Control of male<i>Aedes albopictus</i>Skuse (Diptera: Culicidae) using boric acid sugar bait and its impact on female fecundity and fertility (2017)

Aedes albopictus (Skuse) is a serious threat to human health as a vector of arboviruses such as dengue, chikungunya (Lambrechts et al. 2010, Gubler 1998), and Zika (Wong et al. 2013). Adult Ae. albopictus are controlled primarily via ground and aerial ultra-low volume applications of insecticides. The chemical insecticides may potentially pollute the environment, and mosquito populations gradually develop resistance to them (Waits et al. 2017). The future for mosquito control requires safer and more effective alternatives to replace and improve currently used adulticides. Male and female mosquitoes require carbohydrates after emerging for energy, survival, and reproduction (Yuval 1992, Foster 1995). Adult Ae. albopictus mosquitoes, like other mosquito species, need regular sugar meals (Muller et al. 2010a, Xue et al. 2010). Attractive toxic sugar bait (ATSB) is a novel method for adult mosquito control. This control method takes advantage of mosquito sugar-feeding behavior by mixing insecticides with their food (Lea 1965, Xue et al. 2013). The ingredients in ATSB are fruit juice (attractive), 10% sugar (feeding stimulator), and 1% boric acid (stomach toxin), and adult male and female mosquitoes usually die 24–48 h after ingestion of TSB or ATSB (Xue and Barnard 2003, Xue et al. 2008). The ingredients of toxic sugar bait (TSB) are usually 10% sugar and 1% boric acid, without fruit juice as an attractant. When applied to foliage, TSB or ATSB was successful in controlling Ae. albopictus in both semi-field conditions (Xue et al. 2006) and residential communities in St. Augustine, FL (Naranjo, et al. 2013). ATSB or TSB can be targeted to males when timed to their emergence. Male mosquitoes typically emerge at least 12 h earlier than females (Xue and Barnard 1997) and feed only on sugar for nutrition and survival (Xue et al. 2010). This early emergence time and complete dependence on sugar for survival suggests male mosquitoes can be affected by TSB or ATSB. If TSB or ATSB exposure is directed to this early emergence time, it may have an indirect effect on female mosquito fecundity and fertility. In this study, we tested the hypothesis that mosquito reproductive capacity is negatively affected if male Ae. albopictus are exposed to TSB before females emerge. After a brief exposure period to TSB, male and female mortality were analyzed to see if the exposure period only affected males. Next, reproductive effects of females exposed to TSB were analyzed by counting the number of eggs laid and the hatch rate. Each assessment of TSB was compared to a 10% sucrose control. The Ae. albopictus strain was obtained from the USDA, ARS, Center for Medical, Agricultural, and Veterinary Entomology, in Gainesville, FL and reared in the insectary at Anastasia Mosquito Control District (AMCD) in St. Augustine, according to the method described by Gerberg et al. (1994). The adult mosquitoes were fed a 10% sucrose solution ad libitum, and were maintained in a photoperiod of 14:10 (L:D) h at 80% relative humidity and temperature of 27º C. The toxic sugar bait (TSB) formulation was prepared by adding boric acid at 1% W/V to a 10% sucrose solution. The TSB (treatment formulation) was mixed and heated at 60º C until the boric acid completely dissolved followed by cooling to room temperature. The control formulation was a 10% sucrose solution, and both treatment and control formulations were soaked in cotton balls and fed to mosquitoes. Four hundred pupae were collected and equally distributed at 200 each between treatment and control groups. The pupae were housed inside a screened cage with either TSB (treatment) or 10% sucrose in reverse osmosis water (control). The treatment and control groups were provided their respective solutions after pupae began to emerge to adults, and the time of feeding was noted. Most of the males emerged and fed at the same time. The treatment and control group solutions were replaced with a 10% sucrose solution after TSB exposure for 24 h. After 96 h, the mosquitoes were fed chicken blood by gently holding a chicken's feet in the cage for at least 15 min. The procedure was in accordance with protocol # AC 2005 approved by the animal care and use committee at the Anastasia Mosquito Control District. Brown oviposition filter papers (25 × 8 cm) were placed in a black ovicup (200 ml) with 100 ml infusion water for the treatment and control group after 48 h, respectively. The blood-fed and ovipositing mosquitoes had access to a 10% sugar solution via a cotton strip. The egg papers were collected from ovitraps and dried at room temperature. The numbers of eggs from the treated and control groups were counted under a microscope. The hatch rate was calculated by comparing the number of hatched larvae between treatment and control groups. Hatch rate was calculated as: [% hatch rate = total of larva ×100/ total of eggs]. This experiment was repeated three times with different cycles of mosquitoes from the same colony in identical laboratory conditions. All statistical analyses were performed with SPSS 16.0 statistical software. Comparisons of the data between treatment and control groups were performed using the two-tailed Student's t test. Hatch rate comparisons between treatment and control groups were performed using the Chi-square test. Differences between treatment and control groups were considered statistically significant at p ≤ 0.05. The number of male and female mosquitoes that emerged from the water to be exposed to the TSB were calculated to determine the overall health of each repetition. The emerging number of male mosquitoes (t=0.354, df=4, P=0.741) and female mosquitoes (t=0.158, df=4, P=0.882) were not significantly different between treatment and control groups (Table 1). This indicates that there were no significant health abnormalities between each cycle of mosquitoes used from the colony. Mosquito mortality was calculated by counting the number of dead male and female mosquitoes at 24 and 48 h post-exposure. The mean number (51, 102) of dead males exposed to boric acid bait (treatment) was significantly higher than in control mosquitoes (5, 12) at 24 h(t=6.850, df=4, P=0.002) and 48 h (t=20.893, df=4, P=0.000), respectively. The mean number (3, 8) of dead female mosquitoes exposed to boric acid bait (treatment) was not significantly different, compared to the control (2, 9) at 24 h (t=1.000, df=4, P=0.374) and 48 h (t=0.802, df=4, P=0.468), respectively (Table 2). These results indicate that only males suffered significant population reduction following exposure to boric acid baits. Also, minimal loss of females to TSB at 48 h indicates that little to no females ingested TSB during the exposure period. Eggs were counted between the treatment and control groups to determine if early male exposure to TSB reduced the capacity of the population to reproduce. The mean number (1,070) of eggs produced by females exposed to TSB was significantly reduced (t=10.834, df = 4, P=0.0001) compared to the control (1,858, Table 3). Next, the egg hatch rate was analyzed by comparing the percentage hatch rate between treatment and control groups to get a rate of hatch reduction. Hatch reduction rates indicate that females laid non-fertilized eggs and potentially mated with males that were compromised due to TSB exposure. The mean egg hatching rate (54%) was significantly reduced in females exposed to TSB (χ2= 60.004, df=1, P=0.000) compared to the control (72%, Table 4). Current control strategies against Ae. albopictus include trapping, chemical/biological insecticide treatment, and public health education for source reduction and prevention of mosquito bites. The major problems with several kinds of insecticides currently used for mosquito control are their persistence in the field, the rapid development of insecticide resistance, and their impact on non-targeted species. The majority of current mosquito control methods focus on females, while male mosquitoes are seldom targeted. In reality, the males contribute substantially to many aspects of female behavior and physiology and indirectly influence parasite transmission through their effects on the female (Klowden, 1999). This study on the control of male mosquitoes presents a new alternative strategy to control mosquito populations. It is evident from this study that the male mosquitoes emerged earlier than female mosquitoes, and the emerging male mosquitoes fed on sugar for nutrition and survival. The TSB significantly reduced male survival under laboratory conditions with a morality rate at 91.3% at 48 h. On the other hand, females were not exposed to TSB to the same extent as males because they emerged later, and they suffered little to no mortality. Based on this study, we suspect that a lower abundance of male mosquitoes may reduce the mating rate with females due to a high mortality caused by the exposure to TSB or ATSB. The female mosquitoes in this study still laid eggs, and some of their eggs hatched while others could not due to a lack of insemination. Therefore, the fecundity and fertility (number of total laid eggs and the egg hatch rate) of female mosquitoes in the treated group was significantly reduced. The mean egg production was reduced by 42.4%, and the mean egg hatching rate was reduced by 23.9%, compared with the control groups. The higher mortality of males in the population is what likely contributed to the decrease in fecundity and fertility. Several experiments have been conducted using TSB around mosquito larval habitats to control adult mosquitoes and significantly reduced the mosquito population (Muller et al. 2010b, Hossain et al. 2014). Treatments with ATSB or TSB around mosquito larval habitats may kill the early emerged male mosquitoes and potentially impact female fecundity. This new strategy of targeting males with ATSB or TSB as a means to reduce subsequent generations of mosquitoes is similar to sterile insect technology (SIT). This method may be environmentally safe and economical and has the potential for controlling mosquitoes by the treatment of mosquito breeding sites. The study was only conducted in the laboratory, but future studies should include semi-field and field tests using this approach to target male mosquitoes. We gratefully acknowledge the financial support from the Shanghai youth physician training program, as well as the assistance of Christopher Bibbs, Charolette M. Hall, Xia Liao, You Peng, and Tayler Katsma during the course of this study at the Anastasia Mosquito Control District.