Final Report. Efficacy Evaluation of Insecticidal Ovitraps for Control of Domestic Mosquito Vectors of Dengue, Chikungunya, and Zika Viruses

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1 Final Report Efficacy Evaluation of Insecticidal Ovitraps for Control of Domestic Mosquito Vectors of Dengue, Chikungunya, and Zika Viruses FDACS Contract #23581 John Smith and Taylor Thrall August 27, 2017 Abstract The aim of this study was to evaluate the efficacy of the BG-GAT, In2Care, CDC-AGO, and Springstar Trap-N-Kill ovitraps for control of Aedes aegypti and Aedes albopictus in outdoor screen enclosures. The most significant findings were: 1) black 5-gal buckets and bromeliad plants, Aechmea fasciata, generally provided more attraction for oviposition than did the ovitraps; 2) the CDC-AGO consistently trapped more than 50% of the released gravids; 3) the In2Care and BG-GAT produced the greatest amount of larval/pupal mortality compared to the control (72% and 52%, respectively); 3) the In2Care provided effective autodissemination of pyriproxyfen; and, 4) very few adult cohorts emerged from oviposition sites placed near the In2Care compared to other ovitraps evaluated. Introduction Ovitraps have been used for many years to detect container-breeding Aedes (Fay and Perry 1965, and Fay and Eliason 1966). More recently, these traps have been exploited for target delivery of insecticides (Chan 1973, Chan et al. 1977, and Zeichner and Perich 1999). According to the World Health Organization (2016) lethal ovitraps incorporate an insecticide on an oviposition substrate; autocidal ovitraps allow oviposition, but prevent adult emergence; and, sticky ovitraps capture mosquitoes as they land. As gravid mosquitoes visit these traps, the insect and/or eggs are treated by contacting a residual pesticide i.e., an insecticide, sticky adhesive, or oil. Insecticide contaminated mosquitoes can transfer lethal dosages to other breeding sites (i.e., autodisseminate), thereby killing the progeny of other container-inhabiting mosquitoes. 1

2 Research Questions 1. How attractive are the BG-GAT, In2Care, CDC-AGO, and Springstar Trap-N-Kill ovitraps compared to other man-made and natural water-holding containers to Ae. aegypti and Ae. albopictus? 2. Will lethal/autocidal/sticky ovitraps effectively control Ae. aegypti and Ae. albopictus, and if so, which of the most popular ovitraps are most effective? 3. Will ovitrap insecticide-contaminated mosquitoes disseminate a lethal dose to other mosquito production sites, and if so, what is the impact on developing mosquito populations? Objectives 1. Quantify the attractiveness of the four ovitraps and surrounding natural and man-made containers to Ae. aegypti and Ae. albopictus. 2. Measure change in cohort Ae. aegypti and Ae. albopictus populations when exposed to ovitraps in environments with multiple water-holding containers. 3. Assess impact of insecticides transferred by gravid mosquitoes from autocidal ovitraps to nearby production sites. Mosquito Containment Plan Materials and Methods Prior to project initiation, the Florida State University Environmental Health and Safety Department required development of a plan to insure mosquito rearing and releases were conducted in a contained manner. The attached plan was developed by the P.I. in consultation with the Department and campus administrators for these purposes (Attachment I). Study Site These studies were conducted in a tall pine forest on the northeast side of the Florida State University Panama City (FSU PC) campus during the spring and summer of The experimental site was mechanically cleared of undergrowth to facilitate positioning screen enclosures. Weeds inside enclosures were controlled by string and blade trimming and with the same methods on the outside except to include occasional spraying with the herbicide, glyphosate. An MMX CO2-baited mosquito trap was placed at the center of the experimental site for mosquito escape detection. 2

3 Screen Enclosures Five 12'X24'X8' (300 sq. ft.) model #70591 Shelter Logic greenhouses were assembled and covered with 14X16 mesh nylon screen. The screen was seamed with Gorilla Clear Repair Tape applied with a heat gun, roller, and reinforced at stress points with staples. 12'X4'X8' vestibules with two Velcro doors were installed in each enclosure. Sand bags and 2"X4" lumber were used to seal the screen at the ground perimeter (Fig. 1). Fig. 1. Drone photograph of five 300 sq. ft. screen enclosures used for ovitrap study. Mosquito Rearing A University of Notre Dame strain of Aedes aegypti was colonized at FSU PC in Percival incubators operating at 28 C and 12/12 light/dark cycle and 60-80% R.H. Eggs were hatched (1-2 days) in water preconditioned with a 3:2 part liver powder and Brewer s yeast mixture diluted at 50 ml of mixed powder to 1 L deionized water. Larvae were fed ca. 1 ml each day until pupation. Pupae were placed in small plastic bowls inside 1 ft 2 Bioquip Model 1450B cages for adult emergence. Adults began to emerge 8 days after egg set and were maintained on 10% sugar-water soaked cotton. Three days later, a water-warmed lamb-skin condom filled with citrated chicken blood was provided for three days to produce gravids. 100 gravids were sorted, counted, and placed in each of five 1 ft 2 cages for release in the outdoor screen enclosures. 3

4 Environmental Monitoring Onset HOBO UX or U data loggers were deployed near the center location of the outdoor screen enclosures to record temperature and humidity. Onset Hoboware Pro software was used to graphically display the data. Supplemental graphs of rainfall amounts and rate were obtained from the FSU WeatherSTEM station located on campus. Experimental Design The following treatments were tested in five trials (Fig. 2): 1. BG-GAT treated with bifenthrin at 1 fluid oz. per gallon applied at 1 gal./1000 sq. ft. or 7.9 ml/300 in 2 applied to the inside surface of the upper clear plastic chamber; 2. In2Care with the growth hormone, pyriproxyfen, and the fungal insecticide, Beauveria bassiana; 3. CDC-AGO with black, sticky adhesive styrene cylinder positioned in the trap entry; 4. Springstar Trap-N-Kill (TNK) with dichlorvos; and, 5. Negative control, i.e., water-holding plastic bottom of an untreated In2Care trap. Fig. 2. Ovitraps from left to right; TNK, CDC-AGO, In2Care, BG-GAT, and Control. The treatments were randomly assigned and placed individually in the center of the five screen enclosures. After each trial, treatments were rotated clockwise to the next enclosure so that each treatment was tested in all enclosures. Additional oviposition sites consisting of a 1- pint mason jar, 5-gallon black plastic bucket, used tire, and bromeliad plant (Aechmea fasciata) were also placed in each enclosure about 3 feet from the near left, far left, far right, and near right corners in relation to the entry door (Fig. 3). These containers were rotated one position 4

5 clockwise when moved to the next enclosure after each trial. Oviposition sites assigned to the In2Care were rotated with the In2Care to minimize pyriproxyfen contamination. Fig. 3. Oviposition containers placed in screen enclosures along with treatments. From left to right: 5-gal. plastic bucket, Aechmea fasciata (bromeliad plant), 1 pint mason jar, and used tire. The black plastic bowl in the center was deployed as a negative control treatment. Ovitrap trials were conducted by adding infusion water to treatment and oviposition sites and inoculating each with 20 lab-reared 3 rd instar Aedes aegypti larvae contained in 200 ml of deionized water supplied with larval food. Infusion water was prepared by collecting dried laurel (Quercus laurifolia) and/or live oak (Quercus virginiana) leaves, placing 1.2 lbs. in a 12 in. diameter 5 gal buckets, filling with water to 10 in. level, and covering with lids to steep outdoors for 7 days. The following amounts of infusion water were added along with the 200 ml of water containing the larvae to the ovitraps and other oviposition sites: BG-GAT 1.8L; CDC-AGO 9.8 L; In2Care-3.3L; Trap-N-Kill-filled to drain holes; Control-0.8L; Mason Jar-¼ full; Tire-0.8L; Bucket-4.8L; and, Bromeliad-40 ml. A cohort of 100 gravid Aedes aegypti were released into each enclosure by placing the Bioquip holding cages inside the enclosures and opening the tops. The cages were left inside the enclosures with a cup of cotton-soaked sugar water. Two days post-adult release and larval inoculation, adults were recovered by aspiration and transferred into labelled screened cardboard cups supplied with sugar-water soaked cotton balls. Larvae and pupae were recovered by pouring the water from each container through a size 30 soil sieve or in a large plastic pan and then through the sieve for the bromeliad and treatments with drain holes. Separate sieves and pans were assigned for each treatment. The sieved larvae/pupae were transferred with deionized water-filled squirt bottles to labelled 5

6 screened plastic cups. Adult and larval/pupal holding containers were placed in laboratory incubators. Adult survival and mortality were recorded daily until all died or 21 days had transpired. Larval/pupal survival and mortality were recorded daily until adult emergence. Egg numbers were estimated after larval/pupal removal by wiping all oviposition sites with labelled 6.0 X 6.25 sections of Kimberly Clark Professional Food Service Towels and viewing under a dissecting microscope. Statistical Analysis All datasets were tested for normality with PROC Univariate (SASPC ver. 9.4). Log +1 transformations normalized distributions except for live adult recovery. Actual means were analyzed for these data; however, all other means or totals were analyzed as log-transformed with actual means or totals presented in charts. Variance was analyzed with PROC GLM or ANOVA for unbalanced or balanced datasets, respectively. Statistical comparisons of treatment means were performed with Tukey s Multiple Comparison Test at p<0.05. All datasets were presented with graphs or charts. Results/Discussion Significant progress was made despite a three-month delay in contract execution and an unanticipated P.I. illness. Eight Aedes aegypti ovitrap trials were completed. Trials 1-3 served to refine techniques. This study reports on trials 4-8. Aedes albopictus trials will be scheduled for late summer and fall, This will complete all project objectives. Environmental Data Temperature, rainfall amounts, and rates for trials 4-8 are provided in Figs Generally, temperatures ranged from mid-70s to low-80s for lows and low to mid-90s for highs. 1.8 and 0.35 rainfall occurred during Trials 5 and 7, respectively. The other trials were dry or 6

7 had minor rain (<0.11 ). The humidity sensor (available for Trial 8 only) measured relative humidity from 50% - 90%. Fig. 4. Trial 4: 5/31/17 6/2/17 Temperature Fig. 5. Trial 4: 5/31/17 6/2/17 Rainfall gauge and rate Fig. 6. Trial 5: 6/19/17 6/21/17 Temperature 7

8 Fig. 7. Trial 5: 6/19/17 6/21/17 Rainfall gauge and rate Fig. 8. Trial 6: 6/22/17 6/24/17 Temperature 8

9 Fig. 9. Trial 6: 6/22/17 6/24/17 Rainfall gauge and rate Fig. 10. Trial 7: 6/29/17 7/1/17 Temperature 9

10 Fig. 11. Trial 7: 6/29/17-7/1/17 Rainfall gauge and rate Fig. 12. Trial 8: 7/5/17 7/7/17 Temperature and relative humidity Fig. 13. Trial 8: 7/5/17 7/7/17 Rainfall gauge and rate 10

11 Ovitrap Attraction There was no significant difference (p>0.05) in the average number of Aedes aegypti eggs collected from all containers placed within the five ovitrap enclosures (Fig. 14). Oviposition was found to be highly variable by container (Fig. 15). In the BG-GAT enclosure, the bucket had significantly (p<0.05) more eggs than the BG-GAT (i.e., treatment) and the jar, although it was not significantly (p>0.05) different than the plant or tire. For the control, the plant and bucket had significantly (p<0.05) more eggs than the jar, although there was no significant difference (p>0.05) compared with the control treatment and tire. There was no significant difference (p>0.05) among the control, tire, or jar. For the TNK, the egging strip inside the TNK and the plant had significantly (p<0.05) more eggs than the jar or TNK treatment (i.e., eggs surrounding the water edge of the TNK container not including the strip). There was no significant difference (p>0.05) in eggs deposited on the strip, plant, bucket, or tire. There was also no significant difference (p>0.05) in eggs on the bucket, tire, and jar, nor any significant difference (p>0.05) between the jar and TNK treatment. For the In2Care, the bucket had significantly (p<0.05) more eggs than the plant or jar, although there was no significant difference (p>0.05) between it and the tire or In2Care treatment. The tire had significantly (p<0.05) more eggs than the jar. There was no significant difference (p>0.05) in the tire, In2Care treatment, and plant. For the CDC-AGO, the bucket and plant had significantly (p<0.05) more eggs than the CDC-AGO treatment and jar, but no significant difference (p>0.05) in the tire. There was no significant difference (p>0.05) in eggs in the tire, CDC-AGO treatment, and jar. Fig. 14. Mean number of Aedes aegypti eggs collected within treatment enclosure. 11

12 Fig. 15. Aedes aegypti oviposition by ovitrap enclosure and container. Different letters within ovitrap enclosure represent significant differences at p<0.05. Larval/Pupal Mortality There were significantly (p<0.05) more dead larvae recovered from all containers within the BG-GAT and TNK enclosures compared to the CDC-AGO (Fig.16); however there was no significant difference when comparing BG-GAT and TNK with the Control and In2Care. There was also no significant difference among the CDC-AGO, Control, and In2Care. Fig. 16. Larval mortality by ovitrap enclosure. 12

13 Significantly (p<0.05) more dead pupae occurred in the In2Care enclosure compared to the CDC-AGO and Control (Fig. 17). There were no significant differences (p>0.05) among the In2Care, BG-GAT, and TNK or the BG-GAT, TNK, CDC-AGO, and Control. Fig. 17. Pupal mortality by ovitrap enclosure. Significantly (p<0.05) more dead larvae and pupae combined occurred in the In2Care (72%) and the BG-GAT (52%) compared to the Control and CDC-AGO (Fig. 18). There was no significant difference (p>0.05) among the In2Care, BG-GAT and TNK. There was also no significant difference (p>0.05) between the TNK and Control, or the Control and CDC-AGO. The TNK had significantly (p<0.05) more dead larvae and pupae than the CDC-AGO. Fig. 18. Larval + pupal mortality by ovitrap enclosure. 13

14 A somewhat similar picture emerged when examining larval and pupal mortality in the individual ovitraps excluding data from the surrounding oviposition containers (Figs ). Significantly (p<0.05) more dead larvae occurred in the TNK and BG-GAT compared with the Control and CDC-AGO (Fig. 19). The In2Care was not significantly different (p>0.05) from the TNK and BG-GAT or the Control and CDC-AGO. A Fig. 19. Larval mortality by ovitrap. The In2Care, BG-GAT, and CDC-AGO produced significantly (p<0.05) more pupal mortality than the TNK; however, this mortality was not significantly different (p>0.05) than the Control (Fig. 20). Pupal mortality in the control was also not significantly (p<0.05) different than in the TNK. A Fig. 20. Pupal mortality by ovitrap. The BG-GAT produced significantly (p<0.05) more dead larvae and pupae (combined) than the CDC-AGO and Control; however, there was no significant difference (p>0.05) among the BG-GAT, In2Care, and TNK (Fig. 21). The latter two traps did not produce significantly different larval and pupal mortality than the Control or CDC-AGO. 14

15 Fig. 21. Larval + Pupal mortality by ovitrap. Cohort Post-Recovery Growth Stage Survival The number and survival of larvae, pupae, and adults recovered from the cohorts inoculated in all oviposition containers and ovitraps showed similar patterns of progression (Fig. 22). The most notable difference was in the comparative number and survival of adults. Adult numbers increased substantially in the Control, BG-GAT,CDC-AGO, and TNK enclosures before dropping off. Very few adults emerged in the In2Care enclosure, and those that did, did not survive long as compared to the other ovitrap enclosures. Also, pupal numbers dropped to zero at 4 days post recovery (i.e., 6 days post inoculation) for all ovitraps except the In2Care. Pupae in the In2Care enclosure diminished at day 3. 15

16 Fig. 22. Larval, pupal, and adult survival by ovitrap enclosure (N=5). Note: error bars are within the width of the markers. 16

17 Released/Recovered Adult Survival/Mortality There were no significant differences (p>0.05) in the number of gravid adults recovered after two days post-release among the ovitrap enclosures (Fig. 23). The average recovery per release was 10-20% with the In2Care enclosure having a greater range extending near 30%. Fig. 23. Live adult recovery by ovitrap enclosure two days post-release. There were significant differences (p<0.05) in the number of dead adults recovered (Fig. 24). Significantly (p<0.05) more were collected in the CDC-AGO compared to the other enclosures. The BG-GAT collected significantly (p<0.05) more adults than the TNK and Control. There were no significant differences between the BG-GAT and the In2Care or the TNK and Control. Important considerations here are that the CDC-AGO has a sticky board to capture mosquitoes and the BG-GAT has a catch screen beneath the bifenthrin-treated surface. The other traps did not have mechanisms for retaining mosquitoes, and thus relied on adults dying on the water surface. Fig. 24. Dead adult recovery by ovitrap enclosure two days post-release. 17

18 Comparisons of the percent adult survival by days post recovery between the control and the ovitrap treatments are presented in Fig. 25. From 1-12 days post recovery, adults had 10-20% higher survival in the In2Care enclosure compared to the Control. Survival in the BG- GAT was similar to the Control for 1-10 days; thereafter, it ranged ca % higher until day 21. Adults exposed to the CDC-AGO survived 7-14% greater than the Control at days Survival in the TNK was similar to the Control with minor deviations. 18

19 Fig. 25. Comparative survival of gravid Aedes aegypti in five ovitrap enclosures (N=5). Note: Error bars that do not appear are within the width of the markers. 19

20 Conclusions & Discussion Ovitrap Attraction There were no significant differences in the total number of eggs deposited in all containers among any of the ovitrap enclosures or the control. Upon closer examination of each container, oviposition was found to be highly variable. More frequently, the bucket and plant had significantly more eggs deposited than the ovitraps and jar. When in abundance, these containers may serve as more attractive oviposition sites than the ovitraps. Ovitrap Efficacy When placed in areas with man-made and nature containers, the In2Care and BG-GAT produced significant higher levels of combined larval and pupal mortality compared to the control. A similar level of mortality was observed in the TNK; however, it was not significantly different than the control. The CDC-AGO enclosure yielded larval and pupal mortality in the same range as the control. This trap was not designed to control larvae/pupae in the same way as the insecticidal traps. Upon closer examination of larval/pupal mortality in the ovitraps (not including the other containers in the enclosures), the BG-GAT was the only trap that produced mortality significantly higher than the control. Mortality in the In2Care and TNK was not significantly different than the BG-GAT or the Control. As expected, the CDC-AGO produced the least mortality which on the same range as the Control. Autodissemination and Adult Mosquito Impacts Much fewer adults were generated from cohorts placed in the In2Care enclosure compared to the other ovitraps suggesting the pyriproxyfen was effectively autodisseminated. Adult survival among released gravids on the day of recovery was found to be equivalent (i.e., no significant difference) in all of the ovitrap enclosures. The greatest number of dead adults were recovered, as expected, in ovitraps designed to collect them (i.e., CDC-AGO and BG-GAT). The CDC-AGO consistently collected on average over half of the mosquitoes released into the enclosures. This was significantly more than the other traps. The greatest daily survival of released/recaptured adults compared to the control occurred in the In2Care extending from day 1-12 followed by the BG-GAT extending from day Literature Cited Chan KL. In: Vector Control In Southeast Asia. Yow Cheong C, Kai Lok C, Beng Chuan H, editor. Singapore: Sen Wah Press; The eradication of Aedes aegypti at the Singapore Paya Lebar International Airport; pp

21 Chan, K.L., Kiat, N.S. & Koh, T.K An autocidal ovitrap for the control and possible eradication of Aedes aegypti. Southeast Asian Journal of Tropical Medicine and Public Health, 8, Farenhorst, M Protocol for evaluation of the In2Care mosquito trap against Aedes mosquitoes. In2Care BV, Wageningen, The Netherlands. 11 pp. Fay, R.W. and Eliason, D.A A preferred oviposition site as a surveillance method for Aedes aegypti. Mosquito News, 26, Fay, R.W. and Perry, A.S Laboratory studies of ovipositional preferences of Aedes aegypti. Mosquito News, 24, Zeichner, B.C and Perich M. J Laboratory testing of a lethal ovitrap for Aedes aegypti. Medical and Veterinary Entomology 13: Acknowledgments We appreciate assistance provided by the following individuals and companies in support of this study. James Clauson, Director and Mike Riles, Entomologist, Beach Mosquito Control District (BMCD) reviewed and provided input on this study. BMCD employees, Lee Duke, Phillip Harvey, Kyle Pridgen, Travis Kern, and Cody Vinson assisted in relocating three of the greenhouse frames and assembling two new frames at the experimental site. Dani Carter of Gorilla Glue Inc. provided 19 rolls of clear repair tape for seaming the screen enclosures. Ted Worster of Univar, Inc. provided the In2Care units and sachets. Rebecca Heinig of Springstar Inc. provided the Trap-N-Kill ovitraps and refills. Jennifer McCaw from Biogents AG provided BG-GAT traps at discounted pricing. Manuel Amador provided the CDC-AGO traps and an ample supply of adhesive inserts. Marit Farenhorst of In2Care BV provided several helpful suggestions incorporated into the experimental design. Lastly, and most importantly, the authors would like to thank FDACS for funding. 21