments. Under interspecific co nditions, median developm ent time of both male (F 1,8 = 18.375, p = 0.003) and female (F 1,7 = 26.940, p < 0.001) A. albopictus was significantly shorter than that of O. japonicus. Similarly, the mean estimated finite rate of increase was significantly greater for A. albopictus than O. japonicus (F 1,8 = 11.016, p = 0.011). However, there was no difference in survivorship between the two species under interspecific conditions 76

PAGE 77

(F 1,8 = 3.240, p = 0.110). While the difference in developmen t times may simply be the result of intrinsic metabolic differences between the sp ecies, when coupled with the higher mean estimated rate of finite incr ease it may suggest that perhaps A. albopictus is able to forage and acquire resources more efficiently or employs different feeding be haviors that are more effective in this type of larval habita t. Ho et al. (1973) suggested that perhaps the higher content of proteinases in the gut of A. albopictus facilitates a more efficient feeding style, which ultimately allows the species to develop faster th an other container-inhabiting mosquitoes. These results imply that while A. albopictus may have a slight competitive advantage over O. japonicus the two will likely continue to coexist in containers in areas where their distributions overlap. However, these findings should be view ed in context with field observations of co-occurrences, as well as seas onal distributions, habitat preferences, and overwintering behaviors, as they may ultimately influence the community structure of the artificial containers in which these species coexis t. In addition to these ecological consequences, these findings may potentially have epidemiological implications, particularly with respect to LaCrosse encephalitis virus, of which both A. albopictus and O. japonicus are suspected vectors. Their continued coexistence in containers in LaCrosse endemic regions may be important in epizootic, or potentially even epidemic, transmi ssion of the disease, although this will require further investigation. Because larval competiti on has been linked to greater infection and dissemination rates of dengue and Sindbis viruses for A. albopictus (Alto et al. 2005), similar effects are possible with respect to arboviruses circulating in ar eas in which this species is sympatric with O. japonicus. Although this experiment was conducted in the field under manipulated but ecologically realistic conditions, it is important to note that va riations in resource leve l, type, or frequency 77

PAGE 78

78 (Braks et al. 2004), temperature (Lounibos et al. 2002), sun expos ure, container type (Livdahl and Willey 1991), larval density, and season (T eng and Apperson 2000) may influence larval competition differently among these mosquito sp ecies. Similarly, while interspecific larval competition is likely an important factor infl uencing the survivorship, growth, reproductive success, and population performance of mosquitoes in container environments with limited resources, other factors such as predati on (Griswold and Lounibos 2005, 2006), intraguild predation (Edgerly et al. 1999) apparent compe tition mediated by shared enemies (Munstermann and Wesson 1990, Blackmore et al. 1995, Juliano 1998), habitat alteration (Bertness 1984), and differences in foraging behavior (Yee et al. 2004) may also be important and warrant further research with respect to inter actions between these two species.

PAGE 79

Table 3-1. Means ( SE) of population growth correlates for A. albopictus and O. japonicus Means followed by letters that are not commonly shared are significantly di fferent by pairwise comparisons ( p < 0.05). Density species treatments Aedes albopictus Ochlerotatus japonicus Response 10:0 50:0 25:25 10:0 50:0 25:25 Mean survivorship .94 (.04)a .704 (.038) b .728 (.061)b .78 (.049) .524 (.096) .584 (.052) Median female development time (d) 15.4 (.51)a 29.6 (2.91)b 17.5 ( .71)a 20.8 (2.84)a 32.8 (2.85)b 35.4 (3.08)b Median male development time (d) 12.8 (.56)a 15.2 (.58)b 12.7 (.37) a 14.8 (.52)a 20.3 (1.22)ab 25.9 (2.70)b Median female wing length (mm) 2.71 (.092)a 2.51 (.053)ab 2.41 ( .056)b 3.55 (.060)a 3.05 (.108)b 3.08 (.10)b 1.162 (.007)a 1.093 (.008)b 1.120 (.007)c 1.135 (.007)a 1.071 (.011)b 1.076 (.007)b 79 79

PAGE 80

0.00 0.20 0.40 0.60 0.80 1.00 1.20 TreatmentSurvivorshi p A. albopictus O. japonicus b a b 10:0 50:0 25:25 Survivorship 80 Figure 3-1. Mean survivorship (propo rtion of the original number of larvae surviving to adulthood) of A. albopictus and O. japonicus (SE). Lower case letters indicate significant differen ces among competition treatments resulting from pairwise comparisons ( p < 0.05) for A. albopictus. Analysis of variance did not indicate a significant difference in survivorship among treatments for O. japonicus. 80

PAGE 81

0 5 10 15 20 25 30 35 40 45 Treatment A. albopictus D evelopment time ( d O. japonicus a a b A B B 10:0 50:0 25:25 Develo p ment time ( d ) 81 Figure 3-2. Means of median ti me to adulthood for female A. albopictus and O. japonicus (SE). Lower case and upper case letters indicate significant differences among competition tr eatments resulting from pairwise comparisons (p < 0.05) for A. albopictus and O. japonicus respectively. 81

PAGE 82

0 5 10 15 20 25 30 35 Treatment A. albopictus D evelopment time ( d O. japonicus 25:25 a AB b a B A 10:0 50:0 Develo p ment time ( d ) 82 Figure 3-3. Means of median time to adulthood for male A. albopictus and O. japonicus (SE). Lower case and upper case letters indicate significant differences among competition tr eatments resulting from pairwise comparisons (p < 0.05) for A. albopictus and O. japonicus respectively. 82

PAGE 83

0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 Treatment A. albopictus O. japonicus a B b B a b A Wi ng length (m m Wing length (mm) 83 10:0 50:0 25:25 Figure 3-4. Means of median wing lengths of A. albopictus and O. japonicus adult females (SE). Lower case and upper case letters indicate significant differences among competition tr eatments resulting from pairwise comparisons (p < 0.05) for A. albopictus and O. japonicus respectively. 83

PAGE 84

1.00 1.02 1.04 1.06 1.08 1.10 1.12 1.14 1.16 1.18 Treatment A. albopictus O. japonicus a c b A B B 10:0 50:0 25:25 Figure 3-5. Mean estimates of population performance ( , an estimate of the finite rate of increase for the cohort) for female A. albopictus and O. japonicus adults (SE). Lower case and upper case le tters indicate signifi cant differences among competition treatments resulting from pairwise comparisons ( p < 0.05) for A. albopictus and O. japonicus respectively. 84 84

PAGE 85

CHAPTER 4 INTERSPECIFIC COMPETITION BETWEEN OCHLEROTATUS ATROPALPUS AND OCHLEROTATUS JAPONICUS Introduction Within its native range in Asia, Ochlerotatus japonicus larvae are found in a wide variety of natural and artificial containers; however ro ck pools are the preferre d habitat (LaCasse and Yamaguti 1950, Tanaka et al. 1979). This species has colonized a similar ecological niche since its introduction to the United States in 1998 via used tire shipments (Peyton et al. 1999, Andreadis et al. 2001) from Japan (Fonseca et al. 2001) As an invasive species, the likelihood of O. japonicus to become established and propagate in this niche depends partly on resource availability and its ability to compete with ecologically similar resident species. Interspecific competition is instrumental in determining the outcome of an introduction regardless of whether it promotes or limits the spread of an invader, and may only be avoi ded if an invader is filling a vacant niche by exploiting a previously unoccupied habitat or unused resource (Williamson 1996). The effects of interspecific competition due to severe crowding and limited resources among the larvae of container-inhabiting mosquitoes have been well documented (e.g. Livdahl and Willey 1991, Barrera 1996, Juliano 1998, Braks et al. 2004, Juliano et al. 2004, Costanzo et al. 2005), however the majority of these i nvestigations centered around the invasion of Aedes albopictus. Such studies have demonstrated both the success (OMeara et al 1995, Juliano et al. 2004) and failure (OMeara et al. 1989, Lounibos et al. 2003) of invasive species to spread. Interspecific competition may potentially im pact the overall populat ion performance of container-inhabiting species by a ffecting growth, survivorship, a nd reproductive success (Juliano 85

PAGE 86

and Lounibos 2005). Under conditions of severe inte rspecific competition, such effects may lead to the decline or elimination of a resident species following the introduction of a competitively superior invader (e.g. Juliano 1998). Due to the propensity of O. japonicus to inhabit rock pools, the indigenous mosquito most likely to be affected by the inva sion of this species is the North American rock pool mosquito, O. atropalpus The two species are highly sympatric, with distributions that overlap in parts of the eastern United States. Frequent and abundant collections of O. japonicus larvae co-occurring with O. atropalpus in rock pools provides a natural setting for interspecific larval resource competition (Andreadis et al. 2001), which some have speculated may limit its invasion success (Juliano and Lounibos 2005). However, recent fiel d studies have indicated that competitive displacement of O. atropalpus by O. japonicus may be occurring in rock pools in New Jersey (Scott et al. 2001a), North Carolina (B. Harris on personal communication) and Virginia (see previous chapter). Because of the sp ecialized primary larval habitat of O. atropalpus in rock pools, the distribution of this species tends to be sparsely and irregularly distributed despite its large geographical range. Such conditions may promot e the localized decline or extinction of this species in areas where these two sp ecies co-occur, particularly if O. japonicus is a superior competitor. However, as noted in Lounibos (2002), O. atropalpus has expanded its distribution by occupying discarded tires and has itself been consider ed an invasive species. The decline and potential competitive exclusion of O. atropalpus suggest that the competitive superiority of O. japonicus may be important in the inva sion success of this species. While interspecific larval resource competiti on is a likely mechanism for such ecological processes, no formal research has been c onducted to confirm such speculation. Thus an investigation of larval competitive interactio ns between these two species was proposed. 86

PAGE 87

Comparisons were made in the laboratory between species for intraand interspecific effects of larval density on survivorship, developm ent time, body size, and population growth. Materials and Methods The experiment was conducted in an inse ctary at the Florida Medical Entomology Laboratory in Vero Beach, Florida under controlled conditions of 25.5 0.001 C, 86.7 0.08% RH, and a 12L:12D photoperiod, from October to November 2006. Ambient temperature was monitored hourly for the duration of the experi ment with a single Onset HOBO data logger. The O. japonicus and O. atropalpus used in this experiment were harvested from eggs obtained from colonies maintained at the Headlee Research La boratory Mosquito Resear ch and Control Unit at Rutgers University in New Brunswick, New Jersey. The O. japonicus colony originated from larval collections from a horse farm in New Egypt, Ocean County, New Jersey in 2001, while the O. atropalpus colony originated from larvae coll ected in 1993 from Monmouth, Salem, Cumberland, and Burlington Counties, New Jersey (L. McCuiston personal communication). To investigate interand intraspecific la rval competition between these two species, the development of larvae in surroga te rock pools in different density species combinations was monitored. Surrogate rock pools were constructe d within plastic planters (91.4 x 18 x 12 cm) using a fast setting, high streng th concrete mix consisting of co arse sand aggregate, and cement (Sakrete ). Approximately 15 kg of concrete mixed with 1.6 l water was poured into each of six planters, and within each, five individual indentations (7 x 11.5 cm) were made using glass canisters to create the rock pools. Field surveys in Fairfax County, Virginia indicated that these species occur in rock pools of similar shape and size in nature. Once dry, each planter was completely flooded with water for 24 hours to ensure structural reliability. A randomized complete block design was used for the experi ment, with five dens ity combinations of O. 87

PAGE 88

japonicus:O.atropalpus (20:0, 60:0, 0:20, 0:60, and 30:30) as treatments and individual planters as blocks. Each planter contained one replicate of each density species composition combination in a separate rock pool, for a total of six replicates per tr eatment and 30 individual rock pools. Two months prior and up to the star t of the experiment, surrogate rock pools were flooded with water to allow for any potentially to xic chemicals to leech out of the cement in an effort to reduce or prevent larval mosquito mortality. On 19 October, each planter was randomly labeled with a number, and each of its rock pools, was randomly labeled with a letter corres ponding to one of the five treatments. Food consisted of fallen pin oak leaves ( Quercus palustris ) that had been collected in Fairfax, VA, washed, and dried at room temperature for one week prior to quartering, weighing, and sorting. Four days prior to the start of the experiment, 1.0 g of leaves was added with 300 ml of distilled water to each of the 30 rock pools. The appropria te water level was marked in each rock pool, and was checked every five days for evaporation and refilled with distil led water as necessary. Each planter was covered with fiberglass screen (0.5 mm) and secured with a large rubber band to prevent entry of other macrof auna. In the laboratory, eggs of O. japonicus and O. atropalpus were synchronously hatched (Novak and Shroyer 1978), and 24 hours later larvae were counted into aliquots of 20, 30, and 60. Within one hour afte r counting, the larvae we re distributed into appropriate rock pools. Each rock pool was monitored daily for the presence of pupae, which were collected and housed singly in sealed 10 dram (36.7 ml) vials containing water from their respective rock pool. Each vial was labeled with the appropriate plante r and treatment identifier and placed in a rack until adult eclosion. Upon emergence, adults were killed by freezing before scoring by container, 88

PAGE 89

species, sex, and day of emergence. The experime nt ended on 26 November when the final adult emerged. Data Analysis Population Growth Correlates To quantify the effects of interand intraspecific competition on O. japonicus and O. atropalpus the mean survivorship, median developmen t time of males and females, and median body size at adulthood of females were analyzed by one-way ANOVA was used with Tukeys honestly significantly different (HSD) tests performed post-hoc for pairwise comparisons of means in SPSS (SPSS 2002) with species-density treatments as independent variables. Survivorship was calculated as the proportion of adults that emer ged from the initial cohort of first instar larvae. Development time was calculated as the number of days from hatching to adult emergence. Adult body size was estimated from the length of one wing, which was removed from each female and measured under a dissectin g microscope with an ocular micrometer (Packer and Corbet 1989). Median rather than mean development time and wing length were calculated for each species and treatment becau se of the non-normal distributions of these variables within cohorts. Composite Index of Population Performance Survivorship, female development time a nd wing length were used to calculate a composite index of mosqu ito population performance ( ), which is an analog of the finite rate of increase as defined by Juliano (1998): 89

PAGE 90

where N 0 is the initial number of females (assumed to be 50% of the cohort), A X is the number of females eclosing on day x w X is the mean wing length of females eclosing on day x and f(w X ) is a function relating egg pr oduction to wing length. D is the time from adult eclosion to reproduction, taken as 8 days for O. atropalpus and 12 days for O. japonicus (see below). Values of greater than one indicate that the population is increasing, appr oximately equal to one that the population is stable, and less than one that the population is decreas ing. If no individuals survive to reproduction, equals zero (Lonard and Juliano 1995, Grill and Juliano 1996). D for both species was determined experimentally under controlled conditions of 26 C and 12L:12D photoperiod in an insectary at the Florida Medical Entomology Laboratory in Vero Beach, Florida. In the laboratory, eggs were hatched by flooding with water, and cohorts of approximately 100 larvae hatching within the same 24-hour period were grouped together and placed in plastic trays within separate 0.028 m 3 (30.5 cm x 30.5 cm x 30.5 cm) cages. Each cohort was fed 100 mg of an artificial diet consis ting of one part Brewers yeast and one part lactalbumen every other day. Beginning two days after emergence, adult females of each cohort of O. japonicus were offered a bloodmeal from a restrain ed chicken placed within the cage daily for one hour. As O. atropalpus females can mature their firs t egg batch without blood (OMeara and Craig 1970, OMeara and Krasnick 1970), cohor ts of this species were not offered a bloodmeal. Cotton soaked in a 20% sucrose solu tion was provided as a source of carbohydrates for adults of both species at all times. U pon visual inspection immediately following the 90

PAGE 91

bloodfeeding opportunity, those female s that appeared to have fe d to repletion were removed from the cage using a mouth aspirator and placed singly in 12-dram plas tic vials containing a 2.54 cm by 7.62 cm strip of wet seed germination paper to serve as an oviposition substrate (Steinly et al. 1991). The germination paper was ch ecked daily for the presence of eggs, and was replenished with water if necessa ry. The date of first ovipositi on for each female was recorded, from which the average time from adult eclosion to oviposition for O. japonicus was calculated to be 12 days, with a range of 4 to 17 days (N = 144), while that for O. atropalpus was calculated to be 8 days, with a range of 4 to 17 days (N = 153). Regressions relating female wing length to fecundity were obtained from Lounibos et al. (unpublished data), who used individuals orig inating from the same colonies as these experiments, as follows: O. japonicus : f(w X ) = 53.078 w X 113.91 (r 2 = 0.319, N = 79, p < 0.001) O. atropalpus : f(w X ) = 66.148 w X 150.28 (r 2 = 0.526, N = 74, p < 0.001), where w X is the wing length in millimeters on day x. For analyses of for O. japonicus no transformation yielded da ta that met assumptions of normality and homogeneous variance. Therefore, randomization ANOVA (Manly 1991, 1997) was used to analyze as a randomized complete block desi gn, with three different density species composition combinations as treatments and six different planters as blocks. Following ANOVA, pairwise comparisons of all treatme nt means were conducted using randomization methods (Manly 1991, 1997) at an experimentwise = 0.05, using the sequential Bonferroni 91

PAGE 92

method to correct for experimentwise e rror (Rice 1989). Because all values of for O. atropalpus were zero, no comparis ons could be performed. Results Survivorship to Adulthood Mean survivorship to adulthood of O. atropalpus was affected by treatment but that of O. japonicus was not (Table 4-1, Figure 4-1). Mean survivorship of O. atropalpus varied significantly among treatments (F 2,15 = 3.79, p = 0.047), and was significantly higher in the 20:0 treatment than the 60:0 treatme nt. Mean survivorship of O. atropalpus in interspecific treatments was not significantly different fr om either intraspecific treatment. With respect to individual density species composition combinati on treatments, mean survivorship of O. atropalpus was higher than that of O. japonicus in all instances, except the 60:0 treatment. There was no significant difference in survivorship between the two species under interspecific conditions (F 1,10 = 1.456, p = 0.225). Developmental Time Median time from hatch to adulthood was not affected by density treatment for either sex of O. japonicus (Table 4-1, Figures 4-3, 4-4). Median development time of female (F 2,15 = 8.184, p = 0.004), but not male (F 2,15 = 1.484, p = 0.263), O. atropalpus was significantly affected by treatments, and was significantly shorter in the 20:0 treatment than the 60:0 or 30:30 treatments (Table 4-1, Figure 4-1), which were not different from one another. With respect to individual density treatments, median development times of female O. japonicus were shorter than those of O. atropalpus in all instances, except 20:0 treatments. Median development times of males of O. atropalpus were shorter than those of O. japonicus in all instances, except the interspecific treatment. There was no significant difference between male (F 1,10 = 0.328, p = 0.581), or 92

PAGE 93

female (F 1,10 = 0.995, p = 0.342) development time of O. japonicus and O. atropalpus under interspecific conditions. Female Wing Length Median wing lengths of both O. japonicus and O. atropalpus females were significantly affected by density treatments (F 2,14 = 18.522, p < 0.001; F 2,15 = 39.474, p < 0.001). For both species, median wing length was significantly greater for females from the 20:0 treatment (Table 4-1, Figure 4-1). Estimated Finite Rate of Increase ( ) The mean estimated finite rate of increase of O. japonicus was significantly affected by density treatments (F 2,14 = 3.87, p = 0.058), with less than one for both the 60:0 and 30:30 treatments, indicating populati on decline. Pairwise comparis ons of means showed that of O. japonicus from the 20:0 treatment was significantly higher than that from the interspecific treatment; no other comparisons were si gnificant (Table 41, Figure 4-1). For O. atropalpus was zero in all instances, indica ting that no individuals were ab le to reproduce autogenously (Table 4-1, Figure 4-1). Discussion This experiment indirectly supported predictio ns that interspecifi c competition between larvae of O. japonicus and O. atropalpus contributed to the decline and potential displacement of the latter species in some rock pool communities. However, results indicate that interspecific larval competition was not detectably different from intraspecific competition for either O. japonicus or O. atropalpus as there was no significant differe nce in the mean survivorship, median development time, or median wing length fo r either species under interor intraspecific conditions of the same mosquito density. The mean estimated finite rate of increase, , was zero 93

PAGE 94

in all instances for O. atropalpus indicating that no individual s were able to reproduce autogenously, while that of O. japonicus did not significantly cha nge between intraand interspecific treatments of the same mosquito densities. This suggests be tter overall population performance for this species than O. atropalpus across all experimental treatments. The values for O. atropalpus were a direct result of the zero fecundity values resulting from the small size of emergent O. atropalpus females from all density species combination treatments. Considering the autogenous reproduction of O. atropalpus (OMeara and Craig 1970, OMeara and Krasnick 1970), in which the fecundity of this species depe nds on nutrient reserves obtained in the larval stage, this result is not surprisi ng. Fecundity of O. japonicus also appears to be affected, as the median wing lengths of females from this e xperiment, which ranged from approximately 2.0 2.5 mm, were noticeably smaller than those from the previous field competition experiment with A. albopictus where they were approximately 1 mm longer (Figures 3-4, 4-4). An attempt was made to simulate the ecological conditions experienced by these species in nature; however the somewhat equivocal results as evidenced by the declining or zero estimated finite rates of increase for both species suggest that perhaps thes e experimental conditions were excessively stressful. It has been demonstrated that the type and qua ntity of detritus in a container can influence microorganism popula tions and communities (Walker et al. 1991, Kaufman et al. 2001), and therefore can influe nce mosquito population performance (Lounibos et al. 1993, Walker et al. 1997). Furthermore, inte rspecific differences in feeding behavior are common among container-inhabiting mosquitoes (Y ee et al. 2004); larvae may browse on hard surfaces or filter fluid to gather microorganisms (Merritt et al. 1992), and the efficiency of these feeding modes may depend on habitat structure and complexity. The use of increased or multiple levels of food, a different food source, or pulse delivery of food in this experiment may have 94

PAGE 95

provided experimental conditions more comparable to those in nature in which interspecific competition might be expressed. Furthermore, it is possible that the cement from which the rock pools were constructed imposed some toxic e ffect on the mosquito larvae, although it is important to note that there was no observable difference in the survivorship of O. japonicus in this experiment of the previous field competition experiment of this species with A. albopictus (Figures 3-1, 4-1). Although it is evident that experimental conditio ns were stressful for both species, and thus interspecific interactions between these two speci es as they occur in nature may have been obscured, it is possible that O. japonicus may have a competitive advantage resulting from more efficient feeding behaviors or be tter resistance to st arvation. Although the latt er was not directly demonstrated in this experiment in terms of differences in survivorship, it is interesting to note that male O. atropalpus on average developed fa ster than that of O. japonicus but the reverse applied to females, indicating the need for females of the former species to lengthen larval development to accumulate nutrient reserves for egg production. This suggests that the autogenous reproduction of O. atropalpus (OMeara and Craig 1970, OMeara and Krasnick 1970) may render this species more sensitiv e to competitive stress. Condition-specific competition, wherein competitive superiority varies with the abiotic envi ronment, is known to occur among various life stages of container-inhabiting mosqu itoes (Barrera 1996, Daugherty et al. 2000, Costanzo et al. 2005), and may be an im portant factor in understanding the competitive outcomes among O. atropalpus and O. japonicus These results emphasize the importance of e xperimental conditions and suggest that multiple factors other than interspecific larval resource competition may be important in determining the current abundance and distribution of th ese species. It has been demonstrated 95

PAGE 96

that larvae of O. japonicus can survive at low temperatures for extended time periods (Scott 2003), an ability that appears to allow this spec ies to hatch and/or begin development before other mosquitoes in the early sp ring, and apparently overwinter in the larval as well as the egg stage in temperate areas, even where the surface of larval habitats freeze completely (Kamimura 1976, Scott 2003, B. Harrison pe rsonal communication). Ochlerotatus japonicus has been found as larvae in every month of the year within its natural range (N akata 1962) and in North Carolina (B. Harrison personal communication) and in all months except Fe bruary in New Jersey (Scott 2003). This cold tolerance of O. japonicus may confer an ecologica l advantage in obtaining resources over O. atropalpus which diapauses in the egg stag e (Hedeen 1953) and emerges later in the season. Furthermore, it may facilitate as ymmetric intraguild predation of newly hatched O. atropalpus larvae by fourth instar O. japonicus ; however the importance of this ecological process in structuring co ntainer-inhabiting mosquito communities has yet to be demonstrated in nature (Edgerly et al. 1999). Predation is a prominent feat ure of rock pools in North America with predacious diving beetles of the family Dytiscidae playing a si gnificant role in regul ating the numbers of O. atropalpus (Shaw and Maisey 1961, James 1964a,b). Larvae and adult Laccophilus maculosus are the most efficient predators of this rock pool mosquito because of their habit of crawling about on the bottom and sides of the pool where they came in contact w ith the bottom-feeding larvae of O. atropalpus (James 1964b). Hydra oligactis will capture O. atropalpus killing but not ingesting young larvae and paralyzing later stages, thereby limiting the abundance of this species (James 1964b). If O. atropalpus and O. japonicus tend to occupy different spaces within a rock pool (i.e., at the bottom, surface, or in the water column), select ive predation (Griswold 96

PAGE 97

and Lounibos 2005, 2006) by these diving beetles may be important in facilitating the invasion of the latter species in these habitats and should be investigated further. The tendency of O. atropalpus larvae to frequently congregate under leaves and other organic debris at the bottom of their rock pool ss (Hedeen 1953) has been shown to result from a negative phototropic reaction (Beach and Craig 1 979). Because fully exposed rock pools tend to be more flood prone, O. atropalpus may have adapted this beha vior in response to larval mortality associated with such environmental conditions. While this reaction may promote a preference of this species for areas of lesser concentrations of light, under enough selective pressure, this protective mech anism could potentially allow O. atropalpus to inhabit rock pools fully exposed to the sun, therefore partitioning, at least to some extent, rock pool habitats with O. japonicus, which is less frequently found under such conditions (B. Byrd personal communication, previous chapter). These experimental findings a ppear ambiguous with respect to the nature of interspecific larval resource competition between O. atropalpus and O. japonicus because of the stressful experimental conditions; however, a slight competitive advantage for O. japonicus seems likely. Numerous mechanisms for the perceived reduction in numbers of O. atropalpus in rock pool communities have been proposed here, however it is important to note that variations in temperature (Lounibos et al. 2002), habitat (Ber tness, 1984, Livdahl and Willey 1991), larval density, season (Teng and Apperson 2000), and oviposition attraction and repellency (Maire 1985, Zahiri et al. 1997) may also influence la rval competition differently among mosquito species and warrant further research with respec t to interactions between these two species. Finally, field observations of co-occurrences of these species, seasona l distributions, habitat preferences, and overwintering beha viors should be made on a larg e geographical scale, as they 97

PAGE 98

98 may ultimately influence the community structure of the rock pools in which these species coexist.

PAGE 99

Table 4-1. Means ( SE) of population growth correlates for O. japonicus and O. atropalpus Means followed by letters that are not commonly shared are significantly di fferent by pairwise comparisons ( p < 0.05). Density species treatments Ochlerotatus japonicus Ochlerotatus atropalpus Response 20:0 60:0 30:30 20:0 60:0 30:30 Mean survivorship .53 (0.09) .575 (0.107) .317 (0.057) .575 (0.107)a .26 (0.049)b .432 (0.076)ab Median female development time (d) 13.5 (0.55) 18.25 (1.22) 18.6 (1.22) 12.8 (0.95)a 21.1 (2.35)b 21.7 (1.60)b Median male development time (d) 12.4 (0.75) 13.5 (0.56) 13.9 (0.64) 11.7 (1.26) 13.3 (0.73) 15.3 (2.18) Median female wing length (mm) 2.48 (0.018)a 2.02 (0.064)b 2.05 (0.069)b 2.02 (0.022)a 1.65 (0.051)b 1.60 (0.031)b a 1.082 (0.007)a 0.490 (0.22)ab 0.349 (0.22)b 0 0 0 99 a Multiple comparisons of mean among treatments for O. japonicus were conducted using randomization methods at an experimentwise = 0.05, using the sequential Bonferroni method. 99

PAGE 100

0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 Treatment O. atropalpus Sur vivorshi p O. japonicus ab a b 20:0 60:0 30:30 Survivorship 100 Figure 4-1. Mean survivorship (propo rtion of the original number of larvae surviving to adulthood) of O. japonicus and O. atropalpus (SE). Lower case letters indicate significant differen ces among competition treatments resulting from pairwise comparisons ( p < 0.05) for O. atropalpus Analysis of variance did not indicate significant differences in survivorship among treatments for O. japonicus. 100

PAGE 101

0 5 10 15 20 25 Treatment O. atropalpus D evelopment time ( d O. japonicus a b b 20:0 60:0 30:30 Development time (d) 101 Figure 4-2. Means of median ti me to adulthood for female O. japonicus and O. atropalpus (SE). Lower case letters indicate significant differences among comp etition treatments resulting from pairwise comparisons ( p < 0.05) for O. atropalpus Analysis of variance did not indicate a significant variation in survivorship among treatments for O. japonicus 101

PAGE 102

0 2 4 6 8 10 12 14 16 18 20 Treatment O. atropalpus D evelopment time ( d O. japonicus 20:0 60:0 30:30 Development time (d) 102 Figure 4-3. Means of median time to adulthood for male O. japonicus and O. atropalpus (SE). Analysis of va riance did not indicate significant variations in development time among treatments for males of either species. 102

PAGE 103

0.00 0.50 1.00 1.50 2.00 2.50 3.00 Treatment O. atropalpus O. japonicus aB b B b A Wi ng length (m m Wing length (mm) 103 20:0 60:0 30:30 Figure 4-4. Means of median wing lengths of O. japonicus and O. atropalpus adult females (SE). Lower case and upper case letters indicate significant differences among competition tr eatments resulting from pairwise comparisons (p < 0.05) for O. atropalpus and O. japonicus respectively. 103

PAGE 104

-0.20 0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 Treatment O. atropalpus O. japonicus 20:0 60:0 30:30 AB B A Figure 4-5. Mean estimates of population performance ( , an estimate of the finite rate of increase for the cohort) for female O. japonicus and O. atropalpus adults (SE). Upper case letters indicate si gnificant differences among competition treatments resulting from pair wise comparisons ( p < 0.05) for O. japonicus. Analysis of variance did not indicate a significant difference in survivorship among treatments for O. atropalpus The line at = 1 is where the populat ion is being replaced, neither increasing nor decreasing, and the line at = 0 is where no individuals survive to reproductive age. 104 104

PAGE 105

CHAPTER 5 CONCLUSIONS The establishment of an invasive species in a new region depends on its ability to compete with resident species that occupy a similar ecological niche (Williamson 1996). In mosquito container communities, interactions among invasive and resident species may determine invasion success (OMeara et al. 1995, Juliano et al. 2004) or serve as a barr ier to invasion (Rosen et al. 1976, OMeara et al. 1989). Invasion success and spread may be associated with forms of competition that result in either coexistence with resident species or their decline and elimination (Juliano 1998). Both types of competitive outcomes were observed during the establishment of O. japonicus in Fairfax County, Virginia. The propensity of O. japonicus immatures to inhabit rock po ols was associated with, as predicted, what appears to be the localized decl ine and possible displacem ent of the native rock pool mosquito, O. atropalpus Investigations on the causes of this decline were impeded by both infrequent collections of O. atropalpus from rock pools and stressful conditions used in laboratory experiments to determine the effects of interand intraspecific larval competition on the population performance of O. atropalpus and O. japonicus Although no strong conclusions could be made, results suggest that interspe cific competition between the two species is probable, and likely to favor the success of O. japonicus over O. atropalpus in the resourcelimited conditions expected in rock pool habitats. The autogenous reproduction of O. atropalpus which renders this species more vulnerable to conditions of limited reso urces because nutrient reserves for egg production must be acquired during development (Telang and Wells 2004), may disadvantage this species in larval competition. While rock pools were clearly the preferred habitat of O. japonicus this species was frequently collected from various types of artificial containers as well. Unlike the competitive 105

PAGE 106

exclusion of O. atropalpus that appears to have result ed from the invasion of O. japonicus in rock pools, resident mosquito species, namely A. albopictus and O. triseriatus, appear to coexist with O. japonicus in artificial containers in Fairfax County, Virginia. While O. triseriatus may be a superior larval competitor, the infrequent co llection of this species from artificial containers suggests that its coexistence with O. japonicus in such habitats may be facilitated by speciesspecific differences in habitat pr eference, with a suspected prefer ence of the former species for treeholes. Results of experiment s on interactions of O. japonicus with A. albopictus in artificial containers support the coexistence of these two species in this habita t. Although interand intraspecific effects of larval density on the population performance of these two species in a field experiment indicated A. albopictus to be the superior competitor, the lack of a significant difference between intraand interspecific larval resource competition on the population performance of O. japonicus suggests that the two species should be able to coexist in artificial container habitats in nature. The superior population performance of A. albopictus in the presence of O. japonicus compared to the same larval density of conspecifics suggests that intraspecific competition may be more important than interspecific competition for regulating population growth of this species in container habitats in Vi rginia. This is supported by high larval densities and intras pecific mean crowding of A. albopictus observed in the field, which was much higher than the interspecifi c mean crowding of this species by O. japonicus or by any other co-occurring species in fiel d samples. Because of their potentials as vectors of pathogens, the coexistence of A. albopictus and O. japonicus may have practical c onsequences, thus their involvement in arboviral transmission cycles, such as that of LaCrosse encephalitis virus, should be scrutinized further in areas of co-occurrence. 106

PAGE 107

Species-specific differences in seasonality appe ar to have influenced the interactions of O. japonicus with other species in container communities in Fairfax County, Virginia that resulted in the decline of O. atropalpus and coexistence of this species with A. albopictus. The ability of O. japonicus to overwinter in the larval stage allows this species to resume development earlier in the spring, while other cont ainer-inhabiting aedine species remain in the egg stage. This difference in life hist ory strategies gives O. japonicus a developmental head start over A. albopictus, and the consequent co -occurrence of older O. japonicus with younger A. albopictus larvae may have favored O. japonicus during competition between these species, and allowed it to persist later into the season despite the high abundance of A. albopictus during this time (Figure 2-3). On the other ha nd, the co-occurrence of older O. japonicus larvae with early hatchlings of O. atropalpus has likely exacerbated the asymmetry of interspecific larval resource competition between these two species, which may have contributed to the local decreases in numbers and distribution of th e native rock pool mosquito. The impact of interspecific larval resource competition on O. japonicus and O. atropalpus in rock pools warrants further investigation, but will require more ecologically realistic experimental conditions than thos e used here. Future studies s hould also focus on the role of cohort structure in density-dependent interspecific competition between O. japonicus and resident container-inhabiting mosquitoes, as such research would likely provide more insight into the interspecific interactions among these species as they occur in nature. The roles of other ecological processes in structuring mosquito container communities should also be investigated, particularly the prey preferen ces of predators and the responses of prey species to these predators. Furthermore, the tolerance of these species to varying environmental conditions, particularly temperature, and di fferences in foraging behaviors should be explored. Finally, to 107

PAGE 108

fully appreciate the ecological processes operating in mosquito container communities, and to observe any significant change s that may occur therein following the invasion of O. japonicus continued monitoring of all life st ages of these species over seve ral years would be necessary. 108

PAGE 109

109 APPENDIX A DESCRIPTIVE STATISTICS AND INFORMATION: CHAPTER 2 Table A-1. Descriptive statistics and inform ation for mosquito species collected in a survey of natural and artificial containe r habitats in Fairfax County, Virginia in 2006. Species Container types a No. pos. containers b (%) No. of larvae (%) Mean. no. larvae/container c Mean (SE) water vol. (l) of pos. containers Range of water vol. (l) of pos. containers A. albopictus B, D, F, P, R, R, RH, V, O 91 (47.6) 4437 (51.0) 48.76 (6.92) 1.37 (0.32) 0.01 21.0 A. punctipennis P, R, T, O 7 (3.7) 17 (0.2) 2.43 (0.84) 5.35 (3.8) 0.06 28.0 C. pipiens B, D, F, P, R, T, V, O 51 (26.7) 1073 (12.3) 21.04 (5.47) 2.20 (0.68) 0.02 28.0 C. restuans F, P, R, T, O 36 (18.8) 1198 (14.3) 33.28 (10.01) 1.99 (0.79) 0.06 28.0 O. atropalpus R 4 (2.0) 74 (0.9) 18.5 (15.26) 2.04 (1.04) 0.45 5.0 O. hendersoni T, TH, V, O 10 (5.24) 50 (0.6) 5.0 (1.6) 0.98 (0.29) 0.15 -2.5 O. japonicus B, D, F, P, R, T, TH, O 58 (30.4) 1553 (17.9) 26.78 (5.13) 1.93 (0.58) 0.015 28.0 O. triseriatus P, R, T, TH, V, O 17 (8.9) 203 (2.3) 11.94 (4.93) 0.87 (0.23) 0.03 2.5 O. signifera F 1 (0.5) 28 (0.3) 0.15 T. rutilus F, T, TH, V, O 5 (2.6) 60 (0.7) 12.0 (10.0) 0.63 (0.47) 0.08 2.5 a Container types include birdbaths (B), 55-gall on drums (D), flower pot saucers (F), plas tic such as tarps or garbage bags (P), rock pools (R), tires (T), treeholes (TH), cemetery vases (V), or other miscel laneous artificial containers (O). b Number of and percent positive containers was dete rmined from a total of 191 containers sampled. c Calculations of mean number of larvae per container were based only on positive containers. 109

PAGE 110

APPENDIX B HABITAT COMPARISONS: RANK ORDERS OF MOSQUITO SPECIES Table B-1. Rank orders of immature mosquito abundances used for habitat comparisons of rock pools, tires, small and large artif icial containers. Kendalls coeffi cient of rank correlation, was used for comparisons. Rock pools Tires Small artificial a Large artificial b All artificial No. collected Rank No. collected Rank No. collected Rank No. collected Rank No. collected Rank A. albopictus 242 2 1190 1 2620 1 1553 1 4174 1 A. punctipennis 14 6 3 9 3 9 C. pipiens 169 4 216 3 214 3 590 2 904 3 C. restuans 184 3 488 2 455 2 559 3 1014 2 O. atropalpus 74 5 O. hendersoni 22 6 27 6 14 8 41 7 O. japonicus 1296 1 66 5 167 5 81 5 248 4 O. triseriatus 4 7 140 4 58 4 125 4 183 5 O. signifera 28 7 28 8 T. rutilus 3 7 3 7 3 9 3 9 110 a Small artificial containers include those with less than or equal to 1 l of fluid volume. b Large artificial containers in clude those with more than 1 l of fluid vol ume; this included 12 of 29 tires sampled. 110

PAGE 111

111 LIST OF REFERENCES Aliabadi, B.K. and S.A. Juliano. 2002. Escape form gregarine parasites affects the competitive impact of an invasive mos quito. Biol. Invasions 4: 283-297. Alto, B.W., L.P. Lounibos, S. Higgs and S.A. Juliano. 2005. Larval competition differentially affects arbovirus infections in Aedes mosquitoes. Ecology 86: 3279-3288. Andreadis, T.G., J.F. Anderson, L.E. Munstermann, R.J. Wolfe and D.A. Florin. 2001. Discovery, distribution, and abundance of the newly introduced mosquito Ochlerotatus japonicus (Diptera: Culicidae) in Connecticut USA. J. Med. Entomol. 38: 774-779. Apperson, C.H. B.A. Harrison, T.R. Unnasch, H.K. Hassan, W.S. Irby, H.M. Savage, S.E. Aspen, D.W. Watson, L.M. Rueda, B.R. Engber and R.S. Nasci. 2002. Host-feeding habits of Culex and other mosquitoes (Diptera: Culicidae) in the borough of Queens in New York City, with characters and tec hniques for identification of Culex mosquitoes. J. Med. Entomol. 39: 777-785. Barrera, R. 1996. Competition and resistance to starvation in la rvae of container-inhabiting Aedes mosquitoes. Ecol. Entomol. 21: 117-127. Beach, R.F. and G.B. Craig. 1979. Photoinhibition of diapause in field populations of Aedes atropalpus Environ. Entomol. 8: 392-396. Bdhomme, S., and P. Agnew, C. Sidbore and Y. Michalakis. 2005. Pollution by conspecifics as a component of intraspecific competition among Aedes aegypti larvae. Ecol. Entomol. 30: 1-7. Beier, J.C., W.J. Berry and G.B. Crai g. 1982. Horizontal distribution of adult Aedes triseriatus (Diptera: Culicidae) in relation to habitat stru cture, oviposition, and other mosquito species. J. Med. Entomol. 19: 239-247. Bertness, M.D. 1984. Habitat and community modification by an introduced herbivorous snail. Ecology 65: 370-381. Blackmore, M.S., G.A. Scoles and G.B. Craig. 1995. Parasitism of Aedes aegypti and Ae. albopictus (Diptera: Culicidae) by Ascogregarina spp. (Apiomplexa: Lecudinidae) in Florida. J. Med. Entomol. 32: 847-852. Braks, M.A.H., N.A. Honrio, L.P. Lounibos, R. Loureno-de-Oliveira and S.A. Juliano. 2004. Interspecific competition between two invasive species of container mosquitoes in Brazil. Ann. Entomol. Soc. Am. 97: 130-139. Brooks, J.L. and S.I. Dodson. 1965. Predation, body size, and composition of the plankton. Science 150: 28-35.

PAGE 112

Caldwell, N.D., R.R. Gerhardt and C. J. Jones. 2005. First collection of Ochlerotatus japonicus japonicus in the state of Tennessee. J. Am. Mosq. Control Assoc. 21: 322-324. Chagin, K.P. and P.I. Kondratiev. 1943. Vectors of autumnal (Japanese) encephalitis in Soviet Far East and their Control. Med. Parasit. And Parasitic Dis. 12: 34-44. Christophers, S.R. 1960. Aedes aegypti, the yellow fever mosquito Cambridge University Press, Cambridge., UK. 739 pp. Clark, G.G. and G.B. Craig. 1985. Oviposition behavior of Aedes triseriatus and Aedes hendersoni on the Delmarva Peninsula. J. Am. Mosq. Control Assoc. 1: 526-528. Coquillett, D.W. 1902. Three new species of Culex Can. Entomol. 34: 292-293. Costanzo, K.S., K. Mormann and S.A. Juliano. 2005. Asymmetrical competition and patterns of abundance of Aedes albopictus and Culex pipiens (Diptera: Culicidae). J. Med. Entomol. 42: 559-572. Craven, R.B., D.A. Eliason, D.B. Francy, P. Reiter, E.G. Campos, W.L. Jakob, G.C. Smith, C.J. Bozzik, G.G. Moore, G.O. Maupin a nd T.P. Monath. 1988. Importation of Aedes albopictus and other exotic mosquito species into the Un ited States in used tires from Asia. J. Am. Mosq. Control Assoc. 4: 138-142. Daehler, C.C. 2001. Two ways to be an invader, but one is more suitable for ecology. Bull. Ecol. Soc. Am 81: 101-2. Darsie, R.F. and R.A. Ward. 2005. Identification and Geographica l Distribution of the Mosquitoes of North America, North of Mexico Univ. Press of Florida, Gainesville, FL. USA. 383 pp. 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). J. Med. Entomol. 37: 364-372. Edgerly, J.S. and T.P. Livdahl. 1992. Density-depende nt interactions within a complex life cycle: the roles of cohort structure and mode of recruitment. J. Anim. Ecol. 61: 139-150. Edgerly, J.S., M.S. Willey and T. Livdahl. 1993. The community ecology of Aedes hatching: implications for a mosquito in vasion. Ecol. Entomol. 18: 123-128. Edgerly, J.S., M.S. Willey and T. Livdahl. 1999. Intr aguild predation among treehole mosquitoes Aedes albopictus Ae. aegypti and Ae. triseriatus (Diptera: Culicidae), in laboratory microcosms. J. Med. Entomol. 36: 394-399. Effler, P.W., L. Pang, P. Kitsutani, V. Vorndam, M. Nakata, T. Ayers, J. Elm, T. Tom, P. Reiter, J.G. Rigau-Perez, J.M. Hayes, K. Mills, M. Napier, G..G. Clark and D.J. Gubler for the 112

PAGE 113

Hawaii Dengue Outbreak Investigation Team. Dengue Fever, Hawaii, 2001-2002. 2005 Emerg. Infect. Dis. 11: 742-749. Focks, D.A., S.B. Linda, G.B. Crai g, W.A. Hawley and C.B. Pumpuni. 1994. Aedes albopictus (Diptera: Culicidae) a statis tical model of the role of temperature, photoperiod, and geography in the induction of egg diapause. J. Med. Entomol. 31: 278-286. Fonseca, D.M. S. Campbell, W.J. Crans, M. Mogi, I. Miyagi, T. Toma, M. Bullians, T.G. Andreadis, R.L. Berry, B. Pagac, M.R. Sardelis and R.C. Wilkerson. 2001. Aedes ( Finlaya ) japonicus (Diptera: Culicidae), a ne wly recognized mosquito in the United States: analyses of genetic variation in the United States and putative source populations. J. Med. Entomol. 38: 135-146. Foss, A.F. and R.G. Dearborn. 2001. Preliminary faunistic survey of mosquito species (Diptera: Culicidae) with a focus on population densitie s and potential breedi ng sites in greater Portland, Maine. Technical Report No. 42. Maine Depart ment of Conservation, Maine Forest Service, Forest Health and Monito ring Division, Augusta, Maine. 36 pp. Fox, G.A. 1993. Failure-time analysis: emergence, flowering, survivorship, and other waiting times. In: S.M. Scheiner and J. Gurevitch (eds.), Design and Analysis of Ecological Experiments pp. 253-289. Chapman and Hall, NY. Gallitano, S., L. Blaustein and J. Vonesh. 2006. First occurrence of Ochlerotatus japonicus in Missouri. J. Vector Ecol. 30: 347-348. Gerhardt, R.R., K.L. Gottfried, C.S. Apperson, B.S. Davis, P.C. Erwin, A.B. Smith, N.A. Panella, E.E. Powell and R.S. Nasci. 2001. First isolation of LaCrosse virus from naturally infected Aedes albopictus Emerg. Infect. Dis. 7: 807-811. Ghent, A.W. 1963. Kendalls tau coefficient as an index of similarity in comparisons of plant or animal communities. Can. Entomol. 95: 568-575. Godsey, M.S., M.S. Blackmore, N.A. Panella, K. Burkhalter, K. Gottfried, L.A. Halsey, R. Rutledge, S.A. Langevin, R. Gates, K.M. Lamonte, A.M. Lambert, R.S. Lanciotti, C.G.M. Blackmore, T. Loyless, L. Stark, R. Oliveri, L. Conti and N. Komar. 2005. West Nile virus epizootiology in the southeastern United States, 2001. Vector-Borne Zoonot. 5: 82-89. Graham, A.C. and J. Turmel. 2001. Distribution records of Vermonts first introduced mosquito species, Ochlerotatus japonicus (Diptera: Culicidae). In: Proceedings of the 47 th Annual Meeting of the Northeastern Mosquito Control Association December 3-5, 2001, Salem, MA. Grill, C.P. and S.A. Juliano. 1996. Predicting species interactions based on behaviour: predation and competition in container-dwelling mo squitoes. J. Anim. Ecol. 65: 63-76. 113

PAGE 114

Griswold, M.W. and L.P. Louni bos. 2005. Does differential predation permit invasive and native mosquito larvae to coexist in Florida? Ecol. Entomol. 30: 122-127. Griswold, M.W. and L.P. Lounibos. 2006. Predator identity and additive effects in a treehole community. Ecology 87: 987-995. Gubler, D.J. 1997. Dengue and dengue hemorrhagic fe ver: its history and re surgence as a global public health problem. In: D.J. Gubler and G. Kuno (eds.), Dengue and Dengue Hemorrhagic Fever pp 1-22. CABI International, NY. Haddow, A.J. 1960. Studies on the biting habits and medical importance of East African mosquitoes in the genus Aedes. I. Subgenera Aedimorphus, Banksinella and Dunnius B. Entomol Res. 50: 759-779. Harrison, B.A., P.B. Whitt, S.E. Cope, G.R. Payn e, S.E. Rankin, L.J. Bohn, F.M. Stell and C.J. Neely. 2002. Mosquitoes (Diptera: Culicidae) colle cted near the Great Dismal Swamp: new state records, notes on certain species, and a revised checklist for Virginia. Proc. Entomol. Soc. Wash. 104: 655-662. Hawley, W.A. 1988. The biology of Aedes albopictus J. Am. Mosq. Control Assoc. 4 (Suppl.): 1-40. Hawley, W.A., C.B. Pumpuni, R.H. Brady and G.B. Craig. 1987. Aedes albopictus in North America: probable introduction in used ti res from Northern Asia. Science 236: 1114-1116. Heardon, M., C. Skelly and P. Weinstein. 1999. Impr oving the surveillance of mosquitoes with disease-vector potential in New Zealand. NA Public Health Rep. 6: 25-28. Hedeen, R.A. 1953. The biology of the mosquito Aedes atropalpus Coq. J. Kansas Entomol. Soc. 26: 1-10. Ho, B.C., K.L. Chan and Y.C. Chan. 1973. The biology and bionomics of Aedes albopictus (Skuse). In: Y.C. Chan, K.L. Chan, and B.C. Ho (eds.), Vector Control in Southeast Asia pp. 125-143. Proceedings, 1 st Southeast Asian Ministers for Education Organization Workshop, Singapore. 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. J. Med. Entomol. 26: 615-623. Holick, J., A. Kyle, W. Ferraro, R.R. Delaney and M. Iwaseczko. 2002. Discovery of Aedes albopictus infected with West Nile virus in south eastern Pennsylvania. J. Am. Mosq. Control Assoc. 18: 131. Holt, R.D. and J.H. Lawton. 1994. The ecological c onsequences of shared natural enemies. Ann. Rev. Ecol. Syst. 25: 495-520. 114

PAGE 115

Hurlbert, S.H. 1969. A coefficient of in terspecific association. Ecology 50: 1-9. Ibaez-Bernal, S., B. Briseo, J.-P. Mutebi, E. Argot, G. Rodriguez, C. Martinez-Campos, R. Paz, P. de la Fuente-San Roman, R. Tapia-Conyer and A. Flisser. First record in America of Aedes albopictus naturally infected with dengue viru s during the 1995 outbreak at Reynosa, Mexico. Med. Vet. Entomol. 11: 305-309. James, H.G. 1964a. Insect a nd other fauna associated w ith the rock pool mosquito Aedes atropalpus (Coq.). Mosq. News 24: 325-329. James, H.G. 1964b. Predators of Aedes atropalpus (Coq.) (Diptera: Culicidae) and of other mosquitoes breeding in rock pools in Ontario. Can. J. Zoolog. 43: 155-159. Juliano, S.A. 1998. Species introduction and replacement among mosquitoes: interspecific resource competition or apparent competition? Ecology 79: 255-268. Juliano, S.A. and L.P. Lounibos. 2005. Ecology of invasive mosquitoes: effects on resident species and on human healt h. Ecol. Lett. 8: 558-574. Juliano, S.A., G.F. OMeara, J.R. Morrill a nd M.M. Cutwa. 2002. Desiccation and thermal tolerance of eggs and the coexistence of competing mosquitoes. Oecologia 130: 458-469. 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? O ecologia 139: 583-593. Kamimura, K. 1976. On the Japanese species of the family Culicidae. In: M. Sasa (ed.), Science of Mosquitoes (Japanese). pp 150-188. Hokuryukan, Tokyo, Japan. Kaufman, M.G., S.N. Bland, M.E. Worthen, E.D. Walker and M.J. Klug. 2001. Bacterial productivity and fungal biomass responses to feeding by larval Aedes triseriatus (Say) (Diptera: Culicidae). J. Med. Entomol. 38: 711-718. Kesavaraju, B. and S.A. Juliano. 2004. Differential behavioral responses to water-borne cues to predation in two container dwelling mosquitoes. Ann. Ento mol. Soc. Am. 97: 194-201. Kitching, R.L. 2000. Food Webs and Container Habitats: the Natural History and Ecology of Phytotelmata Cambridge University Press, NY. 431 pp. Kitron, U.D., D.W. Webb and R.J. Novak. 1989. Oviposition behavior of Aedes triseriatus (Diptera: Culicidae): prevalence, intensity, and ag gregation of eggs in oviposition traps. J. Med. Entomol. 26: 462-467. 115

PAGE 116

Knight, K.L. 1968. Contributions to the mosquito fauna of Southeast Asia. IV, Species of the subgroup Chrysolineatus of Group D, Genus Aedes, Subgenus Finlaya Theobald. Contrib. Amer. Entomol. Inst. 2(5): 1-45. Koenekoop, R.K. and T.P. Li vdahl. 1986. Cannibalism among Aedes triseriatus larvae. Ecol. Entomol. 11: 111-114. Koenraadt, D.J.M and W. Takken. 2003. Canniba lism and predation among larvae of the Anopheles gambiae complex. Med. Vet. Entomol. 17: 61-66. Koenraadt, C.J.M., S. Majambere, L. Heme rik and W. Takken. 2004. The effects of food and space on the occurrence of cannibalism and predation among larvae of Anopheles gambiae s.l Entomol. Exp. Appl. 112: 125-134. Kramer, L.D. and K.A. Bernard. 2001. West Nile virus in the western hemisphere. Curr. Opin. Infect. Dis. 14: 519-525. LaCasse, W.J. and S. Yamaguti. 1950. Mosquito Fauna of Japan and Korea. Parts I and II. Mosquito Survey data on Japan and their applica tion in the control of mosquito-borne diseases. Off. Surg., HQ 1 Corps APO 301 (Japan). Laird, M., L. Calder, R.C. Thornton, R. Syme P.W. Holder and M. Mogi. 1994. Japanese Aedes albopictus among four mosquito species reaching Ne w Zealand in used tires. J. Am. Mosq. Control Assoc. 10: 14-23. Linthicum, K.J., V.L. Kramer, M.B. Madon, K. Fujioka and the Surv eillance-Control Team. 2003. Introduction and potential establishment of Aedes albopictus in California in 2001. J. Am. Mosq. Control Assoc. 19: 301-308. Livdahl, T.P. 1982. Competition within and betw een hatching cohorts of a treehole mosquito. Ecology 63: 125-136. 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. Lloyd, M. 1967. Mean Crowding. J. Anim. Ecol. 36: 1-20. Lounibos, L.P. 1981. Habitat segregation among Afri can treehole mosquitoes. Ecol. Entomol. 6: 129-154. Lounibos, L.P. 2002. Invasions by insect vectors of human disease. Ann. Rev. Entomol. 47: 233266. Lounibos, L.P., N. Nishimura and R.L. Escher. 1 993. Fitness of a tree hole mosquito: influences of food type and predation. Oikos 66: 114-118. 116

PAGE 117

Lounibos, L.P., G.F. OMeara, R.L. Escher, N. Nishimura, M. Cutwa, T. Nelson, R.E. Campos and S.A. Juliano. 2001. Testing predictions of displacement of native Aedes by the invasive Asian tiger mosquito Aedes albopictus in Florida, USA. Biol. Invasions 3: 151-166. Lounibos, L.P., G.F. OMeara, N. Nishimura, and R.L. Escher. 2003. Interactions with native mosquito larvae regula te the production of Aedes albopictus from bromeliads in Florida. Ecol. Entomol. 28: 551-558. Lounibos, L.P., S. Surez, Z. Mendez, N. Ni shimura, R.L. Escher, S.M. OConnell and J.R. Rey. 2002. Does temperature affect the outcome of larval competition between Aedes aegypti and Aedes albopictus ? J. Vector Ecol. 27: 86-95. Maire, A. and R. Langis. 1985. Oviposition responses of Aedes ( Ochlerotatus) communis (Diptera: Culicidae) to la rval holding water. J. Med. Entomol. 22: 111-112. Manly, B.F.J. 1991. Randomization and Monte Carlo Methods in Biology Chapman and Hall, London. 292 pp. Manly, B.F.J. 1997. RT: a program for randomizati on testing. Version 2. 1. West Incorporated, Cheyenne, WY. Merritt, R.W., R.H. Dadd and E.D. Walker. 1992. Feeding behavior, natural food, and nutritional relationships of larval mosquitoes. Ann. Rev. Entomol. 37: 349-376. Mitchell, C.J., M.L. Niebylski, N. Karabatsos, D. Martin, J.-P. Mutebi, G.B. Craig and M.J. Mahler. 1992. Isolation of easte rn equine encephalitis from Aedes albopictus in Florida. Science 257: 526-527. Miyagi, I. 1971. Notes on the Aedes (Finlaya) chrysolineatus subgroup in Japan and Korea (Diptera: Culicidae). Trop Med. 13: 141-151. Moore, C.G. 1999. Aedes albopictus in the United States: current status and prospects for further spread. J. Am. Mosq. Control Assoc. 15: 221-227. Moore, C.G. 2005. Exotic and Invasive Vectors of Public Health Importance in the United States. Database. Arthropod-bor ne and Infectious Diseases Laboratory and Dept. of Environmental and Radiological Health Scien ces, Colorado State University, Fort Collins, CO. Munstermann, L.E. and D.M. Wesson. 1990. First record of Ascogregarina taiwanensis (Apiomplexa: Lecudinidae) in North American Aedes albopictus J. Am. Mosq. Control Assoc. 6: 235-243. Nakata, G. 1962. Taxonomical and ecological studies on Japanese mosquitoes. Sanitary Injurious Insects 6: 43-173. 117

PAGE 118

Nasci, R.S., S.G. Hare and F.S. Willis. 1989. Inte rspecific mating between Louisiana strains of Aedes albopictus and Aedes aegypti in the field and laboratory. J. Am. Mosq. Control Assoc. 5: 416-421. Nawrocki, S.J. and W.A. Hawley. 1987. Estimation of the northern limits of distribution of Aedes albopictus in North America. J. Am. Mosq. Control Assoc. 3: 314-317. Novak, R.J. and D.A. Shroyer. 1978. Eggs of Aedes triseriatus and A. hendersoni : a method to stimulate optimal hatch. Mosq. News 38: 515-521. Oliver, J., R.G. Means and J.J. Howard. 2003. Geographica l distribution of Ochlerotatus japonicus in New York State. J. Am. Mosq. Control Assoc. 19: 121-124. OMeara, G.F. and G.B. Crai g. 1970. Geographical variation in Aedes atropalpus (Diptera: Culicidae). Ann. Entomol. Soc. Am. 63: 1392-1400. OMeara, G.F. and G.J. Krasnick. 1970. Dietary and genetic control of the expression of autogenous reproduction in Aedes atropalpus (Coq.) (Diptera: Culicidae). J. Med. Entomol. 7: 328-334. OMeara, G.F., L.F. Evans, Jr. and M.L. Womack. 1997. Colonization of rock holes by Aedes albopictus in the southeastern United States. J. Am. Mosq. Control Assoc. 13: 270-274. OMeara, G.F., V.L. Larson, D.H. Mook and M.D. Latham. 1989. Aedes bahamensis : its invasion of south Florida and association with Aedes aegypti. J. Am. Mosq. Control Assoc. 5: 1-5. OMeara, G.F., L.F. Evans, A.D. Gettman and J.P. Cuda. 1995. Spread of Aedes albopictus and decline of Ae. aegypti (Diptera: Culicidae) in Florid a. J. Med. Entomol. 32: 554-562. Packer, M.J. and P.S. Corbet. 1989. Size vari ation and reproductive success of female Aedes punctor (Diptera: Culicidae). Ecol. Entomol. 1: 297-309. Peyton, E.L., S.R. Campbell, T.M. Candeletti, M. Romanowski and W.J. Crans. 1999. Aedes ( Finlaya ) japonicus japonicus (Theobald), a new introduction in to the United States. J. Am. Mosq. Control Assoc. 15: 238-241. Pielou, E.C. 1977. Mathematical Ecology. John Wiley and Sons, New York, NY. 385 pp. Qualls, W.A. and G.R. Mullen. 2006. Larval survey of tire-breeding mosquitoes in Alabama. J. Am. Mosq. Control Assoc. 22: 601-608. Rathcke, B.J. 1976. Competition and coexistence w ithin a guild of herbi vorous insects. Ecology 57: 76-87. 118

PAGE 119

Reeves, W.K. and J.A. Korecki. 2004. Ochlerotatus japonicus japonicus (Theobald) (Diptera: Culicidae), a new invasive mosquito for Geor gia and South Carolina. Proc. Entomol. Soc. Wash. 106: 233-234. Reinert, J.F. 2000 new classifi cation for the composite genus Aedes (Diptera: Culicidae: Aedini), elevation of subgenus Ochlerotatus to generic rank, reclassifica tion of the other subgenera, and notes on certain subgen era and species. J. Am. Mosq. Control Assoc. 16: 175-188. Reiter, P. 1983. A portable, battery-po wered trap for collection of gravid Culex mosquitoes. Mosq. News 43: 496-498. Reiter, P. and D. Sprenger. 1987. The used tire trade: a mechanism for the worldwide dispersal of container breeding mosquitoes. J. Am .Mosq. Control Assoc. 3: 494-501. Rhymer, J. and D. Simberloff. 1996. Extinction by hybridization and introgression. Ann. Rev. Ecol. Syst. 27: 83-109. Ribiero, J.M.C. and A. Spielman. 1986. The saty r effect: a model predicting parapatry and species extinction. Am. Nat. 128: 513-528. Rice, W.R. 1989. Analyzing tables of statistical tests. Evolution 43: 223-225. Richardson, D.M., W.J. Bond, W.R.J. Dean, S.I. Higgins, G.F. Midgely, S.J. Milton, L.W. Powrie, M.C. Rutherford, M.J. Samways and R.E. Schulze. 2000. Invasive alien species and global change: a South African perspective. In: H.A. Mooney and R.J. Hobbs (eds.), Invasive species in a changing world. pp. 303-349. Island Press, Washington DC. Roppo, M.R., J.L. Lilya, F.A. Maloney a nd W.J. Sames. 2004. First occurrence of Ochlerotatus japonicus in the state of Wash ington. J. Am. Mosq. Control Assoc. 20: 83-84. Rosen, L., L.E. Rozeboom, W.C. Reeves, J. Saug rain and D.J. Gubler. 1976. A field trial of competitive displacement of Aedes polynesiensis by Aedes albopictus on a Pacific atoll. Am. J. Trop. Med. Hyg. 25: 906-913. Ross, R. 1911. The Prevention of Malaria John Murray, London. 651 pp. Ruesink, J.L., I.M. Parker, M.J. Groom and P.M. Kareiva. 1995. Reducing the risks of nonindigenous species introduc tions. BioScience 45: 465-477. Sakai, A.K., F.W. Allendorf, J.S. Holt, D.M. Lodge and J. Molofsky. 2001. The population biology of invasive species. A nn. Rev. Ecol. Syst. 32: 305-332. Sardelis, M.R. and M.J. Turell. 2001. Ochlerotatus j. japonicus in Frederick County, Maryland: discovery, distribution, and vect or competence for West Nile virus. J. Am. Mosq. Control Assoc. 17: 137-141. 119

PAGE 120

Sardelis, M.R., J.D. Dohm, B. Pagac, R.G. Andre and M.J. Turell. 2002a. Experimental transmission of eastern e quine encephalitis virus by Ochlerotatus j. japonicus (Diptera: Culicidae). J. Med. Entomol. 39: 480-484. Sardelis, M.R., M.J. Turell and R.G. Andre. 2002b. Laboratory transmission of LaCrosse virus by Ochlerotatus j. japonicus (Diptera: Culicidae). J. Med. Entomol. 39: 635-639. Sardelis, M.R. M.J. Turell and R.G. Andre. 2003. Experimental transmission of St. Louis encephalitis virus by Ochlerotatus j. japonicus J. Am. Mosq. Control Assoc. 19: 159-162. SAS Institute Inc. 1989. SAS/STAT Users Guide SAS Institute, Cary, NC. Sasa, M. and K. Kamimura. 1971. Index and co nsideration on taxonomy of the Japanese Mosquitoes. In: M Sasa (ed.), Progress in Medical Zoology I (Japanese). p. 1-47 Gakutsusho Shuppankai, Tokyo. Sasa, M., Y. Nakahara, N. Ushiroku, H. Hashimoto, A. Uno, T. Ogino, T. Miyachi, F. Yokomizo, S. Koyama, A. Akagi, K. Yamaguc hi, C. Saito and H. Kumazawa. 1947. Studies on mosquitoes (7). Species of lowlands and m ountains, observations in the Okayama District. Med. Biol. (Japanese) 11: 152-154. Savignac, R., C. Back and J. Bourassa. 2002. Biological Notes on Ochlerotatus japonicus and other mosquito species new to Quebec. Abstract. In: The Abstract Book of A Joint Meeting: 68 th Annual Meeting of the American Mosquito Control Association and the West Central Mosquito & Vector Control Association pp 21-22. February 16-21, 2002, Denver, CO. American Mosquito Control Association, Eatontown, NJ. Schaffner, F., S. Chouin and J. Guilloteau. 2003. Fi rst record of Ochlerotatus (Finlaya) japonicus japonicus (Theobald, 1901) in metropolitan France. J. Am. Mosq. Control Assoc. 19: 1-5. Scott, J.J. 2003. The ecology of the exotic mosquito Ochlerotatus ( Finlaya) japonicus japonicus (Theobald 1901) (Diptera: Culicid ae) and an examination of its role in the We st Nile virus cycle in New Jersey. Ph.D. Dissertation. Rutger s, The State University of New Jersey, New Brunswick, NJ. Scott, J.J. and W.J. Crans. 2003. Expanded Polystyrene (EPS) floats for Ochlerotatus japonicus (Theobald) surveillance. J. Am. Mosq. Control Assoc. 19: 376-381. Scott, J.J., F.L. Carle and W.J. Crans. 2001a. Ochlerotatus japonicus collected from natural rockpools in New Jersey. J. Am. Mosq. Control Assoc. 17: 91-92. Scott, J.J., S.C. Crans and W.J. Crans. 2001b. Use of an infusion-baited gr avid trap to collect adult Ochlerotatus japonicus. J. Am. Mosq. Control Assoc. 17: 142-143. 120

PAGE 121

Shaw, F.R. and S.A. Maisey. 1961. The biology and distribution of the rockpool mosquito, Aedes atropalpus (Coq.). Mosq. News 24: 12-16. Scholl, P.J. and G.R. DeFoliart. 1977. Aedes triseriatus and Aedes hendersoni : vertical and temporal distribution measured by ov iposition. Environ. Entomol. 6: 355-38. Simberloff, D. 1996. Impacts of introduced specie s in the United States. Consequences: Nat. Implic. Environ. Change 2: 13-22. Sinsko, M.U. and P.R. Grimstad. 1977. Habitat sepa ration by differential vertical oviposition of two treehole Aedes in Indiana. Environ. Entomol. 6: 485-487. Slaff, M. and C.S. Apperson. 1989. A key to th e mosquitoes of North Carolina and the MidAtlantic states. N. C. State Univ. Agric. Ext. Serv. Publ. AG. 412: 1-38. Sokal, R.R. and F.J. Rohlf. 1981 Biometry, 2 nd ed. W.H. Freeman and Company, San Francisco, CA. 859 pp. Soper, F.L. and D.B. Wilson. 1943. Anopheles gambiae in Brazil 1930 1940 The Rockefeller Foundation, NY. 262 pp. Sprenger, D. and T. Wuithiranyagool. 1986. The discovery an d distribution of Aedes albopictus in Harris County, Texas, USA. J. Am Mosq. Control Assoc. 2: 217-219. SPSS or Macintosh, Rel. 11.0 2002. SPSS Inc., Chicago, IL. Steinly, B.A., R.J. Novak and D.W. Webb. 1991. A new method for monitoring mosquito oviposition in artificial and natural containers. J. Am. Mosq. Control Assoc. 7: 649-650. Sucharit, S., K. Surathin and S.R. Shrestha. 1989. Vectors of Japanese encephalitis virus (JEV): species complexes of the vectors. Southeast Asian J. Trop. Med. Public Health 20: 611-621. Sucharit, S., W. Tumrasvin, S. Vutikes and S. Viraboonchai. 1978. Interacti on between larvae of Aedes aegypti and Aedes albopictus in mixed experimental popul ations. Southeast Asian J. Trop. Med. Publ. Health 9: 93-97. Sunahara, T. and M. Mogi. 2002. Priority effects of bamboo-stump mosquito larvae: influences of water exchange and leaf litter input. Ecol. Entomol. 27: 346-354. Tabachnick, W.J. 1991. Evolutionary genetics a nd arthropod-borne disease. The Yellow fever mosquito. Am. Entomol. 37: 14-24. Takashima, I. and N. Hashimoto. 1985. Getah virus in several species of mosquitoes. Trans. R. Soc. Trop. Med. Hyg. 79: 546-550. Takashima, I. and L. Rosen. 1989. Horizontal and vertical transmission of Japanese encephalitis virus by Aedes japonicus (Diptera: Culicidae). J. Med. Entomol. 26: 454-458. 121

PAGE 122

Tanaka, K., K. Mizusawa and E.S. Saugstad. 1979. A revision of the adult and larval mosquitoes of Japan (including the Ryukyu Ar chipelago and the Ogasawara Islands) and Korea (Diptera: Culicidae). Contrib. Am. Entomol. Inst. 16(ii-vii): 1-987. Telang, A. and M.A. Wells. 2004. The effect of larval and adult nutrition on successful autogenous egg production by a mosquito. J. Insect Phys. 50: 677-685. Teng, H.J. and C.S. Apperson. 2000. Development and survival of immature Aedes albopictus and Aedes triseriatus (Diptera: Culicidae) in the laborat ory: effects of density, food, and competition on response to temperat ures. J. Med. Entomol. 37: 40-52. Theobald, F.V. 1901. A Monograph of the Culicidae of the World. Vol. I British Museum of Natural History, London. Thielman, A. and F.F. Hunter. 2006. Establishment of Ochlerotatus japonicus (Diptera: Culicidae) in Ontario, Canada. J. Med. Entomol. 43: 138-142. Trpis, M. 1970. A new bleaching and decalcifyin g method for general use in zoology. Can. J. Zoolog. 48: 892-893. Tsai, T.W. and J.C. Lien. 1950. A new species of Aedes ( Finlaya) found in Taiwan. J. Med. Assn. Formosa 49: 177-183. Turell, M.J., M.L. OGuinn, D.J. Dohm and J. W. Jones. 2001. Vector competence of North American mosquitoes (Diptera: Culicidae) fo r West Nile virus. J. Med. Entomol. 38: 130134. Walker, E.D., D.L. Lawson, R.W. Merritt, W.T. Morgan and M.J. Klug. 1991. Nutrient dynamics, bacterial populations, and mosquito productivity in tree hole ecosystems and microcosms. Ecology 72: 1529-1546. Walker, E.D., M.G. Kaufman, M.P. Ayres, M. H. Riedel and R.W. Merritt. 1997. Effects of variation in quality of leaf detritus on growth of the eastern tree-hole mosquito, Aedes triseriatus (Diptera: Culicidae). Can. J. Zool. 75: 706-718. Werner, B.G. 2001. Arbovirus Surveillance and Testing in Massachusetts, 2001. In: Proceedings of the 47 th Annual Meeting of the Northeastern Mosquito Control Association December 3-5, 2001, Salem, MA. White, D.J., L.D. Kramer, P.B. Backenson, G. Lukacik, G. Johnson, J. Oliver, J.J. Howard, R.G. Means, M. Eidson, I. Gotham, V. Kulaseke rea, S. Campbell, the Arbovirus Research Laboratory and the Statewide West Nile Viru s Response Teams. 2001. Mosquito surveillance and polymerase chain reaction detection of West Nile virus, New York State. Emerg. Infect. Dis. 7: 643-649. 122

PAGE 123

Widdel, A.K., L.J. McCuiston, W.J. Crans, L.D. Kramer and D.M. Fonseca. 2005. Finding needles in the haystack: single copy microsatellite loci for Aedes japonicus (Diptera: Culicidae). Am. J. Troop. Med. Hyg. 73: 744-748. Williams, C.B. 1937. The use of logarithms in the interpretation of certain entomological problems. Ann. Appl. Biol. 24: 404-414. Williamson, M. 1996. Biological Invasions Chapman and Hall, New York, NY. 256 pp. Yee, D.A., B. Kesavaraju and S.A. Juliano. 2004. Interspecific differences in feeding behavior and survival under food-limited conditions for larval Aedes albopictus and Aedes aegypti (Diptera: Culicidae). Ann. En tomol. Soc. Am. 97: 720-728. Young, C.L.E., J.A. Beery, R.E. Sheffe r and K.M. Rand. 2004. First record of Ochlerotatus japonicus (Diptera: Culicidae) in St. Joseph Count y, Indiana. Great Lakes Entomologist 37: 196-197. Zahiri, N., M.E. Rau and D.J. Lewis. 1997. Oviposition responses of Aedes aegypti and Ae. atropalpus (Diptera: Culicidae) females to waters from conspecific and heterospecific normal larvae and from larvae infected with Plagiorchis elegans (Trematoda: Plagiorchiidae). J. Med. Entomol. 34: 565-568. 123

PAGE 124

BIOGRAPHICAL SKETCH Jennifer Star Armistead was born in Cocoa Beac h, Florida, the second of three children. As the child of a military officer, she spent her child hood in various parts of the United States until her father retired and settle d in Arlington, Virginia. Shortl y after, Jennifer began her undergraduate degree at George Mason University where she studied biology. During this time, she also developed a passion for entomology, wh ich led her to study th e population ecology of butterflies on Andros Island, the Bahamas for her senior research project. Shortly after graduation, Jennifer was married to her now-husband of nearly five years, Paul Johnson. She workws as a biologist for the Disease Carry ing Insects Program of the Fairfax County Department of Health in Virginia, where she was involved in the surveillance and management of mosquitoes, ticks, and the dis eases these arthropods vector. It wa s from her experiences in this position that she decided to pursue a master of sc ience degree in entomology at the University of Florida. As a distance education student, Jennif er was able to continue her employment while taking Web-based courses and conducting her thesis research in Virginia Her graduate studies have emphasized medical entomology, vect or ecology, and inva sion biology. Upon her graduation, Jennifer will matriculat e at the Bloomberg School of Public Health at the Johns Hopkins University in Baltimore, Maryland to co mplete her graduate studies in the field of public health with a focus on vector-borne diseases. 124


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 E20101112_AAAADS INGEST_TIME 2010-11-12T19:02:43Z PACKAGE UFE0020148_00001
AGREEMENT_INFO ACCOUNT UF PROJECT UFDC
FILES
FILE SIZE 34288 DFID F20101112_AACJKB ORIGIN DEPOSITOR PATH armistead_j_Page_095.QC.jpg GLOBAL false PRESERVATION BIT MESSAGE_DIGEST ALGORITHM MD5
d40de5ae31961211c76ef2d7ae2a0bdc
SHA-1
255d1cedc338a3a00a39bf9c386f50a4898a79d7
36404 F20101112_AACJJM armistead_j_Page_086.QC.jpg
d05f3d53439c930048aa0c4d48b3c25a
ccae2fb663c3d32bd6822e7cacbc33e2bb8d417e
16809 F20101112_AACJIY armistead_j_Page_078.QC.jpg
7db3b6e07832ac7f1724562093e6df6f
fcf44054f495b659f0729ba65a69b57b050d8c38
8667 F20101112_AACIFW armistead_j_Page_012thm.jpg
90daa6dcd323805841de2ce307dcac70
be7a34998388831bcdf47dfdd2627d7eb85eddf4
145535 F20101112_AACIGK UFE0020148_00001.mets FULL
1dcaa8bb4307aa342e26417683c3ba6f
0ac87d5694a1aeec9287376b215f836e088593a6
8477 F20101112_AACJKC armistead_j_Page_095thm.jpg
0f4cc5481f93b9b8fdc15b4cba74ccff
8a00679387a777ecac6afa059dfa21f606c7c05a
9010 F20101112_AACJJN armistead_j_Page_086thm.jpg
2ac4e788e8971b3f77fb5ec848f2b5a0
9ac9f70406f6620d4d4d4821dc06a67f013f3aad
13002 F20101112_AACJIZ armistead_j_Page_079.QC.jpg
f8abd3c27295981341a539fb1ca792fe
13d52dd103d2905b805e8c1be6d7b54b448179cc
105350 F20101112_AACIFX armistead_j_Page_092.jp2
83aad1e7864cddc87f5d1d1904e54e20
7ed597875d40c2971d4278d9d0b97a3dc2bb8611
111655 F20101112_AACIHA armistead_j_Page_014.jpg
42364562e12a01b3dc1e1ffaa18fded1
acd97c52c54761aa80941f742d847264eded80c5
35539 F20101112_AACJKD armistead_j_Page_096.QC.jpg
5e85900787a92954631776194348f7df
1604a3b7369881afd210da476b12b7a63019e55e
36514 F20101112_AACJJO armistead_j_Page_087.QC.jpg
7e650412054bf0b5e13ea19ca8e07902
7b20c06e850fee718952d762e985e43d93dbaabd
122443 F20101112_AACIFY armistead_j_Page_115.jpg
5beec89af2c29f844037719d7d25435f
894c082a8912a36c92a4020c31bfbcc4996bf20b
112640 F20101112_AACIHB armistead_j_Page_015.jpg
bde2f9bb845b2aa11ee75d668da187d6
6ab3f4cab83c3a057244b7b252f5545d9bccd64b
8561 F20101112_AACJKE armistead_j_Page_096thm.jpg
0bf02e46e42c631168c77181f5425f88
793fb25343d1942e609031ebcb7b817edddb9f48
34238 F20101112_AACJJP armistead_j_Page_088.QC.jpg
a08cab748ca8600f3402dda73be2ad0a
c885fe763d59b4acd69d6368d844dcc6f163bf06
36994 F20101112_AACIFZ armistead_j_Page_120.QC.jpg
3fde5265b016be88cc0e0b9726fcea75
2506fe3e452e320eedd18d59319085e3ee25f2fa
108965 F20101112_AACIHC armistead_j_Page_016.jpg
ae05c13eab6441b13d78d985b6f7384d
37bca8bb85b30cc6c4098fc9df26f20fb0a93c81
25424 F20101112_AACIGN armistead_j_Page_001.jpg
54dcda4cd9de5661eed66ae3dcebb194
f69e71683881e18bb1aa507370f7d2c244852704
35573 F20101112_AACJKF armistead_j_Page_097.QC.jpg
eadd8c4c40687c240f50c23a5df85ca3
23676ea8cada3a412e2e1b85f7ca58e8cc63fc43
26951 F20101112_AACJJQ armistead_j_Page_089.QC.jpg
62bb947da9b0cf57a71f154e535c15d8
90d0dcf85f1332452607117b24065744adcf6267
107613 F20101112_AACIHD armistead_j_Page_017.jpg
ed56fc6cc7b5fb14e3f28240561ac850
f9562fc11a816188f308c3c8aa3b5be1d6eb7688
4231 F20101112_AACIGO armistead_j_Page_002.jpg
43ec1c0d76a6f4e7d49470f070ea9dfb
40eee05b355fd66ad2ff732e4d6186788dc552d8
8823 F20101112_AACJKG armistead_j_Page_097thm.jpg
b3bb80cb402936cad362f7b39c6b2707
52875bc1d38f8b8c117a044238e62f6767a477f2
6837 F20101112_AACJJR armistead_j_Page_089thm.jpg
c9d0e18bb0bbae3b14eb368b0ff8791e
45e980819bdd04ad2065fc56f9a150cdd32a84a7
88987 F20101112_AACIHE armistead_j_Page_018.jpg
473be9ae5e178d0cfdd2e41438e099ca
1f4c0e9d52fb1073936e82b10f3833616e5de5f0
13850 F20101112_AACIGP armistead_j_Page_003.jpg
45dec4149d96908d5fa9cd59e94f720d
2d53e839eb7672e7c8217f3250d17dff227d247b
3046 F20101112_AACJKH armistead_j_Page_098.QC.jpg
58f3e4f487f5280ab09824c420877314
3ad9518c7a395f8441bcde1b8cf3dd8c925d2dec
33585 F20101112_AACJJS armistead_j_Page_090.QC.jpg
838a7d08441973b5f4921680c6000f21
c43e529ba613258204fc29d043c0c21145449fb5
107074 F20101112_AACIHF armistead_j_Page_019.jpg
54cc28f85d775dfd68826803bc5201b1
02d5ab1ec44ac7ffbc64d44ccfeffec3c867f40c
58502 F20101112_AACIGQ armistead_j_Page_004.jpg
645104321b67d8743137a4534098bd53
5fe88dfa509dfeeb7d7446e029070cd5f7a99e04
8199 F20101112_AACJJT armistead_j_Page_090thm.jpg
3890b6d1667832ff1deb172c415c7664
f8113ba361f14435aa418f13aa43a83d3c5bb71d
122702 F20101112_AACIGR armistead_j_Page_005.jpg
cd49aa98f8a9ea465d8ce5343f37e68e
0f44f6a7140516614726f03e452dfbcca4907203
910 F20101112_AACJKI armistead_j_Page_098thm.jpg
7eec670474e066e52adb5ac91dcb0373
43ae19c1927490a4925c134c1808109bffeaf69a
29603 F20101112_AACJJU armistead_j_Page_091.QC.jpg
a6685af6fe851a47183fb3cffc577671
cb544f0292b7bfae6374fef8aefae22d99c20d20
113079 F20101112_AACIHG armistead_j_Page_020.jpg
620992b81a4fcac646c5922e94f33a63
b3ce9f3cc5e5bc42b32863c1dfafe9304584d3d3
89401 F20101112_AACIGS armistead_j_Page_006.jpg
b5de052fd490b864ccfec139ebac936e
12b2b775ace37adb8c7f948a5cbec2638116d647
3608 F20101112_AACJKJ armistead_j_Page_099thm.jpg
1d401eb5ec70e8717002b76830f6ea90
841176622cb60c59aae9441d8323d72497ff0e3a
7499 F20101112_AACJJV armistead_j_Page_091thm.jpg
6be7670f65174ba60ae3c1c4e4193891
2b7d74845b57b2da264b5f53a197b5d60b4e3e89
106624 F20101112_AACIHH armistead_j_Page_021.jpg
2154dc79ab3c8d9d69a3a9e2a05c1b97
4be9411a0dbd3bcfd884fd941d3594130eff88d7
138721 F20101112_AACIGT armistead_j_Page_007.jpg
af1068fcab6edf00fb8018cd693f6ab9
312337ea0c666a1d52357eadea70af2232f36e4b
7751 F20101112_AACJKK armistead_j_Page_100.QC.jpg
9d332ffb04284ee87d300ea7b778f51b
dd0e3379b154a037633726ba836f86d3d479e91a
7983 F20101112_AACJJW armistead_j_Page_092thm.jpg
95d69d0a7342a4b27e815ac78e9964f3
ddacc70d8a734cf2bcb9f085cf1e4ab8e35d2ad4
105375 F20101112_AACIHI armistead_j_Page_022.jpg
8c57d4d2cef65abda91c6e2bae7aba72
d9b5b8a80033ac106726419a2cc70c024fba7401
151639 F20101112_AACIGU armistead_j_Page_008.jpg
a0881e77ceaea966c8bebca6cca28fdb
2182ba073a2a3d4e5b07d2aef6900711aa9c0067
19626 F20101112_AACJLA armistead_j_Page_109.QC.jpg
b688b628bcbb698515909124aa0243e6
d4d59ec67c973e4d58ff3265363d2c8981c1828f
2773 F20101112_AACJKL armistead_j_Page_100thm.jpg
9beff07c427a15f6e5033cf224b86298
8cb92d26609e105ed4f6c5773fea2ae44080bd5d
29961 F20101112_AACJJX armistead_j_Page_093.QC.jpg
305e0daca79d24280facd4d970c21f61
377b26d63b83f6a3f9b9e2f86c96a4622944abd7
110694 F20101112_AACIHJ armistead_j_Page_023.jpg
4df1ce98991b7e634a51240b3e4ef0ff
784061de691a3e62368999b98392a47017966076
52692 F20101112_AACIGV armistead_j_Page_009.jpg
3a8e0006804d026b2f2bb241a45e4ac2
ea5b70fb9899930c8f43a183792a3cc0e12586b3
4924 F20101112_AACJLB armistead_j_Page_109thm.jpg
dbc2f164b3db83f619780e0bc2adfc29
698e6b1ad84561c8d399e1a383ecb8fcbb5b3577
7007 F20101112_AACJKM armistead_j_Page_101.QC.jpg
1548ca3acb7e9151040900fd11bfb6e7
2e5d9fa0f38e61f0c4de87c3d6725e85f870f9a3
7854 F20101112_AACJJY armistead_j_Page_093thm.jpg
ca5bb5a619ab5e4dfd3e83cc75864949
57637ddf5627e2fa68f124a65d16e2c9d86b043c
105383 F20101112_AACIHK armistead_j_Page_024.jpg
d1220efaa64ab34227fa24c54d7f8725
d2ff3cd3306af8ca8cd9d3bfa493a2e7dcd37efc
87712 F20101112_AACIGW armistead_j_Page_010.jpg
36c2506f77269ffe50d10790ae6281f7
f3bfaf1ba29b202f6cd7ba73c055044b35662a09
14995 F20101112_AACJLC armistead_j_Page_110.QC.jpg
c1fd399e95dec8bc020ef406c92de791
9cdbe7ee88edaf3775bd2cd7c94aeade34471187
2353 F20101112_AACJKN armistead_j_Page_101thm.jpg
70417b2025e00b49240e03ec9a574a6e
44a854c843a3870d10cf47ec936d0ec5defe9525
36929 F20101112_AACJJZ armistead_j_Page_094.QC.jpg
c4a7891542f7fd672bee9f89da8a0a61
a77d1d14892e55c0296510561af5393d4c9f3abd
111329 F20101112_AACIIA armistead_j_Page_040.jpg
7615ba5bb767b56627f24d0f6382f2c6
4c7e909878050ae8ecf28aa065fcf1009f48fe0f
107783 F20101112_AACIHL armistead_j_Page_025.jpg
7878a488d2ff53edf601496f63d760ef
8911b33ac852dc32c70c106592d28a5bf6641597
108922 F20101112_AACIGX armistead_j_Page_011.jpg
434b250233f95144648f40cd48b8979c
76d6864ec92ce4eb0ec5249da71c2aa82611bf48
4271 F20101112_AACJLD armistead_j_Page_110thm.jpg
528517e589ad39b9bea8e2a14d3590ab
43e8edee22d3ba0ee63f17967b2128e11a46b920
6136 F20101112_AACJKO armistead_j_Page_102.QC.jpg
457023231d90ecad408a158fdb017434
6151449b14bca6f2e7400849ba12b7b833b53bd7
108397 F20101112_AACIIB armistead_j_Page_041.jpg
229be22043eba314d9e90c244b5772f0
fa229444fe3626b5a424ece5d7e6165824e2b233
106043 F20101112_AACIHM armistead_j_Page_026.jpg
fca6db358d471b99c3cd07c8d98133f4
7ab30b4975ef83b79f218ef1bbf4cba20397c9c6
110381 F20101112_AACIGY armistead_j_Page_012.jpg
b0ab4f8936c12c7890bef9fcd3331bb5
0bc86e998979e74eda68fbc42a3f8a047d8e799a
34195 F20101112_AACJLE armistead_j_Page_111.QC.jpg
0f7407356b8ebf58d92b04bf8ff69666
5f700cad42088886fc38ddb9bdce070f5321229a
1970 F20101112_AACJKP armistead_j_Page_102thm.jpg
68ce4e99ea975d4844064ea060ae14b4
3d236842e49eb92a814cc6493d36b2c5b6c17a1f
113038 F20101112_AACIIC armistead_j_Page_042.jpg
c1dd2c799a65a04187e0381658a8eaf5
167986c45715db5b66e9f37e3f9925dfd09c2ea4
105252 F20101112_AACIHN armistead_j_Page_027.jpg
9e72ba74ff8289c8ab8959cbdaba25ab
a61a67f5d12d0333a56b5255597b40ac48a1e6b8
110412 F20101112_AACIGZ armistead_j_Page_013.jpg
e966299b96d809ca34acd000cdab4b62
277a034c81b55fd63051433223808e179d4aa8ad
37486 F20101112_AACJLF armistead_j_Page_112.QC.jpg
9ddd42de7e47a17276d5b914d21a6b59
5a05e610767c6f8380b64f1753c5b0983918f100
6556 F20101112_AACJKQ armistead_j_Page_103.QC.jpg
3c976b6d5c43e53f76cccc437bd13356
cfa404b2d80157ed50f6e619297625a09788d5fe
60751 F20101112_AACIID armistead_j_Page_043.jpg
fc380a2e8cb111883bc1cde610326e22
4621c66bed77c63ff7d8423058bf005e08f2d77b
106233 F20101112_AACIHO armistead_j_Page_028.jpg
2438e637d4031d8b71c710fea473d922
29ce3264ed033d142be5d9048d0f015384d18ca0
9165 F20101112_AACJLG armistead_j_Page_112thm.jpg
5bdb1d5dbb1145d80320588257742b60
bc810e116c4b4c8961df5e1b5454778a8416f528
2183 F20101112_AACJKR armistead_j_Page_103thm.jpg
ccbd046a31e84472b48210a3584d5186
e073523bc5e01ea4c3d15992032fea1ed7f61d00
32248 F20101112_AACIIE armistead_j_Page_045.jpg
a2c7e13202f410711355e37faad70da2
fb2b7a47176ea58f4a2290e8a6506847ca12c1c3
108595 F20101112_AACIHP armistead_j_Page_029.jpg
989a9d6e23d752648daf32a8dfacb16a
2a3826171fbbd663e8cf2741bc7b38a4f184ba38
37442 F20101112_AACJLH armistead_j_Page_113.QC.jpg
9c1361d49eeebeb3baf0ada051ea7039
b8e97efe786c23942ddd11708f51220431a3f513
9280 F20101112_AACJKS armistead_j_Page_104.QC.jpg
61287369ac5ca97168321504e95765d9
80bc5dd52acc897888422b922e52810b5d755588
39769 F20101112_AACIIF armistead_j_Page_046.jpg
7db1b4a6970f68459f337823ceb796aa
88bdd6e32a4ad2e7d231a3af0051717471741243
106995 F20101112_AACIHQ armistead_j_Page_030.jpg
c51917c4e1aaeb307302339d068a8cd1
a5889187ea9afe70687483823d3737131d15c7a6
9134 F20101112_AACJLI armistead_j_Page_113thm.jpg
b4f5fbf98592827b4faa5e4c5cef664d
915753fbf2d7671171a9f6a0db654976ad07051b
2719 F20101112_AACJKT armistead_j_Page_104thm.jpg
da51dafb38284b919be2c1c96eb29fd9
7e8ca1bcde8a19532b6177b8a54e1b68ec792fe7
27152 F20101112_AACIIG armistead_j_Page_047.jpg
dcf11607c82b2427499ee4f97684e859
3f059a116d521e5b15adeeede7c914aa99bd67d5
109624 F20101112_AACIHR armistead_j_Page_031.jpg
20d9ae7279c4c8c097686f31aa087c84
732b5a442a4a8b0b8ad4acce745c11ea082d7a22
34469 F20101112_AACJKU armistead_j_Page_105.QC.jpg
224519e40a0b01b32fd0c54f88c249db
44d0a18c4333747582ae8d36d651ee370aaf99e9
113624 F20101112_AACIHS armistead_j_Page_032.jpg
644a50ffc125146dd7600ecfefee32c0
4655cc723240d920983d8034b0a13dd45309c116
36887 F20101112_AACJLJ armistead_j_Page_114.QC.jpg
b208507435871665ed04c1656784be51
093a63b1cc953b30d9edca5a416db78294af30d3
8663 F20101112_AACJKV armistead_j_Page_105thm.jpg
ea013ae01d25af5a5f8e2fe2cd5a5c1b
913d1a1124996eda4076cd3ebb0c0b8ce42a7cbe
36234 F20101112_AACIIH armistead_j_Page_048.jpg
d0d20a893a62bd32f03dc66d64ff84e2
57b793fc59a881718d4580389afbb000dc0dbefa
107843 F20101112_AACIHT armistead_j_Page_033.jpg
df801464623b116c7fda480c05d1d06f
a3bb890386f9bec969253644bd8313db2da67e34
35662 F20101112_AACJLK armistead_j_Page_115.QC.jpg
651182fa7570ea516ae16752fae2026f
02a0e6d47cec77b1dd08d3e4a90681e8cfff3917
34703 F20101112_AACJKW armistead_j_Page_106.QC.jpg
1e46f4a0244c6eedeb8b76190d6a9f14
5d3522eb6a92913728b1213be4874d36d2edb7b8
30167 F20101112_AACIII armistead_j_Page_049.jpg
4e51c50ef3e61e7e6ae040e76115f797
7bf056a162c139f44c9b98b6c36fea9fdd0c67bf
110699 F20101112_AACIHU armistead_j_Page_034.jpg
9864b829089e2253e87e02f1ce46d1ff
26e4e19a7331857428e8c705f5e8fc5ce27fe9ea
34170 F20101112_AACJLL armistead_j_Page_117.QC.jpg
331b959f9737099a5f8248a85f6c254a
7efaaea820214adc70ebed80b433af346dc339c8
8550 F20101112_AACJKX armistead_j_Page_106thm.jpg
0db2872136de97ffceea7b1cad377271
0e1383dff5af827058913296f6ff50275e38d607
23123 F20101112_AACIIJ armistead_j_Page_050.jpg
ff4a1887dfb15c6c7e363f49bc98c4f1
5fa7f317884177f6640f945346b6c06c526bbf80
110861 F20101112_AACIHV armistead_j_Page_035.jpg
ecc7d58e7d630b59e0f71afeef757d73
61bef896352d203b698d92b63dac278f8b9603ae
8690 F20101112_AACJLM armistead_j_Page_117thm.jpg
cb195706c9ebe529f626b159bfd24d95
0ec093e204a78af91daf0f7b13a82218b8f52e5f
6487 F20101112_AACJKY armistead_j_Page_108.QC.jpg
36876f547f03936736a4621db33604b7
aec223b6680dad00be4f92a228dbf4d60acbbeb8
24653 F20101112_AACIIK armistead_j_Page_051.jpg
7a3f0dc1f76a7ef96242da06c4d5dc6d
750ad0b69bebefc24b7e442635cba088da6fb20a
116303 F20101112_AACIHW armistead_j_Page_036.jpg
2803bb2ed684750761704684a140d905
92d7aef140a302fbf4e2ebf06ed731ff14765e51
34549 F20101112_AACJLN armistead_j_Page_118.QC.jpg
ecbcc8ba71c484252ad3b869dbde5c9e
49fbc743722a4b1c4fa4d804c0f4acdb365dfabe
1747 F20101112_AACJKZ armistead_j_Page_108thm.jpg
12fa2d8443095d3f78b0d47c33bfd389
e26bc0108a63824a762149163ec254c7a1c0afd7
23445 F20101112_AACIIL armistead_j_Page_052.jpg
a1bf9efd7ac9fca63362620b70212113
b5710993df25dcdb6cd74f81102f06760a60d9bd
112161 F20101112_AACIHX armistead_j_Page_037.jpg
3a9d324b56ad3c0b593816d54163e03c
c62602c7f70a3b7e9512be41725e98a9d693dd45
110887 F20101112_AACIJA armistead_j_Page_067.jpg
51185c991c8119c9497f464edb5f9155
c54de82286fe2217aaa7c251f34b41466c396139
8765 F20101112_AACJLO armistead_j_Page_118thm.jpg
fe43aa486ca2fe94c105da54674dfe24
1b9e4f002eaa8d26397c36d717d0d244aad86f36
36478 F20101112_AACIIM armistead_j_Page_053.jpg
b297cd87ca4240b55b75636a19c6c076
cb868b7aa57c1eee5b441e29e0a706ed90b8c43d
114085 F20101112_AACIHY armistead_j_Page_038.jpg
f3261be1a33e8a2b1284bc31140a2b9a
0c3d9da035bd556782f8d3f94d1af6e1e11ebe9c
105742 F20101112_AACIJB armistead_j_Page_068.jpg
ad6cb37a578c2c8e386b9ea7e894c573
51a10bf2d5e5e3b03dee55bd9d090b3d5031740d
36792 F20101112_AACJLP armistead_j_Page_119.QC.jpg
a114312555c7cbba591c243178fe8b9b
42fc4754c2d71ab2626394948708f68f349402fe
50122 F20101112_AACIIN armistead_j_Page_054.jpg
4a9f133b495e305bc15665a1dcbb7016
e5d1856eb3c1e17c7d50b853855b9358060c52a1
115662 F20101112_AACIHZ armistead_j_Page_039.jpg
4adbd4338cf44d5ecec7e525ceca12f6
b2e8050076143767c3909c344a46cc6b48ddc1ea
107152 F20101112_AACIJC armistead_j_Page_069.jpg
5ce2adb2015ff472f99ea3b52d69919b
5936f0481d60a3b67ef1280297561bb7729db8f3
8967 F20101112_AACJLQ armistead_j_Page_120thm.jpg
e764724a1949c9ff04b9f1a671c4e1a3
6991f5565d9d06c742e54991479e58655db6f5e5
24228 F20101112_AACIIO armistead_j_Page_055.jpg
c18cf2401420184baf7af2982957c19a
d9e89bd695b2c1ca89443d3b255aa010bc23efa8
110129 F20101112_AACIJD armistead_j_Page_070.jpg
08348272733a11cb0d15a34c25c91f1b
159a210f985f40eb76471dbcbef09c25385888dd
38176 F20101112_AACJLR armistead_j_Page_121.QC.jpg
d9a95ac4bc2f85fbc7ea097cc9e71c60
d227b77ae84b99e1a37bd8f6e8ce4f71cd7975ee
18901 F20101112_AACIIP armistead_j_Page_056.jpg
ad972c1c209d7c8a1fb9198bde2de425
7532eb415263a25b2b2a5903d61840abe4d05fc0
105634 F20101112_AACIJE armistead_j_Page_071.jpg
e4437d243730a8b8b325f99c6695e997
8c301bd3ff7e4d373dd751f5a7058fdff7b1a369
9113 F20101112_AACJLS armistead_j_Page_122thm.jpg
c369f0cb2dbd6929e546eb25aacb5e1f
800c4789db90e28d79a4704a08a7088438bbe7af
22979 F20101112_AACIIQ armistead_j_Page_057.jpg
e6e933da0a50ab03e262b61a4f0a9b4e
7ce0971502d540dc0c81bc1a08ac484d2c7a8535
88105 F20101112_AACIJF armistead_j_Page_072.jpg
3b152a485eacdbfc3cecd2e82156907a
fdf087aec89939ed5b3c493c53f9647aa49e8f92
18647 F20101112_AACJLT armistead_j_Page_123.QC.jpg
28a773dae41e84b6077c3849070ee41c
4e8d67a15147d36511de62ace9e787978efa2f8e
29113 F20101112_AACIIR armistead_j_Page_058.jpg
1dd590cf13ab8fb730900b75184cf9ba
7c50fd8e80d2be9881aea2dd7496b457378be06e
97984 F20101112_AACIJG armistead_j_Page_073.jpg
2bb166b18c22436b84f2ba8e3af5aed8
27a48eff56d0fd6d97d841d4adaa168b921b791a
4575 F20101112_AACJLU armistead_j_Page_123thm.jpg
119e065f27ccb3dc739777d28f9f9baa
4068330ed99b04da1b13fa2b88b1232af46ad4c6
21563 F20101112_AACIIS armistead_j_Page_059.jpg
40c49770c1f60ac55ef7c1d8a2450f73
ff8bccae4eaaa92e13c8521cb96687f1fc09fe1e
96128 F20101112_AACIJH armistead_j_Page_074.jpg
2dccd261504cbb233282abbcf1b94ccd
9897cf170c6c3c2eda9b04240142ffe3c5d88c42
27985 F20101112_AACJLV armistead_j_Page_124.QC.jpg
fda9b199db2e8dab437ba171500a602f
c8c108ff3d62d836308a09eca4172b932532586a
30464 F20101112_AACIIT armistead_j_Page_060.jpg
28b4ee323cc1185bf0822cf06c534fe0
7ee29152c327dadcc2f8d8d0880179178a3831db
20436 F20101112_AACIIU armistead_j_Page_061.jpg
43949772f53bd579a81ab2b9bd6a1bf2
1c4b8b228313f18de1ba2f4ce8ffceffde313a1e
107137 F20101112_AACIJI armistead_j_Page_075.jpg
ebd293d96795f300ec28458c87a5a8f8
dba85a4de913127e28c635ed372053e94c23b731
19938 F20101112_AACIIV armistead_j_Page_062.jpg
5d42a520d38e3ac1b134bd17c0127177
d22b33b3769f119d42438c5f69e795a8dcd6afec
113301 F20101112_AACIJJ armistead_j_Page_076.jpg
89e0160eecc4f4e2e137fd74d2e20478
81f1bdc6dd86725c0e2b7062d8b9de02ce8c55a6
34842 F20101112_AACIIW armistead_j_Page_063.jpg
1c37c796007be4ab708fdf6217853f7b
6f2392f0ef3cac5d3b589e74cb2db97e65f2cb2a
109738 F20101112_AACIJK armistead_j_Page_077.jpg
4c3c4146df9b7bddc0efd31a7145d806
e02399c60874cd213cc6a20f01d3d74a9779e5da
35609 F20101112_AACIIX armistead_j_Page_064.jpg
67e0f98d20b1e781cc1d6c6771f9f0f5
c966d2cf19a669e1bd11c4dd12eec27dd7a79cac
107017 F20101112_AACIKA armistead_j_Page_095.jpg
6fec518db7b97585a61ea710ce67e779
dde032e7a981042bd3653d5dfb751bd7957b866e
51310 F20101112_AACIJL armistead_j_Page_078.jpg
ade318033e49e9049221acb71748f924
e8ab6b95304a9ae1a538c57cf52523111dac1d50
35950 F20101112_AACIIY armistead_j_Page_065.jpg
e1b3e27f97ac8df450fec7069419b507
8f3553944f9be96c8f5ee78d74c3a65fbc6ec666
108128 F20101112_AACIKB armistead_j_Page_096.jpg
b54cf9d09d6b1015f81aa4ade15986b6
2f97ff50f8dbb57c0127a57fb60a3c0679701630
37564 F20101112_AACIJM armistead_j_Page_079.jpg
22ddcdb6da794967e63eec0d68e65063
4deb5dc30d610591be91f53f4c90020ef802769e
105513 F20101112_AACIIZ armistead_j_Page_066.jpg
5fd527032a1a8dc1f00c9f7dee4ef800
3f2b87e18878e243b56a27572c010e36456bb9ed
109585 F20101112_AACIKC armistead_j_Page_097.jpg
f160eac482e00b9ee1efe2595c48028d
ecb702dabe0bd669ee295fed1e2259694e7fa405
21471 F20101112_AACIJN armistead_j_Page_080.jpg
d6115d48d65ab1afea0cac27173b8edc
955c0d4e25f5ec4c8f0ece0c9d66748892e1e42e
8082 F20101112_AACIKD armistead_j_Page_098.jpg
db52d1844fb236f1a5a6fda267ddfafe
2235c4a46b7e2754af7a7a38d475273d97411294
19516 F20101112_AACIJO armistead_j_Page_081.jpg
85d1a65d6d7d396116a4698f74489aff
9de7cffcc7cad200267c8cbe43e4f805c6269ab5
44235 F20101112_AACIKE armistead_j_Page_099.jpg
5ee987cc9a4c53c76701db8d197d337a
a88241b571413ca63df41e3ee9336dd7f61c8a93
19524 F20101112_AACIJP armistead_j_Page_082.jpg
916aa2207a083e182bf3ddeac794b62f
de06c47b1d88a59f4776525b9892b53fd14cb626
22787 F20101112_AACIKF armistead_j_Page_100.jpg
45427d0b2dcaee1c0b425f2025216c5e
457d988d35d3d2236b846a76c54349b528070119
19919 F20101112_AACIJQ armistead_j_Page_083.jpg
fe0d8ed9771e5556e4ebf72e324cb3b8
7ce3ac0f19a156030a1548a9c5df7eb155f0fbfc
20400 F20101112_AACIKG armistead_j_Page_101.jpg
b4c53d6d2ca94a1a4ae9383503b1144f
510bb94e118e63de8045861289a1c3fd5799a280
21298 F20101112_AACIJR armistead_j_Page_084.jpg
8ef6b54b8205643f06ff85014a2f8f09
a0f95f316cb006e0752b249fa22b3ba821615233
17844 F20101112_AACIKH armistead_j_Page_102.jpg
564fa4b18010421328aed605caee507b
d54940e0beac9b442cf131a2409fd2212682de77
111629 F20101112_AACIJS armistead_j_Page_086.jpg
ed337198e40e35ceca2b68e284bb6650
ef63bf732acd6efe29a8691d8ca504c7c5bb86f9
19088 F20101112_AACIKI armistead_j_Page_103.jpg
ccb90f0ba0ee47231105a1adb7db494a
64db7dca5dc0d30255ab0c2f146753e88c6a160f
111221 F20101112_AACIJT armistead_j_Page_087.jpg
ec3ff933642aa0b4100d51de5d47f44e
7d053114746e58956c401263efe0b69546612643
105237 F20101112_AACIJU armistead_j_Page_088.jpg
bc593487e477bb30a2123fb821e97853
dafb06ab1b86f579b8b4d88e94147f650fe71908
31448 F20101112_AACIKJ armistead_j_Page_104.jpg
277196274ef85ffe6c28edf8b5552703
93e967af726f9b0e485171c2ca8a5e3d3fd8ca0f
79125 F20101112_AACIJV armistead_j_Page_089.jpg
e8c7c1527147e9468bf0c462a223547e
3b54fe61f8aed858fbbee7e76f84f3e956c664aa
106777 F20101112_AACIKK armistead_j_Page_105.jpg
b462ae3a2a45ca6315020232093b140a
279482d1935ee4e281108378ba0456bc8364632f
103649 F20101112_AACIJW armistead_j_Page_090.jpg
d8c7935cbbd2e3c023628b123e73e14e
6b3431e329c1292ba69e2bcd24dcb922a6e42080
127514 F20101112_AACILA armistead_j_Page_122.jpg
c6ed268a713fe8ea81d81749b84ed35d
44df5f1572ba0ef46f3f2cddf63694bce531d28c
108676 F20101112_AACIKL armistead_j_Page_106.jpg
3a8963ba4261dfe4e5a24577bdf22d94
5d9cd6b26c139fb774e8f21d7f20df0c80a8b3dd
91575 F20101112_AACIJX armistead_j_Page_091.jpg
55c9b72e6ff5bef168bfef28c9261716
40ed39fb18f7878057550f61d5194b842f028b2f
66388 F20101112_AACILB armistead_j_Page_123.jpg
8a78f99d9c0b5287da515630e026fb6b
d6ab244699c32ae32ee39c345fed145c39117b0b
112369 F20101112_AACIKM armistead_j_Page_107.jpg
c0d060c1e6715f519182545046e7b7f3
d744a1c213988fe3da8d244c14fefbe60bf0c834
99134 F20101112_AACIJY armistead_j_Page_092.jpg
63f4905b9bb8630c90033b6261c9ebc3
eda825e96aa61442125e32c107bf3332c7f1ae2c
86372 F20101112_AACILC armistead_j_Page_124.jpg
14e678c76edb023bd2466a7099718b84
77d5b95d97b23719d1cf52db57cdf95c1d251e13
18229 F20101112_AACIKN armistead_j_Page_108.jpg
4bfcdd58bf228e2f554376ae30110cc0
497f9f1c9de635c3ee23a17f47317f1fdf85a35e
94017 F20101112_AACIJZ armistead_j_Page_093.jpg
c333a76c6afb4b69c8a7974cbe2fee38
0de7e62bf11c0f54de6b3ebbc2ec97e4dc451447
62468 F20101112_AACIKO armistead_j_Page_109.jpg
89b374398e2f593ede8cad3900d34557
7a95f64ceaf1bcf5374847708e8d2aba70a81da5
24520 F20101112_AACILD armistead_j_Page_001.jp2
4d8a54c9dfb7abfbb8c8f466e07c5b94
c807b6f4dc397ddfb82caddcb3ef13736906eee7
43171 F20101112_AACIKP armistead_j_Page_110.jpg
0f8c0c6e32f421e13778d8969daa33c6
5ea127e63daeac67bc23649dc91aa7d7068a23cc
5646 F20101112_AACILE armistead_j_Page_002.jp2
5d62e87ec087a9e4d4a47759b360caa1
dc68964dedf7933b46b9621cc46af888ed695071
120547 F20101112_AACIKQ armistead_j_Page_111.jpg
dfca03552f1be9cf93adc6e4fa0a9698
060f0f14bfb3fd00a4a5ae99dffb774135c0110b
13801 F20101112_AACILF armistead_j_Page_003.jp2
1d2f7fc603f508a743a41da396586be0
f58141e03ff2bcffb9cc1cb5468e43e4a9a84b18
130048 F20101112_AACIKR armistead_j_Page_112.jpg
82c91364aa3b170c938da5d324c52fba
596d80d26851728aec041926355e6396a9e8c154
60714 F20101112_AACILG armistead_j_Page_004.jp2
4b73fbb12823b0079cb5824da0bc4ade
f330e8948f464acead7374b96cb521df46c1bd78
134412 F20101112_AACIKS armistead_j_Page_113.jpg
65d3926a86ccdc75e50eeb6a32a2ec17
435479895d59a78f009e5f49cd1b995cc24b89f6
1051960 F20101112_AACILH armistead_j_Page_006.jp2
caf486272724ef7a3cdca0a78dd39209
0a36dbb036b858ca75d24abe31f72136f3897213
129085 F20101112_AACIKT armistead_j_Page_114.jpg
ef6ec160143d9f60703ed75d35a9a8fb
93017c06bcfcdb298afc0efe8ae97e730c2cabc1
1051958 F20101112_AACILI armistead_j_Page_007.jp2
1aa0b4657188e136550642aa1a76dd52
4ff124272abaea2f30d9514afaa0e5ccc5e0578c
112826 F20101112_AACIKU armistead_j_Page_116.jpg
34355f841ccdb9e3eecd1c10b3e53d28
43e5db7fd5ade88fd1cc7d1893ceef8b7726202b
F20101112_AACILJ armistead_j_Page_009.jp2
f3133a01c86eee357e44bb4ee2fdcaca
11e756d6f9b9cad6478150daa06298aae51c8575
117900 F20101112_AACIKV armistead_j_Page_117.jpg
f917101ed668243016909b2d7dbaebc9
421dad05bba39028151c8b6f14acefca02a67bbb
117583 F20101112_AACIKW armistead_j_Page_118.jpg
517f5c0112129cf3dab562c5039dce5c
8b0f533d69506e9798d2bb74c8257329d5b892e0
93940 F20101112_AACILK armistead_j_Page_010.jp2
cf3087a6c3cc09fc07110c519a990d11
694c69af41b09850d417b5cd801644cf75123cde
128306 F20101112_AACIKX armistead_j_Page_119.jpg
cec6de4923e7c9e40e57875c3e940dab
bffba50f88da2a3bc7bd5f93eac40a26d1a50bfc
119597 F20101112_AACIMA armistead_j_Page_029.jp2
5e188d7539c181c61d5bfb8ec168791d
353f0ef5118785b6056cb9329b1ce3b7dc8e9283
118323 F20101112_AACILL armistead_j_Page_011.jp2
689e895696ab5542d417062cfff23e7e
9697dcb12290b61221aa82845e8389072380ae79
130847 F20101112_AACIKY armistead_j_Page_120.jpg
0d067b584d6aeebe15b09170a87628df
0f3fadfc6aa605607957a63388b8bd8c738b93bf
113075 F20101112_AACIMB armistead_j_Page_030.jp2
386cca1ff05208d4344a61b68896bcd3
5504eddceb0e6cda67ae7f1406f9f247a3555759
117210 F20101112_AACILM armistead_j_Page_012.jp2
be5b0f1767f01ae98cac3799296c370e
22e3d83755578479f6d43a2e34def17e1451573d
123925 F20101112_AACIKZ armistead_j_Page_121.jpg
a8925ec628439cc951b304e0e795b962
1e6b7189d3a8bf5dea7041b2d71d53e2eecd43ce
118829 F20101112_AACIMC armistead_j_Page_031.jp2
88a4669cf91e09d3fcf82a5b6f1881fe
a8f0f4a93ddb9a76d193eaabbca51e9f53504b01
119413 F20101112_AACILN armistead_j_Page_013.jp2
0fc00bd388de5c464826a7ea4812779a
6b1ea21397e5c82813205b917ce3ed7d2724964b
121723 F20101112_AACIMD armistead_j_Page_032.jp2
87c9999bb326b6dfedf8e55972b82f7e
94a6c682db9b2d17cd1b5d987fd90a6c9f4a3054
121608 F20101112_AACILO armistead_j_Page_015.jp2
b66fc29101c34c93381e7c515de30fd6
a0c5028cd67ba0e2f55149ea9e28c27c78c4b22a
117818 F20101112_AACIME armistead_j_Page_033.jp2
5afd044f06472d06dc5982764f787dc6
4c4d6a7912dec06aec52964b28fc07897c5d2e91
116330 F20101112_AACILP armistead_j_Page_017.jp2
7e57e0f31b2578d1a9fff7afd01a94aa
b1f750f858dfd3205e3c15fff78885bb7d04b5d8
122757 F20101112_AACIMF armistead_j_Page_034.jp2
f73da9efb813084e342a71ae8f6b7600
7e6d324a51bb888edbe79e8be11d56055d94aa77
96993 F20101112_AACILQ armistead_j_Page_018.jp2
ee4923f9990f366139a754b7fdda5851
081a43fb2e7b2f5053326164fd06b9313752db2e
118496 F20101112_AACIMG armistead_j_Page_035.jp2
f34e820d5e0f2338a46c10f61ab90710
9884f5b40f15723b011d089bdd5faad27581091f
113679 F20101112_AACILR armistead_j_Page_019.jp2
b7101074a2b16e208648ecdbf51a9409
dc38c7feedee1e00a2e20b1b7b4cca22c9a6bae3
124367 F20101112_AACIMH armistead_j_Page_036.jp2
4e1bb386d2bade157b86c1120219cda9
3d172df9c35af7d0a5efc7c3db210437aa4f5204
122233 F20101112_AACILS armistead_j_Page_020.jp2
5e7741a620673feea6868546cdbdc63a
61c89d109e84e8cac3531942d637390b9c96c03b
122153 F20101112_AACIMI armistead_j_Page_037.jp2
765079225af64213e95fd83ced386372
f85416b309e59844ab061c3310176fd25116aaa1
114159 F20101112_AACILT armistead_j_Page_021.jp2
98c21e0f74d355fe2e54e80c4ae67773
43e50d9ca5e83eb3f2324a42fe8f73c972b0d764
123969 F20101112_AACIMJ armistead_j_Page_038.jp2
ee8d29b4199dd488e57d8a4968ed445c
e79f6f87a1c66c37c994bc8645658da7a46a399c
113186 F20101112_AACILU armistead_j_Page_022.jp2
6a3e7ad941d3ba4effbf6e4180953bac
8b8628856ddbde0b3151dee9f58bb435a2bb8efc
125623 F20101112_AACIMK armistead_j_Page_039.jp2
61e7cce8cf6032890a8fa053b24cedb4
382130a9466ee7f8dd78b247c5e0966f1eb9b41e
113964 F20101112_AACILV armistead_j_Page_024.jp2
85a60c9b91d0a602b11d68d9adc80cdf
07a1dc7d8e4a248e286ae647f55f89e87a3bcbc7
116634 F20101112_AACILW armistead_j_Page_025.jp2
82a30efdb0bda5db67c9672592b17f7b
6f0c3344361f790f00dc90df1e656a5bf19175e7
242547 F20101112_AACINA armistead_j_Page_056.jp2
37bb1b961f0bd57d5b8e7a7ba11bd076
0fc65020a6885110dc25cd84d2074850d0f6b5f6
119291 F20101112_AACIML armistead_j_Page_040.jp2
2e770b168243cec92b1994ac7816745d
2edba767879291b485cf09e7b68fbf04e4fd2875
114846 F20101112_AACILX armistead_j_Page_026.jp2
f6a3dd19f7a17c21a8acafe3e3cb93ab
ac242c7a0e31513771f200a5a1c6e84e6483a686
308187 F20101112_AACINB armistead_j_Page_057.jp2
eda748f2d9f9785baf9a737889f219eb
8eb1c400fabbc3f7a1e8d22722d80fba748fd805
117035 F20101112_AACIMM armistead_j_Page_041.jp2
b3f66a186c3052881601db67cc505df8
7435d8f37a6b0c5adc34b0690d9f484a7529d1e0
114720 F20101112_AACILY armistead_j_Page_027.jp2
b69eaea4a92a5e973b8560003381bd42
6423207a3e8b0e95509eb79a2e6dcc1852d45a42
36217 F20101112_AACINC armistead_j_Page_058.jp2
41a3e3349f81c11446a14130216c99a0
e7b04dd63eb69a3d21e6892e2af6ae3a311317a0
122374 F20101112_AACIMN armistead_j_Page_042.jp2
fe3c813838e10a2beae5de1b60b333b5
878bf091fff73e8ad6e5b920dd2c02fabdcab00b
114618 F20101112_AACILZ armistead_j_Page_028.jp2
1b6388e089aa2377f5efc40b9552870f
2629c65c8337e19950374d4b50e956902d4961f6
24204 F20101112_AACIND armistead_j_Page_059.jp2
7c7c5f8b9ca058148fb376958e268b45
10db8fd6a0e8277c831e00880d088905f220a594
64469 F20101112_AACIMO armistead_j_Page_043.jp2
d940e85d0c83164d33f0da9f0ce9e0d3
bd44d49dd30934c933f9957dfebb6f136f813cec
453481 F20101112_AACINE armistead_j_Page_060.jp2
1cacc4026969acbfcea1de13bdb01e63
c2f7405b5fcde93680166870385e1c9eb19e83f2
491262 F20101112_AACIMP armistead_j_Page_044.jp2
9f0294a09ecb706749d3e65200afb8af
fda9121c54412c2fe17e64a334cad4e3a52abc39
265731 F20101112_AACINF armistead_j_Page_061.jp2
4a9292e647685852de637464a64dd405
6445eab2a27d289a32f7b27b9bc3f5cd67efbfa5
49966 F20101112_AACIMQ armistead_j_Page_045.jp2
9a8bd43b8f067de20a88b4276b47ef05
f2d23db9cc72a9ab1202f470a97ef21e1ee65363
258834 F20101112_AACING armistead_j_Page_062.jp2
24d07b9bd2d23b931a0de8499ca44344
881c4887cc64220681b8d497ec1d51176bcf3a93
62280 F20101112_AACIMR armistead_j_Page_046.jp2
e30f03c64fc2ad7f16f093aaf57d4d9b
5e274f22b5c9897bb5b093801ebe7329aa3c7535
45924 F20101112_AACINH armistead_j_Page_063.jp2
68c333e8a8b871df4c8beb9d52028228
de4f66cb33e6c69fbfefb3500652691b95592c6d
41622 F20101112_AACIMS armistead_j_Page_047.jp2
ae83da2ee585239b838470c73abacc90
97f517764464a66e12181b55b4176d6cfa86f2e6
48871 F20101112_AACINI armistead_j_Page_064.jp2
7f4971a9477947ee0c80abe26d142762
9f13f6ae8e3558940fc644ba0cec5dde2133f031
57919 F20101112_AACIMT armistead_j_Page_048.jp2
1d37c66ca874d95105c9bf73d13f037b
263053f783696f7dc6930e8ce211071bab6b2d3c
47993 F20101112_AACINJ armistead_j_Page_065.jp2
73c20191a9b4d8b8dd61e2df4b8674a3
d9db391ac8a943ff2ec6a15555c97d477e3d97a2
454408 F20101112_AACIMU armistead_j_Page_049.jp2
1b893a2457790d00c27dcdb628aca7a9
415ea22fe663080004c4486e1b68b232988e696c
113068 F20101112_AACINK armistead_j_Page_066.jp2
ce33e5a6ad47ed1ee64b3590221446bf
6d884d3a59649abee70d4a49ded040730da60422
34564 F20101112_AACIMV armistead_j_Page_050.jp2
b281e68bd0704d212c9db4792c3a3f27
6b9092e6558b7ed7916faa7ac995b7edcceec23a
119581 F20101112_AACINL armistead_j_Page_067.jp2
b488c88986b3f10f54545cf3e531946d
0a7e8010ac3327f30d955639ca522748fa357149
335410 F20101112_AACIMW armistead_j_Page_051.jp2
e48bb7ded2c6da2de14ee8073ac80ec9
bd2762a15ec9f955040fe9ee00367c235797a6e2
58451 F20101112_AACIMX armistead_j_Page_053.jp2
64ccc8a39562ab486a3233f27c5fdb0a
1fc72705fb33a788564287da12afc6d2567dbe76
32126 F20101112_AACIOA armistead_j_Page_082.jp2
698343423b52eedf9c126afa361941fc
2f7064b1c1e517d51846986ff3f10b7691fd8d8e
113646 F20101112_AACINM armistead_j_Page_068.jp2
ef1fa2d317e47a2c281ba3b324062930
c640921263bef5260c444ee71eaec67931b176cc
443087 F20101112_AACIMY armistead_j_Page_054.jp2
ec2a1fb901c798d40319b05836cb20a9
9250ee3fb9c2aa00ee37c09fea72f26daa49a345
33447 F20101112_AACIOB armistead_j_Page_083.jp2
00781eff6e7e3eb76dcab1320fb2ca03
5e92ff59e73251c1e3b28e315f374f8bcee094d7
115897 F20101112_AACINN armistead_j_Page_069.jp2
ea6a7b24ce65dcc4ec053f87bd0847a7
e0c6d1f94897ae7bb0dba16528da72b6cdbe59f3
338984 F20101112_AACIMZ armistead_j_Page_055.jp2
7ab4c202dae600eb450247ab03575abb
7f6f7c57fd459f569725b2476d25be9aa1a35b07
35492 F20101112_AACIOC armistead_j_Page_084.jp2
c1ebec9e1b3d9bf74526b525692900bd
6529d70d2e6888e37736767bbcf5eb4af4f86840
118504 F20101112_AACINO armistead_j_Page_070.jp2
1034fd1f30b7c3436aa1af6e51f34e07
0d97195dcb35d7c1f7c72f79fd8a7a8960454c44
106760 F20101112_AACIOD armistead_j_Page_085.jp2
8a8146468b9cb6ec53069d34ebbfdeee
a901a4fb4c4a52bc573dde78e99f38a5e27c1bc0
111872 F20101112_AACINP armistead_j_Page_071.jp2
886842204bf132cf4bb6c54449b2ed6b
ee55084a3a7aed1bc3cb785938aad581a5122287
120217 F20101112_AACIOE armistead_j_Page_086.jp2
6c01c8784af9022a8e17df46126c3ae2
f89adfae9ab1cb9fc1d9dc939af85e45182c6b16
973606 F20101112_AACINQ armistead_j_Page_072.jp2
49228521610cc6e1180085c471482c85
41c438fb50ef31d99affaa1efcd4f913a261f210
118503 F20101112_AACIOF armistead_j_Page_087.jp2
0ffb0f55142875bde9870b42140bd57a
5910e2ec1c91f1030993d871d26c65fa3aa6e404
104941 F20101112_AACINR armistead_j_Page_073.jp2
d49be577ee8d6aaadfa70b9eeef4c92c
2f4c5e6b2496aa7af96e55a0204a0d69a5c128e9
112887 F20101112_AACIOG armistead_j_Page_088.jp2
ebee38fefe3390ab97432ebe050b076b
d5a9d12757ff2558c3ef30b840f390b90b072cad
104234 F20101112_AACINS armistead_j_Page_074.jp2
cdd718aa3cb0de79eab6b559349dfd7d
f129901746fc56605c06fa707f4f0a5b7bd9f970
86011 F20101112_AACIOH armistead_j_Page_089.jp2
21daecaba6db3181bbff9b4e73dee7fc
16b7a53b052ee6965da5b87e66f7ee6f01101d7a
112938 F20101112_AACINT armistead_j_Page_075.jp2
5eff1654916c945411d908fb7fb37562
8aa23690819fb8421f424aec79064501da46d8ab
1051966 F20101112_AACIOI armistead_j_Page_090.jp2
d6bf6d8b7b823a8c05845553cd167651
9f9d354c74a59f418fa3aac5e48659f9cb490a83
122235 F20101112_AACINU armistead_j_Page_076.jp2
6c4d4d55346bd77fd93c609b16061788
f600c90310fc295d25122614f35c4c6dfce75a38
97987 F20101112_AACIOJ armistead_j_Page_091.jp2
644b47860d97291e9bd58dae68dbbb95
669828b97d3d4935cc24b6aa30d73f67517d6b7b
117938 F20101112_AACINV armistead_j_Page_077.jp2
d95e6c8a6522554952e1a10c375757da
f2306cd10224bffc0e13d03885fa9274af0fc9b7
101098 F20101112_AACIOK armistead_j_Page_093.jp2
747c19077ba1215a5c69162a07c2ac83
572b81992f0f3e042fa7a9241ac1a3f9bd8b6f5d
55161 F20101112_AACINW armistead_j_Page_078.jp2
1e0ae408162a3eddee7a95e8bc41982f
3b93a12ba0b4a832128e90869fe9be6105dd03f0
123475 F20101112_AACIOL armistead_j_Page_094.jp2
ed62e89573b308b795a2532a75d15853
7e882b4be2c5dc0421ec28fe20f7036289d881a8
56204 F20101112_AACINX armistead_j_Page_079.jp2
b9fa62d9dc9f5c96dfce06e447f1d262
569f443ebc49f2bd278f6dea8e1fc74beecde3d2
65237 F20101112_AACIPA armistead_j_Page_110.jp2
b457b8d52b31ac470349e18334ae2c44
1f4a35ba91d380f827e34121b45bed999dae7760
116430 F20101112_AACIOM armistead_j_Page_095.jp2
90b93b20862bd232003d79dcba1bdca0
e7f862b6140d151d8225026eeaaa3c616c0aecae
37099 F20101112_AACINY armistead_j_Page_080.jp2
e37658628c78521efbb05004d1adb08f
eb5d14a6dd2483bc2cfba39fd7febe6e21206f9a
127607 F20101112_AACIPB armistead_j_Page_111.jp2
0c79e830074e45a302981093ad1c6471
0917cfa0df6dbf5ca97cad26fb0976af8fe538d7
32231 F20101112_AACINZ armistead_j_Page_081.jp2
d1d30cb4107bba46044626f6435194bf
6ce5b15ce303515bdea161e89cbc6f99674445d0
134529 F20101112_AACIPC armistead_j_Page_112.jp2
6bd1942adb9da13c60f5c2e5dc5c275e
22d86f24f0fe5aabbf51222d365a5c5797a0e525
116449 F20101112_AACION armistead_j_Page_096.jp2
a50e76fe22b9b2830fe7561eca5a0c2d
225126515a0eeabf59c22f702ad675b42dcf9d6f
131768 F20101112_AACIPD armistead_j_Page_114.jp2
d294eb26473a9f10f6807dab9793d28c
ea0944d7eb3584ee6a7ba88344db971c181e8e71
118953 F20101112_AACIOO armistead_j_Page_097.jp2
fbeeb588eb1cb903e50edcfa29354676
7decb2b533409a7963b2c7e36784328a84719f55
130368 F20101112_AACIPE armistead_j_Page_115.jp2
6d830cbc135da8c37e0cd98909ad4e17
d2c5577fe11ed250aac7f6a2e9aea7f96095f3bb
9803 F20101112_AACIOP armistead_j_Page_098.jp2
60b7c1fe9351a179b63e383d3ea0ac25
ee156e30d4e6b8385adf58714865b4b315fdc670
119769 F20101112_AACIPF armistead_j_Page_116.jp2
249e7ca62c0b9079c545c01442feac43
e31342ddb37a65b7729c042440ccc91949f5965e
65951 F20101112_AACIOQ armistead_j_Page_099.jp2
7032de66ae3201f63fb7ab9b15d1fd32
cf8273ec9053be08398994ccdcac7c3fbf442549
127709 F20101112_AACIPG armistead_j_Page_117.jp2
46cf1bf76d76e380e62f2b99cf1973f9
4681acd749cbd09be4c0b702e81296bb7578c6e2
38787 F20101112_AACIOR armistead_j_Page_100.jp2
593a3cacdc53495d8dedea6801dde82e
627bf2a24e2ab3860184a4cedc5c7a2b97a41484
124416 F20101112_AACIPH armistead_j_Page_118.jp2
af64e18cabe1a4ea30896cd20e75c058
c095635eff54986a9adea76842996decfd0cd6b7
35099 F20101112_AACIOS armistead_j_Page_101.jp2
d3f7e345af5ba0961e7f9ab889312cc8
e79be7d7e860bfd5deb9a9db0c802d4fbc1e2b95
132615 F20101112_AACIPI armistead_j_Page_119.jp2
de376d4314d94f52f1e6b8f47b1baf84
d668f8dcc83b068da7339d0c64224f8bc72711a6
27834 F20101112_AACIOT armistead_j_Page_102.jp2
c840bfb897854002a0ce48bebfc5889d
aae7afd6ce7f21fa5ba2547590c2bb48fc3a3087
140712 F20101112_AACIPJ armistead_j_Page_120.jp2
81af50c84b638038a9fdcd5ebbd8c3dc
54462faec18beec88189d679fec3500e123fa0eb
32873 F20101112_AACIOU armistead_j_Page_103.jp2
5d3652c1cd3afbed9e8c332eb427560b
c002f9508971c38dd793dd3c47708a589d488efd
131616 F20101112_AACIPK armistead_j_Page_121.jp2
40fd2a9cfeee5321e5d478ae32c8d77e
b36aa1bd08da4704788320f423499666f3f6f460
52707 F20101112_AACIOV armistead_j_Page_104.jp2
040b44c8e5175d0af122f270d038e047
0a554ada31b23a53f63264826bc4fceb0c3ed7f6
130135 F20101112_AACIPL armistead_j_Page_122.jp2
88caea4dea7f1bb485bb5661527a488d
566b1b5e324196aff4a01079306f256e13b8a0f6
115204 F20101112_AACIOW armistead_j_Page_105.jp2
73b79c8bfa0d2b02d63fb0dfb08c45ec
bfa31fca89e7d00e27483220d173de77934602a4
67278 F20101112_AACIPM armistead_j_Page_123.jp2
5bb0311622301ee9ae8029d68fb6f263
f0b80e67ef2f1a32078a24e7bc75c72b0939d116
118021 F20101112_AACIOX armistead_j_Page_106.jp2
4f252438e4011e4204a7068b5a16cccc
1492340f7f8efec5f5946bd4e7a5bf572fa4c6c9
1053954 F20101112_AACIQA armistead_j_Page_014.tif
274fa09d2342b6475abd98e937b1e1d5
751752e1f966c421085cf8a2f9359fb8dc34f71c
90620 F20101112_AACIPN armistead_j_Page_124.jp2
f05bc2a8f932d1293fdf9042b54b3c84
018bf3c2500db5f378ebc5a27c739ef3e0b7bb52
121976 F20101112_AACIOY armistead_j_Page_107.jp2
2adca083d10737e626126a9fbee9dae2
f2757e33f0e69938b98aa525ecb4cfaef2c0346e
F20101112_AACIQB armistead_j_Page_015.tif
561b9dd0e0d2824762770018e2fcc4ff
577ad8a522cdc83caf85412b4580e79d42d9e3ee
20766 F20101112_AACIOZ armistead_j_Page_108.jp2
c88d11e7ed50bef3f8760a86d9465a8a
e0d7294d6b1a18eb31dc8dc880d31e3f81c1571f
F20101112_AACIQC armistead_j_Page_016.tif
01b6b55651f37b2f63490759719449ca
8668da5d7d4c2edd41652a477ce3296c4544fd5d
F20101112_AACIPO armistead_j_Page_001.tif
28d76b43ebcc88e6e5e0e2315e9f3a8e
505c755886082469858f38238de414684219dae2
F20101112_AACIQD armistead_j_Page_017.tif
e1ed3c6ef9918dd1d204978293ab4166
824c41d72c23f100dee04d1349e007af8c240193
F20101112_AACIPP armistead_j_Page_003.tif
ba1c7215ca12b3d44ee75ac65f2fc280
702297f36812d45ba6411fb4f84a199f1c30c99d
F20101112_AACIQE armistead_j_Page_018.tif
e7fdffa51a0caf806bac1b5260dc1c4a
e8245f612ad062941e7f53b56589df0d74024d89
F20101112_AACIPQ armistead_j_Page_004.tif
afdecad740bc919215e4c4f39dc1e677
845b707322679e4ae28f14e85be657f07e958109
F20101112_AACIQF armistead_j_Page_019.tif
6f87780f8624a6936381d7aae883035d
a6b3a6cc2abde226d2cdf5803cfffb231b5f5048
25271604 F20101112_AACIPR armistead_j_Page_005.tif
8247593284504b733b4da571ab180fd3
30c7015f0a38c8325d8a0c25a17d7479c963571c
F20101112_AACIQG armistead_j_Page_020.tif
f11e88241bc41db7b06da1e94135503e
4be11f048e71c82692564b38519104b0c89b354b
F20101112_AACIPS armistead_j_Page_006.tif
5c98b98e07686720e6bd9de64f4e947f
34ee62903ba9c9dfc061da7899db584f1dc8f349
F20101112_AACIQH armistead_j_Page_021.tif
c867bbc52b7b07bd9f76ad230b1419a4
ae45b7141665beed94cedc8dec6d380f5eb49057
F20101112_AACIPT armistead_j_Page_007.tif
a5ce796bb00ddb97b743ff880b4e7d20
74b02cd3992fe2d3c26df4ec0d110dba600ce0d4
F20101112_AACIQI armistead_j_Page_022.tif
72b8ffc6edb38151c0d7404c333f2c1c
759de7c7fdcd528ae721b122a31f4fc488637338
F20101112_AACIPU armistead_j_Page_008.tif
68540d96fa8c8695faf106b3980a4b92
93746d1e05aeb89e0699bf7849675e615664df14
F20101112_AACIQJ armistead_j_Page_023.tif
c4b8d167cd7f2c1b3ab0d408b0738628
d062749018ce20e2a90732fa4a9d9a813df166a4
F20101112_AACIPV armistead_j_Page_009.tif
715742724d1063663482321fc4f11bfe
759ec807032212e47d0f0f16edaf14dd283e4364
F20101112_AACIQK armistead_j_Page_024.tif
f14dbee196814316a56c816f4540cd3d
98dbec734d2ee0f8631600840c257d30c54f0e07
F20101112_AACIPW armistead_j_Page_010.tif
0b922246f470de7f8bd57429e3e46f88
54265ebe3a7d040a0cc3884f1d2e6ba7cc28380b
F20101112_AACIQL armistead_j_Page_025.tif
de305be5ce2ca8efc29a70eeb73fbee6
5d754f2b788c060e607bd25f09c635d568948e77
F20101112_AACIPX armistead_j_Page_011.tif
92f0c09514d784dccce755218b3d3ba0
5634d770a801ef30d7f09befa36701327bf846dc
F20101112_AACIRA armistead_j_Page_042.tif
872fc6f3356b3ee1d05b3a7980273fc7
fd72e569ac763dc28efbb3d9c186163fcd46e936
F20101112_AACIQM armistead_j_Page_026.tif
800e552ac2ae692b63298dc7a4dd0bfb
ec0e269c8c3d9a4e8e56ff1b67a6cd7ec95fead2
F20101112_AACIPY armistead_j_Page_012.tif
87e5e0a26e2b235650e7f92f96b0bc3c
59e1766c99525243232d7e0912e7a93e63494903
F20101112_AACIRB armistead_j_Page_043.tif
cc9963962e2ba6f0cc04275cd08f6082
df06b587bf63862d1c2ef305ec94b2c0245929ba
F20101112_AACIQN armistead_j_Page_027.tif
45674fe44124ca777e785327c9061f27
210e08afe96437ba4dee7eaca9d0cfbcea5dfda6
F20101112_AACIPZ armistead_j_Page_013.tif
e7f3fe36d0838614b98f1a878d1a9ed2
4ef59cc290cf2e5a1f5bcf2349f3e7db74e8c68d
25265604 F20101112_AACIRC armistead_j_Page_044.tif
87a10cf366d1841c12730809bfa3773a
0fd976b83acb10b84816260fe328c3f50eaaeec7
F20101112_AACIQO armistead_j_Page_028.tif
5629ef175bd104f8b136dd8425265d5f
140b97d0a5cd5490457347a6431f08932a9a017c
1054428 F20101112_AACIRD armistead_j_Page_045.tif
25ba2a5f2ac4be4ba6e3fd99f40ff8c6
2fd7edf79e387d35e37d832db31e82c788630d70
F20101112_AACIRE armistead_j_Page_046.tif
a11f2bb124813ff478d8638481b12fe6
ac47a284c9406b8cad497396dea958b0967796ee
F20101112_AACIQP armistead_j_Page_029.tif
86e4b376e2d1d959b8acf279037fe3fd
75311a3690ed6b8be72556ffb77624dcecd4274e
F20101112_AACIRF armistead_j_Page_047.tif
8d00b2789dd41b6775758ae48666d973
721731f182922f2bbefe7f57a88abd9a03e7e809
F20101112_AACIQQ armistead_j_Page_031.tif
261a05fabe83c6acee7391b274510dd6
b9c48fb1a0bc56c2a619210442f12905144d2e01
F20101112_AACIRG armistead_j_Page_048.tif
07a8a5c7cae6d69524ac2e443f052ce3
e26a49cd12643539365684b6bdbdf27f585a2e3d
F20101112_AACIQR armistead_j_Page_032.tif
6d952485afc02210d8ca55c6e62565c2
aa02979bd552180ec6440a422b2b16c2e9fafd1b
F20101112_AACIRH armistead_j_Page_049.tif
7fb10a582ee59d4f9ae5d7cf40c92bf3
7e8eed2e7f1d55591037ec7af05f623ab07d5233
F20101112_AACIQS armistead_j_Page_033.tif
db4996086e8d49663374b3a6c14be53a
7952e2b538c78b8622e14bdcd3b9a455b195f34c
F20101112_AACIRI armistead_j_Page_050.tif
81ec2ad4702fcadb386fcb7915d9ba93
fbe181f48316da691eae1b892a31dc8fb2a663b8
F20101112_AACIQT armistead_j_Page_035.tif
ca7c002f4fd72699ada51e620220e9f7
1edeba04e95bf158841fe1472c89c11dbc47e98e
F20101112_AACIRJ armistead_j_Page_051.tif
10d93f1834d3ba22fa84d6c10d4c661d
d163868428d8e311498fdb34f337148d7b619391
F20101112_AACIQU armistead_j_Page_036.tif
86e2d5fa46a4d2b8c3cdc9a46ccf0619
2705919a72d61408dda848e33bced3fe4d298148
F20101112_AACIRK armistead_j_Page_052.tif
22c4ecb7631c326092884ddaff7e5915
ce0c979cf895b949460bd5e568afbfecce47d4ba
F20101112_AACIQV armistead_j_Page_037.tif
5bcbe4eb8f1626f45722959d7ce727c8
a68e63b2c1aa0e57c36e2796a4184334a4b9d6ef
F20101112_AACIRL armistead_j_Page_053.tif
c37bb5e347506743b2086c324360fc7e
88590b64a648172446b1efa21abd24e6b99ab8d8
F20101112_AACIQW armistead_j_Page_038.tif
ba7285496218a5a179b4cf2a82f0ad12
af3e95a621ebf12200493401acb709cf9d43c443
F20101112_AACISA armistead_j_Page_068.tif
01b47a14901dae340cd8e65e908e5652
ac9f4f54758da6de669e5d6107b43e5233c37d21
F20101112_AACIRM armistead_j_Page_054.tif
fa2adb580da29c7165372c810b4f9d47
89bc3d8ea255cc1a8f3616afa7d378f2bb345337
F20101112_AACIQX armistead_j_Page_039.tif
47d5ccf7d5c201aeb85d2e8a5f01f825
c9b60c79ca93cfbd665acf7b55b5ccb8e671073b
F20101112_AACISB armistead_j_Page_069.tif
037cd307a28f461c14e0fe2af49d47f8
bd6a7fb3e9bca351230856486fce285178d9ef9d
F20101112_AACIRN armistead_j_Page_055.tif
c5fd59b40b2b6633c9032d50c7757af2
19744a12f3f51ae2104a3270157c9c97a3091752
F20101112_AACIQY armistead_j_Page_040.tif
6123e956b34336cb8dca184c108162a9
bd608c80581def6144f483b6f4564a9c58724123
F20101112_AACISC armistead_j_Page_070.tif
99783b4e48be0248119b5c2513f86ce1
a8ce48603aa3126c1a335655fbd44af60f2c05bb
8425398 F20101112_AACIRO armistead_j_Page_056.tif
ce81b3e122df9ecb510f8f8ccba338dd
167a0f31994c99179c4771db102a72d816455aec
F20101112_AACIQZ armistead_j_Page_041.tif
034980f460cc8b35253600b43e5d9b4d
14b02498ae737375b76cf54067e752192241b460
F20101112_AACISD armistead_j_Page_071.tif
dc3dd7b5a6561be66676917d4e11a2cb
2988275be59e051eeec1850001a78b84bf1168a7
F20101112_AACIRP armistead_j_Page_057.tif
7b636fd4702f236b519acbd74cc99a11
85fa780da094cbe3d39e49f24818956f02a62093
F20101112_AACISE armistead_j_Page_072.tif
6c60981fe574ca605ac4470cbaef0a31
34ad5a90ab046571de6cd031c939a23688af83d0
F20101112_AACISF armistead_j_Page_074.tif
9485050b71aa8df62aa29f0baf1bcc0d
d41b8f373a945a7e5ed987a0336fd50a6572e866
F20101112_AACIRQ armistead_j_Page_058.tif
e8f0082ecf7f70f7ba11feb4be3cc33e
7fbada311288f7b93d8863dbd345a7419af7ade6
F20101112_AACISG armistead_j_Page_075.tif
42596e2ed99b965839a15932fad144f3
a732fac47732ef85633cc38c867bc907d2703a09
F20101112_AACIRR armistead_j_Page_059.tif
655470ef8212ef9f995379724f5e7f4d
ff42f55c05a0f6cfa906cecd6407c40e3b2c83c9
F20101112_AACISH armistead_j_Page_076.tif
078f2fa04305c34241c3836180283720
e4c6483a00b9f3a567c951a0582b4b5022b2d482
F20101112_AACIRS armistead_j_Page_060.tif
7dacf5db50c913ef3a382ccadb4529ed
4becec3daa2ae7aa5949ce207f1271434e20bf23
F20101112_AACISI armistead_j_Page_077.tif
8f4de10bff7a885d9797a4c3dba8b1de
49642095126524c4791f6df117479ea1b2b1bfb6
F20101112_AACIRT armistead_j_Page_061.tif
41ab5da4966ac9bead5e74eba6fc7ffb
0fa61889a2df7e54a7405a0de26e5bfc192920ef
F20101112_AACISJ armistead_j_Page_078.tif
a9d67dfec27d26affa1c9d312288c795
3db333f137620ff4af9314c82cafbc9cdae284a0
F20101112_AACIRU armistead_j_Page_062.tif
830f00a3c1eb5ff61fcbf648a0250323
d3996a995141432632774b56a9a33462aa48329c
F20101112_AACISK armistead_j_Page_079.tif
f76229a7d33ef5b35cf925a1c318033e
53db2194d60338811423ab7490e4ccccae08e8b2
F20101112_AACIRV armistead_j_Page_063.tif
1165f2d143014e6998e6cf179a1f0d0a
80c22db8f8a8b2ea18826ff05bb2e8c36ec137e7
F20101112_AACISL armistead_j_Page_080.tif
b927c251c15dc61f07170501afb2e11c
11b6f55ef6cb6b5a6cba3e0416802d2320a57e28
F20101112_AACIRW armistead_j_Page_064.tif
7eaee0ebdf8b6eeaa69d493956e7d390
aad008183c0760a3a9de9f88f9230e6e8854004f
F20101112_AACISM armistead_j_Page_081.tif
4df171c37c5635a7f37c38796735bb0a
55737c0066eb24ac4ae07de050f75a84d3ec52a1
F20101112_AACIRX armistead_j_Page_065.tif
0061e910a1cc25e5dd95b6e4c9a9b1a3
5a281f1974da42333eac3c8994c0fbfff93061ad
F20101112_AACITA armistead_j_Page_095.tif
1b4dfffaff3d14945ce91e7b02b45aa0
5348d4f475a553f81735fb5d19c216d0776b8b9b
F20101112_AACISN armistead_j_Page_082.tif
5b349324c7d93d8f581b27e3b4981af4
255fb11295aaba8213d57e199d8db9363fc951f6
F20101112_AACIRY armistead_j_Page_066.tif
7027840187a9e029d953062b0d72354c
1543c9a20cc370c6d8612623519ac49d8a80f6b0
F20101112_AACITB armistead_j_Page_096.tif
e39563fa3d4992b84a04c8d807c2490f
284c2c07ba2e67d7bcd0b3d4c11430d6e0d3a68e
F20101112_AACISO armistead_j_Page_083.tif
c1a28fd422ac969c70341c015a4d0b4b
953943021af1f4950f64ec418c63741e4a5fa651
F20101112_AACIRZ armistead_j_Page_067.tif
0265662acf2a4c02f95af76628267c7d
a2200f75d5683fc5cd4055ac67d792d86102763e
F20101112_AACITC armistead_j_Page_097.tif
77eb3baf1c2e44bd6efd13a97ec51cfb
c2097ca5112358aef8e0d65f1a76e9740f529ebd
F20101112_AACISP armistead_j_Page_084.tif
57d85df4501576377a84affb5220cbf2
1619dfae37213a467354638ea7bc5b29df12e866
F20101112_AACITD armistead_j_Page_098.tif
e15aa9f7fccecff2c2eba5a9ef6e7331
1bbf8d11653eaab1e6fab3712b2b5f054ae270cf
F20101112_AACISQ armistead_j_Page_085.tif
0980187ac5d43685532511f49962cbc3
b449223f47659fbb053f49b9e41101bec48bd7e6
F20101112_AACITE armistead_j_Page_099.tif
48a5d95351f6d512a0a3b5e4c0c4d7bf
00bf14860019d23ff7092d7b4d89455db1e7391b
F20101112_AACITF armistead_j_Page_100.tif
ebc3d345c46d521486d716bc27b39a1d
0d1161449ce55121889f157ff3569890ff4dc435
F20101112_AACISR armistead_j_Page_086.tif
5c5266377877bca5ea1536df323bbd1f
858bf262bca3d1b94de75503e36047d7aa70bec2
F20101112_AACITG armistead_j_Page_101.tif
83c4b95b71294db1a194716d41784fdd
f067d39909041b00e8f063ef2affee16fd26f675
F20101112_AACISS armistead_j_Page_087.tif
9835b5fd12879b6399f1ea6fe7a2d265
5cf905eff875ab3f103da54e168c45519ee8ed0a
F20101112_AACITH armistead_j_Page_102.tif
248cabe8f8704d6602fa86b2b80ad08a
b39956932ab1e25d43335b32d7b029ce27cc433c
F20101112_AACIST armistead_j_Page_088.tif
4867cdad90e92e8ba2a6382d42532f19
be69f8fe1b452ed6af3f2dda76f9ee0b1a02dd60
F20101112_AACITI armistead_j_Page_103.tif
99d1ba3d122c507d7003646fb66e8111
dbc08b7b4d6252fbe5f07df16f2bca16d52f3684
F20101112_AACISU armistead_j_Page_089.tif
31fe973e1e40967398025e4aec9b99fe
1c86d17a4a3bdeef759a0bc5c4fc2fb2d5244441
F20101112_AACITJ armistead_j_Page_105.tif
4971ba6471aa784ba4963b1604464688
106c7d060c6c1ac133aa53f039c968333113b4e4
F20101112_AACISV armistead_j_Page_090.tif
d242118a8e8fe414c47fb2012f73892b
55e111dcfb14b2be230869c3af53ed85949f44cb
F20101112_AACITK armistead_j_Page_106.tif
61a20cbaac69090351333f8d8609d7ea
a2f0c4f11dfbb5250796a95ef99e03a0ff7a7cb6
F20101112_AACISW armistead_j_Page_091.tif
f56db11895ef4a2d506062a2f2747be0
c385ad78fd92b9d8c48ba9ee9ceedfdf84ce4fad
F20101112_AACITL armistead_j_Page_107.tif
56d102fe1f0c8e8d491db45ac6903b76
3b2ac9496cf0f74dae923432f62f4d284a622537
F20101112_AACISX armistead_j_Page_092.tif
b7a47f60e69881e04b6beb9eb574326b
0166da3e8d3fb725085e1f58429243fca54113f0
F20101112_AACIUA armistead_j_Page_123.tif
67a6bf8e641d53fb20945c5eb039e37b
82b9fb31e47131345d1aa8aed54e457ec25a1a26
F20101112_AACITM armistead_j_Page_108.tif
cbf40ab3df44528f5c2a50421590ac23
0dda90c3cea75cf211b51f1e7a0e60db3fc7f7ce
F20101112_AACIUB armistead_j_Page_124.tif
63ce18c8f91348d195005b683196b509
bb21493ee094ede57f79b6538f0149b433def36e
F20101112_AACITN armistead_j_Page_109.tif
bc613666a61be961dc75f7bb70da5aaa
6f680ed8541ef6d41a6c4a6145296fe2cf01b2b5
F20101112_AACISY armistead_j_Page_093.tif
b712387a405fcf0836ce37ce9524071a
2399ef416ec2bc2434282882ffe2607964528ea6
7898 F20101112_AACIUC armistead_j_Page_001.pro
33f039ba79f2aa3acf7a7dec41995289
cb1454ad3bc28dbfbd5faa9c5d566baad23cd2dc
F20101112_AACITO armistead_j_Page_110.tif
18efff0140360a21a0d282a16eee7a4d
3b668bb614be729261602fc67da8207eda7f12c7
F20101112_AACISZ armistead_j_Page_094.tif
ca88318234e5d7c759db86d640f1982c
03b98805827e92e664d93f892a3d34ce953de600
1020 F20101112_AACIUD armistead_j_Page_002.pro
cc401fc774a65e13165065ed3b63474a
112982ebd0472d43b74743733a4dce1866744d1c
F20101112_AACITP armistead_j_Page_111.tif
9105424e6bdd721323d42789533248d6
7c6050571924c9bf7ec54bb812daaec97cfb22ee
5011 F20101112_AACIUE armistead_j_Page_003.pro
95f18f5c79d6fe26cc7435a5da2b7dab
706fc774a6eac4d068dc8e60f133bd6c5ff622af
F20101112_AACITQ armistead_j_Page_113.tif
796fe874145ebaf9f64a3f14c3cb30f9
5cfe08b932dfc1fe9650edbad581b4f093a2d942
27117 F20101112_AACIUF armistead_j_Page_004.pro
3706c388a8bd4cb53a06d3dd99a0df1a
c1b3fe2e510e5dc6a24cc8edc7b3b240342bb032
F20101112_AACITR armistead_j_Page_114.tif
0869cde99fa13673d3bdea8bbe08feb4
310152a86a522c95990114777c63e08f6386ee69
95877 F20101112_AACIUG armistead_j_Page_005.pro
5c7ec8149fd6b163d5f893423e1581ca
f8c2e5c0162dc3c5d881820c71e7b0d801904d12
2245 F20101112_AACJAA armistead_j_Page_038.txt
0b63c8aaea6c35c4df8e9bdbfee63300
8ba3c736399dfb4a8c2769b4e33f802433c5b8cf
64222 F20101112_AACIUH armistead_j_Page_006.pro
da07238860bbafcaa7671d964593b1a7
b5a52b185b7cb70b8b7c8f627090bebcdfdebdf6
F20101112_AACITS armistead_j_Page_115.tif
1e1d2a1f9ab1b6922fce46242bee18f0
3eb215e03b4110ed8e11f26ab9faff40bb319dcd
2275 F20101112_AACJAB armistead_j_Page_039.txt
05988d10eba527fab06e626e2254e69b
afe872d6c953eace854ca75d8cee877f7761937c
68127 F20101112_AACIUI armistead_j_Page_007.pro
f22b9bf2aff2b5389a91555ed6cdd23d
2a80133244af296905541bb565d287bcd4241b7d
F20101112_AACITT armistead_j_Page_116.tif
3d5e408e738d55e775234be2d9eba8e2
6de09dce7e7584cc85f8440c3f3a313502c25895
2188 F20101112_AACJAC armistead_j_Page_040.txt
b2cf34b0cdbd7856c8dfcd67621028c5
993ff39bd443e903daa62187d874c766faf63741
75429 F20101112_AACIUJ armistead_j_Page_008.pro
e68d6591bfd3cb1c3ea45401a4249bc8
d0afef99c96df874c121ff065b3babb455797a88
F20101112_AACITU armistead_j_Page_117.tif
dfc548cef0986c2f54562ae1662e5bd5
573cd2919a16673c60095593a20969a411739d35
2135 F20101112_AACJAD armistead_j_Page_041.txt
abc2654acaced42da6631bd88457f719
52308512724193d1967f7b3945c2c67d1e1eaf6f
22998 F20101112_AACIUK armistead_j_Page_009.pro
44a1ad51c7e98f42ba4ac3826a4e142c
882ad8197505f1823811e4adea39d610f80990bb
F20101112_AACITV armistead_j_Page_118.tif
9abdab87028af80be12426393c0f989d
8bca82729a557705ffa745fd15631c91c06df564
2234 F20101112_AACJAE armistead_j_Page_042.txt
def64eb0ba13e20a9049df5c8d1f28ba
2fbf9479836860512d19a2d90d8fb46e05f6fbfe
40758 F20101112_AACIUL armistead_j_Page_010.pro
4967bbfdc3eafad600f98e1961ebf9e1
98e65ce10da49424e2b58115411f3538d50b130d
F20101112_AACITW armistead_j_Page_119.tif
94b4c416a4086835bfb956a89ce8dd5a
24d9e7a1270588ba38de7aa37905932cacb3f771
1137 F20101112_AACJAF armistead_j_Page_043.txt
c8d493f1298955ad521e766ba15547e1
e30fa075e5d6c223a58a261280fcdb6eea5e9cfb
54795 F20101112_AACIUM armistead_j_Page_011.pro
f259c329b8d6c2a019370340735e2216
54b8f9b9d6e1b8921566263c9b2a7c4ea9e7e9ed
F20101112_AACITX armistead_j_Page_120.tif
aeb52325dd10cf69df3ef992d6677db2
b8401fe2cfd0e03f9e9bec46c0c001f6fe522642
1338 F20101112_AACJAG armistead_j_Page_044.txt
fc48e7df115d002b389218566494806f
ec898a2964b7729d77c5f3664f545907c1623680
53633 F20101112_AACIVA armistead_j_Page_025.pro
f656f30e2625f47fe1159e798d413200
26f9a8986808881b026f5ae159e01b26e55dac57
54071 F20101112_AACIUN armistead_j_Page_012.pro
6c74ab570a36ac9dba0339751998bd0f
e72ebdc8806e87862601b146ded0649179cb8206
F20101112_AACITY armistead_j_Page_121.tif
60190fd4a6c078c68bba81906e9f8e7c
db06b721b8b393757b715e5d7dee1c20c6bc6086
1179 F20101112_AACJAH armistead_j_Page_045.txt
b120e77afc91cbf16cb716f213b5c30e
578389cf045292bdc1b0e53780f67a06202dd2aa
51671 F20101112_AACIVB armistead_j_Page_026.pro
697b920844d6078faff04ae51092aa17
769da77636d2fc9ca57ee41fd8900e082f475d9a
54732 F20101112_AACIUO armistead_j_Page_013.pro
478549738af35773c561b67653b6b36f
7ad72d7d599e0df372ecc8a75c9ac1723807855c
F20101112_AACITZ armistead_j_Page_122.tif
010f94255577f21a9f38b175b95f24f0
fdb43d2c06703168d6381b4c2583c8e7f8977aad
1393 F20101112_AACJAI armistead_j_Page_046.txt
76a5a3ef05dffdcb8378400be35d5378
2b4581a88f512b35c8300618c4dd0def2c3da2ed
50974 F20101112_AACIVC armistead_j_Page_027.pro
78df65999c3e0ebfaddd42b3f208de2d
2bee2c4e13deea2d54918987aa039b8d4b5aabdf
54940 F20101112_AACIUP armistead_j_Page_014.pro
7061dbd847aabccf7a849318b1c97155
aeb051a3272aa6c9af65981d5b8d7eb1cf7578aa
1150 F20101112_AACJAJ armistead_j_Page_047.txt
78348051ab2e971372ff021136e05584
805a7d7382e02e9ad5ac3a9bc99a081148ee341c
52956 F20101112_AACIVD armistead_j_Page_028.pro
1706a47e041c6ab7dd6a7bd84fc142e0
9b426cc7dda9925aea3ccc7984130618c3783c87
55460 F20101112_AACIUQ armistead_j_Page_015.pro
bcebf047df922c0e0da858987c2a048e
9bef12ad4442abac9ef01692a6931127b40d4229
1574 F20101112_AACJAK armistead_j_Page_048.txt
cf3ce022abfd3992e9205f8d2f46a469
fed95343e50700589eac4faad6295fab6aed1a56
55413 F20101112_AACIVE armistead_j_Page_029.pro
cdae2917ce5dbbd474c80285d18201c8
01900461569ecbfe9e08c41fe35bab66caa61e45
52748 F20101112_AACIUR armistead_j_Page_016.pro
6c79afcfa43f9fef40d61fa560330c51
2320f1d2afe876fb8ef5cf33667fc9d56d29389f
1376 F20101112_AACJAL armistead_j_Page_049.txt
8821982828c9130d883ef33b0ba77131
45eb4b32a278bdbb15948e4403d905db2d5735f7
54416 F20101112_AACIVF armistead_j_Page_030.pro
ca71a7abb18b42607c8d962c83463fb8
3276df5ddabbca028555a64e35c30ebecc4be362
53436 F20101112_AACIUS armistead_j_Page_017.pro
1d3a916e39dcca524e94c1c4a4c3141d
c67b28fd7a4db63a574ea9fade12c86802ef1fae
1443 F20101112_AACJBA armistead_j_Page_065.txt
99d5611ff0268d4aa95c3f7e0ec2b344
e4258c20410dd4997942dae74a56c48c2c71fa4b
1070 F20101112_AACJAM armistead_j_Page_050.txt
12b8e2cf8063a817abcc4c5b7e819941
860d6d4e369077a871e34d85f6763d5fb53d221e
54043 F20101112_AACIVG armistead_j_Page_031.pro
29fd9bb25f72942186cbba10fa3d8581
704006bcf5baa626249f3ad3428a5977aa488425
2102 F20101112_AACJBB armistead_j_Page_066.txt
041f55ebae2693210dc0cbf0bd028cc4
0532981f2b7487b115e7e5685536e8aa9bdbebcf
1209 F20101112_AACJAN armistead_j_Page_051.txt
a4e2a182285ae3610cc3d09fa9012a7b
a5465319acdae08a040ac6c88be208cec6292b03
56078 F20101112_AACIVH armistead_j_Page_032.pro
96ee2d377429fe6df4b9820b7ccfc7cf
b5803f119159c88cd2e03b81eeca029248eada76
44564 F20101112_AACIUT armistead_j_Page_018.pro
3efa43fc4412f6365a75c00d28cb84ac
5df3d5c9bc9f7bb14cee65ad15da9f92f32c6a18
2176 F20101112_AACJBC armistead_j_Page_067.txt
a283b6caec5fd2b5d8bd940366eb17ae
13a2fddd93e1a71dd1d6abcd8271146455b7ae65
777 F20101112_AACJAO armistead_j_Page_052.txt
f7522d0738d7b55f90525ad86ed057cf
04c3872fb8d86d31b9290a94b84c367b21df7764
53433 F20101112_AACIVI armistead_j_Page_033.pro
0a36afe3817c375f5032bce22dd4e436
e7ba6143be23add866817e6612bcfe08584cbd00
51805 F20101112_AACIUU armistead_j_Page_019.pro
df2358a3ee18b8b1bba1708ca576c506
9b59357daf6f152877dc21eb0e095f8e73720ede
2057 F20101112_AACJBD armistead_j_Page_068.txt
fc552e1f129107bd814f85d811899974
91761590209554e8e9341041b3bf887b25c1a28a
1225 F20101112_AACJAP armistead_j_Page_053.txt
d4cf75aac22f24f330bc59fa17a663fe
c85b2ed3050962a8c4bb24dc3a8cfd4d9cf1bb7b
55734 F20101112_AACIVJ armistead_j_Page_034.pro
e36b2196d376aa7417ad55e2e3a2a404
2076d87796a873719647ab0d59d49eb577fc182d
56515 F20101112_AACIUV armistead_j_Page_020.pro
7a76b97d82d61bc6bcad939a248963e6
455f1c80ea6cb3a1ac69f3c8b42c01aa6ee1b128
2122 F20101112_AACJBE armistead_j_Page_069.txt
726f111ac43f406e0a7b0e4f0a28546d
f233d6bffeb4cfd98490653b5472ee76a42ffcb0
529 F20101112_AACJAQ armistead_j_Page_054.txt
a1d5faa67e55a0561a8de27d65eaf614
209a3f2573ce5f235ddd04654308aaf53014d386
54987 F20101112_AACIVK armistead_j_Page_035.pro
966f8f6ac205540ee0900bec3d3deb40
f57dd88436f3d9630fbbf39e6c0aef3f3fb9c69b
52968 F20101112_AACIUW armistead_j_Page_021.pro
2b06186f39724f75440812c671a56ed7
4ea22206b9a4b58b6c81ee58650ec6abee86fd95
2144 F20101112_AACJBF armistead_j_Page_070.txt
8b50580d57e75a523117c9dda8e85162
51e10086b3afc2afb90906f6dcbdd990e4004f10
1075 F20101112_AACJAR armistead_j_Page_055.txt
28eb96dd753d7bd8efac763092cc8c3b
242bbddbe95b19b3ef237112b46f158abeb7190c
58113 F20101112_AACIVL armistead_j_Page_036.pro
870c339f450d44e304a9552b40915b65
acff35c60d294c0aaa7210d20aa1ca434fb2751b
52139 F20101112_AACIUX armistead_j_Page_022.pro
b845de4af1e2b3a3af4aa81a781a88aa
ea5ce9828c04bf7c2bb3b4161583b7113f920669
2068 F20101112_AACJBG armistead_j_Page_071.txt
e83fc1ad8b1741f3d3b2259272ccea28
7b9a9a56c83e5506af541d6ed330922ca793f7eb
15476 F20101112_AACIWA armistead_j_Page_052.pro
4a01f2a58bca830b95f0da4a4a892091
bc705da9ad73036af259fa917432f9226b824543
701 F20101112_AACJAS armistead_j_Page_056.txt
9182057bc95609f57e83172864984e62
ff461e26604bad504e428f0e4d6c65c18d8b8b94
56910 F20101112_AACIVM armistead_j_Page_037.pro
710680935a7039ce1235d4a49a248658
6d8e27df50464d76f97c7f77d834879e20d52a95
54542 F20101112_AACIUY armistead_j_Page_023.pro
53ac5f0196ea60ead14fcde297144044
35fed62ec902d7c165d4c461de5a86e44c48c271
1774 F20101112_AACJBH armistead_j_Page_072.txt
e8f34bb9a60ebbb77be5a35b85259702
6bca9acd02a92711836553fab6eaa0e523b9ae04
26893 F20101112_AACIWB armistead_j_Page_053.pro
b1af6e0a8bf10c6d925295e8cb9b70c4
8d0188812b726bee7ead7c740c577ccd797dd8c2
840 F20101112_AACJAT armistead_j_Page_057.txt
38eab6a76e676effeb27540702ab0475
742bf61c85b895c9060113b51e4837e35f5d4640
57404 F20101112_AACIVN armistead_j_Page_038.pro
a04d2316dad70398dcfd7e9e738ec5f6
680ab22a0aeec4c284bbf516ac85b4b1e0170790
51580 F20101112_AACIUZ armistead_j_Page_024.pro
0fa213733873b6b54d79bfa7b158969b
1a5270c8fd0c6c0e1e8e977b91f9e41def3469c8
1896 F20101112_AACJBI armistead_j_Page_073.txt
ed01bfce566f498605bc06fe1f51c94b
089262f0b57b5e943a5f9349e1deb92a86abff8f
8619 F20101112_AACIWC armistead_j_Page_054.pro
cc15fbcdc06027e81c124687367f1fca
628e73ed4d7eaa99cf8bb10ff2e713cb7464a6e5
398 F20101112_AACJAU armistead_j_Page_059.txt
4bde8cd0e317c9f76f0a31dbfecbdc79
ded15b8e8825f5be04710de86b0a1030361585ea
55915 F20101112_AACIVO armistead_j_Page_040.pro
60405b69200820693601b55ec6112b08
af9e970845b851dbf470582a709fc8b1f32612e5
1937 F20101112_AACJBJ armistead_j_Page_074.txt
8f42b0c31c2ab7e1f91c68993cc0a5a8
17a6808a25cda7c397647715285fe0a270c6b51a
14672 F20101112_AACIWD armistead_j_Page_055.pro
5a0a0dc70b9cb57fa77bd86467f8001a
106febd20e43a26f150905f098254055c8978802
811 F20101112_AACJAV armistead_j_Page_060.txt
b037c21a3b0c2f6a5acdd687168ef84f
d8243d2dd4127cbaa87352d753aa4b261665d787
54253 F20101112_AACIVP armistead_j_Page_041.pro
fe88b09b1003c124aa4535516b69fb9e
00f6d842004b09c0cb352dbc80bac3badf96a1a2
2104 F20101112_AACJBK armistead_j_Page_075.txt
76c78e194ccb9959b22b05415e991096
98b72fd6c765e9f2a01982cde02c15796cd53f83
8409 F20101112_AACIWE armistead_j_Page_056.pro
a2695e4082a6485452d96d36d8bd64b3
49fd705fe1fe045f09a3217fea57e7fbf2cd316d
765 F20101112_AACJAW armistead_j_Page_061.txt
f8fa5a28f4b9c0cad40b22afeba58973
9f6e34cda2a96c6bf7152348b010cfabc4cecc2c
57150 F20101112_AACIVQ armistead_j_Page_042.pro
2cea6b4429c6501661e45d32af3c8c28
73c07ea73a7a298b8e3dbaf4ea1d1d3c8c6535b8
2241 F20101112_AACJBL armistead_j_Page_076.txt
88365e19b1f27e5eb4da52cc4e273b5d
e6b26998391f31c444a726c443cdccd66b48ab44
13940 F20101112_AACIWF armistead_j_Page_058.pro
6096b634b434b3b1c75bcb39e669e269
41ed3c921be024971727e0781d1bd315b3790869
425 F20101112_AACJAX armistead_j_Page_062.txt
b09e3bd0773c370ab25d60a4b69eff70
84ca85979794e0710d9038c2485220a95bed08a5
28718 F20101112_AACIVR armistead_j_Page_043.pro
5eab5d81186b61017a0edcae904e22c1
17d0c46151618b5a90f83b6eedcc88a8bd64eded
2172 F20101112_AACJBM armistead_j_Page_077.txt
a1390f59bf083fa06e5d751dd5230eea
89d5a92bdbafd89eea38f1ae54786ea29b3cdec5
8253 F20101112_AACIWG armistead_j_Page_059.pro
b63d2e043caadf030c3ca281fa30222f
cd940a97e862f8b9c092a2c5ea92d73d0c69b96b
1991 F20101112_AACJAY armistead_j_Page_063.txt
9732719aae9ad2205a4bbba13e1fb86f
ba423dc1ff2a76e60531450822ae49c20f8ac36b
26408 F20101112_AACIVS armistead_j_Page_044.pro
ed5c35d5e66d4689bd0eff799b00697f
d3ecccf3ae72aae47144bcda1e1685b558b0a1c1
F20101112_AACJCA armistead_j_Page_094.txt
51fad9c478809f90a7bd57db6bb2d866
c4dee2ba3cde9687b7dce2315fa6c4474180e8cc
939 F20101112_AACJBN armistead_j_Page_078.txt
bad2ebf0d4eb0bf92852c016228f1919
68307e0bf294ac6cf7b4267157c7c960b1541621
17879 F20101112_AACIWH armistead_j_Page_060.pro
6bde696b82dd1f36a403f7dd5483514b
08b2b230cfb5320974d72d66ca2d8c4a65eaf04a
1805 F20101112_AACJAZ armistead_j_Page_064.txt
755fc05df2059d97d97cb4a1298b5570
7c238413bbf20c3a6015eafd902a618c9b636dec
25165 F20101112_AACIVT armistead_j_Page_045.pro
3b19d2bada2062156a5d8e03d853d572
24c2192d8dd7a998940c2b219401fa82ea38ec33
2112 F20101112_AACJCB armistead_j_Page_095.txt
627bac47404fb8958b0595d6f1367ff3
3ab1243bcd7dbfeb4d38012c43b0c9b647cdec0b
1175 F20101112_AACJBO armistead_j_Page_079.txt
c30c2f85277e26e76a1878169eb8dd1a
3b2b989179796ef404ad783cb1cf274451464c9c
11202 F20101112_AACIWI armistead_j_Page_061.pro
0a8bb151aad2f0e306f8ebe4cd19c2e9
1442c6183b9476b1a4f463867203470ad5f8d079
2088 F20101112_AACJCC armistead_j_Page_096.txt
021173b5d3c208e1263c3478d8210dbc
7674ffc8b0770f1ed0efd895f5677322399c81f6
689 F20101112_AACJBP armistead_j_Page_080.txt
841275b1030e0a533241e18e7aae6762
a2754e8c48baf9e44012c61be6151ed4096e1a4c
9195 F20101112_AACIWJ armistead_j_Page_062.pro
84504c2da76588bc674433d3864908e2
8cb4db15c21412db49df64457f8f73f9b1a5e649
30763 F20101112_AACIVU armistead_j_Page_046.pro
535ad8fc95f849f59d484442156c9d27
7998aaa6c38a1362c9fc43b170884f8ced025147
2151 F20101112_AACJCD armistead_j_Page_097.txt
7037da11c126ee1a512241bc2ae1ae80
64fbec4c6acc4bf9badfb0ec311ff2184986427f
530 F20101112_AACJBQ armistead_j_Page_082.txt
e35df10e409671e933b18d1bb9c331b5
c3ab462c3fc1cecff91e81d7859d02374fc8465c
29146 F20101112_AACIWK armistead_j_Page_063.pro
597ff61832979ccac938cc7295dc8441
67dc4a6a2bd5a888b1571c16e91c4c09827422bd
19490 F20101112_AACIVV armistead_j_Page_047.pro
f7d26764e95f754dd21239aaec411bb0
05edc489aaade46045a953651aab70a70a713904
120 F20101112_AACJCE armistead_j_Page_098.txt
2cf69a0a66766fc45bd37eb997a99529
79e1cfa56bb34eddbd280f0e0121aa7dc6254a34
619 F20101112_AACJBR armistead_j_Page_083.txt
14a7bf4cd6c65492ab432a8b15c96457
3819e4f6b2b55b108969a8355407c6b0c50e55d7
26066 F20101112_AACIWL armistead_j_Page_064.pro
8d6440e8a10d35eb2a76a1e591f3369e
a00ca9408ffc703c23eb043a1348d8b50071e94e
29261 F20101112_AACIVW armistead_j_Page_048.pro
a9d256b6e5e2f83417860d2d1f2816d0
3cdbe4a11b182ae121e324e24de4ee40b0b867e4
1367 F20101112_AACJCF armistead_j_Page_099.txt
6c2fccd35b6b77ff12de9a25f8561e46
7a188c10dce7e2ecc4c2d33e0d1cc017f44cbdea
14594 F20101112_AACIXA armistead_j_Page_080.pro
413a6ed4fff0de07bbf0d8df22ed4491
76a72cb8e672e6a3d48504769732f605d27e28fa
677 F20101112_AACJBS armistead_j_Page_084.txt
3ed332938577db93289fc071a10c45f5
076a9a6e0f9667321d880f27992252cc3e11a2bd
21263 F20101112_AACIWM armistead_j_Page_065.pro
8e334ce9fa2b0ac8a963f925f7a9f58d
4decdac48d8db22c4910a919dfb9365917e624ed
26333 F20101112_AACIVX armistead_j_Page_049.pro
15339852c7971a591d8acca24e3057b3
efb8aca005d7ea5ab81bd78f3a1e4cfb827d9022
675 F20101112_AACJCG armistead_j_Page_100.txt
f10fff70525a786b6d69dcb490902291
8aa3135172293c1440886b8dc99d3dae8f186532
10743 F20101112_AACIXB armistead_j_Page_081.pro
7db908210f5195303093eda8ad2308e0
951b6eae75738a1809f81d5c1e0312ff94cb641e
2008 F20101112_AACJBT armistead_j_Page_085.txt
7878ac358734c4cba6515f24e6fbb83d
bed7ee3b396fc8cf85f43c54729af15bcf6a6bc1
55455 F20101112_AACIWN armistead_j_Page_067.pro
014b7478d38402935aad33a0338538ac
194270162c282e8d919a7008391cd5983c547534
17463 F20101112_AACIVY armistead_j_Page_050.pro
81cec0e2b3a921859f96836d1d466fb0
0e67862064d1322122673509d0a89df7d698a8f9
558 F20101112_AACJCH armistead_j_Page_101.txt
620e664a7ffe13e934ff15400f4415d5
edcb42d3e005fb7315ef004e173d96780f4e8676
10731 F20101112_AACIXC armistead_j_Page_082.pro
bc8a4dd57bf2423f872dd75ffa66ea08
445bc960847a686c6201cd7f07ecaa26fa9d6311
2177 F20101112_AACJBU armistead_j_Page_086.txt
c63de575c2b5db3bd54f432e6c1f5427
51db17b468173ea2c09e4a97b7f30a0d5f452c4b
52111 F20101112_AACIWO armistead_j_Page_068.pro
190ddb739cd9d4c0730c63551355da03
39840dd28ee85f4b4652268701e24225fb2d7ef2
19755 F20101112_AACIVZ armistead_j_Page_051.pro
f8f1bfe0da1ea8d035be641e3348c7e6
5b8c3093df845e97c0193132cc0c685a7c15ba01
400 F20101112_AACJCI armistead_j_Page_102.txt
4b665fb3884c455666da0328a8af0e04
69f5abbee0433bc28b23c5e586c4ca4637042009
12347 F20101112_AACIXD armistead_j_Page_083.pro
8f693070d4b8bff044fbd6661cf7ed10
77c647ea73200ea87cfa239c441a6db62265147d
2157 F20101112_AACJBV armistead_j_Page_087.txt
0dc1c081c260ea14bb88f7c74b5d027b
3490f78781aa9883500ce1afc0b12052cb2bd2c8
52804 F20101112_AACIWP armistead_j_Page_069.pro
ad1c3835757f1c970b714dc20e125f34
3ee45dc3fddc05e94181b9403ba5cfd2e3365326
556 F20101112_AACJCJ armistead_j_Page_103.txt
3d3ee1711856974c62cd7ea6b264655e
03abba1acb89d778dfc65b27ba892f91b3b60e95
14270 F20101112_AACIXE armistead_j_Page_084.pro
5ca0f4bf49337822d8b7979a2ea9e757
beecb034f7a06c347f9e752b86a24293d1b29622
2058 F20101112_AACJBW armistead_j_Page_088.txt
e2b6edec135577c1bc65eadf08f48a75
0a4521bf64dee7d9a6db595c54754e303d91764e
54653 F20101112_AACIWQ armistead_j_Page_070.pro
5a3f526a3b14346fc19d12470fc6724b
9b75e17d6949d100fecb714da5ff2bdf512ece16
1162 F20101112_AACJCK armistead_j_Page_104.txt
1712b08f2503933386ead9c41f406ea5
6501d605ee0e8d7f77b040bc2144246f863f4ca9
48250 F20101112_AACIXF armistead_j_Page_085.pro
872460258cc5605b82d0f52b20e43e9d
59838977beb4cf1efa26b90c84cd96d3ae8ba43c
2025 F20101112_AACJBX armistead_j_Page_090.txt
df4c7df5d06a5395d6c55c1abca5d7a6
c82a7d1e385fb3cef6e48e839647d2ad14a8a888
51181 F20101112_AACIWR armistead_j_Page_071.pro
5100fbb35d6fd07cc086f6fe541c6430
0447273fa8d4d2bd998ec0d38084c61d29b97710
1236 F20101112_AACJDA armistead_j_Page_123.txt
7401f9d142bf324aa5feed9e4f33739d
e36c9e93048cb922aa9dbebc5f27c13c4150891d
2162 F20101112_AACJCL armistead_j_Page_105.txt
b2938d1453db722f9961acbfe7c3a241
87461fd6abe63acbd4297369cae6e1b49610d852
F20101112_AACIXG armistead_j_Page_086.pro
82defc1232fb0041e1c0caa935695e68
3b40460762dbbb60dc9547c53174dec249f4d0a4
1795 F20101112_AACJBY armistead_j_Page_091.txt
e07f3ce266c18d09eaa11c64aa4b172c
e9a8bfbe06ac5e6f0a1931a6d7d4d0e339b957f3
41606 F20101112_AACIWS armistead_j_Page_072.pro
fbc3972a5bdcf3b5301e4f2648601135
4c7d18923ee5542fdfc4f6af4be9ceab0afd3fd9
2146 F20101112_AACJCM armistead_j_Page_106.txt
bf5d4b20d494774329422e7b72eba104
fae267a1b47786cad6835e7488eff8f460d6a442
53963 F20101112_AACIXH armistead_j_Page_087.pro
c47cfc2372858808ec5ab2fc56028ec5
6fe4894f7670fa62215669883d946b9b7ccc9748
1959 F20101112_AACJBZ armistead_j_Page_092.txt
5d12f997bd1b3312fcd183520f57474b
04efdfc0842d0230026cde01b947cc9ae9c21122
47311 F20101112_AACIWT armistead_j_Page_073.pro
89ae86343f5a60058e8f91c320dfd77c
6a95c30a6d81c7da99a37f59882cc1897e32919f
F20101112_AACJDB armistead_j_Page_001thm.jpg
4c6cd4e8c0ed277d7f2bc92d94919b66
c3bd4bab8bfea6d6ef2ffb6afe7afb1fb662c006
2218 F20101112_AACJCN armistead_j_Page_107.txt
90dbf3d8b4d1a584137a496e55e8c76a
58a2d98021c9f02d779c98a42c75305b9001613a
52277 F20101112_AACIXI armistead_j_Page_088.pro
45872bc172b528666674b12f8ff0805d
8abd1ead2cbeaa3382eace421f1e50e3178a7db2
47330 F20101112_AACIWU armistead_j_Page_074.pro
243e4497335adc570ebd76c8ef941dd9
c6e5b9ae7ceade5ea00126dfb14c68b676c064bc
924421 F20101112_AACJDC armistead_j.pdf
50a923d7c259f60a27171f1f906a4861
b9255f7252c0feafef54206e47049b5121a78506
307 F20101112_AACJCO armistead_j_Page_108.txt
404cd661b42462152ec3f44cea44a13d
4eb923260422565a67966ac9efbd078183ff226e
37459 F20101112_AACIXJ armistead_j_Page_089.pro
db204e8b1b5534e1745e4501d9fc5aed
b9a6f82dd1ef851b24a4e7a16f9f8be09a8b0fd0
10355 F20101112_AACJDD armistead_j_Page_053.QC.jpg
cf4390057725c69175fe169ed8d1e57a
48b2fda0b9117844deb867fbd0d30d9af409f889
2392 F20101112_AACJCP armistead_j_Page_109.txt
30230416c17c890ac493bf8e3040c625
c8734e9c45bd590f3ec0f024d41cec142ee39e16
48630 F20101112_AACIXK armistead_j_Page_092.pro
23127dafd7ea255079ae0b1d6efe2811
faf606d562773220ce192d1d0cc8437af4ce712f
52140 F20101112_AACIWV armistead_j_Page_075.pro
5d239059d52646c0e9d18c82414e63c2
471601f9063ca10de544f2c6c757b8d8ff5aa08e
8421 F20101112_AACJDE armistead_j_Page_021thm.jpg
4c1a15ca8a9e94fdf53aefe27ab5d05b
412f9e867774f3f558847b9a3b81f20c0ea788bf
2166 F20101112_AACJCQ armistead_j_Page_110.txt
c2b90e70a63223bbe18d9cd40b79bed1
3f95abc498cfb5aa2ce22cab33a3f89021d52f0a
46328 F20101112_AACIXL armistead_j_Page_093.pro
ce1e47a2edc1cf70b3dd460ab32da6a8
6d668a396d5e6fe1c8b4750a2e5f899116268239
56892 F20101112_AACIWW armistead_j_Page_076.pro
ca7066c4b0370633e14a7f931f960af2
44267b8f1f6a1142df24171d969b480f3f6e84c1
35192 F20101112_AACJDF armistead_j_Page_017.QC.jpg
b28fe425fe67711e0db88934f4271369
7dbae3f59f14785c4e96c5072267e3d04dbe1c3e
2426 F20101112_AACJCR armistead_j_Page_111.txt
284760b240eb44bdca2ffdee7301bcac
d22412f8ce494a3c406e006c2eee868f1b92cbe5
57336 F20101112_AACIXM armistead_j_Page_094.pro
bd6e065e5f4ea82515cb290758b7977e
6e2f4eb5276319a95f0d2ca3c344bb312e8f8142
55223 F20101112_AACIWX armistead_j_Page_077.pro
3c7d3e7ef880424b2eabc6529eccdaf8
d8e747dc4d4e8107a318e75bf7381ee4af015ec6
4243 F20101112_AACJDG armistead_j_Page_078thm.jpg
5314d6dc7a687b19bcc512ca71aa24c3
023e66497116e353a6337fae2415f38b137b92b4
7704 F20101112_AACIYA armistead_j_Page_108.pro
d3797febe8cf251e871271439e548a1b
32e82e6558e378471f9e2b981e95968e38caca99
2567 F20101112_AACJCS armistead_j_Page_112.txt
1be8c5035e5d896d2f203c86b04a3cae
ef1bdb00fab4101d8f9d98473e2a95f08d18be52
53685 F20101112_AACIXN armistead_j_Page_095.pro
5f297f9ce2ecfa4208fd659df35baa3b
d79d80d9a164853ca9ee4421f995c0bf0058c93c
23696 F20101112_AACIWY armistead_j_Page_078.pro
7685728a1e6cbb579af136f5ed64da7d
10acc7a45c2773b2dd368460eb72a3d2421d0fe9
2396 F20101112_AACJDH armistead_j_Page_061thm.jpg
d85b0f70d1c9da98d606a69f08d62ae7
12bee2d7dc14a5af876cc1fec205494a26d7b0da
39773 F20101112_AACIYB armistead_j_Page_110.pro
84b383a734306adb088f1878100aad63
d7a1c0fdef3a01ee57b94bc16af634733d923a6d
2506 F20101112_AACJCT armistead_j_Page_114.txt
2a21049d3924cf6ee54d308e805c8079
7e28d2e29931015cb8da821cfcc8c679b1d537af
53216 F20101112_AACIXO armistead_j_Page_096.pro
ecbde1bfbf08837790504739b3bbe036
acb56ddbd73e9bc716c4250fb9d3e34ac749825f
23581 F20101112_AACIWZ armistead_j_Page_079.pro
62608d1a194283253efaa305b49c38d1
cd8ad0b67cacbe51653e9936d1048cb970fb0e24
34669 F20101112_AACJDI armistead_j_Page_021.QC.jpg
1f10f1e0a3e0017f5ed62a7dbc88873f
04722364e68aaaec00fb543ffaf387157d42e02c
61092 F20101112_AACIYC armistead_j_Page_111.pro
ad43fed06ab73e9145649bd29e858215
e334d9c1a45146ef055086089305a73c1c4c0964
2428 F20101112_AACJCU armistead_j_Page_115.txt
cb705d4edb4b29a2f5ed7cbede8805e0
3357120f718fd8215667472fe2c5764cc7dcd1fe
54589 F20101112_AACIXP armistead_j_Page_097.pro
c39bdf977d8cfe7048c0ff2b2cdf2500
ef39584baf941850e5516d7a932b2a494e8119d0
8625 F20101112_AACJDJ armistead_j_Page_115thm.jpg
83ce36fae7bea8beea8f38b4f751ffff
76dc2f247b46d7bd8c0f7b48f090c5d3266ca358
64908 F20101112_AACIYD armistead_j_Page_112.pro
d6842acc4f7bef9f1e48ae53785c66ac
f050c36a90da39edfbd3d3ed1cd85d9dc92d0780
2242 F20101112_AACJCV armistead_j_Page_116.txt
80ddf010b1838c418b5b2708cd856714
3f7a87371f1adaed17cddff90b0d59386990ea4e
2853 F20101112_AACIXQ armistead_j_Page_098.pro
629176b3c1a198af4f85436da7a6367e
72c4323d78b3d624e7d7c62a3f357792245010e7
8185 F20101112_AACJDK armistead_j_Page_074thm.jpg
3d5f811d4453f4278c13d28c61b5929a
4ae7ec7d6066ad8bd8741c8cb8aa1bca141ba345
69528 F20101112_AACIYE armistead_j_Page_113.pro
d8aea6de00ed97e41a4e88e4e2d11cb0
7b5f992a5b44df51a23f66c47df63869c38f1132
2418 F20101112_AACJCW armistead_j_Page_117.txt
d5edc35864f478f0adf8d9351ad261bf
7c10e819fc754b0f374ab99bd8ba016ae4c6039d
29085 F20101112_AACIXR armistead_j_Page_099.pro
2957132d3b379588227478becf35a8a5
7c685195636397d87f86840d725f75a852bae645
33452 F20101112_AACJEA armistead_j_Page_116.QC.jpg
3ce5d3f0ff1eb8ba1970a91ee09c83f0
e5bc49f54294b758e339f8e517d1651a5f744624
37488 F20101112_AACJDL armistead_j_Page_036.QC.jpg
fae60b6d8959e9e2e91b4a41713f2423
64c939a156d87811d1a7bb2498cb038ca907056a
63039 F20101112_AACIYF armistead_j_Page_114.pro
3b2d6e33f9b5c0f56494655eac634217
d54b3075baec95d0d6b4ef95eea8654ba1f0938c
2334 F20101112_AACJCX armistead_j_Page_118.txt
cf5a5241e99ecfd9330103b35f2aacb5
d85bfa53cb761cd9b0287b2b27769224253c2455
15033 F20101112_AACIXS armistead_j_Page_100.pro
87aab578ded4008a3e75941957550f42
f90c0f297d92e2e7f73b4107ef1fd4c7b218d12b
2346 F20101112_AACJEB armistead_j_Page_056thm.jpg
8224214518da44092223cecfb0253bc2
045724e3496ebfda17e251a3ffac4bc5c29e0b69
1281 F20101112_AACJDM armistead_j_Page_003thm.jpg
557004634d627b90fe7d68656e6b4599
22ee3b043b7b1b95fc40b901c8132dcf30d82728
61164 F20101112_AACIYG armistead_j_Page_115.pro
19b3b2a61c9debd2a3810b3d84cfcfdc
db14dbc622ec3c16dafbe4a237d3f648005c9b2a
2510 F20101112_AACJCY armistead_j_Page_119.txt
9927e4e4d568e9cd080486442466ddd8
ac478f1c0dbf1a4a60bffaffb55bc5873be31d1c
11779 F20101112_AACIXT armistead_j_Page_101.pro
31535e7e8a03398b053e65bc6f1f9518
6f9a4f165c1116e88431acba4089f8647430e276
534 F20101112_AACJDN armistead_j_Page_002thm.jpg
27e0e583048267172654cb82c53aaf50
73628deb7c570f8ac9d5e3d213cb60e9896b4773
56193 F20101112_AACIYH armistead_j_Page_116.pro
40bcb3b0e5a7faf9435ce5db6ded91c6
5bbdb56c38ddd73935bcef0c88e54f3d5de2db85
2488 F20101112_AACJCZ armistead_j_Page_121.txt
e2655fdd320980ce51a1ed2688ee52ec
c335882d858991effb9c71e115aaf65690a84483
7881 F20101112_AACIXU armistead_j_Page_102.pro
bf1a80d09265c089ad4dfdac2e62e714
79eabebb0f2a051b6f4ceb3ee8a4cd98d5ec1afc
2629 F20101112_AACJEC armistead_j_Page_053thm.jpg
e0ce805bbe5105e578fdaf3c7867cb41
fe49869c18d9878d320279fc570920ef69ecf344
9098 F20101112_AACJDO armistead_j_Page_076thm.jpg
a3224180936358612494c1aee95959d4
d3a6444113ac1a37c51a4774f2de69bc797dc0f8
60731 F20101112_AACIYI armistead_j_Page_117.pro
4ac53cd4c3664c070709822cf8704e9c
2d4f229f82a4a24f92a5bbe1ed70381ce35ed4fb
11243 F20101112_AACIXV armistead_j_Page_103.pro
2945f6b120ddc54506b16c07c7bd9010
c15b505b664c8278cf645f1048f9853fc3253d5e
9614 F20101112_AACJED armistead_j_Page_114thm.jpg
5c0ad9f41fbc561e36d56cf77bcd18d8
6190e745e438e74f95cab73cf0ad9c7bcdcd0555
8895 F20101112_AACJDP armistead_j_Page_107thm.jpg
494af6ec0874f0bed6fb4e82a9027990
c320d6c7a6f4d2372e9094f3e5a0ef5f2233c6f6
58676 F20101112_AACIYJ armistead_j_Page_118.pro
10f462133e1866082ec57097667fa264
dd13e696c06c5cbd1d77573bbf9f6d8633fbbffc
9095 F20101112_AACJEE armistead_j_Page_111thm.jpg
ec254f998a0d0940f2097dbf8f66395a
d44fdf4587e7d593517691a1d7045458c41f9d93
14638 F20101112_AACJDQ armistead_j_Page_099.QC.jpg
bc92a0a08ecf1c99703fbf6839a9dda3
71b10676c041d710523943aadda972ffe15ab898
63401 F20101112_AACIYK armistead_j_Page_119.pro
9d22b312b32b5579e3b22d8ee9227235
7aae7389759e2f0509db43716f5120366cf53a2a
8937 F20101112_AACJEF armistead_j_Page_116thm.jpg
e83b47551e7fec04f132b8f52ed0e9c7
23553b712d4a513ebba07034141035fcd91aeccc
7008 F20101112_AACJDR armistead_j_Page_061.QC.jpg
c6b8be5c6e28bde5a63d8dbce69f05ff
9b5ccbcbe4b66a3e71e4eb321cc609f327617396
66078 F20101112_AACIYL armistead_j_Page_120.pro
379b07ee830c9a9c52b8bbfcbe9c3818
1906ea52fbf71db0a80777c41009daf189964cbd
26185 F20101112_AACIXW armistead_j_Page_104.pro
52701a57f5ca61891f983ad637822705
949a30dda8299bb2d8b765eba362c2ce5b0907d1
5395 F20101112_AACJEG armistead_j_Page_006thm.jpg
0f81babcd7ed8a9524f6f80643019477
7e0bd5b77332571fd326df3a346c8bc79a2fb727
2160 F20101112_AACIZA armistead_j_Page_011.txt
1e7ec8f88b9be4d0d0c85ec585c2393c
13475d30b82bcb7b8f01bed89c12d3254f804d17
8528 F20101112_AACJDS armistead_j_Page_088thm.jpg
c47ab0b08dcb4614b8186d518d7336ce
0b9fc5ff3c76372f89250e516af11e59ce319639
62991 F20101112_AACIYM armistead_j_Page_121.pro
d967a68f6b44a20a9d4c59b1f00c3cff
e4e3fb465b1466c2f6c7dae7d8312c076bb6c59f
52915 F20101112_AACIXX armistead_j_Page_105.pro
62de2cb0420f0871cedd0682ec1c633e
000c3bcb136624e9d30bda61d2b440884a23e871
8836 F20101112_AACJEH armistead_j_Page_071thm.jpg
99bb6eb9b9b9e009223a00452d58e36c
24ad4f3cc663371e60f4cd4f820ea13d1b41363c
2217 F20101112_AACIZB armistead_j_Page_012.txt
b400a8024325397bea6403a1a5e8f7a9
2bec11a1101b13bd9604628eb4e63f6e9fd5fa2a
8847 F20101112_AACJDT armistead_j_Page_036thm.jpg
3c337c31d48d2af41980b80d4cc5f34f
4104a8f8d88cc9718f5b399596226a4f0dc469da
62357 F20101112_AACIYN armistead_j_Page_122.pro
4fed3f764b817345ea5916d55c96d2ef
42233c572e57985991d8d002f6913ae93b58ee7e
54764 F20101112_AACIXY armistead_j_Page_106.pro
ed93d9a790eee9d098327e2666c88bff
ad7b79fd4a843138e4c0332498965878d32f1d3c
8377 F20101112_AACJEI armistead_j_Page_066thm.jpg
6b7b42ef805baf1d91a7245120a8aa77
f78e15b190a1c2554f8b94e78f06d1bf779a7ae0
2148 F20101112_AACIZC armistead_j_Page_013.txt
f1e399796bc9375bbaeb1dd0bf3e2621
356753ae3891f622142ee70c940df2c52b095c71
8700 F20101112_AACJDU armistead_j_Page_016thm.jpg
2d04dfb4b838c304d7f07a6d9e0622fb
655358d965580b08fadbb418020636fdb4315666
30780 F20101112_AACIYO armistead_j_Page_123.pro
e9170124d1cf691c42e0976bdef7f46d
bcd12548a08fb003629640157f5a284e1d336bd0
56523 F20101112_AACIXZ armistead_j_Page_107.pro
b7c3ceb9031cb02c5849ffdd547f534d
30f6937be2268151e0b498b9e15f27fff215a906
35381 F20101112_AACJEJ armistead_j_Page_028.QC.jpg
7758afb8e2148e9ee19a412f5dda169f
527a892550beb7941c79917b6d6e803c49bb1884
2152 F20101112_AACIZD armistead_j_Page_014.txt
7f185bfefb3aaa144317aefa033fd9b6
41356427a43fb688adfc21fd4f07b76ecb1ccd1c
20902 F20101112_AACJDV armistead_j_Page_006.QC.jpg
2508fe1066948a6d195e8f3117b0313f
72007293c4451270d66fdf94246fe45403845d4f
40857 F20101112_AACIYP armistead_j_Page_124.pro
d29243ab8feec482be00fc2e8f98da37
8916c5e9529689aaeb2a1d61f4c0e42f355aacf2
9011 F20101112_AACJEK armistead_j_Page_087thm.jpg
cb497a98ab6bb1d8d5f060466a93d7b3
73748f061468ff9653327e316f54d1351f131721
2203 F20101112_AACIZE armistead_j_Page_015.txt
b3bd763dec1218063627ea6f13002244
e244aaaeef3975b4042e681f65693d8419814b6f
35805 F20101112_AACJDW armistead_j_Page_067.QC.jpg
516ccdf9bb1322caa07c08b47c25eede
6ec083bcc0f783edcb6f89c04b5e173222481edd
498 F20101112_AACIYQ armistead_j_Page_001.txt
d3b71d3dffa99bd74c909f66d7dba64c
488674e64cc6bebf6ed05d7cbc2a0db4e55cf02a
8780 F20101112_AACJEL armistead_j_Page_051.QC.jpg
b0910a7dcecf6b8bbd1e0c5e96df53f0
1ef6f6542b6c205c324637a02e81d6f8009f7172
2076 F20101112_AACIZF armistead_j_Page_016.txt
d69b056f3481c4494a54ccd930543964
90b637cf98c74c369b4b5697b170458660fd0882
8903 F20101112_AACJDX armistead_j_Page_015thm.jpg
3603ef040d9df57591f47c258bbeafa7
61fe5d883b6aa7285903b6a1e7920f9d68ff382c
95 F20101112_AACIYR armistead_j_Page_002.txt
e4bfaead04819453bbaaac8ffcd37668
e0048dc5251636b4b8fb36722e1200446b762e1f
37795 F20101112_AACJFA armistead_j_Page_007.QC.jpg
8914f4a4f9ddd6ca746e2756acc1e652
2228c1cae3c7fd74cf31a9d19051529f2c598282
8804 F20101112_AACJEM armistead_j_Page_119thm.jpg
451a0ec67c4678c5ea606e2518497b7a
bc6b21f1dcdf5b4efd64ef428b531c01bca33384
F20101112_AACIZG armistead_j_Page_017.txt
620862124d574ca00982883f46ff79a0
d8a462f24ffb0735fb53c46ba92766c5cca1ddd5
F20101112_AACJDY armistead_j_Page_032thm.jpg
16c24dbe97d955d8c58b082819ef8534
3103a17b95377539f3ef4f1956c77534128219ee
250 F20101112_AACIYS armistead_j_Page_003.txt
2d666216e7a63020a2036bb83587cc5b
b19b94a3a13d8aa5c96038afa438c74400436b9c
8949 F20101112_AACJFB armistead_j_Page_007thm.jpg
de8cac95f20e2a1f7d2bda1210498570
155a824f87efdc2249b73f06e98baf299c405db8
35383 F20101112_AACJEN armistead_j_Page_033.QC.jpg
d160b9cb5052b29bfd02d3982fc796da
acac1bdcb4626b8e2c1fd61cfce1c6714e918ca7
1767 F20101112_AACIZH armistead_j_Page_018.txt
e7c458930dea8e29af3e0a7b46544327
b6879195f699a728221379bec49252ebbac6a90c
3214 F20101112_AACJDZ armistead_j_Page_059thm.jpg
0c841c4aed451502a61a322cb4b21da7
a1f9d97c2ca3299363453ceb49d624d30a27812a
1081 F20101112_AACIYT armistead_j_Page_004.txt
3f1f1ca4057ffd5b805cb7d20a9e774b
c1bd99c29a39dce2c938868beb659a4843e4cde8
40562 F20101112_AACJFC armistead_j_Page_008.QC.jpg
0a42df9b10c6e6f5b80a2748096fa0f0
e3dec9bec32aa4929058993ae613bcea3d7a935e
12642 F20101112_AACJEO armistead_j_Page_064.QC.jpg
86dc209fdcea71c17cbf83b8d3367e72
2bc9d10b2ac83463a9f86e76a583cc249cbf0605
2090 F20101112_AACIZI armistead_j_Page_019.txt
9b09228015852f4b7bf39ecd038413a5
1ccaae57388b0e7605e0ca91e18280de93acb340
3972 F20101112_AACIYU armistead_j_Page_005.txt
46708209e46ffcc5da11ad4802fa2c44
cf163e9ba5d0a2de3010ca3b1e2d5903a6092803
8570 F20101112_AACJEP armistead_j_Page_031thm.jpg
7815b70c9d9ca3ec5335f5a14c464ebf
2dae18ddd41a45953da02ae68e9b3d983f676c0c
2216 F20101112_AACIZJ armistead_j_Page_020.txt
e15ce04183f8361607a9cf6f0deca545
ce162d775b64dbd16cf997c79636c254d3d5e2e3
2607 F20101112_AACIYV armistead_j_Page_006.txt
a76e832c4f90edc64cf27ba7297a3041
23289ca89bef03e29868a371eedb871d92e9d928
9878 F20101112_AACJFD armistead_j_Page_008thm.jpg
7d3bb31e9d16d2a7ed6f3da6957d5e65
4f08b39f7002646d18275d0693e406c58a27f46d
4074 F20101112_AACJEQ armistead_j_Page_064thm.jpg
18df0bd147c139268ce6238aafde2abb
4dad284c28c86edec566f0497322191d9790b095
2137 F20101112_AACIZK armistead_j_Page_021.txt
f6cfdb81f7feb0da52e2176d5000061e
9c8d9bcde2db1dec287cf8613ed0c500e020b877
2776 F20101112_AACIYW armistead_j_Page_007.txt
d7381984b502bc97561f9958779517bb
f6a34aacdc2a9e3ee8781ab9463d0486e5dcabcf
15361 F20101112_AACJFE armistead_j_Page_009.QC.jpg
51e3eb877331d3f7066c6e9b67c608fb
709ca65850322999b945830b8aef1c55f4445dfd
32437 F20101112_AACJER armistead_j_Page_073.QC.jpg
ba294f477e94ee7b06e311311adde9a3
ee3e44ff6d7d93cce990fead780c0c68eb72d604
2049 F20101112_AACIZL armistead_j_Page_022.txt
0660e41de85bb0d685e89ffd6e84da5d
3e8164ce2a156f6d97e5fbb722c67adc8a310385
3724 F20101112_AACJFF armistead_j_Page_009thm.jpg
44c0346c6552edc48c50ed896096ad7b
134b4c12e7ff18c4cd4508db84c585e800cc920a
36641 F20101112_AACJES armistead_j_Page_107.QC.jpg
acb63aa77b83bb07ae0ebbe78339a97d
8fe20ebaca3f9d202f8f2f746f805133b94c5dc3
F20101112_AACIZM armistead_j_Page_023.txt
7dca3e1667486c54775b70944aefdf66
92dcbbcbdbdd0db7d4d61dc98fc2a3dd55abd222
3049 F20101112_AACIYX armistead_j_Page_008.txt
188649cfa89550ef41de8e42aab2617b
a5bf0cb9a07e7b451a48f287a1c9d1b5116fa586
27751 F20101112_AACJFG armistead_j_Page_010.QC.jpg
f37e93ba742722e5a3a183e103215b8c
7e012e23e7d23c87ecb7a1fe1bfa299bc90574ea
2069 F20101112_AACIZN armistead_j_Page_024.txt
f21af707724a385bc75bae0b6b1e0f3b
70991ea60f1f753f3d705285f8d6420ab4ed033d
931 F20101112_AACIYY armistead_j_Page_009.txt
af56014cde5c12df95ec846c375b5bcb
49cbcac5723d3ae2ea482641e6cb5ab11305b088
6834 F20101112_AACJFH armistead_j_Page_010thm.jpg
acf4402127a9c145ba137b943b96abb3
c1f2c46ebcc09de69012b74a92789dbb68bfe4f6
6922 F20101112_AACJET armistead_j_Page_124thm.jpg
a6ffdaac5f0bab1823bd94dc2a28ded5
32ffebe9cdacbbf9d53c9bc1fe9410b67eb77619
2105 F20101112_AACIZO armistead_j_Page_025.txt
80256a790ddbefe7a70e44dd34dd9947
2a39fbb7110fbe87a0157ff5eddb5b8392638201
F20101112_AACIYZ armistead_j_Page_010.txt
5eb88b3c486b76df12f0242eb4bd4d46
85070c77c3f6c8b40d1da989bcf63481a486aac4
34863 F20101112_AACJFI armistead_j_Page_011.QC.jpg
52e537dddcfa560c5af09fa82236aa07
2fe66dacf0c47adfff3cc244b48a23da3d34b257
188566 F20101112_AACJEU UFE0020148_00001.xml
fbca8068c002960bcd412c4c8287b0bb
75d03cad4cc189cd616bff919d92071d1af81ee3
2001 F20101112_AACIZP armistead_j_Page_027.txt
2100d4bb3097da7348ea819023722372
b5cfb44ad0b0994bf75927106c420a9344637afc
8684 F20101112_AACJFJ armistead_j_Page_011thm.jpg
804f47d6cd46be19e50b4ba359b110e3
4114380aeb3f91e2bf096a329452b1cbdf8bfdc3
8243 F20101112_AACJEV armistead_j_Page_001.QC.jpg
33b404303ab2a8e0e12bd38fddbef351
d92f70de79c154d7fde427244f35c19be9cc59b0
2118 F20101112_AACIZQ armistead_j_Page_028.txt
2e99d4acbd4fd0989f2297c7655f7c3f
7a41d11a9a865a6f7007a1cdae801894b7e0725f
36105 F20101112_AACJFK armistead_j_Page_012.QC.jpg
35b40af0aee593f32e777e932580fa27
c9924c00782d9df3aba4f744c2d7844ec9a56636
1217 F20101112_AACJEW armistead_j_Page_002.QC.jpg
53b4dea214613e1707108127f72f4094
6ea1aa3e2e05a3c6a5368bec3b2d26ef48863d1a
2170 F20101112_AACIZR armistead_j_Page_029.txt
cd8f7e7c95f1e91f7f6d2c5965d4245a
864e580a198ebd68135725f24945397c4cb3501e
35171 F20101112_AACJGA armistead_j_Page_024.QC.jpg
c6b4ebff90ac3c1cb56de63ef244b962
073f5a0aefe4f4e7e2744f533c9b661c3caeaafa
35791 F20101112_AACJFL armistead_j_Page_013.QC.jpg
164f4c48338d1e4caf0af031c429b0e5
f0c1fd02f5c81e4a6e4fe683a6f5b137d6313463
18589 F20101112_AACJEX armistead_j_Page_004.QC.jpg
cb25a05f0669e6e29efd30c9afa699b3
2c85f1dec8ea7681c9b44d1d5813523b7bd3fcb1
2149 F20101112_AACIZS armistead_j_Page_030.txt
31514a525ea7635c1a670ebcea20c9bf
a8bf89d05e77a7d57b91c344bcc776d5cde92a45
8674 F20101112_AACJGB armistead_j_Page_024thm.jpg
11e1970d19a203b3dcbfead2d96c6e52
7aa5be9ee11525e13b8ac93a92b03da862758846
8839 F20101112_AACJFM armistead_j_Page_013thm.jpg
36f67ed76866895be12992a12d7d3244
d85965b6359eea07d38ad218449ee84beb96cec1
4788 F20101112_AACJEY armistead_j_Page_004thm.jpg
148ccc21a0948cd851ad86718321e700
6ba879eb8856aa8f167f5558b34b0c09414e9495
2139 F20101112_AACIZT armistead_j_Page_031.txt
6014d56287455b6b9138589c6353c88a
bb1b6165e906cb00aa22c1c1b8efa60e63590f9e
34336 F20101112_AACJGC armistead_j_Page_026.QC.jpg
e6600741bcc75f3cf44fc406b88da780
2cb245bd3847fcbe27a826ae57e4b97955f2f8ce
35957 F20101112_AACJFN armistead_j_Page_014.QC.jpg
e5696309817142e7f88af0af6298cd14
617f71cf60ce982bfcbe5f9393c281c298e5de5f
6792 F20101112_AACJEZ armistead_j_Page_005thm.jpg
83e3c6bb0326941a1c31d4fc18d33df2
93c48151e0a4e57b58195afeee190f5c82cabe7c
2193 F20101112_AACIZU armistead_j_Page_032.txt
70856cc2bd4be018602b68f2bb2a2c63
55fab05f5d4168e2d7f97f0780809d15731e4a2c
8623 F20101112_AACJGD armistead_j_Page_026thm.jpg
484117b67c4069650f34aa3b03a5b933
34b7f84d96709a402eadaf5c3c8f4e83a687bfdd
9070 F20101112_AACJFO armistead_j_Page_014thm.jpg
843ff83fb5d245dd609f3b4361c508b0
2c8eddf40e67c152b55ede181ec90dc9243bf174
2100 F20101112_AACIZV armistead_j_Page_033.txt
2bb536ab7d094013b9cd34c5e2723ae9
e03454535ea70b3cf781201e2b43ebd73952cf26
36574 F20101112_AACJFP armistead_j_Page_015.QC.jpg
022b3348e4b2a9e8936e22e127efdb10
334e2b6b7bb79a19938a62fb02ba5a05d1ddd76c
2185 F20101112_AACIZW armistead_j_Page_034.txt
30d63ce0d8767f961e00eba0f7f0f272
da29161d69c5e3e50cbdb425a0ebfb09e9779520
34523 F20101112_AACJGE armistead_j_Page_027.QC.jpg
92e4cb603c3345baf809646d3fc1a9ab
9aef9208eb34895b77dfbb3e128101697eafe1b7
35002 F20101112_AACJFQ armistead_j_Page_016.QC.jpg
2548fe863759e5d7ad66d5b187609691
ff6feab65002e33f9ca82b07a4cdf3af3c7450fd
2156 F20101112_AACIZX armistead_j_Page_035.txt
50a50cc47a6ae1daabf91eb5e1af48ca
72aa885bb9aad3e002dabd3d8a430847200d10c0
8630 F20101112_AACJGF armistead_j_Page_027thm.jpg
0afec26e3a7491e1130649d5f20ac0e0
4990bc460bac85b544a734678f763e396b0ed8a8
8526 F20101112_AACJFR armistead_j_Page_017thm.jpg
1667024e6a062dee205672fdca451946
a2b6019a33fd9f99c3400dc235da9b8cab3ace74
8330 F20101112_AACJGG armistead_j_Page_028thm.jpg
7b88d690ca680d462fe7e7a494250fd5
4ddb36fab86956364aa9b4e878464eb75dc19d19
28951 F20101112_AACJFS armistead_j_Page_018.QC.jpg
5914ca033c4489d4c0b3f1bdb6fb1386
9c706bb09496ad98c79d9e56df842c50a33edaa5
2316 F20101112_AACIZY armistead_j_Page_036.txt
31c49b36e185b1bf1363db1063c98d6e
b16d22951f2eda649bd77c11c5a4f49f6b54ad08
36371 F20101112_AACJGH armistead_j_Page_029.QC.jpg
93e11f87dba824d9fc15310685583451
339d6a75ac17e408deec4ec8226bd3a86dcad168
7059 F20101112_AACJFT armistead_j_Page_018thm.jpg
d0bc58303db86c5501bc20bc95cbf4d4
0bed5d379c5eb07f0f3b3c150c13b870b43113b1
2225 F20101112_AACIZZ armistead_j_Page_037.txt
534bc859f488f4ef52b1443ddeba280c
0595d6abe97be7b009836cfd501bed83907db874
8809 F20101112_AACJGI armistead_j_Page_029thm.jpg
f88c5fd7e6bd801ffc54dbd54d6418f5
3dcb535f2f0525cceef5ef1d71cd6d869577e545
34651 F20101112_AACJFU armistead_j_Page_019.QC.jpg
2fec9ca572ed0add7bf6350d5100d80e
d4a5c512e3f71ad94b9624c6d2a43995fec6b058
36136 F20101112_AACJGJ armistead_j_Page_030.QC.jpg
10bb62153430c5630f95dc2ab391324c
858ceb493e7793ad5dc2542b4c4e999d17ed3310
37062 F20101112_AACJFV armistead_j_Page_020.QC.jpg
eab3710d0e454d145f180af1ea1fbf80
1ae6022d2f4288e7d76a98963f283f2ab0a582d1
8598 F20101112_AACJGK armistead_j_Page_030thm.jpg
7946e5c2158ca8ee73a6f213117a494d
47173508a33ffc3fc217a5e2320ceba450ffd0ed
9081 F20101112_AACJFW armistead_j_Page_020thm.jpg
14826d867748205acb4776e76b221175
71afe1680887a72294fe54ad0a830a84b7d9cbfe
36995 F20101112_AACJGL armistead_j_Page_032.QC.jpg
7ad4ba0081d9aad1016bea42db60498d
1f5bf7c1251f5a7dd0925fb200bc4ca5e6cb1de2
34000 F20101112_AACJFX armistead_j_Page_022.QC.jpg
1087b60ac79103203a97bbcf38259be8
3d0ff7ffaaa879ff89a46126d1956c9bbf237598
F20101112_AACJHA armistead_j_Page_041thm.jpg
46f817a50d1e5fc9c0c9726c719ee5f0
0aedf06570666b056adbf136e0ac03f20880762a
8891 F20101112_AACJGM armistead_j_Page_033thm.jpg
4ab1d2282e0a5bf8dedd3ae7b8ecb2e0
fe8a5179e89b730da1ec7c73ccdd3bd04126fefc
8395 F20101112_AACJFY armistead_j_Page_022thm.jpg
78cee7eb12922fa71a08dae2b4a03889
fdd1c5824d3aa66c3800f001c1e228a93a79712a
36538 F20101112_AACJHB armistead_j_Page_042.QC.jpg
dc23fffe7a01788422eaac9caf913089
ca162f35f83ede0f4f64274350f40157bfbbc44b
36515 F20101112_AACJGN armistead_j_Page_034.QC.jpg
32443542cc6196ac7d2e88815d14221a
d9056dcfd33fc00dc1a033fa0ad328623eb7f00d
36326 F20101112_AACJFZ armistead_j_Page_023.QC.jpg
a1c7061916b90c9fdc92a73e7a9f8b9b
eb637efdafe8b870ee145481251ef27be7e13168
9063 F20101112_AACJHC armistead_j_Page_042thm.jpg
29d06a3f2d8ea14baca6be20c813fe4d
fff8d18a572a91c48f53cd658b2278fbe3fd096a
8854 F20101112_AACJGO armistead_j_Page_034thm.jpg
e2323bb7d92044fa6193907237bbd37f
bc7fb360ea1ede056d39a5d6a19c9b0c7f78f551
19971 F20101112_AACJHD armistead_j_Page_043.QC.jpg
a0a57204a6a198bae1b650813f7bf9ba
cc302d837b88126d311b5c699405bf5ef333cc8e
36905 F20101112_AACJGP armistead_j_Page_035.QC.jpg
62e71f082efa21ccb584f87648827098
3f0f77b947d9d77f32592e9e7b3fd18130b6b4bf
9671 F20101112_AACJHE armistead_j_Page_044.QC.jpg
eb3bf8b0d192e9d21888135d2a352ce4
f90d39dadbb0dd7f2fef35e7b5b31b0c45bd503f
8818 F20101112_AACJGQ armistead_j_Page_035thm.jpg
9b3f86d275c04814a8f2eb12e76795ac
9781a118f4521175fda8c386b10e2dbe3aa29608
35779 F20101112_AACJGR armistead_j_Page_037.QC.jpg
2dce68383f75fefa51a4c6fd7c25b78f
b8a2fa2e0a3a6d4544152f1717041ff4c2c63422
2888 F20101112_AACJHF armistead_j_Page_044thm.jpg
612ccd49c4dfcd3056c072b50d6fa4a1
4ab8c1058c53c25a002caa0fae2e00995c3d18e0
8883 F20101112_AACJGS armistead_j_Page_037thm.jpg
a2511948e7cef7ca52d9855769f153f2
820693c9b4cb48625483bb6cedb7bbc03ed718a6
9296 F20101112_AACJHG armistead_j_Page_045.QC.jpg
b2b07f567dc338c24f782562472ba919
3d93bb319ec79c237a5578874bd13e22625ff9ba
37104 F20101112_AACJGT armistead_j_Page_038.QC.jpg
1aa1f05efd363e4990ff6e3bee5e7d53
5a02bde9669dc8d4a6e85f843d3047c0a03caa45
2519 F20101112_AACJHH armistead_j_Page_045thm.jpg
55e6b1ec5b248df23fc1100f89bacbe2
9aaf472ae87951b4a73cc6d337835265e6bd74c5
9208 F20101112_AACJGU armistead_j_Page_038thm.jpg
b68c2c5ab56220171486416e8e516fd9
e405f374d420bcf88d25c48fb092d83f0a4b9874
10795 F20101112_AACJHI armistead_j_Page_046.QC.jpg
26ed71ac52be51e723bc8f2317dd36ca
693d6efcd2ad5f5c3b5c686765b9e14000893b95
37581 F20101112_AACJGV armistead_j_Page_039.QC.jpg
74f2f962b06af6cf8cbb21a1d664d9a0
445867c33c941f5044c6e5e203c90fb001a6d39f
50627 F20101112_AACIEH armistead_j_Page_066.pro
7dd83bfeda14b77ddda9fdf871a8a2a4
93a5baf510acbb5d430d11ffe87fa3fe20264c85
2966 F20101112_AACJHJ armistead_j_Page_046thm.jpg
0879e33b6d05ea71e17a87d2a345ffc4
e17a3efe2096654b6bc1b4358bd75e8dbf836bb1
9157 F20101112_AACJGW armistead_j_Page_039thm.jpg
8ec37af8d263469348913e1012012656
61dc460f215400a3bcfcc5628a0042261ef27368
1051909 F20101112_AACIEI armistead_j_Page_008.jp2
281268fea63e0cd47f4381e81f872e23
dccd0e2e4b18f05b518c7df4b2653ddff6dd2522
2339 F20101112_AACJHK armistead_j_Page_047thm.jpg
8e378dff86270ec29e44c8e3eaaf95f2
2b61dad2af1d24c74c888e53ed9352495b1b5406
36360 F20101112_AACJGX armistead_j_Page_040.QC.jpg
0f62fdb23b7abf5241732325a5a40241
ca402354af46e566259b231871a8aa3b8f46980f
4153 F20101112_AACJIA armistead_j_Page_058thm.jpg
1b840119f18978770c4c41377fc7595e
caf127e7867b7216189378bf46d7fb3a96ef4a20
144707 F20101112_AACIEJ armistead_j_Page_113.jp2
93c34d32ae99cab1a33ea23685041c88
f928a912af23ec4d7e7fa19ffd12c69999b62254
10400 F20101112_AACJHL armistead_j_Page_048.QC.jpg
02242049ee97d1fd44f3c520d7d92c9f
f82a12b798a55b168a4792d9a2e87c604a0b6af2
8786 F20101112_AACJGY armistead_j_Page_040thm.jpg
9506643e83f51a461e19c4f97d9daafa
15839c669ea743bb244f75586162ec27f5d67195
8428 F20101112_AACJIB armistead_j_Page_059.QC.jpg
cd5f075371df3dc0c8bf970255d7bb84
9407e77132c468d8bf0ccf6c685d1aacfbc856dd
11991 F20101112_AACIEK armistead_j_Page_057.pro
80683b61d50d704091c4ed2b3557e2ee
a8b74a7b35d1c3a80d4e292f555d16c6d4f5b470
2925 F20101112_AACJHM armistead_j_Page_048thm.jpg
463645ac9f3621151a8adcb2ade95eaa
83cc451efb670d235b9c762d4ea8db1c23262a6a
35105 F20101112_AACJGZ armistead_j_Page_041.QC.jpg
c0495fcf8ec57271ed32ffc37da39222
320fe0b7d9af219f408305e3edbef430f84db91f
99423 F20101112_AACIFA armistead_j_Page_085.jpg
f5bb1aa909a1e0f04e95cad05fc46a07
b9334897f557b104cace48472cddd3e6f39b8b4c
3334 F20101112_AACJIC armistead_j_Page_060thm.jpg
a2dbfed8f80e2e469636d8c14d268845
3a48c89b473b384947e99dfc6b93e1d60016ec3c
2475 F20101112_AACIEL armistead_j_Page_122.txt
226ac155dba4515a001520d12a171631
f79c2a555a3f44a95c8769734445f35c1197f376
9037 F20101112_AACJHN armistead_j_Page_049.QC.jpg
0ace3b9a03bced7fca6391669e3fc3d1
6f97ed189c6cc740b0a2ab788809a2f0a4de4ea1
F20101112_AACIFB armistead_j_Page_112.tif
ca578a3f3f1ddb17c3c237afe69b67fe
85d4f2741b56da835385e4c46aee9cbdb1b93877
7151 F20101112_AACJID armistead_j_Page_062.QC.jpg
c6890dbf2584f4575759d973c9f1b812
3bce661567bc3cec521b6c2483bc0ab25c6d975f
119131 F20101112_AACIEM armistead_j_Page_023.jp2
9c4eb317a2ff9a76bfcd2ae0599f59df
fe7287955b4d0cf34a99bee09424541dbbc00e7f
2637 F20101112_AACJHO armistead_j_Page_049thm.jpg
b6cca85cf1125ba8453a78dc69802091
5bd9317a3e30c6eb3048e076ee69d1d4ca6c9782
32526 F20101112_AACIFC armistead_j_Page_092.QC.jpg
0f2ec14ccd9542196753ac801530e0a4
3dfd9441cc8abf67f3dc9eace6e492017e3831ea
2481 F20101112_AACJIE armistead_j_Page_062thm.jpg
8eb86cb42bc31490cb9ea3d919fb126b
f1ec599397bcce8d78ceef2c1dadb27b945e95db
118272 F20101112_AACIEN armistead_j_Page_016.jp2
e8d57f9678770712bdb605734168be60
4621596af47888fbd1d266e7ff62a0d479a1a280
6959 F20101112_AACJHP armistead_j_Page_050.QC.jpg
1f4cede751debe7d1b717a0b9d98cac2
bcc9bfa6e55cbaf3fc3894eaec9820c3b5091937
9295 F20101112_AACIFD armistead_j_Page_060.QC.jpg
a6b333779e48a978a0bb944c26e700c4
49c1818005078136dd43921daff40b5afd0ec423
12382 F20101112_AACJIF armistead_j_Page_063.QC.jpg
9999cdf1a2ff1fd8a0041105c55f49bc
54026b4967c93b3e99ce09995060eb15efdd23bd
F20101112_AACIEO armistead_j_Page_104.tif
917befaff49762ec0ac9d586f8be6487
62b277cf79d63447def392983ff29a323e8b139b
2034 F20101112_AACJHQ armistead_j_Page_050thm.jpg
d8c428901dff877b023b5ffc35f28abc
305209fd4b71f5164739dd9367ca9a0eea80269f
1624 F20101112_AACIEP armistead_j_Page_124.txt
9ae544940c216d7b9b800bd3324f529b
0eb8d48bcfb567b01f6c017c4651f080aafd33df
2403 F20101112_AACJHR armistead_j_Page_051thm.jpg
283bec276ad3eb7bb9b2db8d4edcc477
0ecb51d91137c2df5f41fc35f7fe348b0d888837
36769 F20101112_AACIFE armistead_j_Page_031.QC.jpg
4c9d89891b863f7636459849aa764fe0
776f20650de2ff9c3fdceaac35524fc12b24986e
12936 F20101112_AACJIG armistead_j_Page_065.QC.jpg
f1a9a5875f2e23f7693632731c269577
c65a5b7973260bd732a0dd72f1006d49a000af02
F20101112_AACIEQ armistead_j_Page_030.tif
f1fee773ed5d674e69cbbdb38d342ed6
a9ba8f7d869153b1b84f9435f3d0ea5fdbe15bd8
6751 F20101112_AACJHS armistead_j_Page_052.QC.jpg
da3bb965e8799519f50f4e30565894e2
6b65d333e86c7d5bae30420695a3e340e8a842ef
115768 F20101112_AACIFF armistead_j_Page_094.jpg
5b9c61db90b7a1105a86856765188c59
d2dce2528518b4dd98860e14671151c42a745b79
4102 F20101112_AACJIH armistead_j_Page_065thm.jpg
8bb5ada388a9f5862995a368b516c8b7
a22dbd786a514bd282808900f2be379f2696e571
F20101112_AACIER armistead_j_Page_034.tif
4f58cf693bd28b661cfff8eae2913907
8e062664ac0a8c8a6999bdbf66801b5717c5bc15
1917 F20101112_AACJHT armistead_j_Page_052thm.jpg
72e174e96ed24727f6026c23c522da22
b9bbc75f8bdaffc5054dd37e7b76feef16239463
8860 F20101112_AACIFG armistead_j_Page_067thm.jpg
eaf90c8687aea02d0370f94ddf69f741
355446632771ee37e01c00b3cecd5bcce6c6aad7
34301 F20101112_AACJII armistead_j_Page_066.QC.jpg
5aad8e84993fc219e33f35c68127e6c9
47021471da4f38403c8dfd14a65f9e1500584699
1540 F20101112_AACIES armistead_j_Page_089.txt
ab011646b98ad24d974d8e2c20f79e58
22672fc23b9e1905c1d77e8c4a5d0e05f31b00d0
19126 F20101112_AACJHU armistead_j_Page_054.QC.jpg
2169dbc09a8a54f929312bc225ccd326
cfe605fd3a588548a27a03035168ed2a4c283fae
626 F20101112_AACIFH armistead_j_Page_058.txt
97087b62f19dcd680f1332197183355f
a42c9e90daa796e40212e25a96bfc365292f488a
34119 F20101112_AACJIJ armistead_j_Page_068.QC.jpg
5df733e6eb0a7a8eb8893940026f0a00
1cf67fcacdbe99b398dfc67fdde184c4504cb5c1
2760 F20101112_AACIET armistead_j_Page_113.txt
bb12efa5899c8c03e43dc4722baa6c0a
6daafe9eb613ac17923c8a1dec94ab8bf1f7f06b
6071 F20101112_AACJHV armistead_j_Page_054thm.jpg
ffd0eeeb03af01f6e5bc82e5da949298
06435ce962812604eb7dbcd33d317dfe36bef69d
3403 F20101112_AACIFI armistead_j_Page_003.QC.jpg
76fda6a97d024e27c9ce949b15e030b3
92c3af73b154f9ca5415f7acd9263bda0461b21b
8348 F20101112_AACJIK armistead_j_Page_068thm.jpg
a6652ff605f63679ea1b5e9ab2f4159f
ab68d4aa37c84f3c3787c36f0e7bebcffc0eaadf
2550 F20101112_AACIEU armistead_j_Page_080thm.jpg
b368fbf4a14351aee8b20893893d13b3
20f2682b042c87d52793ca20d283d8b0fd76e771
8386 F20101112_AACJHW armistead_j_Page_055.QC.jpg
3c932c65832f33deeb7d2b03531d3cfd
6b810c196c8a7de5eb123b05945bedb7b796ebae
3479 F20101112_AACJJA armistead_j_Page_079thm.jpg
b016d91504b39e163ba2cae34b1687b9
4fff1a6081cb41d5136369c3b10c19af04aafc81
4701 F20101112_AACIFJ armistead_j_Page_043thm.jpg
6897a5d61d755e0b2023e689851c3bc9
cb9e6d9aba08fe0e6846ddc23a13502cb62c9ff4
34790 F20101112_AACJIL armistead_j_Page_069.QC.jpg
215bfb7956b3ce632463014d28641737
c2707ce7d38960646254a2d247971d9e2242705b
F20101112_AACIEV armistead_j_Page_073.tif
9db7de9feead5f192f4b49d958bb46cf
8bf48240576429f8e84ecba85aac97d8205d87d6
2997 F20101112_AACJHX armistead_j_Page_055thm.jpg
a347dd28069811e2f8a1363227d1a271
6c81aa453449bcbe5af04140edacf5bdc86e8084
7000 F20101112_AACJJB armistead_j_Page_080.QC.jpg
0b228fb6a99594d6b758367aab519dbb
b075cd73f4f89b555894bd64dab18f7463cd58e4
50143 F20101112_AACIFK armistead_j_Page_109.pro
5a524e85851544af45382f8b1da112d7
fea54f291bf2d04abd3815d6a2df009b32750425
F20101112_AACJIM armistead_j_Page_069thm.jpg
c076fac086d5dfc98b4c5e01da747f44
b973c0ecd155984f702398d9d593cd6b64767cfe
9031 F20101112_AACIEW armistead_j_Page_025thm.jpg
b6c80eaed703ed4b5a27e8d69a3da006
cffc567038bfccf347966697e015b17c3e4a5c06
7883 F20101112_AACJHY armistead_j_Page_057.QC.jpg
604a3b8fcd0d190c944697050768ca50
6408b71adec9c7cf75069eeab09cb113c6cd0fc9
6520 F20101112_AACJJC armistead_j_Page_081.QC.jpg
1732c1e092fff3658d0078ccf8d3fadf
e6ce9b7a4c576abcf347871c66e7c8dae2e58cfe
35408 F20101112_AACJIN armistead_j_Page_070.QC.jpg
758870e627d48f9739093648e9419c71
dfcec2aa15203d4948cc18e0dd5b9cdaab4ec362
6352 F20101112_AACIGA armistead_j_Page_056.QC.jpg
fd4df4914ffe4b094b6bbd2e7510859f
a687db0cf35bf1141ac1ce5ce1fd4131ab365fe6
1051918 F20101112_AACIFL armistead_j_Page_005.jp2
ea715a12616e523eec0c682c51ddaf3b
5bea2a7b757217028d349435f920538ad63a900b
58181 F20101112_AACIEX armistead_j_Page_039.pro
fdf853fd59b60616c9eef858ed2949c1
2fb0ed93833135fe74af2c034daafba522858def
11096 F20101112_AACJHZ armistead_j_Page_058.QC.jpg
c1731c9ad542ba7305ddfb68e46b6fd4
3554c3298d56ca11c397e6742f45b94114de4456
2240 F20101112_AACJJD armistead_j_Page_081thm.jpg
b2c00fea82bb83686942a1adf3ea406b
94d55216fac08eaa57bdd41549679b69844245fd
8816 F20101112_AACJIO armistead_j_Page_070thm.jpg
267ec685b0c10e41f685656747ddb04e
8e7cf0c696d983297f665a87c1b9554e540b033f
35469 F20101112_AACIGB armistead_j_Page_122.QC.jpg
1f4b58a39b2f038237a0995ad701bbdd
20b08bc0a22f2591218a7c7c265b8b6cc0718dc7
4056 F20101112_AACIFM armistead_j_Page_063thm.jpg
76666ccd1b8b15fe2e59ec5cb9e91624
567eb227131070a5b953cc08345149a79d2cc26b
2621 F20101112_AACIEY armistead_j_Page_120.txt
e205b416eb74676566db560260b83e2a
f964b7b5bee54d7943070d2f96190edaa458d2cb
6477 F20101112_AACJJE armistead_j_Page_082.QC.jpg
eb145599677ff848a2e4739929cd6b08
b31ccdc6260c1e1d2ffff953cf4a0707507eac34
34893 F20101112_AACJIP armistead_j_Page_071.QC.jpg
11d6a9f358e546deebebf2437e9d77bd
cb1007e506e6e52a08acfbaa72672b9d143dfd5e
32182 F20101112_AACIGC armistead_j_Page_044.jpg
278249433fa8b937baccd9a17c3301c1
b5786634665d5550a9cea34e87c93214577ed485
8250 F20101112_AACIFN armistead_j_Page_047.QC.jpg
78184e0ca1e2fda6ac61edcc5f9697be
7547d683630e46a55745a49a9bc1c012d5065720
36668 F20101112_AACIEZ armistead_j_Page_076.QC.jpg
d1866a0d3bae8252bd859c6ba8d1d31d
0e3d823eec2a343b38b348d5b6ea1d16d8c6315a
2285 F20101112_AACJJF armistead_j_Page_082thm.jpg
0bfdf77972cdd7af66d955e336357c52
0444c362184a8b73445b630279a2e1809d46eea2
28052 F20101112_AACJIQ armistead_j_Page_072.QC.jpg
40c2449e7e39a2cfec44e4ebd3a33a80
1e8ecbd1a6652abf205ae7ec3a2618eb87ab3f59
F20101112_AACIGD armistead_j_Page_121thm.jpg
7b2abf0a430509fabe4b89fd051aebae
252b74139f541cde68f6bb15aa6dddfbcbd1c26e
1877 F20101112_AACIFO armistead_j_Page_093.txt
9b59c501ec0fd79fcd38da35edc36482
d2dfd6826a02000e8f69ce454b119184b8939e1c
6882 F20101112_AACJJG armistead_j_Page_083.QC.jpg
0690832d2928201622e648d6613c768a
98c0be2f6a15eb5c96d54718828fe03e61367eb3
7864 F20101112_AACJIR armistead_j_Page_072thm.jpg
ca47ef977d4b59fe618ae97d8538b4f9
916477023cf9563f8045b2fd421d4a58711b6ca4
34725 F20101112_AACIGE armistead_j_Page_025.QC.jpg
e268dca66a280ad2cc175ee13ab45c2b
9942d079b1d40e55f34dc2e469e6f894f66a9334
48666 F20101112_AACIFP armistead_j_Page_090.pro
de612da0e9b28f1bcffa02fbb5fe40a7
15e3fb3d812fd5ea3c3d4182b2b558423404b013
8052 F20101112_AACJIS armistead_j_Page_073thm.jpg
1ccd3ffadb0f2ce2ba1dc883ce1b8877
4e11fe31cd1d1f9f80d5d845820595f0b24f4bc0
2035 F20101112_AACIFQ armistead_j_Page_026.txt
e85079e907db4ade6b12ab8cc0cc3c4b
e8eea87e8b621b6d0cfe48e1fb93bb0eed1ea792
2221 F20101112_AACJJH armistead_j_Page_083thm.jpg
fdf19c81868bcff1d784ca80c6f8b2cf
dd5000ba7a0ee5268a77d79f9fe679c31326713c
32549 F20101112_AACJIT armistead_j_Page_074.QC.jpg
83616169aebe7c6ebaf40cb98063e4da
315e61535f7114e4297b022493fb1a6c481b1c46
544 F20101112_AACIFR armistead_j_Page_081.txt
60f69153b217a9dff785dd21015408bf
acf2bf00e1e603c324f16f1d17441e269e1904c7
44210 F20101112_AACIGF armistead_j_Page_091.pro
318ea8770ec945d004a377d78bc12c7b
707fbf1ddd57397567b959264dbf1fb85de2c840
7162 F20101112_AACJJI armistead_j_Page_084.QC.jpg
cdd59115aafdc7b808ef4580e5732db3
4928dcd9ed6a01905b8aebad7bcf331071255b7d
35585 F20101112_AACJIU armistead_j_Page_075.QC.jpg
d08bf4ca4578c63fd0fbfd53961a82a9
71852f9583541908dd7401a3eeb225514cf1d63e
97050 F20101112_AACIFS armistead_j_Page_109.jp2
1a7f441ee60a02c0047c30efcec0f759
98805f818fa5dc5374473cc4b973a9842cc7395f
8952 F20101112_AACIGG armistead_j_Page_023thm.jpg
a078acea774fbde40388080af9c20f58
35c2b8601f7c89b1c9a57dcdf17c2a70dac1a28a
2543 F20101112_AACJJJ armistead_j_Page_084thm.jpg
f8f1230625e4130ff5fb9750b20bb770
e051d30d73049ac23b04aaad24afe9d53976b9f1
8810 F20101112_AACJIV armistead_j_Page_075thm.jpg
5be934e200a124a066b56ba3a844068e
704d6a4fea818330ea03c8b3742425697d15a905
119737 F20101112_AACIFT armistead_j_Page_014.jp2
595368377dd4693f629dbea19c0fc93d
d91ad72f6d1f34d922ecd41da55180834ef73b8a
2634 F20101112_AACIGH armistead_j_Page_057thm.jpg
a08644330f1b36b170d356d5ec5db2cf
5cd77dd020df37c6c5f0fa2e78bccf44503998fb
32618 F20101112_AACJJK armistead_j_Page_085.QC.jpg
28a48a4cee133e830bfeedcb9f7a4bd5
d597361e9e2ae19c8ff1f8e0cc72f333e84febf0
35907 F20101112_AACJIW armistead_j_Page_077.QC.jpg
9452467c74f2dbb679675a7a657d02fd
b57d8f48e92fd74c118e65a8677627f95545da84
35367 F20101112_AACIFU armistead_j_Page_052.jp2
32bc2faef6af78389d0de06002086a62
7edbe9a697d52e4d20b10e46433e3e8ab67f1936
26517 F20101112_AACIGI armistead_j_Page_005.QC.jpg
2c69a81d3e775035938349f3881c6d02
70c7620ebfa81b4be5abf74684281f9de2fe8af9
9151 F20101112_AACJKA armistead_j_Page_094thm.jpg
a6663b942e093905463dd1945b11efaf
037533cea265ea68eeac6d2ff5857a5fc99db692
8089 F20101112_AACJJL armistead_j_Page_085thm.jpg
4fa1d4281512cc17e85f7fa2c225a63e
36e533d14e2e988ab6f095e1e729d3fc30e10aa9
8651 F20101112_AACJIX armistead_j_Page_077thm.jpg
aa35773ef77b6362ce5bf466850d771f
8a9516bf632bb16f245788c031921d1ad07cc629
8726 F20101112_AACIFV armistead_j_Page_019thm.jpg
50860349d4e2a5bdf9b1697eeaeb708e
fe05eed81b318baa7239daa54671752c84a12f82
F20101112_AACIGJ armistead_j_Page_002.tif
04d8e2caa8dbee1470b8646365e9b734
b237c0133da405194bb55fc2b3c70e43433fce72


Permanent Link: http://ufdc.ufl.edu/UFE0020148/00001

Material Information

Title: Interactions of Invasive Species in Mosquito Container Communities in Virginia
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: UFE0020148:00001

Permanent Link: http://ufdc.ufl.edu/UFE0020148/00001

Material Information

Title: Interactions of Invasive Species in Mosquito Container Communities in Virginia
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: UFE0020148:00001


This item has the following downloads:


Full Text





INTERACTIONS OF INVASIVE SPECIES IN MOSQUITO CONTAINER COMMUNITIES
IN VIRGINIA





















By

JENNIFER S. ARMISTEAD


A THESIS PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE

UNIVERSITY OF FLORIDA

2007

































2007 Jennifer S. Armistead

































To my husband Paul, whose love, friendship, patience and encouragement are heaven-sent, and
to my father Gary, for showing me that in all things nature there is something marvelous.









ACKNOWLEDGMENTS


I would like to thank my advisor, L. P. Lounibos, for sharing his expertise and providing

direction for my research, as well as for his confidence, patience, and flexibility in working with

me from a distance. I am grateful to my committee members, G. F. O'Meara for his insight in

working with Ochlerotatus atropalpus, and J.R. Arias for sharing his knowledge and ingenuity

and for graciously allowing me to use his equipment and laboratory space. I would like to thank

R. Escher, N. Nishimura, and M. Reiskind for their technical assistance during my time at the

Florida Medical Entomology Laboratory, and at the Fairfax County Department of Health in

Virginia I am appreciative of J. Frescholtz and J. van der Voort for their assistance in the field,

and A. Joye for his help with GIS. I am grateful to L. McCuiston for providing the 0. atropalpus

and O. japonicus eggs used in this research. I would also like to thank B. Harrison and J. Scott

for engaging in discussions with me regarding O. japonicus.









TABLE OF CONTENTS


page

A C K N O W L E D G M E N T S .................................................................................... .....................4

ABSTRAC T ..................................................... 10

CHAPTER

1 INTRODUCTION AND LITERATURE REVIEW ............................................................12

In v a sio n B io lo g y ............................................................................................................... 12
O chlerotatusjaponicus ........................................ ......................... ................ 15

2 FIELD ASSESSMENT OF INTERSPECIFIC INTERACTIONS AMONG INVASIVE
AND NATIVE CONTAINER-INHABITING MOSQUITOES................. ............. .....19

Introduction....................................................................19
M materials and M methods ........................ .. ........................ .. .... ........ ....... .. 2 1
O v ip o sition T rap s ................................................................ 2 1
N natural and Artificial Containers ............................................................................. 23
A dult Surveillance ...... ................. ....... .... .................... .... .............. 24
D ata A analysis ................................................... 24
O v ip o sition T rap s ................................................................24
N natural and Artificial Containers ............................................................................. 25
A dult Surveillance ......... ...... ........................................ .... .............. 27
R e su lts ......... ...................... ................. ................................... 2 8
O v ip o sition T rap s ................................................................2 8
N natural and Artificial Containers ............................................................................. 29
Interspecific A association s ............................................................. .....................30
H habitat C om prisons ................. .... ............................................ ........ ...... .. 3 1
Aedes albopictus and Ochlerotatus japonicus ........................................................31
Ochlerotatusjaponicus and Ochlerotatus triseriatus ...........................................34
Ochlerotatus atropalpus and Ochlerotatusjaponicus ..........................................34
A dult Surveillance ...................................... ..................... ..... ......... ...... 35
D discussion ................................... ...................................... ................ 36

3 INTERSPECIFIC COMPETITION BETWEEN AEDES ALBOPICTUS AND
OCHLOEROTA TUS JAPONICUS .......................... .................................. ............... 66

Introduction .......... ................................ ...............................................66
M materials and M methods ........................ .. ........................ .. .... ........ ........ 69
D ata A n a ly sis .................................................................................................................... 7 1
P population G row th C orrelates ........................................ ...........................................7 1
Composite Index of Population Performance.................. .......................... 72
R esu lts ............................... ......................... ............................ 74









Survivorship to A adulthood ........................................... .................. ............... 74
D evelopm mental Tim e ............................................ .. .. ........... ......... 74
F em ale W ing L ength .............. .................................... .. .................. .... ........... 75
Estimated Finite Rate of Increase ()')........................... .......................75
D discussion .................................... ..................................... ................. 75

4 INTERSPECIFIC COMPETITION BETWEEN OCHLEROTATUSATROPALPUS
AND OCHLEROTATUS JAPONICUS ............................................................. ...............85

Introduction .......... ................................ ...............................................85
M materials and M methods ........................ .. ........................ .. .... ........ ........ 87
D ata A n aly sis .............................................................. ................ 8 9
P population G row th C orrelates .............................................................. .....................89
Composite Index of Population Performance.................. .......................... 89
R e su lts ...................................................................... ........ .. ......... ...... .... 9 2
Survivorship to A adulthood ........................................... .................. ............... 92
D evelopm mental Tim e ............................................ .. .. ........... ......... 92
F em ale W ing L ength .............. .................................... .. .................. .... ........... 93
Estimated Finite Rate of Increase ( ')...................................................... ................93
D iscussion................................................................... 93

5 CONCLUSIONS ................... ......... .. ...... ... ..................105

APPENDIX

A DESCRIPTIVE STATISTICS AND INFORMATION: CHAPTER 2 .............................109

B HABITAT COMPARISONS: RANK ORDERS OF MOSQUITO SPECIES ....................110

L IST O F R E F E R E N C E S ............................................... ................................... ..................... 11

B IO G R A PH IC A L SK E T C H .............................................................................. ... ............ 124









LIST OF TABLES


Table page

2-1 Frequency of occurrence of A. albopictus, 0. hendersoni, and 0. triseriatus in ovitraps
set at three sites and two heights in Fairfax, Virginia in 2006........................................44

2-2 Frequency of occurrence, by month, of A. albopictus, 0. hendersoni, and 0. triseriatus
in ovitraps in Fairfax County, Virginia in 2006.................................... ..................45

2-3 Monthly abundance (mean number of eggs per trap) ofA. albopictus, 0. hendersoni, and
0 triseriatus collected in ovitraps ......... ................................................... .............. 46

2-4 Coefficients of association (C8) for the most abundant species in 191 artificial and
natural container samples from Fairfax County, Virginia in 2006 ..................................47

2-5 Intra- and interspecific mean crowding of the most abundant mosquito species collected
from artificial and natural containers in Fairfax County, Virginia in 2006....................48

2-6 Species rank abundances compared for different container habitats sampled in Fairfax
County, Virginia from May September 2006. ..................................... ............... 49

2-7 Number of larvae and average instar number for A. albopictus and O. japonicus
collected in Fairfax County, Virginia by month, May September 2006.......................50

2-8 Tests for significant heterogeneity of monthly instar distributions ofA. albopictus and 0.
japonicus from June August 2006, based on log-rank statistics.................. ............... 51

2-9 Least square means (SE) for intraspecific crowding (transformed by logo (x + 1))
among larvae of A. albopictus and O. japonicus June August 2006. ........................ 52

2-10 Frequencies of collection ofA. albopictus, O. japonicus, and 0. triseriatus adult
females in C02-baited CDC light traps and gravid traps, 2004 2006...........................53

3-1 Means (SE) of population growth correlates for A. albopictus and O. japonicus ...............79

4-1 Means (SE) of population growth correlates for O. japonicus and 0. atropalpus................99

A-i Descriptive statistics and information for mosquito species collected in a survey of
natural and artificial container habitats in Fairfax County, Virginia in 2006................09

B-l Rank orders of immature mosquito abundances used for habitat comparisons of rock
pools, tires, sm all and large artificial containers .......................................... ..................110









LIST OF FIGURES


Figure page

2-1 Map of Fairfax County, Virginia showing locations of study areas that were sampled or
censused repeatedly. ......................... ...... .................... .. .... ................. 54

2-2 Proportion of mosquito-positive containers containing A. albopictus, 0. japonicus, or
both A. albopictus and 0. japonicus May September 2006........................................55

2-3 Monthly abundance (mean number of mosquitoes collected per container) of A.
albopictus and 0. japonicus (+SE) from 91 mosquito-positive artificial containers ........56

2-4 Seasonal occurrences (proportion of species-positive containers per month) of A.
albopictus and 0. japonicus collected from artificial containers in 2006 ......................57

2-5 Monthly instar distributions of A. albopictus from May September 2006 in Fairfax,
V irginia ......................................................... ................................. 58

2-6 Monthly instar distributions of O. japonicus from May September 2006 in Fairfax,
V irginia ......................................................... ................................. 60

2-7 Interspecific mean crowding ofA. albopictus by O. japonicus and 0. japonicus by A.
a lb op ictu s ...................................... ..................................................... 6 0

2-8 Intraspecific mean crowding (density of conpspecifics encountered per unit resource, a)
of A. albopictus and 0. japonicus by month........................................... ...............61

2-9 Metamorphic success of A. albopictus and 0. japonicus collected from containers in
which the two species co-occurred. .............................................................................62

2-10 Mean weekly abundance of A. albopictus collected in (A) CO2-baited light traps and
(B) gravid traps over time, from 2004 through 2006, in Fairfax County, Virginia..........63

2-11 Mean weekly abundance of O. japonicus collected in (A) C02-baited light traps and
(B) gravid traps over time, from 2004 through 2006, in Fairfax County, Virginia..........64

2-12 Mean weekly abundance of 0. triseriatus collected in (A) CO2-baited light traps and
(B) gravid traps over time, from 2004 through 2006, in Fairfax County, Virginia..........65

3-1 Mean survivorship (proportion of the original number of larvae surviving to adulthood)
of A. albopictus and 0. japonicus (SE) ........................................ ....................... 80

3-2 Means of median time to adulthood for female A. albopictus and 0. japonicus (SE)..........81

3-3 Means of median time to adulthood for male A. albopictus and 0. japonicus (SE).............82

3-4 Means of median wing lengths of A. albopictus and 0. japonicus adult females (+SE) ........83









3-5 Mean estimates of population performance (', an estimate of the finite rate of increase
for the cohort) for female A. albopictus and O. japonicus adults (+SE) .......................84

4-1 Mean survivorship (proportion of the original number of larvae surviving to adulthood)
of O. japonicus and 0. atropalpus (SE) .............. ............................................... 100

4-2 Means of median time to adulthood for female O. japonicus and 0. atropalpus (SE).......101

4-3 Means of median time to adulthood for male O. japonicus and 0. atropalpus (SE)..........102

4-4 Means of median wing lengths of O. japonicus and 0. atropalpus adult females (+SE) .....103

4-5 Mean estimates of population performance (V', an estimate of the finite rate of increase
for the cohort) for female O. japonicus and 0. atropalpus adults (+SE) ........................ 104











ABSTRACT OF THESIS PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE
REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE



INTERACTIONS OF INVASIVE SPECIES IN MOSQUITO CONTAINER COMMUNITIES
IN VIRGINIA

By

Jennifer S. Armistead

May 2007

Chair: L. Philip Lounibos
Major: Entomology and Nematology

The success of an invasive species to become established in a new region often depends on

its interactions with ecologically similar resident species. Introductions of disease vectors,

particularly mosquitoes, are of significant importance as their invasions may have ecological and

epidemiological consequences. Interactions of a recent invasive mosquito with resident species

in containers in Virginia were evaluated through field surveys and controlled experiments.

In my study, sampling of larvae from natural and artificial containers, trapping of adults,

and ovitraps were used to confirm and quantify co-occurrences and potential interactions of

Ochlerotatusjaponicus with resident mosquitoes in these habitats. Frequent and abundant

occurrences of O. japonicus in rock pools were associated with the possible decline and

displacement of native 0. atropalpus. Laboratory evaluation of the effects of larval resource

competition on the population performance of these two species suggest that interspecific

competition is probable and likely to favor the success of O. japonicus over 0. atropalpus.

Autogenous reproduction of 0. atropalpus, which requires a lengthened period of larval









development to obtain nutrient reserves for egg development, may disadvantage it in larval

competition in conditions of limited resources.

In my study, field collections of 0. japonicus from artificial containers inhabited by larvae

of resident mosquitoes demonstrated their coexistence in these habitats. A field experiment that

measured inter- and intraspecific effects of larval density on the population performance of A.

albopictus and 0. japonicus indicated the former species to be a superior competitor. However,

the ability of O. japonicus to perform equally well in the presence of absence ofA. albopictus

suggests these two species will be able to coexist in artificial container habitats in nature. High

larval densities, intraspecific mean crowding, and superior population performance of A.

albopictus under inter- rather than intraspecific conditions suggest that intraspecific competition

may be most important in regulating population growth of this species in container habitats.

Interactions of O. japonicus with resident container-inhabiting mosquitoes appear to be

influenced by species-specific differences in seasonality. The ability of O. japonicus to

overwinter as larvae allows it to resume development earlier in the spring than both A. albopictus

and 0. atropalpus. Consequent co-occurrence of older O. japonicus with early hatchlings of A.

albopictus and 0. atropalpus may favor the more recent invader during competition.

Infrequent collections of O. japonicus in ovitraps suggest that use of this technique may be

improved in alternative macrohabitats where this species is most abundant. While trapping of

adults over a three-year period indicated no changes in the frequency or abundance of A.

albopictus, significant population declines of both O. japonicus and 0. triseriatus were observed

over this period. Continued monitoring of all life stages of these species over several years will

be necessary to observe any significant population changes or identify ecological processes at

work since the invasion of O. japonicus.









CHAPTER 1

INTRODUCTION AND LITERATURE REVIEW

Invasion Biology

Biological invasions are occurring at an alarming frequency worldwide, the impacts of

which threaten biodiversity, ecosystem functioning, resource availability, national economies,

and human health (Ruesink et al. 1995, Simberloff 1996). Nonindigenous organisms, including

invertebrates, vertebrates, plants, bacteria, and fungi, are spreading into new regions at

unprecedented rates, becoming established in all but the most remote areas of the planet.

Historically, some of the most important biological invasions have involved dispersal of disease

vectors, particularly mosquitoes (Diptera: Culicidae), a trend that has continued at an increasing

rate over the past century (reviewed by Lounibos 2002, Juliano and Lounibos 2005). The most

recent successful invasions of mosquitoes have resulted from human transport of immature

stages. The most notable invasions and range expansions in the United States have been by

mosquitoes that occupy container habitats (Lounibos 2002), which is the topic of my study.

The patterns and processes related to the introduction, establishment, spread, and impact of

non-native species is the focus of invasion biology (Williamson 1996). The term invasive refers

to an introduced species that has increased and spread (Daehler 2001, Richardson et al. 2000),

with the potential to impact native species and ecosystems, or human activities (Juliano and

Lounibos 2005). Common characteristics of invasive species are thought to include an r-selected

life history strategy, range expansion, high rate of population increase, ability to compete for

resources and habitat with native species, repeated introductions, and associations with humans

(reviewed by Sakai et al. 2001). The three most successful species of invasive mosquitoes, Aedes

aegypti, A. albopictus and the Culexpipiens Complex, seem to have been favored by propagule

(or invasion) frequency and previous invasion success (Lounibos 2002). A retrospective review









of life history characteristics of successful invasive mosquitoes by Juliano and Lounibos (2005)

revealed that desiccation-resistant eggs and occupying human-dominated habitats are

significantly associated with invasive status, while larval habitat, autogeny, and diapause are not;

however data are absent for many species.

Invasive organisms may impact native species through biotic interactions, such as

predation, parasitism and competition, as well as ecosystems by affecting ecological processes,

such as water and nutrient cycling (Williamson 1996). Invasions may also have genetic and

evolutionary consequences, resulting in short-term changes, such as hybridization or

introgression (Rhymer and Simberloff 1996), or long-term changes from genetic drift or natural

selection (Williamson 1996, Sakai et al. 2001). Multiple processes will likely act in a single

invasion system, and they may either facilitate invasion success and spread, which is often

indicated by the decline or elimination of ecologically similar species (Juliano 1998), or act as

barriers to invasion (Rosen et al. 1976, O'Meara et al. 1989).

Many ecological processes associated with invasive species have been shown to play

important roles in structuring the mosquito communities of container habitats, including

interspecific competition, predation, and parasitism. Interspecific competition may be defined as

any negative negative effect between two species or a negative zero effect where one species

affects another but is not reciprocally affected. Interspecific competition is unavoidable unless

the invader is filling a vacant niche by exploiting a previously unoccupied habitat or unused

resource (Williamson 1996). Interspecific competition among mosquitoes encompasses resource

competition (Ho et al. 1989, Livdahl and Willey 1991, Daugherty et al. 2000, Juliano et al. 2004,

Costanzo et al. 2005), chemical interference (Sunahara and Mogi 2002, Bedhomme et al. 2005),

mating interference (Ribiero and Spielman 1986, Nasci et al. 1989), and hatching inhibition









(Edgerly et al. 1993). Predation is a prominent feature of many container habitats utilized by

mosquitoes. Differences in behavioral responses to water-borne cues from predation (Kesavaraju

and Juliano 2004) are thought to result in selective predation on invasive (Grill and Juliano 1996,

Griswold and Lounibos 2005a,b) or native (Lounibos et al. 2001) species. Intraguild predation,

in which older immature stages prey upon newly hatched larvae of conspecifics or

heterospecifics, is also known to occur in mosquito communities (Edgerly et al. 1999, Koenraadt

and Takken 2003, Koenraadt et al. 2004). This type of predation is often facilitated by

differences in seasonality, in which one species hatches earlier in the year than the other (Teng

and Apperson 2000). Apparent competition caused by shared gregarine parasites has been

investigated in interactions of invasive mosquitoes with multiple resident species with varying

results (Juliano 1998, Aliabadi and Juliano 2002).

In addition to effects on resident species or ecosystems, invasive mosquitoes are of

particular concern because they may also impact human or vertebrate animal health. While

interactions with resident species usually occur among aquatic larvae, it is the terrestrial adult

phase of mosquitoes that is most likely to impact human health. Invasive mosquitoes in the New

World have been associated with human disease outbreaks of yellow fever (Tabachnick 1991),

dengue (Gubler 1997), and malaria (Soper and Wilson 1943, Lounibos 2002), and may be

associated with the transmission of Eastern equine, LaCrosse, and West Nile encephalitis viruses

(reviewed by Lounibos 2002, Juliano and Lounibos 2005). Human health may be affected by

invasive mosquitoes in three ways (Juliano and Lounibos 2005): simultaneous introduction of a

novel vector and novel pathogen (Tabachnick 1991, Gubler 1997), acquisition of a native

pathogen by a novel vector (Soper and Wilson 1943, Gubler 1997, Lounibos 2002), or

independent introductions of a novel vector and a novel pathogen (Ross 1911, Kramer and









Bernard 2001). Simultaneous or independent introductions of novel vectors and novel pathogens

may create new public health threats due to the high susceptibility of the host population,

whereas if a novel vector becomes involved in an existing disease transmission cycle, it alters the

nature of an existing public health threat by changing the transmission rate due to differences in

vector efficiency (Juliano and Lounibos 2005).

Ochlerotatusjaponicus

In 1998, Ochlerotatusjaponicus, a container-inhabiting mosquito native to eastern Asia

(Tanaka et al. 1979), was detected independently in light trap collections in New York and New

Jersey (Peyton et al. 1999) and human biting collections in Connecticut (Andreadis et al. 2001).

The introduction of O. japonicus is presumed to have occurred via tire shipments (Peyton et al.

1999), a mechanism which accounted for multiple interceptions of larvae of this species in New

Zealand (Heardon et al. 1999, Laird et al. 1994). Since its arrival in 1998, O. japonicus has

spread throughout the eastern United States with reports from Alabama (Qualls and Mullen

2006), Georgia and South Carolina (Reeves and Korecki 2004), Indiana (Young et al. 2004),

Maine (Foss and Dearborn 2001), Maryland (Sardelis and Turell 2001), Missouri (Gallitano et al.

2006), Ohio and Pennsylvania (Fonseca et al. 2001), Tennessee (Caldwell et al. 2005), Virginia

(Harrison et al. 2002), and Vermont (Graham and Turmel 2001). On the West coast of the United

States, O. japonicus is only known to occur in Washington state (Roppo et al. 2004).

Examination of patterns of genetic diversity using random amplified polymorphic DNA and

sequences of mtDNA from populations spanning the range of O. japonicus in Japan and the

United States revealed distinct genetic signatures in U.S. populations, suggesting multiple

introductions from Japan (Fonseca et al. 2001). In addition to the United States, O. japonicus has

become established in Canada (Savignac et al. 2002, Thielman and Hunter 2006) Belgium

(Widdel et al. 2005), and France (Schaffner et al. 2003).









Ochlerotatusjaponicus was previously known as Aedesjaponicus until revisions of that

genus by Reinert (2000), based on differences in the primary characteristics of male and female

genitalia, elevated the subgenus Ochlerotatus to generic level. Ochlerotatusjaponicus is a

member of the Chrysolineatus subgroup of the subgenus Finlaya. Ochlerotatusjaponicus sensu

lato includes four morphologically similar subspecies that occur throughout most of Japan,

Taiwan, Korea, eastern China, and Russia (Tanaka et al. 1979): 0. japonicus amamiensis

(Tanaka, Mizusawa and Saugstad 1979), 0. japonicusjaponicus (Theobald 1901), 0. japonicus

shintienensis (Tsai and Lien 1950), and 0. japonicus yaeyamensis (Tanaka, Mizusawa and

Saugstad 1979). Only the subspecies O. japonicusjaponicus, which is common in Palearctic

Japan and Korea, has been detected outside its native range (Fonseca et al. 2001); henceforth in

this thesis, 0. japonicusjaponicus will be referred to simply as O. japonicus.

In both the native range of this species and the United States, O. japonicus larvae inhabit a

wide variety of natural (treeholes, rock pools) and artificial containers (buckets, tires, birdbaths),

with rock pools being the preferred habitat (Tanaka et al. 1979, Andreadis et al. 2001, Scott

2003). Surveys have revealed that the species may occur in temporary ground water sources as

well, although this is uncommon (LaCasse and Yamaguti 1950, Andreadis et al. 2001).

Ochlerotatusjaponicus larvae have been detected in containers with varying levels of sun

exposure and water temperature (Oliver et al. 2003). Adults of this species have been shown to

feed on avian and mammalian hosts in the laboratory (Miyagi 1971), although bloodmeal

analyses of wild-caught females have all been mammalian in origin (Scott 2003, Apperson et al.

2002). Ochlerotatusjaponicus will bite humans that encroach on its habitat (Knight 1968), but

are often protracted in their approach (B. Harrison personal communication).









The introduction of O. japonicus into the United States is of considerable interest because

of the vector potential of this species as well as the ecological consequences that may result from

its invasion. Ochlerotatusjaponicus has demonstrated the ability to transmit Japanese

encephalitis (Sucharit et al. 1989, Takashima and Rosen 1989) and Getah (Takashima and

Hashimoto 1985) viruses in the laboratory, although it is not considered an important vector of

either virus in its native range, having only been implicated in the transmission of Japanese

encephalitis virus in Far East Asia (Chagin and Kondratiev 1943). Ochlerotatusjaponicus is also

a competent laboratory vector of eastern equine encephalitis (Sardelis et al 2002a), LaCrosse

(Sardelis et al. 2002b), St. Louis encephalitis (Sardelis et al. 2003), and West Nile (Sardelis and

Turrell 2001, Turrell et al. 2001) viruses. However, in the United States only the latter has been

recovered from wild-caught females of this species (Werner 2001, White et al. 2001, Scott

2003). This combination of a separately introduced novel vector and novel pathogen could

become epidemiologically significant in North America.

Ochlerotatusjaponicus is known to co-occur with numerous resident species in natural and

artificial containers in North America (Andreadis et al. 2001, Scott et al. 2001a, Oliver et al.

2003, Thielman and Hunter 2006). This species has most frequently been recovered from

containers inhabited by A. albopictus, C. pipiens (Andreadis et al. 2001, Gallitano et al. 2006),

Culex restuans (Scott et al. 2001a), 0. atropalpus, and 0. triseriatus (Andreadis et al. 2001,

Oliver et al. 2003), although it has also been known to occur with Anophelespunctipennis (Scott

et al. 2001a), Anopheles quadrimaculatis (Thielman and Hunter 2006), and Culex territans

(Oliver et al. 2003). However, there is currently no information concerning the nature of the

interactions of O. japonicus with these species or the ecological processes that have been









operating during its invasion. Therefore, a study of the interactions of O. japonicus with resident

species in natural and artificial container communities was proposed.

The primary objective of my study was to confirm and assess the interactions of O.

japonicus in mosquito container communities and make predictions concerning the ecological

processes that may be operating as a result of the invasion of this species. This was accomplished

through a field survey of all life stages conducted areas in which this species is known to occur

in various types of mosquito container communities. As rock pools are the species' preferred

habitat, it is predicted that O. japonicus will most likely interact with and impact the native rock

pool mosquito, 0. atropalpus. Furthermore, the tendency of O. japonicus to inhabit artificial

containers suggests that this species will also likely encounter and interact with the Eastern

treehole mosquito, 0. triseriatus, and its closely related sibling species, 0. hendersoni, as well

the now resident invader A. albopictus. Because severe crowding and limiting resources are

frequent in container habitats (Kitching 2000), it is predicted that interspecific competition via

depletion of shared resources will be important in the interactions of the larvae of O. japonicus

and A. albopictus in artificial containers, and 0. atropalpus in rock pools. Therefore, the effects

of interspecific resource competition on the growth, survivorship, and reproductive success of

these species were measured in a series of field and laboratory experiments, in order to determine

the impact of these larval conditions on the overall population growth of these species.









CHAPTER 2


FIELD ASSESSMENT OF INTERSPECIFIC INTERACTIONS AMONG INVASIVE AND
NATIVE CONTAINER-INHABITING MOSQUITOES

Introduction

Naturally occurring throughout Japan, Korea, Taiwan, and China (Tanaka et al. 1979),

Ochlerotatusjaponicus is the most recently recognized mosquito species to invade the United

States, which is thought to have occurred via the international transport of used tires (Peyton et

al. 1999, Lounibos 2002). Initially collected in the summer of 1998 in Connecticut (as reported

by Andreadis et al. 2001), New York and New Jersey (Peyton et al. 1999), 0. japonicus has

since become established throughout the northeastern United States (reviewed by Scott 2003),

and spread south to Georgia (Reeves and Korecki 2004), north into Canada (Savignac et al.

2002, Thielman and Hunter 2006), and west to Missouri (Gallitano et al. 2006). The spread of O.

japonicus on the west coast has so far been limited to Washington State (Roppo et al. 2004).

Distinct genetic profiles of O. japonicus collected from various sites in Japan and throughout its

range in the United States suggest multiple introductions of the species in the U.S. (Fonseca et al.

2001).

Although common and widely distributed within its native range in eastern Asia (LaCasse

and Yamaguti 1950, Tanaka et al 1979), 0. japonicus attracted little scientific attention until its

discovery in North America, where its potential involvement in the transmission of West Nile

virus (Sardelis and Turell 2001) and other endemic arboviruses (Sardelis et al. 2002ab, 2003) has

stimulated significant scientific interest. Knowledge of the biology and surveillance methods for

the collection of O. japonicus has since improved remarkably. Collection records from both

Japan (Sasa et al. 1947) and the United States (Andreadis et al. 2001) indicate that adults are

rarely taken in light traps, carbon dioxide (C02) baited traps, or biting collections. The use of









infusion-baited gravid traps, typically intended to attract mosquitoes of the genus Culex (Reiter

1983), has provided the most consistent and greatest numbers of O. japonicus adults (Scott et al.

200 b). As a container-inhabiting mosquito, oviposition traps are a logical sampling method with

many potential applications, including detection in new areas, routine population surveillance,

and species distribution studies. However this technique has been employed with varying success

in collecting O. japonicus (Andreadis et al. 2001). Modification of the traditional oviposition trap

using blocks of expanded polystyrene as an oviposition substrate in a variety of aquatic habitats

has been indicated as a suitable alternative (Scott and Crans 2003).

Ochlerotatusjaponicus appears to be most easily and consistently collected in large

numbers in the larval stage. Within its native range in Asia, O. japonicus larvae are found in a

wide variety of natural and artificial containers, with rock pools being the preferred oviposition

substrate (LaCasse and Yamaguti 1950, Tanaka et al. 1979). In the United States, O. japonicus

larvae are commonly found in artificial (e.g. automobile tires, bird baths, gutters, flower pots)

and natural (tree holes and rock pools) containers where they feed on organic detritus and

microorganisms, often in close association with human dwellings (Andreadis et al. 2001). Larvae

may be found in shaded or sunny locations (Scott 2003) in clean, clear water containing decaying

leaf litter and algae (Scott et al. 2001a).

The likelihood of an invasive species colonizing a new region depends on its ability to

adapt to new environmental conditions and compete with resident species that occupy a similar

ecological niche. The indigenous mosquito species most likely to be affected by O. japonicus in

the United States include the rock hole mosquito, 0. atropalpus, the Eastern treehole mosquito,

0. triseriatus and its closely related sibling species, 0. hendersoni, as well as the now resident

invader Aedes albopictus. Frequent and abundant collections of O. japonicus larvae co-occurring









with both 0. atropalpus in rock pools and A. albopictus in artificial containers, provide a natural

setting for interspecific larval resource competition (Andreadis et al. 2001) which some speculate

may limit its invasion success (Juliano and Lounibos 2005). However at present, no field

research has been published to confirm this conjecture following the establishment of O.

japonicus in the United States.

To better understand the ecological niche of O. japonicus with respect to the

aforementioned resident container-inhabiting species, a broad-based survey of all life stages was

conducted in Fairfax County, Virginia (latitude 3850' N, longitude 777' W), a suburb of

Washington, D.C., where all of these species are known to co-occur. Sampling of natural and

artificial larval habitats was used to confirm and quantify co-occurrences and potential

interactions of O. japonicus with resident mosquitoes in these habitats. Routine trapping of

adults over a three-year period was used to assess changes in frequency or abundance of the

species of interest. Ovitraps were employed to better understand the seasonality of egg-laying

behavior of these species as well as their tendencies to oviposit at ground level versus in the

canopy, or in residential areas versus disturbed or undisturbed forests. In particular, the current

chapter focuses on the potential interactions of O. japonicus with A. albopictus in artificial

containers and 0. atropalpus in rock pools in an effort to make predictions regarding the

competitive outcomes resulting from the co-occurrence of these species.

Materials and Methods

Oviposition Traps

To monitor the distribution, abundance, and oviposition of O. japonicus and resident

container-inhabiting mosquitoes in Fairfax County, VA, standardized collections of eggs

employed the use of ovitraps. These dark, water-filled receptacles containing a removable

oviposition substrate have been commonly used for these purposes (e.g., Kitron et. al. 1989,









Lounibos et al. 2001). Ovitraps were constructed from black polypropylene cups (400 ml

capacity) filled with 200 ml of distilled water and approximately 1 g of dried pin oak leaves

(Quercuspalustris), one of the most common trees in the study area. An oviposition stick,

constructed from a single tongue depressor covered with 76 lb. seed germination paper (Anchor

Paper, St. Paul, MN), served as an oviposition substrate (Steinly et al. 1991). A hole, 2.5 cm in

diameter, was drilled 2.5 cm from the top of each cup to allow for suspension and drainage of the

ovitrap.

Ovitraps were placed within or on the edge of three separate sites in Fairfax County, VA

(Figure 2-1) from May through September 2006. The study sites included a high-density

residential development, an undisturbed forested streambed, and a rural forest disturbed from

frequent dumping of trash. Ovitraps were secured to trees using bundling twine at two heights,

five at ground level and five at approximately 3m, that were pre-selected randomly at each site

using a numbered 10 m by 10 m grid overlay of aerial photographs of each site. Vertical

stratification was employed to increase the likelihood of encountering native treehole

mosquitoes, namely 0. hendersoni and 0. triseriatus, and to assess how frequently O. japonicus

mosquitoes seek oviposition sites above ground-level. Ovitraps were set at each site

simultaneously for a period of five days each month, after which they were retrieved and the

oviposition stick of each was stored separately in an individual plastic bag for transport to the

laboratory.

In the laboratory, each oviposition stick was examined using a dissecting microscope, and

the number of eggs present on each was counted. Seed germination papers were carefully

removed from the oviposition sticks and allowed to dry for seven days at room temperature to

ensure embryonation, after which they were placed in the bottom portion of a plastic tray (21 cm









high x 12 cm diameter) and flooded with 400 ml of tap water. Non-natural food, comprised of

ground rabbit chow and brewer's yeast in a 3:1 ratio, was added to each tray upon flooding every

other day to ensure larval development. All mosquitoes were identified in the fourth larval instar

using keys (Slaff and Apperson 1989, Darsie and Ward 2005), and mortality at other immature

stages was noted. Because intermittent hatching is a common occurrence among the eggs of

Aedes and Ochlerotatus mosquitoes, those eggs that did not hatch after being submerged for a

period of seven days were dried on the oviposition substrate for an additional seven days before

reflooding. Those eggs that did not hatch after three cycles of drying and flooding were bleached

and examined microscopically to assess their viability (Trpis 1970). Unhatched eggs were

considered viable if the embryo was segmented, possessed ocelli and an eggbreaker

(Christophers 1960).

Natural and Artificial Containers

To assess the potential for interactions in nature among the larvae of container-inhabiting

mosquitoes in Fairfax, Virginia, a field survey of such habitats was conducted from May through

September 2006 throughout the area (Figure 2-1). Although a few tree holes and ornamental

bromeliad plants were sampled, rock pools were the only natural containers sampled in large

numbers during the study. Only a single river system, the Potomac River and associated

tributaries, were sampled for rock pool mosquitoes. Containers were only sampled once, and the

entire aquatic contents of each were removed using a turkey baster or siphon, measured, and

concentrated by filtering through a fine mesh (small enough to retain first instars) sieve. Samples

were transported to the laboratory in 18-ounce (532.32 ml) plastic Whirl-pak bags. Those

containers containing water but no mosquitoes were noted. The geocoordinates of each site were

obtained with a global positioning system, and the degree of sun exposure (none, partial, or full)

and container type were recorded.









In the laboratory, the mosquitoes from each container were examined separately in a 34.3 x

24.4 cm plastic tray. Fourth instars were identified to species and counted. All other mosquito

immatures were counted, sorted by stage and placed in separate 21 x 12 cm cylindrical growth

chambers (BioQuip Products, Inc., Rancho Dominguez, CA) containing 400 ml of tap water and

food, consisting of three parts ground rabbit chow and one part brewer's yeast. Any predatory

species present, such as Toxorhynchites rutilus, were removed and stored separately. Mosquitoes

collected from the field as pupae were identified upon emergence as adults while all others were

identified as fourth instars. Mortality at all immature stages was noted.

Adult Surveillance

Adult mosquitoes were collected with carbon dioxide (C02)-baited CDC light traps and

gravid traps set at 70 pre-selected trapping sites located throughout Fairfax County (Figure 2-1)

from May through October over a three-year period from 2004 to 2006. One of each type of trap

was set weekly at each site. Traps were set in the early afternoon and retrieved the following

morning. All mosquitoes collected were sorted, counted and identified to the species level using

keys and a dissecting microscope.

Data Analysis

Oviposition Traps

G-tests (Sokal and Rohlf 1981) were used to test for heterogeneity among the frequencies

of mosquito species collected at each site. Pairwise comparisons between species were

conducted as necessary with additional G-tests using the Bonferroni adjustment to correct for

experimentwise error. G-tests were also used to compare the frequency of occurrence of each

individual species by height. Again, pairwise comparisons between sites were conducted for each

species with G-tests using the Bonferroni adjustment to correct for experimentwise error. Due to

non-normal distributions not remediable by standard transformations, Friedman's two-way









analysis of variance and Cochran's Q test were used to compare mean abundance per trap and

frequency of occurrence, respectively, for each individual species among months.

Natural and Artificial Containers

G-tests (Sokal and Rohlf 1981) were used to detect significant differences in frequency of

occurrence of each individual mosquito species, including A. albopictus, 0. japonicus, Culex

pipiens, C. restuans, 0. triseriatus, and 0. hendersoni, by container type, fluid volume, and

exposure to the sun. The coefficient of association (C8), which ranges from +1 to -1, as described

by Hurlbert (1969) was used to quantify co-occurrences of the six most abundant species

collected during the container habitat survey. This metric quantifies frequencies of co-

occurrence based on species presence-absence data. Positive associations between species may

reflect a common habitat preference or interspecific attraction, while negative associations may

result from different habitat preferences or interspecific repulsion. Statistical significance of C8

values were assessed with a corrected X2 formula (Pielou 1977) for approximating a discrete

distribution. Fisher's exact test was applied in those cases with cell totals less than or equal to

five. Only samples from which one or more larvae or pupae were identified were included in

calculations.

To quantify frequencies of encounter among mosquitoes in containers as a function of

habitat size, Lloyd's (1967) indices of mean intra- and interspecific crowding were used per unit

resource (Rathcke 1976), in this case container fluid volume. The mean crowding of one species

upon itself and each of the other species per unit resource was calculated. Interspecific mean

crowding of species x by y per unit resource a was quantified as E(xy,/a,)/1 x,, and intraspecific

mean crowding by the same formula but substituting (x, 1) fory,. Mean inter- and intraspecific

crowding were calculated per month to assess the potential for larval competition between and









among O. japonicus and A. albopictus. A two-way repeated measures-analysis of variance, with

species, month, and interactions as effects was used to detect significant differences in

interspecific mean crowding between the two species over time. Only those months in which the

two species co-occurred more than five times were considered. One-way repeated measures-

ANOVAs were used to detect differences in intraspecific crowding among months for each

species. To compare differences in intraspecific crowding among O. japonicus larvae in artificial

containers and rock pools, a two-way repeated measures ANOVA was used with container type,

month, and interaction as effects. Interspecific crowding data were logo transformed and

intraspecific crowding data were logo (x + 1) transformed to meet assumptions of normality and

homoscedacity. All analyses were performed with PROC MIXED in SAS (SAS 1989).

The large number of samples from both artificial and natural containers permitted a

comparison of the mosquito community structure of these habitats. Kendall's coefficient of rank

correlation, tau (c), was used to estimate the degree of similarity of rank order abundance of

mosquito species in containers in Fairfax County, Virginia (Ghent 1963). The ranked abundances

of species in small vs. large artificial containers, and in rock pools versus tires and artificial

containers were compared. Only those species occurring at least once in both habitats were

considered in the comparisons.

Other analyses focused on potential interactions between A. albopictus and 0. japonicus.

Instar distributions were compared using a Kolmogorov-Smirnov two-sample test, and the

average instar number for each species was calculated per month. Heterogeneity of instar

distributions of O. japonicus and A. albopictus for the months of June, July, and August was

assessed with the life table procedure (PROC LIFETEST) in SAS (SAS 1989). The significance









of heterogeneities was assessed with Wilcoxon and Log-rank tests, which were then used to

construct z-statistics for post-hoc multiple comparisons among groups (Fox 1993).

Metamorphic success of O. japonicus and A. albopictus was calculated per month as

Williams' means (Williams 1937, Haddow 1960), denoted by Mw, where logo (Mw+1) =

[Xloglo(n,+l)]/N, where n, is the number of pupae per total number of immatures in a series of N

container samples. Mw is obtained by subtracting one from the antilog quantity [Xlog(n+l)]/N.

Williams' mean is frequently used as a measure of central tendency in entomological collections

with many zero values (Haddow 1960, Lounibos 1981, Lounibos et al. 2001). Metamorphic

success of the two species was compared using a two-way repeated measures ANOVA with

species, month, and interaction as effects. Only those months in which the two species co-

occurred more than five times were considered. One-way repeated measures-ANOVAs were

used to detect differences in metamorphic success among months for each species. Metamorphic

success of O. japonicus in rock pool and artificial containers was also compared using a two-way

repeated measures-ANOVA, with container type, month, and interaction as effects. All

metamorphic success data were arcsine square root transformed, to best meet assumptions of

normality and homogeneity of variance. Analyses were performed with PROC MIXED in SAS

(SAS 1989)

Adult Surveillance

The frequency of collection for A. albopictus, 0. japonicus, and 0. triseriatus adult

females was calculated per week for 2004 through 2006. Frequencies of collection were analyzed

for each species using two-way repeated measures ANOVAs, with year, trap type, and

interactions as effects. Weekly abundances of adult female A. albopictus, 0. japonicus, and 0.

triseriatus collected in C02-baited CDC light traps and gravid traps were calculated as arithmetic









means (average number collected per trap night) as well as Williams' means, denoted as logo

(Mw+1) = [Xloglo(n,+l)]/N, where n, is the number of individuals collected in each ofN trap

nights. To meet assumptions of normality and homoscedacity, a logo (x + 1) transformation was

applied to Williams' means before analyzing trends in abundance over time, from 2004 through

2006, for each species by linear regression. All analyses were performed with SPSS (SPSS

2002).

Results

Oviposition Traps

Ochlerotatusjaponicus was detected only once in a single ovitrap set at ground level at the

disturbed forest site in June, and was not included in any subsequent analyses. Aedes albopictus,

0. hendersoni, and 0. triseriatus were the only other species collected. The frequencies of

collection were homogeneous among all three species at the undisturbed forest site (G = 2.282, p

= 0.319, df = 2), but were heterogeneous at both the residential (G = 21.06, p < 0.001, df= 2)

and disturbed forest sites (G = 25.575, p < 0.001, df = 2), however A. albopictus was the only

species detected at the former site (Table 2-1). Only A. albopictus and 0. triseriatus were

collected at the disturbed forest site, but pairwise comparisons did not indicate a significant

difference in the frequency of occurrence between these two species (Table 2-1). The

frequencies of occurrence among all three species in ovitraps placed above ground were

homogeneous (G = 0.59, p = 0.744, df = 2), but were heterogeneous at ground level (G = 30.11,

p < 0.001, df = 2). Pairwise comparisons indicated that A. albopictus was collected more

frequently in ovitraps at ground level than either 0. hendersoni or 0. triseriatus (Table 2-1).

G-tests for heterogeneity for the presence ofA. albopictus indicated no significant

difference in the frequency of collection of this species by site (G = 3.206, p = 0.201, df = 2).

The frequency of occurrence of 0. triseriatus (G = 12.468, p = 0.002, df = 2) and 0. hendersoni









(G = 7.284, p = 0.026, df = 2) was heterogeneous among sites, however upon exclusion of those

sites where these species were never detected (residential and disturbed forest, respectively),

these tests were not significant. The frequency of occurrence ofA. albopictus was significantly

greater in ovitraps at ground level than above ground (G = 22.55, p < 0.001 df= 1). There was

no significant difference in the frequency of occurrence of 0. triseriatus (G = 0.445, p = 0.505,

df = 1) or 0. hendersoni (G = 0.002, p = 0.966) by ovitrap height.

The frequency of collection ofA. albopictus was significantly different among months (Q

= 25.217, p < 0.001, df = 4), with the highest frequencies of collection occurring in July and

August (Table 2-2). There was a significant difference among months for the frequency of

collection of 0. hendersoni (Q = 24.8, p < 0.001, df = 4); a comparison of only the two months

in which this species was collected was also significant (Q = 6.0, p = 0.008, df = 1). The

frequency of collection of 0. triseriatus was significantly different among months (Q = 26.4, p <

0.001, df =4), with the highest frequency of collection occurring in July (Table 2-2). Mean

abundances of these three species followed trends similar to their monthly egg frequencies

(Table 2-3).

Natural and Artificial Containers

Overall, 191 containers (131 artificial and 61 natural) were sampled for the presence of

larvae and pupae during the study period, of which 134 were positive for mosquitoes. These

included rock pools, tires, flowerpot saucers, tarpaulins, drainpipes, French drains, birdbaths,

cemetery vases, trashcans, drums, and other small miscellaneous artificial containers. The

following 10 species were collected, in order of frequency of occurrence: A. albopictus, 0.

japonicus, Culex pipiens, C. restuans, 0. triseriatus, 0. hendersoni, Anopheles punctipennis, T.

rutilus, 0. atropalpus, and Orthopodomyia signifera. Descriptive statistics and collection

information for each of these species are provided in Appendix A. Ochlerotatus atropalpus was









collected from only four containers, all of which were rock pools. Anopheles punctipennis was

collected only from rock pools, while T rutilus and 0. signifera were collected only from

artificial containers. These three rare species have not been included in any subsequent analyses

because they were collected in very few samples.

Ochlerotatusjaponicus was the only species to occur more frequently in rock pools than

artificial containers (G = 5.98, p = 0.015, df = 1) while A. albopictus (G = 139.15, p < 0.001, df=

1), C. pipiens (G = 25.6, p < 0.001, df = 1), C. restuans (G = 14.73, p < 0.001, df = 1), 0.

hendersoni (G = 24.95, p < 0.001, df = 1), and 0. triseriatus (G = 29.4, p < 0.001, df = 1) were

collected significantly more frequently from artificial containers. Aedes albopictus (G = 27.63, p

< 0.001, df = 1), C. restuans (G = 8.16, p = 0.004, df = 1), O. japonicus (G = 83.65, p < 0.001, df

= 1), and 0. triseriatus (G = 10.03, p < 0.001, df = 1) were collected more frequently from

containers that were at least partially shaded, while there was no significant difference for C.

pipiens (G = 0.39, p = 0.84, df = 1), 0. atropalpus (G = 2.09, p = 0.15, df = 1), or 0. hendersoni

(G = 3.29,p = 0.070, df = 1).

The mean fluid volume (SE) of containers sampled was 2.42 1, with a median of 0.3 1, and

range of 0.01 120 1. Aedes albopictus (G = 35.37, p < 0.001, df = 1), O. japonicus (G = 7.99, p

= 0.005, df = 1), and 0. triseriatus (G = 4.61, p = 0.032, df = 1) were sampled more frequently

from containers containing less than 1 1 of water, while there was no significant difference for 0.

hendersoni (G = 0.22, p = 0.637, df = 1), C. pipiens (G = 3.19, p = 0.07, df = 1), or C. restuans

(G = 2.01, p = 0.156, df = 1).

Interspecific Associations

Using the Cs index of association, the 15 pairings from containers sampled in Fairfax

County, Virginia revealed seven positive, six negative, and two zero associations (Table 2-4).

Four of the positive associations were significant and those included pairings between C. pipiens









and C. restuans, A. albopictus and 0. triseriatus, A. albopictus and 0. hendersoni, and 0.

hendersoni and 0. triseriatus. There were only two significant negative associations, that of O.

japonicus and 0. triseriatus, and 0. japonicus and A. albopictus. Cs indices were recalculated

excluding rock pools, to determine if this container type contributed disproportionately to those

associations involving species commonly found in rock pools. However, this exclusion did not

have any affect on the magnitude or direction of the associations.

Mean intra- and interspecific crowding was calculated for the six most abundant mosquito

species collected from 191 containers censused from May through September (Table 2-5). Aedes

albopictus and C. restuans both encountered a higher density of conspecifics than any other

mosquito species. 0. japonicus, 0. triseriatus, and C. pipiens encountered a higher density of A.

albopictus than any other species or themselves, while 0. hendersoni encountered a higher

density of 0. triseriatus than any other species or themselves.

Habitat Comparisons

Kendall's coefficient of rank correlation tau (c) was positive in all three comparisons of

rank abundance (Table 2-6), but only the comparison of small and large artificial containers was

significant. These results indicate that while the rank order of abundances of mosquito species

occurring in rock pools and artificial containers are similar, they are not significantly similar.

Ochlerotatusjaponicus was the most abundant species found in rock pools, while A. albopictus

was the most abundant species found in all four subclasses of artificial containers considered for

this analysis (see Appendix B). Although only collected from rock pools, 0. atropalpus ranked

fifth in abundance in this habitat (see Appendix B).

Aedes albopictus and Ochlerotatusjaponicus

Aedes albopictus and 0. japonicus occurred either alone or together in 97.75% of those

containers sampled from May through September that were positive for mosquito immatures.









Aedes albopictus was collected from approximately half of these containers (50.75%) in the

absence of O. japonicus, which was collected from 26% of these containers in the absence of A.

albopictus. The two species occurred together in 21% of these containers. Co-occurrenes of these

two species occurred more frequently in medium-to-large sized (G = 3.67, p = 0.05, df =1),

shaded (G = 29.56, p < 0.001, df = 1) artificial containers (G = 17.65, p < 0.001, df= 1)

containing less than 1 1 of water (G = 9.23, p = 0.002, df = 1). Aedes albopictus was most

frequently sampled in the absence of O. japonicus in September, while the highest proportion of

containers positive for only O. japonicus were sampled in July (Figure 2-2). The two species

were found together most frequently in June (Figure 2-2). Aedes albopictus was consistently

present in greater abundance than O. japonicus in artificial containers in all months except May

(Figure 2-3). Ochlerotatusjaponicus was most frequently collected from artificial containers in

July while A. albopictus was most frequent in August (Figure 2-4).

The average instar number, or age, of A. albopictus larvae was 2.77, ranging among

months from 2.67 to 3.65, while that of O. japonicus was 3.86, ranging from 3.17 to 4.19 (Table

2-7). A comparison of the overall instar distributions of the two species from May through

September by Kolmogorov-Smimov two-sample test indicated the two were significantly

different (D = 18.130, p < 0.001). Significant heterogeneity among monthly instar distributions

was detected from a log-rank test for both A. albopictus (X2 = 13.46, p < 0.001, df = 2) and 0.

japonicus (X2 = 24.22, p < 0.001, df= 2). Paired comparisons showed that the significant

heterogeneity of instar distributions of A. albopictus was largely attributable to that of July,

which was significantly different from June and August (Table 2-8). Paired comparisons for

instar distributions of O. japonicus indicated highly significant differences among all months

(Table 2-8), however graphical comparisons revealed no obvious differences (Figure 2-6. It









should be noted that pupae comprised the greatest proportion of O. japonicus sampled in each of

the months considered (Figure 2-6).

Investigation of the temporal dynamics of co-occurrences of A. albopictus and 0.

japonicus indicated that interspecific mean crowding was not significant affected by species

(F1,24 = 0.46, p = 0.561) or month (F2,22 = 0.39, p = 0.8839). Mean crowding of O. japonicus by

A. albopictus was always more than that ofA. albopictus by O. japonicus, and was highest for

both species in August (Figure 2-7). Intraspecific mean crowding was significantly different

among months for both A. albopictus (F2,30 = 76.51, p < 0.001) and O. japonicus (F2,29 = 9.36, p

< 0.001). While intraspecific mean crowding of O. japonicus was greatest in July, A. albopictus

encountered the greatest density of conspecifics per unit resource the following month in August

(Figure 2-8), pairwise comparisons of least square means indicated that intraspecific competition

was lower for both species in June than July or August (Table 2-9). Because O. japonicus was

collected frequently in high densities from both artificial containers and rock pools, intraspecific

mean crowding for this species was calculated for each habitat as 33.36 and 109.48, respectively.

However, analysis by two-way repeated measures-ANOVA did not indicate that intraspecific

crowding of this species was effected by container type (F1,23 = 1.77, p = 0.1962) or month (Fi,18

= 0.15,p = 0.6989).

In containers where the two species co-occurred, the metamorphic successes of A.

albopictus and O. japonicus, measured as Williams Mean (Mw) number of pupae collected per

total number of immatures, were found to be 0.1234 and 0.1133, respectively. However,

metamorphic success of was not effected by species (F1,24= 0.33, p = 0.5721) or month (F2,22

0.24, p = 0.7871). Metamorphic success was greatest for 0. japonicus in June, while there

appeared to be no difference among months for that ofA. albopictus (Figure 2-9). Repeated









measures-ANOVAs indicated that there were no significantly differences in the metamorphic

success ofA. albopictus (F2,30 = 0.33, p = 0.5620) or O. japonicus (F2,29 = 1.63, p = 0.2135)

among months. While the metamorphic success of O. japonicus was higher in rock pools

(0.1479) than artificial containers (0.1047), this difference was not found to be significant among

container type (F1,26 = 2.09, p = 0.1599) or month (F1,25 = 1.15, p = 0.2930).

Ochlerotatusjaponicus and Ochlerotatus triseriatus

Ochlerotatus triseriatus was only collected from 15 containers during the course of the

census, most of which (73.7%) were sampled in August. Because 0. triseriatus occurred with O.

japonicus in only five of these containers, analysis of the potential for interactions between these

species was rather limited. In addition to the significant negative association of these species as

indicated by the C8 index (Table 2-4), it was found that 0. triseriatus encountered more

conspecifics (34.47) than O. japonicus (19.21) than conspecifics per unit resource, however this

was much less than the interspecific mean crowding of 0. triseriatus by A. albopictus (136.7).

Ochlerotatusjaponicus encountered only 12.85 0. triseriatus compared to 139.3 A. albopictus,

and 127.56 conspecifics per unit resource (Table 2-5). Finally, the metamorphic success of O.

triseriatus was found to be 0.59, while that of O. japonicus was only 0.01 in containers in which

the two species co-occurred.

Ochlerotatus atropalpus and Ochlerotatusjaponicus

Because 0. atropalpus was collected on only four occasions, and only twice with O.

japonicus, an assessment of the potential for interactions of these species was limited. While O.

atropalpus was recovered exclusively from rock pools exposed fully to the sun, 0. japonicus was

only collected from those that were at least partially shaded. A non-significant coefficient of

association (Cs) of zero was calculated for the two species when all container types were

considered. When only rock pools were included, the coefficient decreased slightly to -0.04, but









was still non-significant. These findings are not surprising considering the limited collections of

0. atropalpus from any container habitat. No effort was made to determine the metamorphic

success or interspecific mean crowding of these two species because they only co-occurred

twice.

Adult Surveillance

The frequency of collection of A. albopictus was affected by trap type (F1,19 = 69.439, p <

0.001) but not by year (F2,38 = 1.984, p =0.151), with a higher frequency of collection of this

species in C02-baited CDC light traps than gravid traps in all years (Table 2-10). The frequency

of collection of O. japonicus was affected by both trap type (F1,22 = 75.034, p < 0.001) and year

(F2,44 = 71.504, p < 0.001), with a higher frequency of collection of this species in gravid traps

(Table 2-10). Pairwise comparisons indicated significantly different frequencies of collection of

O. japonicus among all years, with a decline in frequency of collection in both C02-baited CDC

light traps and gravid traps with each subsequent year from 2004 to 2006 (Table 2-10). The

frequency of collection of 0. triseriatus was also affected by both trap type (F1,2 = 9.245, p <

0.001) and year (F2,44 = 67.027, p < 0.001), with a higher frequency of collection of this species

in C02-baited CDC light traps in all years (Table 2-10). Pairwise comparisons indicated that O.

triseriatus was collected more frequently in 2004 than 2006 in C02-baited CDC light traps, and

more frequently in 2004 than in 2005 or 2006 in gravid traps (Table 2-10).

Linear regression analysis of the weekly abundance, given as the logo Williams mean

(Mw) number of adult females collected per trap + 1, ofA. albopictus, as collected in both CO2-

baited CDC light traps and gravid traps, over time did not indicate any significant change in

abundance of this species from 2004 through 2006 (Figure 2-10). However, this time series

analysis indicated that the mean abundances of both O. japonicus (Figure 2-11) and 0.

triseriatus (Figure 2-12) declined significantly over this period of time.









Discussion

The results of this preliminary assessment demonstrate the coexistence of O. japonicus

with resident container-inhabiting mosquitoes in Fairfax County, Virginia, facilitated by what

appears to be species-specific differences in habitat preference and seasonality. The negative

associations of O. japonicus immatures with A. albopictus and 0. triseriatus seem largely due to

an apparent divergence in container preference, with O. japonicus occurring predominately in

rock pools rather than artificial containers. Furthermore, the limited collection of 0. triseriatus

from artificial containers suggests that perhaps this species prefers an alternative habitat, such as

treeholes. Findings from the oviposition survey were similar. A wide distribution and high

abundance ofA. albopictus across macrohabitats was observed throughout the study period, with

this species occurring more frequently at residential and disturbed habitats than other resident

container-inhabiting mosquitoes, a trend similar to that which has been observed in Florida

(Lounibos et al. 2001). Spatial and temporal distributions of 0. hendersoni and 0. triseriatus

were not consistent with those observed in other areas, but this may be due to the collection

effort of this study. Ochlerotatus triseriatus is known to seek out oviposition sites at ground level

(Scholl and DeFoliart 1977) while 0. hendersoni prefers to oviposit above ground level (Sinsko

and Grimstad 1977, Clark and Craig 1985), even up to 9 m (Beier et al. 1982). However, these

results suggest that there is no significant difference in the frequency of oviposition of either

species at ground level or above ground. Whilee the period of oviposition activity of O.

triseriatus typically extends beyond that of 0. hendersoni into late summer (Scholl and DeFoliart

1977), the results of this study suggest a similar seasonality for these two species.

While O. japonicus was only detected once throughout the oviposition study, possibly

implying a univoltine population of this species in Fairfax County, Virginia, oviposition by and

hatching of viable eggs from wild-caught females of this species collected from May through









October in previous years (personal observation, unpublished data) suggests this is an artifact.

While these findings are likely a reflection of inappropriate macrohabitat placement of ovitraps,

it should be noted that others have had limited success with this collection technique for 0.

japonicus in the past (Andreadis et al. 2001). Ovitraps would perhaps have been more useful in

collecting O. japonicus in different macrohabitats, particularly near rock pools where this species

is most abundant.

Findings from the survey of artificial containers suggest that interspecific competition may

be occurring in these habitats where O. japonicus coexists with resident mosquito species in the

presence of limited resources; however this study suggests that the invasion of O. japonicus may

not result in competition in artificial containers that leads to displacement. This is supported by

the abundances and co-occurences of O. japonicus with A. albopictus and 0. triseriatus, the two

aedine mosquitoes this species is most likely to encounter in artificial containers in Fairfax

County, Virginia. Metamorphic success of O. japonicus was not significantly different from that

of A. albopictus, nor were there any significant differences in interspecific crowding of the two

species among months. However, A. albopictus encountered a higher density of conspecifics

than O. japonicus per unit resource, suggesting that intra- rather than interspecific interactions

may be more important in regulating the population growth of this species in artificial containers.

Similarly, there appeared to be greater crowding of O. japonicus by conspecifics than by A.

albopictus (Figures 2-7, 2-8). Furthermore, intraspecific crowding and metamorphic success of

O. japonicus in artificial containers and rock pools, where it is often the only species present,

were similar. While these findings suggests that intraspecific competition may be just as

important as interspecific competition for this species, it is possible that there has been selection

among O. japonicus since arriving in this area to avoid competition in artificial containers with









A. albopictus; however the plausibility of such selection is currently unknown due to the paucity

of data regarding seasonal patterns and abundance of O. japonicus since its arrival in northern

Virginia. Such speculation may be supported further by differences in the seasonal abundances

and frequencies of occurrence of the two species in artificial containers.

The success of O. japonicus in artificial containers in relation to A. albopictus seems to be

facilitated by differences in seasonality and instar distributions. Ochlerotatusjaponicus was

collected most frequently and in greater abundance early in the season in contrast to A.

albopictus, which was most active in later months. Activity of O. japonicus during this time also

happened to coincide with the greatest metamorphic success of this species, and was the only

time when the interspecific mean crowding of O. japonicus on A. albopictus was higher than that

ofA. albopictus on O. japonicus. The different instar distributions ofA. albopictus (Figure 2-5)

and 0. japonicus (Figure 2-6) promoted the early season metamorphic success of the latter

species in artificial containers, with the presence of older instars of O. japonicus in May, June,

and July perhaps giving this species a head start over A. albopictus. This suggests the capacity of

this species to overwinter in such habitats in the larval stage, an observation that has been made

by others in the species native range (LaCasse and Yamaguti 1950), and the United States in

New Jersey (Scott 2003) and North Carolina (B. Harrison personal communication). However,

no data regarding the induction or termination of diapause in O. japonicus are currently

available. Furthermore, the disparity in the age of O. japonicus and A. albopictus larvae, with the

average instar of O. japonicus consistently higher than that ofA. albopictus throughout all

months of the study, may provide a competitive advantage to O. japonicus in containers with A.

albopictus, allowing the former species to persist later into the season despite the high abundance

ofA. albopictus. The importance of cohort structure in density-dependent intraspecific









competition has been documented for 0. triseriatus, whose early hatching larvae experienced

higher survivorship, faster development time, and higher per capital growth rate, than cohorts that

hatched later (Livdahl 1982, Edgerly and Livdahl 1992). Such an advantage may be due to the

size-efficiency of the early cohort (Brooks and Dodson 1965), who as large filter feeders are

more efficient and can exploit a wider range of food particles than smaller competitors, or even

be cannibalistic (Koenekoop and Livdahl 1986), however evidence of the latter may have been

an artifact of simple experimental conditions (Edgerly and Livdahl 1992). Furthermore, egg -

larva interactions between larvae of early hatching cohorts and eggs of later cohorts are a known

form of interference competition among container-inhabiting mosquitoes, in which the presence

of feeding older larvae delayed hatching (Edgerly et al. 1993). The impact of interspecific larval

resource competition, particularly among different instars, on the population growth of these two

species needs to be explored experimentally to reveal the complexities of community dynamics.

This study revealed somewhat different findings for interactions between O. japonicus and

0. triseriatus. In contrast to A. albopictus, 0. triseriatus had five times the metamorphic success

of O. japonicus in containers in which these species co-occurred, suggesting that this species is

superior in larval resource utilization. However, the abundance and of 0. triseriatus and co-

occurrences with O. japonicus were low in these collections. Although outside the scope of this

study, interspecific interactions with 0. triseriatus may be important for 0. japonicus in

treeholes, as these species have been collected from such habitats in Connecticut (Andreadis et

al. 2001), and should be considered in future research. Experimental studies should be viewed in

the context of field observations of the frequency of interspecific interactions, seasonal

distributions, and overwintering behaviors, as these life history traits may ultimately influence

the community structure of treeholes in which these species may coexist. Aedes albopictus is









known to be superior to 0. triseriatus larvae in competition (Livdahl and Willey 1991), which

coupled with the high interspecific mean crowding of 0. triseriatus by A. albopictus

demonstrated in this study, suggests that interactions between these two species may be most

important in determining the success of 0. triseriatus populations in artificial containers in

Fairfax County, Virginia. It would be of great value to compare these findings with that from

similar studies conducted in areas where A. albopictus does not occur, as well as in eastern Asia

where the native ranges of these species overlap; however such data are currently unavailable.

The only comparable survey, conducted by Andreadis et al. (2001) in Connecticut shortly after

the invasion of O. japonicus, indicated that this species ranked fourth in overall abundance

(9.4%) in a tire survey, in which it was severely outnumbered by 0. triseriatus. The potential for

larval resource competition between these two species in areas where A. albopictus does not

occur seems likely, and results of this study suggest that 0. triseriatus will likely be the superior

competitor under such conditions.

Broad-based surveillance of adult females of A. albopictus, 0. japonicus, and 0. triseriatus

with C02-baited CDC light traps and gravid traps provided a limited perspective of the

population trends of these species over time in Fairfax, Virginia. The adult population of A.

albopictus did not alter in frequency or abundance during the three year surveillance period,

which leads one to speculate that perhaps this species has been unaffected by the introduction of

O. japonicus in the area. On the contrary, the adult populations of both O. japonicus and 0.

triseriatus declined during the surveillance period, although this trend seemed to be somewhat

more severe for the former species. While these observations are likely attributable to annual

variations in environmental conditions (i.e. temperature and rainfall), they reflect the local

decline, or possibly the eventual extinction, of O. japonicus populations following the









introduction and initial expansion of this species throughout the area. This phenomenon in which

an invading species reaches a peak of density and then declines is often referred to as boom-and-

bust (Williamson 1996). Selective pressures from predation, competition, or lack of availability

of accessible resources may promote this type of invasion trend (Williamson 1996). Additional

years of consistent surveillance will be required to fully appreciate these trends with respect to

the invasion success of O. japonicus.

The successful establishment of O. japonicus in Fairfax County, Virginia appears to be

associated with a population decline and potential displacement of 0. atropalpus in local rock

pools. The limited collection of this native rock pool mosquito while surveying these habitats is

cause for concern as this species was once abundant throughout the area; in fact the type-form

given by Coquillett (1902) was from nearby Plummer's Island in Montgomery County,

Maryland. A similar pattern of decline for 0. atropalpus has been observed in rock pool

communities of Connecticut (Andreadis et al. 2001) and North Carolina (B. Harrison personal

communication), and the absence of this species in rock pools has been noted in New Jersey

(Scott et al. 2001a), however 0. atropalpus is more common in tires than in rock pools in this

area. It is important to note two major flooding events, heavy rains and a hurricane, that occurred

during this study limited rock pool collections in late June and early July, and again in early

September. Although no conclusions could be based solely on the limited co-occurrence of these

species during this study, these findings suggest that interspecific larval resource competition

with O. japonicus may have had profound affects on populations of 0. atropalpus in areas where

these two species co-occur.

Differences in the overwintering strategies of these species have probably facilitated the

decline of 0. atropalpus, which is known to diapause in the egg stage rather than as larvae,









whereas O. japonicus may diapause in either stage (LaCasse and Yamaguti 1950, Kamimura

1976, Scott 2003, B. Harrison personal communication). Similar to that observed for A.

albopictus and O. japonicus in artificial containers, differences in the cohort structures of these

two species in rock pools may exacerbate larval competitive outcomes or promote hatching

inhibition, intraguild predation, or cannibalism. The tendency of 0. atropalpus to inhabit rock

pools fully exposed to the sun, contrary to the preferences of O. japonicus, may allow this

species to persist in this habitat, albeit probably in small numbers as highly flood prone rock

pools tend to be in the more sunny locations (O'Meara et al. 1997). Survival of 0. atropalpus in

an environment frequently subjected to flooding may be facilitated by seasonal variation in

ovipositional behavior, deposition of different types of eggs, or variable delays before hatching.

It is possible that this finding is an artifact of sampling effort; therefore the rock pool

microhabitat preferences of these species with respect to sun exposure should be researched

further. Furthermore, as noted in Lounibos (2002), 0. atropalpus has expanded its distribution

by occupying artificial containers, primarily tires, and has itself been considered an invasive

species both in the United States and abroad. Such tendencies may allow this species to persist

through dispersal to new areas where O. japonicus does not occur, or may allow it to avoid

competition to some extent in areas where the two species co-occur. However, it is interesting to

note that 0. atropalpus was not recovered from any tires during this study.

In conclusion, the invasion of O. japonicus seems to be associated with the possible

displacement 0. atropalpus, possibly through interspecific resource competition, in part of this

species' rock pool habitat. In artificial containers, 0. japonicus larvae are most likely to interact

with A. albopictus, particularly late in the season, during the months of August and September,

in small-sized shaded containers. The success of O. japonicus in artificial containers is most









likely attributed to the earlier seasonal appearance, older age, and capacity of this species to

complete development in the presence of resident species. While surveillance cannot reveal any

detrimental effects of O. japonicus on resident artificial container inhabiting mosquitoes,

monitoring over subsequent years should continue to observe population trends. While this study

focused predominantly on the potential interspecific interactions of O. japonicus with resident

mosquito species in artificial containers and rock pools, other factors, particularly predators, may

be influencing the structure of these communities and should be investigated. Selective

preference of predators (Griswold and Lounibos 2005, 2006) and differential responses of prey

species to predators (Holt and Lawton 1994) have been shown to influence interspecific

interactions of container-inhabiting mosquitoes. Furthermore, the tolerance of these species to

varying environmental conditions, intraguild predation, or differences in foraging behavior may

be important in their interspecific interactions.









Table 2-1. Frequency of occurrence ofA. albopictus, 0. hendersoni, and 0. triseriatus in ovitraps set at three sites and two heights in
Fairfax, Virginia in 2006.
Frequency (no. pos. trapsa/total)
Site A. albopictus 0. hendersoni 0. triseriatus
Residential 0.1957a Ob Ob
Undisturbed
forest 0.2128 0.1087 0.1304
Disturbed
forest 0.3478a Ob 0.1739a

Height
Ground 0.4265a 0.0588b 0.1471b
Above
ground 0.0857 0.0571 0.0571
aFrequencies were calculated as the number of positive traps per total number of traps set at each site from May September 2006.
Lower case letters indicate significant differences among sites resulting from pairwise comparisons with G-tests (df = 1, p = 0.05),
using the Bonferroni method to adjust for experimentwise error.









Table 2-2. Frequency of occurrence, by month, of A. albopictus, 0. hendersoni, and 0. triseriatus in ovitraps in Fairfax County,
Virginia in 2006.
Frequency (no. pos. trapsa/total)
Species May June July August September
A. albopictus O.la 0.2222ab 0.4231b 0.3929b 0.1481a
0. hendersonib 0 0.0370a 0.2692b 0 0
0. triseriatus Oa 0.lab 0.3b 0.0333a 0.0333a
aFrequencies were determined as the number of positive traps per total number of traps set each month.
bFrequencies of collection of 0. hendersoni were compared only for June and July; differences were significant at c = 0.01 with
Cochran's Q-test, df = 1. Lower case letters indicate significant differences among sites resulting from pairwise comparisons with
Cochran's Q test (df = 1, p = 0.05), using the Bonferroni method to adjust for experimentwise error.









Table 2-3. Monthly abundance (mean number of eggs per trap) ofA. albopictus, 0. hendersoni, and 0. triseriatus collected in
ovitraps.
Species Abundance (mean no. (+SE) /trap) a
May June July August September
A. albopictus 1.0(0.93)a 1.19 (0.66)ab 11.92 (5.85)b 6.39 (2.73)b 0.67 (0.49)a
O. hendersonib 0 0.15 (0.15) 4.08 (2.17) 0 0
0. triseriatus Oa 0.22 (0.13)ab 8.69 (4.24)b 0.79 (0.79)ab 0.15 (0.15)a

Total rainfall (cm) 5.61 35.61 9.04 2.62 16.03
aAbundance was determined as the mean number of eggs collected per trap, based on all ovitraps set each month.
bMean abundances of 0. hendersoni were compared only for June and July; differences were significant at c = 0.05 with Friedman's
two-way analysis of variance, df = 1. Lower case letters indicate significant differences among sites resulting from pairwise
comparisons with Friedman's two-way analysis of variance test (df = 1, p = 0.05), using the Bonferroni method to adjust for
experimentwise error. All other pairwise comparisons were non-significant.










Table 2-4. Coefficients of association (C8) for the most abundant species in 191 artificial and natural container samples from Fairfax
County, Virginia in 2006. Parentheses enclose C8 values that exclude rock pools.
Coefficient of association (Cs)
Species C. restuans C. pipiens 0. triseriatus 0. japonicus 0. hendersoni
A. albopictus -0.056 -0.015 0.067*** -0.238*** 0.037**
(-0.207***)
0. hendersoni 0 -0.272 0.232** 0
(0)
O. japonicus 0.075 0.042 -0.048**
(0.105) (0.133) (0)
0. triseriatus -0.122 -0.140

C. pipiens 0.638***

** p < 0.01, *** < 0.001 by X2; all other interspecific associations are non-significant.









Table 2-5. Intra- and interspecific mean crowding of the most abundant mosquito species collected from artificial and natural
containers in Fairfax County, Virginia in 2006. Mean crowding is defined as the mean density of species encountered by
species x per liter volume. Values in bold indicate intraspecific mean crowding.
Mean crowding
(density of species y encountered by species x per liter volume)
Species y
Species x A. albopictus 0. hendersoni 0. japonicus 0. triseriatus C. pipiens C. restuans
A. albopictus 395.80 3.013 53.01 31.59 351.88 57.37
0. hendersoni 40.73 32.49 18.06 56.27 1.77 5.74
0. japonicus 139.3 9.03 127.56 12.85 51.56 83.06
0. triseriatus 136.7 11.90 19.21 34.47 25.93 39.26
C. pipiens 1098.43 2.48 37.56 12.96 126.98 52.33
C. restuans 20.16 2.51 57.41 4.33 34.05 116.60
aInterspecific mean crowding was determined from only those containers in which both species co-occurred.









Table 2-6. Species rank abundances compared for different container habitats sampled in Fairfax County, Virginia from May -
September 2006. Kendall's c was used as an index of similarity.
Container habitat No. of samples No. of species T tsa P
Small artificial
containers 97 7
containers 97 7 0.714 8.101 < 0.001
Large artificial
containers 33 9
Tires 29 7
r s 59 7 0.200 0.537 0.709
R ock pools 59 7 .......................
All artificial containers 130 9
0.467 1.917 0.055
Rock pools 59 7
aSignificance of Kendall's c was tested by calculating the test statistic, t, which makes use of a normal approximation to test the null
hypothesis that the true value of c = 0: ts = T / sqrt [( 2(2n + 5)) /( 9n(n 1))], where n is the number of data pairs.









Table 2-7. Number of larvae and average instar number for A. albopictus and O. japonicus collected in Fairfax County, Virginia by
month, May September 2006.
A. albopictus O. japonicus
Average instar Average instar
Month No. larvae number (SE) No. larvae number (SE)
May 5 3.0 (0.71) 16 3.13 (0.49)
June 204 2.67 (0.08) 98 4.18 (0.13)
July 574 2.94 (0.05) 1836 3.67 (0.04)
August 3427 2.69 (0.02) 998 4.19 (0.04)
September 227 3.64 (0.05) 2 4.0 (1.0)
Total 4437 2.77 (0.02) 2950 3.86 (0.03)










Table 2-8. Tests for significant heterogeneity of monthly instar distributions of A. albopictus and O. japonicus from June August
2006, based on log-rank statistics.
Paired comparisons with z-test
A. albopictus O. japonicus
July August July August
Month z P z P z P z P

June 3.66 < 0.001 0.203 0.416 4.86 < 0.001 3.06 0.001
July 2.34 0.01 4.61 < 0.001
Test 2 df P 2 df P
Log-rank 13.46 2 < 0.001 24.22 2 < 0.001
Wilcoxon 16.46 2 < 0.001 45.94 2 < 0.001









Table 2-9. Least square means (SE) for intraspecific crowding (transformed by logo (x + 1)) among larvae ofA. albopictus and 0.
japonicus June August 2006. Means followed by lower case letters that are not commonly shared are significantly
different by pairwise comparisons (p < 0.05) with the Bonferroni adjustment for experimentwise error.
Least square means (SE)
Species June July August
A albopictuss 0.6489 (0.1928)a 1.6176 (0.2164)b 1.8092 (0.1163)b
O. japonicus 1.2286 (0.3570)a 2.9186 (0.2558)b 2.3577 (0.2408)ab









Table 2-10. Frequencies of collection ofA. albopictus, 0. japonicus, and 0. triseriatus adult females in C02-baited CDC light traps
and gravid traps, 2004 2006. Means followed by letters that are not commonly shared are significantly different by
pairwise comparisons (p < 0.05) with the Bonferroni adjustment for experimentwise error.
Mean (+SE) frequency of collection (no. pos. traps/totala)
CO2-baited CDC light traps Gravid traps
Species 2004 2005 2006 2004 2005 2006
A. albopictus 0.423 (0.40) 0.433 (0.066) 0.327 (0.054) 0.064 (0.013) 0.046 (0.012) 0.061 (0.012)
O.japonicus 0.213 (0.20)a 0.137 (0.016)b 0.046 (0.008)c 0.454 (0.034)a 0.281 (0.367)b 0.186 (0.023)c
0. triseriatus 0.241 (0.30)a 0.176 (0.27)ab 0.135 (0.23)b 0.094 (0.015)a 0.046 (0.009)b 0.046 (0.12)b
aMean frequency of collection was determined from 23 weekly frequencies (the number of positive traps per total traps set each week)
for each species.






















































* Trap Site
I Oviposition Site
A Artificial Container Site
A Rock Pool Site


S 25 5 10 Miles



Figure 2-1. Map of Fairfax County, Virginia showing locations of study areas that were sampled
or censused repeatedly. Trap sites include both a C02-baited light trap and a gravid
trap.




54











N=2
1.00
O A. albopicuts Q 0. japonicus O Both
0.90

S0.80

p 0.70
N = 64
8 0.60
SN=30
0 N = 20
"| 0.50- 2

1 0.40 N 6
E N=6
S0.30-

0.20

S0.10

0.00 ----
May June July August September

Month

Figure 2-2. Proportion of mosquito-positive containers containing A. albopictus, 0. japonicus, or both A. albopictus and 0. japonicus
May September 2006. Total numbers of mosquito-positive containers sampled are indicated at the top of each histogram
bar.












S- A. albopictus O 0. japonicus
80

S70

60

50
50

40-

S30 -

S20

10 -

0
May June July August September
Month


Figure 2-3. Monthly abundance (mean number of mosquitoes collected per container) ofA. albopictus and 0. japonicus (+SE) from
91 mosquito-positive artificial containers.










0.70

O A. albopictus U 0. japonicus
0.60- N = 81 N =22


S0.50


>
S0.40


a 0.30


0.20


0.10 -


0.00
May June July August September
Month


Figure 2-4. Seasonal occurrences (proportion of species-positive containers per month) ofA. albopictus and 0. japonicus collected
from artificial containers in 2006. The total numbers of species-positive containers sampled from May September are
indicated in the figure legend.













August
n = 53, n2


3427






M


M


September
n1 = 5, n2 = 227


0.80 -

0.60 -

0.40 -

0.20 -

0.00
0.80

0.60

0.40

0.20 -

0.00
0.80

0.60
0.40
0.20
0.00


July
n, = 14, n2


I II


III IV


P


Stage


Figure 2-5. Monthly instar distributions of A. albopictus from May September 2006 in Fairfax, Virginia. Total numbers of positive
containers (ni) sampled and larvae collected (n2) are indicated for each month.


May
n, = 3, n2 = 5


June
n, = 18, n2


1.00


- 4I -


- -


Stage












May
n = 1, n2 8


1.00

0.80

0.60

0.40

0.20

0.00

0.80

0.60

0.40

0.20

0.00

0.80

0.60

0.40

0.20

0.00


August
n1 = 25, n2 = 998


September
n = 1, n2=2


- 4 II


Iv


July
n, =21, n2 = 1836


II


Stage


III

Stage


IV


June
n= 10,n2 = 98


4 -


a -









Figure 2-6. Monthly instar distributions of O. japonicus from May September 2006 in Fairfax, Virginia. Total numbers of positive
containers (ni) sampled and larvae collected (n2) are indicated for each month.


I] A. albopictus by 0. japonicus

6 0. japonicus by A. albopictus


May


June July August


September


Month


Figure 2-7. Interspecific mean crowding ofA. albopictus by O. japonicus and 0. japonicus by A. albopictus. Interspecific mean
crowding was calculated by month as the density of species encountered by species x per unit resource a, in this case,
container volume. Interspecific mean crowding was quantified as E(xy,/a,)/E x,.











300


0 A. albopictus Q 0. japonicus
250


200
C.)

9 150



| 100 -


50 -



0 J l y ,
May June July August September

Month

Figure 2-8. Intraspecific mean crowding (density of conpspecifics encountered per unit resource, a) ofA. albopictus and 0. japonicus
by month. Intraspecific mean crowding was quantified as E[xi(xi 1)/a,]/E x1, where a is container volume.












SA. albopictus


August


S0. japonicus


September


Figure 2-9. Metamorphic success ofA. albopictus and 0. japonicus collected from containers in which the two species co-occurred.
Numbers of samples for each month are indicated above each histogram bar.


n=8


0.3 -



0.25



0.2 -



0.15



0.1



0.05


May


June


July
Month


I











0.7


0.6

0.5
+
0.4

0.3

m 0.:

0.1

0(
-o

E 0.:




0.1


o 0.1


0.C


0.C


70-
A
0- Y =6E-05x + 0.2372
r2 0.0002 *
p =0.904 4

0- *


20-
.
** *


1 0 _
0- *** 4. *



0 -

30- =:
** **


B
5- Y =0.001x + 0.0647
r2 0.0043 *
0- p= 0.6 *


5-


0-
S* .

*

** *
5 -
S** *
** *
0_ a _______________s __


2004


2005


2006


Year
Figure 2-10. Mean weekly abundance ofA. albopictus collected in (A) C02-baited light traps and (B) gravid traps over time, from
2004 through 2006, in Fairfax County, Virginia. Williams means (WM) were transformed by logio(x + 1) to meet
assumptions of normality and homogeneity of variance.











0.20-
A
0.16 Y = -0.008x + 0.1156
r2 = 0.4960
S*p < 0.001
S0.12 -
c* *

0.08 *
S ** **

6 ** 0*
0.04 ** **
S* *
*
0 0.00 *
o B
.04 Y =-0.0015x + 0.2611
S0.40 r2 0.3241

Sp < 0.001
0.30


to 0.20

***
0.10-


0.00 *
2004 2005 2006

Year
Figure 2-11. Mean weekly abundance of O. japonicus collected in (A) C02-baited light traps and (B) gravid traps over time, from
2004 through 2006, in Fairfax County, Virginia. Williams means (WM) were transformed by loglo(x + 1) to meet
assumptions of normality and homogeneity of variance.










0.30-
A
0.25- Y = -0.006x +0.1345
r2= 0.1386
0.20- p = 0.002
+

0.15


0.10


0.05 -


B
E Y = -0.002x + 0.0368
S0.08- r2 0.1166
Sp = 0.004

S0.06
Cd
o
t 0.04 .


0.02- ** *
204* ** -
0.00 ,
2004 2005 2006

Year
Figure 2-12. Mean weekly abundance of 0. triseriatus collected in (A) C02-baited light traps and (B) gravid traps over time, from
2004 through 2006, in Fairfax County, Virginia. Williams means (WM) were transformed by logio(x + 1) to meet
assumptions of normality and homogeneity of variance.









CHAPTER 3

INTERSPECIFIC COMPETITION BETWEEN AEDES ALBOPICTUS AND OCHERLOTATUS
JAPONICUS

Introduction

Aedes albopictus and Ochlerotatusjaponicus are two of the most recently recognized

exotic mosquito species to become established in the United States. Aedes albopictus was

introduced into the United States from Japan by way of tire shipments (Hawley et al. 1987,

Reiter and Sprenger 1987), which led to its establishment in Texas in 1985 (Sprenger and

Wuithiranyagool 1986). State and local mosquito surveillance records indicate that it has since

spread rapidly, becoming established across much of the eastern United States from southern

Florida to New Jersey, Illinois, Indiana, and Ohio (Moore 1999). The westward spread of this

invader has been much slower, presumably due to the drier summers in this region (Nawrocki

and Hawley 1987). Aedes albopictus has been intercepted and destroyed on the west coast in

California (Linthicum et al. 2003) and Washington (Craven et al. 1988).The invasion success and

rapid spread of A. albopictus in the United States has been attributed to its generalized habitat

and food requirements, ability to live in human-dominated habitats (Hawley 1988), desiccation

resistant eggs (Focks et al. 1994, Juliano et al. 2002), and superior larval competitive ability

(Juliano 1998, Juliano et al. 2004).

The introduction of O. japonicus into the United States was initially reported by Peyton et

al. (1999) from light trap collections from New York and New Jersey in August and September

1998. However, an archival search by Andreadis et al. (2001) revealed that this species was

actually first detected one month earlier in Connecticut. Ochlerotatusjaponicus has since been

detected along the East coast with reports as far south as Georgia (Reeves and Korecki 2004),

north as Maine (Foss and Dearborn 2001), and west as Missouri (Gallitano et al. 2006), from









what appear to be multiple introductions from Japan (Fonseca et al. 2001). Ochlerotatus

japonicus appears to have become established on the west coast in Washington (Roppo et al.

2004), and has recently been detected in Mississippi and Nevada (Moore 2005). Like A.

albopictus, the used tire trade is the suspected mechanism of introduction of O. japonicus into

the United States (Peyton et al. 1999, Lounibos 2002).

The current US distributions of these two species overlap considerably, although O.

japonicus appears to be more cold tolerant (Tanaka et al. 1997) than A. albopictus as evidenced

by the former species more northerly native range. It has been predicted that the range of A.

albopictus may eventually expand northward as far as the -50C isotherm, as it does in Asia,

however at such latitudes populations would likely not overwinter (Nawrocki and Hawley 1987).

Furthermore, both species are container-inhabiting mosquitoes commonly found in water-filled

artificial container habitats such as automobile tires, bird baths, and flower pot saucers; however

rock pools are the preferred habitat of O. japonicus in its native range (Tanaka et al. 1979). The

aquatic larvae of both species feed on microorganisms and particulate matter in the water column

as well as on leaves and other organic detritus (Merritt et al. 1992).

Severe crowding and limiting resources are frequent in these habitats, thus it is likely that

larval resource competition, inter- or intraspecific, may have important affects on the growth,

survivorship, and reproductive success of these species (Juliano and Lounibos 2005); therefore

larval conditions may have a significant impact on overall population growth. Those species that

can maintain positive population growth under interspecific conditions of greater density or

lower resource availability than a competitor are considered to have a competitive advantage.

Such a competitive advantage is even greater if one species can maintain positive population

growth under conditions that result in negative population growth for a competitor.









The role of interspecific competition in structuring communities of container-dwelling

mosquitoes has been well documented, perhaps best so for A. albopictus and A. aegypti in the

southeastern United States, where interspecific larval competition (Barrera 1996, Juliano 1998,

Braks et al. 2004) was the probably cause of the decline in range and abundance of the latter

species throughout most of this area (O'Meara et al. 1995, Juliano et al. 2004, Juliano and

Lounibos 2005). Aedes albopictus has also been indicated experimentally to be a superior larval

competitor to 0. triseriatus (Livdahl and Willey 1991) and Culexpipiens (Costanzo et al. 2005).

While the invasion of an introduced species may negatively impact native or other introduced

species as a result of interspecific larval competition, the effects of other interactions such as

predation, habitat alteration, or apparent competition mediated by shared enemies should also be

considered when assessing interspecific interactions.

Understanding the invasion dynamics of A. albopictus and 0. japonicus is important not

only because of the ecological consequences resulting from their interactions with native

container-inhabiting mosquitoes, but also because these species may be of epidemiological

significance. The invasion and establishment of A. albopictus and 0. japonicus in the United

States is cause for concern because of potential involvement of these species in the transmission

cycle of human arboviruses. In its native range A. albopictus is a known vector of dengue virus,

which has been isolated from wild-caught individuals of this species in Mexico (Ibafiez-Bernal et

al. 1997). This species was also implicated as the vector in the 2001 outbreak of dengue in

Hawaii (Effler et al. 2005). Wild-caught A. albopictus in the United States have also been

recovered infected with eastern equine encephalitis (Mitchell et al. 1992) and LaCrosse

encephalitis viruses (Gerhardt et al. 2001).









Although O. japonicus is not considered an important disease vector in its native range in

Asia, it may transmit Japanese encephalitis virus (Takashima and Rosen 1989) and has also been

indicated as a competent experimental vector of eastern equine encephalitis (Sardelis et al.

2002a), LaCrosse encephalitis virus (Sardelis et al. 2002b), and St. Louis encephalitis viruses

(Sardelis et al. 2003). Both A. albopictus and 0. japonicus have shown to be competent vectors

of West Nile virus in the laboratory (Turell et al. 2001), and wild-caught adults of both species

have been recovered infected with this virus (Holick et al. 2002, Scott 2003, Godsey et al. 2005).

The demonstrated ability of these species to be infected by and transmit numerous arboviruses

indicates that their introductions and competitive interactions in the United States may have

important public health consequences (Lounibos 2002).

While considerable data exist regarding the interspecific interactions and competitive

outcomes of A. albopictus with numerous other container-inhabiting mosquito species, there are

no comparable reports on O. japonicus. As A. albopictus and 0. japonicus are known to

frequently co-occur in container habitats, an investigation of larval competitive interactions

between these two species was proposed. An experiment designed similar to those of Juliano

(1998) and Braks et al. (2004), with the exception that only a single resource level was

implemented, was conducted to measure the performance of larvae of Virginia populations of

these species competing for resources under field conditions in Fairfax, Virginia. Comparisons

were made between species for intra- and interspecific effects of larval density on survivorship,

development time, body size, and population growth.

Materials and Methods

The experiment was conducted in a heavily forested streambed located directly behind the

Fairfax County Department of Health in Fairfax, Virginia (latitude 38050'57" N, longitude









77019'W) from August to October 2006. Routine surveillance data collected by the Department

of Health indicated that both A. albopictus and 0. japonicus were commonly detected on this

property as both larvae in artificial containers and as adults in CO2-baited CDC light traps and

gravid traps. The A. albopictus and 0. japonicus used in this experiment were the first generation

progeny of gravid individuals collected from Fairfax, Virginia.

Inter- and intraspecific larval competition was investigated by monitoring the

development of larvae at different densities in 400-ml black polypropylene cups (10.5 cm in

height, 6.5 cm base diameter). Field surveys of container habitats in the area indicated that these

species co-occur in containers of similar shape and size in nature. Three density combinations of

A. albopictus:O. japonicus (10:0, 50:0, 0:10, 0:50, and 25:25) were evaluated using a completely

randomized block design. One replicate of each combination was placed at each of five

experimental sites, spaced approximately 30m apart, for a total of five replicates per treatment

and 25 cups. Although both species have been found in containers with varying sun exposure

(i.e., none, partial, and full exposure), all five sites used were completely shaded to maintain

experimental consistency.

On 10 August, each cup was randomly labeled with a unique number and letter

corresponding to one of the five treatments and sites, where they were secured to plastic stakes to

prevent toppling. Food consisted of fallen pin oak leaves (Quercuspalustris) that had been

collected, washed, and dried at room temperature for one week prior to quartering, weighing, and

sorting. To allow for the leaves to soak and be colonized by microorganisms, four days prior to

the start o the experiment 1 g was added to each of the 25 cups containing 200 ml of distilled

water. Each container was covered with fiberglass screen (0.5 mm) and secured with a rubber

band to prevent entry of other macrofauna and detritus. In the laboratory, eggs ofA. albopictus









and 0. japonicus were synchronously hatched (Novak and Shroyer 1978), and 24 hours later

larvae were counted into aliquots of 10, 25, and 50. Within one hour after counting, the larvae

were distributed into appropriate cups.

Each container was monitored daily for the presence of pupae, which were collected and

housed singly in sealed 50 ml vials containing water from their respective field cup. Each vial

was labeled with the appropriate site and treatment identifier before being secured to a plastic

stake at the field site with a rubber band. Upon emergence, adults were killed by freezing before

scoring by container, species, sex, and day of emergence. The experiment ended on 11 October

when the final adult emerged. Ambient temperature was monitored hourly for the duration of the

experiment with three Onset HOBO data loggers located in the middle and at either end of the

experimental area. The average (SE) hourly ambient temperature recorded was 18.770.060C,

with a range of 7.76C to 29.730C (n = 4197). For two days of the experiment (31 August and 1

September), the area was subjected to intense wind and rain due to Hurricane Ernesto. To

prevent damage to experimental apparatus and loss of data, the cups at each experimental site

were successfully covered and secured with a tarpaulin during this time.

Data Analysis

Population Growth Correlates

To quantify the effects of inter- and intraspecific competition on A. albopictus and 0.

japonicus, the mean survivorship, median development time of males and females, and median

body size at adulthood of females were analyzed by one-way ANOVA, followed by Tukey's

honestly significantly different (HSD) post-hoc tests for pairwise comparisons of means in SPSS

(SPSS 2002). Survivorship was calculated as the proportion of adults that emerged from the

initial cohort of first instar larvae. Development time was calculated as the number of days from









hatching to adult emergence. Adult body size was estimated from the length of one wing, which

was removed from each female and measured under a dissecting microscope with an ocular

micrometer (Packer and Corbet 1989). Median rather than mean development time and wing

length were calculated for each species and treatment because of the non-normal distributions of

these variables within cohorts.

Composite Index of Population Performance

Survivorship, female development time and wing length were used to calculate a

composite index of mosquito population performance (X'), which is an analog of the finite rate of

increase as defined by Juliano (1998):




In (1/No) Af/(w)
A'= exp I

wee( f t w


where No is the initial number of females (assumed to be 50% of the cohort), Ax is the number of

females closing on day x, 1, is the mean wing length of females closing on day x, andfil ) is

a function relating egg production to wing length. D is the time from adult eclosion to

reproduction, taken as 14 days for A. albopictus (Livdahl and Willey 1991) and 12 days for 0.

japonicus (see below). Values of V' greater than one indicate that the population is increasing,

approximately equal to one that the population is stable, and less than one that the population is

decreasing.

D for 0. japonicus was determined experimentally under controlled conditions of 26C

and 12h: 12h light:dark in an insectary at the Florida Medical Entomology Laboratory in Vero

Beach, Florida. Ochlerotatusjaponicus eggs used for this experiment were obtained from the









colony maintained at the Headlee Research Laboratory Mosquito Research and Control Unit at

Rutgers University in New Brunswick, New Jersey. This colony originated from larval

collections from a horse farm in New Egypt, Ocean County, New Jersey in 2001 (L. McCuiston

personal communication).

In the laboratory, eggs were hatched by flooding with water, and cohorts of larvae

hatching within the same 24-hour period were grouped together and placed in plastic trays within

separate 0.028 m3 (30.5 cm x 30.5 cm x 30.5 cm) cages. Each cohort was fed 100 mg of an

artificial diet consisting of one part Brewer's yeast and one part lactalbumen every other day.

Beginning two days after emergence, adult females of each cohort were offered a bloodmeal

from a restrained chicken placed within the cage daily for one hour. Cotton soaked in a 20%

sucrose solution was provided as a source of carbohydrates for adults at all times. Upon visual

inspection immediately following the bloodfeeding opportunity, those females that appeared to

have fed to repletion were removed from the cage using a mouth aspirator and placed singly in

12-dram plastic vials containing a 2.54 cm by 7.62 cm strip of wet seed germination paper

(Anchor Paper, St. Paul, MN) to serve as an oviposition substrate (Steinly et al. 1991). The

germination paper was checked daily for the presence of eggs, and was replenished with water if

necessary. The date of oviposition for each female was recorded, from which the average time

from adult eclosion to oviposition for O. japonicus was calculated to be 12 days, with a range of

4 to 17 days (N= 144).

A regression relating female wing length to fecundity for A. albopictus was obtained

from Lounibos et al. (2002):

f(wx) = 78.02 wx 121.240 (r2 = 0.713, N= 91,p < 0.001)









where wx is the wing length in millimeters on day x, while that for 0. japonicus was

obtained from Lounibos et al. (unpublished data), who used individuals originating from the

same colony from Rutgers University mentioned previously, as follows:

f(wx) = 53.078 wx 113.91 (r2 = 0.319, N= 79,p < 0.001),

where wx is the wing length in millimeters on day x.

For analyses of V' for A. albopictus and 0. japonicus a one-way ANOVA was used with

Tukey's honestly significantly different (HSD) tests performed post-hoc for pairwise

comparisons of means (SPSS 2002).

Results

Survivorship to Adulthood

Mean survivorship to adulthood of A. albopictus was affected by treatment but that of O.

japonicus was not (Table 3-1, Figure 3-1). Mean survivorship of A. albopictus varied

significantly among treatments (F2,12 = 7.442, p = 0.008), and was significantly higher in the 10:0

treatment than the 50:0 or 25:25 treatments, which were not different from one another. With

respect to individual density treatments, mean survivorship ofA. albopictus was consistently

higher than that of O. japonicus.

Developmental Time

Median time from hatch to adulthood of both sexes was significantly affected by density

treatment for both males (F2,12 = 7.560,p = 0.008) and females (F2,12 = 19.114,p < 0.001) of A.

albopictus. Comparisons of means for A. albopictus showed that development time was

significantly shorter for both males and females in the 10:0 and 25:25 density treatments (Table

3-1). Median development time of both male (F2,12 = 7.09, p = 0.009) and female (F2,12 = 10.194,

p = 0.003) 0. japonicus was significantly affected by treatments; however significant differences

in pairwise comparisons differed between the sexes in this species (Table 3-1). With respect to









individual density treatments, median development times of both male and female A. albopictus

were consistently faster than those of O. japonicus (Figures 3-2, 3-3).

Female Wing Length

Median wing lengths of both A. albopictus and 0. japonicus females were significantly

affected by density treatments (F2,12 = 4.837, p = 0.029; F2,12 = 9.584, p = 0.003). For both

species, median wing length was significantly greater for females from 10:0 treatments; however

for A. albopictus this difference was only significant in comparison to females from the 25:25

density treatment (Table 3-1, Figure 3-4).

Estimated Finite Rate of Increase (,')

The mean estimated finite rate of increase of both species was significantly affected by

density treatments (Table 3-1, Figure 3-5), although more pronounced for A. albopictus (F2,12

23.585, p < 0.001) than O. japonicus (F2,12 = 16.366, p < 0.001). Comparisons of means showed

that V' ofA. albopictus was significantly higher for the 10:0 treatment than the 25:25 treatment,

which was significantly higher than that of the 50:0 density treatment (Table 3-1). For O.

japonicus, V' of the low-density treatment was significantly higher than either of the high-density

treatments. With respect to individual density treatments, mean estimated finite rates of increase

ofA. albopictus were consistently higher than those of O. japonicus.

Discussion

It is common opinion that the invasion success and spread of non-native species is

enhanced by superiority in interspecific competition, particularly when similar species and

limited resources are encountered (Williamson 1996). It has been demonstrated that interspecific

larval resource competition plays an important role in structuring the mosquito communities of

artificial container habitats (Juliano and Lounibos 2005). Given the results of this experiment, A.

albopictus does appear to be a superior competitor to O. japonicus; however the non-significant









impact of interspecific larval resource competition on population performance suggests that O.

japonicus will be able to coexist with A. albopictus in artificial container habitats in nature.

Continued coexistence ofA. albopictus and 0. japonicus in artificial containers is

supported by the relatively high survivorship of both species, which were not significantly

different under interspecific (25:25 treatment) versus intraspecific conditions (50:0 treatment) of

the same mosquito density. Furthermore, the estimated finite rate of increase, V', remained

greater than one in all species/density treatments, indicating a population increase for both

species under all experimental conditions (Table 3-1). However, the mean V' for A. albopictus

was actually significantly higher under interspecific conditions than intraspecific conditions of

the same mosquito density (Figure 3-5). This appears to be due to the median development time

of females from interspecific density treatments being significantly shorter than those from

intraspecific treatments of the same mosquito density (Figure 3-2). These findings suggest that

intraspecific competition may be more important for regulating A. albopictus population growth

in container habitats than interspecific competition with O. japonicus.

While neither species appeared to be detrimentally affected under interspecific conditions

with respect to population performance, A. albopictus may have a slight competitive advantage

over O. japonicus. This is supported by the consistently higher survivorship, shorter

development time, and higher finite rate of increase ofA. albopictus compared to O. japonicus

across density treatments. Under interspecific conditions, median development time of both male

(Fi,s = 18.375, p = 0.003) and female (F1,7 = 26.940, p < 0.001) A. albopictus was significantly

shorter than that of O. japonicus. Similarly, the mean estimated finite rate of increase was

significantly greater for A. albopictus than O. japonicus (Fi,8 = 11.016, p = 0.011). However,

there was no difference in survivorship between the two species under interspecific conditions









(Fi,8 = 3.240, p = 0.110). While the difference in development times may simply be the result of

intrinsic metabolic differences between the species, when coupled with the higher mean

estimated rate of finite increase it may suggest that perhaps A. albopictus is able to forage and

acquire resources more efficiently or employs different feeding behaviors that are more effective

in this type of larval habitat. Ho et al. (1973) suggested that perhaps the higher content of

proteinases in the gut ofA. albopictus facilitates a more efficient feeding style, which ultimately

allows the species to develop faster than other container-inhabiting mosquitoes.

These results imply that while A. albopictus may have a slight competitive advantage over

O. japonicus, the two will likely continue to coexist in containers in areas where their

distributions overlap. However, these findings should be viewed in context with field

observations of co-occurrences, as well as seasonal distributions, habitat preferences, and

overwintering behaviors, as they may ultimately influence the community structure of the

artificial containers in which these species coexist. In addition to these ecological consequences,

these findings may potentially have epidemiological implications, particularly with respect to

LaCrosse encephalitis virus, of which both A. albopictus and 0. japonicus are suspected vectors.

Their continued coexistence in containers in LaCrosse endemic regions may be important in

epizootic, or potentially even epidemic, transmission of the disease, although this will require

further investigation. Because larval competition has been linked to greater infection and

dissemination rates of dengue and Sindbis viruses for A. albopictus (Alto et al. 2005), similar

effects are possible with respect to arboviruses circulating in areas in which this species is

sympatric with O. japonicus.

Although this experiment was conducted in the field under manipulated but ecologically

realistic conditions, it is important to note that variations in resource level, type, or frequency









(Braks et al. 2004), temperature (Lounibos et al. 2002), sun exposure, container type (Livdahl

and Willey 1991), larval density, and season (Teng and Apperson 2000) may influence larval

competition differently among these mosquito species. Similarly, while interspecific larval

competition is likely an important factor influencing the survivorship, growth, reproductive

success, and population performance of mosquitoes in container environments with limited

resources, other factors such as predation (Griswold and Lounibos 2005, 2006), intraguild

predation (Edgerly et al. 1999) apparent competition mediated by shared enemies (Munstermann

and Wesson 1990, Blackmore et al. 1995, Juliano 1998), habitat alteration (Bertness 1984), and

differences in foraging behavior (Yee et al. 2004) may also be important and warrant further

research with respect to interactions between these two species.









Table 3-1. Means (+SE) of population growth correlates for A. albopictus and 0. japonicus. Means followed by letters that are not
commonly shared are significantly different by pairwise comparisons (p < 0.05).

Density species treatments


Response

Mean survivorship

Median female development
time (d)

Median male development time
(d)

Median female wing length
(mm)

k'


10:0

.94 (.04)a


15.4 (.51)a


12.8 (.56)a


2.71 (.092)a

1.162 (.007)a


Aedes albopictus

50:0

.704 (.038)b


29.6 (2.91)b


15.2 (.58)b


2.51 (.053)ab

1.093 (.008)b


25:25

.728 (.061)b


17.5 (.71)a


12.7 (.37)a


2.41 (.056)b

1.120 (.007)c


10:0

.78 (.049)


20.8 (2.84)


14.8 (.52)a


3.55 (.060)

1.135 (.007


Ochlerotatus japonicus

50:0

.524(.096)


a 32.8 (2.85)b 3


a 20.3 (1.22)ab 2


a 3.05 (.108)b

)a 1.071 (.011)b 1.


25:25

584 (.052)


5.4 (3.08)b


5.9 (2.70)b


.08 (.10)b

076 (.007)b










1.20


SA. albopictus 0 0. japonicus


1.00


0.80


+ a


+ b


3.60


0.40


0.20


0.00 +


10:0


50:0


25:25


Treatment

Figure 3-1. Mean survivorship (proportion of the original number of larvae surviving to adulthood) of A. albopictus and O. japonicus
(+SE). Lower case letters indicate significant differences among competition treatments resulting from pairwise
comparisons (p < 0.05) for A. albopictus. Analysis of variance did not indicate a significant difference in survivorship
among treatments for 0. japonicus.









*A. albopictus


B


B


A


* a


10:0 50:0 25:25


Treatment
Figure 3-2. Means of median time to adulthood for female A. albopictus and O. japonicus (SE). Lower case and upper case letters
indicate significant differences among competition treatments resulting from pairwise comparisons (p < 0.05) for A.
albopictus and O. japonicus respectively.


0 O. japonicus












SA. albopictus O 0. japonicus


B




* a


10:0


0OA


* a


50:0


25:25


Treatment

Figure 3-3. Means of median time to adulthood for male A. albopictus and O. japonicus (SE). Lower case and upper case letters
indicate significant differences among competition treatments resulting from pairwise comparisons (p < 0.05) for A.
albopictus and 0. japonicus respectively.


I I I I












*A. albopictus


O 0. japonicus


O A


SB


4.00


3.50 -


3.00 -


2.50 -


2.00 -


1.50 -


* ab


25:25


Treatment

Figure 3-4. Means of median wing lengths of A. albopictus and O. japonicus adult females (+SE). Lower case and upper case letters
indicate significant differences among competition treatments resulting from pairwise comparisons (p < 0.05) for A.
albopictus and 0. japonicus respectively.


+ a


1.00 -


0.50 -


0.00 -


10:0


50:0









1.18

1.16

1.14

1.12

1.10

1.08


+ a


*A. albopictus 0 0. japonicus


A


+


+b


B


< B


1.06

1.04

1.02

1.00


10:0


50:0


25:25


Treatment

Figure 3-5. Mean estimates of population performance (V', an estimate of the finite rate of increase for the cohort) for female A.
albopictus and O. japonicus adults (+SE). Lower case and upper case letters indicate significant differences among
competition treatments resulting from pairwise comparisons (p < 0.05) for A. albopictus and 0. japonicus respectively.











CHAPTER 4

INTERSPECIFIC COMPETITION BETWEEN OCHLEROTATUS ATROPALPUS AND
OCHLEROTATUS JAPONICUS

Introduction

Within its native range in Asia, Ochlerotatusjaponicus larvae are found in a wide variety

of natural and artificial containers; however rock pools are the preferred habitat (LaCasse and

Yamaguti 1950, Tanaka et al. 1979). This species has colonized a similar ecological niche since

its introduction to the United States in 1998 via used tire shipments (Peyton et al. 1999,

Andreadis et al. 2001) from Japan (Fonseca et al. 2001). As an invasive species, the likelihood of

O. japonicus to become established and propagate in this niche depends partly on resource

availability and its ability to compete with ecologically similar resident species. Interspecific

competition is instrumental in determining the outcome of an introduction regardless of whether

it promotes or limits the spread of an invader, and may only be avoided if an invader is filling a

vacant niche by exploiting a previously unoccupied habitat or unused resource (Williamson

1996).

The effects of interspecific competition due to severe crowding and limited resources

among the larvae of container-inhabiting mosquitoes have been well documented (e.g. Livdahl

and Willey 1991, Barrera 1996, Juliano 1998, Braks et al. 2004, Juliano et al. 2004, Costanzo et

al. 2005), however the majority of these investigations centered around the invasion of Aedes

albopictus. Such studies have demonstrated both the success (O'Meara et al. 1995, Juliano et al.

2004) and failure (O'Meara et al. 1989, Lounibos et al. 2003) of invasive species to spread.

Interspecific competition may potentially impact the overall population performance of

container-inhabiting species by affecting growth, survivorship, and reproductive success (Juliano









and Lounibos 2005). Under conditions of severe interspecific competition, such effects may lead

to the decline or elimination of a resident species following the introduction of a competitively

superior invader (e.g. Juliano 1998).

Due to the propensity of O. japonicus to inhabit rock pools, the indigenous mosquito most

likely to be affected by the invasion of this species is the North American rock pool mosquito, 0.

atropalpus. The two species are highly sympatric, with distributions that overlap in parts of the

eastern United States. Frequent and abundant collections of O. japonicus larvae co-occurring

with 0. atropalpus in rock pools provides a natural setting for interspecific larval resource

competition (Andreadis et al. 2001), which some have speculated may limit its invasion success

(Juliano and Lounibos 2005). However, recent field studies have indicated that competitive

displacement of 0. atropalpus by O. japonicus may be occurring in rock pools in New Jersey

(Scott et al. 2001a), North Carolina (B. Harrison personal communication), and Virginia (see

previous chapter). Because of the specialized primary larval habitat of 0. atropalpus in rock

pools, the distribution of this species tends to be sparsely and irregularly distributed despite its

large geographical range. Such conditions may promote the localized decline or extinction of this

species in areas where these two species co-occur, particularly if O. japonicus is a superior

competitor. However, as noted in Lounibos (2002), 0. atropalpus has expanded its distribution

by occupying discarded tires and has itself been considered an invasive species.

The decline and potential competitive exclusion of 0. atropalpus suggest that the

competitive superiority of O. japonicus may be important in the invasion success of this species.

While interspecific larval resource competition is a likely mechanism for such ecological

processes, no formal research has been conducted to confirm such speculation. Thus an

investigation of larval competitive interactions between these two species was proposed.









Comparisons were made in the laboratory between species for intra- and interspecific effects of

larval density on survivorship, development time, body size, and population growth.

Materials and Methods

The experiment was conducted in an insectary at the Florida Medical Entomology

Laboratory in Vero Beach, Florida under controlled conditions of 25.50.0010C, 86.70.08%

RH, and a 12L: 12D photoperiod, from October to November 2006. Ambient temperature was

monitored hourly for the duration of the experiment with a single Onset HOBO data logger. The

O. japonicus and 0. atropalpus used in this experiment were harvested from eggs obtained from

colonies maintained at the Headlee Research Laboratory Mosquito Research and Control Unit at

Rutgers University in New Brunswick, New Jersey. The O. japonicus colony originated from

larval collections from a horse farm in New Egypt, Ocean County, New Jersey in 2001, while the

0. atropalpus colony originated from larvae collected in 1993 from Monmouth, Salem,

Cumberland, and Burlington Counties, New Jersey (L. McCuiston personal communication).

To investigate inter- and intraspecific larval competition between these two species, the

development of larvae in surrogate rock pools in different density species combinations was

monitored. Surrogate rock pools were constructed within plastic planters (91.4 x 18 x 12 cm)

using a fast setting, high strength concrete mix consisting of coarse sand aggregate, and cement

(Sakrete). Approximately 15 kg of concrete mixed with 1.6 1 water was poured into each of six

planters, and within each, five individual indentations (7 x 11.5 cm) were made using glass

canisters to create the rock pools. Field surveys in Fairfax County, Virginia indicated that these

species occur in rock pools of similar shape and size in nature. Once dry, each planter was

completely flooded with water for 24 hours to ensure structural reliability. A randomized

complete block design was used for the experiment, with five density combinations of O.









japonicus:O.atropalpus (20:0, 60:0, 0:20, 0:60, and 30:30) as treatments and individual planters

as blocks. Each planter contained one replicate of each density species composition

combination in a separate rock pool, for a total of six replicates per treatment and 30 individual

rock pools. Two months prior and up to the start of the experiment, surrogate rock pools were

flooded with water to allow for any potentially toxic chemicals to leech out of the cement in an

effort to reduce or prevent larval mosquito mortality.

On 19 October, each planter was randomly labeled with a number, and each of its rock

pools, was randomly labeled with a letter corresponding to one of the five treatments. Food

consisted of fallen pin oak leaves (Quercuspalustris) that had been collected in Fairfax, VA,

washed, and dried at room temperature for one week prior to quartering, weighing, and sorting.

Four days prior to the start of the experiment, 1.0 g of leaves was added with 300 ml of distilled

water to each of the 30 rock pools. The appropriate water level was marked in each rock pool,

and was checked every five days for evaporation and refilled with distilled water as necessary.

Each planter was covered with fiberglass screen (0.5 mm) and secured with a large rubber band

to prevent entry of other macrofauna. In the laboratory, eggs of O. japonicus and 0. atropalpus

were synchronously hatched (Novak and Shroyer 1978), and 24 hours later larvae were counted

into aliquots of 20, 30, and 60. Within one hour after counting, the larvae were distributed into

appropriate rock pools.

Each rock pool was monitored daily for the presence of pupae, which were collected and

housed singly in sealed 10 dram (36.7 ml) vials containing water from their respective rock pool.

Each vial was labeled with the appropriate planter and treatment identifier and placed in a rack

until adult eclosion. Upon emergence, adults were killed by freezing before scoring by container,









species, sex, and day of emergence. The experiment ended on 26 November when the final adult

emerged.

Data Analysis

Population Growth Correlates

To quantify the effects of inter- and intraspecific competition on O. japonicus and 0.

atropalpus, the mean survivorship, median development time of males and females, and median

body size at adulthood of females were analyzed by one-way ANOVA was used with Tukey's

honestly significantly different (HSD) tests performed post-hoc for pairwise comparisons of

means in SPSS (SPSS 2002) with species-density treatments as independent variables.

Survivorship was calculated as the proportion of adults that emerged from the initial cohort of

first instar larvae. Development time was calculated as the number of days from hatching to adult

emergence. Adult body size was estimated from the length of one wing, which was removed

from each female and measured under a dissecting microscope with an ocular micrometer

(Packer and Corbet 1989). Median rather than mean development time and wing length were

calculated for each species and treatment because of the non-normal distributions of these

variables within cohorts.

Composite Index of Population Performance

Survivorship, female development time and wing length were used to calculate a

composite index of mosquito population performance (X'), which is an analog of the finite rate of

increase as defined by Juliano (1998):












In (1/No) AfJ(w)
A' = exp I

D+ X



where No is the initial number of females (assumed to be 50% of the cohort), Ax is the number of

females closing on day x, 1i is the mean wing length of females closing on day x, andfil ) is

a function relating egg production to wing length. D is the time from adult eclosion to

reproduction, taken as 8 days for 0. atropalpus and 12 days for O. japonicus (see below). Values

of V' greater than one indicate that the population is increasing, approximately equal to one that

the population is stable, and less than one that the population is decreasing. If no individuals

survive to reproduction, V' equals zero (Leonard and Juliano 1995, Grill and Juliano 1996).

D for both species was determined experimentally under controlled conditions of 26C

and 12L: 12D photoperiod in an insectary at the Florida Medical Entomology Laboratory in Vero

Beach, Florida. In the laboratory, eggs were hatched by flooding with water, and cohorts of

approximately 100 larvae hatching within the same 24-hour period were grouped together and

placed in plastic trays within separate 0.028 m3 (30.5 cm x 30.5 cm x 30.5 cm) cages. Each

cohort was fed 100 mg of an artificial diet consisting of one part Brewer's yeast and one part

lactalbumen every other day. Beginning two days after emergence, adult females of each cohort

of O. japonicus were offered a bloodmeal from a restrained chicken placed within the cage daily

for one hour. As 0. atropalpus females can mature their first egg batch without blood (O'Meara

and Craig 1970, O'Meara and Krasnick 1970), cohorts of this species were not offered a

bloodmeal. Cotton soaked in a 20% sucrose solution was provided as a source of carbohydrates

for adults of both species at all times. Upon visual inspection immediately following the









bloodfeeding opportunity, those females that appeared to have fed to repletion were removed

from the cage using a mouth aspirator and placed singly in 12-dram plastic vials containing a

2.54 cm by 7.62 cm strip of wet seed germination paper to serve as an oviposition substrate

(Steinly et al. 1991). The germination paper was checked daily for the presence of eggs, and was

replenished with water if necessary. The date of first oviposition for each female was recorded,

from which the average time from adult eclosion to oviposition for 0. japonicus was calculated

to be 12 days, with a range of 4 to 17 days (N = 144), while that for 0. atropalpus was calculated

to be 8 days, with a range of 4 to 17 days (N = 153).

Regressions relating female wing length to fecundity were obtained from Lounibos et al.

(unpublished data), who used individuals originating from the same colonies as these

experiments, as follows:

O. japonicus:

f(wx) = 53.078 wx 113.91 (r2 = 0.319, N= 79,p < 0.001)

0. atropalpus:

f(wx) = 66.148 wx 150.28 (r2 = 0.526, N = 74,p < 0.001),

where wx is the wing length in millimeters on day x.

For analyses of V' for 0. japonicus, no transformation yielded data that met assumptions of

normality and homogeneous variance. Therefore, randomization ANOVA (Manly 1991, 1997)

was used to analyze V' as a randomized complete block design, with three different density -

species composition combinations as treatments and six different planters as blocks. Following

ANOVA, pairwise comparisons of all treatment means were conducted using randomization

methods (Manly 1991, 1997) at an experimentwise c = 0.05, using the sequential Bonferroni









method to correct for experimentwise error (Rice 1989). Because all values of V' for 0.

atropalpus were zero, no comparisons could be performed.

Results

Survivorship to Adulthood

Mean survivorship to adulthood of 0. atropalpus was affected by treatment but that of O.

japonicus was not (Table 4-1, Figure 4-1). Mean survivorship of 0. atropalpus varied

significantly among treatments (F2,15 = 3.79, p = 0.047), and was significantly higher in the 20:0

treatment than the 60:0 treatment. Mean survivorship of 0. atropalpus in interspecific treatments

was not significantly different from either intraspecific treatment. With respect to individual

density species composition combination treatments, mean survivorship of 0. atropalpus was

higher than that of O. japonicus in all instances, except the 60:0 treatment. There was no

significant difference in survivorship between the two species under interspecific conditions

(F,io = 1.456, p = 0.225).

Developmental Time

Median time from hatch to adulthood was not affected by density treatment for either sex

of O. japonicus (Table 4-1, Figures 4-3, 4-4). Median development time of female (F2,15 = 8.184,

p = 0.004), but not male (F2,15 = 1.484, p = 0.263), 0. atropalpus was significantly affected by

treatments, and was significantly shorter in the 20:0 treatment than the 60:0 or 30:30 treatments

(Table 4-1, Figure 4-1), which were not different from one another. With respect to individual

density treatments, median development times of female O. japonicus were shorter than those of

0. atropalpus in all instances, except 20:0 treatments. Median development times of males of O.

atropalpus were shorter than those of O. japonicus in all instances, except the interspecific

treatment. There was no significant difference between male (F,o10 = 0.328, p = 0.581), or









female (F1,10 = 0.995, p = 0.342) development time of O. japonicus and 0. atropalpus under

interspecific conditions.

Female Wing Length

Median wing lengths of both O. japonicus and 0. atropalpus females were significantly

affected by density treatments (F2,14 = 18.522, p < 0.001; F2,15 = 39.474, p < 0.001). For both

species, median wing length was significantly greater for females from the 20:0 treatment (Table

4-1, Figure 4-1).

Estimated Finite Rate of Increase (,')

The mean estimated finite rate of increase of O. japonicus was significantly affected by

density treatments (F2,14 = 3.87, p = 0.058), with V' less than one for both the 60:0 and 30:30

treatments, indicating population decline. Pairwise comparisons of means showed that V' of O.

japonicus from the 20:0 treatment was significantly higher than that from the interspecific

treatment; no other comparisons were significant (Table 4-1, Figure 4-1). For 0. atropalpus, V'

was zero in all instances, indicating that no individuals were able to reproduce autogenously

(Table 4-1, Figure 4-1).

Discussion

This experiment indirectly supported predictions that interspecific competition between

larvae of O. japonicus and 0. atropalpus contributed to the decline and potential displacement of

the latter species in some rock pool communities. However, results indicate that interspecific

larval competition was not detectably different from intraspecific competition for either O.

japonicus or 0. atropalpus, as there was no significant difference in the mean survivorship,

median development time, or median wing length for either species under inter- or intraspecific

conditions of the same mosquito density. The mean estimated finite rate of increase, V', was zero









in all instances for 0. atropalpus, indicating that no individuals were able to reproduce

autogenously, while that of O. japonicus did not significantly change between intra- and

interspecific treatments of the same mosquito densities. This suggests better overall population

performance for this species than 0. atropalpus across all experimental treatments. The V' values

for 0. atropalpus were a direct result of the zero fecundity values resulting from the small size of

emergent 0. atropalpus females from all density species combination treatments. Considering

the autogenous reproduction of 0. atropalpus (O'Meara and Craig 1970, O'Meara and Krasnick

1970), in which the fecundity of this species depends on nutrient reserves obtained in the larval

stage, this result is not surprising. Fecundity of 0. japonicus also appears to be affected, as the

median wing lengths of females from this experiment, which ranged from approximately 2.0 -

2.5 mm, were noticeably smaller than those from the previous field competition experiment with

A. albopictus, where they were approximately 1 mm longer (Figures 3-4, 4-4).

An attempt was made to simulate the ecological conditions experienced by these species in

nature; however the somewhat equivocal results as evidenced by the declining or zero estimated

finite rates of increase for both species suggest that perhaps these experimental conditions were

excessively stressful. It has been demonstrated that the type and quantity of detritus in a

container can influence microorganism populations and communities (Walker et al. 1991,

Kaufman et al. 2001), and therefore can influence mosquito population performance (Lounibos

et al. 1993, Walker et al. 1997). Furthermore, interspecific differences in feeding behavior are

common among container-inhabiting mosquitoes (Yee et al. 2004); larvae may browse on hard

surfaces or filter fluid to gather microorganisms (Merritt et al. 1992), and the efficiency of these

feeding modes may depend on habitat structure and complexity. The use of increased or multiple

levels of food, a different food source, or pulse delivery of food in this experiment may have









provided experimental conditions more comparable to those in nature in which interspecific

competition might be expressed. Furthermore, it is possible that the cement from which the rock

pools were constructed imposed some toxic effect on the mosquito larvae, although it is

important to note that there was no observable difference in the survivorship of O. japonicus in

this experiment of the previous field competition experiment of this species with A. albopictus

(Figures 3-1, 4-1).

Although it is evident that experimental conditions were stressful for both species, and thus

interspecific interactions between these two species as they occur in nature may have been

obscured, it is possible that O. japonicus may have a competitive advantage resulting from more

efficient feeding behaviors or better resistance to starvation. Although the latter was not directly

demonstrated in this experiment in terms of differences in survivorship, it is interesting to note

that male 0. atropalpus on average developed faster than that of O. japonicus, but the reverse

applied to females, indicating the need for females of the former species to lengthen larval

development to accumulate nutrient reserves for egg production. This suggests that the

autogenous reproduction of 0. atropalpus (O'Meara and Craig 1970, O'Meara and Krasnick

1970) may render this species more sensitive to competitive stress. Condition-specific

competition, wherein competitive superiority varies with the abiotic environment, is known to

occur among various life stages of container-inhabiting mosquitoes (Barrera 1996, Daugherty et

al. 2000, Costanzo et al. 2005), and may be an important factor in understanding the competitive

outcomes among 0. atropalpus and 0. japonicus.

These results emphasize the importance of experimental conditions and suggest that

multiple factors other than interspecific larval resource competition may be important in

determining the current abundance and distribution of these species. It has been demonstrated









that larvae of O. japonicus can survive at low temperatures for extended time periods (Scott

2003), an ability that appears to allow this species to hatch and/or begin development before

other mosquitoes in the early spring, and apparently overwinter in the larval as well as the egg

stage in temperate areas, even where the surface of larval habitats freeze completely (Kamimura

1976, Scott 2003, B. Harrison personal communication). Ochlerotatusjaponicus has been found

as larvae in every month of the year within its natural range (Nakata 1962) and in North Carolina

(B. Harrison personal communication), and in all months except February in New Jersey (Scott

2003). This cold tolerance of O. japonicus may confer an ecological advantage in obtaining

resources over 0. atropalpus, which diapauses in the egg stage (Hedeen 1953) and emerges later

in the season. Furthermore, it may facilitate asymmetric intraguild predation of newly hatched O.

atropalpus larvae by fourth instar 0. japonicus; however the importance of this ecological

process in structuring container-inhabiting mosquito communities has yet to be demonstrated in

nature (Edgerly et al. 1999).

Predation is a prominent feature of rock pools in North America with predacious diving

beetles of the family Dytiscidae playing a significant role in regulating the numbers of O.

atropalpus (Shaw and Maisey 1961, James 1964a,b). Larvae and adult Laccophilus maculosus

are the most efficient predators of this rock pool mosquito because of their habit of crawling

about on the bottom and sides of the pool where they came in contact with the bottom-feeding

larvae of 0. atropalpus (James 1964b). Hydra oligactis will capture 0. atropalpus, killing but

not ingesting young larvae and paralyzing later stages, thereby limiting the abundance of this

species (James 1964b). If 0. atropalpus and 0. japonicus tend to occupy different spaces within

a rock pool (i.e., at the bottom, surface, or in the water column), selective predation (Griswold









and Lounibos 2005, 2006) by these diving beetles may be important in facilitating the invasion

of the latter species in these habitats and should be investigated further.

The tendency of 0. atropalpus larvae to frequently congregate under leaves and other

organic debris at the bottom of their rock poolss (Hedeen 1953) has been shown to result from a

negative phototropic reaction (Beach and Craig 1979). Because fully exposed rock pools tend to

be more flood prone, 0. atropalpus may have adapted this behavior in response to larval

mortality associated with such environmental conditions. While this reaction may promote a

preference of this species for areas of lesser concentrations of light, under enough selective

pressure, this protective mechanism could potentially allow 0. atropalpus to inhabit rock pools

fully exposed to the sun, therefore partitioning, at least to some extent, rock pool habitats with O.

japonicus, which is less frequently found under such conditions (B. Byrd personal

communication, previous chapter).

These experimental findings appear ambiguous with respect to the nature of interspecific

larval resource competition between 0. atropalpus and 0. japonicus because of the stressful

experimental conditions; however, a slight competitive advantage for 0. japonicus seems likely.

Numerous mechanisms for the perceived reduction in numbers of 0. atropalpus in rock pool

communities have been proposed here, however it is important to note that variations in

temperature (Lounibos et al. 2002), habitat (Bertness, 1984, Livdahl and Willey 1991), larval

density, season (Teng and Apperson 2000), and oviposition attraction and repellency (Maire

1985, Zahiri et al. 1997) may also influence larval competition differently among mosquito

species and warrant further research with respect to interactions between these two species.

Finally, field observations of co-occurrences of these species, seasonal distributions, habitat

preferences, and overwintering behaviors should be made on a large geographical scale, as they










may ultimately influence the community structure of the rock pools in which these species

coexist.









Table 4-1. Means (+SE) of population growth correlates for O. japonicus and 0. atropalpus. Means followed by letters that are not
commonly shared are significantly different by pairwise comparisons (p < 0.05).

Density species treatments

Ochlerotatus japonicus Ochlerotatus atropalpus

Response 20:0 60:0 30:30 20:0 60:0 30:30

Mean survivorship .53 (0.09) .575 (0.107) .317 (0.057) .575 (0.107)a .26 (0.049)b .432 (0.076)ab

Median female development 13.5 (0.55) 18.25 (1.22) 18.6(1.22) 12.8 (0.95)a 21.1 (2.35)b 21.7 (1.60)b
time (d)

Median male development time 12.4 (0.75) 13.5 (0.56) 13.9 (0.64) 11.7 (1.26) 13.3 (0.73) 15.3 (2.18)
(d)

Median female wing length 2.48 (0.018)a 2.02 (0.064)b 2.05 (0.069)b 2.02 (0.022)a 1.65 (0.051)b 1.60 (0.031)b
(mm)

ha 1.082 (0.007)a 0.490 (0.22)ab 0.349 (0.22)b 0 0 0


aMultiple comparisons of mean V' among treatments for 0. japonicus were conducted
experimentwise c = 0.05, using the sequential Bonferroni method.


using randomization methods at an









* 0. atropalpus


a


20:0


O 0. japonicus





+


60:0


Treatment


Figure 4-1. Mean survivorship (proportion of the original number of larvae surviving to adulthood) of O. japonicus and 0. atropalpus
(+SE). Lower case letters indicate significant differences among competition treatments resulting from pairwise
comparisons (p < 0.05) for 0. atropalpus. Analysis of variance did not indicate significant differences in survivorship
among treatments for 0. japonicus.


0.80 -

0.70 -

0.60 -

0.50 -

0.40 -

0.30 -

0.20 -

0.10 -

0.00


ab


30:30


* b