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Surveillance of Aedes albopictus (Skuse) (Diptera

Permanent Link: http://ufdc.ufl.edu/UFE0024716/00001

Material Information

Title: Surveillance of Aedes albopictus (Skuse) (Diptera Culicidae) Suburban and Sylvatic Populations Using Traps and Attractants in North Central Florida
Physical Description: 1 online resource (193 p.)
Language: english
Creator: Obenauer, Peter
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: aedes, albopictus, attractants, distribution, habitats, oviposition, traps, triseriatus, vertical
Entomology and Nematology -- Dissertations, Academic -- UF
Genre: Entomology and Nematology thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: A variety of attractants, traps and sampling methods have been developed to lure and capture mosquito species based on host and oviposition preference. Aedes albopictus (Skuse) is an invasive mosquito known to inhabit suburban and sylvatic habitats of north central Florida, but their vertical distribution within these environments remains unknown. A series of laboratory and field experiments comparing traps, attractants and surveillance methods were designed to ascertain host-seeking and oviposition height preferences between suburban and sylvatic Ae. albopictus populations. The response of Ae. albopictus to the BG-Sentinel , Omni-directional Fay-Prince and Mosquito Magnetregistered trademark X traps was evaluated in four suburban and four sylvatic sites. Trap captures indicate that Ae. albopictus were attracted to traps placed at 6 m; however, the majority (87%) were captured at 1 m. Although no significant differences were detected between trap collections, more Ae. albopictus were captured using the BG-Sentinel trap. Infusion oviposition attractiveness was evaluated in field cages, laboratory bioassays and a dual-port olfactometer to investigate the role north central Florida plant detritus may play in oviposition response. Aedes albopictus demonstrated a stronger oviposition response to containers with plant-based infusions compared to water alone. Results varied among infusion experiments, but oak-pine infusions were the most effective at eliciting an oviposition response from Ae. albopictus. Ovitraps at 1 and 6 m containing oak-pine and oak were evaluated in four suburban and four sylvatic sites. Although no significant differences were detected among infusion treatments, more eggs were laid in ovitraps containing infusions compared to those with only water. Aedes albopictus eggs were collected at 6 m, but the majority (81%) of eggs were collected at 1 m. Furthermore, the majority of eggs were collected in suburban sites, while sylvatic sites comprised less than 14% of the total capture. The BG-Sentinel trap, an oak-pine baited gravid trap, an aspirator and human landing-counts were evaluated to determine their efficacy at detecting the presence of Ae. albopcitus in suburban and sylvatic habitats. Although no method or device was superior at detecting sylvatic populations, the BG-Sentinel trap collected significantly more Ae. albopictus in suburban habitats as compared to the other three surveillance techniques evaluated.
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 Peter Obenauer.
Thesis: Thesis (Ph.D.)--University of Florida, 2009.
Local: Adviser: Kaufman, Phillip Edward.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2010-02-28

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0024716:00001

Permanent Link: http://ufdc.ufl.edu/UFE0024716/00001

Material Information

Title: Surveillance of Aedes albopictus (Skuse) (Diptera Culicidae) Suburban and Sylvatic Populations Using Traps and Attractants in North Central Florida
Physical Description: 1 online resource (193 p.)
Language: english
Creator: Obenauer, Peter
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: aedes, albopictus, attractants, distribution, habitats, oviposition, traps, triseriatus, vertical
Entomology and Nematology -- Dissertations, Academic -- UF
Genre: Entomology and Nematology thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: A variety of attractants, traps and sampling methods have been developed to lure and capture mosquito species based on host and oviposition preference. Aedes albopictus (Skuse) is an invasive mosquito known to inhabit suburban and sylvatic habitats of north central Florida, but their vertical distribution within these environments remains unknown. A series of laboratory and field experiments comparing traps, attractants and surveillance methods were designed to ascertain host-seeking and oviposition height preferences between suburban and sylvatic Ae. albopictus populations. The response of Ae. albopictus to the BG-Sentinel , Omni-directional Fay-Prince and Mosquito Magnetregistered trademark X traps was evaluated in four suburban and four sylvatic sites. Trap captures indicate that Ae. albopictus were attracted to traps placed at 6 m; however, the majority (87%) were captured at 1 m. Although no significant differences were detected between trap collections, more Ae. albopictus were captured using the BG-Sentinel trap. Infusion oviposition attractiveness was evaluated in field cages, laboratory bioassays and a dual-port olfactometer to investigate the role north central Florida plant detritus may play in oviposition response. Aedes albopictus demonstrated a stronger oviposition response to containers with plant-based infusions compared to water alone. Results varied among infusion experiments, but oak-pine infusions were the most effective at eliciting an oviposition response from Ae. albopictus. Ovitraps at 1 and 6 m containing oak-pine and oak were evaluated in four suburban and four sylvatic sites. Although no significant differences were detected among infusion treatments, more eggs were laid in ovitraps containing infusions compared to those with only water. Aedes albopictus eggs were collected at 6 m, but the majority (81%) of eggs were collected at 1 m. Furthermore, the majority of eggs were collected in suburban sites, while sylvatic sites comprised less than 14% of the total capture. The BG-Sentinel trap, an oak-pine baited gravid trap, an aspirator and human landing-counts were evaluated to determine their efficacy at detecting the presence of Ae. albopcitus in suburban and sylvatic habitats. Although no method or device was superior at detecting sylvatic populations, the BG-Sentinel trap collected significantly more Ae. albopictus in suburban habitats as compared to the other three surveillance techniques evaluated.
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 Peter Obenauer.
Thesis: Thesis (Ph.D.)--University of Florida, 2009.
Local: Adviser: Kaufman, Phillip Edward.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2010-02-28

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0024716:00001


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1 SURVEILLANCE OF AEDES ALBOPICTUS (SKUSE) (DIPTERA: CULICIDAE) SUBURBAN AND SYLVATIC POPULATION S USING TRAPS AND ATTRACTANTS IN NORTH CENTRAL FLORIDA By PETER JOSEPH OBENAUER A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2009

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2 2009 Peter J. Obenauer

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3 To Kathy, Lauren, Alexandra and my mom

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4 ACKNOWLEDGMENTS I greatly appreciate the United States Na vy, specifically the Navy Medicine Manpower, Personnel, Training and Education Commands (NMMPTEC) Duty Under Instruction program for providing me this great opportu nity to pursue a Doctor of Philosophy degree in entomology. Mrs. Patricia Edwards and the NMMPTEC comma nd staff have been extremely supportive and gracious while Ive been in school I also thank Dr. Andreas Ro se of BioGents for providing BG-Sentinel traps used in this study. I thank my major advisor, Dr. Phillip E. Kaufman for his guidance and support throughout this project. He provided me office space, vehi cles, materials, lab use, as well as financial support to various professional meetings. He ha s been instrumental in guiding me through these past three years and was especially patient while I analyzed my research data. It has been an honor to be his first Ph.D. student. I am indebted to the members of my graduate committee, Dr. Daniel Kline, Dr. Sandra Allan, Dr. Phil Lounibo s and Dr. Ellis Greiner for their tremendous support, attention and considera tion of my research. I especi ally thank Dr. Sandra Allan for providing statistical insights, additional equipment and lab space. I am grateful for her patience and assistance with my research; she always ke pt her office door opened, never once turning me away. I gratefully acknowledge Jimmy Pitzer for his countless hours of editing this dissertation and providing valuable suggesti ons regarding my research. I consider him a good friend and scientist. In addition, many thanks to the followi ng people for assistance w ith collection of data throughout this project: Kathy Oben auer, Lauren Hurst, Catherine Zettel, Derek Puckett, Bart Bass, Amanda Kushner and Ryan Larson. Special thanks to Lois Wood for assistance with researching and purchasing materi als used in this research. Special thanks to my fellow shipmates, Dr David Hoel and Dr. Craig Stoops, for their helpful suggestions regarding my research. I de eply appreciate their encouragement and support

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5 during the last three years. In addition, I want to recognize CDR Michael Mann (retired) and CDR Bill Kanour (retired) for encouraging me to pursue a Ph.D. I consider them both great mentors. I thank the staff of San Felasco Hammock Pres erve State Park and private landowners who granted me the privilege to conduct research on their property. These people include: Dr. Jerry Hogsette, Dr. Faith Oi, Dr. Don Dickson, Dr. D on Hall, Dr. Kenneth Dodd, Dr. Amy Simone and Mr. Bubba Grainer. The professors in the Entomology and Nematol ogy department are an exceptional group of scientists. I especially thank Dr. Don Hall, Dr. Jerry Hogsette and Dr. Je rry Butler (retired) for their scientific insights and wisdom. They always treated me like a professional, rather than a graduate student and for that I am extremely gr ateful. I will miss our weekly conversations. I thank Debbie Hall for her assistance with all of my administrative requirements. I am indebted to the personnel and staff at USDA-ARS-CMAVE Gainesville, FL. I especially thank Erin Vrzal w ho educated me on mosquito rear ing, infusion preparation and the use of the olfactometer. I also thank Dr. John Sivinski for providing screened cages and Mr. Charlie Stuhl with their set-up. I thank my wife, Kathy, for her loving support du ring these stressful and challenging years. She is my best friend and I owe her more than words can describe. I thank my mom, Patricia Andrews, for providing loving suppo rt throughout my life. In reme mbrance of my father, Peter James Obenauer, I thank him for th e lifelong sacrifices he made to provide me the opportunity to attain higher education. I am confident that I ha ve made him proud. Finally, I thank God for this great life, I am truly blessed!

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6 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4 LIST OF TABLES................................................................................................................. ..........9 LIST OF FIGURES................................................................................................................ .......10 ABSTRACT....................................................................................................................... ............13 CHAPTER 1 INTRODUCTION AND LITERATURE REVIEW OF AEDES ALBOPICTUS SKUSE.....15 Introduction to Aedes albopictus ............................................................................................15 Taxonomy and Distribution.............................................................................................16 Habitat........................................................................................................................ .....18 Bionomics...................................................................................................................... .........19 Host Preference, Host Seeking and Biting Behavior.......................................................19 Mating and Flight Range.................................................................................................22 Diapause and Photoperiod...............................................................................................22 Oviposition, Fecundity and Longevity............................................................................24 Medical Importance............................................................................................................. ...26 Dengue......................................................................................................................... ....26 La Crosse virus................................................................................................................29 West Nile virus................................................................................................................30 Competition and Displacement...............................................................................................31 Surveillance Devices........................................................................................................... ...33 Host Seeking Traps and Attractants................................................................................34 Ovitraps and Gravid Traps..............................................................................................35 Control Measures............................................................................................................... .....36 Biological and Cultural Control......................................................................................37 Chemical Control.............................................................................................................39 Research Objectives............................................................................................................ ....40 2 HOST-SEEKING HEIGHT REFERENCES OF AEDES ALBOPICTUS WITHIN SUBURBAN AND SYLVATIC LOCALE S IN NORTH CENTRAL FLORIDA...............42 Introduction................................................................................................................... ..........42 Materials and Methods.......................................................................................................... .45 Site Selection................................................................................................................. ..45 Traps and Baits................................................................................................................46 Trapping Scheme.............................................................................................................48 Statistical Analysis..........................................................................................................50 Results........................................................................................................................ .............50 Aedes albopictus ..............................................................................................................51

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7 Other Mosquito Species..................................................................................................51 Discussion..................................................................................................................... ..........53 3 OVIPOSITON RESPONSE OF AEDES ALBOPICTUS TO INFUSIONS USING COMMON NORTH CENTRA L FLORIDA PLANTS.........................................................69 Introduction................................................................................................................... ..........69 Materials and Methods.......................................................................................................... .72 Infusions...................................................................................................................... ....72 Mosquitoes..................................................................................................................... .73 Laboratory Cage Bioassays.............................................................................................74 Olfactometer Bioassays...................................................................................................75 Field Cage Bioassays.......................................................................................................76 Statistical Analysis..........................................................................................................78 Results........................................................................................................................ .............79 Laboratory Cage Bioassays.............................................................................................79 Olfactometer Bioassays...................................................................................................80 Field Cage Bioassays.......................................................................................................80 Discussion..................................................................................................................... ..........81 4 EFFICACY OF INFUSION-BAITED OVITRAPS AT TWO HEIGHTS TO MONITOR AEDES ALBOPICTUS IN NORTH-CENTRAL FLORIDA SUBURBAN AND SYLVATIC LOCALES................................................................................................94 Introduction................................................................................................................... ..........94 Materials and Methods.......................................................................................................... .97 Site Selection................................................................................................................. ..97 Baited Ovitraps................................................................................................................97 Infusions...................................................................................................................... ....98 Trapping, Collecting and Egg Identification...................................................................99 Statistical Analysis........................................................................................................101 Results........................................................................................................................ ...........101 Aedes albopictus ............................................................................................................102 Other Mosquito Species................................................................................................103 Discussion..................................................................................................................... ........104 5 EFFICACY OF FOUR SURVEILLAN CE TECHNIQUES TO DETECT AND MONITOR AEDES ALBOPICTUS IN NORTH CENTRAL FLORIDA SUBURBAN AND SYLVATIC HABITATS............................................................................................122 Introduction................................................................................................................... ........122 Materials and Methods.........................................................................................................125 Site Selection.................................................................................................................125 Surveillance Methods....................................................................................................126 Surveillance and Collection Scheme.............................................................................129 Statistical Analysis........................................................................................................130 Results........................................................................................................................ ...........131

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8 Aedes albopictus ............................................................................................................131 Other Mosquito Species................................................................................................133 Discussion..................................................................................................................... ........134 6 FUTURE DEVEVLOPMENT AND APPLICATION OF TRAPS AND ATTRACTANTS TO MONITOR AEDES ALBOPICTUS POPULATIONS.....................153 Introduction................................................................................................................... ........153 Traps and Attractants.......................................................................................................... ..154 APPENDIX A Host-seeking height preferences of Aedes albopictus within suburban and sylvatic locales in north central Florida utilizing three types of traps...............................................157 B Mean monthly temperatures ( C) for suburban and sylvatic lo cales near Gainesville, FL (May-September 2007).........................................................................................................159 C Mean monthly light intensity for suburban and sylvatic locales near Gainesville, FL (May September 2007)......................................................................................................160 D UNIVERSITY OF FLORIDA HEALTH CENTER INSTITUTIONAL REVIEW BOARD #36-2007................................................................................................................161 LIST OF REFERENCES.............................................................................................................166 BIOGRAPHICAL SKETCH.......................................................................................................192

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9 LIST OF TABLES Table page 2-1 Mosquitoes captured at 1 and 6 meter heights in suburban and sylvatic locales between May September 2007 in Gainesville, Florida. ................................................61 2-2 Numbers (mean SE) of the nine most common female mosquitoes collected in a trapping period from three types of traps at 1 and 6 meter heights in suburban and sylvatic locales between May September 2007 in Gainesville, Florida..........................62 3-1 Oviposition response of Aedes albopictus to six infusions and a well water control in indoor cages................................................................................................................... ....92 3-2 Upwind response of 4-d-old gravid Aedes albopictus to 500 L infusion concentrate compared to well water control inside a dual-port olfactometer for 10 min.....................92 3-3 Oviposition response of Aedes albopictus to three concentrations of hay infusions and well water in outdoor cages, Gainesville, Florida, June 2007....................................93 4-1 Total abundance of Culicidae eggs captur ed using ovitraps baited with infusions or well water and suspended at 1 and 6 m he ights in suburban and sylvatic locales between May October 2008 in Gainesville, Florida.....................................................121 5-1 Total mosquitoes collected by four surv eillance methods in suburban and sylvatic locales, Gainesville, Florida, May September, 2008....................................................149 5-2 Mean (SE) of the six most common mosquitoes co llected using the BG Sentinel and CDC gravid traps in suburban and sylva tic locales in Gainesville, Florida....................151 5-3 Mean (SE) of the six most common mosquitoes collected using human landingcounts and a vegetative aspirator in suburba n and sylvatic locales in Gainesville, Florida........................................................................................................................ ......152 A-1 Total Aedes albopictus captured by trap within trial (time period), height and locale between May September 2007 in Gainesville, Florida.................................................157

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10 LIST OF FIGURES Figure page 2-1 A typical residential backyard used for a suburban site (A) and a typical sylvatic site (B) in San Felasco Hammock Preserve Stat e Park, Gainesville, Florida used to collect Aedes albopictus .....................................................................................................63 2-2 Traps used to evaluate host-seeking height of Aedes albopictus in suburban and sylvatic locales. ............................................................................................................ ....64 2-3 Mosquito Magnet-X trap positioned at 6 m in height using an interwoven nylon rope that was attached to a 25 mm metal loop, Sa n Felasco Hammock State Preserve State Park, Gainesville, Florida..................................................................................................65 2-4 Seasonal distribution of Aedes albopictus captured in 2007 and pr ecipitation (cm) for suburban and sylvatic locales in Gainesville, Florida........................................................66 2-5 Mean capture rates of the nine most commonly trapped mosquitoes at 1 m and 6 m heights in sylvatic and suburban lo cales between May September 2007 in Gainesville, Florida. ........................................................................................................ .67 2-6 White arrow denotes tree-hole supporting Aedes albopictus larvae in San Felasco Hammock Preserve State Par k, Gainesville, Florida.........................................................68 3-1 Aedes albopictus adults feeding on a suspended sa usage casing containing bovine blood.......................................................................................................................... ........87 3-2 Laboratory cage containing tw o black 156 ml cups, containing either an infusion or a well water control used to de termine oviposition preference............................................87 3-3 Triple-cage dual port olfact ometer (side-view) used in testing oviposition response, USDA-ARS-CMAVE, Gainesville, Florida......................................................................88 3-4 Circular outdoor screened cages us ed in oviposition trials, USDA-ARS-CMAVE, Gainesville, Florida........................................................................................................... .88 3-5 Interior area of outdoor screened cag e demonstrating ovitrap placement and the nylon curtain fabric covering, USDA-AR S-CMAVE, Gainesville, Florida.....................89 3-6 Mean upwind response of gravid Aedes albopictus to a 500 uL concentrate of infusion placed inside a dual-port olf actometer for 10 min at the USDA-ARSCMAVE, Gainesville, Florida...........................................................................................89 3-7 Aedes albopictus female exhibiting a ovipositiona l behavioral response to an oviposition infusion while in an olfactometer. ................................................................90

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11 3-8 Mean 48-hr oviposition response of 100 Aedes albopictus females released into field cages with ovicups containing infusion treatments and a well water control at USDA-ARS-CMAVE, Gainesville, Flor ida, June September 2007. ............................91 4-1 Mosquito ovitraps construc ted from 473 ml steel cans and inverted pie dishes. A detached hook allowed for easy removal of substrate paper (A) and once attached to the cover, (B) the trap could be suspended......................................................................113 4-2 Ovitrap suspended at 6 m in San Felasc o Hammock Preserve St ate Park, Gainesville, Florida........................................................................................................................ ......113 4-3 Eggs recovered from ovitraps unde r a 10X dissecting microscope: A) Aedes albopictus B) Ochlerotatus triseriatus C) Orthopodomyia signifera D) Toxorhynchites rutilus rutilus ..........................................................................................114 4-4 Weekly precipitation an d seasonal distribution of Aedes albopictus and Ochlerotatus triseriatus eggs recovered from ov itraps placed in suburban and sylvatic locales in 2008, Gainesville, Florida. .............................................................................................115 4-5 Percent of Aedes albopictus eggs recovered from infusion baited-ovitraps placed in four suburban and four sylv atic sites between May Oc tober 2008 in Gainesville, Florida. n = 20 trapping periods (1 week each)..............................................................116 4-6 Mean (SEM) number of Aedes albopictus eggs recovered from 1 m and 6 m suspended ovitraps containing plant-derived infusions and a well water control. ........117 4-7 Mean (SEM) number of Aedes albopictus eggs recovered from ovitraps suspended 1 and 6 m in suburban and sylvatic locales in Gainesville, Florida. Traps were operated between May October 2008............................................................................118 4-8 Mean (SEM) number of Ochlerotatus triseriatus eggs recovered from 1 m and 6 m suspended ovitraps containing plant-derive d infusions and a well water control. Traps were placed in suburban and sylvat ic locales between May October 2008 in Gainesville, Florida. .......................................................................................................119 4-9 Mean (SEM) number of Ochlerotatus triseriatus eggs recovered from ovitraps suspended 1 and 6 m in suburban and sylvatic locales in Gainesville, Florida. Traps were operated between May October 2008. ................................................................120 5-1 Two suburban residential sites used for Aedes albopictus collection in Gainesville, Florida........................................................................................................................ ......142 5-2 A typical sylva tic site used for Aedes albopictus collection in San Felasco Hammock Preserve State Park, Ga inesville, Florida.........................................................................142 5-3 A BG-Sentinel trap used to lure and collect mosquitoes...............................................143

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12 5-4 A CDC Gravid trap model 1712 containi ng oak-pine infusion and placed on the forest floor in San Felasco Hammock Pres erve State Park, Gainesville, Florida............143 5-5 A large mosquito aspirator displaying the inside catch bag (A) was operated by the author to collect resting mosquitoes fr om sylvatic and suburban locales (B).................144 5-6 The author performing mosquito landingcounts using a hand-held aspirator in San Felasco Hammock Preserve State Park, Gainesville, Florida..........................................144 5-7 Composition of the nine most commonly collected mosquito species using four surveillance techniques in suburban and sylvatic locales between May September 2008 in Gainesville, Florida.............................................................................................145 5-8 Likelihood of detection of four surveillance methods on dates when Aedes albopictus were recovered by at least one of the sampling methods. Sampling occurred in suburban and sylvatic locales between May September 2008 in Gainesville, Florida........................................................................................................................ ......146 5-9 Seasonal abundance of female Aedes albopictus in Gainesville, Florida suburban and sylvatic locales between May September 2008, as measured with four trap collection techniques........................................................................................................147 5-10 Comparative efficiency of four sampling devices in capturing female Aedes albopictus in Gainesville, Florida suburban and sylvatic locales between May September 2008...............................................................................................................148

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13 Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy SURVEILLANCE OF AEDES ALBOPICTUS (SKUSE) (DIPTERA: CULICIDAE) SUBURBAN AND SYLVATIC POPULATION S USING TRAPS AND ATTRACTANTS IN NORTH CENTRAL FLORIDA By Peter J. Obenauer August 2009 Chair: Phillip E. Kaufman Major: Entomology and Nematology A variety of attractants, trap s and sampling methods have been developed to lure and capture mosquito species based on host and oviposition preference. Aedes albopictus (Skuse) is an invasive mosquito known to inhabit suburban and sylvatic hab itats of north central Florida, but their vertical distribution with in these environments remains unknown. A series of laboratory and fiel d experiments comparing traps, attractants and surveillance methods were designed to ascertain host-see king and oviposition height preferences between suburban and sylvatic Ae. albopictus populations. The response of Ae. albopictus to the BGSentinel, Omni-directional Fay-Prince and Mosquito Magnet X traps was evaluated in four suburban and four sylvatic site s. Trap captures indicate that Ae. albopictus were attracted to traps placed at 6 m; however, the majority (87%) were captured at 1 m. Although no significant differences were detected be tween trap collections, more Ae. albopictus were captured using the BG-Sentinel trap. Infusion oviposition attractiveness was evaluated in field cages, laboratory bioassays and a dual-port olfactometer to investigate the role no rth central Florida plant detritus may play in oviposition response. Aedes albopictus demonstrated a stronger oviposition response to

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14 containers with plant-based infusions compared to water alone. Results varied among infusion experiments, but oak-pine infusi ons were the most effective at eliciting an oviposition response from Ae. albopictus. Ovitraps at 1 and 6 m containing oak-pi ne and oak were evaluated in four suburban and four sylvatic sites. Although no significant differences were detected among infusion treatments, more eggs were laid in ovitr aps containing infusions compared to those with only water. Aedes albopictus eggs were collected at 6 m, but the majority (81%) of eggs were collected at 1 m. Furthermore, the majority of eggs were collected in suburban sites, while sylvatic sites comprised less than 14% of the total capture. The BG-Sentinel trap, an oak-pine baited gravid tr ap, an aspirator and human landingcounts were evaluated to determine their efficacy at detecti ng the presence of Ae. albopcitus in suburban and sylvatic habitats. Although no method or device was s uperior at detecting sylvatic populations, the BG-Sentinel trap collected significantly more Ae. albopictus in suburban habitats as compared to the other thre e surveillance techniques evaluated.

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15 CHAPTER 1 INTRODUCTION AND LITERATURE REVIEW OF AEDES ALBOPICTUS SKUSE Introduction to Aedes albopictus Throughout history, mosquitoes have been res ponsible for causing some of the greatest scourges affecting mankind. They are vectors of numerous pathogens including the causal agents of malaria, dengue, yellow fever, filariasis and viral encephalitides, as well as a host of diseases affecting animals (Harwood and James 1 979). To a lesser degree, mosquitoes inflict numerous and painful bites to humans and anim als, negatively affecting animal production, causing areas to become unusable for recreation and restricting economic progress (Foster and Walker 2002). The state of Florida holds a reputation for having an abundanc e of mosquitoes. Of the approximately 3,200 mosquito species found throughout the world, 81 currently occur in Florida (Day 2005). Floridas mild climate, along with its numerous swamps, ponds and canals provide ideal breeding habitats for many mosquito specie s. Historically, Florida has been adversely affected by yellow fever, dengue and malaria. Thes e diseases were quickly associated with the state, discouraging early settlements and hamp ering economic development (Patterson 2004). Early visitors to the state quickly coined it t he devils property in response to the large numbers of mosquitoes (Patterson 2004). The Asian tiger mosquito, Aedes albopictus (Skuse), is an exotic mosquito introduced in the late 1980s to north central Fl orida and has since become a seriou s nuisance. It is responsible for the majority of complaints received by ma ny vector control offi cials in residential neighborhoods (Kelly Etherson, pers. comm.). Unlike most mosquitoes, Ae. albopictus is diurnal, preferring to feed dur ing daylight hours, a time when many people are active. Its

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16 biology and close association with human dwellings make standard mosquito control practices unattractive and difficult to employ (Hoel 2005). Aedes albopictus reputation as being a severe nuisance is equally matched by its ability to vector at least 23 arboviruses, including dengue (Moore and Mitchell 1997). Though dengue is not endemic to Florida, past out breaks have occurred. The state experienced a severe outbreak in 1922 that caused an estimated 200,000 cases (Patterson 2004). In 1934, a smaller epidemic affected the city of Miami, resulting in an estimated 15,000 cases (Florida Coordinating Council on Mosquito Control 1998). While Ae. albopictus was not implicated in either of these outbreaks, they are a reminder that vector-borne diseases can arise when certain epidemiological conditions are met. The arrival of Ae. albopictus into the United States almost 25 years ago stimulated great interest and resulted in numerous studies to ascertain additional information on its biology and its influence on other native and non-native mosquito populations and disease transmission cycles. Due to its demonstrated ability to colonize a variety of enviro nments, surveillance and control strategies continue to ev olve in order to manage this persis tent pest. Information gained since the accidental introduction of Ae. albopictus coupled with improved su rveillance techniques, have assisted in preventing th e arrival of other exotic mos quitoes, as well as forseeing the potential importation for othe r non-native organisms (Lounibos 2002). Furthermore, the application of these surveillan ce strategies cannot only prevent future mosquito introductions, but may help limit the spread of other inva sive organisms (Juliano and Lounibos 2005). Taxonomy and Distribution Skuse (1894) originally described Ae. albopictus as Culex albopictus from specimens collected in Calculta, India and named it the banded mosquito of Bengal due to their striking black and white stripes. It has since been renamed the Asian tige r mosquito and is in the order

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17 Diptera, suborder Nematocera, family Culicidae, genus Aedes subgenus Stegomyia group scutellaris and subgroup albopictus (Huang 1970). Aedes albopictus is believed to have orig inated in forest-fringe ar eas of Southeast Asia and gradually expanded throughout the Oriental region to include China, Japan, New Guinea and most of the islands in the Indian and Pacifi c Oceans (Hawley 1988). Its current range has expanded with reports from Belgium, France, It aly and Switzerland in Eu rope, Israel, Cameroon, Equatorial Guinea and Nigeria in Africa (Gratz 2004), Spain (Aranda et al. 2006) and recently in Lebanon and Syria (Haddad et al. 2007). It has also become a widespread pest in the United States, as well as Central and South America. Sp reading to at least 28 c ountries within the past two decades, it is considered one of the most inva sive mosquitoes in the world (Benedict et al. 2007). The rapid spread of Ae. albopictus throughout the world has been associated with the importation of used tires (Rai 1991) facilitated by ship tr ansportation (Lounibos 2002). Inspections of seagoing freight co ntainers have revealed that Ae. albopictus has been the most frequently collected mosquito among Japanese tire casings (Craven et al. 1988). Historically, the successful establishment of Ae. albopictus has been greatly influenced by countries that have a high volume of sea traffic and ports that are cl imatically similar to each other (Tatem et al. 2006). It has been proposed that Ae. albopictus was introduced to North America in used tires imported from northern Asia, most likely Japan (Hawley et al. 1987, Rai 1991). An increase in the importation of ornamental plan ts, specifically lucky bamboo ( Dracaens spp.) has further contributed to the spread Ae. albopictus (Madon et al. 2002). The first report of Ae. albopictus being trapped and identified in North Ameri ca occurred in June of 1983 in a Memphis, TN

PAGE 18

18 cemetery refuse dump (Reiter and Darsie 1984 ). The first established population of Ae. albopictus was discovered in August of 1985 in di scarded tires in Harris County, Texas (Sprenger and Wuithiranyagool 1986). By 1988, Ae. albopictus had spread throughout much of the southeast and was reported east of the Mississippi river and as far north as Illinois, Ohio, Maryland and Delaware (Hawley 1988). Past pr edictions have limited the distribution of Ae. albopictus to the -5 C isotherm line (Nawrocki and Hawley 1987). However, potential changes in the current weather patterns in the United States may extend this line further north, especially if there is an increase in summe r temperatures (Alto and Juliano 2001). In July 1986, Ae. albopictus was discovered in Duval County, Florida in a used tire lot with the majority of larvae occurring next to a wooded ravine (Peacock et al. 1988). Within three years, Ae albopictus was detected in Central Florida and by 1992 it had been recovered in 64 of the states 67 counties (OMea ra et al. 1993). It has conti nued to spread rapidly throughout the eastern USA (Moore et al.1988, 1990, Moore 1999) and has since established throughout the 25 states extending from Texas and Florida in the south to New Jers ey and Nebraska in the north (OMeara 2005). The Western U. S. has not experienced such a rapid invasion of Ae. albopictus and this may be due to the regions dr ier environment (Moore and Mitchell 1997). Habitat Aedes albopictus utilizes a wide range of containers, the two most typical being natural and man-made containers (Hawley 1988). The most common man-made containers are tires, bird baths, buckets, bowls for pets, clogged rain gutters and flower vases; while natural containers include tree holes, rock pools, bamboo stumps and tank bromeliads (Hawley 1988, OMeara et al. 1995, Ali and Nayar 1997). The state of Florida offers a variety of environments that can sustain populations of Ae. albopictus thus it has been collected from tree holes in urban, suburban, rural, and sylvan areas (OMeara et al. 1993). Although considered a woodland

PAGE 19

19 species found in rural ha bitats, it has successfully adapted to the urban environment (Reiter and Darsie 1984). Once introduced to a new area, it is believed that Ae. albopictus will first exploit disturbed habitats that include scrap yards, tires and discarded containers, before entering and becoming part of the local fauna (Rai 1986). In suburban habitats, Ae. albopictus often prefers man-made structures that contain areas with plentiful vegetation (Estrada-Franco and Craig 1995), while those that have been cleared of vegetation contain substantially fewer numbers (pers. obs.). It is not surprising that cemeteries are premier breeding grounds for Ae. albopictus providing four basic resources: suga r (flowers), blood meal (potential visitors), shelter (trees and grass) and water-filled containers (flower vases) (Vezzani 2007). Unlike Ae. aegypti Ae. albopictus is more exophilic and utili zes a wider variety of natura l breeding sites (Gould et al. 1970). Researchers in Thailand discovered that Ae. albopictus were present in both rural and suburban habitats, and while Ae. aegypti was absent at elevations between 1000 and 1700 m above sea level, Ae. albopictus was still present (Pant et al. 1973). Investigations into the composition and habitat preference of mos quitoes in Malaysia revealed that Ae. albopictus is one of the most common active mosquitoes at ground le vel during the daytime, but can move into the forest canopy as high as 17 m during the ev ening (Rudnick 1965, Rudnick and Lim 1986). Bionomics Host Preference, Host Seek ing and Biting Behavior Aedes albopictus is opportunistic with strong anthr opophilic tendencies, but will feed on a wide range of mammals and some birds (Estrada -Franco and Craig 1995). Field studies in North America and Thailand have demonstrated that Ae. albopictus will attempt to feed on: cats, rats, rabbits, deer, horses, pigs, buffalo, dogs, boobies and chickens (Ponlawat and Harrington 2005, Savage et al. 1997, Tempelis et al. 1970, Sullivan et al. 1971), while laboratory studies have shown it to feed on rabbits, mice and rats, althoug h humans were the preferred host (Del Rosario

PAGE 20

20 1963). Analysis of blood meals in Potosi, MI revealed that Ae. albopictus fed on mammals 64% of the time and on birds 16% of the time (Savag e et al. 1997). Although considered a generalist feeder, Ae. albopictus has been shown to be host specific in parts of southern Thailand, feeding on humans 100% of the time (Ponlawat and Harr ington 2005). However, Ri chards et al. (2006) analyzed blood meals from Ae. albopictus in suburban neighborhoods of North Carolina and determined that feeding was based on host a bundance, preferring to feed on dogs and cats relative to humans. Different environments (urban vs. rural) are most likely the reason for hostfeeding preferences (Sullivan et al. 1971). Further investig ations have concluded that Ae. albopictus feeds on an array of hosts depending on the season and microhabitat (Niebylski et al. 1994). Although Ae albopictus is a diurnal mosquito, peaks in feeding activity can be observed an hour after sunrise and an hour before sunset (Ho et al. 1973, Sullivan et al. 1971, Hassan et al. 1996). These peaks may vary slightly depending on climatic and habitat variations (Hawley 1988). Generally, the majority of feeding o ccurs from 0630 to 0730 and from 1630 to 1830, with the least activity occurring be tween 1130 and 1430 (Ho et al. 1973). Odors, visual cues and sound are some of the most important cues that blood-feeding insects use in seeking a specific host (Lehane 2005) Host-seeking is stimulus-response behavior that is governed by the physiologi cal state of the mosquito that includes age, reproductive status and diapause (Bowen 1991). Mosquitoes are at tracted to potential hosts by chemical and physical cues. The most common chemical cu es emitted by animals are expired breath (carbon dioxide), as well as epidermal secretions and bacterial products which produce secondary cues, such as octenol and lactic acid (Clements 1999, Day 2005). These emissions form host odor plumes that help guide the mosquito to its ta rgeted host (Day 2005). As a mosquito nears its

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21 host, heat emanating from the host becomes an important stimulus (Lehane 2005). Relative humidity plays an important role on the behavi or of host-seeking mos quitoes. Takken et al. (1997) confirmed that the ultimate succe ss in locating a host for sub-species of Anopheles gambiae was determined by a combination of high humidity and skin odor. Aedes albopictus targets humans by detecting plumes of carbon dioxide, heat, moisture and organic chemicals emitted from the body (Mogi and Yamamura 1981, Estrada-Franco and Craig 1995, Clements 1999). In addition, Ae. albopictus like other diurnal mosquitoes, use visual cues such as bright colors, patterns, UV reflectance and movement to target their hosts (Allan et al. 1987). Many commercial and experimental traps designed for diurnal mosquitoes use black and white backgrounds, ostensively, to enhance attracti on. The ability to resp ond to these contrasting colors, enable diurnal mosquitoes to discri minate mammalian hosts from their background environment (Gibson and Torr 1999). Kusakabe and Ikeshoji (1990) demonstrated that a combination of heat, noise and a black sheet was a highly attractive stimulus to female and male Ae. albopictus Mosquitoes have specific receptors throughout the body that allow for the detection of particular cues from a host. For example, water vapor is detected by receptors on the antennae, while carbon dioxide is detected by sensilla basiconica on the maxillary papli (Kellogg 1970). The compound eye, the primary visual organ in mo squitoes and most insects, provides sensory input to discriminate pattern and form, move ment, light intensity, and contrast and color (Lehane 2005). Mosquito attraction can vary between humans, due to factors such as body mass, host cues, and genetics (Clements 1999). For example, in a human attractiveness study, one out of ten individuals was found to be attrac tive, while another was repellent (McKenzie 2003). Attraction may also be influenced by ones blood type. Shirai et al. (2004) demonstrated that Ae. albopictus

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22 was slightly more attracted to subjects with blood group O than to blood groups B and AB, and was significantly more attracted to blood group O than to blood group A subjects. Research in Japan determined that Ae. albopictus was attracted to human bait fr om a distance of 4-5 m, but that an increase in wind velocity could str ongly affect the range of attraction (Mogi and Yamamura 1981). Mating and Flight Range Aedes albopictus like most mosquitoes, mate while swarming. Swarms are initiated by males that emerged and have completed sexual ma turation prior to that of females (Clements 1999). In nature, Ae. albopictus will normally mate in swarms 0.30 0.91 m from the ground for an average of 7 9 seconds (Gubler and Bh attacharya 1972). Under controlled conditions, males can inseminate 6.7 females during their lifetime (Ali and Rozeboom 1973). Mating occurs between 48 and 72 hours after eclosi on, usually in the vicinity of hosts, which is believed to insure a high rate of insemina tion (Gubler and Bhattacharya 1972). A dispersal study by Niebylski and Craig ( 1994) demonstrated that when marked Ae. albopictus were released, the majority were recaptured within 100 m of the release site. Similar results were obtained by a mark-release study by Maciel-De-Freitas et al. (2006) using Rbmarked eggs. Although some eggs were recovered as far as 1,000 m away from release sites, the majority (81%) were found w ithin 100 m of the area. Th ese finding demonstrate that Ae. albopictus has a very short flight range, perhaps c onstrained by the availability of water-holding containers in a particular ar ea (Nieblylski and Craig 1994). Diapause and Photoperiod Diapause is a biological mechanism utili zed by most temperate-zone insects whereby metabolism is lowered and a dormant stage is initia ted to survive periods of cold, heat or other environmental challenges (Saunders 1987). Phot operiod and temperature are the most common

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23 factors affecting endocrine changes in mosqu itoes that induce diapause (Bowen 1991). In temperate regions, Ae. albopictus overwinters as an egg due to photoperiodicity-induced diapause, while subtropical and tropical stra ins remain unaffected (Hawley et al. 1989). Decreasing photoperiod and lowe r temperatures are the ke y variables triggering egg diapause in temperate Ae. albopictus (Hanson and Craig 1995a, Estrada-Franco and Craig 1995). Hanson and Craig (1994, 1995a) determined that eggs of temperate strain Ae. albopictus could survive temperatures of -12 C. They also suggested that lower ambient temperatures could be survived if they are protected wi th a layer of snow. This ability has allowed eggs from temperate strains of Ae. albopictus to develop and survive at lower le thal temperature thresholds than tropical strains (Hanson and Crai g 1995b). Imported eggs of Ae. albopictus most likely came from temperate regions of Asia, as current Nort h America strains posses temperate diapause and are resistant to cold temperatur es (Hawley et al. 1987). Egg su rvivability has been shown to differ based on geographical locatio n in the United States. Hawl ey et al. (1989) demonstrated that eggs from northern strains of Ae. albopictus had higher survivorship to cold temperatures than did the southern strains. Nu trition may also play a role as nutritionally deprived larvae give rise to adults that lay eggs with a higher incidence of diapause (Pumpuni et al. 1992). The ability of Ae. albopictus to colonize different geographi cal locations may be largely influenced by diapause expressi on. Lounibos et al. (2003) determ ined that diapause expression within Ae. albopictus populations were positively correlated with latitude. Their study demonstrated that while diapause was strongly ex pressed in populations in Illinois, the incidence was reduced in South Florida, and virtually ab sent in Brazil. Thus, depending on location, the expression of varying degrees of diapau se may be advantageous; facilitating Ae. albopictus to evolve in temperate, subtropical and tropical areas worldwide (Lounibos 2002).

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24 Oviposition, Fecundity and Longevity Once Ae. albopictus has acquired a blood meal, the dur ation of gonotrophic cycle lasts about 5 days in the field (Mor i and Wada 1977) and 3-4 days in the lab (Del Rosario 1963). Selection of an appropriate oviposit ion site is a critical factor for mosquitoes that ensures the survivability of their offspri ng (Bentley and Day 1989). Mosqu itoes respond to an array of visual and chemical stimuli to determine the best site for development of their larvae (Gubler 1971). Numerous studies on several mosquito sp ecies have determined that while chemical compounds may serve as oviposition attractants or re pellents, they are not the only factors that influence oviposition site preference. Combinat ions of external physi cal factors, such as substrate moisture and pool brightness (surface refection), are also important components in oviposition site preference (Bentley and Day 1989). Aedes albopictus tend to respond to visual stimuli, but olfaction and contact chemoreception ar e also very important in selecting oviposition sites (Gubler 1971). Field and laborato ry experiments have concluded that Ae. albopictus will oviposit significantly more eggs in contai ners with fermenting white oak leaves ( Quercusa alba L.) (Trexler et al. 2003b) or maple leaves ( Acer buergerianum ) (Dieng et al. 2002b, 2003) when compared to well water alone In laboratory studies, Ae. albopictus is preferentially attracted to conditioned or larval water over deionized wate r for oviposition (Gubler 1971, Thavara et al. 1989). Based on laboratory experiments, Ae. albopictus normally oviposits an average of 63 eggs in a single gonotrophic cycle and approximately about 280 eggs throughout its reproductive life (Gubler 1970a, Gubler and Bhattach arya 1971). However, if larvae are reared in an high density environment, fecundity can be significantly re duced (Moore and Fisher 1969). In the field, oviposition generally occurs from 0800 to 1900, with the majority of eggs deposited in the late afternoon between 1500 and 1700 (Tsuda et al. 1989, Hassan et al. 1996). However, oviposition

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25 periods may vary within geographic locations as they are influenced by environmental conditions such as light intensity, humidity, temperature an d wind velocity (Tsuda et al. 1989). Studies in North Carolina revealed that Ae. albopictus exhibits diurnal periodic ity in oviposition behavior, ovipositing a small portion of eggs (less th an 10%) in the morn ing, between 0800 and 1100, increasing throughout the day, with peak ovipos ition occurring between 1300 and 1600 (Trexler et al. 1998). An Ae. albopictus female will normally deposit her mature eggs at multiple oviposition sites, while flying in a lateral and vertical motion above the water surface (Rozeboom et al. 1973, Hawley 1988) Field and laboratory studi es have determined that Ae. albopictus prefers to oviposit in cont ainers that are dark-colored (Yap 1975, Yap et al. 1995). Once eggs have been laid, hatching depends on photoperiod, temperature and the timing of floods (Hawley 1988). Temperature and humidity levels play a vital ro le in the survivorship of adult mosquitoes (Estrada-Franco and Craig 1995). Laborator y experiments have shown that female Ae. albopictus can survive between 59 to 84 days when he ld a at temperatures between 15.5 C and 22 C at low or high humidity levels (Hylton 1969). Prior studies in the Philippines produced similar results with females averaging 87 days and males averaging 65 days (Del Rosario 1963). However, in nature this number is much lowe r as Mori (1979) determin ed the average female longevity was between 2.9 and 7.8 days, while ma les survived between 6.6 and 7.8 days. As cited in Hawley (1988) a mark a nd release study in Hawaii showed that some adults survive as long as 21 days in the wild, but the majority we re recaptured in about 10 days. The ability of Ae. albopictus to prosper in a range of temperatures and humidities are important factors that allowed establishment in a wide range of climates (Hylton 1969).

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26 Medical Importance Laboratory studies and field collections have determined that Ae. albopictus is a competent vector of at least 23 arboviruse s (Moore and Mitchell 1997). Worl dwide, it has been found to be naturally infected with: dengue (DEN), Japanese encephalitis Potosi, Keystone, Tensaw, eastern equine encephalitis, and recently with La Crosse (LAC ) and West Nile (WN) viruses (Moore and Mitchell 1997). Tesh et al. (1976) demonstrated that Ae. albopictus was not only susceptible to oral infection by chikungunya (C HIK), but could also re plicate the virus once infected. This was recently confirmed when Ae. albopictus was implicated as the primary vector for CHIK outbreaks in Italy, India and islands throughout the Indian O cean (Reiter et al. 2006, Borgherini et. al. 2007, Rezza et al. 2007). Aedes albopictus is a competent vector of the Ro ss River (RR) and yellow fever (YF) viruses, though this is observed under experiment al conditions, rather than in nature (Tesh and Gubler 1975). In addition to tr ansmitting numerous viruses to man, Ae. albopictus can vector dog heartworm, Dirofilaria immitis a potential concern for many pe t owners (Nayar and Knight 1999). Dengue With the exception of malaria, probably no othe r vector-borne disease affects the health and welfare of more people than dengue. De ngue fever (DF) and dengue hemorrhagic fever (DHF) are caused by four antigenically distin ct virus serotypes known as DEN-1, DEN-2, DEN3 and DEN-4 that are classified in the genus Flavivirus family Flaviviridae (Westaway and Blok 1997). Classic DF produces a wide range of sympto ms that include extreme malaise, severe pain in the muscles, back, limbs and sometimes a rash (George and Lum 1997). Dengue fever and DHF are among the most important reemerging in fectious diseases affecting countries both economically and socially, especially in developi ng tropical regions where certain localities have

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27 become hyperendemic with multiple virus sero types (Gubler 2002, 2005, Ooi et al. 2006). Over a 100 countries report active DF transmission, while DHF has become established in most of the Americas and Southeast Asia, maki ng it one of the leading causes of pediatric mortality (Gratz 1999). It has been estimated that 50 million cas es of dengue infection occur yearly, with approximately 2.5 billion people at risk (World Health Organization 1997). While the majority of dengue outbreaks have been concentrated in Southeast Asia and the Pacific Rim, over 1 million cases in the Americas were reported to Pan American Health Organization between 1980 and 1990 (Estrada-Franco and Craig 1995). The tran sportation of mosquitoes and their eggs to new countries, uncontrolled urbani zation and ecologic ch anges within environments all have contributed to the global increa se in the number of DF and DHF cases (Gubler 1997). Potential changes in climate due to global warming in conjunction with a rapid growth of the human population may also contribute to the spread of DF, increasing at risk tr ansmission rates from the current 35% to 60% by 2085 (Hales et al. 2002, Gubler et al. 2001). The transmission of DEN by mosquitoes was first documented by Graham (1903). Additional work by Bancroft (1906) demonstrated that Ae. aegypti transmitted dengue among infected human subjects. In 1906, the United States Army Medical Corps sent two young officers, Asburn and Craig, to the Philippines to investigate DEN outbreak at Fort William McKinley. Although they implicated the wrong vect or, both officers demonstrated the following etiology of DF: 1) DEN was most likely tran smitted by mosquitoes, 2) DEN was not caused by bacteria or a protozoan, but by so mething ultramicroscopic in si ze, 3) DEN was not contagious and 4) DEN infected patients gained immunity to the disease (Ashburn a nd Craig 1907). Further research in the 1920s by other US Army resear chers demonstrated that DEN was principally transmitted by Ae. aegypti and that Ae. albopictus was a secondary vector (Siler et al. 1926,

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28 Simmons et al. 1931). The long suspicion that Ae. albopictus transmitted DEN was confirmed when Rudnick and Chan (1965) isolated DEN type 2 from wild populations in Singapore. Although there may be other species of Aedes that are capable of transmitting dengue, epidemiologic and experimental obs ervations have reaffirmed that Ae. aegypti Ae. albopictus and Ae. polynesiensis Marks of the subgenus Stegoymia are responsible for the majority of dengue outbreaks in the world (Rodhain and Rosen 1997). Aedes albopictus has been shown to transmit DEN transovarially more readily than Ae. aegypti This may play a role in maintaining the virus in the field, thus serv ing as a potential reservoir fo r endemic DEN (Rosen et al. 1983, Mitchell et al. 1987). Implicating Ae. albopictus in DEN outbreaks is normally conducted by local mosquito surveillance. Ali et al (2003) used spatial analysis to de monstrate a strong correlation between households reporting DEN illness and the presence of Ae. albopictus larvae within the house or an adjoining neighbors house. Ou tbreaks of DEN may involve both Ae. albopictus and Ae. aegypti especially where urban areas are adjacent to forested areas, where Ae albopictus is most likely the primary forest vector (Rudnick 1965) or maintenance v ector in rural areas (Gratz 2004). It is widely believed that during World War II Ae. albopictus was responsible in the transmission of DEN in Japan (M ori 1979). Major DF outbreaks where Ae. albopictus has been implicated as the primary vector include: 1942-44 Japan (Hotta 1998), 1943 Honolulu, Hawaii (Usinger 1944, Gibertson 1945), 1978 Solomon islands Guadalcanal, Santa Cruz Islands (Elliot 1980), 1978 China (Wufang et al. 1989), 1980-1985 Ha inan island China (Tang et al. 1988) and 1976-1977 Seychelles (Metselaar et al. 1980). Th e most recent outbreak of DEN in the United States occurred in 2001-2002 in Hawaii on the is land of Maui. This outbreak was caused by

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29 DEN-1 and was transmitted by Ae. albopictus as Ae aegypti had been eliminated from Maui in the 1940s (Effler et al. 2005, Hayes et al. 2006). Urban populations of Ae. albopictus have exhibited greater su sceptibility to endemic dengue virus than sylvatic stra ins (Moncayo et al. 2004), as well as significantly influencing infection and transmission rates within sp ecific DEN serotypes (Gubler and Rosen 1976, Mitchell et al. 1987, Vazeille et al 2003). There have been no reports of dengue transmitted by Ae. albopictus in the continental United States. Howe ver, seven locally acquired cases of DEN were confirmed in southern Texas in 1995, but Ae. aegypti was determined as the vector (Rawlings et al. 1998). Further i nvestigations revealed that ther e was a link between the lack of air-conditioners in homes and patients who test ed positive for DEN, de monstrating that dengue may be associated more with economic status than with environmental infl uences (Reiter et al. 2003). However, the 2002 Hawaii outbreak demonstrat es the possibility of future scenarios that could occur in the United States when n ecessary epidemiological factors are met. La Crosse virus La Crosse virus (LAC) is an arbovirus be longing to the family Bunyaviridae, genus Orthobunyavirus which can cause encephalitis, particularly in children under six years of age, making it an important public health problem in the United States (Yuill 1984). The virus is endemic throughout Minnesota, Wisconsin, Illinois, Indiana, Ohio, and fairly recently in Tennessee, West Virginia and North Carolina (Estrado-Franco and Craig 1995, Jones et al. 1999). The virus is transmitted prim arily by the bite of infected Ochlerotatus triseriatus (Say). Serological surveys suggest that forest mammals, specifically eastern chipmunks ( Tamias striatus ), gray squirrels ( Sciurus carolinensis ), cottontail rabbits ( Sylvilagus floridanus ) and red foxes ( Vulpes fulva ) are reservoirs for the virus (Yuill 1984). Laboratory studies have shown that Ae. albopictus is capable of transmitting LAC to mice (Grimstad et al. 1989) and transovarially

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30 during the first gonotrophic cycle (Tesh and Gubl er 1975, Cully et al. 1992). The potential for LAC to infect field populations of Ae. albopictus was observed by Kitron et al. (1998) when a chipmunk, which tested positive for the virus, was co llected in close proximity to mosquito traps used for Ae. albopictus surveillance. This observation was affirmed by Gerhardt et al. (2001) when vertically infected Ae. albopictus were collected in the field. There is accumulating evidence showing a close association between LACinfected children, residences with tree holes and residences with high populations of Ae. albopictus (Erwin et al. 2002). Hughes et al. (2006) suggested that although Ae. albopictus does not amplify the virus as well as Oc. triseriatus it may prove to be a more efficient bridge vector to humans due to its ur ban-suburban distribution and its aggressive anthro pophagic behavior and, ther efore, warrants concern. West Nile virus West Nile virus (WN) is an arbovirus virus be longing to the family Flaviviridae and is antigenically similar to Japanese encephal itis, St. Louis encephalitis, and Murray Valley encephalitis (Hayes and Gubler 2006). West N ile virus was first reported in the Western Hemisphere in the summer of 1999 when the New York City area recorded several unexplained cases of encephalitis in humans (CDC 1999). It has since spread throughout North America, Latin America, and the Caribbean (Hayes and G ubler 2006). Laboratory trials revealed that Ae. albopictus was a capable vector of WNV, and was ab le to infect and disseminate the virus (Turell et al. 2001). Sardelis et al. (2002) determined that Ae. albopictus strains from Texas, Maryland and Hawaii were all highly efficient vectors of WN and were able to transmit the virus within 13 days after taking an infectious blood meal. The potential for transmission by Ae. albopictus in the field was confirmed in Pennsylvani a (Holick et al. 2002). The Philadelphia Department of Public Health confirmed that two Ae. albopictus collected in September 2000 (part of a sample pool); tested positive for WN by the reverse transcription-polymerase chain

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31 reaction (RT-PCR) method. Although the primar y vector of WN in Pennsylvania is Culex pipiens (L.), the susceptibility of Ae. albopictus to the virus makes it a li kely bridge vector to humans by introducing WN to wild mammals such as cottontail rabbits where dissemination to human-biting species is more lik ely (Tiawsirisup et al. 2004). Competition and Displacement The introduction of a mosquito species into non-native regions can have detrimental impacts on the local fauna and may dramatically alter disease transmission cycles (Juliano and Lounibos 2005). This is especially important if it displaces another competent disease vector. Although introduced mosquitoes are usually prob lematic, displacement of one species may benefit eradication or control programs if th ey are ecologically homologous (DeBach 1966). This was demonstrated by Gubler (1970b) a nd Rozeboom (1971) in caged studies where Ae. albopictus quickly displaced Ae. polynesiensis a known vector of bancro ftian filariasis. The high reproductive rate a nd longer life span of Ae. albopictus were believed to be the primary factors for displacement (Gubler 1970b). Aedes albopictus has proven to be a versatile mos quito, capable of adapting to many environments. The rapid dissemination of Ae. albopictus throughout much of the South Pacific occurred during World War II. Perhaps the best-known example of Ae. albopictus displacing other native species occurred on the island of Guam. Swezey (1942) conducted the first extensive mosquito survey of Guam and found five species of mosquitoes: Cx. quinquefasciatus Say, Ae. guamensis (Farner and Bohart), Ae. aegypti Ae. pandani (Stone) and Ae. oakleyi (Stone). However, a severe dengue epidemic among military personnel at the time prompted further investigation by Bohart and Ingram (1946). Surveys during 1944-45 added four additional mosquito species to the list of native fauna and implicated non-indigenous Ae. aegypti for the 1944 epidemic. Prior to 1944, there were no reports of Ae. albopictus on Guam. The first

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32 report of Ae. albopictus in Guam was made in 1944 by LCDR Weathersby, an entomologist with the Third Marine Division, who found cast skins and larvae in Ylig Bay (Hull 1952). This was verified in an extensive survey conducte d during 1948-1949 by Reeves and Rudnick (1951). Although Ae. albopictus was widely distri buted over Guam, Ae. aegypti virtually had been eliminated as no larvae were detected on the island. A subsequent survey by Hull (1952) also found Ae. albopictus to be the most commonly collected mosquito, while Ae. aegypti was not found. Since its arrival to the United States, Ae. albopictus has dramatically displaced populations of Ae. aegypti throughout the Southeast. It is believed that interspecific competition between these species has contributed to the sharp decline in Ae. aegypti (Juliano et al. 2004). Reports in early 1990 from Mobile, AL, noted that sightings of Ae. aegypti were extremely rare (Hobbs et al. 1991). In 1991, Ae. albopictus larvae were collected throughout sites in northern Florida previously occupied by Ae. aegypti (OMeara et al. 1992). By 1994, Ae. albopictus had become the dominant Aedes container-inhabiting mosquito in northern Florida an d had colonized containers as far south as parts of so uthern Florida (OMeara et al. 1995). Field studies in Florida and Brazil have shown Ae. albopictus as a superior larval competitor to Ae. aegypti, especially when exploiting food resour ces, enabling it to give rise to more adults, thereby contributi ng to a decline in the number Ae. aegypti (Juliano 1998, Braks et al. 2004). In addition, resistance to starvation and overcrowding with in artificial containers has been shown to affect larval performance, wh ich may contribute to displacement between these species (Barrera 1996). While many countries have observed a displacement of Ae. aegypti by Ae. albopictus the opposite holds true for much of Sout heast Asia. The distribution of Ae. albopictus has

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33 substantially decreased over the past 100 years in Malaysia Bangkok, Calcutta and parts of Indonesia, while that of Ae. aegypti has expanded (Hawley 1988). Chan et al. (1971) proposed this phenomenon may be due in part to rapid urbanization and the destruction of vegetative habitats that are frequently used as breeding and resting sites by Ae. albopictus. Surveillance Devices An integral part of an effective mosquito control program is to maintain an active surveillance component. Surveillan ce provides information on the type of vectors in a particular area, their frequency of occurren ce, changes in density levels, their distribution and important epidemiological parameters (Chan 1985). This is especially important for countries that are both susceptible to invasive mosquitoes and lack survei llance programs. It is in this situation where the introduction of a new mosqu ito species often goes unnoticed (Lounibos 2002). Furthermore, once thresholds have been established, surveilla nce is the primary tool used to measure the effectiveness of control efforts. Surveillance pr ograms often utilize traps, such as the standard New Jersey light trap, to collect crepuscular and nocturnal host-seeking mosquitoes (Reinert 1989). While effective in capturing many differen t species, light trap s are ineffective in capturing day-flying Stegomyia spp. (Service 1993). For this reason, surveillance of other diurnal host-seeking mosquitoes such as Ae. albopictus poses a challenge. Human-landing rates and bite counts have b een used as a quick method to ascertain mosquito distribution in a part icular area and to determine patterns of host-seeking activity (Schmidt 1989). However, this method is labor-intensive and may place samplers at risk for acquiring a mosquito-borne pathogen. Therefor e, numerous other surveillance methods have been developed to capture host-seeking and gravid Stegomyia mosquitoes. These methods include traps baited with host-related semioche micals and / or visually attractive colors and patterns.

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34 Host Seeking Traps and Attractants The use of visual attractants, specifically black and white patterns, have been a proposed method of attracting mosquitoes (Haufe 1964). Fay (1968) first described using a daytime mosquito trap to target resting Ae. aegypti Further development of this configuration by Fay and Prince (1970) resulted in a box-like trap w ith contrasting black and white sides. The contrasting colors were employed as the standard visual attracta nts in the duplex cone trap and biand Omni-directional Fay-Prin ce traps and have been shown us eful in monitoring populations of Ae. albopictus (Freier and Francy 1991, Jensen et al. 1994). Mosquitoes display a host-seeking re sponse to increasing gradients of CO2 (Service 1993). In the laboratory, wind tunnels have demonstrat ed that mosquitoes will fly upwind towards a filamentous plume of carbon dioxide (Geier et al. 1999). Informa tion gained from these studies has been incorporated into a new generation of mosquito traps known as counterflow traps. Counterflow traps greatly improve capture rates by discharging a plume of carbon dioxide that mimics exhalation of potential hosts (Kline 1999). These traps can be further enhanced with catalytic combustion to produce add itional attractants such as wate r vapor and heat (Kline 2002). Recently, the BG-Sentinel trap has b een shown effective in capturing Ae. aegypti and is an acceptable alternative to human landing counts (Krckel et al 2006). Lab experiments with the BG-Sentinel trap in combination with CO2 also have been successful in capturing Ae. albopictus (Kawada et al. 2007). To lure host-seeking, da ytime feeding mosquitoes, this lightweight, collapsible trap uses visual cues, releases sy nthetic compounds that mimic skin secretions and simulates convection currents that are often created by the human body (Krckel et al. 2006). The application of semiochemical s in traps to increase capture rates may not only serve as a monitoring device, but may be utilized as a control management tool (Kline 2007).

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35 Many attractants are used to supplement traps to increase capture rates. The combination of CO2 and 1-octen-3-ol (octenol), ac t synergistically to attract mo re mosquitoes when compared to single baits (Takken and Klin e 1989). Additional synergisms include blends of ammonia, lactic acid and carboxylic acid (Smallegange et al. 2005). Aedes aegypti have been shown to be highly attracted to lactic acid in combination with other human skin odors, which may explain their anthropophilic behavior (Steib et al. 2001). These attractant s have been successfully used in conjunction with a variety of mosquito traps to capture Ae. albopictus Shone et al. (2003) demonstrated that Fay-Prince trap s supplemented with octenol and CO2 caught significantly more Ae. albopictus than non-baited traps, while Hoel et al. (2007) determined that a combination of lactic acid, CO2 and octenol could significantly increase capture rates in residential areas. Ovitraps and Gravid Traps Ovitraps and gravid traps are important su rveillance tools in assessing mosquito populations, and studying ecological habitats with regard to popul ation dynamics (Service 1993). The type and style of an ovitrap for a specific mo squito is largely based on its biology (Service 1993). To assess the population of Ae. albopictus a variety of containers have been used as ovitraps, ranging from bicycle tires (Pena et al 2004) to ceramic ant tr aps (Mogi et al. 1988). While these serve as adequate ovitraps, some still employ the standard black glass pint jar developed by Fay and Eliason (1966) for Ae. aegypti surveillance. Plas tic ovitraps are now commonly used, as they have been shown to be e qually attractive as glass (Bellini et al. 1996). Furthermore, the addition of hay or leaf infusi ons into ovitraps can significantly increase the number of eggs laid by Ae. albopictus than water alone (Holck et al. 1988). A more recent development in the use of ov itraps includes incorporation of impregnated ovistrips with insecticides and adhesives. Pe rich et al. (2003) transf ormed the typical black

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36 polyethylene ovitrap into a lethal version by treating the ovistrip with deltamethrin. However, this serves as a control measure rather than a surveillance tool. Altern atively, sticky ovitraps, provide an advantage over the st andard ovitrap by assessing the number of females visiting the trap and eliminating the need to identify eggs or resultant offspring (Facchinelli et al. 2007). Studies have shown that sticky ov itraps frequently detect more Ae. aegypti than the standard ovitrap (Ritchie et al. 2003), and have been successfully used in behavioral studies and surveillance programs for Ae. polynesiensis and Ae. albopictus (Russell and Ritchie 2004, Facchinelli et al. 2007). Gravid traps are similar to ovitraps, but capture oviposition-seeking adults rather than eggs. Compared to host-seeking traps, gravid traps are often used in arbovirus surveillance because they capture parous mosquitoes (Service 1993). The first portable gravid trap coined the CDC gravid trap was developed by Reiter (1983). Originally designed to capture Culex mosquitoes due to their oviposition behavior (R eiter 1983), other studies have successfully used them in trapping Ae. albopictus (Burkett et al. 2004). Different commercial gravid traps have since been developed, but traps based on the origin al design by Reiter (19 83) have statistically outperformed other models (Allan and Kline 2004). Control Measures To reduce and maintain mosquito populations at acceptable levels, many mosquito control agencies recommend implementing the following control strategies: sour ce reduction, larvicide and adulticides applications and biological control (Floore 2006). Employing these control strategies is costly; the United States alone spends hundreds of m illions of dollars annually in mosquito control (Foster a nd Walker 2002). To control Ae. albopictus using conventional insecticides poses an even greater challenge, of ten requiring several applications (Peacock et al. 1988). This places additional bur dens on manpower availability and increases control costs for

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37 many mosquito districts. (Peacock et al.1988). Source reduction remains the best control measure to manage populations of Ae. albopictus. However, public complacency often ensues, requiring the use of larvicides a nd adulticides to decr ease populations to manageable levels. Biological and Cultural Control Integrated pest management programs targe ting mosquitoes have employed a wide range of biological agents including pr edacious invertebrates, pathogen ic fungi, bacteria, protozoans, nematodes and viruses. To successfully contro l specific mosquitoes, several biological methods are often used based on characteristics of a part icular species breeding habitat and life stage (Legner 1995). Aquatic predators, such as the mosquito fish, Gambusia affinis have been successfully used to manage populations of Anopheles larvae during malari a control campaigns (Bay et al. 1976). However, this method would probably be considered imp ractical as a control measure for container-mosquitoes such as Ae. albopictus Predacious invertebrates have been explored to control Ae. albopictus. Predatory mosquito larvae, such as Toxorhynchites spp., exploit similar habitats as Ae. albopictus and Ae. aegypti and can be used if they are mass-reared (Estrada-Franco and Craig 1995), though their ca nnibalistic lifestyle would likely limit their effectiveness over large areas. Cyclopoid copepods are some of the most effec tive invertebrate predators in controlling mosquito larvae (Marten and Reid 2007). Marten (1984) first noted success of Mesocyclops leuckatrti pilosa in reducing the number of Ae. albopictus larvae in water jugs. Further experiments utilizing Macroclylops albidus and introducing them into tires determined that that they were effective in eliminating Ae. albopictus (Marten 1990). Field success of Mesocyclops spp. as biological control agents we re demonstrated in eradicating Ae. aegypti in small villages in Vietnam (Nam et al. 1998). However, a preference for cryptic microhabitats by Ae. albopictus may inhibit successful control efforts in the fi eld (Dieng et al. 2002a). Compared to other

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38 biological control agents, an advantage of usi ng copepods to reduce mosquito larvae is their compatibility with many larvicides, such as Bacillus thuringinensis israelensis ( B.t.i .) (Marten 1989). Spore-forming bacteria, B.t.i and efficacious strains of Bacillus sphaericus are the most commonly used non-chemical products to cont rol black flies and mo squitoes (Legner 1995, Lacey 2007). Once B.t.i spores are ingested by mosquito larvae, proteinaceous toxins are produced which bind to the membrane lining of the midgut. These toxins begin to interfere with the osmotic balance within the cell, resu lting in cell lysis (Lacey 2007). Although B.t.i is not used extensively in controlling Aedes albopictus larvae are susceptibility to the toxin in both the lab and field (Lee and Zairi 2006). There has been some concern for mosquito avoidance towards containers treated with B.t.i., but Stoops (2005) demonstrated that more eggs were laid in B.t.i .-containing ovitraps than conditioned water controls. Thus, B.t.i remains a potential tool for large scale Ae. albopictus control. The most effective method for controlling Ae. albopictus in suburban neighborhoods is by eliminating their breeding sites, also known as source reduction. This is best accomplished by educating the public about mosquito habitats a nd encouraging the elimination of discarded manmade containers, often the primary breeding sites for Ae. aegypti and Ae. albopictus For example, the Singapore government has successfully reduced breeding sites for Ae. aegypti and Ae. albopictus within residential sites by employing h ealth education programs and enacting legislation to enforce ordinan ces that ban harboring either species (Chan and Bos 1987). However, during larval surveys, Chan and Couns ilman (1985) noted that residential properties containing tin cans, tree-holes and plants with la rge leaf axils accounted for some of the highest densities of Ae. albopictus Therefore, while reducing manmade containers can be used

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39 effectively against Ae. aegypti and Ae. albopictus the control of the latter may present a greater challenge due to its wider range of habitats and propensity to find sites farther from human habitation (Estrada-F ranco and Craig 1995). Chemical Control To mitigate adult mosquitoes in large areas, Ultra Low Volume (ULV) insecticide sprays are often dispensed from trucks or aircraft, while smaller areas often employ space or residual sprayers around structures. It is economically advantageous to in itiate control when mosquitoes are in the larval stages, as larvae are fairly immobile and usually occupy a smaller area (Floore 2006). While the application of B.t.i is often used for larval control, treating domestic containers with pellets containing Altosid (met hoprene) an insect growth regulator, or Abate (temophos), an organophosphate, has been shown to provide excellent residual control of Ae. albopictus up to 150 days post-application (Nasci et al. 1994). To control adult Ae. albopictus the use of naled (Dibrom) in thermal foggers has been shown to be successful (Peacock et al. 1988). In the laboratory, Ae. albopictus are particularly susceptible to pyrethroid insecticides, specifically to lambda-cyhalothrin (Sulaima n et al. 1991). Field studies in suburban neighborhoods also have demonstrated that adul t control was successful when applying lambdacyhalothrin and bifenthrin as barr ier sprays to vegetation (Trout et al. 2007). Within field cages, the use of boric acid as a foliar sp ray has been successful in controlling Ae. albopictus (Xue et al. 2006). Though many insecticides are ineffectiv e at destroying the egg stage, sodium hypochlorite has demonstrated success as a conven tional ovicide and may assist in preventing an introduction of Ae. albopictus eggs into an area (Domenico et al. 2006). There have been worldwide repor ts documenting resistance in Ae. albopictus to several organochlorines and organophosphates, namely mala thion. In Thailand and Malaysia, the use of malathion in thermal foggers and ULV sprayers was ineffective in controlling Ae. albopictus

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40 compared to Ae. aegypti (Gould et al. 1970, Lam and Tham 1988) Similar observations made in Singapore demonstrated that while Ae. albopictus was susceptible to pyrethroids, it was two to five times more resistant to or ganochlorine insecticides than Ae. aegypti (Ho et al. 1981). Once Ae. albopictus became established in the United States, malathion resistance was detected in larvae (Wesson 1990) and adults (Khoo et al. 1988). Research Objectives In this chapter an attempt has been made to re view past and current literature regarding the bionomics of Ae. albopictus, its medical importance, its effect s on other mosquitoes and methods used to survey and control it. While consid erable amount of resear ch has been conducted on Ae. albopictus since its introduction into the United States, little research has been done in surveying Ae. albopictus in areas over 2 m in hei ght. Ecological research on Ae. albopictus must be continued in order to answer many questions re garding disease transmi ssion, specifically dengue (D. J. Gubler pers. comm.). My hypothesis for this study is to ascertain what surveillance techniques are effective for monitoring adult Ae. albopictus populations in suburban and rural forested sites in Florida. Sp ecifically, we hope to learn more about the activity of host-seeking and gravid Ae. albopictus at 1 m and 6 m in height. Gravid female are attracted to water containing different organic subs trates. Natural and man-made c ontainers that hold water and fallen leaves from local flora may influence ov iposition attractiveness. The influence of the height at which these oviposition sites are found may be an important factor in designing control measures. Therefore, we propose to determine the efficacy of three commercially-available traps and to elicit the influence of trap height in the capture of host-seeking, Ae. albopictus It stands to reason that certain environmen ts may be more attrac tive and conducive over time in sustaining larger Ae. albopictus populations than others. Therefore, we propose the

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41 following four studies to elicit behaviors and ec ological parameters aiding in understanding the vector status of Ae. albopictus within suburban and rural sites: 1) Evaluate three attractant-baited, commer cially-available adult traps in capturing Ae. albopictus at two heights in suburban and rural habitats in north central Florida. 2) Compare four commonly used surveill ance techniques to assess relative Ae. albopictus populations in both suburban and rural habitats in north central Florida. 3) Evaluate the attractiveness expe rimental organic infusions to Ae. albopictus in laboratory assays and field cages. 4) Determine the impact of oviposition trap pl acement height and infusion-bait combinations on trap captures in suburban and rura l habitats in north central Florida.

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42 CHAPTER 2 HOST-SEEKING HEIGHT REFERENCES OF AEDES ALBOPICTUS WITHIN SUBURBAN AND SYLVATIC LOCALES IN NORTH CENTRAL FLORIDA Introduction Aedes albopictus (Skuse) is an invasive mosquito that was introduced into Florida in 1986 (Peacock et al. 1988) and quickly became establishe d throughout most of the state. A daytime feeder, it is a persistent biter on many animals, especially humans, making it a severe nuisance in residential suburban areas. Vector control official s report that Ae. albopictus is primarily responsible for the majority of complaints received from re sidents (Kelly Etherson, pers. comm.). In addition, it is capable of vectoring 23 arboviruses, including La Crosse, West Nile (WN) and dengue (Moore and Mitchell 1997, Gerh ardt et al. 2001, Turell et al. 2001, Rudnick and Chan 1965). Although dengue is not endemic to Florida, it remains a public health concern due to a history of outbreaks. The 2002 dengue epidemic on the Hawaiian island of Maui demonstrates how dengue may spread into the United States and reaffirms that Ae. albopictus is a competent vector (Effler et al. 2005). Recently, Ae. albopictus has been implicated as the primary vector for chikungunya (CHIK) outbreaks in Italy, Indi a and islands throughout in the Indian Ocean including Madagascar and the Se ychelles (Reiter et al. 2006, Bor gherini et al. 2007, Rezza et al. 2007). These outbreaks may have originated in parts of Kenya as early as 2004 (Pialoux et al. 2007). Therefore, tourists probably played an impor tant role by transporting the virus, especially into India and to the Indian Ocean islands, wh ich are popular European tourist destinations (Pialoux et al. 2007). A follow-up by Panning et al. (2008) found that a high percentage of patients returning to Europe in 2006 who tested positive for CHIK antibodies had visited countries experiencing a CHIK outbreak. The pote ntial exists for an outbreak similar to the Italian outbreak to occur in Florida, especially if certain epidemiological conditions are met, such

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43 as the presence of a competent vector. For instance, field collected Ae. albopictus from Palm Beach County infected with the La Runi on chikungunya strain (LR2006-OPY1) have demonstrated infection and dissemination rates as high as 100% (Reiskin d et al. 2008). Given the prevalence and competence of Ae. albopictus in Florida, a scenario similar to that in Italy could occur if an infected i ndividual visits Florida and is subsequently bitten by a local Ae. albopictus. Adult mosquito traps are surveillance tools used not only to asse ss mosquito populations, but also to provide critical information regard ing the potential for disease transmission (Chan 1985). Furthermore, vector control agencies em ploy traps to establish preand post-threshold treatment levels, ensuring that control applicat ions are effective. While adult trapping is primarily conducted at ground level, some studies have set traps above 5 m to identify hostseeking and resting sites for particular species. For example, studies demonstrated that the Mosquito Magnet-X (MM-X) trap placed in tree canopies (7.0 m in height) caught significantly greater numbers of Culex pipiens L. than those placed at gr ound level (Anderson et al. 2004, Anderson et al. 2006). Additionally, traps have be en used to investigate feeding periods. For instance, Ochlerotatus triseriatus Say have been shown to feed at different heights depending on the time of day, feeding at ground level during daylight hour s, and moving into the canopy during the evening (Scholl et al. 1979). Lunds trm et al. (1996) also reported that Ae. cinereus Meigen was not only active at ground leve l, but at canopy height s of 14 and 18 m. Aedes albopictus is commonly observed flying within 1 m fr om the ground and prefer s to land and feed on the lower extremities of humans, especially the legs and feet (Shirai et al. 2002). However, studies in Malaysia by Rudnick (1965) and R udnick and Lim (1986) ha ve shown that while Ae.

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44 albopictus was observed at ground level during the day, they retreated and flew as high as 17 m during the evening. Light traps are ineffective in capturing day flying Stegoymia spp. (Service 1993) and therefore, unreliable for detecting the presence of Ae. albopictus Historically, the most effective way to sample diurnal mosquitoes is to us e human-landing counts (Service 1993), but this method is labor-intensive and may place subjects at risk for acquiring a mosquito-borne disease. Therefore, throughout the past thirty years, a vari ety of traps have been developed to attract and capture diurnal mosquitoes. These traps use visual attractants, mainly black and white patterns, often in combination with other chemical attractants. Studies by Freir and Francey ( 1991) and Jensen et al. (1994) have shown the duplex cone trap and the bi-and Omni-directional Fay-Prince traps (ODFP) to be useful in monitoring populations of Ae. albopictus Another effective Ae. albopictus trap is the MM-X commonly called the pickle-jar trap (Hoel 2005). Recent st udies in Brazil and Australia have shown the BG-Sentinel (BG) trap to be an effective surveillance tool for Ae. aegypti (L.) and an acceptable alternative to human landing counts (Krckel et al. 2006, Williams et al. 2006a). While laboratory studies have proven this trap to be effective in capturing Ae. albopictus (Kawada et al. 2007), no studies have been published on its perf ormance in the field. The addition of CO2, octenol and lactic acid to thes e traps increase their effectivenes s compared to non-baited traps (Shone et al. 2003, Hoel et al. 2007, Kawada et al. 2007). It is known that Ae. albopictus will feed on wide range of hos ts including birds, but prefers mammals, especially in suburban settings (S avage et al. 1997, Ponl awat and Harrington 2005, Richards et al. 2006). Therefore, information gained from the vertical distribution of hostseeking Ae. albopictus would help explain how viruses from infected mosquitoes infect birds,

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45 which then infect mammals, including man. Currently, few data exist concerning the performance of traps at varying heights in capturing Ae. albopictus in different environments. Furthermore, no information exists documenting host-seeking preferences at ground level versus the canopy in Florida. In addition, information a bout the efficacy of the ne wly marketed BG trap for Ae. albopictus in north central Florida is limited. My study has two object ives: 1) determine the effectiveness of the BG trap in capturing Ae. albopictus in both suburban and sylvatic environments in north central Florida, and 2) evaluate the BG, MM-X and ODFP traps at two heights to determine the influence of height on Ae. albopictus host-seeking activity in suburban and sylvatic environments. Materials and Methods Site Selection Tests were conducted from May to September 2007. At suburban locales, four residential properties (N 29 37.837, W 82 27.800; N 29 34.248, W 82 24.644; N 29 39.019, W 82 23.234; N 29 42.481, W 82 24.745) were in or near the city li mits of Gainesville, FL. Suburban locales were selected based on the foll owing criteria: 1) residents that have had frequent complaints of mosquito es biting during the day; 2) s ites had thickly-wooded lots that surround the residential propert y; 3) sites had previously supported populations of Ae. albopictus and; 4) sites were secured to pr event against trap thef t. Suburban sites were separated by at least 3.22 km (2 miles) and contained a mixtur e of shrubs and trees, namely azalea ( Rhondendron spp.), oleander ( Nerium oleander ), Indian hawthorn ( Rahphiolepis indica ), live oak ( Quercus virginiana P. Mill) water oak ( Quercus nigra L.) and longleaf pine ( Pinus palustris P. Mill) (Figure 2-1a). Sylvatic locales (N 29 43.574, W 82 27.252; N 29 44.048, W 82 26.458; N 29 43.574, W 82 27.233; N 29 44.238 W 82 28.138) were disper sed throughout San Felasco

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46 Hammock Preserve State Park, Alachua Co., FL A research and collecting permit (# 02130742) was granted to P. J. Obenauer by the Florida De partment of Environmenta l Protection to collect mosquitoes within the park premises. Persona l observations were made in September 2006 to verify the existence of Ae. albopictus in sylvatic sites. Security and park regulations mandated that all traps be placed at a mi nimum of 40 m away from man-made trails. Traps were placed in forest-fringe areas or areas with large openi ngs in the canopy, these usually included areas around sinkholes and swamps (Fig. 2-1b). Sylvatic locales were separate d by at least 0.8 km and contained a mixture of mature hardwood and pine trees, namely live oak, water oak, laurel oak ( Quercus laurifolia Michx.), longleaf pine and slash pine ( Pinus elliottii Engelm). Traps and Baits The BG Sentinel trap (BG) (BioGents GmbH, Re gensburg, Germany) is a white, lightweight, collapsible, bucket-like device with its upper opening covered with mesh (Fig. 22a). Mosquitoes are drawn into the trap by a 12V DC fan. A black plastic tube (12 x 12 cm) is fitted into the top center of the trap and empties into a catch bag. To lure diurnal mosquitoes, white and black colors are used as visual cues in combination with a synthetic bait that mimics skin secretions (Krckel et al. 2006). The synthetic bait, Agrisense BG Lure (BioGents GmbH, Regensburg, Germany), consists of 2 m of coiled 4.75 mm internal diameter silicon tubing (containing 15 mL of lactic acid), 50 cm of 0.4-mm internal diamet er high-density polyethylene tubing (2 mL of caproic acid), a nd a slow release ammonia acrylic fibrous tablet as described in Williams et al. (2006c). The trap design, in combination with the lure, creates ascending currents that mimic similar conve ction currents created by the hu man body (Krckel et al. 2006). The BG trap was originally designed to trap Ae. aegypti and was to be placed inside or close to residential sites (D. Kline pers. comm.). This would have provided shelter from the elements. However, this study required thes e traps to be kept in outdoor e nvironments over long durations

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47 without shelter. Therefore, to prevent rain from damaging electrical circuits and motor components, an aluminum pan (35.56 cm x 1.90 cm) was attached 30.48 cm above the trap entrance with 2 nylon cords and s ecured to the handles of the trap. Blue smoke #2B (Signal Company, Inc, Spotswood, N.J.) was used to ensure that suction was not obstructed. The Mosquito Magnet X (MM-X) trap (Ame rican Biophysics Corporation (ABC), North Kingston, RI) uses a counter-flow concept that discharges an at tractant plume of carbon dioxide at the trap entrance to attract and capture mo squitoes (Fig. 2-2b) (Kline 1999). The MM-X has been shown more effective in capturing mosquito es than similar models because it produces shorter, but frequent bursts of CO2 (Cooperband and Card 2006). This trap consists of two fans, an 80 mm intake fan and a 40 mm exhaust fan, that are inserted into an oval-shaped, clear PVC shell as described in Hoel (2005). Unlike other mosquito traps, an advantage in using the MM-X trap is that captured insects cannot reach the intake fan and are subsequently rarely damaged. The Omni-Directional Fay-Prince (ODFP) trap (John Hock, Gainesville, FL) uses contrasting black and white metal panels that serve as a visual attractant (Fi g. 2-2c). The trap is 2.7 kg in weight, comprised of four extending panels (40.5 cm X 17.5 cm) set at 90 angles to each other which are used to direct mosquitoes in to the center of the trap, where they are pulled down through an opening by a small fa n (Jensen et al. 1994). A 40 cm2 sheet of white metal set 10 cm above the extended panels covers the fan. All traps were baited with CO2 from a 9 kg (20 lb) compressed gas cylinder with a flow rate of 500 mL/min. A Gilmont Accucal flowmeter (Gilmont Instrument Company, Barrington IL.) was used at every rotation to verify the accuracy of CO2 discharge. Flow rates were regulated using 15-psi single stage regulator equipped with microre gulators and an inline filter

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48 (Clarke Mosquito Control, Roselle, IL). Carbon dioxide flowed from the cylinder to the trap using 6.4 mm diameter black plastic tubing (Clarke Mosquito Control, Roselle, IL and ABC). All traps were baited with Agrisense BG-Mesh Lure (batch # ML066A) (BioGents GmbH, Regensburg, Germany). Lures were replaced after 2 months to ensu re that bait attractant was not degraded by heat and humidity (A. Rose, pers. comm.). Binder clips were used to hook the BG-Mesh Lure to the CO2 outflow area of the ODFP and MM-X traps. Rechargeable gel cell batterie s (Battery Wholesale Distribut ors, Georgetown, TX), that were replaced every 48 hours, pow ered all traps. The BG and MMX traps utilized a 12 V, 12 ampere-hour (A-h) battery, while the ODFP trap used a 6 V, 12 A-h battery. At the start of each trapping period, general purpose duc k tape was inversely folded over with the adhesive side facing out to act as a sticky band a nd attached to the top of traps and power cords to prevent ants from consuming captured mosquitoes. Trapping Scheme Each trap was placed at one height (either 1 m or 6 m) per site and randomized at every collection period. Traps were plac ed underneath trees in shaded ar eas, as Peacock et al. (1988) observed traps placed in shaded areas caught 11% more adult Ae. albopictus than those placed in partial shade. Traps within each site were se t at least 20 m from each other and at least 10 m from residences. Two methods of trap suspension were use d. Traps placed at 1 m were hung from a shepherds hook. Traps placed in tree canopies at heights of 6-7 m required a pulley system to allow the trap to be raised and lowered for coll ection. A tree branch was selected that was 7-8 m in height and capable of supporting all three type s of traps. A modified slingshot method (Novak et al. 1981) was used to place the pulley system into the canopy. An 80 g lead pellet was fitted to a spool containing 9 kg test monofilament lin e and was catapulted from a hand-held slingshot

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49 over the selected branch. A modified system using two ropes was used (Lundstrm et al. 1996) (Fig. 2-3). Once the monofilament line had been placed over the selected branch, it drew a 25 m 6.35 mm diameter interwoven nylon rope that was attached to a 25 mm metal loop. To accurately determine height, a second rope cont aining one-meter markings was inserted through the loop and suspended from the canopy. Because th ese traps differ in the position of the trap entrance (bottom or top entry), trap placement was ad justed to ensure that all trap openings were at either 1m or 6 m in height. Traps were set between 0800 and 1100 and left in place for 48 h (1 trapping period); at which time mosquitoes were collected. Traps were repaired when necessary and were raised or lowered to the selected heights for the subse quent trapping period. F our consecutive trapping periods (2 weeks) took place at each locale (subu rban or sylvatic), at which time traps were removed and moved between suburban or sylvatic locales accordingly. The absence of traps for two weeks between the two environments was de signed to mitigate any negative impact on the mosquito population. Trapping occurred from 16 May 09 June, 13 June 07 July, 11 July 04 Aug., 08 Aug. 01 Sep. and 05 Sep. 29 Sep. for a to tal of 5 trials resu lting in 20 trapping periods per locale. Environmental conditions within each site, in cluding temperature and light intensity were monitored using a HOBO pendant temperature/light data logger with 30 mi n recordings. Collected mosquitoes were anaesthetized at 20 C for 5 min, dispen sed into 1.5 mL plastic Fisherbrand Snap-Cap microcentrifuge tubes (ThermoFis her Scientific, Sawanee, GA) and frozen (-20 C) for later identification to specie s using the dichotomous keys of Darsie, Jr. and Morris (2003).

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50 Statistical Analysis A randomized block design with factorial treat ments was used to test differences in mosquito capture between traps, heights and loca les. All traps were rotated after each trapping period within sites to eliminate location and tr ap bias. Data were transformed with log10 ( n +1) prior to analysis. Trap type, trap height, site within locale and locales were fixed effects in the model. The model also included the trap type and tr ap height interacti on effect (Proc GLM) (SAS Institute 2006). Where interactions were f ound to be significant, we used the interaction error term to calculate p-values. Multiple m ean comparisons were made with the Ryan-EinotGabriel-Welsh (REGW) multiple range test ( =0.05). Results Forty trap periods (20 suburban and 20 sylvatic locales) throughout 5 tr ials resulted in a total capture of 44,525 mos quitoes, representing 26 species from 11 genera (Table 2-1). While inverted tape proved effective at preventing ant access to the traps, occasionally large numbers of ants infested the traps and destroyed the mosqu itoes. Data from these traps were discarded and treated as missing values. In suburban locales, 23 mosquito species were captured at 1-m heights, while only 14 species were captured at 6 m heights. Traps pl aced in sylvatic locales captured 18 and 17 species at 1 and 6 m, respectively. Trap collections fr om suburban sites represented 71% of the total capture. The following nine species comprised 99% of the total collection and were statistically analyzed: Ae. albopictus Ae. vexans (Meigen), Anopheles crucians Wiedemann, Coquilletidia. perturbans Dyar, Cx. nigripalpus Say, Cx. erraticus (Dyar and Knab) Oc. infirmatus (Dyar and Knab), Oc. triseriatus (Say) and Psorophora ferox (von Humboldt).

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51 Aedes albopictus Few Ae. albopictus were captured during May and early June. However, by early July all sites had captured at least one Ae. albopictus Captures of Ae. albopictus peaked in mid-July when a total of 1,503 were coll ected. Trap captures of Ae. albopictus were fewest during May (0.65 0.2). Although Ae. albopictus were captured during all trap ping periods in June (6.5 1.2) and September(7.6 2.1), significantly more were collected during July (16.7 3.7) and August (11.1 2.4) trapping periods (F =29.08, df =4, 425, P <0.001) (Fig. 2-4). Male and female Aedes albopictus (3,768) comprised over 8% of the total mosquito capture (Table 2-1) making this species the se cond most commonly captured mosquito. Males comprised 21.7% of the Ae. albopictus captured. The total number of Ae. albopictus collected is presented by trap within trial, height and locale in Appendix A. No significant difference was detected between trap capture means (Table 2-2). However, signifi cant differences were detected between height (F =120.22, df =1, 425, P <0.001) and locale (F = 500.50, df = 1, 425, P <0.001) (Fig. 2-5). A great er percentage (87%) of Ae. albopictus was captured at 1 m versus traps placed at 6 m, while only 2.2% of this species was captured in sylvatic locales. Sites within locales also proved to be highl y significant with respect to Ae. albopictus collections (F =27.78, df =6, 425, P =<0.0001). Within sylvatic sites, one site accounted for 50% of Ae. albopictus captured. This site was in closer proximity to In terstate 75 and residential areas compared to the other three sylvatic sites. Seventy-five percent of all Ae. albopictus trapped in suburban locales were from two of the sites. Other Mosquito Species The BG trap significantly outperformed the MM-X and ODFP traps at capturing Cx. nigripalpus, Cq. perturbans and Oc. triseriatus (Table 2-2). Culex nigripalpus exhibited an

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52 increased attraction for the MM-X (53.5 14.9) over the ODFP (28.5 10.7) trap (F = 51.99; df = 2, 425; P <0.0001). The most commonly captured mosquito from suburban and sylvatic locales was Cx. nigripalpus (Table 2-1). Significantly more Cx. nigripalpus (66.7%) were trapped at 6 m (F = 40.62, df = 1, 425, P <0.0001) (Fig. 2-4). In addition, significantly more Cx. nigripalpus were captured in suburban locales compared to sylvatic locales (F = 7.49, df = 1, 425, P =0.0064). However, significant differences were detected between the block effect (time of year) (F =258.34, df =4, 425, P <0.0001). The fewest Cx. nigripalpus were trapped in the early part of the trapping season, while over 75% of the capture occurred during the months of August and September. Coquilletidia perturbans were captured in traps at both locales and were the third most commonly captured mosquito (Table 2-1). Thei r population levels did not fluctuate throughout the season as did most other mosquitoes in the study. Significantly more Cq. perturbans were captured in the BG trap (9.3 1.8) compared to the MM-X (3.2 0.8) and ODFP traps (4.8 1.0) (F = 37.45; df = 2, 425; P <0.0001) (Table 2-2). The major ity of this species (85%) were captured in suburban locales (F =37.45, df =1, 425, P <0.0001). In addition, trap placement was highly significant with the majority of Cq. perturbans captured at 1 m (F =10.64, df =1, 425, P =0.0012) (Fig. 2-4). Significantly more An. crucians (F =45.13, df =1, 425, P <0.0001) and Ae. vexans (F =6.54, df =1, 425, P =0.0109) were trapped in sylvatic locales. The BG trap captured significantly more Ae. triseriatus (1.2 0.2) and Ps. ferox (6.0 2.0) compared to either the MM-X (0.6 0.3, 0.7 0.1) or ODFP (0.4 0.1, 1.6 0.7) traps, respectively (Table 2-2). However, the MM-X trap captured significantly more Ae. vexans (8.1 2.6) compared to the BG

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53 (5.4 1.6) and the ODFP (2.5 0.8) traps (F = 13.40; df = 2, 425; P =0.0021) (Table 2-2). There was a significant interaction betw een trap type and height for Ae. vexans (F =6.25, df =2, 425, P =0.0021) and Ps. ferox (F =6.44, df =2, 425, P =0.0018). Although Toxorhynchites rutilus rutilus (Coquillet) were not captured as often (n=24) as compar ed to other species listed, over 91% were trapped with the BG sentinel trap a nd the majority (62%) were captured in sylvatic locales. Discussion Mosquito traps utilize a variety of lures that may be attractive to specific mosquito species (Service 1993). Additionally, mosquito attr action can be influenced by a traps physical appearance (Haufe 1964). Our present study uti lized traps with black and white colors supplemented with lactic acid, caproic acid, ammonia and CO2 to maximize captures of hostseeking Ae. albopictus Results indicate that traps ofte n elicited mosquito species-specific responses that were dependant on trap type and height placement. Furthermore, several species demonstrated a preference for sylv atic or suburban locales. Due to its diurnal feeding patterns, few traps are marketed for Ae. albopictus control or surveillance. Yet it remains a serious disease vector as well as a nui sance in most Florida communities. Worldwide outbreaks of dengue and chikungunya continue to appear, placing additional burdens to develop rapid su rveillance tools to access populations of Ae. albopictus without using humans as bait. Studies have shown that the Mosquito Magnet Pro, Mosquito Magnet Liberty, bidirectional Fay and ODFP traps ar e effective trapping tools for Ae. albopictus (Jensen et al. 1994, Shone et al. 2003, Dennett et al. 2004, Hoel 2005). However, these traps are extremely bulky and some require heavy propane tanks for CO2 production. This study indicates that the BG trap may provide an effective a lternative to these traps in capturing Ae. albopictus regardless

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54 of locality. Although we suppl emented it with a 9 kg CO2 tank, the trap by itself is lightweight, collapsible and can be easily transpor ted. Originally designed to capture Ae. aegypti and to be placed in sheltered urban environments (D. Kline, pers. comm.), the BG trap performed well in less-sheltered, field environments. However, modifications were needed to prevent precipitation from damaging the electrical circuits (Fig. 23A). Although the difference was not significant, the BG trap caught more Ae. albopictus than the MM-X or ODFP traps, regardless of locale. The high percentage (9 8%) (Table 2-1) of Ae. albopictus captured from suburban locales may be due to the availability of breeding sites, hosts or both. While, natural breeding sites (i.e. tree holes) were found supporting Ae. albopictus larvae (Fig. 2-6) and tr aps captured adults at every sylvatic locale, the habitat may have b een less conducive to support large populations. In addition, it is believed that U.S. populations of Ae. albopictus were originally introduced in used tires from Japan (Hawley et al. 1987). Therefore, it stands to reason that Ae. albopictus is more inclined to inhabit residen tial or suburban environments than sylvatic ones. Interstate-75 is located next to San Felasco Ha mmock Preserve State Park. Fifty percent of the sylvatic-captured Ae. albopictus were from the site that was cl osest to the Interstate. It is possible that used or damaged tires or other refuse may have been present along the highwayforest edge, and provided ideal Ae. albopictus breeding sites. This si te was also in closer proximity to residential areas as compared to other sites. Residential areas tend to have numerous artificial containers, such as bird bath s, rain gutters and cans. An increase in the number of man-made breeding sites would support a substantially larger Ae. albopictus population as compared to natural containers, such as tree holes. Furthermore, sprinkler systems commonly found in suburban areas, c ould consistently maintain these containers with water, thus

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55 providing ideal breeding s ites that support active Ae. albopictus populations even during times of drought. Trap captures indicate that while Ae. albopictus were attracted to traps placed at 6 m, the majority (87%) were captured at 1 m. Similar re sults were observed in Japan where dry ice traps placed at 1 m captured significantly more Ae. albopictus than those placed above 1 m (Tsuda et al. 2003). However, the fact that 13% were captured at 6 m in th e current study suggests that this behavior may have disease transmission impli cations and impact how control measures are implemented. Few Ae. albopictus were captured during the early portion of this study. This was due to a severe drought that affected Florida. Total precipitation for Alachua County from March to May 2007 was 11.00 cm (4.62 in), a 15.39 cm (6.05 in) deficit for this period (http://fawn.ifas.uf l.edu/data/reports ). However, the overall population of Ae. albopictus dramatically increased with th e arrival of Tropical Storm Barry on 2 June 2007. By the end of July almost 28 cm of precipitation had fallen in Alachua County causing a dramatic increase in the number of Ae. albopictus (Fig. 2-4). Temperat ure differences were minimal between sylvatic and suburban locales (Appendix B). However, fe wer temperature fluctuations were noted in sylvatic locales versus suburban locales. Light intensity was greater during the month of May and this was likely due to an increase in cloud cover brought on by summer thunderstorms during the June through A ugust period (Appendix C). Aedes albopictus is susceptible to WN and can readily disseminate the virus once infected (Turell et al. 2001, Sardelis et al 2002). It is an opportunistic feeder on a variety of hosts, including birds, thus it may serve as a bridge ve ctor to humans (Turell et al. 2001, Turell et al. 2005, Richards et al. 2006). Many mosquitoes, especially Culex spp. feed on birds while they

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56 are roosting. Peak blood-feeding by Ae. albopictus occurs during two periods, one between 0630 0730 and a second between 1630 1800 (Ho et al. 1973); however, Higa et al. (2000) has observed feeding as late as 2300 0400 in Japa n. Therefore, it st ands to reason that Ae. albopictus would play a greater role in disseminating WN to humans if they were feeding on birds. Furthermore, their short flight range and close association with humans may increase transmission potential if infected birds are nes ting in close proximity to residential areas. However, though the potential exists for Ae. albopictus to transmit WN, it is not considered a significant vector and is ineffec tive in maintaining the virus ov er a long period of time (J. Day, pers. comm.). This is partially due to its st rong preference for mammalian hosts (Richards et al. 2006). Recent studies in Alabama rev ealed that while large numbers of Ae. albopictus were captured, few tested positive for the WN virus (C upp et al. 2007). In addition, those that were positive only occurred during years where there was a high level of transmission. Studies in north central Fl orida have documented that Cx. nigripalpus is one of the most frequently trapped mosquito species (Kline et al. 2006, Alla n et al. 2005). Culex nigripalpus is the principal vector of St. Louis encephalitis vi rus and is known to f eed on a range of hosts including: raccoons, cats, opossums, armadillos, co ws, horses, rabbits, humans and several bird species (Edman 1974). Although it is an opportunistic feeder, it pref ers to feed at night on birds within the confines of vegetative cover or forest canopy (Day and Curtis 1994). Therefore, it is not surprising that we captured a higher proportion Cx. nigripalpus in all traps set at 6 m than at 1 m. The majority of this species (71%) were ca ptured in suburban locales compared to sylvatic ones. This dramatic difference may be due to ho st abundance or more attractive breeding sites in suburban locales. While host location may be a key factor influencing Cx. nigripalpus vertical movements, humidity fluctuations within the fore st strata also may influence their distribution.

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57 Humidity fluctuations are known to influence th eir movement, dispersing from drier areas for more humid ones (Dow and Gerrish 1970). Adult Cx. nigripalpus are often observed during periods of high humidity and calm winds; conditions usually occurring seve ral hours after sunset (Day and Curtis 1994). Several studies of Culex species have shown similar heig ht preferences to my results. Studies in England demonstrated that more Cx. pipiens L. were collected in light traps placed at 5-m than at 2.5-m or 1-m (Hutchinson et al. 200 7). Canopy experiments in Sweden also showed that 36% of Cx. pipiens/torrentium were captured between 12 and 15.5 m (Lundstrm et al. 1996). A similar pattern was observed in Connecticut, where MM-X traps caught more Cx. pipiens at 7.6 m than at 1 m (Anders on et al. 2004). In New Yor k, Darbro and Harrington (2006) found significantly more Cx. restuans Theobald trapped at 9 m than at 1.5 m. Recently, Savage et al. (2008) found chicken-bait ed traps placed at 7.6 m height s in urban areas of Tennessee captured a greater number of Cx. pipiens compared to those placed at 4.1 m. Though they did not find WN infection rates to be significantly different by height all mosquitoes that tested positive (n=11) were from the Cx. pipiens complex. However, all of these locations are situated in more northern latitudes as compared to Florida. Although Cx. pipiens is known to feed on both mammals and birds, northern populations are known to become increasingly ornithophilic with an increase with latitude (Spielman 2001). Therefore, future studies should determine if a change in Cx. nigripalpus host preference also fluctuates with an increase in southern latitudes. This may determine if our results are a reflec tion due to host location or a response to local humidity fluctuations. Kline et al. (2006) reported that Cx. nigripalpus were captured equally using the CDC and MM-X traps. This study demonstrated that the BG trap caught significantly more Cx.

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58 nigripalpus compared to the MM-X and ODFP traps. The BG trap is black and white in appearance, which are the preferred colors used to lure daytime feed ing mosquitoes including Ae. albopictus (Freier and Francy 1991, Jensen et al. 1994). Why the BG trap is luring a nighttime feeder remains a mystery, in so much as each trap contained the same lure and CO2 flow rates. Trap constr uction, suction intake and CO2 emission from the trap may be important factors responsible for capturing more Cx. nigripalpus Compared to the MM-X and ODFP traps, the BG trap has multiple outlets for CO2 emission, houses dual chambers allowing for a push-pull mosquito intake system and a drain hole located at the bottom. However, the MM-X is known to discharge numerous short plumes of CO2, which is reported to increase the attraction of host-seeking mosquitoes (Cooperband and Card 2006). Therefore, future comparison studies using BG traps should analyze CO2 plume structure to determin e if trap design affects CO2 plume emission, thereby in creasing trap captures. The BG trap also captured larger numbers of Cq. perturbans compared to the ODPF and MM-X. Coquilletidia perturbans was collected at 1 and 6 m he ights, but significantly more were trapped at 1 m (Fig. 2-5). In Florida, Cq. perturbans feeds on mammals and birds, although it has a stronger propensity for mammalian blood (Edman 1971). Coquilletidia perturbans is a known bridge vector of eastern equine encephalo myelitis virus (Boromisa et al. 1987) as well a competent vector of WN (Turrell et al. 2005). Furthermore, field populations have tested positive for WN (Cupp et al. 2007). Our height capture results are similar to t hose of a Maryland study by Shone et al. (2006) who demonstrated that Cq. perturbans was captured in greater numbers at 1.5 m than at 5 m heights. In addition, Bosak et al. (2001) demonstrated that Cq. perturbans host seek at different heights throughout the night and fou nd interactions between time of year and height. However,

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59 their studies used traps baited with CO2 and octenol, while we used CO2 and a BG lure, that did not contain octenol. The attracta nts, ammonia, lactic acid and cap roic acid found in the BG lure, may provide a stronger attractant th an octenol alone. Therefore, fu ture trapping of this species should not only include an analys is of blood meals to determine if there is a pattern of host selection within heights, but a comparison between trap lures. Although several mosquitoes in this study were not analyzed in detail, they are worth mentioning due to their apparent se lective environment preference. Orthopodomyia signifera (Coquillett) was captured only in sylvatic locales; th is is not surprising as their larval habitats are cryptic tree holes found predominantly in oak fo rests (Woodward et al. 1998). In contrast, Wy. smithii (Coquillet) and Wy. mitchelli (Theobald) were trapped only in suburban locales. Their larval developmental sites are restricted to tank bromeliads, often used as decorative ornamental plants in many suburban neighborho ods throughout Florida (Frank 1990). Few studies report su ccessful capture of Toxorhynchites spp. adults using mosquito traps. Toxorhynchites larvae are predators of other mosquito la rvae, while adults feed only on nectar (Steffan and Evenhuis 1981). They have been used widely as potential biological control agents to control Cx. quinquesfaciatus Ae. aegypti and Ae. albopictus (Focks et al. 1982, EstradaFranco and Craig 1995, Legner 1995). The numbers of Tx. r. rutilus that we captured were likely attracted to the black circular opening of the BG trap, mistaking it for a natural oviposition site such as a tree-hole (D. Kline, pers. comm.) Past methods have us ed hand-nets to recover adults (Trpis 1973), but this is time c onsuming and not cost effective. Though Toxorhynchites spp. cannot bite and do not pose health threats, trapping adults may be a method by which to determine their presence or popula tion estimates in a given area. Therefore, utilizing the BG trap

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60 as a surveillance tool to capture Ae. albopictus would not only indicat e their presence and provide population estimates, but also could determine if Toxorhynchites spp. are present. This study demonstrated that traps baited with host-seeking attractants are highly effective at trapping a variety of mosquitoes, including Ae. albopictus in sylvatic and suburban locales. The BG trap captured significantly greater numbers of Cq. perturbans Cx. nigripalpus Oc. triseriatus, Oc. infirmatus and Ps. ferox compared to the other traps tested. In addition, it captured large numbers of Ae. albopictus in both locales. Its perf ormance in conjunction with being collapsible and lightweight, make it an attractive tool for ra pid vector assessments. In addition, the placement of these baited-traps at va rious heights identified host-seeking behaviors for a variety of mosquitoes. Future application of semiochemicals in traps, such as the BG lure, to increase capture rates serves not only to enha nce surveillance, but also as a management tool (Kline 2007).

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61 Table 2-1. Mosquitoes captured at 1 and 6 meter heights in subur ban and sylvatic locales from May September 2007 in Gainesville, Florida. Species listed in descending order of the total numbers of each species collected. Locale Suburban Sylvatic Species 1 m 6 m 1 m 6 m Totals (%) Culex nigripalpus 7667 14871 2870 6320 31728 (71.0) Aedes albopictus 3203 482 67 16 3768 (8.5) Coquillettidia perturbans 1050 1140 259 89 2538 (5.7) Ae. vexans 862 170 1275 59 2366 (5.3) Ochlerotatus infirmatus 441 85 800 91 1417 (3.2) Psorophora ferox 623 33 521 21 1198 (2.7) Cx. erraticus 120 146 200 39 505 (1.1) Anopheles crucians 29 6 274 35 344 (0.8) Ae. triseriatus 123 33 86 99 341 (0.8) Cx. salinarius 23 50 9 7 89 (0.2) Mansonia titillans 20 28 2 3 53 (0.1) Ps. columbiae 16 9 6 1 32 (<0.1) Toxorhynchites rutilus 4 5 9 6 24 (<0.1) Cx. quinquefasciatus 16 0 4 2 22 (<0.1) An. quadrimaculatus 16 0 1 1 18 (<0.1) Wyeomyia mitchelii 16 0 0 0 16 (<0.1) An. punctipennis 1 2 9 2 14 (<0.1) Orthopodomyia signifera 0 0 2 9 11 (<0.1) Oc. taeniorhynchus 8 2 0 0 10 (<0.1) An. barberi 1 0 4 2 7 (<0.1) Ps. howardii 2 1 4 0 7 (<0.1) Cx. territans 5 0 0 0 5 (<0.1) Ps. ciliata 2 0 2 0 4 (<0.1) Ae. atlanticus 2 0 1 0 3 (<0.1) Wy. smithii 3 0 0 0 3 (<0.1) Culiseta inornata 0 0 0 2 2 (<0.1) Totals 14253 17063 6405 6804 44525

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62 Table 2-2. Numbers (mean SE) of the nine most common female mosquitoes collec ted in a trapping period fr om three types of traps at 1 and 6 meter heights in suburban and sylvatic locales from May Se ptember 2007 in Gainesville, Florida1. 1 Means within each row followed by the same letter are not signi ficantly different (Ryan-Einot-Gabriel-Welsh multiple range tes t), = 0.05, trap periods = 48 h each; df = 2, 425. 2 Traps were baited with CO2 at a flow rate of 500 mL/min and a BG-Mesh lure. BG = BG-Sentinel ( n = 147), MM-X = Mosquito Magnet X ( n = 150), ODFP = Omni-directional Fay-Prince ( n = 145). Traps2 Species BG MM-X ODFP F P Culex nigripalpus 133.1 38.4a 53.5 14.9b 28.5 10.7c 51.99 < 0.0001 Aedes albopictus 10.2 2.1a 7.4 1.5a 8.0 1.7a 1.13 0.3230 Coquillettidia perturbans 9.3 1.8a 3.2 0.8b 4.8 1.0b 37.45 < 0.0001 Ae. vexans 5.4 1.6b 8.1 2.6a 2.5 0.8c 13.40 0.0021 Ae. infirmatus 4.1 1.3a 2.2 1.2b 3.4 1.0a 6.27 0.0021 Psorophora ferox 6.0 2.0a 0.6 0.3b 1.6 0.7b 23.19 < 0.0001 Cx. erraticus 1.6 0.4a 0.7 0.1ab 1.2 0.3ab 2.95 0.0532 Anopheles crucians 0.6 0.3a 1.0 0.3a 0.7 0.3a 1.73 0.1789 Ae. triseriatus 1.2 0.2a 0.4 0.1b 0.7 0.1b 10.40 < 0.0001

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63 A) B) Figure 2-1. A typical residential backyard used for a suburban site (A) and a typical sylvatic site (B) in San Felasco Hammock Preserve State Pa rk, Gainesville, Flor ida used to collect Aedes albopictus

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64 A) B) C) Figure 2-2. Traps used to eval uate host-seeking height of Aedes albopictus in suburban and sylvatic locales. All traps were baited with CO2 and BG Mesh Lure and placed at 1 m and 6 m (1 m shown): A) BG-Sentinel, B) Mosquito Magnet-X, C) Om ni-directional Fay-Prince trap. B C A

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65 Figure 2-3. Mosquito Magnet-X trap positioned at 6 m in height using an interwoven nylon rope that was attached to a 25 mm metal loop, Sa n Felasco Hammock State Preserve State Park, Gainesville, Florida.

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66 0 5 10 15 20 25 30 35 16 May 9 Jun 13 Jun 7 Jul 11 Jul 4 Aug 8 Aug 1 Sep 5 Sep 29 SepMean number of female Ae. albopictus / trap period0 2 4 6 8 10 12 14 16 18Precipitation (cm) Precipitation (cm) Suburban Sylvatic Figure 2-4. Seasonal distribution of Aedes albopictus captured in 2007 and precipitation (cm) for suburban and sylvatic locales in Gainesvill e, Florida. Precipitation data retrieved from the Florida Automated Weather Network, University of Florida.

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67 0 0.5 1 1.5 Ae. albopictus Ae. vexansAn. crucians Cq. perturbans Cx. erraticus Cx. nigripalpus Oc. infirmatus Oc. triseriatus Ps. feroxLog (mean) number of female mosquitoes / trap night 1-Meter 6-Meter Figure 2-5. Mean capture rates of the nine most commonly trapped mosquitoes at 1 m and 6 m heights in sylvatic and suburban locales between May September 2007 in Ga inesville, Florida. Means within sp ecies with the same letter are not significantly different (Ryan-Einot-Gabriel-Welsh Multiple Range Test). = 0.05, n = 40 trap periods (48 h each). Ae = Aedes An = Anopheles Cq = Coquillettidia Cx .= Culex Oc = Ochlerotatus Ps = Psorophora a b a b a b a b a b a b a b a b a b

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68 Figure 2-6. White arrow de notes tree-hole supporting Aedes albopictus larvae in San Felasco Hammock Preserve State Park, Gainesville, Florida.

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69 CHAPTER 3 OVIPOSITON RESPONSE OF AEDES ALBOPICTUS TO INFUSIONS USING COMMON NORTH CENTRAL FLORIDA PLANTS Introduction Mosquitoes oviposit in a wide range of environments including swamps, salt marshes, snow pools, sewage ponds, small cont ainers and various pl ants and trees that collect rain water. Though all sites are aquatic, many species se lectively oviposit in a particular habitat. The majority of mosqu ito larvae utilize a range of organic detritus from bacteria, protists, algae and aquatic fa una to fallen decaying leaves as a primary food source (Kitching 2000). For example, newly emerged larvae of Aedes albopictus (Skuse) and other container-inhabiting mosquito es primarily utilize fallen leaf litter and other vegetative matter as food substrates. Furthermore, the presence or absence of leaves can significantly affect Ae. albopictus larval development (B arrera 1996) and thus affect their fecundity (Clements 1999). Th erefore, environments that contain an abundance of decaying leaf litter will genera lly support mosquito gr owth, resulting in increased larval development and subse quent adult populations (Dieng et al. 2002b). Olfactory cues are important external stim uli used by mosquitoes, not only to detect hosts, but for oviposition selection as well (Takken and Knols 1999). Oviposition stimulants can include pheromones associated with Culex spp. egg rafts (Starratt and Osgood 1973) and products of natural carbon recy cling due to bacterial digestion of organic materials (Dethier 1947). Bact eria responsible for producing oviposition stimulants have included: Aerobacter aerogenes found in hay infusion (Hazard et al. 1967), Enterobacter cloacae Acinitobacter calcoaceticus and Psychrobacter immobilis

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70 found in mosquito larval-rearing water (Benzon and Apperson 1988, Trexler et al. 2003b), Sphingobacterium mulivaroum found in soil-contaminated cotton towels (Trexler et al. 2003b) and pure cultures of Bacillus cereus and Pseudomonas aeruginosa (Hasselschwert and Rockett 1988). Recently, Ponnusamy et al. (2008) identified carboxylic acids and methyl esters produced by bacteria as responsible for stimulating oviposition behavior in Ae. aegypti L. Specific attractants can be applied in ovitrap s to capture different mosquito species. Many of these attractants are referred to as infusions and usually contain mixtures of fermented organic substances that simulate natural water that contains decaying plant matter or waste (Clements 1999). Water prev iously occupied by mosquito larvae has been effective as an oviposition lure as we ll (Bentley et al. 1976, Thavara et al. 1989, Allan and Kline 1995). Furthermore, traps ba ited with animal waste products such as cattle manure have been us ed to attract ovipositing Culex quinquefasciatus Say and Cx. nigripalpus Theobald (Allan et al. 2005). Although considerable research has been conducted to ev aluate different organic infusions to mediate oviposition in Ae. albopictus many organic compounds remain to be examined. A wide variety of material can be used to manufacture infusions that are attractive to Aedes spp. For example, animal feed pelle ts containing lupin or alfalfa seeds (Ritchie 2001), decayed paper birch (Ben tley and Day 1989), hay (Holck et al. 1988, Reiter et al. 1991) and synthe tic compounds such as 3-methylindole and 4-ethylphenol (Allan and Kline 1995) have all been used with some success. Other oviposition attractants, such as rinse water from giant tiger prawn ( Penaeus monodon ) and carpet

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71 shell ( Paphia undulate ) production facilities have also been shown attractive to Ae. albopictus (Thavara et al. 2004). The development of infusions can produ ce a wide range of responses as many interacting variables may exist. These f actors can include duration of fermentation, ammonia or protein concentration and bacteria levels; all of which may turn an infusion from an attractant to a repellent (Gubler 1971). For example, gravid Ae. aegypti are often attracted to bacteria-associa ted cues present in many eutr ophic habitats. However, an increase in the concentrations of these cues can repel this mosquito and make them less attractive as oviposition sites (Ponnusamy et al. 2008). Furthermore, the type of organic matter may significantly influence the level of attractiveness and larval development rate. For example, the presence of bacteria isolated from oak leaves has been shown to have a positive influence on Ae. albopictus oviposition behavior (Trexl er et al. 2003a), while those of alder leaf extracts have delete rious effects on larvae (David et al. 2000). Aedes albopictus oviposited significantly more eggs in laboratory and field ovitraps containing fermenting white oak leaves ( Quercusa alba L.) (Trexler et al. 1998) and maple leaves ( Acer buergerianum ) than well water alone (Dieng et al. 2002b, 2003). Gravid traps baited with oak leaf infusions have also captured greater numbers of Ae. albopictus when compared to hay infusions (Burke tt et al. 2004). Recently, Santana et al. (2006) determined that ovitraps baited with fermented guinea grass ( Panicum maximum Jacq), infusion collected significantly more Ae. albopictus than did water controls. Many suburban backyards in north central Florida contain a mixture of water oak ( Quercus nigra L.), longleaf pine ( Pinus palustris P. Mill) and St. Augustine grass

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72 ( Stenotaphrum secundatum (Walt.) Kuntze). Natural and artificial containers occurring in these areas often collect rainwater. Subs equently, oak leaves, pine needles and various grasses can often be found in th ese containers. This provide s ideal larval habitats for Ae. albopictus and other container breeders. Many st udies have shown enhanced oviposition on grass and leaf infusions (Trexler et al. 1998, Burkett et al. 2004, Santana et al. 2006), but there is limited information on detritus attr activeness from coniferous trees, such as pines, and mixture of all three infusions as on oviposition attractant or stimulant. We examined infusions using various plan t species as oviposi tion attractants for Ae. albopictus in field and laboratory bioassays The upwind response of gravid Ae. albopictus to these oviposition attractants was al so evaluated. These responses were measured in an olfactometer, a tool commonly used to scr een a range of semiochemicals to determine their potential as insect attractants or repellants (Butler 2007). Our objective in this study was to determine the ovipositi on response among six infusion types: water oak, longleaf pine, St. Augustine grass, water oak-pine mixture, pine-grass mixture and a water oak-grass mixture and a well water control. Materials and Methods Infusions Fallen dry leaves of water oak and needles of longleaf pine trees from the grounds at the University of Florida, Gainesville, FL and St. Augustine grass (bitterblue cultivar) that was cut with a lawnmower at the authors residence near Gaines ville were used to manufacture infusions. Special attention was taken to ensure all leaves and needles were free of foreign organic matter. Fresh-cut St. Augustine grass was placed on a sheet and dried for 4 days under natural sunlight. In fusions were prepared by fermenting 25 g of dried leaves, 2.5 g brewers yeast (MP Bi omedicals, LLC, Solon, Ohio), and 2.5 g

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73 lactalbumin (Sigma-Aldrich, St. Louis, MO) in 2.5 liters of well water, approximating methods of Allan and Kline (1995). Infusions were held at ambient temperature (25-27 C) for 10 days in a sealed plastic bucket. F our individual batches of each treatment were developed to ensure precision of true re plications throughout experimental trials. Infusions were past through sterile gauze dr essing to remove large organic matter and transferred into 150 mL pol ypropylene cups (Fisherbrand, Fisher Scientific, Houston, TX) and frozen at -20C. When used, frozen aliquots were placed in a warm bath for 30 min or until they were melted. Hay infusions used in preliminary experiments were provided by the USDA-ARS Center for Medi cal, Agricultural and Veterinary Entomology (CMAVE) in Gainesville, Flor ida and were developed using the same methods and materials. Mosquitoes Aedes albopictus females used during preliminar y experiments January April 2007 and June 2007 were from colonies esta blished in 2002 and held at the USDA-ARSCMAVE laboratory rearing facility. A second Ae. albopictus colony was established in May of 2007 and subsequently used for laboratory,olfactometer and field cag e experiments. Eggs were collected in ovitraps from Gainesville residences and larv ae reared at the Univ ersity of Florida, Entomology and Nematology Department, Gain esville, FL in environmental growth chambers at 29 1C. Larvae were maintained on finely ground TetraFin goldfish flakes. Approximately 50 larvae were placed in enamel coated trays with 2.5 L of deionized water and administered 1.45 g of food for 6 days. Adults were maintained in aluminum cages (30 x 30 x 30 cm) in a climate controlled room at 25 2C, 75% humidity, and a photoperiod of 12:12 (L:D). A dults were provided 5% sucrose-soaked

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74 cotton balls in plastic cups throughout the study. Blood meal s were provided by placing defibrinated bovine blood in sausage casings (blood sausages). Blood sausages were placed in a 34 C water bath for 5 min, the surface patted dry and suspended from the inside of the cage to facilitate feeding (Fig. 3-1). This procedure was repeated every 20 min until all feeding activity ceased. Black plastic cups (400 ml) containing a strip of #76 seed germination paper (Anchor Paper, St Paul, MN) (15 cm x 4 cm) submerged in 8 cm of well water were used as an oviposit ion site (oviposition cup). Oviposition cups were left in the cage for up to 72 h, at which ti me they were collected, dried and placed in Ziploc bags. Once a week, a couple drops of wa ter were placed on the paper to prevent further desiccation. When a new generation wa s needed, eggs were brushed-off of seed papers and placed in a 40 mL vial of water, shaken vigorously for 30 s and set aside until larval eclosion. This procedure is in accordance with that used by the USDA-ARSCMAVE. Laboratory Cage Bioassays Preliminary trials occurred between January and March, 2007 using the USDA colony of Ae. albopictus while a second set of experiments were conducted from January April 2008 using the Ae. albopictus colony established in 2007. Bioassays were conduced in laboratory cages (30 x 30 x 30 cm) constructed of four Plexiglas sides, a gauze access sleeve at one end and window sc reening at the opposite side (Fig. 3-2). Ovitraps consisted of black plastic cups (156 ml) cont aining a strip of #76 seed germination paper (6 x 4 cm). Infusions were diluted to 10% concentrate using well water. Experiment treatments consisted of the six infusions: water oak, longleaf pine, St. Augustine grass, water oak-pine mixture, pine-grass mixture and a water oak-grass mixture and a well water control. Each tria l consisted of 35 cages (n=5). In addition,

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75 well water was tested separately as a treatment to determine if infusions stimulated increased oviposition. The experime nt was replicated five times. Bioassays were conducted at 26-28 C w ith a 12:12 L:D photoperiod. Two ovitraps were placed in each cage. One ovitrap cont ained either an infusion or the well-water control and the second ovitrap always contai ned a well-water only control. Ovitraps were set 14 cm apart in a cage. The position of tr eatment and control cups (right or left) was noted and alternated between cages to eliminat e bias. Cages were st acked up to four high and completely randomized. Ten previously blood-fed (4-d prior) gravid females from the F10 to F12 generations were aspirated from a chill table and placed insi de the cage. Mosquitoes were allowed to oviposit in the plastic cups for 24 hrs, after which time seed paper was collected. After drying, seed papers were placed into sealable plastic bags. Eggs were counted using a dissecting microscope and recorded into a spreadsheet. Olfactometer Bioassays To determine if infusion treatments elicited an upwind response, gravid Ae. albopictus were tested in a clear acrylic triple-c age dual port olfactometer (Posey et al. 1998) (Fig. 3-3). Trials were conduc ted between 1000 1500 from November 2007 through March 2008 using procedures similar to those described by Allan and Kline (1995). Conditions inside the olfactometer were 28 C with 85% relative humidity. Although three olfactometer cham bers were available, only one chamber could be used at any one time. Water oak, longleaf pine, St. Augustine gra ss, water oak-pine mixture, pine-grass mixture and a water oak-grass mixture a nd well water control were evaluated. A micropipette was used to extract 500 L of 100% infusion concentrate from the top of

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76 each aliquot and placed in the bottom of a 50 x 2.5 mm watch glass set inside separate arms of the olfactometer. A total of 50 gr avid females (4-day post bloodmeal) from F5 through F11 generations (2007 colony) were aspirated from a chill table, transferred to the olfactometer and allowed a 1-hr pretreatme nt acclimatization period. The number of dead mosquitoes were noted and not include d in the experiment. Following acclimation, both arms of the olfactometer were opened, allowing access from the mosquitocontaining chamber. A 1 liter /sec airflow was passed over each treatment infusion and water. Olfactometer runs were conducted for 10 min, after which doors were closed and the numbers of females in each chamber were counted. At the conclusion of three consecutive runs (one run per chamber), mos quitoes were aspirated out of the arms and placed back into the chamber and treatments were randomized to a new chamber, ensuring each treatment was exposed in each chamber. A trial consisted of two olfactometer set-ups, on consecutive days. On day one, three of the six infusion treatments were randomly selected and used fo r three runs (n) on that day. The following set-up consisted of the remaining three treatments. Trials were replicated eight times for a total of 24 observations (n) for each treatment. Field Cage Bioassays Outdoor cage trials were conducted from J une September 2007 using four circular screened cages (2.13 m high x 2.74 m diam) co nstructed of a PVC pipe frame (2.54 cm diam) and screening (18 x 14 mesh). Cages were linearly set and spaced 1.82 m apart in a semi-shaded environment (Fig. 3-4). One Gardenia jasminoides J. Ellis, approximately 1 m in height, was placed in the center of th e cage to provide mosquito resting sites. Two cups containing cotton balls soaked in a 5% su crose solution were placed in the cages to provide a carbohydrate resource. Meterologi cal data (temperature and precipitation)

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77 were acquired from the Florida Automated Weather Network, University of Florida, Gainesville, FL. Oviposition was monitored using ovitraps constructed from an 11 x 9 cm black plastic cup with a 1 cm diameter drainage hol e positioned 5.5 cm from the bottom. Seed germination paper was cut into 10 x 10 cm squares and pressed against the inside surface of each cup. Each ovitrap was filled with 200 mL of either the infusion or well water control. Infusions were generated by first diluting the concentrate to 20% (40 mL) with well water before filling the cups. Six infusions, as identified earlier, were analyzed duri ng this study. To determine the percentage of infusion most preferred by Ae. albopictus we first conducted trials using 10, 20, and 30% standard hay infusions. This was critical as prior laboratory results demonstrated that high infusion concen trations act as repellents, rather than as attractants (S. Allan, pers. comm.). Two tr ials were conducted using four cages each with three single-source infusi on treatments (water oak, long leaf pine and St. Augustine grass) and a well water control (4 cups pe r cage). An additional two trials were conducted similar to previous trials with the three 50:50 infusion mixtures (water oakpine, water oak-grass and pine -grass). Four ovitraps were placed every 90 from the inside screen of the cage (Fig. 3-5). Ovitraps were placed on top of concrete blocks (19.05 cm in height), positioned approximately 1 m from the center of the cag e and 1 m apart, while a 14 x 14 cm nylon curtain fabric was placed over the top of each tr ap. Covers were fitted with a 1 g weight attached to the corners of the fabric a nd a 3.6 kg fishing line. These covers were designed to prevent mosquitoes from premat urely ovipositing within the ovitrap before

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78 fully acclimatizing to the cage. Fishing line was woven through the screen of the cage and secured to the outside frame (Fig. 3-5). Previously blood-fed (3 d prior) Ae. albopictus from F2 through F4 generations (2007 colony) were placed on a chill table to sort for gravid females. One hundred gravid females were released in the center of each cage, and were permitted to acclimatize for 1 h, at which point ovitrap covers were removed by pulling the fishing line. After 48 hrs, the oviposition pa per was collected and eggs were counted using a dissecting microscope. Statistical Analysis A completely randomized design was used in laboratory bioassay cage experiments. Treatment, trial, infusion batc h and a treatment were fixed effects in the model. A paired ttest was first conducted on raw means to determine well water and infusion treatment differences within each cage. The total amount of eggs oviposited in treatments were transformed with log10 ( n +1) and analyzed by analysis of variance (ANOVA), to detect differenc es between fixed effects. A randomize block design was used to an alyze the olfactometer bioassay. Oviposition response was measured as the pe rcentage of mosquitoes that responded positively to the treatment to the overall number of nonresponders. Abbott's correction was used to adjust data for those mosquito es that flew into control arms of the olfactometer (Abbott 1925). Means were square-root transformed and analyzed by ANOVA to detect differences between fixed effects. A blocked design was used to mitigate potential differences caused by aspirating mosquitoes back and forth between runs. Treatment, infusion batch, olfactomet er chamber, block and mosquito generation were fixed effects in the model.

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79 A randomized block design was used for field cage trials. Treatments were randomized in the cage to eliminate positi on bias. Statistical analysis was conducted using methods similar to Allan et al. (2005) To determine infusion attractancy or repellency, the total number of eggs laid on each seed-germination paper was divided by the total number of eggs laid in the respec tive cage (treatment + control). An arcsine transformation was performed on the percenta ge of eggs oviposited in each cup and analyzed by ANOVA. Treatment, cage, trial a nd infusion batch were fixed effects in the model. Grass infusion batch #2 was tainte d with an unknown agent causing a negative response; data from this set was discarded and was not included in the final analysis. All statistical analyses were conducted using the PROC GLM procedure of SAS (SAS 2006). Multiple mean comparisons were made with the Student-Newman-Keuls multiple range test ( =0.05). Results Laboratory Cage Bioassays The total number of eggs oviposited duri ng preliminary experiments using 20% hay infusions was 111.2 17.46 (SD) for the treat ment and 104.2 18 for well water control with no significant differences detected. Based on these observations, a reduction in infusion concentration to 10% was made This change resulted in more Ae. albopictus ovipositing in treatments than the control (person. obs.). Table 3-1 shows that all infusion treatments tested were significantly different from the well water control (paired t -test, P <0.0001). Cups containing pine infusi on had the greatest number of eggs (276.6 17.3) compared to those with water (84.72 7.05). In addition, a greater number of eggs were oviposited in cages that contai ned control cups and an infusion, than those cages with cups containing only well water. In cages that contained two well water

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80 ovitraps, approximately equal numbers of e ggs were oviposited in each ovitrap (102.8 12.0) (114.80 11.40). While no significant di fferences were detected among infusion batches, significant differences were presen t between treatments (F = 2.91; df = 5, 115; P = 0.0163) (Table 3-1). Significantly more eggs were oviposited in cups containing pine (276.6 17.3) than those containing grass ( 212.8 20.3). Significant differences within trial (F = 13.17; df = 4, 115; P <0.0001) and treatment by trial interaction (F = 1.88; df = 20, 115; P = 0.0203) were also detected. Olfactometer Bioassays Aedes albopictus exhibited a significantly strong er upwind response to all infusion treatments, with the exception of pine, when compared to well water (paired t -test, P 0.0009) (Table 3-2). Although not statistical ly different, more mosquitoes responded positively to well water compared to pine treatment. While no significant differences were detected between infusion batches or bl ocks, significant differe nces were detected between mosquito generations (F = 5.53; df = 5, 126; P = 0.0001), with earlier generation mosquitoes more responsive than later generations. Treatment comparisons demonstrated that oak-pine infusions elicited a greater response by Ae. albopictus than pine alone (F = 4.54; df = 5, 126; P = 0.0006) (Figure 36). Though not statistically different, Ae. albopictus exhibited the strongest response to the oak-pine infusion. Once inside the ol factometer port contai ning an infusion, many females exhibited a strong attraction by extendi ng their abdomens into the screen (Figure 3-7). Field Cage Bioassays Significantly more eggs were deposited in 30% hay infusion than 10% and 20% infusion and well water contro ls (F = 5.42; df = 3, 24; P = 0.0054) (Table 3-3).

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81 Regardless of concentration, a greater percenta ge of eggs (23% 35%) were oviposited in cups containing hay infusion as compared to those containing only well water (18%). Significantly more eggs were observed in each of the six infusions than in the well water control (F = 8.68; df = 6, 39; P < 0.0001). The percentage of eggs allocated to treatments ranged from 26 37% compared to 13.4% in the well wate r control. While no significant differences were observed between treatments, a greater percentage of eggs were allocated in cups containing water oak and the water oak-pine mixture as compared to those with pine, grass or grass mixtures (Figure 3-8). Discussion It is known that mosquito oviposition ha bitat selection is based on visual and olfactory cues (Bentley and Day 1989). Aedes albopictus have been shown to be highly attracted to black containers, but olfacti on and contact chemoreception remain important variables in acceptance of oviposition sites (G ubler 1971, Yap et al. 1995). It comes to no surprise that common breeding sites are natu ral and man-made containers that are dark and often contain organic matter. This study is the first to compar e pine, oak and grass infusions to elucidate if they el icit a greater oviposition response for Ae. albopictus These experiments demonstrated that Ae. albopictus is attracted to certain plant infusions and these infusions acted as an attractant and an oviposition stimulant resulting in a greater number of ovipositions compared to water alone. Additionally, this study demonstrated that Ae. albopictus exhibits an increased upwin d response to certain plant infusions. Aedes albopictus demonstrated an oviposition pr eference for pine and oak-pine infusions compared to grass under laboratory co nditions (Table 3-1). Infusions from oak leaves have been shown to elicit a stronge r oviposition response when compared to hay

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82 or grass (Burkett et al. 2004) A lack of eggs oviposited in grass during trial 1 was likely caused by an inaccurate measurement of grass infusion. During the laboratory experiments the greatest number s of eggs were oviposited in the pine infusion (Table 31). These results are contrary to what was observed in the olfactometer and field cages. However, laboratory experiments contained 10% pine compared to field cage and olfactometer experiments that utilized 20% and 100% pine infusi on, respectively. Pine infusion likely contains volatile oils that may have repelled Ae. albopictus at the higher concentrations (Butler 2007). The increased response to oak-pine infu sion in the olfactometer compared to individual component of oak, grass and pine infusions suggests a synergistic effect. There are a number of explanations for infusi on selection variability. The bacteria load and species diversity and ratios were unknown and the infusion mixtures likely had more diversity than single source infusi ons. It is also likely that variation between generations of this newly formed mosquito colony played a larg role in infusi on selection. Future olfactometer research should ut ilize a colonized mosquito po pulation that is not aged by more than a couple of generations as this may assist in delineating in fusion preferences. A greater upwind infusion response was observe d when weather was cloudy and rainy. Insect behavior inside an olfactometer has b een reported to be influenced by a range of factors including humidity, temperature, air quality, flow rate and light (Butler 2007). Therefore, it is possible that barometric pres sure may also influence oviposition behavior, thereby eliciting a greater response during approaching or retrea ting weather fronts. Of all infusions tested in the olfact ometer, only pine acted as an oviposition deterrent (Table 3-2). This was likely due to a pine oil repellent compound known as

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83 nerol (Butler 2007). However, the fact that oak-pine infusion elic ited a greater response compared to other infusions tested, suggests th at pine oil is masked when combined with other infusions, thereby altering or negating its repellent properties Furthermore, oakpine, oak-grass, pine-grass, oak and gr ass elicited a signifi cant upwind response compared to well water. These results are similar to those obtained by Allan and Kline (1995). In their study, both hay infusion and water collected from field and laboratory larval breeding sites attracted a greater number of gravid Ae. albopictus compared to well water. In addition, they observed a 69 and 30% response to hay infusion and well water, respectively. Although a dramatic difference was not observed between the infusions and well water, my results are comparable to th eir results. A likely explanation for this difference is the 10 min response time used in this study compared to 24 h used by Allan and Kline (1995). Many of the non-respondi ng mosquitoes may have moved into the infusion port if provided more time. Although no statistical differences were det ected among the treatments in the field cage experiments, a greater percentage of e ggs were oviposited in containers with oak and oak-pine infusion compared to grass or pine. Similarl y, Burkett-Cadena and Mullen (2007) found that Ae. albopictus did not exhibit an infusi on preference between Bermuda grass ( Cynodon dactylon L.), broadleaf cattail ( Typha latifolia ), soft rush ( Juncus effuses ) or sedge ( Rhynchospora corniculata ) when used in gravid traps. A follow-up study using mulch products including ma nure, pine straw, oak ( Quercus spp.) and cypress also failed to elicit a preference response (B urkett-Cadena and Mullen 2008). There are a number of fact ors that influence attractiveness other than substrate alone. First, the duration of fermentation a nd the organic solute c oncentration of water

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84 can drastically change ovipos ition response. Kramer a nd Mulla (1979) reported that while Culex pipiens quinquefasciatus Say was not attracted to 5 day-old, 1% chicken manure, a positive response was observed on day 6 and the response gradually increased until day 11. Santana et al. (2006) later demonstrated that Ae. albopictus were most attracted to guinea grass infusions fermented from 15 to 20 days compared to those at 30 days. Second, the stage at which the leaves are used may produce different levels of chemical cues. For example, Santana et al. (2006) also demonstrated that Ae. albopictus oviposited more eggs in infusions made from fresh guinea grass leaves than dried leaves. However, in contrast to this study, they did not include lactalbumen during their infusion preparation. Lactalbumen powder is a prescribed i ngredient for many infusions (Reiter 1983, Burkett-Cadena and Mullen 2007) and is adde d to serve as an accelerant for fermentation (S. Allan, pers. comm.). It is possible th at the addition of lactalbumen altered or increased the concentration of bacteria, ther eby causing a masking effect and reducing the ability of Ae. albopictus to demonstrate an infusion preference. Furthermore, it is likely that increased bacterial levels within infusions may be responsible for influencing their responses, increasing thei r level of attractiveness or decreasing it to the point of repellency (Ponnusamy et al. 2008, Gubler 1971). Future development of oviposition at tractants should focus on chemicals mimicking those common to organic infusions, ra ther than standardizi ng traditional ones. The major problems with standardizing organi c infusions are differences in ingredients used in infusion preparation and the rapid microbial and chemical changes that occur during fermentation (Bee hler et al. 1994).

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85 There are three primary reasons for studyi ng potential oviposition stimulants that specifically target Ae. albopictus First, in Florida, Ae. albopictus larvae are found in various man-made containers and have been collected from tree holes in urban, suburban and sylvatic habitats (OMear a et al. 1993). This ability has allowed it to quickly and efficiently establish in a variety of habitats Mapping the local flora distribution using remote sensing may assist mosquito abatem ent districts to quickly and effectively pinpoint potential Ae. albopictus breeding sites. Based on the current study, it would seem apparent that Ae. albopictus may prefer to oviposit in oak and oak-pine habitats compared to grass habitats. However, environmental factors such as temperature, humidity and light, along with physical factors such as site texture, color and reflectance are also known to influence site selection (Bentley and Day 1989). Therefore, future models should incorporate these factors with flora distribution to target and manage breeding sites. Second, analyzing plant detritus may elucid ate some of the facets responsible for influencing interspecific competition between Ae. aegypti and Ae. albopictus Aedes albopictus is a superior larval competitor to Ae. aegypti especially when exploiting food resources (Juliano 1998, Braks et al. 2004). A recent study by Mu rrell and Juliano (2008) determined that Ae. albopictus larvae outcompeted Ae. aegypti in oak and pine detritus and they believed that the detritus type was responsible for altering the interspecific competition between the two species. Additi onally, Reiskind et al. (2009) demonstrated that larval development and survival of Ae. albopictus and Ae. triseriatus (Say) was enhanced when reared in water infused with mixed-leaf detritus, compared to that containing a single leaf species. Aedes albopictus was introduced into Florida in the

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86 1980s and became quickly established througho ut most of the state (OMeara 1995). Floridas local floral abunda nce and variation, coupled with its resultant detritus may help determine where these two species ar e competitively exclusive and where they coexist (Murrell and Juliano 2008). Finally, studying plant infusions for mos quito attractiveness may serve as an important control measure. Currently, a numbe r of gravid traps baited with ovipositional attractants are available, but these tend to target Culex mosquito species. Aedes albopictus employs an oviposition behavior calle d skip oviposition whereby eggs are distributed throughout several sites (Burkett -Cadena and Mullen 2007). This strategy distributes their eggs over an area and may increase the likelihood of egg survival, unlike Culex spp. mosquitoes, which ovipo sit all of their eggs in one location. Therefore, infusion-baited ovitraps that target Ae. albopictus may mitigate this behavior by discouraging them from utilizing less attractiv e breeding sites, thereby concentrating their eggs to just a few containers (Ponnusamy et al. 2008). Furthermore, studies have shown that augmenting sticky ovitraps with infusions increases captures of Ae. albopictus thereby enhancing its sensitivity as a survei llance and potential control tool (Zhang and Lei 2008).

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87 Figure 3-1. Aedes albopictus adults feeding on a suspende d sausage casing containing bovine blood. Figure 3-2. Laboratory cage c ontaining two black 156 ml cups containing either an infusion or a well water control used to determine ovipos ition preference.

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88 Figure 3-3. Triple-cage dual port olfactometer (side-view) used in testing oviposition response, USDA-ARS-CMAVE, Gainesville, Florida. Figure 3-4. Circular outdoor screened cag es used in oviposition trials, USDA-ARSCMAVE, Gainesville, Florida.

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89 Figure 3-5. Interior area of outdoor screened cage demonstrating ovitrap placement and the nylon curtain fabric covering, USDAARS-CMAVE, Gainesville, Florida. Figure 3-6. Mean upwind response of gravid Aedes albopictus to a 500 uL concentrate of infusion placed inside a dual-port olf actometer for 10 min at the USDA-ARSCMAVE, Gainesville, Florida. Means with the same letter are not significantly different (Student-Newman-Keuls Multiple Range Test). = 0.05, n = 24. 0 1 2 3 4 5 6 Infusion type Percent Response (SE) Oak-pine Grass Grass-oak Oak Pine-grass Pine a aa aa b

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90 Figure 3-7. Aedes albopictus female exhibiting a ovipositiona l behavioral response to an oviposition infusion while in an olfact ometer. Note the abdomen extended downward by the mosquito shown in th e yellow circle, a common behavior exhibited by ovipositing mosquitoes. Arrow denotes airflow direction.

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91 Fig. 3-8. Mean 48-hr oviposition response of 100 Aedes albopictus females released into field cages with ovicups containing infusion treatments and a well water control at USDA-ARS-CMAVE, Gainesv ille, Florida, June September 2007. Means with the same letter are not significantly different (StudentNewman-Keuls Multiple Range Test). = 0.05, n = 6 oviposition periods (48 h each) for treatments, n = 12 for control. 0 5 10 15 20 25 30 35 40 45Infusion typePercent of Eggs Allocated to Treatment (SE).. Oak Oak-Pine Grass-oak Pine-grass Grass Pine Control a a a a a a b

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92 Table 3-1. Oviposition response of Aedes albopictus to six infusions1 and a well water control in indoor cages. Infusion n Mean no. eggs in treatment cup (SE)2 Mean no. eggs in control cup (SE) df t P > t Pine 25 276.6 (17.3) a 84.72 (7.06) 24 6.57 0.0001 Oak-Pine 25 259.6 (17.3) a 110.60 (8.80) 24 8.89 0.0001 Oak 24 238.3 (17.8) ab 95.16 (10.7) 23 8.89 0.0001 Pine-Grass 25 230.8 (22.0) ab 86.20 (11.0) 24 8.47 0.0001 Grass-Oak 24 224.6 (15.0) ab 81.80 (8.80) 23 11.10 0.0001 Grass 25 212.8 (20.3) b 57.00 (11.4) 24 6.57 0.0001 Well water 25 102.8 (12.0) NI* 114.80 (11.4) 24 -1.53 0.1390 1 Stock infusion was diluted to 10%. 2 Means in the same column followed by the sa me letter are not significantly different (Student-Newman-Keuls Multiple Range Test) = 0.05. 10 mosquitoes used for each replicate. Bioassays were conducted at 26-28 C with a 12:12 L:D photoperiod. Oviposition period = 24 hr. All mosquitoes we re blood-fed to repletion and held for 3 days prior to testing. *N.I = Not included in analysis. Table 3-2. Upwind response of 4-d-old gravid Aedes albopictus to 500 L infusion concentrate compared to well water cont rol inside a dual-port olfactometer for 10 min. Infusion n % response to treatment % response to well water df t P > t Oak-pine 24 4.85 0.76 1.41 0.25 23 4.815 <0.0001 Grass 24 3.92 0.52 1.37 0.26 23 4.155 <0.0004 Oak-grass 24 3.81 0.68 0.83 0.20 23 3.974 <0.0006 Oak 24 3.28 0.56 1.08 0.26 23 3.795 <0.0009 Pine-grass 24 3.35 0.57 0.83 0.23 23 4.047 <0.0005 Pine 24 1.15 0.78 2.00 0.49 23 -0.730 0.4727 Mean SE of 24 observations of 50 mosquitoes.

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93 Table 3-3. Oviposition response of Aedes albopictus to three concentrations of hay infusions and well water in outdoor cage s, Gainesville, Florida, June 2007. Infusion / Concentration n Mean no. eggs in cup (SE) % of eggs in cup Hay (10%) 8 699.3(77.8) a 25 Hay (20%) 8 650.0(82.9) a 23 Hay (30%) 8 976.4(61.3) b 35 Well water (control) 8 514.7 (106.7) a 18 Means in the same column followed by the sa me letter are not significantly different (Student-Newman-Keuls Multiple Range Test) = 0.05.

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94 CHAPTER 4 EFFICACY OF INFUSION-BAITED OVITR APS AT TWO HEIGHTS TO MONITOR AEDES ALBOPICTUS IN NORTH-CENTRAL FLORIDA SUBURBAN AND SYLVATIC LOCALES Introduction Aedes albopictus (Skuse) utilizes a range of natural and man-made containers for larval breeding sites (Hawley 1988) Its ability to e xploit tires, bird baths, clogged rain gutters, flower vases, tree holes, rock pools, bamboo stumps and tank bromeliads (OMeara et al. 1995, Ali and Nayar 1997) for ovi position sites has enabled it to sustain populations throughout Floridas urban, suburban and sylvatic environments (OMeara et al. 1993). Ovitraps serve as important surveillance tools to monitor th e population dynamics of container-breeding mosquitoes and provide crucial informa tion regarding future larval and adult populations (Service 1993). Fay and Eliason (1966) designed ovitraps from black-painted pint jars containi ng a wooden paddle to survey for Aedes aegypti L., which were subsequently used during the Ae. aegypti Eradication Program. These traps provided an economical and reliable surv eillance tool to m onitor populations of Ae. aegypti distribution and population density thro ughout the southeastern United States (Jakob and Bevier 1969). Ovitraps have also been shown to attract other mosquito species including Ochlerotatus triseriatus (Say), Oc. atropalpus (Coquillett), Oc. mediovittaus (Coquillett), Oc. japonicus japonicus (Theobold), Ae. albopictus and Orthopodomyia signifera (Coquillett) (Pratt and Kidw ell 1969, Williges et al. 2008). Although primarily designed for surveillance, ovitraps have evol ved into a control measure for container-breeding mosquitoes. Chan et al. (1977) first constructed an autocidal ovitrap that successfully controlled Ae. aegypti in urban areas of Singapore.

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95 Another lethal ovitrap was developed by Pe rich et al. (2003) using deltamethrinimpregnated ovistrips. Sticky ovitraps have b een successful at dete cting populations of Ae. aegypti and Ae. albopictus negating the need to rear or count eggs and provides a faster and more direct assessment of visiting adults (Ritchie et al 2003, Facchinelli et al. 2007). During dengue outbreaks, sticky ovitraps have provided valuable information to access Ae. aegypti populations, their infective rate s and potential transmission rates (Ritchie et al. 2004). Further augmenting sticky ovitraps with infusion can increase their sensitivity by attracting more gr avid females (Zhang and Lei 2008). Supplementing ovitraps with hay or leaf infusions has b een shown to increase the number of eggs oviposited by Ae. albopictus compared to water alone (Holck et al. 1988). Furthermore, the type of plant materi al used in an infusion has been shown to influence its degree of attractiveness to Ae. albopictus (Burkett et al. 2004). While previous studies using baited-ovitraps have included white oak leaves ( Quercus alba L.) (Trexler et al. 2003b), maple leaves ( Acer buergerianum ) (Dieng et al. 2002b, 2003) and guinea grass ( Panicum maximum ) Jacq (Santana et al. 2006), there are no published studies describing pine needle s as an infusion bait or its effect on attractiveness when combined with other infusions. A number of studies descri be the effects of vertical stratification on mosquito oviposition. Studies within urban areas in the Republic of Trinidad determined that Ae. aegypti preferred to oviposit in traps placed 1.2 m above ground when compared to ovitraps set at ground level, or elevated to 3.0 and 4.6 m above ground (Chadee 1991). In Sri Lanka, studies demonstrated that Ae. albopictus preferred to oviposit at ground level, with a decrease in oviposition as height increas ed. However, this study also revealed that

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96 Ae. albopictus will oviposit at 7 m and that microenvironmental conditions, such as temperature, humidity and light may play a large role in the attr activeness of breeding areas (Amerasinghe and Alagoda 1984). Recentl y in Singapore, Liew and Curtis (2004) demonstrated Ae. albopictus oviposition above 10 m. In their study, gravid Ae. albopictus marked with rubidium were released at 30 m and eggs recovered above 40 m. Since its introduction over twenty year s ago, there have been no studies on oviposition height preference by Ae. albopictus in suburban and rural environments in the United States. Therefore, it is important to investigate their oviposition height preferences in suburban and sylvatic lo cales as this information may optimize surveillance and assist vector control ag encies in reducing unnecessary insecticide treatments. Past observations found that Ae. albopictus were more prevalent in tree holes in urban areas compared to sylvan sites (OMeara et al. 1993). The most common method used to deter Ae. albopictus from breeding within suburban areas is to remove man-made containers from the surro unding environment. However, if Ae. albopictus oviposits high in the canopy as well as at groun d level, then significan t changes in vector surveillance and control may be warranted. In addition, many suburban backyards in north central Florida may contain natural and artificial containers that collect fallen leaves and pine needles from water oak ( Quercus nigra L.) and longleaf pine ( Pinus palustris P. Mill), potentially creating mo re attractive larval habitats for Ae. albopictus than other mosquito container breeders. There were two objectives for this study: 1) Determine if habitat influences Ae. albopictus oviposition height selection and 2) Determine the effectiveness and

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97 preferences for ovitraps containing oak or an oak-pine mixture compared to those containing water alone in attracting Ae. albopictus at 1 and 6 m heights. Materials and Methods Site Selection This study was conducted in four suburban a nd four sylvatic locales from May to October 2008. Suburban locales were loca ted at the following areas: N 29 37.837, W 82 27.800; N 29 34.248, W 82 24.644; N 29 39.019, W 82 23.234; N 29 42.481, W 82 24.745. Suburban locales met four criteria : 1) residents that have had frequent complaints of mosquitoes biting during the day; 2) sites with thickly wooded lots surrounding the residential propert y; 3) sites that had previo usly supported populations of Ae. albopictus and; 4) sites that were secure to he lp prevent trap theft. Suburban sites were separated by at least 3.22 km (2 miles) a nd contained a mixture of shrubs and trees, namely azalea ( Rhododendron spp.), oleander ( Nerium oleander ), Indian hawthorn ( Rahphiolepis indica ), live oak ( Quercus virginiana P. Mill) water oak and longleaf pine (Figure 3-1a). Sylvatic locales (N 29 43.574, W 82 27.252; N 29 44.048, W 82 26.458; N 29 43.574, W 82 27.233; N 29 44.238, W 82 28.138) were located throughout San Felasco Hammock Preserve State Park ( SFHPSP), Alachua Co., FL. Sylvatic sites contained a mixture of live oak, water oak a nd longleaf pine. A re search and collecting permit (# 02130742) was granted to the auth or by the Florida Department of Environmental Protection to collect mosquito eggs w ithin the park boundaries. Baited Ovitraps A modified ovitrap originally descri bed by Weinbren and OGower (1966) to capture Ae. aegypti was used. Lidless steel cans ( 11 cm high and 7.5 cm in diameter)

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98 were attached at three locations to an invert ed circular aluminum pie dish (7.5 cm base and 12.5 cm outside and 3 cm deep) by bending 12 gauge electrical wires into a closed loop. One of the three wires was bent into a hook, permitting the cover to be opened and closed (Fig. 4-1a). The pie dish served as a cover to prevent leaves or other debris from falling into the trap, possibly altering the in fusion. A 1 cm drain hole was made 7.5 cm above the bottom of the can, to prevent floodi ng. The entire ovitrap was spray-painted with Rust-Oleum flat black paint. To remove any paint odors or contaminants, ovitraps were preconditioned and aged by filling them with well water and letting them sit for 2 wk in a semi-shaded environment prior to the study (Burkett et al. 2004, Allan et al. 2005). Seed germination paper (76#, Anchor Paper, St. Paul, MN) (22 x 8 cm) was pressed against the inside surface of each can acting as a oviposition substrate. Seed paper was selected as it resists bacterial and fungal growth and tearing. Additionally, eggs from Oc. triseriatus and Ae. albopictus could be easily distinguished against the light brown paper (Steinly et al. 1991). To suspend the ovitrap, a 38 mm eyebolt a nd 2 nuts were fitted through the top of the pie dish (Fig. 4-1b). A 168 g fishing weight was attach ed with 12-gauge electrical wire to the bottom of the eyebolt providing ad ditional weight and stab ility. Each ovitrap was filled with 270 mL of either an infusion or deionized water (control). Infusions were generated by first diluting the concentrate to 35% (70 mL) with 200 mL of deionized water before filling the traps. Infusions Infusions were developed by collecting fa llen dry leaves of water oak (Oak) and longleaf pine needles (Pine) from the grounds at the University of Florida, Gainesville, FL. Special attention was take n to ensure all leaves and needles were free of foreign

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99 organic matter. Infusions were prepared by fermenting 120 g of leaves, 7 g brewers yeast (MP Biomedicals, LLC, Solon, Ohio ), and 7 g lactalbumin (Sigma-Aldrich, St. Louis, MO) in 12 liters of well water. Th e mixture was held at ambient temperature between (25-27 C) for 10 days in a sealed plastic bucket, approximating the methods of Allan and Kline (1995). Infusions were pa ssed through a sterile gauze dressing to remove large organic matter and tran sferred into 150 mL polypropylene cups (Fisherbrand, Fisher Scientific, Houston, TX) and frozen at -20 C. When used, frozen aliquots were placed in a warm bath for 30 min or until melted. Four individual batches of Oak and Oak-Pine infusion we re developed to ensure prec ision of true replications throughout the experiment. Subs equent evaluations consisted of the three treatments, Oak, Oak-Pine and a well water control. Trapping, Collecting and Egg Identification Each of the eight sites (4 suburban and 4 sylvatic) wa s partitioned into three stations. Stations were placed 20 m from each other and at least 10 m from residential structures. A total of 48 ovitraps were us ed during a given trapping period, two traps were placed at each of the thre e stations at each of the eigh t sites. At each station, one ovitrap was suspended at 1 m, with the second suspended at 6 m. Ov itraps at each station were baited with two of the three treatments : either an oak, Oak-pine infusion or a well water control. Station treatments were random ized at every collection period to eliminate position or placement bias. At a given site and placement period, the three treatments were represented at each height. Two methods of ovitrap suspension were used. Traps placed at 1 m were suspended from a shepherds hook. Traps plac ed in tree canopies at heights of 6 m required a pulley system (Fig. 2). A modifi ed slingshot method (Novak et al. 1981) was

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100 used to place the pulley system into the ca nopy. An 80 g lead pellet was fitted to a spool containing 9 kg test monofilament line and was catapulted from a hand-held slingshot over the selected branch. Next, a modified system using two ropes was used (Lundstrm et al. 1996). Once the monofilament line had been placed over the selected branch, it drew a 25 m 6.35 mm diameter interwoven nyl on rope that was attached to a 25 mm metal loop. To accurately determine hei ght, a second rope containing one-meter markings was inserted through the loop and suspended from the canopy. Traps were set between 0800 and 1400 and left in place for 1 week (1 trapping period). At this time the contents of each ovitrap were checked and the infusion, water and seed germination paper were replaced. A visual inspection for mosquito eggs and larvae was conducted on each ovitrap and all seed germination papers were placed in a sealed plastic bag for later identification and enumeration. Occasionally, some ovitraps contained mosquito larvae, unknown aquatic Di ptera and springtails (Collembola). All were counted, transferred to mosquito br eeding containers (Bi oquip, Rancho Dominguez, CA) and returned to the laboratory for further identification. To ensu re that old infusion was not present, all ovitraps were thoroughly ri nsed with deionized water to remove any organic matter prior to repl acing the infusion and paper. A second inspection of the egg paper wa s conducted in the laboratory. Seed germination papers were placed on paper towels and allowed to dry for 2 hrs. Eggs were counted and identified to speci es under a dissecting microscope based on their color, size, luster and shape (Kalpage and Brust 1968). To ensure accuracy, 10% of the eggs captured were reared to adults under laborat ory conditions following similar methods of Gerberg et al. (1994) and positive identifica tion was made using dichotomous keys of

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101 Darsie and Morris (2003). In addition, those mosquito larvae that were collected in the field were reared to adults using the same methods. Unknown aquatic Diptera were reared to adults and submitted to the Florida Department of Agriculture, Division of Plant Industry, Gainesville, FL for subsequent identification. Environmental conditions within each site, including temperature and light intensity were monitored using a HOBO pendant temperature/light data logger with 1 h recordings. A total of 20 consecutive trapping periods in each locale were conducted from 15 May to 3 October 2008. Statistical Analysis A nested analysis of variance was used to identify differences in mosquito egg capture due to infusion batch, infusion type, he ight and locale (Proc GLM) (SAS Institute 2006). Data were transformed with log10 (n+1) prior to analysis. Once it was determined that infusion batch was not statistically si gnificant, it was removed from the model prior to the final analysis. Treatment (infusion t ype), location:height were included as fixed effects, while trial was include d as a quantitative effect in the model. The model also included site and the interaction term treatmen t by location:height. In this analysis, the site variable was nested within the location: height variable. Multiple mean comparisons were made with the Ryan-Einot-Gabriel -Welsh (REGW) multiple range test ( =0.05). Untransformed means are presented in all figures. Results A total of 13,276 mosquito eggs were co llected, representing five species that included: Ae. albopictus (Fig. 4-3a), Oc. triseriatus (Fig.4-3b), Culex quinquefasciatus (Say), Or. signifera (Fig.4-3c) and Toxorhynchites rutilus rutilus (Coquillett) (Fig.4-3d) (Table 4-1a). In addition, other arthropods including collembolans and several families

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102 of Diptera including: Phoridae, Psychodidae and Corethrellid ae were collected (Table 42). Thunderstorms and Tropical Storm Fay cau sed a number of trees to fall during this study. Consequently, forest trails became inaccessible and several ovitraps sustained damage, resulting in lost data. For statistical purposes, lost data were treated as missing values. Less than 5 cm of precipitation occurred from 23 May and 13 June 2008 when fewer than 450 mosquitoes eggs were co llected (Fig. 4-4). However, following numerous rain events between 20 June to 18 July, conducive habitats for mosquito breeding were present. The largest rain event occurred in mid August, when Tropical Storm Fay stalled over the area, dr opping over 15 cm of precipitation. Aedes albopictus A total of 5,940 Ae. albopictus eggs were collected duri ng this study. Almost half (48.8%) were oviposited in traps containing an Oak-pine infusion. In suburban sites, 5,288 Ae. albopictus eggs were recovered, 50% from Oakpine baited ovitraps, while 39 and 9% were in Oak and water, respectively (Fig. 4-5). Out of the 652 Ae. albopictus eggs recovered from sylvatic sites, 58% were allocated in Oak-baited ovitraps, while 33 and 7% were recovered from Oak-pineand wate r-baited ovitraps, respec tively (Fig. 4-5). Although no significant differences was detected between Oa k-pine and Oak infusions, significantly more Ae. albopictus eggs were oviposited in tr aps containing infusions than those with only water (F = 7.95; df = 2, 910; P = 0.0004) (Fig. 4-6). A significant interaction was detected between treatment and location:height (F = 4.67; df = 6, 910; P < 0.0001). Significantly more Ae. albopictus eggs were recovered from ovitraps placed in suburban sites at 1 m than any ot her location (F = 76.31; df = 3, 910; P < 0.001) ( Fig. 48). A total of only 13% of eggs were recove red in sylvatic locales at both heights.

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103 Collections of Ae. albopictus eggs fluctuated greatly throughout the trapping period and corresponded with precip itation events (Fig. 4-4). Although several eggs were collected in late May, no eggs were recovered from any ovitraps on 6 June or 13 June. By 4 July mosquito collections had dramati cally increased, culminating in 890 eggs on 15 Aug. A sharp decrease in egg collections occurred on 22 Aug and again on 5 Sep. Other Mosquito Species Ochlerotatus triseriatus eggs were the most recovered mosquito eggs in this study. A total of 6,275 eggs were recovered from ovitr aps at all locales and heights. However, eggs were not recovered from any ovitrap until 4 Jul (Fig. 4-4), with the greatest number (1,457) recovered on 18 Jul. The majority of Oc. triseriatus eggs (45%) were recove red in ovitraps containing Oak-pine infusions, with only 36.7 and 17.7% of eggs from ovitraps containing Oak infusion or water, respectively. Although no significant difference was detected between infusion-containing treatments, significantly fewer eggs were recovered in ovitraps containing only water (F = 5.88; df = 2, 910; P = 0.0029) (Fig. 4-8). Significantly more Oc. triseriatus eggs were recovered from ovitraps pl aced in sylvatic locales at 6 m (17.90 2.97) than those sylvatic locales at 1 m ( 5.4 1.27), or suburban locales at 6 or 1 m (1.29 0.70, 2.63 1.0 respectively) (F = 30.17; df = 3, 910; P < 0.0001) (Fig. 4-9). While no discernable differences in egg collection was detected between suburban locales at 1 and 6 m heights, si gnificantly more eggs were re covered in ovitraps placed in sylvatic locales at 1 m than those placed at the same height in suburban locales. Significant differences were detected among site s, especially in sylv atic sites, whereby more Oc. triseriatus eggs were collected from traps lo cated at sites furthest from any urbanized development, including highways (F = 5.90; df = 12, 910; P = 0.0063).

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104 The remaining mosquitoes, Or. signifera Cx. quinquefasciatus and Tx. r. rutilus comprised less than nine percent of the total egg capture. Orthopodomyia signifera were only recovered in sylvatic habitats and were not detected until 15 Aug. Although no statistical analyses were pe rformed, a greater number of Or. signifera eggs were recovered from ovitraps placed at 6 m than at 1 m (Table 4-1). Culex quinquefasciatus eggs were only recovered in suburban lo cales and only from 23 May 13 June. The majority of Cx. quinquefaciatus eggs were recovered in ovitr aps containing infusions. A total of 39 Tx. r. rutilus eggs were recovered during July and August with the majority collected at sylvatic sites (61%) a nd at 6 m heights (71%) (Table 4-1). Discussion Ovitraps have been designed to survey a nd control a variety of container-breeding mosquitoes, as well as to ascertain their ovi position behavior (Servi ce 1993). In Chapter Three, a range of laboratory infusions was evaluated for effectiveness in attracting Ae. albopictus In this study two of the Chapter 3 ov iposition attractants were selected and added to ovitraps to ac cess field responses of Ae. albopictus at two heights and in two habitats. Four of the five mosquito species collected, Ae. albopictus Oc. triseriatus Tx. r. rutilus and Or. signifera are species commonly collected as larvae in Florida tree holes and their respective abundance (T able 4-1) reflects that of past studies (Lounibos and Escher 2008). In the current study, Ae. albopictus clearly demonstrated an oviposition preference for suburban locales at 1 m compared to other locations or heights, as 86% of eggs were collected at this height. These resu lts are similar to othe r studies at different geographical locations. In Sri Lanka, Amerasinghe and Alagoda (1984) demonstrated

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105 that Ae. albopictus oviposited more often at ground leve l than at 3.5 and 7.0 m. In New Orleans, Schreiber et al (1988) determined that Ae. albopictus distributed its eggs evenly from the ground up to 3 m. Suburban locales accounted for 87% of the total Ae. albopictus eggs collected in this study. The sharp contrast between the number of eggs collected in suburban and sylvatic locales demonstrates the reported propensity for Ae. albopictus inhabiting suburban areas in the U.S. (OMeara et al. 1993 ). This likely is due to the numerous man-made containers available in suburban lo cales compared to sparse tree holes in sylvatic locales. The abu ndance of hosts, including dogs, cats and humans are also likely factors confining its range, as these are prefer red hosts in suburban areas (Richards et al. 2006). Aedes albopictus is believed to have originat ed from forest-fringe areas of Southeast Asia (Hawley 1988). Adult Ae. albopictus collections from Thailand and Malaysia have shown it to fa vor rural jungle-like areas (Pant et al. 1973, Rudnick and Lim 1986) and suggests a significant habitat shif t for more suburban locations such as the ones in north central Flor ida. The introduction of Aedes albopictus into the U.S. was likely the combination of importing used ti res from Northern Asia and lucky bamboo (Craven et al.1988, Madon et al. 2002). Many exotic insects quickly become esta blished when they overcome geographic barriers or exploit a habitat niche, freque ntly becoming a genetic ally unique population from their origin (Andrewartha and Bi rch 1954). If suburban and sylvatic Ae. albopictus populations in this study are related, future research shoul d encompass the use of DNA

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106 satellites to determine potential genetic diffe rences between populations, as genetic drift, are known to create local differentiation in other U.S. populations (Rai 1986). Burkett et al. (2004) demonstrated that Oak leaf infusions elicited a stronger Ae. albopictus oviposition response over sta ndard hay infusions. Although, Ae. albopictus did not prefer one infusion type over anothe r when evaluated in the laboratory (see Chapter 3), Oak and Oak-pine infusions were strong oviposition attractants in the field. Although both infusions were not statistically different, more Ae. albopictus eggs were recovered in ovitraps containing Oak-pine infusions. With refinement, perhaps differences between these two infusions would become apparent. The significant preference for one treatment in one locale compared to others may be due to other oviposition factors such as light intensity, temp erature and humidity, rather than infusion type. Williams et al. (2006b) demonstrated that placement of sticky ovitraps during the dry or wet season a nd time of day significantly influenced Ae. aegypti collections. It is also possible that the concen tration of Oak trees or lack of pine trees in the sylvatic locales may used in this st udy have interacted with the infusion, causing more Ae. albopictus to be attracted to ovitraps cont aining Oak infusion rather than Oakpine. While the ovitrap cover greatly reduced th e entrance of foreign debris, it did not eliminate all fauna. Occasionally, tree frogs, paper wasps and beetles were found inside the ovitrap, perhaps altering the attractiveness of some traps. As discussed in previous chapters, the addition of lactalbumen may have increased the concentration of bacteria, resulting in a masking effect that reduced the ability of Ae. albopictus to demonstrate an infusion preference. It is known that increased bacterial levels can change the

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107 attractiveness of mosquito oviposition sites (Ponnusam y et al. 2008). Repeated experimentation with natural tree hole wa ter or fermented oak leaf water without lactalbumin are necessary to determine if th is is a natural resp onse or due to other commonly used infusion ingredients. Eggs collected from sylvatic locales de monstrate that infusion-baited ovitraps provide a sensitive method to survey Ae. albopictus even in areas where their numbers are low. Despite competing with natura l containers, such as tree holes, over 600 Ae. albopictus eggs were collected in sylvatic locale s using infusion-baited ovitraps in 2008. In contrast, 90 adults were collected with host-seeking traps with 40 trap nights (48 h each) in the same area during 2007 (see Chap ter 2). Although comparisons between these two trapping method studies must be vi ewed with cautioned as they cannot be statistically compared, they do provide insigh ts for future use of either method as a detection tool for Ae. albopictus especially in areas with low population densities. Ovitraps can provide a low cost alternativ e to conventional surveying methods (adult trapping and tree hole larval collections) for Ae. albopictus when a fast turnaround in sampling is not a neccessity. For example, the cost of many diurnal mosquito traps ranges from $100 to $350. In addition, supplementing these traps with CO2 from compressed cylinders or dry ice adds costs and may increase trap weight constraints making them difficult to use under field conditi ons, especially in th ickly wooded areas. Although ovitraps are affordable, eggs must be reared-out to larvae or adults, as certain egg characteristics can only be viewed with advanced microscopy. This is timeconsuming and may not be as e fficient as collecting adults.

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108 Controlling adult Ae. albopictus is another potential use for ovitraps. While recovering eggs from ovitraps is primaril y a surveillance techni que, adding a sticky adhesive can provide a means of capturing fema le adults visiting the traps (Facchinelli et al. 2007). Furthermore, baiting ovitraps with infusions would increase trap captures (Zhang and Lei 2008). Tree-holes are nutrient rich habitats containing microbial communities and organic substrates such as leaf and fungal de tritus, which are attractive breeding sites for Oc. triseriatus (Kaufman et al. 2008). Furthermor e, the abundance of bacteria from these tree-holes, primarily Flavobacterium spp., is believed to be an important food source for Oc. triseriatus larvae (Xu et al. 2008). The high proportion of Oc. triseriatus eggs recovered in this study is not surp rising, as it is the most abundant mosquito occupying Florida tree-hole communities (L ounibos 1983). Furthermore, its observed preference for sylvatic locales is likely due to the considerable concen tration of tree-holes found in forested areas and its propensity to feed on chipmunks a nd deer (Nasci 1982). Lounibos et al. (2001) re ported similar results in south Florida, where Ae triseriatus was frequently collected in undisturbed habitats compared to Ae albopictus Mosquito oviposition site selection is considered species dependent, whereby the interaction of chemical and physical factors influence spec ific species to oviposit in a particular environment (Bentley and Day 1989). Unlike Ae. albopictus female Oc. triseriatus were likely influenced by a number of non-odor ovipos ition cues. For example, Oc triseriatus is attracted to dark background colors for oviposition sites, specifically wavelengths in the blue ra nge (Williams 1962). Field studies have determined Oc. triseriatus oviposition attraction for dark colors was a stronger factor

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109 than organic matter in selection of ovipositi on sites (Loor and DeFoliart 1969). Ovitrap construction was a likely fact or in attracting gravid Oc. triseriatus even without an infusion. During oviposition site selection, Oc. triseriatus selects black colored containers with horizontal openings, organic matter and rough textured surfaces (Wilton 1968). These physical characteristics are co mmonly found in natural tree-holes. The ovitrap used in this study was selected fo r their mimicry of tree-holes, which likely contributed to our tr apping success. While Oc. triseriatus comprised the majority of mosquito eggs collected, more eggs were recovered from ovitraps containing only water as compared to Ae. albopictus (Figs. 4-5 and 4-7). Sim ilar to this study, Loor and DeFoliart (1969) determined that Oc. triseriatus were most attracted to ovitraps possessing a black interior and containing organic matter. Although a significant difference was detected between water and in fusion-baited ovitraps in the present study, oviposition site selection by Oc. triseriatus may have been based more strongly on light or color than criteria used by Ae. albopictus Ochlerotatus triseriatus clearly demonstrated a pr eference for ovitraps at 6 m (Fig. 4-9). Similarly, adults have shown a strong preference for inhabiting the tree canopies up to 27 m (Novak et al. 1981). In contrast, Scholl and DeFoliart (1977) reported that Oc. triseriatus oviposition had predominantly occurred at ground level. However, their study took place in Wiscons in, where a closely related species, Oc. hendersoni (Cockerell) coexists with Oc. triseriatus Compared to Oc. triseriatus Oc. hendersoni occupies and oviposits in the upper ca nopy of forests, creating a vertical stratification between the speci es (Sinsko and Grimstad 1977). Therefore, it is possible that Oc. triseriatus will prefer to oviposit at incr eased heights in the absence of Oc.

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110 hendersoni Aedes hendersoni possesses similar adult phys ical characteristics as Ae. triseriatus and is known to exist in northern coun ties of Florida (Darsie, Jr. and Morris 2003). This study took precautions by reari ng over 1,300 eggs to adults and did not recover any Ae. hendersoni in our collections. Additionally, no Ae. aegypti were captured, and while small populations may exist in Gainesville (J. Butler pers. comm.), there occurrence is rare in north Florida, w ith populations mainly confined to sourthern Florida (Lounibos et al. 2001). In addition, these two species were not collected in any host-seeking traps in the same area during th e previous year (Obe nauer et al. 2009). Therefore, because all eggs were not rear ed out, the possibility remains that some samples may have contained these species although this woul d be unlikely to significantly impact our conclusions. The presence of Tx. r. rutilus eggs recovered in this study was consistent with their egg-laying habits. Common oviposition sites for Tx. r. rutilus are numerous artificial and natural containers, es pecially tree-holes (Focks 2007). Toxorhynchites spp. also tends to oviposit in well-shaded sylvat ic environments (Steffan and Evenhuis 1981), which may explain the disproportional number of eggs recovered in our sylvatic sites. Furthermore, most Tx r rutilus eggs in this study were co llected from ovitraps at 6 m (Table 4-1). Although a few studies, cited by Steffan and Evenhuis (1981), describe the preference of other Toxorhynchites spp. to oviposit at canopy he ights, there is no record describing this behavior by Tx .r. rutilus Therefore, future studies should determine if Tx. r. rutilus has a propensity for ovipositing at heights greater than 1 m. The fact that Cx. quinquefaciatus rafts were recovered from our ovitraps is not surprising as they have shown a preference fo r small containers, such as jars (OMeara

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111 1989). However, it is interesting that more Culex spp. egg rafts were not recovered in our ovitraps as compared to similar studie s. In addition, the low number of Culex spp. egg rafts recovered in our ovitra ps likely reflects the weak in fusion and the location of ovitraps off the ground. Ovitraps have historically been plac ed at ground level to survey for Ae. aegypti (Service 1993). However, this makes them susceptible to being tipped or damaged by wandering animals. Ants and snails are al so commonly found consuming mosquito eggs that have been deposited on ovitraps (pers. obs.). Furthermore, other Stegomyia mosquitoes such as Ae. aegypti have shown oviposition preference for ovitraps set just above 1 m (Chadee 1991). Therefore, suspended ovitraps provide an alternative method to standard surveillance pract ices, thereby increasing their efficiency. Future studies should be aimed at comparing infusion and l eaf-baited ovitraps at similar heights to elucidate if differences exist. Di eng et al. (2003) also noted that Ae. albopictus prefers to oviposit in larger containers rather than sm aller ones; thus, increa sing the ovitrap size, specifically a larger diameter, may re nder the ovitrap more effective. Further research is need ed to identify the volatiles and compounds acting as oviposition attractants or stimulants, especia lly in our Oak-Pine infusion. Oviposition semiochemicals that act as attractants a nd stimulants for mosquitoes are not only important for surveillance purposes, but may serve as a control measure. By augmenting ovitraps with infusion compounds, their se nsitivity can be in creased and their effectiveness for monitoring a nd detecting populations of co ntainer-breeding mosquitoes, especially invasive species, can be consid erably enhanced (Allan and Kline 1995, Zhang and Lei 2008).

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112 In summary, this study demonstrated th at infusion-baited ovitraps captured significantly more Ae. albopictus eggs than those baited wi th only water. Although no statistical difference was detected among infu sion types, a greater percentage of eggs were recovered in ovitraps cont aining an Oak-pine infusion. Aedes albopictus clearly demonstrated an oviposition preference for ovi traps placed at 1 m compared to 6 m in suburban locales. This height preference was not detected in sylvatic locales, indicating that while more eggs were captured in ovitrap s at 1 m, other environmental variables may have influenced gravid females to select hi gher oviposition sites. Furthermore, drier sylvatic sites may ha ve forced gravid Ae. albopictus to be more opportunistic, seeking higher oviposition sites compared to lower sites in wetter environments.

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113 A B Figure 4-1. Mosquito ovitraps cons tructed from 473 ml steel cans and inverted pie dishes. A detached hook allowed for easy removal of substrate paper (A) and once attached to the cover, (B) the trap could be suspended. Figure 4-2. Ovitrap suspended at 6 m in San Felasco Hammock Pr eserve State Park, Gainesville, Florida.

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114 Figure 4-3. Eggs recovered from ovitrap s under a 10X dissecting microscope: A) Aedes albopictus B) Ochlerotatus triseriatus C) Orthopodomyia signifera D) Toxorhynchites rutilus rutilus Photos taken by L. Buss and P. J. Obenauer. All photos shown were taken with Auto-Montage Pro version 5.02. A B C D

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115 0 5 10 15 20 25 30 3523M ay 30-May 6-J u n 13J un 20-Jun 27-Jun 4Ju l 11 Jul 18-Jul 25-Jul 1A ug 8-A ug 15-Aug 22A ug 29 A u g 5-Sep 12-Sep 19S e p 26-S e p 3-OctDate Mean no. of eggs / week. 0 2 4 6 8 10 12 14 16 18 20cm Precipitation Ae. albopictus Oc. triseriatus Figure. 4-4. Weekly precipita tion and seasonal distribution of Aedes albopictus and Ochlerotatus triseriatus eggs recovered from ovitraps placed in suburban and sylvatic locales in 2008, Gainesville, Florida. Pr ecipitation was measured at the Dept. of Agronomy Forage Research Unit in Gain esville, Florida and retrieved from the Florida Automated Weather Network, University of Florida. A B C D

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116 0 10 20 30 40 50 60 70 SuburbanSylvatic Environment% of Eggs Oak-Pine Oak Water Figure 4-5. Percent of Aedes albopictus eggs recovered from infusion baited-ovitraps placed in four suburban and four sylvatic sites fr om May October 2008 in Gainesville, Florida. n = 20 trapping periods (1 week each).

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117 0 2 4 6 8 10 12InfusionEggs per ovitrap Oak-Pine Oak Water a a b Figure 4-6. Mean (SEM) number of Aedes albopictus eggs recovered from 1 m and 6 m suspended ovitraps containing plant-derive d infusions and a well water control. Traps were placed in suburban and sylvat ic locales between May October 2008 in Gainesville, Florida. Means with the same letter are not significantly different (RyanEinot-Gabriel-Welsh Multiple Rang Test). = 0.05, n = 20 trap periods (1 wk each).

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118 0 5 10 15 20 25LocaleEggs per ovitrap Suburban 1 m Suburban 6 m Sylvatic 1 m Sylvatic 6 ma bb b Figure 4-7. Mean (SEM) number of Aedes albopictus eggs recovered from ovitraps suspended 1 and 6 m in suburban and sylvatic locales in Gainesville, Florida. Traps were operated between May October 2008. M eans with the same letter are not significantly different (Ryan-Einot-Gabriel-Welsh Multiple Rang Test). = 0.05, n = 20 trap periods (1 wk each).

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119 0 2 4 6 8 10 12 InfusionEggs per ovitrap Oak-Pine Oak Watera a b Figure 4-8. Mean (SEM) number of Ochlerotatus triseriatus eggs recovered from 1 m and 6 m suspended ovitraps containing plant-derive d infusions and a well water control. Traps were placed in suburban and sylvat ic locales between May October 2008 in Gainesville, Florida. Means with the same letter are not significantly different (RyanEinot-Gabriel-Welsh Multiple Rang Test). = 0.05, n = 20 trap periods (1 wk each).

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120 0 5 10 15 20 LocaleEggs per ovitrap Suburban 1 m Suburban 6 m Sylvatic 1 m Sylvatic 6 ma b c bc Figure 4-9. Mean (SEM) number of Ochlerotatus triseriatus eggs recovered from ovitraps suspended 1 and 6 m in suburban and sylvatic locales in Gainesville, Florida. Traps were operated between May October 2008. Means with the same letter are not significantly different (Ryan-Einot-Gabriel-Welsh Multiple Rang Test). = 0.05, n = 20 trap periods (1 wk each).

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121Table 4-1. Total abundance of Culicidae eggs captured using ovitraps baited with infu sions or well water and suspended at 1 an d 6 m heights in suburban and sylvatic locales between May October 2008 in Gainesville, Florida. Oak-Pine Oak Control (Water) Suburban Sylvatic Suburban Sylvatic Suburban Sylvatic Species 1 m 6 m 1 m 6 m 1 m 6 m 1 m 6 m 1 m 6 m 1 m 6 m Aedes albopictus 2549 133 153 68 1917 191 213 169430 68 30 19 Ochlerotatus triseriatus 246 288 686 1635 207 14 429 1656177 0 147 790 Orthopodomyia signifera 0 0 7 506 0 0 19 00 0 8 127 Culex quinquefasciatus 258 12 0 0 56 0 0 014 8 0 0 Toxorhynchites rutilus 2 1 7 5 2 12 4 71 2 2 1 Total 3055 434 853 2214 2182 217 665 1832622 78 187 937 Table 4-2. Total abundance of adult Collum bola and immature Diptera collected using ovitraps baited with infusions or well wat er and suspended at 1 and 6 m heights in s uburban and sylvatic locales between May October 2008 in Gainesville, Florida. Oak-Pine Oak Control (Water) Suburban Sylvatic Suburban Sylvatic Suburban Sylvatic Family 1 m 6 m 1 m 6 m 1 m 6 m 1 m 6 m 1 m 6 m 1 m 6 m Collumbola 105 10 8 76 8 18 14 6 12 8 2 4 Corotherillidae 41 0 41 0 0 0 95 0 0 0 0 0 Phoridae >10800 >2700 > 2600 >2200 >3700 >2900 >1700 >2400 7 28 32 44 Psychodidae 108 21 11 19 33 40 32 7 4 0 0 1

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122 CHAPTER 5 EFFICACY OF FOUR SURVEILLANCE TECHNIQUES TO DETECT AND MONITOR AEDES ALBOPICTUS IN NORTH CENTRAL FLORID A SUBURBAN AND SYLVATIC HABITATS Introduction A variety of methods and devices have been developed to survey mosquito populations and to adequately sample their numbers in different environments. Holck and Meek (1991) describe two forms for collecting adult mosquitoes, a pa ssive form, whereby mosquitoes are collected using stationary light, carbon dioxi de-baited traps or non-attractant devices, and an active form, whereby the investigator physically searched a nd captured mosquitoes by mechanical means. Mosquito diversity depends largely on ecologica l factors such as breeding sites, altitude, vegetative fauna and habitat types, such as urba n and sylvatic areas; all of which are important considerations when sampling mosquitoes (Me ndoza et al. 2008). However, many mosquitoes utilize a range of diverse habitats, especia lly those that border on a habitat interface. Aedes albopictus (Skuse) is believed to originate from Southeast Asia, occupying urban, suburban, rural and forest-edged environments (Hawley 1988). Similarly, adult populations in Florida have been detected in suburban and sylv atic habitats (O Meara et al. 1993). Different collecting techniques are used to sample mosquitoes depending on their biology and developmental state. Service (1993) descri bes a number of sampling methods used to survey adult mosquitoes including: light, colored patterns and CO2-baited traps for host-seeking mosquitoes; resting boxes and back pack aspirations for resting mosquitoes; and gravid traps baited with attractants for ovipositing mosquitoes. For example, mosquitoes aloft are generally host-seeking and can be collected using traps baited with an asso rtment of attractants such as carbon dioxide and lactic acid (L ehane 2005). Once a female mosquito has fed, it shifts its searching behavior towards locating suitabl e resting sites for blood digestion and egg

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123 development. Upon egg maturing, gravid mosquitoes become attracted to an array of chemical and visual oviposition cues (Bentley and Day 1989) Therefore, measured interpretation of adult Ae. albopictus captures should be made depending on the sampling method used in each habitat. Mosquito light traps effectively attract most nocturnal and crepuscular mosquitoes, but are ineffective against most diurnal species, such as Ae. aegypti L and Ae. albopictus (Service 1993). Therefore, adult surveillance a nd population estimates of most Stegomyia have relied on ovitraps, visual attractants, human-landing counts, sticky traps and aspira tor collections (Focks 2004). Human-landing counts have been shown to be a significantly more effective method of surveying Ae. aegypti than traps specifically designed fo r diurnal mosquitoes (Jones et al. 2003, Schoeler et al. 2004). Numerous mosquito abatem ent districts have long used this surveillance technique to quickly ascertain mosquito abunda nce, species composition, and effectiveness of adulticides (Schmidt 1989). In addition, this tech nique is especially important when determining infection rates and vector capacity of a part icular mosquito species (Service 1993). Although this is a proven, sensitive method to survey Ae. aegypti and Ae. albopictus many view the technique to be labor-intensive, expensive and potentially dangerous for the collector, especially in endemic disease areas (Focks 2004). Furt hermore, human attractiveness and collection efficiency vary among individuals making it difficult to develop repeatable standards for this technique. To attract host-seeking Ae. albopictus a variety of traps in cluding the BG-Sentinel (Meeraus et al. 2008), Omni-directional FayPrince (Jensen et al. 1994), and Mosquito Magnet-X have incorpor ated contrasting colors, patterns an d semiochemical attractants (Hoel 2005). Comparisons between these traps have show n that the BG-Sentinel trap is an effective

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124 trap at collecting Ae. albopictus in north central Florida suburban and sylvatic habitats (Chapter 2, Obenauer et al. 2009). Gravid traps have primarily b een used to collect ovipositing Culex mosquitoes (Reiter 1983). However, Ae. albopictus have also been recovered with baited plant infusions (Burkett et al. 2004, Burkett-Cadena and Mullen 2007). Furtherm ore, gravid traps are an equally effective method of collecting Ae. albopictus as the commonly used CO2-baited, Centers for Disease Control and Prevention (CDC) style light traps (Burke tt et al. 2004). Sweep nets and aspirators have long been used to collect resting adult mosquitoes (Service 1993). However, Holck and Meek (1991) determined that sweep nets provided a faster and more consistent method to sample resting mosquitoes as compared to aspirators. Nonetheless, both methods provide information on mosquito dispersal rates, diurnal resting sites, adult emergence and distribution (Mullen 1971). Although past studies document th e successful use of the CDC backpack aspirator to collect Ae. aegypti within indoor environments (Clark et al. 1994, Schoeler et al. 2004), few studies exist de monstrating its use in sampling Ae. albopictus in an outdoor environment. Aedes albopictus exhibits exophilic behavior and rarely seeks hosts inside dwellings (pers. obs.). In a ddition, it prefers urban vegetate d areas, as reduced numbers are collected in areas clea red of foliage (pers. obs.). Ponl awat and Harrington (2005) successfully collected Ae. albopictus from vegetation around the perimeter of homes in Thailand using a large custom made aspirator. Their study also dem onstrated the use of aspirators for successful collection of blood-fed Ae. albopictus a task for which few alternatives exist. Results from Chapters 2 and 4 of this dissertation document that although Ae. albopictus were found in sylvatic habitats significantly greater numbers were collected from suburban habitats. Our objective in this study was to ev aluate the efficacy of the BG-Sentinel trap, a

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125 plant derived infusion-baited gravid trap, human -landing counts and vegetation aspiration to detect adult populations of Ae. albopictus in suburban and sylvatic habitats. Currently, no published study exists comparing these common ly used surveillance methods in capturing Ae. albopictus Sampling adult mosquito populations in these two Florida habitats may assist mosquito control agencies in asse ssing mosquito abundance in a partic ular area of the state. In addition, information gained from this study may be used to detect, prevent or reduce the likelihood of Ae. albopictus from becoming established in other countries. Materials and Methods Site Selection This study was conducted in suburban and sylv atic locales from May to September 2008. All sites in this study were independent from thos e described in Chapters 2 and 4. Four suburban sites were located at the following areas : N 29 36.957, W 082 25.780; N 29 40.383, W 082 22.656; N 29 40.322, W 082 23.630; N 29 42.25, W 082 23.658. S uburban locales were selected based on the following criteria: 1) re sidents that have had frequent complaints of mosquitoes biting during the day; 2) site s had thickly-wooded lo ts that surrounding the residential property; 3) sites had previously support ed populations of Ae. albopictus and; 4) sites were secured to prevent against tr ap theft. Suburban sites were separated by at least 3.22 km (2 miles) and contained a mixture of shrubs and trees, namely azalea ( Rhondendron spp.), oleander ( Nerium oleander ), Indian hawthorn ( Rahphiolepis indica ), live oak ( Quercus virginiaina P. Mill), water oak ( Quercus nigra L.), laurel oak ( Quercus laurifolia Michx.) and longleaf pine ( Pinus palustris P. Mill) (Fig. 5-1a). One site contained large numbers of tank bromeliads, namely Aechmea fasciata (Lindley) Baker, Neoregelia spectabilis (Moore) and Bilbergia spp. (Fig. 5-1b).

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126 Four sylvatic sites (N 29 43.267, W 82 26.725; N 29 44.669, W 82 28.099; N 29 44.287, W 82 27.354; N 29 43.848, W 82 27.297) (Fig.5-2) were dispersed throughout San Felasco Hammock Preserve State Park (SFMPSP), Alachua Co., FL. A research and collecting permit (# 02130742) was granted to the author by the Florida Department of Environmental Protection to collect mosquitoes within the park. Surveillance Methods The BG-Sentinel (BG) trap, (BioGents GmbH Regensburg, Germany) was selected for this study based on its performance in previous experiments (Chapter 2, Obenauer et al. 2009). The white, collapsible bucket-shaped trap has a mesh-like covered opening and contains a black plastic tube (12 x 12 cm) that is inserted at the top of the trap, which empties into a catch bag (Fig. 5-3). Mosquitoes are draw n into the trap by a 12-V DC fan. To lure diurnal mosquitoes, white and black colors are used as visual cues in combination with a synthetic bait that mimics skin secretions (Krckel et al. 2006). The synthetic bait, Agrisense BG-Mesh Lure (BioGents GmbH, Regensburg, Germany), consists of 2 m of coiled 4.75 mm internal diameter silicon tubing (containing 15 mL of lactic acid), 50 cm of 0.4-mm internal diameter high-density polyethylene tubing (2 mL of capro ic acid), and a slow release am monia acrylic fibrous tablet as described in Williams et al. (2006c). The trap design, in combination with the lure, creates ascending currents that mimic similar convectio n currents created by the human body (Krckel et al. 2006). Carbon dioxide was supplied from a 9 kg (20 lb) compressed gas cylinder with a flow rate of 500 mL/min using 6.4 mm diameter bl ack plastic tubing (Clarke Mosquito Control, Roselle, IL). The tubing was placed inside the trap with the opening placed near the lure pocket and secured using a white Velcro strap loca ted in the housing unit. A Gilmont Accucal flowmeter (Gilmont Instrument Company, Barringt on, IL) was used at every rotation to verify the accuracy of CO2 discharge.

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127 The BG trap was originally designed to trap Ae. aegypti inside or close to protected residential sites (D. Kline pers. comm.). However, this study required that these traps be kept in unprotected, outdoor environments over long dura tions. Therefore, to prevent rain from damaging electrical circuits and motor component s, an aluminum pan (35.56 cm x 1.90 cm) was attached 30.48 cm above the trap entrance with 2 nylon cords and secured to the handles of the trap. The BG trap was suspended 1 m from the ground using a shepherds hook. The CDC Gravid trap model 1712 (John Hock, Gain esville, FL) (Fig. 5-4) was used to lure gravid Ae. albopictus As described in Reiter (1983), gravid mosquitoes are attracted to the trap, which contains an oviposition medium in the pan. Moquitoes are collected in the upward current created by a fan located inside th e black PVC tubing (30.5 cm in le ngth x 7.62 cm wide). This trap utilizes a 6 V, 12 amperehour (A-h) battery (Battery Whol esale Distributors, Georgetown, TX) to power the motor. To maximize visual attractiveness, green Rubbermaid 439 pans (22 cm wide X 34 cm long X 17 cm deep) (Rubbermaid Commercial Products Winchester, VA), were spray-painted wi th black gloss Krylon Fusion paint (Krylon Products Group, Cleveland, OH). To remove any paint odors or contaminants, trap pans were preconditioned and aged by filling them with well water and letting them sit for 2 wk in a semi-shaded environment prior to the study (Burkett et al. 2004, Alla n et al. 2005). To prevent rains from flooding the trap, 0.60 cm holes were drilled into either side of the trap pan, approximately 6 cm from the bottom. Traps were placed on the ground. The infusion used in the gravid traps was developed by collecting fallen dry leaves of water oak and longleaf pine needle s from the grounds at the Univer sity of Florida, Gainesville, FL. Special attention was taken to ensure all leaves and needle s were free of foreign organic matter. The infusion was prepared by fermenting 60 g of oak leaves, 60 g of pine needles, 7 g

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128 brewers yeast (MP Biomedicals, LLC, Solon, Ohio), and 7 g lactalbumin (Sigma-Aldrich, St. Louis, MO) in 12 liters of well water and held at ambient temperature between (25-27 C) for 10 days in a sealed plastic bucket (Allan and K line 1995). After 10 days, the infusion was passed through gauze netting to remove particulate matt er and 1.5 L of infusion was transferred to 2 L plastic bottles and frozen until needed. To each 1.5 L infusion, 0.5 L of deionized water was added, generating a 75% infusion concentration. A large aspirator (Fig. 5-5a), originally desi gned and built by David Evans to sample saltmarsh mosquitoes in the Everglades (G.F. O Meara pers. comm.) and later modified by Laura Harrington (Ponlawat and Harringt on 2005), was used to collect resting mosquitoes. The aspirator was powered by a 12 V, 12 A-h battery, which enables a large fan to funnel mosquitoes through the aspirator and into a mesh catch bag. When in use, the battery was secured in a backpack and carried by the operator (Fig. 5-5b). Habitat at each site was sampled continuously for 10 min, paying special attention to tree-holes tree stumps, vegetation, man-made containers and other ground debris. The catch bag was replaced at every site to prevent intermixing of mosquitoes. Humanlanding-counts were performed by coll ecting mosquitoes landing on the author using a mechanical flashlight aspirator (Hau sherrs Machine Works, Toms River, NJ). Mosquitoes were aspirated into a new collecting tube at each site. Collections were conducted for 5 min at two locations within each site to eliminate bias. Before collecting mosquitoes, the surrounding vegetation was stirred up while the author exhaled vigorously as prescribed by Schmidt (1989) and Slaff et al. (1996). To attr act mosquitoes, the author sat in a collapsible chair, rolled-up his pant legs approximately 5 cm above the knee and lowered his socks below the ankles, as these regions are most attractive to Ae. albopictus (Robertson and Hu 1935, Shirai

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129 et al. 2002). With the exception of the hands, face and portions of the legs and ankles, all other extremities were covered by a Bugout mosquito jacket (Rattlers Brand, Inc, Osceola, Iowa) (Fig.5-6). Approval to collect mosquitoes from the author was granted by the University of Florida Health Science Center Institutional Review Board Agreement IRB # 36-2007 (Appendix D). Surveillance and Collection Scheme Surveillance was conducted twice weekly between 0800 and 1100, for two consecutive weeks at which time traps and aspiration samp ling rotated to the ne xt locale (suburban or sylvatic). The absence of surveying for two weeks between the two e nvironments was designed to mitigate any collection impact on the mosquito population. At each site, four surveillance methods were utilized in the following order at the start of the 48 hr trap operation period: vegetation aspirations, human-landing counts and pl acement of the gravid and BG traps. Traps were placed underneath trees in shaded areas and were set at least 20 m from each other and at least 3 m from any dwelling. Traps were opera ted for 48 h (1 trapping period) afterwhich mosquitoes were collected. Adhesive tape was att ached at the base of th e gravid trap catch bag and at the top of shepherd hooks to prevent ants from consuming captured mosquitoes. Gravid trap infusion and adhesi ve tape were replaced at each trapping period. Surveillance occurred from 14 May 07 June, 11 June 05 July, 09 July 02 Aug., 06 Aug. 30 Aug. and 03 Sep. 27 Sep. for a total of 5 trials and 20 trap ping periods (40 trap nights) per locale. Temperature and precipitation were measured at the Department of Agronomy Forage Research Unit in Gainesville, Florida with data retrieved from the Florida Automated Weather Network, University of Florida. Collected mo squitoes were immobilized at -20 C for 5 min, dispensed into 1.5 mL plastic Fisherbrand Snap-Cap microcentrifuge tu bes (ThermoFisher

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130 Scientific, Sawanee, GA) and frozen (-20 C) for la ter identification to spec ies using the keys of Darsie and Morris (2003). Statistical Analysis Differences between Ae. albopictus collection techniques were evaluated using a randomized complete block design to test for differences within and between suburban and sylvatic habitats. All data were analyzed in th ree ways and with combined captures of male and female mosquitoes as the response variable. Data were first analyzed by a presence/absence test to determine the most sensitive collection technique; that technique that documented the collection of at least one Ae. albopictus Sample periods in which no Ae. albopictus were collected at a site, by any method, we re excluded from this analysis. The second analysis compared collection me thod efficacy over time. Because landing counts and aspirations were conducted for 10 mi n and traps were run for 48 hrs, a time conversion was applied. To equilibrate trap co llections to a 10-minute period, mosquito captures from traps were divided by the value 144 min/tr ap period. This value was determined by the following formula: TC10 = TP DLH DAY. Where TC10 = estimated trap capture in 10minute exposure equivilent. The variable TP = 6, to reflect the six 10 min time periods in one hour. DLH = 12 and represents the 12 da ylight hours of diurnal activity for Aedes albopictus which usually lasts from 0630 to 1830 (Ho et al. 1973). DAY = 2 which encompasses the two trapping days in a collection period. Data from the presence/absence test and me thod efficacy comparison were examined using an analysis of variance (ANOVA) model to detect differences betw een the fixed effects, locale (suburban or sylvatic), collection method and tria l. The model also in cluded the locale and collection method interaction. Wh ere interactions were found to be significant, we used the interaction error term to calculate p-values. St atistical analyses were conducted using PROC

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131 GLM (SAS Institute 2006). Mu ltiple means comparisons were made with the Ryan-EinotGabriel-Welsh (REGW) multiple range test ( =0.05). A third analyses used a paired t-test ( =0.05) to determine diffe rences in the collection efficiency between the 2-day BG and gravid traps and a separate analysis between the short-term landing counts and aspirator collections. Data were analyzed using this method because collection methods contained different length ex posures. These analyses were conducted with the six most commonly captured mosquitoes. Only sites that contained paired samples within the collections were analyzed. Results A total of 73,849 mosquitoes, representing 29 spec ies from 12 genera were captured (Table 5-1). The following six species composed 93.7% of the total collection and were subsequently analyzed: Ae. albopictus Ae. vexans (Meigen), Culex nigripalpus Say, Cx. quinquefasciatus Say, Ochlerotatus infirmatus (Dyar and Knab) and Psorophora ferox (von Humboldt) (Fig. 5-7). More mosquito species were collected in suburban locales than in sylvatic locales with 25 and 22 species, respectively. The number of mosquito sp ecies collected in each locale was dependent on the surveillance method used. The BG trap collected 25 mosquito species, compared to 22, 15, and 10 using the aspirator, gravid traps and landing-counts, respectivel y (Table 5-1). Data from two trapping periods (18 Aug 23 Aug) we re lost due to flooding by Tropical Storm Fay. Furthermore, landing counts and vegetative aspira tions were not conducted on days with periods of heavy rain. Lost data from these days were treated as missing values. Aedes albopictus A total of 5,066 Ae. albopictus were collected with females co mprising 68% of the capture. Aedes albopictus was the fifth most common mosquito collected and represented 6.9% of the entire mosquito capture (Fig. 57). Those collected in suburban locales using all four sampling

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132 tools represented over 97% of the total Ae. albopictus captured with a daily mean of 15.8 2.27 in suburban locales compared to 0.48 0.07 in sylv atic locales. One suburban site accounted for 47% (2,368) of all Ae. albopictus captured. Aedes albopictus were collected in approximately equal numbers from the four sylvatic sites, ranging from 22 43 tota l specimens for the 5 trapping periods. Aedes albopictus was the second most commonly collected mosquito from gravid traps and landing-counts (Table 5-1) The BG trap accounted for over 85% of all Ae. albopictus captured and was significantly more effective at detecting the presence of Ae. albopictus compared to the other three techniques (F = 40.04; df = 3, 492; P <0.0001) (Fig. 5-8). Locale was highly significan t (F = 98.09; df = 1, 492; P <0.0001) with ne arly twice as many Ae. albopictus detected in suburban locales (0.62 0.03) compared to sylvatic locales (0.31 0.03). Trial (time of yea r) was also significant with more Ae. albopictus captured during trials three, four and five compared to the two early season trials (F = 40.31; df = 4, 492; P <0.0001) (Fig. 5-9). All surveillance methods performed similarly in detecting a large Ae. albopictus population increase in mid-July, a peak in early August and a decrease in late September (Fig. 5-9). Following conversion to 10 min in tervals, significantly more Ae. albopictus were collected with landing-counts than other methods (F = 15.22; df = 3, 496; P <0.0001) (Fig. 5-10). In addition, an interaction effect was detected between sampling method and locale. Within the suburban locale, significantly more Ae. albopictus were captured using landing counts (4.14 0.73) as compared to the aspi rator (2.32 0.45), BG (0.38 0.46) and gravid trap methods (0.12 0.01) (F = 24.43; df = 3, 302; P <0.0001). However, in the sylvatic environment no differences were observed between samp ling methods (Fig. 5-10).

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133 In suburban locales significantly more Ae. albopictus were collected with BG traps (54.8 7.79) as compared to gravid (2.01 0.34) traps (t = -6.920; df = 74; P 0.0001); while more mosquitoes were collected using landing-counts (4.00 0.71) than the as pirator (2.3 0.45) (t = -3.17; df = 74; P = 0.0022). No differences were dete cted between sampling methods at the sylvatic locale (Table 5-3). Other Mosquito Species Ochlerotatus infirmatus was the most abundant species collected, comprising 36% of all mosquito specimens. The BG trap collected more Oc. infirmatus (202.8 58.8) than all other techniques combined (Tables 5-2 and 5-3). The aspirator collected significantly more Oc. infirmatus compared to landing-counts in suburb an and sylvatic locales (Table 5-3). Aedes vexans was collected with every sampling technique except gravid traps (Tables 5-2, 5-3). Significantly more Ae vexans were collected with the aspirator than with landing-counts in suburban (t = 4.92; df = 74; P 0.0001) and sylvatic (t = 3.619; df = 60; P 0.0006) locales. Over 96% of Cx nigripalpus were collected with the BG trap. The aspirator was significantly more effective than the landing-counts at sampling th eir population in suburban (t = 1.974; df = 74; P 0.0521) and sylvatic locales (t = 2.498; df = 60 P 0.0153). Culex quinquefasciatus was the dominant species co llected in gravid traps, representing more than 80% of the total collection. Furthermore, 99% of Cx. quinquefasciatus were collected in suburban locales. Gravid traps (32.3 4.50) placed in suburban areas captured significantly greater numbers of Cx. quinquefasciatus compared to the BG trap (1 5.0 2.59) (t = -4.756; df = 74; P 0.0001) (Table 5-2). The aspirator collect ed significantly (t = 2.922; df = 74; P 0.0046) more Cx. quinquefasciatus in suburban locales as co mpared to landing-counts. Psorphora ferox was the third most commonly colle cted mosquito species. Landingcounts and aspirator collections were equally eff ective in both locales. However, significantly

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134 greater numbers of Ps. ferox were collected in BG traps (97.8 40.3) (103.9 30.3) compared to gravid (0.03 0.03) (0.00 0.00) traps in suburba n and sylvatic locales, respectively (Table 52). Mosquito distribution patterns in this study were similar to those observed in previous studies (Chapter 2, Oben auer et al. 2009). Ochlerotatus triseriatus (Say) was collected 79% of the time in sylvatic locales, while Wy. mitchellii was only collected at tw o suburban sites, with one site accounting for 55% (542) of the total capture. Ochlerotatus fulvus pallens (Ross), Oc. sollicitans (Walkeri), Uranotaenia sapphirina (Osten Sacken) and Cx. coronator (Dyar and Knab) were additional species collected that were not trapped in previous studies (Chapter 2). Uranotaenia sapphirina was recovered at one sylvatic si te and was only collected using the aspirator. Culex coronator commonly found throughout Mexi co and Central and South America, has moved further north and has recently been added as a new Florida species record (Smith et al. 2006). Discussion The rapid introduction of Ae. albopictus to many countries within the last 20 years has driven considerable research efforts to devel op effective surveillance tools for detecting its presence in order to halt its spread. Aedes albopictus is a potential health threat even to countries that normally do not have the endemic pathogens it is capable of vectori ng. This was evident when it was recently incriminated as the prim ary vector responsible for chikungunya (CHIK) outbreaks in Italy (Rezza et al. 2007). Aedes albopictus is a versatile mosquito, feeding on a range of hosts, ovipositing in numerous types of natural and man-made c ontainers and occupying a number of diverse habitats (Hawley 1988). Th erefore, its behavior and biology may vary dependent on habitat type, complicating trad itional collection methods to survey adult populations.

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135 Many mosquito trapping studi es compare and evaluate tr aps based on the number of collected mosquitoes. However, the majority of traps used by mosquito abatement districts are designed to target host-seeking mosquitoes, potentially neglecting other mosquitoes. This study utilized four methods to sample populations of Ae. albopictus based on their adult life history. The BG trap and landing counts targeted host-seeking Ae. albopictus while gravid traps collected females searching for oviposition si tes. Aspirator collections of resting Ae. albopictus provide, perhaps, the most unbiased sampling me thod as recently eclosed, host-seeking, bloodfed and gravid mosquitoes can be captured. Therefore, comparisons between collection techniques should be approached with caution, as the aim of this study was to detect the presence of Ae. albopictus and rate the techniques based on th eir performance in different habitats. Results from this study demonstrated that surveillance techniques used to sample populations of Ae. albopictus performed differently depending on habitat type. For example, the BG trap was no more effective at collecting Ae. albopictus than gravid traps in sylvatic locales, while its performance drastically increased on ce placed in suburban locales. Landing-counts were specific to collecting da y-time biting mosquitoes, as, Ae. albopictus Oc. infirmatus Ps. ferox and Wyeomyia mitchellii (Theobald) comprised over 97% of the catch (Table 5-1). Low Ae. albopictus populations in sylvatic locales was the like ly reason for this variation. In addition, gravid traps placed in sylvatic habita ts offered prime oviposition targets for Ae. albopictus whereas, availability of oviposit ion sites in suburban backyards was much greater. Although sylvatic locales contained a number of tree-holes, many rema ined dry throughout the summer when this study was conducted. Therefore, subur ban habitats likely provided more abundant and stable breeding areas for Ae. albopictus resulting in decreased gravid trap captures relative to the adult mosquito population size.

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136 Aedes albopictus were likely attracted to the visual and olfactory cues presented by gravid traps used in this study. Pans were shiny and black, a known color to be attractive for ovipositing Ae. albopictus (Yap et al. 1995). In addition, the oak-pine infusion may have increased the attraction of gravid females. Oakpine infusion has been shown to be oviposition attractant and stimulant (Chapter 3 and 4). Sim ilarly, Burkett et al. ( 2004) demonstrated that oak-baited infusions used with black gravid traps were attractive to Ae. albopictus Results from this Ae. albopictus collection comparison are similar to studies with Ae. aegypti In Thailand, researchers dete rmined that landing-counts were still more effective at collecting adult Ae. aegypti when compared to Omni-Direc tional Fay-Prince, sticky or CDC Wilton traps (Jones et al. 2003). Sc hoeler et al. (2004) also determin ed that no trap tested was an acceptable alternative to backp ack aspiration or human landing collections. Of the total mosquitoes collected in their study, 73% were co llected via backpack as pirator, followed by 23% with human-landing methods. In Australia, simila r results were observed when the BG trap was compared to 10 min samplings conducted with a CDC backpack aspirator (Williams et al. 2006a). Although their study determined the BG trap collected significantly more female Ae. aegypti compared to the CDC aspirator, both devices proved equally effectiv e at collecting when males were included in the total. However, unlike Ae. aegypti Ae. albopictus is an exophilic mosquito, preferring to feed a nd rest outside of dwellings (Hawley 1988), potentially making collections more challenging due to various ou tdoor environmental influences. This study demonstrated that Ae. albopictus can be successfully collected w ith an aspirator in suburban and sylvatic locals. Of a ll collection methods, the aspirator wa s the second most effective sampling technique during 10 min intervals (Fig. 5-10).

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137 Although no significant differences in Ae. albopictus collections were found between the BG trap and the other three techni ques when placed in sylvatic locales, the BG trap collected the greatest number of Ae. albopictus when placed in environmen ts with a high density of Ae. albopictus (Table 5-1). The effectivene ss of the BG trap at collecting Ae. albopictus is further supported by my results as presented in Chapter 2 of this dissertation. Culex quinquefasciatus were the most frequently caught mos quito in gravid traps. The fact that more Cx. quinquefasciatus were collected with gravid tr aps (32.32 4.50) compared to BG traps (15.0 2.59) demonstrates that the gravid trap is a superior survey tool for this species. Similar results were reported by Kline et al. (20 06). Studies comparing gr avid traps with other host-seeking traps report similar re sults. Burkett et al. (2004) de monstrated gravid traps baited with an oak infusion caught significantly more Cx. quinquefasciatus than the Mosquito Magnet, CDC trap, and miniature blacklight traps. While hay is a common ingredient used to create infusions to attract Culex spp. (Reiter et al. 1991), others have used leaves from red oak ( Quercus rubura ) (Burkett et al. 2004), live oak and laurel oak (Allan et al. 2005). Results from this study also demonstrate that water oak leaves and longleaf pine needle s provide an effective alternative to the common hay infusion. The abundance of the floodwater mosquitoes, Ae. vexans, Oc. infirmatus and Ps. ferox that were captured during this study were likely due to heavy spring rains in the Gainesville area between February and April 2008. During this tim e, the area received a little over 22 cm of precipitation. Many Gainesville residents compla ined of large numbers of biting mosquitoes by early April (Kelly Etherson, pe rs. comm.). These mosquitoes would have oviposited by midApril, setting the stage for a nother large emergence by June.

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138 Each surveillance technique evaluated in this study had advantages and disadvantages in sampling Ae. albopictus The BG trap was the most effective tool at capturing a range of mosquito species, including larg e numbers of male and female Ae. albopictus Furthermore, unlike landing counts and aspirator co llections, which can vary between operators, the BG trap is objective and could serve as a standard (Williams et al. 2006a). However, traps were susceptible to periodic mechanical malfunctions and required batteries, lures and CO2 canisters. The current cost of a BG trap is approximately $290.00 (Bioquip, Rancho Dominguez, CA) and does not include daily ancillary costs. The CDC gravid trap is easy to operate, can be transported to the fi eld and only requires a 6 V battery. However, the trap collected only 15 mosquito species compared to 25 and 22 with the BG and aspirator, respectiv ely. In addition, it was suscep tible to periodic mechanical malfunctions and was occasionally vandalized, presumably by ground-dwelling mammals. Furthermore, preparation of large volumes of infusion required throughout the trapping season created additional weight and storage issues. The aspirator was quick and effective, aspira ting 22 mosquito speci es from brush, treeholes and various other containers. The aspira tor may provide an important tool for future studies that investigate host preference, as many of the Ae. albopictus collected by this method had recently blood-fed (pers. obs.). However, the aspirator was cumbersome to operate, particularly in thickly-wooded areas. Budge t constraints may be an additional factor. Depending on the material used, a custom-made aspira tor, such as the one used in this study, can costs as much as $800.00. Occasional mechanical problems and a 12 V battery were additional drawbacks.

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139 Human landing-counts provided a quick and e ffective weekly assessment of the major biting species and were the most effective method for sampling Ae. albopictus within a 10 min period. Compared to the cost of other techni ques, landing-counts were the most economical. However, landing-counts provided the fe west mosquito species (n = 10). This study demonstrated that the BG trap was an effective surveillanc e device in detecting Ae. albopictus but success was dependent upon the length of operation (Fig. 5-9). The BG trap used white and black colors for visual attracti on in combination with a synthetic bait that mimicked skin secretions (Krckel et al. 2006). The addition of CO2 in this study was designed to maximize its effectiveness. However, despite these added host-seeking cues, it was still not as effective as landing counts duri ng a 10 min period (Fig. 5-10). Techniques used to capture Ae. albopictus in this study provide potentially different applications for research. For example, in the present study, nulliparous females were consistently collected using the BG trap and landing-c ounts (data not shown). However, to test for infected mosquitoes or to conduct a blood meal analysis, vegetative as pirations and, to some extent, gravid traps, would likely provide the most effective technique as they target previously blood-fed females. Results of this study demonstrate that selecting a sampling device to survey Ae. albopictus populations should not only be based on the aim of a study, but also the habitat settings as well. Sampling mosquito field populations are known to produce bias among collection techniques (Service 1977). When habitats we re not considered, the BG trap was significantly more effective at detecting Ae. albopictus (79% of total colletions) than ot her methods (Fig. 5-8). However, while significant differences we re detected among surveillance methods in suburban habitats, neither technique was more effective at collecting Ae. albopictus in the sylvatic habitat (Table 5-

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140 2, 5-3). Similarly, aspirator co llections demonstrated that Ae. albopictus could be detected with the same effectiveness as landing counts when habitat was not a consid eration (Fig 5-8). Furthermore, the aim of aspirato r collections was to collect res ting mosquitoes, one of the more challenging and time-consuming pr ocesses due to mosquito di spersal and preferences for specific habitats (Service 1977). In addition, we aspirated for mosquitoes from various containers found within sites (i.e tree-holes, bromeliads, vegeta tion, man-made containers etc.) and did not standardize th ese resting sites based on type or dimensions. Culex and Anopheles species are known to select their resting site s based on size and shape (Burkett-Cadena et al. 2008). Therefore, targ eted surveying of Ae. albopictus resting sites would help elucidate preferences for types of resti ng containers within habitats. This study also demonstrated that collection method efficiency is often based on several variables. For instance, if su rveillance was required to be conducted in a short time period, landing-counts were the most time-effi cient surveillance method for detecting Ae. albopictus (Fig. 5-10). However, this was strictly base d on overall captures and did not compensate for habitat differences. These results may affect the manner in which future Ae. albopictus surveillance is conducted, especially in areas where it has been recently introduced. Based on our results, the BG trap would likely be the choi ce for detecting the presence of Ae. albopictus in suburban habitats. However, if Ae. albopictus were introduced into sylva tic areas, a multi-facetted approach may be required, employing all availa ble collecting techniqu es, including infusionbaited ovitraps. The introduction of Ae. albopictus into other countries is likely to take place along sea ports (Tatem et al. 2006) with disper sal into less populated areas. Recently, in response to the CHIK outbreak in Italy, the U. S. Navy employed several BG traps around bases

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141 as part of a surveillance plan to monitor Ae. albopictus population levels (C. Stoops, person. comm.). However, this plan is designed to track populations over a long period. If a scenario required a rapid mosquito assessment, landing-co unts would be the most time-efficient method. While recent advances in mosquito attractants have been made, no attractant has worked as effectively as human baits for anthropophagic mosquito surveillance (Service 1993). Evaluating new host attractants and incorpor ating them into traps targeting diurnal mosquitoes should continue in order to increase their sensitivity and captures, an d to eliminate unnecessary human landing-counts. Future survei llance comparison studies of adu lt mosquito populations should be conducted over an extended period of time to comp ensate for seasonal changes, as variation in mosquito behavior, sampling techniques and en vironmental conditions are known to influence sampling (Bidlingmayer 1985).

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142 A B Figure 5-1. Two suburban residential sites used for Aedes albopictus collection in Gainesville, Florida. Most backyards contained azal eas and pine trees (A), while some (B) contained various ornamentals such as bamboo and numerous tank bromeliads. Figure 5-2. A typical sylv atic site used for Aedes albopictus collection in San Felasco Hammock Preserve State Park, Gainesville, Florida.

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143 Figure 5-3. A BG-Sentinel trap used to lure and collect mosquitoes. Figure 5-4. A CDC Gravid trap model 1712 cont aining oak-pine infusion and placed on the forest floor in San Felasco Hammock Pres erve State Park, Gainesville, Florida.

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144 A B Figure 5-5. A large mosquito aspirator displaying the inside catch bag (A) was operated by the author to collect resting mosquitoes fr om sylvatic and suburban locales (B). Figure 5-6. The author performing mosquito land ing-counts using a hand-held aspirator in San Felasco Hammock Preserve State Park, Gainesville, Florida.

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145 Culex nigripalpus 17% Psorophora ferox 20% Aedes vexans 9% Ae. albopictus 7% Other 2% Oc. canadensis 2% Anopheles quadrimaculatus 1% Cx. quinquefasciatus 5% Wyomyia mitchellii 1% Ochlerotatus infirmatus 36% Figure 5-7. Composition of the nine most comm only collected mosquito species using four surveillance techniques in suburban and sylvatic locales between May September 2008 in Gainesville, Florida.

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146 0 10 20 30 40 50 60 70 80 90 100Collection Techniques % (SEM) Detection BG Gravid Landing-Counts Aspirator Figure 5-8. Likelihood of de tection of four surveillance methods on dates when Aedes albopictus were recovered by at least one of the sampling methods. Sampling occurred in suburban and sylvatic lo cales between May September 2008 in Gainesville, Florida. Means with the same letter are not significantly different (RyanEinot-Gabriel-Wesh Multiple Rang Test). = 0.05. BG = BG Sentinel trap baited with CO2 at a flow rate of 500 mL/min and a BG-Mesh lure and operated for 48 h ( n = 148), Gravid = CDC Gravid trap baited wi th a 75% oak-pine infusion and operated for 48 h ( n = 148), Landing-counts = human mosqu ito landing counts conducted for 10 min ( n = 144), Aspirator = vegetative aspiration conducted for 10 min ( n = 145). a b c c

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147 0.01 0.1 1 10 1001 4 M a y 7 J u n 1 1 J u n 5 J u l 9 J u l 2 A u g 6 A u g 3 0 A u g 3 S e p 2 7 S e pTrialMean number of female Ae. albopictus / trap period0 5 10 15 20 25Precipitation (cm) Total Precipitation (cm) BG Gravid Landing-Counts Aspirator Figure 5-9. Seasonal abundance of female Aedes albopictus in Gainesville, Florida suburban and sylvatic locales between May September 2008, as measured with four trap collection techniques. Precipitation data retrieved from the Florida Automated Weather Network, University of Florida.

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148 0 0.5 1 1.5 2 2.5 3 3.5 Collection Techniques Mean Captured / 10 min / 12 hrs BG Gravid Landing-Counts Aspirator Figure 5-10. Comparative effi ciency of four sampling devices in capturing female Aedes albopictus in Gainesville, Florida suburban and sylvatic locales between May September 2008. Means with the same lette r are not significantly different (RyanEinot-Gabriel-Wesh Multiple Rang Test). = 0.05. BG = BG Sentinel trap baited with CO2 at a flow rate of 500 mL/min and a BG-Mesh lure ( n = 129), Gravid = CDC Gravid trap baited with a 75% oak-pine infusion ( n = 129), Landing-counts = human mosquito landing counts ( n = 126), Aspirator = vegetative aspiration ( n = 124). Traps operated for 48h = 1 trap peri od, while landing-counts and aspirations were 10 min = 1 collection period. Traps we re converted to 10 min comparatives by dividing total mosquito collec tion in 1 trap period by 144. a b c c

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149Table 5-1. Total mosquitoes coll ected by four surveillance methods1 in suburban and sylvatic locale s, Gainesville, Florida, May September 2008. Surveillance method BG trap Gravid trap Landing-counts Aspirator Mosquito species Suburban Sylvatic Suburban Sy lvatic Suburban Sylvatic Suburban Sylvatic Aedes albopictus 4,273 72 167 47 319 6 176 6 Ae. vexans 3,317 2,132 0 1 11 2 347 813 Ochlerotatus canadensis 1,125 149 0 0 5 6 30 8 Oc. fulvus pallens 0 1 0 0 0 0 0 1 Oc. infirmatus 15,245 9,113 38 9 388 249 1,391 493 Oc. triseriatus 88 242 2 52 2 1 9 70 Oc. sollicitans 7 0 0 0 0 0 11 0 Anopheles barberi 0 6 0 2 0 0 0 0 An. crucians 43 166 0 1 0 0 1 4 An. punctipennis 5 51 0 0 0 0 0 0 An. quadrimaculatus 81 689 2 23 0 1 1 2 Coquillettida perturbans 166 44 0 0 0 0 2 0 Culiseta inornata 1 0 0 0 0 0 0 0 Culex coronator 15 2 0 0 0 0 0 2 Cx. erraticus 42 61 13 0 0 0 28 17 Cx. nigripalpus 6,352 5,523 48 133 1 2 92 128 Cx. restuans 14 0 14 0 0 0 3 2 Cx. salinarius 11 0 0 0 0 0 0 0 Cx. quinquefasciatus 1,140 6 2,587 11 0 0 15 3 Mansonia titilans 2 15 0 0 0 1 0 0 Orthopodomyia signifera 0 0 0 9 0 0 0 1 Psorophora ciliata 18 17 0 0 0 0 0 1 Ps. columbiae 11 0 0 0 0 0 2 2 Ps. ferox 7,361 6,749 2 0 133 34 228 58 Ps. howardii 40 120 0 0 0 0 1 2 Toxorhynchites rutilus 6 13 1 1 0 0 0 0

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150Uranotaenia sapphirina 0 0 0 0 0 0 0 5 Wyeomyia mitchelii 942 0 5 0 20 0 9 0 Wy smithii 60 0 0 0 0 0 0 0 Total species 25 20 11 11 8 9 17 19 Total mosquitoes 40,365 25,171 2,879 289 879 302 2,346 1,618 1Surveillance methods included the BG Sentinel (BG) trap baited with CO2 at a flow rate of 500 mL/min and a BG-Mesh lure, CDC gravid trap = Gravid trap baited w ith a 75% oak-pine infusion, human landing-c ounts and a vegetative aspirator. Total collection periods = 40 (48 hrs for traps, 10 min for landing-counts and aspirations).

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151 Table 5-2. Mean (SE) 1 of the six most common mosquitoes collected using the BG Sentinel a nd CDC gravid traps in suburban and sylvatic locales in Gainesville, Florida. Mosquito species Locale type n BG2 Gravid2 df t p Aedes albopictus Suburban Sylvatic 75 60 54.8 7.79 1.0 0.22 2.01 0.34 0.75 0.21 74 59 -6.920 -1.280 <0.0001 0.2057 Ae. vexans Suburban Sylvatic 75 60 44.0 16.8 35.2 15.1 0.00 0.00 0.00 0.00 74 59 2.629 2.320 0.0104 0.0238 Ochlerotatus infirmatus Suburban Sylvatic 75 60 202.8 58.8 130.3 35.2 0.50 0.25 0.10 0.46 74 59 3.442 3.704 0.0010 0.0005 Culex nigripalpus Suburban Sylvatic 75 60 84.2 32.6 73.4 20.7 0.62 0.38 1.93 0.57 74 59 2.561 3.487 0.0125 0.0009 Cx. quinquefasciatus Suburban Sylvatic 75 60 15.0 2.59 0.1 0.05 32.32 4.50 0.18 0.07 74 59 -4.756 -0.962 <0.0001 0.3402 Psorphora ferox Suburban Sylvatic 75 60 97.8 40.3 103.9 30.3 0.03 0.03 0.00 0.00 74 59 2.425 3.434 0.0178 0.0011 1 Paired t -test, = 0.05. 2 Traps were the BG Sentinel (BG) trap baited with CO2 at a flow rate of 500 mL/min and a BG-Mesh lure, CDC gravid trap = Gravid trap baited with a 75% oak-pine infusion, n = number of trapping periods (48 hr each) between May and September, 2008.

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152Table 5-3. Mean (SE) 1 of the six most common mosquito es collected using human landing-coun ts and a vegetative aspirator in suburban and sylvatic locales in Gainesville, Florida. Mosquito species Locale type n Landing-counts2 Aspirator2 df t p Aedes albopictus Suburban Sylvatic 75 60 4.00 0.71 0.08 0.04 2.30 0.45 0.08 0.04 74 60 -3.172 0.000 0.0022 1.0000 Ae. vexans Suburban Sylvatic 75 60 0.15 0.06 0.03 0.02 4.59 0.92 12.40 3.41 74 60 4.920 3.619 <0.0001 0.0006 Ochlerotatus infirmatus Suburban Sylvatic 75 60 5.16 1.39 3.40 0.76 18.50 6.33 7.27 2.10 74 60 2.346 2.625 0.0217 0.0110 Culex nigripalpus Suburban Sylvatic 75 60 0.01 0.01 0.03 0.03 1.22 0.61 1.83 0.72 74 60 1.974 2.498 0.0521 0.0153 Cx. quinquefasciatus Suburban Sylvatic 75 60 0.00 0.00 0.00 0.00 0.20 0.07 0.05 0.03 74 60 2.922 1.762 0.0046 0.0832 Psorphora ferox Suburban Sylvatic 75 60 1.77 0.80 0.47 0.15 3.04 1.30 0.90 0.46 74 60 1.055 1.173 0.2948 0.2453 1 Paired t -test, = 0.05. 2 Techniques included human landing-counts (lan ding-counts) and a vegetative aspirator (a spirator) each perfor med for 10 min, n = collection periods.

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153 CHAPTER 6 FUTURE DEVEVLOPMENT AND APPLICAT ION OF TRAPS AND ATTRACTANTS TO MONITOR AEDES ALBOPICTUS POPULATIONS Introduction Aedes albopictus (Skuse), also known as the Asian tiger mosquito, is one of the most invasive mosquitoes, having become established in at least 28 countries w ithin the past 20 years (Benedict et al. 2007). A daytime-feeder with a propensity to feed on humans, Ae. albopictus has become a severe nuisance in many countries where it has become established. Capable of vectoring at least 23 arboviru ses (Moore and Mitchell 1997), it did not receive serious consideration as a potential health threat until it was rece ntly implicated for dengue and chikungunya outbreaks in Hawaii (Effler et al. 2 005) and Italy (Rezza et al. 2007), respectively. Its ability to exploit natural and man-made containers in suburban and sylvatic habitats and its diurnal activity has made Ae. albopictus difficult to control and monitor with traditional tactics. Recently, a renewed interest in Ae. albopictus control strategies ha s been undertaken in the form of a cooperativ e agreement between the USDA and the Center for Vector Biology at Rutgers University New Jersey Experiment Station (CVBRUNJES) to develop and use integrated pest management (IPM) techniques to control this persistent pest. Additionally in February 2009, CVBRUNJES hosted the first in ternational symposium on the Asian tiger mosquito (http://vectorbio.rutgers.edu ). The development and use of traps and attractants targeting Ae albopictus is not only important for popul ation monitoring during control applications, but also necessary to prevent thei r introduction. Furthermore, the effects of this invasive species on native mosquito fauna are still being assessed. To track long-term fluctuations in Ae. albopictus populations, these new surveillance methods should be incorporated in to a broader plan that includes remote sensing. In Florida,

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154 geographic information system (GIS) is being used to monitor Ae. albopictus populations and their effect on Ae. aegypti L. populations (Britch et al. 2008). Aedes albopictus oviposition may be influenced by local flora and other biotic a nd abiotic factors. Leaf detritus collected in containers may influence larval success and may exclude competi ng species (Murrell and Juliano 2008). Finally, if an outbreak of dengue or CHIK occurred in Florida, implicating Ae. albopictus as the primary vector, GIS could be used to li nk potential clinical cases with known sites that produce a greater number of Ae. albopictus potentially increasing the risk of transmission (Ali et al. 2003). Traps and Attractants The development of traps to target diurnal mo squitoes has enabled scientists and vector biologists to better understand their feeding habits and habitat pref rences (Obenauer et al. 2009). Furthermore, combining host-seeking and oviposi tion traps can effectively determine mosquito height preferences. For example, results from Chapter 2 and 4 were sim ilar in that 87 and 81% of adults and eggs were recovered at 1 m, sugge sting a similar, but not exclusive pairing of hostseeking and oviposition activity area s. The future use of semiochemicals to target and control Ae. albopictus is a pratical approach th at should should undergo furt her consideration. Kline (2007) describes mass trapping systems, mating disr uption and chemicals that act as attractants and adulticides, as the most applicable uses for semiochemicals in insect vector control programs. Aedes albopictus represents one the most appropri ate mosquito candidates for these strategies. Generally, Ae. albopictus does not fly further than 100 m from its breeding site (Maciel-De-Freitas et al. 2006), pr oviding a situation where mass tr apping could be an effective management tool. Furthermore, during our studi es (Chapters 2 and 5) a surprising number of males (at least 20% of specimens) were collected in traps. Therefore, th e close association of localized male and female emergences and beha vior may allow the use of lures and traps to

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155 interrupt mating behavior by mass-trappping ma les. Finally, results from our studies demonstrate that adult Ae. albopictus are strongly influenced by host-seeking semiochemical lures, as well as naturally-derived oviposit ion products. Mass-trapping programs have been extremely successful in controlling tsetse flie s in Africa, sometimes reducing populations by 70% (Kline 2007). Although control ling mosquito populations using semiochemical-baited traps may be impratical for certain species, such as salt-marsh mosquitoes, it may be appropriate for those with shorter fli ght ranges, such as Ae. albopictus Over the past twenty years, a number of se miochemicals have been identified from plant infusions that strongly stimulate mosquito oviposition. Two semiochemicals, 4-methyphenol from decaying paper birch (Bentley et al. 1979) and 3-methylindole from Bermuda grass (Millar et al. 1992), are known oviposition stimulants for Oc. triseriatus (Say) and Cx. quinquefasciatus (Say), respectively. Recently, carboxylic ac ids and methyl esters, isolated from bamboo infusions, were reported as semiochemicals cap able of stimulating oviposition in gravid Ae. aegypti (Ponnusamy et al. 2008). Therefore, isola ting the semiochemicals found in our oak-pine infusion would help elucidate the exact chemicals or compounds responsible for oviposition stimulation in Ae. albopictus Future use of mosquito sp ecific oviposition-stimulating kairomones in ovitraps may greatly reduce the reliance on adulticides and larvicides. Several commercial ovitraps are currently being marketed. One ovitrap, called OakStump (SpringStar LLC, Woodinville, WA) has b een designed for residential yards and includes an oviposition pheromone and subs trates to attract and capture gravid Culex mosquitoes These traps are attract ive to many homeowners as they contain no insecticides, are easy to use and are relatively affordable. Furt hermore, lethal augmentation of these with the addition of an insect-capturing adhesive would serve as a control measure, as well as an

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156 epidemiological tool used duri ng disease outbreaks (Ritchie et al. 2004). The primary control strategy used to reduce Ae. albopictus populations is similar to that used to control Ae. aegypti ; breeding site source reduction. Failed control practices such as using ultra-low-volume space sprays to control adult Ae. aegypti during dengue epidemics demonstrates the need to develop more efficient control strategies (Gubler 2005). As with many Stegomyia mosquitoes, collection dynamics and control strategies for Ae. albopictus can vary depending on whether they occupy suburban or sylvatic areas (Reiter 2007).

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157 APPENDIX A HOST-SEEKING HEIGHT PREFERENCES OF AEDES ALBOPICTUS WITHIN SUBURBAN AND SYLVATIC LOCALES IN NORTH CENTRAL FLORIDA UTILIZING THREE TYPES OF TRAPS Table A-1. Total Aedes albopictus captured by trap within trial (time period), height and locale between May September 2007 in Gainesville, Florida. Trap Trial Height (m) Suburban Total (Mean SE) Sylvatic Total / (Mean SE) BG 1 2 3 4 5 1 6 1 6 1 6 1 6 1 6 23 (2.90 0.78) 1 (0.16 0.16) 279 (31.00 7.60) 36 (4.60 2.20) 755 (107.90 30.5) 94 (11.90 4.20) 497 (71.0 22.60) 75 (10.70 4.20) 303 (37.80 12.30) 38 (45.40 2.30) 0 0 4 (0.50 0.32) 0 7 (0.87 0.51) 1 (0.13 0.13) 1 (0.16 0.16) 4 (0.57 0.30) 4 (0.50 0.18) 2 (0.28 0.18) MM-X 1 2 3 4 5 1 6 1 6 1 6 1 6 1 6 19 (2.70 1.50) 6 (0.85 0.26) 119 (14.80 3.80) 16 ( 2.20 1.10) 454 (56.70 16.50) 47 (5.90 1.70) 377 (47.10 13.80) 49 (6.10 2.30) 175 (21.80 7.90) 22 (3.14 1.80) 1 (0.14 0.14) 0 15 (2.00 0.65) 0 6 (0.75 0.49) 0 6 (0.75 0.36) 0 6 (0.75 0.49) 5 (0.71 0.42) ODFP 1 2 1 6 1 6 9 (1.28 0.71) 1 (0.12 0.12) 295 (32.7 10.2) 59 (8.4 5.0) 0 0 6 (0.75 0.49) 1 (0.12 0.12)

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158 3 4 5 1 6 1 6 1 6 414 (51.70 21.80) 68 (8.50 3.70) 188 (31.30 10.00) 34 ( 4.86 1.42 257 (36.70 22.20) 18 (2.25 0.94) 8 (1.14 0.67) 1 (0.17 0.17) 4 (1.00 0.40) 2 (0.25 0.25) 4 (0.53 0.18) 1 (0.16 0.16) BG = BG-Sentinel ( n = 147), MM-X = Mosquito Magnet X ( n = 150), ODFP = Omnidirectional Fay-Prince ( n = 145). Trap Periods = 40 (48 hours = 1 trap period). All traps were baited with CO2 and BG Mesh Lure

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159 APPENDIX B MEAN MONTHLY TEMPERATURES ( C) FOR SUBURBAN AND SY LVATIC LOCALES NEAR GAINESVILLE, FL (MAY-SEPTEMBE R 2007), USING DATA RETRIEVED FROM HOBO PENDANT TEMPERATURE/LIGHT DATA LOGGER 0 5 10 15 20 25 301 426 M a y 279 Ju n 10-23 June 2 4 J u n 7 J ul 8 J ul 2 1 Jul 22 Jul 4 Aug 5 A u g 1 8 Aug 19 A ug 1 Se p 2 Sep 15 Sep 1 6 Se p 29 SepDate Temperature ( C) Suburban Sylvatic

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160 APPENDIX C MEAN MONTHLY LIGHT INTENSITY FO R SUBURBAN AND SYLVATIC LOCALES NEAR GAINESVILLE, FL (MAY SEPTEMBER 2007), USING DATA RETRIEVED FROM HOBO PENDANT TEMPERATURE LIGHT DATA LOGGER 0 10 20 30 40 50 60 7014-2 6 May 27-9 J un 10 -2 3 J u ne 24 J un 7 Jul 8 Ju l 21 J ul 2 2 J ul 4 Au g 5 A u g 1 8 Aug 19 A ug 1 Sep 2 S ep 1 5 Sep 16 S e p 29 S epDateLumens (m2) Suburban Sylvatic

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161 APPENDIX D UNIVERSITY OF FLORIDA HEALTH CENT ER INSTITUTIONAL REVIEW BOARD #362007

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162 APPENDIX D. Continued.

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163 APPENDIX D. Continued.

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164 APPENDIX D. Continued.

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165 APPENDIX D. Continued.

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192 BIOGRAPHICAL SKETCH Peter Joseph Obenauer was born in Pittsfiel d, Massachusetts. He spent most of his childhood years traveling throughout Central and S outh America. In 1983, he and his family settled in Northern Virginia. He received a B.S. in biology and secondary education from Longwood University, Farmville, Virginia in 1996 and an M.S. in etomology and plant pathology in 1998 from the University of Tenness ee, where he worked with Dr. Charles Pless studying the biological mechanisms of resistance of the tobacco aphid, Myzus nicotianae He received a commission as a Lieutenant (juni or grade) in the Medical Service Corps in March 1998. After attending Officer Indoctrinati on School, he was assigned to the Navy Disease Vector Ecology and Control Cent er, Bangor, WA in September 1998. He deployed to Nicaragua in October 1998 to serve in Operation Build H ope, following devastating floods from Hurricane Mitch. While in Nicaragua, he worked with the local ministry of health to curtail the spread of malaria and dengue. In September 2000, he was a ssigned to the Preventi ve Medicine Unit, 1st Force Service Support Group (FSSG), 1st Medical Battalion, Camp Pendleton, CA. In January 2003 he deployed to Kuwait in support of Op eration Enduring Free dom /Operation Iraqi Freedom. He completed his tour at Camp Pendleton as the Company Commander of H & S Company and transferred to Naval Environmen tal and Preventive Medicine Unit No. 6, Pearl Harbor, Hawaii in November 2003. In 2004, he ag ain deployed with the Marines and served with 1st Marine Division, CSSG-11 in Ramadii, Iraq in support of Operation Iraqi Freedom II. Following the devastating tsunami that struck Banda Aceh, Indonesia in 2005, he deployed to the region to provide force health protection. Lieutenant Commander Obenauer was sele cted for Duty Under Instruction by the Medical Service Corps and began his Ph.D. progr am in August 2006. The author is an active member of the Entomological Society of Ameri ca, the Society for Vect or Ecology, the Florida

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193 Entomological Society, and the American Mosqu ito Control Association. Upon graduation, he will be assigned to the Naval Medical Research Unit No. 3 (NAMRU-3), Cairo, Egypt. He and his wife, Kathy have two child ren, Lauren and Alexandra.