Journal oF THE FlorIDa MoSQuITo ConTrol aSSoCIaTIon

Journal oF THE FlorIDa MoSQuITo ConTrol aSSoCIaTIon

VOLUME 67, 2020

ISSN 1055-355X (print)ISSN 2638-6054 (online)

Journal of the florida Mosquito Control assoCiation

EDITORIAL STAFF

Rui-De Xue, Editor, Anastasia Mosquito Control District, 120 EOC Drive, St. Augustine, FL, 32092. [email protected]

Seth C. Britch, Assistant Editor, USDA-ARS, Center for Medical, Agricultural, and Veterinary Entomology, 1600 SW 23rd Dr., Gainesville, FL 32608. [email protected]

Derrick Mathias, Assistant Editor, University of Florida/IFAS, Florida Medical Entomology Laboratory, 200 9th St. SE, Vero Beach, FL 32962. [email protected]

Eva Buckner, Assistant Editor, University of Florida /IFAS, Florida Medical Entomology Laboratory, 200 9th St. SE, Vero Beach, FL 32962. [email protected]

New manuscripts of articles, operational or scientific notes, and annual meeting abstracts and page proofs should be sent to the Editor by e-mail attachment at [email protected]. The Editor will assemble the manuscripts to the assistant editor or subject editor to conduct the peer review process.

Copyright ©2020 by The Florida Mosquito Control Association, Inc.

Printed by E.O. Painter Printing Company, P.O. Box 877, De Leon Springs, FL 32130

i

Table of ConTenTs

Articles

Updated county mosquito species records for Northwest FloridaJohn P. smith, taylor J. taylor, cami i. Adams, richard A. tennant, Jr., eric cope, Jimmy Walsh, Ben Nomann, caleb Horn, Fred Johnson, Gail Hilson, charles Golden, Gary O’Neal, John Kozak, Keith Armstrong, and les conyers . . . . . . . . . . . . 1

Gravid infusion water comparison for collection of the West Nile virus vector Culex quinquefasciatus sayNicholas Acevedo, caroline efstathion, Dena Autry, Vindhya s. Aryaprema, rui-De Xue, and Whitney A. Qualls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

surveillance of Aedes aegypti after resurgence in downtown st. Augustine, Northeastern FloridaDaniel Dixon, Dena Autry, James Martin, and rui-De Xue . . . . . . . . . . . . . . . . . . . . . . 15

the effects of infusion water and fermentation time on mosquito and non-target organisms collected in the cDc’s autocidal gravid ovitraplagan J. Mullin, Daniel Dixon, and rui-De Xue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

Field evaluations of attractive toxic sugar bait station and vegetation spray applications for control of Aedes aegypti in Key largo, FloridaDiana P. Naranjo, lin Zhu, Kristopher l. Arheart, Douglas Fuller, Gunter c. Muller, rui-De Xue, and Whitney A. Qualls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28

earth fill increases efficacy and longevity of λ-cyhalothrin residual insecticide treatment of HescO® blast wall geotextile seth c. Britch, James e. cilek, erica J. lindroth, robert l. Aldridge, Frances V. Golden, Joshua r. Weston, Jason D. Fajardo, Alec G. richardson, Jessika s. Blersch, and Kenneth J. linthicum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36

effect of the combined nozzle oritentrion and truck speed on efficacy of Ultra-low-Volume spray against caged Aedes albopictus in urban Gainesville, FloridaYongxing Jiang, Muhammad Farooq, James cilek, and Alec G. richardson . . . . . . . . 51

scieNtiFic AND OPerAtiONAl NOtes

increased water hardness in catch basins treated with spinosad (Natular® Xrt) extended release tablets lawrence J. Hribar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60

Nuisance Psychoda alternata (Diptera: Psychodidae) developing in potted plants at a commercial nursery Matteo Pallottini, c. lee Bloomcamp, roberto M. Pereira, and Philip G. Koehler . . . . . 64

comparison of the cDc light trap and the DYNAtrAP Dt 2000 for collection of mosquitoes in semi-field and field settings Nicholas Acevedo, caroline efstathion, rui-De Xue, and Whitney A. Qualls . . . . . . . 69

A new laboratory colonization of Aedes aegypti after reemergence and unsuccessful eradication in st. Augustine, Florida rui-De Xue, christopher s. Bibbs, Daniel Dixon, and Dena Autry . . . . . . . . . . . . . . . 73

ii Journal of the Florida Mosquito Control Association, Vol. 67, 2020

Continued

Laboratory evaluation of boric acid sugar baits against irradiated Aedes aegypti

Vindhya S. Aryaprema, Kai Blore, Jedidiah Kline, Robert L. Aldridge,

Kenneth J. Linthicum, and Rui-De Xue. . . . . . . . . . . . . . . . . . . . …………………….……76

Efficacy of commercial attractive toxic sugar bait station (ATSB) against Aedes

albopictus

Vindhya S. Aryaprema, Edward Zeszutko, Courtney Cunningham, Emad I. M. Khater

and Rui-De Xue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .….…… 80

Characterization and efficacy of VectoBac®WDG applications targeting container- inhabiting

mosquitoes using Unmanned Aerial Vehicles

Keira J. Lucas, Peter Brake, Sara Grant, Leanne Lake, Rachel B. Bales,

Richard Ryan, Nate Phillips, and Patrick Linn. . . . . . . . . . . . . . . . . . . . . . . . . . . . . ….…… .84

.

SUBMITTED ABSTRACTS OF THE 91ST ANNUAL MEETING

Mosquito community composition and seasonal distribution in Northeastern

Florida

Bryan V. Giordano, Lindsay Y. Campbell, Sue Bartlett, Caroline Efstathion,

Rui-De Xue, and Randy Wishard. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . …...91

Ornamental bromeliads of local botanical gardens serve as breeding sites for

pyrethroid-resistant disease vector mosquito species in Collier County, Florida

Alexandria Watkins, Cameron Cole, Emory Babcock, and Keira J. Lucas . . . . . . . . … .. .91

Surveillance report of Aedes aegypti and Aedes albopictus in St. Johns County, 2019

Presented by Steve Smoleroff. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . … .. . .92

Trap modification to increase the capture rate of Aedes aegypti

Presented by Lea Bangonan. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ……. . .. .93

Vector competence of Chinese Aedes aegypti and Aedes albopictus for transmission of

Zika virus

Xiao-Xia Guo. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . …….. . .93

Effects of irradiation on the blood-feeding activity of Aedes aegypti

Presented by Courtney A. Cunningham. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ……… . .94

Host avidity and Deet repellency in irradiated Aedes aegypti

Rui-De Xue and Kenneth J. Linthicum . . . . . . . . . . . . . . . . . . . . . . . . . . ………. . . . . . .. .. 94

Comparing the efficacy of autocidal gravid ovitraps and In2Care traps on Aedes

aegypti and Aedes albopictus in St. Johns County, Florida

Presented by Dena Autry. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . …… … . .95

Bioefficacy of the SAFI product (CUSO4.5H2O) as a larvicide against mosquito

genera—Aedes, Culex, and Anopheles—as a function of concentration (PPM),

time and application

Presented by Kai Blore. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . … . . .…….. 95

Laboratory evaluation of boric acid sugar bait against the Puerto Rico resistant

strain of Aedes aegypti

Presented by Mandi Pearson. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .…....... .96

Continued

table of contents iii

effects of barrier treatment and ground UlV application of adulticides on the honey bee, Apis melliferaYongxing Jiang, Hussein sanchez-Arroyo, robert Horsburgh, Philip Koehler, and rui-De Xue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96

evaluating the efficacy of the truck-mounted super duty A1 mist sprayer against Aedes aegypti and Aedes albopictus in urban environments in lee county Kara tyler-Julian, rachel Morreale, David Hoel, and Aaron lloyd . . . . . . . . . . . . . . . 97

Artificial membrane feeding techniques for mosquito mass rearing steven stenhouse, rachel Morreale, Aaron lloyd, David Hoel, and t. Wayne Gale . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97

sentinel chicken surveillance methodologies and best practices Milton sterling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98

Virtual medical insect collection of institute of Microbiology and epidemiology Ming-Yu Wu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98

More data, more problems: how cMcD is managing its ever-expanding surveillance program rebecca Heinig, Peter Drake, Nate Phillips, and Keira A. lucas . . . . . . . . . . . . . . . . . 99

enhancing resources and capacity of entities that do mosquito control in texas Whitney A. Qualls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99

editors’ acknowledgements. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100

instruction for contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Back cover

iv

2018-2019 THE FMCA PRESIDENTIAL ADDRESS

ANDREA LEAL

Florida Keys Mosquito Control District, 503 107th Street Gulf, Marathon, FL 33050

W e l c o m e to the 91st An-nual Meeting of the Florida Mosquito Con-trol Association in beautiful St. Augustine, Florida. I would like to thank all of those that have worked so hard to bring

this meeting together, both program and venue.

I’d like to start by reminding everyone of the mission statement of our association: The mission of the FMCA is to promote effective and environmentally sound control of disease-transmitting and pestiferous mosquitoes and other arthropods of public health importance, develop and enhance public interest, aware-ness, and support for the control of mosqui-toes, and provide for the scientific advance-ment of members through our meetings, training and education. This is the perfect reminder of why each and every one of us are in this particular field. How many of us grow-ing up said, “I want to be in mosquito control?” While the majority of us may not have started out on this particular path, at some point, we all developed the same passion for this field. What each and every one of us does on a daily basis is extremely important and we are all here for the same reason...to protect public health. I want to thank you all for your com-mitment and dedication to that noble cause.

Current Board Members of FMCA are the following: Andrea Leal (President), Don-nie Powers (President-Elect), James Clauson (Vice President), Wayne Gale (Immediate Past President), Austin Horton (Northwest Representative), Peter Jiang (Northeast Representative), Katie Williams (Southwest Representative), Sherry Burroughs (South-east Representative), John Magee (Com-missioner Representative), Robert Santana (Industry Representative), and Barry Alto (Representative At Large). If you are inter-ested in becoming more involved with the FMCA, I encourage you to join one of our many committees. We are always looking for more volunteers!

FMCA has had a very busy year in 2019. First, on the management side, 2019 was a transitional year for the administration of FMCA. We have recently hired Integrum Consulting, LLC for our association man-agement. Additional areas of focus for the Board and management are website/event registrations, finances, electronic voting and updating Bylaws/Policies and Proce-dures. On the education side, FMCA had very successful participation at the Dodd Short Courses, Aerial Short Courses, Talla-hassee Days, AMCA Truck Day, and AMCA Washington Days. Additionally, please check out our publications on our website: BuzzWords, Wing Beats, and Journal of the Florida Mosquito Control Association.

I hope you all enjoy the meeting and be sure to mark your calendars for FMCA’s up-coming events.

1

UPDATED COUNTY MOSQUITO SPECIES RECORDS FOR NORTHWEST FLORIDA

JOHN P. SMITH1,2,3, TAYLOR J. TAYLOR1,4, CAMI I. ADAMS1,5, RICHARD A. TENNANT, JR.1,2,6, ERIC COPE2,7, JIMMY WALSH2,

BEN NOMANN1, CALEB HORN1, FRED JOHNSON1, GAIL HILSON1, CHARLES GOLDEN1, GARY O’NEAL1, JOHN KOZAK2,

KEITH ARMSTRONG2, AND LES CONYERS1

1Florida State University, 4750 Collegiate Drive, Panama City, FL 32405

2Florida A&M University, John A. Mulrennan Sr., Public Health Entomology Research and Education Center, 4000 Frankford Av., Panama City, FL 32405

3Current address and to whom correspondence should be addressed: Public Health Entomology Services, LLC,

8205 Grand Palm Blvd., Panama City Beach, Florida 32408

4Current address: Pasco County Mosquito Control District, 2308 Marathon Rd., Odessa, FL 33556

5Current address: South Walton County Mosquito Control District, 774 N. County Hwy 393, Santa Rosa Beach, FL 32459

6Current address: Mosquito Surveillance Services, LLC, 100 Dogwood Lane, Crestview, FL 32536

7Current address: Bay County Mosquito Control, 4800 Fire Tower Rd, Panama City, FL 32404

Guest Editor: Nathan D. Burkett-Cadena

ABSTRACT

This report updates the mosquito species composition for Santa Rosa, Okaloosa, Walton, Holmes, Washington, Jackson, Calhoun, Liberty, Gadsden, Leon, Wakulla, Jefferson, Madison, and Taylor Counties, through collections made in a centralized surveillance program operated from 2002-2020 in northwest Florida. 91 county species re-cords were documented. The most notable discoveries included finding Mansonia titillans (Walker) in eleven of the fourteen surveyed counties, Psorophora horrida (Dyar and Knab) in nine, Anopheles perplexens Ludlow in eight and Cu-lex erraticus (Dyar and Knab) and Uranotaenia lowii Theobald in seven. Psorophora mathesoni Belkin and Heinemann and Aedes japonicus japonicus (Theobald) were found in six new counties. Culex pilosus (Dyar and Knab) was found solely in Calhoun and Liberty Cos., while Culex peccator Dyar and Knab and Culex tarsalis Coquillett were recovered in Calhoun Co. and Santa Rosa Co., respectively. Mansonia titillans, Cx. erraticus, Cx. tarsalis and Ae. j. japonicus are known arbovirus vectors, thus increasing the disease risk in this region.

Key Words: mosquitoes, surveillance, species composition, distribution, northwest Florida

INTRODUCTION

County-level mosquito species distribu-tions in Florida have been published by

Darsie and Morris (2003). Since then, sev-eral new introductions and county range expansions have been reported. Anopheles grabhammi (Theobald) and Aedes condoles-

2 Journal of the Florida Mosquito Control Association, Vol. 67, 2020

cens (Dyar and Knab) were discovered in Monroe County (Darsie et al. 2002 and Darsie 2003). Culex declarator Dyar and Knab was found in Indian River and Mon-roe Counties (Darsie and Shroyer, 2004). In northwest Florida, Culex coronator Dyar and Knab was originally discovered in Oka-loosa, Santa Rosa, Washington and Wal-ton Counties and later recovered in Bay and Holmes Counties (Smith et al. 2006, and Smith 2008). Additional surveillance found this species in all remaining Florida counties except Gulf, Franklin and Monroe Counties (Connelly et al. 2016). In 2009, collections of Culex erraticus (Dyar and Knab) were reported from Walton County (Vander Kellen et al. 2012). Aedes pertinex (Grabham) was discovered in 2011 in In-dian River County (Shroyer et al. 2015) and Culex interrogator (Dyar and Knab) was originally found in Broward, Indian River, Okeechobee, and Citrus Counties (Shin et al. 2016). Aedes japonicus japonicus (Theo-bald) was first reported in a collection from Okaloosa Co. in 2012 and later in Bay, Leon, Santa Rosa and Walton Coun-ties (Riles et al. 2017). Larvae and adults of the tropical mosquitoes Culex (Melanoco-nion) panocossa and Aedeomyia squamipennis (Lynch Arribalzaga) were discovered near Homestead, FL in Miami-Dade County (Blosser and Burkett-Cadena 2017, and Burkett-Cadena and Blosser 2017). This

article documents additional records from surveillance conducted in 14 of 18 north-west Florida counties from 2002-2020.

MATERIALS AND METHODS

From 2002 through 2020, mosquito sur-veillance was conducted in Santa Rosa, Oka-loosa, Walton, Holmes, Washington, Jackson, Calhoun, Liberty, Gadsden, Leon, Wakulla, Jefferson, Madison, and Taylor Counties in northwest Florida (Table 1). Mosquito Mag-net X (MMX) traps a.k.a. “counterflow or pickle-jar traps” (Woodstream Corporation, Lancaster, PA) supplemented with 200 cc/min. compressed carbon dioxide (CO2) gas were operated year-round once per week during crepuscular and nocturnal hours to capture host-seeking mosquitoes at 12 sites per county except for Leon County. Leon County submitted collections from BG Sen-tinel traps (Biogents USA, Moorefield, WV) supplemented with octenol (Woodstream Corporation, Lancaster, PA), BG Lure (Bio-gents USA, Moorefield, WV), and dry ice at varying locations within the county.

Container-breeding mosquitoes were also surveyed by deploying one sixteen-ounce black ovicup (4imprint USA, Osh-kosh, WI) supplied with red velour paper strip (Hygloss Products, Inc., Wallington, NJ 07057) or seed germination paper (Anchor Paper Co., St. Paul MN, 55101) at each MMX

Table 1. County, years, and funding source for mosquito surveillance.

County Years Funding Source

Calhoun 2004, 2015-2019 County and StateGadsden 2017-2020 County and StateHolmes 2004, 2015-2019 County and StateJackson 2004, 2017-2019 County and StateJefferson 2017-2019 StateLeon 2016-2020 CountyLiberty 2004, 2015-2019 County and StateMadison 2017-2019 StateWalton 2002-2011 CountyOkaloosa 2002-2019 CountySanta Rosa 2002-2020 CountyTaylor 2017-2019 StateWakulla 2015-2019 County and StateWashington 2002-2007, 2015, 2017-2019 County and State

Smith et al.: County mosquito species records 3

sites during 2017-18 in all counties except for Leon, Okaloosa, Santa Rosa, and Walton.

Adult mosquito traps were operated de-pending on available funding. The most intensive surveillance occurred 2017-2019 when the Florida Legislature funded the program through a Florida Department of Health (FDOH) – Florida State University (FSU) contract. The most consistent surveil-lance was in Santa Rosa and Okaloosa Coun-ties where the program was continuously funded by the counties for 18 and 16 years, respectively. Other counties provided fund-ing on an intermittent basis (Table 1). Santa Rosa, Gadsden, and Leon Cos. continued surveillance and/or identification services in 2020.

RESULTS

Ninety-one new county mosquito species records were documented from fourteen NW Florida counties (Tables 2 & 3). Mul-tiple collections of these species were made at several sites within each county. Table 3 provides collection details for specific sites with the most specimens collected. The greatest number of new county records were reported from Washington, Santa Rosa and Liberty Counties with sixteen, eleven, and ten, respectively. Okaloosa, Jefferson, and Calhoun Counties each had eight. The re-maining counties ranged from one to seven new county records. Including these new records, the total known species for most counties ranged from 43-48. Based on our surveillance and review of the published literature for northwest Florida counties, Jackson and Leon Counties have the great-est known diversity with 57 and 53 species, respectively. The most notable observations in county range distributions included Man-sonia titillans (Walker) in eleven of the four-teen surveyed counties, Psorophora horrida (Dyar and Knab) in nine, Anopheles perplexens Ludlow in eight and Cx. erraticus and Ura-notaenia lowii Theobald in seven. Psorophora mathesoni Belkin and Heinemann and Ae. j. japonicus (Theobald) were found in six new counties. Culex pilosus (Dyar and Knab) was found solely in Calhoun and Liberty Cos.,

while Culex peccator Dyar and Knab and Cu-lex tarsalis Coquillett were recovered in Cal-houn Co. and Santa Rosa Co., respectively.

Ovitrap surveillance in Holmes, Wash-ington, Jackson, Calhoun, Liberty, Gadsden, Wakulla, Jefferson, Madison, and Taylor Counties found only Aedes albopictus, Aedes triseriatus, and Ae. j. japonicus. Aedes aegypti was not collected in any of the surveyed northwest Florida counties by either adult traps or ovitraps.

DISCUSSION

In Florida, arboviruses that cause West Nile virus, Eastern equine encephalitis, and Venezuelan equine encephalitis, annually circulate among competent mosquito vectors and vertebrate reservoirs and are transmit-ted to humans and equine as dead-end hosts (Florida Department of Health 2019). This study documented in several northwest Flor-ida counties the first occurrence of Mn. titil-lans, Cx. erraticus, Ae. j. japonicus, and Cx. tarsa-lis that serve as vectors of these diseases. The first three species were found in 79%, 50%, and 43% of the 14 counties surveyed. This is a significant expansion of the recorded coun-ty species composition, thus increasing our knowledge of the distribution of vectors and mosquito-borne disease in this region.

Some species collected in this surveil-lance program could be easily overlooked because of similarity to more commonly col-lected species. Culex tarsalis and Ps. horrida can be easily confused with Cx. coronator and Ps. ferox, respectively. Connelly and O’Meara (2008) provide a helpful checklist of char-acteristics to aid in the identification of the Culex. Harrison and Whitt (1996) provide a similar checklist for the Psorophora.

Many of the species recovered in this program were likely present much earlier, but not detected due to the lack of surveil-lance throughout much of this region. More species could have been recovered by in-cluding additional surveillance methods be-yond host-seeking adult traps and ovitraps such as: larval surveys, light trapping, resting box collections, and other adult aspiration methods.


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2


Mosquito surveillance is the foundation necessary for building and maintaining in-tegrated mosquito control programs (EPA 2017). Rural counties that dominate most of northwest Florida do not have resources to provide surveillance without supplanting con-trol operations. Faced with this choice, most forego or provide minimal surveillance. Deter-mining species composition and quantifying population levels effectively aids in targeting/prioritizing control efforts and assessing pro-gram efficacy. The welfare of Florida citizens and visitors would benefit greatly if sustained support for comprehensive surveillance was provided throughout the State.

ACKNOWLEDGEMENTS

This work was financially supported in part through FDOH contract #CODNV made possible through appropriations from the Florida 2017 and 2018 Legisla-tures. It would not have been possible with-out the cooperation and support of the county mosquito control programs. The di-rectors (listed below) and their staff greatly assisted in locating trap sites and provid-ing needed information. Santa Rosa, Oka-loosa, N. Walton, and Leon Cos. provided sole financial support for the surveillance program in their jurisdictions. Washington, Holmes, Liberty, Jackson, and Calhoun Cos. provided financial support in the interven-ing years before it was supported by FSU through the Florida Legislature. The state appropriations received in 2017 and 2018 were a huge boost to the ten most rural county programs. Dr. Randy Hanna, FSU-PC Dean, and Kathy Mears, FSU Chief Leg-islative Affairs Officer, are recognized for supporting the FSU budget requests that made this possible. We also acknowledge the support of Banyon Pelham and the of-fice staff in FSU-PC Contracts and Grants and the FDOH contract manager, Reneeka Rogers. We are indebted to the FSU-PC electrical engineering faculty, Drs. Geoffrey Brooks and Shafiul Islam, and their students who greatly assisted with trap hardware and software repairs and enhancements. Lastly, appreciation is extended to the reviewers

of this manuscript who made helpful sug-gestions for improvements. The directors are: Parrish Barwick – Jefferson Co., Da-vid Brazille & Lee Duke – Holmes Co., Al Cleveland & Amanda Baker – Washington Co., Jace Ford – Calhoun & Liberty Co., Ste-phen Ford – Liberty Co., Tommy Harkrider, Jr. – Jackson Co., Cindy Halsey & Scott Hen-son – Okaloosa Co., Keith Hussey & Tony Gomillion – Santa Rosa Co., Brenda Hunt – N. Walton Co., Padraic Juarez-Wakulla Co., Glen Pourciau – Leon Co., Jamison Spen-cer – Gadsden Co., Cheryl White – Taylor Co., Jamie Willoughby – Madison Co.

REFERENCES CITED

Blosser EM, Burkett-Cadena ND. 2017. Culex (Melanoco-nion) panocossa from peninsular Florida, USA. Acta Trop 167: 59-63.

Burkett-Cadena ND and Blosser EM. 2017. Aedeomyia squamipennis (Diptera: Culicidae) in Florida, USA, a new state and county record. J Med Entomol 54: 788-792.

Connelly CR, Alto BW, O’Meara GF. 2016. The spread of Culex coronator (Diptera: Culicidae) throughout Florida. J Vec Ecol 41: 195-199.

Connelly CR, O’Meara GF. 2008. Is it Culex tarsalis or Culex coronator? Buzz Words Sep/Oct.

Darsie RF Jr, Vlach JJ, and Fussell EM. 2002. New addi-tion to the mosquito fauna of United States, Anoph-eles grabhamii (Diptera: Culicidae). J Med Entomol 39: 430-431.

Darsie RF Jr. 2003. First report of Ochlerotatus condole-scens (Dyar and Knab) (Diptera: Culicidae) in the United States. Proc Entomol Soc Wash 105: 1067-1068.

Darsie RF Jr, Morris CD. 2003. Keys to the Adult Fe-males and Fourth Instar Larvae of the Mosquitoes of Florida (Diptera: Culicidae). Tech Bull FL Mosq Control Assoc 1: 1–159.

Darsie RF Jr, Shroyer DA. 2004. Culex (Culex) declarator, a mosquito species new to Florida. J Am Mosq Control Assoc 20:224-227.

EPA. 2017. Success in Mosquito Control: An Integrated Approach. https://www.epa.gov/mosquitocontrol/success-mosquito-control-integrated-approach

Florida Department of Health. 2019. Mosquito-Borne Dis-ease Guidebook. Ch. 2. http://www.floridahealth.gov/diseases-and-conditions/mosquito-borne-diseases/guidebook.html.

Harrison BA, Parker BW. 1996. Identifying Psorophora horrida females in North Carolina (Diptera: Culici-dae). J Am Mosq Control Assoc 12:725-727.

Riles M, Smith JP, Burkett-Cadena ND, Rutledge-Con-nelly CR. 2017. First Record of Aedes japonicus In Florida. J Am Mosq Control Assoc 33:340-344.

Shin D, O’Meara GF, Civana A, Shroyer DA, Miqueli E. Culex interrogator (Diptera: Culicidae), a mosquito species new to Florida. J Vec Ecol 41:316-319.

Shroyer DA, Harrison BA, Bintz BJ, Wilson MR, Sither CB, Byrd BD. 2015. Aedes pertinax, a newly recog-nized mosquito species in the United States. J Am Mosq Control Assoc 31: 97-100.


Smith JP. 2008. Spread, larval habitat, seasonal abun-dance and vector status of Culex coronator: A new invasive vector species in Florida. Report to the FL Dept of Ag and Cons Srvs. http://www.freshfrom-florida.com/content/download/3174/19965/Cor-onator%20Final%20FDACS%20Report.pdf

Smith JP, Walsh JD, Cope EH, Tennant Jr. RA, Kozak III JA, and Darsie Jr. RF. 2006. Culex coronator Dyar and

Knab: a new Florida species record. J Am Mosq Con-trol Assoc 22: 330-332.

Vander Kelen PT, Downs JA, Burkett-Cadena ND, Ot-tendorfer CL, Hill K, Sickerman S, Hernandez J, Jinright J, Hunt B, Lusk J, Hoover V, Armstrong K, Unnasch RS, Stark LM, and Unnasch TR. 2012. Habitat associations of Eastern Equine Encephalitis transmission in Walton County, Florida. J Med Ento-mol 49(3): 746-756.

10

GRAVID INFUSION WATER COMPARISON FOR COLLECTION OF THE WEST NILE VIRUS VECTOR CULEX

QUINQUEFASCIATUS SAY

NICHOLAS ACEVEDO, CAROLINE EFSTATHION, DENA AUTRY, VINDHYA S. ARYAPREMA, RUI-DE XUE, AND WHITNEY QUALLS

Anastasia Mosquito Control District, 120 EOC Drive, St. Augustine, FL 32092

Guest Editor: Yongxing Jiang

ABSTRACT

Gravid traps are an important tool in mosquito surveillance for the collection of gravid female mosquitoes that can be screened for arboviruses. The type of infusion water used is vital in targeting certain mosquito species, espe-cially the West Nile vector, Culex quinquefasciatus Say. The gold standard of infusions is a whey protein mixture but is expensive and time consuming to make. The current study compared a cattail infusion to whey protein to determine if it is as effective at collecting Cx. quinquefasciatus as the current standard. If as attractive, the cattail infusion could be a more economical and less time-consuming option for use in WNV surveillance. Three sites were used to evaluate the efficacy of cattail infusion water with whey protein mixture. Each site had a trap with 100% whey protein, one with 100% cattail infusion and one with 50% cattail water infusion. All 9 traps were operated for 24 hours for seven trap nights. Collected mosquitoes were identified, speciated, and physiological stage was assessed. There was no statistically significant difference in the total number of mosquitoes, gravid female, and total number of females amongst the three types of infusions, at any of the sites for the collection of Cx. quinquefasciatus. These results demonstrate that a cattail infusion can be used as a more economical and less labor-intensive alternative to whey protein for the collection of Cx. quinquefasciatus.

Key Words: Culex quinquefasciatus, West Nile virus, gravid traps, surveillance

INTRODUCTION

Arbovirus surveillance is an important component of an integrated mosquito man-agement plan. West Nile virus (WNV) is one of the most important arboviruses due to its distribution and incidence in the United States (Petersen et al. 2013). Therefore, it has become routine for programs to screen for the virus using molecular testing of mos-quito pools. The major vectors of WNV, Culex quinquefasciatus, Cx. pipiens L and Cx. nigripalpus Theobald (Lustig et al, 2018), can be surveilled using collection methods that target the egg laying behavior of these species. Mosquito control districts programs have relied on the Reiter-Cummings gravid traps for collection of gravid mosquitoes for WNV surveillance. Gravid traps function by attracting gravid female mosquitoes with an attractive infusion water. When a mosquito lands to oviposit, she gets sucked through an opening in the trap by a fan and is retained

in a capture box where she can later be iden-tified and tested for viruses.

The most important part of the gravid trap is the infusion water used to attract the ovi-positioning mosquitoes. There is con-tention on best practices due to resource availability and local vector composition. Previous comparisons have been made, with varying success but ultimately no clear defi-nition of a best practice (Allan et al. 2005). Culex mosquitoes prefer water with high or-ganic material, so an infusion using whey protein has been developed as the gold standard in infusion water (Burkett-Cadena and Mullen 2008). To be able to produce this standard for infusion water, mosquito controls have tested and revisited the idea of what the “ideal” infusion water recipe may be. Time is a factor that can easily be overlooked when assessing the feasibility of preparing an infusion water. Some standards of making infusion water that targets WNV vectors requires investing in supplies such as

Acevedo et al.: Infusion water and collection of gravid mosquitoes 11

whey protein, brewer’s yeast, hay, and many more products. These ingredients have both an economical and time associated-cost to prepare the infusion. For a fiscally and/or time constrained mosquito control program these costs become a disadvantage for us-ing the whey protein infusion. This study was to investigate if the natural flora located within a wide geographic region may pro-vide a cheaper and less time-consuming way to make an infusion water for WNV surveil-lance.

Cattails (Typha species) are among the most common aquatic plant and have a wide geographic distribution (Bansal et al. 2019). Due to this fact, cattails are widely distribut-ed and easily accessible. The project objec-tive was to test an infusion water made with cattails and determine if this infusion water is as attractive or more than the infusion wa-ter in targeting gravid WNV vector mosqui-toes, including our primary vector Cx. quin-quefasciatus.


The sites selected for this study were cho-sen based on the historical abundance of Cx. quinquefasciatus. Three locations were select-ed: Valencia (29° 53’ 35.7’’ N 81° 18’ 59.148’’ W), Ribera” (29° 52’ 36.444’’ N 81° 18’ 38.232’’ W) and Cartwheel Bay Avenue (30° 3’ 36.468’’ N 81° 32’ 4.452’’ W). Valencia is an urban tourist area located in downtown St. Augustine, FL. The traps at Valencia were placed near flooded sewer drains and had daily foot traffic from university students in the area. These sewer drains were confirmed to be the emergence site of Cx. quinquefas-ciatus. Riberia is in an area containing a wa-ter treatment facility and a number of horse stables in St. Augustine, FL. The emergence site of Cx. quinquefasciatus at Riberia was not confirmed but suspected to be around the water treatment facility. The area where the traps were placed at Cartwheel Bay (pine forest) would occasionally flood the location for the emergence site was unidentified.

This experiment took place from June to July, 2019 and consisted of seven trap nights. The whey protein infusion water was pre-

pared following Burkett-Cadena and Mullen (2008) protocol. The infusion water being tested was a cattail infusion that consisted of 4 lbs of fresh cattails (including the roots, leaves, and seed pods) in 40 L of water from the retention pond located on the Anasta-sia Mosquito Control District (AMCD) fa-cility, and was left to ferment in a 32 gallon plastic garbage can (2.7 kg). Once the whey and cattail infusion waters were prepared, the solutions would ferment for 5 days prior to deployment in the field. The 50% cattail infusion water was made by taking 9 L and mixing with 9 L of DI water. A new infusion batch, both whey and cattail would be made every week.

Each evaluation site had three Reiters-Cummings gravid traps (BioQuip, Rancho Dominguez, CA) modified for a 6-volt bat-tery positioned 9 m apart. At each site the three infusions were evaluated using a Latin shift design and rotated weekly. The traps were set and collected in the field during the early morning. Upon retrieval, the cap-ture boxes in each trap would be placed in a cooler to help preserve the state of each specimen for later identification. The speci-mens were frozen and then identified to species and physiological state (gravid, non-bloodfed, bloodfed, male) when possible, via microscopy.

The data was analyzed using IBM® SPSS®

Statistics Version 20 software package. Once tested, the data sets by the Shapiro-Wilk nor-mality test, all the comparisons were carried out by non-parametric (Kruskal-Wallis or Mann-Whitney) tests where appropriate.

RESULTS & DISCUSSION

A total of 16 species in five genera were captured (Table 1). The highest percentage of mosquitoes collected by each infusion at each site was Cx. quinquefasciatus (Figure 1). There were no significant differences in any of the observed parameters among the three sites (Table 2). All data were then pooled to compare the efficacy of the three infusion waters in sampling mosquitoes. None of the parameters showed significant differences among the three infusions indicating that


both the cattail infusions are as effective as whey protein infusion in sampling mosqui-

toes. Considering the physiological condi-tions of female Cx. quinquefasciatus, the num-ber of blood-fed females collected showed a significant difference among the three sites (χ2=14.21, P = 0.001) with the Cartwheel site collecting higher numbers of bloodfed mosquitoes than both Riberia (U=95.0, P = 0.002) and Valencia (U=77.0, P=0.001). It was not influenced by the type of the infu-sion (P>0.05 for all) but by the trap posi-tion of Cartwheel (χ2=10.94, P = 0.004) and Valencia (χ2=9.81, P = 0.007). Positional influence on gravid females was significant only at Valencia (χ2=6.843, P = 0.033) while it has a significant influence on non-gravid mosquitoes at both Cartwheel (χ2=8.931, P = 0.011) and Valencia (χ2=8.191, P = 0.017).

In this study we demonstrated that cat-tail infusion water used in gravid traps was as attractive to Cx. quinquefasciatus mosquitoes as the standard whey protein infusion water. The results show that both cattail infusions (100% cattail and 50% cattail/50% water) could be used as there is no difference in at-

Table 1. Total number of mosquitoes and species col-lected during testing period.

Mosquito species

Study Site

Cartwheel Bay Ribera Valencia

Aedes aegypti 0 12 60Ae. albopictus 32 20 4Ae. infirmatus 2 2 0Ae. taeniorhynchus 0 2 0Anopheles crucians 6 1 2An. quadrimaculatus 4 0 0Culiseta melanura 2 4 0Culex coronator 0 7 0Cx. erraticus 16 0 3Cx. nigripalpus 22 28 8Cx. peccator 4 0 2Cx. quinquefasciatus 1252 519 561Cx. salinarus 1 0 0Cx. territans 25 0 0Uranotaenia lowii 1 0 0Ur. sapphirina 1 7 2Totals 1368 602 642

Figure 1. Average number of Cx. quinquefasciatus (male and females combined) trapped by infusion type throughout the evaluation period.

Cartwheel Riberia ValanciaEvaluation Site

Cattail 50%Cattail 100%Whey 100%

Ave

rage

Cul

ex q

uinq

uefa

scia

tus

trap

ped

80

70

60

50

40

30

20

10

0

Acevedo et al.: Infusion water and collection of gravid mosquitoes 13

traction to the mosquitoes sampled in this study. The significant differences in mos-quito physiological stage that was collected was due to trap position. This is in part ex-plained by the close proximity of the gravid traps at the Cartwheel site near the AMCD sentinel chicken coup. The chickens serve as an attractant and may have increased the number of mosquitoes visiting the site, bloodfeeding, and then looking to oviposit. Thus, resulting in the increase in the num-ber of blood-fed mosquitoes collected com-pared to the other two locations.

Our findings support previous work com-paring whey protein infusion to cattail infu-sions (Allan et al. 2005, Dixon et al. 2019). Al-lan et al. (2005) compared the whey protein infusion to a cattail (Typha species) infusion that found both infusions to attract similar numbers of gravid Cx. quinquefasciatus. Ad-ditionally, when evaluating the gravid trap for collection of eastern equine encephalitis vectors, a whey and cattail infusion was used. This study found no significant difference in the number and abundance of species collected (Dixon et al. 2005). For resource limited or developing mosquito controls, the cattail infusion can provide great benefit in maintaining and developing an arbovirus surveillance program. The cattail is readily available during peak mosquito season and can be harvested with little to no cost.

The cattail infusion water is a low cost, easy to make alternative to the whey protein infusion water and could be a viable option for resource limited mosquito control pro-grams. Further investigations should be car-ried out using the cattail infusion over the course of the mosquito season to optimize the role of this infusion in arbovirus surveil-lance.

REFERENCES CITED

Allan SA, Bernier UR, Kline DL. 2005. Evaluation of oviposition substances and organic infusions on col-lection of Culex in Florida. J Am Mosq Control Assoc 21:268-273.

Bansal S, Lishawa SC, Newman S, Tangen BA, Wilcox D, Albert D, Anteau MF, Chimney MJ, Cressey RL, DeKeyser E, Elgersma KJ, Finkelstein SA, Freeland J, Grosshans R, Klug PE, Larkin DJ, Lawrence BA, Linz G, Marburger J, Noe G, Otto C, Reo N, Richard

Tabl

e 2.

Ave

rage

num

ber

of m

osqu

itoe

s co

llect

ed b

y th

e th

ree

type

s of

infu

sion

wat

er a

t th

e th

ree

diff

eren

t stu

dy s

ites

thro

ugh

out t

he

eval

uati

on p

erio

d.

Car

twh

eel

(Mea

n ±

SD

) R

iber

ia(M

ean

± S

D)

Vale

nci

a(M

ean

± S

D)

Cat

tail

50

%C

atta

il

100%

Wh

ey p

rote

in

100%

Cat

tail

50

%C

atta

il

100%

Wh

ey p

rote

in

100%

Cat

tail

50

%C

atta

il

100%

Wh

ey p

rote

in

100%

Tota

l57

.00

± 51

.96

74.8

8 ±

110.

374

.0 ±

112

.62

29.1

4 ±

23.5

140

.00

± 31

.43

16.8

6 ±

11.8

822

.57

± 17

.14

46.5

± 3

1.55

31.7

1 ±

25.7

7C

x. q

iun.

mal

es16

.00

± 11

.55

33.8

5 ±

67.1

129

.67

± 45

.42

7.1

4 ±

5.27

14.0

0 ±

12.7

1 4

.00

± 3.

11 8

.29

± 5.

3822

.67

± 20

.29

13.7

1 ±

10.7

7C

x. q

iun.

fem

ales

34.8

6 ±

45.8

532

.57

± 41

.11

38.6

7 ±

58.5

917

.14

± 18

.98

21.2

9 ±

17.5

910

.57

± 10

.52

9.4

3 ±

10.5

518

.67

± 8.

4115

.57

± 15

.39

Oth

er s

peci

es 6

.0 ±

4.5

1 8

.14

± 7.

19

2.0

± 2

,76

4.8

6 ±

6.15

4.7

1 ±

4.15

2.2

9 ±

3.3

5.7

1 ±

5.74

5.1

7 ±

8.66

2.4

3 ±

1.9


J, Richardson C, Rodgers L, Schrank AJ, Syedarsky D, Travis S, Tuchman N, Windham-Myers L. 2019. Typha (Cattail) invasion in North American wet-lands: biology, regional problem, impacts, ecosys-tem services, and management. Wetlands 39:645-694.

Burkett-Cadena ND, Mullen GR. 2008. Comparison of infusion of commercially available garden products for collection of container-breeding mosquitoes. J Am Mosq Control Assoc 24:236-243.

Dixon D, Auty D, Xue RD 2019. Evaluation of multiple trap types for the capture of vector mosquitoes of eastern equine encephalitis virus in St. Johns county, Florida. J F Mosq Control Assoc. 66:11-19.

Lusting Y, Sofer D, Bucris ED, Mendelson F. 2018. Sur-veillance and diagnosis of west nile virus in the face of Flavivirus cross-reactivity. Front Microbiol 9:2421.

Petersen LR, Brault AC, Nasci RS. 2013. West nile virus review of the literature. J Am Med Assoc 310:308-315.

15

SURVEILLANCE OF AEDES AEGYPTI AFTER RESURGENCE IN DOWNTOWN ST. AUGUSTINE, NORTHEASTERN FLORIDA

DANIEL DIXON1,2, DENA AUTRY2, JAMES MARTIN3, AND RUI-DE XUE2

1Current Address: United States Department of Agriculture, Agricultural Research Services, 1700 SW 23rd Dr, Gainesville, FL 32608

2Anastasia Mosquito Control District, 120 EOC Drive, St. Augustine, FL 32092

3Quantitative Disease Ecology & Conservation Lab, Department of Geography, University of Florida, Gainesville, FL, 32611

Subject Editor: Derrick Mathias

ABSTRACT

Aedes aegypti is an anthropophilic vector of several arboviruses, including yellow fever, Dengue virus, Chikungunya virus, and the infamous Zika virus. In 2016, Zika virus was spreading rapidly throughout Brazil and mosquito control districts expected Zika virus would be imported to Florida and vectored by endemic Aedes aegypti. Aedes aegypti often takes advantage of cryptic oviposition sites and therefore circumvents conventional control and surveillance strategies used by mosquito control practitioners. The objective of this study was to find Ae. aegypti breeding sites in the tourist district of Saint Augustine, FL, using a door-to-door on-foot approach. Mosquito control technicians, biologists and interns worked to inspect and treat each property for Ae. aegypti. Additionally, residents were informed about Ae. ae-gypti and its public health risk factors. In total, Anastasia Mosquito Control District inspected 1199 of the 1995 parcels in downtown Saint Augustine (60% coverage) in three months. Artificial containers were found at 1,099 of the homes inspected, and Ae. aegypti were found at 120 homes in the area. Each property where mosquito larvae and/or adults were detected was treated using source reduction, larvicides and adulticides. Residents were educated about this project and Ae. aegypti via small flyers, door hangers, pamphlets and/or verbal communication. This study provided insight into the location of Ae. aegypti breeding sites in the tourist district of Saint Augustine, FL, which will facilitate future control efforts.

Key Words: Aedes aegypti, surveillance, trap, habitat, dengue vector

INTRODUCTION

Aedes aegypti (L.) efficiently vectors a number of arboviruses, including Zika virus, yellow fever virus, dengue virus, and chikun-gunya virus, each of which may cause severe morbidity, lifelong health complications and sometimes death (Braak et al. 2018). Howev-er, Zika virus became a notable global health concern in 2016 because of its association with Guillain-Barre syndrome, rare cases of me-ningoencephalitis, and birth defects includ-ing microcephaly and eye issues (blindness, optic neuritis, and intraretinal hemorrhages) (Koppolu and Raju, 2018). In addition, Zika virus is sexually transmitted and can persist in sperm for the first few weeks or up to six months post-infection (Mead et al. 2018). Finally, there is still no vaccine or antiviral

treatment for Zika virus, leaving little to no medical preventative practices and only vec-tor control to contain its spread (Poland et al. 2019). In 2015, there were 62 symptomatic Zika virus disease cases in the United States and 10 symptomatic cases in U.S. territories (CDC, 2019). In 2016, the Zika virus caseload increased to 5,168 symptomatic U.S. cases and 36,512 symptomatic cases among U.S. territories (ibid). With the combination of a large spike in Zika virus transmission and no available vaccine or antiviral treatment, pre-venting an outbreak through vector control and public outreach became a major goal of the mosquito control district in Saint Johns County, FL.

Despite the urgency to control Ae. aegypti and prevent the emergence of Zika virus in Saint Augustine, little information was avail-


able on vector density and oviposition-site locations for targeted treatment of the area. Aedes aegypti oviposits in artificial containers such as bird baths, bottles, paint buckets, discarded tires, and even bottle caps (CDC, 2017). Along with artificial containers, bro-meliads were recently identified as both a harborage and oviposition site for Ae. aegypti (Wilke et al. 2018; Wilke et al 2019). Peri-do-mestic mosquitoes rest underneath brome-liad leaf axils to prevent dehydration in the heat (Muzari et al. 2014), feed from brome-liad flowers and extrafloral nectaries (Xue et al. 2018), and oviposit in the water collected between bromeliad leaf axils (Pittendrigh, 1948; O’Meara et al. 2003; Mocellin et al. 2009; Xue et al. 2018). Along with brome-liads, container-inhabiting mosquitoes are also found in man-made structures, such as wells (Russel et al. 1992), rain barrels (Chris-tophers, 1960), roof gutters (Montgomery and Ritchie, 2002), and construction sites (Liang et al. 2018).

Aedes aegypti has a dynamic history in Saint Johns County that was heavily driven by the invasion of Aedes albopictus (Skuse). Aedes aegypti was detected in Saint Augustine and most of Florida around the early 1900s when dengue fever outbreaks were occur-ring throughout the state (Rey, 2014). Aedes albopictus was first detected in discarded tires in northeast Florida (Duval County) around 1986 (O’meara et al. 1995) and began dis-placing Ae. aegypti in Saint Johns County when it invaded the area in 1989 (ibid). By 1994, Ae. albopictus was found throughout much of Florida and displaced Ae. aegypti in many locations (ibid). This pattern of Ae. albopictus displacement of Ae. aegypti also occurred in other regions within the Unit-ed States and elsewhere (Lounibos, 2007; Kaplan et al. 2010; Lounibos and Kramer, 2016). In 2016, Ae. aegypti was detected in Saint Augustine after 12 years of absence while dipping larvae from a tire near a busy tourist site, which prompted an increase in Ae. aegypti surveillance in the area so that targeted treatments based on mosquito loca-tion could be implemented.

In Saint Augustine, FL, the Anastasia Mos-quito Control District (AMCD) is still devel-

oping a program focused on the surveillance and control of Ae. aegypti, which is especially important because Saint Augustine attracts numerous international tourists. In 2016, AMCD first started its door-to-door mosqui-to surveillance and control campaign with the goal of eradicating Ae. aegypti from major tourist areas. A similar campaign was tried in Brazil in the early 1930s. There the strat-egies for eradication were to identify areas that favored the spread of yellow fever (e.g., areas infested with Ae. aegypti) and survey the human population through testing and au-topsies (Löwy, 2017). During this campaign, weekly inspections were made at Aedes-infest-ed areas with fines for non-compliant resi-dents (ibid). Mosquito inspectors were heav-ily supervised, which was key to the success of the program (ibid), and in the 1940s, Ae. aegypti was deemed “eradicated” in Brazil. However, Ae. aegypti control efforts were not standardized throughout the Americas (Kot-sakiozki et al. 2017; Löwy, 2017), and these non-uniform standards allowed Ae. aegypti to re-invade Brazil from nearby countries like Venezuela and the United States (ibid). The 2016 eradication efforts by the Anasta-sia Mosquito Control District involved the identification of Aedes habitats, inspection, and treatment of the area one day per week for 3 – 4 hours. However, the eradication pe-riod was limited to just three months, con-fined to just the downtown area, and little information was collected about Aedes ovipo-sition and resting sites. In 2016, Ae. aegypti was detected in only six locations in down-town Saint Augustine, whereas Ae. albopictus and Culex quinquefasciatus Say were detected throughout the rest of the downtown area.

Biological larvicides are often used in an integrated mosquito management program to limit the application of adulticides. How-ever, controlling Ae. aegypti has proven to be difficult due to their propensity to oviposit in a variety of cryptic breeding sites, as well as their egg resilience, insecticide resistance, and their close association with humans (Re-iter, 2007; Viennet et al. 2016). Due to these challenges, improvements are needed to ef-fectively control Ae. aegypti with integrated pest management practices. In 2017, AMCD

Dixon et al.: Aedes aegypti surveillance 17

continued the door-to-door Ae. aegypti con-trol campaign begun in 2016, but with the goal of implementing higher resolution surveillance of Ae. aegypti compared to the 2016 efforts by inspecting every property, business, and street in the downtown Saint Augustine area.

This article focuses only on the surveil-lance improvements for the 2017 Ae. aegypti eradication campaign in Saint Augustine. Surveillance was conducted through in-dividual parcel inspections on foot, going through the back and front of each prop-erty, business, or abandoned lot to search for larval and adult Ae. aegypti. Data were collected from each inspected parcel in the downtown area concerning container den-sity, larval and adult mosquito presence, and human housing characteristics (air condi-tioning and screened windows/entrances). These data were used to map locations that could potentially act as Ae. aegypti habitat and therefore glean information on new and existing areas where control efforts could be better focused, making control of Ae. aegypti more efficient. Close attention was paid to areas that had Ae. aegypti larvae and/or adults to identify potential hot-spot areas for future container management and insecticide application. Efforts were made to control the population while conducting surveillance during the 2017 campaign, but no data are provided on the success or fail-ure of control practices.


Data concerning 1,995 parcels was gath-ered from the Saint John’s County Apprais-er’s Office, and addresses were collated into excel sheets for teams to refer to in the field. The following information about each par-cel was collected by field teams: Number of containers, presence of larvae and adults and number of bromeliads. Additionally, the presence of barriers (screened windows/doors and air conditioning) to the inside of homes and businesses was documented be-cause they are recommended by the CDC to prevent blood feeding activity from Ae. aegypti (CDC, 2019). The spatial distribu-

tion of containers was analyzed using a map produced on ARC-GIS (Redlands, CA). The surveillance study was conducted for a to-tal of three months from May to August in 2017. Each parcel was examined for 5 - 10 minutes depending on the size of the prop-erty and time needed for larval source re-duction and insecticide application. Adult mosquitoes were treated with DUET® di-luted 50% with Orchex oil (Clarke®, St. Charles, IL) using a Longray Handheld Pio-neer ULV fogger (PestGoAway®, Hayward, CA). Artificial containers were treated with Natular® DT tablets (Clarke, St. Charles, IL) and Sustain MBG (AllPro®, Northville, MI) according to the specifications on the insecticide labels. Team investigations and treatments of parcels were only done once with no return inspections, which was due to the large number of parcels and limited personnel for inspections and treatments. Each team was supplied with a backpack to hold all supplies and PPE and included: Pasteur pipets and turkey basters for larval collections from artificial containers, whirl packs for transporting a minimum of 10 live larvae from positive parcels back to the laboratory for analysis, markers and pens to record data into log sheets for each parcel, and binders for holding the data sheets for each team. Collected larvae were brought back to the lab, separated by address, and identified to species after adult emergence on a lab bench at room temperature and/or in an insectary at 28°C and 80% relative hu-midity. Residents were educated throughout the program about Ae. aegypti and Zika virus from Mosquito control experts and the local Department of health using door-hangers, pamphlets, and in-person conversation.

RESULTS

From May through August 2017, field teams inspected 1,199 out of the 1,995 par-cels assigned to them, which was a 60% cov-erage of the downtown Saint Augustine area. Some inspections were more exhaustive than others due to access limitations at some properties. For breeding site inspections, a container was defined as any object visible


to technicians that could hold at least 2.5 mLs (or one bottlecap) of water. Field teams found containers on 91% (1,088) of the par-cels that were inspected (Table 1). Container density and location was mapped using ARC-GIS to determine the distribution of homes that are likely breeding sites for Aedes aegypti (Figure 1). Bromeliads were also detected in downtown Saint Augustine. Specifically, 11% (132) of the parcels investigated had at least 1 bromeliad, and 54% (71) of the bromeliad-positive sites had more than 10 per parcel. If more than 10 containers and/or bromeliads were found at a parcel, it was considered a major potential breeding site for Ae. aegypti. Larvae were detected at 19.5% (235) of the parcels that were inspected. These were col-lected and raised to adulthood for species identification. Only a portion of the larvae collected were identified due to limitations in employees and resources. For a sample of 154 larval positive parcels, Aedes aegypti was identified at 78% (120) of them and their locations were tracked on Google maps® (Google, San Francisco, CA) (Figure 2).

The presence/absence of barriers (screened windows/doors and air condition-ing) to the inside of homes and businesses was also documented because they are rec-ommended from the CDC to prevent feed-ing activity from Aedes aegypti (CDC, 2019). Screened windows/doors were found at 48.8% (585) of the inspected parcels and air conditioning was found at 86.9% (1,042).

DISCUSSION

In the summer of 2017, Anastasia Mos-quito Control District field teams inspected

approximately 60% of the parcels in down-town Saint Augustine. Container distribu-tion based on density and location were mapped for future reference as potential Ae. aegypti oviposition sites. Bromeliad density and location were also documented for fu-ture surveillance and larval control. Houses and buildings with barriers to prevent mos-quito entry, such as screened windows/doors and air conditioning, were found on at least 48% of the parcels inspected. The major-ity of mosquitoes collected from downtown Saint Augustine during the surveillance pe-riod were either Cx. quinquefasciatus or Ae. aegypti. Ae. albopictus was not detected from door-to-door container inspections through-out the study duration.

One of the major findings from this re-search was the number of houses with a high density of water-holding artificial containers. Each parcel containing 10 or more contain-ers may represent a threat to human health due to the likelihood of attracting gravid female Ae. aegypti, although there are no universally accepted thresholds for larval in-dicators above which arbovirus transmission is likely to occur (CDC 2017). Nevertheless, high container density may increase the like-lihood of Ae. aegypti establishment, which could lead to continued spread of this spe-cies and ultimately pathogen transmission and incidence of human disease. Important-ly, parcels with a high density of containers were spread throughout the study area, with some such parcels adjacent to one other in small clusters. The latter situation is relative-ly easy to treat, but when target parcels are spread out, greater effort and expense are required to reach them for chemical appli-cation. Another potential breeding site for Ae. aegypti are bromeliads, which were also found throughout the downtown area. Bro-meliad density varied among parcels from complete absence to low (1-8 bromeliads) or high (>10) densities. Although bromeliads are eye-catching and hardy, they potentially act as harborages for Ae. aegypti and other mosquito species by providing shade, nec-tar through floral and extra-floral nectaries, and water collected in leaf axils, which func-tion as oviposition sites (Wilke et al. 2018).

Table 1. Downtown Surveillance of Ae. aegypti – param-eters and data

Start date May 2017End date August 2017Total acres 825Number of Parcels 1995Parcels Inspected 1199Parcels inspected with containers 1088Parcels inspected with bromeliads 132Parcels inspected with larvae 235Parcels inspected with Aedes aegypti 120


Regarding the latter, studies examining the use of bromeliads as breeding sites for Ae. aegypti have had conflicting results. Multiple studies in Brazil have shown that Ae. aegypti

oviposit in leaf axils, but not to a degree that is biologically significant (Maciel-de-Freitas et al. 2007; Mocellin et al. 2009; Santos et al. 2011). Additionally, when bromeliads

Figure 1. Container distribution in downtown Saint Augustine. The map shows the distribution of containers in the parcels inspected throughout downtown Saint Augustine. Container density varied from 0 to greater than 10, represented by circles with a gradient of color temperature and size as shown in the legend.


were targeted for control during dengue outbreaks, Mocellin et al. (2009) suggested that bromeliads should not be the focus of intervention strategies. However, Wilke et al. (2018) surveyed bromeliads in Miami dur-ing a Zika virus outbreak and showed that bromeliads may have led to the proliferation

of Ae. aegypti in that city. In addition, previ-ous studies found Ae. albopictus, Ae. aegypti, and other mosquitoes that vector pathogens breeding in bromeliads from Saint Augus-tine (Bibbs et al. 2018; Xue et al. 2018). Con-sidering that bromeliads in Miami and Saint Augustine act as potential oviposition sites

Figure 2. Distribution of Ae. aegypti in parcels inspected throughout downtown Saint Augustine. Below is a satellite image of downtown Saint Augustine with blue map pins inserted at addresses where Ae. aegypti larvae were captured. The black scale bar below is 1000 feet.


for Ae. aegypti and Ae. albopictus, they warrant continued surveillance and treatment for container-inhabiting mosquitoes that could vector Zika virus to Florida residents.

One of the caveats to door-to-door surveil-lance strategies is the time and labor costs as-sociated with regular inspections. Aedes mos-quitoes in urban and suburban locations are elusive because they deposit eggs in hard to reach, or cryptic, oviposition sites. Examples of cryptic oviposition sites include corrugat-ed extension spouts (Unlu et al. 2014), gut-ters, exposed septic tanks, storm drains, and old sprinkler heads (CDC, 2017). In order to detect these sites, field teams needed to per-form a thorough search of each parcel. This area was relatively small compared to other areas throughout the county, but due to a high density of parcels (at least 2000) and limited accessibility to sections of property, the detection of all mosquito oviposition sites, especially cryptic ones, was not feasible. If Saint Augustine had been in a mosquito-borne disease epidemic, 90% of the homes would have needed to be inspected within a one-week period (CDC, 2017) to collect the surveillance data needed to contain patho-gen transmission. For perspective, it took field teams in this study three months to cov-er 60% of the total downtown area. Thus, a door-to-door strategy may be more effective in isolated areas with a smaller concentra-tion of homes where time can be given for thorough inspections of each parcel.

AMCD personnel also faced multiple challenges when trying to perform door-to-door inspections. Weather events, like mas-sive rain or extreme heat, prevented field teams from inspecting homes for multiple days throughout the summer. Additionally, a small number of homeowners refused in-spections of their home and/or made their parcels inaccessible. Finally, Saint John’s County experienced multiple mosquito out-breaks and two hurricanes during the 2016 – 2017 fiscal year which limited resources and manpower for urban mosquito control in downtown Saint Augustine. To circumvent these issues, new strategies that allow for rapid and thorough detection of container density should be developed.

The downtown Ae.aegypti surveillance project revealed that just over half the par-cels in downtown Saint Augustine may func-tion as breeding sites for Ae. aegypti, and a portion of them may function as major hot spots for Ae. aegypti activity. Despite the data compiled during this investigation, some questions remain unanswered. What is the most effective toolset available to mosquito control districts to effectively control Ae. aegypti where parcel density is high? What factors led to the resurgence of Ae. aegypti in downtown Saint Augustine, and can this be prevented from occurring in the rest of Saint Johns County? New methodologies are currently under development to prevent further spread of Ae. aegypti, such as lethal ovitraps, incompatible insect technique, and sterile insect technique. The coupling of new control methods with thorough surveillance strategies may make these new technologies more efficient for mosquito abatement and disease prevention.

ACKNOWLEDGMENTS

We thank M. Clark, M.K. Gaines, R. Weaver, C. Bibbs, and other AMCD employ-ees for partial participation in this project, and the residents for allowing us to use their properties to conduct the surveillance.

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Braack L, Gouveia de Almeida AP, Cornel AJ, Swane-poel R, de Jager C. 2018. Mosquito-borne arbovi-ruses of African origin: Review of key viruses and vectors. Parasit Vectors 11(1):29.

Centers for Disease Control and Prevention. 2017. Sur-veillance and control of Aedes aegypti and Aedes al-bopictus in the United States. https://www.cdc.gov/zika/pdfs/Guidelines-for-Aedes-Surveillance-and-Insecticide-Resistance-Testing.pdf. [accessed April 22, 2020].

Centers for Disease Control and Prevention. 2019. Zika virus statistics and maps. https://www.cdc.gov/zika/reporting/index.html. [accessed April 22, 2020].

Christophers, RC. 1960. Aedes aegypti the yellow fever mosquito: Its life history, bionomics, and structure. Cambridge: Cambridge University Press.

Kaplan L, Kendell D, Robertson D, Livdahl T, Khat-chikian C. 2010. Aedes aegypti and Aedes albopictus in


Bermuda: Extinction, invasion, invasion and extinc-tion. Biol Invasions 12(9):3277-3288.

Koppolu V, Shantha Raju T. 2018. Zika virus outbreak: A review of neurological complications, diagnosis, and treatment options. J Neurovirol 24(3):255-272.

Kotsakiozi P, Gloria-Soria A, Caccone A, Evans B, Schama R, Martins AJ, Powell JR. 2017. Tracking the return of Aedes aegypti to Brazil, the major vector of the dengue, chikungunya and Zika viruses. PLoS Negl Trop Dis 11(7):e0005653.

Liang S, Hapuarachchi H, Rajarethinam J, Koo C, Tang C, Chong C, Ng L, Yap G. 2018. Construction sites as an important driver of dengue transmission: Im-plications for disease control. Bmc Infect Dis 18:382

Lounibos L. 2007. Competitive displacement and re-duction. J Am Mosq Control Assoc 23(2):276.

Lounibos LP, Kramer LD. 2016. Invasiveness of Aedes aegypti and Aedes albopictus and vectorial capacity for chikungunya virus. J Infect Dis 214(5):453-458.

Löwy I. 2017. Leaking containers: Success and failure in controlling the mosquito Aedes aegypti in Brazil. Am J Public Health 107(4):517-524.

Maciel-de-Freitas R, Marques WA, Peres RC, Cunha SP, Lourenço-de-Oliveira R. 2007. Variation in Aedes aegypti (Diptera: Culicidae) container productivity in a slum and a suburban district of Rio de Janeiro during dry and wet seasons. Memórias do Instituto Os-waldo Cruz 102(4):489-496.

Mead P, Duggal N, Hook S, Delorey M, Fischer M, McGuire D, Becksted H, Max R, Anishchenko M, Schwartz A, et al. 2018. Zika virus shedding in se-men of symptomatic infected men. N Engl J Med 378(15):1377-1385.

Mocellin MG, Simões TC, Nascimento TFSd, Teixeira MLF, Lounibos LP, Oliveira RLd. 2009. Bromeliad-inhabiting mosquitoes in an urban botanical garden of dengue endemic Rio de Janeiro - are bromeliads productive habitats for the invasive vectors Aedes aegypti and Aedes albopictus? Memórias do Instituto Os-waldo Cruz 104:1171-1176.

Montgomery B, Ritchie S. 2002. Roof gutters: A key container for Aedes aegypti and Ochlerotatus notoscrip-tus (Diptera: Culicidae) in Australia. Am J Trop Med Hyg 67(3):244-246.

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23

THE EffECTS Of INfUSION WATER TYPE AND fERMENTATION TIME ON MOSQUITO AND NON-TARGET

ORGANISM COLLECTED IN THE CDC’S AUTOCIDAL GRAVID OVITRAP

LAGAN J. MULLIN, DANIEL DIXON1 AND RUI-DE XUE


Current Address: USDA/CMAVE, 1600-1700 SW 23rd Dr. Gainesville, FL 32608

Guest Editor: Caroline Efstathion

ABSTRACT

The Autocidal Gravid Ovitrap (AGO) is being marketed as an alternative mosquito control tactic to potentially harmful spraying. One problem is the high number of non-target organisms captured with AGOs that is not pres-ent in other gravid traps utilized by mosquito control districts. We tested three different infusion water types with a tap water control in AGOs to determine if they would reduce non-target capture rates. However, all three infusion water types captured more non-target organisms than the tap water control, and the infusion water did not have a significant effect on the number of Aedes aegypti or Ae. albopictus mosquitoes collected. Fermentation time (the date) had an effect on non-target organism capture rates in the AGO traps, but weather conditions may have confounded the fermentation effect. Despite capturing a low number of mosquitoes, this trap was attractive to pest species such as Lucilia sericata. Overall, this trap was ineffective at capturing high rates of Aedes mosquitoes, but it may function as a passive pest control trap with future design modifications.

Key Words: Autocidal gravid ovitrap, Aedes aegypti, Aedes albopictus, infusion water, non-targets

INTRODUCTION

Aedes aegypti Linn. and Ae. albopictus Skuse are nuisance mosquitoes found in Flor-ida and much of the United States that are known vectors of Dengue, Zika, Yellow Fever, Chikungunya and other arboviruses (WHO, 2012). These mosquitoes prefer to feed on people and rest near residential properties. In addition, Aedes aegypti and Ae. albopictus lay their eggs in bottles, plastic buckets, trash cans, and other artificial containers or orna-mental plants associated with human housing (Barerra et al. 2006; Wilke et al. 2018; Hawley, 1988; Delatte et al. 2008; Wong et al. 2011).

One of the major obstacles to controlling Ae. aegypti is its propensity to oviposit in cryp-tic breeding sites (under housing structures with collected water, small bottle caps, hid-den artificial containers, etc). Springstar de-veloped the Biocare Autocidal Gravid Ovitrap (AGO), which captures and kills gravid Aedes females, as a way to control mosquitoes that are searching for these cryptic oviposition

sites. The trap is based off the CDC Autocidal Gravid Ovitrap (Mackay et al. 2013), which consists of a five-gallon black bucket filled with infusion water that is modified to hold a capture chamber laced with a glue board. AGO traps were successful in Puerto Rico with a reported 60-80% reduction in female Ae. aegypti when used as part of an area-wide mosquito management strategy (Barrera et al. 2014). Ideally, autocidal gravid ovitraps will utilize the most effective infusion water to capture an abundance of a target mosquito species while minimizing bycatch. Previous studies determined that ovitrap capture rates for mosquitoes can be enhanced through the use of hay infusions rather than just water (Reiter et al. 1991; Trexler et al. 1998; Pon-nusamy et al. 2010). For Ae. aegypti and Ae. albopictus, alfalfa (Montgomery et al. 2017), orchard grass (unpublished suggestion), and live oak (Ponnusamy et al. 2010) are the sug-gested infusion water substrates for ovitraps. However, the CDC Autocidal Gravid Ovitrap uses hay as its infusion water substrate (Bar-


erra et al. 2014) which captures a high abun-dance of non-target organisms (Dixon et al. unpublished).

The purpose of this study was to compare the capture rates of mosquitoes and non-tar-get organisms in AGO traps with alfalfa, or-chard grass, hay and tap water infusions. The objective was to determine which infusion collected the lowest number of non-target organisms. This was a two-fold approach as both the type of infusion water substrate and its fermentation time were assessed. We hy-pothesize that both non-target and mosquito capture-rates will be highest after two weeks then begin to decline as the infusion water becomes too fermented to attract organisms. It is also expected that the most effective in-fusion-water substrate will be the hay utilized in the Springstar Biocare AGO trap.


Autocidal Gravid Ovitraps were obtained from the company SpringStar (Woodinville, WA, U.S.A.) through an SBIR grant from the NIH (Grant # 2R44AI115782-02) for testing the effectiveness of CDC AGO traps in the field. Infusion water was produced using 24 grams of the orchard grass, Springstar hay, or alfalfa and 8.5 liters of tap water. The control infusion was tap water only. These traps were deployed at the Evergreen Cemetery in St. Augustine Florida (29.894304, -81.335801) after each infusion set fermented in the buckets for one week.

Following a random block design with 4.6 meters between each trap and each block (no Latin shift rotations), nine traps filled with the different infusion water types and nine tap water controls were left in the field continuously for 6 weeks. The traps were ar-ranged so one of each infusion water type and the tap water control were in different groups (9 groups of 4 traps). The tops of the AGO traps were replaced and brought back to the Anastasia Mosquito Control District (AMCD) Base station once a week for those six weeks to identify and record the number of mosquitoes and non-target organisms captured from each respective infusion wa-ter type.

Data was recorded in an excel spread-sheet to analyze mosquito and non-target col-lections in each AGO trap. A Kruskal-Wallis test was used to detect significant differences across the treatment conditions compared to the controls with a p-value ≤ 0.05. This was followed up with a non-parametric compari-son of all pairs using the Steel-Dwass Method. A generalized linear mixed model (GLMM) was used to determine if fermentation time influenced capture rate. Weather data was collected using Weather Underground (The Weather Company, San Francisco, CA) for the zip code of the study site. All statistical analyses were conducted through JMP soft-ware (Cary, NC, U.S.A.).

RESULTS

Overall, the infusions did not collect the number of mosquitoes that were expected. The number of Ae. aegypti and Ae. albopictus collected were very low and not statistically significant from the tap water control in all the infusions tested (Figure 1). However, the number of Culex captured with the Orchard grass and SpringStar Hay was greater than the control (χ2

(3) = 12.1, P = 0.0071), but the orchard grass and springsSar hay collections of Culex were not significantly different from each other. In a similar fashion, the three infusions caught more non-targets than the control (χ2

(3) = 36.4, P < 0.0001), but were not significantly different from each other.

The dataset for mosquitoes was Binomial-ly distributed while the dataset for non-target organisms was Poisson distributed. Accord-ing to the GLMM, the most important fac-tor for the collection of mosquitoes (Figure 2) was the date (P < 0.0479, χ2

(5) =11.18, AICc = 217.3), specifically for weeks two through three of the study. For non-target organisms (Figure 3), the most important factors were the date (P < 0.0001, χ2

(5) =382.4) followed by the date and infusion (P < 0.0001, χ2

(15) =168.64), and finally the infusion (P < 0.0001, χ2

(3) =168.3) all with an AICc = 3442.6. The data collected from Weather Underground indicated that the rainfall during week three was 10.72 cm, week one was 5.49 cm, and the remaining weeks were all under 1.5 cm.

Mullin et al.: Impact of infusion water on non-target collection 25

DISCUSSION

Our study showed that all three infusion water types captured more non-target organ-

isms than the tap water control, but the infu-sion water did not have a significant effect on the number of Ae. aegypti or Ae. albopic-tus mosquitoes collected. According to the

Figure 1. Effect of Infusion water type on mosquito and non-target organism capture rates. The X-axis shows the 3 different infusion water types (Orchard Grass, Springstar Hay, and Alfalfa) and the control (tap water). The Y-axis shows the average organism abundance. Error bars represent standard error (SE) of the mean.

Aver

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Figure 2. Effect of infusion water fermentation time on mosquito collections. The x-axis represents each week of the study. The primary y-axis (left side) represents mosquito abundance while the secondary y-axis (right side) represents rainfall in centimeters. The weekly rainfall is depicted as an area graph in the background in transparent grey. Each bar pattern represents a different infusion water type as shown in the legend above. Error bars represent standard error (SE).

Aver

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GLMM, fermentation time (the date) had an effect on non-target organism capture rates in the AGOs, but weather conditions may have confounded the fermentation ef-fect. Our results suggest that the infusion wa-ter types tested did not improve the efficacy of the CDC AGO.

As mentioned above, weather can be a major factor in field testing results. During week three of the study, just over 10.16 cm of rain was reported on Weather Underground which could explain the sudden decrease in populations of both mosquitoes and non-tar-gets during that time. This decrease could have been attributed to an increase in alter-native oviposition sites for mosquitoes and a possible dilution effect to the infusion water after the rain.

A large abundance of the mosquitoes captured were not Ae. aegypti and Ae. albop-ictus, which were the main target species of AGOs. The capture rates of Ae. aegypti and Ae. albopictus were quite low and not signifi-cantly different from each other. A majority of the mosquitoes captured were actually Culex (species not determined), Psorophora ferox, Ae. taeniorhynchus, and “other mosqui-toes” some of which were unidentifiable. In addition to an abundance of non-target

mosquitoes captured by the AGOs, on some trapping days an excess of over 200 Lucilia sericata were found in the AGOs. This was noticed in two different treatments, the al-falfa and orchard grass treatments. This is potentially due to the rotten fish smell ex-uded by the infusion water in the traps. Lu-cilia sericata favor rotting flesh as a breeding site (Liu et al. 2016), therefore, the odorants coming from AGO infusions may function as attractants for this livestock pest species. Other non-target species captured by this trap ranged across multiple families and or-ders of organisms: Anolis carolinensis, Anolis sagrei, Lucilia sericata, Camponotus floridanus, Dyscinetus morator, Caenurgina erechtea, Cam-paea perlata, as well as various Chironomidae species.

The main goal of this project was to re-duce the biological impact of AGOs on non-target organisms. We can surmise from this study that infusion water substrate types were not one of the main attractants of Aedes mos-quitoes to the AGOs in this study. However, the infusion water types were more attractive to non-target organisms. Although the Aedes mosquito capture rate was very low, AGOs have the potential to be modified as passive pest control traps. There are a vast array of

Figure 3. Effect of infusion water fermentation time on non-target collections. The x-axis represents each week of the study. The primary y-axis (left side) represents non-target organism abundance while the secondary y-axis (right side) represents rainfall in centimeters. The weekly rainfall is depicted as an area graph in the background in transparent grey. Each bar pattern represents a different infusion water type as shown in the legend above. Error bars represent standard error (SE).

Aver

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Mullin et al.: Impact of infusion water on non-target collection 27

agricultural and medical pest species that af-fect people. These species include Spodoptera frugiperda (moth), Anoplophora glabripennis (ant), Lucilia sericata (fly), and many others. Continued work with AGOs may facilitate a change in function of these traps to target other major medical and agricultural nui-sance pests. Along those same lines, contin-ued assessment of AGOs might divulge new practices to prevent any impact they might have on beneficial non-target organisms.

REFERENCES CITED

Barrea R, Amador M, Acevedo V, Hemme RR, Felix G. 2014. Sustained, area-wide control of Aedes aegypti using CDC autocidal gravid ovitraps. Am J Trop Med Hyg, 91: 1269-1276.

Barrera R, Amador M, Clark GG 2006. Ecological Fac-tors Influencing Aedes aegypti (Diptera: Culicidae) Productivity in Artificial Containers in Salinas, Puer-to Rico. Journal of Medical Entomology, 43: 484-492.

Delatte H, Dehecq JS, Thiria J, Domerg C, Paupy C, Fontenille D. 2008. Geographic Distribution and Developmental Sites of Aedes albopictus (Diptera: Culicidae) During a Chikungunya Epidemic Event. Vector-Borne and Zoonotic Diseases, 8: 25-34.

Hawley WA. 1988. The biology of Aedes albopictus. J Am Mosq Control Assoc Suppl, 1, 1-39.

World Health Organization. (2012). Global Strategy for Dengue Prevention and Control, 2012–2020 WHO, Ge-neva 2012.

Liu W, Longnecker M, Tarone A, Tomberlin J. 2016. Re-sponses of Lucilia sericata (Diptera: Calliphoridae) to com-pounds from microbial decomposition of larval resources (Vol. 115).

Mackay AJ, Amador M, Barrera R. 2013. An improved autocidal gravid ovitrap for the control and surveil-lance of Aedes aegypti. Parasites & Vectors, 6: 225.

Montgomery BL, Shivas MA, Hall-Mendelin S, Edwards J, Hamilton NA, Jansen CC, et al. 2017. Rapid Sur-veillance for Vector Presence (RSVP): Development of a novel system for detecting Aedes aegypti and Aedes albopictus. PLOS Neglected Tropical Diseases, 11: e0005505.

Ponnusamy L, Xu N, Boroczky K, Wesson DM, Abu Ayyash L, Schal C, et al. 2010. Oviposition responses of the mosquitoes Aedes aegypti and Aedes albopictus to experimental plant infusions in laboratory bioas-says. J Chem Ecol, 36: 709-719.

Reiter P, Amador MA, Colon N. 1991. Enhancement of the CDC ovitrap with hay infusions for daily moni-toring of Aedes aegypti populations. J Am Mosq Control Assoc, 7: 52-55.

Trexler JD, Apperson CS, Schal C. 1998. Laboratory and field evaluations of oviposition responses of Aedes al-bopictus and Aedes triseriatus (Diptera: Culicidae) to oak leaf infusions. J Med Entomol, 35: 967-976.

Wilke ABB, Vasquez C, Mauriello PJ, Beier JC. 2018. Ornamental bromeliads of Miami-Dade County, Florida are important breeding sites for Aedes aegypti (Diptera: Culicidae). Parasit Vectors, 11: 283.

Wong J, Stoddard ST, Astete H, Morrison AC, Scott TW. 2011. Oviposition site selection by the dengue vec-tor Aedes aegypti and its implications for dengue con-trol. PLoS Negl Trop Dis, 5: e1015.

28

Field evaluations oF attractive toxic sugar bait station and vegetation sPraY aPPlications For

control oF aedes aegYPti in KeY largo, Florida

Diana P. naranjo1,2, Lin Zhu1, KristoPher L. arheart1, DougLas FuLLer3, gunter C. MuLLer4, rui-De Xue5, Whitney a. QuaLLs1,5

1Department of Public health sciences, university of Miami Miller school of Medicine, Miami, FL 33136, usa

2Vysnova Partners inc, Washington, DC 20036, usa

3Department of geography and regional studies, university of Miami, Coral gables, FL 33124, usa

4Malaria research and training Center, Faculty of Medicine, Pharmacy and odontostomatology, university of sciences, techniques and technology of Bamako, Bamako, Mali, BP 1805, Bamako, Mali

5anastasia Mosquito Control District, saint augustine, FL 32080, usa

guest editor: amy junnila

ABSTRACT

Aedes aegypti and Aedes albopictus, vectors of many arboviruses including Zika, dengue, and chikungunya, are dif-ficult to control with traditional methods. We tested two novel approaches utilizing attractive toxic sugar baits (ATSB) against Ae. aegypti in the upper Florida Keys. Residential sites on the island of Key Largo were systematically selected using Google maps. Sites received either bait stations or vegetation spray application with ATSB. An untreated control site was selected to monitor mosquito populations. Adult and egg counts were monitored through baited Biogents-Sentinel and oviposition traps. The treatment evaluation lasted 28 days following a 14-day pre-treatment evaluation. Treatment efficacy was evaluated using regression models to estimate the percent reduction of mosquitoes over time. Post-treatment, Ae. aegypti mosquito populations were reduced by 81% and 74% at days 7 and 28 (p<0.05) at the bait station site, while mosquito populations at the spray treatment site for the same period (7 to 28 days) were reduced by 66% and 82% (p<0.05), respectively. Treatment and time had no significant effect on the proportion of eggs collected after the application of the ATSB treatments. This is the first residential field trial against the Zika vector, Ae. aegypti, in South Florida that demonstrated successful reduction of female and males using both ATSB stations and vegetation spray treatments. The findings suggest that 1) ATSB stations and vegetation spray applications can reduce populations of Ae. aegypti in residential and semi-tropical areas at least up to 28 days and 2) Ae. aegypti female mosquitoes in South Florida feed on sugar, and their sugar-feeding behavior can be exploited to enhance control strategies.

Key Words: sugar-feeding, integrated vector management, urban vectors, Aedes aegypti

INTRODUCTION

Aedes aegypti (Linnaeus) (Skuse) serves as primary vector of Zika (ZIKV), dengue, and chikungunya (Ioos et al. 2014). Vector control has been the only resource available to protect the human population against Ae. aegypti vectored arboviruses. However, vec-tor control of this species has been difficult

(Barreto et al. 2011, Gubler 2011, Naranjo et al. 2014) due partly to its survival mechanism and high adaptability to the environment. Aedes aegypti eggs survive from 2 to 12 months (Faull and Williams 2015), even at tempera-tures of 10°C (Waldock et al. 2013). Larval development occurs in many different habi-tats, from man-made to natural, through a skip oviposition pattern for dispersal (Reiter

Narangjo et al.: Attractive toxic sugar bait against Aedes aegypti 29

2007). For instance, both water bottle caps and tree holes, are suitable for Aedes aegypti development in residential areas if filled with water from 3 to 5 days (Reiter 2007). In addi-tion, focal control with insecticides is costly, may result in community opposition, and a loss of control due to insecticide resistance. Source reduction is rarely effective as it re-lies on high levels of community participa-tion (Reiter 2007, Troyo et al. 2008, Unlu et al. 2013). Therefore, when the environment favors Aedes-vector populations with suitable climate, home-to-home control becomes fu-tile (Reiter 2007,Troyo et al. 2008).

Attractive toxic sugar bait (ATSB) is a novel method to deliver insecticides to mos-quitoes. Even though blood-feeding studies argue that sugar-feeding is unimportant for female Ae. aegypti (Scott et al. 2000, Edman et al. 1992, Harrington et al. 2001), there is growing evidence that sugar enhances the survivorship and fitness of female Ae. aegypti mosquitoes (Gary and Foster 2006, Qualls et al. 2016). ATSB can enhance integrated vector management approaches by targeting the sugar-feeding behavior of mosquitoes. Laboratory and field trials have demonstrat-ed that Aedes albopictus Skuse can be lured to an attractive toxic sugar bait infused with oral toxins such as boric acid (Naranjo et al. 2013, Junnila et al. 2015).

ATSB can be adapted to different set-tings both indoors and outdoors (Revay et al. 2014, Qualls et al. 2015), which is important due to the behavioral adaptability and peri-domestic preferences of Ae. aegypti and Ae. albopictus (Bonizzoni et al. 2013, Rodrigues et al. 2015). In field trials, ATSB as bait sta-tions and spray treatments decimated Aedes, Culex, and Anopheles species from arid areas in Mali (Qualls et al. 2015), and sub-tropical environments in Florida (Revay et al. 2014, Qualls et al. 2014). Individual-based models demonstrated the effectiveness of ATSB sta-tions in controlling An. gambiae Giles mos-quitoes and malaria parasite transmission in typical African village settings (Zhu et al. 2015). Three field experiments of vegeta-tion spray application of ATSB, with boric acid and eugenol, significantly reduced Ae. albopictus and other vector-populations in

residential areas of St. Augustine, FL (Nara-njo et al. 2013, Revay et al. 2014, Qualls et al. 2014). The reduction in Ae. albopictus with ATSB control persisted under warm and rainy conditions for at least 21 days in com-parison to control sites (Naranjo et al. 2013).

Implementation of ATSB methods in key regions of the U.S. that serve as ports of en-try and export for Aedes may reduce arbovi-rus risk. Sub-tropical and tropical Southern U.S. counties sustain Aedes populations year round (Monaghan et al. 2016). Moreover, these areas also serve as destinations for trav-elers worldwide. The confluence of infected travelers from Zika-endemic regions, suit-able climate, and the presence of infected mosquito-vectors, resulted in local transmis-sion in both Miami and Texas. At of the end of 2016, there were 216 locally acquired Zika cases in Florida and 6 in Texas (Centers for Disease Control and Prevention 2016 a, b). These recent outbreaks raise concerns about how to sustainably control Zika vectors in the tropical environments of the U.S. Hence, our motivation to test approaches utilizing ATSB to control Ae. aegypti in residential ar-eas of South Florida.


This study was conducted in residential areas of the island of Key Largo, FL locat-ed in the upper Florida Keys (25.086515 N latitude, -80.447281 W longitude). The total surface area of the city is 53 km2. Key Largo temperatures range from 24 to 32° C from May through November; rains and high humidity are a common daily occurrence during the wet season from May-November. From December through April, tempera-tures fluctuate between 18 and 29° C, and the atmospheric humidity decreases. Adult mosquitoes tend to be more active from May through September.

The study sites were systematically se-lected on the island of Key Largo, FL us-ing Google maps. Eligible study sites were selected using criteria based on the built environment. Eligibility was met if the resi-dential block of houses was surrounded by 3 or 4 streets, and by other residential blocks.


Streets and roads have shown to provide an ecological barrier against contamination across sites, meaning that the vector pres-ence is significantly contained within city blocks (Barbu et al. 2013, Coffin 2007). The same barrier applies to Ae. aegypti, as Hemme and colleagues detailed in the study of the effect of highways and dispersal patterns of this species (Hemme et al. 2010).

The selection criteria for study sites re-duced the chance that mosquitoes would disperse between blocks. The distance be-tween any two study sites was > 500 m. Nine eligible study sites were initially identified and after the first field visit, we further nar-rowed it to seven sites. Of these, one site was excluded per resident request. The fi-nal sample size was six study sites. One site was used as the control, two sites received bait station treatment, and three sites re-ceived spray treatment around the perim-eter vegetation. The perimeter and area of all study sites was measured, and a 14-day pretreatment population abundance evaluation was conducted. This study was

conducted from mid-July through Septem-ber of 2016.

All non-flowering vegetation in the pe-rimeter of the three sites selected for the vegetation spray were treated with C/S Pi-lot ATSB Mosquito Bait Concentrate (Wes-tham Co.; Dallas, TX; active ingredients 0.4% sesame oil and 0.2% cinnamon oil) using a 4-gallon pump-up sprayer (West-ward Parts Services Ltd.). Per label instruc-tions, a 32-ounce pouch was mixed with 64 ounces of water and applied as a barrier treatment to the non-flowering vegetation (Terminix All Clear® C/S Pilot Mosquito Bait Concentrate; Dallas, Texas, Westham Co. 2016). Given that the mean flight height of Aedes species is approximately 1 m, vegetation was sprayed no higher than 1 m above the ground (Bidlingmayer et al. 1981, Bellini et al. 1997). Both the top and bottom surfaces of the leaves were sprayed to protect the active ingredient from rain events.

Bait stations used for this study were 2nd generation prototypes developed by West-

Figure 1. Google earth images displaying a sample configuration of the bait station treatment with attractive toxic sugar baits (ATSB) in Key Largo, FL.


ham Co. that measured 28 cm x 22 cm. The ATSB Bait Station (Westham Ltd.; Tel Aviv, Israel) was fabricated from a white, layered, laminated sheet (polyethylene and poly-ethylene terephthalate). The bait stations were fused to a membrane made of styrene-ethylene butylene styrene and specially de-signed so that mosquitoes could sugar-feed. Several crossing seams created 16 cells which were filled with 4 ml of the ATSB containing 0.04% sesame oil and 0.2% cinnamon oil as the active ingredients, microencapsulated with 1.5 beta-cyclodextrin, all parts in weight volume percent. A thick polyethylene pane provided support for the bait stations, which were attached to wood stake and inserted in the ground to be about 25 centimeters off the ground. Six bait stations were placed ev-ery 10 m around one of the Biogent (BG) Sentinel traps (Biogents AG, Regensburg, Germany) following a recommended config-uration in consideration of both flight range of Aedes mosquitoes and past ATSB configu-ration studies (Muller et al. 2011, Qualls et al. 2016, Zhu et al. 2015) (Figure 1).

For all the evaluations, mosquito popu-lations were monitored using entomologi-cal methods. These included the use of CO2

and lure-baited BG Sentinel traps and non-baited oviposition traps (ovi-traps). Two BG Sentinel traps were set in backyards and in vegetative areas at each site for 24 h every 7 days. The battery-operated traps were set between 7 am and 9 am on Mondays and collected after a 24-hour period. The nets were retrieved from the traps and trapped mosquitoes were transferred to a cooler for transportation. Mosquitoes were then moved to the lab freezer for later identifica-tion to species and gender.

Ovi-traps were placed out in the field and left at the same locations for the duration of the project. There were three ovi-traps at each study site, which were placed separate from each other and from the BG-Sentinel traps by at least 40 m. The ovi-traps were emptied and then filled with distilled water at each visit every 7 days. Oviposition papers were placed at the first visit and removed, and replaced at each subsequent visit. The removed oviposition papers were placed in

individual plastic bags and transferred to the laboratory for later inspection and egg counting under the microscope.

Excluding planning, the field trial time-line included a 7-day site location selection period, 14 days of pre-treatment evaluation, and 28 days post-treatment monitoring. At post-treatment days 10 to 14, cumulative rainfall reached 5 cm. Additionally, at 10-days post-treatment, 20 m of treated vegeta-tion at one study site were removed by land-scape workers.

Consent was obtained from the Florida Keys Mosquito Control District for this ento-mological evaluation. To inform the commu-nity, we prepared flyers with the description of the trial prior to treatment applications and included our contact information at the University of Miami.

Collections of Ae. aegypti and other mos-quito species by trap (gender and species) and eggs were recorded. A generalized lin-ear model (GLM) and a negative binomial distribution was used for the analysis. The GLM helped estimate the means at each treatment site and day. Means served as the baseline to calculate percent changes in the number of eggs and adult Ae. aegypti over the three-week treatment evaluation period. The means were compared by time and treatment application to the control site. The main analysis was conducted with female Ae. aegypti counts, however, we con-ducted secondary analyses with male and females together, and males alone to check for the consistency of results. To assess ran-dom error in the estimates, an alpha of 0.05 and 95% confidence interval was used for the analyses. The data was analyzed using descriptive analysis, and proc glimmix for the GLM model and planned comparisons using the percent change as our outcome. Software use was SAS Statistical Software v9.3 (SAS Institute Inc. Cary, NC).

RESULTS AND DISCUSSION

During the study, there were 924 female and 289 male Ae. aegypti collected, in the BG Sentinel traps. The total catches of Ae. albop-ictus were 19 females with no males collected


during the study period. In addition, 1,053 fe-male Aedes taeniorhynchus (Wiedemann) were collected. There were 1,155 Aedes eggs collect-ed in 21 ovitraps throughout the evaluation.

Overall, the control populations of fe-male Ae. aegypti were relatively stable yield-ing pre-treatment means of 34 ± 13.3 SE and post-treatment means of 21.25 ± 8.6 SE. There were no significant differences in the control collections before and after the ATS-Bs evaluation (p>0.05). After the ATSB bait station and vegetation spray applications populations declined significantly com-pared to pre-treatment populations (Figure 2). The pre-treatment mean of 19.5 ± 5.9 SE Ae. aegypti females per trap decreased to 2 ± 0.9 SE at the bait station application site with an overall 90% reduction. At the vegeta-tion spray application site, the pre-treatment mean decreased from 26.9 ± 6.9 SE to 7.3 ± 1.9 SE resulting in a 72% reduction in Ae. ae-gypti female populations. When comparing pre-treatment collections at the bait station

application site to post-treatment collections at days 7, 14, 21, and 28 (days 21, 28, 35, 42 in Fig. 2), the percent reduction in Ae. ae-gypti populations was 81%, 96%, 89%, and 74%, respectively. All these changes were sta-tistically significant (p<0.001). When com-paring the vegetation spray application site pre-treatment collections to post-treatment collections, the percent reductions at day 7, 14, 21, and 28 (days 21, 28, 35, 42 in Fig. 2) were 66% (p=0.003), 44% (p=0.09), 91% (<0.001), and 82% (<0.001), respectively.

Area, perimeter, or site characteristics did not confound the relationship of treatment and time on the total mosquito population and therefore were excluded from the analy-sis. The results from the male counts tested alone, and the female and male counts test-ed together were consisted with female Ae. aegypti results. Our analysis accounted for missing data. We missed 5.3% (3/56) obser-vations of adult catches and 5.6% (7/126) of oviposition papers.

Figure 2. Unadjusted and adjusted means for pre-treatment of female Aedes aegypti mosquitoes at the vegeta-tion spray and bait station applications, and the Control site at pre-treatment day 14 and post-treatment days 21-42


Overall, there were significantly fewer Ae. aegypti mosquitoes after the applica-tion of both ATSB bait station and vegeta-tion spray methods in comparison to pre-treatment numbers. The female mosquito data, and the combined male and female data demonstrated a significant decrease in mosquitoes at the treatment sites. Signifi-cant population reductions by both treat-ments persisted throughout the study, up to 28 days post-treatment. It is consistent with results from previous field trials which demonstrate that ATSB applications result-ed in reductions that lasted for 21 days in tropical settings (Naranjo et al. 2013, Xue et al. 2011). Previous caged experiments in tropical environments comparably showed that boric acid sugar baits applied to plant foliage resulted in estimates of 80 to 100% mortality in Ae. albopictus populations within 48 h (Xue et al. 2006). Additionally, in drier environments in Israel, Ae. aegypti popula-tions collapsed 4-days post ATSB treatment and dropped steadily for 27 days (Junnila et al. 2015).

During the post-treatment time, the sprayed vegetation was partially removed at one site by landscapers for a new home construction. Removal of the treated veg-etation could explain why the population reduction was not significant at day 14 post-treatment at the vegetation spray treatment sites. Yet, even after intense rain events, the spray treatment on vegetation resulted in significant overall control of adult Ae. aegypti populations. Despite field challenges, such as executing optimum ATSB bait station configuration, the bait station treatment successfully controlled Ae. aegypti popula-

tions in two residential sites. Only six bait stations were needed to show a reduction in the number of mosquitoes as measured by BG-Sentinel traps at the application sites compared to the pre-treatment controls.

The effect of treatment did not have a significant impact (p=0.103) on the egg population. The interaction between treat-ment and control site was also not significant (p=0.660) (Table 1). Although, the propor-tion of eggs, measured through ovi-traps, decreased only immediately after treatment at day 28 (day 7 post-treatment; p < 0.05). Our findings are similar to other trials where multiple comparisons of different trapping methods in China showed Aedes adult moni-toring with BG-Sentinel traps was more rep-resentative of measuring Aedes populations than ovi-traps (Li et al. 2016).

Our study suggests that female Ae. aegypti mosquitoes will feed on sugar in the field, which is contrary to the common belief that they feed only on blood if given the option (Scott et al. 2000). In Thailand, mark and re-capture trials showed that released and wild females fed unfrequently on natural sugar sources (Edman et al. 1992). Such results are challenged by the speed in which sug-ar is processed in the mosquito gut and by not having the same collection method for males to perform fair comparisons (Edman et al. 1992). Diptera, Culicidae. Qualls et al. (2016) demonstrated that in urban environ-ments in Ecuador, more Ae. aegypti females than males sugar fed on marked natural sugar sources from a distance of up to 60 m away from the marked sugar sources. Results of our study provide further evidence that female Ae. aegypti feed on sugar in the field,

Table 1. Egg collections means and standard errors (SE) per treatment time at the Control and ATSB bait station and ATSB vegetation spray treatment sites.

Day Control (SE) Bait Station (SE) Vegetation Spray (SE)

Pre-treatment 7 2.7 (2.4) 4.6 (3.1) 4.0 (2.3)14 2.3 (2.1) 3.5 (2.6) 4.2 (2.1)21 24.3(19.9) 1.67 (1.1) 7.9 (4.0)

Post-treatment 28 22.7 (18.6) 4.2 (2.8) 7.0 (3.6)35 39.0 (31.8) 16.5 (9.6) 15.4 (7.3)42 40.7 (33.1) 10.8 (6.3) 18.0 (8.5)

*There were 7 missing observations of egg counts in oviposition traps from a total of 126 collections.


that sugar-feeding behavior can be targeted in integrated vector management programs of Aedes. ATSB presented in the field as ei-ther a bait station or a vegetation spray treat-ment can be used to reduce the numbers of these mosquitoes.

Regarding limitations, this study was not a randomized control trial, therefore it may be prone to selection bias. To minimize bias, the sites were systematically selected based on the rule that a block of houses had to be surrounded by three to four streets. This sampling approach reduced the number of eligible sites. The active ingredients and ap-plication method of ATSB have been further modified to withstand rain events and can contain a variety of environmentally friendly active ingredients as oral toxins (Muller and Schlein 2011). However, additional testing is needed to achieve optimal configurations and prototypes in tropical settings. The bait station membrane lost integrity in the envi-ronmental conditions during the field study and further research is needed to optimize the bait membrane.

This is the first field residential trial of ATSB against the important Zika vector, Ae. aegypti in South Florida that demon-strates successful population reductions us-ing both ATSB bait stations and vegetation spray treatments. The island of Key Largo provides highly suitable conditions for sur-vival of Ae. aegypti and our field trial sug-gests that if ATSB is employed routinely, it will limit the survival of this species. ATSB continued to reduce Ae. aegypti populations under rainy and tropical conditions. Reduc-tion in the population of vector mosquitoes in residential areas also reduces vector-human contact. This is an important con-sideration regarding Zika transmission in residential areas in South Florida and other tropical regions.

ACKNOWLEDGEMENTS

We would like to acknowledge the com-munity of Key Largo, FL and the Florida Keys Mosquito Control District for their support in conducting this research study. This research was conducted under a grant

66664-3616 mosquito research funding number 2016-03, from Westham Co. (Dal-las, Texas) to the University of Miami Miller School of Medicine, Department of Public Health Sciences.

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36

EARTH FILL INCREASES EFFICACY AND LONGEVITY OF λ-CYHALOTHRIN RESIDUAL INSECTCIDE TREATMENT OF

HESCO® BLAST WALL GEOTEXTILE

SETH C. BRITCH1,2*, JAMES E. CILEK1,3†, ERICA J. LINDROTH3, ROBERT L. ALDRIDGE2, FRANCES V. GOLDEN2, JOSHUA R. WESTON3, JASON D. FAJARDO3, ALEC G. RICHARDSON3, JESSIKA S. BLERSCH4,

AND KENNETH J. LINTHICUM2

1Co-senior authors of this work

2US Department of Agriculture, Agricultural Research Service Center for Medical, Agricultural, and Veterinary Entomology, 1600 SW 23rd Drive,

Gainesville, FL 32608

3Navy Entomology Center of Excellence, Box 43, 937 Child Street, Jacksonville, FL 32212

4Camp Blanding Joint Training Center, Florida Army National Guard, 5629 State Road 16W, Starke, FL 32091

Subject Editor: Rui-De Xue

*Corresponding author: Seth C. Britch USDA-ARS-CMAVE, 1600 SW 23rd Dr., Gainesville, FL 32608

[email protected]

ABSTRACT

The prevention of vector-borne disease to protect the health and readiness of United States forces in the field continues to be a high priority for the US Department of Defense. Previous studies have demonstrated that the risk of human contact with disease-vector mosquitoes and other biting flies can be reduced by apply-ing an insecticide to perimeters of military materials such as camouflage netting or HESCO blast protection wall geotextile already in place around troops in the field. In this study we investigated whether residual pes-ticide efficacy will persist in the presence of earth fill that is required for operational use of HESCOs, using a warm temperate field site in north Florida. Results from laboratory bioassays measuring mosquito mortality and field collections of natural mosquito populations indicated superior efficacy and greater longevity of pesticide treated geotextile exposed to soil fill. These findings not only support immediate implementation of this technique in US military field scenarios, but also provide evidence that HESCO technology currently used in natural disaster flood control could be leveraged to protect civilian personnel from emerging floodwater mosquitoes.

Key Words: Military operational entomology, passive control, deployment, barrier treatment, flood control, natu-ral disaster

†The views expressed in this article are those of the authors and do not necessarily reflect the official policy or position of the Department of the Navy, Department of Defense, nor the U. S. Government. All authors are employees of the U.S. Government. This work was prepared as part of their official duties. Title 17, U.S.C., §105 provides that copyright protection under this title is not available for any work of the U.S. Government. Title 17, U.S.C., §101 defines a U.S. Government work as a work prepared by a military Service member or employee of the U.S. Government as part of that person’s official duties.

Britch et al.: Earth fill enhances residual treatment 37

INTRODUCTION

Arthropod-borne diseases such as ma-laria or leishmaniasis transmitted to humans from the bites of infected mosquitoes or sand flies, respectively, continue to pose sig-nificant threat to United States (US) military personnel worldwide (Kitchen et al. 2009; Stoops et al. 2013; Garcia et al. 2017). Lack-ing effective or approved vaccines for these and other high-risk arthropod-borne patho-gens such as Zika, chikungunya, or dengue viruses the best strategy for reducing disease risk is to reduce contact between humans and arthropod vectors (Scott and Morri-son 2004; Eisen et al. 2009; World Health Organization 2014). Strategies designed to reduce contact with disease vectors may also improve morale and reduce impact of nui-sance populations of mosquitoes and filth flies on US military missions.

One method to reduce risk of human contact with infected mosquitoes or sand flies is to create a protective barrier around personnel in the field by applying residual insecticides to existing vegetation or perim-eter structures (Frances 2007; Xue 2008; Britch et al. 2010, 2011, 2018). A common perimeter structure used worldwide by the US military is the HESCO MIL® blast wall system (Szabó et al. 2011; HESCO Bastion, Inc. 2019), that consists of a welded steel cage lined with a durable nonwoven poly-propylene geotextile (Müller and Saathoff 2015) packed with earth fill removed from the surrounding terrain (Figure 1). Such pe-rimeters can be exploited as targets for con-trol because mosquitoes or sand flies have been observed to rest there (Britch et al. 2018) and HESCO walls typically surround outdoor locations where personnel are pres-ent in US military expeditionary installa-

Figure 1. A HESCO perimeter at a US military outpost in southern Iraq, 2009. The HESCO wall is seen on the left and in the far background. A narrow walkway separates this structure from tents and other living areas to the right.


tions. Furthermore, preliminary testing of resting surface affinity of laboratory colonies of disease vector Phlebotomus papatasi sand flies revealed that they preferentially rest on HESCO geotextile, compared to other com-mon US military field materials such as cam-ouflage netting, plywood, or concrete (M. L. Aubuchon, unpublished data).

A recent study (Britch et al. 2018) dem-onstrated that HESCO geotextile treated with λ-cyhalothrin may be effective against mosquitoes and sand flies for up to 45 days during exposure to warm temperate condi-tions in the field. Additional data from a related field study showed that HESCO geo-textile treated with λ-cyhalothrin substantial-ly reduced host-seeking natural populations of Phlebotomus spp. sand flies, compared with untreated HESCO geotextile in a dry hot environment in western Kenya (SCB, KJL, unpublished data). Although the residual efficacy of this treatment on HESCO geotex-tile material has been demonstrated against mosquitoes and sand flies in the laboratory and field, those prior studies did not inves-tigate efficacy of λ-cyhalothrin applied to HESCO packed with earth fill as they would be in a US military field scenario (Britch et al. 2018). It is possible that the presence of soil could alter moisture content of the geo-textile or facilitate transfer of microbes or dissolved soil chemistry constituents into the geotextile fabric, potentially affecting (posi-tively or negatively) the pesticide itself or the capacity of the material to retain an effective residual treatment against mosquitoes.

Another prominent scenario where pe-rimeters of HESCO MIL cells are used is in natural disaster response to create flood-resistant berms. Modular units of these cells were used in 2005 to successfully reinforce levees around New Orleans in the few days between Hurricane Katrina and Hurricane Rita (Gibson 2006; US Army Corps of Engi-neers 2006). Following an extreme weather event such as a hurricane, these berms are often placed adjacent to flood prone areas of potential habitat for immature floodwater mosquitoes. If these structures were treated with insecticides they could provide a first line of residual protection against newly

emerged adult mosquitoes as they leave their immature habitat to seek their first blood meals. A residual insecticide treatment on earth filled HESCO flood control berms would need to remain effective despite per-sistent contact with moist soil.

In this study we investigated the relative performance of soil-filled HESCO MIL cells compared with unfilled units treated with λ-cyhalothrin for reducing natural popula-tions of disease vector mosquitoes in a warm temperate field site in north Florida. We hy-pothesized that the presence of earth fill in HESCO MIL cells could affect (positively or negatively) the longevity and/or residual ef-ficacy of the pesticide treatment, providing key data to guide future military operational or natural disaster response use.


We designed small simulated HESCO MIL perimeters to study the effects of soil fill on the capability of HESCO geotextile treated with a residual pesticide to reduce penetration of a finite protected space by natural populations of disease vector mos-quitoes. These perimeters were similar to but larger than the HESCO enclosures used by Britch et al. (2018). Each perimeter con-sisted of 22 woodland green geotextile 0.9 m (3 ft) x 0.9 m (3 ft) x 1.2 m (4 ft) HESCO MIL cells (HESCO Bastion, Ltd.; Charles-ton, SC) arranged in two tiers of 11 cells to enclose a protected 1.8 m (6 ft) x 1.8 m (6 ft) x 2.5 m (8 ft) interior space, with a gap near one corner for access (Figure 2). We constructed doors to cover the gap in each perimeter by breaking down extra HESCO MIL cells into sets of two 0.9 m x 1.2 m pan-els joined vertically along their short sides (Figure 2). All HESCO perimeters were se-cured and stabilized using steel hog rings linking the upper and lower tiers and steel cables threaded through the HESCO steel mesh connected to large steel pins screwed into the ground.

We identified 16 sites for the HESCO pe-rimeters evenly distributed across two study plots – 8 sites in a southern plot centered on 29.888190° N, 82.045735° W and 8 in a


northern plot centered on 29.890552° N, 82.045720° W – in freshwater swamp land east of a remote dirt road parallel to the western boundary of Camp Blanding Joint Training Center (CBJTC), Starke, Florida (Figure S1; Supplementary Materials avail-able at https://www.ars.usda.gov/cmave/mfru/HESCO). The centroids of the two plots were separated by approximately 500 m. A controlled burn had been conducted in the area several years earlier causing con-siderable emergent shrubs, forbs, and other natural vegetation from 0.5-3.0 m in height throughout and between the plots. Due to the presence of standing water throughout the study area, we situated the 16 perimeter

sites on available fragments of slightly elevat-ed, dry ground that separated all perimeter sites in each plot by approximately 9-12 m.

Prior to installation of the perimeters we conducted mosquito surveys that confirmed both study plots were situated in areas highly productive for natural populations of medi-cally important mosquito species. From 18-27 July 2016 (six 24 h collection periods) we suspended US Centers for Disease Control and Prevention (CDC) style suction traps (J. W. Hock Co., Gainesville, FL) baited with 1.3 kg dry ice and light 1.2 m from the ground at the 4 cardinal directions around the approx-imate boundary of each plot (L1-L8; Figure 3). We identified all collections to species

Figure 2. One of 16 two-tiered HESCO perimeters under construction at Camp Blanding Joint Training Center, Starke, FL (left hand image). Access door made of two stacked side panels from a broken-down HESCO MIL cell is visible at left front of this perimeter. Right hand image shows placement of a CDC light trap baited with dry ice to sample mosquito populations inside a soil filled HESCO perimeter.


from these 8 traps using the taxonomic keys of Darsie and Morris (2003).

We randomly assigned 4 of the 8 perim-eter sites in each plot to situate soil-filled perimeters, with the other 4 sites in each plot for unfilled perimeters. In each plot, we randomly designated 2 soil-filled and 2 un-filled perimeters for treatment with residual pesticide. Therefore, across the southern and northern plots combined we created 4 experimental classes of HESCO perimeters: soil-filled/untreated (N=4), soil-filled/resid-ual insecticide treated (N=4), unfilled/un-treated (N=4), and unfilled/residual treated (N=4) (Figure 3).

On 27 July 2016 we constructed 4 unfilled and untreated HESCO MIL perimeters, 2 in the southern plot H-1 and H-5 (Figure 3A); and 2 in the northern plot H-9 and H-16 (Fig-

ure 3B). On 6 August 2016 we constructed 4 soil-filled and untreated HESCO perimeters: 2 in the southern plot H-3 and H-8 and 2 in the northern plot H-12 and H-13. The soil had been trucked in and stored in heaps at the two plots for this experiment from rou-tine CBJTC Environmental Department for-estry operations over the previous several months and was similar to the ambient undif-ferentiated sand and clay soil (Dearstyne et al. 1991) observed at the site. We used a front-end loader to transfer soil into HESCO cells, first constructing and filling the lower 11-cell layer, then carefully constructing, filling, and anchoring the upper 11-cell layer in each soil-filled HESCO perimeter. We constructed the 4 unfilled treated HESCO MIL perimeters (H-2 and H-7 in the southern plot; H-11 and H-15 in the northern plot) on 11-12 August

Figure 3. Detailed views of (A) the southern and (B) the northern experimental plots. Outside CDC traps are indicated with small white triangles labeled L1-L4 (southern plot) and L5-L8 (northern plot). HESCO perimeters filled with soil and treated perimeters are indicated with labels. Insets in (A) show close up aerial views of an un-filled (inset-upper) and soil-filled (inset lower) HESCO perimeter.

BA


2016 and the 4 soil filled and treated HESCO MIL perimeters (H-4 and H-6 in the southern plot; H-10 and H-14 in the northern plot) on 18-19 August 2016, after the residual treat-ment was applied to individual HESCO MIL cells on 9 August 2016 (see below).

We conducted two separate trials with the HESCO MIL perimeters, each lasting several months. In Trial 1, we treated HES-CO MIL cells individually on 9 August 2016 with λ-cyhalothrin (Demand CS; Syngenta, Greensboro, NC) at the maximum label rate of 0.8 fl oz/1000 ft2 (23.7 ml/93 m2) in water using a backpack mist blower (SR-450; STIHL Inc., Virginia Beach, VA) before they were assembled into the 8 treated (4 soil-filled and 4 unfilled) perimeters as de-scribed above. The sides of each HESCO MIL cell that would be facing other cells in these perimeters were not treated. However, both the outward and inward facing sides of each cell were all treated and clearly marked before assembly. For Trial 2, we treated the inward and outward facing sides of the 8 finished HESCO MIL treated perimeters in place on 9 November 2016 with the same sprayer, formulation, and application rate.

We used 2 methods to measure the rela-tive effect of soil on the longevity and efficacy of the treated HESCO MIL geotextile. First, we placed a CDC trap baited with light and CO2 (dry ice) for 24 hr in the center of the protected space in all 16 HESCO perimeters to periodically survey the overnight penetra-tion of the perimeters by natural populations of medically important mosquitoes. Second, at the time of mosquito collection we cut ~15 cm x 15 cm samples of geotextile from the inside and outside facing surfaces of each pe-rimeter and exposed them to laboratory colo-ny Culex quinquefasciatus females in glass tube bioassays at the USDA Agricultural Research Service Center for Medical, Agricultural, and Veterinary Entomology (CMAVE) following the methods and colony description of Al-dridge et al. (2012, 2013) and Britch et al. (2009, 2010, 2011). We stored each cutting separately in labeled re-sealable bags to mini-mize opportunity for cross-contamination of samples. We collected the first geotextile cuttings on 18 August 2016 from all perim-

eters before soil fill was introduced. We set a minimum benchmark of 80% mortality in bioassays to determine residual efficacy of the pesticide treatment of HESCO perimeters (Britch et al. 2018).

We also deployed the 8 outside CDC traps (L1-L8; Figure 3) situated at the 4 cardinal di-rections around each of the two study plots in synchrony with the periodic CDC collections within the perimeters to verify presence of host seeking mosquitoes in the study area. We recorded local meteorology throughout the study from a permanent weather station ~2.4 km south of the southern plot at a separate research site in comparable habitat, supple-mented during periods of malfunction by Na-tional Climate Data Center Climate Data On-line (https://www.ncdc.noaa.gov/cdo-web/) from other weather stations within 20 km of the research site.

Statistical Analysis

Our hypotheses were that (a) insecticide-treated HESCO perimeters regardless of the presence of soil would have lower CDC light trap collection numbers and higher bioassay mortality than untreated perimeters, thus demonstrating protection of the interior space by the presence of the residual pes-ticide, and (b) treated HESCO perimeters with soil would show significantly different (larger or smaller) CDC light trap collec-tions or bioassay mortality for a significantly different (longer or shorter) duration than treated perimeters without soil.

To compensate for inevitable organic dif-ferences between no fill and soil filled HES-CO perimeters yet still be able to investigate comparative efficacy of these two classes, we calculated the percent reduction in collec-tions in treated HESCO perimeters, com-pared with untreated HESCO perimeters for the “no soil” and the “soil filled” classes separately using the formula:

mean number of mosquitoes

(HESCO untreated )


(HESCO treated )


(HESCO untreated )

–

x 100


Using this formula, we could then directly compare the normalized reductions for no soil and soil to determine the effect of soil on efficacy and longevity of the residual in-secticide treatment against natural mosquito populations. Initially, mean percent reduc-tion data from Trials 1 and 2 were subjected to goodness-of-fit tests using the Kolmogorov-Smirnov and Bartlett tests. Results of these tests showed that datasets conformed to non-normal and heteroscedastic behavior (even after data transformation). Hence, the non-parametric Kruskal-Wallis test was applied. Following the hypothesis test, an optimal post hoc multiple-comparison test was conducted for each of the factors and interactions to identify the specific pairwise combinations of levels of each factor and interaction contrib-uting to the overall variability. Post hoc tests included Tukey multiple-comparison (Tukey, 1949, 1953), Newman-Keuls multiple-range (Newman, 1939), Duncan multiple-range (Duncan, 1951, 1955), and Scheffe multiple-contrast tests (Scheffe, 1953, 1959). Statistical analyses were performed using Intel Visual Fortran Compiler XE 2013 (Intel Corpora-tion, Santa Clara, CA). Differences from these analyses were considered significant at P ≤ 0.05. Non-transformed means are pre-sented in tables and figures.

RESULTS

Light trap collections of natural mosquito popula-tions

Across all traps inside and outside HESCO perimeters we collected 17 species of mosqui-toes in Trial 1 (total 19,400 specimens; includ-ing the 6 pre-trial survey collections) and 19 species in Trial 2 (59,863 specimens) (Table S1). Overall mosquito abundance across the 8 CDC light traps placed outside of HESCO perimeters was significantly greater, compared with traps inside the HESCOs regardless of presence of soil (data not shown). Meteorolog-ical conditions were generally mild through-out both trials (Figure S2) with temperatures rarely exceeding the low 80s °F (27-29 °C) and very little precipitation. Humidity was variable-high, frequently exceeding 80 %RH in both trials.

Field percent reduction efficacy results are shown in Figs. 4 and 5 for Trials 1 and 2, respectively. The majority of Trial 1 fell dur-ing a period of consistently high mosquito biting pressure (i.e., mean outside CDC trap collections >100; Figure 4). Therefore, Trial 1 efficacy data based on relative reductions in field collections are potentially informa-tive from the time of treatment forward for several weeks, although bioassay data on cut-tings suggest that the treatment was weak from the outset (see HESCO fabric bioassays results below and Figure 6 ). Bioassay data from Trial 2 suggest a much stronger residu-al treatment from the outset (Figure 7). Un-fortunately, the majority of Trial 2 from the time of treatment (9 November 2016) for-ward occurred during winter months mak-ing it potentially difficult to resolve percent reduction levels against low natural popula-tion numbers (Figure 5) when the treatment was recent and at its strongest as shown by bioassays.

Using the summary data in Table S1 we initially restricted field efficacy analyses to species where at least ~1,000 specimens were collected in each trial, which generally indicated high biting pressure for that spe-cies early in the treatment period when re-sidual efficacy in bioassays was highest (Figs. 6 and 7). Past study (Britch et al. 2018) has shown that low biting pressure (i.e., low col-lection numbers) in the earlier sample peri-ods when the residual treatment is strongest makes efficacy analysis inconclusive.

For Trial 1, Aedes atlanticus Dyar and Knab, Anopheles crucians Wiedemann, Coquil-lettidia perturbans (Walker), Culex erraticus (Dyar and Knab), Culex nigripalpus Theo-bald, and Culex salinarius Coquillett fit this benchmark and were included in analyses. Collections for Culiseta melanura (Coquil-lett), though overall much lower than 1,000 individuals, were consistent across the peri-od of Trial 1 so were included also.

For Trial 2, Ae. atlanticus, An. crucians, Cq. perturbans, and Cx. erraticus exceeded 1,000 individuals as in Trial 1, but Cs. melanura and Psorophora columbiae (Dyar and Knab) were also high. However, only 3 of these 6 species, Ae. atlanticus, An. crucians, and Cx. erraticus,


had substantial collections in the first 100 days post-treatment and analysis was restrict-ed to these species for Trial 2. Also, though low overall, Culex restuans Theobald had high collection numbers in the first 100 days post-treatment and was included in the Trial 2 analysis.

In Trial 1, considering efficacy of the HESCO perimeters against all collected spe-cies combined, no soil and soil filled treated perimeters overwhelmingly showed reduc-tion in trap counts – represented by bars above the x-axis – compared with untreated perimeters up through day 57 post-treatment (Figure 4). Collections from days 16, 37, and 57 post treatment indicate that soil filled treated HESCO perimeters showed a greater (days 37 and 57) or approximately equivalent (day 16) reduction in trap counts compared with no soil treated perimeters. The day 23 collections showed inferior performance of soil filled treated perimeters compared with

no soil treated perimeters yet were still more effective than untreated soil filled perimeters. Finally, collections from days 30 and 71 post treatment show that populations were higher in the soil filled treated perimeters – repre-sented by bars below the x-axis – compared with untreated soil filled perimeters.

If we separate percent reduction efficacy by species (Figure S3B-H), however, a spec-trum of results emerges from Trial 1. For ex-ample, soil filled treated perimeters mostly outperformed no soil treated perimeters up to and including day 37 against Ae. atlanti-cus (Figure S3B), An. crucians (Figure S3C), and Cx. salinarius (Figure S3G). In contrast, efficacy was more varied for Cq. perturbans (Figure S3D), Cx. erraticus (Figure S3E), Cx. nigripalpus (Figure S3F), and Cs. melanura (Figure S3H) over the same time period. Also, the day 30 relative abundance of mos-quitoes in soil filled treated perimeters when considering all collected species (Figure 4)

Figure 4. Trial 1 CDC light trap collection data for all species collected across the sample period; data for focal species across the sample period re shown in Supplementary Materials Figure S3, available at https://www.ars.usda.gov/cmave/mfru/HESCO. The upper section of each graph shows the local biting pressure indicated by collec-tion means (with bars for standard errors of the mean, SEM) across the 8 outside CDC traps. The lower section shows the percent reduction in collections in treated HESCO perimeters compared to untreated ones, separated by whether soil was present. Bars below the zero line (negative values) signify that more mosquitoes were collected in treated perimeters – i.e., less control than untreated perimeters.


is actually driven by only three species, An. crucians, Cx. erraticus, and Cs. melanura which were to a greater or lesser extent asymmetri-cally more abundant in the soil filled treated perimeters. Conversely, on day 30 Cx. nigri-palpus were less abundant in the soil filled treated perimeters and asymmetrically abun-dant in the no soil treated perimeters.

In Trial 2, the consistent and relatively su-perior percent reduction efficacy of the soil filled treated HESCO perimeters compared with no soil treated perimeters is appar-ent when considering all collected species through day 164 post treatment (Figure 5). One exception is day 71 collections where traps in soil and no soil treated barriers col-lected more mosquitoes – i.e., bars below the x-axis – than either class of untreated barrier (Figure 5). Due to seasonally low populations early in the treatment period of Trial 2, only a few species (Figure S4B-E) can be exam-ined individually to determine their contri-bution to the pattern in Figure 5. Unlike Tri-al 1, Ae. atlanticus collections (Figure S4B) in Trial 2 were not uniformly lowest in the soil filled treated HESCO perimeters. However, except for the day 127 collections, soil filled

treated perimeters in Trial 2 performed best against An. crucians up to and including day 164 (Figure S4C). Although showing strong efficacy on day 127 against Cx. erraticus (Fig-ure S4D) and Cx. restuans (Figure S4E) the remaining periodic samples up to day 164 post treatment do not suggest that soil filled treated HESCO perimeters provided a more efficacious relative reduction compared with no soil treated HESCO.

Spring transitioned into summer be-tween the days 189 and 218 sample periods in Trial 2, which corresponds to the large jump in collection numbers shown in Figure 5. Although combined collections on day 218 suggest that no soil and soil filled treated perimeters were less efficacious – i.e., bars below the x-axis – than untreated perimeters (Figure 5), subsequent collections on days 253, 281, and 344 indicate high or equiva-lent relative efficacy of soil filled treated HESCO perimeters. However, these periods are also punctuated by asymmetrically low efficacy of the soil filled treated HESCO on days 309 and 372 (Figure 5), largely driven by numbers from the An. crucians collections (Figure S4C).

Figure 5. Trial 2 CDC light trap collection data for all species collected across the sample period; data for focal species across the sample period are shown in Supplementary Materials Figure S4, available at https://www.ars.usda.gov/cmave/mfru/HESCO. See Figure 4 legend for details.


HESCO fabric bioassays

Bioassay results are shown in Figs. 6 and 7 for Trials 1 and 2, respectively. In each figure, the first graph shows data from inside and outside geotextile samples combined and the second graph separates the inside and outside efficacy data. Curves for treated sam-ples are black and the control sample curves are blue and generally clustered near zero. Bioassay efficacy data for both trials show a general trend of decline in efficacy over time, yet also show variation, sometimes ex-

treme, between sample periods that indicate an apparent loss then restoration of efficacy. This phenomenon has been observed pre-viously in pesticide treated material studies (Britch et al. 2010, 2011, 2018) and is likely a result of uneven treatment and/or uneven weathering that becomes evident as samples are cut from sequentially adjacent sections of treated surface. The phenomenon may be exaggerated when the initial treatment itself is weak, as seen in Trial 1 where the highest recorded efficacy was only ~85% mortality (Figure 6B) and even then only for one sam-

Figure 6. Mean percent Culex quinquefasciatus mortality following 24 h exposure in laboratory bioassays to insec-ticide-treated (black lines) and untreated (blue lines) HESCO geotextile samples from Trial 1, separated by no soil (dashed lines) and soil present (solid lines). Graph (A) presents combined mortality data across geotextile samples from both inside and outside the perimeters; graph (B) separates data from samples collected inside (not bold) and outside (bold lines) the HESCO perimeters.

B

A


ple period (day 16). The Trial 1 treatment was carefully applied at maximum label rate but the pesticide batch was not fresh and it is possible that light rainfall that same day had diluted some of the treatment on the HESCO geotextile before it had fully dried. The Trial 2 treatment was applied with a fresh batch of pesticide and no meteorologi-cal challenges to the proper drying and fix-ing of the treatment to the geotextile fabric,

resulting in much less inter-sample variation in efficacy and overall much longer duration of the treatment (Figure 7).

DISCUSSION

In this study we filled a major gap in our understanding of the capabilities of residual treatments targeting disease vector insects applied to a prominent US military material

Figure 7. Mean percent Culex quinquefasciatus mortality following 24 h exposure in laboratory bioassays to HES-CO geotextile samples from Trial 2. See Fig. 6 legend for details. Graph (A) presents combined mortality data across geotextile samples from both inside and outside the perimeters; graph (B) separates data from samples col-lected inside (not bold) and outside (bold lines) the HESCO perimeters.

A

B


used pervasively worldwide. In prior work we established that residual pesticides such as λ-cyhalothrin on HESCO geotextile could substantially reduce mosquito and sand fly entry into protected perimeters, but these investigations were conducted in a non-op-erational configuration – i.e., the HESCO blast protection walls were not filled with soil. In the present study we mirrored earlier investigations but with the improved condi-tion that treated geotextile was exposed to soil fill. We sought to determine whether soil filled treated HESCO perimeters would still have lower CDC trap collection numbers and higher bioassay mortality than untreat-ed perimeters, and whether longevity of the treatment would be affected by the presence of soil. We found that for the majority of col-lected species across most post-treatment sample periods, when bioassays indicated the treatment was still active, treated HES-CO material exposed to soil fill was as good as or better than no soil treated HESCO at reducing entry of mosquitoes into the pro-tected area.

Two major patterns emerge from the bioassay portion of this experiment: (i) the superior efficacy (amplitude and duration) of treated geotextile exposed to soil fill and (ii) the superior efficacy of treated material on the inside of the perimeter. These major patterns are less obvious in Trial 1 (Figure 6) possibly because of the exaggerated noise associated with the poor initial treatment, but the patterns are highly visible across the entire bioassay data set from Trial 2 (Figure 7). In Trial 1, bioassays on treated material not exposed to soil were consistently less ef-ficacious (maximum just under 20% mortal-ity at day 16, Figure 6A) than treated mate-rial exposed to soil (maximum 70% at day 16). In Trial 2 within just over 1 month post treatment, treated HESCO geotextile not ex-posed to soil dropped from just under 90% mortality on day 2 to just over 15% mortality on day 34; whereas, treated material in the presence of soil remained above 50% mor-tality up to day 164 (Figure 7A)

In a previous study (Britch et al. 2018) we set an arbitrary minimum efficacy bench-mark of 80% mortality in bioassays for re-

sidual pesticide treatment of HESCO pe-rimeters. Bioassays on samples from Trial 2 revealed that inner surfaces of soil filled treated HESCO perimeters continued to provide at least 85% mortality up to day 189 post treatment (Figure 7B). Toxicity of out-side treated surfaces, however, substantially lost efficacious control within 2 months post treatment (Figure 7B). This pattern is also visible in the later (days 37-71) samples in Trial 1 where interior locations of both no soil and soil filled treated geotextile exceed-ed efficacy of the outside locations (Figure 6B). However, this pattern in Trial 1 is ob-scured early in the trial period by the high sample-to-sample variability in efficacy possi-bly due to the poor initial treatment (Figure 6B).

The periodic efficacy as shown by field collections in the soil filled treated perim-eters late in the Trial 2 sample period long after bioassays indicate zero efficacy intro-duces the possibility that the field collec-tions are not reliable indicators of efficacy, being perhaps more driven by spatial or mi-croclimate effects. However, the combina-tion of a high biting pressure and a waning or absent treatment will naturally lead to in-conclusive results. On the other hand, the trend in field collections from Trial 2 show-ing more consistent superior or equivalent efficacy of soil filled treated perimeters early in the sample period up to day 160 – despite lower field populations than Trial 1 early in the sample period – followed by a trend of less consistent efficacy later in the sample period (Figure 5), point to a rea-sonable match with the bioassay results that show a fall to zero efficacy between days 189 and 218 (Figure 7). Even with the poor ini-tial treatment in Trial 1, we see more con-sistent superior or equivalent efficacy of soil filled treated perimeters up to day 57 in field collections (Figure 4) which nearly co-incides with the loss of efficacy in bioassays between days 37 and 71 (Figure 6).

One key finding of this investigation is the heterogeneity of the residual treatment efficacy across mosquito species in the field environment. Implementation of residual treatment of HESCO geotextile should thus


be carefully tempered by surveillance of lo-cal populations so that the relative likely contribution of the residual treatment in the local integrated vector management (IVM) program is understood. Adjustments should be made in other parts of the IVM program to support increased vigilance and control of species less prone to control from the perimeter treatment such as Cx. erraticus or Cx. restuans as observed in Trial 2. In particular, prominent worldwide high-threat species such as Aedes aegypti and Aedes albopictus should be monitored closely for susceptibility to control with a residual bar-rier such as the one investigated herein.

Processes underlying the increased per-formance of the residual treatment when soil is present could include the buffering effect of soil moisture on temperature. The high specific heat capacity of water means that moistened material should take longer to heat up and longer to cool down than dry material. In addition, with soil present the material may have some level of mois-ture present for longer intervals which could make inundation with rainfall less of a shock to the chemistry of the residual formulation. Unfilled HESCO cells allow weathering to take place on both sides of the cloth, and there is increased mechani-cal action in unfilled HESCO because air currents can shake, fold, and abrade the fabric otherwise held still by the pressure of the soil fill. Although both treated and untreated soil-filled HESCO perimeters will have a warm, moist, and still-air microcli-mate in the interior protected space poten-tially attractive to resting mosquitoes, the treated soil filled HESCO also has a control measure: more mosquitoes may enter a soil filled perimeter, but may also be more likely to rest on the favorable, but toxic, surface. Additionally, we observed narrow (3-9 cm) vertical spaces between HESCO MIL cells in perimeters with no soil fill that allowed light and possibly CO2 from the CDC traps to project through the perimeters, possi-bly enhancing collections compared to soil filled HESCO perimeters that had no such gaps. The physical presence of the HESCO perimeters themselves seemed to act as a

barrier to mosquito trap entry when com-pared to perimeter traps. Other processes underlying the increased efficacy of treated HESCO when soil is present could be the chemistry of the soil itself which may en-hance the adhesive properties of the re-sidual formulation, thus compelling future studies to consider efficacy across a variety of soil type exposures.

These findings provide deployed US military personnel responsible for mosquito control specific and attainable methodology to enhance management of mosquito vec-tors of malaria, dengue, Zika, yellow fever, and chikungunya in endemic settings. In particular, the technique of residual treat-ment of soil filled HESCO will also provide immediate mitigation for units that have been unable to adequately reduce malaria vectors because of nearby but physically in-accessible habitat where continuous mosqui-to production may occur. Likewise in non-military scenarios, these findings provide emergency management personnel an ad-ditional tool for control of mosquito vectors and other biting flies after extreme weather events. HESCO barriers can be placed and treated prior to anticipated flood events to help protect residents from large numbers of mosquitoes that may emerge after hurri-canes and heavy rains. Reduction of natural populations of disease vector mosquitoes following HESCO treatments is certainly not expected to be absolute and will likely vary by species but should be considered one lay-er in a multi-faceted pest and vector manage-ment system. This study was conducted in a warm temperate region using one soil type with one residual pesticide treatment formu-lation and application technology targeting mosquitoes, but should be repeated in other key militarily relevant environments includ-ing hot arid, hot tropical, Mediterranean, and cool temperate regions with additional soil types with more combinations of for-mulations and spray equipment, and in the presence of other important disease vectors such as sand flies. Post natural disaster flood-ing presents risks to public health worldwide and should also be included in design of fu-ture studies on treated HESCO geotextile.


ACKNOWLEDGEMENTS

We thank Camp Blanding Joint Training Center (CBJTC) Environmental and DPW for permission to establish and access field sites and for lending soil; the 388th Engineer-ing Company (US Army Reserve) for provid-ing and operating earth moving equipment to fill HESCO MIL cell perimeters; and C. McDermott and E. Moore for expert assis-tance in the field and lab. For expert pro-duction of colony insect specimens we thank H. Brown, T. Carney, K. Kern, B. Smith, C. Swain, D. Kline, J. Urban, H. Furlong, J. Hogsette, R. TenBroeck, W. Delaney, D. Johnson, C. Taylor, C. Geden, and R. White at the USDA-ARS-CMAVE insectaries. This research was supported by the US Depart-ment of Agriculture (USDA)—Agricultural Research Service and with a US Department of Defense (DoD) Defense Health Program DHP 6.715_I_15_J9_1124 grant to J. Cilek. Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or en-dorsement by USDA, US Navy, DoD, or the Florida Army National Guard. Data in this study have been added to the Mobile Pesti-cide App operational entomology decision support system database (https://ars.usda.gov/saa/cmave/PesticideApp; Britch et al. 2014). The USDA is an equal opportunity provider and employer.

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51

EFFECT OF THE COMBINED NOZZLE ORIENTATION AND TRUCK SPEED ON EFFICACY OF ULTRA-LOW-VOLUME SPRAY AGAINST CAGED AEDES ALBOPICTUS IN URBAN

GAINESVILLE, FLORIDA†

YONGXING JIANG1, MUHAMMAD FAROOQ2, JAMES CILEK3, AND ALEC G. RICHARDSON3

1Department of Public Works, City of Gainesville Mosquito Control, Gainesville, FL 32609

2Anastasia Mosquito Control District, St. Augustine, FL 32092

3Navy Entomology Center of Excellence, Jacksonville, FL 32212

Subject Editor: Rui-De Xue

ABSTRACT

A field study was conducted to evaluate the combined effect of nozzle orientation and vehicle travel speed on droplet dispersion and mosquito mortality of an adulticide applied from a truck mounted ULV sprayer in the City of Gainesville, Florida. Three multi-block areas with dense, medium, and sparse vegetation were selected for the study. Aqua-Reslin® was applied in each area in the following treatment combinations: a) horizontal nozzle at 24 km/h travel speed, b) 45° upward orientation (standard) at 16 km/h, and c) 22.5° upward orientation at 24 km/h. Caged, three to five day old Aedes albopictus females were used in all evaluations. Spray deposition was de-termined at various locations inside each application area using Florida Latham Bonds droplet impingers. There was a significant difference in 24-h mortality among the 3 nozzle angle and speed treatment combinations, but not in the interaction between those combinations and application distance. The 22.5° nozzle combination resulted in the greatest mosquito mortality (88.3%) while the 45° combination resulted in the least mortality (63.1%). A significant difference in 24-h mortality among the 3 vegetation densities and application distances occurred with no significant interaction among these two parameters. The greatest Ae. albopictus mortality was recorded in the sparse (91.4%) and the lowest in the medium vegetation area (72.2%) at the maximum rate of 0.0015 lb./acre. Adulticide deposition was not significantly different among vegetation levels, but was significantly different among the distances and interactions of those parameters.

Key Words: Adulticide efficacy, Aqua-Reslin, London Fogger 18-20, permethrin, vegetation density

INTRODUCTION

Ground application of ultra-low-volume (ULV) adulticides has been the standard method to combat pestiferous and disease transmitting mosquitoes worldwide for more than 45 years (Bonds 2012). As a key compo-nent of Integrated Mosquito Management (IMM), ground application of ULV has been studied extensively, however, control effi-cacy has varied greatly. Mount (1998) has

discussed a number of factors that could af-fect the control efficacy of ground applied ULV adulticides including droplet size, me-teorology (e.g. wind speed and direction, temperature, relative humidity, atmospheric stability and turbulence), vegetation, and structural obstacles (such as homes, solid walls or board fences). Bonds (2012) fur-ther discussed the effectiveness of adulticide application timing and whether or not the aerosol plume actually contacted mosqui-

†Mention of a trademark, vendor, or proprietary product does not constitute a guarantee or warranty of the product by the US Navy and does not imply its approval to the exclusion of other products that may also be suit-able. The views expressed in this article are those of the author and do not necessarily reflect the official policy or position of the Department of the Navy, Department of Defense, nor the U.S. Government.


toes directly. However, no reviews or studies have discussed or tested the potential effect of nozzle discharge direction on the effec-tiveness of ULV ground applications in con-trolling adult mosquitoes. The current com-mon practice of ULV application in Florida, and elsewhere, is to orient the spray nozzle at an upward arc of 45° (Teske et al. 2015). To date, no published data are available to support the fact that this angle is the optimal one in terms of control efficacy.

Jiang and Farooq (2016) compared the efficacy of truck mounted ULV sprayer noz-zle discharge direction at 45° upward and 0° horizontal in urban Gainesville, Florida against caged Aedes aegypti (L.). Results in-dicated that a horizontally oriented nozzle outperformed a 45° upward nozzle in three out of four field trials. Recently, Farooq et al. (2017) reported from an open field study that the greatest mortality of caged adult Ae. aegypti occurred when the nozzle angle was positioned horizontally, followed by a 30° downward angle, while a 45° angle showed the least efficacy. The above two studies point out that nozzle discharge direction could have an actual impact on the efficacy of a ULV spray.

Mount (1998) believed that when an ef-fective insecticide dose and appropriate at-omization are maintained for a designated swath, dispersal speed (vehicle speed) is not a factor affecting efficacy. However, the most recent study reported by Farooq et al. (2018) showed that 32 km/h travel speed provided the best ULV spray dispersal as indicated by complete mortality of caged female Ae. aegypti up to 91 m from the spray line com-pared with 8 and 16 km/h. Farooq et al. (2018) further discussed that movement of a vehicle creates an air vortex behind it that strengthens with increasing travel speed. This vortex helps mix the spray with air, resulting in higher probability of drop-lets contacting flying insects. Moreover, the speed of the induced air increases with an increase in travel speed (Farooq et al. 2018). The induced air deflects the spray plume towards the ground and suppresses upward spray movement resulting in better efficacy. Therefore, the objective of this study was

to evaluate the effectiveness of a combina-tion of the best nozzle orientation and truck speed for ULV adulticide application against caged Ae. albopictus (Skuse) in an urban resi-dential area of Gainesville, Florida.


Study sites

Previous studies indicated that landscape and housing density have an impact on the effectiveness of ULV spray (Mount et al. 1968; Pant et al. 1971). In order to test the impacts, three communities, namely Rid-geview [29° 41ʹ 02.7ʺ N, 82° 20ʹ 54.7" W], Iron Wood [29° 41ʹ 48.3" N, 82° 18ʹ 16.8" W], and Lamplighter [29° 40ʹ 33.7" N, 82° 15ʹ 36.1" W] located in Gainesville, Florida were selected for the testing sites. Each com-munity was classified as dense, medium and sparse based on visual estimation of vegeta-tion cover, age of the houses and width of the street. The Ridgeview community was built in the 1960’s that consisted of mixed dense vegetation of landscaped palms, ornamental plants, Southern live oaks, and pine trees. Houses were characteristically terraced with shared backyards along a two lane-street without sidewalks. Most front yards were covered with heavy ornamental plants such as holly, Indian hawthorn, and evergreen azaleas. The Iron Wood community was built in the late 1980’s with a medium dense vegetation of low shrubs, Southern live oak, and palm trees. These terraced houses are mostly single-family detached homes with shared backyards, two lane-streets and side-walks. Most of front yards are covered by turf grasses and flower beds. Lamplighter is a sin-gle or double-wide mobile home community built in the 1970s with very few shrubs and little to no vegetation present in the front or shared backyards.

Mosquitoes

Mosquitoes used in this study were ob-tained from an insecticide-susceptible USDA strain of Ae. albopictus, reared at Gainesville Mosquito Control headquarters at 26.6 °C, 85 ±5% RH, and a photoperiod of 14:10 (L:

Jiang et al.: Impact of nozzle orientation & truck speed on Aedes albopictus 53

D). Larvae were fed with a 3:2 mixture of bovine liver powder (MP Biomedicals, LLC, OH) and brewer’s yeast (MP Biomedicals, LLC, OH). Adult mosquitoes were supplied with 10% sugar water, and 3-5 days old non-blood-fed females were used in all evalua-tions.

Test product

Aqua-Reslin® (20.0% permethrin AI, 20% PBO, Bayer Environmental Science) a synergized permethrin water-based perme-thrin was used for this study. The formula-tion was diluted with water at the ratio of 1:2 and the flow rate is 142 ml/min which is the maximum rate of 0.0015 lb./acre.

Field study

Field studies were conducted following the methods of Farooq et al. (2017) with minor modifications. Briefly, Aqua-Reslin®, mixed with a fluorescent dye (1, 3, 6, 8-py-rene tetra sulfonic acid tetra sodium salt, Spectra Colors Corporation, Kearny, NJ) at 8,000 ppm, was used. The spray mixture was applied with a truck-mounted ULV London Fogger 18-20 (London Foggers, Minneapo-

lis, MN) at a flow rate of 142 ml/min. This equipment produced spray droplets with a volume median diameter (Dv0.5) of 15.9 mi-crons and DV0.9 of 30.4 microns. Treatments included a) standard nozzle orientation 45° upward at a travel speed of 16 km/h (45° combination), b) 22.5° upward at 24 km/h (22.5° combination), and c) 0° (horizontal) at 24 km/h (0° combination) . Effective-ness of each application combination was assessed by determining Ae. albopictus mor-tality 24 h post-application, spray deposition quantification, and droplet size spectra.

Figure 1 illustrates the field layout, ULV spraying direction, and relative position of bioassay cages. Two rows of bioassay cages were placed at least 15 m apart, and up to 90 m perpendicular to the spray line. Each row contained six cages with 25 female Ae. albopictus per cage, were positioned at 0, 15, 30, 45, 60, and 90 m from the line of appli-cation. Alongside, and 1 m away from each treatment cage, a Florida Latham Bonds spinner (model 319; John W. Hock Compa-ny, Gainesville, FL) using two 3 mm × 75 mm acrylic rods was deployed to collect sprays for assessing droplet size characterization and deposition. One of the 2 rods was used for

90 m

15 m

Spraying direction

Row A

1

2

3

4

5

6

1

2

3

4

5

6

30 m

Row B

15 m

Figure. 1. Field layout illustrating relative position of mosquito sentinel cages and spraying direction. Figure 1. Field layout illustrating relative position of mosquito sentinel cages and spraying directions.


droplet size and the other for quantification of adulticide deposition. Cages and spinners were suspended 1.5 m above ground. On the first day, both rows of samplers in three veg-etation levels were randomly assigned to be sprayed with one of the three nozzle orienta-tion and travel speed combinations to make 1 replication of all treatments. The 3 replica-tions were made on 3 different days at least 2 weeks apart and treatments sequentially rotated. The same spray time sequence for sites on each day was maintained. Tempera-ture, RH, wind speed, and wind direction were recorded 1.5 m above the ground.

Control cages were deployed well out and upwind of the spray zone and placed in the same environment for 15 min, then col-lected before application. Spray cages and rods were placed in the field immediately before spraying. Cages were removed from all stations 15 min post-spray, supplied with 10% sugar solution, transferred to the labo-ratory, and maintained under normal room conditions until the 24 h mortality count was taken. Along with cages, spinner rods were also removed from the field. One rod from each location was preserved for measure-ment of droplets and the other stored in a pre-labeled, re-sealable plastic bag. All rods were then stored in a cool and dark environ-ment and transported to the US Navy En-tomology Center of Excellence laboratory where they were stored in a refrigerator for later droplet size measurements and deter-mination of deposition.

Droplet size on rods was determined us-ing the DropVision system (Leading Edge Associates Inc., Fletcher, NC) and droplet distribution parameters (DV0.1, DV0.5, and DV0.9) were determined. The DV0.1, DV0.5, and DV0.9 are the droplet diameters (µm) where 10, 50, and 90% of the spray volume is con-tained in droplets smaller than these diam-eters (ASTM Standard E1620, 2004). Adul-ticide deposition on rods was measured using the methods described by Farooq et al. (2009). Rods were washed inside a plas-tic bag using 25 ml of deionized water. Fluo-rescence readings of the solution were de-termined using a spectrofluorophotometer (Shimadzu, Model RF5000U, Kyoto, Japan)

and converted to the amount of dye on the slide using calibrations developed from a set of standardized fluorescence concentra-tions. The amount of dye in each sample was then divided by the effective sampling area of 63 cm2 to calculate dye deposition (ng/cm2). Using the ratio of dye and active ingre-dient (AI) in the spray tank, deposition was converted to AI deposition (ng/cm2).

Statistical analysis

Statistical analysis was conducted with In-tel® Visual Fortran Composer XE 2013. The Kolmogorov-Smirnov test (Smirnov 1939) showed that all datasets were non-normal and the Bartlett test (Bartlett 1937) showed non-homogeneity of variances. Therefore, mean 24 h Ae. albopictus mortality, adulticide deposition, and Dv0.5 data were subjected to a 3-way nonparametric Kruskal-Wallis analysis (α = 0.05) that was used to assess differences among the nozzle combinations (i.e. 45° up-ward at 16 km/h, 22.5° upward at 24 km/h, and 0° horizontal at 24 km/h-factor 1), veg-etation levels (dense, medium, and sparse-factor-2), and application distances (0, 15, 30, 45, 60, and 90 m-factor 3), as well as the interactions between the nozzle combina-tion × vegetation, the nozzle combination × distance interaction, the vegetation type × distance interaction, and nozzle combina-tion × vegetation type × distance interaction. Subsequent Tukey multiple-comparisons tests were conducted to identify those nozzle combination treatments that were signifi-cantly different from each other. Differences from all analyses were considered significant when P < 0.05.

RESULTS

Effect of Nozzle Orientation and Speed on Mortality

There was a significant difference in 24-h mortality of caged Ae. albopictus among the 3 nozzle/speed combinations (P = 0.0096) and distances (P = 0.0212) but not the inter-action between combination and distance (P = 0.5863) (Figure 2). The 22.5° combination resulted in the greatest mortality (88.3%)


and 45° combination resulted in the least mortality (63.1%) (Table 1). There was no significant difference between 22.5° combi-nation and 0°combination in terms of 24-h mortality. At every distance, mortality from 45° combination was significantly lower than 22.5° and 0° combinations. At distance 0 m, 22.5° and 0° combinations resulted in 100% mortality; at distances of 15, 30, and 60 m the 22.5° combination slightly outperformed 0° whereas, at 45 m, the 0° combination slight-ly outperformed the 22.5° combination but these differences were not significant (Fig-ure 2).

Effect of Vegetation Density on Mosquito Mortality

There was a significant difference in 24-h mosquito mortality among the 3 vegetation levels (P = 0.0319) and distances (P = 0.0212) but no significant difference among the in-

teractions of vegetation level and distance (P = 0.5863). The greatest mortality (91.4%) was recorded in the sparse and the lowest in medium vegetation density (72.2%) (Table 1). At 0 m, no significant difference in 24-h mortality among the three vegetation levels occurred. At 15 m, Ae. albopictus mortality in the sparse vegetation community remained close to 100% but significantly lower mor-tality occurred in the medium and dense vegetation communities (Figure 3). At 30 m, mortality in sparse vegetation areas de-creased to 80% while in medium and dense locations mortality was further reduced to 60% and 40%, respectively.

Effect of Nozzle Orientation and Travel Speed Combination on Adulticide Deposi-tion

Adulticide deposition was not signifi-cantly affected by nozzle orientation and

Figure 2. Comparison of 24-h mortality among the combinations of angle and speed and distances.

Table 1. Mean mosquito mortality from nozzle angle combinations in different vegetation densities.

Vegetation Density

Mortality, % (Mean ± SD) from angle combinations

0° 22.5° 45° Average

Dense 74.9 ± 23.2 90.4 ± 23.4 59.7 ± 45.0 75.0 ± 30.5Medium 86.6 ± 15.3 96.5 ± 6.5 33.5 ± 41.9 72.2 ± 21.2Sparse 100.0 ± 0.0 78.1 ± 29.7 96.1 ± 7.4 91.4 ± 12.4Average 87.2 ±12.8 88.3 ± 19.9 63.1 ± 31.4 79.5 ± 21.4


travel speed combinations (P = 0.2066), or interaction of these combinations with distance (P = 0.2806). Deposition was sig-nificantly affected by application distance (P < 0.0001) (Figure 4). Overall, 0° com-bination resulted in the greatest average deposition, followed by 22.5° and 45° combinations. At 0 m, significantly higher deposition occurred from 0° combination than from 22.5° and 45° combinations,

whereas at the remaining distances, there were no significant differences in deposi-tion between nozzle/speed combinations (Figure 4).

Effect of Vegetation Density on Adulticide Deposition

Deposition on rods was not significantly different among the 3 vegetation densities (P = 0.3594) but was significantly different

Figure 3. Comparison of 24-h mortality among the vegetation levels and distances.

Figure 4. Comparison of deposition among the angel combinations and distances.


among sampling distances (P < 0.0001) and interaction of vegetation density and dis-tance (P = 0.0337). Generally, deposition was greatest (27.5 ng/cm2) for medium and lowest (23.6 ng/cm2) for sparse vegetation (Figure 5). Also, deposition was greatest at 0 m and lowest at 30 m, and highest for 0° combination at 0 m and lowest at 45° combi-nation at 30 m (Figure 4).

DISCUSSION

Farooq et al. (2017, 2018) demonstrat-ed that nozzle orientation or travel speed alone resulted in a significant impact on adulticide efficacy in an open field. Over-all, horizontal nozzle spraying with a truck mounted ULV sprayer achieved the great-est efficacy against adult Ae. aegypti, fol-lowed by a 30° downward nozzle angle while a 45° angle upward showed the least effectiveness (Farooq et al. 2017). Jiang and Farooq (2016) showed that a ULV nozzle oriented horizontally outperformed those upward at 45° in three out of four field tri-als, although those differences were not statistically significant. In addition, Farooq et al. (2018) recently reported that a trav-el speed of 32 km/h achieved the highest efficacy against caged Ae. aegypti followed

by 16 km/h and then 8 km/h. Our study found that a ULV nozzle oriented at 22.5° at a travel speed of 24 km/h resulted in the greatest mortality against Ae. albopictus whereas, the standard nozzle orientation of 45° at speed of 16 km/h (10 mph) re-sulted in the least mortality. These results confirmed that by changing nozzle orien-tation and travel speed together one can significantly improve adulticide efficacy. Farooq et al. (2017) explained that when spray nozzles are oriented at 45° upward, most spray material remains above the mos-quito fly zone, so no droplets <40 µm would descend to the space 1.4 m above ground (which is habitat for most humans and mos-quitoes) before traveling 100 m in a hori-zontal direction. By setting the nozzle ori-entation lower than 45° upward, enhanced mixing of spray into the air by the truck wake may have also resulted in an increase of spray efficacy. Likewise, improvement in application effectiveness with increased travel speed is due to the resultant combi-nation of two physical phenomena. First, the induced air movement due to vehicle travel occurs in an opposite direction and increases with an increase in travel speed. The induced air deflects the spray plume towards the ground and suppresses upward

Figure 5. Comparison of deposition among the vegetation levels and distances.


droplet movement resulting in better effi-cacy. Second, movement of a vehicle creates an air vortex behind it that strengthens with increasing travel speed. This vortex helps to better mix the spray with air, resulting in a higher probability of droplets contacting flying insects (Farooq et al. 2018). At an up-ward nozzle of 45°, the spray cloud is gener-ally not expected to interact with the vortex behind the vehicle.

Vegetation and housing density is anoth-er factor that has a significant impact upon control effectiveness as reported by many authors. Taylor and Schoof (1971) obtained twice the level of kill for 3 species of mosqui-toes exposed to 95% malathion aerosols in an open area compared with those exposed in moderately dense wooded areas. Andis et al. (1987) stated that the average mortality of caged Ae. aegypti was 95.5% and 49% in open and sequestered locations, respectively. Lin-ley and Jordan (1992) obtained greater per-cent kills of caged Culex quinquefasciatus Say exposed to aerosols of malathion, naled, and resmethrin plus piperonyl butoxide in open compared with vegetated terrain. Results from our study showed that 0° and 22.5° combinations resulted in 75 and 90% mor-tality of Ae. albopictus, respectively in dense vegetation, compared with 60% from the standard 45° combination. Comparing our results with those reported by Taylor and Schoof (1971), Andis et al. (1987), and Lin-ley and Jordan (1992) indicated that adjust-ing nozzle orientation increased mortality in vegetation.

In summary, significant improvement in performance of a ULV applied adulticide was achieved when the combination of an optimal nozzle orientation was paired with an increase in travel speed. Importantly, Fa-rooq et al (2017) pointed out that this ap-plication optimization does not require a structural change, takes only a few minutes to accomplish, and has a significant impact on spray efficacy.

ACKNOWLEDGMENTS

This study was supported in part by a grant from the Deployed War-Fighter Pro-

tection Research Program, funded by the US Department of Defense through the Armed Forces Pest Management Board. The au-thors thank Josh Weston and Jason Fajardo of the US Navy Entomology Center of Excel-lence, Jacksonville, FL, and Jarod Lloyd, Jus-tin Baker, Cason Bartz and Karen St. Pierre of Gainesville Mosquito Control, Gainesville, FL, for their support during different parts of the study.

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Farooq M, Fulcher A, Xue RD, Smith ML, Anderson JL, Richardson SR, Knapp JA. 2017. Effect of nozzle orientation on dispersion of Aqualuer 20-20 sprayed by a truck-mounted ultra-low-volume sprayer against caged Aedes aegypti. J Am Mosq Control Assoc 33: 209-214.

Farooq M, Gibson-Corrado J, Knapp JA, Xue RD, Smith ML, Briley AKC, Richardson AR and Cilek J. 2018. Effect of travel speed on dispersion of Aqualuer 20-20 sprayed by a truck-mounted ultra-low-volume sprayer against caged Aedes aegypti. J Am Mosq Control Assoc 34: 70-74.

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60

OPERATIONS NOTE

INCREASED WATER HARDNESS IN CATCH BASINS TREATED WITH SPINOSAD (NATULAR XRT)

EXTENDED RELEASE TABLETS

LAWRENCE J. HRIBAR

Florida Keys Mosquito Control District, 503 107th Street, Marathon, Florida 33050

[email protected]

Subject Editor: Seth Britch

ABSTRACT

In response to apparent lack of efficacy of spinosad treatments of storm drain catch basins in Marathon, Flor-ida, we investigated water quality parameters where drains had been treated with Natular® XRT extended release tablets. An analysis of water samples from these sites revealed that alkalinity and water hardness differed significant-ly between treated and untreated drains. However, when tested in a semi-field environment protected from runoff, differences in alkalinity were not associated with spinosad treatment, whereas water hardness increased over time in replicates treated with Natular XRT. Water quality may be a reason for poor larval control rather than product failure or resistance. Future work will investigate whether changes in water hardness associated with spinosad treat-ment may impact the efficacy of this larvicide at reducing adult emergence in field environments.

Key Words: spinosad, water hardness, pH, alkalinity, nitrate, nitrite, ammonia

The Florida Keys Mosquito Control District (FKMCD) includes Natular® XRT (Clarke, St. Charles, IL) extended release tablets in its larvicide arsenal. The active in-gredient of Natular XRT is spinosad 6.25%, a mixture of spinosyns A and D derived from bacterial fermentation that may be ingested by larvae or absorbed by direct contact caus-ing muscle spasms and eventually paralysis or death. Spinosad is a useful addition to the mosquito control toolbox, although it has a broader range of nontarget effects than oth-er bacterial-derived larvicides (Lawler 2017). According to the product label, Natular XRT tablets may be placed into storm drain catch basins and allowed to dissolve into the stand-ing water, providing up to 180 d of long-term mosquito control.

During 2017 we observed an apparent lack of efficacy in drains treated with Natu-lar XRT tablets in FKMCD. We hypothesized that water chemistry in the treated locations could have interfered with the action of the

formulation because water quality can af-fect solubility of spinosyns (Thompson et al. 1999). We conducted two experiments to investigate (1) whether water quality pa-rameters in drains treated with Natular XRT tablets in the field differed from those in untreated drains, and (2) whether Natular XRT tablets themselves in a semi-field envi-ronment may be associated with changes in any water quality parameters identified in Experiment 1.

For Experiment 1 we selected two park-ing lots, one at the Government Center (GC; 9 drains present) and one at the Home De-pot (HD; 16 drains present) in Marathon, Florida. We randomly selected 3 drains at GC and 8 drains at HD to be treated with Natular XRT tablets at the label rate, leaving the remaining 6 and 8 drains, respectively, as untreated controls. At 84 d post treatment we sampled water from all 25 drains and used aquarium testing reagents (Aquarium Pharmaceuticals, Inc., Chalfont, PA) to mea-

Hribar: Water hardness and spinosad 61

sure pH (hydrogen ion concentration), al-kalinity (calcium carbonate concentration), hardness (calcium and magnesium concen-tration), and three indicators of decompo-sition of organic matter or fertilizer runoff (nitrite, nitrate, and ammonia) in each drain water sample. Prior to analysis, we log-trans-formed (log(x+1); GWMAP 1999) alkalin-ity, hardness, nitrite, nitrate, and ammonia data, and data for pH were backtransformed into hydrogen ion concentration (Fiorica 1968). We pooled data across the two sites and conducted an analysis of variance (SY-STAT 2009) to investigate differences in wa-ter quality parameters between treated and untreated control drains. The findings from Experiment 1, that is, identification of water quality parameters significantly different be-tween treated and untreated control sites, were used to guide the selection of specific parameters tested in Experiment 2.

For Experiment 2 we set up a static semi-field system with six covered 5-gallon plastic buckets (Leaktite, Leominster, MA) at the Marathon FKMCD facility. Each bucket re-ceived 3 gallons of distilled water, and we then collected a 6 ml water sample from each bucket. We designated three buckets for immediate treatment with one Natular XRT tablet each and three buckets as un-treated controls. We collected a 6 ml water sample from each of the six buckets daily for seven continuous days after the initial treat-ment day. Each water sample was tested im-mediately after collection for water quality parameters identified as significantly associ-ated with Natular XRT treatment in Experi-ment 1.

Analysis of variance on data from drain water samples in Experiment 1 revealed sig-

nificant differences (P < 0.05) in alkalinity and hardness in water collected in treated compared to untreated control drains (Ta-ble 1). We tested water samples from Experi-ment 2 for changes in these two parameters and found that alkalinity did not vary over the week, whereas general water hardness in-creased (R2=0.9706) over the 7 d experiment (Figure 1). In a separate analysis (data not shown) we found that water quality param-eters only significantly differed between the GC and HD sites in hydrogen concentration (pH), possibly attributable to different mo-tor vehicle activities and consequent runoff solutes at the two sites (Alam et al. 2017).

The chemical composition of the Natu-lar XRT tablet formulation is proprietary information but combined results from Ex-periments 1 and 2 suggest that the larvicide product application itself could have con-tributed to the observed differences in wa-ter hardness between treated and untreated control drains across the two study sites. Al-though both alkalinity and water hardness significantly differed between treated and untreated control drains in the field, the al-kalinity did not significantly differ in treated buckets in the semi-field study. However, the buckets were protected from runoff and introduction of other material during the course of the study that may have, in con-trast, affected this water quality parameter at the field sites.

Water quality in storm drain catch basins may be an important consideration in de-signing operational larvicide programs that include the active ingredient spinosad. It is known that solubility of spinosyns in water decreases as pH increases (Thompson et al. 1999, Cleveland et al. 2002, Liu and Li 2004,

Table 1. Analysis of variance of water quality parameters between treated and untreated control drains.

ParameterTR1

Mean ± SEUT1

Mean ± SE F Ratio P Value

Alkalinity 1.99 ± 0.01 1.52 ± 0.06 6.118 0.022Hardness 1.86 ± 0.07 1.35 ± 0.07 5.248 0.032Nitrite 0 ± 0 0.03 ± 0.01 1.394 0.251Nitrate 0.12 ± 0.04 0.3 ± 0.4 0.228 0.638Ammonia 0.27 ± 0.02 0.22 ± 0.01 0.026 0.875Hydrogen 75.12 ± 3.14 65.81 ± 2.53 0.048 0.829

1TR, Treated; UT, Untreated


Adak and Mukherjee 2016). Mosquitoes are more likely to be found in catch basins with relatively low pH and high total suspended solids, carbon, and nitrogen (Butler et al. 2007, Gardner et al. 2013). Catch basins are designed to retain water and they also col-lect and retain debris, which may have an effect on performance of larvicides (Harbi-son et al. 2016). In this study we show that water hardness may be affected by the pres-ence of the larvicide product itself. However, further work needs to be done to investigate whether changes in water hardness impact the efficacy of this spinosad formulation to inhibit adult emergence from treated stand-ing water.

ACKNOWLEDGMENTS

The author thanks H. Murray and C. Pruszynski for collecting water samples from field sites.

REFERENCES CITED

Adak T. and Mukherjee I. 2016. Investigating role of abiotic factors on spinosad dissipation. Bull Environ Contam Toxicol 96:125–129.

Alam MZ, Anwar AHMF, Sarker DC, Heitz A, and Roth-leitner C. 2017. Characterising stormwater gross pollutants captured in catch basin inserts. Sci Total Environ 586:76-86.

Butler M, Ginsberg HS, LeBrun RA, Gettman AD, and Pollnak F. 2007. Natural communities in catch ba-sins in southern Rhode Island. Northeast Nat 14:235-250.

Cleveland CB, Bormett GA, Saunders DG, Powers FL, McGibbon AS, Reeves GL, Rutherford L, and Balcer JL. 2002. Environmental fate of spinosad. 1. Dissi-pation and degradation in aqueous systems. J Agric Food Chem 50:3244- 3256.

Fiorica, V. 1968. A Table for Converting pH to Hydro-gen Ion Concentration [H+] Over the Range 5 – 9. Department of Transportation, Federal Aviation Ad-ministration, Office of Aviation Medicine.

Gardner AM, Anderson TK, Hamer GL, Johnson DE, Varela KE, Walker ED, and Ruiz MO. 2013. Terrestri-al vegetation and aquatic chemistry influence larval mosquito abundance in catch basins, Chicago, USA. Parasites Vectors 6:9. http://www.parasitesandvectors.com/content/6/1/9.

GWMAP [Ground Water Monitoring and Assessment Program]. 1999. Data Protocol for the Ground Water Monitoring and Assessment Program. Min-nesota Pollution Control Agency, Environmental Outcomes Division, Environmental Monitoring and Analysis Section, Ground Water and Toxics Monitor-ing Unit, St. Paul, Minnesota.

Harbison JE, Henry M, Corcoran PC, Zazra D, and Xam-plas C. 2016. Small-scale trials suggest increasing ap-plications of NatularTM XRT and NatularTM T30 larvi-cide tablets may not improve mosquito reduction in some catch basins. Environ Health Perspect 10:31-34.

Lawler SP. 2017. Environmental safety review of metho-prene and bacterially-derived pesticides commonly

Figure 1. Change in general water hardness (GH) in Experiment 2. Data were plotted as degrees of carbonate hardness (°dKH) against time; °dKH may be converted to parts per million by multiplying by 17.848.

Hribar: Water hardness and spinosad 63

used for sustained mosquito control. Ecotoxicol Envi-ron Saf 139:335-343.

Liu S, and Li QX. 2004. Photolysis of spinosyns in sea-water, stream water and various aqueous solutions. Chemosphere 56: 1121-1127.

SYSTAT. 2009. SYSTAT 13 for Windows. SYSTAT Soft-ware, Inc.

Thompson GD, Hutchins SH, and Sparks TC. 1999. De-velopment of spinosad and attributes of a new class of insect control products. In: E. B. Radcliffe, W. D. Hutchison & R. E. Cancelado [eds.], Radcliffe’s IPM World Textbook, URL: https://ipmworld.umn.edu, University of Minnesota, St. Paul, MN. (Accessed April 8, 2020)

64

NuisaNce Psychoda alterNata (diPtera: Psychodidae) develoPiNg iN Potted PlaNts at a

commercial Nursery

Matteo Pallottini1, C. lee BlooMCaMP2, RoBeRto M. PeReiRa3*, and PhiliP G. KoehleR3

1department of Chemistry, Biology and Biotechnology, University of Perugia, Via elce di Sotto 8, 06132 Perugia (PG), italy

e-mail: [email protected]

2Syngenta Professional Solutions, 8518 SW 98th ave., Gainesville Fl 32608 e-mail: [email protected]

3entomology and nematology department, University of Florida, 1881 natural area dr., Gainesville, Fl 32611

e-mail: [email protected], [email protected].

*Corresponding author: Roberto M. Pereira e-mail: [email protected]

Subject editor: derrick Mathias

Abstract

Moth flies (Psychoda alternata Say) were reported emerging in large numbers from potted plants at a com-mercial nursery near Fort White, Columbia County, FL and causing an annoyance and potentially a public health nuisance at neighboring residences. The distribution of its fly immature stages in the soil of recently re-potted plants was investigated. Two species of plants from the commercial nursery were selected, soil samples were taken at different depths and positions and each soil sample was extracted using a technique for nematode extraction from soil. Larvae and pupae of P. alternata moth flies were identified in the samples. Psychoda alternata is commonly found breeding in trickling filters and this is the first record of it being an important nuisance pest in newly potted plants.

Key Words: drain fly; trickling filter fly; nuisance; soil

Several species of flies are known to be nuisance pests at commercial nurseries and greenhouses (Tilley et al. 2011; Cloyd 2015), however, Psychoda alternata Say (Psychodi-dae, Psychodinae) have not been reported as a pest problem in potted plants. Recently, moth flies were reported to be emerging in large numbers from potted plants at a com-mercial nursery near Fort White, Colum-bia County, Florida causing annoyance and potential health problems for neighboring homeowners.

Psychoda alternata is usually called “drain fly”, due to its tendency to live and repro-duce in shallow and polluted water (El Bardicy et al. 2009), or “trickling filter fly” because it is commonly found breeding in

trickling filters (Fair 1934). The adults are dark grey, 2.5 to 4.5 mm long, the body and the wings are characterized by a dense cover-ing of long hair and leaf-shaped wings which are held roof-like over the body (Fair 1934; El Bardicy et al. 2009; Yones et al. 2014). Psychoda alternata do not bite, but they can represent a real problem when emerging in enormous numbers. The flies can be car-ried by the wind up to one mile and pen-etrate through the window screens of nearby structures (Headlee 1919; Fair 1934; den Otter 1966; El Bardicy et al. 2009) causing nuisance and public health concerns. Asth-ma caused by P. alternata has been reported in many parts of the world (Ordman 1946; den Otter 1966; Phanichyakarn et al. 1969;

Pattottini et al.: Psychoda alternata in commercial nursery 65

Gold et al. 1985). Cases of urogenital myiasis caused by P. alternata have been related to poor human hygienic conditions (den Otter 1966; Hira et al. 1997; Yones et al. 2014; Saa-dawi et al. 2017), and ocular myiasis has also occurred (Kamimura 1967). Psychoda alterna-ta is also known to be forensically significant, and recently was identified in human cadav-ers (Lindgren et al. 2015). Therefore, P. al-ternata has the potential to be a significant public health problem for residences near commercial nurseries.

The County Health Department, Florida Department of Agriculture and Consumer Services, and Mosquito Control District were contacted by the affected homeowners to in-vestigate the problem. A large commercial nursery bordered the affected houses and no other sources of these moth flies were lo-cated in this rural area. The nursery owner observed that 10-14 days after re-potting new plants large numbers of 2-4 mm sized moth flies would be seen on the foliage of some plants. An inspection of the ground under pots placed on a weed barrier indicated no larvae.

The main objective was to determine whether moth flies were developing in plant pots at this commercial nursery and to de-termine the distribution of immature moth fly stages in the media of recently re-potted plant species that were observed to have adult flies associated with their vegetation.

Two species of recently re-potted plants from the commercial nursery were selected. Camellia (Camellia japonica) were propagat-ed at the same nursery and then re-potted in 26.5-liter pots 7 days before collection. Gar-denia (Gardenia jasminoides) were purchased from another nursery as small plants and then re-potted in 26.5-liter pots 14 days be-fore collection. Three containers for each of the two species were returned to the labora-tory for analysis during March 2019.

The soil used for re-potting all plants was composed of 55% pine bark (1.3 cm), 25% pine bark (2.2 cm), and 20% Canadian peat. Additives to the media mix were fertilizer (7.12 kg/m³ Nutricote, Arysta Life Science, Cary, NC), iron humate (5.93 kg/m³), do-lomite (2.67 kg/m³), and synthetic gypsum

100 (1.48 kg/m³). Bifenthrin granules (1.78 kg/m³) were added to the potting media to comply with the USDA imported fire ant quarantine (APHIS 2018). The plants were irrigated twice per week, as needed, to wet the medium to the bottom of the pot, with-out runoff.

Twelve samples (150 ml each) of media were collected the day after bringing the plants to the laboratory. Samples were taken at 8-days after re-potting (DARP) for Camel-lia and 15-DARP for Gardenia. To establish the distribution of insect larvae and pupae in the potting medium surrounding the roots of potted plants, twelve media samples were removed from each pot at 5-cm incre-ments from the surface (0-5 cm) to the bot-tom (25-30 cm). Six samples were from the perimeter of the medium, and six were from the center. There were 72 samples total with 36 for 8-DARP Camellia and 36 for 15-DARP Gardenia.

Samples were examined using a tech-nique commonly applied for nematode extraction from soil. This technique was tested prior to use in the experiment and produced good results in the extraction of small fly larvae from soil. This technique may be useful for extraction and quantifica-tion of other small insect larvae from soil samples. Each 150-ml sample was washed thoroughly into a sieve (2-mm mesh) to re-move the bigger fraction of the media fol-lowed by passing media through a smaller sieve (37-μm mesh). The smaller fraction was transferred into a 100-ml centrifuge tube and centrifuged for 5 min at 3500 rpm. The supernatant was removed, and sugar solution (454 g sucrose/liter of water) was added to the remaining sample and re-centrifuged for 5 min at 3500 rpm. At this stage, the supernatant was filtered through a 25-μm mesh sieve and transferred into 50-ml tubes. The collected material was pre-served in 70% isopropyl alcohol and exam-ined under a stereo microscope to identify and count fly larvae and pupae. Taxonomic keys by Quate (1955) were used.

The effects of depth and position (pe-riphery or center) on the distribution of moth fly larvae and pupae were analyzed us-


ing a two-way ANOVA, and means were com-pared using Student’s t-test (P<0.05) in JMP Statistical Analysis Software (SAS Institute, Cary, North Carolina, USA). Before analy-sis, data were transformed using square-root transformation to normalize the data distri-bution.

Four hundred and fourty-four larvae and pupae of Psychoda alternata were found in the samples (Figure 1). Psychoda alternata to-taled 60% of all insect pupae in all samples, 56% of the insect pupae in 8-DARP Camel-lia, and 81% of the pupae in 15-DARP Gar-denia. Only mature larvae were found in the

Figure 1. Distribution (mean +/- std. error) of Psychoda alternata larvae and pupae at 5 to 30 cm depth in (top) 8-DARP Gardenia and (bottom) 15-DARP Camellia pots at a commercial nursery near Fort White, Columbia County, Florida in March 2019.

Pattottini et al.: Psychoda alternata in commercial nursery 67

samples. Considering the duration of the life cycle and the short time of the pupal period (20-40 h, El Bardicy et al. 2009), egg deposition may have occurred soon after re-potting, with only one generation occur-ring inside the pots without an overlapping generation. In other studies in which popu-lations of P. alternata were stable with over-lapping generations (Ali et al. 1991, Ali and Kok-Yokomi 1991), pupae were found in low percentages, between 8 and 23%, due to the short duration of the pupal stage.

In 8-DARP Camellia (Figure 1, top), num-ber of P. alternata larvae (F=1.60; p=0.20) were not associated with layer depth in the pots while pupae were more abundant in the first three layers (0-15 cm) (F=5.11; p= 0.003), with a significant difference in rela-tion to deeper layers. In 15-DARP Gardenia (Fig 1, bottom), pupae were not associated with depth (F=1.00; p=0.44), while larvae were more abundant (F=3.37; p=0.02) deep-er in the pot soil, with a significant difference in relation to the upper layers, although the overall larval abundance was very low com-pared to the numbers of P. alternata pupae.

In 8-DARP Camellia, pupae were signifi-cantly more abundant (F=6.27; p=0.02) in the exterior perimeter of the potting soil compared with the central area of the pots, whereas larvae were found in similar num-bers at the perimeter and the central area of the pots (F=2.23, p=0.15). In 15-DARP Garde-nia, both larvae (F=1.00; p=0.44) and pupae (F=1.86; p=0.19) were uniformly dispersed in the media, showing no preference for either the perimeter or central area of the pots.

Psychoda alternata has not been previ-ously reported as an important nuisance pest in newly potted plants. For both spe-cies of sampled plants, the potting medium was identical, and provided all the necessary requirements for P. alternata development regardless of plant species. Moreover, the bifenthrin treatment applied to the potting medium, as part of the fire ant quarantine protocol to prevent establishment and move-ment of fire ant colonies, was ineffective for control of the P. alternata, potentially indicat-ing some level of pesticide resistance in this insect population.

In order to complete its development, P. alternata needs high relative humidity, oxygen, decaying organic material and mi-croorganisms for larval development, lo-cations for pupation, and for the imagoes to hatch (den Otter 1966; El Bardicy et al. 2009). Psychoda alternata is a principal mem-ber of the invertebrate grazing fauna com-munity which inhabits the biological filters of sewage treatment plants (Learner 2000), but it can inhabit other environments with decaying organic material and sufficient moisture (Haseman 1907; Turner 1925; Saunders 1928; Redborg et al. 1983). Ali and Kok-Yokomi (1991) and Ali et al. (1991) reported massive emergences of the species at a turf cultivation facility in Florida, where nutrient-rich wastewater was used for irriga-tion. In addition to these other locations, P. alternata has the potential to be a significant problem in commercial nurseries, although it appears to be a pest in the newly-placed media of re-potted plants, completing only one generation per pot.

Due to the nursery industry practices of replotting plants, the potential synchronous emergence of very high populations of these flies may represent a problem that mosquito control districts may be called upon to re-solve and remediate. Additionally, if large populations of these flies move through densely populated areas, this may represent a potential health problem, especially for the elderly and other vulnerable segments of the population.

REFERENCES CITED

Ali A, Kok-Yokomi ML, Alexander JB. 1991. Vertical distribution of Psychoda alternata (Diptera: Psychodi-dae) in soil receiving wastewater utilized for turf cul-tivation. J Am Mosq Control Assoc 7: 287-289.

Ali A, Kok-Yokomi ML. 1991. Preliminary population assessment of Psychoda alternata (Diptera: Psychodi-dae) in soil irrigated with wastewater for turf cultiva-tion. Fla Entomol 74: 591-596.

APHIS. 2018. Labels Available for Use in IFA Quaran-tine (March 2018). [Internet]Available from United States Department of Agriculture - Animal and Plant Health Inspection Service [accessed April 25, 2019].

https://www.aphis.usda.gov/plant_health/plant_pest_info/fireants/downloads/IFA_QuarantineL-ables.pdf.

Cloyd RA. 2015. Ecology of fungus gnats (Bradysia spp.) in greenhouse production systems associated


with disease-interactions and alternative manage-ment strategies. Insects 6: 325-332. doi: 10.3390/in-sects6020325.

den Otter CJ. 1966. A physical method for permanent control of Psychoda pests at wastewater treatment plants. J Water Pollut Control Fed 38(2): 156-164.

El Bardicy S, Tadros M, Yousif F, Hafez S. 2009. Predatory activity of Psychoda alternata Say (Diptera: Psychodi-dae) larvae on Biomphalaria glabrata and Lymnaea natalensis snails and the free-living larval stages of Schistosoma mansoni. Aust J Basic Appl Sci 3: 4503-4509.

Fair GM. 1934. The trickling filter fly (Psychoda alterna-ta), its habits and control. Sewage Work J 6: 966-981.

Gold BL, Mathews KP, Burge HA. 1985. Occupational asthma caused by sewer flies. Am Rev Respir Dis 131: 949-952. doi: 10.1164/arrd.1985.131.6.949.

Haseman L. 1907. A monograph of the North Ameri-can Psychodidae, including ten new species of an aquatic psychodid from Florida. T Am Entomol Soc 33: 299-333.

Headlee TJ. 1919. Practical application of the methods recently discovered for the control of the sprinkling sewage filter fly (Psychoda alternata). J Econ Entomol 12: 35-41.

Hira PR, Hall MJR, Hajj B, Al-Ali F, Farooq R, Muzairai IA. 1997. Human myiasis in Kuwait due to Oestrus ovis, Psychoda species and Megaselia species. Med Princ Pract 6: 129-136. doi: 10.1159/000157540.

Kamimura K. 1967. A case of human ocular myiasis due to the moth fly, Psychoda alternata. Med Entomol Zool 18: 305-306. doi: 10.7601/mez.18.305.

Learner MA. 2000. Egression of flies from sewage filter-beds. Water Res 34: 877-889. doi: 10.1016/S0043-1354(99)00190-6.

Lindgren NK, Sisson MS, Archambeault AD, Rahlwes BC, Willett JR, Bucheli SR. 2015. Four forensic en-

tomology case studies: records and behavioral ob-servations on seldom reported cadaver fauna with notes on relevant previous occurrences and ecology. J Med Entomol 52: 143-150. doi: 10.1093/jme/tju023.

Ordman D. 1946. Bronchial asthma caused by the trick-ling sewage filter fly (Psychoda): inhalant insect al-lergy. Nature 157: 441.

Phanichyakarn P, Dockhorn RJ, Kirkpatrick CH. 1969. Asthma due to inhalation of moth flies (Psychoda). J Allerg 44: 51-58.

Quate LW. 1955. A revision of the Psychodidae (Dip-tera) in America north of Mexico. Univ Calif Publ Entomol 10: 103-273.

Redborg KE, Hinesly TD, Ziegler EL. 1983. Rearing Psy-choda alternata (Diptera: Psychodidae) in the labora-tory on digested sewage sludge, with some observa-tions on its biology. Environ Entomol 12: 412-415.

Saadawi WK, Shaibi T, Annajar BB. 2017. A human case of urogenital myiasis caused by Psychoda sp. larvae in Tripoli, Libya. Ann Parasitol 63: 69-71. doi: 10.17420/ap6301.88.

Saunders LG. 1928. Psychoda alternata Say breeding in the sea. Entomologist 61: 209.

Tilley LAN, Croft P, Mayhew PJ. 2011. Control of a glass-house pest through the conservation of its natural enemies? An evaluation of apparently naturally con-trolled shore fly populations. Biol Control 56: 22-29. doi: 10.1016/j.biocontrol.2010.09.003.

Turner CL. 1925. The Psychodidae (moth-like flies) as subjects for studies in breeding and heredity. Am Nat 57: 545-549.

Yones DA, Bakir HY, Hameed DA. 2014. Human Uro-genital Myiasis Caused by Psychoda Species Larvae: Report of Five Cases and Morphological Studies. J Adv Parasitol. 1: 12-20. doi: 10.14737/journal.jap/2014/1.2.12.20.

69

COMPARISON OF THE CDC LIGHT TRAP AND THE DYNATRAP® DT2000 FOR COLLECTION OF MOSQUITOES IN

SEMI-FIELD AND FIELD SETTINGS

NICHOLAS ACEVEDO, CAROLINE EFSTATHION, RUI-DE XUE, AND WHITNEY A. QUALLS


Guest Editor: M. Farooq

ABSTRACT

The CDC light trap has been the standard used by mosquito control programs to conduct mosquito and arbo-virus surveillance. For the last two decades, this trap has been used with little to no modifications to its original de-sign. Recently, new traps that utilize different light sources, modified designs, and attractants have been developed and evaluated against the CDC light trap. A semi-field and field comparison of the Dynatrap® (Model DT2000) against the CDC light trap was conducted at Anastasia Mosquito Control District. The DT2000 varies from the CDC light trap with a UV light, trapdoor/fan mechanism, and Atrakta lure which is a combination of lactic acid, ammonia, and hexanoic acid. Overall, the DT2000 collected 56% (327/600) of the Ae. aegypti released in the semi-field cage, compared to 18.5% (111/600) collected by the CDC light traps. These findings suggest that the DT2000 outperformed the CDC light trap in collecting Ae. aegypti. In the field, the DT2000 collected nine target mosquito species while the CDC light trap collected four target species. The DT2000 averaged 109 ± 97.46 mosquitoes and the CDC light trap averaged 8 ± 4.64 mosquitoes. The DT2000 presented functional limitations in the field as an electrical outlet was required. Study findings suggest that where an electrical outlet is available, the DT2000 may be an alternative to the CDC light trap for mosquito surveillance.

Key Words: mosquito surveillance, CDC light trap, mosquito traps, Aedes aegypti

For decades, mechanical traps have served as one of the primary means by which mosquito surveillance is conducted (Kline, 2006). Additionally, these traps have provid-ed many useful ways to survey blood-feeding vectors in the genera Aedes, Anopheles, and Culex. These mosquito genera are vectors of important diseases such as West Nile vi-rus, malaria, yellow fever, and Zika virus (Turell, 2001).

To monitor these vector species, the CDC light trap has been used for years with little to no modifications to its original design. This design as shown in Figure 1A includes a black circular plastic piece that covers a cylindrical clear plastic housing unit, which holds a standard incandescent bulb, a wire mesh, and a small computer fan with motor. A funnel attaches to the housing unit which has threading at the end for attachment of a capture bottle. This CDC light trap is pow-ered by a 12-volt battery. As a shift from this original CDC light trap design, Dynatrap® (Dynamic Solutions Worldwide, Milwaukee,

WI) has produced the DT2000 (Figure 1B). This trap comes with components that dis-tinguish it from the CDC light trap, which include a UV light, trapdoor/fan mecha-nism, and Atrakta® lure (Dynamic Solution Worldwide LLC, Milwaukee, WI), a combi-nation of lactic acid, ammonia, and hexa-noic acid. Though the DT2000 is intended for homeowner use, this trap may have a po-tential role in operational mosquito surveil-lance. Therefore, Anastasia Mosquito Con-trol District (AMCD) of St. Johns County, FL conducted a semi-field and field evaluation of the DT2000 in comparison with CDC light trap.

A study was carried out in a semi-field setting to compare the Dynatrap® DT2000 with the CDC light trap. The study was con-ducted in two 6m x 12m screened enclo-sures located on the AMCD complex. The DT2000 was hung in one screened enclo-sure and the CDC light trap was hung in the second screened enclosure. Both traps were hung at 0.9m from the ground by a


shepherd’s hook. The DT2000 was powered using an outdoor outlet and the CDC light trap was powered by a 12-volt battery. The traps were both baited with the Atrakta® lure. Two hundred 5-7-day old female Ae. aegypti were aspirated into transport cages and released in two screened enclosures by placing and opening transport cages at a distance of 7m from the traps. The con-tainers for each trap were removed at 24 hours from initial release, the collection bags were placed in a freezer, and then the mosquitoes were counted to determine the number of Ae. aegypti recaptured. Three replications were conducted. A Student’s t-test was used for the comparison of the col-lections from two traps.

Overall, the DT2000 collected 56% (327/600) of the Ae. aegypti released in the semi-field cage, compared to 18.5% (111/600) collected by the CDC light trap. These findings suggest that the DT2000 out-performed the CDC light trap in collecting Ae. aegypti (t=3.812, df=4, P=0.0189). After the successful evaluation in the semi-field cages showing difference in performance of the two traps, a field evaluation was con-ducted.

Field evaluations were conducted to compare the traps in terms of mosquito abundance and diversity. The design of the field test was similar to the semi-field test with these traps placed one at each study site and rotated twice for a total of three replica-tions. The two areas chosen had power out-lets, however to maximize suitable mosquito trapping habitat, several extension cords were used for the trap to function at the se-lected field sites. The traps were operated for 24 hours and collections were brought back to AMCD and stored in the freezer un-til the samples were processed. Mosquitoes were counted and speciated. All non-target arthropods were identified to order. A Stu-dent’s t-test was used for the comparison be-tween the collections by two traps.

Originally, the field testing of the DT2000 was to be conducted in a swampy forested area in St. Augustine, FL. However, sev-eral limitations quickly arose. To allow the DT2000 to operate without a power outlet, a wire power converter was hooked up to a 12V car battery. Upon returning the follow-ing day, the battery was drained. During the following weeks, several attempts were made to enable the DT2000 to work without a pow-er outlet. These attempts included switching wire configurations, adding an additional car battery, and also trying deep-cycle batter-ies in place of car batteries (Figure 2).

Unfortunately, the battery modification outcomes were the same. After monitoring the power output of the DT2000 every hour, it was found that the power of the deep-cy-cle batteries would begin to waiver at about 4 hours and would be depleted by 5 hours. Following the continued battery failures, it was determined that the DT2000 required

A

B

Figure 1. A) The CDC light trap and B) the DT2000 trap displayed in the semi-field cages during the initial comparison on recapture rate of Aedes aegypti.

Acevedo et al.: CDC light trap and DYNAtrap comparison 71

a power outlet for field use. The two areas chosen had power outlets, however to maxi-mize suitable mosquito trapping habitat, sev-eral extension cords were used for the trap to function at the selected field sites.

Table 1 shows the total number by spe-cies collected by two traps in the field evalu-ation and the numbers were not different from each other (t=1.0, df=16, P=0.3129). Overall, the DT2000 collected nine target mosquito species while the CDC light trap

collected four target species. The collections with the DT2000 averaged 109 ± 97.46 mos-quitoes (n=3 trap nights) and the CDC light trap averaged 8 ± 4.64 mosquitoes (n=3 trap nights). Anopheles crucians was the outlier with an average of 99 ± 81.8 collected per trap night for the DT2000 and 5 ± 2.3 for the CDC light trap. For all other species, the DT2000 averaged 11 ± 3.4 mosquitoes per trap night with the CDC light trap av-eraging 3 ± 1. These numbers are very low for mosquito trap evaluations and suggest that further evaluations of the DT2000 trap is necessary in a more productive mosquito location.

Table 2 shows the total numbers of three non-target orders in the CDC light traps, compared to the four non-target orders cap-tured in the DT2000. The DT2000 had sig-nificantly higher non-target collection than CDC light trap (t=1.3738, df=6, P=0.2186). The number of Lepidoptera specimens col-lected in the DT2000 compared to the CDC light trap warrants further investigation to figure out whether this trap is a suitable tool for mosquito surveillance due to the impact on non-targets. Replacing the attractant used with the DT2000 with octenol may re-duce the number of non-targets collected.

Dynatrap® recently marketed a new DT160 trap which may have potential in mosquito surveillance. Though smaller and slightly different than the DT2000, the DT160 operates with a 12V battery which helps it surpass any power limitations. The price of one of these traps is around $50 which is less expensive than the CDC light trap. For resource limited mosquito control programs, these traps could be used as a tool for surveillance to better direct control in the field and thus should be investigated further.

Figure 2. The picture demonstrates the attempts to utilize a battery with the DT2000 to allow for placement and evaluation in field settings.

Table 1. Total number of mosquito species collected by the DT2000 and CDC Light Traps.

Species DT2000 CDC

Aedes aegypti 1 0Anopheles crucians 296 14Culex erraticus 6 1Culex salinaris 5 3Culex quinquefasciatus 1 0Culiseta melanura 16 5Coquillettidia petrubans 1 0Psorophora columbiae 1 0Mansonia sp. 1 0Totals 328 23

Table 2. Total number of non-targets by insect order col-lected by the DT2000 and CDC Light Traps.

Non-target Order DT2000 CDC

Coleoptera 41 1Diptera 73 17Mecoptera 8 0Lepidoptera 340 25Totals 462 43


This comparison study would not have been possible without the generosity and co-operation of Karen McKenzie as well as her team at Dynamic Solutions Worldwide, LLC for providing the traps for evaluation. This is a research report only and specific mention of any commercial products does not imply endorsement by AMCD.

REFERENCES CITED

Kline DL. 2006. Traps and Trapping Techniques for Adult Mosquito Control. J Amer Mosq Control Assoc, 22:490–496

Turell M J, O’Guinn ML, Dohm DJ, Jones JW. 2001. Vector Competence of North American Mosquitoes (Diptera: Culicidae) for West Nile Virus. J Med Ento-mol, 38:130-134.

73

A NEW LABORATORY COLONIZATION OF AEDES AEGYPTI AFTER REEMERGENCE AND UNSUCCESSFUL

ERADICATION IN ST. AUGUSTINE, FLORIDA

RUI-DE XUE, CHRISTOPHER S. BIBBS, DANIEL DIXON, AND DENA AUTRY


Guest Editor: Whitney Qualls

ABSTRACT

After unsuccessful eradication attempts against Aedes aegypti (L.) following a sudden re-emergence in St. Au-gustine, Florida in early 2016; a new locally acquired colony strain of Ae. aegypti was established at the Anastasia Mos-quito Control District (AMCD) in June 2017. Aedes aegypti adults were maintained in cages at the AMCD insectary. Larval and adult mosquitoes were collected from downtown St. Augustine, Florida. Female mosquitoes at 5-7 days old were fed upon the exposed forearm of human volunteers in the 1st and 2nd generations. Mating was observed in a large cage and confirmed with eggs deposited on wet filter paper in ovicups. Over 90% egg hatch was observed in the laboratory. The new colony strain of Ae. aegypti has been cataloged at the USDA, Center for Medical, Agricul-tural, and Veterinary Entomology facility in Gainesville, FL and is being used to further research and control this species across North Florida.

Key Words: Aedes aegypti, colonization, eggs, blood meal, integrated mosquito management

Aedes aegypti (Linn.) is a major vector of dengue virus, chikungunya virus, yellow fever virus, and Zika virus. Before the mid-1980’s Ae. aegypti was the primary container-inhabiting vector mosquitoes in St. Augus-tine and throughout northeastern Florida (Betts 1994). After the introduction of Aedes albopictus (Skuse) in the mid-1980’s (Smith et al. 1990), Ae. aegypti gradually disappeared from Northern and Northeastern Florida (O’Meara et al. 1995).

Saint Augustine, one of the oldest cit-ies in North America, is a tourist attraction in Northeastern Florida; hosting a signifi-cant number of temporary residents and vacation-travelers, creating an environment susceptible to travel-related cases of dengue, chikungunya, yellow fever, and Zika (Smith et al. 2018). Consequently, surveillance and control of Ae. aegypti is a cornerstone for lo-cal public health and mosquito control pro-grams.

In 2011, a resurgence of Ae. aegypti was de-tected in the urban areas of Jacksonville and Northeastern Florida (Wright et al 2015). St. Johns County shares borders with the city of Jacksonville, FL (Duval County), therefore

leading to increased surveillance by Anas-tasia Mosquito Control District (AMCD) of container-inhabiting mosquitoes since 2011 in order to detect the presence of Ae. aegypti in St. Augustine. On 15 of Feb, 2016, a re-surgent population of larval Ae. aegypti was collected for the first time from used tires and cisterns in downtown St. Augustine, FL. In response to this detection, AMCD launched a city-wide surveillance grid and eradication program focused on the down-town area surrounding the positive sample sites. As a result, 9 hot spots were located in the surrounding area by BG traps with BG lures (Biogents AG, Regensburg, Germany). These hotspots inflated to cover all of the downtown municipality by July 2016.

From April 2016 to August 2017, an erad-ication survey program was developed for the attempted eradication of Ae. aegypti from downtown St. Augustine. Initial program elements relied on traditional methods through systematic visitation of all streets, businesses, and residences by personnel in combination with integrated mosquito man-agement for inspection, source reduction, treatment when hotspots were detected, and


education of all local residents. Downtown St. Augustine was divided into five routes and assigned two technicians per route (1 from the Department of Health and 1 from AMCD) for inspection, education, and con-trol of mosquitoes on a weekly basis.

In the beginning of the eradication sur-vey, a few adult Ae. aegypti were collected from George Street and Spanish Street in the heart of downtown St. Augustine. These two thoroughfares host tourist attractions and historic preservations, such as the old-est schoolhouse, local businesses, churches, and semi-enclosed courtyards of Spanish architecture. All areas of interest were sur-veilled using manual source reduction, sam-pling, and overnight trapping with BG traps combined with BG lure. Relatively few adults were captured in traps, however a high num-ber of human landings were confirmed to be Ae. aegypti from the BG trap sites, likely due to the traps failing to compete against natural human odors from numerous visi-tors and residents along the affected streets (Owino et al 2014). To overcome the weaker long-range attraction of the BG lure, CO2 was added in combination with BG lure lead-ing to an 80% increase in female Ae. aegypti. This result was similar to a study by De Azara, et al. (2013) using BG lure in combination with CO2 for increased collection of Ae. ae-gypti by BG trap in Manaus, Brazil.

The resurgent Ae. aegypti population spread and was detected by dual baited BG traps outside of St. Augustine city limits and many areas of St. Johns County, Florida in 2017. Concomitantly, the eradication of Ae. aegypti was considered to have failed. Since 2018, AMCD collaborated with USDA/CMAVE, University of Florida, Mosquito-Mate, and SpringStar to explore and adopt novel IPM strategies and tools to control Ae. aegypti, such as SIT (sterile insect technique) male release, release of Wolbachia-infected male mosquitoes, and mass deployment of Autocidal Gravid Oviposition traps for tar-geted control of Ae. aegypti.

Alongside the eradication survey taking place in 2016, AMCD was collaborating with University of Florida and USDA / CMAVE to release irradiated 1952 Orlando strain Ae. ae-

gypti within St. Augustine Beach for control of reemerged Ae. aegypti mosquitoes. The ex-perimental control method generated con-cerns from local residents about the release and infestation of the Orlando strain into St. Augustine. Therefore, it was a critically im-portant objective to establish a local colony of Ae. aegypti for public acceptance and sup-port.

In June 2017, mosquito larvae were aspi-rated from a variety of containers: bromeli-ads, tires, stagnant pools, cisterns, and other artificial water impoundments throughout downtown St. Augustine, FL. The larvae were transported to the insectaries at AMCD via Nasco WHIRL-PAK sample bags. Larvae were transferred into 4 oz soufflé cups and placed inside a receptacle with a saturated sugar water cotton ball for newly emerged adults. The soufflé cups were given 0.1 gram of ground cat food for larval consumption. Every day the collected larvae were moni-tored for pupation and adult emergence. Emerged adults were removed, sorted based on species and sex, and transferred to rear-ing cages (Bug Dorm 2, BioQuip, Rancho, Dominguez, CA 90220) using mouth aspira-tors.

Within the first generation, adult female mosquitoes were not receptive to blood meals offered as part of standard operating procedure at AMCD, therefore a human vol-unteer exposed their forearm to feed adult mosquitoes until engorgement. An oviposi-tion source (vol. 0.5L) with an ovistrip was placed in the cage (30.5 cm W x 45.7 cm L, BioQuip), but no eggs were collected after 5 days. A second attempt to collect eggs, involved 45 blood engorged females and 50 males transferred to a larger, collapsible rearing cage for mating and oviposition (Mega View Science, 60 x 60 x 60 cm). A black oviposition container (1 L) with ovis-trip and reverse osmosis water (250 mL) was placed into the cage for egg collection. With-in 3 days a total of 2,050 eggs were collected from the 50 engorged female mosquitoes after multiple blood meals. The eggs (ca. 98%) were hatched in a cup (1 L) of osmo-sis water (200 mL). After the F1 generation, the F2 generation of Ae. aegypti adults were

Xue et al.: New colonization of Aedes aegypti 75

weened onto blood meals used as part of AMCD’s standard insectary procedures. Ae-des aegypti colonies were fed on 10% sucrose soln. ad. libitum from a 150 mL receptacle and cotton wick. The colony was maintained in the AMCD insectary at 80% RH and 26 °C in a 14:10 L:D photoperiod. Adult female mosquitoes were provided blood weekly via restricted live chicken (AMCD animal care manual approved in 2005), and oviposition substrates were collected 72-96 hours post blood meal.

Observations during the establishment of the Ae. aegypti AMCD strain led to a hy-pothesis that newly emerged male and fe-male adult mosquitoes lacked the appropri-ate space in the initial colony cage, as there were no observations of mating activity. The female mosquitoes would feed, but not on live chickens. After transferring male and fe-male mosquitoes to the larger cage (45.7 cm L x 30.5 cm W x 30.5 cm H), males and fe-males were observed engaging in courtship and successfully copulating. The gravid fe-male mosquitoes subsequently oviposited on an ovistrip in the containers provided. The 2nd generation of Ae. aegypti were weened by first offering a human arm, but rather than allowing to feed, mosquitoes were left in an active host-seeking state and the chicken blood meal was provided. This process was repeated until mosquitoes fed without the need for a human volunteer. The estab-lished colony actively feeds on restricted live chicken and lays sufficient eggs to maintain itself.

To facilitate the new control methods and to establish trust and understanding

among St. Johns County residents, the new local strain of Ae. aegypti mosquitoes needed to be scaled up to meet the research and production needs. Consequently, the colony was transferred to our collaborators, USDA/Center for Medical, Agricultural, and Vet-erinary Entomology, Gainesville, FL and the University of Florida for mass production and SIT treatment. In return, the SIT treat-ed mosquitoes are returned to St. Augustine to release against the naturalized population of Ae. aegypti.

REFERENCES CITED

Betts RR. 1994. Aedes albopictus and Aedes aegypti: Spe-cies domination in St. Johns County. J Florida Mosq Control Assoc 65:17-19.

De Azara TM, Degener CM, Roque RA, Geier M, Wiras AE. 2013. The impact of CO2 on collection of Aedes aegypti (Linnaeus) and Culex quinquefasciatus Say by BG-Sentinel traps in Manaus, Brazil. Mem Inst Oswal-do Cruz 108:229-232.

O’Meara GF, Evans LF Jr, Gettman AD, Cuda JP. 1995. Spread of Aedes albopictus and decline of Aedes ae-gypti (Diptera: Culicidae) in Florida. J Med Entomol 32:554-562.

Owino EA, Sang R, Sole CL, Pirk C, Mbogo C, Torto B. 2014. Field evaluation of natural human odors and the biogent-synthetic lure in trapping Aedes aegypti, vector of dengue and chikungunya viruses in Kenya. Parasites & Vectors 7:451.

Smith JP, Loyless TM, Mulrennan JA Jr. 1990. An update on Aedes albopictus in Florida. J Am Mosq Control Assoc 6:318-320.

Smith M, Dixon D, Bibbs CS, Autry D, Xue RD. 2018. Diel patterns of Aedes aegypti (Diptera: Culicidae) af-ter resurgence in St. Augustine, Florida as collected by a mechanical rotator trap. J Vector Ecology 44:201-204.

Wright JA, Larson RT, Richardson AG, Cote NM, Stoops CA, Clark M, Obenauer PJ. 2015. Comparison of BG-Sentinel trap and oviposition cups for Aedes ae-gypti and Aedes albopictus surveillance in Jacksonville, Florida.. J Am Mosq Control Assoc 31:26-31.

76

Laboratory evaLuation of boriC aCiD Sugar baitS againSt irraDiateD aeDeS aegypti

Vindhya S. aryaprema1, Kai Blore1, Jedidiah Kline², roBert l. aldridge², Kenneth J. linthicum2, and rui-de Xue1

1anastasia mosquito control district, 120, eoc drive, St. augustine, Florida, uSa

uSda-center for medical, agricultural & Veterinary entomology, gainesville, Florida, uSa

Subject editor: eva Buckner

Abstract

The use of attractive toxic sugar baits (ATSBs) is a new paradigm in mosquito control. ATSBs kill both female and male mosquitoes attracted to sugar feed on a sugary solution containing a toxic substance such as boric acid. The impact of boric acid sugar baits on irradiated and non-irradiated Aedes aegypti was evaluated in the laboratory to determine any difference in mortality between the two groups. The mortality rates from the toxic sugar baits in both irradiated with 50 Gy and non-irradiated groups were highly significantly different, compared to those of corresponding control groups (t=6.916, p <0.0001 and t=6.451, p<0.01, respectively). Irradiated Ae. aegypti were not significantly different in mortality rates from non-irradiated counter parts after 24 and 48 h exposure to toxic sugar bait (t=0.576, p=0.578 and t=0.642, p=0.535 respectively). There was no significant difference in mortality caused by toxic sugar baits between the two sexes (t=0.595, p=0.869 and t=0.169, p=0.869 for irradiated and non-irradiated groups, respectively). The mean percent mortality after 48 h of exposure to toxic sugar baits was 43.3% for irradiated mosquitoes and 38.6% for non-irradiated mosquitoes. Mortality rates were significantly higher during second 24 h period in both irradiated and non-irradiated groups (t=-6.612, p<0.01 and t=-5.278, p<0.01 respectively). The study suggests that the toxic sugar bait approach could not be used within a sterile insect technique program to reduce the wild male population to increase the chances of wild females mating with irradiated males.

Key Words: radiation, sterile insect technique, Aedes aegypti, boric acid sugar bait, mortality

Aedes aegypti Linn. is an important mosqui-to vector species which transmits arboviruses causing diseases, such as yellow fever, dengue, chikungunya and Zika viruses in humans (Beaumier et al. 2014, Chippaux & Chip-paux 2018, Guerbois et al. 2015, Honório et al. 2018). Vector control consists primarily of the removal of immature development habi-tats augmented by targeted adulticide appli-cation. Ubiquitous and cryptic breeding sites combined with the domiciliary resting behav-ior of the species have made density reduc-tion challenging. Moreover, rapid evolution of insecticide resistance in Ae. aegypti popula-tions has become a major problem. This justi-fies the development of novel and enhanced control strategies (Deming et al. 2016) that are environmentally friendly, sustainable, and cost-effective (WHO 2012). Sterile insect

technique (SIT) is one such control strategy that has been of increasing interest (Alphey et al. 2010, Chung et al. 2018, https://www.iaea.org/topics/sterile-insect-technique ). Sterile insect technique involves the mass-rearing of mosquitoes and, release of sterile males.

Attractive-toxic sugar baits (ATSBs) that kill both female and male mosquitoes are also considered to be a promising strategy, which can be added in integrated vector management (IVM) programs for mosquito control (Fiorenzano et al. 2017). Boric acid has shown to be an effective active ingredient for the ATSB (Xue & Barnard 2003, Bhami & Das 2015 behind Xue & Barnard 2002). Boric acid sugar baits applied as a foliar or surface space spray have previously demon-strated effective control of several mosqui-to species in field trials (Beier et al. 2012,

Aryaprema et al.: SIT and boric acid sugar bait against mosquitoes 77

Fiorenzano et al. 2017, Maia et al. 2018, Qualls et al. 2012, Qualls et al. 2015, Xue et al. 2006, 2011). However, SIT and ATSB control methods work in different ways. The SIT slowly reduces the fecundity of the wild population (Alphey et al. 2010) while the application of ATSB produces quick mortality of both males and females of tar-geted vector populations (Xue et al. 2006). Additionally, although SIT is species-specif-ic, the ATSB impacts a wide range of target and non-target organisms alike given the generic effect of some active ingredients.

The purpose of this study was to deter-mine if ATSB could be used to reduce the wild male population within a SIT program to increase the chances of wild females mat-ing with irradiated males. The impact of ATSB on adult female Ae. aegypti was also evaluated, because it is possible for some irradiated females to also be released with the sterile males. A laboratory experiment was conducted with colonized Ae. aegypti (St. Augustine strain 2016) provided by the USDA-Center for Medical, Agricultural & Veterinary Entomology, Gainesville, Flori-da. Male and female Ae. aegypti pupae were irradiated with 50 Gray (Gy) by γ-radiation using a Gammator M (Radiation Machin-ery Corp., Parsippany, NJ) containing a ce-sium-137 source that generated 8.8Gy/min. The radiation doses applied to the pupae were 0 and 50 Gy, with the 0 Gy acting as a control. Radiation doses were checked with alanine films applied to petri dishes with pupae for every dose. Two hundred 5-7- day-old irradiated and non-irradiated males and females were mouth-aspirated into each of several separate cages (29 cm L x 29 cm W X 29 cm H,). The experiment was carried out with one treatment group and one control group, each with cages of irradiated males, irradiated females, non-ir-radiated males and non-irradiated females.

Each treatment cage was provided with a cup of cotton wool swabs (Cloud Mar-keting Inc. Rancho Cucamonga, CA) satu-rated with 5% sugar solution + 1% boric acid (Sigma-Aldrich, Co. USA) while the control cages were provided only with 5% sugar solution. Dead mosquitoes in cages

were hand-picked and counted 24 and 48 h after the exposure to the sugar solutions (control and treatment). The remaining live mosquitoes were counted after 48 h to enumerate the total number in each cage and the percent mortalities were calculated based on the totals summed across two col-lection days. Three replicates of the experi-ment were carried out in three different times. Each replicate was at least one week apart to minimize any environmental bias.

Data analysis was done using SPSS (IBM® SPSS® statistics, version 20). Nor-mal distributions of the data sets were con-firmed using the Shapiro-Wilk normality test. Means of percent mortalities between different groups after 48 h and at two 24 h periods were compared appropriately using independent sample t-test. The level of sig-nificance was maintained at p<0.05.

Both irradiated and non-irradiated Ae. aegypti had statistically significant differenc-es in mortality rates after exposure to boric acid sugar bait (treatment) for 48 h com-pared to the corresponding control group (t= 6.916, p<0.0001 and t=6.451, p<0.01 respectively). The difference in mortality rates between irradiated and non-irradiat-ed mosquitoes exposed to boric acid sugar bait was not significantly different at both 24 (t=0.576, p=0.578) and 48 h (t=0.642, p=0.535) exposure periods (Figure 1). Further, no difference in mortality rates between the two sexes was documented (t=0.477, p>0.05 and t=0.511, p>0.05 for irradiated and non-irradiated groups, re-spectively). Mean percent mortalities after 24 and 48 h of boric acid sugar bait expo-sure were 5.89 ± 1.30 (SE) and 37.01 ± 4.52 (SE) respectively for irradiated mosquitoes and 4.76 ± 1.45 (SE) and 33.87 ± 5.32 (SE) respectively for non-irradiated mosquitoes. There were highly significant differences in mortality rates between the two time peri-ods with a higher mortality after the second 24 h period (t=-6.612, p<0.01 for irradiated mosquitoes and t=-5.278, p<0.01 p<0.01 for non-irradiated mosquitoes (Table 1).

The results indicate that irradiated Ae. aegypti were as susceptible as non-irradiated Ae. aegypti when exposed to boric acid sugar


baits, and there was no statistical difference in susceptibility between males and females independent of irradiation. The results also suggest that significant mortality from boric acid sugar baits could be achieved 48 h post exposure. However, for the release of irra-diated Ae. aegypti to be successful in control-ling population densities, the released mos-quitoes must disperse rapidly among the wild population so that they have a higher probability of mating with wild females within their life span (White et al. 2010). A requirement of SIT is that the release ratio is sufficiently large to overcome the natu-ral rate of population increase, so that the trend in population size is downward follow-ing the first release (Dunn & Follett, 2017).

Therefore, the application of boric acid sugar baits or ATSB and the release of ir-radiated male Ae. aegypti in the same area at the same time would not be complementary. Incorporation of the two techniques into an integrated vector management program will require considerable thought and planning to implement them both effectively. Further studies with field released irradiated and wild are suggested as the findings with colonized mosquitoes are not always indicative of what will be found with field mosquitoes.

We would like to acknowledge the support of Daniel A. Hahn and Chao Chen, University of Florida, Gainesville Florida, for supporting the research by exposing the mosquitoes in their radiator.

Figure 1. Mean (+ SE) mortality (%) of irradiated and non-irradiated adult Ae. aegypti after exposure to boric acid sugar baits at 24h and 48h in the laboratory.

Aryaprema et al.: SIT and boric acid sugar bait against mosquitoes 79

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Table 1. Comparison of mean ( ± SE) percent mortality between irradiated and non-irradiated adult Ae. aegypti after exposure to boric acid sugar baits for 24 and 48 h

Mean ± SE

First 24 h Second 24 h After 48 h

Irradiated-treated 5.89 ± 1.30 37.01 ± 4.52 43.32 ± 4.68Irradiated-control 1.11 ± 0.45 1.20 ± 0.18 2.31 ± 0.57Non-irradiated-treated 4.76 ± 1.45 33.87 ± 5.32 38.61 ± 5.63Non-irradiated-control 1.36 ± 0.51 0.26 ± 0.18 1.63 ± 0.63

80

EFFICACY OF COMMERCIAL ATTRACTIVE TOXIC SUGAR BAIT STATION (ATSB) AGAINST AEDES ALBOPICTUS

VINDHYA S. ARYAPREMA, EDWARD ZESZUTKO, COURTNEY CUNNINGHAM, EMAD I. M. KHATER AND RUI-DE XUE

Anastasia Mosquito Control District, 120 EOC Drive, St. Augustine, FL, USA

Guest Editor: Gunter C. Muller

ABSTRACT

The use of toxic sugar baits is a new paradigm in mosquito control. A commercial product of attractive toxic sugar bait station (Spartan Mosquito Eradicator) contains a toxic sugar bait with sodium chloride as the active ingredient and yeast as an attractant. We studied the efficacy of the device against adult Aedes albopictus Skuse. The study composed of a laboratory and a field component with treatment and control cohorts. The treatment in the laboratory experiment resulted in nonsignificant mortality of adult mosquitoes compared with untreated mosquitoes. Neither laboratory nor field components of the study showed significant evidence that the commercial product could reduce the abundance of Ae. albopictus in the natural environment. The device may need to be improved and further evaluation conducted.

Key Words: attractive toxic sugar bait, Aedes albopictus, sodium chloride, efficacy

The use of toxic sugar baits (TSB) targeting sugar feeding behavior of the mosquito is an expanding technology in the field of mosquito control (Fiorenzano et al. 2017). Lea, in 1965 pioneered the method with malathion in a sucrose solu-tion formulating the first mosquito toxic sugar bait (TSB) which was fed to Aedes aegypti (Lea 1965). Since then, differ-ent toxic substances including boric acid (Blore et al 2018, Qualls et al. 2015, Xue et al. 2006, Xue & Barnard 2003), euge-nol (Qualls et al 2014), chlorfenapyr and tolfenpyrad (Stewart et al. 2013) and iver-mectin (Maia et al. 2018) have been evalu-ated against a number of adult mosquito vectors. Adult mosquitoes have easy access to sugar sources, like floral and extra flo-ral nectaries, rotted fruits and damaged fruits in the environment (Bidlingmayer 1973, Foster 1995). Toxic sugar baits have thus been supplemented with suitable at-tractants that attract adult mosquitoes for the bait in spite of the availability of natural sugars (Qualls et al. 2014). Since the control of mosquito populations with traditional insecticides are becoming less effective due to the development of resis-tance (Deming et al. 2016), the ATSBs may

be an important alternative option. New ATSB products are therefore, considered to be introduced to the market.

Spartan Mosquito Eradicator is a device with an ATSB released to the market target-ing control of adult mosquito populations. The commercial device is a plastic tube (5 cm D x 27 cm H) containing a product of 11.48% sodium chloride (active ingredient-the toxic substance), 0.18% yeast (the at-tractant) and 88.34% sucrose as a dry pow-der indicated on the label. The tube lid has 6 small holes of ~3 mm diameter through which the mosquitoes are supposed to go in and feed on the dissolved product. It is de-signed to hang on trees or structures in the environment. The purpose of this study was to evaluate the effectiveness of the commer-cial product in reducing population densi-ties of Aedes albopictus (Skuse,1894), an im-portant vector of arboviral diseases, such as dengue, zika and chikungunya (Kumari et al. 2011, McKenzie et al. 2019, Monteiro et al. 2019, Paupy et al. 2012, Sivan et al. 2016) that is geographically well distributed over the globe (Kraemer et al. 2015, Paupy et al. 2009).

The study was carried out from Octo-ber to December 2019 in the laboratory and

Aryaprema et al.: Attractive toxic sugar bait against Aedes albopictus 81

field. The commercial products were pur-chased from online and shipped to AMCD by BioOpus LLC for evaluation. The labora-tory study was carried out in three mosquito bug-dorms (BugDorm-2120 insect rearing tent, MegaView Science Co., Ltd. Taiwan) each containing 5-7 day old, 100 female and 100 male Ae. albopictus obtained from the insectary of the Anastasia Mosquito Control District (AMCD). One bug-dorm was pro-vided with a Spartan Mosquito Eradicator tube with the original product dissolved in water (treatment bug-dorm) as per the man-ufacturer’s guidelines to make a solution of 450 ml. Once dissolved the actual propor-tion of active ingredient in the solution was 1% and the proportion of sucrose was 8%. The control bug-dorm was thus provided with a Spartan Mosquito Eradicator tube containing only 8% sucrose solution and the other bug-dorm was provided with two Spartan Mosquito Eradicator tubes, one with the dissolved product and the other with 8% sucrose solution to give the mosquitoes a choice (choice bug-dorm). Number of dead mosquitos in each bug-dorm (in both the tube and the dorm) was counted at 24h in-tervals for 72 hours. Temperature and rela-tive humidity (RH) of the three laboratory replicates ranged from 18.2 °C -24.6 °C and RH: 56.1%–63.9% respectively.

The field study was carried out at two lo-cations with large tire piles which are known to have high abundance of Ae. albopictus. One location was used as the control site and the other as the treatment site rotating bi-weekly to minimize any bias characterized to the loca-tion. Five tubes were placed at each site, the distance between each tube was 4 meters; the tubes at the control site had the 8% sucrose solution only and the tubes at the treatment site had the dissolved Spartan product. Weekly mortality counts in each tube were recorded. One BG Sentinel trap (without CO2) was set out weekly at each site for 24 hours and col-lected mosquitoes were identified and count-ed. The study was carried out for 8 weeks.

Notable control mortalities, mainly in males, were observed for all laboratory repli-cates in spite of all possible remedial measures (Figure 1). Most of the dead mosquitoes were found in the bug-dorm and comparatively very few inside the tubes. It indicates that the mosquitoes were dying due to deprivation of water/sugar (desiccating) as they were not able to enter the devices through the very small holes. Comparatively low female mortal-ity (Figure 1) was likely due to the generally higher survival fitness of females. Mortalities in both males and females were lower in choice bug-dorms than in control bug-dorms (Figure 1). Each preferential bug-dorm having two

Figure 1. Cumulative mortality of Aedes albopictus exposed to Spartan Mosquito Eradicator in comparison to control mortality at different time periods under laboratory conditions


hydrated Spartan Mosquito Eradicators likely have been more saturated with water vapor than control bug-dorms, thus allowing better survival of mosquitoes.

In the field study, dead mosquitoes were found only once in one treatment tube (2 Ae. albopictus and 1 Anopheles quadrimaculatus). Ae.

albopictus collected in BG traps did not show any evidence of reduction in abundance in both males and females (Figure 2). Reductions in the numbers collected in the last two repli-cates were found in both control and treated sites and could be attributed to environmen-tal conditions. Furthermore, very high abun-

Figure 2. Pre-treatment and post-treatment BioGent Sentinel trap collections of Aedes albopictus males and fe-males during the field study

Aryaprema et al.: Attractive toxic sugar bait against Aedes albopictus 83

dance of Ae. albopictus were casually observed at both sites during each replicate except the last one. Numbers of Ae. albopictus males and females collected by BG traps did not show any significant difference between control and treatment sites (Mann-Whitney U=27, p=0.573 and U=28.5 and p= 0.709, respectively).

Both laboratory and field components of our study show that the Spartan Mos-quito Eradicator is not effective in reduc-ing abundance of Ae. albopictus. To compete with many alternative sugar sources in the natural environment the product should be more attractive and the device should be modified so that the mosquitoes can reach the product easily and feed on it. A separate study should be carried out to evaluate the effectiveness of the active ingredient at the concentration (1% sodium chloride) used in the dissolved product.

ACKNOWLEDGEMENTS

Authors would like to acknowledge K. Blore for rearing mosquitoes, R. Weaver, for the technical help during the study, J. Hainze for reviewing /editing the manuscript, and BioOus, LLC for providing the commercial products for the testing. This is a research re-port only and does not mean that AMCD en-dorses any commercial product.

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84

CHARACTERIZATION AND EFFICACY OF VECTOBAC® WDG APPLICATIONS TARGETING CONTAINER-INHABITING

MOSQUITOES USING AN UNMANNED AERIAL VEHICLE

KEIRA J. LUCAS1*(CO), PETER BRAKE1(CO), SARA GRANT1, LEANNE LAKE2, RACHEL B. BALES1, RICHARD RYAN1, NATE PHILLIPS1, PATRICK LINN1

1Collier Mosquito Control District, Naples, FL

2Valent Biosciences, Libertyville, IL

(CO)Contributed equally to this work with: Keira J. Lucas, Peter Brake

Subject Editor: Seth Britch

ABSTRACT

Application of Bacillus thuringiensis israelensis (Bti)-based liquid larvicide for the control of container inhabiting mosquito species, such as Aedes aegypti, is typically performed through the use of portable sprayers, truck-mounted mist systems, or manned fixed and rotary-wing aircraft. Recently, unmanned aerial vehicles (UAV) have provided a new avenue for control material applications. Here we report the characterization and efficacy of Wide Area Larvi-cide Sprays (WALS™) applications of Vectobac® WDG using UAV technology. Collier Mosquito Control District’s PrecisionVision 13 UAV was outfitted with the PrecisionVision Liquid Application System using four flat-fan TeeJet nozzles capable of producing fine/extra fine droplets for WALS applications of Vectobac WDG at a rate of 0.5 lb/A. Droplet characterization and mortality assays indicated that we achieved nearly 100% efficacy within 30-40 ft swaths. Furthermore, semi-field tests indicated delivery of the control material with six to seven adjacent swaths of 30 ft to open bioassay containers at the desired application rate within a 1 A treatment block, which was supported by reduction of natural populations of container inhabiting mosquitoes in the treatment area.

Key Words: Aedes aegypti, Bacillus thuringiensis israelensis (Bti), containers, larvicide, Unmanned Aerial Vehicles (UAV)

Unmanned aerial vehicle (UAV) tech-nology represents a novel tool for mosquito control operations. The potential for UAV usage has rapidly expanded to different operational activities, including enhanced larval and mosquito surveillance methods (Hass-Stapleton et al. 2019), habitat map-ping (Hardy et al. 2017, Carrasco-Escobar et al. 2019) and precise application of control materials. Small-scale applications of control materials using UAVs has successfully been used to target agricultural pests and patho-gens (Qin et al. 2016, Hunter et al. 2019, Wang et al. 2019, Xiao et al. 2019), as well as adult mosquito populations (Li et al. 2016). Recently, the Collier Mosquito Control Dis-trict (the District) began incorporating UAVs into their Integrated Pest Management Pro-gram, including the adoption of the Preci-sionVision 13 (PV13) UAV (Leading Edge

Aerial Technologies, Fletcher, NC) outfitted with the PrecisionVision Liquid Application System (Leading Edge Aerial Technologies, Fletcher, NC).

Application of Bacillus thuringiensis is-raelensis (Bti)-based liquid larvicide for the control of container inhabiting mosquito species is typically performed through the use of portable sprayers, truck-mounted mist systems, or manned fixed and rotary-winged aircraft. Here we report the characterization and efficacy of the proprietary Wide Area Larvicide Spray (WALS™, Valent Biosci-ences, Libertyville, IL) applications of the Bti-based water dispersible granule, Vecto-bac® WDG (Valent Biosciences, Libertyville, IL), using UAV technology in targeting con-tainer-inhabiting mosquito species. The Dis-trict’s PV13 UAV Liquid Application System was equipped with four flat-fan TeeJet noz-

Lucas et al.: UAV applications of VectoBac WDG 85

zles (#800067) capable of producing fine/extra fine droplets for WALS applications of a 12% VectoBac WDG suspension in water at an application rate of 0.5 lb/A (0.6 kg/Ha). A flow rate of 40 oz/min (1.2 L/min), at 10 mph (16.1 km/h) and an application height of 30 ft (9.1 m) was suitable for delivery of 0.5 lb/A at a theoretical swath of 30 ft.

Initial calibrations and swath character-izations included the use of VectoBac WDG mixed with food-grade red dye (FD&C Red Number 40 Granular) at a rate of approxi-mately 1 oz/gal (7.8 ml/L). The system was calibrated and flow-checked to achieve 40 oz/min of the 12% VectoBac® WDG suspension. For droplet characterization and application efficacy, two sampling lines were established – one setup to assess droplet distribution into the wind (narrowest swath) and another tak-ing into account cross wind (widest swath).

The “into wind” format included a 75 ft (22.9 m)sampling line placed perpendicu-lar to the wind with UAV applications occur-ring toward the wind. Twenty-five card sam-pling stations were set at 3 ft (1 m) intervals. At each station, one 7 x 9 cm Kromekote® card (CTI Paper USA Inc, Sun Prairie, WI) was secured to a CD Jewel Case and placed flat on the ground. A larval assay cup (8 oz soup container; Good Start Packaging, Bed-ford, NH) was placed every 6 ft (1.8 m) start-ing at 15 ft (4.8 m) to 69 ft (21 m) for a total of 10 cups across 54 ft (16.5 m). Three rep-licate single-pass flights at the 36 ft (11 m) station with an application rate of 0.5 lb/A were performed using the PV13 UAV – after each replicate, cards and larval assay cups were replaced. Applications were made prior to sunrise – between 6:04 AM and 6:38 AM – to maximize ground deposition of small droplets. Kromakote cards were analyzed for droplet measurements using BacDrop™ (Va-lent Biosciences, Libertyville, IL) and larval assay cups were brought back to the District’s

laboratory for mortality assays. Larval assays were performed by adding 100 mL of deion-ized water and approximately 20 late-2nd and early-3rd instar laboratory reared Aedes aegypti L. larvae (Orlando 1952 Strain) to each as-say cup. Non-treatment assay cups, placed in untreated area at the District headquarters, supplemented with 100 mL of deionized wa-ter were used as controls. Larval mortality was scored at 0 hr, 1 hr, 24 hr, and 48 hr post-treatment, and determined by the absence of movement upon disturbance. Percent mor-tality was determined by dividing the number of dead larvae by the total number of larvae and multiplying by 100, and an average was produced between the three replicates.

The “cross wind” design included a 200 ft (61 m) sampling line placed parallel to ex-pected wind pattern with UAV applications occurring perpendicular to the card line. Forty sampling stations were established starting from 0 to 200 ft at 5 ft (1.5 m) inter-vals along the sampling line. At each station one Kromekote card secured to a CD Jewel Case was placed flat on the ground as de-scribed above. A larval assay cup was placed every 10 ft (0.9 m) starting at 20 ft (6.1 m) to 120 ft (36.6 m) for a total of 10 cups across 100 ft (30.5 m). Three replicate single-pass flights commenced as described above; how-ever, the PV13 UAV crossed the card line at the 30 ft station to allow drift of the prod-uct down the sampling line – after each replicate, cards and larval assay cups were replaced. Applications were made prior to sunrise – between 7:05 AM and 7:46 AM – to maximize ground deposition of small drops. Droplet analysis and larval assays were per-formed as described above.

The “into the wind” design, representing the narrowest swath, resulted in an average droplet median diameter (Dv0.5) of 216.67 ± 4.78 μm across all three replications (Ta-ble 1, Figure 1A). Droplet size was greatest

Table 1. Droplet Characterization.

N Dv 0.1 (um) Dv 0.5 (um) Dv 0.9 (um) Mean Total Droplets

Mean Drop Density (cm2)

Into Wind 3 150.33 ± 6.13 216.67 ± 4.78 285 ± 15.12 190.39 ± 43.89 3.07 ± 0.72Cross Wind 3 131.33 ± 9.74 235.67 ± 22.95 332.33 ± 25.95 203.75 ± 36.13 3.78 ± 0.18


Figure 1. Droplet characterization and larval mortality for into wind (A-D) and cross wind (E-H) designs. Red dashed line indicates flight line. Data represent three replicates and are shown as mean ± SEM. (A) Average Vol-ume Median Diameter (VMD), (B) average number of droplets, and (C) average droplet density, and (D) larval mortality across collection stations. (E) Average VMD, (F) average number of droplets, (G) average droplet density, and (D) larval mortality across collection stations of cross wind characterization.

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near the flight line (36 ft station); however, droplets were relatively larger in size from 30-75 ft (9.1-22.9 m) compared to 0-24 ft (0-7.3 m) collection stations (Figure 1A), which may represent drift of the product to chang-ing wind direction or variable flow between the spray system booms. Average droplet number and droplet density followed a similar pattern (Figure 1B-C). Larval assays displayed mortality of greater than 90% be-tween the 45-69 ft (13.7-21 m) stations, indi-cating an effective swath of approximately 24 ft on average based on mortality rates at 24 hr post-treatment (Figure 1D). Mortality was not observed in the non-treatment control cups. The narrowest effective swath may be larger than captured in mortality assays, as the drift pattern appears to have extended beyond the last larval assay cup placed at the 69 ft station (Figure 1A-D). Temperature averaged 78.6 °F (25.9 °C) with an average relative humidity of 95% and a wind speed of 1.7 mph (0.76 m/s) NE.

Likewise, the “cross wind” format re-sulted in a Dv0.5 of 235.67 ± 22.95 μm across all three replications (Table 1, Figure 1E), with the greatest number of droplets accu-mulating between the 30-90 ft (7.1-27.4 m) stations (Figure 1F-G). Droplet size was also largest near the flight line (30 ft station) with droplets extending beyond the 200 ft station (Figure 1E). Larval assays displayed

mortality of greater than 90% between the 40-80 ft (12.1-24.3 m) stations, indicating an effective swath of 40 ft on average based on mortality rates at 24 hr post-treatment (Fig-ure 1H). Mortality was not detected in the non-treatment control cups. Together these results suggest that the UAV liquid larvicide spray system equipped with the four flat-fan TeeJet nozzles were sufficient to deliver 0.5 lb/A at an effective droplet spectrum rec-ommended for aerial WALS applications of a 12% suspension of VectoBac WDG. Tem-perature averaged 78.0 °F (25.6 °C) with an average relative humidity of 95%, and a wind speed of 1 mph (0.45 m/s) NE.

We then conducted two semi-field tri-als to determine efficacy within a treatment block using two different design methods. The first semi-field trial was performed by placing 10 larval assay cups within a 1 A treat-ment block located within an open area at the District headquarters (Figure 2A). Three replicate multi-swath treatments were per-formed using the PV13 UAV with an applica-tion rate of 0.5 lb/A with the 12% suspension of VectoBac WDG at an application height at 10 ft above canopy (40-50 ft above ground) and a speed of 10 mph. After each replicate treatment of 6 adjacent swaths, cups were replaced. Control cups were placed in an untreated area at District headquarters dur-ing trials. Applications were made prior to

Figure 2. (A) A flight map showing six 30 ft swaths and bioassay sentinel cup placements. (B) Percent mortality for each cup placement at 24 hr post treatment. Graphical analysis was performed using GraphPad Prism 8. Data represent three replicates and are shown as mean ± SEM.

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sunrise – between 6:45 AM and 7:10 AM – to maximize ground deposition of small drops. Temperature averaged 78.1 °F with an av-erage relative humidity of 96% and a wind speed of 3 mph (1.3 m/s) ENE. Treatment was manually offset to account for drift. Larval assay cups were brought back to the District’s laboratory for mortality assays as described above. Nearly 100% efficacy was achieved within 24 hr post-treatment when compared to non-treatment controls, with the exception of the final replicate display-ing reduced efficacy in cups 4-6 (Figure 2B). Reduced efficacy in the final replicate is like-ly to have been caused by an increase in wind speeds and change in wind direction toward the end of the field trials.

The second semi-field trial was per-formed in conjunction with an operational treatment of a 1 A treatment block in an ur-ban industrial park known to harbor large number of container inhabiting mosquitoes on September 26, 2019. The habitat desig-nated for treatment contained several larval habitats including tires, pallets, paint buck-ets, storage containers, PVC and large metal pipes, and trash bins. The habitat contained abundant garbage and debris, with emer-gent vegetation. Because of its high produc-tion rate of container-inhabiting mosquito species, the industrial park is routinely on rotations for larvicide treatment using Vec-toBac WDG from the District’s Buffalo Tur-bine sprayer (Buffalo Turbine, Springville, NY); however, treatment to this area had not been made in over 6 wk prior to UAV appli-cations. No adulticide treatments had been performed in the area in 2019. Ten larval as-say cups were placed in the 1 A treatment block to simulate containers in cryptic habi-tats (Figure 3A). In addition, pre-treatment larval dips, human landing rates1, and BG-trap (Biogents AG, Regensburg, Germany) data were collected (Figure 3C-F).

A single replicate multi-swath application was made at the industrial park using the PV13 UAV at a rate of 0.5 lb/A of the 12%

1Landing rates were performed as defined by CMCD SOP#12 (effective date: January 15, 2018) in accor-dance with F.S. Chapter 388 and 5E-13.

suspension of VectoBac WDG at an applica-tion height of 30 ft above canopy/buildings (75 ft [22.9 m] above ground). The applica-tion of 7 adjacent swaths was made prior to sunrise – at 7:11 AM – to maximize ground deposition of small drops and minimize dis-ruption to industrial park employees. Tem-perature was 71° F with a relative humidity of 93% and a wind speed of 1 mph NE. Larval assay cups were brought back to the District’s laboratory for mortality assays as described above. Control cups were placed in untreat-ed area at the District headquarters during trials. Nearly 100% efficacy was achieved within 24 hr (t = 60.45, df = 9, P < 0.0001) and 48 hr (t = 64.29. df = 9, P < 0.0001) post-treatment in larval assay cups (Figure 3B) compared to non-treatment controls.

Post-treatment larval dips, landing rates, and BG-trap data were collected each week for four weeks following VectoBac WDG treatment at the industrial park. Larval den-sity appeared lowest at 2-wk post-treatment (t = 2.672, df = 8, P = 0.0283) when compared to pre-treatment dips, with larval density reestab-lishing by 4-wk post-treatment (Figure 3C). By 1-wk post-treatment, the percent of positive containers in the area reduced by 60% and remained low during the duration of surveil-lance activity in the area (Figure 3D). Larvae inhabiting tires were most difficult to control, possibly due to the difficulty of the material to penetrate the standing water hiding within the tires (data not shown). Human landing rates (Ae. aegypti) were significantly reduced by 1-wk post-treatment (t = 7.780, df = 4, P = 0.0015) and remained low for 3 wk (2-wk: t = 8.795, df = 4, P = 0.0009; 3-ws: t = 4.908, df = 4, p = 0.0080) compared to pre-treatment land-ing rates, with the population reestablishing within 4-wk post-treatment (Figure 3E). Trap collections using BG-Sentinel traps baited with CO2 and BG-lure depicted a decreased trend in the container-inhabiting species A. aegypti and Culex quinquefasciatus (Say) by 2-wk post-treatment, with the population re-establishing within 3-wk post-treatment (Fig-ure 3F). Culex nigripalpus (Theobald), previ-ously identified in containers in the area, also depicted a decreased trend. Comprehensive data for larval dips, human landing rates, and


BG-Sentinel traps were not collected for a non-treatment control site; however, weather patterns and larval habitat (water-holding containers) remained consistent throughout the study.

This study indicated that the District’s PV13 UAV equipped with the PrecisionVi-sion Liquid Application System was suitable for delivery of a 12% VectoBac WDG suspen-sion using the WALS application strategy. Four tee-jet nozzles capable of producing fine/extra fine droplets, a flow rate of 40 oz/min, at 10 mph, and either 6 or 7 adjacent swaths of 30 ft effectively delivered 0.5 lb/A of VectoBac WDG into cryptic containers and reduced juvenile and adult container-inhabiting mosquito populations for up to 21 days. For adequate control of container-inhabiting mosquitoes using the PV13 UAV and VectoBac WDG, the District will plan to conduct multi-swath applications every 14-21 d in areas suitable for UAV use.

Although these results with our UAV system are highly encouraging and point to broader future adoption of UAVs in operational vector control, tank capacity of the larvicide system, acreage needing treatment, labor available, and applicator skill must be taken into con-sideration when deliberating incorporation of UAVs in a mosquito control operation. For ex-ample, the District’s current PV13 application system has a tank capacity of 2.5 gal (9.5 L) which supports delivery of VectoBac WDG to 5 A (2 Ha) per tank requiring returns and refills for larger areas. Also, the operator must main-tain visual line of sight with the UAV either directly or via communication with another employee. Both of these aspects often require additional labor. Furthermore, regulations set by the Federal Aviation Administration (FAA) and the Florida Department of Agriculture and Consumer Services (FDACS) require UAV operators conducting mosquito control pesti-cide applications to obtain a Part 107 Remote

Figure 3. (A) 1 A flight map showing seven 30 ft swaths and cup placements within industrial park. (B) Percent mortality at 0 hr, 1 hr, 24 hr, and 48 hr post-treatment. (C) Average larvae per container (Density Index), (D) percent positive containers (Container Index), (E) average human landing rate, and (F) BG-trap collections deter-mined weekly for 4 wk post-treatment. Graphical and statistical analysis were performed using GraphPad Prism 8. Data represent three replicates and are shown as mean ± SEM where appropriate. A two-tailed student’s t-test was performed to indicate statistical significance where appropriate; * P < 0.05; ** P < 0.01; *** P < 0.001.

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Pilot Certification, a Public Health Pest Appli-cator License, and an Aerial Pesticide Applica-tor License.

ACKNOWLEDGMENTS

The authors thank the Collier Mosquito Control District (CMCD) Board of Com-missioners and all the employees at CMCD who participated in sample collection and technical assistance. We also thank C. Royals (Valent Biosciences) for initiating the col-laborative work with Valent Biosciences to perform spray system calibration and char-acterization.

REFERENCES CITED

Carrasco-Escobar G, Manrique E, Ruiz-Cabrejos J, Saa-vedra M, Alava F, Bickersmith S, Prussing C, Vinetz JM, Conn JE, Moreno M, Gamboa D. 2019. High-accuracy detection of malaria vector larval habitats using drone-based multispectral imagery. PLoS Negl Trop Dis 13:e0007105.

Hardy A, Makame M, Cross D, Majambere S, Msellem M. 2017. Using low-cost drones to map malaria vec-tor habitats. Parasit Vectors 10:29.

Hass-Stapleton EJ, Barretto MC, Castillo EB, Clausnitzer RJ, Ferdan RL. 2019. Assessing mosquito breeding sites and abundance using unmanned aircraft. J Am Mosq Control Assoc. 35(3): 228-232.

Hunter JE, Gannon TW, Richardson RJ, Yelverton FH, Leon RG. 2019. Integration of remote weed-map-ping and an autonomous spraying unmanned aerial vehicle for site-specific weed management. Pest Man-ag Sci. doi: 10.1002/ps.5651.

Li CX, Zhang YM, D YD, Zhou MH, Zhang HD, Chen HN, Tian Y, Yang WF, Wu XQ, Chu HL, Zhao TY. 2016. An unmanned aerial vehicle-mounted cold mist spray of permethrin and tetramethylflutherin targeting Aedes albopictus in China. J Am Mosq Control Assoc. 32(1): 59-62.

Qin WC, Qiu BJ, Xue XY, Chen C, Xu ZF, Zhou QQ. 2016. Droplet deposition and control effect of in-secticides sprayed with an unmanned aerial vehicle against plant hoppers. J Crop Prot 85:79–88.

Wang G, Lan Y, Qi H, Hewitt A, Han Y. 2019. Field evalu-ation of an unmanned aerial vehicle (UAV) sprayer: effect of spray volume on deposition and the control of pests and disease in wheat. Pest Manag Sci.75(6): 1546-1555.

Xiao J, Chen L, Pan F, Deng Y, Ding C, Liao M, Su X, Cao H. 2019. Application method affects pesticide efficiency and effectiveness in wheat fields. Pest Man-ag Sci. doi: 10.1002/ps.5635.

91

SUBMITTED ABSTRACTS OF THE 91ST ANNUAL MEETING

MOSQUITO COMMUNITY COMPOSITION AND SEASONAL DISTRIBUTION IN NORTHEASTERN FLORIDA

BRYAN V. GIORDANO1, LINDSAY CAMPBELL1, SUE BARTLETT2, CAROLINE EFSTATHION3, RUI-DE XUE3, AND RANDY WISHARD4.

1University of Florida, IFAS – Florida Medical Entomology Laboratory, Vero Beach, Florida 32962

2Volusia County Mosquito Control, New Smyrna Beach, Florida 32168

3Anastasia Mosquito Control, St. Augustine, Florida 32092

4City of Jacksonville Mosquito Control, Jacksonville, Florida 32218

In the current work, we have compiled five years of mosquito surveillance data from Volusia County located in northeastern Florida. We will assess mosquito abundance and spe-cies diversity over time and identify trends resulting from trap type. Seasonal distributions of vector species of eastern equine encephalitis virus and West Nile virus will be presented. These data can be used to better inform mosquito control personnel of species of interests, optimal trapping methodologies, and seasonal peaks in mosquito abundance.

ORNAMENTAL BROMELIADS OF LOCAL BOTANICAL GARDENS SERVE AS BREEDING SITES FOR PYRETHROID-

RESISTANT DISEASE VECTOR MOSQUITO SPECIES IN COLLIER COUNTY, FLORIDA

ALEXANDRIA WATKINS1,2*, CAMERON COLE3, EMORY BABCOCK1,4,5, AND KEIRA J. LUCAS1

1Collier Mosquito Control District, 600 North Road Naples, FL 34104

2Florida Gulf Coast University, Fort Myers, FL 33965

3Naples Botanical Garden, 4820 Bayshore Drive, Naples, FL 34112

4School of Public Health and Tropical Medicine, Tulane University, 1440 Canal Street New Orleans, LA 70112

5CDC Southeastern Center of Excellence in Vector Borne Disease, 2055 Mowry Road, Gainesville FL 32611

The Naples Botanical Garden located in Collier County, Florida attracts over 220,000 visitors and tourists each year. The gardens house a collection of plants from around the world, and includes a featured area for over 100 species of exotic and native bromeli-


ads. Ornamental bromeliads have previously been investigated to distinguish them as a breeding site for mosquitoes due to their “tank” that can serve as a safe-haven for mos-quito eggs and larvae. Recent studies in Miami-Dade County have revealed Aedes aegypti successfully utilize tank-type bromeliads as breeding sites and is the dominant species found in ornamental bromeliads. In 2018, we identified that the dominant species of mosquito found in bromeliads within residential areas of Collier County are Wyeomyia mitchellii, with some bromeliads harboring disease vectors such as Ae. aegypti, Ae. albopic-tus, and Culex quinquefasciatus. Given the relationship between disease vector mosquito species and bromeliads, the Naples Botanical Gardens was investigated for species di-versity and resistance in their large-tanked bromeliads. A survey of mosquito species in bromeliads of the Naples Botanical Gardens indicated that neither Wyeomyia spp. nor Ae. Aegypti were the dominant species found in the gardens. Instead, the most abundant spe-cies found were Cx. quinquefasciatus, Ae. albopictus, and Cx. nigrapalpus. With the ongoing threat of vector borne disease, and the abundance of vector mosquitoes and the heavy tourist traffic in the gardens, resistance testing was performed on the most abundant species in order to assess each materials ability to successfully target these species in the event of a disease outbreak.

SURVEILLANCE REPORT OF AEDES AEGYPTI AND AEDES ALBOPICTUS IN ST. JOHNS COUNTY, 2019

PRESENTED BY STEVE SMOLEROFF


While Ae. aegypti has been absent in North-East Florida since the early 1990s, its re-appearance around 2013 alongside the introduction of the Zika virus to the Americas in 2016 prompted diligent surveillance of Aedes mosquitoes. While Aedes aegypti and Aedes albopictus are competent vectors of yellow fever, chikungunya, and dengue, moni-toring the populations of these mosquitoes became inevitably more important so that treatment efforts could be carried out in specific high risk areas. Historic downtown St. Augustine and its surrounding areas were chosen for extensive control and surveillance efforts because of its diverse population of tourists and travelers from around the world. These tourists are active outdoors sightseeing, dining, and doing tourist activities where they are at risk of getting bitten by Ae. aegypti and Ae. albopictus mosquitoes. Surveillance of Aedes mosquitoes over the last few years has been achieved mainly with the use of BG Sentinel 2 traps which are baited with BG lure and dry ice. A total of 12 traps are placed once a week in previously selected sites used within and around the downtown Saint Augustine area. In addition to the BG traps, ovi cups are placed at each BG site to detect the presence of gravid Aedes mosquitoes. With these surveillance techniques combined high risk “hot spots” can be identified for treatment efforts. In addition to the regular trapping sites, additional BG traps and ovi cups are frequently placed in random spots around the county to actively be on lookout for new populations of Ae. aegypti mosqui-toes.

Annual Meeting Abstracts 93

TRAP MODIFICATION TO INCREASE THE CAPTURE RATE OF AEDES AEGYPTI

PRESENTED BY LEA BANGONAN


Surveillance of Aedes aegypti is a priority for mosquito control districts because it is a vector of several arboviruses. Unfortunately, this species is not efficiently captured by the most commonly used mosquito traps, such as light traps or gravid traps. Its cryptic nature and avoidance of the most common trap types complicates efforts at reducing popula-tions. Our objective was to see if targeting multiple physiological states of Ae. aegypti by modifying traps could increase capture rates. We modified three traps, the AGO bucket, BG bowl, and the CDC gravid trap. Specific modifications were different for each trap, but all traps were modified to contain a fan and a BG lure. Traps were first tested in a screened enclosure where 200 female Ae. aegypti, half of which were gravid, were released inside to test whether traps functioned after modifications. Traps were then tested in the field at three sites, each site having one of each trap type. Traps were run for 24 hours for nine trap nights. Collected mosquitoes were identified to Genus and species and separated into non-gravid and gravid females. All three traps functioned after modification and captured both host seeking and gravid females in the screen enclosures. The BG bowl collected the most non-gravid and gravid females in both the screen enclosures and the field. Modifica-tion of current traps could help increase capture rates of Ae. aegypti for surveillance efforts.

VECTOR COMPETENCE OF CHINESE AEDES AEGYPTI AND AE. ALBOPICTUS FOR TRANSMISSION OF ZIKA VIRUS

XIAO-XIA GUO

State Key Laboratory of Pathogen and Biosecurity, Institute of Microbiology and Epidemiology, Beijing 100071, China

E-mail address: [email protected]

Zika virus (ZIKV) has become a serious threat to global health since the outbreak in Brazil in 2015. Additional Chinese cases have continuously been reported since the first case of laboratory-confirmed ZIKV infection in China on February 6, 2016. Aedes aegypti and Aedes albopictus are the important vector for ZIKV. This study shows that two strains of Ae. aegypti from China exhibit high levels of midgut infection and highly disseminated infection of salivary glands and ovaries. Both strains can transmit ZIKV to infant mice bitten by infec-tious mosquitoes. Moreover, the results provide the first evidence of transovarial transmis-sion of ZIKV in mosquitoes. The study indicates that the two Ae. aegypti strains are not only effective transmission vectors but also persistent survival hosts for ZIKV during unfavorable inter-epidemic periods. Also, Ae. albopictus can infect and successfully transmit zika virus to infant mice. Transovarial transmission was observed during the first gonotrophic cycle. This function as a reservoir of infection has epidemiological implications that further enhance the risk of potential future outbreaks.


EFFECTS OF IRRADIATION ON THE BLOOD FEEDING ACTIVITY OF AEDES AEGYPTI

PRESENTED BY COURTNEY A. CUNNINGHAM


The St. Augustine strain of female Aedes aegypti Linn. has been irradiated by different doses in the laboratory. The irradiated female mosquitoes have been offered blood through a membrane and the percentages of partial and full blood engorgement have been deter-mined and counted. The results showed that the increase of irradiation doses decrease the percentage of full blood engorgement, compared with the group of untreated control. The current used radiation dose at 50gy for the sterilization of male and female Ae. aegypti still has about 10% successful blood feeding. The release irradiated females for control of a population of mosquitoes are still need to be addressed.

HOST AVIDITY AND DEET REPELLENCY IN IRRADIATED AEDES AEGYPTI

RUI-DE XUE

Anastasia Mosquito Control District, 120 EOC Dr., St. Augustine, FL 32092

KENNETH J. LINTHICUM

USDA/CMAVE, 1600-1700 SW 23rd Dr., Gainesville, FL 32608

AMCD has collaborated with USDA/Center for Medical, Agricultural, and Veterinary Entomology, and University of Florida’s Department of Entomology and Nematology to re-lease irradiated male Ae. aegypti mosquitoes to part of Anastasia Island and downtown of St. Augustine to control natural populations of Ae. aegypti since 2017. During the experimental releases, several local residents asked us whether any irradiated female mosquitoes escaped when separating the females from males before release and if the irradiated females are still biting people and if the Deet repellent still works for the irradiated female mosquitoes. We used 100 irradiated 5-7 day old female mosquitoes in a USDA standard cage for repel-lent testing and 100 non-irradiated females in other USDA testing cage. A volunteer used a garden glove to protect hand and exposed untreated forearm to each cage for 1 minute and the number of mosquitoes landed and probed on forearm was counted and recorded. Then, the volunteer’s forearm treated by 15% Deet repellent exposed to each cage for three minutes and the number of mosquitoes landed and probed was counted and recorded. The testing was conducted /exposed at 30 minute interval until the bite on skin by mosquitoes was confirmed. The results showed that the host avidity (attacking/ min) in irradiated mos-quitoes was significantly lower than the attacking rate in the non- irradiated mosquitoes, and the Deet repellent (15%) on volunteer forearm provided 1.5-2 hrs longer time than against the non-irradiated female mosquitoes. The experiment was repeated three times at different week.


COMPARING THE EFFICACY OF AUTOCIDAL GRAVID OVITRAPS AND IN2CARE TRAPS ON AEDES AEGYPTI AND

AEDES ALBOPICTUS IN ST. JOHNS COUNTY, FLORIDA

PRESENTED BY DENA AUTRY

Anastasia Mosquito Control District, St. Augustine, FL 32092

This field study compared the efficacy of the In2care Mosquito Trap and CDC’s Auto-cidal Gravid Ovitrap (AGO). Mosquito Control programs are seeking cost and labor effec-tive treatment plans for the cryptic breeding Aedes mosquitoes. This study consisted of one control and two treatment sites, and each treatment site had either 100 In2care Mosquito Traps or 100 CDC’s Autocidal Gravid Ovitraps. Each site was monitored using BG-Sentinel 2 mosquito traps and Oviposition cups. Raw data imply both sets of traps were effectively working; post surveillance shows an increase in Aedes populations.

BIO-EFFICACY OF THE SAFI PRODUCT (CUSO4.5H2O) AS A LARVICIDE AGAINST MOSQUITO GENERA—AEDES, CULEX

AND ANOPHELES AS A FUNCTION OF CONCENTRATION (PPM), TIME, AND APPLICATION

PRESENTED BY KAI BLORE


A pilot study was conducted to evaluate the efficacy of SAFI product (CuSO4 · 5H2O) as a larvicide against three mosquito genera. Concentrations ranging between 5–100ppm were tested in both lab and semi-field conditions against third instar Aedes aegypti, Culex quinquefas-ciatus and Anopheles quadrimaculatus larvae to determine LC50 values at 72h. Four replicates of each were conducted. Under lab conditions, 10 larvae of each species were introduced into separate 100mL of treated water with mortality observed at 24, 48 and 72h. For the semi-field condition, 25 Ae. aegypti larvae were introduced into 5-gallon plastic buckets kept outside under a tented canopy and filled with 9000mL of treated water. For Cx. quinquefasciatus and An. quadrimaculatus, 200 larvae were introduced into 250 gallon concrete pools filled with 200 gallons of treated water. Mortality was observed at 24, 48 and 72h. Study results indicate that concentrations below 5ppm show no observable difference in larval mortality from the control. Furthermore, larval mortality was higher with increasing concentration and time. Of the three species, Cx. quinquefasciatus was most susceptible to the larvicide. The SAFI product was much less toxic to Ae. aegypti (72h LC50lab=28ppm; LC50semi-field=34ppm) than both Cx. quinquefasciatus (72h LC50lab=4ppm; LC50semi-field=14ppm) and An. quadrimaculatus (72h LC50lab=4ppm; LC50semi-field=15ppm) for lab and semi-field conditions respectively. The study demonstrates effectiveness of SAFI product at low concentrations as a potential larvicide. Additional studies are necessary to confirm its effectiveness and further define parameters for real-world application.


LABORATORY EVALUATION OF BORIC ACID SUGAR BAIT AGAINST THE PUERTO RICO RESISTANT STRAIN OF

AEDES AEGYPTI

PRESENTED BY MANDI PEARSON


This study evaluated the effects of toxic sugar bait (TSB) against the resistant Puerto Rico strain of Aedes aegypti in the laboratory. Puerto Rico and Orlando 1952 Ae. aegypti strains were aspirated into eight bug dorms, each containing approximately 100 mosquitoes. Each strain had two female and two male dorms; one bug dorm of each sex received the TSB (1% boric acid, 5% sugar) and the others received the control solution (5% sugar). Mortality counts were taken at 24, 48, and 72 hours. Cumulative mortalities of each strain were compared using a Student’s t-test. No significant differences were found between the cumulative average mortality between the Puerto Rico and Orlando 1952 strains at any time interval for both males and females.

EFFECTS OF BARRIER TREATMENT AND GROUND ULV APPLICATION OF ADULTICIDES ON

THE HONEY BEE, APIS MELLIFERA

YONGXING JIANG 1, HUSSEIN SANCHEZ-ARROYO2, ROBERT HORSBURGH3, PHILIP KOEHLER2, AND RUI-DE XUE4

1City of Gainesville Mosquito Control, Gainesville, FL 32601

2Department of Entomology & Nematology, University of Florida

3Bureau of Plant and Apiary inspection, Florida Department of Agriculture and Consumer Services

4Anastasia Mosquito Control District, St. Augustine, FL 32092

As the number of backyard beekeepers surged in the past decade in Florida, so have the concerns of the impact of mosquito adulticide applications on backyard honey bees. The goal of this study was to determine the effects of routine applications of barrier treatments and truck-based ultra-low volume (ULV) treatments on honey bees under conditions that re-flect actual field exposure in Gainesville, Florida. Caged bees (Apis mellifera) and mosquitoes (Aedes albopictus) were directly exposed to bifenthrin (TalstarP®, AI 7.9%, FMC Corporation, Philadelphia, PA) as a barrier treatment at the maximum rate (1fl.oz./gallon). 24-h mortal-ity of caged bees and mosquitoes was 96.7%, 78.3%, 80% and 91.7%, 73.3%, 58.3%, respec-tively, at 0, 10 and 20 ft. distance from application site. Laboratory leaf bioassay results show that 24-h mortality of bees and mosquitoes decreased as the leaf sample distance increased (0, 10 and 20 ft.) and also decreased over time (0, 1, 2 and 3 wk.). When bee hives were placed in a residential backyard (150 ft. from spray line) and exposed to permethrin (Aqua-Kontrol 30-30, Univar Environmental Sciences, Austin, Texas) via ULV application at the medium application rate of 0.00175 lbs /acre, no significant differences were observed in the number of dead bees per hive, mortality of caged bees and mosquitoes, and total weight of bees per hive between the treatment and control. Results suggest that different mosquito application techniques may have different effects on honey bee survivorship.


EVLUATING THE EFFICACY OF THE TRUCK-MOUNTED SUPER DUTY A1 MIST SPRAYER AGAINST AEDES AEGYPTI

AND AEDES ALBOPICTUS IN URBAN ENVIRONMENTS IN LEE COUNTY

KARA TYLER-JULIAN, RACHEL MORREALE, DAVID HOEL, AND AARON LIOYD

Lee County Mosquito Control District, Lehigh Acres, FL 33971

Container-breeding mosquitoes present a unique challenge to mosquito control in ur-ban environments. Due to the cryptic nature of their breeding habitat, targeted larvicide treatments have not been feasible or effective against these mosquitoes. Traditionally, adul-ticide applications have been the main method of managing these species in urban environ-ments. The recent development of Wide-Area Larvicide application technologies provides mosquito control entities with a new ability to broadcast larvicides through neighborhoods and yards allowing droplets to penetrate into these cryptic habitats. Lee County Mosquito Control District tested the efficacy of a new truck-mounted A1 Mist Sprayer using Vecto-bac WDG and Altosid SR-20 larvicides in two urban environments in the summer of 2019. Runway trials were conducted to characterize droplets and larval mortality. Larval bioassays showed 100% mortality of susceptible third instar Aedes aegypti up to 350 feet downwind of the A1 sprayer using Vectobac WDG at the mid-level RPM. Field trials were then conducted using larval bioassays in a residential location representing various real-world challenges. BG traps were set and collected twice per week to monitor adult populations in the test and control areas. The field larval bioassays also resulted in 100% larval mortality using Vecto-bac WDG in all conditions. BG trap counts revealed 60- 67% control of Ae. aegypti and 33% control of Ae. albopictus populations by Vectobac WDG. Altosid SR-20 was less effective when used in this context, exerting 27% control over Ae. aegypti and 37% control over Ae. albop-ictus. Populations of Ae. aegypti and Ae. albopictus are suppressed using these larvicides in a Wide-Area Larvicide application and this effect was seen for two weeks following application.

ARTIFICIAL MEMBRANE FEEDING TECHNIQUES FOR MOSQUITO MASS REARING

STEVEN STENHOUSE, RACHEL MORREALE, AARON LIOYD, DAVID HOEL, AND T. WAYNE GALE


The mass rearing of insects is an essential component of any sterile insect technique (SIT) program. Mosquito SIT programs, such as that currently being developed by Lee County Mosquito Control District to control Aedes aegypti, similarly require considerable numbers of adult males in order to be successful. However, the mass rearing of hematopha-gous arthropods presents unique challenges. The use of live animals for blood feeding is expensive and time consuming, particularly for a mass rearing program. Furthermore, there is the risk that host animals can serve as sources of pathogen introduction into the mosquito colony. To overcome some of these difficulties, we set about developing and modifying a number of artificial feeding methods in order to move away from the use of live vertebrate hosts. We ultimately developed an artificial membrane feeding method that is simple to


prepare and use. Our method uses commercially available food-grade pork blood loaded onto borosilicate watch glasses and covered with Parafilm-M membranes. These are placed on heated glass dishes filled with an absorbent clay material. This material helps to retain heat and also aids in clean-up. With this simple set up we have been able to efficiently feed multiple mosquito cages simultaneously while also attaining egg yields comparable to those obtained from vertebrate host feedings.

SENTINEL CHICKEN SURVEILLANCE METHODOLOGIES AND BEST PRACTICES

MILTON STERLING


Mosquito-borne disease monitoring is among the arsenal of surveillance tools being uti-lized in an integrated mosquito management program. Because no single surveillance tool is best, there is a necessity to integrate and contemplate information from all sources. The sentinel chicken surveillance program is an essential epidemiological means that is used for monitoring WNV, SLEV, EEEV, and other arboviruses. It provides accurate data on the time and location of viral transmission, and allows for uninterrupted arbovirus detection activity throughout the season.

This presentation provides an overview of the sentinel chicken surveillance methods and best practices as it relates to mosquito-borne disease surveillance. Topics include program methods and tools utilized in serum collection, serum data analysis and interpretation, and the importance of sentinel chicken monitoring for vector populations.

VIRTUAL MEDICAL INSECT COLLECTION OF INSTITUTE OF MICROBIOLOGY AND EPIDEMIOLOGY

MING-YU WU

State Key Laboratory of Pathogen and Biosecurity, Institute of Microbiology and Epidemiology, Beijing 100071, China

E-mail address: [email protected]

Medical Insect Collection of Beijing Institute of Microbiology and Epidemiology is the largest medical insect museum with the most complete insect species in Asia. With the comprehensive collection of generations of taxonomists nationwide, and exchanges with collectors at home and abroad, it has accumulated more than 4500 species and two million specimens, including almost 1200 rare and valuable type specimens. There are 12 major species of mosquitoes, midges, blackflies, horseflies, flies, fleas, sucking lice, ticks, mites, sandflies, bedbugs, cockroaches, and also collections of rodents and other host animals of the medical insects. Standardized sorting preservation and digitized description of insect specimens will be conducted in order to establish Virtual Medical Insect Collection.


MORE DATA, MORE PROBLEMS: HOW CMCD IS MANAGING ITS EVER-EXPANDING SURVEILLANCE PROGRAM

REBECCA HEINIG, PETER BRAKE, NATE PHILLIPS, AND KEIRA LUCAS

Collier Mosquito Control District, 600 North Road, Naples, FL 34104

Due to upgrades in our surveillance coverage, District size, and technological capabili-ties, Collier Mosquito Control District is generating a much higher volume and diversity of data than ever before. As a result, we’ve had to get smarter about how we record, maintain, and analyze those data. In this presentation, I’ll talk about some of the methods we’re utiliz-ing to manage our multiple data streams, including software enhancements and task auto-mation, as well as new tools we’re developing to interact with the data and turn them into actionable information.

ENHANCING RESOURCES AND CAPACITY OF ENTITIES THAT DO MOSQUITO CONTROL IN TEXAS

WHITNEY A. QUALLS

Anastasia mosquito Control District, St. Augustine, FL 32092

Hurricanes Harvey, Irma, and Maria in 2017 resulted in severe weather disasters through-out the Gulf Coast regions, Florida, and Puerto Rico causing a combined 265 billion dollars in damages, being three of the top six costliest hurricanes in history. In response, the United States Centers for Disease Control and Prevention activated its cooperative agreement to aid in response, recovery, preparation, mitigation, and other expenses related to these three hurricanes. Areas impacted by the three hurricanes were invited to apply for funds under the 2017 Hurricane Crisis Cooperative Agreement (Hurricane Crisis CoAg) to build capac-ity in several public health areas directly related to Harvey, Irma, and Maria. The Texas De-partment of State Health Services under the Hurricane Crisis CoAg received >$6,000,000 to use towards increasing capacity and to aid in recovery for vector control projects related to Hurricane Harvey. Funds have been used to increase State and Local Capacity. At the local level funds have been contracted to local health departments and mosquito control districts throughout jurisdictions impacted by Hurricane Harvey. A total of 28 contracts to entities that perform mosquito control have been awarded. The presentation focused on projects, statuses, and challenges for the Hurricane Harvey impacted jurisdictions in Texas that were awarded funds.

Editorial Acknowledgements The following scientists have provided valued assistance in reviewing articles for this issue.

Names followed by an asterisk (*) are of individuals who have reviewed two or more

manuscripts. A special thank you is given to these scientists by the editors. The editors also

acknowledge N. Burkett-Cadena, C. Efstathion, M. Farooq, Y. Jiang, A. Junnila, G. Muller,

and W. Qualls as the Guest Editors for 1 manuscript’s peer-review and editing process. Also,

the editor thanks C. Hall for editorial assistant and proof reading.

R. Aldridge*

V. Aryaprema*

M. Aubuchon

C. Bibbs*

K. Blore*

C. Boohene

S. Britch

E. Buckner

N. Burkett-Cadena

J. Cilek

R. Connelly

D. Dixon

C. Efstathion*

A. Faraji

M. Farooq*

E. Haas-Stapleton

K. Hung

N. Indelicato

Y. Jiang

A. Junnila

E. Khater

D. Kline

P. Koehler

C. Lesser*

A. Lloyd

K. Lucas

D. Mathias

B. McGregor

G. Muller

E. Norris

P. Obenauer

W. Qualls*

M. Rile

S. Smoleroff

T. Su

I. Unlu

K. Williams

A. Wilke

RD Xue*

100

JOURNAL OF THE FLORIDAMOSQUITO CONTROLASSOCIATION

INFORMATION FOR CONTRIBUTORS

The Journal of the FMCA encourages the submission of unpublished manuscripts in the field of biology and control of mosquitoes and mosquito-borne diseases.

Manuscripts in MS Word or Rich Text Format should be sent to the Editor, JFMCA, Dr. Rui-De Xue, Anastasia Mosquito Control District, 120 EOC Drive, St. Augustine, FL 32092, USA by e-mail attachment at [email protected]. Each manuscript will be sent to 2 or 3 authorities for peer review. Their comments and recommendations re-main anonymous and are forwarded to the authors. The Editorial Board of the Journal serves as an adjudication panel for resolving conflicts between authors and the editors. Manuscripts require double space throughout, including references, and indent paragraphs. A title page containing the corresponding author’s complete mailing address, telephone and fax numbers, and e-mail address should be included, as well as the name and affiliations of all co-authors. Each article must be accomplished by an abstract not longer than 3% of the paper and a short title of not more than 40 letters to serve as a running head. Five im-portant key words are required. The paper should be divided as follows: abstract, key words, introduction, material and methods, results, discussion, acknowledgments, and references cited. References should conform to the style presented in this issue.

Tables should be used sparingly and self-explanatory. Each table should be double spaced on its own page and all acronyms should be explained in a footnote. Only high quality, computer-generated graphs will be accepted. Figure keys should be included on the figure itself. Images should be high-quality graphics files with sharp focus and good contrast.

The journal accepts the submission of operational notes or scientific notes. The notes may contain 1 or 2 tables and or illustrations, with acknowledgements included in the last paragraph of the text. There should be an abstract and key words. No section headings are needed.

Following peer review, authors are required to submit their revised manuscript in elec-tronic format. Authors are expected to read proofs carefully, make corrections, answer que-ries, and return proofs promptly to the editors.

FLORIDA MOSQUITO CONTROLASSOCIATION

The mission of the FMCA (www.floridamosquito.org) is to promote effective and envi-ronmentally sound control of disease-transmitting and pestiferous mosquitoes and other ar-thropods of public health importance, develop and enhance public interest, awareness, and support for the control of mosquitoes, and provide for the scientific advancement of mem-bers through our meetings, training, and education. The FMCA is a non-profit, technical, scientific, and educational association and publishes the Journal of The Florida Mosquito Control Association in the furtherance of these objectives.

BOARD OF DIRECTORS, 2019-2020 OFFICERS

Donnie Powers, PresidentFMC Corporation, (205) 641-1157

James Clauson, President-ElectBeach Mosquito Control District, Panama City Beach, FL 32407, (850)233-5030

Christopher Lesser, Vice PresidentManatee County Mosquito Control District, Palmetto, FL 34221, (941)981-3895

Andrea Leal, Immediate Past PresidentFlorida Keys Mosquito Control District, 5224 College Road, Stock Island, Key West, FL 33040

Martin O’neil, Executive [email protected], 1-866-Go4-FMCA

REgIOnAl DIRECTORS

Austin Horton, northwest RegionGulf County Mosquito Control, 1001 10th Street, Port St. Joe, FL 32456, (850)227-1401

Peter Jiang, Ph.D. northeast RegionCity of Gainesville Mosquito Control, 405 NW 39th Avenue, Gainesville, FL 32169

Katie Williams, Southwest RegionManatee County Mosquito Control District, 2317 2nd Avenue West, Palmetto, FL 34221, (941)722-3720

Sherry Burroughs, Southeast RegionIndian River Mosquito Control District, 5655 41st Street, Vero Beach, FL 32967-1905

Wendy DeCorah, Industry RepresentativeADAPCO, 550 Aero Lane, Sanford, FL 32771, (407) 328-6576

Barry Alto, Member-at-largeUniversity of Florida/FMEL, 200 9th Street SE, Vero Beach, FL 32962, (772)778-7200

John Magee, Commissioner RepresentativeSouth Walton Mosquito Control District, P.O. Box 1130, Santa Rosa Beach, FL 32459, (850) 267-2112

Documents

Journal oF THE FlorIDa MoSQuITo ConTrol aSSoCIaTIon