TECHNICAL BULLETIN OF THE FLORIDA CONTROL ASSOCIATION

VOLUME 10, 2016 TECHNICAL BULLETIN OF THE FLORIDA MOSQUITO CONTROL ASSOCIATION

VOLUME 10, 2016 FLORIDA MOSQUITO CONTROL ASSOCIATION, INC. ORGANIZED IN 1922

The Florida Mosquito Control Association, Inc. is a non-profit, technical, scientific, and educational association of mosquito control, medical, public health, and military biologists, entomologists, engineers, and lay persons who are interested in the biology and control of mosquitoes or other· of public health importance.

TECHNICAL BULLETIN OF THE FLORIDA MOSQUITO CONTROL ASSOCIATION

EDITOR-IN-CHIEF: James E. Cilek, Ph.D. E-mail: [email protected]

ASSISTANT EDITOR: Jonathan F. Day, Ph.D. E-mail: [email protected]

ASSISTANT EDITOR: Nathan D. Burkett-Cadena, Ph.D. Email: [email protected]

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CORRESPONDENCE

Communications relating to membership, change of address, and other Association matters should be sent to the Executive Director at: [email protected]. Communications relating to suggested content of future volumes of the Technical Bulletin should be addressed to the Editor-In-Chief. The Technical Bulletin of the Florida Mosquito Control Association is published by the Florida Mosquito Control Association, Inc.

Printed by the E. O. Painter Printing Company P.O. Box 877 DeLeon Springs. FL 32130 ARBOVIRUS SURVEILLANCE AND MOSQUITO CONTROL WORKSHOP

A volume of selected papers from: The 11th workshop, March 25-27, 2014, and the 12th workshop, March 24-26, 2015, and Anastasia Mosquito Control District and its Collaborating Organizations

Edited by: Rui-De Xue

Sponsored by: Anastasia Mosquito Control District St. Johns County, St. Augustine, Florida and USDA-ARS, Center for Medical, Agricultural, and Veterinary Entomology, Gainesville, Florida TABLE OF CONTENTS Introduction Rui-De Xue...... 1 Review Paper Host-seeking and blood-feeding behavior of Aedes albopictus Rui-De Xue...... 2 Research Articles Arbovirus surveillance report in St. Johns County, Florida, 2008-2014 James H. R. Weaver, Catherine Lippi, Mohamed F. Sallam, Marcia K. Gaines, and Rui-De Xue...... 14 Spatial analysis of arbovirus transmission in St. Johns County, Florida Mohamed F. Sallam, Catherine Lippi, and Rui-De Xue...... 18 Relationship between citizen knowledge, vegetation coverage, and frequency of requests for mosquito control service in St. Johns County, Florida Claudia A. Davidson, Jodi M. Scott, Tahjim H. Hossain, John C. Beier, and Rui-De Xue...... 30 Mosquito composition and impact of trapping sites on floodwater mosquitoes, Aedes vexans in Xinjiang, China Mei-De Liu, Yan-De Dong, Gui-Lin Zhang, Zhong Zheng, Rui-De Xue, and Tong-Yan Zhao...... 39 Lunar phase impact on Coquillettidia perturbans and Culex erraticus host seeking in northern Florida Yong-Xing Jiang...... 44 Field evaluation of three commercial mosquito traps and five attractants in northeastern Florida Ali Fulcher, Rachel Shirley, Michael L. Smith, Jodi M. Scott, and Rui-De Xue...... 50 Field evaluation of Mosquiron 0.12CRD against Culex quinquefasciatus in storm drains, downtown St. Augustine, Florida Ali Fulcher, Rui-De Xue, Jodi M. Scott, Michael L. Smith, Marcia K. Gaines, and James H.R. Weaver...... 54 Population reduction of mosquitoes and biting midges after deployment of mosquito magnet traps at a large golf course adjacent to salt marsh habitats in St. Augustine, Florida Rui-De Xue, Whitney A. Qualls, and Dan L. Kline ...... 59 Sublethal doses of attractive toxic sugar bait mixed with the growth regulator, pyriproxifen did not effect survival or fecundity of Aedes albopictus Codi Anderson, Jodi M. Scott, Ali Fulcher, Gunter C. Muller, and Rui-De Xue. . . . . 64 Evaluation of power breezer and misting citronella against Aedes albopictus Emily H. Thompson, Jodi M. Scott, Ali Fulcher, Michael L. Smith, Phil Koehler, and Rui-De Xu...... 70 barrier spray against Aedes albopictus around an urban cemetery, St. Augustine, Florida Christopher Bibbs, Codi Anderson, Michael L. Smith, and Rui-De Xue...... 76 Laboratory evaluation of seven insect repellents against the lone star tick, Amblyomma americanum Jodi M. Scott, Ali Fulcher, John M. Henlzer, and Rui-De Xue ...... 81 Laboratory and field evaluation of OFF! CLIP-ON mosquito repellent device containing metofluthrin against the lone star tick,Amblyomma americanum (Acari: Ixodidae) Rui-De Xue, Jodi M. Scott, Ali Fulcher, Whitney A. Qualls, John M. Henlzer, Marcia K. Gaines, James H.R. Weaver, and Mustapha Debbou...... 85

Operational and Scientific Notes Evaluation of Talent® UV light traps compared with CDC light traps with or without dry ice to collect fresh and salt water mosquitoes in northeast Florida Michael L. Smith, Whitney A. Qualls, and Rui-De Xue...... 91 Effects of leaf washing on the persistence of a sugar bait – mixture to control larval Aedes albopictus Jodi M. Scott, Ali Fulcher, Whitney A. Qualls, Gunter C. Muller, and Rui-De Xue. . . . 93 Field comparison of thermal fog application of sumithrin and barrier spraying of Talstar against Aedes albopictus in residential yards, St. Augustine, Florida Jennifer Gibson, Rui-De Xue, and Michael L. Smith ...... 96 Workshop Programs Program for the eleventh Arbovirus Surveillance and Mosquito Control Workshop, AMCD, St. Augustine, Florida, March 25-27, 2014 ...... 99 Program for the twelfth Arbovirus Surveillance and Mosquito Control Workshop, AMCD, St. Augustine, Florida, March 24-26, 2015 ...... 105

INTRODUCTION

During 2014 and 2015 the Anastasia Mos- chikungunya outbreak and transmission quito Control District (AMCD) of St. Johns risk assessments. Randy Gaugler from Rut- County, Florida held its eleventh and twelfth gers University, New Jersey was the domestic annual Arbovirus Surveillance and Mosquito guest speaker and presented a summary of Control Workshops at District headquarters activities from the Center for Vector Biology in St. Augustine, Florida. Both workshops located on the main campus. Scott Ritchie, were jointly sponsored by the AMCD and the from Australia, gave the international guest USDA, Center for Medical, Agricultural, and speaker presentation on his work with honey Veterinary Entomology (CMAVE) in Gaines- cards for arbovirus detection. Scientists from ville, FL. These workshops were designed USDA/CMAVE, Navy Entomology Center of to facilitate the exchange of information Excellence, universities, as well as mosquito regarding mosquito-borne diseases, review control professionals from industry, state and recent research and developments in arbo- local governmental agencies all provided pre- virus and mosquito surveillance, mosquito sentations that included the latest updates on control, and to offer unique training oppor- mosquito surveillance and control methods tunities for mosquito control professionals. as well as control products and equipment. The eleventh workshop was held from A total of 40 continuing education cred- March 25-27, 2014, and included 66 presen- its (CEU) were provided to workshop at- tations divided into 9 sessions. Due to the tendees between 2014 and 2015. We are outbreak of eastern equine encephalitis virus especially appreciative of the speakers and (EEE) in the USA in 2013, the prevention and contributors who gave presentations in control of this disease was the theme for this these workshops and to those who submit- workshop. Thomas Unnasch from Univer- ted manuscripts for this volume, as well as sity of South Florida, Tampa, was the keynote the organizations/companies who provided speaker and presented some new insights into partial funding for the workshops. We thank the ecology of eastern equine encephalitis vi- those who reviewed manuscripts prior to rus transmission in the southeastern United publication, including Lisa Drake*, Whitney States. Scientists from Mali, Malaysia, Saudi Qualls*, Mohamed Sallam*, Ali Fulcher*, Arabia, China, Brazil, and Israel, USDA/ Jennifer Gibson*, Michael L. Smith, John CMAVE, Navy Entomology Center of Excel- C. Beier*, Barry Tyler, Larry Hribar*, Peter lence, universities, as well as mosquito control Jiang*, Gunter Muller*, Edith Revery, James professionals from industry, state and local Cilek, Sandy Allan, Seth Britch, Muhammad governmental agencies provided presenta- Farooq, Phil Kaufman, Tianyun (Steven) Su, tions that included updates on mosquito sur- Andrew Li, Aaron Lloyd, Gregg Ross, Rich- veillance and control and review of new mos- ard Weaver, Donald Barnard, Dan Kline, Jer- quito control products and equipment. ry Hogsette, Jodi Scott*, Christopher Bibbs*, The twelfth workshop was held from and Charolette Hall* (* indicates reviewers March 24-26, 2015, and included 73 presen- who reviewed more than one manuscript). tations divided into 7 sessions. Due to the We also acknowledge the support and en- outbreak of chikungunya in the Caribbean couragement of the AMCD Board of Com- basin in 2014, the prevention and control missioners, administrative office, District of this disease was the theme for this work- staff, and industry. shop. John Beier from University of Miami Rui-De Xue, Ph.D. Miller School of Medicine was the keynote Director, Anastasia Mosquito Control speaker and provided information on the District

1 HOST SEEKING AND BLOOD FEEDING BEHAVIOR OF AEDES ALBOPICTUS

RUI-DE XUE Anastasia Mosquito Control District 500 Old Beach Road, St. Augustine, FL 32080

ABSTRACT. Aedes albopictus is a vector of related to transmission of pathogens and has dengue and chikungunya viruses. In recent driven the pathway of research to identify and years, this species has expanded its range to develop attractants and repellents for preven- the Americas, Europe, and Africa. Host seek- tion and control of mosquitoes. This paper ing and blood feeding is one of the most im- reviews some of the progress made on Ae. al- portant behaviors mosquitoes possess. Un- bopictus behavior as it relates to finding and derstanding both behaviors in Ae. albopictus successfully feeding on a suitable host. may result in more effective control of this species from a nuisance standpoint, as well II. OLFACTORY STRUCTURES as pathogen transmission. The topics dis- cussed in this review include: olfaction, adult Visual, thermal, and olfactory stimuli all ecdysis (as it relates to first host seeking at- contribute to host seeking and blood feed- tempt), frequency of host seeking and blood ing behavior in female mosquitoes (Bowen feeding, biting cycle and blood feeding pat- 1991). The internal, external ultrastructure tern, feeding location and biting behavior, (as well as possible biological features) of host preference, multiple blood feeding be- virtually all olfactory sensilla on the anten- havior, blood meal size, artificial blood feed- nae of Ae. aegypti (L.) and Anopheles stephensi ing, and a few other miscellaneous factors Liston have been summarized in a review that have been reported to influence host by Sutcliffe (1994). He found that small seeking and blood feeding behavior in this and large sensilla coeloconica, ampullace- species. ae, grooved pegs, and trichodea existed on mosquito antennae, as well as capitate pegs Key Words: host seeking, blood feeding, on mosquito palps. Sensilla coeloconica blood meal, Aedes albopictus and ampullaceae may respond to humid- ity and temperature while trichodea appear to have a sensory function that may distin- I. INTRODUCTION guish oviposition site-related compounds, The Asian tiger mosquito, Aedes albopictus essential oils, or fatty acids associated with (Skuse), is an important vector of dengue human skin and certain repellents (Sutcliffe and chikungunya viruses and a competent 1994). Grooved pegs were also reported to vector of several other arboviruses including have an olfactory function that responded yellow fever, Ross River virus, West Nile virus, to ammonia, acetone, acetic acid, anisole, La Crosse virus, and eastern equine encepha- and lactic acid. Capitate pegs on mosquito litis virus (Mitchell 1991, Ali & Nayar 1997, palpi responded to carbon dioxide(Sutcliffe Gratz 2004). A considerable body of work has 1994). Subsequently, the type, distribution, arisen since its invasion into the Americas and and fine structure of sensillae on the surface Europe in the middle of the 1980’s (Hawley of the antenna of Ae. albopictus was studied 1988, Francy et al. 1990, Rai 1991, Bonizzoni using scanning and transmission electron et al. 2013). Host seeking and blood feed- microscope methods by Huang et al. (1991), ing in Ae. albopictus is a major part of female Xue et al. (1991), and Seenivasagan et al. mosquito behavior and a requirement for re- (2009). One of the olfactory structures on production. This critical behavior is directly the antenna of Asian tiger mosquitoes was

2 Xue: Host Seeking and Blood Feeding Behavior of Aedes albopictus 3 sensilla trichoidea that possessed a pore at 2.5 days for an Ae. albopictus population from their tip with many pores through the hair Nagasaki, Japan, but this time increased to wall. Other structures such as trichodea were 3.5-4.5 days when adults were produced by numerously distributed along all flagella seg- rearing larvae at high densities (Mori 1979). ments of the antenna and consisted of long or short hairs with sharp or blunt tips. In ad- IV. FREQUENCY OF HOST SEEKING dition, long and short types of grooved peg AND BLOOD FEEDING sensillae were also observed on the antenna. Sensilla coeloconicae were observed in the Host seeking activity of Ae. albopictus, terminal flagellum ofAe. albopictus while as with any other species, is stimulated by sensilla chaeticae were distributed through- certain chemical odors and compounds out the body surface and revealed greater (Huang et al. 1991b, Hao et al. 2008, Guha variation in morphology and morphomet- et al. 2014). Klowden and Briegel (1994) re- ric parameters (Seenivasagan et al. 2009). ported that host seeking was inhibited dur- The four types of sensillae from antenna of ing the gonotrophic cycle (as measured with Ae. albopictus are chactica (19%), trichodea an olfactometer). Furthermore, Fukumitsu (71%), grooved pegs (9%), and coeloconica et al. (2012) reported that the concentration (1%), based on their morphological charac- of a neurotransmitter, dopamine, declined ters, thickness of hair wall, pore density, and in the head of Ae. albopictus as host-seeking dendrite number, respectively. Interestingly, increased. Huang et al. (1991) found that olfactory sen- The behavioral sequence of host seeking sillae, in mosquitoes, may be stimulated by and blood feeding in Ae. albopictus is similar host odors before any host seeking behavior to other species of mosquitoes; these steps occurs. include, orientation, landing, probing, and feeding as described by Hocking (1971), Ed- III. ADULT ECDYSIS TO FIRST HOST man and Spielman (1988), Bowen (1991), SEEKING and Takken (1991). Also, the frequency of host seeking and blood feeding depends After adult emergence, juvenile hor- largely upon longevity and number of gono- mone secretion plays an important role in trophic cycles. Generally, female Ae. albopic- the physiological regulatory process in most tus live from 4 to 8 weeks in the laboratory female mosquitoes before host seeking and at 25° C but may survive up to 3-6 months blood feeding (Huang and Xue 1991, Clem- outdoors (Hawley 1988, Xue et al. 2010). A ents 1999, Meola and Readio 1988, Klowden female can take about 1 week to complete 1990, Hansen et al. 2014). However, in Ae. a gonotrophic cycle (the interval between aegypti, host-seeking was either independent successive oviposition) (Hawley 1988) and of juvenile hormone concentration or the may complete 4 to 8 gonotrophic cycles dur- sensitivity threshold was very different than ing her lifetime, according to a laboratory that of the ovaries (Bowen 1991) a process study by Fu et al. (1982). In order to com- that may be similar for Ae. albopictus. plete these numbers of gonotrophic cycles, Several workers have observed that the Ae. albopictus females would need to seek time from adult Ae.albopictus emergence to out hosts and feed at least 4 to 8 times (Fu the beginning of host seeking and blood et al. 1982). However, this may not be abso- feeding varied, depending on mosquito lute, because under laboratory conditions, a strain. del Rosario (1963), Gubler and Bhat- Shenzhen strain of Ae. albopictus fed on a hu- tacharya (1971), Hien (1976), and Huang man subject twice during one gonotrophic and Gao (1991) reported that this period was cycle and 10 times during 7 gonotrophic cy- about 2-3 days, at 24-29° C, for strains from cles (Liu Yang unpubl data). Field data from Calcutta, Vietnam, Philippines, and Shang- Liu (1965) showed that the females, in their hai. The median time from emergence to study, had usually completed 1-2 gonotro- host seeking and blood feeding at 25° C was phic cycles in their lifetime, according to the 4 Technical Bulletin of the Florida Mosquito Control Association, Vol. 10, 2016 number of observable follicular dilations. host and protective behavior, spatial repel- But interpretation of field data must be ap- lents (Hao et al. 2008, 2013), and other mis- proached with caution as the number of fol- cellaneous factors (Xue et al. 1995, Xue and licular dilations is often fewer than the actu- Debboun 2014). al number of gonotrophic cycles completed. For example, no more than 3 dilations were V. BITING CYCLE AND BLOOD observed from field collectedAe. albopictus FEEDING PATTERN even though those individuals were known to have completed 4 or 5 gonotrophic cycles Kawada and Takagi (2004) used a pho- (Liu, 1965). Moreover, if longevity of this toelectric sensor to record the nocturnal be- mosquito species is based on parity status havior of Ae. albopictus and found that host (i.e. gonotrophic cycles) the data may un- seeking was positively correlated with increas- derestimate age (Mori and Wada 1977). ing light intensity where the threshold inten- Time between oviposition events can sity was >10 lux (approximately 1 foot candle) vary with geographic strains of Ae. albopictus. (Kawada et al. 2005). However, these authors Gubler and Bhattacharya (1971) reported added that complete darkness during the day- that a Vietnam strain of this mosquito spe- time deactivated host seeking behavior. Ear- cies oviposited every 4.6 days at 26° C and lier, Hawley (1988) stated that Asian tiger mos- from 3-5 days at 25° C for a Shanghai and quitoes primarily sought hosts and blood fed Guangxi strain from China (Li, 1991). These during the day and rarely at night. Costanaza latter authors also noted that oviposition in- et al. (2015) also found that host seeking ac- terval time was protracted at 20º C to at least tivity varied with light levels (0 to 440 lux). 10 days and considerably shortened to 4.5-6 Consistent biting activity in their laboratory days at 30° C. studies occurred at a photoperiod of 12L:12D. The numbers of blood meals, and fre- Kawada and Takagi (2004) further observed quency of host seeking, were negatively cor- that there were two peaks of diel activity in fe- related with body size in Ae. albopictus (Haw- male Ae. albopictus one at 1000 to 1200 and ley 1988, Farjana and Tuno 2013). Moreover, another from 1400 to 1600 h. Under complete several authors subsequently observed that darkness, biting activity did not peak, but un- frequency of host seeking and blood feeding, der a constant light regime, biting peaked at during a gonotrophic cycle, depended on a 2200-2300 (Huang and Gao 1991a, Xue 1991). variety of factors that included, mosquito The USDA Gainesville, Florida strain, reared age (Xue et al. 1995), body size (Xue & Bar- under different photoperiod regimes in the nard 1996, 2012, Farjana and Tuno 2013), laboratory, did not show temporal significant blood meal size and mating status (Klowden, differences in blood feeding frequency. Also 1988, Lee and Klowden 1999, Barnard and short photoperiods did not affect blood-feed- Xue 2009), parity and time of day (Xue and ing activity of this strain (Table 1). Fu (1990) Barnard 1996), carbohydrate availability and also reported that outdoor biting activity fatigue (Xue and Barnard 1999, Xue and peaked in early morning (7-9 am) and late Debboun 2014), host availability, species of afternoon (5-8 pm) in strains from Shanghai,

Table 1. Effects of photoperiod (at 25° C) on Aedes albopictus first gonotrophic feeding success using baby chickens under laboratory conditions (Xue unpubl data)1.

Photoperiod regime (h)2 No. tested Blood fed (% + SD) L:D=LL 195 74.9 + 6.2 L:D=14:10 400 66.8 + 2.0 L:D=12:12 240 64.2 + 4.0 L:D=08:16 240 59.2 + 4.0 L:D=DD 240 65.5 + 7.6 1Sugar water (3%) was provided when rearing adults. Six day old mosquitoes used in study, 1 hour blood feeding time. 2Photoperiod regime of fourth instar larvae in rearing chamber: L=light, D=dark, LL=continuous light, DD=continuous darkness. Xue: Host Seeking and Blood Feeding Behavior of Aedes albopictus 5 Fujian, Jiangsu, Guangxi and Henan, China. A ally bite the head, feet, and other exposed study by Delatte et al. (2010) also found that skin, according to this author’s observations Ae. albopictus exhibited a bimodal daily feed- (Xue unpubl). Although Ae. albopictus are ing activity in La Reunion where 89% of the usually outdoor biting species, biting fe- mosquito population was exophagic and 87% males can be collected from indoors. were exophilic. Most female Ae. albopictus take 1-2 min- Peak biting activity of Ae. albopictus also ap- utes to completed engorgement. Some pears to vary with location and habitat. A clear strains required 3.6 minutes to engorge bimodal pattern (early morning and late after- on a human arm (Soekiman et al. 1984 in noon) of feeding activity has been observed Hawley 1988). Fu (1990) found this species in Asian countries, such as China, Japan, took 20-30 seconds from probing to abdo- Philippines, and Singapore (Ho et al. 1973). men distention at 23-33°C and RH 68-95%. In southern Brazil, biting activity occurred The complete feeding period from probing during early morning, early evening, and late to engorgement averaged 104.14 seconds night with peaks at 6 am and 10 pm but the (range 70-160 seconds). The temperature greatest activity was between 4-5pm (Margues and humidity change during experiments & Gomes 1997). Interestingly, sugar feeding did not significantly influence feeding per- and host seeking rhythm in Ae. albopictus, re- sistence (Fu, 1990). Hien (1976) also found corded in the laboratory using remote sensors, that biting persistence of Ae. albopictus varied discovered that the evening sugar feeding pe- according to strain, age, body size, host be- riod was similar to the evening host seeking havior, and environmental conditions. period (Yee and Foster 1992). In the laboratory, an indoor population VII. HOST PREFERENCE showed only a single broad period of activ- ity in the afternoon (Wang, 1962). However, Host preference in Ae. albopictus can di- the biting activities of a laboratory Shanghai rectly affect vector competence and trans- strain of Ae. albopictus showed that females mission risk of mosquito-borne disease would feed on a host anytime during 24 pathogens. Determination of host prefer- hours regardless of long or short photoperi- ence by mosquito vectors is usually obtained ods (Huang & Gao 1991a). Also, the USDA by a variety of baits or traps (Takken Gainesville laboratory strain of this species and Verhulst 2013). Blood meal source iden- will also seek, bite, and feed on human hosts tification in Asian tiger mosquitoes can be anytime during a 24 hour period but has ear- obtained by a variety of laboratory methods, ly morning and late afternoon biting peaks such as sera precipitin (Tempelis 1975) agar (Xue and Barnard 1996, 1997). gel-precipitin tests (Sivan et al. 2015), direct In summary, laboratory and field studies or indirect enzyme-linked immunosorbent have shown, without a doubt, that most strains assays (ELISA) using antisera (Ponlawat and of Ae. albopictus are bimodal daytime biters Harrington, 2005), and polymerase chain with a small peak in the early morning and a reaction (PCR) (Richards et al 2006, Egizi et larger peak in the late afternoon. Those strains al 2013). that do not show this bimodality are probably In the laboratory, there are two methods highly inbred laboratory colonies reared un- that can be used to determine host prefer- der artificial light regime conditions. ence of female Ae. albopictus. Olfactometers are often used to test host odors from dif- VI. FEEDING LOCATION AND BITING ferent . Using this method, Miyagi BEHAVIOR (1972) and Gubler (1970) determined that Ae. albopictus preferred feeding on mam- Female Ae. albopictus prefer to bite ex- mals, including mice/rats, guinea pigs, dogs, posed human skin primarily around the cows, and humans. These authors also found ankles and knees (Robertson and Hu 1935). that chickens, snakes, turtles, and frogs can For chickens, this mosquito species will usu- be fed upon by this mosquito species (Miyagi 6 Technical Bulletin of the Florida Mosquito Control Association, Vol. 10, 2016 1972, Gubler 1970). A second method em- cats, horses, rats, or swine were detected in ployed by Hess et al. (1968) used forage ra- those mosquitoes. tios for determination of host preference ob- The majority of blood meals (83.2%) tained by precipitin tests. His results showed from Ae. albopictus in rural and semi-urban a mammal forage ratio >10 times that of areas of Singapore were from humans with birds, but female Ae. albopictus could feed a small percentage from shrews, swine, dogs, on birds when mammals were unavailable. cats, turtles, and multiple hosts in rural set- Konishi’s (1989) laboratory experimental tings (Kek et al. 2014). However, in that same results showed that Ae. albopictus preferred study blood meals from Asian tiger mosqui- to feed on humans compared with canines toes in the urban areas were entirely from when exposed concurrently. However, the humans. Faraji et al. (2014) reported that authors acknowledge that feeding pattern Ae. albopictus from Mercer County, New Jer- in the laboratory and field were influenced sey fed on mammalian hosts with over 90% primarily by host availability. of their blood meals derived from humans In Japan and rural Singapore (Kim et al. (58%) and domestic pets (23% cats and 15% 2009, Sawabe et al. 2010, Kek et al. 2014) Ae. dogs). This mosquito species fed on humans albopictus reportedly fed on mammals. In the significantly more in suburban than in ur- USA, this species preferred mammals and ban areas and cat-derived blood meals were birds (Passeriformes, Columbiformes, and greater in urban habitats while were no avi- Ciconiiformes) as well as rats, dogs, humans, an-derived blood meals were detected . In a rabbits, deer, squirrels, opossums, raccoons, central North Carolina suburban landscape bovine, (Savage et al. 1993, Niebylski et al. study, 7% of blood meals from Ae. albopictus 1994, Faraji et al. 2014, Valerio et al. 2010, were avian hosts while 83% were mammali- 2010a). Asian tiger mosquitoes in Hawaii, an that included humans (24%), cats (21%), also preferred to feed on mammals, includ- and dogs (14%) (Richards et al. 2006). ing dogs, cows, humans, cats, mongeese, Blood from Asian tiger mosquitoes was ex- pigs, and horses (Tempelis 1975, Tempe- clusively human (100%) in urban zones lis et al. 1970). In Missouri and New Jersey from Spain and southern Thailand and 95% Savage et al. (1993) and Faraji et al. (2014), from Cameroon (Munnoz et al. 2011, Pon- respectively, found only a small percentage lawat and Harrington 2005, Kamgang et al. of blood meals from birds in their field col- 2012, respectively). The results show that the lections. Sullivan et al. (1971) reported that Ae. albopictus populations in the New World Ae. albopictus commonly fed on a variety of are opportunistic feeders that use a variety hosts including humans, buffalo, pigs, dogs, of hosts from cold-blooded to warm-blooded and chickens on the island of Koh Samui, animals (Delatte et al. 2010) This fact has Thailand with an even higher percentage the potential for this mosquito species to be- of human feeding detected by sandwich-B come involved in the transmission cycle of enzyme-linked immunosorbent assays (Pon- indigenous arboviruses (Savage et al. 1993). lawat & Harrington 2005). These authors Conversely, feeding preference may also ranked the blood feeding pattern of Ae. al- limit the vector potential of Ae. albopictus for bopictus (top 4 host species) as follows: (1) some arboviruses but the high mammalian humans, (2) bovine, (3) swine, and (4) cat, affinity of this mosquito species suggests that rat, chicken (equal numbers detected). They it can be an efficient vector of mammal-and concluded that nonhuman hosts provided a human-driven zoonoses such as dengue, La significant source of the blood meals forAe. Crosse, and chikungunya viruses. albopictus in Thailand. However, precipitin tests, ELISA, and PCR were used to investi- VIII. MULTIPLE BLOOD-FEEDING gate host-feeding patterns of 172 blood-fed BEHAVIOR Asian tiger mosquitoes collected from Po- tosi, Missouri, during the summers of 1989- Aedes albopictus egg development is initi- 1990. In that study, no blood meals from ated by the blood meal (Xue et al. 2009). Xue: Host Seeking and Blood Feeding Behavior of Aedes albopictus 7 This developmental process is linked to cies possessed a high susceptibility to several the type of host, frequency, and volume arboviruses, including dengue, in the labo- of blood that a mosquito can obtain. It is ratory. Also, Xue and Barnard (1996) found known that anti-mosquito behavior by a that multiple feeding and host-seeking be- host can adversely influence the amount of haviors were negatively correlated with body blood that mosquitoes will have available size. Small body size tended to have more to mature eggs (Walker and Edman, 1985). contact with a host due to low nutrition Other factors related to the mosquito her- (Xue and Barnard 1996). self (i.e. body size, age, and pathogen in- In the laboratory, Ae. albopictus must fection), may result in reduced fecundity or feed on blood at least once for egg matura- failure of egg maturation (Klowden 1988, tion. About 12.5% of a strain from Vietnam Xue and Edman 1991). For example, fe- required two blood meals to successfully male Ae. albopictus blood fed on guinea pig oviposit their first batch of eggs. Gubler and and human hosts produced significantly Bhattacharya (1971) found that females in more eggs compared with other species. Fe- the later gonotrophic cycles required two cundity in mosquitoes that took a double blood meals, but never exceeded 22% of blood meal (chicken and guinea pig), a tri- the population. Mori (1979) reported the ple blood meal (3 separate guinea pigs), or number of blood meals required was de- mixed blood meals (chickens, guinea pigs, pendent on mosquito strain, nutrition, host and humans) produced significantly more behavior, and environmental conditions. eggs than mosquitoes fed once or twice Klowden and Briegel (1994) also observed on a single chicken. Also, triple-feeding or that if Ae. albopictus was allowed to blood- mixed feeding (3 meals on 3 host types) feed to repletion, its host-seeking behav- decreased the gonotrophic dissociation ior was inhibited during that gonotrophic frequency in those mosquitoes (Xue et al. cycle. 2008, 2009). Gonotrophic dissociation oc- In laboratory studies, Fu (1982, 1990) curs when eggs fail to mature after a blood found that about 13-50% of engorged Ae. meal has been obtained [Xue et al. 2009]. albopictus were able to take a second blood Boreham (1976) suggested that mul- meal from a mouse or human arm (Table tiple blood-feeding in vector mosquitoes 2). Furthermore, when Fu (1990) dissected increased the probability of pathogen trans- ovaries of females that had taken a second mission. Because Ae. albopictus will engage in blood meal 60, 75, and 100 hours after their multiple blood-feeding its’ risk of pathogen first blood meal they had produced about 98 infection and transmission is greater than to 102 eggs/female while 84.4% of those in- species that do not possess this ability. In- dividuals had already laid some eggs. In the deed, Shroyer (1986), Lu (1990), Mitchell field, 5% gravid females were collected from (1991), Rai (1991), and Gratz (2004) have human legs in biting collections whereas found that Asian strains of this mosquito spe- 37.8% gravid females were collected from

Table 2. Mean percentage of secondary blood meals from a Aedes albopictus Guangzhou, China strain under labora- tory conditions (from Fu 1990). Mice Human arm

No. tested Secondary feeding (%) No. tested Secondary feeding (%) Time after initial blood meal (h) 12 198 10.0 87 23.0 24 199 9.5 94 37.6 36 200 8.5 NAa NA 48 199 6.0 66 50.0 60 200 17.5 NA NA 72 100 4.0 71 12.7 aNA=not available 8 Technical Bulletin of the Florida Mosquito Control Association, Vol. 10, 2016 human hosts indoors (Fu, 1982). In that 2.) some host seeking gravid individuals field study, 8.3% of collections contained may have fed more than once before com- gravid females that had obtained a second ing to bite the collector. Fu’s result (1982) blood meal during the 24 h observation pe- from laboratory experiments and Li’s obser- riod. In that same study, second blood meals vations from field studies (1991) have pro- by Ae. albopictus were observed in gravid in- vided further evidence that gravid Ae. albop- dividuals at each hourly sample interval dur- ictus can take a second blood meal. Hawley ing biting cycles. (1988) noted that while female Asian tiger Other field studies where multiple blood mosquitoes can take a second blood meal, it feeding by Ae. albopictus has been reported was not necessary for successful egg laying. involve mixed blood meals from avian and However, Hawley further commented that if non-avian hosts (Richards et al. 2006) or the female did not have enough energy re- humans and other mammals (Delatte et serves in the larval stage then a second blood al. 2010). Ponlawat and Harrington (2005) meal may be essential for egg development. found that 3.8% of Ae. albopictus populations contained a mixture of swine-human blood IX. BLOOD MEAL SIZE while <1% of blood meals were dog-human or cat-human. Multiple host blood meals Blood meal size is dependent upon have also been reported from this mosquito mosquito strain, body size, age, host spe- species collected from rural settings in Sin- cies and defense behavior, and environ- gapore (Kek et al. 2014). Also, Tempelis et mental conditions (Klowden 1988, Xue & al. (1970) reported 0.3% mixed blood meals Edman 1991). The mean blood meal size in an Ae. albopictus population from Hawaii. in a Malaysian strain of Ae. albopictus was Molecular analysis (cytochrome b) of blood 2.6 mg (range 1.5-4.2mg) (from Hawley meals from individuals of this species collect- 1988). In a Vietnam strain, blood meal size ed from 2 South Carolina zoological parks was 0.2 mg to 2.5 mg (Hien 1976) and in revealed that the mosquitoes readily fed on a Chinese strain 1.23-1.83 mg (Fu 1990). the captive animals, as well as the local hu- Blood meal size from a Japanese strain was mans and wildlife, resulting in mixed blood 1.2-1.6 ul on unrestrained dogs with 45.2- meals (Tuten et al. 2012). Multiple blood 64.3% of the mosquitoes engorging (Koni- feeding by Ae. albopictus was reported in ev- shi 1989, Konishi and Yamanishi 1984). ery possible combination from the animals Blood meal size in Ae. albopictus also affects and humans present on La Reunion during host seeking activity. Barnard and Xue the chikungunya epidemic (Delatte et al. (2009) state that there was a curvilinear re- 2010). Therefore, the frequency of multiple lationship between blood meal volume in blood-feeding in Ae. albopictus should be fur- partially fed Ae. albopictus and host avidity ther studied in the laboratory and field set- with a threshold between 0.8 and 1.0 mg tings. that reduced host seeking. Hawley (1988) discussed the phenom- enon of multiple feeding in Ae. albopictus X. ARTIFICIAL BLOOD FEEDING as a result of his field discovery during gut dissections of gravid host seeking females. Laboratory colonization of Ae. albopictus Since, then several investigators have dis- is important and necessary for providing re- sected the ovaries of this species from their search material for a variety of R&D projects. human biting collections and found varying In addition, mass rearing of this species for proportions of gravid females. Defining the use in conventional sterile insect release or relationship of parity and number of blood RIDL (release of carrying a dominant meals is confounded by two factors. These lethal) programs for population replace- factors have been pointed out by Hawley ment require a different mass feeding sys- (1988) and are, 1.) some gravid Ae. albopic- tem for adult to egg production. Currently, tus develop eggs without blood feeding and colonization of any blood feeding mosquito Xue: Host Seeking and Blood Feeding Behavior of Aedes albopictus 9 requires vertebrate blood to optimize egg cent blood-free protein diet, using a mem- production. Wind tunnel studies have shown brane feeding system, was evaluated by Pitts that carbon dioxide, water vapor, warmth, (2014). In that study, Ae. albopictus accepted and adenosine triphosphate (ATP) as stim- the diet and produced eggs in greater num- uli that induce host seeking and blood feed- bers than cohort females fed with whole hu- ing activity (Klun et al. 2013). Generally, Ae. man blood. This implied that a readily avail- albopictus is a difficult species to rear when able blood free diet could be utilized in the using artificial techniques (Lyski et al. 2011). laboratory for rearing this species. Elimina- However, this has not deterred a variety of tion of handling blood, reduced animal cost, workers from trying. Usually sausage casings, and consistency of diet should potentially be or lambskin condoms containing warm bo- advantageous to mosquito rearing and mass vine, or other animal, blood have been used rearing facilities (Pitts 2104). for artificially feeding several species of mos- quitoes, including Ae. albopictus. Interesting- XI. OTHER INFLUENCING FACTORS ly, there are instances this mosquito species has preferred sausage casings over lambskin. Host seeking and blood feeding behav- Ultrastructural analysis revealed that sausage ior in Ae. albopictus is a complex behavioral casings have a textured surface while lamb- process. Friend and Smith (1977) have pre- skin membranes do not. viously reviewed several factors affecting Parafilm membrane stretched and feeding by bloodsucking insects. Since that pressed into fiberglass window screen to time, several additional studies have been form a packet for holding warmed blood to conducted on this species and are as follows. feed Ae. albopictus colonies have also been Host seeking and blood feeding in Ae. albop- used successfully by Tseng (2003). Ooi et al ictus is influenced by human physiological (2005) found that cattle skin was the most fa- differences, host species, and host defensive vorable membrane to use after comparative behavior. Shirai et al. (2004) reported that tests with the skin of chicken, fish, and salt human subjects with blood group O attract- sausage. A 32-46% increase in blood feeding ed more Asian tiger mosquitoes than other was observed when Ae. albopictus were fed us- blood groups (i.e. B, AB, and A) but was ing horizontal or vertical blood packets (Ly- only significantly more attractive than blood ski et al. 2011). Also, use of bovine collagen group. sausage membranes in a vertical feeding po- Xue (unpubl) found that host defensive sition will increase the number of engorged behavior significantly affected blood feeding females and may be an additional factor success in Ae. albopictus where the percent- influencing feeding success ofAe. albopictus age of blood-feeding mosquitoes on a re- (Lyski et al. 2011). A novel system by Deng et strained host (chicken) was greater than an al. (2012) consisted of a collagen membrane unrestrained one in laboratory studies (Ta- casing filled with pathogen-free guinea pig ble 3). Also, Xue and Barnard (1996) found blood warmed by a heating device that yield- that host attacking behavior of Asian tiger ed the same fecundity, survival rate, and egg mosquitoes was influenced by age and body hatchability when compared with live guinea size. Large bodied, older females exhibited a pigs. Luo (2014) also used this method and higher frequency of biting humans than did found that Ae. albopictus fed on whole pig younger, small body ones. blood with ATP achieved an engorgement Temperature remains a main factor rate of 84% compared with 51% from live influencing host seeking and blood feed- mice. ing activity in Ae. albopictus. A Chinese Other studies have shown that blood strain of this species will feed in a tem- serum and bovine serum albumin, not he- perature range of 12-35° C in the labora- moglobin, may replace vertebrate blood tory. Fu (1990) found that feeding fre- in artificial diets for mass mosquito rear- quency was increased when temperature ing (Gonzales et al. 2015). However, a re- increased, but a thermal threshold was 10 Technical Bulletin of the Florida Mosquito Control Association, Vol. 10, 2016 Table 3. Effect of temperature on mean percent blood-feeding by Aedes albopictus on restrained and unrestrained chickens under laboratory conditions (Xue unpubl data). Restrained chickens Unrestrained chickens Temperature (°C) No. tested Feeding (%) No. tested Feeding (%) 15 148 3.4 ± 1.2 300 0.7 ± 1.2 20 149 35.7 ± 10.4 280 5.7 ± 2.1 25 151 76.7 ± 3.1 250 1.7 ± 2.7 27 150 69.3 ± 5.0 250 3.0 ± 0.6 30 150 87.3 ± 5.0 150 7.3 ± 2.3 t=3.414, df=4, P<0.05 (between restrained and unrestrained hosts).

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Sutcliffe, J.F. 1994. Sensory bases of attractancy: mor- Xue R. D. and J.D. Edman, 1991. Multiple blood feed- phology of mosquito olfactory sensilla- a review. J. ing by mosquitoes and influencing factors, a review. Am. Mosq. Control Assoc. 10(2 Pt 2):309-315. Cont. Blood Sucking Diptera Ins. 3:137-155. Takken, W. 1991. The role of olfaction in host-seeking Xue, R. D. and D.R. Barnard. 1997. Diel patterns of pu- of mosquitoes: a review. Insect Sci. Appl. 12:287-295. pation, emergence, and oviposition in a laboratory Takken, W. and N.O. Verhulst. 2013. Host preferences population of Aedes albopictus, J. Am. Mosq. Control of blood-feeding mosquitoes. Annu. Rev. Entomol. Assoc. 13:205-207. 58:433-453. Xue, R. D., D.R. Barnard, and C. E. Schreck 1995. Influ- Tempelis, C.H. 1975. Host feeding patterns of mosqui- ence of body size and age of Aedes albopictus on hu- toes, with a review of advances in analysis of blood man host attack rates and the repellency of DEET. J. meals by serology. J. Med. Entomol. 11:635-653. Am. Mosq. Control Assoc. 11:50-53. Tempelis, C.H., R.O. Hayes, A.D. Hess, and W.C. Xue, R. D. and D.R. Barnard. 1996. Human host avidity Reeves. 1970. Blood feeding habits of four species of in Aedes albopictus: influence of mosquito body size, mosquito found in Hawaii. Am. J. Trop. Med. Hyg. age, parity, and time of day, J. Am. Mosq. Control 19:335-341. Assoc. 12:58-63. Tseng, M. 2003. A simple parafilm M-based method for Xue, R.D. and D.R. Barnard. 1999. Effects of partial blood-feeding Aedes aegypti and Aedes albopictus (Dip- blood engorgement and pretest carbohydrate avail- tera: Culicidae). J. Med. Entomol. 40:588-589. ability on the repellency of DEET to Aedes albopictus. Tuten, H.C., W.C. Bridges, K.S. Paul, and P.H. Adler. J. Vector Ecol. 24:111-114. 2012. Blood-feeding ecology of mosquitoes in zoos. Xue, R.D., A. Ali,, and D. R. Barnard. 2008. Host species Med. Vet. Entomol. 26:407-416. diversity and post-blood feeding carbohydrate avail- Valerio, L., F. Marini, G. Bongiorno, L. Facchinelli, M. ability enhance survival of female and fecundity in Pombi, B. Caputo, M. Maroli, and T.A. Della. 2010. Aedes albopictus (Diptera: Culcidae). Exp. Parasitol. Blood-feeding preferences of Aedes albopictus (Dip- 119:225-228. tera: Culicidae) in urban and rural settings within Xue, R.D., D.R. Barnard, and A. Ali. 2009. Influence of the province of Rome, Italy. Parassitologia 50:103- multiple blood meals on gonotrophic dissociation 104. and fecundity in Aedes albopictus. J. Am. Mosq. Con- Valerio, L., F. Marini, G. Bongiorno, L. Facchinelli, M. trol Assoc. 25:504-507. Pombi, B. Caputo, M. Maroli, and T.A. Della. 2010a. Xue, R.D. and D.R. Barnard. 2009. Partial blood meal, Host-feeding patterns of Aedes albopictus (Diptera: Cu- carbohydrate availability, and bloodfeeding post- licidae) in urban and rural contexts within Rome prov- ponement effects on human host avidity and DEET ince, Italy. Vector-Borne Zoonotic Dis. 10:291-294. repellency in Aedes albopictus. J. Am. Mosq. Control Walker, E.D. & J.D. Edman. 1985. The influence of Assoc. 25:431-435. host defensive behavior on mosquito (Diptera: Cu- Xue, R.D., D.R. Barnard, and G.C. Muller. 2010. Effects licidae) biting persistence. J. Med. Entomol. 22:370- of body size and nutritional regimen on survival in 372. adult Aedes albopictus (Diptera: Culicidae). J. Med. Wang, K.C. 1962. One year observation on the ecology Entomol. 47:778-782. of Aedes albopictus (Skuse) in Foochow. Acta Ento- Xue, R. D., and D.R. Barnard. 2012. Mating status and mol. Sinica 11:357-362 (in Chinese with English body size in Aedes albopictus (Diptera: Culicidae) af- summary). fect host finding and deet repellency. Florida Ento- Wiwatanaratanabutr, I., S. A. Allan, K. J. Linthicum, and mol. 95:268-272. P. Kittayapong. 2010. Strain-specific differences in Xue, R.D. and M. Debboun. 2014. Fatigue from mul- mating, oviposition, and host-seeking behavior be- tiple host-seeking activity of Aedes albopictus and its tween Wolbachia-infected and uninfected Aedes albop- effects on blood feeding behavior and DEET repel- ictus. J. Am. Mosq. Control Assoc. 26:265-273. lency. J. Am. Mosq. Control Assoc. 30:116-118. Xue R. D. 1991. Studies on the olfactory behavior of Yee, W.L. and W.A. Foster. 1992. Diel sugar-feeding and Periplaneta americana, Musca domestic and Aedes al- host-seeking rhythms in mosquitoes (Diptera: Culic- bopictus, Chin. J. Vector Biol. Control, 2 (Supple- idae) under laboratory conditions. J. Med. Entomol. ment):5-9 (in Chinese with English summary). 29:784-791. ARBOVIRUS SURVEILLANCE REPORT IN ST. JOHNS COUNTY, FLORIDA, 2008-2014

JAMES H.R. WEAVER, CATHERINE LIPPI, MOHAMED F. SALLAM, MARCIA K. GAINES, AND RUI-DE XUE Anastasia Mosquito Control District 500 Old Beach Road, St. Augustine, FL 32080

ABSTRACT. Arbovirus surveillance is a core programs with the goals of alleviating the function of the Anastasia Mosquito Control risk of disease transmission and mitigating District in St. Johns County, Florida. This disease burden. In the last decade, local con- agency largely relies on the use of sentinel cerns have increased over mosquito-borne chicken flocks to monitor annual incidence diseases since the first record of West Nile of arbovirus transmission. Environmental virus (WNV) in 1999 in New York (Hayes and arbovirus surveillance data for the years et al., 2005) and its eventual spread to St. 2008-2014 were analyzed. Total monthly Augustine in 2001 (Connelly et al. 2012). rainfall and mean monthly temperature In addition to public health concerns, the were found to have significant relationships presence of mosquito-borne diseases can with eastern equine encephalitis seroconver- have significant economic impact. St. Johns sions in sentinel chicken flocks, while only County’s local economy is primarily driven temperature was found to have a significant by tourism. However, in spite of the recent influence on West Nile virus seroconversion. national economic downturn, the county is The identification of environmental and considered one of the fastest growing areas temporal patterns that influence transmis- in the United States (SJC TDC, 2015). In sion of arboviruses is vital to the formation light of this fact, AMCD continues to stress of effective vector management programs the importance of their arbovirus surveil- for the District lance program as a critical part of control activities that provides economic and public Key Words. West Nile virus, eastern equine health benefits for the county. encephalitis virus, sentinel chicken, vector Florida mosquito control arbovirus surveil- mosquitoes lance, as administered by most local programs, continues to serve as an early warning system for mosquito-borne viral diseases. This pro- I. INTRODUCTION gram is part of the “Florida Sentinel Chicken Arbovirus Surveillance Network” established St. Johns County lies in northeastern in 1978 (O’Bryan and Jefferson, 1991). The Florida with a total area of 1,588 km2 (609 sq. Surveillance Network is based on using senti- miles) and is located between the St. Johns nel chicken flocks to monitor epizootic virus River to the west and Atlantic Ocean to the transmission by infected mosquitoes. Anastas- east. As a result, there is great diversity in tia Mosquito Control District participates in salt and fresh water habitats throughout the this program where data from chicken viral County that provides developmental habi- seroconversion rates, together with mosquito tats for approximately 42 mosquito species. surveillance data, provide local public health Eleven of these mosquito species are known and mosquito control officials the needed in- vectors of mosquito-borne pathogens. With- formation to assess the frequency and intensity in this jurisdiction, the Anastasia Mosquito of epizootic virus transmission. Control District (AMCD) oversees the entire Moreover, consistent and appropriate county’s mosquito surveillance and control mosquito collection data, coupled with ar-

14 Weaver et al.: Arbovirus Surveillance Report in St. Johns County, Florida, 2008-2014 15 boviral surveillance, provides the necessary ture were obtained for the time period 2008- justification for the application of vector 2014 from NOAA weather station records control measures for AMCD. Previously, Xue (http://www.weathersource.com). and Qualls (2008) reported arbovirus surveil- Human Surveillance. Human blood sam- lance results for St. Johns County from 2001 ples obtained by local physicians, and other through 2007. In this report, we present the health related professionals, were sent to results and analysis of AMCD’s mosquito ar- the FDOH Virus Laboratory in Tampa and bovirus surveillance program for West Nile tested for WNV, EEE, and Saint Louis en- (WNV), eastern equine encephalitis (EEEV), cephalitis virus. Positive results were shared and Highlands J (HJ) viral diseases in St. by St. Johns County Department of Health Johns County from 2008 through 2014. with AMCD to determine appropriate local vector control measures. II. MATERIALS AND METHODS Other Animal Surveillance.Dead birds re- ported by local residents to AMCD were Sentinel Chicken Surveillance. Sixty 21-week referred to the St. Johns County Health De- old chickens were distributed over 10 loca- partment who sent the remains to the FDOH tions in the county at the beginning of April Tampa Laboratory for detection of WNV. through December every year to monitor Local veterinary reports and clinical samples arbovirus activity. Approximately 2.0 ml of from horses and dogs with suspected WNV blood was taken from each chicken’s wing or EEE infections were tested by the Florida vein once a week. Blood samples were kept Department of Agricultural and Consumer in labeled vacutainers (Fisher Scientific) and Services (FDACS) Arbovirus Laboratory in transported back to the laboratory at the Kissimmee, Florida. This agency shared test AMCD Base Station in St. Augustine Beach results with St. Johns County FDOH and where they were centrifuged at 4,375 RPM AMCD. for 15 minutes. Samples were then placed Statistical analysis. Simple logistic regres- in a labeled and sealed plastic bag, shipped sions were used to determine if significant to the Florida State Department of Health correlations (a=0.05) existed between sen- (FDOH) Virus Laboratory in Tampa, Flori- tinel chicken seroconversion events, mean da and tested for WN, EEE, and HJ viruses. monthly temperature, and total monthly Blood samples were sent to the Laboratory rainfall using the statistical software pro- on a Monday and results reported to the gram R (ver. 3.13) (R Core Team 2012). District by the end of the same week. Once a chicken seroconverted it was removed, de- III. RESULTS AND DISCUSSION stroyed, and replaced with a new bird at its respective location. A total of 389,899 mosquitoes were col- Mosquito Surveillance. Population abun- lected from CDC light traps from 2008 to dance of adult mosquito vectors were moni- 2014 with an average of 5,026 mosquitoes tored by AMCD using CDC light traps (John collected per site annually. Forty mosquito W. Hock Company, Gainesville, FL) baited species were collected during the study pe- with octenol (BioSensory, Inc., Putnam, CT) riod, including Culiseta melanura Coquillett, at 41 permanent locations in the County. Culex nigripalpus Theobald, and Cx. quinque- Traps were placed outdoors at the beginning fasciatus Say, the known vectors of EEEV, HJ of March through the last week of November. virus, SLE, or WNV. Traps were suspended 1 m above ground sur- The only human case of arbovirus infec- face by a shepherd’s hook and operated for tion in St. Johns County from 2008-2014 was 18-20 h using a 12-v battery. Mosquito collec- a WNV asymptomatic blood donor reported tions were transported from the field to the by FDOH in September 2014. During 2008- AMCD facility and identified to species us- 2014 there were 184 positive WNV, 113 posi- ing the taxonomic keys of Darsie and Ward tive EEE, and 34 positive HJ sentinel chick- (2005). Monthly mean rainfall and tempera- ens reported within the County (Fig. 1). No 16 Technical Bulletin of the Florida Mosquito Control Association, Vol. 10, 2016

Figure. 1. Annual totals of positive sentinel chicken arbovirus seroconversions for West Nile, eastern equine encephalitits, and Highlands J virus (2008-2014), St. Johns County, Florida. arboviral positive dead birds were reported December with the majority of cases occur- during this time period. There were 5 con- ring in September (Fig. 2). Eastern equine firmed EEE horse cases from 2008 to 2014. encephalitis seropositive sentinel chickens West Nile virus seropositive sentinel were reported from March through Novem- chickens were reported from May through ber with most seroconversions occurring

Figure. 2. Monthly totals of positive sentinel chicken arbovirus seroconversions for West Nile and eastern equine encephalitits virus (2008-2014), St. Johns County, Florida. Weaver et al.: Arbovirus Surveillance Report in St. Johns County, Florida, 2008-2014 17 in June (Fig. 2). Highlands Jay infection of geographical sampling resolution can be sentinel chicken flocks was recorded from strengthened through the adoption of novel April through September. Most seroconver- mosquito arbovirus surveillance technolo- sions occurred during July. Eastern equine gies incorporating geographic information encephalitis horse cases occurred in March systems (GIS) and molecular diagnostics. through July. The average rainfall from 2008 to 2014 IV. ACKNOWLEDGEMENTS was 116.9 cm. In 2009, the average rainfall was 155.1 cm, which was the most rain re- We thank all employees of AMCD and ceived in a year during this 7 year period. To- former employees, Whitney Qualls and Ali tal monthly rainfall did have a significantly Fulcher, who partially coordinated and par- positive relationship with EEE serocon rates ticipated in this program. We are thankful in sentinel chicken flocks β( = 0.09, SE = for the support from the property owners 0.04, p = 0.028, α = 0.05). It appeared that who allowed us to use their property to place rainfall may have had a slight negative cor- the sentinel chickens. relation with WNV (β = -0.07, SE = 0.04, p = 0.06, α = 0.05) and HJ (β = -0.01, SE = 0.06, p VI. REFERENCES CITED = 0.82, α = 0.05) seroconversion in sentinel flocks but these relationships were not statis- Connelly, C.R., J.F. Day, and W.J. Tabachnick. 2012. tically significant. West Nile Virus. Institute of Food and Agricultural The average temperature from 2008 to Sciences (IFAS), University of Florida, Gainesville, 2014 was 21.0°C. The highest monthly aver- FL. Pub. #ENY-642. Darsie, R.F. and R.A. Ward. 2005. Identification and Geo- age temperature was recorded at 21.7°C in graphical Distribution of the Mosquitoes of North America, 2012. For the 2008-2014 time period, the North of Mexico. University of Florida Press, Gaines- incidence of WNV in sentinel chickens was ville, FL. Hayes, E.B., J.J. Sejvar, S.R. Zak, R.S. Lanciotti, A.V. negatively correlated with average monthly Bode, and G.L. Campbell. 2005. Virology, pathol- temperature (β = -0.13, SE = 0.02, p = 1.78e-8, ogy, and clinical manifestations of West Nile virus α = 0.05) while EEE seroconversions were disease. Emerg. Infect. Dis. 11:1174-1179. O’Bryan, P.D. and H.J. Jefferson. 1991. The year of the positively correlated with temperature (β = chicken: the good and bad of a sentinel chicken 0.11, SE = 0.02, p = 4.71e-6, α = 0.05). There flock during the 1990 Florida SLEV epidemic. J. was no significant relationship between HJ Florida. Mosq. Control Assoc. 62:59-63. R Core Team. 2012. R: A Language and Environment for and temperature (β = 0.07, SE = 0.04, p = Statistical Computing. Version 3.13. R Foundation 0.05, α = 0.05). for Statistical Computing, Vienna, Austria. ISBN Arbovirus transmission is influenced by a 3-900051-07-0. SJC TDC. (St. Johns County Tourist Development complex system of environmental, host, and Council) 2015. Tourism in St. Johns County. St. vector interactions (Tabachnick, 2010). De- Johns County, Florida, Government. http://www. spite the complex nature of this system, the co.st-johns.fl.us/TDC [Accessed 2 June 2015]. Tabachnick, W.J. 2010. Challenges in predicting cli- combination of surveillance methods used mate and environmental effects on vector-borne by AMCD provides the basic framework nec- disease episystems in a changing world. J. Exp. Biol. essary to protect the health and well being 213:946-954. Xue, R.D. and W.A. Qualls. 2008. Arbovirus surveillance of citizens and tourists alike. However, we in St. Johns County, Florida. Tech. Bull. Florida suggest that future agency surveillance and Mosq. Control Assoc. 8:27-30. SPATIAL ANALYSIS OF ARBOVIRUS TRANSMISSION IN ST. JOHNS COUNTY, FLORIDA

MOHAMED F. SALLAM1,2,3, CATHERINE LIPPI1, AND RUI-DE XUE1 1Anastasia Mosquito Control District 500 Old Beach Road, St. Augustine, FL 32080

2Department of Entomology and Nematology, University of Florida Gainesville, FL 32611

3Department of Entomology, College of Science Ain Shams University Cairo, Egypt

ABSTRACT. The arbovirus surveillance pro- I. INTRODUCTION gram conducted by Anastasia Mosquito Con- In the past decade, the adoption of Geo- trol District is an early warning monitoring graphic Information System (GIS) technolo- system designed to identify the factors affect- gies by public health agencies has promoted ing the transmission risk of vector borne dis- an increased awareness of the spatial and eases. The sporadic frequency and intensity temporal distributions of disease. A power- of eastern equine encephalitis virus (EEEV) ful tool in the management of community and West Nile virus (WNV) transmission in level diseases, geospatial methods have been St. Johns County prompted the use of spatial successfully used to model disease transmis- pattern analysis to understand the geograph- sion dynamics and produce spatial predictive ic distribution of both diseases in the County maps of disease distribution (Dambach et al. using Geographic Information System (GIS) 2012). Geographic Information Systems have technology. The significance of GIS applica- also proven useful for studying the spatial and tion in surveillance programs stem from its temporal distribution patterns of mosquito- potential to determine the spatial distribu- borne diseases and their vectors (Kulkarni et tion patterns and directional trends of dis- al. 2010, Abdel-Dayem et al. 2012, Sallam et eases and to produce transmission probability al. 2013). The interactions between environ- risk maps. Data from 2008-2014 on frequency mental variables, hosts, and disease vectors and intensity of EEEV, WNV sentinel chicken are often complex, so the ability to accurate- seroconversions and subsequent mosquito ly model these parameters at the local level vector abundance of Culiseta melanura, Culex would provide valuable information for pub- nigripalpus, and Cx. quinquefasciatus were used lic health management decisions, policy for- in analyses. Geographically, mosquito vector mation, surveillance, and disease mitigation. density and frequency/intensity of disease The Anastasia Mosquito Control District transmission was randomly distributed. This (AMCD) currently provides all mosquito was also confirmed by logistical analysis that surveillance and control in St. Johns County, indicated a significantly reduced dependency Florida, with the goals of mitigating disease of EEEV and WNV chicken serconversion re- burden and minimizing transmission risk. St. cords with vector density. Johns County lies in the northeastern part of Florida, USA with a total area of 1,588 km2 Key Words. eastern equine encephalitis vi- (609 sq. miles). The County is positioned rus, West Nile virus, Culiseta melanura, Culex between the St. Johns River to the west and nigripalpus, Culex quinquefasciatus, Geograph- the Atlantic Ocean to the east (Figure 1). As ic Information System a result, there is great diversity in saltwater,

18 Sallam et al.: Spatial Analysis of Arbovirus Transmission in St. Johns County, Florida 19

Figure 1. Culex quinquefasciatus collection sites within 5 km buffer zones around sentinel chicken traps. freshwater, and vegetative habitat features are known vectors of several pathogens, no- throughout the County, providing develop- tably arthropod-borne viruses (Lord and Day mental habitats for about 43 mosquito species 2001, Sardelis et al. 2001, Blackmore et al. (Naranjo et al. 2014). Eleven of these species 2003, Cupp et al. 2003). Public health con- 20 Technical Bulletin of the Florida Mosquito Control Association, Vol. 10, 2016 cern has continued to surface over mosquito- II. MATERIALS AND METHODS borne diseases since the first domestic case of West Nile virus (WNV) was recorded in New Sentinel Chicken Surveillance. Sixty, 21- York in 1999 (Hayes et al. 2005). West Nile week old chickens were distributed over 12 virus was first reported in St. Johns County in locations in the County at the beginning of 2001 (Connelly et al. 2012). April through December every year during Arbovirus surveillance in Florida serves 2008-2014 to monitor arbovirus activity. Ap- as an early warning system for potential proximately 2.0 ml of blood was taken from outbreaks of mosquito-borne viral diseases. each chicken’s wing vein once a week. Blood Established in 1978, the Arbovirus Surveil- samples were kept in labeled vacutainers lance Network is the primary system for (Fisher Scientific) and transported back to monitoring mosquito-borne diseases in the laboratory at the AMCD base station in the state of Florida (O’Bryan and Jefferson St. Augustine Beach where they were centri- 1991). This network is largely reliant on fuged at 4,375 RPM for 15 minutes. Samples the use of sentinel chicken flocks to moni- were then placed in a labeled and sealed tor endemic virus transmission. In St. Johns plastic bag, shipped to the Florida State De- County, sentinel chicken arboviral surveil- partment of Health (FDOH) Virus Labora- lance is a core function of AMCD. Sentinel tory in Tampa, Florida where blood samples chicken seroconversion records and mos- were tested for WNV, and EEEV. Samples quito surveillance data are collected by the were sent to the Lab on Mondays and results District that, in turn, aids public health and reported to AMCD by the end of the same mosquito control officials in assessing the week. Once a chicken seroconverted it was incidence and transmission risk of endemic removed, destroyed, and replaced with a virus transmission. new bird at its respective location. Data driven conclusions drawn from Mosquito Surveillance. Population abun- surveillance data provide the necessary jus- dance of adult host seeking mosquito vec- tification for the application of larvicides tors were monitored by AMCD using CDC and adulticides. Prior to our study, there light traps (John W. Hock Company, Gaines- has been no attempt to quantify the spatial ville, FL) baited with dry ice at 68 permanent distribution of WNV and EEEV in St. Johns locations in the County. Traps were placed County. In this report, we present a spatial outdoors at the beginning of March through analysis of the AMCD’s mosquito arbovirus the last week of November during 2008-2014. surveillance program for EEEV, WNV, and Traps were suspended 1 m above ground sur- abundance of vectors in St. Johns County face by a shepherd’s hook and operated for for the years 2008–2014. The delineation of 18-20 h using a 12-v battery. Mosquito col- geographic distribution patterns and spa- lections were transported from the field to tial direction trends of both diseases and the AMCD facility for further identification their vectors may help to gain insight into to species level using the taxonomic keys of their spatial characteristics. Indeed, mos- (Darsie and Ward 2005). quito abundance has been addressed in Abundance data of WNV Culex quinque- previous studies by other authors and have fasciatus Say and Cx. nigripalpus Theobald, been identified as a major predictor in the and EEEV vectors, Culiseta melanura (Co- likelihood of epizootics (Scott et al. 1983, quillett), were extracted from AMCD field Anderson et al. 1999, Lord and Day 2001). records within a 5 km buffer zone around Also, we plan on comparing EEEV and each sentinel chicken trap using the geopro- WNV seropositive sentinel chicken records cessing package in ArcGIS (ver. 10.0). Buffer with that of vector population density in or- zones were used as an indication of proba- der to test the hypothesis that intensity of ble mosquito transmission within their flight EEEV and WNV transmission are correlated range around sentinel chicken traps (De- with the density of the mosquito vectors in Meillon 1934, Nayar and Sauerman Jr 1973) St. Johns County. (Figures 1-2). In addition, the study area was Sallam et al.: Spatial Analysis of Arbovirus Transmission in St. Johns County, Florida 21

Figure 2. Culiseta melanura collection sites within 5 km buffer zones around sentinel chicken traps. categorized into AMCD operational route lance and control activities conducted by zones (Figure 3) previously extracted from AMCD field personnel. In addition, these St. Johns County GIS division. This was operational route zones were coupled with done in order to provide data driven guid- the risk maps produced in order to accu- ance risk maps for the operational surveil- rately identify the focal risk areas. 22 Technical Bulletin of the Florida Mosquito Control Association, Vol. 10, 2016

Figure 3. Map of St. Johns County showing operational route zones and open water bodies.

Spatio-Statistical Analysis. Parameters of viational ellipse (SDE) analyses in this pack- EEEV and WNV seroconversion data (fre- age were used to summarize the spatial pat- quency and intensity), and mosquito vector terns of the above parameter values around density were analyzed using spatial statistics their mean. The SDE creates a polygon to tools in ArcGIS (ver. 10.0). The standard de- summarize the spatial directional distribu- Sallam et al.: Spatial Analysis of Arbovirus Transmission in St. Johns County, Florida 23 tion which may show dispersion, central ported from the County for EEEV and WNV tendency, and directional trends. Therefore, antibodies, respectively. A total of 389,899 SDE was used to map the directional distri- mosquitoes were collected from CDC light bution trend of mosquito vectors and spread traps with an annual average of 5,026 per of EEEV and WNV transmission. site. Forty mosquito species were collected Spatial autocorrelation analysis, using during the study period including EEEV and Global Moran’s I Index, was used to identify WNV bridge vectors and other non-anoph- the distribution pattern of mosquito vectors, eline mosquitoes that represented 68.43% EEEV, and WNV sentinel chicken serocon- of the total mosquitoes collected. Culiseta version. The spatial autocorrelation pattern melanura, Cx. nigripalpus, and Cx. quinquefas- may be random, clustered, or dispersed (Lee ciatus represented 1.01%, 5.91%, and 2.72%, and Wong 2001). The distribution pattern is respectively of the total non-anopheline predicted by comparing values of neighbor- mosquitoes collected during the study. ing sampling sites for: 1.) mosquito vector The SDE analysis indicated that density density and 2.) frequency and intensity of of Cs. melanura, Cx. nigripalpus, and Cx. quin- EEEV and WNV seroconversion records. A quefasciatus and intensity records of EEEV, strong positive spatial autocorrelation (clus- WNV were randomly distributed and showed tered) is indicated when the neighboring a directional trend about the mean; this is sampling points have similar values. Where- verified by the ellipsoid polygons in Figures as, if the neighboring sampling points have 4-6. SDE analysis revealed that a random dissimilar values (dispersed), then it indi- distribution pattern highlighted the habitat cates strong negative spatial autocorrelation. suitability for these mosquito vectors within The value of Moran’s I Index is between -1 the ellipsoid polygons. These results were and 1. The ‘Z’ score value is calculated to test further confirmed by Moran’s I index values whether the observed clustering or dispers- where these same parameters were found ing is significant. When the Z score indicates to be insignificant P( >0.05) confirming the statistical significance, a positive Moran’s I findings generating by the SDE polygons. index indicates tendency toward clustering Because we sampled adult host seeking mos- while a negative value indicates tendency to- quitoes, these individuals were most likely ward dispersion. When the Z score value is newly emerged from their developmental not significantly different from 0, there is no habitats within the risk polygons. Accord- spatial autocorrelation and the pattern does ingly, this reflects the probable abundance not appear to be significantly different from of larval habitats for these vectors within the a random distribution. The null hypothesis polygons. Similarly, extracted data on mos- (Ho) for the current analysis was that there quito vectors around sentinel chicken traps is no spatial clustering of EEEV, WNV, with reflected the flight range of these individu- abundance of the mosquito vectors in St. als and the availability of their host(s) as a Johns County. source for blood meals. Although adult host Logistic regression R statistical software seeking mosquito vectors were collected out- (ver. 3.13) was used to assess the relationship side the risk polygons (which may have been between frequency of sentinel chicken sero- infective with either EEEV or WNV) larval conversion records and the abundance of habitats within the ellipsoid polygons are their corresponding mosquito vectors (i.e., worthy of surveillance and should be evalu- Cs. melanura, Cx. nigripalpus, and Cx. quinque- ated for control compared with other areas. fasciatus) sampled within a 5 km buffer zone The random distribution of seropositive around the chicken flocks. records of EEEV and WNV indicated virus circulation inside the mosquito vectors rather III. RESULTS AND DISCUSSION than virus amplification inside bird host(s). Although the spatial analysis potentially pre- From 2008 through 2014 there were 113 dicted the directional distribution pattern of and 184 sentinel chicken seroconversions re- infective mosquito vectors, and their flight 24 Technical Bulletin of the Florida Mosquito Control Association, Vol. 10, 2016

39.4%

42%

61%

Figure 4. Statistical distribution pattern of WNV and Culex quinquefasciatus in St. Johns County showing standard deviational ellipsoids. range, during host seeking, the number of tributed to lack of consistent distribution data parameters used in this analysis prevented us on reservoir host(s), which represents the from highlighting virus amplification inside source of infection for uninfected mosquito their reservoir host(s). This limitation is at- vectors within their flight range. Sallam et al.: Spatial Analysis of Arbovirus Transmission in St. Johns County, Florida 25

42% 51%

39.4%

Figure 5. Statistical distribution pattern of WNV and Culex nigripalpus in St. Johns County showing standard deviational ellipsoids.

The GIS spatial analysis predicted high risk area of the County, respectively. These areas areas for mosquito bites for Cs. melanura, Cx. ni- were found to represent AMCD operational gripalpus, and Cx. quinquefasciatus that covered route zones N01-N03, C04-C08, S01-S03 for 806.67 km2 (50.80%), 825.12 km2 (51.96%), the three mosquito vectors, respectively. Fur- and 961.16 km2(60.53%) of the total land thermore, the amount of land under EEEV 26 Technical Bulletin of the Florida Mosquito Control Association, Vol. 10, 2016

51%

45%

20.29%

Figure 6. Statistical distribution pattern of EEEV and Culiseta melanura in St. Johns County showing standard deviational ellipsoids. and WNV transmission risk from probable C04-C08, S01 for EEEV and N02, N03, C04- infective mosquito vectors represented 45% C09, S01, S02 for WNV. Overlapping between and 42%, respectively, of the total area of St. polygons produced for high transmission risk Johns County. These risk areas were found to areas shown in Figs. 4-6 show the likelihood represent operational route zones N02, N03, of probable association between pathogen, Sallam et al.: Spatial Analysis of Arbovirus Transmission in St. Johns County, Florida 27 vector, and reservoir host. In this context, (Scott et al. 1983, Anderson et al. 1999, the overlapping areas under risk of probable Lord and Day 2001). We found that east- infective mosquito bites should be targeted ern equine encephalitis virus seroconver- for EEEV and WNV surveillance and control sion rates were significantly correlated with activities rather than the entire county. the density of Cs. melanura (β = 0.001, SE = The predicted distribution developed by 0.000, p = 1.10e-7, α = 0.05) (Figure 7). Also, SDE polygons demonstrated the relative ex- the WNV serocons were positively correlated panded distribution pattern of Cx. quinque- with the abundance of Cx. quinquefasciatus (β fasciatus compared with the other two vec- = -0.002, SE = 0.000, p = 5.26e-10, α = 0.05) tors. This reflected either a wide flight range and Cx. nigripalpus (β = 0.003, SE = 0.000, p of this mosquito or the availability of suitable = 1.10e-14, α = 0.05) (Figure 8). Surprisingly, larval habitats and/or availability of hosts as even though correlations were significant a blood meal source. However, the param- (i.e. P<0.05) between seroconversion rates eters used in our analysis prevented us from and vector abundance, in our study, the de- addressing actual flight range away from pendency of seroconversion on mosquito their larval habitats. Further investigations vector density was considerably reduced be- are needed to include more parameters on cause both values of (β) and (p) were close larval developmental sites in proximity to to zero which indicated that those relation- wild reservoir host populations. ships were random. These results highlight In previous studies, the density of mos- the important contribution that other culi- quito vectors played a major role in predict- cine bridge vector(s) may have in the disease ing the transmission of arboviral diseases transmission cycle. Moreover, our findings

Figure 7. Annual abundance of eastern equine encephalitis virus seropositive sentinel chicken records, Culiseta melanura density during 2008-2014. 28 Technical Bulletin of the Florida Mosquito Control Association, Vol. 10, 2016

Figure 8. Annual abundance of West Nile virus seropositive sentinel chicken records, Culex. quinquefasciatus, and Cx. nigripalpus density during 2008-2014. were confirmed by the evident slight shifting ecological niches for mosquito vectors, sea- in SDE polygons between mosquito vectors sonal abundance of animal reservoirs such and their diseases (Figures 4-6). Previous as birds, amphibians, reptiles, and possible studies confirmed thatCs. melanura, Cx. ni- bridge vector(s). Additional information is gripalpus, and Cx. quinquefasciatus are compe- needed from these parameters in order to tent vectors in transmitting EEEV and WNV shed light on virus amplification and char- (Anderson et al. 1999, Blackmore et al. 2003, acterize various habitats suitable for mosqui- Cupp et al. 2003) in comparison with other to vectors and reservoir host(s). Although mosquito vectors. However, the contribution some disease surveillance methods provide of other culicine bridge vectors such as Cx. more information than others, combining restuans Theobald, Cx. erraticus (Dyar and traditional operational surveillance pro- Knab), Aedes vexans (Meigen), and Uranotae- grams with GIS technologies and modeling nia lowii Theobald, for EEEV transmission packages will enhance the ability of public (Chamberlain et al. 1954, Cupp et al. 2003, health agencies to maximize the benefits of Cohen et al. 2009, Estep et al. 2013) and Cx. practical application of disease surveillance salinarius Coquillett for WNV (Sardelis et al. data. Developing real time data driven risk 2001, Blackmore et al. 2003) should be con- maps will help in the decision making pro- sidered. The contribution of other bridging cess by prioritizing control activities, at the vectors exacerbates the complexity of virus District level, while increasing control effec- circulation in nature. The variation of feed- tiveness, plus reducing labor and associated ing preference by bridge vectors may sustain cost especially during periods of arboviral virus circulation in other reservoir host(s) outbreaks. such as amphibians and possibly reptiles (Cupp et al. 2003, Cupp et al. 2004). IV. ACKNOWLEDGEMENTS In summary, AMCD arbovirus surveil- lance showed that the overall distribution We would like to express our deep grati- of EEEV, WNV and their mosquito vectors tude to Mr. Richard Weaver for his consis- were confined to certain areas of St. Johns tent help in supporting us with the necessar- County. This may be attributed to possible ily data for the current work. Also, we extend Sallam et al.: Spatial Analysis of Arbovirus Transmission in St. Johns County, Florida 29 our thanks to all employees in AMCD and bined remote sensing techniques to detect environ- mental variables influencing malaria vector densi- former employees especially Dr. Whitney ties in rural West Africa. Int. J. Health. Geogr. 11:8. Qualls and Ali Fulcher. Moreover, authors Darsie, R. F., and R. A. Ward. 2005. Identification and Geo- extend their gratitude to the two anonymous graphical Distribution of the Mosquitoes of North America, North of Mexico. University of Florida Press, Gaines- reviewers for their valuable comments that ville, FL. potentially helped in improving the read- DeMeillon, B. 1934. Studies on insects of medical im- ability and the significance of the current portance in South Africa. South African Inst. Med. Res. VI:249-308. investigation. Estep, L. K., C. J. W. McClure, P. Vander Kelen, N. D. Burkett-Cadena, S. Sickerman, J. Hernandez, J. Jin- right, B. Hunt, J. Lusk, V. Hoover, K. Armstrong, L. VI. REFERENCES CITED M. Stark, G. E. Hill, and T. R. Unnasch. 2013. Risk of exposure to eastern equine encephalomyelitis vi- Abdel-Dayem, M. S., B. B. Annajar, H. A. Hanafi, and rus Increases with the density of northern cardinals. P. J. Obenauer. 2012. 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Utilization of com- rus, Turlock (Bunyaviridae). J. Med. Entomol. 20: 625-640. RELATIONSHIP BETWEEN CITIZEN KNOWLEDGE, VEGETATION COVERAGE, AND FREQUENCY of REQUESTS FOR MOSQUITO CONTROL SERVICE IN ST. JOHNS COUNTY, FLORIDA

CLAUDIA A. DAVIDSON1, JODI M. SCOTT2, TANJIM H. HOSSAIN3, JOHN C. BEIER1, 3, AND RUI-DE XUE2 1Department of Public Health Sciences, Miller School of Medicine University of Miami, Miami, FL 33136

2Anastasia Mosquito Control District 500 Old Beach road, St. Augustine, FL 32080

3Leonard and Jayne Abess Center for Ecosystem Science and Policy University of Miami, Coral Gables, FL 33146

ABSTRACT. Customer service requests play cation programs should include information an important role in mosquito control pro- on the associations of plants with mosquito grams by providing mosquito surveillance behavior, such as resting and sugar feeding, information and by allowing the opportu- to better inform the public and aid in in- nity for inspectors to educate the public on creasing their knowledge base as they relate mosquitoes and mosquito borne diseases. In to homeowner control practices. this study, we assessed differences in knowl- edge, attitude, mosquito control practices Key Words. Service request; Vector Control (KAP), and vegetation characteristics be- Management System; KAP survey tween frequent service requesters and non- frequent service requesters by conducting paper, vegetation, and entomological sur- I. INTRODUCTION veys. There were no significant differences in customer knowledge about mosquitoes Although the majority of mosquito-relat- between the two groups. However, there was ed issues in the United States arise from them a general lack of knowledge about mosquito being a nuisance, there remains an imminent resting and sugar feeding behavior in >90% threat of the emergence and re-emergence of of participants. Perceptions of mosquito in- mosquito-borne diseases. In 2002, 4,156 cases festation and vegetative coverage were de- of the West Nile Virus were reported in the termined to be driving factors for frequent United States; in 2005, 25 cases of dengue fe- service requests. Frequent service requesters ver were reported in Texas; and between 2009 reported being bothered or bitten by mos- and 2010, 90 cases of dengue fever cases in quitoes a few days a week or daily more of- Key West, Florida were reported (O’Leary et ten than non-frequent; demonstrating that al. 2004; CDC 2007, CDC 2013). By the end perceptions of the severity of mosquito in- of 2014, there have been 11 locally acquired festation are associated with service request chikungunya cases in Florida, and though it volume. Vegetation coverage on residential rarely results in death, the disease can be de- properties of frequent service requesters was bilitating to those infected (CDC 2014). 13% higher than non-frequent service re- Mosquito control programs, like Anas- questers and identified vegetation coverage tasia Mosquito Control District (AMCD) of as another potential factor driving service re- St. Johns County, Florida, use surveillance quest volume. We conclude that future edu- methods to monitor nuisance and vector

30 Davidson et al.: Relationship Between Citizen Knowledge, Vegetation Coverage, and Frequency 31 capable mosquitoes to reduce and prevent mosquito population abundance (Day and the spread of mosquito-borne diseases (Day Shaman 2011). and Shaman 2011). Despite the efforts of Understanding residents’ knowledge and these programs, an increase in the incidence attitude, toward mosquito behavior and ecol- of mosquito-borne diseases is still of major ogy on residential properties is essential for public health concern. It is imperative that developing and/or improving mosquito con- these programs continue to conduct applied trol approaches. Therefore, the purpose of research, as well as continued surveillance this study is to: 1) determine if there are dif- to produce new or improved approaches for ferences in knowledge, attitude, and control mosquito control. practice of residents in regards to mosquitoes The District uses the Vector Control between residents; 2) determine if service re- Management System (VCMS) to manage quests are related to vegetation on properties; service requests made by residents of St. 3) identify major vegetation density on resi- Johns County. Service requests entered dential properties of service requesters; and into VCMS play a major role in supporting 4) determine the major species of mosquitoes surveillance efforts (AMCD 2013, Weaver and abundance in residential properties. This et al. 2013). This method of surveillance study will provide feedback to AMCD and has proven to be successful as there is a lessons learned can be used as a tool by the correlation between the volume of service District to continue to protect public health requests received and the density of mos- by revising educational materials, and serving quitoes (Morris and Clanton 1981, 1992). as a catalyst for discussion on future studies The software allows for service requests to involving vegetation, mosquito behavior, and be linked with mosquito abundance, along control. with information of their location in the county. The VCMS system relies on people II. MATERIALS AND METHODS to make service requests; however, some residents make more frequent requests Study site selection and sampling. Data col- than others. Moreover, there seems to be lection was conducted in St. Johns County, a disconnect in the knowledge, attitude, Florida between June 3 and July 31, 2013. St. and control practices between residents Johns County, with a population of approxi- that call in service requests frequently and mately 202,000, covers 609 square miles residents who do not (AMCD staff pers. of northeastern Florida (US Census 2010, comm.). Weaver et al. 2013). The VCMS is an inte- In addition, over the last few years, grated database and mobile field data col- AMCD staff has observed that residents who lection system used by AMCD for mosquito frequently put in service requests seem to control (Weaver et al. 2013). The software have more vegetation on their property than assists with management of the District’s residents without service requests (AMCD mosquito borne disease and surveillance staff pers. comm.). The density of vegetation data; integrating mosquito management on residential properties is important be- activities (service requests, inspection, and cause mosquitoes rely on sugar from plants spraying); and monitoring employee activi- for energy (Hocking 1953, Nayar and Van ties. Using the VCMS program, four years’ Handel 1971, Van Handel 1965) and veg- worth of service requests was assessed to cat- etation for resting sites (Allan et al 2009). egorize and identify residential properties. Energy gained from plants is used to sup- Service requests were grouped as 1, 2, 3, 4, port their host seeking behavior in flight and >4 requests by household within each and fecundity, increasing the probability of year. The data was filtered by deleting mul- disease transmission (Muller et al. 2011, Yu- tiple repeat entries that were not different val 1992). Therefore, the method in which requests, this allowed for a more accurate a residential property owner manages his/ description of the frequency of calls over the her agricultural landscape is correlated with past four years. 32 Technical Bulletin of the Florida Mosquito Control Association, Vol. 10, 2016 Residential properties that had frequent was collected, as it was not necessary for the service requests were selectively sampled completion of this study. from the VCMS database. Requests had to Vegetation survey. After each question- meet three criteria: 1) be a residential prop- naire was administered, consent was request- erty in which an occupant has made >3 re- ed to survey their property and measure quests in 2012; 2) multiple requests had to vegetation growth. This measurement was have the same address but different dates of taken with the use of a 1,000 ft tape-measur- entry; and 3) properties could not be farm ing wheel (#PSMW48, Lufkin, Sparks, MD). lands, apartment complexes, or greater than All vegetation above ground level was mea- 43,560 sq ft. If occupants were not available sured by taking the circumference or perim- at the pre-selected household, then the next eter and calculating its area in square feet. pre-selected household was visited until con- Tree canopies and grass coverage were not sent was granted. measured as investigators were only inter- Residential properties that were not fre- ested in non-grass vegetation that covered quent service requesters or non-service re- the soil surface. Circumference of trees was questers, were selected at random as control measured at their base and included in the sites. Control sites also had to meet three crite- survey. To calculate vegetation coverage of ria: 1) residential properties had to be within the property, measurements were summed the same neighborhood as a study site, either and divided by the total square footage of on the same street or the next street over; 2) the property. Square footage of each prop- they were not identified as having >3 service erty was obtained from the St. Johns County requests a year between 2009-2012; and 3) Property Appraiser’s Office website. Photos properties could not be farm lands, apart- of vegetation were taken on each property. ment complexes, or a larger than≥43,560 sq Frequently occurring plants on each prop- ft. If occupants of a household were not avail- erty were identified by Ali Fulcher, at AMCD, able or refused to participate, neighboring and Keith Fuller, at the St. Johns County Ag- houses were visited until consent was granted. riculture Center. Study sites identified with these crite- Entomological survey: In addition to ria and sampling techniques resulted in 24 the vegetation survey, consent to check par- residential homes within 4 zip codes: 32080, ticipants’ yards for adult mosquitoes and 32082, 32084, and 32086. Of these study mosquito larval habitats were also obtained sites, six houses were randomly selected upon completion of the questionnaire. To from each zip code, three of which were determine the presence of adult mosquitoes, identified as frequent service requests from landing rate counts (LRC) were conducted the VCMS database and three randomly se- by volunteers. Landing rate counts counts lected control sites. All houses were visited consisted of volunteers exposing the surface during the evening hours (4pm-6pm) for 1.5 from their elbow to their hand, and count- weeks: July 8-16, 2013. ing how many mosquitoes landed, within Paper-based KAP survey. A member of the three minutes at each property. Mosquitoes research team administered a knowledge, were allowed to land, but were not allowed attitude, and practice (KAP) questionnaire to take a blood meal. Landing rate counts to one consenting adult (≥18 years old) were conducted in areas identified as pos- from each household. The questionnaire sible resting areas (shaded vegetated areas). was designed to elicit resident’s knowledge Each property was also checked for con- on mosquito behavior, mosquito-borne dis- tainers or plants with standing water for the ease, attitudes towards and practices used in presence or absence of larvae or pupae. Lar- mosquito control. Their responses were as- vae and pupae were not collected. Containers sumed representative of the household. All found with standing water were emptied by questionnaires were asked face-to-face and surveyors. Adult mosquitoes were collected most questions were asked as open-ended with the use of a BioGents sentinel (BGS) questions. No demographic information trap with BGS lure and were set up for 24 hrs Davidson et al.: Relationship Between Citizen Knowledge, Vegetation Coverage, and Frequency 33 on each property. The BGS trap was chosen knowledge score than those who self-rated as due to its ability to attract various container being not very knowledgeable. ovipositing mosquitoes such as Aedes spp. Overall, 92% of the participants were and Culex spp. Mosquito collections were able to identify at least one source of mos- taken within a day or two of administering quito larval habitat such as standing water, the questionnaire. Collected mosquitoes marshes, swamps, or damp/moist areas. were brought back to the lab and kept fro- More than 75% of the participants were able zen in a standard freezer until they could be to list at least one mosquito-borne disease, sexed, identified to species, and counted. such as malaria, dengue, West Nile virus, Data analysis. SPSS (ver. 21) was used to eastern equine encephalitis.. When asked assess differences in vegetation coverage, where mosquitoes generally rested as adults, knowledge, attitude, and practice between only 25% of participants were able to identify frequent and non-frequent service request- vegetation as a resting area. Specific knowl- ers. Chi-square tests were used to test for edge of non-blood meal sources for mosqui- statistical significance. Mean differences of toes was low, with only one responder report- vegetation coverage, mosquito collection, ing knowledge that plants are a meal source knowledge, and practice scores were calcu- for mosquitoes. There were no significant lated using an independent t-test. differences between non-frequent service re- questers and frequent service requesters in III. RESULTS overall knowledge scores or specific knowl- edge about mosquito behavior and disease Paper-based survey. Twenty-four partici- (Table 1). pants completed the survey, 12 of which Attitude. A majority of the participants were from households that had requested reported being bothered or bitten by mos- services frequently and 12 from households quitoes daily or a few days a week with 71% that had not requested services frequently. reporting being bothered or bitten daily dur- Each participant answered every question ing the summer months (Table 2). An addi- on the survey. tional 8% reported being bothered or bitten Knowledge. An equal number of frequent a few days a week. There was a significant as- service requesters and non-frequent service sociation (χ2, p = 0.019) between service re- requesters (4 each) scored as being very quest status and reporting how often they are knowledgeable of mosquitoes. The remain- bothered or bitten by mosquitoes. On aver- ing frequent and non-frequents service re- age, frequent service requesters reported be- questers (8 each) scored as not being very ing bothered or bitten by mosquitoes more knowledgeable about mosquitoes (Table 1). often than non-frequent service requesters. Participants who rated their propensity of When asked where they were more likely to knowledge as 4-5, on average, had a higher be bothered by mosquitoes, over 80% of the

Table 1. Responses to the open-ended questions on mosquito knowledge.

Non-frequent service Frequent service requesters requesters Overall Question n % n % n % Mosquito breeding sources 10 83.3 12 100.0 22 91.7 Mosquito resting area 2 16.7 4 33.3 6 25.0 Mosquito meal sources 1 8.3 0 0.0 1 4.2 Mosquito sugar feed on plants 1 8.3 0 0.0 1 4.2 Mosquito borne diseases 10 83.3 9 75.0 19 79.2 Non-frequent service Frequent service Average Knowledge Score requesters requesters Overall Mean (SD) 2.00 (1.04) 2.08 (0.67) 2.04 (0.85) 34 Technical Bulletin of the Florida Mosquito Control Association, Vol. 10, 2016 participants reported being bothered out- Vegetation survey. Vegetation type and cov- doors on their property. When asked what erage varied between residential properties time of the day they were outdoors on their (Table 4). While not significant, frequent property, most participants reported being service requester properties had 13% great- outdoors mostly in the evening hours and er vegetation coverage than non-frequent multiple times to most of the day (80%). services requesters (Figure 1). When com- More than 80% of participants reported paring mean differences between these two great concern towards reducing mosquito groups within each zip code, no significant larval habitats, scoring 4 or 5 on a five point differences were found in 3 out of the 4 scale (Table 2). On average, frequent service zip codes with. In zip code 32080 there was requesters reported spending more time a significant (p < 0.05) difference in mean outdoors (22.75 hours) than non-frequent vegetation coverage between the two groups service requesters (18.67 hours). (Figure 2). The more vegetation coverage Practice. Most participants reported that the more service requests. they did something to reduce larval habitats, Entomological survey. There were no sig- including all of the frequent service request- nificant differences between frequent and ers (Table 3). Participants varied in their re- non-frequent service requesters in the aver- sponses on the measures they take to reduce age number of mosquitoes trapped (Figure larval habitats, with emptying standing water 4). The majority of the mosquitoes caught as one of the most frequent methods used. in the BGS traps were container ovipositing Participants also varied in their responses on mosquitoes, of which 70% were Aedes albop- the personal protection measures used to ictus (Skuse). Average landing rate counts, protect themselves and their family, with the conducted 3 months post-initial survey, were use of mosquito repellent as one of the most less than or equal to one, with the exception frequently used methods. On average, fre- of one home, which had an average count quent service requesters had a significantly of 5.7 mosquitoes. Approximately half of the higher practice score (p = 0.023) than non- houses surveyed were positive for containers frequent service requesters (Table 3). with standing water. However, there were no

Table 2. Survey mean responses (SD) to open-ended questions about attitude towards mosquitoes.

Non-frequent Frequent service service requesters’ n requesters n Overall n Question (%) (%) (%) How often were you bothered or bitten by mosquitoes?* Daily or few days a week 7 (58.3) 12 (100.0) 19 (79.2) Few days a month or fewer 5 (41.7) — 5 (21.8) Where on your property you were most likely to be bothered Outside around house 8 (66.7) 12 (100.0) 20 (83.3) Inside Home 1 (8.3) — 1 (4.2) Everywhere 2 (16.7) — 2 (8.3) Not sure 1 (8.3) — 1 (4.2) What time of the day outdoors were you bothered? Morning 2 (16.7) 1 (8.3) 3 (12.5) Afternoon 1 (8.3) 1 (8.3) 2 (8.3) Evening 3 (25.0) 2 (16.7) 5 (21.8) Multiple times/All day 6 (50.0) 8 (66.7) 14 (58.3) How concerned are you about reducing mosquitoes? 0-3 score, not very concerned 2 (16.7) 1 (8.3) 3 (12.5) 4-5 score, very concerned 10 (83.3) 11 (91.7) 21 (87.5) Time spent outdoor on property per week 18.67 (13.4) 22.75 (14.6) 20.7 (13.9) Percentages are column % out of 12 within each group and out of 24 for overall *significant difference in response between groups (p < 0.05) Davidson et al.: Relationship Between Citizen Knowledge, Vegetation Coverage, and Frequency 35

Table 3. Survey responses to open-ended questions about practices for protection against mosquitoes.

Non-frequent ser- Frequent service vice requesters requesters Overall Question (n) (n) (n) Do you do anything to reduce mosquito breeding sources? No 3 0 3 Yes 9 12 21 What measures do you take to reduce sources? Self app. of store bought pesticide 1 4 5 Emptying standing water 8 11 19 Call county services (AMCD) 3 4 7 Other 1 2 3 What personal protection measures do you take to protect you and your family? Mosquito repellent 7 6 13 Citronella candles 1 4 5 Wearing long sleeves and long pants 0 1 1 Alter outdoor activities 4 4 8 Other 1 3 4 Non-frequent ser- Frequent service Practice score vice requesters requesters Overall Mean (SD) 2.92 (1.44) 4.25 (1.22) 3.58 (1.47) significant differences in the presence or ab- were able to identify mosquito larval habi- sence of containers with standing water be- tats and at least one mosquito-borne disease. tween the two groups. However, collectively, there was a lack of knowledge about mosquito resting and feed- IV. DISCUSSION ing behavior with only six individuals cor- rectly identifying some kind of vegetation There were no significant differences in as a mosquito resting area. Only one indi- overall or specific knowledge about mosqui- vidual correctly identified plants as a meal toes between the two groups, where most source. The other residents reported that participants reported not being very knowl- mosquitoes generally rested on humans and edgeable about mosquitoes. Most residents animals and that they feed on human and/

Table 4. Commonly found plants on St. Johns County residential properties.

Scientific name (family) Common name Plant description Bromelia spp. (Bromeliaceae) Bromeliads – Native to tropical America – Flowering plant – Cleft calyx that can collect water and become a breeding source for mosquitoes Polystichum munitum (Dryopteridaceae) Sword fern – Found in temperate and tropical regions Wood fern – Non-flowering Plumbago spp. (Plumbaginales) Leadwort – Native to South Africa – Flowering ornamental plant Callicarpa spp. (Verbenaceae) Beauty berry – Native to east North America and Southeast Asia – Flowering and fruit producing plant Tradescantia spp. (Commelinaceae) Spiderwort – Native throughout Gulf Coast region – Invasive species, used as ornamental plant Ruellia spp. Petunias – Native from South America (Acanthaceae) – Flowering plant *p < 0.05 the difference between frequent and non-frequent 36 Technical Bulletin of the Florida Mosquito Control Association, Vol. 10, 2016 clothing, and altering daily activities around dawn and dusk. Although these methods have been repeatedly proven to be effective in protecting the public health and the envi- ronment (WHO 2014), it is not sufficient in educating the public on mosquito behavior. As expected, frequent service requesters reported being bothered or bitten by mos- quitoes more often than non-frequent ser- vice requesters. In addition, frequent service requesters tended to spend more time out- doors and to take a number of measures to protect themselves and their families from nuisance and disease carrying mosquitoes; more so than non-frequent service request- ers. This supports their belief that they are Figure 1. Average vegetation coverage overall be- tween frequent service requesters and non-frequent bothered or bitten by mosquitoes more of- service requesters (±SE). ten than the non-frequent requesters are bitten. or animal blood for their main meal source. We found that there was a mean differ- The beliefs of the majority of the interview- ence of 13 % higher vegetation seen in the ees, could be due to lack of education and frequent service request residence, the lack dissemination about mosquito behavior. By of significance was probably due to the ex- reviewing educational materials provided tremely small sample size and/or the meth- by national and local institutions, it was ob- od used to measure vegetation coverage on served that there was a constant emphasis residential properties. Previous work with on disease information and control/preven- vegetation coverage utilized a grid method tion practices for the layman that included in which each grid size is dependent on the reducing standing water, wearing protective plant shrubs, trees, or flowering. Environ-

Figure 2. Average vegetation coverage by zip codes between frequent service requesters and non-frequent ser- vice requesters (±SE). Davidson et al.: Relationship Between Citizen Knowledge, Vegetation Coverage, and Frequency 37 ing and feeding behavior of adult mosqui- toes. To be comprehensive, future educa- tional materials for mosquito control and disease prevention should include this in- formation. Vegetation was not found to be a driving factor for service request calls. Veg- etation cannot be ruled out. Previous stud- ies have shown that mosquitoes do rely on vegetation for resting sites and plant sugars for fecundity and flight (Muller et al. 2001, Yuval 1992). Future research should ex- Figure 3. Average mosquitoes collected over 24-hour pand the experimental sample size of sur- collection period with BioGents sentinel traps baited vey participants for a more accurate analysis with BioGents lure, between frequent service request- of the correlation of vegetation and service ers and non-frequent service requesters (±SE). requests. mental factors such as seasonality of plants and the density of tree canopies, which may V. ACKNOWLEDGMENTS affect the behaviors of mosquitoes, were not taken into consideration (Daubenmire Thanks to A. Fulcher, M. Smith, and K. 1959). Fuller for their assistance during this study. Although vegetation types varied across All human subjects were informed before all 24 houses, similarities were seen in the administering surveys based on the District- occurrence of a number of plants that can approved protocol for using human sub- support mosquito development, feeding and jects. resting, such as the Bromelia spp., Polystichum munitum, Ruellia spp., and Callicarpa spp. VI. REFERENCES CITED Further studies should investigate mosquito preferences for types of vegetation, as well Allan, S.A., D.L. Kline, and T. Walker. 2009. Environ- mental factors affecting the efficacy of bifenthrin- as their uses for them for feeding or resting. treated vegetation for mosquito control. J. Am. The major species of mosquito caught in Mosq. Control Assoc. 25: 338-346. BGS traps was Ae. albopictus. This maybe due AMCD (Anastasia Mosquito Control District). 2013. About AMCD [Internet]. http://www.amcdsjc.org/ to the anthropogenic changes to landscape about-amcd/operations/district.aspx [Accessed Au- vegetation and artificial uncovered contain- gust 28, 2013]. ers, in which this species has been proven to CDC (Center for Disease Control and Prevention). 2007. Dengue hemorrhagic fever- U.S.- Mexico bor- thrive in. In addition, the time of year that der, 2005 [Internet]. Available from the CDC Mor- trapping occurred (i.e. a summer month bidity and Mortality Weekly Report. 56 (31):785-9 with high temperature and precipitation) http://www.cdc.gov/mmwr/preview/mmwrhtml/ mm5631a1.htm [Accessed August 29, 2013]. provided ideal abiotic factors for develop- CDC (Center for Disease Control and Prevention). ment of Ae. albopictus (Paupy et al. 2009). 2013. West Nile virus neuroinvasive disease inci- To our best of knowledge, our study is dence by state [Internet]. Available from CDC http://www.cdc.gov/westnile/statsMaps/prelimi- unique in its assessment of key factors (e.g., naryMapsData/incidencestatedate.html [Accessed KAP and vegetation) driving the volume of August 29, 2013]. mosquito control service requests. We con- CDC (Center for Disease Control and Prevention). 2014. Chikungunya virus in the United States [In- clude that knowledge and practice did not ternet]. http://www.cdc.gov/chikungunya/geo/ vary greatly by service request volume, but united-states.html [Accessed September 1, 2014]. residents perceptions of how often they are Daubenmire, R. 1959. A canopy coverage method of vegetation analysis. Northwest Sci. 33: 43-64. bothered/bitten was a driving factor for Day, J. F. and J. Shaman. 2011. Mosquito-borne arbovi- service request volume. Overall, residents ral surveillance and the prediction of disease out- were proficient in their general knowledge breaks. In: Flavivirus Encephalitis. Dr. Daniel Ruzek (Ed) [Internet]. Available from: In Tech. http:// about mosquito larval habitats and disease; www.intechopen.com/download/get/type/pdfs/ however, they lacked knowledge about rest- id/20865 [Accessed August 30, 2013]. 38 Technical Bulletin of the Florida Mosquito Control Association, Vol. 10, 2016 Hocking, B. 1953. The intrinsic range and speed of Paupy, C., B. L. Delatte, V. Corbel, and D. Fontenille. flight of insects. Trans. R. Ent. Soc. Lond. 104: 223- 2009. Aedes albopictus, an arbovirus vector: from the 345. darkness to the light. Microb. Infect. 11:1177-1185. Morris, C. D. and K. B. Clanton. 1981. Significant asso- U.S. Bureau of the Census, Population Estimates Pro- ciations between mosquito control service requests gram (PEP). Updated annually. http://www.census. and mosquito populations. J. Am. Mosq. Control As- gov/popest/index.html. Census Bureau, 2010 Cen- soc. 5:36-41. sus of Population, Public Law 94-171 Redistricting Morris, C. D. and K. B. Clanton. 1992. Comparison of Data File. Updated every 10 years. http://factfind- people who request mosquito control services and er2.census.gov [Accessed November 1, 2013]. their non-requesting neighbors. J. Am. Mosq. Con- Van Handel, E. 1965. The obese mosquito. J. Physiol. trol Assoc. 8:65-68. 181:478-86. Müller, G. C., R. D. Xue, and Y. Schlein. 2011. Differen- Weaver, J. H. R., J. D. Phillips, M. K. Gaines, R. D. Xue, and tial attraction of Aedes albopictus in field to flowers, J. M. Bequette. 2013. Analysis of Anastasia Mosquito fruits and honeydew. Acta Tropica. 118:45-49. Control District’s service requests. Wing Beats 24:34-39 Nayar, J. K., and E. Van Handel. 1971. The fuel for sus- WHO (World Health Organization). 2014. A global brief on tained mosquito flight. J. Insect. Physiol. 17:473-481. vector-borne diseases. Available from the WHO. http:// O’Leary, D. R., A. A. Marfin, S. P. Montgomery, A. M. apps.who.int/iris/bitstream/10665/111008/1/ Kipp, J. A. Lehman, B. J. Biggerstaff, V. L. Elko, P. WHO_DCO_WHD_2014.1_eng.pdf?ua = 1 [Accessed D. Collins, J. E. Jones, and G. L. Campbell. 2004. March 28, 2014]. The epidemic of West Nile virus in the United States Yuval, B. 1992. The other habit: sugar feeding by mos- 2002. Vector-Borne Zoonotic Dis. 4:61-702. quitoes. Bull. Soc. Vect. Ecol. 17:150-156. Mosquito species composition and impact of trapping sites on floodwater mosquitoes, Aedes Vexans in xinjiang, China

MEI-DE LIU1, YAN-DE DONG1, GUI-LIN ZHANG2, ZHONG ZHENG2, RUI-DE XUE3, AND TONG-YAN ZHAO1 1State Key Laboratory of Pathogen and Biosecurity Institute of Microbiology and Epidemiology, Beijing 100071, P.R. China

2Center Disease Control and Prevention of Xinjiang Command Urumuqi, China

3Anastasia Mosquito Control District 500 Old Beach Road, St. Augustine, FL 32080

Abstract. Mosquito species composition nually inundating the residential area and and impact of mosquito trapping sites on a ma- adjoining semi-desert land. After the flood jor species of floodwater mosquitoAedes vexans, water recedes to its normal level, many shal- was investigated by using Mosquito Magnet low ponds, lowland puddles, water holes, Liberty Plus (MMLP) traps baited with octenol and swamps become ideal production sites in Beiwan, Xinjiang. A total of six trapping for floodwater mosquitoes. Previous studies, sites inside (three) and outside (three) human have shown that extremely high floodwater occupied areas were selected for the study. Six mosquito production occurs creating a seri- species of mosquitoes, Aedes vexans, Anopheles ous health threat for humans and livestock messeae, Ae. cyprius, Coquillettidia richiardii, Culex (Lu and Liu, 1990; Ma and An, 1994; Liu modestus, and Cx. pipiens pipiens were collected and Zhang et al., 2005) including potential during the study. The results indicated that Ae. alphavirus transmission in Xinjiang (Li and vexans was the major species and represent- Liang et al., 1992; Li and Karabatsos et al., ed 97.14% of the total collection. Mosquito 1995). Therefore, it is of critical importance Magnet Liberty Plus traps also significantly to determine the species composition and reduced the number of mosquitoes in the resi- seasonal abundance of floodwater mosqui- dential areas. We found that MMLP traps were toes in this region. Furthermore, we wanted suitable tools for the collection of floodwater to determine if commercial mosquito traps mosquitoes in Xinjiang where extremely high could be used in residential areas to reduce floodwater mosquito production occurred as local floodwater mosquito populations in well as provide protection of humans and live- Beiwan, Xinjiang. stock from host seeking mosquitoes. II. Materials And Methods Key Words: Mosquito Magnet® Liberty Plus, surveillance, Aedes vexans Study area. The study area is a residential area enclosed by a dike in the wilderness of Beiwan, Xinjiang (western China) on the I. INTRODUCTION northern side of the Irtysh River (Figure 1). In this area, there is plentiful vegetation Beiwan is located near the Chinese bor- coverage with shrubs, woods, and reeds. The der with Kazakstan and is situated along the northern part of the trapping site is a semi- flood lands of the Irtysh River, Xinjiang in desert area covered with scanty vegetation western China. River water levels rise an- on dry sand. The human residential area is

39 40 Technical Bulletin of the Florida Mosquito Control Association, Vol. 10, 2016

Figure 1. Diagrammatic sketch of the study area and trap sites. located in the transition belt between these number of mosquitoes and Ni the number of two different landscapes. species “i”. Chi square test was performed to Sampling locations. A total of six trapping compare the Pi of different mosquito species sites were selected for the study. Three trap- between sites and species (P = 0.05) ping sites were located along the outside of the dike in the residential area and were; III. Results riverside (A site), semi-desert (B site), and a middle zone (C site) between sites A and A total 251,475 mosquitoes representing B. Three trapping sites inside the residential six species was collected from MMLPs and area were located along the dike, riverside included Aedes vexans (Meigen), Ae. cyprius (D site), semi-desert (E site), and a middle Ludl., Anopheles messeae Falleroni, Coquillet- zone (F site) between sites D and E. tidia richiardii (Ficalbi), Culex modestus Fical- Mosquito Magnet® Liberty Plus traps bi , and Cx. pipiens pipiens L. (Table 1). Chi (MMLP) baited with octenol was provided square test results revealed that a significant by the former American Biophysics Corpo- Table 1 Composition of mosquitoes trapped with the ration, USA. In each trapping site, the same Mosquito Magnet Liberty Plus traps in Beiwan, China. MMLP was operated in the field for two days. Species Pi(%) QTY Mosquitoes in each trap were collected ev- ery eight hours (a total of 6 collections) and Aedes vexans 97.14 244,279 Ae. cyprius 1.30 3,280 brought back to the laboratory for species Anopheles messeae 0.53 1,342 identification and total counts. Collections Coquillettida richiardii 0.80 2,007 were made during the peak flood season Culex modestus 0.09 229 (July and August). Cx. pipiens pipiens 0.13 338 Total 100.00 251,475 Statistical analysis. The proportion of each Pi = proportion of each species; QTY = number of mos- species (Pi) in MMLP collections was calculat- quitoes ed as Pi (%) = (Ni*100)/N, where N is total χ2 = 254902, p<0.001 Liu et al.: Mosquito Species Composition and Impact of Trapping Sites on Floodwater Mosquitoes 41 statistical difference existed between the between those 2 latter collection sites. The proportion of each species (χ2 = 254902, proportions of Cx. modestus and Cx. pipiens p < 0.001) with Ae. vexans as the dominant pipiens collected from the C site area were species (97.14% of total catch). The other higher than those from the A site and B site. 5 species made up about 2.86% of the total collection. IV. Discussion The proportionth of Ae. vexans, An. messeae,Cx. modestus, and Cx. pipiens pipiens Aedes vexans is a major nuisance species caught from the inside human residential in northern China (Liang, 1997). Moreover, area were higher than those caught outside Japanese B encephalomyelitis virus has been (Table 2). The proportion of Ae. cyprius and isolated from this species in China (Li and Li- Cq. richiardii collected from inside human ang et al., 1992; Zhang and Shi et al., 1999). the residential area were lower than those Our study showed that Ae. vexans still was the caught from outside the area. Also there was major species and represented 97% of the a significant difference between the propor- total MMLP collections. Because this species tion of mosquito species outside and inside is one of the more proliferate floodwater the residential area (χ2 = 16.486, p < 0.01). mosquitoes, it poses a great health threat to There was a significant difference in the residents and livestock in this area. Although proportion of each species between trap- few numbers of An. messeae were collected in ping sites inside the residential area (χ2 = MMLPs, this species is a suspected vector of 392.86, p < 0.001) (Table 3). Culex pipiens malaria (Tong and Li et al., 1996; Lu, 1999). pipiens from the D site inside the residential Additionally, Cx. modestus and Cx. pipiens pipi- area were higher than the other areas. How- ens were collected from traps in low abun- ever, the proportion of Ae. vexans from the dance and are suspected vectors of West Nile D site area was lower than those collected virus (Hayes, 1998). from the F site and E site areas. Also, similar The proportion of Ae. vexans and An. mes- proportions of Ae. vexans, Ae. cyprius, and An. seae in the riverside were the same as in the messeae were collected from the F site and E semi-desert environments, which also was site areas. No Cx. pipiens pipiens was collected higher than those in the transition zone be- from all three sites. tween these two types of environments. The There was a significant difference in species of mosquitoes. Ae. vexans flourishes the proportion of each species between the in flood environments (O’Malley, 1990). The three trapping sites in the outside residen- flood environment in Beiwan accounted for tial area (χ2 = 6201.936, p < 0.001) (Table 4). the predominance and high proportion of Aedes vexans and An. messeae collected from Ae. vexans in the local mosquito community. the C site were lower than those collected We noticed that density of Cq. richiardii, in from sites A and B; however, the proportion traps from one of our sites, decreased with of Ae. vexans and An. messeae were similar increasing distance from the larval habitat as

Table 2 Composition of mosquitoes trapped with the Mosquito Magnet Liberty Plus inside and outside a residential area in Beiwan, China. Within residential areas Outside residential areas Species Pi(%) QTY Pi(%) QTY Aedes vexans 97.83 76,190 96.83 168,089 Ae. cyprius 1.26 983 1.32 2,297 Anopheles messeae 0.56 438 0.52 904 Coquillettida richiardii 0.01 4 1.15 2,003 Culex modestus 0.16 121 0.06 108 Cx. pipiens pipiens 0.18 144 0.11 194 Pi = proportion of each species; QTY = number of mosquitoes χ2 = 16.486, p < 0.01 42 Technical Bulletin of the Florida Mosquito Control Association, Vol. 10, 2016 Table 3. Proportions of mosquito species at different sample sites from Mosquito Magnet Liberty Plus traps in resi- dential areas close to the wilderness, Beiwan, China. D site F site E site Species Pi(%) QTY Pi(%) QTY Pi(%) QTY Ae. vexans 93.99 9,014 98.40 24,557 98.35 42,619 Ae. cyprius 1.71 164 1.07 267 1.27 552 An. messeae 2.46 236 0.30 74 0.30 128 Cq. richiardii 0.00 0 0.02 4 0.00 0 Cx. modestus 0.88 84 0.01 3 0.08 34 Cx. pipiens pipiens 0.96 92 0.21 52 0.00 0 Note: Pi = proportion of each species; QTY = number of mosquitoes D site, the riverside site in residential area; F site, the semi-desert one in residential area; E site, is located between F site and D site. χ2 = 392.86, p < 0.001 observed by other authors (Anno and Tak- and Nicolescu et al., 1990). The trapping agi et al., 2000 and Leonardo and Rivera et sites with different amounts of vegetation al., 2005).The larvae of Cq. richiardii can only were significantly influencing the collection be found in reeds from river environments of species and amount of floodwater mos- in China (Lu, 1999). This may explain why quitoes in the study area. the proportion of Cq. richiardii decreased Our study site, located in the transition with an increase in trapping distance from zone between the river and semi-desert en- the river. vironments, undoubtedly influenced which The proportion of An. messeae collected mosquito species were present and their from inside and outside the human resi- relative abundance. The proportion of Ae. dential area were not different. This may cyprius, An. messeae, Cx. modestus, and Cx. be caused by the Anopheles mosquitoes. The pipiens pipiens at the trapping sites near the proportion of Cx. modestus and Cx. pipiens river were higher than those at the trapping pipiens were higher in the human residential sites in the center of the human residential area than outside, but the proportion of Ae. area and near semi-desert land. However, in cyprius and Cq. richiardii in the outside areas the semi-desert region side, the proportion were higher than those in the residential of Ae. vexans, Ae. cyprius, An. messeae, and Cq. area. The species variation from different richiardii at the sites located in the residential trapping sites and environments obtained area center were the same as those sites be- from our study were similar with other re- ing close to the semi-desert region. There is ports from different environments (Ciolpan plentiful vegetation (such as: reeds, weeds,

Table 4. Proportions of mosquito species at different sample sites from Mosquito Magnet Liberty Plus traps located in different wilderness areas outside residential areas, Beiwan, China. A site C site B site Species Pi(%) QTY Pi(%) QTY Pi(%) QTY Ae. vexans 96.65 87,880 96.36 34,076 97.52 46,133 Ae. cyprius 0.56 512 2.76 977 1.71 808 An. messeae 0.56 512 0.36 128 0.56 264 Cq. richiardii 2.18 1,984 0.05 16 0.01 3 Cx. modestus 0.00 4 0.23 80 0.05 24 Cx. pipiens pipiens 0.04 32 0.25 88 0.16 74 Note: A site, the riverside outside of residential area; B site, the semi-desert part site outside residential area; C site, is located between A site and B site. χ2 = 6201.936, p < 0.001 Liu et al.: Mosquito Species Composition and Impact of Trapping Sites on Floodwater Mosquitoes 43 shrubs, pygmy trees and some arbors.) which uation against mosquitoes in malaria’s area of the Republic of Korea.” Korean J. Parasitol (40): 45-54. might provide good resources and resting Ciolpan, O. and G. Nicolescu, et al. (1990). Investigatii sites for adult mosquitoes. asupra faunei de tîntari (Diptera: Culicidae) din Our results tell us that surveillance of padurea Cernica, potential vectori pentru arboviru- the mosquito density and species composi- suri. Analele Banatului, Stiintele Naturii 2:356-360. Hayes, C. (1998). West Nile fever. In: The Arboviruses: Ep- tion is important to present good protec- idemiology and Ecology. T.P. Monath (ed.) CRC Press, tion for people and livestock. Human-baited Boca Raton, Florida,. Kline, D. L. (2006). Traps and trapping techniques for bed nets had previously been used for adult adult mosquito control. J. Am. Mosq. Control Assoc. mosquito surveillance in this area (Liu and 22:490-6. Zhang et al., 2005). Our study found that Ae. Leonardo, L. and P. Rivera, et al. (2005). A study of the environmental determinants of malaria and schis- vexans was the major species in the area (Pi tosomiasis in the Philippines using remote sensing of this species was 99.5%) while Ae. cyprius and geographic information systems. Parassitol. and An. messeae were also collected. In addi- 47:105-114. Li, Q. and G. Liang, et al. (1992). Survey of arboviruses tion, the Mosquito Magnet® Traps with oc- in Xinjiang. Chin J. Exp. Clin. Virol. 6:247. tenol collected a great number of floodwa- Li, Q. and N. Karabatsos, et al. (1995). Further iden- ter mosquitoes and this method may reduce tification of some arboviruses isolated in Xinjiang, China in recent years. Chin. J. Exp. Clin. Virol. the nuisance problem in Beiwan where an 9:367-370. extremely high density of mosquitoes occurs Liang, G. (1997). The arbovirus research situation in during the flood seasons. Mosquito Magnet China. Chin. J. Zool. 13:61-63. Liberty Plus traps are an efficient surveil- Liu, B. and G. Zhang, et al. (2005). Investigation of the trends of growth and decay of Aedes vexans in the lance tool for mosquito population and daytime and at night in the uninhabited region of could be considered for use as control mea- Beiwan in Xinjiang province. Chin. J. Hyg. Insecti- cides and Equipment 11:103-105. sures to reduce the mosquito population in Lu, B. (1999). Progress of studies of mosquito vectors in the residential area (Burkett and Lee et al., China. Chin. J. Vect. Biol. Control 10:161-165. 2002; Kline, 2006). Lu, B. and Z. Liu (1990). Medical Entomology. Beijing, China, Beijing Science Publishing House. Ma, D. and J. An (1994). Survey of mosquito composi- V. References cited tion and ecology in the A’letai district of Xinjiang. Chin. J. Vect. Biol. Control 5:48-49. Anno, S. and M. Takagi, et al. (2000). Analysis of rela- O’Malley, C. M. (1990). Aedes vexans (Meigen): An old tionship between Anopheles subpictus larval densities foe. Proc. NJ Mosq. Control Assoc. 1:90-95. and environmental parameters using Remote Sens- Tong, S. and B. Li, et al. (1996). Study on the surveil- ing (RS), a Global Positioning System (GPS) and lance of vivax malaria in the Yili Area. Xinjiang. En- a Geographic Information System (GIS). Kobe J. dem. Dis. Bull. 11:72-74. Med. Sci. 46:231-243. Zhang, H. and H. Shi, et al. (1999). Isolation of Japa- Burkett, D. and W. Lee, et al. (2002). “Late season com- nese encephalitis virus from four species of Aedes mercial mosquito trap and host seeking activity eval- mosquito in Yunnan Province. Virol. Sin. 14:32-35. LUNAR PHASE IMPACT ON COQUILLETTIDIA PERTURBANS AND CULEX ERRATICUS HOST SEEKING IN NORTHERN FLORIDA

YONGXING JIANG Mosquito Control Section Department of Public Works, City of Gainesville 405 NW 39th Ave, Gainesville, FL 32609

ABSTRACT. The host seeking periodicity atmospheric moisture, wind, moonlight, and of Coquillettidia perturbans and Culex erraticus mosquito physiological age may influence (two important mosquito bridge vectors of the host seeking behavior of mosquitoes on eastern equine encephalitis virus in northern any given night. Therefore, it is important to Florida) were determined by using program- have some understanding of the prevailing mable collection bottle rotary traps. Results meteorological conditions and how these showed that both species host seeking activ- conditions modify the behavior of different ity peaked 1 hour after sunset while Cx. er- mosquito species, and consequently the ef- raticus continued that level of activity for an ficacy of controlling those species. During additional two hours. On full moon nights, 2008-2009, CDC light traps were placed both species displayed peak host seeking in four northern Florida counties (Leon, activity 1-2 hours longer than that observed Madison, Holmes and Washington) where during non-full moon nights. High relative positive eastern equine encephalitis (EEEV) humidity significantly increasedCx. erraticus equine or sentinel chicken sera were report- abundance in CDC light traps, as well as host ed to determine what mosquito species were seeking activity but not Cq. perturbans. in those areas. Coquillettidia perturbans (Walk- er) and Culex erraticus (Dyar and Knab) were KEY WORDS. Coquillettidia perturbans, Culex the dominant species in all collections and erraticus,host seeking pattern, lunar phase, may have been involved in virus transmis- humidity sion. Both species have been incriminated as competent EEEV bridge vectors in North America (Howitt et al. 1949, Boromisa et al. I. INTRODUCTION 1987, Cupp et al. 2003, 2004). McNeel (1932), in an observational Knowledge of temporal host seeking study, found that Cq. perturbans host seeking patterns of mosquitoes is important for un- activity was greatest at dusk. Snow and Pick- derstanding mosquito-host contact, as mos- ard (1957) presented data that indicated quitoes will only bite hosts that are available Cq. perturbans host seeking activity peaked during the period of their host seeking activ- between 1930-2000 h. However, results from ity (Williams 2005). The time of host seek- these earlier observational studies were not ing by mosquitoes delineates periods when in agreement with a recent study conducted females are active and the risk of pathogen by Bosak et al. (2001). These authors re- transmission greatest for vector species (Rei- vealed that significantly moreCq. perturbans sen et al. 1997). Therefore, determination host seeking activity occurred during the of the host seeking time (or biting rhythms) night period (between 2200-0100 h) com- exhibited by vector and nuisance mosquito pared with early evening or morning hours. species is essential to decide when adulti- Because of the inconsistencies between the cides can be most effectively applied. Obvi- previous studies there is a need to re-exam- ously, climatic factors such as temperature, ine the host seeking pattern of this species

44 Jiang: Lunar Phase Impact on Host Seeking in Northern Florida 45 in northern Florida especially in areas where central stainless steel rod extended from un- EEEV is prevalent. derneath the rotator trap and inserted into As stated earlier, Cx. erraticus was also one a tripod lawn sprinkler stand that supported of the more abundant species collected in the unit and CDC trap 1.5 meter above the light traps from EEEV sites and may be an ground. The first collection started at 1943 important bridge vector in the area. To this h (1 hour before sunset) and ended after author’s knowledge, host seeking periodic- the collection at 0639h (sunrise). Because ity of this species has not been reported in each rotator trap only contained 8 bottles, the scientific literature. Therefore, the aim two traps were used to achieve a total of 10 of this study was to determine the temporal hourly collections. In order to synchronize host seeking activity for Cq. perturbans and sunset/sunrise collections, the programma- Cx. erraticus and identify some of the me- ble timer was adjusted accordingly to reflect teorological factors that may affect timing daily time changes. A separated CDC light of this behavior. Obtaining this information trap was set up about 50 m apart from the will provide data-driven guidance on when CBR traps at the edge of the swamp in order adulticides should be most effectively ap- to compare species composition between plied. the two collecting methods. CDC and CBR traps were baited with 5 lbs of dry ice con- II. MATERIALS AND METHODS tained in a one gallon cooler jug. A small hole was drilled at the bottom of jug so the

Study site. Field studies were conducted CO2 could be delivered to the traps directly. at Leon County’s solid waste management Traps were operated for nine nights during center,7550 Apalachee Parkway, Tallahassee, July 1 through August 8, 2009. Mosquito Florida (30.25.362 N, 084.08.956 W). This lo- collections were transported to the labora- cation is one of Leon County Mosquito Con- tory, anesthetized, identified to species, and trol’s permanent sentinel chicken sites that counted. The number of Cq. perturbans and is used to monitor mosquito-borne diseases Cx. erraticus trapped in each hourly bottle such as EEEV and West Nile virus in Leon collection were summed and an average County. This site consisted of a large fresh number of mosquitoes per hour calculated water swamp (6.1 ha) covered with cattails for each species. (Typha spp.), arrowhead weeds (Sagittaria Meteorological data. On each collection longiloba), pickerelweed (Pontederia cordata), day, temperature, relative humidity, and a large forested wood lot, and office build- wind speed were obtained from a www.wun- ings. Seropositive EEEV chickens have been derground.com weather station located reported from this site for the last five years about 2 miles from the study site. Moon (http://www.floridahealth.gov/diseases- phase data were obtained from www.moon- and-conditions/mosquito-borne-diseases/ connection.com. For this study, the night of surveillance.html). a full moon and the night following a full Mosquito collection and processing. Pro- noon were considered as “full moon nights”. grammable collection bottle rotator traps (Criterion for this determination was based (CBR) (Model 1512, John W Hock Compa- on the moon phase calendar where the ny, Gainesville, FL) were used to determine night following a full noon is closer to a full temporal host seeking periodicity of Cq. per- moon than the night before a full moon). turbans and Cx. erraticus. The CBR is a device A total of four full moon night collections that segregates collections into distinct time were made in this study, (two nights in July periods by a programmable timer to which and two nights in August). The remaining any mechanical mosquito trap, such as a sample nights were considered as non-full CDC light trap, is attached. In this study, a moon nights. standard CDC light trap was mounted onto Statistical analysis Mosquito collections the screen holder located on the top of the were summarized on the basis of the num- CBR unit and secured with thumb screws. A ber of females caught per trap per hour. To 46 Technical Bulletin of the Florida Mosquito Control Association, Vol. 10, 2016 determine if temperature, relative humidity, cies (15 species from CDC traps vs. 12 species and/or wind speed impacted the host seek- from CBR traps) and higher numbers of mos- ing behavior of Cq. perturbans and Cx. errati- quitoes (2,984 female mosquitoes from CDC cus, one way analysis of variance was used to trap collection vs. 1,705 female mosquitoes analyze these relationships using STATIS- from CBR collection) were collected by the TICA software (StatSoft Inc. 2001) (P < 0.05). CDC traps. In terms of percentage, Cq. pertur- Hourly mosquito counts were log-transformed bans collections from CDC traps were slightly [log (n + 1)] to normalize the distribution and greater than CBR traps (40.7% vs. 37.1%); in relative abundance prior to analysis. contrast, a higher percentage of Cx. erraticus was collected in CBR traps than CDC trap III. RESULTS (57.1% vs. 48.4%), respectively. Host seeking periodicity. Figure 1 shows the Mosquito collection. Mosquito collection temporal host seeking patterns of Cq. pertur- started from July 1 and ended on August 8, bans and Cx. erraticus. Overall, Cq. perturbans 2009. During this 10 week collection period, host seeking activity started to rise at sunset a total of 9 nightly collections were made. A (SS) then peaked 1 hour after sunset (SS+1), total of 2,984 female mosquitoes represent- followed by a gradually reduction in activ- ing 16 species were collected by the CDC light ity through the night until one hour before trap, and 1,705 mosquitoes representing 12 sunrise. species were collected by the CBR traps. Ae- Culex erraticus peaked at sunset and sus- des albopictus (Skuse), Ae. infirmatus (Dyar and tained a higher level of host seeking activity Knab), Ae. triseriatus (Say), Ae. vexans (Meigen), for an additional two hours (SS+2), followed Anopheles crucians Wiedemann, Cq. perturbans, by a sharp reduction in host seeking activity 3 Cx.erraticus, Cx. nigripalpus Theobald, Pso- hr after sunset (SS+3). Activity then gradually rophora columbiae (Dyar and Knab) and Ps. cili- slowed down throughout the night. Host seek- ata (Fabricius) were collected in both types of ing activity was lowest 3 hours before sunrise. traps. The composition of Cq. perturbans and Host seeking periodicity at full-moon and Cx. erraticus was 40.7%, 48.4% in CDC traps non-full nights. Figure 2 shows the temporal and 37.1%, 57.1% in CBR traps, respectively. pattern of host-seeking activity for Cq. per- Compared with the CBR, more mosquito spe- turbans and Cx. erraticus as influenced by lu-

Figure 1. Overall host seeking patterns of Coquillettida perturbans andCulex erraticus. SS, sunset time; SR, sunrise time. *indicates that significantly P<0.05( ) differences between the number of mosquitoes collected at SS+2 and SS+3 hr. Jiang: Lunar Phase Impact on Host Seeking in Northern Florida 47

Figure 2. Comparison of host seeking patterns of Coquillettida perturbans (Cq.) and Culex erraticus (Cx.) under dif- ferent moon phase nights. SS, sunset time; SR, sunrise time. * *indicates significant differences P( <0.01) between the number of mosquitoes at the non-full and full moon nights. nar phases. Both species delayed their peak P<0.088; respectively). Temperature and host seeking activity at least 1 hour on full wind speed did not significantly effect Cx. er- moon nights than non-full moon nights ac- raticus (P<0.48; P<0.263; respectively). How- companied by an increase in the number of ever, significant P<( 0.01) higher numbers of mosquitoes trapped during this extended Cx. erraticus were trapped at higher humidity activity period. In terms of Cq. perturbans, its (85-95%) than low humidity and indicated peak activity was one hour later at full moon that this meteorological factor altered Cx. er- nights (SS+2) than that of non-full moon raticus host seeking behavior. nights (SS+1).The number of mosquitoes collected at SS time and SS+1 were not much IV. DISCUSSION different (Figure 2), however, the numbers collected at SS+3, SS+4, SS+5 and SS+6 were Information regarding the precise tim- much higher (no statistical significantly dif- ing of host seeking by Cq. perturbans and Cx. ferences) at the full-noon nights than that of erraticus is essential for the optimal timing the non-full moon nights. Furthermore, Cq. of adulticide missions. This study clearly in- perturbans extended its activity by two hours dicated that Cq. perturbans host seeking ac- on full-moon nights (SS to SS+3) compared tivity started at sunset and sustained a peak with non-full moon nights (i.e. SS to SS+1). level of activity for at least two hours. This Culex erraticus, on the other hand, exhib- is in contrast to that reported by Snow and ited a two hour delay in peak host seeking Pickard (1957) where peak host seeking oc- activity on full-moon nights (SS+2) compared curred between 1930-2000 h and Bosak et al. with non-full moon nights (SS) (Figure 2). (1987) from 2200 to 0100 h. It is likely that Significantly more mosquitoes P( <0.05) were mosquitoes react to light intensity crepuscu- trapped at SS+1, SS+2 and SS+4 on full moon lar changes (sunset, sunrise) to initiate their nights than non-full moon nights. flight and host seeking rather than actual Host seeking periodicity under different me- “clock time” because the periods of sunrise/ teorological conditions. Host seeking activity sunset change over the seasons. Moreover, of Cq. perturbans was not significantly influ- temporal host seeking by Cq. perturbans may enced by changes of temperature, relative vary in different geographic regions and the humidity, or wind speed (P<0.362; P<0.463; use of crepuscular light intensity changes 48 Technical Bulletin of the Florida Mosquito Control Association, Vol. 10, 2016 may be a more appropriate indicator of host captured in mouse-baited traps were collected seeking flight behavior. between 2200 and 0400 h. They compared the Although Cx. erraticus dispersal behavior collections made during the four lunar quar- (Estep et al. 2010), host feeding preference ters and found out that neither the extent of (Oliveira et al. 2011; Burkett-Candena et al. host seeking activity nor its timing was affected 2012; Mendenhall et al. 2012) and general by moon phase. The impact of lunar phase on bionomics (Robertson et al. 1993) have the host seeking patterns of mosquitoes may been extensively studied, temporal host vary by species, physiological age, and meteo- feeding periodicity has not been reported. rological conditions (Provost 1959). This is the first report to describe accurately It is important to understand ambient the timing of host seeking behavior of Cx. meteorological conditions as they affect erraticus in northern Florida, as well as the the behavior of various mosquito species. influence of meteorological conditions, Temperature, relative humidity, and wind such as moon phase and relative humidity, speed affect mosquito behavioral periodici- on host seeking behavior. ties as permissive factors. This study showed Coquillettidia perturbans and Cx. erraticus that there was no correlation between host are considered crepuscular-nocturnal spe- seeking periodicity and the meteorological cies; however, their host seeking patterns parameters recorded during collections for have similarity and differences. For exam- Cq. perturbans. However, relative humidity ple, both species consistently commenced had a significantly P( < 0.01) positive im- host seeking at sunset, either continued or pact on Cx. erraticus host seeking patterns. slightly increased its host seeking activity for A statistically significant increase in the the next two hours. However, Cq. perturbans number of Cx. erraticus collected during reduced its host seeking activity gradually at periods of high relative humidity (85-95%) SS+2, whereas, Cx. erraticus host seeking ac- was reported in this study. Day and Curtis tivity abruptly dropped at SS+2. (1989) reported a significant positive cor- Many mosquito species have their greatest relation between blood feeding by Cx. ni- period of flight activity during twilight (Mitch- gripalpus and relative humidity. Dow and ell, 1982; Nelson and Spadoni, 1972; Reisen et Gerrish (1970) also reported a positive cor- al. 1983). Moonlight can simulate twilight con- relation between host seeking of Cx. nigri- ditions by increasing nocturnal illumination. palpus in baited traps and humidity with a This study also illustrates that Cq. perturbans 4.8% increase in mosquito catches for each and Cx. erraticus significantly their increased 1% increase of relative humidity. Evidently, activity and an extended activity period un- the meteorological factors such as humidity der the full moon phase (Figure 2), which is had influence onCx. erraticus host seeking consistent with early studies. Antonipulle et behavior but not Cq. perturbans. The impact al. (1958) studied the host seeking behavior was more species-specific, as in the case of of Mansonia uniformis (Theobald) using cattle- Cx. erraticus in this study. baited traps. Analysis of the catches by hour The precise timing of an adulticide showed that during each lunar phase, the spray application designed to intersect highest approach rates occurred during the with maximum adult mosquito flight be- periods of full moonlight. Ribbands (1946) havior is critical for effective mosquito also found that the number of Anopheles fu- control. This study reveals the accurate nestus Giles entered man-baited huts was sig- timing of the crepuscular/nocturnal flight nificantly correlated with the times of twilight patterns of Cq. perturbans and Cx. erraticus, and of moonlight. Enter rates were low during two potential EEEV bridge vectors in North moonless periods of the night and highest at Florida. Results from this study could be the full moon period. However, exactly the op- providing the optimal time for controlling posite results were reported by Chadee and Ti- the two species of mosquitoes and reduc- kasingh (1989). Their studies showed that over ing the risk of EEE virus transmission in 70% mosquito Culex caudelli Dyar and Knab North Florida. Jiang: Lunar Phase Impact on Host Seeking in Northern Florida 49 V. ACKNOWLEDGEMENTS persal distances of Culex erraticus in a focus of east- ern equine encephalitis virus in the southeastern United States. J. Med. Entomol. 47:977-986. The author would like to thank Leon Howitt, B. F., H.R. Dodge, L. K. Bishop, and R. H. Gor- County Mosquito Control for helping to rie. 1949. Recovery of the virus of eastern equine identify mosquito trap locations and agree- encephalomyelitis from mosquitoes (Mansonia per- turbans) collected in Georgia. Science 110:141-142. ing to use their sentinel chicken sites for this McNeel, T. E. 1932. Observations on the biology of study. Mansonia perturbans (Walker) (Diptera: Culicidae). Proc. NJ Mosq. Exterm. Assoc. 19:91-96. Mitchell, L. 1982. Time-segregated mosquito collec- VI. REFERENCES CITED tions with a CDC miniature light trap. Mosq. News 42: 12-18. Antonipulle, P., H. V. David, and M. D. R. Karunaratne. Mendenhall, I. H., S. A. Tello, L. A. Neira, L. F. Castillo, 1958. Biology and control of Taeniorhynchus (Manso- C. B. Ocampo, and D. M. Wesson. 2012. Host prefer- nioides) uniformis Theobald, the chief vector of rural ence of the arbovirus vector Culex erraticus (Diptera: filariasis in Ceylon. Bull. World Health Org. 19:285- Culicidae) at Sonso Lake, Cauca Valley Department, 295. Colombia. J. Med. Entomol. 49: 1092-1102. Boromisa, R. D., R. S. Copeland, and P. R. Grimstad. Nelson, R. L. and R. D. Spadoni. 1972. Nightly patterns 1987. Oral transmission of eastern equine encepha- of biting activity and parous rates of some California lomyelitis virus by a northern Indiana strain of Co- mosquito species. Proc. Calif. Mosq. Control Assoc. quillettidia perturbans. J. Amer. Mosq. Control Assoc. 40:72-76. 3:102-104. Oliveira, A., C. R. Katholi, N. D. Burkett-Cadena, H.K. Bosak, P. J., L. M. Reed, and W. J. Crans. 2001. Habitat Hassan, S. Kristensen and T. R. Unnasch. 2011. Tem- preference of host-seeking Coquillettidia perturbans poral analysis of feeding patterns of Culex erraticus in (Walker) in relation to birds and eastern equine central Alabama. Vector-borne Zoonotic Dis. 11:413- encephalomyelitis virus in New Jersey. J. Vect. Ecol. 421. 26:103-109. Provost, M. W. 1959. The influence of moonlight on Burkett-Cadena, N. D., H. K. Hassan, M. D. Eubanks, E. light-trap catches of mosquitoes. Ann. Entomol. W. Cupp, and T.R. Unnasch. 2012. Winter severity Soc. Am. 52:261-271. predicts the timing of host shift in mosquito Culex er- Reisen, W. K., H. D. Lothrop, and R. P. Meyer. 1997. raticus. Biol. Letters doi: 10.1098/rsbl.2012.0075: 1-3. Time of host-seeking by Culex tarsalis (Diptera: Cu- Chadee, D. D. and E. S. Tikasingh. 1989. Diel biting licidae) in California. J. Med. Entomol. 34:430-437. activity of Culex (Melanoconion) caudelli in Trinidad, Reisen, W.K., M.M. Milby, W.C. Reeves, R.P. Meyer, and West Indies. Med. Vet. Entomol. 3:231-237. M.E. Bock. 1983. Population ecology of Culex tarsa- Cohen, S. B., K. Lowoczko, D. B. Huddleston, and E. lis (Diptera: Culicidae) in a foothill environment Moody. 2009. Host feeding patterns of potential vec- of Kern County, California: temporal changes in tors of eastern equine encephalitis virus at an epi- female relative abundance, reproductive status and zootic focus in Tennessee. Am. J. Trop. Med. Hyg. survivorship. Ann. Entomol. Soc. Am. 76:800-808. 81:452-456. Ribbands, C. R. 1946. Moonlight and house-haunting Cupp, E. W., K. J. Klingler, H. K. Hassan, L. M. Viguers, habits of female anophelines in West Africa. Bull. and T. R. Unnasch. 2003. Transmission of eastern Entomol. Res. 36:395-417. equine encephalomyelitis virus in central Alabama. Robertson, L. C., S. Prior, C.S. Apperson, and W.S. Irby. Am. J. Trop. Med. Hyg. 68:495-500. 1993. Bionomics of Anopheles quadrimaculatus and Cupp, E. W., D. Zhang, X. Yue, and M. S. Cupp. 2004. Culex erraticus (Diptera: Culicidae) in the Falls Lake Identification of reptilian and amphibian blood Basin, North Carolina: seasonal changes in abun- meals from mosquitoes in an eastern equine en- dance and gonotrophic status and host-feeding pat- cephalomyelitis virus focus in central Alabama. Am. terns. J. Med. Entomol. 30:689-698. J. Trop. Med. Hyg. 71:272-276. Snow, W. E. and E. Pickard. 1957. Correlation of vertical Day, J. F. and G. A. Curtis. 1989. Influence of rainfall on and horizontal flight activity ofMansonia perturbans Culex nigripalpus (Diptera: Culicidae) blood-feeding with reference to marked changes in light intensity. behavior in Indian River County, Florida. Ann. En- Ann. Entomol. Soc. Am. 50:306-311. tomol. Soc. Am. 82:32-37. Stat Soft, Inc. 2004. STATISTICA, Version 7. www.stat- Dow, R. P. and G .M. Gerrish. 1970. Day-to-day change soft.com. in relative humidity and the activity of Culex nigri- Williams, C. R. 2005. Timing of host-seeking behavior of palpus (Diptera: Culicidae). Ann. Entomol. Soc. Am. the mosquitoes Anopheles annulipes sensu lato Walker .63:995-999. and Coquillettidia linealis (Skuse) (Diptera: Culici- Estep, L. K., N. D. Burkett-Cadena, G. E. Hill, R. S. Un- dae) in the Murray River Valley, South Australia. nasch, and T. R. Unnasch. 2010. Estimation of dis- Aust. J. Entomol. 44:110-112. FIELD EVALUATION OF THREE COMMERCIAL MOSQUITO TRAPS AND FIVE ATTRACTANTS IN NORTHEASTERN FLORIDA

ALI FULCHER, RACHEL SHIRLEY, MICHAEL L. SMITH, JODI M. SCOTT, AND RUI-DE XUE Anastasia Mosquito Control District 500 Old Beach Road, St. Augustine, FL, 32080

ABSTRACT. Three commercial mosquito acid, octenol, ammonia, or fatty acids) col- traps and five attractants were evaluated in lect a variety of mosquito species (Kline et northeastern Florida. The combinations al. 1991, 2006, Ritchie et al. 2006, Farajol- of traps and attractants were BG Senti- lahi et al. 2009, Hoel et al. 2009, Cilek et al. nel (BGS) + BG Lure, Mosquito Magnet X 2011, Xue and Smith 2013). Recently, Xue (MMX) + CO2, Mos-Hole + naphtha, MMX et al. (2015) found the novel Mos-HoleTM + octenol, Mos-Hole + BG Lure, BGS + oc- trap, baited with naphtha, was an effective tenol, and BGS + Lurex3. The MMX trap tool for surveillance of anthropophilic mos- paired with CO2 collected the greatest num- quitoes when compared with BioGents Sen- ber of mosquitoes and the MMX trap paired tinelTM (BGS) and MMX traps. In this paper with octenol trapped the least amount com- we report on additional field evaluations we pared with the rest of the trap/attractant conducted regarding the attractiveness of combinations. BGS, MMX, and Mos-Hole traps baited with

either CO2, naphtha, octenol, lactic acid, or a Key Words. BG Sentinel trap, Mos-Hole trap, commercial BG Lure™ to collect adult mos- Mosquito Magnet X trap, BG lure, octenol quitoes in St. Johns County, Florida.

II. MATERIALS AND METHODS I. INTRODUCTION Field trials were conducted in St. Johns Mosquitoes are highly dependent on County during June and July 2013. The loca- their olfactory capabilities to host seek (An- tion of the study site was off of County Road ton and Rospars 2004). Semiochemicals 214 (29.87.13N, 81.36.98W). This low-lying

(e.g. octenol and CO2) and physical char- hardwood habitat contained several hun- acteristics (such as convection currents and dred used tires (16 m W × 133 m L × 4 m warmth) can signal the presence of a host. H) surrounded by agricultural land with Historically, mosquito traps have been bait- shallow ditches. BGS (white cover version) ed with a variety of attractant substances that (BioGents, Regensburg, Germany), Mos- take advantage of the host seeking instinct Hole (Lnd E-TND Co. Ltd., Hanam City, by mimicking a blood meal source or chemi- Gyeongi-Do, Republic of Korea), and MMX cals exuded by animal hosts. Generally, from (Woodstream Corportation, Litiz, PA) traps the perspective of the Anastasia Mosquito were used in this study and powered by 12v Control District (AMCD), there are no traps batteries. The Mos-Hole trap was a dark blue or attractants that are universally attractive unit placed on the ground surface similar to to all 43 species of mosquitoes found within how BGS traps are positioned. its jurisdiction. The following five attractants were test- A number of studies have reported that ed: BG Lure (ammonia, lactic acid, and fatty Mosquito Magnet® X traps (MMX) with acids, BioGents, Regensburg, Germany), liq-

CO2 and other attractants (including lactic uid naphtha (Lnd E-TND Co. Ltd., Hanam

50 Fulcher et al.: Field Evaluation of Three Commercial Mosquito Traps and Five Attractants 51 ® City, Gyeongi-Do, Republic of Korea), CO2 ANOVA using JMP ver. 11.1 software (SAS (dry ice block from local supplier), octenol Institute, Inc. 2013) after log transformation (BioSensory, Willimantic, Connecticut) and of trap count data. Differences were consid- Lurex3 (ammonia and lactic acid, Wood- ered significant atP ≤ 0.05. A Tukey-Kramer stream Corportation, Litiz, PA). A total of HSD multiple mean test was used to estab- seven trap/attractant combinations were lish genera differences within trap/lure evaluated (Table 1). Liquid naptha is a flam- combinations. mable liquid hydrocarbon that when com- bined with air, produces heat and CO2 that III. RESULTS AND DISCUSSION was specifically designed for the Mos-Hole trap (Xue et al. 2015). The liquid was con- A total of 1,207 mosquitoes from 15 spe- tained in a polyethylene bottle and placed cies were collected during the study period on the bottom surface inside the trap. Also representing 8 genera. Overall mosquito a mesh pouch was sewn into the Mos-Hole composition and abundance in collections trap to mimic the pouch of the BGS, so that were as follows: Aedes albopictus (Skuse) both traps were able to contain attractants. (27.9%), Culex nigripalpus Theobald (26.6%), The MMX trap was suspended from a shep- Ae. infirmatus (Dyar and Knab) (26.5%), Culi- herd’s hook so that the trap opening was seta melanura (Coquillett) (4.0%), approximately 0.5m from the ground sur- ferox (von Humboldt) (3.2%), Ps. columbiae face. Carbon dioxide was dispensed to this (Dyar and Knab) (3.0%), Ae. atlanticus (Dyar trap from a pressurized gas cylinder through and Knab) (1.8%), Anopheles quadrimacu- 5 mm silicon tubing and controlled using latus Say (1.6%), An. crucians Wiedemann a flow-meter. BG Sentinel and MMX traps (1.6%), Ae. triseriatus (Say) (0.8%) Ae. fulvus plus naphtha and MMX plus Lurex3 were pallens (Ross) (0.2%), Ps. howardii Coquillett not evaluated in this study, because they had (0.1%), rutilus (Coquillett) been evaluated earlier by Xue et al. (2015). (0.1%) and Cx. quinquefasciatus Say (0.1%). Traps were set up in the shade in a linear Of the total mosquitoes collected 0.7% were fashion, 10 m apart, and >15m from the unable to be identified due to damage. closest the tire pile. Traps were rotated and There was no statistical difference when rebaited daily after collection. After each 24- traps were compared with total mosquitoes hour trapping period, the labeled collection collected (F = 2.984, P = 0.055) as well as bags were transported to the AMCD labora- attractants and mosquito totals (F = 1.778, tory, frozen to -20° C, and identified to spe- P = 0.138). Overall, there was a significant cies using the taxonomic keys of Darsie and difference in mosquito abundance between Ward (2004). the seven trap/attractant combinations (F = Statistical analysis. Mean mosquito abun- 6.240, P = 0.004). The MMX trap paired with dance from each trap/lure combination was CO2 collected the greatest number of mos- separately pooled by genera (Aedes, Culex, quitoes followed by BGS+Lurex3, BGS+BG and Psorophora) and subjected to one-way Lure, Mos-Hole+naphtha, Mos-Hole+BG

Table 1. Mean number of adult mosquitoes from three genera collected by BG Sentinel (BGS), Mos-Hole, and Mosquito Magnet X (MMX) traps and five attractants from St. Johns County FL1 Trap Culex Aedes Psorophora

MMX + CO2 84.7a 39.7b 7.3c BGS + Lurex3 4.0c 23.0bc 6.0c BGS + octenol 2.0bc 12.0c 1.5c BG + BG Lure 2.3c 6.6c 2.0c Mos-Hole + BG Lure 2.0c 11.7c 2.0c Mos-Hole + naphtha 8.3c 10.9c 1.5c MMX + octenol 1.0c NA NA 1Means followed by the same letters in a row are not significantly different P( >0.05). 52 Technical Bulletin of the Florida Mosquito Control Association, Vol. 10, 2016

Lure, BGS+octenol, while the MMX trap paired with CO2 was the overall best combina- paired with octenol trapped the least amount tion. Hoel et al. (2007) reported similar results of mosquitoes. where un-baited Mosquito Magnet Pro traps No significant difference existed between caught the largest number of floodwater spe- each of the 15 mosquito species trapped by cies. Moreover, these authors also found that the 7 trap/attractant combinations. However, their traps, when baited with octenol and lactic there were differences (F = 3.589, P = 0.0001) acid, actually decreased collection abundance. between abundance of the three genera with- In studies by Xue et al. (2008) and Zhu et al. in trap/attractant combination (Table 1). (2014) dry ice baited MMX traps were very ef-

MMX traps plus CO2 collected significantly fective for collecting floodwater mosquitoes more Aedes and Culex mosquitoes than Pso- from multiple field sites rophora. However, trap abundance for the rest MMX traps remain effective tools for mos- of the trap/attractant combinations were no quito surveillance because the units intake different between genera. entrance, while positioned at the bottom, Previously, Xue et al. (2015) found that takes advantage of the natural tendency of Mos-Hole traps baited with naphtha were an mosquitoes to up. Moreover, MMX traps effective tool for surveillance and control for are ideal for research purposes when mosqui- anthropophilic mosquitoes compared with toes need to remain alive during collection. BGS+BG Lure and MMX traps baited with The trap’s collection chamber is able to hold Lurex3. That study did not find differences a water-soaked cotton ball for moisture and in the number of individuals, per genera, nutritional requirements if this is required by from BGS traps baited with naphtha. Xue the trapped mosquitoes. The BGS and Mos- et al. (2015) also reported that Mos-Hole Hole traps have fairly small mesh collection traps baited with BG Lure collected greater bags. Mosquito specimens in these traps tend numbers of Aedes mosquitoes than BGS traps to be easily desiccated and damaged by the baited with naphtha. We noticed the same air velocity of their intake fans. trend for Mos-Hole traps baited with either In summary, continuing studies are war- BG Lure or naphtha in our study (Table 1). ranted that evaluate the effectiveness of We also found that the MMX trap plus traps without attractive substances and/or octenol was least effective. This contradicted combining additional attractants in order to the results from Xue et al (2010) who report- determine additive or synergistic effects on ed that the same trap/attractant combination trap abundance and species composition. collected more floodwater mosquitoes than Comparative studies should consider evalua- similar traps baited with Lurex3 and a CO2 tion with traps that are currently considered sachet. Xue et al. (2010) also found Lurex3 the gold standard for mosquito surveillance and octenol were effective attractants when in order to increase the effectiveness of op- combined with MMX traps or the Mosquito erational programs. Magnet® Pro, in order “to improve the collec- tion and possibly the management of flood- IV. ACKNOWLEDGMENTS water mosquitoes”. Although the BGS trap plus Lurex3, or BG Lure, did not collect as The authors are grateful to Claudia Da- many total mosquitoes (as MMX plus CO2) vidson, Tanjim Hossain, Richard Weaver, they captured the second highest number of and Kay Gaines for their technical help. Psorophora. Our data suggests that the BGS This is a research report only and does not trap/Lurex3 combination targeted floodwa- endorse any of the commercial products in- ter species more than expected because the volved or mentioned in this report. attractant sachet (which contained compo- nents of human odor) should have drawn more anthropophilic mosquitoes to the trap. IV. REFERENCES CITED Out of all seven different trap and at- Anton, S. and J. P. Rospars. 2004. Quantitative analysis tractant combinations tested, the MMX trap of olfactory receptor neuron projections in the an- Fulcher et al.: Field Evaluation of Three Commercial Mosquito Traps and Five Attractants 53

tennal lobe of the malaria mosquito, Anopheles gam- Kline, D.L. 2006. Traps and trapping techniques for biae. J. Comp. Neurol. 475:315–326. adult mosquito control. J. Am. Mosq. Control Assoc. Cilek, J. E., C. F. Hallmon, and R. Johnson. 2011. Semi- 22:490–496. field comparison of the BG Lure, nonanal, and 1-oc- Ritchie, S.A., P. Moore, M. Carruthers, C. Wolliams, B. ten-3-ol to attract adult mosquitoes in northwestern Montgomery, P. Foley, S. Ahboo, A.F. Van Den Hurk, Florida. J. Am. Mosq. Control Assoc. 27:393-7. M.D. Lindsay, B. Cooper, N. Beebe, and R.C. Russell. Darsie, R. F. and R. A. Ward. 2004. Identification and Geo- 2006. Discovery of a widespread infestation of Aedes graphical Distribution of the Mosquitoes of North America, albopictus in the Torres Strait, Australia. J. Am. Mosq. North of Mexico. University Press of Florida, Gaines- Control Assoc. 22:358-365. ville, FL. SAS Institute, Inc. 2013. JMP®, Version 11.1. Cary, NC. Farajollahi, A., B. Kesavaraju, D. C. Price, G. M. Wil- Xue, R. D., W. A. Qualls, D. L. Kline, and T. Y. Zhao.

liams, S. P. Healy, R. Gaugler, and M. P. Nelder. 2009. 2010. Evaluation of Lurex3™, octenol, and CO2 sa- Field efficacy of BG-Sentinel and industry-standard chets as baits in mosquito magnet pro traps against traps tor Aedes albopictus (Diptera: Culicidae) and flood water mosquitoes. J. Am. Mosq. Control Assoc. West Nile virus surveillance. J. Med. Entomol. 26:244–245. 46:919–925. Xue, R. D. and M. L. Smith. 2013. Field evaluation of Hoel, D. F., D. L. Kline, and S. Allan. 2007. Evaluation of octenol, Lurex3, BG-Lure, and octenol+Lurex3 carbon dioxide, 1-octen-3-ol, and lactic acid as baits combination as baits in BG Sentinel traps to collect in Mosquito Magnet Pro traps for Aedes albopictus in Aedes albopictus. Tech. Bull. Fl. Mosq. Control Assoc. north central Florida. J. Am. Mosq. Control Assoc. 9:21–23. 25:47-57. Xue, R. D., M. L. Smith, H. Yi, and D. L. Kline. 2015. Hoel, D.F ., D. L. Kline, and S. Allan. 2009. Evaluation Field evaluation of a novel Mos-Hole trap and naph- of six mosquito traps for collection of Aedes albopictus tha compared with BG Sentinel trap and Mosquito and associated mosquito species in a suburban set- Magnet X trap to collect adult mosquitoes. J. Am. ting in north central Florida. J. Am. Mosq. Control Mosq. Control Assoc. 31:110-112. Assoc. 25:47-57. Xue, R. D., M. A. Doyle, and D. L. Kline. 2008. Field eval- Kline, D. L. 1994. Olfactory attractants for mosquito uation of CDC and Mosquito Magnet X traps baited

surveillance and control: 1-octen-3-ol. J. Am. Mosq. with dry ice, CO2 sachet, and octenol against mosqui- Control Assoc. 10(2 Pt 2):280-287. toes. J. Am. Mosq. Control Assoc. 24:249–252. Kline, D. L., D. A. Dame, and M. V. Meisch. 1991. Zhu, L., A. Fulcher, T. Hossain, C. Davidson, J.C. Beier, Evaluation of 1-octen-3-ol and carbon dioxide as and R. D. Xue. 2014. Body size, blood feeding activ- attractants for mosquitoes associated with irrigated ity and fecundity of Psorophora howardii, Psorophora rice fields in Arkansas. J. Am. Mosq. Control Assoc. ciliata and Psorophora ferox (Diptera: Culicidae). J. 7:165-169. Med. Entomol. 51:382-6. FIELD EVALUATION OF MOSQUIRON 0.12CRD AGAINST CULEX QUINQUEFASCIATUS IN STORM DRAINS, DOWNTOWN ST. AUGUSTINE, FLORIDA

ALI FULCHER, RUI-DE XUE, JODI M. SCOTT, MICHAEL L. SMITH, MARCIA K. GAINES, AND JAMES H. R. WEAVER Anastasia Mosquito Control District 500 Old Beach Road, St. Augustine, FL 32080

ABSTRACT. A controlled release formula- Treatment of individual storm drains by tion of the chitin synthesis inhibitor noval- mosquito control district personnel can be uron (Mosquiron 0.12 CRD) was evaluated laborious, time consuming, and costly. Many in downtown St. Augustine, FL storm drains controlled release larvicide formulations are for 8 weeks. Results suggested that the treat- expensive. Some sink to the bottom and are ments provided effective control of larval buried in sediments, while others may be Culex quinquefasciatus in storm drains that washed away during heavy storm and rainfall were cleaned of debris prior to treatment. events. Anastasia Mosquito Control District Synchronizing treatment with municipali- currently uses Bacillus thuringiensis israelen- ties’ drain debris cleaning schedules may im- sis (Bti) liquid in downtown St. Augustine prove the effectiveness of this formulation storm drains to achieve successful control. against mosquito larvae. However, this formulation has a limited treatment window where application must Key Words. Culex quinquefasciatus, noval- occur before fourth instar development be- uron, , storm drain cause feeding slows down as they approach later instars. The other challenges of Bti for- mulations include short residual life and the I. INTRODUCTION adverse impact that organic matter, in the drain, has on product efficacy. In Florida, the number of suburban and Past treatment of storm drain habitats urban storm drains has been steadily in- by AMCD in downtown St. Augustine has creasing due to continued growth associated focused on thermal fogging and toxic bait with residential and infrastructural develop- stations to control adult mosquitoes (Müller ment. As the number of storm drains have et al. 2010, Xue et al. 2012, Xue et al. 2013). increased, their water and debris holding Controlling larval stages in storm drains be- capacity have also increased as a result of re- fore they become adults has advantages over cent National Pollutant Discharge Elimina- adult control. The ideal profile of a larval tion System (NPDES) regulations (Metzger control product is that it would be reasonable 2004). As older infrastructure is replaced in cost, provide long residual efficacy, and with NPDES compliant ones, a subsequent remain in the application location through increase in potential larval production sites significant rainfall events. With an increase in may be inevitable (Su et al. 2003a). An in- storm drains with higher capacities, new larvi- crease of these new storm drains in close cide products that provide controlled release proximity to humans increases the challeng- of the active ingredient are critically needed. es faced by the Anastasia Mosquito Control Therefore, we report here on the evaluation District (AMCD). of a novel controlled release larvicide formu- West Nile virus vectors, like Culex quinque- lation, Mosquiron® 0.12 CRD, (novaluron fasciatus Say, thrive in storm drains and will 0.12%AI) against mosquito larvae in down- readily feed on humans (Moulis et al. 2013). town storm drains in St. Augustine, Florida.

54 Fulcher et al.: Field Evaluation of Mosquiron 0.12Crd Against Culex quinquefasciatus 55 Since 2003, various formulations of no- ment storm water drainage lines, inflow, or valuron have been evaluated against Culex outflow, according to the storm water system species in laboratory and field studies (Su map provided by the City of St. Augustine. et al. 2003b). Interestingly, application No Bti treatments were made in the study rates that achieved <80% control appear to sites by AMCD or adjacent storm drains a be species dependent (Arredondo-Jiminez week prior to, or during, the study period. and Valdez-Delgado, 2006). The mode of One CRD was placed in each of the treat- action of novaluron is that of a chitin syn- ment storm drains according to the label. thesis inhibitor within the class of insect The CRDs were cylindrical in shape, 5-10 cm growth regulators. Early studies found low- length by 1-2 cm diameter, and weighed 13.8 er concentrations of novaluron primarily g/each. A 200 to 350 ml water sample was re- resulted in inhibition emergence of adults, moved weekly from each drain using a white while higher levels primarily produced lar- plastic handheld-dipper (12.7 cm diameter val mortality (Su et al. 2003b, 2014). The 5.1 cm deep, Clarke ABC Dipper with Tele- World Health Organization recommend- scoping Handle, St. Charles, IL). Samples ed novaluron for mosquito larval control from each drain were individually placed into (WHO 2006) and also approved it for use clear plastic mosquito emergence containers in drinking water. (Bioquip, Rancho Dominguez, CA), labeled Mosquiron CRDs have been evaluated by site location and date, then transported to with success in multiple water bodies, but the AMCD insectary. If non-target organisms only one earlier study has explored the were present in samples they were placed in laboratory activity and field efficacy of this separate containers, with their sample water, product in storm drains or catch basins in and any adverse effects recorded during the North America (Su et al. 2014). The product experiment. label does not allow treatment of water di- Mortality in aquatic stages was recorded rectly connected to natural water bodies but every 48 hours and adult mosquito inhibi- does permit the treatment of catch basins or tion weekly through 14 days. If no larvae storm drains. Residual activity of novaluron were present in storm drain samples then depends on water characteristics, such as ten laboratory-reared Cx. quinquefasciatus volume and presence of organic debris. Mos- early third instar larvae were added and re- quiron CRDs are designed to deliver waxy corded as above. Larvae in each emergence particles containing novaluron in a short pe- container were maintained in AMCD’s in- riod of time, while the active ingredient is re- sectary (12L:12D, 26.6°C, >70% RH) and leased from the particle and adheres to the weekly fed approximately 0.03 g of ground surfaces of the storm drain and sediments dog biscuits. for continual release. At post treatment week 4, retreatment was necessary due to heavy rains that re- II. MATERIALS AND METHODS sulted in a considerable decrease in noval- uron efficacy. In this trial 2, each of the eight Twelve storm drains in downtown St. Au- treatment drains (control included) were gustine were selected for this study that con- treated. Four additional untreated drains tained approximately six inches (15.2 cm) of were used as controls. During trials 1 and 2 standing water containing mosquito larvae weekly precipitation, water level, and vegeta- previously determined by dipper samples. tion/debris contents were recorded for each All drains were similar in size and structure. drain. The City of St. Augustine staff also col- Eight drains were used in the study. Four lected salinity, dissolved oxygen, and pH in drains were utilized for novaluron treat- the drains for the study from April to Sep- ment. The remaining drains were untreated tember 2014. controls located at a minimum of 30 meters Statistical analysis ANOVA and a subse- from the first treatment site. No untreated quent Student’s t test were run with JMP® control sites were connected to the treat- statistical software (SAS Institute Inc. 2013). 56 Technical Bulletin of the Florida Mosquito Control Association, Vol. 10, 2016 III. RESULTS AND DISCUSSION emergence inhibition despite the later rain events (Figure 1). When larvae were found Average water temperature in the storm in higher numbers during sampling, emer- drains was 23.2° C; salinity averaged 12.4 gence inhibition was significantly greater parts per thousand; dissolved oxygen aver- (P = 0.03). Similarly, when we focused on aged 0.41 mg/l; and the average pH was 6.8. trial two, lower numbers of larvae in the Salinity was the only measure that varied. drain samples exhibited higher emergence One week after treatment, novaluron pro- inhibition and was statistically significant duced an average of 73% adult inhibition (P = 0.02). of Cx. quinquefasciatus (Figure 1). Efficacy The City provided a storm drain clean- was affected by rainfall after week 1, which ing schedule that allowed us to track which appeared to have washed the CRD compo- drains had been cleaned during our study. nents out of the drain before they had time For drains cleaned most recently, the mean to adhere to the sides of the drains. Adult emergence inhibition was higher than those emergence was reduced to 23 and 33% for not cleaned recently or upon initiation of weeks 2 and 3, respectively, but rose again to the study (P = 0.04). Lower levels of vegeta- 78% at week 4. At that time, efficacy was un- tion and debris in drains resulted in statisti- acceptable so drains were retreated on week cally significant higher levels of emergence 4. Retreatment on week 4 provided 98% inhibition (P = 0.04). Wax from the noval- emergence inhibition in week 5 samples and uron treatment was observed in multiple remained between 68 and 84% for the dura- treated drains post treatment on the sides tion of the study. of the structures and on debris (especially Overall, there was a statistically signifi- mulch chunks). When debris was minimal, cant difference in treatment versus control or non-existent, the wax appeared to adhere in adult emergence inhibition (P = 0.002). more to the sides of the drains thereby in- There was no significant difference between creasing residual efficacy of the formulation. treatments and controls in trial one (P = The challenges to the study were the signifi- 0.05), while a statistically significant differ- cant rain events. ence was found in trial two between treat- We did not observe differences in non- ment and controls (P = 0.004). Retreatment target mortality between treatments and on week 4 appeared to have boosted the controls. Similarly, Su et al. (2003b) found

Figure 1. Weekly mean percent adult emergence inhibition of Culex quinquefasciatus after treatment of storm drains by Mosquiron 0.12CRD (novaluron) at wks 0 and 4 and accompanying precipitation levels during the study. Fulcher et al.: Field Evaluation of Mosquiron 0.12Crd Against Culex quinquefasciatus 57 the novaluron 10% EC formula safe for non- able support and collaboration. Also, we targets in outdoor mesocosms at the appli- thank Barry Tyler (Pest Alto Environmental cation rate used for mosquito control. The Products Inc., Guelph, ON, Canada) for pro- majority of non-targets in our study were viding partial funding and product samples. copepods and our observations were also The assistance of Tom Downey, Ken Daniels, confirmed by Su et al. (2003b) and Arredon- Emily Thomson, and Rick Stockley in the do-Jiminez and Valdez-Delgado (2006) that field during this evaluation was appreciated. these crustaceans are not adversely affected by novaluron. Although we did not collect V. REFERENCES CITED larvivorus fish from the storm drains, Paiva et al (2014) found 2% novaluron did not af- Arredondo-Jimenez, J. I. and K. M. Valdez-Degado. 2006. Effect of novaluron (Rimon 10EC) on the fect these predators. In addition, Tawatsin et mosquitoes Anopheles albimanus, Anopheles pseudo- al. (2007) did not observe negative impacts punctipennis, Aedes aegypti, Aedes albopictus, and Culex on fish when treating forCx. quinquefasciatus quinquefasciatus from Chiapas, Mexico. Med. Vet. Entomol. 20:377-387. with 10% EC novaluron in polluted water. Jambulingam, P., C. Sadanandane, N. Nithiyananthan, Our average emergence inhibition of Cx. S. Subramanian, and M. Zaim. 2009. Efficacy of no- quinquefasciauts with the CRD formulation valuron against Culex quinquefasciatus in small- and medium-scale trials, India. J. Am. Mosq. Control As- of novaluron was not as high as reported soc. 25:315-322. by Arredondo-Jiminez and Valdez-Delgado Metzger, M.E. 2004. Managing Mosquitoes in Storm water (2006) and Su et al. (2003b) who used a Treatment Devices. University of California, Division of Agriculture and Natural Resources. Publication 8125. 10% EC formulation. Other studies, such as Miladin, S. 2004. Mosquito management going under- Jambulingam et al. (2009), involving differ- ground. Wing Beats. 15:10-12. ent formulations of novaluron found high Moulis, R. A., H. B. Lewandowski, Jr, J. D. Russell, J. L. Heusel , L.F.A.W. Peaty, D.G. Mead and R. Kelly. emergence inhibition for Cx. quinquefascia- 2013. West Nile Virus activity in Chatham County, tus in wells. Additionally, Mulla et al (2003) Georgia in 2011. Wing Beats. 24: 23-27. found that 10% EC and technical material Mulla, M. S., U. Thavara, A. Tawatsin, J. Chompoosri, M., Zaim, and T. Su. 2003. Laboratory and field eval- had long term activity in controlling Ae. ae- uation of novaluron, a new acylurea insect growth gypti in storage containers. However, our regulator, against Aedes aegypti (Diptera: Culicidae). second treatment with the CRD formulation J. Vector Ecol. 28:241-254. Müller, G. C., A. Junnila, W. A. Qualls, E. E. Revay, D. L. produced greater inhibition and may have Kline, S. Allan, Y. Schlein and R.D. Xue. 2010. Con- benefited from the previous application. trol of Culex quinquefasciatus in a storm drain system In summary, Mosquiron CRDs can be an in Florida using attractive toxic sugar baits. Med. Vet. Entomol. 24:346-351. effective control agent against the produc- Paiva, C. N., J. W. Lima, S. S. Camelo, C.de.F Lima, and tion of adult mosquitoes in recently cleaned L. P. Cavalcanti. 2014. Survival of larvivorous fish storm drains over the course of multiple used for biological control of Aedes aegypti (Diptera: Culicidae) combined with different larvicides. Trop. weeks. As the City of St. Augustine contin- Med. Intl. Health. 19:1082-6. ues to follow Best Management Practices for Rey, J. R., G. F. O’Meara, S. M. O’Connell, and M. M. urban storm water runoff, working in con- Cutwa-Francis. 2006. Factors affecting mosquito pro- duction from storm water drains and catch basins in junction with AMCD in reducing the abun- two Florida cities. J. Vector Ecol. 45:384-390. dance of Cx. quinquefasciatus in these systems SAS Institute, Inc. 2013. JMP, Version 11.1 Cary, NC. will benefit the general public, at large, by Strickman, D and J. T. Lang. 1986. Activity of Culex quin- quefasciatus in an underground storm drain in San An- reducing the nuisance and risk of disease tonio, Texas. J. Am. Mosq. Control Assoc. 2:379-381. transmission from this species. Su, T., J. P. Webb, R. P. Myer and M. S. Mulla. 2003a. Spatial and temporal distribution of mosquitoes in underground storm drain systems in Orange Coun- IV. ACKNOWLEDGEMENTS ty, California. J. Vector Ecol. 28: 79-89. Su, T, M. S. Mulla, and M. Zaim. 2003b. Laboratory and field evaluations of novaluron, a new insect growth This is a research report only, AMCD regulator (IGR), against Culex mosquitoes. J. Am. does not endorse any commercial products Mosq. Control Assoc. 19:408-418. mentioned. We would like to thank John Re- Su, T., M. L. Cheng, A. Melgoza, and J. Thieme. 2014. Lab- oratory and field evaluations of mosquiron 0.12CRD, a gan, Todd Grant, Reuben Franklin, Glabra new formulation of novaluron, against Culex mosqui- Skipp, and Jeanne Moeller for their invalu- toes. J. Am. Mosq. Control Assoc. 30:284-290. 58 Technical Bulletin of the Florida Mosquito Control Association, Vol. 10, 2016 Tawatsin, A., U. Thavara, P. Bhakdeenuan, J. Chom- public health importance. Sixth Edition. World Health poosri, P. Siriyasatien, P. Asavadachanukorn, and Organization, Geneva, Switzerland. 29. M. S. Mulla. 2007. Field evaluation of novaluron, a Xue, R. D., M. L. Smith, W. A. Qualls, and M. K. Gaines. chitin synthesis inhibitor larvicide, against mosquito 2012. Thermal fog of Anvil 10+10 ULV against Culex larvae in polluted water in urban areas of Bangkok, quinquefasciatus in storm drains in St Augustine, FL. Thailand. Southeast Asian J. Trop. Med. Public Wing Beats. 23:27-29. Health. 38:434-441. Xue R. D., G. C. Muller, W. A. Qualls, M. L. Smith, J. WHO [World Health Organization]. 2006. Pesticides M. Scott, J. Lear, and S. E. Cope. 2013. Attractive and their application for the control of vectors and pests of targeted sugar baits: field evaluations and potential for use in mosquito control. Wing Beats. 24:13-22. POPULATION REDUCTION OF MOSQUITOES AND BITING MIDGES AFTER DEPLOYMENT OF MOSQUITO MAGNET® TRAPS AT A GOLF COURSE ADJACENT TO SALTMARSH HABITATS IN ST. AUGUSTINE BEACH, FLORIDA

RUI-DE XUE, WHITNEY A. QUALLS¹ AND DANIEL L. KLINE2 Anastasia Mosquito Control District 500 Old Beach Rd, St. Augustine, FL 32080

2Department of Public Health Sciences Miller School of Medicine University of Miami, Miami, FL 33124

2Center for Medical, Agricultural, and Veterinary Entomology Gainesville, FL 32608

ABSTRACT. A two-year study was conducted Mosquito Magnet Liberty Plus trap, Mosqui- to evaluate the effectiveness of Mosquito Mag- to Magnet X traps, biting midges net® Liberty Plus (MMLP) traps to reduce mosquito and biting midge abundance in a St. Augustine Beach, FL golf community bor- I. INTRODUCTION dered by salt marsh habitat. These traps were placed in residential backyards on the fringe Mosquito traps combined with attractants of the salt marsh for the purpose of reducing have been evaluated by several researchers the number of mosquitoes and biting midges and remain the standard for adult mosquito entering the central residential area. These surveillance (Allen et al. 2009; Chaves et al. traps collected a total of 35,265 mosquitoes 2014; Jackson et al. 2012). Recently, many from 9 genera where Aedes taeniorhynchus commercial mosquito traps have integrated (28%), and Anopheles crucians (27%) were attractants into their units for the express the two major species. Mosquito Magnet X purpose of providing control of adult mos- (MMX) traps were used to monitor mosquito quitoes (Kline 2006, Xue et al. 2008). These and biting midge population abundance in types of traps have attracted homeowner the residential backyards. These traps collect- attention due to safety concerns regarding ed 8,998 mosquitoes from 5 genera with Culex pesticide-based mosquito control technol- nigripalpus (75%) as the major species. More ogy. A variety of traps and lures that emit than one million biting midges were collect- host cues have been marketed for the gener- ed from both residential areas with Culicoides al public. A prime example is the Mosquito furens (95%) being the dominant species. Magnet® mosquito trap series (Woodstream Mosquito and biting midge abundance from Corp., Lititz, PA) that has been marketed the MMX traps in residential backyards were worldwide to consumers as a convenient, ef- significantly reduced by 59 and 23%, respec- fective method to control adult mosquitoes tively, compared with trap abundance from outdoors. the residential areas with MMLP traps in the Mosquito Magnet traps have been evalu- adjacent salt marsh. However, mosquito land- ated against a variety of nuisance mosquitoes ing rates were not significantly reduced in the (Henderson et al. 2006; Jackson et al. 2012; residential areas. Kitau et al. 2010; Smith et al. 2010) and bit- ing midges (Cilek and Hallmon 2005; Lloyd Key Words. Aedes taeniorhynchus, Anopheles et al. 2008). However, these traps have not crucians, Culex nigripalpus, Culicoides furens, been evaluated for their effectiveness at re-

59 60 Technical Bulletin of the Florida Mosquito Control Association, Vol. 10, 2016 ducing major mosquito and biting midge mosquitoes landing on both forearms for 3 populations in large residential/recreation- minutes at the time of MMLP collection. al areas (such as golf club communities) In order to monitor temporal mosquito with surrounding salt marsh habitat. and biting midge abundance in the treat- The purposes of this study were to: (1) ment and control sites, a MMX trap was determine species composition of mosqui- placed in each of two backyards in all areas. toes and biting midges in the salt marsh Traps were suspended by a shepherd’s hook habitat and central residential area in a large and baited with dry ice (Xue et al. 2008). golf course community, (2) evaluate wheth- Distance between MMX traps in each area er Mosquito Magnet Liberty Plus traps can was about 1.5 km while the distance between reduce population abundance of the major MMX traps and MMLP traps ranged from mosquito and biting midge species encoun- 1-3 km. MMX traps were turned on a Thurs- tered in the residential areas, compared with day and collected 24 h later on Friday. Mos- the salt marsh habitat, and (3) determine quito and biting midge collections from all whether traps can reduce human landing MMLP and MMX traps were transported to rate counts of mosquitoes in the residential the laboratory for species identification and area. counting under a 10-40X-zoom dissection microscope. II. MATERIALS AND METHODS Statistical analysis. All statistical analyses were performed using SAS (SAS Institute This study was conducted from July to 2001). Prior to analyses, mean trap counts October, 2004-2005, at the Marsh Creek were log (x+1) transformed then subjected Golf Course on Anastasia Island, St. Augus- to PROC MIXED with a repeated measures tine Beach, Florida. This golf course covers analysis of variance (ANOVA). A χ2 test was 9,333 acres that includes a residential com- performed on mosquito collections to de- munity with about 400 homes. The habitat termine if differences existed between treat- surrounding the golf course is primarily ment and control areas (P<0.05). salt marsh with low-lying grassy areas that will retain rainwater for days at a time. The III. RESULTS central residential area and residential back- yards adjacent to the salt marsh were each A total of 35,265 mosquitoes represent- divided into 2 sections. In one area, 16 Mos- ing 27 species from 9 genera were collected quito Magnet Liberty Plus (MMLP) traps from all MMLP traps from backyard resi- (American Biophysics Corporation), baited dences adjacent to the salt marsh. The spe- with octenol, were placed at 500 m intervals cies composition is presented in Table 1. The in central residential backyards. The same major species of mosquitoes in this area was number of traps, and set up, were placed in Aedes taeniorhynchus (Wiedemann) (28%), the backyard of homes adjacent to the salt Anopheles crucians Wiedemann (27%), Culex marsh. Residences with MMLP traps will be nigripalpus Theobald (12%), Psorophora co- referred to as “treatments” and were oper- lumbiae (Dyar and Knab) (9%), Aedes sollici- ated from Monday through Thursday for tans (Walker) (6%), and Ae. infirmatus (Dyar 4 nights per week. Controls consisted of and Knab) (5%). Total abundance from all homes in each residential area without traps. MMLP traps in the central residential sec- Treatment and control areas were switched tion totaled 8,998 mosquitoes and contained back and forth weekly, within each residen- 10 species from 6 genera. The dominant spe- tial area, during the 6 wk study. In order to cies in these traps was Cx. nigripalpus (75%) obtain additional information of mosquito (Table 2). Collections of all MMX traps from abundance in the central residential section residential areas adjacent to the salt marsh with MMLP traps, landing rate counts (LRC) (with MMLP traps) totaled 27,072 mosqui- were conducted (2 m away from each trap) toes and 495,780 biting midges (95% Culi- by a volunteer who recorded the number of coides furens and 5% C. mississippiensis) while Xue et al.: Population Reduction of Mosquitoes and Biting Midges 61 Table 1. Species composition of saltmarsh and freshwa- central residential and salt marsh areas with ter mosquitoes collected by Mosquito Magnet Liberty Plus traps baited with octenol in residential backyards MMLP traps were not significantly different adjacent to salt marshes and collected by Mosquito from LRCs in areas without traps (Table 3). Magnet X traps baited with dry ice in central residen- tial areas, Marsh Creek golf course, St. Augustine, FL 2004-2006a. IV. DISCUSSION

Central Our study found that Mosquito Magnet Salt marsh side residential area Species (%) (%) Liberty Plus traps significantly reduced mos- quito and biting midge populations in the Aedes albopictus 0.03 0.03 Ae. atlanticus 2 0.1 central residential area of the Marsh Creek Ae. canadensis 0.001 0 Golf Course. Similar population reduction Ae. fulvus pallens 0.09 0 has been observed by other in trap out stud- Ae. infirmatus 5 3 ies where Mosquito Magnets have been eval- Ae. mathesoni 0.001 0 uated (Cilek and Hallmon 2005, Kline 2002, Ae. mitchellae 0.09 0 2007, Collier et al. 2006, Qualls and Mullen Ae. sollicitans 6 0 2007, Lloyd et al. 2008, Li et al. 2010). Un- Ae. taeniorhynchus 28 4 fortunately, Mosquito Magnet Liberty traps Ae. triseriatus 0.001 0 Ae. vexans 0.02 0 placed in residential backyards adjacent to Anopheles atropos 2 0 the salt marsh or within the central residen- An. crucians 27 6 tial of the golf course did not result in a sig- An. punctipennis 1.3 0 nificant reduction of landing rate counts, An. quadrimaculatus 2.6 0.7 compared with areas without MMLPs. It is Culex erraticus 0.2 0 possible that the sites where landing rates Cx. nigripalpus 12 75 were conducted were too close (2 meters) Cx. quinquefasciatus 3 3 Culiseta melanura 1.2 3 to the MMLP sites where more mosquitoes Coquillettidia perturbans 0.1 0 may have been present. However, MMLPs Mansonia dyari 0.08 0 did collect a large number of mosquitoes Ms. titillans 0.02 0 that had significantly reduced the number Psorophora ciliata 0.001 0 of mosquitoes in those areas. Ps. columbiae 9 5 The deployment of a linear array of Mos- Ps. cyanescens 0.002 0 quito Magnet traps in residential backyards Ps. ferox 0.5 0 Ps. howardii 0.08 0 that are adjacent to salt marsh areas may be aχ² = 4260.81, df = 26, P<0.01 considered to be an environmentally friend- ly method to reduce the nuisance mosquito and biting midge problems, especially in sen- sitive habitats. This control method could be easily adopted and accepted by the residents 45,579 adult mosquitoes and 2,195,533 bit- who live by salt marshes. ing midges were collected from MMX traps in the same area without MMLP traps. V. ACKNOWLEDGEMENTS Mosquito and biting midge collections from MMX traps in MMLP residential back- The authors wish to thank Alex Santoro yards adjacent to the salt marsh were signifi- and James Wynn for their assistance on the cantly reduced by 59 and 23%, respectively, project. We also thank A. Grant and K. McK- compared with trap abundance in similar enzie from the former American Biophys- areas without MMLPs. This trend continued ics Corporation for providing the Mosquito in the central residential area where MMLP Magnet Liberty Plus and Mosquito Magnet- traps provided about a 63% reduction in X traps. We also the May Management Com- the number of mosquitoes in this area pany and many residents in Marsh Creek to compared with the same area without traps allow us to use their properties during the (Table 2). Unfortunately, mosquito LRCs in study. This is a research report only and 62 Technical Bulletin of the Florida Mosquito Control Association, Vol. 10, 2016 Table 2. Total numbers of mosquitoes collected by MMX traps baited with dry ice in the central residential areas between treated areas with Mosquito Magnet Liberty Plus (MMLP) traps and untreated control areas (without MMLP traps), Marsh Creek, St. Augustine, Florida 2004-2006a. Species Central area in treated section Central area in untreated control section Aedes albopictus 1 2 Ae. atlanticus 4 5 Ae. infirmatus 166 122 Ae. taeniorhynchus 138 199 Anopheles crucians 226 310 An. quadrimaculatus 18 41 Culex nigripalpus 2,703 4,049 Cx. quinquefasciatus 100 190 Culiseta melanura 14 259 Psorophora columbiae 105 346 Total 3,475 5,523 aχ² = 231.081, df = 9, P < 0.01

Table 3. Mean human landing rate counts of Aedes taeniorhynchus (primarily) between Mosquito Magnet Liberty Plus traps-treated section and untreated (control) section, Marsh Creek, St. Augustine, Florida 2004-2005a. Treated Untreated control Central residential area Central residential area (MMX site) Years salt marsh area salt marsh area (MMX site) in treated section in untreated control section 2004 0.52 1.36 0.88 1.00 2005 2.02 1.83 2.13 2.25 Total 2.54 3.19 3.02 3.25 aχ² = 1.005, df = 3, P>0.05 mention of specific products does not indi- Henderson, J.P., R. Westwood, and T. Galloway. 2006. An assessment of the effectiveness of the Mosquito cate endorsement by the AMCD. Volunteers Magnet Pro model for suppression of nuisance mos- gave informed consent prior to participa- quitoes. J. Am. Mosq. Control Assoc. 22:401-407. tion in the study and this research study was Jackson, M.J., J.L. Gow, M.J. Evelyn, T.J. McMahon, T.J. Howay, H. Campbell, J. Blancard, and A. Thielman. conducted according to protocol number 2012. An evaluation of the effectiveness of a com- AMCD 10-13-2005 as approved by the An- mercial mechanical trap to reduce abundance of astasia Mosquito Control Board of Commis- adult nuisance mosquito populations. J. Am. Mosq. Control Assoc. 28:292-300. sioners for use of human subjects in opera- Kitau, J, H. Pates, T.R. Rwegoshora, D. Rwegoshore, J. tional projects. Matowo, E.J., Kweka,, F.W. Mosha, K. McKenzier, and S.M. Magesa. 2010. The effect of Mosquito Mag- net Liberty Plus trap on the human mosquito biting VI. REFERENCES CITED rate under semi-field conditions. J. Am. Mosq. Con- trol Assoc. 26:287-294. Allen, R.A., C.N. Lewis, M.V. Meisch, and D.A. Dame. Kline, D.L. 1999. Comparison of two American Bio- 2009. Comparison of three residential mosquito physics mosquito traps: the professional and a new traps against riceland mosquitoes in southeast Ar- counterflow geometry design. J. Am. Mosq. Control kansas. J. Am. Mosq. Control Assoc. 25:110-112. Assoc. 15:276-282. Chaves, L.S., G.Z. Laporta, and M.A. Sallum. 2014. Ef- Kline, D.L. 2002. Evaluation of various models of pro- fectiveness of Mosquito Magnet in preserved area on pane powered mosquito traps. J. Vect. Ecol. 27:1-7. the coastal Atlantic rainforest: implication for ento- Kline, D.L. 2006. Traps and trapping techniques for mological surveillance. J. Med. Entomol. 51:915-924. adult mosquito control. J. Am. Mosq. Control Assoc. Cilek, J.E .and C.F. Hallmon. 2005. The effectiveness 22:490-496. of the Mosquito Magnet trap for reducing biting Kline, D.L. 2007. Semiochemicals, traps/targets and midge (Diptera: Ceratopogonidae) populations in mass trapping technology for mosquito manage- coastal residential backyards, J. Am. Mosq. Control ment. J. Am. Mosq. Control Assoc. 23(2S):241-251. Assoc. 21:218-221. Li, C.X., Y.D. Dong, X.L. Zhang, C. Chen, S.P. Song, B. Collier, B.W., M.J. Perich, G.J. Boquin, S.R. Harrington, Deng, T.Y. Zhao, and R.D. Xue, 2010. Evaluation of and M.J. Francis. 2006. Field evaluation of mosquito octenol and lurex as baits in Mosquito Magnet Pro control devices in southern Louisiana. J. Am. Mosq. traps to collect vector mosquitoes in China. J. Am. Control Assoc. 22:444-450. Mosq. Control Assoc. 26:449-451. Xue et al.: Population Reduction of Mosquitoes and Biting Midges 63

Lloyd, A.M., D.L. Kline, J.A. Hogsette, P.E. Kaufman, and 3-ol for collecting Aedes albopictus and other urban S.A. Allan. 2008. Evaluation of two commercial traps mosquitoes. J. Am. Mosq. Control Assoc. 23:131-136. for the collection of Culicoides (Diptera: Ceratopogo- Xue, R.D., M.A. Doyle, and D.L. Kline. 2008. Field nidae). J. Am. Mosq. Control Assoc. 24:253-262. evaluation of CDC and Mosquito Magnet® X traps Qualls, W.A. and G.R. Mullen. 2007. Evaluation of the baited with dry ice, sachet, and octenol against mos- Mosquito Magnet Pro trap with and without 1-octen- quitoes. J. Am. Mosq. Control Assoc. 24:249-252. SUBLETHAL DOSES OF AN ATTRACTIVE TOXIC SUGAR BAIT MIXED WITH THE INSECT GROWTH REGULATOR PYRIPROXYFEN DID NOT EFFECT SURVIVAL OR FECUNDITY OF AEDES AEGYPTI

CODI S. ANDERSON, JODI M. SCOTT, ALICE P. FULCHER, GUNTER C. MULLER¹, AND RUI-DE XUE Anastasia Mosquito Control District 500 Old Beach Road, St. Augustine, FL 32080

1Department of Microbiology and Molecular Genetics, IMRIC Kuvin Centre for the Study of Infectious and Tropical Diseases Faculty of Medicine, Hebrew University, Jerusalem, Israel 91120

ABSTRACT. Addition of the insect growth laayoune et al. 2013). In addition, regulator, pyriproxyfen (0.5%), into an at- and eugenol have been reported to be orally tractive toxic sugar bait solution containing effective against several species of mosqui- a sublethal dose of eugenol (0.0125%) or toes (Xue and Barnard 2003, Qualls et al. boric acid (0.1%) had no significant effect 2014, Xue et al. 2006). Interestingly, Ali et al. on the survival or fecundity of adult Aedes (2006) reported that sub-lethal doses of bo- aegypti compared with sugar baits without ric acid affected survival, blood feeding, and these active ingredients. fecundity in Aedes albopictus (Skuse) when orally ingested. A recent laboratory study by Key Words. Aedes aegypti, pyriproxyfen, euge- Fulcher et al. (2014) reported effective con- nol, boric acid, attractive toxic sugar bait trol of adult and larval mosquitoes in runoff water from plants previously sprayed with an attractive sugar bait (ASB) and the insect growth regulator (IGR) pyriproxyfen. In ad- I. INTRODUCTION dition, Chism and Apperson (2003) dem- Female and male mosquitoes feed on onstrated 38% emergence inhibition when plant nectars as carbohydrate sources in pyriproxyfen-treated Ae. albopictus oviposited order to obtain the necessary energy re- in untreated larval habitats. Therefore, we quirements to survive. Indeed, Marshall et decided to investigate the effects of sub-le- al. (2013) found, in laboratory studies, that thal ATSBs containing boric acid or eugenol some female Anopheles species will feed on in combination with pyriproxyfen on surviv- sucrose daily. A number of authors have re- al, fecundity, and fertility of the dengue and ported on the evaluation of a variety of plant chikungunya vector, Aedes aegypti (L.). sugar sources that could be incorporated into toxic baits thereby exploiting a mos- II. MATERIALS AND METHODS quito’s sugar feeding behavior (Muller et al. 2008, Muller et al. 2010, Beier et al. 2012) Mosquitoes. The Ae. aegypti colony used in Studies of attractive toxic sugar baits our study, was obtained from the USDA, Cen- (ATSB), during the past decade, have fo- ter for Medical, Agricultural, and Veterinary cused predominantly on integrating topi- Entomology, Gainesville, FL. Oviposition pa- cally active such as , per containing eggs of this species was cut , or into into 2 cm strips and placed into larval rearing oral formulations for adult mosquito con- pans (28 cm × 56 cm) containing 2,500 ml of trol (Allan 2011, Muller et al. 2010, Khal- reverse osmosis water (RO). This method was

64 Anderson et al.: Sublethal Doses of An Attractive Toxic Sugar Bait 65 utilized to produce a maximum of 500 mos- solution were 0.1%, 0.05%, 0.025%, and quito larvae per pan. Mosquito larvae were 0.0125%. Green food coloring was not add- given 3 mg of 3:2 ratio of bovine liver pow- ed to these solutions. All mosquitoes were der and brewer’s yeast (ICN Biomedical, Inc., fed about 12 ml of each solution through OH). While in the pupal stage, mosquitoes cotton balls placed on top of the tulle mesh. were placed into 15 cm × 15 cm × 5 cm Ziploc Cotton balls remained on the mesh as adult brand sandwich containers (SC Johnson & mortality was recorded at 24, 48, and 72 Son Inc., Racine, WI) and moved into rear- hours. The experiment was repeated three ing cages (Bioquip Products Inc., Compton, times. Based on the results of the eugenol CA) to emerge. Cotton balls saturated with bioassays, 0.0125% was selected as the sub- 10% sucrose solution in 266 ml clear plastic lethal concentration for incorporation into cups (SOLO®, Dart Container Corporation, ATSBs (Figure 1). Mason, MI) were provided ad libitum. Adult ATSB/IGR bioassays. Boric acid (0.1%) mosquitoes were utilized in bioassays once and eugenol (0.0125%) ATSBs were sepa- they reached 5-7 days of age. rately mixed with 0.5% pyriproxyfen (P) Attractive sugar bait (ASB). Mango fruit (NyGuard® [10% AI] IGR Concentrate, pulp was combined with white cane sugar Minneapolis, MN) and 0.5% P with ASB only, at a 1:1 ratio with approximately 0.8% lime as the final formulations for testing. Con- juice. The mixture was stirred and heated to trol formulations consisted of ASB without 100° C and allowed to cool to 26° C, produc- a toxicant (negative control) and ATSB 1% ing mango syrup. To prepare the attractive boric acid (positive control). Approximately sugar bait (ASB), the syrup was mixed with 70-80, five to seven-day old male and female reverse osmosis water at a ratio of 1:3. Green mosquitoes were mouth aspirated into modi- food coloring (McCormick brand) was add- fied 5 L buckets with a 15 cm hole cut into ed (0.5%) to the formulation in order to vi- the side covered by a fabric sleeve adhered sualize ingestion by the mosquitoes (Müller with glue (Qualls et al. 2014). This sleeve et al. 2010). Also about 0.08% Agricultural allowed for continuous access to the mos- Poly Control 2 Sticker Spreader (Brewer quitoes, with minimal escapees. The bucket International, Vero Beach FL) was added opening was covered with Wal-Mart brand to the ASB solution because this substance tulle mesh secured with rubber bands. Each was previously used during previous field ap- treatment was assigned randomly to each of plications of ATSB (JM Scott unpubl. data). five buckets. Baits were poured into unsalted Attractive toxic sugar bait was prepared by natural collagen sausage casings, and then adding toxicant to the ASB (percentages de- tied with a balloon (single) knot. Bait “sau- termined below). sages” were placed on top of the tulle mesh, ATSB sub-lethal determination. As reported and mosquitoes were allowed to feed for 48 by Ali et al. (2006), we selected 0.1% boric hours. Adult mortality was recorded at 24 acid as the sub-lethal concentration for test- and 48 h. ing this chemical. Because biologically active Fecundity. Males and females (70-80 in- sub-lethal concentrations of eugenol have dividuals, five to seven-day old) were placed not been reported, we determined this con- together in each bucket (to ensure mating centration is a series of ASB serial dilution occurred) and presented with bovine blood bioassays. The following procedures were (36° C) for 48 hours administered in the same followed: nine to twelve female Ae. aegypti fashion as the bait sausages. After this time, (5-7 days old) were mouth aspirated into in- blood-fed individuals were mouth aspirated dividual 1 L glass mason jars, with the mouth from the buckets into oviposition cages (that of the jars covered with Wal-Mart brand tulle consisted of inverted 266 mL clear plastic mesh secured with the metal bands of the cups with a 1 cm hole in the base to facilitate jar lid. A total of 25 jars were used for each transfer) at the rate of one mosquito per cup. of 3 repetitions. Negative controls were fed Ten cups were used per treatment including ASB only. Eugenol concentrations in ASB controls. This hole was stoppered with cot- 66 Technical Bulletin of the Florida Mosquito Control Association, Vol. 10, 2016

1(F = 8.757, df = 4,45, P < 0.001)

Figure 1. Average percent mortality of adult Aedes aegypti to various concentrations of eugenol when mixed with an attractive sugar bait (ASB) solution after ingestion at 24, 48, and 72 h1. ASB without eugenol is an untreated control. ton and utilized throughout the experiment and will be referred to as “hatch containers”. to administer sucrose solution (10%) to the Larval food was added and lids left unsealed mosquitoes. The mouth of the cup was cov- on one corner in order to allow for ventila- ered with tulle and secured into place with a tion. Hatch containers were observed daily rubber band. A 2 cm × 6 cm strip of germina- for pupae. Pupae were counted and trans- tion paper was placed loosely into the mouth ferred to clear plastic 266 ml plastic cups of the inverted cup by stretching the rubber through a 0.5 cm hole in the tulle mesh cov- band to the side and sliding the paper inside ering the aperture of the cup. There were to rest on the mesh. 50 cups per repetition. When not in use, The oviposition cages were placed at a the hole was covered with tape. Emerging 45° angle into larval rearing pans (28 cm × adults were provided with a cotton ball satu- 56 cm) that contained 1 cm of reverse osmo- rated with 10% sucrose solution, ad libitum, sis water to allow the oviposition paper to be placed on top of the mesh. Successful emer- dampened. Pans were placed into an incuba- gence was counted by subtracting the dead/ tor and maintained at 26.6° C and 14:10 L:D. un-emerged pupae from total pupae. Adult This study was repeated three times with 10 mortality was recorded at 48 h post initial cups (females) per control/treatment and oviposition and observed for ten days. 50 cups total per repetition. Mosquitoes Statistical analysis. Analysis of data used were allowed to oviposit for 48 hours. After JMP statistical software (SAS Institute, Inc. this time, egg papers were collected, num- 2012). Mean adult survival was assessed ber of eggs counted, and allowed to dry for through chi square analysis to determine sig- five days allowing for egg embryonation. nificant differences between formulations. Fertility. Eggs were hatched in 400 ml of Blood feeding, and total egg data were sub- water in 15 cm × 15 cm × 5 cm plastic Zip- jected to ANOVA to determine significant loc® brand sandwich containers with lids differences between formulations. For all Anderson et al.: Sublethal Doses of An Attractive Toxic Sugar Bait 67 analyses, differences were considered signifi- for this study, we found that 0.1% produced cant when P <0.05. significantly greater mortality compared with 0.0125% selected for our sublethal evalua- III. RESULTS AND DISCUSSION tions (Figure 3). These results were similar with those of Qualls et al. (2014). However, Adult Survival. The ASB+P 1% boric acid Hao et al. (2008, 2012) found that eugenol at formulation caused significantly greater higher concentrations significantly reduced mortality of Ae. aegypti at 48 h post ingestion, blood feeding in adult mosquitoes and might compared with the mortality caused by oth- act as a repellent. Perhaps, this may be the er formulations (Figure 2). Overall, none of reason, in our study, why eugenol mixed with the other ATSBs significantly affected mor- pyriproxyfen did not result in any significant tality or survival of females, 10 days after ovi- mortality compared with controls. position, even though the 0.1% boric acid Fecundity and fertility. No significant dif- formulation resulted in >16% mortality (Fig- ference was observed between the total num- ure 3). However, Ali et al. (2006) reported ber of eggs laid between ATSBs and controls that a 0.1% boric acid sugar bait significantly (Figure 4). This result was similar with the reduced the survival rate of Ae. albopictus report that sublethal concentrations of boric compared with an untreated control. We be- acid sugar bait significantly reduced fecun- lieve that this may be caused, in our study, dity of Ae. albopictus (Ali et al. 2006), but the by lower boric acid concentration in the ASB reduction of number of eggs in our experi- after mixture with pyriproxyfen. ment was not significant, compared with the In our initial bioassay identification of a other baits. As stated earlier, this may have eugenol sub-lethal concentration suitable been caused by the addition of pyriproxyfen

124 h (χ² = 11.753, df = 4, P<0.01) and 48 h (χ² = 17.108, df = 4, P<0.01)

Figure 2. Average percent mortality of adult Aedes aegypti from ingestion of an attractive sugar bait (ASB) mixed with either boric acid or eugenol with 0.5% pyriproxyfen (P) after 24 and 48 h. ASB without boric acid or eugenol is an untreated control1. 68 Technical Bulletin of the Florida Mosquito Control Association, Vol. 10, 2016

1(F = 0.269, df = 4,20, P > 0.05)

Figure 3. Average percent mortality of Aedes aegypti at 10 days following oviposition after feeding on attractive sugar baits (ASB) containing either boric acid or eugenol with pyriproxyfen (P). ASB without boric acid or eugenol is an untreated control1. to the ATSB that reduced the concentration 0.5% pyriproxyfen did not significantly af- of boric acid. However, Chism and Apperson fect survival of Ae. aegypti. Also, mixing pyri- (2003) did not observe any adverse effects on proxyfen with boric acid or eugenol as baits fecundity even when gravid Ae. albopictus were at the concentrations used in this study did exposed to pyriproxyfen-treated surfaces. not reveal vertical transfer nor produce any In our study, egg hatchability consid- significant impact on fecundity by this insect erably varied between ATSBs. In one rep- growth regulator. etition of the experiment, complete larval emergence of all eggs occurred. In anoth- IV. ACKNOWLEDGEMENTS er repetition, three of 25 containers with eggs did not produce larvae. In other rep- Thank you to the fellow interns and An- etitions, only a single was recovered astasia Mosquito Control District employees from eggs produced from females that fed who volunteered in assisting with this proj- upon ASB+P (0.5%), ASB+P+(0.0125%) ect and to AMCD for the opportunity to con- eugenol, and ASB+P+(0.1%) boric acid. duct the study. Larvae in another container, from another repetition, died in the third instar stage V. REFERENCES CITED (ASB+P (0.5%). We believe that the con- Ali, A. R. D. Xue, and D. R. Barnard. 2006. Effects of tainers were possibly contaminated by an sublethal exposure to boric acid sugar bait on adult unknown contaminant, or contamination survival, host-seeking, blood feeding behavior, and reproduction of Aedes (Stegomyia) albopictus. J. Amer. was the result of sucrose solution cotton Mosq. Control Assoc. 22:464-468. balls where eggs had been oviposited dur- Allan, S. A. 2011. Susceptibility of adult mosquitoes to ing the evaluation. insecticides in aqueous sucrose baits. J. Vect. Ecol. 36:59-67. In summary, sublethal concentrations of Beier, J. C., G. C. Muller, G. U. Weidong, K. L. Arheart, boric acid or eugenol in an ASB mixed with and Y. Schlein. 2012. Attractive toxic sugar bait Anderson et al.: Sublethal Doses of An Attractive Toxic Sugar Bait 69

1(F = 0.773, df = 4,20, P > 0.05)

Figure 4. Total number of eggs laid by Aedes aegypti after ingestion of attractive sugar baits (ASB) containing either boric acid or eugenol with pyriproxyfen (P). ASB without boric acid or eugenol is an untreated control1.

(ATSB) decimates populations of Anopheles malaria impacts on nontarget organisms in Morocco. Envi- vectors in arid environments regardless of local ron. Entomol. 42:1040-1045. availability of favored sugar-source blossoms. Malar. Marshall, J.M., M.T. White, A.C. Ghani, Y. Schlein, G.C. J. 11:31. Muller, and J.C. Beier. 2013. Quantifying the mos- Cheng, S. S., J. Y. Liu, K. H. Tsai, W.J. Chen, and S. T. quito’s sweet tooth: modeling the effectiveness of at- Chang. 2004. Chemical composition and mosquito tractive toxic sugar baits (ATSB) for malaria vector larvicidal activity of essential oils from leaves of dif- control. Malaria J. 12:291-304. ferent Cinnamomum osmopheloeum provenances. J. Muller, G .C., V. D. Kravchenko, and Y. Schlein. 2008. Agri. Food Chem. 52:4395-400. Decline of Anopheles sergentii and Aedes caspius popu- Chism, B. D. and C. S. Apperson. 2003. Horizontal lations following presentation of attractive toxic transfer of the insect growth regulator pyriproxifen () sugar baits stations in an oasis. J. Am. to larval microcosms by gravid Aedes albopictus and Mosq. Contr. Assoc. 24:147-149. Ochleotatus triseriatus mosquitoes in the laboratory. Muller, G. C., A. Junnila , W. A. Qualls, E.E. Revay, D. L. Med. Vet. Entomol. 17:211-220. Kline, S. A. Allan, Y. Schlein, and R. D. Xue. 2010. Fulcher, A., J. M. Scott, W. A. Qualls, G. C. Muller, and Control of Culex quinquefasciatus in a storm drain R.D. Xue. 2014. Attractive toxic sugar baits mixed system in Florida using attractive toxic sugar baits. with pyriproxyfen sprayed on plants against adult Med. Vet. Entomol. 24:346-351. and larval Aedes albopictus (Diptera: Culicidae). J. Qualls, W. A., G. C. Muller, E. E. Revay, S. A. Allan, K. Med. Entomol. 51:896-899. L. Arheart, J. C. Beier., M. L. Smith, J. M. Scott., V. Hao, H., J. Wei, J. Dai, and J. Du. 2008. Host-seeking D. Kravchenko, A. Hausmann, Z .A. Yefremova, and and blood-feeding behavior of Aedes albopictus (Dip- R. D. Xue. 2014. Evaluation of attractive toxic sugar tera: Culicidae) exposed to vapors of geraniol, citral, bait (ATSB) – barrier control of vector and nuisance citronellal, eugenol, or anisaldehyde. J. Med. Ento- mosquitoes and its effect of non-target organisms mol. 45:533-539. in sub-tropical environments in Florida. Acta Trop. Hao, H., J. Sun, and J. Dai. 2012. Preliminary analysis 131:104-110. of several attractants and spatial repellants for the SAS Institute, Inc. 2012. JMP®, Version 10.1. Cary, NC. mosquito, Aedes albopictus using an olfactometer. J. Xue, R. D. and D. R. Barnard. 2003. Boric acid sugar Ins. Sci. 12:1-10. bait kills adult mosquitoes (Diptera: Culicidae). J. Khallaayoune, K., W. A. Qualls, E. E. Revay, S. A. Al- Econ. Entomol. 96: 1559-1562. lan, K. L. Arheart, V. D. Kravchenko, R. D. Xue, Y. Xue, R. D., D. L. Kline, A. Ali, and D. R. Barnard. 2006. Schlein, J. C. Beier, and G. C. Muller. 2013. Attrac- Application of boric acid to plant foliage for adult tive toxic sugar baits: control of mosquitoes with the mosquito control. J. Amer. Mosq. Control Assoc. 22: low-risk active ingredient and potential 297-500. EVALUATION OF POWER BREEZER AND MISTING CITRONELLA AGAINST AEDES ALBOPICTUS

EMILY H. THOMSON¹, JODI M. SCOTT1, ALI FULCHER1, MICHAEL L. SMITH1, PHIL KOEHLER2, AND RUI-DE XUE1 1Anastasia Mosquito Control District 500 Old Beach Rd, St. Augustine, FL32080

2University of Florida, Entomology and Nematology Department, University of Florida, Gainesville, FL 32611

ABSTRACT. An industrial high-velocity viruses, are leading global public health misting fan, with and without a 1% citro- concerns (Kilpatrick et al. 2006, Mitchell nella mixture, was evaluated for repelling 1991). With the advancement of wide- host seeking Aedes albopictus. Human partici- spread human transportation, these dis- pants were utilized as targets and arranged eases and their vectors are no longer en- in a horizontal line 5, 8, 10, and 15 meters demically confined. In addition, as human from the fan while mosquitoes were released populations grow, especially in developing from the opposite end of the testing arena. countries, the risk of mosquito-borne ill- Control evaluations were conducted without ness is becoming more threatening (Kil- wind or citronella. Overall, no significant patrick et al. 2006). Aedes albopictus (Skuse) difference in participant preference by Ae. is an important vector of several arboviral albopictus was observed in treatments or con- pathogens and one that is competent in trols at any of the distances except at 8 m. transmitting many diseases in high density At 15 m, significantly more mosquitoes were urban and suburban settings because of collected from participants with the fan on widely available larval developmental ar- (and no mist) compared with the control eas in close proximity to human habitation (fan off). When the fan was misting 1% cit- (Mitchell 1991). ronella, more mosquitoes landed on partici- A recent report claiming that an oscil- pants as distance from the fan unit increased lating table fan was capable of deterring but no statistical significant difference be- mosquito bites at a backyard gathering by tween the control and treatment groups disrupting a mosquito’s ability to locate occurred. In summary, the wind velocity potential hosts has provided additional generated by the misting fan itself, or the cit- interest in non-chemical and natural re- ronella mixture, did not significantly reduce pellent options (Broad 2013). Fans have host seeking Ae. albopictus and may not be a commonly been used to deter pest in- practical method of protection from biting sects, such as cockroaches and fruit in mosquitoes for personal or public outdoor homes and businesses (Kolbe 2003). The events based upon the results of our study. American Mosquito Control Association describes most mosquitoes as weak fliers, Key Words. Misting fan, Aedes albopictus, only capable of flying at 0.45 to 0.67 me- citronella, host seeking ters per second, and recommends placing a large fan on a household deck or patio to provide a “low tech solution” to reduce I. INTRODUCTION mosquito bites (AMCA, 2014). Manipulat- ing wind as a means of repellency is gain- Mosquito-borne illnesses, such as West ing more public attention and remains Nile, chikungunya, dengue, and yellow relatively unstudied.

70 Thomson et al.: Evaluation of Power Breezer and Misting Citronella Against Aedes albopictus 71 Citronella oil is a widely recognized of the fan to prevent mosquito bites when and commonly utilized . misting a 1% citronella oil at multiple dis- Recent studies have debated the efficacy tances from the machine. of citronella to repel mosquitoes (Tawat- sin et al. 2001, Yang and Ma 2005, Hao et II. MATERIALS AND METHODS al. 2008, Gross and Coats 2015). Multiple studies have hailed the success of combin- Power Breezer® machines (Breezer ing wind currents with vapor or mists of Holdings LLC, Deerfield Beach, Florida) the repellent, DEET (Hoffman and Miller were acquired from the manufacturer 2002, Hoffman and Miller 2003), the spa- for testing. These industrial fans were tial repellent, metofluthrin in a clip-on fan equipped with a 380 L (100 gal) tank and device (Xue et al 2012, Revay et al 2013), locking rear casters to secure the fan in or geraniol and citric acid in ALLCLEAR place. Another feature that was utilized in Backyard Mosquito Misters (Revay et al this study was the atomizer capable of pro- 2013a) for use in reducing mosquito bites. ducing a fine mist from the solution in the However, there is lack of evidence that com- tank. The unit was evaluated at Anastasia mercial mechanical fans misting citronella Mosquito Control District (AMCD) dis- can repel the container-inhabiting mos- trict headquarters in a 12.2 × 18.3 m metal quito, Ae. albopictus. Our study objectives garage previously cleaned and sealed for were to 1) determine the effectiveness of use as the experimental arena. The Power a high powered commercial misting fan to Breezer was situated in the northwest cor- deter Ae. albopictus from host-seeking with ner of the garage and oriented with the wind currents at multiple distances from fan-facing the opposing southeastern cor- the machine, and 2) test the effectiveness ner. A 24 m line was measured from the

�Figure 1. Orientation and average wind velocity at multiple distances of the Power Breezer machine during experimental trials. 72 Technical Bulletin of the Florida Mosquito Control Association, Vol. 10, 2016 fan to the opposite corner of the building tive one was conducted in April of 2014, with lines marked with blue painters tape while objective two was conducted in June at 5 m, 8 m, 10 m, and 15 m (Figure 1). 2014. During each objective, new partici- For each distance, six points were marked pants were used for controls and treat- 0.6 m from one another that represented ments based on availability. where each participant would stand for Statistical analysis All data were ana- evaluation. lyzed by ANOVA using JMP® software (SAS Average wind speed of the Power Institute Inc. 2013). Overall, host seek- Breezer was determined at its highest ing abundance of Ae. albopictus between setting using a handheld SKYWATCH® participants was compared within pooled Xplorer 3 anemometer (JDC Electron- controls and pooled treatment groups to ics SA, Yverdon-les-Bains, Switzerland). determine if differences occurred. The Speeds were recorded at angles left, right, average number of mosquitoes able to lo- and center of the machine at the 5 m, 10 cate participants in control (no wind) and m, and 15 m distances. At 8 m, only wind treatment wind trials (with and without cit- speed at the center point was measured. ronella) at each distance from the Power Average wind-speeds are listed in Figure Breezer were compared. Significant differ- 1. The garage bay door closest to the unit ences were determined at p <0.05. remained open to alleviate potential pres- sure buildup in the building generated by III. RESULTS the high-powered fan. The misting unit flow rate averaged 100 ml/min for each No significant difference in the num- ten-minute trial. ber of Ae. albopictus recovered between par- Two hundred, caged, adult 5-7 day ticipants at each sample distance occurred old laboratory reared female Ae. albopic- with the exception of 8 m where signifi- tus were brought to the testing area and cantly more mosquitoes were collected (15 placed at the opposite corner, 24 m from m: F=0.1757, p=0.9620; 10 m: F=1.2236, the fan unit. For each test, mosquitoes p=0.4005; 8 m: F=4.6032, p=0.0451; 5 m: were allowed to acclimate to the new en- F=1.9314, p=0.2223) (Figure 2). During the vironment for up to 30 minutes. Six par- first objective, there were no observed differ- ticipants (3 males, 3 females) were used ence between the treatments and controls in the study and did not rotate during at 5, 8, 10 meters from the Power Breezer evaluations. Testing began at the farthest fan (5 m: F=2.2321, p=0.1660, 8 m: F=0.0440, distance from the fan (15 m). Mosquitoes p=0.8381, 10 m; F=2.4806, p=0.1463). At 15 were released and given ten minutes to ac- m significantly more mosquitoes were col- tively host seek participants. Mosquitoes lected from the treatment than the control that came to the participants, during that (F=23.416, p=0.0007) (Figure 2). time were killed and counted. At the end When the fan was misting 1% citronel- of the evaluation period total mosquito la, statistically more mosquitoes landed on landings were recorded. New mosquitoes participants as distance from the fan unit were used for each distance tested. Each increased (F=18.2826, p=0.0003) (Figure objective was evaluated only one time. 3). There were no observed difference Objective one, evaluated the fan on between treatments and control at either without mist, while in the second objective distance from the Power Breezer fan (10 the fan applied a 1% citronella oil in wa- m: F=2.5966, p=0.1382 and 15 m: F=0.2062, ter mist. Testing distances were reduced to p=0.6595). the 10 m and 15 m points in the citronella trials due to insufficient landing numbers IV. DISCUSSION in previous trials at the 5 m and 8 m dis- tances. Control trials were conducted with The significant differences that we the fan off and without citronella. Objec- found between the participants at 8 m in Thomson et al.: Evaluation of Power Breezer and Misting Citronella Against Aedes albopictus 73

Figure 2. Mean (±SE) host seeking Aedes albopictus collected at multiple distances from a Power Breezer. Treat- ment consisted of fan on with no mist, control consisted of the fan off. control and treatment groups and at 15 study results. In addition, trial participants m for the wind without citronella trial were also allowed to actively bend, squat, may be attributable to a variety of random and physically exhale carbon dioxide in variables within the testing arena. Time order to attract mosquitoes. At 15 m, par- interval between tests for each distance ticipants were only 9 m from the mosquito and increased temperature in the partially release site where the weakest winds far- enclosed space may have influenced the thest from the fan occurred. Low wind

Figure 3. Mean (±SE) host seeking Aedes albopictus collected at multiple distances from a Power Breezer. Treat- ment consisted of fan on with 1% citronella mist, control consisted of the fan off. 74 Technical Bulletin of the Florida Mosquito Control Association, Vol. 10, 2016 velocity from the fan may have facilitated any commercial products. Volunteers gave host-seeking behavior by enhancing dis- informed consent prior to participation persal of a participant’s kariomones mak- in the study. This study was conducted ac- ing the mosquitoes to be more attractive cording to protocol number AMCD-10-13- to their host. 2005 as approved by the AMCD Board of Hoffman and Miller (2003) analyzed Commissioners for use of human subjects the relationship of wind velocity with mos- in the operation of projects. quito host seeking and flight behavior. The authors reported that wind likely had the VI. REFERENCES CITED greatest effect on host odor dilution for host seeking mosquitoes. This mechanism out- AMCA (American Mosquito Control Association). weighed the relationship of higher wind ve- 2014. Frequently asked questions [Internet]. locity, that can create intolerable flying con- Mount Laurel, NJ http://www.mosquito.org/ faq#fast [Accessed April 15, 2015] ditions for the mosquito. The high velocity Broad, W. J. 2013. A low-tech mosquito deterrent. The wind stream produced by the Power Breezer New York Times. [Internet]. http://www.nytimes. exceeded the 0.45 m/s flying capability of com/2013/07/16/science/a-low-tech-mosquito-de- terrent.html?_r = 0 [accessed February 25, 2015]. mosquitoes at every test distance. But we Gross, A. D. and J. R. Coats. 2015. Can green chemis- found that wind alone did not significantly try provide effective repellents? In: Debboun M., deter host seeking mosquitoes as pointed Frances S. P., Strickman D.A., eds. Insect Repellents Handbook. Second Edition. Boca Raton, Florida, out by Snow (1977) and Hoffman and Miller United States: CRC Press. pp. 75-90. (2002). We found that the wind stream from Hao, H., J. Wei, J. Dai, and J. Du. 2008. Host-seeking the Power Breezer appeared to produce a and blood-feeding behavior of Aedes albopictus (Diptera: Culicidae) exposed to vapors of gera- boundary layer of calmer conditions lower niol, citral, citronellal, eugenol, or anisaldehyde. to the ground and may have facilitated favor- J. Med. Entomol. 45: 533-539. able conditions for mosquito up wind flight Hoffman, E. J. and J. R. Miller. 2002. Reduction of mosquito (Diptera: Culicidae) attacks on a human to participants after host location. subject by combination of wind and vapor-phase In an earlier study, Hoffman and Miller DEET repellent. J. Med. Entomol. 39: 935-938. (2002) focused on manipulating wind cur- Hoffman, E. J. and J. R. Miller. 2003. Reassessment of the role and utility of wind in suppression of mos- rents while combining a DEET mist and quito (Diptera: Culicidae) host finding: stimulus found this treatment provided temporary dilution supported over flight limitation. J. Med. protection from mosquitoes. These results Entomol. 40:607-614. Kolbe, W. A. 2003. No hot air. Technology. are in contrast with our study with citronella. 31(6) http://www.pctonline.com/article/-Annual-Fly- The differences in both studies may be attrib- Control-Issue--No-Hot-Air. [Accessed 20 December utable to the chemicals used and possibly con- 2014] Kilpatrick, M., L. D. Kramer, M. J. Jones, P. P. Marra, centrations. Most laboratory studies that test and P. Daszak. 2006. West Nile virus epidemics in repellency of citronella use concentrations North American are driven by shifts in mosquito ranging from 10-30%, and often include 5% feeding behavior. PloS Biology. 4:e82 Mitchell, C. J. 1991. Vector competence of North and vanillin to enhance repellency (Tawatsin et South American strains of Aedes albopictus for cer- al. 2001, Yang and Ma 2005, Hao et al. 2008) tain arboviruses: a review. J. Am. Mosq. Control while we only applied 1% citronella. In sum- Assoc. 7:446-452. Revay, E. E., A. W. A. Qualls, R. D. Xue, and G. C. mary, wind velocity generated by the Power Muller 2013a. Evaluation of a portable backyard Breezer fan itself, or with the citronella mix- mosquito mister against Culex pipiens and Aedes ture, did not significantly reduce host seeking albopictus in Israel. Tech. Bull. Florida Mosq. Con- trol Assoc. 9:24-28. Ae. albopictus and may not be a practical meth- Revay, E. E., A. Junnila, R. D. Xue, D. L. Kline, U. R. od for preventing mosquito bites for personal Bernier, V. D. Kravchenko, W. A. Qualls, N. Ghat- during public outdoor events. tas, and G. C. Müller 2013. Evaluation of com- mercial products for personal protection against mosquitoes. Acta Trop. 125:226-230. V. ACKNOWLEDGEMENTS SAS Institute Inc. 2013. JMP®, Version 11. Cary, NC. Snow, W. F. 1977. The height and direction of flight This is a research report only and of mosquitoes in West African savanna, in relation to wind speed and direction. Bull. Entomol. Res. does not imply AMCD endorsement of 67:271-279. Thomson et al.: Evaluation of Power Breezer and Misting Citronella Against Aedes albopictus 75

Tawatsin, A., S. D. Wratten, R. R. Scott, U. Thavara, (metofluthrin) against Aedes albopictus and Aedes and Y. Techadamrongsin. 2001. Repellency of vol- taeniorhynchus (Diptera: Culicidae) in northeast- atile oils from plants against three mosquito vec- ern Florida. J. Med. Entomol. 49:652-655. tors. J. Med. Entomol. 26:76-82. Yang, P., and Y. Ma. 2005. Repellent effect of plant Xue, R. D., W. A. Qualls, M. A. Smith, M. K. Gaines, essential oils against Aedes albopictus. J. Vect. Ecol. J. H. Weaver, and M. Debboun. 2012. Field evalu- 30:231-234. ation of the Off! Clip-on mosquito repellent BIFENTHRIN BARRIER SPRAY AGAINST AEDES ALBOPICTUS AROUND AN URBAN CEMETERY, ST. AUGUSTINE, FLORIDA

CHRISTOPHER BIBBS, CODI ANDERSON, MICHAEL L. SMITH, AND RUI-DE XUE Anastasia Mosquito Control District 500 Old Beach Road, St. Augustine, FL 32080

ABSTRACT. Talstar® Professional (7.9% abundant and invasive anthropophilic mos- bifenthrin, AI) was applied by a Stihl SR quitoes commonly encountered in urban 420 gas powered backpack sprayer to the environments (Lounibos et al. 2001). The perimeter vegetation of an 11 ha cemetery ubiquity of this species, especially when in in order to control the source of adult association with human environs, poses Aedes albopictus into the surrounding resi- significant problems as the result of its suc- dential neighborhoods. Prior to, and after cessful ability to colonize cryptic oviposi- treatment, BG Sentinel traps baited with tion sites and rapidly produce large popu- BG-Lure were used to monitor adult abun- lations. Moreover, Ae. albopictus is a known dance in the cemetery while egg produc- vector of multiple arboviruses, including tion was monitored using black oviposition chikungunya (CHIKV), dengue, and the cups. Surveillance continued for six weeks recent Zika virus (Derraik and Slaney post treatment with no additional treat- 2015, Ngoagouni et al. 2015, Wilson and ments applied to the site. Adult Asian ti- Chen 2015). In 2014, the Florida Depart- ger populations were significantly reduced ment of Health confirmed locally acquired in the cemetery one week after treatment cases of CHIKV from several counties. (97.75%) compared with areas with no Cemeteries provide a good example of control measures. Population control de- cryptic environments suitable for Ae. al- clined, thereafter, to about 43% at four bopictus production and harborage. These weeks post treatment and was still signifi- areas are often large sites with static water- cantly reduced despite a total rainfall of holding containers such as flower urns built 12.7 cm (with a peak of 6.4 cm) during the into, or mounted on, head stones. This en- study period. The trend in egg reduction vironment often contains extensive patches from oviposition traps mirrored that of of high density vegetation such as bamboo the adult mosquito population. We found thickets that can create small containers for that the barrier treatment was an effec- mosquito larvae when pruned back (Wash- tive method for controlling Ae. albopictus burn and Hartmann 1992, Johnson and where other methods, especially source re- Sukhdeo 2013). This situation can lead to duction, have limited utility. highly localized, but heavy, outbreaks as adult Ae. albopictus host seek during the Key Words. Aedes albopictus, barrier spray, day. Source reduction is often a principle bifenthrin, cemetery, residential area method in eliminating oviposition and lar- val production habitat for this species but may be ineffective in some instances. I. INTRODUCTION Therefore, the best approach for build- ing a repertoire of effective management The Asian tiger mosquito, Aedes albop- strategies to reduce Ae. albopictus popula- ictus (Skuse), is considered one of the most tions and/or the transmission risk of arbo-

76 Bibb et al.: Bifenthrin Barrier Spray Against Aedes albopictus Around An Urban Cemetery 77 viral infection is through integrated vector residential housing with high human activ- management (IVM). One method, with de- ity. The property was chosen for its abun- cades of usage, is barrier spraying (Trout dant larval sites for which source reduc- et al. 2007, Qualls et al. 2013). The Anas- tion education has had little impact. Larval tasia Mosquito Control District (AMCD) developmental habitats in the cemetery is familiar with this method and has used included dense bamboo thickets, flower it to successfully control adult mosquitoes urns, exposed crypts, and ground mainte- (Qualls 2013). Other techniques,such as nance storage areas overloaded with tire thermal fogging with adulticides, have piles. These habitats were routinely irri- been reintroduced into AMCD operation- gated as part of normal ground mainte- al programs for control of adult container- nance. Mosquito landing rate counts were inhabiting mosquitoes in order to bolster conducted in the cemetery for three min- the options for IVM (Xue et al. 2013). utes in 18 locations to identify where the But just having the technologies available majority of Ae. albopictus activity occurred. for use is not enough. Chemical applica- From this information, four locations were tion methods, either singular or in com- chosen for the study and monitored from bination, must be continually re-evaluated June 1 through July 25, 2015. for their applicability to contemporary Surveillance. Two BG Sentinel™ traps problems of vector abatement in order to (BGS) baited with a standard Biogents® stay one step ahead of emergent disease BG-Lure (Biogents AG, Regensburg, Ger- threats like CHIKV, dengue, and Zika vi- many) were placed at least 50 m apart in ruses. However, any tactics for long-term each location (Figure 1). Traps were op- control of Ae. albopictus populations would erated continuously for 48 h per week for be welcomed. two weeks prior to treatment. In addition, As mentioned earlier, pesticides ap- five 500 ml black oviposition cups, lined plied to vegetation as a residual barrier for with velour oviposition paper, were placed adult mosquito control can provide several in the same four locations and allowed to versatile approaches for mosquito man- collect eggs for 48 h per week during the agement. A study by Cilek (2008) showed same period. After treatment, both surveil- that bifentrhin applied to the perimeter lance methods were continued in the same vegetation of a public park provided ef- frequency for six weeks. Rainfall was re- fective (>70%) control against adult Ae. al- corded weekly. Collections from BGS traps bopictus and Culex quinquefasciatus Say, for and ovicup papers were transported to the six weeks, in residual leaf bioassays. Using lab. Captured mosquitoes were identified this approach we tested the efficacy of in- to species and eggs from ovipapers were secticide barrier treatments to reduce Ae. counted using a light dissecting scope. albopictus populations in a cemetery sur- Barrier treatment. The outer perimeter rounded by an urban residential network vegetation and subsequent fence line of the of neighborhoods. These neighborhoods entire 11 ha site was treated with Talstar® shared a fence line perimeter with the Professional (7.9% bifenthrin AI, Philadel- cemetery and have experienced consider- phia, PA) at a dilution rate of 11.4-L water able annoyance from host seeking Ae. al- to 88.7 ml bifenthrin. Treatment was applied bopictus originating from the latter area. using a Stihl SR 420 gas powered backpack sprayer (Andreas Stihl Ag & Co. KG, Waib- II. MATERIALS AND METHODS lingen, Germany) at a 3.3 L flow rate while walking the area at 6.4 kph. Application Test Site: A local 11 ha cemetery (lati- height was at 3 m with a 60 m penetration tude 29.893712, longitude -81.337268) in depth for a total treatment area of 6400 m2. St. Johns County was targeted as a major The pesticide application was carried out source of Ae. albopictus production. This by a certified State of Florida Public Health area was surrounded on all sides by urban Pest Control applicator. 78 Technical Bulletin of the Florida Mosquito Control Association, Vol. 10, 2016

Figure 1. Surveillance layout of the 11 ha cemetery used as a test site (latitude 29.893712, longitude -81.337268) St. Johns County, FL. Each location contained two BG Sentinel traps (baited with BG-Lure) and five oviposition cups.

Statistical analysis Percent population III. RESULTS AND DISCUSSION reduction was calculated using a propor- tion derived by T/(T+P) where T = the Aedes albopticus populations were sig- population for a given week of treatment nificantly reduced by 97.75% during week and P = the population for the two weeks one; 65.64% during week two; 58.72% for of pre-treatment sampling. Statistical soft- week 3; and 43.39% at week 4 (Figure 2). ware JMP® version 10.1 (SAS Institute, Population levels at weeks 5 and 6, post Inc. 2012) was used to run a paired t-test treatment, were no longer significantly dif- on mean adult and egg surveillance data to ferent than pretreatment levels. Egg abun- determine differences in population level dance after treatment was also significantly from the treatment compared with pre- reduced (t = 4.7154, df = 3, p < 0.0181) for treatment level per week for 6 weeks. the first four weeks. Ethics statement. Those volunteers who Despite such a large source site, at 11 participated in landing rate counts were ha, the perimeter barrier spray still pro- briefed on the study procedures and made vided successful control of the resident aware of the health risks involved by their Ae. albopictus population without addi- participation. Written informed consent tional adulticide or larvicidal treatment. was received from volunteers prior to the Previously, this area served as an impor- start of the study. Volunteer consent is doc- tant source of adult Asian tiger mosquito umented under the AMCD IRB Protocol for neighborhoods surrounding it. The #10.13.2005 and approved for use of hu- cemetery is a largely unmanaged prop- man subjects by the Board of Commission- erty compounded by dense bamboo pri- ers presiding over the AMCD. vacy screens along the perimeter. Addi- Bibb et al.: Bifenthrin Barrier Spray Against Aedes albopictus Around An Urban Cemetery 79

Figure 2. Weekly mean precipitation and abundance of adult Aedes albopictus from BG Sentinel (BG AVG) and mosquito egg oviposition cups (EGG AVG) in an 11 ha cemetery before (week 1-2) and after (week 3-8) treatment of perimeter vegetation with Talstar Professional (bifenthrin). Vertical lines at each data point are standard errors. tionally, tire piles and human generated AMCD does not endorse any commercial debris that included artificial containers mentioned in this report. expanded the available sources. As stated earlier, traditional, methods of source re- VI. REFERENCES CITED duction and custodial education of cem- Cilek, J. E. 2008. Application of insecticides to vegeta- etery employees on identifying and remov- tion as barriers against host-seeking mosquitoes. J. ing urban mosquito sources were met with Am. Mosq. Control Assoc. 24:172-176. resistance. Moreover, relatively few roads Cilek, J. E. and C. F. Hallmon. 2008. Residual effec- tiveness of three pyrethroids on vegetation against for truck access into the cemetery limited adult Aedes albopictus and Culex quinquefasciatus in the effectiveness, and increased the cost of screened field cages. J. Am. Mosq. Control Assoc. any larviciding or adulticiding effort often 24:263-269. Derraik, J. G. and D. Slaney. 2015. Notes on Zika vi- requiring multiple visits over a season. Bar- rus—an emerging pathogen in the South Pacific. rier spraying with Talstar Professional has Aust. N. Z. J. Public Health 39:5-7. shown, in this case, to be the most effective Johnson, B. J. and M. V. K. Sukhdeo. 2013. Succes- sional mosquito dynamics in surrogate treehole means of reducing large urban sources of and ground-container habitats in the northeast- adult Ae. albopictus. ern United States: where does Aedes albopictus fit in? J. Vector Ecol. 38:168-174. Lounibos, L. P., G. F. O’Meara, R. L. Escher, N. Nishimura, M. Cutwa, T. Nelson, and S. A. Julia- IV. ACKNOWLEDGEMENTS no. (2001). Testing predictions of displacement of native Aedes by the invasive Asian tiger mosquito Appreciation is extended to the custo- Aedes albopictus in Florida, USA. Biological Inva- dians of the cemetery site who collaborat- sions, 3:151-166. Ngaogouni C., B. Kamgang, E. Nakoune, C. Paupy, ed with the test evaluation. The operation- and M. Kazanji. 2015. Invasion of Aedes albopictus al technicians of the Anastasia Mosquito (Diptera: Culicidae) into central Africa: what conse- Control District are thanked for their col- quences for emerging diseases? Paras. Vectors 8:191. Qualls, W. A., M. L. Smith, and R. D. Xue. 2013. Suc- laboration during this large barrier mis- cessful applications of barrier treatments using sion. This is a research report only. The bifenthrin against mosquitoes in St. Johns County, 80 Technical Bulletin of the Florida Mosquito Control Association, Vol. 10, 2016 Florida, from 2006-2009. Tech. Bull. Florida Mosq. in California tree holes? J. Med. Entomol. 29:995- Control Assoc. 9:33-37. 1005. SAS Institute, Inc. 2012. JMP, Version 10.1. Cary NC. Wilson, M. E. and L. H. Chen. 2015. Dengue: update in Trout, R. T., G. C. Brown, M. F. Potter, and J. L. Hub- epidemiology. Curr. Infect. Dis. Rep. 17:457. bard. 2007. Efficacy of two pyrethroids insecticides Xue, R. D., J. H. Weaver, M. L. Smith, W. A. Qualls. applied as barrier treatments for managing mos- 2013. Field evaluation of thermal fog application quito (Diptera: Culicidae) populations in suburban of sumithrin products against adult mosquitoes in residential properties. J. Med. Entomol. 44:470-477. northeast Florida. Tech. Bull. Florida Mosq. Control Washburn, J. O. and E. U. Hartmann. 1992. Could Aedes Assoc. 9:38-41. albopictus (Diptera: Culicidae) become established LABORATORY EVALUATIONS OF SEVEN INSECT REPELLENTS AGAINST THE LONE STAR TICK AMBLYOMMA AMERICANUM

JODI M. SCOTT, ALI FULCHER, JOHN M. HENLZER, AND RUI-DE XUE Anastasia Mosquito Control District 500 Old Beach Road, St. Augustine, FL 32080

ABSTRACT. In laboratory bioassays, BioUD, mendations on how to avoid tick bites. In ad- Bio Block-Organic Outdoor, Bio Block- dition, AMCD’s field technicians frequently Organic Pest Control, Bio Blocker-Organic encounter these ticks when conducting Insect Repellent, OFF! Botanicals Insect Re- fieldwork. Topical and barrier insect repel- pellent, OFF! Deep Woods Insect Repellent, lents offer an inexpensive mode of protec- and Repel Insect Repellent Sportsman Gear tion that imposes the fewest limitations on Smartinsect repellents were applied to post- human activities (Staub et al. 2002). With er paper and assessed by four volunteers for the possible transmission of tick-borne efficacy against unfed nymphalAmblyomma pathogens, such as Ehrlichia chaffeensis, E. americanum ticks. Assessment times of 10 and ewingii, E. muris, Southern Tick-Associated 120 minutes post application were chosen Rash Illness (STARI), and Francisella tula- to mimic freshly applied repellent and sug- rensis (Mixson et al. 2006, CDC 2015), as gested duration of effectiveness against ticks well as the growing concerns over DEET as determined by the product manufactur- (N,N-diethyl-3-methylbenzamide) based ers. Significant differences between repel- products on the market (such as, smell, lents existed when both application periods possible bioaccumulation, and damage to were compared but not between volunteers. plastics and some clothing materials) as re- At 10 min post application, Bio Block Pest ported by Katz et al. (2008) and Semmler et Repel was the most effective product at repel- al. (2011), alternative tick repellents should ling ticks (85%) and Repel the least effective be evaluated. Therefore, a laboratory study (30%). At 120 min, BiteBlocker-Insect Repel, was conducted to determine the repellent OFF! Deepwoods, and Bio Block-Organic effectiveness of several commercially avail- Outdoor provided ≥55% protection from able non-DEET products against lone star ticks. Efficacy at 120 min, when measured at ticks. 50% or greater repellency, did not reflect du- ration times listed on the BioUD (20%), and II. MATERIALS AND METHODS OFF! Botanicals (30%) product labels. Insect repellents. Seven DEET and non- Key Words: Insect repellents, DEET, ticks, DEET commercially available products were Amblyomma americanum chosen to provide diversity of active ingredi- ents that may be repellent to A. americanum (Table 1). HOMS products were supplied I. INTRODUCTION to AMCD for evaluation of efficacy by the manufacturer, while other repellents were Amblyomma americanum (L.), the lone purchased at local pharmacies or hardware star tick, is a commonly encountered tick in stores. HOMS Bio Block Organic Outdoor northeastern Florida. Annually, Anastasia and HOMS Bio-Block Organic Pest Control Mosquito Control District (AMCD) receives are designed for premise treatment while multiple customer complaints about this the rest of the products that we tested were tick species and is often asked for recom- labeled for application to human skin.

81 82 Technical Bulletin of the Florida Mosquito Control Association, Vol. 10, 2016 Human volunteers. Male (2) and female (2) volunteers were utilized in these evalu- ations. Ages of the volunteers ranged from 24-55 years old and signed consent forms under AMCD’s IRB protocol#10-13-2005 (as approved by the AMCD Board of Commis-

Formulation sioners for studies involving use of human subjects). Ticks. Non-blood fed, nymphal A. america- Pump spray, Lotion Pump spray, Pump spray Aerosol Spray Concentrate No longer available Pump Spray Pump spray, Lotion, Travel pen Lotion, Travel Pump spray, num were obtained from a laboratory colony maintained by USDA/ARS, Tick and Bit- ing Fly Research Unit, Knipling-Bushland U.S. Livestock Insect Research Laboratory, Kerrville, TX. New ticks were used by each volunteer during each repellent evaluation. After individual volunteers evaluated repel- lency, the ticks were collected by scotch tape and disposed of. Duration of protection Bioassays: The bioassay utilized a modi- 120 minutes 120 minutes Not listed Outdoor-Monthly 480 minutes Indoor-Monthly, Outdoor-Weekly Indoor-Monthly, 120 minutes fication of human skin test evaluations used by Semmler et al. (2011). White post- er paper (1 meter x 1 meter) was used as a substratum for all repellent applications. Poster boards were taped onto individual tables (183 cm x 76 cm x 74 cm) located one meter from each other and designated as either repellent treated poster boards or untreated (control) posterboards. Three Insect Repellent

Brand Name different concentric circles were drawn onto each poster paper: 15.5 cm outer circle, 14.5 cm middle circle, and 5.5 cm inner circle. Following label instructions for application, repellents were painted in- between the 15.5 cm and 14.5 cm circles BioUD® OFF!-Deep Woods, Insect Repellent OFF!-Deep Woods, Bio Block® -Organic Outdoor Repel®-Insect Repellent, Sportsman Gear Smart Bio Block® -Organic, Pest Control BiteBlocker®-Organic, Insect Repellent OFF!-Botanicals® and allowed to dry for ten minutes. After ten minutes, 5 ticks were placed with for-

1 ceps onto the center of the 5.5 cm circle. Volunteers sat in a chair at each table and placed their arms to the left and right of the outer 15.5 cm circle, ensuring no con- tact with repellent. Ticks were given 3 min to locate volunteers. Successful location Active Ingredients

25% was recorded when ticks crossed the 5.5 cm 2 center circle and came into contact with 2-undecanone 7.75% DEET Soybean Oil 20%, Citric Acid 4%, Cedar Oil 1% Picardin 15% Soybean oil 12%, Geranium 3%, Caster oil 1%, Lemon Grass 0.5% Soybean Oil 30%, Citric Acid 4%, Cedar Oil 1% p-Menthane-3,8-Diol 10% 14.5 cm middle circle. At the end of three minutes, ticks were collected with scotch tape, and the next volunteer evaluated the same repellent using new ticks. Each repellent was evaluated in this manner. A Brand name formulations are subject to change; labels should always be read to ensure exact ingredients. Brand name formulations are subject to change; labels should always be read ensure N,N-diethyl-3-methylbenzamide

1 2 secondary evaluation, 120 minutes after initial application, was conducted which HOMS, LLC SC Johnson & Sons Table 1. Manufacturer, active ingredient (%), commercial product, formulation and manufacturer’s stated duration of protection. active ingredient (%), commercial product, formulation and manufacturer’s 1. Manufacturer, Table Manufacturer HOMS, LLC WPC Brands, Inc. HOMS, LLC HOMS. LLC SC Johnson & Sons Scott et al.: Laboratory Evaluations of Seven Insect Repellents Against The Lone Star Tick 83 followed the previously outlined bioassay. Effectiveness guidelines (EPA 2015). At 120 Ticks on the control poster board followed min, the efficacy of BioUD (20%), and OFF! the same bioassay procedure as the treated Botanicals (30%), measured at 50% or great- ones. er repellency, did not reflect labeled duration Statistical analysis. The statistical software times listed on their labels. We believe these JMP version 11.1.1 (SAS Institute Inc. 2013) studies have provided several options, for the was used for data analysis. Statistical sig- general public and our field technicians, on nificance was determined through ANOVA what commercially repellents are efficacious (P<0.05). Post hoc analysis was conducted and available for protection against nymphal to separate the means of repellents using host seeking A. americanum. Tukey HSD.

IV. ACKNOWLEDGEMENTS III. RESULTS All volunteers gave informed consent re- Our study focused on insect repellents garding the possible risks of acquiring tick- that offered a wide variety of active ingredi- borne diseases during the laboratory trials. ents and evaluated their efficacy when mim- This is a research report only, use or men- icking freshly applied at 10 min and at 120 tion of trade names does not constitute an min post application to mimic the label du- official endorsement by Anastasia Mosquito ration of effectiveness listed by most of the Control District. manufacturers. Significant differences ex- isted between products but not between vol- unteers, at both post application time periods V. REFERENCES CITED (F = 5.1137, df10,21 = 31, P = 0.0008, and F = Achee, N. L., M. J. Bangs, R. Farlow, G. F. Killeen, S. 4.0310, df10,21 = 31, P = 0.0034, respectively) (Table 2). At 10 min post application, Bio Lindsay, J .G. Logan, S. J. Moore, M. Rowland, K. Sweeney, S. J. Torr L. J. Zwiebel, and J. P. Grieco. Block Pest Repel was the most effective prod- 2012. Spatial repellents: from discovery and devel- uct at repelling ticks (85%) while Repel was opment to evidence-based validation. Malaria Jour- the least effective (30%). At 120 min, Bite nal 11:164 doi: 10.1186/1475-2875-11-164. Carroll, J. F., J. P Benante, M. Kramer, K. J. Lohmeyer, Blocker Insect Repel, OFF! Deep woods, and and K. Lawrence. 2010. Formulations of deet, picari- Bio Block-Organic Outdoor provided ≥55% din, and IR3535 applied to skin repel nymphs of the protection from ticks and was consistent with Lone Star tick (Acari: Ixodidae). J. Med. Entomol. 47:699-704. their respective labeled protection rates as CDC (Center for Disease Control and Prevention). listed in the EPA Insect Repellents Use and 2015. Tickborne disease of the U.S. http://www.cdc. gov/ticks/diseases/.[Accessed 8 July 2015]. Childs, J. E. and C. D. Paddock. 2003. The ascendancy of Amblyomma americanum as a vector of pathogens affecting humans in the United States. Annu. Rev. Table 2. Mean percent repellency (SD) of seven in- Entomol. 48:307-377. sect repellents against non-blood fed Amblyomma EPA (Environmental Protection Agency). 2015. Insect americanum nymphs at 10 min and 120 min post ap- repellents: use and effectiveness. http://cfpub.epa. plication. gov/oppref/insect/search_results.cfm?Rangetime =&hidSelected=3&ProductName=&Ingredient=nul Repellents 10 min1 20 min1 l&Company=null&Registration=&Submit=Search#. [Accessed 1 July 2015]. None 0 (0)c 5 (10)b Gratz, N. G. 1999. Emerging and resurging vector-borne BioUD 50 (35)abc 20 (16)ab diseases. Annu. Rev. Entomol. 44:51-75. Repel 30 (25)bc 50 (26)ab Katz, T. M., J. H. Miller, and A. A. Herbert. 2008. Insect BioBlock-Pest Control 85 (19)a 50 (12)ab repellents: historical perspectives and new develop- OFF! Deep Woods 80 (28)ab 65 (25)a ments. J. Am. Acad. Dermatol. 58:865-871. Mixson, T. R., S. R. Campbell, J. S. Gill, H. S. Ginsberg, BioBlock-Outdoor 70 (26)ab 55 (25)a M. V. Reichard, T. L. Schulze, and G. A. Dasch. 2006. OFF Botanicals 70 (26)ab 30 (35)ab Prevalence of Ehrlichia, Borrelia, and Rickettsial agents BiteBlocker-Insect Repel 80 (16)ab 65 (19)a in Amblyomma americanum (Acari: Ixodidae) collect- 1Means within each time-period, between products, fol- ed from nine states. J. Med. Entomol. 43:1261-1268. lowed by a different letter are statistically different (Tukey Moody, R. P. 1989. The safety of diethyltoluamide insect HSD). repellents. J. Am. Mosq. Control Assoc. 262:28-29. 84 Technical Bulletin of the Florida Mosquito Control Association, Vol. 10, 2016 Osimitz, T. G. and R. H. Grothaus. 1995. The present Staub, D., M. Debrunner, L. Amsler, and R. Steffen. safety assessment of DEET. J.Am. Mosq. Control As- 2002. Effectiveness of repellent containing DEET soc. 11:274-278. and EBAAP for preventing tick bites. Wild. Environ SAS Institute Inc. 2013. JMP statistical software. Version Med. 13:12-20. 11.1.1 Cary, NC. Xue, R. D., A. Ali, and J. F. Day. 2007. Commercially Semmler, M., F. Abdel-Ghaffer, K. A. S. Al-Rasheid, and available insect repellents and criteria for their use. H. Mehlhorn. 2011. Comparison of the tick repel- In: Insect Repellents, Principles, Methods, and Uses, ed. lent efficacy of chemical and biological products by M. Debboun, S.P. Frances, and D. Strickman. origination from Europe and the USA. Parasitol. CRC Press. Boca Raton, FL, USA, Chapter 25, 405- Res. 108:899-904. 415 pp. LABORATORY AND FIELD EVALUATION OF OFF! CLIP- ON MOSQUITO REPELLENT DEVICE CONTAINING METOFLUTHRIN AGAINST THE LONE STAR TICK, AMBLYOMMA AMERICANUM (ACARI: IXODIDAE)

RUI-DE XUE¹, JODI M. SCOTT¹, ALICIE FULCHER¹, WHITNEY A. QUALLS¹, JOHN M. HENLZER¹,², MARCIA K. GAINES¹, JAMES H.R. WEAVER¹, AND MUSTAPHA DEBBOUN³ 1Anastasia Mosquito Control District 500 Old Beach Road, St. Augustine, FL 32080

2Guana Tolomato Matanzas Research Reserve, Environmental Education Center, Florida Department of Environmental Protection 505 Guana River Road, Ponte Vedra Beach, FL 32082

3Mosquito Control Division, Harris County Public Health & Environmental Services, Houston, TX 78234

ABSTRACT. Choice tests were conduct- operating compared with those wearing ed in the laboratory to determine the the device in the off position, regardless repellency of the of sitting or walking slowly through a tick metofluthrin (Off!® Clip-on™ Mosquito infested area. Repellent, AI 31.2%) to lone star ticks, Amblyomma americanum. In this study, the Key Words. Amblyomma americanum, tick device’s fan unit was disabled to allow eval- repellent, metofluthrin, personal protec- uation of the active ingredient as a passive tion, Off Clip-on Mosquito Repellent spatial repellent. We found that 57% of the ticks were significantly repelled from hands that held the Off! Clip-on refill, I. INTRODUCTION compared with 27% of volunteer hands without it (control). Field trials conducted Tick-borne diseases in humans and in northeastern Florida with volunteers ei- animals are a growing world-wide concern ther sitting or walking while wearing the and problem (Gratz 1999). A common tick repellent device in areas infested with A. in the southeastern US is the lone star tick, americanum were also evaluated. Seated Amblyomma americanum (L.) and has been volunteers, with the device attached to reported to be a potential vector of hu- their waist, with the fan running repelled man monocytic ehrlichiosis as well as sev- 89% of host-seeking ticks when compared eral other emerging tick-borne pathogens with persons similarly placed without the (Childs and Paddock 2003). Anastasia fan on. In a second field trial, volunteers Mosquito Control District clientele, and that walked slowly through the naturally our field staff, have experienced numerous infested area with the repellent fan run- encounters with this pest tick species after ning provided 28% protection from quest- visiting some of the state parks in St. Johns ing ticks compared with similar volunteers County, Florida (Xue, pers. comm.). Tick without the fan on. Our overall results control using an acaricide is very difficult demonstrated that fewer numbers of lone in Florida state parks because these areas star ticks were found on persons with the are considered conservation lands where Off! Clip-on Mosquito Repellent device pesticide applications are prohibited.

85 86 Technical Bulletin of the Florida Mosquito Control Association, Vol. 10, 2016 Therefore, personal protective measures from a local convenience store. This de- rely on topical application of repellents as vice consists of a plastic dispenser contain- the most effective protection against host ing a replaceable cartridge with metofluth- seeking ticks (Carroll et al. 2010). rin (AI 31.2%). This product is designed Most common commercially available to operate by turning on a fan powered repellent products contain different con- by two 1.5 volt AA alkaline batteries to as- centrations of N, N-diethyl-3-methylben- sist in the release of the active ingredient zamide (DEET) as the active ingredient into the air surrounding the individual. (Xue et al. 2007). Due to concerns about According to the label, the device can be DEET’s potential toxicity and safety (Osim- worn by adults or children for protection itz and Grothaus 1995), including possible against mosquitoes for up to 12 h of opera- health risks associated with repeated use of tion. high concentrations (Moody 1989), spatial Laboratory test. Static, non-fan-mediated repellents have been suggested as alter- activity of metofluthrin was evaluated in natives. Theoretically, an effective spatial laboratory cage bioassays. Each cage was repellent should provide an envelope of constructed from a plastic Sterilite® box protection that prevents contact between (13 × 13 × 21.5 cm; L × W × H, Sterilite, host and pest while preventing pathogen Townsend, MA) and served as an open transmission if disease vectors are present airflow chamber (OAC) for choice evalua- (Achee et al. 2012). Most arthropod spatial tions (Figure 1). A clear vinyl tube (3.5 cm repellent products release an active ingre- outer diameter × 30 cm) was connected to dient as a vapor or smoke. Both dispersal the OAC through a 3.5 cm diameter hole methods often use a heat source, or fan, on the side. The tube was then connect- to produce the protection zone. Metoflu- ed to a 1,000 ml polystyrene disposable thrin, a vapor-active pyrethroid, has been storage bottle (Corning®, Corning, NY) identified as an effective spatial mosquito through a 3.5 cm diameter hole in the cen- repellent (Ujihara et al. 2004, Kawada et ter of the bottle lid. The base of the bottle al. 2005). Several novel methods of apply- had been removed to allow insertion of a ing spatial repellents against mosquitoes volunteer’s hand. have included impregnated paper (Argue- Laboratory-reared (non-blood fed) ta et al. 2004; Lucas et al. 2007) and plastic adult A. americanum were used in the study strips (Kawada et al. 2008). Recently, Off!® from a colony maintained by the USDA/ Clip-on™ Mosquito Repellent was intro- ARS, Tick and Biting Fly Research Unit, duced as a new commercially-available spa- Knipling-Bushland U.S. Livestock Insect tial repellent device; this product has been Research Laboratory, Kerrville, TX. For tested against mosquitoes and provided ef- each test, five ticks were placed into the fective protection in northeastern Florida vinyl tube, using feather tip forceps, and (Xue et al. 2012). To date, Off! Clip-on has secured with fabric mesh held in place by not been evaluated (nor its active ingredi- rubber bands to ensure that no ticks es- ent) for personal protection against ticks. caped the tube. Ticks were allowed 2 min Here we report the results of laboratory to acclimate in the tube before it was at- and field evaluations conducted in north- tached to the OAC and polystyrene bottle. eastern Florida, to determine the repel- A volunteer then placed one hand into lency of Off! Clip-on Mosquito Repellent the end of the polystyrene bottle and ticks against host-seeking adult and nymphal A. were given 5 min to move to their final americanum. position. After five minutes, ticks located between the hand, on the mesh, and up II. MATERIALS AND METHODS to 15 cm from the hand were counted as “host seeking”. Ticks were given a 2-min Spatial repellent device. Off! Clip-on Mos- rest period then the assay repeated but quito Repellent devices were purchased with the volunteer holding an OFF! Clip- Xue et al.: Laboratory and Field Evaluation of Off! Clip-On Mosquito Repellent Device 87

Figure 1. Choice tube laboratory bioassay for evaluating adult female Amblyomma americanum response to a hu- man hand with and without Off! Clip-on Mosquito Repellent cartridge containing metofluthrin. on refill while placing their hand in the Three volunteers served as treatments, polystyrene chamber. Ticks were allowed with their device fan turned on for 30 min. 10 min to locate the volunteer’s hand. Af- During the same time period, the three ter 10 min, ticks located on the mesh next remaining volunteers had an Off! Clip-on to the hand, and within 15 cm of the hand, device attached to their waist but without were counted as attracted to the host. For the fan operating (referred to as “static all tests, the percentage responding was device”) and served as controls. All stages calculated. Three volunteers conducted of ticks that climbed upon the volunteers three sessions each providing three repli- clothing were collected by scotch tape by cations with an N = 9. New ticks were used the end of the observation period. All vol- for each replication. unteers then relocated to a new testing Field test. Field trials were conducted site in the same area and switched treat- at Guana State Park (permit# 2012-3-12) ments, (i.e. previous control volunteers in South Ponte Vedra, Florida (30° 07” switched to serve as the treatment and the 56.37”N and 81° 22” 40.44 W). This land previous treated volunteers now served as parcel is a conservation area where large the control). This scenario was repeated 6 populations of lone star ticks occur. Six times on one day during June 2012. Meteo- human volunteers (four males and two rological data (wind direction, speed, air females) who each signed a consent form temperature, and relative humidity) were under protocol No. 445-96, as approved collected hourly with a small hand-held by the University of Florida, Institutional digital meter (Skywatch Xplorer, N Tech, Review Board (IRB-01) participated in the USA, Holmen, WI). trials. Volunteers wore white clothing that In the second field trial at the same fully covered their body from head to toe. state park, six volunteers (3 females and Each person had one Off! Clip-on device 3 males) participated. Three volunteers attached to their waist and sat on a chair wore devices, attached to their waist, with- 15 m away from the nearest volunteer. out operating the fan while the remaining 88 Technical Bulletin of the Florida Mosquito Control Association, Vol. 10, 2016 three volunteers wore devices where the 2 ticks were recovered from only one of fan was operated. Volunteers wore the pre- the six volunteers with operating devices viously mentioned white suits from the first for a mean of 0.33 ticks. In addition, 2-6 trial and walked at a pace covering 30 m ticks were noticed around the feet of the for 15 min. Each volunteer was positioned three sitting volunteers while the fan was 15 m from the next volunteer. Ticks were on but they did not climb onto either per- removed from volunteers by the end of son. During this part of the field study, each time period. After the 15 min collec- 89% protection was provided by the clip- tion period, volunteers relocated to a new on device operating against questing ticks test site and either switched on or off their for volunteers who were siting. Wind speed fans, to provide a comparison to their pre- during the trial was recorded at 0-9 km/h vious treatment assignment. These field with air temperature ranging between 25 trials were repeated six times on one day and 28° C (70% RH). from 0800 to 1500 during late May, 2012. At the beginning of the walking field tri- Data analyses. Mean percent response al, numerous ticks were initially observed data from the laboratory study were sub- on the feet and legs of all volunteers. jected to an analysis of variance using JMP However, those with the repellent device version 11 (SAS Institute, Inc. 2013) on turned on noticed that those ticks either non-transformed data, to assess statistical left or dropped off each person while the significance between controls and treat- device was operated for a few minutes. A ments on pooled data across all volun- mean of 7.3 ticks per person per 15 min teers. Tukeys HSD was then conducted on were observed on volunteers with the this dataset to determine if differences ex- device off and 5 ticks per person per 15 isted (P<0.05). Field trial repellency data min on persons with an operating device. was evaluated in a split-plot design. The The percent protection from questing A. data for walking and sitting were analyzed americanum afforded to walking individu- separately. Mean tick counts were trans- als by operating OFF! Clip-on device was formed using square root (x + 1) prior to 28 % compared with persons with a non- analyses and Student’s t-test performed to operational device. In this field trial, there determine differences (P<0.05). were no significant differences in the num- bers of ticks recovered between volunteers when using, or not using, the fan compo- III. RESULTS nent. During this study wind speed was re- corded at 0-3.6 km/h with air temperature Laboratory test. There was statistical sig- ranging from 26–28° C (70% RH) in the nificance between the treatment and the morning of the second field trial. control (F1,16 = 9.7826, P = 0.0065). Fifty- seven percent of ticks were repelled from IV. DISCUSSION the volunteer holding the clip on refill, while 27% ticks were repelled from the vol- Host-seeking ticks can approach a host unteer without holding the clip on refill. by walking or they may sit and wait for There was no statistical difference between the host to make contact, a process called volunteers in these trials (F2,15 = 0.3439, p questing. Usually questing is the primary = 0.7144). host seeking behavior for ticks rather than Field test. Only one tick species, A. ameri- directly approaching a host. Therefore, canum, was collected in the field trials. A walking is an ideal method to evaluate tick total of 98 ticks were collected including repellents for those species that primarily 6 females, 3 males, and 89 nymphs. At utilize questing to acquire hosts. Because 30 min, a mean of 3 ticks were recovered few ticks actively move toward a host, and from seated volunteers holding the clip most ticks walk/approach a host very slow- on devices. During the same time period, ly, sitting for 15 min periods may be a less Xue et al.: Laboratory and Field Evaluation of Off! Clip-On Mosquito Repellent Device 89 effective method for a repellent evaluation of trade names does not constitute an of- against these organisms. ficial endorsement by Anastasia Mosquito We found that operating an Off! Clip- Control District. on mosquito repellent device containing metofluthrin proved to be more effective (89%) for protecting inactive (sitting) VI. REFERENCES CITED hosts from climbing ticks than it was for hosts that were actively walking (28%). Achee, N. L., M. J. Bangs, R. Farlow, G. F. Killeen, S. Lindsay, J. G. Logan, S. J. Moore, M. Rowland, K. This effect was most likely influenced by Sweeney, S. J. Torr, and J. P. Grieco. 2012. Spatial entering tick harborage areas and the repellents: from discovery and development to tick’s ability for host location through its evidence-based validation. Malaria Journal 11:164 doi: 10.1186/1475-2875-11-164. questing behavior. To provide more ef- Argueta, T. B. O., H. Kawada, and M. Takagi. 2004. fective protection for individuals walking Spatial repellency of metofluthrin-impregnated in tick infested habitats, the Off! Clip-on multilayer paper against Aedes albopictus under outdoor conditions, Nagasaki, Japan. Med. Ento- device needs to be improved by possibly mol. Zool. 55:211-216. increasing the concentration of metofluth- Carroll, J. F., J. P. Benante, M. Kramer, K. J. Lohm- rin, fan speed, and/or wearing the device eyer, and K. Lawrence. 2010. Formulations of deet, picaridin, and IR3535 applied to skin repel on the lower part of legs rather than on nymphs of the lone star tick (Acari: Ixodidae). J. the waist. Med. Entomol. 47:699-704. Although the Off! Clip-On Mosquito Childs, J. E. and C. D. Paddock. 2003. The ascendan- cy of Amblyomma americanum as a vector of patho- Repellent was designed to primarily repel gens affecting human in the United States. Ann. mosquitoes, we observed that a certain Rev. Entomol. 48:307-377. amount of effectiveness against crawling A. Gratz, N. G. 1999. Emerging and resurging vector- borne diseases. Ann. Rev. Entomol. 44:1-75. americanum on clothing can be achieved. Kawada, H., E. A. Temu, J. N. Minjas, O. Matsumoto, Moreover, we found that the Off! device T. Iwasaki, and M. Takagi. 2008. Field evaluation provided effective protection from crawl- of spatial repellency of metofluthrin-impregnated plastic strips against Anopheles gambiae complex in ing ticks especially when people sit out- Bagamoyo Coastal Tanzania. J. Am. Mosq. Control doors. Assoc. 24:404-409. The Off! Clip-on Mosquito Repellent Kawada, H., Y. Maekawa, and M. Takagi. 2005. Field trial on the spatial repellency of metofluthrin-im- system is easy to operate and can be at- pregnated plastic strips for mosquitoes in shelters tached on a belt, purse, or placed on a flat without walls (beruga) in Lombok, Indonesia. J. surface for protection against host-seeking Vect. Ecol. 30:181-185. Kawada, H., Y. Maekawa, Y. Tsuda, and M. Takagi. arthropods (Xue et al. 2012). The active 2004. Trial of spatial repellency of metofluthrin- ingredient, metofluthrin, can last for up to impregnated paper strip against Anopheles and Cu- 12 h (for mosquitoes) and used multiple lex in shelters without walls in Lombok, Indonesia. J. Am. Mosq. Control Assoc. 20:434-437. times but must be refilled within 14 days Lucas, J. R., Y. Shono, T. Iwasaki, T. Ishiwatari, N. after opening according to the manufac- Spero, and G. B. Benzon. 2007. U.S. Laboratory turer. However, the actual longevity of the and field trials of metofluthrin (SumiOne) ema- nators for reducing mosquito biting outdoors. J. active ingredient in this device (measured Am. Mosq. Control Assoc. 23:47-54. in hours or days) as personal protection Moody, R. P. 1989. The safety of diethyltoluamide against host-seeking ticks has not been insect repellents. J. Am. Mosq. Control Assoc. 262:28-29. evaluated. Osimitz, T. G. and R. H. Grothaus. 1995. The present safety assessment of DEET. Mosq. Control Assoc. 11:274-278. V. ACKNOWLEDGEMENTS SAS Institute, Inc. 2013. JMP. Version 11. Cary, NC. Ujihara, K., T. Mori, T. Iwasaki, M. Sugano, Y. Sho- We thank the volunteers who partici- no, and N. Matsuo. 2004. Metofluthrin: a potent new synthetic pyrethroid with high vapor activity pated in the partial or whole field trials. against mosquitoes. Biosci. Biotechnol. Biochem. All volunteers gave informed consent re- 68:170-174. garding the possible risks of getting tick- Xue, R. D., A. Ali, and J. F. Day. 2007. Commercially available insect repellents and criteria for their borne diseases during the field trials. This use. In: Insect Repellents, Principles, Methods, and is a research report only. Use or mention Uses. M. Debboun, S. P. Frances, and D. Strickman 90 Technical Bulletin of the Florida Mosquito Control Association, Vol. 10, 2016 (eds). CRC Press, Boca Raton, FL, USA, chapter taeniorhynchus (Diptera: Culicidae) in northeast- 25, pp. 405-415. ern Florida. J. Med. Entomol. 49:652-655. Xue, R. D., W. A, Qualls, M. L. Smith, M. K. Gaines, Zollner, G. and L. Orshan. 2011. Evaluation of a J. H. Weaver, and M. Debboun. 2012. Field evalu- metofluthrin fan vaporizer device against phle- ation of the Off! Clip-on mosquito repellent botomine sand flies (Diptera: Psychodidae) in a (metofluthrin) against Aedes albopictus and Aedes cutaneous leishmaniasis focus in the Judean Des- ert, Israel. J. Vect. Ecol. 36(suppl.): s157-165. Operational Note

EVALUATION OF TALENT UV LIGHT TRAPS COMPARED WITH CDC LIGHT TRAPS, WITH OR WITHOUT DRY ICE, TO COLLECT FRESH AND SALT WATER MOSQUITOES IN NORTHEAST FLORIDA

MICHAEL L. SMITH, WHTINEY A. QUALLS¹, AND RUI-DE XUE Anastasia Mosquito Control District 500 Old Beach Road, St. Augustine, FL 32080

1Department of Public Health Sciences Miller School of Medicine, University of Miami Miami, FL 33124

ABSTRACT. The Talent UV light trap was trap has been developed for the surveillance compared with the CDC light trap, baited of adult mosquitoes but has not been evalu- with or without dry ice, to determine the ated for the collection of saltmarsh mos- best trapping method to collect saltmarsh quitoes. The objective of the study was to and freshwater mosquitoes on Anastasia Is- compare the Talent UV light trap with the land and southern St. Johns County, Florida. gold standard CDC light trap in order to de- Seven species of mosquitoes were collected termine the best trapping method to collect from a saltmarsh on Anastasia Island, of saltmarsh and freshwater mosquitoes in An- which Aedes taeniorhynchus was the major astasia Mosquito Control District. species (90%). Seven species of mosquitoes Six Talent UV traps (model MSTRS), were collected from freshwater habitats in modified from a standard CDC light trap, southern St. Johns County, where Culex quin- were supplied by J. W. Zhu (Iowa State Uni- quefasciatus was the dominate species (81%). versity, Ames, Iowa) and compared with 6 Overall, the CDC trap collected significantly standard CDC light traps (John W. Hock more mosquitoes than the UV trap. For both Company, Gainesville, FL). The Talent UV traps, the addition of dry ice considerably in- traps were modified by adding a 190 mm creased mosquito abundance in collections. (L) × 63 mm (W) × 32 mm (H) UV light bar that produced a light wavelength of 325 nm. Key Words. UV light trap, CDC light trap, Both traps were placed in a random com- dry Ice, Aedes taeniorhynchus, Culex quinque- plete block design separated by 30 meters in fasciatus a residential community on Anastasia Island, St. Augustine, FL for the collection of salt- Insect suction traps, with and without at- marsh mosquitoes. The same experimental tractants, are major tools used for surveillance design was used to evaluate the collection of of adult mosquito populations (Kline 2006). freshwater mosquitoes in a companion study Evaluation of new traps and attractants for in the southern part of St. Johns County. All use within operational mosquito surveillance traps were operated 4 nights per week over programs continue to be an active area of re- a period of three weeks. Each trap was ran- search in the field of medical entomology. domly assigned a location during each trap Mosquitoes perceive ultraviolet light (UV) night. During the first field trial, Talent traps (Allan 1994) and traps have been evaluated without dry ice were compared with dry ice- that use this wavelength for illumination (e.g. baited CDC light traps (Table 1). During the Allan 1994, Li et al. 2015). The Talent UV second field trial, Talent traps were baited

91 92 Technical Bulletin of the Florida Mosquito Control Association, Vol. 10, 2016 Table 1. Mean number (±SE) of mosquitoes collected by Talent UV traps and CDC traps baited with dry ice or without dry ice from saltmarsh and freshwater habitats, northeastern Florida1. Saltmarsh Freshwater Trap type Dry Ice No Dry Ice Dry Ice No Dry Ice CDC Trap 73 ± 0.5 91 ± 4.3 46 ± 13.0 38 ± 12.7 Talent UV Trap 57 ± 3.0 25 ± 8.3 19 ± 6.3 32 ± 4.3 1Saltmarsh habitat mosquito collections consisted of 90% Aedes taeniorhynchus: χ² = 16.999, df = 2, P < 0.01; freshwater collec- tions consisted of 81% Culex quinquefasciatus: χ² = 23.491, df = 2, P < 0.01. with dry ice while the CDC traps were oper- traps baited with dry ice were significantly ated without dry ice. In the third field trial, more than similar baited Talent traps. Re- Talent traps and CDC traps were each oper- gardless of what habitat they were collected ated without dry ice. One gallon Igloo con- in. tainers purchased from local suppliers were In our study, both light traps collected used to dispense the dry ice from one pound mosquitoes equally without the addition blocks. Traps were placed 1.5 meters above of dry ice from the freshwater habitat but ground using a Shepard’s hook. this relationship changed considerably (i.e. Trap data collected from saltmarsh and 2-fold increase) when dry ice was added. It freshwater areas were separately analyzed is well known that carbon dioxide is an at- using non-parametric Kruskal-Wallis χ² tests tractant that considerably increases adult for each trap type and attractant combina- mosquito abundance in CDC light trap col- tion. Differences were determined at P < lections (Newhouse et al 1966, Kline 2006). 0.05. We also found that the CDC light trap out- A total of seven mosquito species were performed the Talent trap in the saltmarsh collected from the saltmarsh habitat. The habitat, regardless if both traps were baited species composition (and percent abun- with dry ice. These results are similar to what dance in total collection) were: Aedes taenio- Li et al. (2015) found in Elkton, St. Johns rhynchus (Weidemann) (90%), Anopheles cru- County, FL. In summary, the CDC light trap cians Weidemann (7%), and Culex nigripalpus currently remains the best adult mosquito Theobald (2%) with Ae. sollicitans (Walker), surveillance trap available for the species we Cx. erraticus (Dyar and Knab), Ae. triseriatus collected in our study. (Say), and Masonia dyari Belkin, Hinemann, We thank Catherine Lippi for assisting in and Page making up the remainder of the data analysis. This is a research report only collection. Seven species of mosquitoes were and does not reflect the endorsement of the collected from freshwater habitats includ- Anastasia Mosquito Control District for any ing Cx. quinquinfasciatus Say (81%), An. cru- of the products mentioned in this study. cians (12%), Ae. atlanticus (Dyar and Knab) (3%), and Psorophora ferox (von Humbolt) REFERENCES CITED (3%) with the remainder of the collection Allan, S. A. 1994. Physics of mosquito vision-an over- represented by Ae. triseriatus, Culiseta melan- view. J. Am. Mosq. Control Assoc. 10:2661-2671. ura (Coquillett), and Coquillettidia perturbans Kline, D. L. 2006. Mosquito population surveillance (Walker). techniques. Tech. Bull. Florida Mosq. Control Assoc. 7:2-8. In the freshwater habitat, mean abun- Li, C. X., M. L. Smith, A. Fulcher, P. E. Kaufman, T. Y. dance of mosquitoes (trap/night) from Zhao, and R. D. Xue. 2015. Field evaluation of three both traps were not significantly different new mosquito light traps against two standard light traps to collect mosquitoes (Diptera: Culicidae) and in the absence of dry ice (Table 1). But un- non-target insects in northeast Florida. Florida En- baited CDC light traps collected significantly tomol. 98:114-117. more mosquitoes than unbaited Talent traps Newhouse, V. F., R. W. Chamberlain, J. G. Johnson, and W. D. Sudia. 1966. Use of dry ice to increase mosqui- in the saltmarsh habitat. We also found that to catches of the CDC miniature light trap. Mosq. the mean number of mosquitoes from CDC News 26:30-35. Operational Note

EFFECTS OF LEAF WASHING ON THE PERSISTENCE OF A SUGAR BAIT-PYRIPROXYFEN MIXTURE TO CONTROL LARVAL AEDES ALBOPICTUS

JODI M. SCOTT¹, ALI FULCHER¹, WHITNEY A. QUALLS², GUNTER C. MULLER³, AND RUI-DE XUE¹ 1Anastasia Mosquito Control District 500 Old Beach Road, St. Augustine, FL 32080

2Department of Public Health Sciences Miller School of Medicine, University of Miami Miami, FL 33124

3Department of Microbiology and Molecular Genetics IMRIC, Kuvin Centre for the Study of Infectious and Tropical Diseases Faculty of Medicine, Hebrew University Jerusalem, Israel 91120

ABSTRACT. The effect of adding the insect this technique has been proven to be safe for growth regulator, pyriproxyfen, to an attrac- non-target insects when applied to non-flow- tive sugar bait (ASB) was evaluated in semi- ering plants or incorporated in bait stations field bioassays against larvalAedes albopictus. (Qualls et al. 2012, Revay et al. 2013). Treatments were applied to croton “Petra” However, rainfall currently limits the ef- potted plants while control plants were fectiveness of ATSBs as barrier treatments sprayed with ASB only. Once a week, for six against adult mosquitoes. In order to turn weeks, plants were “washed” with water to this limitation into an advantage, we re- simulate rainfall and the runoff collected for port here on the addition of pyriproxyfen adult emergence inhibition bioassays. Adult to a commercial attractive sugar bait (ASB) inhibition in the ASB/pyriproxyfen treat- formulation to order to control Aedes albop- ment was about 90% for the first two weeks ictus (Skuse) larvae. Pyriproxyfen, by itself, of the study then gradually decreased from is an effective larvicide that inhibits adult about 80% in week three to about 30% at emergence according to laboratory bioas- week six. says reported by Ali et al. (1995). This active ingredient has also provided effective larval Key Words. Aedes albopictus, pyriproxyfen, at- control of Ae. albopictus when applied by tractive sugar bait, larvicide, croton “Petra” truck-mounted equipment ULV equipment in northeastern Florida (Scott et al. 2013 and Previous studies have shown that attrac- Doud et al. 2014). A recent laboratory study tive toxic sugar baits (ATSB) sprayed on by Fulcher et al. (2014) reported effective plants will kill adult Aedes, Anopheles, and control of adult and larval mosquitoes from Culex species (Xue et al. 2006, 2011, 2013, plants sprayed with an ASB and pyriproxyfen Muller et al. 2010, Naranjo et al. 2013, Revay mixture. However, it was unclear whether et al. 2013, Hossain et al. 2014). Attractive natural precipitation on ASB pyriproxyfen- toxic sugar bait is a highly effective method treated plants would affect the efficacy and of controlling adult mosquitoes by exploit- persistence of Ae. albopictus larval control. ing their sugar feeding behaviors. Moreover, Therefore, the objective of this study was to

93 94 Technical Bulletin of the Florida Mosquito Control Association, Vol. 10, 2016 determine the impact of leaf washing on ef- ter and maintained in the insectary. Larvae ficacy and persistence of ASB/pyriproxyfen were fed every three days with 0.03 g ground applications on plants for larval Ae. albopictus dog biscuits. Emergence in each dish pan in a semi-field trial. was monitored daily until all larvae/pupae Eleven, 25 cm dia. croton, Codiaeum varie- had died or emerged as adults. Experiments gatum “Petra” (L.) Rumph ex. A. Juss, plants were repeated three times. If adult mortality were purchased from Home Depot, St. Au- exceeded 30% in control group that test was gustine, FL. Plants were washed off and al- either excluded or repeated. Percent emer- lowed to dry before application to the leaves. gence inhibition was calculated as described A commercially available attractive sugar in Scott et al. (2013). bait concentrate was mixed with water 1:4 Mean percent adult emergence inhibi- ratio (Westham Innovations LTD, Tel Aviv, tion values were pooled for the nine treat- Israel) to which 1% NyGuard® ED (10% ment plants and two control plants. Data pyriproxyfen AI, provided by Navy Entomol- were then arcsine square root transformed ogy Center of Excellence) was then added to to fit the basic assumptions of ANOVA in the bait solution. The mixture was applied JMP version 9 statistical software prior to to nine plants using a 5.6 L pump-up sprayer analysis. Student’s paired t- tests were used to (ACE half-gallon polypropylene sprayer) in determine differences in treatments for each sweeping motions over the plants until com- week compared with controls (P > 0.05). plete coverage occurred but not to the point Adult inhibition in the ASB/pyriproxy- of runoff. This technique simulated barrier fen treatment was about 90% for the first applications used in field environments. Two two weeks of the study. After this time, effica- control plants were sprayed with ASB only cy gradually decreased to about 80% at week in the same manner as treated plants. Only 3 then about 50% during weeks 4 and 5 with one ATSB (and ASB for control) application 30% adult inhibition at week 6. was made to plants during the study. After Our data suggested that runoff from a 24 hour drying period, 200ml of reverse pyriproxyfen/ASB treated vegetation can osmosis water, (GE SmartWater™ Reverse provide about 90% adult inhibition of Ae. Osmosis Filtration System, Fairfield, CN) albopictus for at least 2 weeks after applica- was applied by a handheld sprayer over the tion. The results from this study may serve surfaces of the leaves while clock-wise rotat- as a future model for individuals who want ing the plants to simulate rainfall. The run- to combine sugar baits with IGRs for opera- off was collected into an 11.4-L (40 cm × 31.5 tional larval control in areas where barrier cm × 15.2 cm) plastic dish pan (Sterilite Cor- applications may also be useful for adult poration, Townsend, MA) and transferred to mosquito control. 946 ml all-purpose-calibrated (APC) plastic The authors would like to thank Mike containers (Ace Hardware, St. Augustine, Smith for technical help. This is a research FL). Plant washes were conducted once a report only and the mention of any products week for six weeks. does not imply Anastasia Mosquito Control Aedes albopictus larvae, maintained in the District endorsement. Anastasia Mosquito Control District, were used in bioassays. This insecticide-suscep- REFERENCES CITED tible laboratory colony was originally ob- Ali, A., J. K. Nayar, and R. D. Xue. 1995. Comparative tained from the USDA, Center for Medical, toxicity of selected larvicides and insect growth reg- Agricultural, and Veterinary Entomology, ulators to a Florida laboratory population of Aedes Gainesville, Florida. Mosquito larvae were albopictus. J. Am. Mosq. Control Assoc. 11:72-76. Doud, C. W., A. M. Hanley, K. C. Chalaire, A. G. Rich- reared at 27° C; 80% RH; 12:12 L:D photo- ardson, S. C. Britch, and R. D. Xue. 2014. Truck- period and used the procedures described mounted area-wide application of pyriproxyfen tar- by Gerberg et al (1994). Ten late third to geting Aedes aegypti and Aedes albopictus in northeast Florida. J. Am. Mosq. Control Assoc. 30:291-297. early fourth instars were placed into each Fulcher, A., J. M. Scott, W. A. Qualls, G. C. Muller, and APC plastic container containing runoff wa- R. D. Xue. 2014. Attractive toxic sugar baits mixed Scott et al.: Effects of Leaf Washing On The Persistence of A Sugar Bait-Pyriproxyfen Mixture 95

with pyriproxyfen sprayed on plants against adult Revay, E. E., G. C. Müller, W. A. Qualls, D. L. Kline, D. and larval Aedes albopictus (Diptera: Culicidae). J. P. Naranjo, K. L. Arheart, V. D. Kravchenko, Z. Yefre- Med. Entomol. 51:896-899. mova, A. Hausmann, J. C. Beier, Y. Schlein, and R. Gerberg et al. 1994. Manual for mosquito rearing and ex- D. Xue. 2013. Control of Aedes albopictus with attrac- perimental techniques. Bulletin 5. Amer. Mosq. Control tive toxic sugar baits (ATSB) and potential impact Assoc., Lake Charles, LA pp 102. on non-target organisms in St. Augustine, Florida. Hossain, T. T., A. Fulcher, C. Davidson, J. C. Beier, and Parasitol. Res. 113:73-79. R. D. Xue. 2014. Evaluation of boric acid sugar baits SAS Institute, Inc. 2007. JMP. Version 9, Cary, NC. sprayed on plants against the salt marsh mosquito, Scott, J. M., W. A. Qualls, M. K. Gaines, R. D. Xue, C. Aedes taeniorhynchus (Diptera: Culicidae). Florida Doud, and G. B. White. 2013. Efficacy of ground Entomol. 97:1865-1868. ULV application of Nyguard (10% pyriproxifen) Muller, G. C., A. Junnila, W. A. Qualls, E. E. Revay, D. L. against Aedes albopictus larvae in St Augustine, Flor- Kline, S. A. Allan, Y. Schlein, and R. D. Xue. 2010. ida. Tech. Bull. Florida Mosq. Control Assoc. 9: 48- Control of Culex quinquefasciatus in a storm drain 52. system in Florida using attractive toxic sugar baits. Xue, R. D., G. C. Muller, W. A. Qualls, M. L. Smith, J. Med. Vet. Entomol. 24:346-351. M. Scott, J. Lear, and S. E. Cope. 2013. Attractive Naranjo, D. P., W. A. Qualls, G. C. Muller, D. M. Samson, targeted sugar baits: field evaluations and potential D. Roque, T. Alimi, K. Arheart, J. C. Beier, and R. use in mosquito control. Wing Beats. 24:13-18. D. Xue. 2013. Evaluation of boric acid sugar baits Xue, R. D., D. L. Kline, A. Ali, and D. R. Barnard. 2006. against Aedes albopictus (Diptera: Culicidae) in tropi- Application of boric acid baits to plant foliage for cal environments. Parasitol. Res. 112:1593-1587. adult mosquito control. J. Am. Mosq. Control Assoc. Qualls, W. A., R. D. Xue, E. E. Revay, S. A. Allan, and 22:497-500. G. C. Muller. 2012. Implications for operational Xue, R. D., G. C. Muller, D. L. Kline, and D. R. Barnard. control of adult mosquito production in cisterns 2011. Effect of application rate and persistence of bo- and wells in St. Augustine, FL using attractive sugar ric acid sugar baits applied to plants for control of baits. Acta. Tropica. 124:158-161. Aedes albopictus. J. Am. Mosq. Control Assoc. 27:56-60. Operational Note

FIELD COMPARISON OF THERMAL FOG APPLICATION OF DUET® AND BARRIER SPRAYING OF TALSTAR® AGAINST AEDES ALBOPICTUS AT A RESIDENTIAL PROPERTY IN ST. AUGUSTINE, FLORIDA

JENNIFER GIBSON, RUI-DE XUE, AND MICHAEL L. SMITH Anastasia Mosquito Control District 500 Old Beach Road, St. Augustine, FL 32080

ABSTRACT. The effectiveness of separate imported and 11 locally-acquired cases of area-wide ground thermal fog applications CHIKV. Because Ae. albopictus is quite prev- of DUET® (sumithrin/) and alent in St. Johns County, understanding barrier spraying of vegetation with Talstar® and controlling this potential vector species (bifenthrin) to control adult Aedes albopictus within local residential areas is becoming in- populations was compared at a large resi- creasingly important. dential property with dense vegetation and Prior to the 1970’s, area-wide ground multiple larval developmental sites in St. thermal fogging was a mainstay and highly Augustine, Florida. The thermal fog treat- effective method for adult mosquito control, ment completely eliminated this mosquito especially in the US. Later, this methodol- pest through one week post treatment from ogy was replaced in favor of ultra low vol- a pretreatment landing count that aver- ume technology due to a number of factors aged 24/person/3 min. Prior to the barrier including a rapid spike in the cost of diesel treatment, the average landing rate count fuel used as a carrier to deliver the pesticide. was 28/person/3 min. After application, However, compared with ULV application, landing rate counts were reduced to zero thermal foggers showed more consistency through three weeks. in droplet dispersal across distances as far as 90 m (Britch et al. 2010). Additionally, ther- Key Words. Aedes albopictus, thermal fog, bar- mal fog application of pesticides provided rier spray, sumithrin, prallethrin, bifenthrin greater mortality against the West Nile virus vector Culex quinquefasciatus Say (Britch et al. In 1985, the Asian tiger mosquito, Aedes 2010). Recently, Anastasia Mosquito Control albopictus (Skuse) became established in District (AMCD) has re-introduced thermal Houston, Texas after arriving in the USA, as fogging into its operational adult control eggs, in automobile tires imported from Ja- program (Xue et al. 2013). pan (Benedict et al. 2007). Since then, this During the middle 2000’s, barrier appli- mosquito has rapidly spread throughout the cation of residual pesticides to vegetation for southeastern parts of the USA, including the purpose of controlling adult mosquitoes Florida, and has become the most abundant gained considerable interest among mosqui- and invasive container-inhabiting mosquito to control programs, an interest that contin- in the State (Lounibos et al. 2001). Accord- ues to this day (Trout et al. 2007, Qualls et ing to the Centers for Disease Control and al. 2013). By applying residual pesticides to Prevention, Ae. albopictus is a known vector mosquito harborage/resting sites (e.g. veg- of multiple arboviruses including chikun- etation), mosquito programs can selectively gunya (CHIKV). In 2014, the Department target adult mosquito species such as Ae. al- of Health confirmed that Florida had 425 bopictus, a daytime-biting species, at relatively

96 Gibson et al.: Field Comparison of Thermal Fog Application of Duet and Barrier Spraying of Talstar 97 low cost (Bengoa et al. 2013). Anastasia Mos- application rate was 30 meters per 3 minutes quito Control District has used insecticide at a spray height of 3 meters. Landing rate barrier applications to perimeter vegetation counts were conducted 24 hours, 1 week, 2 to successfully control adult mosquitoes weeks, and 3 weeks after treatment. around golf courses and schools (Qualls et Mean landing rate counts were subjected al. 2013). to analysis using JMP® software (SAS Insti- The objective of our study was to com- tute, Inc. 2007). Student’s t-test was used pare the effectiveness of an area-wide ther- to compare pre- and post treatment LRC’s mal fog application of DUET® with that of within each treatment (i.e. thermal fog and a barrier application of Talstar®, against the barrier). container-inhabiting mosquito, Ae. albopic- Our results showed that there was a sig- tus, at a large local residential property. nificant difference between LRCs pre and Pre-treatment landing rate counts (LRC) post for the thermal fog treatment after 24 were conducted by three volunteers for hours (t = 2.15, P < 0.05). This application three minutes in three different locations completely eliminated the Ae. albopictus pop- approximately 35 meters apart at a 2.7 hect- ulation through 1 week post treatreatment are County St. Johns County property (lati- from a pretreatment LRC that averaged 24/ tude 29.93049, longitude -81.343876). The person/3 min. For the barrier application, property was chosen due to its numerous lar- pre- and post LRCs were also significantly val developmental sites and dense vegetation different after 24 hours (t = 2.15, P < 0.05). that are known habitat locations of adult Ae. The pre-treatment LRC averaged 28 Ae. al- albopictus. The study took place from 0900 to bopictus/person/3 min, then after applica- 1100 during October 2014 when peak sea- tion, LRCs were reduced to zero through 3 sonal populations of this species occur in St. weeks post treatment. Johns County. Targeting an urban mosquito vector for DUET (active ingredient: 5% sum- control, like Ae. albopictus, can be challeng- ithrin® and 1% prallethrin) was applied ing due to high population densities of the at maximum label rate along the outside pest often produced from numerous cryptic perimeter of the property and domicile us- larval habitats. Determining adult harbor- ing a LongRay® TS-35A thermal fogger age sites to target control measures can be (ADAPCO, Sanford, FL). The travel distance helpful in designing the scenario for barrier at which treatment was applied at was 11 m application. For example, one study showed per minute at a spray height of 3 meters. that Ae. albopictus had high mortality in leaf Wind direction and speed were monitored bioassays after exposure to Talstar-treated during application. At 30 minutes, and 24 wax myrtle plants (Cilek and Hallmon 2008). hours after treatment, landing rate counts Plant identification can provide an indica- were conducted again for three minutes in tor for harborage sites and is only one of the same locations used for pre-treatment. the many variables that integrated mosqui- Landing rate counts were conducted weekly to management programs need to address thereafter and continued until there was a when considering barrier application effec- significant increase in post treatmentAe . al- tiveness. Past studies by Cilek (2008) have bopictus LRC. shown that some pyrethroid insecticides, Once Ae. albopictus LRCs rebounded applied as barrier treatments to vegetation, after two weeks, a residual barrier applica- can last up to 6 weeks in controlling adult tion was administered, at maximum label mosquitoes. Our results suggest that the bar- rate, to perimeter vegetation of the property rier treatment was a better alternative for with Talstar® (active ingredient: 7.9% bifen- controlling Ae.albopictus in dense vegetation thrin) using a Birchmeier Rec 15 backpack because of its longer lasting effects on the sprayer. The sprayer was calibrated to deliver population in the field. However, we believe 43.3 ounces per minute with a mixture of 3 that by utilizing a combination of methods, gallons of water to 3 ounces of Talstar. The such as thermal fogging and barrier spray- 98 Technical Bulletin of the Florida Mosquito Control Association, Vol. 10, 2016 ing, mosquito control districts may be able J. D. Kerce, J. J. Becnel, U. R. Bernier, and J. W. Prid- geon. 2010. Evaluation of ULV and thermal fog mos- to limit costs in areas where consistent adult quito control applications in temperate and desert control is needed but may be difficult to environments. J. Am. Mosq. Control Assoc. 26:183- achieve. Furthermore, AMCD will continue 197. Cilek, J. E. 2008. Application of insecticides to vegeta- to judiously use thermal fogging and barrier tion as barriers against host-seeking mosquitoes. J. treatments as only one of the tools in its in- Am. Mosq. Control Assoc. 24:172-176. tegrated mosquito management program in Cilek, J. E. and C. F. Hallmon. 2008. Residual effec- order to prevent the outbreak of mosquito- tiveness of three pyrethroids on vegetation against adult Aedes albopictus and Culex quinquefasciatus in borne diseases in St. Johns County. screened field cages. J. Am. Mosq. Control Assoc. Special thanks go to the owners of the 24:263-269. Lounibos, L. P., G. F. O’Meara, R. L. Escher, N. Nishimu- residence that allowed us conduct this study ra, M. Cutwa, T. Nelson, and S. A. Juliano. 2001. on their property. We are also thankful for Testing predictions of displacement of native Aedes the support of Ali Fulcher, Cat Smith and by the invasive Asian tiger mosquito Aedes albopictus in Florida, USA. Biol. Invasions 3:151-166. the AMCD volunteers who helped conduct Qualls, W. A., M. L. Smith, and R. D. Xue. 2013. Suc- the landing rate counts. cessful applications of barrier treatments using bi- fenthrin against mosquitoes in St. Johns County, Florida, from 2006-2009. Tech. Bull. Florida Mosq. REFERENCES CITED Control Assoc. 9:33-37. SAS Institute, Inc. 2007. JMP®. version 11. Cary, NC. Benedict, M. Q., R. S. Levine, W. A. Hawley, and L. P. Trout, R. T., G. C. Brown, M. F. Potter, and J. L. Hub- Lounibos. 2007. Spread of the tiger: global risk of in- bard. 2007. Efficacy of two pyrethroid insecticides vasion by the mosquito Aedes albopictus. Vector-borne applied as barrier treatments for managing mos- Zoonotic. Dis. 7:76-85. quito (Diptera: Culicidae) populations in sub- Bengoa, M., R. Eritja., and J. Lucientes. 2013. Labora- urban residential properties. J. Med. Entomol. tory tests of the residual effect of on 44:470-477. vegetation against Aedes albopictus. J. Am. Mosq. Con- Xue, R. D., Weaver, J. H., M. L. Smith, and W. A. Qualls. trol Assoc. 29:284-288. 2013. Field evaluation of thermal fog application Britch, S. C., K. J. Linthicum, W. W. Wynn, T. W. Walk- of sumithrin products against adult mosquitoes in er, M. F. Farooq, V. L. Smith, C. A. Robinson, B. B. northeast Florida. Tech. Bull. Florida Mosq. Control Lothrop, M. Snelling, A. Gutierrez, H. D. Lothrop, Assoc. 9:38-41. The Eleventh Arbovirus Surveillance and Mosquito Control Workshop Sponsored by AMCD and USDA/CMAVE St. Augustine, Florida March 25-27, 2014

PROGRAM

Tuesday (March 25, 2014) Panel Session: Moderator: Dr. Rui-De Xue, Director, AMCD, St. Augustine, FL 8:30 am Welcome & Introduction Mrs. Catherine Brandhorst, Chairperson, AMCD Board of Commission- ers Dr. Ken Linthicum, Center Director, USDA/CMAVE Mr. Neil Wilkinson, President, Florida Mosquito Control Association 8:40 am Keynote Speaker: New insights into the ecology of eastern equine en- cephalitis virus transmission in the southeastern USA ... Dr. Thomas Unnasch, Distinguished USF Health Professor and Depart- ment Chair, University of South Florida, Tampa, FL 9:20 am Dengue vector control in Malaysia Dr. Abu Ahmad, Professor, Universiti Sains, Malaysia 9:45 am The transgenic mosquitoes and its release for dengue control in Brazil Dr. Aldo Malavasi, Director, Medfly and Mosquito Facility, Brazil 10:10 am Overview of dengue vaccine study in China Dr. Qiang-Ming Sun, Visiting Professor, Yale University, New Haven, CT 10:35 am Mosquitoes and plants—the principle and use of plants in mosquito control Dr. Rui-De Xue, Director, AMCD, St. Augustine, FL 10:45 am Break

Moderator: Dr. Ken Linthicum, Center Director, USDA/CMAVE, Gainesville, FL 11:00 am Real time monitoring of the dengue vector online: cost benefit of a Bra- zilian technology Dr. Alvaro E. Eiras, Professor, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil 10:20 am Historical perspective of dengue fever and its management control strat- egy in Saudi Arabia Dr. Abdul Aziz Althbyani, Assistant Professor, University of Tabuk, Saudi Arabia 11:40 am Challenges for the malaria vector control strategies in three ecological zones (West, Central and East Africa)

99 100 Technical Bulletin of the Florida Mosquito Control Association, Vol. 10, 2016 Dr. Seydou Doumbia, Professor and Co-Director of MRTC/FMPOS, University of Sciences, Techniques and Technology of Bamako, Mali 12:00 pm An update about Clarke’s new products for mosquito control Mr. Frank Clarke, Vice President, Clarke, Kissimmee, FL 12:10 pm Lunch break (provided by Clarke)

Programs and Associations: Moderator: Dr. Gary Clark, Research Leader, USDA/CMAVE, Gainesville, FL 1:00 pm The International Center of Excellence in Research of Vector-borne Diseases in Mali Dr. Traore Sekou F., Professor and Co-Director of MRTC/FMPOS, Uni- versity of Sciences, Techniques and Technology of Bamako, Mali 1:20 pm Overview of USDA/CMAVE program Dr. Ken Linthicum, Center Director, USDA/CMAVE, Gainesville, FL 1:40 pm USDA/Rutgers University’s area wide management program of (http:// asiantigermosquito.rutgers.edu/) Dr. Gary Clark, Research Leader, USDA/CMAVE, Gainesville, FL 2:00 pm Update on the American Mosquito Control Association Dr. Roxanne Connelly, President, AMCA, Professor, University of Florida/IFAS/FMEL, Vero Beach, FL 2:15 pm Overview of the Society of Vector Ecology Dr. Dan. Kline, Vice President, SOVE, Research Entomologist, USDA/ ARS/CMAVE, Gainesville, FL 2:30 pm Update on the Florida Mosquito Control Association Mr. Neil Wilkinson, President, FMCA, Florida Gulf Coast University, Ft. Myers, FL 2:45 pm Chemical and biological threats and vulnerabilities in industry and aca- demia Mrs. Bridgette Frost, FBI Special Agent, Jacksonville, FL 3:05 pm. Break

Arbovirus/Dengue Outbreak in Martin County, FL: Moderator: Dr. John Beier, Professor, University of Miami, Miami, FL 3:20 pm Arbovirus surveillance in Florida, 2013 Stephanie M. Moody-Geissler, Arbovirus Surveillance Coordinator, Bu- reau of Epidemiology, FLDOH, Tallahassee, FL 3:40 pm Dengue outbreak in Martin County, Florida in 2013 Mrs. Karlette Peck, Martin County Department of Health, Stuart, FL 4:00 pm FDACS’s entomological investigation on the 2013 outbreak of dengue fever in Martin County Dr. Peter Jiang, Medical Entomologist, Bureau of Pesticides, FDACS, Tallahassee, FL 4:15 pm Larval surveillance of dengue fever vectors, and in Hillsborough County, FL, 2011-2012 Dr. Azliyati Azizan, Assistant Professor, Department of Global Health, University of South Florida, Tampa, FL Program 101 4:35 pm Analysis of sentinel chickens and arbovirus in St. Johns County, FL Mr. Richard Weaver, Data Manager, AMCD, St. Augustine, FL 4:45 pm WNV in Jacksonville, Florida, 2013 and activity pattern of vector mosquitoes Ms. Marah Clark, Entomologist, Jacksonville Mosquito Control, FL 5:00 pm Cooperative projects to control in Florida and Thailand Drs. Kenneth Linthicum and Seth C. Britch, USDA/CMAVE, Gainesville, FL 5:20 pm End of the session 6:00 pm Dinner and Lecture (Holiday Isle, Beach Blvd, St. Augustine Beach City): Highlights of the Oldest City, City Commissioner, St. Augustine, FL Wednesday (March 26, 2014)

Moderator: Dr. Dan Kline, Research Entomologist, USDA/CMAVE, Gainesville, FL 8:30 am Sex, Y, and the control of mosquito-borne infectious diseases Dr. Jake Tu, Professor, Virginia Tech University, Blacksburg, VA 8:50 am Gene silencing as a strategy for vector control Dr. James Becnel and Mr. Al Estep, USDA/CMAVE, Gainesville, FL 9:10 am Update about research on carbonic anhydrases in mosquitoes Mr. D. Dixon and Dr. Paul Linser, Professor, Whitney Laboratory, Uni- versity of Florida, St. Augustine, FL 9:30 am Integrating molecular tools with Florida mosquito control Dr. Liming Zhao, Research Assistant Professor, University of Florida/ IFAS/FMEL, Vero Beach, FL 9:50 am Environmental assessment of mosquito ecology in Northern Haiti Dr. Whitney A. Qualls, Ms D.M. Samson, and Dr. John Beier, University of Miami, FL 10:10 am Expanding integrated vector management to promote healthy environ- ments Dr. John Beier, Professor, University of Miami, Miami, FL 10:30 am Break

Attractants/Traps/Repellents: Moderator: Dr. Uli. Bernier, Research Chemist, USDA/CMAVE, Gainesville, FL 10:40 am Developing new insecticides and repellents for skin and cloth Dr. Uli. Bernier, Research Chemist, USDA/CMAVE, Gainesville, FL 11:00 am Evaluation of spatial repellents for military use LT Marcus McDonough, Navy Entomology Center of Excellence, Jack- sonville, FL 11:15 am Plant flower attraction for Aedes albopictus Dr. Dan. Kline, Research Entomologist, USDA/CMAVE, Gainesville, FL

11:35 am Field assessment of yeast and oxalic acid generated CO2 for mosquito surveillance LT James Harwood, Navy Entomology Center of Excellence, Jackson- ville, FL 102 Technical Bulletin of the Florida Mosquito Control Association, Vol. 10, 2016 11:50 am Five light trap comparison for collection of mosquitoes and non-target insects Dr. Chun-Xiao Li, Visiting Scientist, Department of Entomology and Nematology, University of Florida, Gainesville, FL 12:00 pm An update about ADAPCO’s new technology Mr. Derek Wright, ADAPCO, Sanford, FL 12:15 pm Lunch Break (provided by ADAPCO)

Larval Habitats & Control: Moderator: Dr. Jerry Hogsette, Research Entomologist, USDA/ARS/CMAVE, Gainesville, FL 1:00 pm Florida’s Indian River Lagoon: mosquito control within an estuary in distress Mr. Doug Carlson, Director, Indian River Mosquito Control District, Vero Beach, FL 1:20 pm Vertical distribution of container-inhabiting mosquitoes in a LaCrosse virus endemic area Mr. Michael Riles, Dr. Bruce Harrison, and Dr. Brian Byrd, University of Western North Carolina, NC 1:30 pm Acoustic techniques for mosquito control in Houston, TX Mr. Herbert Nyberg, New Mountain Innovations, Inc., Niantic, CT 1:50 pm The use of cree carrying larvicides for possible control of mosquito larvae Mr. John Olsen, President, Cree Industries Inc., FL 2:10 pm Aerial larviciding in the Florida Keys Mr. Mike Doyle, Director, Florida Keys Mosquito Control District, Key West, FL 2:30 pm Evaluation of Mosquiron CRD against in downtown storm drains, St. Augustine Mrs. Ali Fulcher, Biologist, AMCD, St. Augustine, FL 2:40 pm A durable dual action lethal ovitrap for control of container breeding mosquitoes Drs. Roberto Pereira and Phil Koehler, Department of Entomology and Nematology, University of Florida, Gainesville, FL 3:00 pm Wildlife lighting- protect wildlife through responsible lighting practices Mr. Mike Hudon, Research Entomologist, Indian River Mosquito Con- trol District, Vero Beach, FL 3:20 pm Break

Adult Control: Moderator: Dr. Jake Tu, Professor, Virginia Tech University, Blacksburg, VA 3:40 pm Primarily testing and formulations of an attractive targeted sugar bait against Aedes aegypti and Aedes albopictus Ms. Jodi Scott, Education Specialist, AMCD, St. Augustine, FL 3:50 pm Hand thermal fogging comparison study Mrs. Ali Fulcher, Biologist, AMCD, St. Augustine, FL Program 103 4:00 pm Sticky traps against Aedes aegypti Ms. Emily Thompson AMCD, St. Augustine, FL and Ms. Jodi Scott, Depart- ment of Entomology and Nematology, University of Florida, Gainesville, FL 4:10 pm Exploring new thermal fog and ultra-low volume technologies to im- prove indoor control of the dengue vector, LT James Harwood, Navy Entomological Center of Excellence, Jacksonville, FL 4:25 pm The knight stick: a new trap for stable flies Dr. Jerry Hogsette, Research Entomologist, USDA/ARS/CMAVE, Gainesville, FL 4:45 pm Attractive targeted sugar baits (ATSB) Mrs. Julie Lear, Marketing Director, Universal Pest Solutions, Dallas, TX 4:55 pm Attractive toxic sugar bait stations for malaria vector control in Mali Mr. Mohamed M. Traore, University of South Carolina, Colombia, SC 5:10 pm Aerial spray to control Aedes aegypti in Manatee County, FL Mr. Mark Latham, Director, Manatee County Mosquito Control District, FL 5:30 pm End of the session

Thursday (March 27, 2014)

Programs, Education, & Legislation: Moderator: Mr. Neil Wilkinson, Florida Gulf Coast University, Ft. Myers, FL 8:30 am Overview of the school program at Lee County Mosquito Control District Mr. Neil Wilkinson, Florida Gulf Coast University, Ft. Myers, FL 8:50 am Efficacy of a new fly bait containing Mr. Casey Parker, Drs. Roberto Pereira and Phil Koehler, Department of Entomology and Nematology, University of Florida, Gainesville, FL 9:00 am ATSB and pyriproxfen on plants against adult and larval Aedes aegypti Dr. Rui-De Xue, Director, AMCD, St. Augustine, FL 9:15 am FLDACS update – progress on rulemaking Mr. Michael Page, Chief, Bureau of Entomology and Pest Control, FL- DACS, Tallahassee, FL 9:30 am Overview of Mosquito Research Foundation Mr. James Clauson, Director, Beach Mosquito Control District, Panama City, FL 9:45 am Program overview of Volusia County Mosquito Control Mr. James McNelly, Director, Volusia Mosquito Control District, New Smyrna Beach, FL 10:00 am South Walton County Mosquito Control program Mr. Ben Brewer, Director, South Walton County Mosquito Control Dis- trict, Santa Rosa Beach FL 10:20 am Break 104 Technical Bulletin of the Florida Mosquito Control Association, Vol. 10, 2016 Surveillance, Control, & Other: Moderator: Mr. Michael Page, Chief, Bureau of Entomology and Pest Control, FLDACS, Tallahassee, FL 10:35 am Large scale evaluation of a new barrier spray machine and barrier treat- ment Mr. Mike Smith, Biological Technician, AMCD, St. Augustine, FL 10:45 am Early population outbreak of mosquitoes and control response in St. Johns County, FL Mrs. Kay Gaines, Supervisor, AMCD, St. Augustine, FL 10:55 am Evaluating the temporal effects of Nuvan Prostrips for filth fly control LCDR Shani Gourdine, Navy Entomological Center for Excellence, Jacksonville, FL 11:10 am Evaluation of DeltaGard G as an acaricide LT Mattew Yans, Navy Entomological Center for Excellence, Jackson- ville, FL 11:25 am Database software with smart phone for operational mosquito control Mr. Tim Morris, Mobisoft Infotech, Houston, TX 11:40 am Spot weather detectors & recorder and possible for mosquito control operation Dr. William R. Eisenstadt, Professor, Department of Electrical and Com- puter Engineering, University of Florida, Gainesville, FL 12:00 pm Update about products from UNIVAR Mr. Jason E. Conrad, Industry Specialist, UNIVAR 12:10 pm Lunch break (provided by UNIVAR)

END OF WORKSHOP The Twelfth Arbovirus Surveillance and Mosquito Control Workshop Sponsored by AMCD and USDA/CMAVE St. Augustine, Florida March 24-26, 2015

PROGRAM

Tuesday, March 24, 2015

Panel Session: Moderator:Dr. Rui-De Xue, Director, AMCD, St. Augustine, FL 8:30 am Welcome & Introduction Ms. Vivian Browning, Chairperson, AMCD Board of Commissioners Dr. Ken Linthicum, Center Director, USDA/CMAVE, Gainesville, FL Ms. Sue Bartlett, President of the FMCA, New Smyrna, FL 8:40 am Keynote Speaker: Epidemic of chikungunya and dengue fever and re- search response and focus from the University of Miami Dr. John C. Beier, Professor and Section Chief, University of Miami, Miller School of Medicine, Miami, FL 9:20 am Guest Speaker: Overview of Rutgers University’s Center for Vector Biology Dr. Randy Gaugler, Distinguished Professor and Director, Center for Vector Biology, Rutgers University, NJ 9:50 am Dengue virus and potential vaccine investigation Dr. Scott Michael, Professor, Florida Gulf Coast University, Ft. Myers, FL 10:20 am The 4th International Forum for Surveillance and Control of Mosquitoes and Mosquito-borne Diseases, Guangzhou, China, (May 25-28, 2015) and AMCD’s new complex Dr. Rui-De Xue, Director, AMCD, St. Augustine, FL 10:30 am Break

Moderator: Dr. Ken Linthicum, Center Director, USDA/CMAVE, Gainesville, FL 11:00 am Spatial Repellents for Control of Vector-borne Disease: The Current Status Dr. Nicole Achee, Associate Professor, University of Notre Dame, IN 11:20 am Progress and prospective of attractive toxic sugar baits against mosqui- toes and sand Flies Dr. Gunter Muller, Visiting Professor, Hebrew University, Jerusalem, Israel 11:40 am Extreme weather events and impact on vector-borne diseases and agri- culture Dr. Kenneth Linthicum, Center Director, USDA/CMAVE, Gainesville, FL 12:00 pm An update on Clarke’s new products for mosquito control Mr. Frank Clarke, Vice President, Clarke, Kissimmee, FL 12:10 pm Lunch break (provided by Clarke)

105 106 Technical Bulletin of the Florida Mosquito Control Association, Vol. 10, 2016 Programs & Associations: Moderator: Dr. Dan Kline, Research Entomologist, USDA/CMAVE, Gainesville, FL 1:00 pm ATSB in the marketplace, commercial and consumer update Ms. Laura Uggerholt, Universal Pest Control 1:20 pm FLDACS program update Ms. Adriane N. Tambasco, Medical Entomologist, FLDACS/Division of Agricultural Environmental Services, Tallahassee, FL 1:40 pm Update on the American Mosquito Control Association in 2015 Dr. Ken Linthicum, President, AMCA and Center Director, USDA/ CMAVE, Gainesville, FL 1:55 pm Overview of the Society of Vector Ecology Dr. Dan Kline, President-Elect, SOVE and Research Entomologist, USDA/ARS/CMAVE, Gainesville, FL 2:10 pm Update on the Florida Mosquito Control Association Mrs. Sue Bartlett, President, FMCA and Operations Manager, Volusia Mosquito Control, New Smyrna, FL 2:25 pm Overview of the Florida Entomological Society Dr. Cindy McKenzie, President, Florida Entomological Society and Re- search Entomologist, USDA/USHRL, Ft. Pierce, FL 2:40 pm Malaria vector mosquito’s resting behavior and movement in day time Dr. Gunter Muller, Visiting Professor, Hebrew University, Jerusalem, Israel 3:00 pm Break

Arbovirus: Moderator: Dr. Randy Gaugler, Distinguished Professor and Director, Rutgers University, NJ 3:20 pm Arbovirus surveillance in Florida, 2014: the year of chikungunya Dr. Andrea Bingham, Arbovirus Surveillance Coordinator, Bureau of Epidemiology, FLDOH, Tallahassee, FL 3:40 pm Long-term health impacts of chikungunya virus infection in Florida residents Ms. Katie Kendrick, Epidemiologist, Bureau of Epidemiology, FLDOH, Tallahassee, FL 4:00 pm West Nile virus outbreak in Volusia County, Florida in 2014 Mr. James McNelly, Director, Volusia Mosquito Control, New Smyrna, FL 4:20 pm Vector competence of Florida mosquitoes for chikungunya and dengue viruses Dr. Barry Alto, Assistant Professor, University of Florida/IFAS, Florida Medical Entomology Laboratory, Vero Beach, FL 4:40 pm Arbovirus surveillance by sentinel chickens and a novel technique in St. Johns County Miss Jennifer Gibson, Biological Technician, AMCD, St. Augustine, FL 5:00 pm West Nile virus in Jacksonville, Florida, 2014 and daily activity pattern of vector mosquitoes Ms. Marah Clark, Entomologist, Jacksonville Mosquito Control District, FL Program 107 5:20 pm End of the session 6:00 pm Dinner and Lecture (Holiday Isle, Beach Blvd., City of St. Augustine Beach): Multi-rotor unmanned aerial systems: new tool for mosquito control Dr. Randy Gaugler, Distinguished Professor and Director, Rutgers Uni- versity’s Center for Vector Biology, NJ

Wednesday, March 25, 2015

Biology & Ecology: Moderator: Dr. Nicole Achee, Associate Professor, University of Notre Dame 8:20 am Update on the Acoustic Techniques for Mosquito Control Mr. Herbert Nyberg, New Mountain Innovations, Inc., Niantic, CT 8:35 am Mosquito gut ecosystem and potential impact on control action Dr. Jiannong Xu, Associate Professor, Department of Biology, New Mexico State University, Las Cruces, NM 8:55 am Population dynamics of the floodwater mosquito in St. Johns County, Florida Miss Emily Thomson, Biological Technician, AMCD, St. Augustine, FL 9:05 am Aedes japonicus japonicus (Theobald): a new emerging vector in north- west Florida Mr. Mike Riles, Entomologist, Beach Mosquito Control District, Panama City Beach, FL 9:20 am Update on research on carbonic anhydrases in Aedes aegypti Mr. D. Dixon and Dr. Paul Linser, Whitney Laboratory, University of Florida, St. Augustine, FL 9:35 am Mosquitoes and non-target insects and attractive target sugar baits Ms. Jodi Scott, Visiting Scientist, AMCD, St. Augustine, FL and Ph.D. candidate, Department of Entomology and Nematology, University of Florida, Gainesville, FL 9:50 am Mosquitoes associated with suburban backyards and dog kennels near Gainesville, FL Mr. Chris Holderman, Ph.D. candidate, Department of Entomology and Nematology, University of Florida, Gainesville, FL 10:05 am Comparison of target and non-target mortality rates from residual pesti- cide on HESCO material Mr. Robert L. Aldridge, Mr. Thomas T. Dao, Dr. Seth C. Britch and Dr. Kenneth J. Linthicum, USDA/CMAVE, Gainesville, FL 10:20 am Indoor and outdoor sugar feeding behaviors of dengue vector in Ecuador Drs. Whitney Qualls and John Beier, Professor, University of Miami, Miami, FL 10:35 am Break

Attractants/Traps/Repellents: Moderator: Dr. James Becnel, Research Entomologist, USDA/CMAVE, Gainesville, FL 10:40 am Current research on mosquito repellents for clothing and skin Ms. Natasha Agramonte and Dr. Ulrich Bernier, USDA/CMAVE, Gaines- ville, FL 108 Technical Bulletin of the Florida Mosquito Control Association, Vol. 10, 2016 11:00 am Various approaches for the control of blood-feeding insects Dr. Lisa L. Drake, Department of Biology, New Mexico State University, Las Cruces, NM 11:20 am A multiyear study of oviposition patterns in suburban residential back- yards Dr. Dan Kline, Research Entomologist, USDA/CMAVE, Gainesville, FL 11:40 am Evaluation of thermal cell mosquito repellents against several species of mosquitoes Mr. Christopher Bibbs, Education Specialist, AMCD, St. Augustine, FL 12:00 pm An update on ADAPCO’s new products Mr. Derek Wright, ADAPCO, Sanford, FL 12:10 pm Lunch break (provided by ADAPCO)

Pesticides, Larval &Adult Control: Moderator: Dr. Jerry Hogsette, Research Entomologist, USDA/ARS/CMAVE, Gainesville, FL 1:00 pm Ground application of larviciding and adulticiding at AMCD Mrs. Kay Gaines, Supervisor, AMCD, St. Augustine, FL 1:15 pm New formulations of larvicide and adulticide for mosquito control …. Mr. David Sykes, All Pro Vector Group, Madison, FL 1:25 pm Effects of residual IGRs on Aedes aegypti and Aedes albopictus Ms. Kristen Donovan, Undergraduate Research Assistant, Dr. Roberto Pereira and Dr. Phil Koehler, Associate Research Scientist and Professor, Entomology Department, University of Florida, Gainesville, FL 1:40 pm Malaria transmission along the Niger river in a Sudan savanna area of Mali Dr. Sekou F. Trtaore, Professor and Director of MRTC Entomology, University of Sciences, Techniques and Technology of Bamako, Mali 1:50 pm Program Overview: Collier Mosquito Control District Mr. Jim Stark, Executive Director, Collier Mosquito Control District, Naples, FL 2:10 pm Evaluation of larvicides and pupacides against Aedes aegypti and Culex quinquefasciatus Miss Jennifer Gibson, Biological Technician, AMCD, St. Augustine, FL 2:20 pm Public Health Pesticide Inventory and new insecticides Dr. Karl Malamud-Roam, Public Health Pesticides Program Manager, IR-4 Project Headquarters, Princeton, NJ 2:40 pm Possible impact of mosquito control pesticides on coral reef health in Florida Dr. Cliff Ross, Associate Professor, Department of Biology, University of North Florida, Jacksonville, FL 3:00 pm Evaluation of Cimi-Shield Knock-Out Bed Bug Eliminator against adult house flies Dr. Jerry Hogsette, Lead Scientist and Research Entomologist, USDA/ ARS/CMAVE, Gainesville, FL 3:20 pm Break Program 109 Insecticide Resistance: Moderator: Dr. Whitney Qualls, University of Miami, Miami, FL 3:40 pm Resistance mechanisms in the Akron strain of Anopheles gambiae isolated in Benin, West Africa Drs. J. R. Bloomquist, A. D. Gross, D. R. Swale, F. Tong, and T. D. An- derson, Department of Entomology and Nematology, Emerging Patho- gens Institute, University of Florida, Gainesville, FL 4:00 pm Characterization of a pyrethroid resistant atrain of from Puerto Rico Dr. James J. Becnel, Mr. Alden Estep, Mr. Neil Sanscrainte and Ms. Jes- sica Louton, USDA/CMAVE, Gainesville, FL 4:20 pm Survey of Aedes aegypti in Florida to determine resistance levels and mechanisms Mr. Alden Estep, Dr. James J. Becnel, and Mr. Neil Sanscrainte, USDA/ CMAVE, Gainesville, FL and Ms Christine Waits, Navy Entomology Cen- ter of Excellence 4:40 pm Comparison of pyrethroid resistance in adult and larval Culex pipiens pallens in five field populations from China Ms. Hui Liu, Research Associate, Department of Vector Biology and Control, Jiangsu Provincial CDC, Nanjing, China 5:00 pm Testing for insecticide resistance in Aedes alboptictus strains from St. Augustine, Florida Ms. Christy Waits, Navy Entomology Center for Excellence, Jacksonville, FL, Mrs. Ali Fulcher, AMCD, Mr. Alden Estep, Mr. Alec Richardson, Navy Entomology Center for Excellence, Jacksonville, FL, Dr. Jimmy Becnel, USDA/CMAVE, and Dr. Rui-De Xue, AMCD 5:20 pm Vector epidemiology and challenge of malaria control in Mali Dr. Seydou Doumbia, Professor and Director, MRTC Entomology, Uni- versity of Sciences, Techniques and Technology of Bamako, Mali 5:40 pm End of the session

Thursday, March 26, 2015

Technology & Program: Moderator: Ms. Sue Bartlett, Operations Manager, Volusia Mosquito Control, New Smyrna, FL 8:30 am SMACK those mosquitoes: a “Sentinel Mosquito Arbovirus Capture Kit” (SMACK) for monitoring arboviruses in the field Dr. Scott Ritchie, Professor, James Cook University, Cairns, Australia 9:00 am Overwintering of eastern equine encephalitis virus in North America Dr. Nathan Burkett-Cadena, Assistant Professor, University of Florida/ IFAS/FMEL, Vero Beach, FL 9:20 am Durable Dual-action Lethal Ovitraps (DDALO) for management of the dengue vector and other container-breeding mosquitoes Ms. Casey Parker, Graduate Research Assistant, Drs. Roberto Pereira and Phil Koehler, Associate Research Scientist and Professor, Department of Entomology and Nematology, University of Florida, Gainesville, FL 9:35 am Overview of the IGR-pyriproxyfen through auto-dissemination against dengue vector mosquitoes Mr. Mike Banfield, CEO, Spring Star, Inc. Seattle, WA 110 Technical Bulletin of the Florida Mosquito Control Association, Vol. 10, 2016 9:50 am Database software with smart phone for operational mosquito control in AMCD Mr. Richard Weaver, Data Manager, AMCD and Mr. Tim Morris, Mobi- soft Infotech, Houston, TX 10:05 am GeoWorld Outdoors, Inc. GeoMosquito Mapping Solution Mr. Daniel Finch, GeoWorld Outdoors, Inc, Kenosha, WI 10:20 am Mobile application with a focus on pest activity, material target pests, and e-content Mr. Adam Holt, Service Pro, St. Augustine, FL 10:30 am Break

Surveillance & Other: Moderator: Dr. Nathan Burkett-Cadena, Assistant Professor, UF/IFAS/FMEL, Vero Beach 10:50 am Population surveillance of Aedes albopictus detected by BGS traps in St. Johns County, FL Mr. Mike Smith, Biologist, AMCD, St. Augustine, FL 11:00 am Development of a rapid detection method for arbovirus surveillance in Florida Dr. Limin Zhao, Research Assistant Professor, University of Florida/ IFAS/FMEL, Vero Beach, FL 11:20 am Understanding IGRs and their potential for controlling bed bugs Ms. Brittany Delong, Graduate Research Assistant, Drs. Roberto Pereira and Phil Koehler, Associate Research Scientist and Professor, Depart- ment of Entomology and Nematology, University of Florida, Gainesville, FL 11:40 am Mosquito population surveillance and GIS Dr. Mohamed F. Sallam, Visiting Scientist, Department of Entomology and Nematology, University of Florida, Gainesville, FL 12:00 pm Intact Immunoglobulin traffic from blood meal to hemoplymph: a new look at mosquito control via host immunization Dr. Paul Linser, Professor, Whitney Laboratory, University of Florida, St. Augustine, FL 12:20 pm Update on products from UNIVAR Mr. Jason E. Conrad, Industry Specialist, UNIVAR 12:30 pm Lunch break (provided by UNIVAR)

END OF WORKSHOP

2:30 pm New AMCD facility ground breaking