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Comparitive Behavioral Analysis of Oviposition Behavior in Aedes and Culex Mosquitoes and the Impact of Pathogen Infecti...

Permanent Link: http://ufdc.ufl.edu/UFE0024842/00001

Material Information

Title: Comparitive Behavioral Analysis of Oviposition Behavior in Aedes and Culex Mosquitoes and the Impact of Pathogen Infection on Oviposition Behavior
Physical Description: 1 online resource (142 p.)
Language: english
Creator: Zettel Nalen, Catherine
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: aedes, culex, cuninpv, edhazardia, oviposition, vavraia
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: COMPARITIVE BEHAVIORAL ANALYSIS OF OVIPOSITION BEHAVIOR IN AEDES AND CULEX MOSQUITOES AND THE IMPACT OF PATHOGEN INFECTION ON OVIPOSITION BEHAVIOR By Catherine M. Zettel Nalen August 2009 Chair: Name Sandra A. Allan Major: Entomology and Nematology The pathogen transmission cycle relies upon at least one oviposition event for pathogen transmission. A series of high resolution recordings allowed for a detailed analysis and comparison of oviposition behaviors in four species of mosquitoes, and a dual choice oviposition bioassay analyzed the effect of pathogen-infected larvae on oviposition substrate selection in gravid females. Aedes albopictus (Skuse) and Ae. aegypti (Linnaeus) are major vectors of dengue fever, chikungunya virus, and Eastern equine encephalitis. Culex quinquefasciatus Say and Culex tarsalis Coquillett are both capable vetors of West Nile virus, and several other viruses in the Japanese encephalitis complex. Details of the similarities and differences in oviposition behavior of these species are largely unknown. Digital video recording and behavioral software were used to compare oviposition behavior between Ae. albopictus and Ae. aegypti. Average number of behaviors and durations of behaviors were compared, transition matrices constructed, and time budgets created for each species. Major differences were observed in the mean number of eggs laid by Ae. aegypti (10.2 eggs) and Ae. albopictus (5.5 eggs), and the amount of time Ae. albopictus spends performing tarsal activities. Ae. albopictus also performed all tarsal waving and grooming behaviors more often than Ae. aegypti. The same behavioral experimental design was used to compare oviposition behavior of Cx. quinquefasciatus and Cx. tarsalis. These was no significant difference in the number of eggs laid, however Cx. quinquefasciatus was observed drinking significantly more often and significantly longer than Cx. tarsalis. Otherwise the two species were very similar in oviposition behavior. Dual-choice oviposition bioassays were conducted using three mosquito species and three associated pathogens: Ae. aegypti and Edhazardia aedis Kudo, Ae. albopictus and Vavraia culicis floridensis (Weiser), and Cx. quinquefasciatus and CuniNPV. Gravid females were given a choice between an oviposition cup containing water and 10 healthy larvae versus water and 10 pathogen-infected larvae. Aedes aegypti oviposited significantly more eggs in the oviposition cup containing 10 healthy larvae. Edhazardia aedis-infected adults also oviposited significantly more often in cups containing 10 healthy larvae. Both Ae. albopictus and Cx. quinquefasciatus did not exhibit any significant preference for either oviposition cup. A detailed oviposition behavior analysis was conducted for four species. Aedes aegypti and Ae. albopictus were quite different in their oviposition behavior, but Cx. quinquefasciatus and Cx. tarsalis were very similar. Both healthy and E. aedis-infected adult Ae. aegypti laid significantly more eggs in substrate containing healthy larvae as opposed to substrate containing uninfected larvae, however Ae. albopictus and Cx. quinquefasciatus showed no preference. By understanding the behavioral steps in oviposition, new strategies for intervention can be developed to break the pathogen transmission cycle and better understand the behavioral changes that come with mosquito parasite or pathogen infection.
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 Catherine Zettel Nalen.
Thesis: Thesis (M.S.)--University of Florida, 2009.
Local: Adviser: Allan, Sandra A.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2011-08-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0024842:00001

Permanent Link: http://ufdc.ufl.edu/UFE0024842/00001

Material Information

Title: Comparitive Behavioral Analysis of Oviposition Behavior in Aedes and Culex Mosquitoes and the Impact of Pathogen Infection on Oviposition Behavior
Physical Description: 1 online resource (142 p.)
Language: english
Creator: Zettel Nalen, Catherine
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: aedes, culex, cuninpv, edhazardia, oviposition, vavraia
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: COMPARITIVE BEHAVIORAL ANALYSIS OF OVIPOSITION BEHAVIOR IN AEDES AND CULEX MOSQUITOES AND THE IMPACT OF PATHOGEN INFECTION ON OVIPOSITION BEHAVIOR By Catherine M. Zettel Nalen August 2009 Chair: Name Sandra A. Allan Major: Entomology and Nematology The pathogen transmission cycle relies upon at least one oviposition event for pathogen transmission. A series of high resolution recordings allowed for a detailed analysis and comparison of oviposition behaviors in four species of mosquitoes, and a dual choice oviposition bioassay analyzed the effect of pathogen-infected larvae on oviposition substrate selection in gravid females. Aedes albopictus (Skuse) and Ae. aegypti (Linnaeus) are major vectors of dengue fever, chikungunya virus, and Eastern equine encephalitis. Culex quinquefasciatus Say and Culex tarsalis Coquillett are both capable vetors of West Nile virus, and several other viruses in the Japanese encephalitis complex. Details of the similarities and differences in oviposition behavior of these species are largely unknown. Digital video recording and behavioral software were used to compare oviposition behavior between Ae. albopictus and Ae. aegypti. Average number of behaviors and durations of behaviors were compared, transition matrices constructed, and time budgets created for each species. Major differences were observed in the mean number of eggs laid by Ae. aegypti (10.2 eggs) and Ae. albopictus (5.5 eggs), and the amount of time Ae. albopictus spends performing tarsal activities. Ae. albopictus also performed all tarsal waving and grooming behaviors more often than Ae. aegypti. The same behavioral experimental design was used to compare oviposition behavior of Cx. quinquefasciatus and Cx. tarsalis. These was no significant difference in the number of eggs laid, however Cx. quinquefasciatus was observed drinking significantly more often and significantly longer than Cx. tarsalis. Otherwise the two species were very similar in oviposition behavior. Dual-choice oviposition bioassays were conducted using three mosquito species and three associated pathogens: Ae. aegypti and Edhazardia aedis Kudo, Ae. albopictus and Vavraia culicis floridensis (Weiser), and Cx. quinquefasciatus and CuniNPV. Gravid females were given a choice between an oviposition cup containing water and 10 healthy larvae versus water and 10 pathogen-infected larvae. Aedes aegypti oviposited significantly more eggs in the oviposition cup containing 10 healthy larvae. Edhazardia aedis-infected adults also oviposited significantly more often in cups containing 10 healthy larvae. Both Ae. albopictus and Cx. quinquefasciatus did not exhibit any significant preference for either oviposition cup. A detailed oviposition behavior analysis was conducted for four species. Aedes aegypti and Ae. albopictus were quite different in their oviposition behavior, but Cx. quinquefasciatus and Cx. tarsalis were very similar. Both healthy and E. aedis-infected adult Ae. aegypti laid significantly more eggs in substrate containing healthy larvae as opposed to substrate containing uninfected larvae, however Ae. albopictus and Cx. quinquefasciatus showed no preference. By understanding the behavioral steps in oviposition, new strategies for intervention can be developed to break the pathogen transmission cycle and better understand the behavioral changes that come with mosquito parasite or pathogen infection.
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 Catherine Zettel Nalen.
Thesis: Thesis (M.S.)--University of Florida, 2009.
Local: Adviser: Allan, Sandra A.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2011-08-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0024842:00001


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1 COMPARITIVE BEHAVIORAL ANAL YSIS OF OVIPOSITION BEHAVIOR IN AEDES AND CULEX MOSQUITOES AND THE IMPACT OF PATHOGEN INFECTION ON OVIPOSITION BEHAVIOR By CATHERINE M. ZETTEL NALEN 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 Catherine M. Zettel Nalen

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3 To my parents and my husband

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4 ACKNOWLEDGMENTS I would like to thank my parents and my husband for their continuing support and encouragement. I also need to thank Dr. Sandy Allan for her guidance, as well as Dr. Phil Kaufman and Dr. Jimmy Becnel for their input Thanks go out to Erin Vrzal, Julie McClurg, and Neil Sanscrainte for their technical support.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ...............................................................................................................4 LIST OF TABLES ...........................................................................................................................7 LIST OF FIGURES .........................................................................................................................9 CHAPTER 1 LITERATURE REVIEW .......................................................................................................13 Medical and Veterinary Importance of Aedes and Culex Mosquitoes ...................................13 Current Vector Status .............................................................................................................15 Aedes aegypti Life Hi story and Biology .........................................................................16 Aedes albopictus Life History and Biology .....................................................................16 Culex quinquefasciatus Biology and Ecology .................................................................17 Culex tarsalis Biology and Ecology ................................................................................17 Role of Chemoreception in Behavior .....................................................................................18 Chemoreception and Oviposition Behavior ....................................................................19 Behavioral Differences Between Aedes and Culex Oviposition Strategies .....................23 Review of Cx. molestus Cx. restuans and Cx. pipens Oviposition Behavior .................24 Review of Ae. aegypti Oviposition Behavior ..................................................................24 Importance of Biological Control ...........................................................................................25 Edhazardia aedis Kudo ...................................................................................................26 Vavraia culicis (Weiser) ..................................................................................................27 Culex nigripalpus Nucleopolyhedrovirus (CuniNPV) ....................................................28 Objectives ............................................................................................................................30 2 A COMPARITIVE ANALYS IS OF OVIPOSITION BEHAVIOR OF AEDES AEGYPTI (LINNAEUS) AND AEDES ALBOPICTUS (SKUSE) .........................................31 Introduction .............................................................................................................................31 Materials and Methods ...........................................................................................................34 Insect Colonies ................................................................................................................34 Behavioral Experimental Design .....................................................................................35 Behavioral Analysis .........................................................................................................36 Data Analysis ................................................................................................................... 38 Results .....................................................................................................................................38 Discussion ...............................................................................................................................42 3 A COMPARATIVE ANALYSIS OF OVIPOSITION BEHAVIOR BETWEEN CULEX PIPIENS QUINQUEFASCIATUS SAY AND CULEX TARSALIS COQUILLETT ..............65 Introduction .............................................................................................................................65 Materials and Methods ...........................................................................................................68 Mosquito Colonies ...........................................................................................................68 Behavioral Experimental Design .....................................................................................69

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6 Video Analysis ................................................................................................................70 Data Analysis ...................................................................................................................71 Results .....................................................................................................................................72 D iscussion ...............................................................................................................................74 4 EFFECT OF INSECT PATHOGENS ON OVIPOSITION SUBSTRATE SELECTION FOR THREE MEDICALLY IMPORTANT MOSQUITO SPECIES .................................101 Introduction ...........................................................................................................................101 Materials and Methods .........................................................................................................105 Insect Colonies ..............................................................................................................105 Aedes aegypti ..........................................................................................................105 Aedes aegypti infected with Edhazardia aedis .......................................................106 Aedes albopictus .....................................................................................................107 Aedes albopictus infected with Vavraia culicis floridensis ...................................107 Culex quinquefasciatus infected with CuniNPV : ...................................................109 Dual Choice Oviposition Bioassay Methods ................................................................109 U ninfected Aedes aegypti adults ............................................................................109 Infected Aedes aegypti adults .................................................................................110 Uninfected Aedes albopictus adults .......................................................................111 Uninfected Culex quinquefasciatus adults .............................................................111 Data Analysis .................................................................................................................111 Results ...................................................................................................................................112 Aedes aegypti ..........................................................................................................112 Aedes albopictus .....................................................................................................112 Culex quinquefasciatus ..........................................................................................113 Discussion .............................................................................................................................113 5 FUTURE RESEARCH .........................................................................................................127 APPENDIX A AEDES CODING SCHEME AS ENT ERED INTO THE OBSERV ER XT .......................129 B CULEX CODING SCHEME AS ENT ERED INTO THE OBSERV ER XT ....................... 130 LIST OF REFERENCES .............................................................................................................131 BIOGRAPHICAL SKETCH .......................................................................................................142

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7 LIST OF TABLES Table page 21 Aedes ae gypti ethogram. ....................................................................................................47 22 Aedes albopictus ethogram. ...............................................................................................48 23 Average number of times behaviors were observed by ovipositing Aedes aegypti and Aedes albopictus females. ..................................................................................................49 24 Average duration of behaviors exhibited by ovipositing Aedes aegypti and Aedes albopictus females. ............................................................................................................50 25 Transition matrix for location of Aedes aegypti during oviposition. .................................51 26 Transition matrix for location of Aedes albopictus during oviposition. ............................51 27 Transition matrix for movement of Aedes aegypti during oviposition. .............................51 28 Transition matrix for movement of Aedes albopictus during ovipos ition. ........................52 29 Transition matrix for tarsal activity of Aedes aegypti during oviposition. ........................52 210 Transition matrix for tarsal activity of Ae. albopictus during oviposition. ........................53 211 Transition matrix for oviposition activity of Aedes aegypti during oviposition. ...............53 212 Transition matrix for oviposition activity of Aedes albopictus during oviposition. ..........54 31 Culex quinquefasciatus ethogram. .....................................................................................79 32 Culex tarsalis ethogram. ....................................................................................................80 33 Comparison of the occurrences of oviposition behaviors between Culex quinquefasciatus and Culex tarsalis .................................................................................81 34 Comparison of duration of oviposition behaviors between female Culex quinquefasciatus and Culex tarsalis .................................................................................82 35 Transition matrix for location of Cule x quinquefasciatus during oviposition. ..................83 36 Transition matrix for location of Culex tarsalis during oviposition. .................................83 37 Tran sition matrix for movement of Culex quinquefasciatus during oviposition. ..............84 38 Transition matrix for movement of Culex tarsalis during oviposition. .............................84 39 Transition matrix for tarsal activity of Culex quinquefasciatus during oviposition. .........85

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8 310 Transition matrix for tarsal activity of Culex tarsalis during ovipos ition. ........................86 311 Transition matrix for oviposition activity of Culex quinquefasciatus during oviposition. .........................................................................................................................87 312 Transition matrix for oviposition activity of Culex tarsalis during oviposition. ...............87 313 Transition matrix for drinking activity of Culex quinquefasciatus during oviposition. ....88 314 Transition matrix for drinking activity in Culex tarsalis during oviposition. ...................88

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9 LIST OF FIGURES Figure page 21 Behavioral setup used for video capture of oviposit ion behaviors of mosquitoes. ...........55 22 A kinematic diagram for location of ovipositing Aedes aegypti. .......................................56 23 A kinematic diagram for movement of ovipositing Aedes albopictus ..............................56 24 A kinematic diagram for movement of ovipositing Aedes aegypti ....................................57 25 A kinematic diagram for movement of ovipositing Aedes albopictus ..............................57 26 A kinematic diagram for tarsal activity of ovipositing Aedes aegypti ..............................58 27 A kinematic diagram of tarsal activity of ovipositing Aedes albopictus. ..........................58 28 A kinematic diagram of oviposi tion activity of Aedes aegypti .........................................59 29 A kinematic diagram of oviposition activity of Aedes albopictus. ....................................59 210 Time budgets f or location of Aedes aegypti and Aedes albopictus during oviposition.. ...60 211 Time budgets for movement of Aedes aegypti and Aedes albopictus during oviposition. .........................................................................................................................61 212 Time budgets for tarsal activity of Aedes aegypti and Aedes albopictus during oviposition. .........................................................................................................................62 213 Time budgets for oviposition activity of Aedes aegypti and Aedes albopictus during oviposition. .........................................................................................................................63 214 A representative visual analysis of Aedes aegypti and Ae. albopictus showing overlappi ng behaviors during oviposition. ........................................................................64 31 Behavioral setup used for video capture of oviposition behaviors of mosquitoes. ...........89 32 A kinematic diagram of location of Culex quinque fasciatus during oviposition. .............90 33 A kinematic diagram of location for Culex tarsalis during oviposition. ...........................90 34 A kinematic diagram of movement for Culex quinquefasciatus during oviposition. ........91 35 A kinematic diagram of movement for Culex tarsalis during oviposition. .......................91 37 A kinematic diagram of tarsal activity for Culex tarsalis during oviposition. ..................93

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10 38 A kinematic diagram of oviposition activity for Culex quinquefasciatus during oviposition. .........................................................................................................................93 39 A kinematic diagram of oviposition activity for Culex tarsalis during oviposition. .........94 310 A kinematic diag ram of drinking activity for Culex quinquefasciatus during oviposition. .........................................................................................................................94 311 A kinematic diagram of drinking activity for Culex tarsalis during oviposition. .............95 312 Time budgets for location of ovipositing Culex quinquefasciatus and Culex tarsalis females. ..............................................................................................................................96 313 Time budgets for movement of ovipositing Culex qui nquefasciatus and Culex tarsalis females. .................................................................................................................97 314 Time budgets for tarsal activity of ovipositing Culex quinquefasciatus and Culex tarsalis females. .................................................................................................................98 315 Time budgets for oviposition activity of ovipositing Culex quinquefasciatus and Culex tarsalis females. .......................................................................................................99 316 Visual representation of oviposition behaviora l sequences of Culex quinquefasciatus and Culex tarsalis ...........................................................................................................100 41 Oviposition responses of uninfected gravid Aedes aegypti in dual choice oviposition assays ...............................................................................................................................123 42 Oviposition responses of Edhazardia aedis infected gravid Aedes aegypti in dual choice oviposition assays. ................................................................................................124 43 Oviposition responses of uninfected gr avid Aedes albopictus in dual choice oviposition assays ............................................................................................................125 44 Oviposition responses of uninfected gravid Culex quinquefasciatus in dual choice oviposition assays ............................................................................................................126

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11 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 COMPARITIVE BEHAVIORAL ANAL YSIS OF OVIPOSITION BEHAVIOR IN AEDES AND CULEX MOSQUIT OES AND THE IMPACT OF PATHOGEN INFECTION ON OVIPOSITION BEHAVIOR By Catherine M. Zettel Nalen August 2009 Chair: Name Sandra A. Allan Major: Entomology and Nematology The pathogen transmission cycle relies upon at least one oviposition event for patho gen transmission. A series of high resolution recordings allowed for a detailed analysis and comparison of oviposition behaviors in four species of mosquitoes, and a dua l choice oviposition bioassay analyzed the effect of pathogen infected larvae on ovipos ition substrate selection in gravid females. Aedes albopictus (Skuse) and Ae. aegypti (Linnaeus) are major vectors of dengue fever, chikungunya virus, and Eastern equine e n cephalitis. Culex quinquefasciatus Say and Culex tarsalis Coquillett are both capabl e vetors of West Nile virus, and several other viruses in the Japanese encephalitis complex. Details of the similarities and differences in oviposition behavior of these species are largely unknown. Digital video recording and behavioral software were used to compare o viposition behavior between Ae. albopictus and Ae. aegypti Average number of behaviors and durations of behaviors were compared, transition matrices constructed, and time budgets created for each species. Major differences were observed in th e mean number of eggs laid by Ae. aegypti (10.2 eggs) and Ae. albopictus (5.5 eggs), and the amount of time Ae. albopictus spends performing

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12 tarsal activities. Ae. albopictus also performed all tarsal waving and grooming behaviors more often than Ae. aegyp ti. The same behavioral experimen tal design was used to compare oviposition behavior of Cx. quinquefasciatus and Cx. tarsalis These was no significant difference in the number of eggs laid, however Cx. quinquefasciatus was observed drinking significantly more often and significantly longer than Cx. tarsalis Otherwise the tw o species were very similar in o viposition behavior. Dual choice o viposition bioassays were conducted using three mosquito species and three associated pathogens: Ae. aegypti and Edhazardia aedis Kudo, Ae. albopictus and Vavraia culicis floridensis ( Weiser) and Cx. quinquefasciatus and CuniNPV. Gravid females were given a choice between an o viposition cup containing water and 10 healthy larvae versus water and 10 pathogeninfected lar vae. Aedes aegypti oviposited significantly more eggs in the o viposition cup containing 10 healthy larvae. Edhazardia aedis infected adults also oviposited significantly more often in cups containing 10 healthy larvae. Both Ae. albopictus and Cx. quinquefa sciatus did not exhibit any sig nificant preference for either o viposition cup. A detailed o viposition behavior analysis was conducted for four species. Aedes aegypti and Ae. albopictus were quite different in their oviposition behavior, but Cx. quinquefas ciatus and Cx. tarsalis were very similar. Both healthy and E. aedis infected adult Ae. aegypti laid significantly more eggs in substrate containing healthy larvae as opposed to substrate containing uninfected larvae, however Ae. albopictus and Cx. quinque fasciatus showed no preference. By understanding the behavioral steps in oviposition, new strategies for intervention can be developed to break the pathogen transmission cycle and better understand the behavioral changes that come with mosquito parasite or pathogen infection.

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13 CHAPTER 1 LITERATURE REVIEW Medical and Veterinary Importance of Aedes and Culex Mosquitoes Mosquitoes are major disease vectors present on every continent except Antarctica. Mosquitoes have long been associated with disease in both people and animals, but it was not until the late 1800s that mosquitoes were discovered to be intermediate hosts of vertebrate parasites (Foster and Walker 2002). Since then, mosquitoes have been labeled as the greatest arthropod threat to human health. M any debilitating pathogens are vectore d by mosquitoes that cause illness in humans such as dengue fever (DEN), encephalitis, filariasis, yellow fever, chikungunya virus (CHIK V) and malaria (Foster and Walker 2002) Malaria is one of the most widespread and abundant diseases causing over one million deaths per year, with 300 500 million cases occurring annually ( Foster and Walker 2002, C enters for Disease Control 2009) With treatments costly or unavailable mosquito control is the primary source of pre vention for most human populations (Foster and Walker 2002). Mosquitoes carrying pathogens are most abundant in tropical, developing regions, but are not absent from temperate and industrialized areas. Many species are adapting and thriving in urban settin gs, living in close proximity to humans (Foster and Walker 2002) In the United States, several mosquitoborne illnesses that are considere d notifiable diseases appear each year, including Eastern equine encephalitis (EEE), Western equine encephalitis (WEE ), St. Louis encephalitis (SLE) (Calisher 1994) and West Nile virus encephalitis (WNV) (Blackmore et al. 2003) Future threats include DEN (Gubler 1998) and CHIK V (Reiskind et al. 2008) Members of the family Culicidae, specifically Culex spp. and Aedes spp., are major disease vectors in the United States and specifica lly Florida (Nayar et al. 2001) In the past, Florida has experienced outbreaks of yellow feve r, malaria, DEN, filariasis and encephalitis (Nayar et al. 2001) and more recently WNV (Blac kmore et al. 2003)

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14 Because of the close proximity of Florida to the Caribbean where many of these illnesses still persist, there is potential for reintroduction into Florida. Today, SLE, EEE, (Nayar et al. 2001), and WNV are present in the state of Florida (Girard et al. 2007) and a low number of isolated dengue cases are reported each year (Gill et al. 2000). Besides vectoring pathogens mosquitoes inflict irriating bites a nd are considered a pest around the world. Severe mosquito emergences can negativ ely impact tourism, recreation, and even affect livestock production (Foster and Walker 2002). Because most mosquitoborne illnesses are prevalent in warmer temperatures, the American Meteorological Society is now considering mosquitoborne illnesses as re presentative of global warming patterns, using both location of disease reports, and the time frames in whi ch the diseases occur as metrics (Epstein et al. 1998). Florida is home to 81 mosquito species (Day 2005). Primary vector s pecies of importance to Fl ori da include the invasive Aedes albopictus (Skuse), Aedes aegypti (Linnaeus), Culex pipi ens quinquefasciatus Say and a secondary vector Culex tarsalis Coquillett (Darsie and Ward 2005). Because the general pattern of pathogen transmission requires an initial bloodmeal for pathogen aquisition pathogen transmission cycles are greatly dependent on at least one oviposition cycle (Weaver and Barrett 2004) The second bloodmeal is essential for transmission of the pathogen, but often a second bloodmeal will not occur before the first oviposition cyc le is complete (Bentley and Day 1989). Understanding the behavioral steps involved in mosquito oviposition provides a critical point in the life cycle where population increase can be reduced through biocontrol agents, toxicants, or attract and kill approaches. Additionally, it provides a weak link in the mosquito life cycle where the mosquito borne pathogen transmission cycle can be broken through intervention.

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15 Current Vector S tatus There are numerous species of Ae des found in the United States, several of which are present in Florida (Darsie and Ward 2005). Two of these, Ae. aegypti and Ae. albopictus are treehole or container inhabiting mosquitoes that opportunistically oviposit in manmade containers such as rain barrels, flower pots, and buckets ( Hawley 1988, Clements 1999) This behavior results in close association with people around both urban and rural developments which increases the risk of exposure to a mosquitoborne illness Many Aedes species are suc cessful vectors and are known to transmit diseases such as yellow fever, DEN and dengue hemorrhagic fever (DHF), CHIK V and LaCrosse encephalitis (Foster and Walker 2002) Today, DEN is considered a constant threat to the Americas with cases annually repo rted in the continental United States, and can be transmitted by both Ae. aegypti and Ae. albopictus (Rai 1991, Gill et al. 2000). Most cases reported in Florida are considered airport cases from people who have traveled to countries where DEN is present Bringing it into the United States from travelling abroad risks triggering an epidemic in Florida and the Southeast ern US (Gill et al. 2000). Aedes albopictus has also been implicated as a minor EEE vector in Florida (Mitchell et al. 1992). Culex tarsali s is a major disease vect or in the Western United States, and vectors viruses such as WNV, SLE, Japanese equine encephalitis (JEE), Venezuelan equine encephalitis (VEE), Llano Seco virus, Turlock virus, Gay Lodge virus, Hart Park virus ( Reisen 1993, Reisen et al. 1993), and potentially Rift Valley Fever (Gargan et al. 1988). Rift Valley f ever is primarily a disease of African ruminants, but also infects humans, and, if introduced to the United States could be detrimental to livestock populations ( Gargan et al. 1988). Culex quinquefasciatus is the primary vector of WNV in the Southeast (Godsey et al. 2005) and a secondary vector of SLE (Meyer et al. 1983).

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16 Aedes aegypti Life History and B iology Aedes aegypti has been in the Western hemisphere for centuries (N elson 1986, Barrett and Higgs 2007) Targeted by the yellow fever eradication program, Ae. aegypti were nearly eradicated from most of the Americas by the mid 1900 s, however, the eradication program was not maintained, leading to the reestablishment of Ae aegypti (Nelson 1986) Aedes aegypti were once found throughout the state of Florida, but since the introduction of Ae. albopictus they have been largely displaced from the northern half of Florida (OMeara et al. 1995) Aedes aegypti are container inha biting mosquitoes and will often proliferate in artificial containers in urban habitats with diurnal mating and host seeking behavior (Gubler and Clark 1995) Aedes aegypti is present globally in tropical and subtropical regions ( Christophers 1960) Fema les produce 100200 eggs after taki ng a complete bloodmeal (Nelson 1986). Females oviposit on damp surfaces located near temporary flood pools, such as manmade containers, tree holes, or puddles. Eggs are laid singly and ar e not all laid at the same site, a behavior commo nly referred to as installment laying (Clements 1999). Development is temperature dependent, and e ggs may embryonate in as little as two days or as long as a week depending on environmental conditions. Eggs can survive desiccation for m onths and resume development once submerged (Nelson 1986, Foster and Walker 2002). Aedes albopictus Life History and B iology Aedes albopictus first e stablished in the United States via Hawaii in the 1800s (Moore and Mitchell 1997) In 1985 Ae. albopictus was found in intercontinental tire shipments in Texas (Sprenger and Wuithiranyagool 1986) and by 1991 had spread to 18 states (Rai 1991). As of 2005, Ae. albopictus was established in 25 states (O Meara 2005). Aedes albopictus is a tree hole i nhabiting mosquito, but larvae are often found in standing water in unused tires, cemetery vases flowerpots, and pet bowls (Hawley 1988, Ali and Nayar 1997). The affinity of Ae.

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17 albopictus for breeding in small containers results in proliferation in urban environme nts Aedes albopictus are anthropophilic and diurnal (Harrington et al. 2001). After taking a bloodmeal, females can produce 100 200 eggs, which are laid on damp surfaces around temporary fl ood pools Aedes mosquitoes lay eggs singly, rather than in a raft and will lay eggs at multiple sites to increase the chance of larval survival (Nasci and Miller 1996). Eggs can survive desiccation for days, and often require a dry period before hatching (Clements 1999). Culex quinquefasciatus Biology and E cology There are approximately 15 species of Culex in the United States, with nine species represented in Florida (Darsie and Ward 2005). Culex pipiens quinquefasciatus is present throughout the state of Florida and up the e ast coast, through western Texas and the Midwest, and north to southern Canada (Darsie and Ward 2005). Culex quinquefasciatus is oft en asso c i ated with birds, but willingly feed on humans and other mammals (Foster a nd Walker 2002). Known for being indoor biters, Cx. quinquefasciatus are nocturnally active for both host feeding and oviposition, and prefer to oviposit in substrate high in organic matter (Allan et al. 2005). Compact egg rafts are deposited in sewage water, drains, gutters, or other fermenting still water oft en found around houses (King e t al. 1960). Development is temperature dependent, but unlike Aedes eggs cannot survive desiccation Eggs hatch approximately 24 hours after deposition (Clements 1999). Culex tarsalis Biology and E cology Culex tarsalis can be found nearly everywhere in th e United St ates with the exception of the e ast coast, but rarely scattered throughout Georgia and north Florida The range extends north to s outhern Canada, and sout h to northern Mexico. Scattered pockets can be found around the Great Lakes, but they are r are east of the Mississippi ( Reisen 1993, Darsie and Ward 2005). Like other Culex species, Cx. tarsalis is crepuscular and noctournal, and prefers to bloodfeed on avian

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18 hosts but will feed oppor tunistically on mammals (Reisen 1993). The specifics of oviposition behavior have not been studied in Cx. tarsalis however, they do lay egg rafts containing up to 190 eggs per batch in water pools exposed to sunlight and often surrounded by abundant vegetation. Larvae can be found in both freshwater and saline flood pools, although water with high organic content is a deterrant (Reisen 1993). Culex tarsalis larvae feed on microfloral blooms, often being the first mosquito to colonize a newly formed pool (Reisen 1993) Culex tarsalis can be autogenous for the first batch of eggs and deposit egg rafts approximately four days after emergence. Autogeny is influenced by genetics, photoperiod, nutrition, and temperature (Reisen 1993) and is common in laboratory colonies. Role of Chemoreception in B ehavior Mosquitoes rely heavily on chemoreception to find a suitable host, as well as to locate appropriate substrate to oviposit (Clements 1999) Chemicals produced by one organism that initiate response in another organism are broadly referred to as semioc hemicals (Matthews and Matthews 1978). Sensory signals that an insect perceives from other organisms can be broken down into two broad categories: allelochemicals and pheromones. Allelochemicals are signals produced by one organism and detected by another organism such as a pl ant, or an insect of a dif ferent species. Allelochemicals can be divided into allomones, which are favorable to the emitter, kariomones, which are favorable to the receiver, or synomones, which are favorable to both (Matthews and Matthews 1978). Pheromones are signals produced and detected between members of t he same species (Flint and Doan 1996). Along with using pheromones to find mates, many anautogenous insects such as mosquitoes rely on semiochemicals for host seeking. Whether mosquitoes are anthropophilic, ornithophilic, or mammalophilic, each relies on semiochemicals to initially locate a host.

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19 Female mosquitoes use both olfactory and visual cues t o locate potential hosts (Bowen 1991). Despite the type of host, mosquitoes can detect odor plumes, whic h act like smoke plumes of odor molecules. Like smoke, odor plumes are mediated by wind speed and wind direction and diffuse into the air (Murlis et al. 1992). Once an odor plume is detected, it can be followed directly back to the host. Mosquito host se eking has been studied extensively, and research has shown that mosquitoes are strongly attracted to compounds such as car bon dioxide, lactic acid, ammonia, carbo xylic acids and 1octen 3ol ( Takken and Kline 1989, Bowen 1991, Smallegange et al. 2005) Sen silla basiconica on the maxillary palps of mosquitoes are largely responsible for detecting carbon dioxide molecules with slight variations in carbon dioxide receptor neurons between species (Grant et al. 1995). Antennal receptors appear to contain receptors for most other host associated compounds (Clements 1999). Chemoreception and Oviposition B ehavior Aedes aegypti are often used as models for mosquito morphology, and research has shown that Ae. aegypti and presumably most other mosquito species, have e volved eight types of sensilla to detect cues from their surroundings (Bowen 1991) Sensilla chaetica contain one neuron and are used for mechanoreception. Sensilla ampulacea contain three neurons and are most likely used for thermoreception. Sensilla coel onica contain three neurons and are used for temperature reception. Sensilla basiconica contain three to five neurons, and are used for olfaction. Sensilla trichodea come in four morphological types, each containing one to two neurons, and are used for olf action as well. The four morphological types are longpointed, short pointed, blunt tipped I, and blunt tipped II. All of the above sensilla are located on the antennae; however are not evenly dispersed. As much as 93% of the sensilla per flagellum can

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20 be olfactory in nature (Bowen 1991). Carbon dioxide reception is isolated to the pegs of the maxillary palps, where thermoreceptors are al so located (McIver and Charlton 1970). Nearly all mosquitoes have complex behaviors associated with oviposition that can be broken down into four basic steps: ranging flight, orientation, encounter, and acceptance (Clements 1999). After a bloodmeal, timing for an ovipositional flight is determined by the optimal combination of rainfall, humidity, temperature, and wind speeds (Bentley and Day 1989). Once flight has been initiated, mosquitoes locate and evaluate potential sites using a combination of olfactory a nd visual cues (Bentley and Day 1989). As a female approaches a potential site, several factors determine whether the site is acceptable or not. These factors vary between species, and can potentially vary between individuals. Temperature, humidity, salinity, and oxygen content are all important components, as well as volatiles e mitted from the water (Clements 1999). Som e mosquitoes detect oviposition pheromones released by previous females or the presence of eggs ( Mboera et al. 2000, Braks et al. 200 7), and are often attracted to water that contains c onspecific larvae ( Wilmont et al. 1987, Zahiri and Rau 1998, Allan et a l. 2005). Different mosquito genera follow different oviposition strategies. Culex lay egg rafts directly on the waters surface. Aedes lay eggs singly just above the water line, s everal Anopheles and Toxorhynchites hover over the water while depositing individual eggs, and Mansonia mosquitoes lay eggs on or underneath aquatic vegetation (Bentley and Day 1989). Regardless of the oviposition strategy, all mosquitoes need to evaluate substrate before ovipositing to ensure appropriate habitats t hat will sustai n larval growth. Compounds in and around substrates can be broken down into five categories established by Clements (1999). Oviposition attractants induce gravid females to orient towards the source. Oviposition stimulants invoke oviposition by the female An oviposition arrestant halts oriented

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21 movement and causes females to remain at the site. An oviposition repellant causes females to orient away from the source. Oviposition deterrents are nonvolatile substances that inhibit oviposition. Nearly any phy sical, chemical, or biological component of an oviposition site can act as one or more of the above properties. All of these categories may be airborne or waterborne signals. Location of an appropriate oviposition site for landing relies partially on sensi lla of the antenna (Davis and Bowen 1994) Once a female deems an oviposition substrate acceptable for landing based on long and short range stimuli, she will make contact with the water several times while in the air, presumably detecting waterborne compo unds with the tarsal receptors. T arsal receptors are generally either short blunt or short sharp sensilla trichodea, which may detect both chem ical compounds in the substrate and oviposition pheromones from previously deposited eggs (Davis and Bowen 1994). Female Ae. aegypti have more tarsal receptors than males, and the pro, meso and meta thoracic legs differ in the numbers of sensilla (Chapman 1982). Hair sensilla are found on the underside of Ae. aegypti tarsi and may be of three morphological types. Each type contains three to five neurons, which may be mixed mechanosensory and chemosensory neurons ( Clements 1999). The pro thoracic tarsi of Culiseta inor nata (Williston) have the greatest number of receptors and the metath oracic tarsi the least (Chapm an 1982). The tarsi of Ae. aegypti have campaniform sensilla ( McIver and Siemicki 1978). The gustatory sensilla, or taste sensilla are located on the mouthparts of mosquitoes, and receptors that respond to molecules in a liquid phase are in the form of uniporous hairs or pegs. These have also been reported from the distal end of the tibiae and tarsi of Cs. inor nata. Each sensilla contains four neurons, capable of detecting salinity and glucose concentrations in water (Owen 1963, Lee and Craig 1983 ). Sali nity and glucose concentrations can also be

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22 detected through uniporous peg sensilla on the mouthparts, which are identical to the gustatory sensilla on the legs. Females of Culex drink from the oviposition substrat e before ovipositing (Wallis 1954, Hudson 1956) It is uncle ar whether this is an additional evaluation of oviposition substrate, or to increase hydrostatic pressure to aid t he release of eggs (Hudson 1956, Weber and Tipping 1990). Wallis (1954) removed all the olfactory sensory organs (palps, antennae, mouthparts) on the heads of Ae. aegypti Ae. polynes i ensis Marks Ae. pseudos cutellaris Theobald, Cx. pipiens Linnaeus Cx. quinquefasciatus Cx. molestus Weidemann Cx. salinarius Coquillett, An. quadrimaculatus Say, An. freeborni Aitken, and An. azt ecus Hoffman, and found that removal of sensory organs had no effect on oviposition. Hudson (1956) conducted experiments involving amputation of the proboscis, palps, or both. Results showed that amputation of the proboscis, palps, and antennae did not alter the choice of substrate, suggesting that drinking did not help disc riminate between sites. Ike shoji (1966) however, found that when the proboscis of Cx. fatigans Weidemann was amputated, oviposition decreased greatly. There are few studies on the role of tarsal receptors Both Wallis (1954) and Hudson (1956) reported that when Ae. aegypti had tarsi amputated and tibia e covered in wax, mosquitoe s could not distinguish between freshwater or a salt solution and there was no difference in the number of eggs laid in freshwater compared to saltwater, suggesting th at tarsi are critical in determining salinity of an oviposition substrate. Aedes aegypti also perform abdominal probing of the substrate while searching for an oviposition site, suggesting there m ay be key receptors on the abdomen for determining an acceptable substrate. Though many Culex species oviposit directly on water, they also appear to contact the water with the end of the abdomen befor e oviposition commences (Wallis 1954, Beament and Corbe t 1981).

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23 An interesting component of mosquito oviposition is the preference for ovipositing in water that contains conspecific larvae, reinforcing the suggestion that larvae provide chemical emanations that female mosquitoes can detect ( Zahiri and Rau 1998, Allan et al. 2005). Larvae under stress, such as when over crowded or starved, appear to produce powe rful repellants (Zahiri and Rau 1998). Originally thought to be airborne t hese larval pheromone s have a low volatility, suggesting they may be detected through tarsal contact with the substrate rather than olfaction (Bentley and Day 1989). The chemical composition of the larval pheromone has not yet been identified. Another pheromonal attractant to Culex species is an oviposition pheromone, identified as [( ) (5R,6S) 6acetoxy 5hexadecanolide], which is present in apical droplets on eggs in egg rafts (Laurence and Pickett 1982) This pheromone seems to have a domino effect, and rapidly attracts females of the same species to lay eg gs in the same substrate (Laurence and Pickett 1982). The effects of this oviposition pheromone can be duplicated in the lab and is being synthesized for gr avid traps in the field (Mboera et al. 2000). Behavioral Differences B etween Aedes and Culex Oviposition S trategies Behavior ally, Aedes and Culex mosquitoes differ when selecting an oviposition site. Aedes aegypti will settle on the substrate, and then fly away. If the water is suitable, they will return to oviposit (Rozenboom et al. 1973). Culex molestus often exhibit a simila r dipping behavior, analyzing several different pools before choosing a site (Hudson 1956). Once Cx. molestus choose a site, they are often observed insert ing the ir proboscis into the water and drinking. Drinking behavior has also been reported in Cx. rest uans Theobald and Cx. pipens (Weber and Tipping 1990). Some theories for this behavior include further discrimination of the site, or to increase hydrostatic pressure to help with the release o f the eggs (Hudson 1956, Weber and Tipping 1990). Unlike the af orementioned species Ae. aegypti do not drink before ovipositing (Hudson 1956) Despite the numerous studies done on different aspects of

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24 oviposition behavior, the literature for a quantitative analysis of behavior between different species is lacking. Re view of C x. molestus Cx. restuans and Cx. pipens Oviposition B ehavior Initial studies of behavior have outlined the general process of Cx. molestus and Cx. restuans oviposition and egg raft formation. Hudson (1956) reported that females would fly forwards and backwards over the substrate before landing. If more than one site is present, females would make contact with each site before choosing a site. Once a female landed on the water, the metathoracic legs were held upwa rd and contact with the water made only by the prothoracic and meso thoracic legs. The meta thoracic legs were lowered so that the tarsi lay horizontally along the surface and the distal tibia was in contact with the substrate. The distal tibia were moved together, sometimes crossing, for ming a pen for the egg raft and the tip of the abdomen wa s lowered into the water. Hudson (1956) did not observe drinking in all studies. Wallis (1954) described the same general pattern, but with drinking occurring before the meta thoracic legs were lower ed. It is unknown how oviposition varies between Culex species and how differences, if they exist, may impact the potential for certain toxicants, pathogens, or repellants to be effective. Review of Ae. aegypti Oviposition B ehavior Aedes aegypti follow a s lightly different behavior pattern when selecting an oviposition substrate. Clements (1999) outlined Ae. aegypti oviposition behavior, beginning with erratic flight patterns over the substrate until a female found a pl ace to settle. Once a female had lande d, she rested on all six legs. Females walk ed to the edge of the water and walk ed along the waterline until a suitable location wa s determined. Once an appropriate location was found, she would orient herself facing away from the substrate and raise d the m etathoracic legs. Aedes aegypti then appears to become inactive. After inactivity, the female w oul d raise and lower her

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25 legs synchronously and the abdomen wa s arched downward to release an egg. Once the egg wa s released, it was detached by moving the abdomen side to side while walking forwards and sideways. Little is documented about Ae. albopictus oviposition behavior, includi ng if and how it differs from Ae. aegypti. A greater understanding of the similarities and dif ferences of these two medically impor tant species can contribute to our understanding of potential avenues a nd short comings of ovipositionbased surveillance and control. Importance of Biological C ontrol Mosquitoes are competent and common vectors for vertebrate pathogens, but are also susce ptible to paras ites and pathogens Many para sites and pathogens are species specific. Because of such high host specificity, associated parasites and pathogens make appropriate and potentially successful bio logical control agents. Two of the most widely us ed biological control agents today are the bacterial agents Bacillus thuringiensis israelensis ( Bti ) and Bacillus sphaericus ( Bsph ), which have been used with great success in larval control. Bio logical control agents are becoming increasingly important du e to the rapid development of resistance to many common insecticides, and growing public concern about the type and number of chemicals used in the environment (Hardy et al. 1983). One of the most well known pesticides is dichlorodiphenyltrichloroethane (D DT) DDT was used in the United States for many years before being outlawed, however is still commonly used in Africa (Hargreaves et al. 2003) DDT has been linked to cancer, and was commonly found in food products including meat, dairy, produce, and grains (Jaga and Duvvi 2001). Many African mosquitoes have developed some level of DDT resistance (Hemingway and Ranson 2000, Hargreaves et al 2003). Mosquitoes have also show n insecticide resistance to many of the organophosphates, carbamates, and py rethroids (Hemingway and Ranson 2000, Weill et al. 2003). Because of the problems

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26 associated with deleterious environmental impact and resistance in the reliance of insecticides alternative methods of control are the subject of much research. Edhazardia aedis Kudo Microsporidia are common parasite s of mosquitoes and can be used as model s for parasite infection, as well as potential bio logical control agents Microsporidia are intracellular spore producing parasites most often parasitizing insects (Hirt et al. 199 9). Edhazardia aedis is an obligate parasite of Ae. aegypti (Becnel and Andreadis 1999). Larvae of Ae. aegypti often ingest spores either after an infected host dies in the larval water, or a host releases spores through excretion. Edhazardia aedis can be transmitted both horizontally and vertically, and has four sporulation sequences (Johnson et al. 1997, Becnel and Andreadis 1999). Once uninucleate spores are ingested by Ae. aegypti larvae, spores infect the midgut epithelium and the epithleuim of the ga stric ceca (Becnel and Andreadis 1999). Spores undergo a m inor asexual reproductive phase, and gam e togenesis follows which results in the formation of uninucleate, pyriform gametes. The pyriform gametes undergo plasmogamy. Diplokaryotic stages result from the plasmogamy, and develop into binucleate spores, known as primary spore s. The primary spores germinate and infect other tissues in the host, such as the oenocytes. Most vertically infected l arvae with high infection rates die before metamorphosis, but low to moderate infection rates may allow for development to adulthood (Becnel and Andreadis 1999). In the adult, E. aedis spores initiate a second asexual reproductive phase, and eventually vegetative stages surround the ovaries of the female. Once the f emale takes a bloodmeal, a second sporulation sequence occurs forming binucleate spores. The resulting binucleate spore is known as the transovarial spore and infects the ovaries The binucleate spores are responsible for transovarial transmission of the parasite by injecting sporoplasms into developing eggs through a polar filament in the spore. In the infected progeny spore development is restricted to

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27 the fat bodies where E. aedis undergoes another asexual reproduction cycle involving diplokaryotic sta ges and ends the life cycle by either meiosis or nuclear disassociation to produce uninucleate spores that are infectious per os (Becnel and Andreadis 1999). Microsporidian parasites often alter the behavior of the host (Becnel and Andreadis 1999, Barnard et al. 2007). The age of infection affects bloodfeeding success as an adult. Larvae infected late in the developmental process fed more successfully as adults than larvae infected as early instars (Koella and Agnew 1997). Behaviorally, E. aedis reduces h ost attraction in host seeking females by nearly 50%, and increases repellen cy effects of DEET (Barnard et al. 2007). Controlled studies have concluded that microsporidia can be potentially successful biological control agents due to high host specificity, safety for non target organisms, and dissemination by both vertical transmission and horizontal transmission ( Becnel 1992, Becnel and Johnson 2000). Reproductive capacity was greatly reduced in adults that had been infected as larvae, regardless of the tr ansmission route (Becnel et al. 1995). Because oviposition is critical for dissemination, understanding the details of oviposition behavior will lead to a greater understanding of E. aedis transmission as well as effects on oviposition. Vavraia culicis (W eiser) Vavraia culicis is a microsporidian species once thought to belong to the genus Pleistospora. It has since been renamed, and V. culicis is now the type species for the genus Vavraia. Vavraia culicis infects 13 mosquito species, including both Culici ne and Anopheline groups It also infects nonmosquito hosts such as Lepidoptera. Spores have been found in at least five mosquito genera and it has a very broad geographic range which includes Africa, Europe, and North America to date. Originally though t to be insect specific, V. culicis has recently been questioned as a potential pathogen of immunosuppre ssed human hosts (Becnel et al. 2005)

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28 A V. culicis isolate was found in five mosquito species during a pathogen survey in Gainesville FL in 1997 (Fukuda et al. 1997). The pathogen was most commonly found in Ae. albopictus and is very phylogenetically and physically similar to the human microsporidian pathogen, Trachipleistophora hominis (Lobo et al. 2006). The isolate has been classified as V. culicis floridensis and was successfully propagated in the lab in both Lepidopteran ( Helicoverpa zea (Boddie) and Spodoptera littoralis Boisduval ) and An. quadrimaculatus hosts (Vavra and Becnel 2007). Vavraia culicis unlike E. aedis has a simple life cycle an d host relationship. Only one uninucleate spore type is produced, and spores are not tissue specific. The pathogen is transmitted horizontally between larval hosts upon the death of an infec ted larva (Becnel and Andreadis 1999). In Cx. quinquefasciatus V. culicis infection alters the reproductive success of the host. Parasitism in Cx. quinquefasciatus causes females to pup ate and emerge earlier than noninfected females (Agnew et al. 1999) Females were also smaller, suggesting reduced fecundity when infec ted. Males were not affected by V. culicis infection (Agnew et al. 1999). It is important to know how V. culicis infection affects mosquito oviposition because it has recently been implicated as a pathogen of human infection. Additionally, V. culicis infection can serve as a model system to understand the behavioral effects of parasite infection on mosquitoes. Finally, because V. culicis decreases fitness and and reduces fecundity in infected mosquitoes, V. culicis may play an important role in the biologic al control of mosquitoes in the future. Culex nigripalpus N ucleopolyhedrovirus (CuniNPV) In recent years, research has been conducted on a newly recognized nucleopolyhedrovirus, CuniNPV (Baculoviridae: nucleopolyhedrovirus). CuniNPV has been found in wild populations of Cx. nigripalpus Cx. quinquefasciatus and Cx. salinarius L aboratory trials have shown nearly all Culex species found in the Southeast susceptible, with

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29 the exception of Cx. territans (Andreadis et al. 2003). Occlusion body proteins are approximately three times the size of proteins from other baculoviruses (Perera et al. 2006) and many gene sequences are distinct for this virus, including gene organization, lack of homologues, and lack of gene conservation (Afonso et al. 2001). As a result CuniNPV is currently being rec lassified as a deltabaculovirus (CuniDBV) (Jehle et al. 2006) but for the purposes of this research, it will be referred to as a nucleopolyhedrovirus CuniNPV is an obligate baculovirus that infects multiple Culex species (P erera et al. 2006, Becnel and White 2007). Baculoviruses are not commonly found in wild mosquito populations, but are highly virulent when infections occur (Becnel et al. 2001). Initial infection begins in the gastric ceca and posterior stomach w hen occlu sionderived virions (ODVs) are ingested. The virus replicates, and budded virions spread the virus to new nuclei within the midgut (Moser et al. 2001). After 2448 hours post inoculation, the majority of nuclei within cells of the gastric ceca and the pos terior stomach are filled with occlusion bodies (Moser et al. 2001). The virus is generally restr icted to the secretion/resorbtion cells of the gast ric cecae and posterior midgut (Moser et al. 2001). Behaviorally, infected larvae become lethargic and remai n at the waters surface despite surrounding disturbances (Moser et al. 2001). Although CuniNPV generally infects larvae, the virus has been isolated from the adult midgut (Becnel et al. 2003). During the larval stages, the virus is spread by horizontal tr ansmission. If the mosquito survives to adulthood, the occlusion bodies are generally shed with the first meconium. It can also be vertically transmitted if an adult dies in a new uninfected aquatic habitat (Becnel et al. 2003). F or high transmission rates to occur, divalent cations must be present in the water, such as magnesium. Calcium acts as a strong inhibitor of the virus, and therefore the virus is common in

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30 pools containing high amounts of sw ine effluent, but uncommon in pools containing dairy cattl e effluent (Becnel et al. 2001), indicating that very specific conditions must be met for transmission to occur Like most baculoviruses, there are two phases: the budded virion stage and the occluded virion stage. Occlusion bodies lack a polyhedron envelope, which is a normal characteristic of baculoviruses. Each virio n contains one nucleocapsid, an intermediate layer, and an outer envelope (Moser et al. 2001). With such high larval mortality rates, CuniNPV should be considered as a potential larval bio log ical control agent, and understanding the relationship between oviposition behavior and virus infected conspecific larvae or larval substrate is key. Mosquitoes have been major disease causing vectors around the world and remain so today. Current emphasi s on biological control agents may provide an alternative to pest control, and disease management is now focusing on vector control E xploiting mosquitoes vulnerability to their own associated parasites and pathogens may provide a new tactic for biologica l control Because many parasites and pathogens are tr ansmitted transovarially, an in depth unders tanding of oviposition behavior and the relationship between oviposition and infection is important to understand. By understanding how both infected mosquitoes and uninfected mosquitoes respond behaviorally to i nfection, we can manipulate conditi ons to improve mosquito control by possibly establishing natural repellants or highly effective larvicides and adulticides. Objectives 1. Perform a comparative behavioral analysis of oviposition behavior between Culex pip i ens quinquefasciatus and Culex tarsalis to determine similarities and differences in oviposition behavior. 2. Perform a comparative behavioral analysis of oviposition behaviors between Aedes aegypti and Ae des albopictus to determine similarities and difference in oviposition behavior. 3. Examine the effect of mosquito pathogens on mosquito oviposition behavior.

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31 CHAPTER 2 A COMPARITIVE ANALYSIS OF OVIPOSITION BEHAVIOR OF AEDES AEGYPTI (LINNAEUS) AND AEDES AL BOPICTUS Introduction (SKUSE) Aedes aegypti (Linnaeus) and Ae. albopictus (Skuse) are analogous mosquito species in life history strategy, biology, and ecology. Both are container inhabiting species that willingly oviposit in rain barrels, buckets, and fl ower pots (Hawley 1988, Clements 1999). Both are diurnal in feeding and oviposition, and even physically appear very similar. Aedes aegypti has been in the new world for centuries, originating in Africa (Nelson 1986, Barrett and Higgs 2007). Aedes albopict us first entered the United States from South East Asia via Hawaii in the 1800s (Moore and Mitchell 1997). In 1985, Ae. albopictus were found in intercontinental tire shipments in Texas (Sprenger and Wuithiranyagool 1986), and by 1991 had spread to 18 sta tes (Rai 1991). As of 2005, Ae. albopictus was established in 25 states (OMeara 2005). Since the introduction of Ae. albopictus an overlap in habi tat has resulted in the displacement of Ae. aegypti by Ae. albopictus everywhere north of Central Florida (O Meara et al. 1995). Many Aedes species are successful vectors and are known to transmit diseases such as yellow fever, dengue (DEN) and dengue hemorrhagic fever (DHF), chikungunya virus (CHIK V), and LaCrosse encephalitis (Foster and Walker 2002). Today, D EN is considered a constant threat to the Americas with cases regularly reported in the continental United States, and can be transmitted by both Ae. aegypti and Ae. albopictus (Rai 1991, Gill et al. 2000). Floridas subtropical climate and close proximity to the Cari bbean, where many diseases persist make it an ideal location for several arbovirus epidemics. Besides vectoring pathogens, mosquitoes inflict irritating bites and are considered a pest species around the world. Severe mosquito emergences can ne gatively impact tourism, recreation, and even affect livestock production (Foster and Walker 2002). Because the general pattern of pathogen transmission requires an initial

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32 bloodmeal to acquire the pathogen, pathogen transmission is greatly dependent on at least one oviposition event (Weaver and Barrett 2004). The second bloodmeal is essential for transmission of the pathogen, but often a second bloodmeal will not occur before the first oviposition cycle is complete (Bentley and Day 1989). Understanding the steps involved in mosquito oviposition provides a critical point in the mosquito life cycle where population supression can occur through the use of biocontrol agents, toxicants, or attract and kill approaches. Additionally, a deeper understanding provide s a weak link in the mosquito life cycle where the mosquitoborne pathogen transmission cycle can be broken through intervention. Aedes aegypti are often used as models for mosquito morphology, and research has shown that Ae. aegypti and presumably most other mosquito species, have evolved eight types of sensilla to detect cues from their surroundings (Bowen 1991). Nearly all mosquitoes have complex behaviors associated with oviposition that can be broken down into four basic steps: ranging flight, orient ation, encounter, and acceptance (Clements 1999). After a bloodmeal, timing for an ovipositional flight is determined by the optimal combination of rainfall, humidity, temperature, and wind speed (Bentley and Day 1989). Once flight has been initiated, mosquitoes locate and evaluate potential sites using a combination of olfactory and visual cues (Bentley and Day 1989). As a female approaches a potential site, several factors determine whether the site is acceptable or not. These factors vary between species and can potentially vary between individuals. Temperature, humidity, salinity, and oxygen content are all important components, as well as volatiles emitted from the water (Clements 1999). Some mosquitoes detect oviposition pheromones released by previous females or the presence of eggs (Mboera et al. 2000, Braks et al. 2007), and are often attracted to water that contains conspecific larvae (Wilmont et al. 1987, Zahiri and Rau 1998, Allan et al. 2005).

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33 Different mosquito genera follow a variety of oviposition strategies. Culex spp. lay egg rafts directly on the waters surface. Aedes spp. lay eggs just above the water line, Anopheles and Toxorhynchites spp. hover over the water while depositing individual eggs, and Mansonia spp. lay eggs on or underneath aquatic vegetation (Bentley and Day 1989). Regardless of the oviposition strategy, all mosquitoes need to evaluate oviposition substrate before ovipositing, ensuring appropriate habitats to sustain larval growth. Location of an appropriate oviposition s ite for landing relies partially on sensilla of the antennae (Davis and Bowen 1994). Once a female identifies an oviposition substrate as acceptable for landing, based on long and short range stimuli, she makes contact with the water several times while in the air, presumably detecting waterborne compounds with the tarsal receptors. Tarsal receptors are generally either short blunt or short sharp sensilla trichodea, which may detect both chem ical compounds in the substrate and oviposition pheromones from pr eviously deposited eggs (Davis and Bowen 1994). Female Ae. aegypti have more tarsal receptors than males, and the pro meso and meta thoracic legs differ in their number of sensilla (Chapman 1982). Campaniform sensilla have been identified from the tars i of Ae. aegypti (McIver and Siemicki 1978) as well as hair sensilla found on the underside of Ae. aegypti tarsi, which may be of three morphological types. Each type may contain mechanosensory or chemosensory neurons (Clements 1999). Salinity and glucose concentrations also can be detected through uniporous peg sensilla on the mouthparts, which are identical to the gustatory sensilla on the legs (Clements 1999). There are few studies on the role of tarsal receptors. Both Wallis (1954) and Hudson (1956) reported that when Ae. aegypti had tarsi amputated and tibiae covered in wax, mosquitoes could not distinguish between freshwater or a salt solution and there was no difference in the

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34 number of eggs laid in freshwater compared to saltwater, suggesting the tarsi are critical in determining salinity of an oviposition substrate. Aedes aegypti also perform abdominal probing of the substrate while searching for an oviposition site, suggesting there may be key receptors on the abdomen for determining an acceptabl e substrate. Although previous research has been conducted on Ae. aegypti oviposition behavior, very little has been done on Ae. albopictus despite its vector status. Much of the basic behavioral research on Ae. aegypti oviposition behavior was conducted with simple visual observation. The recent introduction of Ae. albopictus to the Americas warrants a closer look at the similarities and differences between these species because of their dangerous vectorial capabilities. Because Ae. aegypti and Ae. albopi ctus are so similar in their life history strategies, it was expected that they would be similar in their oviposition behavior as well. The objective of this research is to examine, in close detail, the oviposition behavior of Ae. aegypti and Ae. albopictus and to determine the similarities and differences in ovipositio n behavior between each species. Materials and Methods Insect Colonies A South Florida strain of Ae. aegypti was collected from cemeteries in Tampa, FL in March 2008. Approximately 500 l arvae were reared in plastic pans (35 cm x 48 cm x 6.25 cm) containing 1500 ml well water, and were fed every other day with 50 ml larval slurry made from a 1:1 mix of liver powder and brewers yeast (MP Biomedical, Solon, OH) mixed with well water (10 g dry mi xture per 100 ml water). Once the larvae pupated, they were placed in a plastic cup containing water and set inside a 30 cm x 30 cm x 30 cm P lex iglas and screen cage to emerge. Adults had constant exposure to cotton saturated with a 5% sucrose solution (50 g sucrose in 1000 ml water). Five to seven days after emergence, Ae. aegypti were bloodfed with

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35 defibrinated bovine blood (Cathy Jennings, Ocala, FL) in a sausage casing (4.45 cm, The Sausage Maker Inc., Buffalo, NY). Forty eight hours post bloodfeeding, pieces of seed germination paper (8 cm x 6 cm) (Anchor Paper Company, St. Paul, MN) were placed in 500 ml dark colored plastic cups (Solo Cup Company, Highland Park, IL) filled with 250 ml well water. At 24 hour intervals over the course of three days, the egg papers were removed and allowed to dry at room temperature for approximately 20 minutes. Once dry, the egg papers were placed in a sealed plastic bag to maintain moisture. On the third day, the egg cup was removed. To hatch, egg papers were placed in plastic cups filled with deionized water, and placed under a vacuum for 10 minutes. Cups were removed and egg papers were left in the cups overnight to observe hatch rates. All stages of development were kept in a temperaturecontrolled incubator (Model 818, Precision Scientific, Teynampet India) at 28+ 1 oBehavioral Experimental Design C, with a 14:10 light/dark photoperiod and an 8085% relative humidity. The Ae. albopictus colony was established from a field collection from Gainesville, FL in 2007. All other rearing methods follow th ose for Ae. aegypti A 20 cm x 20 cm x 20 cm Plexigla s chamber with one side consisting of tubular gauze for access was placed within a metal frame, and the frame was draped with a blackout cloth (Duvetyne) on all sides but the front to prevent surrounding disturbances (Fig. 21). Two infrared lights (940 nm, Rainbow CCTV, Costa Mesa, CA) were placed on top of the chamber. Aedes aegypti were filmed with a 5 55mm F1.4 CS lens on a CCD camera (Hi res DNL 46D 1/3 DSP) (Rainbow CCTV, Costa Mesa, CA) mounted on an adjustable tripod (Manfrotto 3021BN Bogen, Ramsey, NJ) Aedes albopictus was filmed with a 50mm F2.8 Telecentric lens (Rainbow CCTV, Costa Mesa, CA) using the same camera. Cameras were set up directly in front of the cham ber.

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36 Ten gravid nulliparous females were added to the chamber using a battery operated aspirator (Hausherrs Machine Works, Toms River, NJ) and allowed to settle for 10 minutes. After 10 minutes, a petri dish (10 cm diameter) painted with flat black pain t was filled with well water (10 ml) and 5 ml pupal emergence water extracted directly from cups in which Ae. aegypti pupae had recently emerged was placed inside the chamber. An 8 cm x 14 cm rectangular piece of seed germination paper (Anchor Paper Compan y, St. Paul, MN) was added to the rear side of the petri dish with striations going vertically. Behavioral Analysis Oviposition behavior was recorded using the design above with an analog camera, and the video was digitized with a real time MPEG encoder (C anopus EMR100), and MediaCruise (Canopus, San Jose, CA) computer software. Each video clip consisted of a single gravid female, and the recording was started when hovering over the substrate was observed or upon first landing. Once oviposition was initiate d, the camera was focused on the mosquito to provide maximum recording detail. Recording was stopped after oviposition was completed and the female left the substrate. Ten videos were obtained and analyzed for both Ae. aegypti and Ae. albopictus Each video clip was analyzed using behavioral analysis software (The Observer XT Version 6.1.4 Noldus Information Technology, Inc., Leesburg, VA). The videos were watched and replayed through the software, and each defined behavior scored using assigned keys on a computer keyboard. Coding schemes for analysis were developed for each species based on the defined behaviors from the ethograms. Behaviors within the coding scheme were classified as point behaviors, which are quick solitary events, or state events whi ch are recorded with durations. These schemes were developed and revised as needed after a p reliminary analysis of videos. Coding schemes for the behavioral software are presented in Appendix 1. After each video clip

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37 was scored, a behavioral analysis, raw nu mber lag sequential analysis, and proportion lag sequential analysis was run in The Observer XT. Through viewing of preliminary videotapes for each species and examination of the literature on Ae. aegypti behavior (Wallis 1954, Hudson 1956, Rozenboom et a l. 1973, Clements 1999), an ethogram for each species was developed (Table s 21 and 22). An ethogram is a catalog of behaviors with an accompanying definition for each behavior and development of the ethogram was based on Boyd and Houpt (1994). Because be haviors associated with mosquito oviposition are not always mutually exclusive, behaviors were separated into four categories: location of the substrate, movement of the subject, tarsal activity, and oviposition activity by the subject Each behavior withi n a category is mutually exclusive, but behaviors between categories can occur simultaneously. These cataloged behaviors formed the basis for the kinematic analysis and detailed behavioral analysis. The proportional lag sequent ial analysis, which is the p roportion of time a certain behavior follows another, was exported to Microsoft Excel (Microsoft Corporation, Redmond, WA). All behaviors for the 10 individuals of each species were averaged. This first order transition matrix summarizes how often each mut ually exclusive behavior (y axis) was followed by another behavior (x axis). In behavioral categories where behavioral information contained sub categories, such as directional behaviors including walk up, walk down, or walk horizontally. T he consolidation of these behaviors resulted in one type of walk behavior being following by a different type of walk behavior, or a self transition. Rows labeled Start Observation mean the following behavior was the first seen at the start of the video, usually the pre determined default behavior, and the column labeled End Observation means the behaviors in that column were the last behaviors seen as the vide o ended. Based on the transition proportions a kinematic diagram

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38 was created to visualize the action patterns of each species. For clarity, b oth the transition matrix and kinematic diagrams are also separated into the previously mentioned four categories. In addition to the ethograms, transition tables, and kinematic diagra ms, the occurrence and duration of each behavior was calculated and averaged. Paired statistical comparisons were made between Ae. aegypti and Ae. albopictus Finally, based on the sum of durations for each behavior, time budgets were created for Ae. aegypti and Ae. albopictus and again assigned to the previously mentioned fo ur categories. Data Analysis Behavioral analyses were exported to Microsoft Excel. Count data were square root transformed and percentage data were arc sine transformed, and transformed data exported to SigmaStat (Systat Softwar e, Chicago, IL) t o test for normality (Sha piro Wilk test). All normal data was analyzed using a paired t test ( P < 0.05). Data that was not distributed normally was analyzed using a Mann Whitney Rank Sum test. In the transition matrix tables, frequencies abo ve 0 were compared by chi square analysis to determine those occurring at greater than expected ( P < 0.05). Results Ethograms were developed for Ae. aegypti (Table 2 1) and Ae. albopictus (Table 2 2) When comparing the shared behaviors of both species, there were several significant differences in both the number of times a behavior was performed, and the durations of the behaviors. Table 23 lists the mean number of times each behavior was performed by each species. Behaviors that were performed significant ly more by Ae. aegypti include number of ABDOMINAL PROBE events ( t =5.088, df=18, P < 0.001), mean number of EGG DEPOSITIONS ( t =3.804, df=18, P =0.001), mean number of STOP events ( T =154.0. df=20, P < 0.001), and mean number of WALK DOWN events ( t =2.436, df=18, P =0.025). Behaviors that were performed significantly

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39 more by Ae. albopictus include the mean number of ABDOMINAL GROOM events ( T =80.00, df=20, P =0.015), ABDOMINAL FAN, which was seen only in Ae. albopictus ( T =55.0, df=20, P < 0.001), mean number of NONE e vents ( t =3.276, df=18, P =0.004), mean number of STILL events ( T =55.0, df=20, P <0.001), mean number of TOTAL TARSAL WAVE events ( t =5.350, df=18, P < 0.001), mean number of SIMULTANEOUS TARSAL WAVE events ( t =5.934, df=18, P < 0.001), mean number of SINGLE TARSAL WAVE events ( t =6.648, df=18, P < 0.001), mean number of TOTAL WALK events ( t =3.867, df=18, P =0.001), and mean number of WALK HORIZONTAL events ( t =6.490, df=18, P < 0.001). There was no significant difference between species for all other behavioral events. A verage durations of all behavioral events for both spec ies are presented in Table 2 4, including average oviposition event time. Behaviors by Ae. aegypti with a significantly longer durations than Ae. albopictus include the mean duration of ABDOMINAL PROBE events ( T =80.00, df=20, P =0.015), the mean duration of NONE events ( t =3.603, df=18, P =0.002), the mean duration of TOTAL WALK events ( t =3.973, df=18, P < 0.001), the mean duration of WALK DOWN events ( T =134.5, df=20, P =0.019), and the mean duration of WALK HORIZONTAL events ( t =3.669, df=18, P =0.002). Behaviors performed by Ae. albopictus that had a significantly longer duration than Ae. aegypti include the mean duration of ABDOMINAL FAN events ( T =55.00, df=20, P < 0.001), the m ean duration of TOTAL TARSAL WAVE events ( t =6.392, df=18, P < 0.001), the mean duration of SIMULTANEOUS TARSAL WAVE events ( t =6.872, df=18, P < 0.001), and the mean duration of SINGLE TARSAL WAVE events ( t =5.199, df=18, P < 0.001). First order transitional matrices for Ae. a egypti and Ae. albopictus provide the basis for development of the kinematic diagrams for Ae. aegypti and Ae. albopictus For both Ae. aegypti

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40 (Table 2 5) and Ae. albopictus (Table 2 6), there was a strong pattern of females observed on the wa ter before being located on the egg paper. Also Ae. albopictus lo cated on egg paper often preceded being located in the OTHER category For movement behaviors of Ae. aegypti (Table 27) and Ae. albopictus (Table 2 8), floating and landing behaviors often preceded walking. For Ae. albopictus jumping and walking often pre ceded being still (Table 2 8). For tarsal activity the behavioral category of NONE often preceded TARSAL WAVING for both Ae. aegypti (Table 2 9) and Ae. albopictus (Table 2 10). Other behavioral patterns observed in Ae albopictus included WING GROOM and ABDOMINAL GROOM before TARSAL GROOM Furthermore, TARSAL GROOM and NONE occured before TARSAL WAVE (Table 2 10 ). In oviposition behaviors, Ae. aegypti often performed STOP behaviors before EGG DEPOSITION (Table 2 11). Differences bet ween species were also seen in oviposition behaviors with significantly more NO PROBE and EGG DEPOSITION occurred before ABDOMINAL PROBE and ABDOMINAL PROBE occurred before STOP in Ae. aegypti (Table 2 11 ). For Ae. albopictus ABDOMINAL FAN often occurred before ABDOMINAL PROBE behaviors (Table 2 12). Kinematic diagrams of the visual representation of action patterns were constructed based on transition tables for Ae. aegypti and Ae. albopictus For both species, females were observed on the water before moving t o the egg paper (Figures 2 2, 23 ). For movement behaviors by Ae. aegypti and Ae. albopictus LAND and FLOAT were often followed by WALK (Figure 24, 2 5). For Ae. aegypti LAND was followed by WALK, which was followed by either FLY STILL or FLOAT. (Figure 2 4 ). Additionally, for Ae. albopictus JUMP was following by STILL, followed by WALK followed again by STILL (Figure 2 5).Tarsal activities for Ae. aegypti did

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41 not follow clear patterns except for NONE followed by TARSAL WAVE (Fi gure 2 6 ). For Ae. albopictus however, stronger pa tterns were observed (Figure 2 7). NONE TARSAL GROOM and ANTENNAL GROOM were often followed by TARSAL WAVE T ARSAL WAVE included different types of tarsal waving as defined in Table 2 1, inc luding ALTERN ATING TARSAL WAVE SIMULTANEOUS TARSAL WAVING and SINGLE TARSAL WAVING such that one tarsal wave episode was often followed by another type of tarsal waving. ABDOMINAL GROOM and WING GROOM were followed by TARSAL GROOM Oviposition behavior sequences fo r Ae. aegypti consisted primarily of NO PROBE followed by ABDOMINAL PROBE STOP EGG DEPOSITION, subsequently followed by ABDOMINAL PROBE (Figure 2 8 ). For Ae. albopictus the primary patterns of oviposition behavior were STOP, followed by EGG DEPOSITION an d ABDOMINAL FAN, followed by ABDOMINAL PROBE an d EGG DEPOSITION (Figure 2 9). Time budgets were created for behaviors within each behavioral category for Ae. aegypti and Ae. albopictus When comparing the two species for location behaviors, Ae. albopictus spent significantly more time on the water ( T =75.00, df=20, P =0.017) (Figure 210) although this difference only represented 2 % of the overall time. For movement behaviors, Aedes albopictus spent significantly more time WALKING (25% compared to 13%) ( t =4. 649, df=18, P < 0.001), and Ae. aegypti spent more time STILL (35% compared to 23%) ( t =4.651, df=18, P < 0.001) (Figure 2 11). For tarsal activities, Aedes aegypti spent more time with NONE (67% compared to 13%) ( T =153.00, df=20, P < 0.001). Females of Ae. albopictus however, spent more time ABDOMINAL GROOMING (1% compared to 0%) ( T =80.00, df=20, P =0.015), TARSAL WAVING (67% compared to 12%) ( t =5.931, df=18, P < 0.001) and a unique behavior, ABDOMINAL FANNING ( T =55.00, df=20, P < 0.001) than Ae. aegypti (Figure 2 12). For

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42 oviposition activity Ae. aegypti spend more time in a STOP (9% compared to 1%)( t =182.00, df=20, P < 0.001) and deposited significantly more eggs ( t =4.68, df=18, P < 0.001) as compared to Ae. albopictus (Figure 2 13) Behavioral sequences for representatives of each species are presented in Figure 2 14 to demonstrate overlapping behaviors. Discussion Clements (1999) outlined the behavioral steps of Ae. aegypti females when selecting an oviposition site, beginning with erratic flight patterns over the substrate until the female found a place to settle. He observed that once a female had landed, she rested on all six legs and walked to the edge of the water and walked along the waterline until a suitable location was determined. Once an appropriate location was found, she would orient herself facing away from the substrate and raise the metathoracic legs. Females would then appear to become inactive. After inactivity, the female would raise and lower her legs synchronously and the abdomen was arched downward to release an egg. Once the egg was released, it was removed by moving the abdomen side to side while walking forwards and sideways. The current study did not contain observations of the same behavioral patterns. Flight patterns were not noted in this st udy, but females did land on the substrate on all six legs. All females in the current study landed with the posterior abdomen facing the water, and walked backwards to the water. Although some females did wave the meta thoracic tarsi before walking to the water, it was not observed in all females. Once at the waters edge, Ae. aegypti females generally kept at least one of the meta thoracic tarsi in contact with the waters surface at all times. Because of previously identified tarsal receptors (Davis and Bowen 1994), this observed behavior could be an additional evaluation of the oviposition substrate, or may be used as a tactile guide to maintain position along the waters edge Another major difference between these studies is that no females were observ ed moving the abdomen in the current study while

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43 depositing an egg. In contrast, the females stopped all movement while the egg was extruded and did not continue movement until the egg deposition was complete. Rozenboom et al. (1973) observed that Ae. aeg ypti would settle on the substrate and fly away before returning to oviposit. This was not observed in the current study, but may be due to laboratory colonization or the lack of multiple sites to choose from. Dipping behavior was documented by Davis and B owen (1994), as were several types of sensilla on the tarsi, including gustatory sensilla, which were suspected to detect chemical compounds and pheromones in the water. Wallis (1954) and Hudson (1956) also observed that when the tibia were waxed over, fe males were unable to determine salinity or glucose concentrations in the water, suggesting that tarsal reception plays a critical role in oviposition site selection or rejection. Aedes albopictus did not maintain tarsal contact with the water while ovipositing, were more likely to travel vertically along the egg pap er, and only walked a very short distance along the horizontal plane of the egg paper. When Ae. albopictus did walk horizontally, no more than a few steps were taken, whereas Ae. aegypti would quickly walk the entire length of the paper multiple times during an oviposition event, as seen by the significant difference in both the number of times and the duration of time Ae. albopictus remained still. Floating on the water was seen in both species, although not often. Eggs were generally not deposited while the female was floating on the water, and she was usually quick to exit the water whether it was by returning to the egg paper or flying away. Because of the erratic, agitated movements while on t he water, I speculate that floating on the water is not intentional, but rather a result of disorientation along the waters edge. Aedes albopictus groomed the abdomen, whereas Ae. aegypti did not; however, this may be due to lower resolution/magnificatio n of the equipment used. A lower power lens was used to

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44 capture the entire egg paper for Ae. aegypti because of the quick movements during oviposition. Because Ae. albopictus were more stationary while ovipositing, a higher magnification could be used. It is likely that Ae. aegypti do groom the abdomen, however the resolution of taped im age was not clear enough to delineate abdominal grooming and tarsal grooming. Grooming however is a common behavior in many insect species (Hlavac 1975) and has been previ ously documented in female Aedes triseriatus Say (Walker and Archer 1988). Goldman et al. (1972) proposed that the purpose of proboscis grooming was to remove particulate matter from sensilla. Walker and Archer (1988) noted several categories of grooming, suggesting that grooming behaviors aided in the clearing of particulate matter from sensory organs, such as proboscis, antennae, and the tip of the abdomen, as well as to smooth scales on locomotory structures such as the wings and legs, to improve aerodyn amicity in flight. Because of the known receptors on the legs of mosquitoes (Davis and Bowen 1994), tarsal grooming may serve to clear particulates from sensilla as well as smooth scales. For a diagram of Aedes grooming behaviors, refer to Walker and Arche r (1988). Aedes aegypti were not observed fanning the abdomen, but it was observed in all Ae. albopictus females. Abdominal fanning did not coincide with tarsal waving and therefore was not a physiological response to the flexing of the meta thoracic legs but rather appears to be of conscious control. Aedes aegypti performed significantly more abdominal probing events than Ae. albopictus however the average duration of each probing event was not significant between the species. This directly correlates w ith significantly more eggs being laid by Ae. aegypti as a bdominal probing always preceded an egg deposition. Previous studies have shown that there are several receptors in the abdomen used during oviposition (Rossignol and McIver 2005).

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45 Abdominal probing may be another way that a female evaluates the substrate, or she may be using mechanoreceptors to find an appropriate groove in the substrate to deposit an egg. The cessation of all movement before egg deposition was seen in all Ae. aegypti females, but was only seen in one Ae. albopictus female. This leads to speculation that the stop observed in the Ae. albopictus female was not an intentional stop behavior, but rather a coincidence. Aedes albopictus do not stop all movement while depositing eggs, as t he metathoracic tarsi are nearly always waving during an egg deposition event. Aedes albopictus waved a single tarsus significantly more times than Ae. aegypti From visual observations during filming, it appeared that Ae. albopictus always had the same t arsus up as the direction she was travelling. Often, ovipositing females would encounter one another, usually resulting in the disruption of oviposition and the disrupted female flying away. Contact is usually first made by the meta thoracic tarsi, and a p ossible explanation for the single tarsal wave in the direction of movement is to disrupt other ovipositing females that may be in the oviposition pathway. Aedes aegypti were very rarely observed performing a single tarsal wave, which could be explained by their shorter oviposition event time, and therefore less chance of encountering another female while ovipositing. Aedes albopictus spent a significantly longer amount of time waving the tarsi during the entire oviposition event. Like Ae. aegypti they are considered opportunistic ovipositors, however this behavior would suggest that they spend more time analyzing the substrate, which also can be supported by their slow, calculated movements during oviposition as opposed to the quick, erratic movements of A e. aegypti Additionally, Ae. aegypti laid nearly twice as many eggs during each oviposition event. This behavioral difference between the two species could contribute to the cause of displacement of Ae. aegypti by Ae. albopictus If a female of each speci es lays eggs in a particular pool, and that pool contains larval predators, the Ae. aegypti

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46 female will lose twice as many progeny as the Ae. albopictus female who has deposited the other half of those eggs in a different location. Because Ae. albopictus appeared to spend more time analyzing the substrate, it may also be the case that Ae. albopictus are more capable of determining nutrient rich larval pools to deposit eggs. Based on the biology of both species, Ae. albopictus prefer to oviposi t in pools containing infusions, created by fermenting vegetation. Aedes aegypti are a more urban species, and are the predominant species in cities such as Tampa and Miami, whereas Ae. albopictus is the predominant species on the outski rts of cities and in the suburba n areas (Phil Lounibos personal comm.). Suburban landscaping and the use of ornamental plants such as bromeliads has provided ideal, nutrient rich habitat for Ae. albopictus whereas water filled container found in cities generally do not contain any fer menting vegetation, and therefore probably do not harbor the nutrients that suburban larval pools do. Superior larval nutrition may also be a contributing factor to the displacement of Ae. aegypti. Though the repertoire of behaviors for both species is ex tensive, both species examined were from one and two year old laboratory colonies. Behaviors of ovipositing females in the wild may be expected to be more discriminatory based on the fact that there is much more external stimulus in nature. Although ovipos ition behavior in wild populations of mosquitoes may vary from those of laboratory populations, the primordial behaviors for oviposition site selection are the same. It would be very difficult to obtain a comprehensive analysis of mosquito oviposition beha vior in the wild, albeit interesting. A greater understanding of the similarities and differences of oviposition behavior of these two medically important species can contribute to our understanding of potential avenues and short comings of oviposition bas ed control and surveillance and provide a baseline for future studies on potential interventions.

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47 Table 2 1. Aedes aegypti ethogram. Behavioral Category Behavior Description Location Egg p aper The subject is making contact with the egg paper with at le ast three legs Other The subject is in a location other than the egg paper, water, or air Water The subject is resting with at least four legs on the waters surface Movement Fly The subject is flying Walk d own The subject orients itself with the abdomen proximal to the water and walks backwards down to the water Walk u p The subject walks in an upward direction perpendicular to the water Walk h orizontally The subject walks along the egg paper parallel to the water Still The subject is stays in one location on the substrate Land The subject stops flying as the legs make contact with the egg paper Float The subject sits on top of the water with at least four legs in contact with the water Tarsal activity None No tarsal a ctivity Tarsal Groom The meta thoracic tarsi are rubbed together, or a meso or pro thoracic leg is placed in between the meta thoracic legs and rubbed Antennal Groom An antennae is rubbed between the prothoracic legs from the distal to the terminal end Alternating Tarsal Wave The meta thoracic tarsi are fanned up and down in opposite directions Simultaneous tarsal w ave The meta thoracic tarsi are fanned up and down parallel to one another Single tarsal w ave One metathoracic tarsi is fanned up and down Wing g room The wings are rubbed from the distal end to the proximal end by the meta thoracic legs Oviposition Abdominal p robe The tip of the abdomen makes quick repeated contact with the substrate Egg d eposition An egg is extruded No p robe No abd ominal activity Stop The subject becomes completely inactive

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48 Table 2 2. A edes albopictus e thogram Behavioral Category Behavior Description Location Egg p aper The subject is making contact with the egg paper with at least three legs Other The subj ect is in a location other than the egg paper, water, or air Water The subject is resting with at least four legs on the waters surface Movement Fly The subject is flying Walk d own The subject orients itself with the abdomen proximal to the water an d walks backwards down to the water Walk u p The subject walks in an upward direction perpendicular to the water Walk h orizontally The subject walks along the egg paper parallel to the water Still The subject is stays in one location on the substrate Land The subject stops flying as the legs make contact with the egg paper Float The subject sits on top of the water with at least four legs in contact with the water Tarsal Activity None No tarsal a ctivity Tarsal g room The meta thoracic tarsi are rubbed together, or a meso or pro thoracic leg is placed in between the meta thoracic legs and rubbed Antennal g room An antenna is rubbed between the prothoracic legs from the distal to the terminal end Alternating tarsal w ave The meta thoracic tars i are fanned up and down in opposite directions Simultaneous tarsal w ave The meta thoracic tarsi are fanned up and down parallel to one another Single tarsal w ave One metathoracic tarsi is fanned up and down Wing g room The wings are rubbed from the d istal end to the proximal end by the meta thoracic legs Abdominal g room The posterior end of the abdomen is rubbed by one or both of the metathoracic legs Abdominal fan The abdomen is held rigid while being raised and lowered in a fanning motion parall el to the substrate Oviposition Abdominal p robe The tip of the abdomen makes quick repeated contact with the substrate Egg d eposition An egg is extruded No p robe No abdominal activity Stop The subject becomes completely inactive

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49 Table 2 3. Average number of times behaviors were observed by ovipositing Aedes aegypti and Aedes albopictus females. Behavior Aedes aegypti Average count ( + SE) Aedes albopictus Average count ( + SE) P Abdominal g room 0.0 ( + 0.0) 2.6 ( + 0.6) 0.015 Abdominal fan 0 .0 ( + 0.0) 3.3 ( + 0.9) <0.001 Abdominal probe 12.4 ( + 1.3) 4.4 ( + 1.1) <0.001 Antennal g room 0.0 ( + 0.0) 3.0 ( + 0.5) 0.168 Egg d eposition 1 0.2 ( + 1.1) 5.5 ( + 0.6) 0.001 Egg p aper 2.2 ( + 0.3) 2 .0 ( + 0.3) 0.613 Float 1.3 ( + 0.3 ) 1.5 ( + 0.3) 0.431 Fly 1.7 ( + 0.3) 1.4 ( + 0.2) 0.185 Jump 0.0 ( + 0.0) 1.0 ( + 1.3) 0.167 Land 1.6 ( + 0.4) 1.3 ( + 0.2) 0.687 No p rob e 3.0 ( + 0.5) 3.6 ( + 0.5) 0.303 None 2.9 ( + 0.5) 8.0 ( + 1.6) 0.004 Other 2.1 ( + 0. 4) 2.3 ( + 0.2) 0.498 Still 2.5 ( + 0.6) 13.1 ( + 1.7) <0.001 Stop 10.4 ( + 1.1) 5.0 ( + 0.5) <0.001 Tarsal g room 3.7 ( + 0.7) 10.4 ( + 4.9) 0.659 Total tarsal wave 2.6 ( + 0.8) 16.9 ( + 6.2) <0.001 Alternating tarsal w ave 1.5 ( + 0.4) 4 .9 ( + 1.6) 0.100 Simultaneous tarsal w ave 1.0 ( + 0.4) 16.8 ( + 3.9) <0.001 Single tarsal w ave 0.7 ( + 0.3) 9.5 ( + 1.5) <0.001 Total w alk 2.5 ( + 0.6) 10.0 ( + 1.9) 0.001 Walk d own 1.1 ( + 0.3) 0.3 ( + 0.2) 0.025 Walk u p 0.5 ( + 0. 2) 0.7 ( + 0.3) 0.148 Walk horizontally 2.4 ( + 0.4) 12.0 ( + 1.9) <0.001 Water 1.3 ( + 0.2) 1.3 ( + 0.2) 0.217 Dip 0.0 ( + 0.0) 0.5 ( + 0.5) 0.386 Note: Asterisks represent statistically significant P values when P < 0.05. N=10 for all be ahviors for each species.

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50 Table 2 4. Average duration of behaviors exhibited by ovipositing Aedes aegypti and Aedes albopictus females. Note: Asterisks represent statistically significant P values when P < 0.05. N=10 for all beahviors for each species. Behavior Aedes aegypti Average Duration sec (+SE) Aedes albopictus Average Duration sec (+SE) P Total o bservation 369.1 ( + 77.0 ) 431.4 ( + 80.2) 0.473 Abdominal g room 0.0 ( + 0.0) 10.8 ( + 2.2) 0.015 Abdominal f an 0.0 ( + 0.0) 33.0 ( + 14.5) <0.001 Abdominal p robe 170.4 ( + 36.1) 178.3 ( + 28.0) 0.616 Antennal g room 0.0 ( + 0.0) 0.6 ( + 0.5) 0.168 Egg paper 317.2 ( + 64.6) 389.5 ( + 83.8) 0.514 Float 12.7 ( + 3.2) 26.5 ( + 7.1) 0.600 Fly 13.2 ( + 5.3) 12.4 ( + 3.2) 0.818 No p robing 171.3 ( + 60.5) 210.2 ( + 81.4) 0.781 No ne 230.6 ( + 40.8) 80.0 ( + 22.3) 0.002 Other 47.4 ( + 23.9) 32.5 ( + 13.0) 0.384 Still 152.9 ( + 58.5) 343.9 ( + 81.0) 0.002 Stop 91.1 ( + 40.9) 19.0 ( + 1.9) 0.384 Tarsal g room 104.8 ( + 43.0) 103.5 ( + 47.2) 0.755 Total tarsal w ave 32.3 ( + 10.1) 213.3 ( + 34.8) <0.001 Alternating tarsal w ave 19.3 ( + 5.8) 46.2 ( + 14.6) 0.301 Simultaneous tarsal w ave 17.2 ( + 1.7 ) 130.8 ( + 25.5) <0.001 Single tarsal w ave 7.6 ( + 1.7) 50.1 ( + 12.1) <0.001 Total w alk 195.5 ( + 37.7) 66.9 ( + 10.0) <0.001 Walk d own 8.5 ( + 3.2) 5.0 ( + 0.7) 0.019 Walk u p 2.0 ( + 0.8) 1.0 ( + 0.7) 0.733 Wa lk h orizontally 186.5 ( + 38.8) 61.8 ( + 8.5) 0.002 Water 6.0 ( + 1.9) 23.5 ( + 6.2) 0.721 Wing g room 7.8 ( + 3.7) 22.0 ( + 13.3) 0.965

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51 Table 2 5. Transition matrix for location of Aedes aegypti during oviposition. Note: Transitions (excluding start and end observations) in bold occurred at greater than expected levels (chi square). Behaviors are defined in Table 2 1. Table 2 6. Transition matrix for location of Aedes albopictus during oviposition. Location Succeeding behavioral event Preceding behavi oral event Other Egg p aper Water End o bservation Other 0% 61.8% 0% 33.2% Egg p aper 66.7% 0% 20.0% 10.0% Water 25.0% 75.0% 0% 0% Start o bservation 25.0% 0% 0% 0% Note: Transitions (excluding start and end observa tions) in bold occurred at greater than expected levels (chi square). Behaviors are defined in Table 2 1. Table 2 7. Transition matrix for movement of Aedes aegypti during oviposition. Movement Succeeding behavioral event Preceding behavioral event Fly Walk Still Land Float End o bservation Fly 0% 0% 0% 27.5% 0% 72.5% Walk 33.6% 27.8% 23.5% 0% 15.0% 0% Still 15.7% 44.1% 0% 40.3% 0% 0% Land 10.0% 69.0% 9.0% 2.0% 0% 0% Float 16.7% 83.3% 0% 0% 0% 0% Start o bservation 0% 0% 25.0% 0% 0% 0% Note: Transitions (excluding start and end observations) in bold occurred at greater than expected levels (chi square). Behaviors are defined in Table 2 1. Location Succeeding behavioral event Pr eceding behavioral event Other Egg Paper Water End observation Other 0.0% 59.4% 15.0% 23.6% Egg paper 37.5% 0% 30.0% 30.0% Water 14.3% 85.7% 0 % 0% Start observation 25.0% 0% 0% 0%

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52 Table 2 8. Tran sition matrix for movement of Ae des albopictus during oviposition. Movement Succeeding behavioral event Preceding behavioral event Fly Walk Still Jump Land Float Dip End observation Fly 0% 0% 0% 0% 12.5% 0% 6.3% 81.25% Walk 1.7% 0% 91.1% 1.1% 0% 3.5% 0% 0% Still 6.0% 84.1% 0% 0% 8.2% 0% 0% 1.8% Jump 0% 0% 100.0% 0% 0% 0% 0% 0% Land 0% 80.0% 15.0% 5.0% 0% 0% 0% 0% Float 33.3 % 66.7% 0% 0% 0% 0% 0% 0% Dip 0% 0% 0% 0% 2.0% 0% 0% 0% Start observation 25.0% 0% 0% 0% 0% 0% 0% 0% Note: Transitions (excluding start and end observations) in bold occurred at greater than expected levels (chi square). Behaviors are defined in Table 2 1. Table 2 9. Transition matrix for tarsal activity of Aedes aegypti during oviposition. Tarsal Activity Succeeding behavioral event Preceding behavioral event None Tarsal g room Wing g room Tarsal w ave End o bservation None 0% 8.3% 2.5% 47.5% 41.6% Tarsal g room 25.7% 0% 21.4% 20.4% 2.5% Wing g room 0% 36.7% 0% 3.3% 0% Tarsal w ave 32.7% 30.8% 3.3% 12.0% 1.3% Start o b servation 25.0% 0% 0% 0% 0% Note: Transitions (excluding start and end observations) in bold occurred at greater than expected levels (chi square). Behaviors are defined in Table 2 1.

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53 Table 2 10. Transition matrix for tarsal activit y of Ae. albopictus during oviposition. Tarsal Activity Succeeding behavioral event Preceding behavioral event None Tarsal g room Abdominal g room Tarsal w ave Wing g room Antennal g room End o bservation None 0% 1.8% 0% 84.5% 0% 1.2% 12.6% Tarsal g room 1.6% 0% 9.2% 70.2% 16.8% 2.0% 0% Abdominal g room 0% 80.0% 0% 20.0% 0% 0% 0% Tarsal wave 22.7% 12.8% 2.3% 55.5% 3.3% 0% 3.3% Wing g room 0% 86.0% 0% 12.7% 0 % 0% 0% Antenn al g room 20.0% 30.0% 0% 50.0% 0% 0% 0% Start o bservation 25.0% 0% 0% 0% 0% 0% 0% Transitions (excluding start and end observations) in bold occurred at greater than expected levels (chi square). Behaviors are defined in Table 2 1. Table 2 11. Transition matrix for oviposition activity of Aedes aegypti during oviposition. Oviposition Succeeding behavioral event Preceding behavioral event Egg deposition No Probe Abdominal probe Stop End observation Egg d eposit ion 0% 4.0% 94.2% 1.8% 0% No p robe 0% 0% 80.2% 0% 19.8% Abdominal p robe 0% 13.0% 0% 82.6% 2.6% Stop 97.8% 0% 2.3% 0% 0% Start o bservation 0% 25.0% 0% 0% 0% Transitions (e xcluding start and end observations) in bold occurred at greater than expected levels (chi square). Behaviors are defined in Table 2 1.

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54 Table 2 12. Transition matrix for oviposition activity of Aedes albopictus during oviposition. Note: Transitions (excluding start and end observations) in bold occurred at greater than expected levels (chi square). Behaviors are defined in Table 2 1. Oviposition Succeeding b ehavioral event Preceding behavioral event Egg d eposition No p robe Abdominal p robe Stop Abdominal f an End o bservation Egg d eposition 44.3% 14.6% 9.6% 1.7% 29.9% 0% No p robe 0% 0% 40.3% 0% 25.4% 31.6% Abdominal p robe 71.2% 12.8 % 0% 4.4% 8.5% 0% Stop 100.0% 0% 0% 0% 0% 0% Abdominal f an 0% 23.8% 72.9% 0% 0% 0% Start o bservation 0% 25.0% 0% 0% 0% 0%

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55 Figure 2 1. Behavioral setup used for video capture of oviposition behaviors of mosquitoes. A) Analog CCD Camera. B) 20 cm x 20 cm x 20 cm cage containing gravid mosquitoes and oviposition substra te. C) LCD Infrared lights. D) R eal time MPEG encoder digitizer. E) Laptop with MediaCruise recording software and Noldus behavioral analysis software. A B C D E

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56 Figure 22. A kinematic diagr am for location of ovipositing Aedes aegypti Boxes represent behavioral states and arrows represent transitions between behaviors. Numbers next to arrows represent percentages of transitions Figure 2 3. A kinematic diagram for movement of ovipositing Aedes albopictus Boxes represent behavioral states and arrows represent transitions be tween behaviors. Numbers next to arrows represent percentages of transitions.

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57 Figure 24. A k inematic diagram for movement of ovipositing Aedes aegypti Boxes repres ent behavioral states and arrows represent transitions between behaviors. Numbers next to arrows represent percentages of transitions. Figure 2 5. A kinematic diagram for movement of ovipositing Aedes albopictus Boxes represent behavioral states and arr ows represent transitions between behaviors. Numbers next to arrows represent percentages of transitions.

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58 Figure 2 6. A kinematic di agram for tarsal activity of ovipositing Aedes aegypti Boxes represent behavioral states and arrows represent transitions between behaviors. Numbers next to arrows represent percentages of transitions. Figure 2 7. A kinematic diagram of tarsal activity of ovipositing Aedes albopictus. Boxes represent behavioral states and arrows represent transitions between behavior s. Numbers next to arrows represent percentages of transitions.

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59 Fig ure 2 8. A kinematic diagram of oviposition activity of Aedes aegypti Boxes represent behavioral states and arrows represent transitions between behaviors. Numbers next to arrows rep resent percentages of transitions. Figure 2 9. A k inematic diagram of oviposition activity of Aedes albopictus. Boxes represent behavioral states and arrows represent transitions between behaviors. Numbers next to arrows represent percentages of transi tions.

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60 Figure 2 10. Time budgets for location of Aedes aegypti and Aedes albopictus during oviposition. Locations marked with an asterisk are significantly different between t test).

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61 F igure 2 11. Time budgets for movement of Aedes aegypti and Aedes albopictus during oviposition. Movements marked with an asterisk are significantly different between t test).

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62 Figure 212. Time budgets for tarsal activity of Aedes ae gypti and Aedes albopictus during oviposition. Tarsal activity behaviors marked with an asterisk are significantly t test).

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63 Figure 213. Ti me budgets for oviposition activity of Aedes aegypti and Aedes al bopictus during ovipos i tion. Ovipostion behaviors marked with an asterisk are statistically different t test).

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64 Figure 2 14. A representative visual analysis of Aedes aegypti (top) and Ae. albopictus (bottom) show ing overlapping behaviors during oviposition as created by The Observer XT behavioral software.

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65 CHAPTER 3 A COMPARATIVE ANALYSIS OF OVIPOSITION BEHAVIOR BETWEEN CULEX PIP I ENS QUINQUEFASCIATUS SAY AND CULEX TARSALIS COQUILLETT Introduction Culex species ran k among the most important vector species in the United States, and specifically Florida (Nayar et al. 2001). Currently these species are associated with St. Louis encephalitis (SLE) Eastern equine encephalitis (EEE) (Nayar et al. 2001), and more recentl y West Nile virus (WNV) which are notifiable illnesses in Florida (Girard et al. 2007). These diseases can infect people, but more often occur in horses, making the control of these diseases economically important due to the high number of race horse bree ders in the state. Rift Valley fever primarily a disease of Afr ican ruminants, but also infecting humans, is also considered a threat for introduction and the viral cycle sustained by these species (Gargan et al. 1988). Diseases in the encephalit id es com plex are often fatal to horses, or causes severe central nerve damage leading to euthanasia (Porter et al. 2003), meaning the control of Culex mosquitoes is important both for health and economics. Besides vectoring pathogen s, mosquitoes inflict irritating bites and are considered a pest around the world. Severe mosquito emergences can negatively impact tourism, recreation, and even affect livestock production (Foster and Walker 2002). Because most mosquitoborne illnesses are prevalent in warmer temperatur es, the American Meteorological Society is now considering mosquitoborne illnesses representative of global warming patterns, using both location of disease reports and the time frames in which the diseases occur as a measure of warming patterns (Epstein et al. 1998). Culex quinquefasciatus Say is present throughout the state of Florida and up the E ast coast, west through Texas and the Midwest, and north to southern Canada (Darsie and Ward 2005). Culex quinquefasciatus is nocturnally active for oviposition, and deposit organized egg rafts in sewage water, drains, gutters, or other fermenting still water often found around houses (King et

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66 al. 1960). While this species is the primary vector of WNV in the Southeast, it is also competent in transmitting several other endemic diseases (Foster and Walker 2002). Culex tarsalis can be found nearly everywhere in the United States with the exception of the East coast and is rarely scattered throughout Georgia and north Florida (Reisen 1993, Darsie and Ward 2005). Li ke other Culex species, Cx. tarsalis is crepuscular and nocturnal in oviposition activity (Reisen 1993). Relatively little is known about oviposition behavior in Cx. tarsalis ; however, they do lay egg rafts containing up to 190 eggs per batch in wat er pool s exposed to sunlight that often are surrounded by abundant vegetation. Culex tarsalis is a vector of pathogens causing WNV, SLE, Japanese equine encephalitis, Venezuelan equine encephalitis (Reisen 1993, Reisen et al. 1993), and potentially Rift Valley f e ver (Gargan et al. 1988). Nearly all mosquitoes begin seeking an oviposition site when gravid, which can be categorized into four basic steps: ranging flight, orientation, encounter, and acceptance of the substrate (C lements 1999). After completion of embryonic development in the eggs rainfall, humidity, temperature, and wind speed determine the timing for an ovipositional flight (Bentley and Day 1989). Once flight has been initiated, visual, olfactory, and chemical cues lead a female to a potential oviposition substrate (Bentley and Day 1989). As a female approaches a potential site, several different factors determine whether the site is acceptable, which will vary between species, and between individuals. Humidity and water volatiles are assessed before landing, and salinity, temperature, and oxygen cont ent are generally assessed upon contact (Clements 1999). Many species s are attracted to water that contains conspecific larvae (Wilmont et al. 1987, Zahiri and Rau 1998, Allan et al. 2005). Additionally i n Culex species an ovipos ition pheromone, identified as [( ) (5R,6S) 6acetoxy 5hexadecanolide], is present in apical droplets on eggs in egg rafts (Laurence and Pickett 1982). This pheromone appears to have a domino effect on

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67 oviposition, and rapidly at tracts females of the same species to lay eggs in the same substrate (Laurence and Pickett 1982). The oviposition pheromone is enhanced in the presence of infusion volatiles (Braks et al. 2007) and used for gravid traps in the field (Mboera et al. 2000). O viposition involved various sensory structures, though not all have been identified. Initial location of an appropriate oviposition site for landing relies partially on sensilla of the antenna (Davis and Bowen 1994). Once a female de termines an oviposition substrate acceptable for landing, she will make contact with the water several times while in the air using the meta thoracic tarsi presumably detecting waterborne com pounds Tarsal receptors are generally short, and either blunt or sharp sensilla trichodea, and may possibly detect both chemical compounds in the substrate, and airborne volatiles such as oviposition pheromones from previously deposited eggs (Davis and Bowen 1994). The gustatory sensilla are located on the mouthparts and have also been reported from the distal end of the tibiae and tarsi of C uliseta i nornata (Williston). These sensilla contain neurons capable of detecting salinity and glucose concentrations in water (Owen 1963, Lee and Craig 1983). The same gustatory sensilla have also been reported from uniporous peg s ensilla on the mouthparts, and are identical to the gustatory sensilla on the legs capable of dectecting s alinity and glucose concentrati ons Females of Culex have been reported to drink from the oviposition substrate before ovipositing (Wallis 1954, Hudson 1956, Weber and Tipping 1990) and it remains unclear whether this is for evaluation of oviposition substrate, or to increase hydrostatic pressure to aid the release of eggs (Hudson 1956, Weber and Tipping 1990). When Hudson (1956) amputat ed the proboscis, palps, or both of Cx. molestus Forskal oviposition substrate choice was not altered, suggesting that drinking did not help discriminate between sites however Ikeshoji (1966) reported that oviposition greatly decreased when the proboscis of Cx. fatigans Weidemann was amputated Though many Culex species oviposit

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68 directly on the water, they also appear to contact the water with the end of the abdomen before oviposition commences (Wallis 1954, Beament and Corbet 1981) however little is known about the extent of this behavior This contact may be for further discrimination of the substrate, or a tactile behavior to locate the next position for the egg in the egg raft. Understanding the steps involved in mosquito oviposition ma y provide a weak link in the life cycle where interventions such as biocontrol agents, toxicants, or attract and kill can be utilized Because the general pattern of pathogen transmission requires an initial bloodmeal to acquire the pathogen, pathogen transmission is greatly dependent on at least one oviposition c ycle (Weaver and Barrett 2004). The second bloodmeal is essential for transmission of the pathogen, but often a second bloodmeal will not occur befor e the first oviposition cycle has been complete d (Bentley and Day 1989). This provides an opportunity for exploitation of the contact of the mosquito with the oviposition site for potential control strategies. The objective of this study was to conduct a detailed comparative analysis of oviposition behavior in two important Culex vectors, Cx quinquefasciatus and Cx tarsalis Materials and Methods Mosquito Colonies A colony of Cx quinquefasciatus was established from collections in Gainesville, FL in 1995. Larvae were reared in plastic pans (35 c m x 4 8 cm x 6.25 cm) containing 1500 ml well water, and were fed every other day with 50 ml larval slurry made from a 1:1 mix of liver powder and brewers yeast (MP Biomedical, Solon, OH) mixed with well water (10 g dry mixture per 100 ml water). Pupae were p laced in a plastic cup containing water and set inside a 30 cm x 30 cm x 30 cm plexiglass and screen cage to emerge. Adults had constant exposure to cotton saturated with 5% sucrose solution (50 g sucrose in 1000 ml water). Seven days after emergence, adul ts were bloodfed on a chicken for up to 15 minutes ( University of Florida,

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69 IACUC No. 469). Five days post bloodfeeding, adults were provided a black egg cup containing 500 ml well wa ter and 2.0 ml hay infusion prepared following Reiter et al. (1991). After 24 hr egg cups were removed, and the egg rafts were set in plastic pans (35 cm x 48 cm x 6.25 cm) with 4 5 egg rafts per pan. All stages of development were kept in a temperaturecontrolled incubator ( M odel 818 Precision Scientific, Teynampet India) at 28+ 1 oBehavioral Experimental Design C, with a 14:10 light/dark photoperiod and an 8085 % relative humidity. Photophase began at 8:00 pm. A C x tarsalis colony was established in 1993 from a colony from Coachella Valley, California All rearing methods follow those of Cx. quinquefasciatus A 20 cm x 20 cm x 20 cm plexiglass chamber with one side consisting of tubular gauze for access was placed under a metal frame, and the frame was draped with a blackout cloth (Duvetyne) on all sides, including over the c amera to simulate nocturnal conditions (Fig. 2 1). Two infrared lights (940 nm, Rainbow CCTV, Costa Mesa, CA) were placed on top of the chamber. Mosquitoes were filmed with a 50mm F2.8 Telecentric lens (Rainbow CCTV, Costa Mesa, CA) on a CCD camera (Hi res DNL 46D 1/3 DSP) (Rainbow CCTV, Costa Mesa, CA) mounted on an adjustable tripod (Manfrotto 3021BN Bogen, Ramsey, NJ). Cameras were set up directly in front of the chamber. Ten gravid females were added to the chamber using a battery operated aspirator ( Hausherrs Machine Works, Toms River, NJ) and allowed to settle for 10 minutes. After 10 minutes, a petri dish (50 cm diameter) painted with flat black paint was filled with well water (50 ml) For Cx. quinquefasciatus 5 ml of 15 day old hay infusion was added to the water and for Cx. tarsalis 1 ml of 15 day old hay infusion was added and the dish and placed inside the chamber.

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70 Video Analysis Oviposition behavior was recorded as described above with analog cameras then digitized with a real time MPEG en coder (Canopus EMR100, Canopus, San Jose, CA.), and MediaCruise computer software (Canopus, San Jose, CA.) Each video clip consisted of an oviposition event by a single gravid female and the recording was started when a female was seen flying close to the su bstrate, or upon first landing. As soon as the female started oviposition behavior, the camera was focused to provide maximum v isualization of the behaviors. Recording was stopped after an oviposition event when the female left the substrate. Ten videos of the entire oviposition sequence were obtained and analyzed for both species Each digitized video clip was analyzed using behavioral analysis software ( The Observer XT Version 6.1.4, Noldus Information T echnology, Inc., Leesburg, VA). Prior to analysis video clips were viewed and behaviors defined for the ethogr ams. These behaviors were then categorized as point events, which were quick, single event behaviors, or state events, which had measurable durations, and a coding scheme dev eloped by the softwa re program. Through an iterative process with several video clips from both species, an appropriate coding scheme was established. The coding scheme developed for these analys es is presented in Appendix 1. The videos were watched and replayed and each defi ned behavior scored in The Observer XT software using assigned keys on a computer keyboard to obtain a time stamp After each video clip was scored, a behavioral analysis, raw number lag sequential analysis, and proportion lag sequential analysis was run i n the behavioral analysis software ( The Observer XT). Recordings for both species were viewed and all behavior al steps associated with oviposition were categorized, compared to known literature, and defined. Behaviors were split into five different catego ries: location of the subject in space, overall body movement of the

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71 subject, tarsal activity of the subject, behaviors directly related to ovposition, and drinking behavior. Each behavior within a category is mutually exclusive of all other behaviors in t he same category; however behaviors between categories may occur simultaneously. These behaviors formed the basis for the subsequent kinematic analysis and detailed behavioral analysis (above). The number of occurrences and durations of each behavior were calculated and averaged. Paired statistical comparisons were made between species for each behavior to determine sig nificant differences between Cx. quinquefasciatus and Cx. tarsalis The proportional lag sequential analysis for each individual was automa tically generated by The Observer XT, and exported to Microsoft Excel (Microsoft Corporation, Redmond, WA). All ten individuals for each species were averaged, and a transition matrix was developed for each species, which quantified how often one behavior followed another The matrix table was used to calculate the transition frequency for each behavior by calculating the percentage one action follows another action. Based on the percentage rates, a kinematic diagram was created to visualize the action patterns of the study subjects. Behaviors that started or ended a sequence were designated as such. Time budgets were created for each species and divided into the previously mentioned five categories. Time spent on each behavior by individual mosquitoes was calculated as the percentage of time within a category. Data Analysis Data from the behavioral analyses were exported to Microsoft Excel. Data was square root trans formed, and transformed data were exported to SigmaStat (Systat Software, Chicago, IL) to test for normality (ShapiroWilk test) All normally distributed data were analyzed using a paired t test ( P < 0.05). Data that was not distributed normally w ere analyzed using a Mann Whitney

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72 Rank Sum test. Data for time budgets were percentages and therefo re were arcsine transformed before means were obtained and compared between species using paired t tests. In the transition matrix tables, frequencies above 0 were compared by chi square analysis to determine those occurring at greater than expected level s ( P < 0.05). Results Ethograms were developed for Cx. quinquefasciatus (Table 3 1) and Cx. tarsalis (Table 3 2). Culex quinquefasciatus sometimes performed a SIMULTANEOUS TARSAL WAVE, which was not seen in Cx. tarsalis U nique behaviors for Cx. tarsalis inc luded an ALTERNATING TARSAL WAVE and a PROBOSCIS FLICK during oviposition. Otherwise, b oth Cx. quinquefasciatus and Cx. tarsalis females were very similar in behaviors exhibited during oviposition (Table 33). Culex quinquefasciatus were observed DRINKING significantly more times than Cx. tarsalis ( T =135.0, df=20, P =0.025), and as a result performed the NOT DRINKING behavior significantly more times ( T =134.0, df=20, P =0.030) Culex tarsalis performed a PROBOSCIS FLICK significantly more often ( T =55.0, df= 20, P < 0.001) which was a behavior not seen in Cx. quinquefasciatus The total duration of oviposition behavior did not differ significantly between Cx. quinquefasciatus and Cx. tarsalis (Table 3 4). Females of Cx. quinquefasciatus spent more time than Cx. tarsalis females in the AIR ( t =2.299, df=20, P =0.034), and DRINKING ( T =135.0, df=20, P =0.026) but less time FLYING (t=3.603, df=18, P =0.007). Transition tables and kinematic diagrams show major action patterns for each species. For both species, signi ficant patterns in location were OTHER followed by WATER and WATER preceded by AIR (Tables 3 5, 36 ). For both species for movement behaviors, significant patterns were seen, such as STILL before LAND, and DIP before DIP (Table 3 7, 38). In the latter cas es, sequential DIP events occurred. The two species differed in other

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73 movement behaviors, with Cx. quinquefasciatus showing a pattern of WALK before STILL and LAND just before FLY (Table 3 7). Additionally, Cx. tarsalis had a pattern of WALK before FLY an d LAND just before WALK (Table 3 8). For tarsal associated behaviors, the only significant pattern similar between the two species was TARSAL LINKING followed by UNLINKING (Table s 39, 3 10). For Cx. quinquefasciatus additional significant patterns were seen with a SINGLE TARSAL WAVE followed by a SINGLE TARSUS RAISED followed by NONE and SIMULTANEOUS TARSAL WAVE followed by BOTH TARSI RAISED (Table 3 9). For Cx. tarsalis, significant patterns seen included SINGLE TARSAL WAVE ALTERNATING TARSAL W AVE a nd SINGLE TARSUS RAISED followed by NONE (Table 3 10). For oviposition behaviors, both species showed similar significant trends with NO PROBING followed by ABDOMINAL PROBE ABDOMINAL PROBE preceding EGG DEPOSITION and EGG DEPOSITION followed by repeated EGG DEPOSITION (Tab les 3 11, 312). For Cx. tarsalis PROBOSCIS FLICK also preceded EGG DEPOSITION. For both species, DRINK occurred repeatedly (Tables 3 13, 314). In both species, major action patterns, classified as having a transition frequency of 50% or higher, for location during oviposition were OTHER followed by WATER f ollowed by AIR (Figure s 3 2, 33). Major movement behavioral patterns seen in Cx. quinquefasciatus were STILL, followed by LAND, followed by STILL, and WALK was always followed by S TILL (Figu re 3 4). In Cx. tarsalis the major movement action patterns were STILL followed by LAND followed by WALK followed by FLY (Figure 3 5). Both species also showed repetitive DIP patterns before LAND Major action patterns of tarsal activity for Cx. quinquefasciatus include SIMULTANEOUS TARSAL WAVE followed by BOTH TARSI RAISED, followed by

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74 a SINGLE TARSUS RAISED, followed by NONE (Figure 3 6). Additionally, TARSAL LINKING was often followed by TARSAL UNLINKING, which was also seen in Cx. tarsalis Culex tarsalis differed slightly in their tarsal activity action patterns, often exhibiting patterns of SINGLE TARSAL WAV E to NONE SINGLE TARSUS RAISED or BOTH TARSI RAISED to an ALTERNATING TARSAL W AVE and TARSAL LOWERING followed by TARSAL LINKING (F igure 3 7 ). Major ovipositional patterns in both species were NO PROBING followed by ABDOMINAL PROB E and repe ated EGG DEPOSITION (Figure s 3 8, 39). ABDOMINAL PROB E followed by EGG DEPOSITION, as well as PROBOSCIS FLICK followed by an EGG DEPOSITION was also observed in Cx. tarsalis Finally, drinking patterns in both species were the same, which was DRINK followed by NO DRINKING, followed by DRINK (Figure s 310, 3 11). Both species were similar in how they budget their time during oviposition. Culex quinquefasciatus spent significantly more time FLYING ( t =2.50, df=18, P =0.022) (Figure 312), and as a result, significantly more time in the AIR ( t =149.00, df=20, P =0.001) (Figure 313). All other behaviors were not significantly different between species (Fi gure 3 14, 315, 316). Figure 317 shows a comparative visual representation of all behaviors over time of each species. Discussion Initial studies reporting on Culex oviposition and egg raft formation were conducted on Cx. molestus and Cx. restuans Theobald Hudson (1956) reported that females would fly forwards and backwards over the substrate before landing Culex molestus often exhibit a dipping behavior, analyzing several different pools before choosing a site (Hudson 1956). Once Cx. molestus chose a site, they were often observed insert ing the proboscis into the water and drinking the substrate Drinking behavior has also been reported in Cx. restuans and Cx. pipens pipi ens Linnaeus (Weber and Tipping 1990). Some theories for this behavior include fur ther

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75 discrimination of the site, or to increase hydrostatic pressure to help with the release of the eggs (Hudson 1956; Weber and Tipping 1990). In the current study, once a female landed on the water, the metathoracic legs were held upward and contact w ith the water was made only by the prothoracic and meso thoracic legs. The metathoracic legs were lowered so that the tarsi lay horizontally along the surface and the distal tibia was in contact with the substrate. The distal tibiae were moved together, somet imes crossing, forming a pen to control egg raft formation and the tip of the abdomen was lowered into the water. Wallis (1954) described the same general pattern, but with drinking occurring before the metathoracic legs were lowered. Hudson (1956) did not observe drinking in all studies. In the current study, dipping behavior of the meta thoracic tarsi was observed, although not by all subjects. This could be explained by the difference in species and long colonization periods, and would be expected to be more frequent in nature as external stimulus cannot be controlled. In the current study, there was no evidence of flying backwards, even when the video was slowed down. Contrary to Hudsons study, upon landing, females in the current study most ofte n only raised one meta thoracic tarsus, not both. The role that tarsal receptors play in oviposition is not known but the reason for raising one or both tarsi could be an additional evaluation of the surrounding. Based on personal observations, females oft en dipped the hind tarsi into the water directly where other females were ovipositing, which leads me to speculate that raising a tarsus above the body may also be a form of protection from other females flying overhead. Drinking was observed in nearly al l vi deos, but occurred more often with Cx. quinquefasciatus and each drinking episode by Cx. quinquefasciatus longer than observed in Cx.

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76 tarsalis Culex quinquefasciatus prefer to oviposit in substrate high in organic matter (Allan et al. 2005), whereas C x. tarsalis prefer to oviposit in newly formed freshwater pools surrounded by vegetation (Reisen 1993). Larvae can be found in both freshwater and saline flood pools, although water with high organic content is a deterrent (Reisen 1993). Culex tarsalis lar vae feed on microfloral blooms, often being the first mosquito to colonize a newly formed pool (Reisen 1993). Although not recorded, Cx. tarsalis took considerably longer to begin oviposition activity than Cx. quinquefasciatus Based on observations and l ife history strategies of these two species, it would appear that Cx. quinquefasciatus relies heavily on gustatory cu es for substrate discrimination and Cx. tarsalis relies heavily on visual cues. Hudson (1956) also reported that females lay the metathora cic tarsi horizontally on the waters surface to guide raft formation. Although not all subjects in the current study linked the meta thoracic tarsi, several did, and those that did not, moved the meta thoracic tarsi back and lined them up so that they wer e parallel, and eggs could be laid between. Females in the current study did not lay their entire meta thoracic tarsi on the waters surface. Distal segments of the tarsi w ere often arched up Wallis (1954) described drinking behavior occurring before th e metathoracic tarsi were lowered, and Hudson (1956) described it after the lowering of the tarsi. Females in the current study drank more than once, and were often observed drinking throughout the oviposition event, supporting the idea that drinking incr eases hydrostatic pressure aiding in the extrusion of eggs. In the case of Culex mosquitoes, drinking most likely functions as both an aid in egg extrusion, as well as a gustatory evaluation of the oviposition substrate. Additionally, understanding drinking behavior better increases the likelihood of developing a control strategy based on ingestion of the substrate. Although the larvicidal bacterium Bacillus thuringiensis israelensis ( B ti) is targeted at

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77 the control of larval mosquitoes, Zahiri and Mulla (2005) reported that the ingestion of B titreated water decreased oviposition, and increased mortality in both male and female adults. By exploiting the drinking behavior of most Culex mosquitoes, developed toxicants or biological control agents could be use d in ovitraps to target multiple life stages of Culex mosquitoes, therefore increasing the success of control strategies. Culex quinquefasciatus and Cx. tarsalis are related species, vectoring many of the same pathogens and often inhabiting similar habita ts in different parts of the United States Because of the analogous relationship, it can be expected that ovi position behavior would be similar. The original intent of this study was to include an additional two species: Cx. salinarius Coquillett and Cx. nigripalpus Theobald, however, due to colony collapse, oviposition behavior analysis was only partially completed with these two species and therefore not included in the current study. Based on visual observations and partial analysis, Cx. salinarius and Cx. nigripalpus were also very similar in their oviposition behavior to each other ; however the oviposition behavior of these two species was slightly different than the behavior of Cx. quinquefasciatus and Cx. tarsalis Culex salinarius took several hours to begin an oviposition event, and would nearly always l and on the substrate, drink, fly away, and rest before returning to the substrate to oviposit. Analysis of Cx. salinarius and Cx. nigripalpus will be completed in the future, and all four species wil l be compared to one another. While research has focused on the oviposition preferences of mosquitoes, little attention has been given to the means by which these substrates are assessed. Very few comparative analyses of oviposition behavior have e ver published. Those that have are dated, but the development of new technology allows us to look closely at insect behaviors that may never have been seen with the unaided eye. By closely examining individual behaviors such as

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78 drinking, new control strategies m ay be developed that would allow for the control of mosquitoes at multiple life stages using a single control strategy. Additionally, these studies provide a baseline for examining the means by which adult females may be able to detect adverse conditions s uch as toxicants, parasites or predators in the environment.

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79 Table 3 1. Culex quinquefasciatus ethogram Category Behavior Definition Location Air The subject is in the air Water The subject has at least four legs contacting the surface of the water Other The subject is in a location other than the water or air Movement Fly The subject is flying through the air Dip The meta thoracic tarsi make contact with the waters surface while the subject is flying in the air Land The subject settles on the waters surface with at least the proand meso thoracic legs. Can also include meta thoracic legs contacting surface. Walk The subject is walking. This can be on any surface Still The subject remains in one location and is not walking Tarsal Act ivity Both tarsi r aised Both meta thoracic tarsi are held above with waters surface, not moving Simultaneous tarsal w ave The meta thoracic tarsi are fanned parallel to one another None The subject is exhibiting no tarsal activity Single tar sal w ave One meta thoracic leg is fanned vertically while the other leg is resting on the surface of the water Single tarsus r aised One meta thoracic leg is held above the waters surface and is not moving Tarsal l inking The meta thoracic tarsi are pla ced on the surface of the water and moved parallel to one another until the distal tarsi contact one another, forming a pen for egg rafts Tarsal l owering The meta thoracic tarsi are lowered from a raised position to laying parallel on the surface of the water Tarsal u nlinking The meta thoracic tarsi are moved away from one another and disconnected from the egg raft after oviposition has ended Oviposition Abdominal p robe The tip of the abdomen makes rapid repeated contact with the oviposition substrate No probing The subject is exhibiting no abdominal activity Drinking The proboscis is inserted into the substrate and the subject ingests the water. This can be detected by movement of the proboscis while inserted into the substrate. Egg d eposition An egg is extruded No d rinking The subject does not have the proboscis inserted into the substrate

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80 Table 3 2. Culex tarsalis ethogram Category Behavior Definition Location Air The subject is in the air Water The subject has at least four legs co ntacting the surface of the water Other The subject is in a location other than the water or air Movement Fly The subject is flying through the air Dip The meta thoracic tarsi make contact with the waters surface while the subject is flying in the ai r Land The subject settles on the waters surface with at least the proand meso thoracic legs. Can also include meta thoracic legs contacting surface. Walk The subject is walking. This can be on any surface Still The subject remains in one location and is not walking Tarsal Activity Alternating tarsal wave Both meta thoracic tarsi are fanned vertically in opposite directions Both tarsi raised Both meta thoracic tarsi are held above with waters surface, not moving Simultaneous tarsal wave The m eta thoracic tarsi are fanned parallel to one another None The subject is exhibiting no tarsal activity Single tarsal wave One meta thoracic leg is fanned vertically while the other leg is resting on the surface of the water Single tarsus raised One meta thoracic leg is held above the waters surface and is not moving Both tarsi raised Both meta thoracic tarsi are held above with waters surface, not moving Tarsal linking The meta thoracic tarsi are placed on the surface of the water and moved par allel to one another until the distal tarsi contact one another, forming a pen for egg rafts Tarsal lowering The meta thoracic tarsi are lowered from a raised position to laying parallel on the surface of the water Oviposition Abdominal probe The tip of the abdomen makes rapid repeated contact with the oviposition substrate. No probing The subject is exhibiting no abdominal activity Drinking The proboscis is inserted into the substrate and the subject ingests the water. This can be detected by moveme nt of the proboscis while inserted into the substrate. Proboscis flick A rapid flexing of the proboscis. Can be flexed up or down, and often occurs during an egg deposition Egg deposition An egg is extruded

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81 Table 3 2 Continued. No drinking The subje ct does not have the proboscis inserted into the substrate Table 3 3. Comparison of the occurrences of oviposition behaviors bet ween C ule x quinquefasciatus and C ule x tarsalis Note: Means within rows were analyzed by paired t tests or Mann Whitney Rank sum tests depending on normality. P values marked with an asterisk represent a statistically sig nificant .05. Average count ( +S E) Behavior Cx quinquefasciatus Cx tarsalis P Abdominal p robe 4.3 ( + 0.6) 2.7 ( + 0.3) 0.063 Air 1.0 ( + 0.0) 1.0 ( + 0.0) 0.369 Alternating tarsal wave 0.0 ( + 0.0) 2.0 ( + 0.0) 0.167 Both tarsi raised 1.0 ( + 0.2) 2.0 ( + 0.0) 1.000 Dip 4.0 ( + 0.2) 3.5 ( + 0.2) 1.000 Drink 10.1 ( + 0.9) 4.7 ( + 0.9) 0.025 Egg d eposition 62.3 ( + 7.2) 76.2 ( + 7.8) 0.214 Fly 1.0 ( + 0.0) 1.0 ( + 0.0) 0.167 Land 1.0 ( + 0.0) 1.0 ( + 0.0) 0.368 No drinking 10.0 ( + 1.4) 5.8 ( + 1.0) 0.030 No probing 5.3 ( + 0.6) 3.8 ( + 0.6) 0.086 None 2.3 ( + 0.2) 2.5 ( + 0.3) 0.563 Other 1.0 ( + 0.0) 1.0 ( + 0.0) 1.000 Proboscis f lick 0.0 ( + 0.0) 11.3 ( + 2.5) <0.001 Si multaneous tarsal wave 1.0 ( + 0.0) 0.0 ( + 0.0) 0.368 Single tarsal wave 1.9 ( + 0.3) 1.6 ( + 0.2) 0.753 Single tarsus raised 2.2 ( + 0.5) 1.8 ( + 0.4) 0.377 Tarsal l inking 1.0 ( + 0.0) 1.0 ( + 0.0) 0.357 Tarsal l owering 1.0 ( + 0.0) 1.0 ( + 0 .0) 0.957 Tarsal u nlinking 1.0 ( + 0.0) 1.0 ( + 0.0) 0.089 Still 1.0 ( + 0.0) 1.0 ( + 0.0) 0.962 Walk 1.0 ( + 0.0) 1.0 ( + 0.0) 0.185 Water 1.0 ( + 0.0) 1.0 ( + 0.0) 0.368

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82 Table 3 4. Comparison of duration (Mean+ SE) of oviposition behaviors between female Culex quinquefasciatus and Culex tarsalis Average duration sec ( + SE) Behavior Cx quinquefasciatus Cx tarsalis P Total duration 1010. 1 ( + 86.5) 950.9 ( + 66.8) 0.595 Abdominal probe 583.1 ( + 45.5) 487.0 ( + 50.0) 0.162 Air 4.6 ( + 0.6) 2.8 ( + 0.5) 0.034 Alternating tarsal wave 0.0 ( + 0.0) 13.8 ( + 12.4) <0.001* Both tarsi raised 3.7 ( + 3.7) 13.3 ( + 13.3) 1.000 Drink 161.1 ( + 80.0) 29.5 ( + 7.4) 0.026 Fly 4.8 ( + 0.6) 2.1 ( + 0.6) 0.007 No drinking 865.3 ( + 122.1) 921.1 ( + 66.4) 0.791 No probing 427.3 ( + 54.0) 463.8 ( + 42.7) 0.546 None 91 6.4 ( + 88.1) 846.5 ( + 108.9) 0.563 Other 19.2 ( + 10.2) 74.7 ( + 63.8) 0.907 Simultaneous tarsal wave 0.5 ( + 0.5) 0.0 ( + 0.0) 0.368 Single tarsal wave 36.4 ( + 6.7) 26.7 ( + 8.6) 0.549 Single tarsus raised 71 .1 ( + 19.6) 69.3 ( + 22.8) 0.610 Still 1003.3 ( + 86.7) 943.0 ( + 71.1) 0.644 Walk 1.9 ( + 1.3) 2.4 ( + 1.9) 0.399 Water 986.0 ( + 86.0) 970.4 ( + 60.8) 0.850 Note: Means within rows were analyzed by paired t tests or Mann W hitney Rank sum tests depending on normality. P values marked with an asterisk represent a statistically sig nificant .05.

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83 Table 3 5. Transition matrix for location of Culex quinquefasciatus during oviposition. Note: Transitions (excluding start and end observations) in bold occurred at greater than expected levels (chi square test). Table 3 6. Transit ion matrix for location of Culex tarsalis during oviposition. Location Succeeding behavioral e vent P receding behavioral e vent Other Air Water End o bservation Other 0% 0% 100.0% 0% Air 0% 0% 0% 100.00% Water 0% 88.89% 0% 11.11% Start o bservation 20.0% 0% 0% 0% N ote: Transitions (excluding start and end observations) in bold occurred at greater than expected levels (chi square test). Location Succeeding behavio ral e vent Precedin g behavioral e vent Other Air Water End o bservation Other 0% 0% 100.0% 0% Air 0% 0% 0% 100.0% Water 0% 100.0% 0% 0% Start o bservation 20.0% 0% 0% 0%

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84 Table 3 7. Transition matrix for movement of Culex quinquefasciatus during oviposition. Not e: Transitions (excluding start and end observations) in bold occurred at greater than expected levels (chi square test). Table 3 8. Transition matrix for movement of Culex tarsalis during oviposition. Movement Succeeding behavioral e vent Preceding beha vioral e vent Still Fly Land Dip Walk End o bservation Still 0% 5. 6% 72.2% 22.2% 0% 0% Fly 0% 0% 0% 0% 0% 100.0% Land 0% 40.0% 0% 0% 50.0% 10.0% Dip 0% 0% 29.0 % 71.0 % 0% 0% Walk 20.0% 60.0% 0% 0% 0% 20.0% Start o bservation 20.0% 0% 0% 0% 0% 0% Note: Transitions (excluding start and end observations) in bold occurred at greater than expected levels (chi square test). Movement Succeeding behavioral e vent Prece ding behavioral e vent Still Fly Land Dip Walk End o bservation Still 0% 10.0% 65.0% 20.0% 5.0% 0% Fly 0% 0% 0% 0% 0% 100.0% Land 0% 88.9% 0% 0% 11.1% 0% Dip 0% 0% 26.5% 73.5% 0% 0% Walk 100.0% 0% 0% 0% 0% 0% Start o bservation 20.0% 0% 0% 0% 0% 0%

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85 Table 3 9. Transition matrix for tarsal activity of Culex quinquefasciatus during oviposition. Note: Transitions (excluding start and end observations) in bold occurred at greater than expected levels (chi square test). Tarsal Activity Succeeding behavioral e vent Preceding behavioral e vent None Single tarsal w ave Simultaneous t arsal w ave Single t arsus r aised Both tarsi r aised Tarsal l owering Tarsal l inking Tarsal unlinking End observation None 3.3% 20.0% 0% 35.0% 0% 5.0% 23.3% 8.3% 5.0% Single tarsal w ave 14.3% 0% 14.3% 71.43% 0% 0% 0% 0% 0% Simultaneous tarsal w ave 0% 0% 0% 0% 100.0% 0% 0% 0% 0% Single tarsus r aised 68.5% 31.5% 0% 0% 0% 0% 0% 0% 0% Both tarsi r aised 0% 0% 0% 100.0% 0% 0% 0% 0% 0% Tarsal l owering 0% 0% 0% 0% 0% 0% 0% 0% 0% Tarsal l inking 16.67% 0% 0% 0% 0% 0% 0% 83.3% 0% Ta rsal u nlinking 0% 0% 0% 14.3% 0% 0% 0% 0% 85.71% Start o bservation 20.0% 0% 0% 0% 0% 0% 0% 0% 0%

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86 Table 3 10. Transitio n matrix for tarsal activity of Culex tarsalis during oviposition. Note: Transitions (excluding start and end observations) in bold occurred at greater than expected levels (chi square test) Tarsal Activity Succeeding behavioral e vent Preceding behavioral e vent None Single tarsal w ave Simultaneous tarsal w ave Alternating tarsal w ave Single tarsus r aised Both tarsi r aised Tarsal l owering Tarsal l inking Tarsal unlinking End obs erv. None 0% 12.5% 0% 5.00% 30.0% 2.50% 22.5% 10. 00 % 5.0% 12.5% Single tarsal w ave 57.59% 0% 0% 0% 35.71% 0% 0% 4.71% 0% 0% Alternating tarsal w ave 5 0.00% 0% 0% 0% 25.0% 25.00% 0% 0% 0% 0% Single tarsus r aised 42.86% 42.86 % 0% 7.14% 0% 0% 7.14% 0% 0% 0% Both tarsi r aised 0% 0% 0% 100.00% 0% 0% 0% 0% 0% 0% Tarsal l owering 14.89% 0% 0% 0% 14.29% 0% 0% 71.43% 0% 0% Tarsal l inking 0% 14.29% 0% 0% 0% 0% 0% 0% 85.71% 0% Tarsal u nlinking 0% 0% 0% 0% 28.57% 0% 0% 0% 0% 71.43% Start o bservation 20.0% 0% 0% 0% 0% 0% 0% 0% 0% 0%

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87 Table 3 11. Transition matrix for oviposition activity of Culex quinquefasciatus during oviposition. Note: Transitions (excluding start and end observations) in bold occurred at greater than expected levels (chi square test). Table 3 12. Transition matrix for oviposition activity of Culex tarsalis dur ing oviposition. Oviposition Succeeding b ehavioral e vent Preceding behavioral e vent No probing Abdominal prob e Egg deposition Proboscis f lick End observation No p robing 0 % 50.50% 18.30% 13.30% 15.40% Abdominal p rob e 17.60% 0% 77.00% 5.30% 0% Egg d eposition 2.20% 0.90% 79.80% 16.90% 0.20% Proboscis f lick 2.40% 1.10% 87.90% 7.60% 1.00% Start observation 20.0% 0% 0% 0% 0% Note: Transitions (excluding start and end observations) in bold occurred at greater than expected levels (chi square test). Oviposition Su cceeding behavioral e vent Preceding behavioral event No probing Abdominal probe Egg deposition End observation No probing 0% 50.60% 36.13% 13.27% Abdominal prob e 19.20% 0% 80.80% 0% Egg deposition 6.10% 2.5% 90.7% 0.80% Start observation 20.0% 0% 0% 0%

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88 Table 3 13. Transiti on matrix for drinking activity of Culex quinquefasciatus during oviposition. Note: Transitions (excluding start and end observations) in bold occurred at greater than expected levels (chi square test). Table 3 14. Transition matrix for drinking activity in Culex tarsalis during oviposition. Drinking Succeeding Behavioral Event Preceding Behavioral Event No drinking Drinking End observation No drinking 0% 76.0% 22.9% Drinking 100.0% 0% 0% Start observation 20.0% 0% 0% Note: Transitions (excluding start and end observations) in bold occurred at greater than expected levels (chi square test). Drinking Succeeding Behavioral Event Preceding Behavioral Event No d rinking Drinking End observation No d rinking 0% 81. 10% 22.90% Drinking 98.44% 0 % 0% Start observation 20.00% 0% 0%

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89 Figure 3 1. Behavioral setup used for video capture of oviposition behaviors of mosquitoes. A) Analog CCD Ca mera. B) 20 cm x 20 cm x 20 cm cage containing gravid mosquitoes and oviposition substrate. C) LCD Infrared lights. D) Real time mpeg encoder digitizer. E) Laptop with MediaCruise recording software and Noldus behavioral analysis software. A B C D E

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90 Fig ure 3 2. A k inematic diagram of location of Culex quinquefasciatus during oviposition. Figure 3 3. A k inematic diagram of location for Culex tarsali s during oviposition.

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91 Figure 3 4. A k inematic diagram of movement for Culex quinquefasciatus during oviposition. Figure 3 5. A k inematic diagram of movement for Culex tarsali s during oviposition.

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92 Figure 3 6. A k inematic diagram of tarsal activity for Culex quinquefasciatus during oviposition.

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93 Figure 3 7. A k inematic diagram of tarsal activity for Culex tarsali s during oviposition. Figure 3 8. A k inematic diagram of oviposition activity for Culex quinquefasciatus during oviposition.

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94 Figure 3 9. A ki nematic diagram of oviposition activity for Culex tarsali s during oviposition. Figure 3 10. A k inematic diagram of drinking activity for Culex quinquefasciatus during oviposition.

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95 Figure 3 11. A k inematic diagram of drinking activity for Culex tarsali s during oviposition.

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96 Figure 3 12. Time budgets for location of ovipositi ng Culex quinquefasciatus and Culex tarsalis females. Locations marke d with an asterisk represent statistically significant t test).

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97 Figure 3 13. Time budgets for movement of ovipositing Culex quinquefasciatus and Culex tarsalis females. Locations marked with an asterisk represent statistically significant t test).

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98 Figure 3 14. T ime budgets for tarsal activity of ovipositing Culex quinquefasciatus an d Culex tarsalis females.

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99 Figure 3 15. T ime b udgets for oviposition activity of ovipositing Culex quinquefasciatus and Culex tarsalis females.

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100 Figure 3 16. Visual representation of oviposition behavioral sequences of Culex quinquefasciat us (top) and Culex tarsalis (bottom) as created by The Observer XT softwar e.

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101 CHAPTER 4 EFFECT OF INSECT PATHOGENS ON OVIPOSITION SUBSTRATE SELECTION FOR THREE MEDICALLY IMPO RTANT MOSQUITO SPECI ES Introduction Three medically important mosquito species fou nd in Florida are the Asian tiger mosquito, Aedes albopictus (Skuse), the yellow fever mosquito, Aedes aegypti (Linnaeus), and the southern house mosquito, Culex pipiens quinquefasciatus Say. Aedes aegypt i and Ae. albopictus are treehole or container inha biting mosquitoes, with a predilection for ovipositing in manmade containers such as rain barrels, flower pots, and buckets. Because both opportunistically oviposi t, they are commonly found in both urban and rural areas around homes, increasing the risk of human exposure to mosquitoborne pathogens Many Aedes species are very successful vectors for yellow fever, dengue fever (DEN) and dengue hemorrhagic fever (DHF), chikungunya (CHIK V), and LaCrosse encephalitis (Nasci and Miller 1996) viruses Culex qui nquefasciatus is the primary vector of West Nile virus (WNV) in the S outheast, and is a secondary vector for several other important dis eases, mainly in the encephalitides complex (Nasci and Miller 1996). With current concerns about pesticide use and subs equent resistance, bio logical control agents are playing an increasing role in vector management. By exploiting mosquitoes vulnerability to their own associated parasites and pathogens, a new arsenal of potential biological control agents may be developed. Since many of these parasites and pathogens are transmitted transovarially, an in depth understanding of oviposition behavior and the relationship between oviposition and infection needs to be studied. By understanding how both infected mosquitoes and uninfected mosquitoes respond behaviorally to infection, we can manipulate those conditions to improve mosquito control, by possibly establishing natural repellants or highly effective larvicides and adulticides.

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102 All mosquitoes have associated parasites and pathogens. Microsporidia are obligate intracellular spore producing parasites found most often parasitizing insects (Hirt et al. 1999). The highly complex microsporidian parasite Edhazardia aedi s Kudo is species specific to Ae. aegypti and is spread both transovarially and horizontally, when larvae consume spores found in the the remains of infected larvae. Uninucleate spores ingested by Ae. aegypti larvae initially infect the midgut epithelium and the epithleuim of the gastric ceca. Most vertically infected larvae with high infection rates die before metamorphosis, but low to moderate infection levels may allow for development to adulthood (Becnel and Andreadis 1999). In adults, an asexual reproductive phase occurs in the oenocytes, and vegetative stages surround the ovaries of the female. Once females ingest a bloodmeal sporulation occurs and the resulting transovarial binucleate spores infect the ovaries allowing for transovarial transmission of the parasite. In the infected progeny, vegetative stages r eplicate in the fat bodies where they undergo asexual reproduction before ending the life cycle through meiosis or nuclear disassociation to produce uninucleate spores that are infectious per os (Becnel and Andreadis 1999). Parasites often alter the behav ior of the host ( Becnel and Andreadis 1999, Barnard et al. 2007). The age of E. aedis infection in Ae. aegypti affects bloodfeeding success as an adult. Larvae infected late in the developmental process fed more successfully as adults than larvae infected as early instars (Koella and Agnew 1997) which demonstrates that E. aedis is capable of altering host behaviors Controlled studies have concluded that microsporidia can be potentially successful bio logical control agents due to high host specificity, saf ety for nontarget organisms, and the ability to be transmitted vertically, which spreads the pathogen, and horizontally, which a m plifies the pathogen (Becnel 1992, Becnel and Johnson 2000) Reproductive capacity was greatly reduced in adults that had been infected as larvae, regardless of the transmission route

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103 (Becnel et al. 1995). Because oviposition is critical for dissemination, understanding the impact of infection on oviposition behavior will enhance the understanding of E. aedis transmission and its potential use for biological control. The microsporidian parasite Vavraia culicis, once thought to belong to the genus Pleistospora, has since been renamed Vavraia Vavraia culicis is capable of infecting 13 mosquito species across five different genera, a nd has recently been implicated as a potential pathogen of immunosuppressed human hosts (Becnel et al. 2005). Vavraia culicis unlike E. aedis has a simple life cycle and host relationship. Only one uninucleate spore type is produced, and spores are not tissue specific. The pathogen is transmitted horizontally between larval hosts upon the death of an infected larva (Becnel and Andreadis 1999). A V. culicis isolate was found during a pathogen survey in Gainesville, Florida in 1997 (Fukuda et al. 1997) wh ere it was most commonly found in Ae. albopictus The isolate has been successfully propagated in the lab in both Lepidopteran ( Helicoverpa zea (Boddie) and Spodoptera littoralis Biosduval) and Anopheles quadrimaculatus Say hosts (Vavra and Becnel 2007). Infection with V. culicis does affect life history parameters of Cx. quinquefasciatus Agnew et al. (1999) found that parasitism in Cx. quinquefasciatus caused females to pupate and emerge earlier than non infected females. Females were also smaller, sugge sting reduced fecundity with infection. Life history parameters of males were not affected by V. culicis infection (Agnew et al. 1999). Because there is a fitness cost to infection and fecundity may be reduced, a better understanding of the effects of V. c ulicis infection on mosquito oviposition is important to optimize its potential use as a bio logical control agent.

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104 Culex nigripalpus nucleopolyhedrovirus (CuniNPV), is a baculovirus present in wild populations of Cx. nigripalpus Theobald, Cx. quinquefasc iatus and Cx. salinarius Coquillett however laboratory trials have shown nearly all Culex species found in the Southeast, except Cx. territans Walker are susceptible (Andreadis et al. 2003). While baculoviruses are not commonly found in wild populations of mosquitoes, they are highly virulent (Becnel et al. 2001). Virus development begins in the posterior midgut and gastric ceca of larvae when occlusions derived virions are ingested. After reproduction in the nuclei of the cells, budded virions spread the virus to new nuclei within the gut (Moser et al. 2001). After 2448 hours post inoculation, the majority of cells of the gastric ceca and the posterior stomach are filled with occlusion bodies (Moser et al. 2001) with the virus generally restricted to the nuclei of the secretion/resorbtion cells of the gastric cecae and posterior stomach. Behaviorally, infected larvae become lethargic and remain at the waters surface despite surrounding disturbances (Moser et al. 2001). Although CuniNPV more commonly infe cts larvae, the virus has been isolated from adult midguts (Becnel et al. 2003). T he virus is spread by horizontal transmission during the larval stages If the mosquito survives to adulthood, the occlusion bodies are generally shed with the first meconium It can also be vertically spread if a newly emerged adult dies in an uninfected aquatic habitat (Becnel et al. 2003). In order for high transmission rates to occur, divalent cations must be present i n the water, such as magnesiusm Magnesium is commonly found in water containing high amounts of effluent from swine farms (Becnel et al. 2001 ), meaning conditions have to be very specific for transmission. Calcium acts as an inhibitor of the virus and prevents transmission (Becnel et al. 2001). With such hig h larval mortality rates, CuniNPV should be looked at as a potential larval

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105 biological control agent, and understanding the relationship between oviposition behavior and virus infected conspecific larvae or larval substrate is key. Materials and Methods I nsect Colonies Aedes aegypti A South Florida strain of Ae. aegypti was collected from cemeteries in Tampa, FL in March 2008. Larvae were reared in plastic pans (35 c m x 48 cm x 6.25 cm) containing 1500 ml well water, and were fed every other day with 50 ml of a slurry made from a 1:1 mix of liver powder and brewers yeast (MP Biomedical, Solon, OH) mixed with well water (10 g dry mixture per 100 ml water). Once the larvae pupated, they were placed in a plastic cup containing water and set inside a 30 cm x 3 0 cm x 30 cm Plexiglas and screen cage to emerge. Adults had constant access to cotton saturated with 5% sucrose solution (50 g sucrose in 1000 ml water). Five to seven days after emergence, Ae. aegypti were bloodfed on a sausage casing (4.45 cm, The Sausa ge Maker Inc., Buffalo, NY ) filled with defibrinated bovine blood (Cathy Jennings, Ocala, FL). Forty eight hours post bloodfeeding, a dark colored plastic cup (Solo Cup Company, Highland Park, IL) containing a piece of seed germination paper (8 cm x 6 cm) (Anchor Paper Company, St. Paul, MN) filled with 250 ml well water was placed in the cage. At 24 hour intervals over the course of three days, the egg papers were removed and allowed to dry at room temperature for approximately 20 minutes whereafter the y w ere placed in a sealed plastic bag to maintain moisture. On the third day, the egg cup was removed from the adult cage To encourage eclosion egg papers were placed in plastic cups filled with deionized water, and placed under a vacuum for 10 minutes. Cups were removed and egg papers were left in the cups overnight to observe hatch rates. All stages of development were kept in a temperature controlled incubator

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106 model 818 (Precision Scientific, Teynampet India) at 28oAedes aegypti infected with Edhazardia aedis C, with a 14:10 light/dark cycle and a n 80% 85% relative humidity range. Edhazardia aedis infected Ae. aegypti were maintained in a lab colony using a modified protocol established by James Becnel at the USDA ARS CMAVE, Gainesville, FL (Becnel an d Undeen 1992). Infected Ae. aegypti larvae were hatched from stock colonies maintained at the USDAARS CMAVE, also of the South Florida strain. Infected mosquitoes were continually reared, and infected eggs collected to maintain infection within the colon y. Infected eggs were placed in a 100 ml petri dish filled with deionized water, left uncovered, and placed under a vacuum for 10 minutes. The vacuum was sealed and turned off, but eggs were allowed to sit in the vacuum sealed container for an additional 10 minutes. Air was then allowed to return to the vacuum. Eggs and larvae were kept in the petri dish for 24 hours to observe hatch rates. Larvae were transferred to plastic cups, each containing 100 ml of water. One hundred larvae were placed into each lar val cup, and a 0.5 ml of larval slurry was added. Larvae were provided one ml of slurry every other day until they reached third instar. Larvae were checked for fat body infection with uninucleate spores by squashing larvae in a drop of deionized water on a slide and observation with phase contrast under a compound microscope at 400x magnification For bioassays, 10 infected larvae per bioassay cage were selected and the remaining infected third instars macerated finely and put into one ml of water. After maceration, spore counts were co nducted using a bright line hemo cytometer. After spore concentration was calculated, the solution was diluted to obtain a final spore count of 1.3 x 104 spores per 100 ml water. The solution was added to larval cups containi ng 100 healthy third instar larvae in 100 ml well water and fed every other day with one ml larval slurry until pupation. Pupae were transferred to 30 cm x 30 cm x 30 cm Plexiglas and screen cages in plastic cups and allowed to

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107 emerge. Individual adults we re checked for infection by placing the adult on a slide with a drop of deionized water and squashing with a slip cover. The presence or absence of binucleate spores was observed with phase contrast through a compound microscope at 400x magnificatio n. Adul ts were maintained with constant access to a 5% sucrose solution. Adults were bloodfed on bovine blood once a week, and eggs were collected as needed. Eggs were stored in a resealable plastic bag. Adults were held as previously described. Aedes albopictus The Aedes albopictus colony was established from a field collection from Gainesville in 2007. All rearing methods for healthy Ae. albopictus follow rearing methods of healthy Ae. aegypti. Aedes albopictus infected with Vavraia culicis floridensis The micr osporidian parasite, Vavraia culicis floridensis was maintained in the laboratory of James Becnel at the USDA ARS CMAVE, Gainesville, Fl (Vavra and Becnel, 2007) using Anopheles quadrimaculatus and Helicoverpa zea Vavraia culicis was alternated in cultur e between An. quadrimaculatus and H. zea every four months to ensure infectivity in mosquitoes. Spores were extracted by macerating infected adult mosquitoes with a handheld tissue grinder with a minimal amount of deionized water added, and the mixture f iltered through a 140 mesh nylon screen sieve to remove large particulates. Spores were then placed on t op of a continuous gradient of equal part HS Ludox (Sigma Aldrich St. Louis, MO) on the bottom with 0.1 ml of 0.5M Nh4CL for each 10 ml of Ludox, and deionized water on top with 0.1 ml of 0.5M NH4Cl for each 10 ml of water. NH4Cl prevents spores from germinating in solution. Gradients were run in a 10,000 RPM centrifuge for 30 min utes Spore pellets were removed from the solution and rinsed in deionized water three times by centrifugation at 10,000 RPM for 30

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108 minutes each. Spores were placed in 10 ml deionized water and stored in a refrigerator at 4oApproximately 400 healthy Ae. albopictus larvae were hatched using the above mentioned vacuu m procedure. After 24 hours, larvae were split into two groups, and each group was transferred to 100 ml deionized water in 120 ml petri dishes. One larval dish was dosed with 0.25 ml purified V. culicis spores at a concentration of 1x10 C until used. 4 1x105Adults were scored for infection by taking a random sample, placing individual adults with a drop of deionized water on a slide, and squashing with a cover slip. The presence or absence of spores was determined under a compound mi croscope at 40x magnfication. Adults were maintained as previously described and eggs were collected as needed. spores/ml The other control dish did not receive spores, and both dishes received 0.10 ml larval slurry. After 24 hours, larvae were transferred to 30 cm x 18 cm x 5 cm ceramic pans filled with 1000 ml well water Larvae were fed eight ml of slurry every other day and reared to adulthood. Culex quinquefasciatus A colony of Culex quinquefasciatus was established from collections in Gainesville, FL in 1995. Larvae were reared in pl astic pans (35 cm x 48 cm x 6.25 cm), and were fed every other day with 50 ml of larval food. Once the larvae pupated, they were placed in a plastic cup and set inside a 30 cm x 30 cm x 30 cm Plexiglas and screen cage to emerge. Adults had constant access to cotton saturated with 5% sucrose solution. Seven days after emergence, adults were bloodfed on a chicken for up to 15 minutes ( University of Florida IACUC No. 469). Five days after the bloodmeal, adults were provided a black egg cup containing 500 ml we ll water and 2.0 ml hay infusion (prepared following Reiter et al. 1991). After 24 hours, egg cups were removed, and the egg rafts were set in plastic pans (35 cm x 48 cm x 6.25 cm) with 4 5 egg rafts per pan. All

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109 stages of development were kept in a temp erature controlled incubator at 28oCulex quinquefasciatus infected with CuniNPV : C, with a 14:10 light/dark cycle and an 80% 85% humidity range. Photophase began at 8:00 pm. Culex nigripalpus nucleopolyhedrovirus (CuniNPV) was supplied by James Becnel a t the USDAARS CMAVE, Gainesville, FL and Cx. quinquefasciatus larvae were infected as described by Moser et al. (2001). Infected Culex nigripalpus mosquitoes were originally field collected from a swine wastewater site in Gainesville, FL, in 1997. CuniNPV field isolates were amplified in the laboratory using Cx. quinquefasciatus and virus was collected and st ored in a solution made up of 5x107 2x108 occlusion bodies /ml and 15mM MgSO4 at 80oC. Groups of 50 infected larvae were frozen with deionized wate r at 80o C and used for culture maintenance. Approximately 3000 healthy Cx. quinquefasciatus larvae (early third instar) were exposed to 100 larval equivalents ( LE ) of virus in solution with 14 mM MgCl2Dual C hoice Oviposition Bioassay Methods and used for bioassays 48 hours post infection Lar vae were fed two grams of larval slurry per tray (~0.7 ug /larva) at the time of exposure. Infected larvae general ly do not survive to adulthood (Becnel et al. 2003). Uninfected Aedes aegypti adults Forty eight hours post bloodfeeding, 10 healthy gravid, nulliparous Ae. aegypti were placed in a 30 cm x 30 cm x 30 cm screen and Plexiglas cag e. Fifteen bioassay cages were used in each trial. Each bioassay cage contained two black 100 ml egg cups (Solo Cup Company, Hi ghland Park, IL) filled with 50 ml well water and placed 20 cm apart. Each egg cup contained one piece (8 cm x 6 cm) of seed germination paper as an oviposition substrate. In 10 out of the 15 bioassay cages per day, one cup contained 10 healthy third inst ars in well water, and the other cup contained 10 E. aedis infected third instars in well water. Larvae were placed in egg cups immediately preceding the start of the bioassay. Because the pathogens

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110 have varying degrees of virulence, adding larvae to the w ater immediately preceding the bioassay minimized the risk of microbial degradation or larval starvation altering the ovipositional response of the females. Five control assays were also conducted each day, with both egg cups containing 10 healthy third instars to verify that there was no discrimination between substrates on any given day. Females were allowed 24 hours to oviposit with a 16:8 light:dark photoperiod. Bioassays began at 11:00 am, and ended at 11:00 am the following day. Using this time frame mimiced the natu ral light dark cycle and exposed the females to two diurnal cycles. Mosquitoes were not provided with sugar solution during the bioassays. After 24 hours, egg cups and papers were removed from the cages, and eggs were counted using a diss ecting microscope. Based on the number of eggs laid in each cage, the percentage of eggs laid on each oviposition substrate was determined. Bioassays were performed on at least three different days, using a new batch of nulliparous gravid females each time for a total of 30 treatment bioassays and 15 control assays. Cages in which females did not lay eggs were not included in the analysis Infected Aedes aegypti adults Edhazardia aedis infected Ae. aegypti adults were also tested for oviposition preference to determine if pathogen infection in the adults would affect oviposit ion substrate choice. Bioassays for infected gravid Ae. a egypti followed the same protocol as above, except that only one gravid female was placed in a cage rather than 10 gravid femal es. It was not possible to obtain infection in 100% of adults, so it was necessary to conduct assays using individual females so that infection could be verified after the assay. After running the bioassays with individual females, they were examined to v erify E. aedis infection. Any females that were found to be

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111 uninfected were not included in the analysis A total of 30 treatment bioassays and 15 control assays were completed. Uninfected Aedes albopictus adults Bioassays with Ae. albopictus followed the same protocol as above, using healthy Ae. albopictus larvae, as well as V. culicis infected larvae as treatments. All other methods and numbers of repitions were the same Although V. culicis infected larvae generally survive to adulthood, fecundity is gre atly reduced with infection. I originally intended to perform the bioassays also with V. culicis infected Ae. albopictus adults, but it was difficult to obtain oviposition. To check fecundity, the ovaries were dissected from 45 infected adult females. Ovar ies were dissected two, three, and four days after bloodfeeding, and egg development graded following Christophers (1911) scale. Eggs developing past stage I were not found in inf ected females, and therefore infected adult bioassay s could not be completed. Uninfected Culex quinquefasciatus adults Bioassays with Culex quinquefasciatus were performed using the above protocol; using 10 healthy Cx. quinquefasciatus larvae and 10 live CuniNPV infected Cx. quinquefasciatus larvae at 48 hours post innoculation as treatments. Egg papers were not placed into egg cups, and 2.0 ml hay infusion (prepared following Reiter et al. 1991) was added to each cup to stimulate oviposition. Egg rafts were counted, and the percentage of total egg raft oviposition determined for e ach substrate. A total of 30 treatment bioassays and 15 control assays were completed. Data A nalysis Data was analyzed using SigmaPlot 11.0 (Systat Software, Chicago, IL). Since results were in proportions, data were ArcSin transformed in Microsoft Excel 2007 and transferred to SigmaPlot 11.0 to test for normality (Shaprio Wilk test, ). Normally distributed data were

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112 analyzed using a SigmaStat paired t test. Data that did not pass normality were analyzed using the Mann Whitney Rank Sum Test. Results Aedes aegypti In two choice oviposition bioassays using an oviposition substrate containing uninfected larvae or E. aedis infected larvae, uninfected Ae. aegypti adults laid a total of 15,603 eggs. There was no significant difference in the percentage of eggs laid in either oviposition cups in the control trials, each contain ing uninfe cted larvae (Figure 1A ) indicating no positional bias in the cage. Uninfected adult Ae. aegypti laid significantly more eggs ( t =7.275; df=58; P < 0.001) in oviposition cups containing uninfected larvae than in oviposition cups containing E. aedis infected l arvae ( F igure1 B ). In total, 9,262 eggs were laid in cups with uninfected larvae, with a mean of 309.73 (60.78%) eggs per cup. The total number of eggs laid in cups containing E. aedis infected larvae was 6,341, with a mean of 211.37 (39.19%) eggs per cup. Edhazardia aedis infected adults laid a total of 1,039 eggs. There was no significant difference in the percentage of eggs laid in two oviposition cups, each containing uninfected larvae (Figure 2 A ) indicating no positional bias for either side of the cages. Adult Ae. aegypti infected with E. aedis laid significantly more eggs in oviposition cups containing uninfected larvae than in oviposition cups containing E. aedis infected larvae (t=1 .055; df=60; P=0.036) (Figure 2B ). A total of 451 eggs were laid i n oviposition cups containing uninfected larvae, with a mean of 19.6 (60.47%) eggs per cup. A total of 588 eggs were laid in oviposition cups containing E. aedis infected larvae, with a mean of 15.03 (39.53%) eggs per cup. Aedes albopictus In two choice oviposition bioassays using uninfected larvae and V. culicis infected larvae, uninfected Ae. albopictus adults laid a total of 1,376 eggs during 30 trials. There was no

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113 significa nt difference in the percentage of eggs laid in two oviposition cups, each containing uninfected larvae (Figure 3A ) indicating no positional bias for either side of the cages. Healthy adult Ae. albopictus s howed no significant difference in the number of eggs laid between oviposition cups containing uninfected larvae and oviposition cups containing V. culicis infected larvae (Figure 3B ). The total number of eggs laid in oviposition cups containing uninfected larvae was 658 eggs, with a mean of 23.93 (51.34%) eggs laid per cup. The total number of eggs laid in oviposition cups contai ning V. culicis infected larvae was 718 eggs, with a mean of 21.93 (48.65%) eggs per cup. Culex quinquefasciatus In two choice oviposition bioassays using an oviposition substrate containing uninfected larvae and and oviposition substrate containing Cuni NPVinfected larvae, uninfected Cx. quinquefasciatus females laid a total of 15,603 eggs. There was no significant diff erence in the pe rcentage of egg rafts laid in two oviposition cups, each containing healthy larvae (Figure 4A ) indicating no positional bias for either side of the cages. Females of C x quinquefasciatus showed no significant difference in the number of egg rafts laid between oviposition cups containing uninfected larvae or Cu niNPVinfected larvae (Figure 4B ). The total number of egg rafts l aid in oviposition cups with uninfected larvae equaled 75 rafts, with a mean of 2.5 (50.52%) egg rafts per cup. The total number of egg rafts laid in oviposition cups containing CuniNPV infected larvae equaled 76 rafts, with a mean of 2.56 (49.48%) egg raf ts per cup. Discussion Based on the theory of natural selection, female mosquitoes that can detect potential predatory threats to their progeny should avoid those predators to maximize survival of the ir progeny. In many instances with mosquito species, this theory appears to hold true. Predator detection in oviposition substrates has been widely studied, however the understanding of the

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114 mosquito detection abilities for pathogens and parasites in oviposition substrates is fairly understudied. Multiple mosq uito predator relationships have been studied involving vertebrate predators such as bluegill sunfish ( Lepomis macrochirus ), (Petranka and Fakhoury 1991), mosquitofish ( Gambusia affinis ) (Angelon and Petranka 2002), and several species of amphibians: Limno dynastes peronii (Mokany and Shine 2003) Rana clamitans (Petranka and Fakhoury 1991) and Bufo spp. (Blaus tein and Kotler 1993). Mosquito and invertebrate predator relationships also have been extensively studied, often with Hemipteran (Notonectidae) preda tors (Blaustein et al. 2004, Blaustein et al. 2005, Chesson 1984, Eitam et al. 2002, Kiflawi et al. 2003), as well as studies with odonate predators (Stav et al. 2000). In all of the above mentioned studies, ovipositing female mosquitoes avoided substrates containing these predators, but whether by visual or chemical detection is unclear. In several studies, predator released kairomones provided the cue for avoidance of ovipositing mosquitoes (Angelon and Petranka 2002, Blaustein et al. 2004, Blaustein et a l. 2005, Mokany and Shine 2003, Petranka and Fakhoury 1991). The chemical identification for many of these kairomones remains unknown. Another highly studied organism in relation to mosquito oviposition is the bacteria, Bacillus thuringiensis var. israele nsis ( B ti) which produces a protoxin and is used extensively in larval mosquito control In a dual choice oviposition study, Stoops (2004) found that in laboratory bioassays, Ae. albopictus preferred to oviposit in water containing Bti rather than in the control water. In field studies, more eggs appeared to be laid in B titreated water, although differences were not statistically significant. This does, however, demonstrate that Ae. albopictus do not avoid Bti treated water, and that adults detect Bti in t he oviposition substrate (Stoops

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115 2004). Previous studies have reported the preference of mosquitoes to oviposit in or on substrate containing high numbers of bacterial colonies (Hazard et al. 1967, Trexler et al. 2003). The effect of Bti on the oviposition behavior of Culex mosquitoes also has been studied, with very different results. Zahiri and Mulla (2005) found that the oviposition response of Cx. quinquefasciatus and the levels of Bti and Bacillus sphaericus ( Bsph ) were inversely related: the higher th e concentration of Bti or Bsph the less likely females were to oviposit on that particular substrate. Expanding on that study, Zahiri and Mulla (2006) also reported that female Cx. quinquefasciatus that did oviposit on substrate containing Bti laid fewer eggs per egg raft and had misshapen rafts. Again, a similar pattern was seen: with the higher the concentration of Bti the less successful the oviposition. The difference in responses between Ae. albopictus and Cx. quinquefasciatus to Bti may result from behavioral differences between the two species. Culex mosquitoes commonly drink from the substrate before ovipositing, possibly to increase hydrostatic pressure for egg extrusion (Hudson 1956), or to evaluate suitability of the substrate with the mouthpar ts (Lee and Craig 1983). In contrast, Ae. albopictus females do not ingest the substrate. The ill formed rafts and decrease in egg numbers by Culex females may result from Bti poisoning or the alteration of oviposition behavior as a result of detection of Bti with her mouthparts. Ecological factors and the risk of exposure may also affect the ability of mosquitoes to detect parasites and pathogens in oviposition substrates. Mokany and Shine (2003) found that Cx. quinquefasciatus did not respond differently between substrates containing syntopic tadpoles and substrates without tadpoles, despite the fact that tadpoles are a common predator of mosquito larvae. However, when comparing those responses to those of Oc hlerotatus australis (Erichson) they reported t hat O c australis significantly avoided substrates containing tadpoles. Mokany and

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116 Shine (2003) proposed that the difference in mosquito response was due to ecological differences between the two species and the likelihood of encountering a particular predator in the natural habitat. Additionally, Culex mosquitoes avoid substrates that contain or once contained a common la rval predator, Gambusia affinis (Angelon and Petranka 2002). Because females avoided pools that once contained fish, but were empty at th e time of oviposition, it is likely that avoidance was based on chemical cues left behind by the fish. Some parasites, and more commonly parasitoids, alter the behavior of their host to benefit the fitness of the parasite or parasitoid (Moore 2002). Merme thid nematodes alter the predator avoidance behavior of larval Ae. aegypti to make the larvae more cryptic and engage in more resting behavior than healthy larvae (Wise de Valdez 200 6). Similarly, Ae. sierrensis (Ludlow) adults infected with the protozoan Lambornella clarki also display behavioral modifications. Because the parasite invades ovaries, females are castrated and unable to develop eggs, however, with parasite infection, the female still engages in oviposition behavior and deposits the protozoan parasites so that the parasite life cycle may continue (Lee 1995). Reeves (2004) reported that female Ae. aegypti were more attracted to water containing conspecific larvae infected with internal symbionts. When testing Ae. aegypti infected with the protozoan Ascogregarina taiwanensis the yeast Candida near pseudoglaebosa, and a trichomycete Smittium morbosum gravid Ae. aegypti significantly preferred oviposition water containing A. taiwanensis infected larvae and Candidainfected larvae. There was no si gnificant preference for S. morbosum or uninfected larvae (Reeves, 2004). Reeves (2004) suggested that becau se all three of these pathogens and parasites infect the larval midgut, it is much less damaging to the larvae, and therefore is undetectable to ovi positing adults. E dhazardia aedis causes an initial gut infection; however the larvae used in this study were in the fat body

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117 infection stage, which may explain detection, as females laid significantly fewer eggs in water containing E. aedis infected larva e. Additionally, A. taiwanensis is a natural parasite of Ae. albopictus and while it is capable of infecting Ae. aegypti a full life cycle cannot be completed within Ae. aegypti hosts (Munstermann and Wesson 1990), which may have impacted the results of Reeves (2004) study. Lowenberger and Rau (1994) reported a significantly reduced oviposition response of adult Ae. aegypti to healthy larvae compared to larvae parasitized by the fluke, Plagiorchis elegans at both low and high infection rates. Parasitize d larvae were quantified by high infection rates and low infection rates D espite the infection rate, Ae. aegypti avoided all parasitized larvae equa lly, suggesting that detection wa s not density dependent. While microsporidia infection rates were not quantified in the current study, results were similar. Lowenburger and Rau (1994) also obtained similar results using larval holding water; with females ovipositing significantly more eggs in water that previously contained healthy larvae compared to water tha t previously contained P. elegans infected larvae. Bacterial quantities were relatively similar, but culturing the bacteria found that there was a significantly larger amount of Flavobacterium species in holding water that previously contained P. elegans i nfected larvae. When both water choices were boiled and treated with antibiotics, females still significantly preferred to oviposit in the water that had contained unparasitized larvae. The authors suggest that the deterrence from parasitized larvae is not a reaction to a naturally occurring organic substance, but rather a reaction to some sort of chemical cue emitted by parasitized larvae (Lowenberger and Rau 1994). In the current study, both Cx. quinquefasciatus and Ae. albopictus were unable to detect th eir respective pathogens in oviposition substrate. Although CuniNPV and V. c ulicis are very

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118 different from each other, neither were species specific. Gravid Ae. aegypti were able to detect the presence of E. aedis but unlike the other two pathogens, E. ae dis is species specific to Ae. aegypti When a pathogen is species specific, it suggests specialization by the pathogen (Poulin and Mouillot 2003). Vavraia culicis infects multiple mosquito genera, as well as multiple insect families, only develops with a single unicellular phase, and the lifecycle is completed in the adult mosquito (Becnel and Andreadis 1999). Edhazardia aedis develops through four sporulation sequences, involving both uninucleate and binucleate spores, and commences in the F 1 progeny (Bec nel and Andreadis 1999). Specialization may suggest a longer evolutionary relationship, and as a result, a longer period that the mosquito has had to develop a counter defense against the pathogen, which in the case of Ae. aegypti and E. aedis appears to include an avoidance mechanism. Adult C x quinquefasciatus mosquitoes did not respond to substrate containing healthy or infected larvae. Ecologically, Cx. quinquefasciatus are more likely to encounter tadpoles, fish, and other natural predators in the wil d because of shared habitat, which would evolutionarily drive the selection for predator avoidance. Specific conditions are needed to support CuniNPV infected mosquitoes. Such conditions are found in pools high in swine effluent that contain high levels of divalent cations (Becnel et al. 2001). A potential explanation for the lack of detection of infected larvae is that swine effluent pools are not a primary breeding site for Cx. quinquefasciatus mosquitoes, and therefore the exposure to CuniNPV infected la rvae may not be enough to evolutionarily drive an avoidance response. Culex mosquitoes will readily oviposit in runoff from swine farms when given the opportunity (Becnel et al. 2001), but the chances of them ovipositing in a pool high in divalent cations such as a swine effluent lagoon is much less

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119 likely than ovipositing in a noninfected pool, and therefore the majority of Culex populations may never be exposed to the virus. Previous studies, in addition to my results, suggest that the ability of ovipos iting females to detect potential threats to their offspring may not be based on a single factor, but rather a combination of host, paras ite, pathogen, environment, and evolution. When comparing microsporidia with nucleopolyhedroviruses, we can clearly see two different modes of infection. Edhazardia aedis and V. culicis are true internal parasites that survive on larval intestinal sugars and deplete nutritional reserves (Riviero et al. 2007). As a result of internal competition, larvae become stressed. Res earch has shown that larvae experience stress in overcrowded conditions or when starved, and stress can be an oviposition deterrent, as in the case of Ae. aegypti (Zahiri and Rau 1998). Baculoviruses generally do not use nutritional reserves from the host. In Lepidoptera, some baculoviruses inhibit ecdysone production in the larvae, which prevents molting prior to death (OReilly and Miller 1989). More recent studies challenged the theory that the lack of molting is the cause of death, but rather it is the degeneration of the malpighian tubules that causes death (Flipsen et al. 1995). If either of these scenarios applied to mosquitoes, there may be no signs of stress from the larvae, but rather normal functioning until death. Larvae infected with CuniNPV are lethargic and respond less to stimuli before dying; however the exac t mechanism of death is unknown. This may be the key to understanding why females cannot detect infected larvae in oviposition substrate. Ecologically speaking, if a mosquito is less lik ely to encounter a predator, parasite, or pathogen in nature, the mosquito will not have developed the necessary defense mechanisms needed to overcome the challenge. Additionally, the length of the relationship may determine the ability to detect potential dangers as species co evolve and counter adapt. In my research, I found

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120 that Ae. aegypti were able to detect E. aedis infected larvae. The infecting microsporidia have highly complex life cycles, are highly pathogenic, and species specific. As a result, A e. aegypti have developed behavioral defense mechanisms against the pathogen. Culex quinquefasciatus was unable to detect CuniNPV, however CuniNPV is not species specific, but is highly pathogenic. Besides not being species specific, CuniNPV needs very spe cific conditions to proliferate. Because Cx. qui nquefasciatus is so widespread and will oviposit in a variety of substrates, the chance of a female encountering a CuniNPV infected pool is low, therefore not driving Cx. quinquefasciatus to develop a defense mechanism. Likewise, Ae. albopictus was unable to detect V. culicis infection in conspecific larvae. Vavraia culicis has a very simple life cycle, low pathogenicity, and like CuniNPV, is not species specific. This low pathogenicity and broad host range ma y not drive Ae. albopictus to develop any defense mechanisms against the microsporidia. As with many parsite infections, there are always tradeoffs between transmission and virulence. Frank (1996) discusses these trade offs and applies several ecological models to explain the complex relationships between parasite genotypes, parasite hosts, and the fitness costs of both the host and the parasite. Simply put, a decrease in virulence increases dispersal rates of the parasites (Frank 1996). Because of the sim ple life cycle and broad host range of V. culicis survival of the microsporidia in nature is more likely, suggesting that the organism may have actually adapted itself to maximize survival. Because Ae. albopictus, and presumably other hosts cannot detect the pathogen, the pathogen has an increased fitness advantage over species specific pathogens such as E. aedis Likewise, CuniNPV has a narrower host range, increasing the likelihood of survival for the virus, although it still inhabits a very limited ecol ogical niche.

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121 Edhazardia aedis is species specific and highly virulent, which decreases the chances of survival and dispersal, although Ae. aegypti is a very widespread and robust species. My results support the use of CuniNPV as a biological control agent because it is undetectable in oviposition substrate, is highly pathogenic, and targets several types of medically important Culex species ( Andreadis et al. 2003). It therefore can be effectively used in ovitraps and would be safe to use on farms where Cu lex mosquitoes are often a major pest as well as a medical threat to both humans and animals (Lord and Rutledge Connelly 2001). My results with E. aedis support the use of this microsporidia for biological co ntrol because infected larvae serve as an ovipos ition deterrent to gravid females Oviposition was not deterred completely, and some females still laid eggs on the infected substrate, which aids in maintaining the pathogen in nature Infection as early instars cause high mortality in larvae however infe ction as later instars supports development to adulthood, which would disseminate the pathogen. Because of Ae. aegyptis preference for ovipositing in tree holes and man made containers, the pathogen would not have to be transmitted in high doses, adding t o its efficacy. Edhazardia aedis can be highly virulent and safe for nontarget aquatic organisms (Becnel 1992) making this method of biological control potentially effective and practical. Vavraia culicis may be a useful bio logical control agent because A e. abopictus and presumably other hosts, cannot detect infected substrate. Kell y et al (1981) reports that V. culicis has fatality rates of 50 60 % in Cx. salinarius Coquillett and Cx. tarsali s Coquillett with low doses, and mortality at a higher doses i n An. albimanus Weidemann Cx. quinquefasciatus Say and Ae. taeniorhynchus (Weidemann) suggesting that although this microsporidia may not exhibit high pathogenicity in Ae. albopictus or Ae. aegypti it would still be useful as a broad range mosquito biological control agent for several pest species (Kelly et al. 1981). Additionally,

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122 V. culicis can be mass produced in Lepidoptera making it easily accessible I found that adult Ae. albopictus do not develop eggs when infected, which would be beneficial from a bio logical control standpoint. However, females still took a bloodmeal, suggesting that although the number of Ae. albopictus produced would decrease, infection would not pevent host feeding by infected Ae. albopictus and therefore would only slow the pathogen transmission cycle. Additional extensive research is need ed to determine the potential for V. culicis to cause microsporidosis in immuno supressed humans.

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123 a a a Uninfected larvae Uninfected larvae. Proportion Eggs Laid ( + SE) 0.0 0.1 0.2 0.3 0.4 0.5 0.6 a a A Proportion Eggs Laid ( + SE) 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 a b B E. aedis-infected larvae Uninfected larvae Figure 41. Oviposition responses of uninfected gravid Aedes aegypti in dual choice oviposition assays to A) two cups containing uninfected larvae (N=15), and B) one cup containing uninfected larvae and one cup containing Edhazardia aedis inf ected larvae (N=30). Means with the same letter are not significantly different (paired t < 0.05).

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124 Uninfected larvae Uninfected larvae. Proportion Eggs Laid ( + SE) 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 a a A Proportion Eggs Laid ( + SE) 0.0 0.2 0.4 0.6 0.8 a b B E. aedis-infected larvae Uninfected larvae Figure 42. Oviposition responses of Edhazardia aedis infected gravid Aedes aegypti in dual choi ce oviposition assays to A) two cups containing uninfected larvae (N=15) and B) one cup containing uninfected larvae and one cup containing E. aedis infected larvae (N=30). Means with the same letter are not significantly different (paired t < 0.05).

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125 Uninfected larvae Uninfected larvae. Proportion Eggs Laid ( + SE) 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 a a A Uninfected larvae V. culicis-infected larvae Proportion Eggs Laid ( + SE) 0.0 0.1 0.2 0.3 0.4 0.5 0.6 a a B Figure 43. Oviposition responses of uninfected gravid Aedes albopictus in dual choice oviposition assays to A ) two cups containing uninfected larvae (N=15), and B ) one cup containing uninfected larvae and one cup containing V. culicis infected larvae (N=30). Means with the same letter are not significantly different (paired t 0.05).

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126 Uninfected larvae Uninfected larvae. Proportion Eggs Laid ( + SE) 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 a a A Uninfected larvae CuniNPV-infected larvae Proportion Eggs Laid ( + SE) 0.0 0.1 0.2 0.3 0.4 0.5 0.6 a a B Figure 44. Oviposition responses of uninfected gravid Culex quinquefasciatus in dua l choice oviposition assays to A ) two cups containing uninfected larvae (N =15), and B ) one cup containing uninfected larvae, and one cup containing CuniNPV infected larvae (N=30). Means with the same letter are not significantly different (paired t test < 0.05).

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127 CHAPTER 5 FUTURE RESEARCH Culex quinquefasciatus and Cx. tarsalis were similar in their oviposition activity with the exception of drinking. Again b y exposing these two species to different substrate compositions, it might be possible to determine if Cx. quinquefasciatus consistently drink more often and for longer than Cx. tarsalis or that was just a result of substrate composition in this study. Be cause Cx. quinquefasciatus prefers substrate high in organic matter, it is possible that drinking may be a key behavior in determining the organic matter content in any given substrate. Detailed Though many behaviors between Ae. aeg ypti and Ae. albopictus were similar or the same, there were some species specific behaviors, such as abdominal fanning. It would be interesting to expose gravid Ae. albopictus females to optimal and less optimal oviposition substrates and observe the fanning behavior to determine if it would change with different substrate compositions. Currently the function of abdominal fanning is a mystery; however by observing how the behavior changes with varying environments, it may be possible to determine whether t he behavior is discriminatory in nature. Another behavior that was observed but not categorized in this study was interactions and conflicts between two ovipositing females. Conflicts usually ended with one female flying away, but it often appeared that a female would intentionally provoke another female by directly contacting her with a leg rather than accidentally bumping into her. Such a behavior would be of particular interest in locations where oviposition sites are limited, which would be likely for t hese container inhabiting species and competition between species may be a factor in the displacement of Ae. aegypti Additionally, by exposing Ae. albopictus females to different substrates, the number of eggs laid per oviposition event can be evaluated t o determine if Ae. albopictus consistantly lay fewer eggs per event, or if that was a factor of substrate composition in this study.

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128 Oviposition behavior studies should be expanded to include more species, and specifically more genera with varying oviposition strategies to understand the steps in oviposition and look for potential interventions in the pathogen transmission cycle. The next step in the dual choice oviposition bioassays is to analy ze water chemisty and volatiles from these oviposition substrates using gas chromatography and mass spectrometry. Several mosquito pheromones have previously been identified; however the detection mechanism of E. aedis infected Ae. aegypti has not been ide ntified. It is possible that infected larvae are secreting some sort of chemical cue specific to E. aedis infection, or it could possibly be a stress pheromone due to the competition for nutritional reserves within the larvae. To determine if the cues are volatiles being emitted from the la rvae or a water based compound, dual choice bioassays could be performed using the larval water that contained uninfected and infected larvae. Following Lowenburger and Raus (1994) study, it would be interesting to cultu re and compare the bacterial content of the larval water of these three species, and treat with antibiotics to determine if Ae. aegypti are responding to bacterial or completely chemical cues. An additional analysis would be the physiological relationshi p between mosquitoes and baculoviruses. Much is known about baculoviruses from Lepidoptera, however the mosquito baculoviruses are dramatically unique. It would be interesting to see if the virus also effects ecdysone production in mosquitoes as it does in Lepidoptera.

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129 APPENDIX A AEDES CODING SCHEME AS ENT ERED INTO THE OBSERV ER XT

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130 APPENDIX B CULEX CODING SCHEME AS ENT ERED INTO THE OBSERV ER XT

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131 LIST OF REFERENCES Afonso, C. L., E. R. Tulman, Z. Lu C. A. Balinsky, B. A. Moser, J. J. Becnel D. L. Ro ck and G. F. Kutish 2001. Genome sequence of a baculovirus pathogenic for Culex nigripalpus J. Virol. 75: 1115711165. Agnew, P., S. Bedhomme, C. Haussy, and Y. Michalakis 1999. Age and size at maturity of the mosquito Culex pipiens infected by the m icrosporidian parasite Vavraia culicis Proc. R. Soc. Lond. B 266: 947952. Angelon, K. A., and J. W. Petranka. 2002. Chemicals of predatory mosquitofish ( Gambusia affinis ) influence selection of oviposition site by Culex mosquitoes. J. Chem. Ecol 28: 797806. Ali, A., and J. K. Nayar. 1997. Invasion, spread, and vector potential of Aedes albopictus in the USA and its control possibilities. Med. Entomol. Zool. 48: 19. Allan, S. A., U. R. Bernier, and D. A. Kline 2005. Evaluations of oviposition substrates and organic infusions on collection of Culex in Florida. J. Am. Mosq. Control Assoc. 21: 268273. Andreadis, T. G., J. F. Anderson C. R. Vossbrink and A. J. Main. 2004. Epidemiology of West Nile virus in Connecticut: A five year analysis of mosq uito data 19992003. Vector Borne Zoonotic Dis. 4: 360378. Andreadis, T. G., J. J. Becnel and S. E. White. 2003. Infectivity and pathogenicity of a novel baculovirus, CuniNPV, from Culex nigripalpus (Diptera: Culicidae) for thirteen species and four gen era of mosquitoes. J. Med. Entomol. 40: 512517. Barnard, D. R., R. Xue M. A. Rotstein and J. J. Becnel. 2007. Microsporidiosis (Microsporidia: Culicosporidae) alters bloodfeeding responses and DEET repellency in Aedes aegypti (Diptera: Culicidae). J. Med. Entomol. 44: 10401046. Barrett, A. D. T., and S. Higgs. 2007. Yellow fever: A disease yet to be conquered. Ann. Rev. Entomol. 52: 209229. Beament, J., and S. A. Corbet. 1981. Surface properties of Culex pipiens eggs and the behaviour of the femal e during eggraft assembly. Physiol. Entomol. 6: 135148. Becnel, J. J. 1992. Safety of Edhazardia aedis (Microsporidia: Amblyosporidae) for nontarget aquatic organisms. J. Am. Mosq. Control Assoc. 8: 256260. Becnel, J. J., and A. H. Undeen. 1992. Infl uence of temperature on developmental parameters of the parasite/host system Edhazardia aedis (Microspora: Amblyosporidae) on Aedes aegypti (Diptera: Culicidae). J. Parasitol 92: 299303.

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132 Becnel, J. J., and T. G. Andreadis 1999. Microsporidia in insects pp. 447501. In M. Wittner (ed.), The Microsporidia and Microsporidosis Am erican Society for Microbiology, Washington D.C. Becnel, J. J., and M. A. Johnson 2000. Impact of Edhazardia aedis (Microsporidia: Culicosporidae) on a seminatural population of Aedes aegypti (Diptera: Culicidae). Biol. Control. 18: 3948. Becnel, J. J., and S. E. White 2007. Mosquito pathogenic viruses: T he last 20 years. Bull. Am. Mosq. Control Assoc. 7: 3649. Be cnel, 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. Becnel, J.J., S.E. White and A.M. Shapiro. 2003. Culex nigripalpus nucleopolyhedrovirus (CuniNPV) infections in adult mosquitoes and possible mechanisms for dispersal J. Invertebr. Pathol. 83: 181183. Becnel, J. J., S. E. White and A. M. Shapiro 2005. Review of microsp oridia mosquito relationships: F rom the simple to t he complex. Folia Parasitol 54: 4150. Becnel, J. J., S. E. White B. A. Moser, T. Fukuda M. J. R otstein A. H. Undeen and A. Cockburn. 2001. Epizootiology and transmission of a newly discovered baculovirus from the mosquitoes Culex nigripalpus and C. quinquefasciatus J. Gen. Virol. 82: 275282. Bentley, M. D., and J. F. Day. 1989. Chemical ecology and behavioral aspects of mosquito oviposition. Ann. Rev. of Entomol. 34: 401421. Blackmore, C. G. M., L. M. Starke, W. C. Jeter R. L. Oliveri, R. G. Brooks L. A. Conti, and S. T. Weirsma. 2003. Surveillance results from the first West Nile virus transmission season in Florida, 2001. Am. J. Trop. Med. Hyg. 60: 141150. Blaustein, L., and B. P. Kotler. 1993. Oviposition habitat selection by the mosquito, Culiseta longiareolata: effects of conspecifics, food and green toad tadpoles. Ecol. Entomol 18: 104108. Blaustein, L., M. Kiflawi, A. Eitam M. Mangel and J. E. Cohen. 2004. Oviposition habitat selection in response to risk of predation in temporary pools: mode of detection and consist ency across experimental value. Oecologia. 138: 300305. Blaustein, L., J. Blaustein and J. Chase. 2005. Chemical detection of the predator Notonecta irrorata by ovipositing Culex mosquitoes. J. Vector Ecol 30: 299301.

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134 Day, J. F. 2005. Host seeking strategies of mosquito vectors. J. Am. Mosq. Control Assoc. 21: 1722. Day, J. F., and G. A. Curtis 1999. Blood feeding and oviposition by Culex nigripalpus (Diptera: Culicidae) before, during, and after a widespread St. Louis encephalitis virus epidemic in Florida. J. Med. Entomol. 36: 176181. Day J. F., and J. D. Edman. 1988. Host location, bloodfeeding, and oviposition behavior of Culex nigripalpus (Diptera: Culic idae): Their influence on St. Louis encephalitis virus transmission in southern Florida. Misc. Publ. Entomol Soc Am 68: 18. Day, J. F., and L. M. Starke. 2000. Frequency of St. Louis encephalitis virus in humans from Florida, USA: 19901999. J. Med. E ntomol. 37: 626633. Eitam, A., Blaustein, L., and M. Mange. 2002. Effects of Anisops sardea (Hemiptera: Notonectidae) on oviposition habitat selection structure in artificial pools. Hydrobiologia. 485: 183189. Epstein, P. R., H. F. Diaz S. Elias G. Grabherr N. E. Graham W. J. M. Martens E. Mosley Thompson and J. Susskind. 1998. Biological and physical signs of climate change: Focus on mosquitoborne diseases. Bull. Am. Meterol. Soc. 79: 409417. Flint, H. M., and C. C. Doan 1996. Understanding semiochemicals with emphasis on insect sex pheromones in integrated pest management programs. In E. B. Radcliffe, W.D. Hutchison, and R. E. Cancelado (eds.), IPM World Textbook. Retrieved November 13, 2009. University of Minnesota, St. Paul, Minnesota. Web site: http://ipmworld.umn.edu/ Flipsen, J. T. M., R. M. W. Manns A. W. F. Kleefsman D. Knebel Morsdorf and J. M. Vlak. 1995. Deletion of the baculovirus ecdysteroid UDP glucosyltransferase gene induces early de generation of malpighian tubules in infected insects. J. Virol 69: 45294534. Foster, W. A., and E. D. Walker 2002. Mosquitoes: Culicidae. pp. 203256. In G. Mullen and L. Durden (eds.), Medical and Veterinary Entomology Academic Press, San Diego, C A Frank, S. A. 1996. M odels of parasite virulence. Q. Rev. Biol 71: 3778. Fukuda, T., O. R. Willis and D. R. Barnard 1997. Parasites of the Asian tiger mosquito and other container inhabiting mosquitoes (Diptera: Culicidae) in Northcentral Florida J Med. Entomol 34: 226233. Gargan II, T. P., G. G. Clark D. D. Dohm M. J. Turell and C. L. Bailey 1988. Vector potential of selected North American mosquito species for Rift Valley fever virus. Am. J. Trop. Med. Hyg. 38: 440 446.

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142 BIOGRAPHICAL SKETCH Catherine Zettel N alen was raised in Rochester, New York. After graduating from RushHenrietta Senior High S chool in 2002, Catherine attended the State University of New York at Oswego with a major in zoology. In 2006, she was awarded a Bachelor of Science degree, and moved to Gainesville, Florida to persue a career working with old world fruit bats. In 2007, she joined the University of Florida full time for a Master of Science degree in entomology and nematology, with a concentration in medical entomology.