Citation
Evaluation of an Autodissemination Station for the Suppression of Dengue Virus and Chikungunya Virus Vectors in Florida

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Title:
Evaluation of an Autodissemination Station for the Suppression of Dengue Virus and Chikungunya Virus Vectors in Florida
Creator:
Kartzinel, Mark A
Place of Publication:
[Gainesville, Fla.]
Florida
Publisher:
University of Florida
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Language:
english
Physical Description:
1 online resource (97 p.)

Thesis/Dissertation Information

Degree:
Master's ( M.S.)
Degree Grantor:
University of Florida
Degree Disciplines:
Entomology and Nematology
Committee Chair:
BURKETT-CADENA,NATHAN DANIEL
Committee Co-Chair:
CONNELLY,CYNTHIA R
Committee Members:
ALTO,BARRY WILMER
Graduation Date:
12/18/2015

Subjects

Subjects / Keywords:
Adult insects ( jstor )
Bioassay ( jstor )
Corn oil ( jstor )
Female animals ( jstor )
Larvae ( jstor )
Life tables ( jstor )
Mortality ( jstor )
Oviposition ( jstor )
Tap water ( jstor )
Velvet ( jstor )
Entomology and Nematology -- Dissertations, Academic -- UF
chikungunya -- dengue -- mosquito
Martin County ( local )
Genre:
bibliography ( marcgt )
theses ( marcgt )
government publication (state, provincial, terriorial, dependent) ( marcgt )
born-digital ( sobekcm )
Electronic Thesis or Dissertation
Entomology and Nematology thesis, M.S.

Notes

Abstract:
Florida is facing the imminent threat of two exotic mosquito-borne viruses: dengue virus (DENV) and chikungunya virus (CHIKV). These viruses are typically transmitted by Aedes albopictus and Aedes aegypti, two exotic mosquitoes that are firmly established throughout Florida. In the absence of FDA approved vaccines, the control of vector populations is the only way to prevent DENV and CHIKV from establishing and spreading in Florida. However, Ae. albopictus and Ae. aegypti are notoriously challenging to control through conventional chemical means, due primarily to difficulties in applying pesticides to their cryptic larval container habitats. A novel strategy for suppressing populations of these species is the autodissemination of insect growth regulators (IGRs), in which adult female mosquitoes are tainted with small amounts of potent IGRs. Later, when the adult females visit oviposition sites, they inadvertently deliver (disseminate) the IGR to larval development sites, suppressing their own population. The following research outlines a series of bioassays and field experiments to first design a novel autodissemination station (ADS) and then test the methodology at locations in southern Florida, using the prototype ADS that attracts females from the surrounding habitat through a combination of visual and olfactory cues mimicking resting sites, oviposition sites, and host animals. The success of this strategy was assessed through emergence inhibition of Ae. albopictus and Ae. aegypti. Field evaluations were conducted at well-characterized locations in Indian River County, St. Lucie County and Martin County, the latter two sites of recent local DENV and CHIKV transmission. At one location in Indian River Co. the ADS was able to achieve pupal mortality of 44.12 plus or minus 7.47 percent in treatment sentinel ovicups as compared to control cups with a pupal mortality of 1.01 plus or minus 1.01 percent (P less than 0.01). However, further field experiments in both St. Lucie Co. and Martin Co. could not reproduce this same level of control. In St. Lucie Co the treatments achieved a mortality range of 1.97 plus or minus 0.81 percent to 5.63 plus or minus 3.60 percent as opposed to the control mortality of 0.00 plus or minus 0 percent to 1.54 plus or minus 0.77 percent (P greater than 0.05). Treatment pupal mortality in Martin Co. ranged from 0.00 plus or minus 0 percent to 28.99 plus or minus 8.20 percent while the control mortality ranged from 0.00 plus or minus 0 percent to 14.68 plus or minus 8.65 percent (P greater than 0.05). The apparent ineffectiveness of the ADS at St. Lucie and Martin Co. locations may have been due to the relative lack of attractiveness of sentinel ovicups when compared with natural larval habitats at those locations. ( en )
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.
Thesis:
Thesis (M.S.)--University of Florida, 2015.
Local:
Adviser: BURKETT-CADENA,NATHAN DANIEL.
Local:
Co-adviser: CONNELLY,CYNTHIA R.
Electronic Access:
RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2016-06-30
Statement of Responsibility:
by Mark A Kartzinel.

Record Information

Source Institution:
UFRGP
Rights Management:
Applicable rights reserved.
Embargo Date:
6/30/2016
Classification:
LD1780 2015 ( lcc )

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EVALUATION OF AN AUTODISSEMINATION STATION FOR THE SUPPRESSION OF DENGUE VIRUS AND CHIKUNGUNYA VIRUS VECTORS IN FLORIDA By MARK A. KARTZINEL A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FO R THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FL ORI DA 2015

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© 2015 Mark A. Kartzinel

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3 ACKNOWLEDGMENTS I would like to thank Dr. Nathan Burkett Cadena who helped me every step of the way through this thesis. I definitely could not have done this without all the time and effort he shared to help me through this process. Also, I would like to thank Dr. Roxanne Connelly and Dr. Barry Alto for graciously agreeing to serve on my committee. I would like to thank my family and friends for all their encouragement and support through this. Lastly, I would like to give thanks and credit to God, my heavenly father, who gave me this opportunity and provided me with the strength to get through it.

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4 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 3 LIST OF TABLES ................................ ................................ ................................ ............ 5 LIST OF FIGURES ................................ ................................ ................................ .......... 7 ABSTRACT ................................ ................................ ................................ ..................... 9 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ .... 11 2 DESIGNING AND IMPLEMENTING AN AUTODISSEMINATION STATION ......... 16 Laboratory Optimization of a Prototype Autodissemination Station ........................ 16 Material and Methods ................................ ................................ .......................... 16 Results ................................ ................................ ................................ ................. 22 Field Implementation of a Laboratory Optimized Autodissemination Station .......... 2 2 Materials and Methods ................................ ................................ ........................ 26 Results ................................ ................................ ................................ ................. 34 Tables ................................ ................................ ................................ ..................... 37 Figures ................................ ................................ ................................ .................... 57 3 DISCUSSION ................................ ................................ ................................ ......... 83 Laboratory Optimization of a Prototype Autodissemination Station ........................ 82 Field Implementation of a Laboratory Optimized Autodissemination Station .......... 87 LI ST OF REFERENCES ................................ ................................ ............................... 93 BIOGRAPHICAL SKETCH ................................ ................................ ............................ 97

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5 LIST OF TABLES Table page 2 1 Insect growth regulator (IGR) formulation, concentration, and transfer medium ................................ ................................ ................................ ............... 37 2 2 Statistical outcomes for landings of male and female Aedes albopictus on different fabric types ................................ ................................ ........................... 38 2 3 Statistical outcomes fro landings of female Aedes albopictus on different fabric types ................................ ................................ ................................ ......... 39 2 4 Statistical outcomes for landings of male and female Aedes albopictus on different stations sizes ................................ ................................ ........................ 40 2 5 Adult emergence and pupal mortality from Esteem ® and Nyguard ® cage bioassay ................................ ................................ ................................ ............. 41 2 6 Statistical outcomes for adult emergence and pupal mortality from Esteem ® and Nyguard ® cage bioassay ................................ ................................ .............. 42 2 7 Adult emergence and pupal mortality from transfer medium cage bioassay ...... 43 2 8 Statistical outcomes for adult emergence and pupal mortality from transfer medium cage bioassay ................................ ................................ ....................... 44 2 9 Adult emergence and pupal mortality from Esteem ® oil concentration cage bioassay ................................ ................................ ................................ ............. 45 2 10 Statistical outcomes for adult emergence and pupal mortality from Esteem® oil concentration cage bioassay ................................ ................................ .......... 46 2 11 Statistical outcomes for adult emergence and pupal mortality from food and water level treatments ................................ ................................ ........................ 47 2 12 Martin County field site arrangement ................................ ................................ .. 48 2 13 Statistical outcomes for adult emergence, pupal mortality, and mortality from Indian River County field site ................................ ................................ .............. 49 2 14 Adult emergence, pupal mortality, and mortality for Esteem ® and PAM ® applica tion at Indian River County field site ................................ ........................ 50 2 15 Statistical outcomes for adult emergence, pupal mortality, and mortality at White City Cemetery ................................ ................................ ........................... 51 2 16 Statistical outcomes for adult emergence and pupal mortalit y at White City Cemetery ................................ ................................ ................................ ............ 52

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6 2 17 Adult emergence at Martin County sites ................................ ............................. 53 2 18 Pupal mortality at Martin County sites ................................ ................................ 54 2 19 Statistical outcomes for adult emergence and pupal mortality at White City Cemetery ................................ ................................ ................................ ............ 55 2 20 Statistical outcomes for adult emergence and pupal mortality at Martin County sites ................................ ................................ ................................ ........ 56

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7 LIST OF FIGURES Figure page 2 1 Fabrics tested for attractiveness in cage bioassay ................................ ............. 57 2 2 Landings on red velvet station ................................ ................................ ............ 58 2 3 Station sizes . ................................ ................................ ................................ ...... 59 2 4 Disposable bioassat cage . ................................ ................................ .................. 60 2 5 Adult mosquito and larvae in water . ................................ ................................ .... 61 2 6 A Whirl pak ® (Nasco) . ................................ ................................ ......................... 62 2 7 Lure housing . ................................ ................................ ................................ ...... 63 2 8 BG Sentinel trap . ................................ ................................ ............................ 64 2 9 Male and female Aedes albopictus landings on different fabric types . ............... 65 2 10 Female Aedes albopictus landings on different fabric types ............................... 66 2 11 Male and Female Ae. albopictus landings on different station sizes ................... 67 2 12 Adult emergence and pupal mortality from Esteem ® and Nyguard ® cage bioassay ................................ ................................ ................................ ............. 68 2 13 Adult emergence and pupal mortality from transfer medium cage bioassay ...... 6 9 2 14 The adult emergence and pupal mortality from Esteem ® oil concentration cage bioassay . ................................ ................................ ................................ .... 70 2 15 The numbers of Aedes spp. eggs deposited in ovicups containing oak leaf infusion or water . ................................ ................................ ................................ 71 2 16 Number of Aedes albopictus and Ae. aegypti captured in BG Sentinel traps at Martin County sites . ................................ ................................ ........................ 72 2 17 Adult emergence and pupal mortality from food and water level treatments ...... 7 3 2 18 Sentinel ovicup tripod (SOT) ................................ ................................ ............... 74 2 19 Ovicup . ................................ ................................ ................................ ............... 75 2 20 Prototype autodissemination station . ................................ ................................ .. 76 2 21 Effects of IGR treated autodissemination station at White City Cemetery .......... 7 7

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8 2 22 Correlation between mortality and egg numbers at White City Cemetery .......... 7 8 2 23 Pupal mortality by site at Martin County locations ................................ .............. 7 9 2 24 Mortality by collection at Martin County locations ................................ ............... 80 2 25 Aedes albopictus and Ae. aegypti females collected by BG Sentinel traps ........ 81

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9 Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science EVALUATION OF AN AUTODISSEMINATION STATION FOR THE SUPPRESSION OF DENGUE VIRUS AND CHIKUNGUNYA VIRUS VECTORS IN FLORIDA By Mark A. Kartzinel December 2015 Chair: Natha n Burkett Cadena Major: Entomology and Nematology Florida is facing the imminent threat of two exotic mosquito borne viruses: dengue virus (DENV) and chikungunya virus (CHIKV). These viruses are typically transmitte d by Aedes albopictus and Aedes aegypti , two exotic mosquitoes that are firmly established throughout Florida. In the absence of FDA approved vaccines, the control of vector populations is the only way to prevent DENV and CHIKV from establishing and spread ing in Florida. However, Ae. albopictus and Ae. aegypti are notoriously challenging to control through conventional chemical means, due primarily to difficulties in applying pesticides to their cryptic larval container habitats. A novel strategy for suppre they inadvertently d eliver (disseminate) the IGR to larval development sites, suppressing their own population. The following research outlines a series of bioassays and field experiments to first design a novel autodissemination station (ADS) and then test the methodology at locations in southern Florida, using the prototype ADS that attracts females from the surrounding habitat through a combination of visual and

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10 olfactory cues mimicking resting sites, oviposition sites, and host animals. The success of this strategy was ass essed through emergence inhibition of Ae. albopictus and Ae. aegypti . Field evaluations were conducted at well characterized locations in Indian River County, St. Lucie County and Martin County, the latter two site s of recent local DENV and CHIKV transmiss ion. At one location in Indian River Co. the ADS was able to achieve pupal mortality of 44.12 ± 7.47 % in treatment sentinel ovi cups as compared to control cups with a pupal mortality of 1.01±1.01% (P< 0.01). However, further field experiments in both St. Lucie Co. and Martin Co. could not reproduce this same level of control. In St. Lucie Co the treatments achieved a mortality range of 1.97±0.81% to 5.63±3.60% as opposed to the control mortality of 0. 00±0% to 1.54±0.77% (P>0.05). Treatment pupal mortality in Martin Co. ranged from 0.00±0% to 28.99±8.20% while the control mortality ranged from 0.00±0% to 14.68±8.65% (P>0.05). The apparent ineffectiveness of the ADS at St. Lucie and Martin Co. locatio ns may have been due to the relative lack of attractiveness of sentinel ovicups when compared with natural larval habitats at those locations.

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11 CHAPTER 1 INTRODUCTION The arboviral pathogens, dengue virus ( DENV ) and chik ungunya virus ( CHIKV ) , are two prevalent potentially epidemic causing viruses now common throughout the Caribbean region and also currently threatening Florida and other Southeastern states . Both DENV and CHIKV are transmitted by the same two mosquito spe cies, Ae des albopictus (Skuse) and Ae des aegypti (Linnaeus), which are already established though this potential epidemic region (Graham et al. 2011; Kuehn 2014). Dengue has long been firmly established in the Caribbean with outbreaks dating back to 1635 (Guzman and Isturiz 2010). In 2013 , 2.4 million cases of DENV were reported throughout the Caribbean, Central and South America. In addition there were 773 travel related DENV cases and 48 cases of local transmission in the continental United States (Kue hn 2014). Since its first introduction to the western hemisphere in 2013 on the island of St. Martin in the Caribbean, CHIKV has spread rapidly throughout the Caribbean. It has now been found in 44 countries and territories across the western hemisphere including the United States. By February 2015, 47 states had reported 2,492 cases of CHIKV, the majority being in Florida, which also had the only 11 cases of local transmission (Peper et al. 2015). The vectors of both CHIKV and DENV, Ae. albopictus and Ae . aegypti , are commonly found across these areas of recent transmission and outbreak (Graham et al. 2011, Kuehn 2014 ) Aedes albopictus and Ae. aegypti are remarkably similar with respect to their biologies and their ability to transmit both dengue and ch ikungunya viruses. Both mosquitoes feed preferentially upon humans in urban areas , and thrive in densely (human) populated areas (Simard et al. 2005 ). Their similarities make these vectors

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12 equally dangerous, but also provide an opportunity for managing bot h species simultaneously. Efforts to prevent the spread of DENV and CHIKV would be greatly aided by a new strategy that can effectively suppress these peri urban mosquitoes. In the absence of FDA approved vaccines for these viruses, control mediated reduc tions in vector populations and limitation of human contact with vectors are the only viable methods to prevent DENV and CHIKV from establishing and spreading in Florida. However, these mosquitoes are notoriously difficult to control using conventional met hods, due to their abilities to find and exploit small larval habitats (Bartlett Healy et al. 2011, Tapia Conyer et al. 2012). Applying larvicides to larval habitats of Ae. albopictus and Ae. aegypti poses a major challenge to effective control, due to t he small size and cryptic nature of the larv al habitats (Russell et al. 2002, Gonzalez and Suarez 1995, Kay et al. 2000, Montgomery and Ritchie 2002, Barrera et al. 2008). The difficulty in locating and controlling these cryptic larval habitats contributes to persistent local DENV transmission and has inhibited the widespread use of chemical means to control these mosquitoes for transmission suppression (Russe l l et al. 2002). Intimate knowledge concerning the behavior of these mosquitoes may prove to be th e key to unlocking this control problem. A retrospective study conducted in Australia by Russel l et al. (2002) examined the correlation of these cryptic larval habitats with DENV infections during the 1993 DENV epidemic at Charters Towers. In this region of Australia the cryptic larval habitats presented themselves in the form of subterranean water containers such as wells and service manholes. Originally, these habitats were not treated by the conventional surface directed pesticide treatments implemented at the time. In turn this study found

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13 a direct correlation between DENV cases and the proximi ty to the subterranean l arval habitats . Aedes albopictus and Ae. aegypti both exhibit skip oviposition behavior, whereby females lay a limited number of eggs in one location before moving to a different location to lay more eggs (Gaugler et al. 2011). Wi th this knowledge in mind, a novel approach to delivering insecticides to cryptic larval habitats is by exploiting their own in conjunction with potent insect growth reg interfere with the metamorphosis of immature stages and can help to sterilize females and decrease spermiogenesis i n males (Hirano et al. 1998, Devine et al. 2009). More specifically, IGRs act by targeting the g rowth and development of insects by mimicking hormones or working antagonistically against hormones present in the insect system. A key advantage of IGRs is their low toxicity to organisms other than insects (i.e. mammals, birds, and reptiles) due to thei r insect specific hormone nature. However, they have been problematic in aquatic environments because they can affect the development of crustaceans. The five main categories of IGRs are ecdysone, juvenile hormone analogues (JHA), anti juvenile hormones, chitin synthesis inhibitors, and triazines (Emden and Service 2004). Pyriproxyfen, a JHA, has been used extensively in mosquito autodissemination research (Caputo et al . 2012, Devine et al. 2009, Gaugler et al. 2011 ). The autodissemination tec hnique man ipulates adult female mosquitoes causing them to transfer per billion) from IGR

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14 Sihuincha et al. 2005). Pyriproxyfen has emerged as the IGR of choice in the autodissemination technique due to its potential for suppressing the emergence of Ae. aegypti and Ae. albopictus at extremely low concentrations (as low as 6 ppb) (Dell and Appers on 2003, Gaugler et al. 2011). Various designs of the autodissemination technique have been tested, with varying levels of suppression achieved. In a Peruvian cemetery, Devine et al. (2009) used treated autodissemination stations in the form of resting sites with pyriproxyfen and achiev ed 42 98% control of Ae. aegypti in surveillance ovicups. In Italy, Caputo et al. (2012), using a similar approach as Devine (2009 ), achieved 50 70% pupal mortality for Ae. albopictus (compared to <2% mortality in controls). Working with Ae. albopictus in a laboratory setting, Gaugler et al. (2011) achieved 100% and 81% emergence inhibition, in caged and small room bioassays, respectively, using an autodissemination station that utilized oviposition site, rather than a resting site. As with other modes of control, the effectiveness of the autodissemination technique may be to a large extent determined by the ecological context (e.g., landscape ecology). For example , in the previously mentioned study , Russel et al. (2002) found that the surface applications of pesticide were ineffective on the subterranean larval habitat demonstrated by the correlation of proximity to a subterranean habitat with DENV infections. In order to construct an optimized ADS, it is necessary to first conduct an extensive series of bioassays that thoroughly explore all aspects of the ADS design from the size and shape of the station to the color and lures incorporated in the station in order to maximize the attractiveness of the ADS to the targeted Aedes species. The following serie s of bioassays allowed for the design and evaluation of the most

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15 desirable characteristics to comprise the novel ADS design. These characteristics were specifically tested and designed to attract resting site seeking (resting) , host seeking, and ovipositi on site seeking (ovipositing) female Ae. aegypti and Ae. albopictus .

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16 CHAPTER 2 DESIGNING AND IMPLEMENTING AN AUTODISSEMINATION STATION Laboratory Optimization of a Prototype Autodissemination Station Materials and Methods Fabric Bioassay : A bioa ssay was conducted to evaluate four different types of fabric that would be used to cover the body of the ADS. This bioassay used an 7.62x8.89cm (3x3.5in) diameter section of PVC pipe as its structure. This material was inexpensive, simple to work with, and easy to obtain. The fabrics tested were chosen solely on their potential hypothesized ability to attract host seeking females through visual cues. The four fabric types were: black velvet, red velvet, faux zebra pattern velvet, and bear fabric (faux b lack fur) (Figure 2 1). The black velvet and bear fabric were chosen for their resemblance to real animal fur, while the zebra patterned fabric was chosen due to its high black and white contrast, hypothesized to be an alluring trait and had previously bee n tested in ovitraps (Hoel et al. 2011). The red velvet was chosen based on observational evidence indicating that female Ae. albopictus and Ae. aegypti are highly attracted to the color red. The experiment was conducted in an insectary at the Universi ty of Florida, Institute of Food and Agricultural Sciences, Florida Medical Entomology Laboratory (FMEL). The mosquito cage, 38.1x35.56x35.56cm (15x14x14in), contained approximately 700 individual laboratory reared Ae. albopictus adults (male and female c ombined). Laborator reared Ae. albopictus were reared from egg papers obtained from blood fed (chickens) lab colonies kept in an insectary (23 ±2 °C) . The egg papers were placed in a pan of tap water along with food (1:1 yeast:lactalbumin). Pupae were

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17 collected and placed in container with tap water in cages which contained sugar and water. Pieces of PVC pipe, 7.62x8.89cm (3x3.5in), were covered with one of the four types of fa bric. E minute period and photographed at 30 second intervals with a Canon EOS Rebel T1i (digital camera) from one side of the station . Stations of different fabric types were rotated out immediately at the end of each 5 minute period and the process repeated four times for each fabric covere d station (10 pictures per trial, 4 trials per fabric type). Using the digital images (Figure 2 2), t he numbers of individual male and female mosquitoes on the station were observed and averages were calculated across the four trials. ADS Size Bioassay : A bioassay was conducted to test the effect of ADS size on visual attraction and visitation by Ae. albopictus , and was conducted in exactly the same manner as the fabric bioassay and under the same conditions. Stations were photographed every 30 seconds f or 5 minute trials and repeated 4 times for each station type: small, 7.62cm (3in), and large, 17.68cm (7in) (Figure 2 3). From the digital images, the total landings for each 30 second observation (male+female) were quantified. IGR Formulation, Transfer Medium, and Concentration Bioassay : A series of bioassays were conducted to explore various options for IGR formulations, transfer mediums, and IGR concentrations within the transfer medium (Table 2 1) . The premise behind these bioassays was to identify the optimum combination of materials that result in the highest mortality in mosquito immatures. The layout for these bioassays consisted of releasing groups of female Ae. albopictus , using an aspirator, into a cylindrical transparent plastic bioassay cha mber 15.24x21.59cm (6x8.5in), which

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18 contained a treated ADS, composed of a short length of PVC pipe 7.62x8.89cm (3x3.5in) covered with IGR treated fabric (Figure 2 4). Female mosquitoes were left in the cages for varying times, either 1 hour or 24 hours d epending on the trial. After the exposure period, the bioassay chambers were placed in a freezer and the females were freeze killed. The IGR exposed females were then placed into cups containing 80mL of water and approximately ten laboratory reared 2 nd 3 r d instar conspecific larvae ( Ae. albopictus ) . L arval cups (Figure 2 5) (6 8 cups per treatment) were supplied with food (1:1 yeast:lactalbumin) ad libitum for 11 15 days (approximately the time it took for the majority of the immature mosquitoes to emerge as adults) and placed in an insectary (23 ±2 °C) . At the end of the allotted time, the number of pupal skins, dead pupae, live pupae, dead larvae, and live larvae in each cup were recorded. Data analysis focused on pupal skins, which indicated emerged adu lts, and dead pupae, which indicated pupal mortality. The larval control cups were identical to the treatment cups except that the freeze killed adult female introduced to the larval cup was not exposed to IGR. The first of these bioassays examined two formulations of pyriproxyfen (IGR): NyGuard ® (manufactured by MGK, Minneapolis, MN.) and Esteem ® (manufactured by Valent USA Corporation, Walnut Creek, CA.) . NyGuard ® is a liquid oil formulation (10% pyriproxyfen by weight), while Esteem ® is a dry powder formulation (35% pyriproxyfen by weight). NyGuard ® (5mL ) and Esteem ® (0.22g) were applied directly to the fabric covered station within the bioassay chamber. Additionally, corn oil was tested as a potential transf er medium for Esteem ® (0.22g Esteem ® /25.04g Corn oil). A similar bioassay was conducted which tested Esteem ® at a 1% formulation with various media: Esteem ® /water (0.22g/25g), Esteem ® /ethanol (0.22g/25g), Esteem ® /corn

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19 oil (0.24g/25g) and Esteem ® with n o medium (0.26g). The media varied from polar substances (water and ethanol) to a non polar substance (corn oil). In exploring polar and non polar media, this bioassay helped to expound upon the nature of the Esteem ® and whether or not any of these subst ances dissolved it or helped preserve it. The last of these bioassays compared 1% and 0.1% concentrations of Esteem ® in glycerine and two different oils. The 1% mixtures were as follows: Esteem ® /corn oil (0.26g/25.52g), Esteem ® /castor oil (0.26g/25.02g), and Esteem ® /glycerin (0.26g/25.01g). The 0.1% mixtures were as follows: Esteem ® /corn oil (0.03g/25.02g), Esteem ® /castor oil (0.03g/25.10g), and Esteem ® /glycerin (0.03g/25.07g). In the previous two bioassays female mosquitoes were left for 24 hours in the bioassay chambers before freezing, but in this last experiment the females were left for just one hour. This change limited the amount of time the mos quitoes were exposed to the IGR in order to test whether or not the adults would pick up enough Esteem ® in a shorter exposure time to still result in high pupal mortalities. Sentinel Larval Transfer Bioassay : A sentinel larval transfer bioassay was conducted to test the transport of IGR contaminated sentinel larva from field sites to the laboratory without opening the containers and risking contamination of other laboratory colonies. Specifically, it determine d the optimum levels of water and food that could support the full development of 10 larvae without opening the container. Four test groups were examined: 200mL high food, 100mL high food, 200mL low food, and 100mL low food. The 200mL treatment groups ha d 200mL of tap water as opposed to the 100mL groups which contained 100mL of tap water. High food groups were supplied with 10mL of food suspension that contained 0.08g of food while low food

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20 groups were given 10mL of food suspension containing 0.04g of f ood (1:1 yeast:albumen). The containers used in this experiment were 532mL (18oz) Nasco Whirl paks ® . Ten 2 nd 3 rd instar laboratory reared Ae. albopictus larvae were placed into plastic cups with either 100mL or 200mL of water and 10mL of high or low food suspension with five replicates for each group. The cups with the larvae and food suspension were left outdoors for three days with the temperature ranging from 13 26 °C . After the 3 days contents of the cups were transferred in the field to the Whirl pa ks ® (Figure 2 6) and returned to the laboratory (FMEL) and left for 12 days in an insectary (23 ±2 °C) . After 12 days, pupal skins, dead pupae, live pupae, dead larvae, and live larvae were counted. Oviposition Lure Bioassay : Incorporating a chemical ovip osition lure into the ADS should increase the number and frequency of visits by gravid mosquitoes, thereby increasing the overall level of control achieved by the ADS. However, there is no commercially available lure for attracting gravid Aedes mosquitoes . Bentley et al. (1979) working with another container inhabiting Aedes species, Aedes triseriatus (Say), found that the odor of P cresol and 4 methylcyclohexanol could elicit a positive chemotactic response. Both of these chemicals are highly toxic and were not used for the prototype ADS. Ponnusamy et al. (2008) isolated several combinations of acids from oak and bamboo leaf infusions that when present in trace amounts in water elicited greater egg laying than pure water. Maximum egg laying was achieve d with 10ng of a 16% nonanoic acid, 83% tetradecanoic acid, and 1% tetradecanoic acid methyl ester mixture in 30 mL of water. The research of Ponnusamy et al. (2008) built upon the use of an

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21 organic attractant like a hay or oak leaf infusion (OLI) (Trexle r et al. 1998). The major drawback with using an OLI is its unreliability and difficulty in mass producing a consistent product. In order to delineate the most efficient processes for the ADS, a series of bioassays were implemented to test the reliabilit y in using an OLI in an ADS system. A small field bioassay was conducted to evaluate the effectiveness of incorporating OLI into the ADS sampling methods. The location of this experiment was an abandoned lot in downtown Vero Beach, FL, with nuisance popu lations of Ae. albopictus . Larval cups were placed in the field and left for either four or five days. Larval cups contained 100mL of tap water, 10mL of food suspension and 10 2 nd to 3 rd instar larvae. OLI cups also contained 5mL of OLI . The OLI was ma de with tap water, dried oak leaves, and yeast and left to age for 2 4 weeks in a closed container indoors. Paper clipped to the inner side of the larval cups were strips of seed germination paper (3.81x10.16cm). These papers served as an oviposition sur face for the female mosquitoes. Preference for OLI or tap water was evaluated by comparing the number of eggs present on each paper within the different treatments. Host Seeking Lure Bioassay : Additional bioassays were conducted to evaluate a chemical lu re that was developed to attract host seeking female mosquitoes in a cost effective formulation. Attractive volatiles used in lure development were compounds previously tested by others including: lactic acid, caproic acid (hexanoic acid), and ammonium bic arbonate ( Braks et al. 2001, Bernier et al. 2000, Logan et al. 2007 ). These compounds were tested for their attractiveness to host seeking Aedes spp. in a combination of 10 mL Hexanoic acid, 130 mL Lactic acid, 260 mL tap water, and 23g

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22 sodium polyacrylat e (a superabsorbent compound used to help keep the lure moist). Approximately 40mL of lure were placed in perforated containers (Figure 2 7). Ammonium bicarbonate (8g) was placed in a separate dry lure chamber with a single opening at the top with a fine m esh netting placed over it. Lures were kept in sealed containers until they were placed in the field. These experimental chemical lures were evaluated using the BG Sentinel trap (manufactured by Biogents, Regensburg, Germany) (Figure 2 8) . BG traps wer e operated for 24 hr periods with either an experimental lure or no lure. Daily captures of Ae. albopictus and Ae. aegypti were compared, with the positions of traps rotated daily to reduce location bias. This bioassay was conducted at a waste tire facili ty at the Vero Beach landfill, a site with an abundance of Ae. aegypti and Ae. albopictus . Statistical analysis : Treatment groups were compared with control groups in bioassay and field evaluations and significant differences were determined using t tests and hoc analysis (R studio). Alpha was set at 0.05 for all comparisons. Results are reported as mean and standard error of mean. Results Fabric Bioassay : The bioassay conducted to explore different types of fabric to cover the b ody of the ADS resulted in significant differences among fabric types in the number of mosquitoes (male and female) landing on treatments (P=<0.01). Significantly more total mosquitoes mosquitoes (male+female) landed on the red velvet fabric stations than zebra pattern or bear fabric covered stations at nearly all time points, but in most cases significant differences were not detected between red velvet and black velv et fabric (Figure 2 9, Table 2 2 ). At 30 sec, the number of total

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23 mosquitoes on red velv et fabric covered stations (7.3 ± 0.5), was not significantly different than on black fabric stations (7.0 ± 1.0; P=0.99), but was significantly greater than zebra pattern fabric (2.0 ±0 ; P=<0.01) and bear pattern fabric (0.5 ± 0.5; P=<0.01). At 150 sec, the number of total mosquitoes on red velvet fabric covered stations (15.0 ± 1.6), was not significantly different than on black fabric stations (10 ± 1.2; P=0.07), but was significantly different for zebra pattern fabric (5.5 ± 1.6; P=<0.01) and bear pattern fabric (0.3 ± 0.3; P=<0.01). At 300 sec, the number of mosquitoes on red velvet fabric covered stations (18.3 ± 0.5), was not significantly different than on black fabric stations (12.8 ± 0.8; P=0.06), but was significantly greater than zebra pattern fabric (6.8 ± 2.5; P=<0.01) and bear fabric (0.3 ± 0.3; P=<0.01). Significantly more female mosquitoes landed on red fabric stations than black or zebra pattern fabric covered stations, at nearly all ti me points (Figure 2 10, Table 2 3 ). At 30 sec, the number of females on re d fabric covered stations (4.8 ± 0.8), was not significantly different than on black fabric stations (6.3 ± 1.3; P=0.46), or zebra pattern fabric (1.8 ± 0.3; P=0.08). At 150 sec, the number of females landing on red fabric covered stations (10.3 ± 1.4), was signif icantly greater than on black fabric stations (5.8 ± 1.0; P=0.04), and zebra pattern fabric stations (4.5 ± 0.8; P=<0.01). At 300 sec the number of females on the red velvet fabric covered stations was significantly greater (12.0 ± 1.00) than on black fabric sta tions (6.8 ± 1.3; P=0.05), and zebra pattern fabric stations (4.5 ± 1.7; P=<0.01). ADS Size Bioassay : This bioassay conducted to explore the size of the ADS size on visual attraction and visitation by Ae. albopictus found almost no significant differences am ong station size in the number of mosquitoes landing on large and small

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24 stations (P=>0.05) for the majority of trials, with one exception (P=0.04). There was no significant difference between mosquitoes landing on the large station with those of the small station at nearly all tim e points (Figure 2 11, Table 2 4 ). At 30 sec, the number of mosquitoes on the large station (19.3 ± 4.0), was significantly greater than on small stations (8.0 ± 1.5; F=6.88 P=0.04). At 150 sec, the number of mosquito landings on lar ge station (8.3 ± 1.8), was not significantly different than the small station (4.8 ± 0.5; F=3.54 P=0.11). At 300 sec, the number of mosquito landings on small station (4.8 ± 0.5), was not significantly different than the small station (2.8 ± 0.8; F=5.05 P=0.07). IGR Formulation, Transfer Medium, and Concentration Bioassay : This series of bioassays conducted to explore various options for IGR formulations, transfer mediums, and IGR concentrations within the transfer medium found significant difference between c ertain treatments and the control (P=<0.01). The bioassay examining the potential of two formulations of pyriproxyfen (IGR): NyGuard ® and Esteem ® , found significant differences between treatments (P<0.01). The control emergence and pupal mortality (emergence=96.67 ±1.92%; mortality=0.00±0%) differed with those of NyGuard ® (emergence=24.70±8.83%; mortality=67.27±7.52%), Corn Oil with Esteem ® (emergence=11.67±10.65%; mortality=81.66±13.21%), and Esteem ® only (emergence=22.50±5.98%; mortality=70.83±3.42 %) (Figure 2 12, Table 5 ). There were significant differences between all treatment groups and the control in both adult emergence (P<0.05) and pup al mortality (P<0.01) (Table 2 6 ). The bioassay examining the potential effectiveness of Esteem ® at 1% form ulations in a variety of mediums (corn oil, ethanol, and water), found significant

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25 differences in pupal mortality (F=13.60, P=<0.01) and adult emergence (F=11.58, P<0.01). The control emergence and pupal mortality (emergence=70.71 ±5.99%; mortality=2.53±1. 90%) differed with some of the treatments: Esteem ® (emergence= 29.56±9.23% ; mortality=45.79±8.77% ), Corn Oil ( emergence=11.41±6.60%; mortality=56.50±8.04%), Ethanol (emergence=58.02±5.28%; mortality=4.40±2.77%) and Water (emergence=63.75±13.84%; mortality= 15.11±13.76% ) (Figure 2 13 ,Table 2 7 ). Pyriproxyfen formulated with corn oil and as powdered Esteem ® resulted in significantly greater pupal mortality (corn oil: P<0.01; Esteem ® : P<0.01) and significantly less adult emergence (corn oil: P<0.01; Esteem ® : P <0.01) than untreated larvae. Pyriproxyfen formulated with ethanol and water did not result in significantly greater pupal mortality (ethanol: P=1.00; water: P=0.75) and adult emergence (ethanol: P=0.85; water: P=0.92) th an untreated larvae (Table 2 8). The bioassay examining the potential for Esteem ® at both 1% and 0.1% concentrations in various mediums, found significant differences in pupal mortality (F=4.0, P<0.01) and adult emergence (F=5.97, P< 0.01) between treatments. The control emergence and pupal mortality (91.30 ±3.36%, 3.70±0%) differed with some of the treatments: Corn Oil 1% (54.07±12.42%, 44.26±13.21%), Corn Oil 0.1% (89.04±2.83%, 6.24±3.26%), Castor Oil 1% (64.51±13.49%, 33.97±12.50%), Castor Oil 0.1% (96.67±2.11%, 0.00±0%), Glycerine 1% (81.45±4.13%, 1.67±1.67%), and Glycerine 0.1% (88.11±3.31%, 5.37±3.75%) (Figure 2 14 , Table 2 9 ). Pyriproxyfen formulated with 1% corn oil resulted in significantly greater pupal mortality (P<0.01) and significantly less adult emergence (P=0.02) than untreated larvae. Pyriproxyfen in all other formulations did not result in significantly greater pupal mortality (corn oil 0.1%:

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26 P=1.00; castor oil 1%: P=0.08; castor oil 0.1%: P=1.00; glycerine 1%: P=1.00; glycerine 0.1%: P=1.00) or adult emergence (corn oil 0.1%: P=1.00; castor oil 1%: P=0.17; castor oil 0.1%: P=1.00; glycerine 1%: P=0.96; glycerine 0.1%: P=1.00) than untreated larvae (Table 2 10 ). Sentinel Larval Transfer Bioassay : The bioassay exploring food suspensions in combination with water levels for immature survival found no difference in either adult emergence (F=0.38, P=0.77) or immature mortality (F=1.55, P=0.24) (Figure 2 17, Table 2 11 ) between treatments. Oviposition Lure Bioassay : The bio assay exploring the attractiveness of OLI over tap water found no difference between OLI and tap water for either of the two egg collection periods (DF=1 Collection 1: F=0.437, P= 0.516 and Collection 2: F=0.074, P=0.787). The average number of eggs laid in OLI baited ovicups (collection 1: 7.4 ±2.8 ; collection 2: 19.8 ±6.9 ) were not significantly greater than Tap Water baited ovicups (collection 1: 9.9 ±2.6 ; collection 2: 17.3 ±6.2 ) (Figure 2 14). Host Seeking Lure Bioassay : The bioassay examining the potent ial of an experimental chemical host lure found significantly greater numbers of Ae. albopictus + Ae. aegypti in the lure baited trap than the unbaited trap (T=<0.01 F=9.07 DF=12) (angular transformation of data). Collections from baited traps ranged from 6 to 354 Ae. albopictus + Ae. aegypti , while control trap collections ranged from 2 to 79 Ae. albopictus + Ae. aegypti (Figure 2 15). Field Implementation of a Laboratory Optimized Autodissemination Station Materials and Methods

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27 Overview : Based on the results from the extensive series of bioassays discussed in the previous section of this thesis, this second chapter explored field evaluations of the experimental ADS with the optimized characters derived from the previous experiments. These experiments demonstrated increased effectiveness of the ADS by incorporating Esteem ® in a 1% solution in corn oil relative to other conditions . This treatment was applied as a coating on the external surfaces of the ADS. The ADS structure was composed o f a 17.78cm (7in) PVC pipe covered in red velvet. The experimental host lure was placed inside the cylindrical station in an effort to increase visitation by Ae. aegypti and Ae. albopictus females. However, as stated in the previous chapter no practical oviposition lure was derived. In order to ascertain the effectiveness and usefulness of the ADS in actual mosquito control efforts, three different types of field experiments were carried out. One criteria for all field experiments was that the field sit es must be located at areas where either Ae. aegypti or Ae. albopictus had high populations. The locations for these experiments included sites in Indian River Co., St. Lucie Co., and Martin Co. The first set of field experiments sought to use short week long tests to determine whether the experimental ADS was effective in reducing adult emergence in targeted species and whether or not proposed sampling methods were practical for field work. The second field site experiment built up the data from the fir st set of field work and attempted to derive the potential range of the ADS of mortality effects on larval populations . The third experiment spanned four sites and was designed to test the ADS in an urban environment .

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28 Field Sites: The single field site i n Vero Beach, Florida (Indian River County) was an abandoned lot located near downtown Vero Beach. Vero Beach has a median household income of $37,051 and a population of 15,749; 20.4% of the individuals are below the poverty level (United States Census Bereau 2015c). The downtown site was characterized by a large population of adult Ae. albopictus . In contrast to the adult population, based on an observational survey during initial visits to the location, there was a distinct lack of larval habitats in the area . Oak trees ( Quercus sp.) shaded large portions of the abandoned lot, which was generally o vergrown with a variety of shrubbery. The single field site in White City, Florida (St. Lucie County) was White City Cemetery, which is in t he Ft. Pierce area . This area of Florida is especially important to mosquito control efforts as it had local tra nsm ission of CHIKV in 2014 (Kendrick et al. 2014). St. Lucie Co. has a median household income of $43,413 and a population of 291,028; 18.4% of the individuals are below the poverty level. (United States Census Bereau 2015a). The cemetery location was well characterized by an abundance of both Aedes albopictus and Aedes aegypti . There were also a large number of larval habitats mostly in the form of water filled vases. Oak trees ( Quercus sp.) were the dominant shade tree at the site. The field sites in M artin County, Florida were located in urban and residential areas. Martin Co. is an area of importance as it was associated with the local transmission of DENV in 2013 (Teets et al. 2014). Martin Co. has a median household income of $49,846 and a populat ion of 153,392; 13% of the individuals are below the poverty level (United States Census Bereau 2015b). Four locations were chosen based

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29 on information provided by local mosquito control , who had already identified populations of Ae. aegypti and Ae. albop ictus at all field sites. Site 1, Site 2, and Site 3 were all at residential locations located in neighborhoods . The yards at all four residential sites appeared to be regularly maintained (i.e. mowed and landscaped). All residential locations had bromeli ads (family Bromeliaceae ) present. Bromeliads are used by both Ae. albopictus and Ae. aegypti during growth and development of their immature stages Site 4 was not in a residential location, but was located at a wooded lot behind a restaurant near a downtown area. Oak trees ( Quercus sp.) were the dominant tree at Site 4 . Transfer Medium Optimization: The first series of one week trials took place at the downtown Vero Beach site . Two characters that made this location ideal for this preliminary field work was the combination of an abundance of Ae. albopictus and a paucity of competing oviposition sites based on an observational survey during initial visits to the location . These e xperiments had a similar purpose to those in chapter 1 aimed at determining the effectiveness of IGR formulations and transfer mediums. However, as data collected from actual field research often varies greatly from laboratory research, some treatments we re repeated from the laboratory bioassays. These short trials in a semi controlled field location were a preliminary assessment of the newly developed ADS before the planned full field trials. For these one sentinel ovicups 2 18 ). The SOT was composed of three 16oz. black stadium cups (Figure 2 19 ). Two of the stadium cups, which served as treatment cups, had a piece of seed germination paper,

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30 approximately 4.45x10.16cm (1.75x4in), paper clipped vertically to the inside of the cup. The seed g ermination paper served as an ov iposition substrate for female mosquitoes. It also aided in the detection of female visitations to the artificial larval habitats by using the eggs numbers as an index. The control cups lacked seed germination paper and instead had three holes, 1.75 1.91cm (11/16 3/4in), drilled in their sides towards the upper lip of the cup. Each hole was covered by a piece of fin e mesh netting hot glued to the exterior of the cup. The opening of the cup was then covered by a rubber glove in order to eliminate possible contamination from adult mosquitoes. All cups contained 10 2 nd 3 rd instar laboratory reared Aedes albopictus lar vae, along with 100mL of tap water, and 10mL of food suspension (obtained from a suspension of 500mL of tap water mixed with 2.0 grams of food (1:1 yeast: lactalbumin) . The SOTs were held together in their tripod by two #32 rubber bands. This tripod confi guration of the larval cups allowed for increased stability and a decreased likelihood of tipping. In this field experiment, 13 SOTs were placed at irregular intervals between stations along the fence line of the site and were left Monday through Friday ( 5 days). The ADS for these trials (Figure 2 20 ) and all subsequent experiments was sign frame and a 30.48x33.02cm (12x13in) piece of plastic corrugated roofing material. These two pieces were held together by zip ties. This arrangement allowed for protection, although somewhat limited, from rain and offered a stable platform for the station itself. The host seeking and resting site seeking portion of the ADS was compr ised of a 17.78x7.62cm (7x 3in) piece of PVC (polyvinyl chloride) pipe covered in a

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31 30.48x30.48cm (12x12in) piece of red velvet. This portion of the station was suspended beneath the roofing of the housing component. The experimental host seeking mosquito lure, composed of a dry and wet portion, was placed inside this portion of the ADS. The wet portion of the l ure contained approximately 40mL of lure (taken from a solution made of 10mL of Hexanoic acid, 130mL of Lactic acid, 260mL of tap water and 23grams of Sodium Polyacrylate). The dry portion of the lure was 8.0 grams of ammonium bicarbonate. On the ground below the host seeking portion of the ADS was a black 16oz. stadium cup filled with 1g of Sodium Polyacrylate mixed with 100mL of either tap wat er or larval water (water taken from 3 rd to 4 th instar larvae developing in a pan) depending on the treatment (this served as an oviposition lure). Around the top edge of the stadium cup a 17.78cm x 5.08cm (7in x 2in) piece of red velvet was stapled to th e inside lip of the cup. This portion of the ADS was the oviposition site seeking portion of the station. In all experiments both parts of the ADS covered in red velvet were the treated portions of the ADS. Based on prior experiments the first of the tr ials conducted used an Esteem ® and corn oil treatment (0.20 grams of Esteem ® and 20grams of corn oil). This first trial did not incorporate the oviposition treatment cup below the host seeking portion of the station (all subsequent trials incorporated the oviposition treatment cup below the host station). The second trial introd uced the oviposition treatment cup and incorporated a dry IGR treatment using 0.25g of Esteem ® on the oviposition treatment cup and 1 gram of Esteem ® on the host seeking station. The third trial tested a liquid pyriproxyfen formulation called NyGuard ® tha t was also tested in the bioassays discussed in chapter

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32 1. The forth trial utilized the Esteem ® IGR formulation and used a dry powder application followed by a spray on oil ( PAM ® Original) ad libitum . Following the PAM ® application trial, the fifth and l ast treatment used an Esteem ® and wax combination. This incorporated a micronized wax powder (MPi Aquabead 519: MicroPowders, Inc) with the average micron ranging from 6.0 8.0 microns. The wax was mixed with the Esteem ® in an effort to use the micronized wax as an adhesive on the waxy carapace of the insects. Incorporating a micronized wax as a substrate for autodissemination has been done in previous work with beetles (Baxter et al. 2008). Preliminary Field Trial: Based on the favorable results from tr ials at the Vero Beach site, t he treatment that was incorporated into the next series of experiments was Esteem ® and PAM ® original. PAM ® Original was applied ad libitum to the ADSs in all cases. One gram of Esteem ® was applied to the host seeking portion of the ADS while the oviposition portion of the ADS had 0.25grams of Esteem ® applied to it. The Esteem ® application was performed in the laboratory. At the field locations the PAM ® spray was applied to both portio ns of each station. The location of this second experiment was the White City Cemetery site. Methods incorporated in this experiment were identical to those used in the previous trials in Vero Beach. Fourteen SOTs were placed throughout a portion of th e cemetery at varying distances from 5 ADSs. Ten laboratory reared Ae. albopictus larvae were placed in each larval cup with a food suspension as described in the methods portion of the previous experiments. The first collection of larvae was placed in t he field on a Monday. The following Friday those first larvae were collected and the second collection of larvae were placed in the field. The next Monday the second collection of larvae were

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33 collected and the third collection of larvae were placed in th e field and collected the following Friday. In total the first and third collections were in the field for 5 days while the second collection was in the field for 4 days. During the two weeks of the experiment, the ADS stations and lures remained t he sam e and were not changed . The main goal of the experiment was to test the range (distance) of effectiveness of the experimental ADS through monitoring of affects on larval populations in SOTs. Full Field Evaluation: The locations of the full field trials were at the Martin Co. sites. The treatment and control sites were rotated over a four week period. Sites 1 and 2 served as treatment sites for weeks one and two, while Sites 3 and 4 served as non treatment (control) sites. During weeks three and four S ites 3 and 4 were treatment sites, while Sites 1 and 2 reverted to non treatment sites (Table 2 12) . During weeks where sites served as non treatment sites, the only research at the sites was the monitoring of adult populations with BG Sentinel Traps bait ed with the experimental lure. During the course of the experiment, 10 SOTs were placed at each experimental location three times on the first Monday (collected the next Friday), the following Friday (collected the following Monday), and the second Monday (collected the last Friday). Thus, the first and third larval collections were in the field 5 days and the second larval collection was out in the field for 4 days. BG Sentinel Traps were left in the field for approximately 24 hour periods from Thursday morning to Friday morning of each week. These traps were baited with the experimental host lure and traps were placed at both treatment and non treatment sites every week.

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34 On the Monday of the first and third week 5 ADSs were placed at each treatment si te (first week: Sites 1 and 2, second week: Sites 3 and 4). The ADS was treated (both host portion and oviposition portion) with Esteem ® applied by gloved hand. One gram of Esteem ® was applied evenly on the host portion of the station while 0.25 grams wa s applied to the oviposition portion of the station. Powder application was performed within the laboratory prior to going to the field. The entire treated portion of the ADS was then coated with an even layer of PAM ® Original cooking spray applied ad li bitum . Statistics : Treatment groups were compared with control groups in bioassay and field evaluations and significant differences were determined using t tests and hoc analysis (R studio). Alpha was set at 0.05 for all compariso ns. Results are reported as mean and standard error of mean. Results Transfer Medium Optimization: The series of one week trials at the Vero Beach location, examining the potential of a variety of transfer medium and IGR formulations, found significant difference between only one treatment ( Esteem ® with PAM ® ) and the control in emergence (F=14.96, P<0.01), pupal mortality (F =13.86, P<0.01), and mortality (pupal mortality and adults which failed to emerge from pupal skins) (F=15.05, P<0.01) (Table 2 13 ). Control pupal mortality ( 1.01±1.01%) was lower than the treatment (44.12 ±7.47%) and control emergence (97.31 ±1.40%) was higher than the treatment (55.52 ±8.06% ) (Table 2 14 ). Preliminary Field Trial: The experiment testing the effects of the ADS with the Esteem ® / PAM ® treatment at White City Cemetery , found no difference between adult emergence (P>0.05), pupal mortality (P>0.05), or mortality (P>0.05) across all three

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35 col lections periods (Table 2 15 ). Collection 1 demonstrated similar control and treatment emergence (99.21 ±0.79%, 93.51±3.60% ) and mortality (0.00 ±0%, 5.63±3.60% ), collection 2 demonstrated similar control and treatment emergence (96.98 ±1.67%, 92.01±4.19% ) and mortality (0.77 ±0.77%, 4.84±4.01% ), and collection 3 also demonstrated similar contr ol and treatment emergence (96.75 ±1.41%, 92.71±2.29% ) and mortality (1.54 ±0.77%, 1.97±0.81% ) (Figure 2 21 ). The experiment found significant difference in the 1 st collection when comparing egg laying with adult emergence (F=6.32, P=0.02) and pupal mortality (F=7.42, P=0.01), but not with the 2 nd collection: adult emergence (F=1.67, P=0.21) pupal mortality (F=0.76, P=0.39), or 3 rd collection: adult emergence (F=0.48 , P=0.49) pupal morta lity (F=0.18, P=0.67) (Table 2 16 ). A linear regression on log transformed data also found no significant pattern between the pupal mortality with the distance to the nearest ADS (T= 1.07 P=0.31 F=1.14 DF=12) (Figure 2 22 ). Full Fie ld Evalutation: The field experiment in Martin Co., examining field effects of the experimental ADS at four locations with 3 collections at each site, demonstrated no difference between adult emergence (P>0.05) and pupal mortality (P>0.05) (Figure 2 23, Ta ble 2 17, Table 2 18, Table 2 19 ) at treatment and control sites. Pupal mortality of Ae. albopictus from sentinel ovicups testing efficacy of an autodissemination station employing pyriproxyfen varied between the collections (collection 1:12.47 ±4.47 %; col lection 2: 1.20 ±0.87 %; collection 3: 3.25 ±1.84 %) (Figure 2 24 ).

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36 No difference was detected in the majority of collections at all sites between egg laying, adult emergence and pupal mortality. The one exception was Site 4 collection 2 (adult emergence: F=6.03, P=0.03; pupal mortality: F=6.03 P=0.03). The adult collections from the BG Sentinel traps varied greatly from week to week (Figure 2 25 ).

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37 Tables Table 2 1 . Insect growth regulator (IGR) formulation, concentration, and transfer medium. The combinations of insect growth regulators (IGR) and transfer mediums at varying concentrations used as treatments in preliminary bioassasys. IGR Medium Concentration Esteem ® None NA Esteem ® Corn Oil 1% and 0.1% Esteem ® Ethanol 1% Esteem ® Water 1% Esteem ® Castor Oil 1% and 0.1% Esteem ® Glycerine 1% and 0.1% NyGuard ® None NA

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38 Table 2 2 . Statistical outcomes for landings of male and female Aedes albopictus on different fabric types. Statistical outcomes (p values) of ANOVA comparing the numbers of adult Aedes albopictus landing on the different fabric types over time (s). The degrees of fr eedom in all cases were 3. Fabric Types 30 60 90 120 150 180 210 240 270 300 Black v. Bear <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 Red v. Bear <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 Zebra v. Bear 0.34 0.37 0.04 0.07 0.05 0.07 0.04 0.06 0.05 0.02 Red v. Black 0.99 0.53 0.04 0.04 0.07 0.23 0.08 0.07 0.02 0.06 Zebra v. Black <0.01 0.12 0.06 0.09 0.11 0.37 0.13 0.21 0.08 0.04 Zebra v. Red <0.01 0.01 <0.01 <0.01 <0.01 0.01 <0.01 <0.01 <0.01 <0.01 F 32.16 12.24 30.03 26.11 24.47 15.28 23.89 21.18 29.74 33.15 P <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01

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39 Table 2 3 . Statistical outcomes fro landings of female Aedes albopictus on different fabric types. Statistical outcomes (p values) of ANOVA comparing the numbers of female adult Aedes albopictus landing on the different fabric types over time (s). The degrees of freedom in all cases were 2. Fabric Types 30 60 90 120 150 180 210 240 270 300 Red v. Black 0.46 0.36 0.08 0.07 0.04 0.13 0.06 0.09 0.15 0.05 Zebra v. Black 0.01 0.23 0.15 0.14 0.23 0.82 0.19 0.57 0.26 0.48 Zebra v. Red 0.08 0.03 <0.01 <0.01 <0.01 0.05 <0.01 0.02 0.01 <0.01 F 7.20 5.19 10.40 11.17 11.09 4.30 10.74 6.37 7.16 8.37 P 0.01 0.03 <0.01 <0.01 <0.01 0.05 <0.01 0.02 0.01 <0.01

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40 Table 2 4 . Statistical outcomes for landings of male and female Aedes albopictus on different stations sizes. Statistical outcomes (p values) of ANOVA comparing the numbers of adult Aedes albopictus landing on the different station sizes over time (s). The degrees of freedom in all cases were 1. 30 60 90 120 150 180 210 240 270 300 F 6.88 0.99 1.59 0.48 3.54 0.51 0.27 0.60 1.29 5.05 P 0.04 0.36 0.26 0.52 0.11 0.50 0.62 0.47 0.30 0.07

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41 Table 2 5 . Adult emergence and pupal mortality from Esteem® and Nyguard® cage bioassay. Adult emergence and pupal mortality of 2 nd 3 rd instar larvae exposed to pyriproxyfen treated adult females in cage bioassays. Females were allowed 24 hour contact time with pyriproxyfen treated fabrics. Treatments included NyGuard ® (an oil based pyriproxyfen formulation), Esteem ® (a powder pyriproxyfe n formulation), Esteem ® mixed with corn oil, and untreated control (N=6). Adult Emergence (% ± SEM) Pupal Mortality (% ± SEM) Control 96.67 ±1.92 0.00 ±0 NyGuard ® 24.70 ±8.83 67.27 ±7.52 Corn Oil with Esteem ® 11.67 ±10.65 81.66 ±13.21 Esteem ® 22.50 ±5.98 70.83 ±3.42

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42 Table 2 6 . Statistical outcomes for adult emergence and pupal mortality from Esteem ® and Nyguard ® cage bioassay. Statistical outcomes (p values) of ANOVA comparing the adult emergence and pupal mortality of 2 nd 3 rd instar larvae exposed to pyriproxyfen treated adult females in cage bioassays. Females were allowed 24 hour contact time with pyriproxyfen treated fabrics. Treatments included NyGuard ® (an oil based pyriproxyfen formulation), Esteem ® (a powder pyriproxyfe n formulation), Esteem ® mixed with corn oil, and untreated control. Global F= 14.45, P=<0.01, DF=3 (emergence) F=18.92, P=<0.01, DF=3 (mortality). Adult Emergence Pupal Mortality Treatment Comparison P P Corn Oil Control <0.01 <0.01 Esteem ® Control < 0.01 <0.01 NyGuard ® Control 0.02 <0.01 Esteem ® Corn Oil 0.87 0.81 NyGuard ® Corn Oil 0.05 0.64 NyGuard ® Esteem ® 0.20 0.99

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43 Table 2 7 . Adult emergence and pupal mortality from transfer medium cage bioassay. Adult emergence and pupal mortality of 2 nd 3 rd instar larvae exposed to pyriproxyfen treated adult females in cage bioassays. Females were allowed 24 hour contact time with pyriproxyfen treated fabrics. Treatments included Esteem ® (a powder pyriproxyfen formulation), Este em ® mixed with corn oil, Esteem ® mixed with ethanol, Esteem ® mixed with water, and untreated control (N=8). Adult Emergence (% ± SEM) Pupal Mortality (% ± SEM) Control 70.71 ±5.99 2.53 ±1.90 Esteem ® 29.56 ±9.23 45.79 ±8.77 Corn Oil 11.41 ±6.60 56.50 ±8.04 Ethanol 58.02 ±5.28 4.40 ±2.77 Water 63.75 ±13.84 15.11 ±13.76

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44 Table 2 8 . Statistical outcomes for adult emergence and pupal mortality from transfer medium cage bioassay. Statistical outcomes (p values) of ANOVA comparing the adult emergence and pupal mortality of 2 nd 3 rd instar larvae exposed to pyriproxyfen treated adult females in cage bioassays. Females were allowed 24 hour contact time with pyriproxyfen treated fabrics. Treatments included Esteem ® (a powder pyriproxyfen fo rmulation), Esteem ® mixed with corn oil, Esteem ® mixed with ethanol, Esteem ® mixed with water, and untreated control. Global F 13.60 and P <0.01 (mortality) F 11.58 P <0.01 (emergence). Pupal Mortality Adult Emergence Treatment Comparison P P Corn Oil Control <0.01 <0.01 EtOH Control 1.00 0.85 Esteem ® Control <0.01 <0.01 Water Control 0.75 0.92 EtOH Corn Oil <0.01 <0.01 Esteem ® Corn Oil 0.80 0.43 Water Corn Oil <0.01 <0.01 Esteem ® EtOH <0.01 <0.01 Water EtOH 0.83 1.00 Water Esteem ® 0.02 0.03

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45 Table 2 9 . Adult emergence and pupal mortality from Esteem ® oil concentration cage bioassay. Adult emergence and pupal mortality of 2 nd 3 rd instar larvae exposed to pyriproxyfen treated adult females in cage bioassays. Females were al lowed 1 hour contact time with p yriproxyfen treated fabrics. Treatments included Esteem ® (a powder pyriproxyfen formulation), Esteem ® mixed with corn oil at 1%, Esteem ® mixed with corn oil at 0.1%, Esteem ® mixed with castor oil at 1%, Esteem ® mixed with castor oil at 0.1%, Esteem ® mixed with Glycerine at 1%, Esteem ® mixed with Glycerine at 0.1%, and untreated control (N=6). Adult Emergence (% ± SEM) Pupal Mortality (% ± SEM) Control 91.30 ±3.36 3.70 ±0 Corn Oil 1% 54.07 ±12.42 44.26 ±13.21 Corn Oil 0.1% 89.04 ±2.83 6.24 ±3.26 Castor Oil 1% 64.51 ±13.49 33.97 ±12.50 Castor Oil 0.1% 96.67 ±2.11 0.00 ±0 Glycerine 1% 81.45 ±4.13 1.67 ±1.67 Glycerine 0.1% 88.11 ±3.31 5.37 ±3.75

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46 Table 2 10 . Statistical outcomes for adult emergence and pupal mortality from Esteem® oil concentration cage bioassay. Statistical outcomes (p values) of ANOVA comparing the adult emergence and pupal mortality of 2 nd 3 rd instar larvae exposed to pyriproxyfen treated adult females in cage bioassays. Females were allowed 24 hour contact time with pyriproxyfen treated fabrics. Treatments included Esteem ® (a powder pyriproxyfen formulation), Esteem ® mixed with corn oil at 1%, Esteem ® mixed with corn oil at 0.1%, Esteem ® mixed with castor oil at 1%, Esteem ® mixed with castor oil at 0.1%, Esteem ® mixed with Glycerine at 1%, Esteem ® mixed with Glycerine at 0.1%, and untreated control. Global F=4.0, P=< 0.01, DF=6 (emergence) and F=5.97, P=<0.01, DF=6 (mortality). Adult Emergence: Pupal Mortality Treatment Comparison P P Castor Oil 1% Castor Oil 0.1% 0.06 0.03 Control Castor Oil 0.1% 1.00 1.00 Corn Oil 0.1% Castor Oil 0.1% 0.99 1.00 Corn Oil 1% Castor Oil 0.1% <0.01 0.02 Glycerine 0.1% Castor Oil 0.1% 0.98 1.00 Glycerine 1% Castor Oil 0.1% 0.77 1.00 Control Castor Oil 1% 0.17 0.08 Corn Oil 0.1% Castor Oil 1% 0.26 0.13 Corn Oil 1% Castor Oil 1% 0.95 0.95 Glycerine 0.1% Castor Oil 1% 0.30 0.11 Glycerine 1% Castor Oil 1% 0.68 0.05 Corn Oil 0.1% Control 1.00 1.00 Corn Oil 1% Control 0.02 <0.01 Glycerine 0.1% Control 1.00 1.00 Glycerine 1% Control 0.96 1.00 Corn Oil 1% Corn Oil 0.1% 0.03 0.01 Glycerine 0.1% Corn Oil 0.1% 1.00 1.00 Glycerine 1% Corn Oil 0.1% 0.99 1.00 Glycerine 0.1% Corn Oil 1% 0.04 <0.01 Glycerine 1% Corn Oil 1% 0.16 <0.01 Glycerine 1% Glycerine 0.1% 1.00 1.00

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47 Table 2 11 . Statistical outcomes for adult emergence and pupal mortality from food and water level treatments. Statistical outcomes (p values) of ANOVA comparing the adult emergence and pupal mortality of 2 nd 3 rd exposed to four food concentration and water level treatments. Treatments included varying levels of tap water and food suspension (1:1 yeast:albumen): 200L (200mL water, 0.04g food), 100L (100mL water, 0.04g food), 100H (100mL water, 0.08g food), and 200H (200mL water, 0.08g food). Global F=0.38, P=0.77, DF=3 (emergence) and F=1.55, P=0.24, DF=3 (mortality). Adult Emergence Mortality Treatment Comparison P P 100L 100H 0.73 1.00 200H 100H 0.98 0.29 200L 100H 0.99 0.83 200H 100L 0.92 0.29 200L 100L 0.89 0.83 200L 200H 1.00 0.75

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48 Table 2 12. Martin County field site arrangement . The arrangement of field sites ( 2 4) in Martin County across four weeks of trials with respect to designation as treatment or control site . Site 1 Site 2 Site 3 Site 4 Week 1 Treatment Treatment Control Control Week 2 Treatment Treatment Control Control Week 3 Control Control Treatment Treatment Week 4 Control Control Treatment Treatment

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49 Table 2 13 . Statistical outcomes for adult emergence, pupal mortality, and mortality from Indian River County field site. Statistical outcomes (p values) of ANOVA comparing the effects of an IGR treated autodissemination station on the adult emergence, pupal mortality and mortality of Aedes albopictus immatures from sentinel ovicup tripods (control and treatment). Treatments included Esteem ® (a powder pyriproxyfen formulation), Esteem ® with PAM ® Original spray, Esteem ® with corn oil (1% formulation), NyGuard ® (an oil pyriproxyfen formulation), Esteem ® mixed with micronized wax. The study was conducted at field sites in Vero Be ach, FL, USA (2015). Adult Emergence Pupal Mortality Mortality P F P F P F Esteem ® trial 1 0.56 0.36 0.51 0.44 0.51 0.44 Esteem ® trial 2 0.13 2.41 0.17 2.00 0.17 2.00 Esteem ® with PAM ® <0.01 14.96 <0.01 13.86 <0.01 15.05 Esteem ® with Corn Oil 0.28 1.31 0.12 2.26 0.12 2.26 NyGuard ® 0.19 1.76 0.23 1.50 0.23 1.50 Esteem ® with Wax trial 1 0.86 0.03 0.50 0.46 0.98 <0.01 Esteem ® with Wax trial 2 0.25 1.35 0.54 0.39 0.54 0.39

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50 Table 2 14 . Adult emergence, pupal mortality, and mortality for Esteem® and PAM® application at Indian River County field site. Adult emergence, pupal mortality, and mortality of Aedes albopictus immatures from sentinel ovicup tripods (control: N=13; treatment: N=26; treatment mortality: N=17) comparing the effects of an IGR treated autodissemination station. Treatment mortality looked at only the ovicups where mortality occurred and excluded all uncontaminated treatment ovicups. The study was conducted at a lot in Vero Beach, FL, USA (2015). Ca tegory Pupal Mortality % Adult Emergence % Mortality % Control 1.01±1.01 97.31±1.40 1.01±1.01 Treatment 44.12±7.47 52.52±8.06 45.25±7.65 Treatment Mortality 62.28±6.53 32.97±7.06 63.89±6.65

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51 Table 2 15 . Statistical outcomes for adult emergence, pupal mortality, and mortality at White City Cemetery. Statistical outcomes (p values) of ANOVA comparing the effects of an IGR treated autodissemination station on the adult emergence, pupal mortality and mortality of Aedes a lbopictus immatures from sentinel ovicup tripods (control and treatment) from three collection periods. The study was conducted at White City Cemetery in Ft. Pierce, FL, USA (2015). WCC Adult Emergence Pupal Mortality Mortality P F P F P F Collection 1 0.28 1.21 0.28 1.22 0.28 1.20 Collection 2 0.41 0.69 0.47 0.53 0.47 0.53 Collection 3 0.24 1.46 0.34 0.92 0.75 0.10

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52 Table 2 16 . Statistical outcomes for adult emergence and pupal mortality at White City Cemetery. Statistical outcomes (p values) of a linear regression comparing the effects of an IGR treated autodissemination station on the adult emergence and pupal mortality Aedes albopictus immatures from sentinel ovicup tripods (control and treatment) when compare d with egg numbers in corresponding sentinel ovicups from three collection periods. The study was conducted at White City Cemetery in Ft. Pierce, FL, USA (2015). WCC Adult Emergence Pupal Mortality P F P F Collection 1 0.02 6.32 0.01 7.42 Collection 2 0.21 1.67 0.39 0.76 Collection 3 0.49 0.48 0.67 0.18

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53 Table 2 17 . Adult emergence at Martin County sites. Adult emergence of Aedes albopictus immatures comparing the effects of an IGR treated autodissemination station from sentinel ovicup tripods (control: N=10; treatment: N=20). The study was conducted at a lot in Martin Co., FL, USA (2015). Emergence Collection 1 Collection 2 Collection 3 Control Treatment Control Treatment Control Treatment Site 1 95.00 ±2.22 95.99 ±1.49 98.61 ±1.39 100.00 ±0 99.00 ±1.00 99.35 ±0.65 Site 2 86.88 ±6.93 66.94 ±8.20 97.64 ±1.58 95.33 ±2.71 100.00 ±0 87.69 ±6.34 Site 3 85.32 ±8.65 89.48 ±3.64 97.22 ±2.78 100.00 ±0 98.00 ±2.00 97.18 ±1.73 Site 4 98.75 ±1.25 90.27 ±5.40 96.39 ±1.82 98.83 ±0.81 97.65 ±1.55 100.00 ±0

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54 Table 2 18 . Pupal mortality at Martin County sites . Pupal mortality of Aedes albopictus immatures comparing the effects of an IGR treated autodissemination station from sentinel ovicup tripods (control: N=10; treatment: N=20). The study was conducted at a lot in Martin Co., FL, USA (2015). Mortality Collection 1 Collection 2 Collection 3 Control Treatment Control Treatment Control Treatment Site 1 2.91 ±2.10 0.63 ±0.63 0.00 ±0 0.00 ±0 1.00 ±1.00 0.65 ±0.65 Site 2 13.12 ±6.93 28.99 ±8.20 1.11 ±1.11 3.61 ±2.68 0.00 ±0 11.09 ±5.88 Site 3 14.68 ±8.65 10.52 ±3.64 1.39 ±1.39 0.00 ±0 0.00 ±0 1.24 ±0.86 Site 4 1.25 ±1.25 9.73 ±5.40 1.39 ±1.39 1.17 ±0.81 2.35 ±1.55 0.00 ±0

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55 Table 2 19 . Statistical outcomes for adult emergence and pupal mortality at White City Cemetery. Statistical outcomes (p values) of ANOVA comparing the effects of an IGR treated autodissemination station on the adult emergence and pupal mortality of Aedes albopictus immatures from sentinel ovicup tripods (control and treatment) spanning four sites and three collections per site. The study was conducted at White City Cemetery in Martin Co., FL, USA (2015). The degrees of freedom in all cases were equal to 1. Adult Emergence Pupal Mortality Site 1 P F P F Collection 1 0.73 0.12 0.19 1.78 Collection 2 0.15 2.23 NA* NA* Collection 3 0.77 0.09 0.77 0.09 Site 2 Collection 1 0.14 2.33 0.21 1.64 Collection 2 0.57 0.33 0.53 0.41 Collection 3 0.19 1.85 0.20 1.75 Site 3 Collection 1 0.61 0.23 0.61 0.23 Collection 2 0.15 2.23 0.15 2.23 Collection 3 0.77 0.09 0.31 1.09 Site 4 Collection 1 0.28 1.20 0.28 1.20 Collection 2 0.17 2.04 0.89 0.02 Collection 3 0.05 4.47 0.05 4.47 *Note: in situations where NA is noted all data points for both control and treatment were equal to zero without any variation across collections

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56 Table 2 20 . Statistical outcomes for adult emergence and pupal mortality at Martin County sites. Statistical outcomes (p values) of linear regression comparing the effects of an IGR treated autodissemination station on the adult emergence and pupal mortality of Aedes albopictus larvae from sentinel ovicup tripods (control and treatment) with egg numbe rs from corresponding ovicups spanning four sites and three collections per site. The study was conducted at White City Cemetery in Martin Co., FL, USA (2015). The degrees of freedom in all cases were equal to 1. Adult Emergence Pupal Mortality Site 1 P F P F Collection 1 0.57 0.34 0.38 0.81 Collection 2 0.03* 2.34* NA* NA* Collection 3 0.51 0.45 0.51 0.45 Site 2 Collection 1 0.33 1.02 0.19 1.93 Collection 2 0.71 0.15 0.56 0.35 Collection 3 0.52 0.43 0.53 0.41 Site 3 Collection 1 0.09 3.29 0.08 3.29 Collection 2 0.87* 15.03 NA NA Collection 3 0.11 2.80 0.51 0.45 Site 4 Collection 1 0.47 0.55 0.47 0.55 Collection 2 0.03 6.03 0.03 6.03 Collection 3 0.70* 15.15 NA NA *Essentially a perfect fit and statistics were unreliable

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57 Figures Figure 2 1. Fabrics tested for attractiveness in cage bioassay. Four fabrics were tested in a bioassay for their attractiveness to Aedes albopictus . A) Bear (Imitation Fur) was tested. B) Zebra Pattern was tested. C) Black Velvet was tested. D) Red Velvet was tested .

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58 Figure 2 2. Landings on red velvet station. Aedes albopictus (males and females) landing on a red velvet station during a time trial.

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59 Figure 2 3. Station sizes. Two autodissemination stations, large 17.68cm (7in) and small 7.62cm (3in), used in a bioassay testing the effects of station size on attractancy to Aedes albopictus .

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60 Figure 2 4. Disposable bioassay cage. A disposable bioassay cage constructed with 21.6x27.9cm (8.5x11in) transparency film and scotch tape with an experimental autodissemination station inside . Tops and bottoms of cages were constructed from corrugated plastic board. Constructed cages measured approximately 15.24x21.59cm (6x8.5in).

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61 Figure 2 5. Adult mosquito and larvae in water. Ten 2 nd 3 rd instar laboratory reared Aedes albopictus larvae were placed in 80mL of water with one freeze killed IGR contaminated adult female.

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62 Figure 2 6. A Whirl pak ® (Nasco). Sentinel ovicup transfer trials, consisting of adult mosquitoes and pupal skins within a Whirl pak ® (Nasco).

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63 Figure 2 7. Lure housing. A) This section of lure housing was designed to hold the 8 grams s eeking lure. The hole at the top of the lure housing was covered with fine mesh netting in the field. B) This section of lure housing served as ost seeking lure and contained water, hexanoic acid, and sodium polyacrylate lure mixture . A B

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64 Figure 2 8. BG BG Sentinel trap manufactured by Biogents, Regensburg, Germany.

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65 Figure 2 9 . Male and female Aedes albopictus landings on different fabric types. Average Aedes albopictus landings (male and female) taken at thirty second intervals for five minute long time trials with a Canon EOS Rebel T1i (N=4). Landings compared 4 fabric groups: Bear (Imitation Fur), Zebra Pattern, Black Velvet, and Red Velvet. 0 5 10 15 20 25 30 60 90 120 150 180 210 240 270 300 Ae. albopictus Landings (Mean SEM) Time in Seconds Red Velvet Black Velvet Zebra Bear

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66 Figure 2 10 . Female Aedes albopictus landings on different fabric types. Average Aedes albopictus landings (female only) taken at thirty second intervals for five minute long time tria ls with a Canon EOS Rebel T1i (N=4). Landings compared 3 fabric groups: Zebra Pattern, Black Velvet, and Red Velvet. 0 2 4 6 8 10 12 14 30 60 90 120 150 180 210 240 270 300 Ae. albopictus Female Landings (Mean SEM) Time in Seconds Red Velvet Black Velvet Zebra

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67 Figure 2 11 . Male and Female Ae. albopictus landings on different station sizes. Ae. albopictus landings (male and female) taken at th irty second intervals for five minute long time trials with a Canon EOS Rebel T1i (N=4). Landings compared 2 station sizes: large, 17.68cm (7in), and small, 7.62cm (3in). 0 5 10 15 20 25 30 60 90 120 150 180 210 240 270 300 Mosquito Landings (Male+Female) (Mean SEM) Time In Seconds AVG Large 7" AVG Small 3"

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68 Figure 2 12 . Adult emergence and pupal mortality from Esteem ® and Nyguard ® cage bioassay. Adult emergence and pupal mortality of 2 nd 3 rd instar larvae exposed to pyriproxyfen contaminated adult females in cage bioassays. Females were allowed 24 hour contact time with pyriproxyfen treated fabrics. Treatments included NyGuard ® (an oil based pyriproxyfen formulation), Esteem ® (a powder pyriproxyfen formulation), Esteem ® mixed with corn oil, and untreated control (N=6). Different letters and numbers denote significant differences among treatments for emergence and mortality. 0 25 50 75 100 0 25 50 75 100 Control Nyguard Corn Oil Powder Mortality (Percent SEM) Emerged (Percent SEM) Treatment Type Emerged Mortality A B 1 2 2 2 B B

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69 Fi gure 2 13 . Adult emergence and pupal mortality from transfer medium cage bioassay. Adult emergence and pupal mortality of 2 nd 3 rd instar larvae exposed to pyriproxyfen contaminated adult females in cage bioassays. Females were allowed 24 hour contact time with pyriproxyfen treated fabrics. Treatments included Esteem ® (a powder pyriproxyfen formulation), Esteem ® mixed with corn oil, Esteem ® mixed with ethanol, Esteem ® mixed with water, and untreated control (N=8). Different letters and numbers denote significant differences among treatments for emergence and mortality. 0 25 50 75 100 0 25 50 75 100 Control Powder Corn Oil EtOH Water Mortality (Percnet SEM) Emergence (Percent SEM) Treatment Emerged Mortality A A A B B 1 1 1 2 2

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70 Figure 2 14 . The adult emergence and pupal mortality from Esteem ® oil concentration cage bioassay. Adult emergence and pupal mortality of 2 nd 3 rd instar larvae exposed to pyriproxyfen contaminated adult females in cage bioassays. Females were allowed 1 hour contact time with pyriproxyfen treated fabrics. Treatments included Esteem ® (a powder pyriproxyf en formulation), Esteem ® mixed with corn oil at 1%, Esteem ® mixed with corn oil at 0.1%, Esteem ® mixed with castor oil at 1%, Esteem ® mixed with castor oil at 0.1%, Esteem ® mixed with Glycerine at 1%, Esteem ® mixed with Glycerine at 0.1%, and untreated con trol (N=6). Different letters and numbers denote significant differences among treatments for emergence and mortality. 0 25 50 75 100 0 25 50 75 100 Mortality (percent SEM) Emergence (Percent SEM) Treatment Emerged Mortality A A A A,B A A,B B 1 1 1 1 1 1,2 2

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71 Figure 2 15 . The numbers of Aedes spp. eggs deposited in ovicups containing oak leaf infusion or water. The numbers of Aedes spp. eggs ( average ± SEM) deposited in ovicups (N=11 13) containing oak leaf infusion or water. Two collections were conducted at field sites in Vero Beach, FL, USA (2015). No significant differences were detected among treatment and control in either collection. 0 5 10 15 20 25 30 Collection 1 Collection 2 Number of Eggs (Mean SEM) Oak Leaf Infusion Tap Water

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72 Figure 2 16 . Number of Aedes albopictus and Ae. aegypti captured in BG Sentinel traps at Martin County sites. N umber of Aedes albopictus and Ae. aegypti captured in BG Sentinel traps, one control trap without a lure and one trap with an experimental host seeking lure. Seven collections were conducted at field sites in Vero Beach, FL, USA (2015). 0 50 100 150 200 250 300 350 Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 Ae. albopictus+ Ae. aegypti Lure Control

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73 Figure 2 17 . Adult emergence and pupal mortality f rom food and water level treatments. Adult emergence and pupal mortality of 2 nd 3 rd instar Aedes albopictus larvae exposed to four food concentration and water level treatments. Treatments included varying levels of tap water and food suspension (1:1 yeas t:albumen): 200L (200mL water, 0.04g food), 100L (100mL water, 0.04g food), 100H (100mL water, 0.08g food), and 200H (200mL water, 0.08g food) (N=5). No significant differences were detected among treatments. 0 20 40 60 80 100 200L 100L 100H 200H Emerged and Mortality (Percent SEM) Treatment Emergence Mortality

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74 Figure 2 18 . Sentinel ovicup tripod (SOT). A sentinel ovicup tripod (SOT) composed of three 0.47L (16oz.) ovicups and used to monitor affects on larval populations. Two of the stadium cups, which served as treatment cups, had a piece of seed germination paper, approximately 4.45x10.16cm (1.75x4in), paper clipped vertically to the inside of the cup. The control cup lacked seed germination paper and instead had three holes, 1.75 1.91cm (11/16 3/4in), drilled in their sides towards the upper lip of the cup. Each hole was covered by a piece of fine me sh netting hot glued to the exterior of the cup. The opening of the control cup was covered by a rubber glove in order to eliminate possible contamination from adult mosquitoes.

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75 Figure 2 19 . Ovicup. A 0.47L (16oz.) ovicup incorporated into field sampli ng and surveillance methods of Aedes albopictus and Ae. aegypti .

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76 Figure 2 20 . Prototype autodissemination station. An autodissemination station shown with essential labeled components. The treated portions of the autodissemination station included the host seeking, rest seeking, an d o viposition seeking portions. The served as weather deterrent housing. Host Seeking Rest Seeking Host L ure Oviposition Seeking Wire Frame Corrugated Roofing (Housing)

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77 Figure 2 21 . Effects of IGR treated autodissemination station at White City Cemetery. The effects of an IGR treated autodissemination station on adult emergence, pupal mortality, and mortality of Aedes albopictus immatures across three collections (A,B,C) showing control (N=14) and treatment (N=28). A) This figure demonstrates the results from the first collection. B) This figure demonstrates the results from the second collection. C) This figure demonstrates the results from the third collection. Means ( ±SEM) represent evaluations conducted at White City Cemetery in Ft. Pierce, FL, USA ( 2015). No significant differences were detected among treatment and control in any collection.

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78 Figure 2 22 . Correlation between mortality and egg numbers at White City Cemetery. The log transformed mortality of Aedes albopictus immatures of both control ovicups and treatment ovicups from sentinel ovicups tripods plotted with respect to the distance to the nearest autodissemination station (ADS). Data taken from a study conducted at White City Cemetery in Ft. Pierce, FL, USA (20 15) exploring the efficiency of an IGR treated ADS. y = 0.0034ln(x) 0.0031 R² = 0.1328 y = 0.041ln(x) + 0.1565 R² = 0.2868 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0 20 40 60 80 100 Log transformed Mortality Distance to Nearest ADS (meters) Control Treatment Log. (Control) Log. (Treatment)

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79 Figure 2 23 . Pupal mortality by site at Martin County locations. The Pupal Mortality of Aedes albopictus immatures (Percent ± mean SEM) (control: N=30; treatment: N=60) across three larval collecti ons from sentinel ovicups tripods testing efficacy of an autodissemination station employing pyriproxyfen from four sites in Martin Co., FL, USA (2015). No significant differences were detected among treatment and control in any collection. 0 5 10 15 20 25 30 S1 S2 S3 S4 Pupal Mortality (Percent mean SEM) Martin Co. Location Control Treatment

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80 Figure 2 24 . Mortality by collection at Martin County locations. Mortality of Aedes albopictus immatures from sentinel ovicups testing efficacy of an autodissemination station employing pyriproxyfen. Means ( ±SEM) (N=80) represent evaluations conducted at four re si dential sites in Martin Co., Fl, USA (2015), across three sampling periods. 0 2 4 6 8 10 12 14 16 18 Collection 1 Collection 2 Collection 3 Mortality (% SEM)

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81 Figure 2 25 . Aedes albopictus and Ae. aegypti females collected by BG Sentinel traps. Aedes albopictus and Ae. aegypti females collected by BG Sentinel traps across four sampling dates at field sites in Martin Co. , FL, USA ( 2015 ) . 0 5 10 15 20 25 30 8/7 8/14 8/21 8/28 Number of female Ae. albopictus + Ae. aegypti Date Site 1 Site 2 Site 3 Site 4

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82 CHAPTER 3 DISCUSSION Laboratory Optimization of a Prototype Autodissemination Station The average landing of males and females at regularly timed intervals proved insightful into which fabric type was more attractive to host seeking females (Figure 2 9 ). The data demonstrated a statistical difference between red velvet and black velvet wit h zebra and bear fabrics when total adults (males+females) were combined (Table 2 2 ). This coincides with what past research has shown that host seeking mosquitoes are generally more attracted to low intensity colors such as blue, black, and red (Allan et al. 1987). However, female only landings were also recorded as this likely plays a greater role in the autod issemination process (Figure 2 10 ). In this part of the analysis, the bear fabric was not included, since landing numbers were minimal and it was exceedingly difficult to differentiate male from female mosquitoes on the bear fabric. In the female only analysis (Table 2 3 ), the results demonstrated an overall greater attractiveness for the red velvet fabric. The hypothesis that the contrasting blac k and white pattern of the zebra fabric would be more attractive was not supported by the data. Likewise Hoel et al. (2011) found that greater numbers of eggs were laid in black ovitraps compared to checkered and striped: black (122.53 ±9.63), checkered (1 01.84±9.53), and striped (84.62±8.17) the black and red velvet. The bear fabric was the least attractive to the mosquitoes, perhaps due to the long fibers of this fabric. For this bioassay it was concluded that the most attractive fabric type tested for Ae. albopictus was the red velvet. This fabric bioassay demonstrated the attractiveness of solid colors, showing that female host seeking Ae. albopictus when offered the four fabri c types tested are most

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83 attracted to an object covered in red velvet. Furthermore, the proportionally long fibers of the bear fabric (faux fur) showed that fabric with these longer fibers were not attractive. This is similar to observations by Kilham and Dalmat (1955) that mosquitoes including Ae. aegypti and Ae. triseriatius would not bite through the denser portions of rabbit fur. Red velvet was the most attractive of the four fabrics tested for host seeking daytime active mosquitoes. In the station s ize bioassay there was an overall similar trend in landings of males a nd females combined (Figure 2 11 ). There was no difference between the landing rates on the larger station with that of the landing rates of the smaller station except in the first time period of 30 seconds (Table 2 4 ). At this one exception the large In contrast to the fabric type bioassay, this experiment did not demonstrate a clear tendency of grea ter attractiveness for either station size. However, the larger of the two stations is the better choice for this ADS design as it provides greater external surface area for mosquito/IGR interaction. In addition, the larger size provides a greater interna l area for placing host lures and resting sites for resting site seeking mosquitoes. The results from the first IGR formulation and transfer medium bioassay showed high emergence in the control group with greatly diminished emergence in the treatments ( Esteem ® , NyGuard ® , Corn Oil) (Table 2 5 and Figure 2 12 ). Also, the pupal mortality in the control group was quite low (0.00 ±0%) , but was as high as 81.66 ±13.21% in the corn oil treatment. The results from this bioassay demonstrated that all treatments w ere effective in reducing adult emergence and causing pupal mortality (Table 2 6 ). Although not an objective of this experiment, it was noted that the

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84 mosquitoes appeared to avoid landing on the NyGuard ® treated station, which was not the case with Esteem ® , making it the IGR of choice for future bioassays. The IGR formulation and transfer medium bioassays were insightful for guiding future experiments. From the previous bioassays , Esteem ® was shown to be the more effective pyriproxyfen formulation. This is supported by other autodissemination studies that also incorporated a powder formulation as the pyriproxyfen formulation in their approach (Wang et al. 2014, Caputo et al. 2012). These experiments demonstrated that the ADS should incorporate a 1% conc entration of Esteem ® /corn oil. The results in the second bioassay (Table 2 7 and Figure 2 13 ) again indicated that c or n o il with Esteem ® at a 1% mixture caused the highest pupal mortality (56.50 ±8.04%) . Ethanol and w ater were highly ineffective media for Esteem ® , possibly degrading the pyriproxyfen, and causing low pupal mortality (15.11 ±13.76% and 4.40 ±2.77%) . Esteem ® powder by itself or with corn oil were the only media in this bioassay that significantly increased mortality, compared to the control (Tab le 2 8 ). However, using Esteem ® by itself in a field setting is not ideal as the powder would not stand up to field conditions as well as Esteem ® with an oil medium. Longevity is crucial to the success of the ADS technique. In the final transfer medium b ioassay, corn oil at a 1% concentration resulted in the highest pupal mortality, followed by castor oil at a 1% concentration (Table 2 9 and Figure 2 14 ). However, only Esteem ® and corn oil at a 1% concentration caused significantly greater mortality comp ared to the control (Table 2 10 ). In the final bioassay ad ult female mosquitoes were exposed to the IGR station for only one hour as opposed

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85 to 24hrs in the previous two bioassays. As expected, the mortalities were lower than previous bioassays for all t reatments . In summary, this series of bioassays indicated that a 1% formulation of Esteem ® in corn oil is the most effective of the fabric treatments tested. Corn oil is a good transfer medium as the non polar nature of the oil will allow for protection of the IGR from external elements. Corn oil is also readily available, safe, and inexpensive. This conclusion was similar to that of Wang et al. (2014) who used an application of Esteem ® and corn oil and tween (a synthetic water and oil soluble liquid) i n their autodissemination station. However, our laboratory studies also showed great variation in the effectiveness of this treatment. Field studies will likely vary to an even greater extent. The OLI proved ineffective in increasing egg numbers in ovic ups as the number of eggs found in ovicups with OLI were not different from ovicups with tap water alone (Figure 2 16 ). This bioassay supported the future use of tap water in the oviposition portion of the ADS rather than OLI. However, in retrospect at t his specific field location the larval habitats were never encountered and were assumed to be subterranean. Perhaps, this lack of preference for OLI over tap water was merely a reaction of this specific population of Ae. albopictus at this specific locati on . Reiskind and Zarrabi (2013) worked specifically with an idea known as natal hab itat preference induction. The concept , when applied to mosquitoes , is that an adult will prefer oviposition locations similar to the environment that it had as an immature . In my bioassay with the OLI, the immature environment for this population may be dramatically different from both the OLI and tap water that would in turn mean a lack of a preference for either

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86 treatment in that neither were ideal oviposition locations. However, Reiskind and Zarrabi (2013) rejected the idea of natal habitat preference induction for Ae. albopictus and rather found that the adults spread their eggs evenly between high and low quality oviposition locations. This suggests that furth er research is necessary on the oviposition lure and that future research should proceed cautiously if incorporating egg laying as an indicator of preference, as it may be an unreliable indicator for Ae. albopictus . BG Sentinel traps baited with the experime ntal host seeking lure collected greater numbers of Ae. albopictus across sample days than a BG Sentinel trap without a lure (Figure 2 17 ). This was an expected outcome as similar combinations of these chemical attract ant s have proven quite capable in pas t experiments. For example Okumu et al. (2010) used a combination of hydrous ammonia, L lactic acid and several carboxylic acids in a synthetic host lure and found no difference between captures of total mosquitoes with their synthetic blend (46.5%) versu s natural human foot odor with carbon dioxide (53.5%). This experimental host lure used in this research proved quite effective in field studies and has potential in a wide variety of research topics , not limited to autodissemination. This host lure is c ost effective and can be easily prepared with accessible ingredients. In the larval habitat enclosure bioassay high rates of emergence were observed across all treatments with corresponding low mortality (Figure 2 15 ). Statistically there was no differen ce be tween any treatments (Table 2 11 ). However, the 100mL low food group boasted zero mortality across all trials and the highest adult emergence. This

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87 testifies to the hardiness of Ae. albopictus larvae especially with respect to their ability to survi ve in areas of food limitation and crowding (Barrera 1996). These bioassays were pivotal in helping to discern the individual elements necessary to derive the methods for constructing and implementing a novel ADS design for field experiments. Field Implem entation of a Laboratory Optimized Autodissemination Station Five different treatments were tested in preliminary field trials. Two of the treatments were tested for two one week trials, Esteem ® (dry application) and Esteem ® with wax (Table 2 13 ). Of the 7 trials only one treatment showed significantly greater mortality compared to the control: Esteem ® with PAM ® Original. In trials of the ADS with Esteem ® and PAM ® , the control ovi cups had 1.01±1.01 % pupal mortality while the treatment ovi cups demonstrated pupal mortality of 44.12±7.47 %. However, if treatment cups that were not contaminated (showing no mortality ) were excluded, the treatment cups exhibited 62.28±6.53 % pupal mortality and 63.89±6.65 % mortality (Table 2 14 ). Mortality was s lightly higher than pupal mortality in a few cases as it include adults that failed to emerge from their pupal skins, which is a documented effect of pyriproxyfen through p upal mortality (or a larval pupal intermediate) and incomplete adult emergence from pupal skins. These mortalities are similar to those reported by Caputo et al. (2012) in their field experiment that achieved mortalities ranging from 50 70% in sentinel cu ps. Due to its effectiveness in raising pupal mortality the Esteem ® and PAM ® treatment was incorporated into all further field evaluations. All three collections from WCC demonstrated high adult emergence between control and treatments and

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88 correspondingly low pupal and mortality (Figure 2 21 ), which was in stark contrast to what was found in preliminary field trials. Across the three collections average treatment adult emergence ranged from 92 94% which was not statistically different than the control, 97 99%. Average treatment pupal mortality ranged from 2 5%, while control was 0 1%, average mortality in the treatment ranged from 2 6% while control was 0 1%. An analysis of the data showed no e ffects on adult emergence or pupal mortality f rom sentinel cups across all thre e collections periods (Table 2 15 ). For the three collection periods at WCC, egg papers were also collected from the treatment cups and egg numbers were counted. During the first collection as might be expected there was a positive linear correlation between mortality and egg numbers and a negative linear correlation between adult emergence and egg numbers. This was an expected outcome, as an increase in egg numbers would suggest greater numbers of visitations to the ovi cups that in turn would increase the likelihood of IGR contamination, thereby increasing mortality. However, in collections 2 and 3 there was no correlation between adult emergence and p upal mortality with egg numbers, which could be due to the deteriorat ion of the IGR treatment on the ADS. However, one of the major objectives of the experiments at WCC was to quantify the relationship between the distance from the nearest ADS and the immature mortality in S OT s. Upon first inspection there appeared to be a possible correlation between the mortality in treatment larval cups and the distance to the nearest ADS (Figure 2 24 ). A linear regression of log transformed mortality found no significant correlation between these variables.

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89 The results from the four locations in Martin Co. were generally similar to those of WCC. Overall, the emergence and mortality between the control and treatment groups appeared fairly similar (Tabl e 2 17 and 2 18 ). Although greater mortality was observed in the treatment at Site 2, no statistically significant difference was detected by ANOVA (Figure 2 25 ). With one exception the results from all four Martin Co. sites showed no significance in all collections between t reatment and control (Table 2 20 ). The one exception was Sit e 4 collection 3, which demonstrated significance in both adult emergence and pupal mortality. However, the treatment had 100% emergence and 0% pupal mortality in all samples and it was actually the control that proved to have significantly lower adult em ergence and higher pupal mortality. The emergence and mortality data from Martin Co. was also analyzed for a correlation between the number of eggs laid on the seed germination papers in the treatment cups and the proportion of adult emergence and pu pal m ortality (Table 2 18 ). No correlation was found between egg laying and the proportion of adult emergence or pupal mortality in the treatment cups. The adult trap collections coincided with the egg papers from the treatment cups demonstrating the strong presence of container breeding mosquitoes at all Martin Co. locations (Figure 2 25 ). Site 3 traps collected the highest numbers of Ae. albopictus and Ae. aegypti over all three collection weeks. With its high populations over a relatively small area, site 3 was an ideal location to demonstrate a working ADS. However, even at this location, the ADS showed no control improvement of these problematic species.

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90 As w ith previous studies, this research was able to demonstrate that an autodissemination approach is a viable strategy for controlling mosquito species that utilize cryptic larval habitats (Caputo et al. 2012, Devine et al. 2012, Gaugler et al. 2011). It is clear that more information must be gathered for future studies to provide a more effective transfer medium for the IGR and ADS. The successful trial using Esteem ® and PAM ® at the field site in Vero Beach demonstrated that this station has potential for c ontrol efforts. However, due to the overwhelming lack of control in all further field experiments, there are elements of this station that must be improved. Species specific behaviors may have been a factor as to why the ADS was only effective at the V ero Beach location. The data from this experiment demonstrated the ability of the ADS impact populations of Ae. albopictus . However, both the locations further south (St. Lucie Co. and Martin Co.) had a higher percentage of Ae. aegypti . While not conclu sive, this evidence may indicate that this ADS design has more potential for Ae. albopictus control than Ae. aegypti . At WCC while the ADS proved ineffective in lowering mortality in all collections, on the first collection there was a positive linear cor relation between egg laying and pupal mortality. This would mean that the more visits (ovipositon) from adult females increased the likelihood of mortality within a larval cup. The only reason this would occur is if females were indeed visiting an ADS an d transferring trace IGR amounts to larval habitats. The idea of oviposition site seeking pressure could be another answer as to why the ADS only worked at some locations. The ADS appeared to work at the Vero Beach location because there were limited ovi

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91 choose the artificial larval habitats in the form of SOTs on which to oviposit. The Vero Beach location was not characterized by a large variety of potential larval habitats. At both the WCC and Martin Co. sites, there was an overwhelming abundance of potential larval habitats. WCC had a large number of vases and potted plants. The Martin Co. sites 1, 3, and 3 all had bromeliads, while site 4 had a great deal of debris that could hold water. Perhaps, the as attractive to females to oviposit on as abundantly natural sites. Unfortunately, this idea was not supported by the egg laying data that demonstrated that eggs were laid across all si tes. Hence, there was no correlation between mortality and egg laying except in the one instance. An explanation at WCC could be that the ADS failed to work due to the size of the population over a large area. At WCC the ADS was dealing with very large mosquito populations coupled with several acres of relatively open field (sparse trees and limited undergrowth). This could mean that the mosquitoes visiting the ADSs were not the same mosquitoes that oviposited at SOTs. Unfortunately, this explanation w ould not work as well with the Martin Co. sites. However, as previously stated, these sites had larger populations of Ae. aegypti and perhaps this species reacted differently from the Ae. albopictus with respect to visitation of the ADS. Another pathway this research could take is a reexamination of the transfer medium. The Vero Beach experiment showed that the station was coated in effective amounts of IGR to effectively increase mortality . However, there could be potential to find an even more effecti ve transfer medium increasing mortality .

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92 In retrospect the methods incorporated into the research for larval and pupal monitoring were slightly flawed. In all cases the larval habitats were left in the field for a maximum of 5 field days. Perhaps this r elatively minimal amount of time did not allow the IGR to build up to toxic levels in the ovicups . A possible way to work around this problem would be to monitor natural larval habitats. Another method would be to incorporate similar methodology as Caput o et al. (2012) who left their artificial larval habitats in the field and collected pupa directly from them, which they then transferred back to the laboratory. If Florida is to have any chance of combating exotic viruses like DENV and CHIKV, it must hav e cost effective strategies to suppress populations of the peri urban mosquitoes that transmit them. Autodissemination is potentially superior to traditional insecticide applications in that mosquitoes deliver the IGR to cryptic larval habitats or properti es that are inaccessible by vector control personnel. However, there is still a great deal of research and data to be collected before a working ADS is derived.

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93 LIST OF REFERENCES Ali A, Nayar JK, RD Xue. 1995. Comparative toxicity of selected larvicides and insect growth regulators to a Florida laboratory population of Aedes albopictus . J. Am. Mosq. Control Assoc. 11: 72 76. Allan, Sandra A., Jonat han F. Day, and John D. Edman. 1 98 7. Visual ecology of biting flies. Annu. Rev. Entomol. 32:297 316. Barrera Roberto. 1996. Competition and resistance to starvation in larvae of container inhabiting Aedes mosquitoes. Ecological Entomology 21:117 127. Barrera R, Amador M, Diaz A, Smith J, Munoz Jordan JL, Y Rosario. 2008. Unusual productivity of Aedes aegypti in septic tanks and its implications for dengue control. Med. Vet. Entomol. 2 2: 62 69. Bartlett Healy K, Healy SP, GC Hamilton. 2011. A model to p redict evaporation rates in habitats used by container dwelling mosquitoes. J. Med. Entomol. 48: 712 716. Baxter, Ian H., Nicola Howard, Clare G. Armsworth, Lucy E. E. Barton, and Chris Jackson. 2008. The potential of two electrostatic powders as the basi s for an autodissemination control method of Plodia interpunctella (Hubner). J. Stored Prod. Res. 44:152 161. Bentley, Michael D., Ivan N. McDaniel, Mitsuyoshi Yatagai, Hai Poong Lee, Reuben Maynard. 1979. p Cresol: an oviposition a ttractant of Aedes tris eriatus . Environ. Entomol. 8:206 209. Bernier, Ulrich R., Daniel L. Kline, Donald R. Barnard, Carl E. Schreck, and Richard A. Yost. 2000. Analysis of human skin emanations by gas chromatography/mass spectrometry. 2. Identification of volatile compounds th at are candidate attractants for the yellow fever mosquito ( Aedes aegypti ). Anal. Chem. 72:747 756. Braks, M. A. H., J. Meijerink, and W. Takken. 2001. The response of the malaria mosquito, Anopheles gambiae , to two components of human sweat ammonia and L lactic acid, in an olfactometer. Physiol. Entomol. 26:142 148. Aedes albopictus in Urban Areas. PLoS Negl. Trop. Dis. 6(8): e1793 . doi:10.1371/journal.pntd.0001793 . Delatte, H., C. Paupy, J.S. Dehecq, J. Thiria, A.B. Failloux, and D. Fontenille. 2008. Aedes albopictus , vector of chikungunya and dengue viruses in Reunion Island: biology and control. Parasite 15:3 13 .

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94 Dell CB, and CS Apperson. 2003. Horizontal transfer of the insect growth regulator pyriproxyfen to larval microcosms by gravid Aedes albopictus and Ochlerotatus triseriatus mos quitoes in the laboratory. Med. Vet. Entomol. 17: 211 220. Devine GJ, Elvira Zamora Perea, Ge rry F. Killeen, Jeffrey D. Stancil, Suzanne J. Clark, and AC Morrison. 2009. Using adult mosquitoes to transfer insecticides to Aedes aegypti larval habitats. Proc. Natl. Acad. Sci. USA 106(28): 11530 11534. Emden, H.F. van and M.W. Service. 2004. Pest and Vector Control. Cambridge, UK, Cambridge University Press. Gaugler R, Suman D, and Y Wang. 2011. An autodissemination station for the transfer of an insect growth regulator to mosquito oviposi tion sites. Med. Vet. Entomol. 26:37 45. Gonzalez R, Suarez M F. 1995. Sewers: The principal Aedes aegypti breeding sites in Cali, Colombia. Am. J. Trop. Med. Hyg. 53:160. Graham, Amanda S., Catherine A. Pruszynski, Lawrence J. Hribar, David J. DeMay, Adriane N. Tambasco, Anne E. Hartley, Edsel M. Fussell, Scott F. Michael, and Sharon Isern. 2011. Mosquito Associated Dengue Virus, Key West, Florida, USA, 2010 . Emerg. Infec t. Dis. 17(11):2074 2075. Guzman, Alfonso and Raul E. Isturiz. 2010. Update on the global spread of dengue. Int. J. Antimicrob. Agents 36S:S40 S42. Hoel, David F., Peter J. Obenauer, Marah Clark, Richard Smith, Tony H. Hughes, Ryan T. Larson, Joseph W. D iclaro, and Sandra A. Allan. 2011. Efficacy of ovitrap colors and patterns for attracting Aedes albopictus at suburban field sites in North Central Florida. J. Am. Mosq. Control. Assoc. 27(3):245 251. Hirano M, Hatakoshi M, Kawada H, and Y Takimoto. 1998. Pyriproxyfen and other juvenile hormone analogues. Crit. Rev. Toxicol. 2:357 394. Itoh T, Kawada H, Abe A, Eshita Y, Rongsriyam Y, et al. 1994. Utilization of bloodfed females of Aedes aegypti as a vehicle for the transfer of the insect growth regulator pyriproxyfen to larval habitats. J. Am. Mosq. Control Assoc. 10: 344 347. Kamal, Hany A. and Emad I.M. Khater. 2010. The biological effects of the insect growth regulators; pyriproxyfen and diflubenzuron on the mosquito Aedes aegypti . J. Egypt. Soc. Parasi tol. 40(3):565 574. Kay BH, Ryan PA, Russell MB, Holt JS, Lyons SA. 2000. The importance of subterranean mosquito habitat to arbovirus vector control strategies in north Queensland, Australia. J. Med. Entomol. 37:840 843.

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95 Kendrick, Katherine, Danielle S tanek, and Carina Blackmore. 2014. Notes from the field: transmission of chikungunya virus in the continental United States Florida, 2014. MMWR Morb Mortal Wkly Rep 2014 63(48):1137. Kilham, Lawrence and Herbert T. Dalmat. 1955. Host vir us mosquito rel ations of S hope fibromas in cottontail rabbits. Am. J. Epidemiol. 61(1):45 54. Kuehn, Bridget M. 2014. Chikungunya virus transmission found in the United States US health authorities brace for wider spread. J. Am. Med. Assoc. 312(8): 776 777. Logan, Jam es G. Michael A. Birkett, Suzanne J. Clark, Stephan Powers, Nicola J. Seal, Lester J. Wadhams, A. Jennifer Modue (Luntz), and John A. Pickett. 2007. Identification of human derived volatile chemicals that interfere with attraction of Aedes aegypti mosquito es. J. Chem. Ecol. 34:308 322. Montgomery BL, Ritchie SA. 2002. Roof gutters: A key container for Aedes aegypti and Ochlerotatus notoscriptus (Diptera: Culicidae) in Australia. Am. J. Trop. Med. Hyg. 67:244 246. Okumu, Fredros O., Gerry F. Killen, Sheil a Ogoma, Lubandwa Biswaro, Renate C. Smallegange, Edgar Mbeyela, Emmanuel Titus, Cristina Munk, Hassan Ngonyani, Willen Takken, Hassan Mshinda, Wolfgang R. Mukabana, Sarah J. Moore. 2010. Development and field evaluation of a synthetic mosquito lure that is more attractive than humans. PLoS ONE 5(1): e8951. doi:10.1371/journal.pone.0008951 bromeliads harboring immature Aedes albopictus and Aedes bahamensis (Diptera: Culicidae) in Florida. J. Vector. Ecol. 20(2):216 224 Peper, Shana M., Benjamin J. Monson, Trevor Van Schooneveld, and Christopher J. Smith. 2015. That which bends up: a case report and literature review of chikungunya virus. J. Gen. Intern. Me d. DOI: 10.1007/s11606 015 3459 3. Ponnusamy, Loganathan, Ning Xu, Satoshi Nojima, Dawn M. Wesson, Coby Schai, and Charles S. Apperson. 2008. Identifi cation of b a cteria and b acteria associated chemcial cues that mediate oviposition site p references by Aedes Aegypti . PNAS 105(27):9262 9267. Reiskind, Michael H. and Ali A. Zarrabi. 2013. Habitat quality favoured over familiarity: a rejection of natal habitat preference induction in the mosquito Aedes albopictus . Ecol. Entomol. 38(1):96 100.

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96 Russell BM , McBride JH, Mullner H, Kay BH. 2002. Epidemiological significance of subterranean Aedes aegypti breeding sites to dengue virus infection in Charters Towers 1993. J. Med. Entomol. 39:143 145. Sihuincha M, Zamora Perea E, Orellana Rios W, Stancil J, Lopez Sifuentes V, et al. 2005. Potential use of pyriproxyfen for control of Aedes aegypti (Diptera: Culicidae) in Iquitos, Peru. J. Med. Entomol. 42:620 630. Simard, Frederic, Elysee Nchoutpouen, Jean Claude Toto, and Didier Fontenille. 2005. Geographic distr ibution and breeding site preference of Aedes albopictus and Aedes aegypti (Diptera: Culicidae) in Cameroon, Central Africa. J. Med. Entomol. y 42(5):726 731. Tapia Conyer R, Betancourt Cravioto M, Mendez Galvan J. 2012. Dengue: an escalating public healt h problem in Latin America. Paediatrics and Internat. Child Hlth. 32:14 17. Teets, Frank D., Moti N. Ramgopal, Kristen D. Sweeney, Amanda S. Graham, Scott F. Michael, Sharon Isern. 2014. Origin of the dengue virus outbreak in Martin County, Flo rida, USA 2 013. Virol. Rep. 1(2):2 8. Trexler, Jonathan D., Charles S. Apperson, and Coby Sch al. 1998. Laboratory and f ield evaluations of oviposition r esponses of Aedes albopictus and Aedes triseriatus (Diptera: Culicidae) to oak leaf i nfusions. J. Med. Entomol. 35 (6): 967 976. United States Census Bereau. 2015a. Census data from St. Lucie County, FL. [Internet] [October 25, 2015] http://www.census.gov United States Census Bereau. 2015b. Census data from Martin County, FL. [Internet] [October 25, 2015] http://www. census.gov United States Census Bereau. 2015c. Census data from Vero Beach, FL. [Internet] [October 25, 2015] http://www.census.gov Wang, Yi, Devi S. Suman, Jacques Bertrand, Limin Dong, and Randy Gaugler. 2014 . Dual treatment autodissemination s tation w ith enhanced transfer of an i nsect growth regulator to mosquito oviposition s ites. Pest Manag. Sci. 70(8):1299 1304.

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97 BIOGRAPHICAL SKETCH Mark Aaron Kartzinel specialized in the field of medical entomology, looking into novel mosquito control techniques. He received a Bachelor of Science in Conservation Biology from Union University in the spring of 2013 and a Mas ter of Science in e ntomology and nematology from the University of Florida in the fall of 2015.