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Interactions Between Age and the Reproductive Fitness of Culex quinquefasciatus (Diptera

Permanent Link: http://ufdc.ufl.edu/UFE0024841/00001

Material Information

Title: Interactions Between Age and the Reproductive Fitness of Culex quinquefasciatus (Diptera Culicidae)
Physical Description: 1 online resource (87 p.)
Language: english
Creator: Larrick, Stephanie
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: culex, fecundity, fertility, reproductive
Entomology and Nematology -- Dissertations, Academic -- UF
Genre: Entomology and Nematology thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: INTERACTIONS BETWEEN AGE AND THE REPRODUCTIVE FITNESS OF Culex quinquefasciatus (DIPTERA: CULICIDAE) This study reports the results of experiments considering the reproductive fitness of adult Culex quinquefasciatus Say as they age chronologically and physiologically. The mean age of mortality following the first gonotrophic cycle for adult female mosquitoes reared in the laboratory was 15 days. Female Cx. quinquefasciatus exhibited age-dependent mortality that decreased with age following the first gonotrophic cycle. Adult Cx. quinquefasciatus females were reared and maintained in the laboratory, blood-fed on day 3 post emergence (PE), allowed to oviposit on day 6 PE, and then blood-fed on days 7-21 PE to assess the impact of age on fecundity (number of eggs laid) and fertility (number of resulting larvae). The fecundity and fertility decreased in respect to increasing gonotrophic cycles and the increased time between oviposition and bloodmeal acquisition. The reduction in fecundity and fertility was attributed to mosquito age at the time of the bloodmeal.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Stephanie Larrick.
Thesis: Thesis (M.S.)--University of Florida, 2009.
Local: Adviser: Connelly, Cynthia Roxanne.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0024841:00001

Permanent Link: http://ufdc.ufl.edu/UFE0024841/00001

Material Information

Title: Interactions Between Age and the Reproductive Fitness of Culex quinquefasciatus (Diptera Culicidae)
Physical Description: 1 online resource (87 p.)
Language: english
Creator: Larrick, Stephanie
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: culex, fecundity, fertility, reproductive
Entomology and Nematology -- Dissertations, Academic -- UF
Genre: Entomology and Nematology thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: INTERACTIONS BETWEEN AGE AND THE REPRODUCTIVE FITNESS OF Culex quinquefasciatus (DIPTERA: CULICIDAE) This study reports the results of experiments considering the reproductive fitness of adult Culex quinquefasciatus Say as they age chronologically and physiologically. The mean age of mortality following the first gonotrophic cycle for adult female mosquitoes reared in the laboratory was 15 days. Female Cx. quinquefasciatus exhibited age-dependent mortality that decreased with age following the first gonotrophic cycle. Adult Cx. quinquefasciatus females were reared and maintained in the laboratory, blood-fed on day 3 post emergence (PE), allowed to oviposit on day 6 PE, and then blood-fed on days 7-21 PE to assess the impact of age on fecundity (number of eggs laid) and fertility (number of resulting larvae). The fecundity and fertility decreased in respect to increasing gonotrophic cycles and the increased time between oviposition and bloodmeal acquisition. The reduction in fecundity and fertility was attributed to mosquito age at the time of the bloodmeal.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Stephanie Larrick.
Thesis: Thesis (M.S.)--University of Florida, 2009.
Local: Adviser: Connelly, Cynthia Roxanne.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0024841:00001


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1 INTERACTIONS BETWEEN AGE AND THE REPRODUCTIVE FITNESS OF Culex quinquefasciatus (DIPTERA: CULICIDAE) By STEPHANIE LARRICK 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 2009

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2 2009 Stephanie Larrick

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3 To my Mom and Brother

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4 ACKNOWLEDGMENTS I thank my supervisory committee chair, Dr. Roxanne Connelly, and members Dr. Cynthia Lord and Dr. Jonathan Day for their support, help and guidance. I would especially like to thank Dr. Jonathan Day for getting me involv ed at Masters Academy of Vero Beach. I gratefully thank the laboratory staff at the Florida Medical Entomology Laboratory for all their help in preparing and running experiments. I thank all the faculty, staff and students at the Urban Entomology Labora tory for giving me a home when I was semi displaced. I would not have made it through without their help. Finally, I would like to thank my family and friends. Their love and support helped me get through this chapter of my life. I thank the Florida Mosquito Control Association Foundation for awarding me the T. Wainwright Miller, Jr. Florida Mosquito Control Association Scholarship. This work was supported by a grant from the National Institutes of Health, Empirical Modeling of Arboviruses in Florida (AI42164).

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................................... 4 LIST OF TABLES ................................................................................................................................ 8 LIST OF FIGURES .............................................................................................................................. 9 ABSTRACT ........................................................................................................................................ 10 CHAPTER 1 INTRODUCTION ....................................................................................................................... 11 2 LITERATURE REVIEW ........................................................................................................... 14 Classificati on and Distribution ................................................................................................... 14 Worldwide Distribution of Culex quinquefasciatus .......................................................... 14 United States Distribution of Culex quinquefasciatus ....................................................... 14 Florida Distribution of Culex quinquefasciatus ................................................................. 15 Morphology ................................................................................................................................. 15 The Egg ................................................................................................................................ 15 The Larva ............................................................................................................................. 15 The Pupa ............................................................................................................................... 15 The Adult .............................................................................................................................. 16 Life Cycle .................................................................................................................................... 16 The Egg ................................................................................................................................ 16 The Larva ............................................................................................................................. 16 The Pupa ............................................................................................................................... 17 The Adult .............................................................................................................................. 17 Emergence .................................................................................................................... 17 Mating ........................................................................................................................... 17 Feeding .......................................................................................................................... 18 Flight range ................................................................................................................... 18 Oviposition ................................................................................................................... 19 Longevity ...................................................................................................................... 19 Body size ....................................................................................................................... 21 Fecundity and Fertility ................................................................................................................ 22 Reproductive Allocation ............................................................................................................. 23 Assaying Bloodmeal Size ........................................................................................................... 24 Mass Rearing ............................................................................................................................... 25 Vector Competence of Culex quinquefasciatus for Saint Louis Encephalitis Virus .............. 27 Vector Competence of Culex quinquefasciatus for West Nile Virus ...................................... 28

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6 3 THE AVERAGE LENGTH OF TIME A FEMALE Culex quinquefasciatus WILL LIVE AFTER A SING LE GONOTROPHIC CYCLE ............................................................. 30 Introduction ................................................................................................................................. 30 Materials and Methods ................................................................................................................ 32 Larval Rea ring ..................................................................................................................... 32 Pupae and Blood feeding..................................................................................................... 32 Oviposition ........................................................................................................................... 32 Analysis ................................................................................................................................ 33 Results .......................................................................................................................................... 33 Discussion .................................................................................................................................... 34 4 THE EFFECT OF THE TIME SPAN BETWEEN OVIPOSITION AND BLOODMEALS ON THE FECUNDITY OF Culex quinquefasciatus .................................. 40 Introduction ................................................................................................................................. 40 Materials and Method s ................................................................................................................ 42 Larval Rearing ..................................................................................................................... 42 Pupae and Blood feeding..................................................................................................... 42 Oviposition ........................................................................................................................... 42 Analysis ................................................................................................................................ 43 Results .......................................................................................................................................... 44 Discussion .................................................................................................................................... 45 5 THE EFFECT OF THE TIME SPAN BETWEEN OVIPOSITION AND BLOODMEALS ON THE FERTILITY OF Culex quinquefasciatus ..................................... 53 Introduction ................................................................................................................................. 53 Materials and Methods ................................................................................................................ 55 Eggs ...................................................................................................................................... 55 Analysis ................................................................................................................................ 55 Results .......................................................................................................................................... 55 Discussion .................................................................................................................................... 56 6 SUMMARY ................................................................................................................................. 62 APPENDIX A HEMATIN STANDARD CURVE ............................................................................................ 66 Introduction ................................................................................................................................. 66 Objective ...................................................................................................................................... 66 Methods ....................................................................................................................................... 66 Results .......................................................................................................................................... 67 Conclusion ................................................................................................................................... 67

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7 B HEMATIN STANDARD CURVE VALIDATION ................................................................. 70 Introduction ................................................................................................................................. 70 Objective ...................................................................................................................................... 71 Materials and Methods ................................................................................................................ 71 Results .......................................................................................................................................... 72 Conclusion ................................................................................................................................... 72 LIST OF REFERENCES ................................................................................................................... 77 BIOGRAPHICAL SKETCH ............................................................................................................. 87

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8 LIST OF TABLES Table page 3 1 Hazard functions for the four mortality models used to fit mortality data. ........................ 36 3 2 Summary of the four mortality models for each replicate. The numbers represent the log likelihood of the data fitting the model. ......................................................................... 37 4 1 Calendar age, in days, of Cx. quinquefasciatus at the time of bloodmeals. ....................... 48 4 2 The sample size of mosquitoes in each time group in each gonotrophic cycle. ................. 49 4 3 Analysis of variance for the mean fecundity between gonotrophic cycles, days between bloodm eals, and the interaction of the two. ........................................................... 50 4 4 Mean standard error and means comparisons for the fecundity of Cx. quinquefasciatus .................................................................................................................... 51 5 1 Analysis of variance for the mean square root transformation of fertility between gonotrophic cycles, days between bloodm eals, and the interaction of the two. ................. 59 5 2 Mean standard error and means comparisons for the fertility (percent hatch) of Cx. quinquefasciatus .................................................................................................................... 60 B-1 Results of hematin expelled versus bloodmeal weight regression analysis. ...................... 74

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9 LIST OF FIGURES Figure page 3 1 Individual mosquito holding chamber. ................................................................................. 38 3 2 Average number of deaths per day and average populati on size over three replicates. ... 39 4 1 Regression of the mean fecundity with respect to the time between bloodmeals and gonotrophic cycle.. ................................................................................................................. 52 5 1 Regression of the percent hatch with respect to the time between bloodmeals and gonotrophic cycle.. ................................................................................................................. 61 A 1 Flow chart of methods. .......................................................................................................... 68 A 2 Standard curve of the abso rbance versus concentration of hematin used to determine the amount of hematin in mosquito feces. ............................................................................ 69 B1 Individual mosquito holding chamber. ................................................................................. 75 B2 Regression of the hematin expelled versus bloodmeal weight. ........................................... 7 6

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10 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 BETWEEN AGE AND THE REPRODUCTIVE FITNESS OF Culex quinquefasciatus (DIPTERA: CULICIDAE) By Stephanie Larrick August 2009 Chair: Roxanne Connelly Major: Entomology and Nematology This study reports the results of experiments considering t he reproductive fitness of adult Culex quinquefasciatus Say as they age chronologically and physiologically The mean age of mortality following the first gonotrophic cycle for adult female mosquitoes reared in the laboratory was 15 days. Female Cx. quinquefasciatus exhibited age -dependent mortality that decreased with age following the first gonotrophic cycle. Adult Cx. quinquefasciatus females were reared and maintained in the laboratory, blood -fed on day 3 post emergence (PE ), allowed to oviposit on day 6 PE and then blood-fed on days 7 21 PE to assess the impact of age on fecundity (number of eggs laid) and fertility (number of resulting larvae). The fecundity and fertility decreased in respect to increasing gonotrophic cycles and the increased time between oviposition and bloodmeal acquisition. The reduction in fecundity and fertility was attributed to mosquito age at the time of the bloodmeal

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11 CHAPTER 1 INTRODUCTION Mosquitoes vector many pathogens that cause diseases in humans and animals. Throughout history humans have been tormented by mosquito -borne diseases. Many historians attribute the fall of Greece to malaria (Gillett 1972) Napoleon lost approximately 33, 000 men in the Mississippi Valley USA to yellow fever (YF) in 1802 leading to the Louisiana Purchase by the United States (Gillett 1972) The construction of the Panama Canal was halted many times due to malaria and YF (Gillett 1972). Florida has had 6 epidemics of YF the firs t in 1857 (Mulrennan 1986). The last YF case reported in Florida was in 1910 (Mulrenna n 1986). Malaria has also plagued Florida. The disease was eliminated from the state due largely to the formation of mosquito control program s throughout the state (Mu lrennan 1986). Malaria and YF are one -host (human to mosquito to human transmission cycles) mosquitoborne d iseases that are no longer a major threat in Florida. The more complex multi -host mosquito -borne disease transmission cy cles involving a n avian amplification host include s eastern equine encephalitis virus (EEEV), Saint Louis encephalitis virus (SLEV), and West Nile virus (WNV) all of which are present in Florida From 2000 through 2007 there were 12 eastern equine encephalitis ( EEE) 1 Saint Louis encephalitis (SLE) and 201 West Nile ( WN ) human cases reported in Florida (CDC 2008). Culex mosquitoes are well known arbovirus ve ctors. Culex mosquitoes were first identified as the vector of SL EV in St. Louis, Missouri in 1933 (Day 2001, Mullen and Durden 2002). Since then there have been 41 reported human outbreak s cause d by SLEV in North America T he most recent was in northeast Louisiana during the summer of 2001 (Jones et al. 2002). Culex quinque fasciatus Say 1823 is a major vector of SLEV from the central Midwestern United States south to Mexico and west to California (Day 2001).

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12 West Nile virus was first detected in the United States (US) during the summer of 1999 (Campbell et al. 2002). T here have been 28,961 human cases with 1,131 deaths caused by WNV in the US (CDC 2009) since the 1999 introduction Culex pipiens (L.) in the norther n US, Culex tarsalis Co quillett in the southwestern and western US, and Cx. quinquefasciatus in the southe rn US are major vec tors of WNV (Hayes et al. 2005). Culex nigripalpus Theobald is the primary vector of WNV in Florida (Day 2001). Saint Louis encephalitis and WN viruses have enzootic transmission cycles where the viruses are seasonally transmitted betwe en avian hosts and susceptible mosquitoes. A bird is infected by a mosquito a nd the virus is amplified (undergoes replication in the circulatory system) in the avian host. Mosquitoes obtain the virus from the infected bird. In the mosquito, the virus go es through the extrinsic incubation period (EIP) d uring which the virus replicates in the midgut After the end of the incubation period the mosquito is able to transmit the virus to susceptible vertebrate hosts. Replication in the mosquito can be acce lerated at higher temperatures leading to increased numbers of infected individuals. T o decrease the amplification of the pathogen, it is important to understand the role that the vector plays in the transmission cycle. One factor that affects the transm ission cycle is the age of the mosquito, both chronological ( calendar age ) and physiological (number of reproductive cycles ). Older mosquitoes have a greater probability of acquiring an arbovirus, completing the EIP, and transmitting the virus to additional vertebrate hosts In addition to understanding how age influences the transmission cycle it is necessary to have an understanding of the reproductive fitness of mosquito vectors. O ld er mosquitoes have decreased reproductive fitness and produce fewer progeny (Akoh et al 1992, Becnel et al 1995, Hogg and Hurd 1995, McCann 2006).

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13 Culex quinquefasciatus is a medium sized brown mosquito that can be found between the latitudes 36N and 36S around the world (Smith and Fonseca 2004, Knight 1978) It is present in all 67 Florida counties (Darsie 2004) In the United States, between 36N and 39N, Cx. quinquefasciatus freely mates with Cx pipiens producing viable offspring capable o f reproducing (Smith and Fonseca 2004, Savage et al 2006) The research reported here was performed in south Florida, where Cx. pipiens does not exist (Darsie 2004, Darsie and Ward 2005) ; therefore, s ub -specific naming will not be used. Culex quinquefas ciatus is a permanent water mosquito preferring nutrient rich organic water (Clements 1999) The adults are nighttime opportunistic feeders, preferring avian hosts. As part of a major research project dealing with the modeling and empirical studies of arboviruses in Florida, the research reported here will be used to help formulate the parameters for population age structure models and will provide improved knowledge about the reproductive fitness of Cx. quinquefasciatus The objectives of this project were: 1 Determine the average length of time a female Cx. quinquefasciatus will live after a single gonotrophic cycle. 2 Determine the effect of the time span between oviposition and bloodmeals on the fecundity of Cx. quinque fasciatus 3 Determine the effect of the time span between oviposition and bloodmeals on the fertility of Cx. quinquefasciatus

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14 CHAPTER 2 LITERATURE REVIEW More than most other living things, the mosquito is a self -serving creature. She doesnt aerate the soil, like ants and worms. She is not an important pollinator of plants, like the bees. She does not even serve as an essential food item for some other animal. She has no purpose other than to perpetuate her species. That the mosquito pla gues human beings is really, to her, incidental. She is si mply surviving and reproducing. (S pielman and DAntonio 2001) Classification and Distribution Culex quinquefasciatus is in the family Culicidae, subfamily Culicinae, and the tribe Culicini. Culex quinquefasciatus belongs to a group of morphologically similar species in the Culex pipiens complex that includes Culex pipiens There has been much debate centering on whether Cx. quinquefasciatus is its own species or a geographical variant of Cx. pipi ens making i t a subspecies (Barr 1957, 1967; Linam and Nielson 1962). Cule x quinquefasciatus has also been known as Culex pungens and Culex pipens fatigans (V inogradova 2000) For the work reported here Cx. quinquefasciatus refers to the mosquito species that occur s in south Florida and do es not overlap with Cx. pipiens World wide Distribution of Culex quinquefasciatus Culex quinquefasciatus is distributed worldwide. It is a tropical and subtropica l species, with a range fr om 36N to 36 S (Smith and Fonseca 2004, Knight 1978). It is found in North America, South America, Africa, South Asia, and A ustralia (Smith and Fonseca 2004). Cule x quinquefasciatus ma tes and produces viable hybrids with its sibling species Culex pipien s pipiens in regions from 36 N to 39N as well as Argentina, and Ma dagascar (Smith and Fonseca 2004, Savage et al 2006). United States Distribution of Culex quinquefasciatus Cule x quinquefasciatus occurs in 27 states: Alabama, Arizona, Arkansas, Calif ornia, Florida, Georgia, Illinois, Indiana, Iowa, Kansas, Kentucky, Louisiana, Maryland, Mississippi,

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15 Missouri, Nevada, Nebraska, New Mexico, North Carolina, Ohio, Oklahoma, South Carolina, Tennessee, Texas, Utah, Virginia, and West Virginia (Darsie and Wa rd 2005). Florida Distribution of Culex quinquefasciatus Culex quinquefasciatus is distributed throughout Florida where it is found in all 67 counties (Darsie 2004). Morphology The Egg Eggs of Cx. quinquefasciatus are laid on permanent water sources in oval rafts containing 100 or more eggs A single egg is elongate and tapers toward the posterior end (Clements 1999). After being laid, viable eggs will darken from white to black in about 2 hours (Guptavanij and Barr 1985). Cule x quinquefasciatus eg gs do not survive desiccation. The Larva The larval body consists of three regions: the head, thorax, and the abdomen. The head contains sensory as well as feeding organs. The abdomen is comprised of 9 segments, the siphon, and the gills (Darsie and Wa rd 2005, Mullen and Durden 2002, Rutledge and Evans 2004). The siphon of Culex mosquitoes is four times longer than it is wide with multiple tufts of hair and pectin spines. Specific to Cx. quinquefasciatus the siphon has four pairs of hair tufts and the aciculae on the saddle are evenly sized (Darsie 2004, Darsie and Ward 2005). Culex quinquefasciatus has a siphon index (length divided by the basal width) of 4.0 5.0 (Darsie 2004, Darsie and Ward 2005). The Pupa During the pupal stage, the insec t completes transformation from larva to adult and no longer eats The pupa is made up of two regions: the cephalothorax (head and thorax) and the

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16 abdomen. Trumpets on the cephalothorax allow the pupa to breathe. Culex pupae can be identified by compari ng length of the setae to the paddle. Cule x quinquefasciatus can be distinguished by a trumpet index (length divided by the basal width) of 5.0 and setae 17 have four or more branches (Darsie 2004, Darsie and Ward 2005). The Adult Culex quinquefasciatus is a brown colored, medium sized mosquito ranging from 3.96 to 4.25 mm in length (Lima et al. 2003). The pale basal bands of the abdomen ar e rounded posteriorly. Another defining characteristic is that the segments of the flagellum have few or no scale s (Sirivanakarn and White 1978). Life Cycle The Egg Culex quinquefasciatus is a permanent water mosquito. They lay their eggs in foul water containing organic compounds such as (but not limited to) sewage pits, ditches, tires, dairy retention ponds, agr icultural retention ponds, storm drains, and neglected artificial containers (Clements 1999, Rutledge and Evans 2004). The eggs are laid in rafts that are held together by a complex combination of tubercles and meshwork (Clements 1999). The eggs stand ve rtically on the surface of the water. At temperatures between 23 and 30C and a relative humidity between 8085%, eggs hatch 24 30 hours after being laid (Bates 1949, Clements 1999, Singh et al. 1975, Gerberg et al. 1970, 1994). The Larva Mosquito larvae are known as wigglers. T he larva hatches directly into the water in which they complete four instars. Culex feeds by filtering organic rich nutrients out of the water column (Clements 1992). The larval development time is dependent on factors includi ng temperature, larval density and food availability (Gerberg et al. 1994, De Meillon et al. 1967a).

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17 Culex quinquefasciatus larval development time at 27 C is between six and eight days (Gerberg et al. 1994), and they stop feeding toward the end of the 4th instar. The male larval growth time is shorter than that of females (Clements 1992). The Pupa The active pupae are known as tumblers. The pupal stage is the non -feeding phase of metamorphosis when the larva develops into the adult. This stage lasts two to three days, depending on the water temperature (Gerberg et al. 1994, Evans 2004). The Adult Emergence Emergence of Cx. quinquefasciatus is asynchronous. In general male mosquitoes emerge first, usually a day before the females (Clements 1992). Culex has more of an emergence overlap between sexes than other genera (Clements 1999) Upon emergence the mosquito must rest and let its cuticle harden. During the time it takes for the females to emerge, the males become sexually mature. To do this, the males must invert their terminalia then rotate it 180 and the antenna fibrillae must become erect. The antenna fibrillae will remain erect for the entire life of the male (Nijhout 1977, Beach 1980, McAlpine et al. 1981). Mating Matting occurs 4 8 to 72 hours after emergence (Lea and Edman 1972). Mating occurs within swarming flights Swarming occurs at dusk and dawn (Nielsen and Nielsen 1962, Jones and Gubbins 1979). After mate selection, the male engages the female with his claspers and the aedeagus is inserted into the vagina (Clements 1999). The male then delivers a sperm packet that is stored in the females spermatheca (Clements 1999). The female will mate o nce and then search for a

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18 blood meal. The males will continue to nectar feed, swarm, and copulate for an average of twenty days in the laboratory but for less time in the wild (Liles 1965, Clements 1999). Feeding Male mosquitoes do not blood feed, but rather nectar feed to meet their energy requirements (Clements 1992, Mullen and Durden 2002). In order to meet the initial energy requirement, the female will also nectar feed. After mating, the female searches for a bloodmeal to fulfill the protein requirement needed to develop viable eggs (Mullen and Durden 2002). Culex quinquefasciatus is an opportunistic feeder, feeding on birds, mammals, and occasionally reptiles (De Meillon and Sebastian 1967, McCray and Schoof 1970, Edman 1974, 1979; Yasuno et al. 1975, Reisen and Boreham 1979). The female us es kairomones to locate a blood meal. Kairomones consist of carbon dioxide olfactory detection, the olfactory detection of secondary attractants such as octenol and lactic acid, visual, and thermal stimulus (Clements 1999). Once in the proximity of the host, the female rests before feeding (Howlett 1910, De Meillon and Sebastian 1967). In regard to visual attractants Cx. quinquefasciatus is more attracted to brown and black than to blue, yellow, and white (Wen et al. 1997). Flight range The flight range of Cx. quinquefasciatus is dependent on food sources (sugar and blood) and oviposition sites (Clements 1999). In several dispersal experiments, it was shown that Cx. quinquefasciatus flew from 0.23 km up to 7 km. The longer distances tr aveled are attributed to prevailing winds and topography (Fussell 1964, Lindquist et al. 1967, MacD onald et al. 1968, Yasuno et al. 1973, 1975; Schreiber et al. 1988, Reisen et al. 1991).

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19 Oviposition Approximately 66 hours after blood feeding, gravid m osquitoes search for an oviposition site (De Meillon et al. 1967b). Female Cx. quinquefasciatus prefer water rich in organic compounds, and respond to oviposition aggregation pheromone or apical droplets that are released from the top of the egg after the y have been laid (Iltis and Zweig 1962). Bruno and Lau rence (1979) showed that in a laboratory setting, gravid Cx. quinquefasciatus females preferred to oviposit in bowls of water previously used for oviposition by other Cx. quinquefasciatus Once a suit able oviposition site has been found, the female will land on the surface of the water and deposit an egg raft. For the female to lay another batch of eggs, she needs to take another bloodmeal. Samarawickrema (1967) found that female Cx. quinquefasciatus can blood feed within two hours of oviposition. Longevity Longevity can be measured in two ways: calendar age and physiological age. Calendar age does not reflect physiological age and vice versa. Both are important in determining population dynamics. Calendar age is measured in days, while the physiological a ge is measured in the number of gonotrophic cycles. To determine the calendar age of wild mosquitoes, daily growth bands on the thoracic apodemes can be counted (McFarland and M agy 1962, Schlein and Gratz 1972, Moore et al. 1986). This technique requires accurate and precise counting so not to give a wrong age range. Daily growth bands in the thoracic apodemes are a way to measure calendar age Moore et al. (1986) measured the thoracic apodemes of wild Culex pipiens s.l. (a sibling species of Cx. quinquefasciatus ) from a single trap night. The oldest mosquitoes they identified with this technique were six days old.

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20 A gonotrophic cycle begins with the acquisition of a bloodmeal and ends with oviposition (Clements 1992). Gonotrophic cycles are not easy to determine. Ovule dissection is a method for determining the number of gonotrophic cycles in some mosquito species (Cheong 1985). This process includes dissecting the ovules and counting the dilations or follicular relics attached to the ovules (Samar awickrema 1967, Cheong 1985). Parity, whether or not eggs have been laid, can be determined by ovary dissections. Before the eggs are laid, the trachea within the ovarioles are tightly coiled A fter oviposition the ovariol trachea uncoils (Akoh et al. 1 992, Gowda and Vijayan 1993, Mullen and Durden 2002). Female mosquitoes that have not completed a gonotrophic cycle are termed nulliparous while those that have completed on e or more gonotrophic cycles are parous (Mullen and Durden 2002). Gowda and Vi jayan (1993) and Samarawickre ma (1967) performed three separate studies of wild Cx. quinquefasciatus to determine the age of specific populations. The age was calculated by the number of ovariole skeins and dilatations in the follicular tubes. The number of dilatations is equivalent to the number of gonotrophic cycles. They found that 4556. 6 % of the females collected were nulliparous. As the gonotrophic cycles increased, the number of females collected decreased. The first gon otrophic cycle represente d 31.735.7% of the population. The second, third, and fourth gonotrophic cycles represented 10.112.1%, 0.9 1.4%, and 0.1 0.3% of the population respectively. There were no Cx. quinquefasciatus collected that had completed five or more gonotrophic cycl es. Culex quinquefasciatus have been reported to live 64 days or l onger in laboratory colonies (Kerdpibule et al. 1981, Oda et al. 2002). Oda et al. (2002) found that at 25C females lived for 64.4 days while males lived for 39. 8 days in colony. At 30C, females lived for 30.1 days whil e males lived for 24.7 days. The increased longevity in the colony mosquitoes is

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21 attributed to controlled environments and a lack of environmental, predatory, and other stresses faced by wild mosquitoes (Clements 1999). Milby and Reisen (1989) tested the daily survivorship of wild Cx. quinquefasciatus by releasing marked mosquitoes. Daily survivorship ranged from 74.2 88.2%. Similar results were found by Reisen et al. (1991) and ElizondoQuiroga et al. (2006). Body s ize Adult m osquito body size can be measured in two ways: wing length or dry weight. Both measurements are affected by larval diet and temperature. For wing length calculations the wing is measured from the tip to the alula (Lyimo and Koella 1992, L ima et al. 2003, McCann 2006). The alula as define d by Harbach and Knight (1980) is a lobe of the posterior margin of the wing bound proximally by the upper calypte r distally by the axillary incision and anteriorly by the base of the anal vein. In wil d Cx. quinquefasciatus wing length can vary from 3.394.22 mm (Lowrie et al. 1989, Lima et al. 2003). The large variation of wing lengths in nature is due to the larval competition for resources, water temperature, and larval crowding. The wing length o f laboratory colonies is generally more uniform due to t he control of variables such as larval diet, air temperature, and water temperature Van Handel and Day (1989) correlated wing length with the amount of protein in the mosquito. In Cx. quinquefasciatus there is a direct correlation between protein and wing length (r2 = 0.92 for females, r2= 0.96 for males). Dry w eight is measured directly after emergence (Shelton 1973, Akoh et al. 1992). Wild Cx. quinquefasciatus tested at varying larval habita t temperatures produced the heaviest adults (2.9 mg) at 20 and 23 C (Shelton 1973). A t emperature increase or decrease in the larval habitat reduced the adult dry weight. A koh et al (1992) tested the impact of larval nutrition on adult body weight. L aboratory Cx. quinquefasciatus larvae fed liver, milk, o r liver and milk powder weighed 3.15 0.02 mg, 1.36 0.03 mg, and 2.45 0.03 mg respectfully.

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22 Fecundity and Fertility Fecundity is defined as the potential reproduction capacity of an organism (Clements 1992), or the number of gametes or eggs formed. Fecundity can be affected by many factors. Lima et al (2003) showed that mosquito body size, as measured by wing length, affected egg production. Culex quinquefasciatus with large wing s had a correspondingly higher rate of egg production. Other studies have shown that for Cx. quinquefasciatus Culex nigripalpus, and Aedes aegypti (L.) large blood meals increase fecundity ( Briegel 1986, Akoh et al. 1992, Lima et al. 2003, McCann 2006). Environmental factors, temperature and humidity also a ffect fecundity. In warm, humid environments, Cx. quinquefasciatus produces more eggs over a life time than do those in cool, less humid environments ( Lambrecht and Fernando 1974). This is thought to be due to the longer lifespan of the mosquito (Lambrecht and Fernando 1974). Strickman (1988) comp ared the daily oviposition rate of wild Cx. quinquefasciatus in two oviposition traps with ambient air temperature. He fo und that at temperatures below 2 C no eggs were laid. It was not until the temperature reached a minimum of 8 C that an average of 1 egg raft per trap was laid. A s the temperature increased, t he number of eggs also increased From 20 22C there was an average of 14.1 rafts laid in the traps. The calendar age of mosquitoes plays an important role in fecundity. Akoh et al (1992) s howed that by keeping the blood meal size consistent, older female Cx. quinquefasciatus laid fewer eggs per egg raf t. At seven days after emergence the mean number of eggs laid by a Cx. quinquefasciatus female was 219.57 6.77. The mean number of eggs 35 days after emerge nce dropped to 139.40 2.67. McCann (2006) blood-fed Cx. quinquefasciatus at four days and fourteen days after emerging. He f ound that the correlation of fecundity to body size in Cx. quinquefasciatus is positive in younger females and declines as they age

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23 Fertility is the number of viable offspring that an organism produces (Lincoln et al. 1982, Clements 1992). Fertility c an be considered in a single reproductive effort or over the lifetime of the individual. To measure fertility, the percent hatch can be calculated. McCann (2006) used the percent hatch to determine the relationship between age and fertility. Culex quinquefasciatus were blood -fed at set intervals after emergence (5 25 days). The eggs from each interval were counted and allowed to hatch. The percent hatch was calculated and compared for each group. A Kruskal Wallis test revealed a significant differenc e in the percent hatch between the age groups, but a scatter plot of the data did not show a correlation between percent hatch and age. Reproductive Allocation The resources a n organism obtains are allocated to three functions: growth, body maintenance, and reproduction (Fox and Czesak 2000). The allocation of reproductive resources is directed either to numerous small progeny or to a few large progeny. Smith and Fretwell (1974) first modeled the optimal progeny size and number. Though their model did not account for all variables, they were able to illustrate that parents must make a decision of how to balance the progeny size and fitness given a finite amount of resources. Since Smith and Fretwells (1974) model new models have been developed accoun ting for more variables that a ffect optimal progeny size and number (CluttonBrock 1991, Roff 1992). The newer models still start with the same assumptions of Smith and Fretwell (1974): there is a balance between progeny size and number and increased pare ntal care leads to an increase in progeny fitness. Allocation of resources occurs over the lifetime of the individual as well as in a single reproductive effort. It has been shown that as an individual ages, their reproductive output (clutch size) decreases (Wel lington 1965, Harvey 1977, 1983; Begon and Parker 1986). The Begon -Parker model shows that for insects that obtain reproductive resources as larva (Lepidoptera, Ephemeroptera, and autogenous mosquitoes ), there is a decline of reproductive

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24 ou tput over time (Begon and Parker 1986). The same is seen in individuals that gather food during their lifetime. Takydromus septentrionalis, the northern grass lizard, was observed laying fewer eggs over the breeding season, but as fewer eggs were laid t he eggs were larger (Ji et al. 2007). The clutch size of Eulophus pennicornis, an ectoparasitoid wasp, decreased as it aged (Bell et al. 2005). The resources that an individual has available for reproduction deplete over a season/ lifetime, and are alloca ted to other functions. Assaying Bloodmeal Size The size of a blood meal affects the fecundity and fertility of mosquitoes (Lima et al. 2003, McCann 2006). In this respect it is important to have an accurate way to measure bloodmeal size. Hematin is an excreted by -product of blood hemoglobin that has been digested by blood feeding arthropods but never entered the hemocoel (Briegel 1980, Mitchell and Briegel 1989, Hurd et al. 1995). Briegel (1980) showed that the amount of hematin excreted by Aedes a e gypti is proportional to the blood meal size. Quantifying the hematin is a noninvasive method in which the mosquito does not have to be anesthetized. To quantify the amount of hematin excreted, the reddish brown pellets excreted by the mosquito were dissolved in 1% lithium carbonate and measured with a spectrometer at 387 nm, which is the absorption peak for hematin. By using a standard curve, the reading is converted into micrograms of blood taken by the mosquito. The method presented by Briegel has b een used successfully by many researchers (Briegel 1980, 1986; Mitchell and Briegel 1989, Hurd et al. 1995, Hogg and Hurd 1995, 1997). McCann (2006) used Briegels method and determine d that the size of a mosquito bloodmeal has a direct effect on fecundity. McCann (2006) constructed a standard curve for hematin concentration from stock solution and measured the mixtures at 387 nm. By using the slope of the regression line, the value for hematin or the blood meal s ize in micrograms was determined.

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25 The accuracy of t his method is not known, because it has not been calibrated against any other method described. Roi tberg and Gordon (2005) developed an assay to determine bloodmeal size where the mosquito was weighe d. Prior to a bloodmeal, mosquitoes were anesthetized by chilling at 5 C for 90 seconds and then weighed to the near est 0.001 mg. Following a bloodmeal, mosquitoes were chilled at 5C for 90 s and weighed again. By subtracting the two weights, the mass of the bloodmeal was ca l culated This determination was use d to correlate body size, blood meal size, and fecundity. The authors found a curvilinear relationship between the blood meal mass and fecundity. One disadvantage of this method is that it would b e impractical to do with a large number of mosquitoes Near infrared reflective spectroscopy was used by Hall et al. (1990) as a means to q uantify the volume of the bloodmeal. The method was tested by administering predetermined amounts of blood via e nema. The mosquitoes were then placed in a scanning reflective monochromator. The reflectance measurements were then graphed against the quantity of blood administered, and the graph showed a direct correlation between the two. An advantage t o this meth od is that the blood meal size can be deter mined to the nearest microliter Disadvantages of this method include anesthetizing the mosquito, giving an enema to a mosquito (invasive method that may cause increased mortality) and high cost of the equipment. Mass Rearing Rearing techniques for Cx. quinquefasciatus have been reported by Singh et al (1975), and Gerberg et al. (1970, 1994). Adult Cx. quinquefasciatus were housed in cages no smaller than 1 x 1 x 1 ft with four mesh sides, a cloth sleeve, and a solid bottom. Adults were maintained on 10% sugar in water, and the water was maintained daily. Approximately three to four days after emergence the females were blood -fed to obtain protein for egg development. All

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26 life stages were kept at a 12: 12 light /dark period with a temperature between 23C and 30 C and 8085% relative humidity. For blood feeding, Wirtz and Rutledge (1980) used defibrinated bovine blood. The blood was funneled into a sausage casing and warmed. The casing was placed inside the cage. The casing was monitored closely so that its contents did not get cold (which would discourage fe eding). A second, and more commonly used method, required a live young chicken (Singh et al. 1975, Gerberg et al. 1970, 1994). In order for the mosquito to feed without disturbance, the chicken must be properly restrained. This also prevents the chicken from eating the mosquitoes. The chicken was left in the cage overnight when necessary. The female Cx. quinquefasciatus requires 48 to 120 hours to digest their bloodmeal This time is dependent on temperature and humidity Oviposition cups can be added to the cages after the digesti on time to encourage oviposition Gerberg et al. (1970, 1994) suggested that sugar water be removed 24 hours before adding oviposition cups to the mosquitos cage The oviposition cups were removed from the cage after a night with the gravid mosquitoes. The eggs were set in a pan so that each larva had at least 1 sq. cm of water surface (Gerberg et al. 1994). A mixture of liver powder and brewers yeast (2:3) was placed in the pans at the same time as the eggs so that the first instars had a food source and were not starved for any period of time ( Gerberg et al. 1970, 1994). The eggs typically hatch ed within 24 to 30 hours The larvae were sustained on a high protein diet throughout their development. The protein came from various sources: ground dog biscuits, ground hog food, liver powder, or b rewers yeast. In the fourth instar the amount of food was increased. The larval stage lasted six to eight days depending on the temperature.

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27 The pupa e were picked daily with a plastic pip ett e s and placed in containers with in a cage. When pupation occurred simultaneously, the cold water method of separation was used (Hazzard 1967). When placed in cold water, the pupa e of Cx. quinquefasciatus float, while any remaining larvae will sink, allowing for easy separation. A fter approximately 36 hours at 27C the adult s emerged (Gerberg et al. 1970, 1994). Vector Competence of Culex quinquefasciatus for Saint Louis Encephalitis V irus Saint Louis encephalitis virus (family Flaviviridae, genus Flavivirus SLEV ) is found in North, Central, and South Americ a. Saint Louis encephalitis virus is maintained in avian populations, and the intrinsic incubation time, the replication time of the virus in a primary host, is between 1 6 days (Mullen and Durden 2002) When a sus ceptible mosquito takes a blood meal from an infected bird, there is a chance it will pick up virus The virus replicates in the mosquito ; this is known as the extrinsic incubation period (EIP) The EIP is temperature dependent, usually taking 1021 days (Mullen and Durden 2002). During the EI P the mosquito may have the chance to oviposit and would be ready to take another blood meal. The mosquit o may not be able to vector SLEV at the time of the second bloodmeal, but there is a possibility of transmitting the virus during subsequent bloodmeals Primary hosts are avian, but secondary transmission is seen in mammals including humans. Humans are the only mammals that known to show pathology after being infected with SLEV (Mullen and Durden 2002). Saint Louis encephalitis is vectored by Culex mosquitoes. In the Midwest United States Cx. quinquefasciatus is the main vector and it is a minor vector in the western United States and Florida (Day 2001). In 2005, Reisen et al. tested the competence of two wild populations of Cx quinquefasciatus from California for SLEV They found that in the southernmost population, 96% of the mosquitoes became infected but only 20% were able to transmit the virus when fed on chickens with 4.3 log10 PFU/ml of virus. The northern population was given a chicken with

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28 2.2 log10 PFU/ml of virus and 65% of them became infected while only 12% were able to transmit. In order for Cx. quinquefasciatus to become a more competent vector, it would have to be more susceptible to the virus at lower titers The southern population needs to be tested at lower virus titers to see if it is still able to become infected and can transmit. Chamberlain and Sudia (1961) and Chamberlai n, Sudia, and Gillett (1959) determined the time it took for Cx. quinquefas ciatus to reach its peak transmission rate when infected with SLEV This was done by orally infecting the mosquitoes and recording the extrinsic incubation time. They found that at 27 C, it took 1926 days for the mosquitoes to reach their peak transmiss ion rate Gowda and Vijayan (1993) found that in the wild, Cx. quinquefasciatus lives an average of 14 days. Given that the mosquitoes only live an average of 14 days in the wild, Cx. quinquefasciatus would not be a good vector at temperatures below 27C because they do not live long enough. Vector Competence of Culex quinquefasciatus for West Nile Virus West Nile virus (Family Flaviviridae, genus Flavivirus WNV) is found i n Africa, Europe, the Middl e East, Asia, and North America (CDC 2009) West Nile virus has a transmission cycle similar to SLEV The amplifying hosts are birds and WNV is maintained in nature by a mosquito-bird -mosquito cycle. West Nile virus (WNV) is vectored by Culex mosquitoes. In the eastern U.S., north of 36N, WNV is vectored by Cx. pipiens and in the southeastern U.S., below 36 it is vectored by Cx. quinquefasciatus and Cx. nigripalpus (Savage et al. 2006, Rutledge et al. 2003). Sardelis et al. (2001) tested the vector competence of two strains of Cx. quinquefasciatus from Florida for WNV They found that in both strains feeding on chickens with 105.57.5 PFU/ml of virus, 91% and 94% became infected. The estimated transmission rate was 20% and respectively. Culex quinquefasciatus are competent and efficient WNV vectors.

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29 T hree wild populations of Cx. quinquefasciatus from Cali fornia were tested for their vector competence of WNV ( Reisen et al. 2005) Each population was assigned a chicken to feed on with 5.0 5.5, or 5.7 log10 PFU/ml of virus All three populations had at least a 40% infection rate, but none of the populations were able to transmit the virus. This data is lower than previously publis hed data by Goddard et al. (2002) in which each wild population was able to transmit the virus. Goddard et al.s (2002) data is older than that of Reisen et al. (2005) and the susceptib ility of the mosquitoes could have changed over that time. Changes in susceptibility need to be monitored in order to make the best predictions about arbovirus activity.

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30 CHAPTER 3 THE AVERAGE LENGTH OF TI ME A FEMALE CULEX QUINQUEFASCIAT US WILL LIVE AFTER A SINGLE GONOTROPHIC CYCLE Introduction As a female mosquito ages, both chronologically and physiologically, the number of progeny, the likelihood of encountering a pathogeninfected host and the risk of mortality increases. It is important to know the population dynamics (birth rates, death r ates, population age composition, etc.) of a species to predict the number of offspring the chance of a mosquito being infected with a pathogen and age -dependent mortality risks To help describe the population dynamics of any biological population at a given time, age -dependent mortality models can be used. The models give a measure for age -dependent mortality and allow further biological fitness calculations to be made. The four main models used by bio logists are Gompertz, Gompertz -Makeham, logistic, and logistic Makeham (Pletcher 1999 a). Age dependency is the change in mortality rate with age. The dependency can increase, decrease, or first increase and then decrease with age. Senescence is first the increase and then the decrease of mortality w ith age. Senescing is associated with decreased reproduction (Kirkwood and Rose 1991). The Gompertz, Gompertz -Makeham, logistic, and logistic Makeham models characterize the parameters for age -specific mortality (Table 3 1). The Gompertz model assumes age dependent mortality. The mortality rate at a certain age ( x ) is determined by the initial mortality rate ( a ) and the mortality increases with age ( b ) (Pletcher 1999a St yer et al. 2007). The logistic model assumes age -dependent mortality that declines as the organism gets older. The mortality rate at x is determined by 3 parameters: a b and the deceleration of mortality s (Pletcher 1999a Styer et al. 2007). As the value of s increases there is a leveling off of mortality at older ages. The logistic models can be reduced to the Gompertz model if the value of s is 0. To account for

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31 deaths caused by factors other than age, an age independent factor ( c ) is added to the Gompertz and logistic models making the Gompertz -Makeham and the logisti c -Makeham models (Pletcher 1999a). The models are considered hierarchical because they can all be reduced to the Gompertz model by eliminating parameters. Styer et al. (2007) conducted two mortality studies to prove the assumption that the mortality of Aedes aegypti changes as they age, or they senesce The first was a large scale (N > 100,000) mortality study in which males and females were caged together and allowed to die naturally. The second, smaller experiment allowed blood -fed and reproductivel y active mosquitoes to die naturally. The logistic model fit best to the data in both experiments. The logistic models factors are age -dependent mortality and the rate mortality decreases with age thus, Ae. aegypti do senesce In the wild, using mark release recapture studies, the daily mortality rates of Cx. quinquefasciatus range d from 11.8 25.8% (Milby and Reisen 1989, Reisen et al. 1991, ElizondoQuiroga et al. 2006) The analysis for the mark release recapture studies assumed age indep endent mortality. Moore et al. (1986) collected Culex pipiens using a CO2 trap, age d the mosquitoes using the daily growth bands on the thoracic apodemes, and found that the oldest mosquitoes were 6 days old. In the laboratory, under ideal climatic condi tions, non-blood -fed nul l iparous Cx quinquefasciatus have been observed living an average of 64.4 days and longer (Oda et al. 2002). In order to determine if female Cx. quinquefasciatus mosquitos senesce after a single gonotrophic cycle a mortality study was done. The average length of time the mosquitoes lived was recorded.

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32 Materials and Methods Larval Rearing Eggs were obtained from a colony of Cx. quinquefasciatus established in 1995 ( Allan et al 2006). For this work reported here the first batch of egg s received is referred to as the 1st generation. The 10th12th generation s w ere used for this experiment. Four egg rafts containing approximately 100 eggs each were placed into plastic pans measuring 36 x 45 x 6.5 cm containing 4 L of tap water. The larvae were provided 20 mg of 1:1 Brewers yeast/liver powder daily as a nutrient source. The temperature of the larval rearing room was 27 C with a light/dark period of 12:12. Once the larval stage was complete, the pupae were removed with plastic pipett e s Pupa e and Blood feeding Pupae were placed in 100 ml dishes with 45 ml of tap water and allowed to emerge in 36 x 36 x 36 cm cages. The adults were maintained on a 10% sucrose solution. Males and females were left in the same cage for 36 hrs prior to the first bloodmeal. Bovine blood (Hemostat Laboratories, D ixon CA) was offered as a blood meal on an artificial membrane three days after emergence (Richards et al. 2009) The mosquitoes were allowed to feed to repletion, usually taking 1 hr, and visually inspected to ensure a full bloodmeal was taken. This was the best noninvasive method to verify a full bloodmeal was consumed (Appendix A and B). Oviposition Immediately after blood feeding, individual mosquitoes were placed in separate 75 ml vials (4 cm diameter x 6 cm deep) (Fig. 3 1). A 10% sucrose solution was provided on cotton wicks. After 3 days, a vial containing 25 ml of tap water was offered to the mosquito for oviposition. The mosquitoes were allowed 24 h to oviposit. Those mosquitoes that did not oviposit were discarded The mosquitoes that did oviposit were maintained in their individual

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33 containers on a 10% sucrose solution until natural death. The mosquitoes were held at a light/dark period of 12:12, 27 C, and 80% relative humidity. Three replications of 95, 106, an d 103 blood -fed mosquitoes, respectively, from subsequent generations were run. Analysis The softwa re WinModest 1.0 (Pletcher 1999b) was used to determine the most suitable mortality model that fit the observed mortality. Age specific hazard, ux, allow s standard mortality models to instantaneously measure mortality data using ux = ln( px) where px is the probability that an individual alive at age x survives to age x+ 1 (Carey 1993). The software fits four hierarchical models to the data along with the calculating the log likelihood (Table 3 1). The best fit model has the largest log likelihood. Each replicate and the combined data had the models fit to it. The loglikelihoods were then compared to see if the models fit the data better when the replicates are co nsidered separately or together. The log-likelihoods from each replicate was summed and compared using 2*(summed log -likelihood -comb ined data log likelihoo d), distributed as chi square. The loglikelihoods of the combined data were then compared to determine what model best fit the data. The difference in the log -likelihoods was multiplied by 2 and distributed as chi squared. Results The models were fit to the data (Table 3 2). Separately, the logistic model was the best fit for each replicate. The models fit best when the data was combined 2= 8.99, p -2= 8.99, p 2=16.39, p logistic -2=16.39, p The Win Modest (Pletcher 199 9 b) program fit the c parameter to 0 for the Makeham models reducing the models to a simpler level and resulting in

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34 the same log -likelihoods for the Gompertz and the Gompertz Makeham, logistic and logistic Makeham From this point forward, only the Gompertz and logistic models were compared. The logistic model was a better fit to the combined data than the Gompertz 2=79.15, p The para meters for the model were a = 5.64 x 106, b = 0.78, s =1.73. The mean age of death after one gonotrophic cycle in each of the 3 replicates was 15.6 2.5 (n=95 ), 15.91 2.1(n=106), and 15.7 2.3 days (n=103). The overall mean age of death after one gonotrophic cycle was 15.74 2.3 days (Fig. 3 2) The decline in the number of deaths at the end of the experiment is largely due to the decline in population size. Discussion The logistic model offered the best fit to the mortality data presented here. F emale Cx. quinquefasciatus exhibit age -dependent mortality which declines at older ages after one gonotrophic cycle The initial mortality rate ( a ) was low with a high er rate of mortality with age (b ) and t he deceleration rate of mortality ( s ) was greater than b This suggests that the mosquitoes had high early life mortality and greater survival later in life. The probability of a mosquito vectoring a pathogen depends on the age of the mosquito when it acquired the pathogen. Since the rate of age dependent mortality declines with age, older mosquitoes are important in the transmissio n of pathogens. If a mosquito acquires a pathogen at a young age and survives the time period before the rate of age -dependent mortality starts to decline then it has a great er chance to vector a pathogen to more hosts. Older mosquitoe s may also be critical in maintaining pathogens in nature over winter or during drought conditions. The mosquitoes in this study showed age -dependent mortality after one gonotroph ic cycle. Variables such as time between bloodmeals and oviposition, time between oviposition

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35 and bloodmeals, and physiological age of the mosquito need to be examined. Those parameters need to be taken into account in order to make better population dyna mic s models In laboratory colonies of Cx. quinquefasciatus supplied with 2% s ucrose solution and kept at 25 C, females lived for an average of 64.4 days and females kept at 30 C lived for an average of 30.1 days (Oda et al. 2002), however these mosquitoe s were not put under the stress of blood feeding or egg laying. The average lifespan for the Cx. quinquefasciatus in the work reported here was 15 days after one g onotrophic cycle, but chronologically the mosquitoes were an average of 21 days old. The re sults from the current study show that when female Cx. quinquefasciatus are subject to these two physiological stresses (blood feeding and egg laying) their average life span decreases. In subsequent experiments for this thesis, the effect of t ime between bloodmeals on fecundity and fertility will be examined. The mosquitoes will be held at different time intervals up to 15 days (mean age of mortality). Knowing that laboratory Cx. quinquefasciatus do senesce, a large number of mosquitoes will have to be blood -fed in the longer time intervals to ensure significant results.

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36 Table 3 1 Hazard functions for the four mortality models used to fit mortality data. Model Hazard Gompertz Gompertz Makeham + Logistic 1 + ( 1 ) Logistic Makeham 1 + ( 1 ) + a, initial mortality rate; b, exponential mortality increase with age; x, age; c, age -independent mortality; s, degree of mortalit y deceleration. (Pletcher 1999 a, Styer et al. 2007)

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37 Table 3 2 Summary of the four mortality models for each replicate. The numbers represent the log likelihood of the data fitting the model. Gompertz Gompertz Makeham Logistic Logistic Makeham Replicate 1(n=95) 231.64671 231.64671 224.38659 224.38659 Replicate 2 (n=106) 291.49190 291.49190 271.83749 271.83749 Replicate 3 (n=103) 260.41765 260.41765 251.45677 251.45677 Combined (n=303 ) 779.06109 779.061 09 739.48815 739.4881 5

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38 Figure 3 1. Individual mosquito holding chamber. 6 cm 6 cm Oviposition Vial Passage Hole Sucrose Container Screened Ventilation Hole 4 cm

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3 9 Figure 3 2. Average number of deaths per day and average population size over three replicates. The columns represent the deaths per day and the points represent the population size. 0 2 4 6 8 10 12 14 16 18 0 15 30 45 60 75 90 105 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23Deaths per Day Population SizeDay

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40 CHAPTER 4 THE EFFECT OF THE TIME SPAN BETWEEN OVIPOSITION AND BLOODMEALS ON THE FECUNDITY OF CULEX QUINQUEFASCIAT US Introduction As defined by Clements (1992), fecundity is the potential reproductive capacity of an organism, or the number of eggs produced Fecundity can be affected by many factors including body size, bloodmeal size, bloodmeal source, climatic conditions, and age. In the laboratory, Cx quinquefasciatus egg rafts contain between 100 and 350 eggs (Mogi 1992). In the wild, Cx. quinquefasciatus egg rafts contain between 30 350 eggs (Chadee and Haeger 1986). Lima et al (2003) showed that female Cx. quinquefasciatus with larger body size s, measured by wingspan, had a higher rate of fecundity than those with smaller body sizes T hey also showed that larger bloodmeals increase fecundity. This has also been seen in several other species (Akoh et al. 1992, McCann 2006). The bloodmeal source plays an important role in fecundity (Ikeshoji 1964). Culex quinquefasciatus that fed on chickens laid two times more eggs than those that fed on humans (Ikeshoji 1964). Culex quinquefasciatus fed on chicken blood for many generations then switched to ra bbi t blood produced 75% fewer eggs (McCray and Schoof 1970). After 5 generations of being fed on rabbit blood, there were 100120 eggs per raft compared to an average of 30 eggs per raft in the first generation (McCray and Schoof 1970). Lambrecht and Fer nando (1974) showed that Cx. quinquefasciatus produced more eggs over a lifetime in warmer, more humid environments. Similarly, Strickman (1988) found that air temperature affects daily ovipositioning. Below 8 C, an average of one egg raft per ovipositio n trap was found compared t o 14.1 rafts per trap at 2022 C. The calendar age of the female mosquito can affect its fecundity. When given the same size bloodmeal, older Cx. quinquefasciatus laid fewer eggs. The mean number of eggs laid 7

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41 days after emerg ence was 219.57 6.77 compared to 139.40 2.56 eggs laid 35 days after emergence (Akoh et al 1992). Fecundity is also dependent on the gonotrophic cycle. The mosquito ages physiologically with each cycle. In Anopheles stephensi Liston, a 24% reduction in fecundity was seen over three gonotrophic cycles (Hogg and Hurd 1995). From the first to the second gonotrophic cycle there was a 33% reduction in fecundity and a 15% reduction in fecundity was seen from the second to the third gonotrophic cycle (Hogg and Hurd 1995). Over the lifetime of an animal, the allocation of resources changes (Williams 1957, Wellington 1965, Harvey 1977, 1983, Begon and Parker 1986). Mosquitoes have a high rate of daily mortality and have increased ea rly life reproduction and lower fitness later in life (Dow 1971, Kirkwood and Rose 1991, ElizondoQuiroga et al. 2006). This is known as the antagonistic pleiotropy hypothesis which states that senescence begins at the time of reproductive maturity and wh en early life reproduction is favored, later -life fitness suffers (Williams 1957). The objective of the work reported here was to assess how the time span between bloodmeals affects the fecundity of Cx. quinquefasciatus Mosquitoes were provided a blood meal at specific intervals after ovipositioning until natural death, and the eggs from each batch were counted. Knowing that fecundity decreases with age and gonotrophic cycle, it is expected that as time between bloodmeals increases, fecundity will decrease. Known factors that affect fecundity such as body size (Lima et al. 2003), bloodmeal size (Akoh et al. 1992, Lima et al. 2003, McCann 2006) and climatic conditions (Lambrecht and Fer nando 1974, Strickman 1988) were controlled to minimize experimental variation The bloodmeal source remain ed constant throughout the experiment.

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42 Materials and Methods Larval Rearing Eggs were obtained from a colony of Cx. quinquefasciatus established in 1995 ( Allan et al 2006). For this work reported here, the first b atch of egg is referred to as the 1st generation. The 16th through 25th generations were used for this experiment. Four egg rafts containing approximately 100 eggs each were placed into plastic pans measuring 36 x 45 x 6.5 cm containing 4 L of tap water. The larvae were provided 20 mg of 1:1 Brewers yeast/liver powder daily as a nutrient source. This allowed us to produce mosquitoes that were similar in body size. The temperature of the larval rearing room was 27 C with a light/dark period of 12:12. Once the larval stage was complete, the pupae were removed. Pupa e and Blood feeding Pupae were placed in 100 ml dishes with 45 ml of tap water and allowed to emerge in 36 x 36 x 36 cm cages. The adults were maintained on a 10% sucrose solution. Male s and females were left in the same cage for 36 hrs prior to the first bloodmeal. Twenty females were removed from the cage and anesthetized O ne wing was removed from each m osquito and measured using Motic Imaging 3.2 ( Motic China Group Co., Ltd. 2001). Bovine blood (Hemostat Laboratories, D ixon CA) was offered as a blood meal on an artificial membrane three days after emergence (Richards et al. 2009). The mosquitoes were allowed to feed to repletion, usually taking 1 hr, and visually inspected to ensur e a full bloodmeal was taken. This was the best noninvasive method to verify a full bloodmeal was consumed (Appendix A, B). Oviposition Immediately after blood feeding, individual mosquitoes were placed in separate 75 ml vials (4 cm diameter x 6 cm deep ). A 10% sucrose solution was provided on cotton wicks. After 3 days, a vial containing 25 ml of tap water was offered to the mosquito for oviposition. The

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43 mosquitoes were allowed 24 h to oviposit. Those mosquitoes that did not oviposit were discarded. In Chapter 3 the average age of death after one gonotrophic cycle was found to be 15 days; therefore 15 groups of 200 mosquitoes were formed. Group 1 was allowed to feed one day after oviposition; group 2, two days etc. up to 15 days between each ovi position and blood feeding. The mosquitoes were allowed to blood feed and lay eggs until their natural death. Throughout the experiment mosquitoes were maintained in their individual containers on 10% sucrose. The mosquitoes were held at a light/dark pe riod of 12:12, 27 C, and 80% relative humidity. The number of eggs from each female was recorded. Bovine blood (Hemostat Laboratories, D ixon CA) was offered as a blood meal on an artificial membrane (Richards et al. 2009) at the designated time after ovip osition The mosquitoes were allowed to feed to repletion, usually taking 1 hr, and visually inspected to ensure a full bloodmeal was taken. This was the best non invasive method to verify a full bloodmeal was consumed (Appendix A, B). These steps were repeated until natural death of all the mosquitoes in the group. At the start of this experiment, t here were 200 mosquitoes in each time group. Analysis The raw data (number of eggs per female) from each gonotrophic cycle at each of the time points wa s tested for normality. Differences in mean fecundity with respect to the days between bloodmeals were tested for significance by a n analysis of variance (ANOVA) A p value of <0.05% was accepted as significant. A Tukey s test was ru n to compare the means The correlation of fecundity with respect to the time between bloodmeals and gonotrophic cycle was determined by a multiple regression. All statistical tests were performed using Excel (Window s ) 2007 version.

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44 Results No significant variation w as found in the wing size (3.09 0.13 mm) All of the raw data were found to be normally distributed (p The calendar age of the mosquitoes at the time of the bloodmeal and the sample size of mosquitoes in each gonotrophic cycle is shown in Tabl e s 4 1 and 4 2, respectively. Culex quinquefasciatus females were significantly affected by the increase in time between bloodmeals (df = 14, F= 256.47, p and the increase in gonotrophic cycles ( df= 4, F= 7.78, p (Table 4 2 ). The intera ction between the two was also significant (df= 18, F= 8.58, p ). The average number of eggs laid in the first gonotrophic cycle ranged from 66 to 69 and decreased to an average of 9 to 20 eggs in the fifth gonotrophic cycle. The mean fecundity for all the females in the time group and a comparison of the means is shown in Table 4 2 There was no difference in the means in any of the time groups for the first gonotrophic cycle. In the second gonotrophic cycle, there was a significant dif ference between the means at 7 and 13 days between oviposition and blood feeding. In t he third gonotrophic cycle a significant difference in the means was seen at 4, 5, 7 8, and 11 days between oviposition and blood feeding. There were significant differences for t he fourth gonotrophic cycle at 3, 6, and 7 days between oviposition and blood feeding. There were no eggs laid when the time exceeded 8 days between oviposition and blood feeding in the fourth gonotrophic cycle. The mosquitoes in the fifth gonotrophic cycle exhibited significant differences between the means at 4 5, and 6 days between oviposition and blood feeding. There were no eggs laid when the time exceeded 6 days between oviposition and blood feeding. The means were also significantly differe nt between gonotrophic cycles within treatment groups A multiple regression analysis of the fecundity with respect to the time between bloodmeals and gonotrophic cycle showed a negative correlation (R2= 0.9 311, p < 0.001) (Fig. 4 2 ). The residual data around the regression line appear ed to be randomly distributed.

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45 Discussion Mosquitoes age chronologicall y and physiologically with each gonotrophic cycle. In order to ensure that individuals thrive mosquitoes follow the antagonistic pleiotrop y hypothesis, in that they have early life reproduction (Dow 1971, Kirkwood and Rose 1991, ElizondoQuiroga et al. 2006). At the time of the first bloodmeal, all of the mosquitoes were three days old, so no significant difference in the means was expected. A s the time between egg laying and blood feeding increased, there were significant decline s in the mean fecundity except in the 1st gonotrophic cycle (Table 4 3) As the gonotrophic cycles increase d there was also a significant decline in the mean fecundit y. The ANOVA between the time span between bloodmeals and gonotrophic cycles showed that the interaction between the two was significant. Gonotrophically, the mosquitoes had aged the same, but by the cale ndar the mosquitoes held longer between egg laying and blood feeding were much older (Table 4 1 ). There is a correlation between fecundity with respect to the time between bloodmeals and gonotrophic cycles As the time between bloodmeals and gonotrophic cycles increase the fecundity decreases (F ig. 4 1 ). Again, this shows how calendar age and gonotrophic cycle affects fecundity. As the time between bloodmeals and gonotrophic cycles increase, the mosquito ages and re allocates its energy to body maintenance rather than reproduction (Williams 197 5, Wellington 1965, Harvey 1977, 1983, Begon and Parker 1986). Regressions are helpful in making correlations and predicting the population dynamics. To use regression models as predicting tool s the residuals need to be examined By examining residual data around the regression line the points appear to be randomly distributed. In order to use this regression model for predictive purposes, nonlinear models would have to be fitted to the data to ensure that the linear model is the best fit.

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46 Culex quinquefasciatus lay between 100350 eggs per raft in the laboratory (Mogi 1992). In the work reported here the average number of eggs per raft in the first gonotrophic cycle was 68.01 15.22 and decreased with each following gonotrophic cycle. Th is may be due to several variables such as bloodmeal source, membrane feeding, and housing them in individual containers The mosquitoes were provided bovine blood on an artificial m e mbrane for 15 generations before the experiment. According to McCray and Schoof (1970), five generations should have been adequate for the mosquitoes to adjust to the blood source. The best way to feed the mosquitoes in individual containers was on an artificial membrane. The mosquitoes were inspected to ensure a f ull bloodmeal was taken. Since all the mosquitoes were fe d using the same method, a full bloodmeal using artificial membrane feeding maybe smaller than when fed using a live animal. The mosquitoes were housed in individual containers and were forced to o viposit in the wate r provided. They may have retained some of their eggs. In future experiments, it is necessary that a proportion of the mosquitoes be dissected to count the eggs retained in each generation. It is good mosquito control practice t o treat areas plagued by Cx. quinquefasciatus early, effective ly and continuous ly ( EPA 2007). The results of the study reported here exemplifies the need for this practice. During adverse conditions, if a mosquito cannot take a bloodmeal immediately af ter oviposition the fecundity would decrease and t he adults would be producing fewer eggs (especially in later gonotrophic cycles ). E arly, effective, and continuous control would help reduce the vector population. This is the first time the in teraction between fecundity and the time between bloodmeals has been reported for Cx. quinquefasciatus and it should promote further research in this area. Consideration should be gi ven to delaying the first blood meal in the time groups to see how

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47 fecundity is affected by the delay. This would help in designing population dynamic s models Other experiments should also be done to test how mosquitoes of varying sizes are affected by the time between bloodmeals.

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48 Table 4 1. Calendar age in days, of Cx. quinquefasciatus at the time of bloodmeals. Gonotrophic Cycle Colony Generation Used Day s b/n bloodmeals 1st 2nd 3rd 4th 5th 16 1 3 7 11 15 19 16 2 3 8 13 18 23 17 3 3 9 15 21 27 17 4 3 10 17 24 31 18 5 3 11 19 27 35 18 6 3 12 21 30 39 19 7 3 13 23 33 19 8 3 14 25 36 20 9 3 15 27 20 10 3 16 29 21 11 3 17 31 22 12 3 18 33 23 13 3 19 35 24 14 3 20 37 25 15 3 21 39

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49 Table 4 2. The sample size of mosquitoes in each time group in each gonotrophic cycle Gonotrophic Cycle Day s b/n bloodmeals 1st 2nd 3rd 4th 5th 1 200 102 61 29 14 2 200 103 54 21 10 3 200 101 47 14 6 4 200 98 31 9 1 5 200 97 25 7 3 6 200 94 16 5 1 7 200 91 11 2 0 8 200 88 9 1 0 9 200 83 7 0 0 10 200 80 4 0 0 11 200 71 5 0 0 12 200 68 4 0 0 13 200 54 3 0 0 14 200 48 3 0 0 15 200 41 1 0 0

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50 T able 4 3 A nalysis of variance for the mean fecundity between gonotrophic cycles, days between bloodmeals, and the interaction of the two. Source of Variation df MS F P value Gonotrophic Cycles 4 9454.681 256.4703 2.71E 35 Days Between Bloodmeals 14 286.8873 7.782182 9.1E 09 Interaction 18 643.7842 8.578902 3.97E 05 Error 38 236.8648 Total 74

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51 Table 4 4 Mean standard error and means comparisons for the fecundity of Cx. quinquefasciatus Gonotrophic Cycle 1 2 3 4 5 1 68.84 16.89a 50.65 14.56b 37.89 11.69e 30.83 6.88 g 20.93 6.37 h 2 69.19 14.82a 51.60 14.13b 37.81 11.52e 29.95 7.15 g 20.3 5.81 h 3 66.27 16.67a 49.89 15.67b 38.39 10. 54e 23.14 5.9 l 20.0 5.61h 4 66.21 14.72a 50.21 15.58b 31.74 9.4f 23.44 5.48 l 15 0.0 m Days 5 67.31 15.31a 50.03 15.68b 30.86 8.81 g 22.86 3.79 l 14.0 1.63 j Between 6 68.99 13.88a 51.47 15.22b 29.95 8.19 g 16.6 2.87 m 9.0 n Bloodmeals 7 67.31 16.06a 32.41 8.59c 19.81 4.76 h 8.5 1.50n 8 69.29 14.20a 32.93 8.44c 18.0 3.49 i 7.0 n 9 69.11 15.28a 33.84 9.62c 20.0 3.51 h 10 67.77 14.73a 32.43 9.44c 20.75 2.38 h 11 68.06 15.19a 32.65 9.20c 12.4 2.42 j 12 67.12 15.46a 33.11 7.33c 13.0 4.06 j 13 69.06 14.51a 28.63 8.92d 12.0 2.45 j 14 67.08 15.82a 27.17 8.99d 14.5 1.5 j 15 68.52 14.71a 27.66 5.96d 12.0 j Means followed by the same letter are not significantly different. A p value of <0.05% was accepted as significant. -, no eggs laid *, only one egg raft was laid

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52 Figure 4 1. Regression of the m ean fecundity with respect to the time between bloodmeals and gonotrophic cycle The bars represent the mean fecundity and the line represents the fitted regression [ y= 6.489x + 31.91, p < 0.001, R2= 0.9 311, F =234.42 with 2 an d 42 degrees of freedom (n=75 )]. 0 10 20 30 40 50 60 70 80 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Mean FecundityDays Between Bloodmeals Gonotrophic Cycle 1 Gonotrophic Cycle 2 Gonotrophic Cycle 3 Gonotrophic Cycle 4 Gonotrophic Cycle 5

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53 CHAPTER 5 THE EFFECT OF THE TIME SPAN BETWEEN OVIPOSITION AND BLOODMEALS ON THE FERTILITY OF CULEX QUINQUEFASCIAT US Introduction Fertility is the number of viable offspring that an organism produces during a unit of time (over a reproductive cycle or over a lifetime) (Lincoln et al 1982, Clements 1992). Fertility in egg laying organisms is measured by percent hatch (=larvae hatched/eggs laid). Fertility can be affected by several factors including air temperature, water temperature, reproductive fitness, bloodmeal source, an d age. To predict the spacial distribution of several Culex species in Japan, Oda et al. (1980, 2002) tested fertility at different air temperatures. Oda et al. (1980) found at 21, 25, and 30 C the hatch rate of Cx quinquefasciatus was 95.9, 87.0, and 94.0%, respectively, while a t the same temperatures Culex molestus Forskal had a 82.1, 93.2, and 7.4% hatch respectively. Oda et al (2002) found that Cx. quinquefasciatus had a 100% hatch rate at both 25 and 30 C. Of the 3 Culex p allens Coquillett strains tested, two had a 100% hatch and one had 91.8% hatch at 25C, while at 30 C the hatch rate was 66.6, 12.5, and 0%. The water temperature plays an important role in fertility (Shriver and Bickley 1964). The percent hatch was cal culated for recently oviposited Cx. quinquefasciatus eggs in water ranging from 45 100 F (Shriver and Bickley 1964). Water temperature for optim al fertility was between 75 85 F. The embryonic development stopped when the eggs were maintained on water coo ler than 60F and warmer than 100 F. De Oliveira et al (2003) tested the reproductive fitness of two different generations (25th and 47th) of Cx. quinquefasciatus that were resistant to Bacillus sphaericus The mean percent hatch of a colony susceptibl e to B sphaericus was 91.2%. The mean percent hatch for the resistant strains was 73.3% for the 25th generation and 83.7% for the 47th generation. The increase in fertility between the generations was attributed to the resistance gene(s). They

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54 concluded that Cx. quinquefasciatus strains resistant to B. sphaericus have a reproductive fitness disadvantage. As with fecundity in mosquitoes fertility is affected by bloodmeal source (McCray and Schoof 1970). Culex quinquefasciatus is primarily an ornithophilic feeder (Sub ra 1981) In the laboratory, Cx. quinquefasciatus fed on chicken blood lay ed about 200 eggs per raft with more than 95% hatch (McCray and Schoof 1970). When switched to rabbit blood, the number of eggs per raft dropped to 30. Af ter 5 ge nerations of being fed on rabbit blood, eggs per raft increased to 100120 with greater than 95% hatch (McCray and Schoof 1970). The calendar age of mosquitoes has been shown to affect fertility while the physiological age has not (McCann 2006, B ecnel et al. 1995). McCann (2006) tested how the time between eclosion and the first bloodmeal affected the percent hatch of Cx. quinquefasciatus. Six groups of mosquitoes were fed at set time intervals after eclosion. The time until the first bloodmeal ranged from 5 25 days post eclosion. Statistical analysis of the data showed a significant difference between the age groups but a scatter plot of the data failed to show a reliable trend. Time elapsed between ec l o sion and the first bloodmeal affects fertility, but a positive or negative correlation between the two could not be made. Becnel et al. (1995) tested the effects of Edhazardia aedis (Kudo), a microsporidian pathogen, on the reproductive fitness of Aedes aegypti Over four gonotrophic cycles survival, fecundity, egg hatch, and percent adult emergence were compared for control and E. aedis infected mosquitoes. In the control group, there was no significant difference in egg hatch between gonotrophic cycles. In the infected group, egg hatch d ecreased with each gonotrophic cycle. Edhazardia aedis causes a decline in the reproductive fitness of Ae. aegypti

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55 No work has been done to test the effect of time between bloodmeals on fertility. To determine the effect of the time span between ovipo sition and bloodmeals on the fertility of Cx. quinquefasciatus mosquitoes of different ages were allowed to blood feed and the larvae were counted. Known factors that affect fertility such as air temperature (Oda et al. 1980, 2002), water temperature (Sh river and Bickley 1964), and bloodmeal source (McCray and Schoof 1970) were controlled to reduce variation. Materials and Methods Eggs The eggs used for this experiment were obtained from the fecundity experiment described in Chapter 4. The eggs were pl aced in petri -dishes fi lled with water and kept at 27C for 36h to allow hatching. The larvae were then counted and percent hatch was calculated by dividing the number of larvae by the number of eggs and multiplying that by 100. Analysis The raw data from each gonotrophic cycle at each of the time points was tested for normality. The percent hatch data from each gonotrophic cycle at each of the time points was square root transformed and tested for normality. Differences in mean fertility and the days between bloodmeals were tested for significance by ANOVA. A p -value of <0.05% was accepted as significant. A Tukeys test was run to compare the means. The percent hatch data was back transformed to display the data. The correlation of fertility with respect to the time between bloodmeals and gonotrophic cycle was determined by a multiple regression. All statistical tests were performed using Excel (Window s ) 2007 version. Results All the data were shown to be normal (p The fertility of Cx quinquefasciatus females was significantly affected by the increase in time between bloodmeals and the increase

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56 in gonotrophic cycles (Table 5 1 ). The interaction between the two was also significant The avera ge percent hatch in the first gonotrophic cycle ranged from 98 to 99 and decreased to an average of 94 to 96 larvae in the fifth gonotrophic cycle. The mean fertility and a comparison of the means are shown in Table 5 2 There was no difference between t he means in any time span for the first gonotrophic cycle. The percent hatch was significantly lower for the fifteen day group between bloodmeals in the 2nd gonotrophic cycle. In the 3rd gonotrophic cycle there were significant differences between percen t hatch, but no clear patterns In the 4th gonotrophic cycle, the percent hatch for 1 through 5 days between egg laying and blood feeding was significantly different than the percent hatch for 6 through 8 days between egg laying and blood feeding. In the 5th gonotrophic cycle, the percent hatch for 1 and 3 days between egg laying and blood fee ding was significantly different than the percent hatch for 2 and 4 through 6 days between egg laying and blood feeding. There were no eggs laid when the time exceeded 8 days between oviposition and blood feeding in the 4th gonotrophic cycle and no eggs laid when the time exceeded 6 days between oviposition and blood feeding in the 5th gonotrophic cycle thus no percent hatch could be calculated In each time span gr oup there was at least one significant difference over the five gonotrophic cycles. A multiple regression analysis of the percent with respect to the time between bloodmeals and gonotrophic cycle showed a negative correlation (R2= 0.4318, p < 0.001) (Fig 4 2). The residual data around the regression line appeared to be grouped at 0% and between 95100%. Discussion This is the first time the interaction between fertility and the time between bloodmeals has been reported for Cx. quinquefasciatus A sig nificant difference between fertility and the time between bloodmeals and gonotrophic cycles was seen (Table 5 1). This is similar to how the time between bloodmeals affects fecundity (Chapter 4).

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57 A regression of the fertility data shows a negative correlation betwe en fertility with respect to the time between bloodmeals and the gonotrophic cycle (R2= 0.4318, p < 0.01) (Fig. 5 1). As the time between bloodmeals and gonotrophic cycles increase the fertility decreases. The calendar age and gonotro phic cycle affects fertility but not as strong as they affected fecundity This is because the numbers for percent hatch are clearly grouped. There is a group that has 0% hatch and a group that has between 95 100% hatch. The two extremes cause d the r s quared value to be low. As the time between bloodmeals and gonotrophic cycles increase, the mosquito ages and re allocates the nutrients used for viable egg production to body maintenance (Williams 1975, Wellington 1965, Harvey 1977, 1983, Begon and Parke r 1986). M osquitoes age physiologically with each gonotrophic cycle As the time between oviposition and bloodmeals increased, the fecundity of Cx. quinquefasciatus decreased (Chapter 4). This is also true for fertility of Cx. quinquefasciatus The fertility did not decline significantly between each gonotrophic cycle in each time group, but overall the fertility declined significantly This is in contrast to what was reported for Ae. aegypti (Becnel et al. 1995) where t he mosquitoes were fed at regul ar time intervals and no significant difference between the percent hatch over four gonotrophic cycles was seen Although t he correlation between fertility with respect for the time span between egg laying and bloodmeals and gonotrophic cycles was not as strong as the correlation between fecundity with respect for the time span between egg laying and bloodmeals and gonotrophic cycles the antagonistic pleiotropy hypothesis was still being exemplified. As the mosquitoes aged both chronologically and physiologically, fewer viable progeny were being produced. The fertility of Cx. quinquefasciatus was affected by the time between oviposition and bloodmeals. Future experiments should focus on obtaining a better correlation between fertility

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58 and t he time of the first bloodmeal as well as between oviposition and blood feeding It would also be interesting to see how the fertility of other mosquito species is affected by changes in time between bloodmeals

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59 Table 5 1. Analysis of variance for the mean square root transformation of fertility between gonotrophic cycles, days between bloodmeals, and the interaction of the two. Source of Variation df MS F P value Gonotrophic Cycles 4 13067.28 16.99375 3.54E 09 Days Between Bloodmeals 14 1714.197 2.22928 0.02 7549 Interaction 18 18 47.739 4 .14247 0.01 7213 Error 38 768.9463 Total 74

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60 Table 5 2. Mean standard error and mea ns comparisons for the fertility (percent hatch) of Cx. quinquefasciatus Gonotrophic Cycle 1 2 3 4 5 1 99.17 3.66 a 98.51 2.02 a 97.39 3.85 b 96.72 3.36 b 96.93 3.30 b 2 98.66 4.4 a 97.83 2.39 a 97.7 2.87 a 96.79 3.45 b 95.23 3.12 c 3 99.14 2.27 a 98.77 2.59 a 96.96 2.98 b 96.83 2.1b 96.09 2.59 b 4 99.57 3.23a 97.89 3.13a 95.83 1.63c 96 2.17b 94.08 0.0* c 5 98.93 3.52 a 97.89 2.51 a 97.2 1.67 b 96.46 2.95 b 94.08 2.21 c 6 98.47 2.83 a 98.43 2.94 a 96.23 2.29 b 94.82 2.89 c 94.94 c Days 7 99.38 3.16 a 98.54 2.83 a 95.97 2.05 b 95.43 2.43 c Between 8 98.55 2.62 a 97.73 2.85 a 96.66 2.01 b 94.46 c Bloodmeal 9 98.86 3.17a 97.90 1.70a 95.51 2.23c 10 99.36 2.99 a 98.1 2.78 a 95.51 2.13 c 11 99.26 2.91 a 97.71 1.97 a 95.44 2.10 c 12 99.31 3.04 a 98.47 2.97 a 96.13 2.63 b 13 99.38 2.95 a 98.69 1.86 a 95.74 2.45 c 14 99.47 3.02a 97.89 2.56a 95.88 2.64c 15 99.24 3.12 a 97.08 2.21 b 95.9 b Means followed by the same letter are not significantly different (p -, zero percent hatch (no eggs laid) *, only one egg raft was laid

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61 Figure 5 1. Regression of the percent hatch with respect to the time between blood meals and gonotrophic cycle The bars represent the data and the line represents the fitted regression [ y= 3.042x + 12.619, p < 0.001, R2= 0.4318, F= 27.36 with 2 a nd 72 degrees of freedom (n=75 )]. 0 10 20 30 40 50 60 70 80 90 100 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15Percent HatchDays Between Bloodmeals Gonotrophic Cycle 1 Gonotrophic Cycle 2 Gonotrophic Cycle 3 Gonotrophic Cycle 4 Gonotrophic Cycle 5

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62 CHAPTER 6 SUMMARY The reproductive fitness of a female mosquito is determined by many factors. An i mportant factor is age, both calendar and physiological. Age also plays a role in the transmission cycle of pathogens vectored by mosquitoes. The reproductive fitness decreases and the probability of a mosquito acquiring and vectoring a pathogen increase s with age (Akoh et al. 1992, Becnel et al. 1995, Hogg and Hurd 1995, McCann 2006). To better understand the age specific reproductive fitness of Culex quinquefasciatus experiments on life expectancy, reproductive fitness, and fecundity were conducted The first objective of my research was to determine the average number of days that a female Cx. quinquefasciatus would live following a single gonotrophic cycle The average day of death for a Cx. quinquefasciatus that had completed a single gonotrophic cycle was 15 days (chronologically 21 days old) The pattern of mortality followed the logistic model that states mortality is age dependent and the rate of dependency declines as the mosquito ages This information was used to determine how long to hold mosquitoes before allowing them to b lood fe e d and the number of mosquitoes to use for subsequent experiments. The information reported here will be useful in deriving parameters for population dynamic s models. In objectives 2 and 3, t he reproductive fitness of Cx. quinquefasciatus was examined as the time between oviposit ion and bloodmeals increased The fecundity decreased as the time between oviposition and blood feeding increase d. The fecundity also decreased between each gonotrophic cycle. The response in fertility was similar but not as significant In essence, overall fewer eggs and more nonviable eggs are being laid as the time between ovipos ition and bloodmeals increases.

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63 The size of the bloodmeal a mosquito takes can affect its reproductive fitness (Akoh et al. 1992, McCann 2006). It was thought that hematin could be used to indirectly measure the bloodmeal size in the study reported here (Appendix A and B). In previously reported studies, a standard curve regression model was used to measure the size of a bloodmeal a mosquito acquired (Bri e gel 1980, 1986; Hogg and Hurd 1995, 1997, McCann 2006). A standard curve was made and the results were similar to previously published data (McCann 2006) The original sta ndard curv e regression model (Briegel 1986) was tested for accuracy in Ae. aegypti but never in any other species. The newly made standard curve was then tested for its accuracy in estimating bloodmeal size in Cx. quinquefasciatus There was no correlation found between bloodmeal weight and the amount of hematin expelled in Cx quinquefasciatus In the experiments reported here, a visual inspection was used to ensure a full bloodmeal. Further studies are needed to find a non invasive method to measure bloodmeal size. In the laboratory, Cx. quinquefasciatus were found to live for 15 days after one gonotrophic cycle and 21 days chronologically. This number is most likel y underestimated because of humidity in the laboratory. The humidity was controlled throughout the experiment, but i n nature mosquitoes can find micro-habitats that meet their high humidity needs (Price 1997) Even so, if a mosquito acquires St. Louis encephalitis virus (SLEV) or West Nile virus (WNV) at the first bloodmeal, 1 5 days is still long enough for the mosquit o to become infective (Hurlbut 1973, Anderson et al. 2008) The long lifespan of Culex quinquefasciatus contributes to making it a good vector of SLEV and WNV. In most modeling of mosquito population s mortality was assumed to be age independent (MacDonald 1952). Styer et al. (2007) and the data report ed here refute the idea of age independent mortality and showed that age -dependent mortality decreases with age. O lder

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64 mosquitoes have a greater probability of biting an infectious host, surviving to be infectious, and transmitting a pathogen because the rate of mortality decreases as the mosquito ages It is then possible that older mosquito es are responsible for increased pathogen transmission More resea rch needs to be done to exami ne whether or not age -dependent mortality is evident in different scenarios in the laboratory, t hen the age dependent mortality in nature needs to be examined. It can be assume d that the antagonist pleiotrop y hypothesis is ta king place and later life fitness is decreasing (Williams 1957) because there are fewer eggs and more non -viable eggs are being laid as the mosquito ages. Even though fewer eggs are being laid, the potential vector population is increasing In the philos ophies of mosquito control l arval mosquito control needs to be timed accordingly, early in the mosquito season to reduce the initial population numbers (EPA 2007). The results of the studies reported here intensify the need for early larval control becau se the first batch es of eggs are being laid by potentially chronological ly and physiological ly older mosquitoes; thus, fewer eggs and more non-viable eggs are being laid initially. By controlling the initial population, and the use of c ontinuous and effective larval control measures the potential vector population would decline. Older mosquitoes have a greater probability of being vectors than younger mosquitoes depending on the age of the mosquito when it acquires the pathogen. St. Louis encepha litis and WNV are able to be transmitted vertically in the labor atory (Goddard et al. 2002, Naya r et al. 1986). Vertical transmission would allow the virus to be maintained in nature over winter or through adverse conditions. Models that do not take vert ical transmission into account need to start adding it as a parameter.

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65 Culex quinquefasciatus is a vector of pathogens that affect humans domestic animals, and wildlife The more we know about its involv ement in the transmission cycle of these pathogens, the more likely it is that we will be able control the spread of the pathogens. The results of the studies reported here provide an insight into the reproductive fitness of Cx. quinquefasciatus and how pathogen transmission may be a ffected by age structure of Cx. quinquefasciatus populations. The results reported here c an be used to set parameters in population models that aid i n a r bovirus outbreak prediction as well as opening up new areas of research.

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66 APPENDIX A HEMATIN STANDARD CUR VE Introduction All insects have a process for voiding waste excreta and feces from their body. In female mosquitoes, waste is voided after adult emergence after sugar feeding, and after each blood meal (Clements 1992). The waste products include excreta and feces (Clements 1992). Excreta are the nitrogenous waste, while feces are the undigested food residuals and other midgut waste (Clements 1992). The feces contain hematin, produced by the digestion of hemoglobin. It has been shown that the input of hemoglobin and output of fecal hematin has an exact stoichiometric (1 to 1) relat ionship (Briegel 1986). In 1980, Briegel introduced the idea that by quantifying the amount of hematin in the feces, the r elative size of the bloodmeal could be estimated To calculate this relationship feces were dissolved in 1% lithium carbonate and the absorbance was read in a spectrophotometer at 387 nm, the absorption peak for hematin. Objective The objective of this experiment was t o c onstruct a regression equation that could be used to calculate the amount of hematin contained in the feces of blood -fed Culex quinquefasciatus females. Methods The hematin standard curve was made as previously described by McCann (2006) following Briegel (1980). Briefly porcine hematin (MP Biomedicals, Irvine, CA) was diluted 1% lithium carbonate (LiCO3) at 1.00 mg/ml, dilutions were made, and absorbance of the solutions was taken (Fig. A 1). D iffering from McCann (2006), who used S -Plus for Windows, the linear regression was made in Excel (Windows 2007) by regressing the absorbance at 387 nm in the hematin concentration.

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67 Results A regression equation was constructed (Fig. A 2 ). The equatio n is y = 13.951x 0.3417 with R2=0.9978, F=18121 with 1 and 40 degrees of freedom. Conclusion To e s timate the bloodmeal size of Cx. quinquefasciatus, feces were collected, dissolved in 1% LiCO3, and the absorbance read at 387 nm in a spectrophotometer. The spectrophotometer reading was used to calculate a regression equation and the hematin concentration of the bloodmeal was calculated. The input of hemoglobin in a bloodmeal and output of fecal hematin have an exact stoichiometric relationship (Briegel 1986) that allows an estimate of bloodmeal size to be calculated from the hematin concentration.

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68 Figure A 1. Flow chart of methods. Stock solution of 1 mg/ml porcine hematin in 1% LiCO3 Stock solution of 1 mg/ml porcine hematin in 1% LiCO3 Stock solution of 1 mg/ml porcine hematin in 1% LiCO3 0 26 g/ml dilutions made at 2.00 g/ml intervals 0 26 g/ml dilutions made at 2.00 g/ml intervals 0 26 g/ml dilutions made at 2.00 g/ml intervals Absorbance read at 387 nm Linear regression made

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69 Figure A 2 Standard curve of the absorbance versus concentration of hematin used to determine the amount of hematin in mosquito feces. For this regression y = 13.951x 0.3417, R2=0.9978, F=18121 with 1 and 40 degrees of freedom (n=42). 0 5 10 15 20 25 30 0 0.5 1 1.5 2Hematin Concentration ( g/ml)Absorbance at 387

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70 APPENDIX B HEMATIN STANDARD CUR VE VALIDATION Introduction To measure the si ze of a mosquito bloodm eal with minimal invasion, the amount of hematin in the feces can be measured (Briegel 1980). The absorbance value of the feces dissolved in 1% lithium carbonate can be used to construct a standardized regression equation that can be used to calculate the approximate size of a mosquito bloodmeal (Appendix A ). Briegel (1980) developed this technique to measure Aedes aegypti (L. ) bloodmeal size The method was verified by giving Ae. aegypti blood enemas containing known amounts of blood and then collect ing and measuring the excreta (Briegel 1980, 1986). Briegel (1986) reported a significan t correlation between the bloodmeal hemoglobin content and the hematin excreted. Briegels method has been used for Anopheles and Culex species without any verificati on (Hogg and Hurd 1995, 1997; McCann 2006) Hogg and Hurd (1995, 1997) examined the effects of Plasmodium spp on the reproductive fitness of Anopheles spp Plasmodium infected blood was fed to the mosquitoes to determine whether the presence of Plasmod ium in a hosts blood influenced the amount of blood taken by a mosquito vector They found that Anopheles stephensi Liston excreted significantly less hematin when mosquitoes fed on Plasmodium infected blood In Anopheles gambiae sensu lato there was no difference in the amount of hematin excreted between mosquitoes feeding on infected and uninfected hosts. To examine how varying levels of Wuchereria bancrofti affect the body size, fecundity (egg production), and blood meal size of Culex quinquefasciatus Lima et al (2003) analyzed these aspects in infected and uninfected mosquitoes They found no significant correlation s for fecundity or body size when compared to bloodmeal size. T hey observed considerable variation

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71 in blood meal size t hroughout their experiments. McCann (2006) similarly tested the effect of blood meal size on the fecundity of Culex nigripalpus Theoba ld and Cx. quinquefasciatus Using the same hematin technique as Briegel (1980) and Lima et al. (2003) he found a signifi cant positive correlation between bloo d meal size and fecundity in both species. The objective of this experiment was to determine whether Briegels (1980) method can be validated using Cx. quinquefasciatus If proven to be an effective method, it will be used throughout this research, and if not individual mosquitoes will be visually inspected to ensure they take a full bloodmeal. Objective The objective of this experiment was t o t est the hematin standard curve regression model as an estimator of blood meal size in Culex quinquefasciatus Materials and Methods Eggs were obtained from a colony of Cx. quinquefasciatus established in 1995 ( Allan et al 2006). For this work reported here, the first batch of egg is referred to as the 1st generation. The 7th generation of mosquitoes was used. Four egg rafts containing approximately 100 eggs each were placed into plastic pans measuring 36 x 45 x 6.5 cm containing 4 L of tap water. The larvae were provided 20 mg of 1:1 Brewers yeast/liver powder daily as a nutrient source. The temperature of the larval rearing room was 27 C with a light/dark period of 12:12. Once the larval stage was complete, the pupae were removed. Pupae were placed in 100 ml dishes with 45 ml of tap water and allowed to emerge in 36 x 36 x 36 cm cages. The adults were maintained on a 10% sucrose solution. Three days after emergence 50 mosquitoes were anaesthetized by chilling them on ice for 2 min. Next, they were weighed on an analytical balance (0. 0001g accuracy) (Fisher Scientific Corp.). The mosquitoes were put in separate 75 ml vials (4 cm diameter x 6 cm deep) (Figure B 1) supplied with 10%

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72 sucrose solution and allowed to recover from the anesthesia for an hour. Bovine blood (Hemostat Laboratories, Dixon, CA) was off e red as a bloodmeal for two hours. After removing the blood source mosquitoes were again anaesthetized and weighed. The mosquitoes were maintained in their individual containers for 3 days to allow bloodmeal digestion and elimination. The feces were then washed from the container with 1 ml of 1% lithium carbonate. The absorbance of the solution was read at 387 nm. The absorbance value was entered into the regression equation from Appendix A to determine the hematin weight Regression analysis was used to determine the relationship between the bloodmeal mass and the calculated hematin weight in Excel (Windows 2007). Results Initially 50 female Cx. quinquefasciatus were weig h ed, but only 29 blood-fed and defecated. The average weight of the unfe d mosquitoes was 2.19 0.27 mg and the average weight after the blood meal was 3.39 0.42 mg The average bloodmeal size was 1.2 0.45 mg while the average calculated hematin weight demonstrated between the size of t he bloodmeal and the amount of hematin expelled (Figure B 2 and Table B 1). Conclusion Female mosquitoes expel hematin in their feces. It has been suggested that the amount of hematin in the feces can be used indirectly to estimate the size of a mos quito blood meal (Briegel 1980, 1986; Hogg and Hurd 1997, Lima et al. 2003, McCann 2006). A standard curve (Appendix A ) was constructed for the present study to calculate bloodmeal size from the hematin contained in the feces. After using the regression e quation from the standard curve, it was found that there was no correlation between the calculated weight of the bloodmeal and the amount of

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73 hematin expelled (Fig. B 1). The experimental protocol was as precise and accurate as possible, but the method cou l d not be validated for Cx. quinquefasciatus Hogg and Hurd (1995, 1997), Lima et al. (2003) and McCann (2006) have used Briegels (1980) method to attempt to make correlations between hematin excretion and body size, egg production, and bloodmeal size. Hogg and Hurd (1995) tested the effect of Plasmodium yoelii oocycsts on egg production over three gonotrophic cycles. In the control groups for the first and second gonotrophic cycles there was a significant correlation between hematin excreted and egg production. In the infected groups and the third gonotrophic cycle there was not a significant correlation between hematin excreted and egg production. Hogg and Hurd (1997) determined there was no significant difference in the amount of hematin excreted between mosquitoes fed on uninfected or Plasmodium falciparum infected hosts. Lima et al. (2003) was unable to determine a significant correlation between hematin excretion and egg production in mosquitoes fed on uninfected blood, egg production in mosqui toes fed on Wuchereria bancrofti infected blood, or body size. McCann (2006) tested the effect of bloodmeal size on the fecundity and found a significant positive correlation between the two. In studies that have used Briegels (1980) standard curve method, some have been successful while others have not. It has been suggested that the amount of hematin excreted is species and strain specific (McCann 2006). Personal observations by Hillary Hurd note that the hematin excretion method is not as a reliable measure of bloodmeal size as other invasive procedures (Lima et al. 2003). The Briegel (1980) method was determined to be unsuitable for this research project. Instead, t he mosquitoes w ere reared to a uniform size blood -fed, and visually inspected to confirm that they ha d taken a full blood meal

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74 Table B -1. Results of hematin expelled versus bloodmeal weight regression analysis. Parameter Coefficients Standard Error P value Intercept 1.188556 0.129179 <0.01 Slope 0.004903 0.031398 0.877078

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75 Figure B 1. Individual mosquito holding chamber. 4 cm 6 cm Screened Ventilation Hole Sucrose Container

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76 Figure B 2. Regression of the hematin expelled versus bloodmeal weight For this regression y=0.184x + 2.816, p < 0.001, R2=0.0009, F=0.024 with 1 and 27 degrees of freedom (n=29). 0 2 4 6 8 10 12 0 0.5 1 1.5 2 2.5Hematin Mass ( g)Blood Meal Mass (mg)

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77 LIST OF REFERENCES Akoh, J. I., F. I. Aigbodion, and D. Kumbak. 1992. Studies on the effect of larval diet, adult body weight, size of blood-meal and age on the fecundity of Culex quinquefasciatus (Diptera: Culicidae). Insect Sci. Applic. 13:177181. Allan, S. A., U. R. Bernier, and D. L. Kline. 2006. Laboratory evaluation of avian odors for mosquito (Diptera: Culicidae) attraction. J. Med. Entomol. 43:225231. Anderson, J. F., A. J. Main, K. Delroux, and E. Fikrig. 2008 Extrinsic incubation periods for horizontal and vertical transmission of West Nile virus by Culex pipiens pipiens (Diptera: Culicidae). J. Med. Entomol. 45:445451. Barr, A. R. 1957. The distribution of Culex p. pipiens in North America. Am. J. Trop. Med. Hyg. 6:153165. Barr, A. R. 1967. Occurrence and distribution on the Culex pipiens complex. Bull. Wld. Hlth. Org. 37:293296. Bates, M. 1949. The natural history of mosquitoes. Macmillian Company, New York, NY. Beach, R. 1980. Physiological changes governing the onset of sexual receptivity in male mosquitoes. J. Insect Physiol. 26:245252. Becnel, J. J., J. J. Garcia, and M. A. Johnson. 1995. Edhazardia aedis (Microspora: Culicosporidae) effects on the reproductive capacity of Aedes aegypti (Diptera: Culicidae). J. Med. Entomol. 32: 549553. Begon, M. and G. A. Parker. 1986. Should egg size and clutch size decrease with age? Oikos. 47:293302. Bell, H. A., G. C. Marris, A. J. Prickett, and J. P. Edwards. 2005. Influence of host size on the clutch size and developmental success of the gregarious ectoparasitoid Eulophus pennicornis (Nees) (Hymenoptera: Braconidae) attacking larvae of the tomato moth Lacanobia oleracea (L. ) (Lepidoptera: Noctuidae). J. Exp. Biol. 208:31993209. Briegel, H. 1980. Determination of uric acid and hematin in a single sample of excreta from blood -fed insects. Experientia 36:1428. Briegel, H. 1986. Protein catabolism and nitrogen partitioning during oogenesis in the mosquito Aedes aeg ypti. J. Insect Physiol. 32:455462. Bruno, D. W. and B. R. Laurence. 1979. The influence of the apical droplet of Culex egg rafts on oviposition of Culex pipens fatigans (Diptera: Culicidae). J. Med. Entomol. 16:300305. Campbell, G. L., A. A. Marfin, R. S. Lanciotti, and D. J. Gubler. 2002. West Nile virus. Lancet. Infect. Dis. 2: 519529.

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78 Carey, J.R. 1993. Applied Biodemography for Biologists with Special Emphasis on Insects. New York: Oxford University Press. (CDC) Centers for Disease Control and P revention. 2008. Arboviral encephalitis cases reported in humans by type, United States, 19642007. CDC, Atlanta, GA. http://www.cdc.gov/ncidod/dvbid/arbor/arbocase.htm .(May 2009) (CDC) Centers for Disease Control and Prevention. 2009 West Nile virus. CDC, Atlanta, Ga. http://www.cdc.gov/ncidod/dvbid/westnile/surv&control.htm .(May 2009) Chadee, D. D., and J. S. Haeger. 1986. A description of the egg of Culex nigripalpus Theobald from Florida (USA), with notes on five egg rafts (Diptera:Culicidae). Mos q. Syst. 18:288292. Chamberlain, R. W. and W. D. Sudia. 1961. Mechanism of transmission of viruses by mosquitoes. Annu. Rev. Entomol. 6:371390. Chamberlain, R. W., W. D. Sudia, and J. D. Gillett. 1959 St. Louis encephalitis virus in mosquitoes. Am. J. Epidemiol 70:221236. Cheong, W. H. 1985. Physiological ageing of mosquito vectors of filariasis and their significance. Sem. Brugian Filariasis. 83 96. Clements, A. N. 1992. The biology of mosquitoes, vol. 1. CABI Publishing, New York, NY. Cleme nts, A. N. 1999. The biology of mosquitoes, vol. 2. CABI Publishing, New York, NY. Clements, A.N. and G.D. Paterson. 1981 The analysis of mortality and survival rates in wild populations of mosquitoes. J. Appl. Ecol. 18:373399. Clutton Brock, T. H. 1991. The evolution of parental care. Princeton University Press, Princeton, NJ. Darsie Jr., R. F. 2004 Distribution of Florida mosquitoes, pp. DISTMAP 1 9. In H. T. Evans, C. D. Morris, R. H. Baker, and W. R. Opp (eds.), Florida mosquito control handbo ok. Florida Mosquito Control Association, Gainesville, FL. Darsie Jr., R. F., and R. A. Ward. 2005 Identification and geographical distribution of the mosquitoes of North America, North of Mexico. University Press of Florida, Gainesville, FL. Day, J. F. 2001. Predicting St. Louis encephalitis virus epidemics: lessons from recent, and not so recent, outbreaks. Annu. Rev. Entomol. 46:111138. De Meillon, B., and A. Sebastian. 1967 Qualitative and quantitative characteristics of adult Culex pipiens fat igans populations according to time, site and place of capture. Bull. World Health Organ. 36:75 80.

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79 De Meillon, B., A. Sebastian, and Z. H. Khan. 1967a The duration of egg, larval and pupal stages of Culex pipiens fatigans in Rangoon, Burma. Bull. World Health Organ. 36:714. De Meillon, B., A. Sebastian, and Z. H. Khan. 1967b Time of arrival of gravid Culex pipiens quinquefasciatus at an oviposition site, the oviposition cycle and the relationship between time of feeding and time of oviposition. Bull World Health Organ. 36:39 46. De Oliveria, C. M., F. C. Filho, J. F. N. Beltran, M. H. Silva Filha, and L. Regis. 2003. Biological fitness of Culex quinquefasciatus population and its resistance to Bacillus sphaericus. J. Am. Mosq. Control Assoc. 19: 125129. Dow, R. P. 1971 The dispersal of Culex nigripalpus marked with high concentrations of radiophosphorus. J. Med. Entomol. 8:353363. Edman, J. D. 1974. Host -feeding patterns of Florida mosquitoes. III. Culex (Culex) and Culex (Neoculex). J. Med. E ntomol. 11:95104. Edman, J. D. 1979. Host -feeding patterns of Florida mosquitoes (Diptera: Culicidae) VI. Culex (Melanoconion). J. Med. Entomol. 15:521525. Elizondo Quiroga, A., A. Flores -Suarez, D. Elizondo -Quiroga, G. Ponce -Garcia, B. J. Blitvich, J. F. Contreras -Cordero, J. I. Gonzalez -Rojas, R. Mercado -Hernandez, B. J. Beaty, and I. Fernandez Salas. 2006. Gonotrophic cycle and survivorship of Culex quinquefasciatus (Diptera: Culicidae) using sticky ovitraps in Monterry, Northeastern Mexico. J. Am. Mosq. Control Assoc. 22:1014. (EPA) United States Environmental Protection Agency. 2007 Joint statement on mosquito control in the United States from the U. S. Environmental Protection Agency (EPA and the U. S. Centers for Disease Control and Prevention (CDC). (http://www.epa.gov/opp00001/health/mosquitoes/mosquitojoint.htm ).(June 2009) Evans, H. T. 2004. Pupae, pp. BPU 1 3. In C. R. Rutledge (ed.), Florida mosquito control handbook. 3rd ed. Florida Mosquito Control Association, Gainesville, FL. Fox, C. W. and M. E. Czesak. 2000. Evolutionary ecology of progeny size in arthropods. Annu. Rev. Entomol. 45:341369. Fussell, E. M. 1964 Dispersal studies on radioactive tagged Culex quinquefasciatus Say. Mosq. News. 24:422426. Gillett, J. D. 1972. The mosquito: its life, activities, and impact on human affairs. Doubleday and Company, Inc., Garden City, New York. Gerberg, E. J., D. R. Barnard, and R. A. Ward. 1970. Manual for mosquito rearing and experimental technique. AMCA B ulletin, no. 5. American M osquito Control Association.

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80 Gerberg, E. J., D. R. Barnard, and R. A. Ward. 1994 Manual for mosquito rearing and experimental technique. AMCA B ulletin, no. 5 revised. American Mosquito Control Association. Goddard, L., A. Roth, W. K. Reisen, and T. W. Scott. 2002. Vector competence of California mosquitoes for West Nile virus. Emerg. Infect. Dis. 8:13851391. Gowda, N. N., and V. A. Vijayan. 1993. Biting density, behavior and age distribution of Culex quinquefasciatus, Say in Mysore City, India. Southe ast Asian J. Trop. Med. Public Health. 24:152156. Guptavanij, P., and R. A. Barr. 1985. Failure of culicine eggs to darken in the field. J. Med. Entomol. 22:228229. Hall, M. H., S. M. Dutro, and M. J. Klowden. 199 0. Determination by new infrared refl ectance spectroscopy of mosquito (Diptera: Culicidae) blood meal size. J. Med. Entomol. 27:767 9. Harbach, R. E. and K. L. Knight. 1980. Taxonomists glossary of mosquito anatomy. Plexus Publishing, Marlton, NJ. Harvey, G. T. 1977. Mean weight and rear ing performance of successive egg clusters of eastern spruce budworm (Lepidoptera: Tortricidae). Can. Entomol. 109:487496. Harvey, G. T. 1983. Environmental and genetic effects on mean egg weight in spruce budworm (Lepidoptera: Torricidae). Can. Entomol 115:11091117. Hazzard, E. I., 1967 Modification of the ice water method for harvesting Anopheles and Culex. Mosq. News. 27:115116. Hayes, E. B., N. Komar, R. S. Nasci, S. P. Montgomery, D. R. OLeary, and G. L. Campbell. 2005. Epidemiology and t ransmission dynamics of West Nile virus disease. Emerging Infect. Dis. 11:11671173. Hogg, J. C. and H. Hurd. 1995. Malaria induced reduction of fecundity during the first gonotrophic cycle of Anopheles stephensi mosquito. Med. Vet. Entomol. 9:176180. Hogg, J. C. and H. Hurd. 1997. The effects of natural Plasmodium falciparum infection on the fecundity and mortality of Anopheles gambiae s. l. in north east Tanzania. Parasitology 114:325331. Howlett, F. M. 191 0. The influence of temperature upon bit ing mosquitoes. Parasitology. 3:479484. Hurd, H., J. C. Hogg, and M. Renshaw. 1995. Interactions between blood feeding, fecundity and infection in mosquitoes. Parasitol. Today 11:411416.

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81 Hurlbut, H. 1973. The effect of environmental temperature upon t he transmission of St. Louis encephalitis virus by Culex pipiens quinquefasciatus. J. Med. Entomol. 10:1 12. Ikeshoji, T. 1964. Fecundity of Culex pipiens fatigans Wied. fed on various amounts of blood and different h osts. World Health Organization report 133.65. Iltis, W. G. and G. Zweig. 1962. Surfactant in apical drop of egg of some culicine mosquitoes. Ann. Entomol. Soc. Am. 55:409415. Ji, X., W. G. Du, Z.H. Lin, and L. G. Luo. 2007 Measuring temporal variation in reproductive output reveals opti mal resource allocation to reproduction in the northern grass lizard, Takydromus septentrionalis. Biol. J. Linn. Soc. 91:315324. Jones, M. D. R., and S. J. Gubbins. 1979. Modification of female circadian flight -activity by a male accessory gland pheromone in the mosquito, Culex pipiens quinquefasciatus. Physiol. Entomol. 4:345351. Jones, S. C., J. Morris, G. Hill, M. Alderman, and R. C. Ratard. 2002 St. Louis encephalitis outbreak in Louisiana in 2001. J. La. State Med. Soc. 6:303306. Kerdpi b ule, V., S. Sucharit, and T. Deesin. 1981 DDT resistant Culex quinquefasciatus: the effect of DDT on the mosquitos size, fecundity, and survival. Southeast Asian J. Trop. Med. Public Health. 12:79 82. Kirkwood, T. B., and M. R. Rose. 1991 Evolution of senescence: late survival sacrificed for reproduction. Philos. Trans. R. Soc. Lond. B. Biol. Sci. 332:1524. Knight, K. L. 1978. Supplement to a catalog of the mosquitoes of the world (Diptera: Culicidae). Thomas Say Foundation. College Park, MD. Koke rnot, R. H. and C. A. Brandly. 1969. Arbovirus studies in the Ohio -Mississippi basin, 19641967. Am. J. Trop. Med. Hyg.18:743749. Lambrecht, F. L. and J. B. Fernando. 1974. Age -grading of Culex pipiens fatigans Wiedemann from different climatic zones in Ceylon. Southeast Asian J. Trop. Med. Public Health. 5:76 79. Lea, A. O., and J. D. Edman. 1972 Sexual behavior of mosquitoes. 3. Age dependence of insemination of Culex nigripalpus and Culex pipiens quinquefasciatus in nature. Ann. Entomol. Soc. Am. 65:290293. Liles, J. N. 1965. Effects of mating or association of sexes on longevity in Aedes a egypti. Mosq. News. 25:434439. Lima, C. A., R. W. Almeida, H. Hurd, and C. M. Albuquerque. 2003. Reproductive aspects of the mosquito Culex quinquefasciatus (Diptera: Culicidae) infected with Wuchereria bancrofti (Spirurida: Onchocercidae ). Mem. Inst. Os wald. Cruz. 98:217222.

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82 Linam, J. H., and L. T. Nielsen. 1962. Notes on the taxonomic separation of adult females of Culex pipiens L. and Culex quinquefasciatus Say. Mosq. News. 22:390393. Lincoln, R. J., G. A. Boxshall, and P. F. Clark. 1982. A Dicti onary of ecology, evolution, and systematic. Cambridge University Press, Cambridge, England. Lindquest, A. W., T. Ikeshoji, B. Grab, B. De Meillion, and Z. H. Khan. 1967. Dispersion studies of Culex pipiens fatigans tagged with 32p in the Kemmendine area of Rangoon, Burma. Bull. World Health Organ. 36:2137. Lowrie Jr., R. C., M. L. Eberhard, P. J. Lammie, C. F. Raccurt, S. P. Katz, and Y. T. Duverseau. 1989. Uptake and development of Wuchereria b ancrofti in Culex quinquefasciatus that fed on Hatian car ries with different microfilaria densities. Am. J. Trop. Med. Hyg. 41:429439. Lyimo, E. O. and J. C. Koella. 1992. Relationship between body size of adult Anopheles gambiae s. l. and infection with the malaria parasite Plasmodium falciparum. Parasitol ogy. 104:233237. MacDonald, G. 1952. The analysis of the sporozoite rate. Trop. Dis. Bull. 49:569586. MacDonald, W. W., A. Sebastian, and M. M. Tun. 1968 A mark release -recapture experiment with Culex pipiens fatigans in the Village of Okpo, Burma. T rop. Med. Parasitol. 62:200209. McAlpine, J. F., B. V. Peterson, G. V. Shewll, H. J. Teskey, J. R. Vockeroth, and D. M. Wood. 1981. Manual of Nearctic Diptera, vol. 1. Monograph No. 27, Research Branch, Agriculture Canada, Ottawa. McCann, S. M. 2006. Senescence and other factors affect fecundity in two species of Culex mosquitoes (Diptera: Culicidae). M. S. thesis, University of Florida, Gainesville. McCray, E. M., and H. F. Schoof. 1970 Laboratory behavior of Culex pipiens quinquefasciatus and the effects of TEMA, METEPA, and apholate upon reproduction. Mosq. News. 30:149155. McFarland, G. C. and H. I. Magy. 1962. Use of determination techniques to locate sources of Culex quinquefasciatus. pp. 8586. In Proceedings, 30th Annual Conference of the California Mosquito Control Association. San Mateo, CA. Milby, M. M., and W. K. Reisen. 1989 Estimation of vectorial capacity: vector survivorship. Bull. Soc. Vector Ecol. 14:47 54. Mitchell, C. J., and H. Briegel. 1989. Fate of the blood meal in force -fed, diapausing Culex pipiens (Diptera: Culicidae). J. Med. Entomol. 26:332341.

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83 Mogi, M. 1992. Temperature and photoperiod effects on larval and ovarian development of New Zealand strains of Culex quinquefasciatus (Diptera:Culicidae). Ann. Entomol Soc. Am. 85:5866. Moore, C. G., P. Reiter, and X. Jin -Jiang. 1986. Determination of chronological age in Culex pipiens s. l. J. Am. Mosq. Control Assoc. 2:204208. Motic China Group Co., Ltd. 2001. Quick start guide, version 3.2. Motic Headquarters, X iamen, China. Mullen, G., and L. Durden. 2002 Medical and veterinary entomology. Academic Press, San Diego, CA. Mulrennan Jr J. A. 1986. Mosquito control its impact on the growth and development of Florida. J. Fla. Med. Assoc. 73:310311. Nayar, J. K., L. Rosin, and J. W. Knight. 1986. Experimental vertical transmission of Saint Louis encephalitis virus by Florida mosquitoes. Am. J. Trop. Med. Hyg. 35:12961301. Nielsen, E. T., and A. T. Nielsen. 1962 Swarming of mosquitoes: laboratory experiments under controlled conditions. Entomol. Exp. Applic. 5:14 32. Nijhout, H. F. 1977. Control of antennal hair erection in male mosquitoes. Biol. Bull. 153:591603. Oda, T., Y. Eshita, K. Uchida, K. Kurokawa, Y. Ogawa, K. Kato, and H. Tahara. 2002. Reproduct ive activity and survival of Culex pipiens pallens and Culex quinquefasciatus (Diptera: Culicidae) in Japan at high temperatures. J. Med. Entomol. 39:185 190. Oda, T., A. Mori, and K. Kurokawa. 1980. Effects of temperatures on the oviposition and hatchin g of eggs on Culex pipiens molestus and Culex pipiens quinquefasciatus. Trop. Med. 22: 167172. Pant, C. P. 1979. Vectors of Japanese encephalitis and their bionomics. Bull. World Health Organ. 79:118. Pletcher, S. D. 1999a Model fitting and hypothesis testing for age -specific mortality data. J.Evol.Biol. 12:430439. Pletcher, S. D. 1999b WinModest 1.0. Demographic Analysis Tool. Rostock Germany: Max Planck Institute for Demographic Research. Price, P. 1997 Insect ecology, 3rd ed. Wiley, New York. Rajagopalan, P. K., M. Yasuno, and G.C. Labrecque. 1973. Dispersal and survival in the field of chemosterilized, irradiated, and cytoplasmically incompatible male Culex pipiens fatigans. Bull. World Health Organ. 48:631635.

PAGE 84

84 Rajkumar, S., and A. Jebanesan. 2004 Ovicidal activity of Solanum trilobatum L. (Solanaceae) leaf extract against Culex quinquefasciatus Say and Culex tritaeniorhynchus Giles (Diptera: Culicidae). Int. J. Trop. Insect Sci. 24: 340 342. Reis en, W. K., Y. Aslam, and T. F. Siddiqui. 1977 Observations on the swarming and mating of some Pakistan mosquitoes in nature. Ann. Entomol. Soc. Am. 70: 988985. Reisen, W. K., and P. R. L. Boreham. 1979. Host selection patterns in Pakistan mosquitoes. A m. J. Trop. Med. Hyg. 28:408421. Reisen, W. K., M. M. Miliby, R. P. Meyer, A. R. Pfuntner, F. Spoehel, J. E. Hazelrigg, and J. P. Webb Jr. 1991. Mark release -recapture studies with Culex mosquitoes (Diptera: Culicidae) in southern California. J. Med. Entomol. 28:35771. Reisen, W. K., Y. Yang, and V. W. Martinez. 2005 Avian host and mosquito (Diptera:Culicidae) vector competence determine the efficiency of West Nile and St. Louis encephalitis virus transmission. J. Med. Entomol. 42:367375. Reisen, W K. and A. C. Brault. 2007. West Nile virus in North America: perspective on epidemiology and intervention. Pest Manag. Sci. 63: 641646. Richards, S. L., C. C. Lord, K. A. Pesko, and W. J. Tabachnick. 2009 Environmental and biological factors influenc e Culex pipiens quinquefasciatus Say (Diptera:Culicidae) vector competence for Saint Louis encephalitis virus. Amer. J. Trop. Med. Hyg. (in press). Roff, D. A. 1992. The evolution of life histories: theory and analysis. Chapman and Hall, New York, NY. Roit berg, B. D., and I. Gordon. 2005. Does the Anopheles blood meal fecundity curve, curve? J. Vector Ecol. 30:8386. Rowland, M. 1989. Changes in circadian flight activity of the mosquito Anopheles stephensi associated with insemination, bloodfeeding, ovi position, and nocturnal light intensity. Physiol. Entomol. 14: 7784. Rutledge, C. R., J. F. Day, C. C. Lord, L. M. Stark, and W. J. Tabachnick. 2003 West Nile virus infection rates in Culex nigripalpus (Diptera: Culicidae) do not reflect transmission rat es in Florida. J. Med. Entomol. 40:253258. Rutledge, C. R., and H. T. Evans. 2004. Larval biology, pp. BLA 1 7. In C. R. Rutledge (ed.), Florida Mosquito Control Handbook. 3rd ed Florida Mosquito Control Association, Gainesville, FL. Samarawickrema, W. A. 1967. A study of the age -composition of natural populations of Culex pipiens fatigans Wiedemann in relation to the transmission of filariasis due to Wuchereria bancrofti (Cobbold) in Ceylon. Bull. World Health Organ. 37:117 137.

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85 Sardelis, M. R., M. J. Turell, D.J. Dohm, and M. L. OGuinn. 2001. Vector competence of selected North American Culex and Coquillettidia mosquitoes for West Nile virus. Emer. Infec. Disea. 6:10181022. Savage, H., M. Anderson, E. Gordon, L. McMillen, L. Colton, D. Charnetzky M. Delorey, S. Aspen, K. Burkhalter, B. J. Biggerstaff, and M. Godsey. 2006. Oviposition activity patterns and West Nile virus infection rates for members of the Culex pipiens complex at different habitat types within the hybrid zone, Shelby County, TN, 2002. (Diptera: Culicidae). J. Med. Entomol. 43:12271238. Schlein, J. and N. G. Gratz. 1972 Age determination of some flies and mosquitoes by daily growth layers of skeletal apodemes. Bull. World Health Organ. 47:7176. Schreiber, E. T., M. S. Mulla, J. D. Chaney, and M. S. Dhillon. 1988. Dispersal of Culex quinquefasciatus from a dairy in southern California. J. Am. Mosq. Control Assoc. 4:300304. Shelton, R. H. 1973. The effect of temperatures on development of eight mosquito species. J. Am. Mosq. Control Assoc. 33: 112. Shriver, D., and W. E. Bickley. 1964 The effect of temperature on hatching of eggs of the mosquito, Culex pipiens quinquefasciatus. Mosq. News. 24: 137140. Singh, K. R. P., R. S. Patterson, G. C. LaBrecque, and R. K. Razdan. 197 5 Mass rearing of Culex fatigans. Bull. World Health Organ. 72:386. Sirivanakarn, S. and G. B. White. 1978 Neotype designation of Culex quinquefasciatus Say (Diptera: Culicidae). Proc. Entomol Soc. Wash. 80:360372. Smith, C. C. and S. D. Fretwell. 1974. The optimal balance between size and number of offspring. Am. Nat. 108:499506. Smith, J. L., and D. M. Fonseca. 2004. Rapid assays for identification of members of Culex (Culex) pipiens complex, their hybrids, and other sibling species (Diptera: Culicidae). Am. J. Trop. Med. Hyg. 70:339345. Spielman, A., and M. DAntonio. 2001. Mosquito: a natural history of our most persistent and deadly foe. Hyperion, New York, NY. Strickman, D. 1988. Rate of oviposition by Culex quinquefasciatus in San Anto nio, Texas, during three years. J. Am. Mosq. Control Assoc. 3:339344. Styer, L.M., J.R. Carey, J.L. Wang, and T.W. Scott. 2007. Mosquitoes do senesce: Departure from the paradigm of constant mortality. Am. J. Trop. Med. Hyg. 76:111117. Sudia, W. D. 1959 The multiplication of St. Louis encephalitis virus in two species of mosquitoes: Culex quinquefasciatus Say and Culex pipiens Linnaeus. Am. J. Hyg. 70:237245.

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86 Subra, R. 1981. Biology and control of Culex pipiens quinquefasciatus Say, 1823 (Diptera, Culcidae) with special reference to Africa. Insect Sci. Application. 1:319 338. (USDA) United States Department of Agriculture. 2005 Techniques for mass rearing Culex quinquefas ciatus. Gainesville, FL. Van Handel, E. 1991. Postvitellogenic metabolism of the mosquito (Culex quinquefasciatus) ovary. J. Insect Physiol. 38:75 79. Van Handel, E., and J. F. Day. 1989. Correlation between wing length and protein content of mosquitoes. J. Am. Mosq. Control Assoc. 5:180182. Varma, M. G. R. and G. B. White. 1989. Mosquito borne virus diseases in geographical distribution of arthropod borne diseases and their principal vectors. Bull. World Health Organ. 89:3554. Vaupel, J.W., J.R. Carey, K. Christensen, T.E. Johnson, A.I. Yashin, N.V. Holm, I.A. Iachine, V. Kannisto, A.A. Khazaeli, P. Liedo, V.D. Longo, Y. Zeng, K.G. Manton, and J.W. Curtsinger. 1998. Biodemographic tr ajectories of longevity. Science. 280:855 860. Vinogradova, E. B. 2000. The Culex pipiens complex, pp. 4 45. In E. B. Vinagradova (ed.),Culex pipiens pipiens mosquitoes: taxonomy, distribution, ecology, physiology, genetics, applied importance and control Pensoft Publishers, Sofia. Wellington, W. G. 1965. Some maternal influences on progeny quality in the western tent caterpillar, Malacosoma pluvial (Dyar). Can. Entomol. 97:1 14. Wen, Y., L. E. Muir, and B. H. Kay. 1997 Response of Culex quinquefascia tus to visual stimuli. J. Am. Mosq. Control Assoc. 13:150152. Williams, G. C. 1957. Pleiotrophy, natural selection, and the evolution of senescence. Evolution. 11: 398411. Windows. 2007. Microsoft Excel. Windows Corporation. R e dmond, WA. Wirtz, R. A., and L. C. Rutledge. 1980. Reconstituted collagen sausage casing for the blood feedin g of mosquitoes. Mosq. Ne ws. 40:287:288. Yasuno, M., P., K. Rajagopalan, S. Russell, and G. C. La Brecque. 1973 Influence of seasonal changes in climate on dispersal o f released Culex pipiens fatigans. Bull. World Health Organ. 48:317321. Yasuno, M., P. K. Rajagopalan, and G. C. La Brecque. 1975. Migration patterns of Culex fatigans around Delhi, India. J. Trop. Med. 17:9196.

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87 BIOGRAPHICAL SKETCH Stephani e Larrick was born an d raised in Salesville, Ohio. She attended Buckeye Trail High School, where she graduated in 2003. Stephanie attended Hiram College until 2007, when she received her Bachelor of Arts degree in biology. She competed in track and fiel d throughout high school and college. Stephanie moved to Flor ida in May 2007 to pursue a Mast er of Science degree in entomology.