Effects of radiation on blood‐feeding activity of <i>Aedes aegypti</i> (Diptera: Culicidae)

Abstract

Aedes aegypti L. is found globally from temperate to tropical urban environments, and it is known as the primary vector of viruses such as Zika, dengue, and chikungunya (Pless et al. 2017). Control of this vector mosquito is a critical component for the prevention and control of vector-borne diseases wherever it is present or at risk of spreading. Sterile insect technique (SIT) is a method of insect population control that exploits mating techniques and insect behavior (Dunn and Follett 2017). Radiation is one method for sterilization of male mosquitoes before release (Dame et al 2009, Bellini et al 2013). Recently, a renewed interest in use of radiation for SIT to control vector mosquitoes has occurred due in part to the increase of pathogens, spread of the vector mosquito, and alternative control strategies such as the incompatable insect technique (IIT) (Dandalo et al. 2017). Mosquitoes irradiated for SIT programs were mass-reared and processed in large numbers, increasing the likelihood that females could be inadvertently released alongside sterile males. Since these unintentionally released females were also irradiated, we evaluated the impact that radiation has on female mosquitoes and their blood-feeding habit. Aedes aegypti eggs collected from St. Augustine, FL were used to start a colony in early 2016, maintained at the USDA-Center for Medical, Agricultural, and Veterinary Entomology (CMAVE), Gainesville, FL insectaries within environmental chambers set to 28 ± 1° C, 70% RH, 12:12 LD interval, and a 30 min crepuscular period at dawn and dusk. Oviposition papers containing eggs were cut into strips, immersed in tap water, agitated for 1 min, and exposed to a 0.3 atm vacuum for 30 min using a vacuum desiccator to encourage egg hatch. Hatched 1st instar larvae (day 1) were reared in 22” x 18” x 3” nesting totes (400–3N, Blue Ridge Thermoforming, Greenville, SC) filled with 3 liters of tap water. The larvae were fed a diet of 2.5 ml of pulverized TetraMin® (77101; Tetra, Blacksburg, VA) administered every other day for three days. On day seven, pupae were strained and separated by sex using a pupal separator (5412; John W. Hock Co., Gainesville, FL). Pupae were separated and manually counted (n=50) into 60 x 15 mm Petri dishes, lined with filter paper cut to fit the bottom dish. Dishes were then checked under a dissecting stereoscope to ensure that all pupae in the dish were the same sex by comparing size and shape of the genital lobe (Harbach and Knight 1980). The water was aspirated from the dishes with a pasture pipette and the dishes were then closed, secured with tape, bundled together, and irradiated by γ-radiation using a Gammator M (Radiation Machinery Corp., Parsippany, NJ) and a cesium-137 source that generated 8.8 gray/min. The radiation doses applied to the female pupae were 0, 30, 50, 65, 85, 100, and 110 gray; with the 0 gray acting as a control. Radiation doses were checked by sending selective Gafchromic film and alanine pellent samples in the dishes to the national standard laboratory at the Texas A & M University's Electron Beam Center. Following irradiation, the dishes containing female pupae were returned to USDA-CMAVE, opened, and rinsed into 118 ml cups with tap water. The dishes containing 50 non-irradiated male pupae were then rinsed into the cups containing the treated female pupae. The soufflé cups were then transferred to 30x30x30 cm cages (BugDorm-1; MegaView Science Co., Taichung, Taiwan). The cages were later collected by Anastasia Mosquito Control District staff and transported to their facilities, where the adults were able to emerge. Cotton balls soaked in a 3% sucrose solution were left in the cages for the mosquitoes to feed ad libitum for 48 h. After 24 h without sucrose, cages were offered blood in a lambskin casing (Trojan Naturalamb, Church & Dwight Co., Inc., Ewing, NJ). Adults were allowed to feed for 15 min, then the blood was removed and the cage was left to rest for 30 min. After the resting period, the cages were put in a freezer (−20° C) and the mosquitoes were dispatched. The dead mosquitoes were then removed from the cages and assessed for blood-feeding. Male mosquitoes were discarded and females were sorted onto white paper. Clear tape was placed over each mosquito to act as a cover that protected the cadaver. Taped mosquitoes were compressed using forceps starting from the proximal thoracic connection of the abdomen and moved outwards to the distal tip of the abdomen. This allowed for any blood that was in the abdominal cavity to escape. Engorgement was qualitatively assessed as fed, where mosquitoes took a full or partial blood meal, or unfed, where mosquitoes had no visible blood in them and had no blood extruded when compressed. This was scored for all seven irradiation groups and the whole experiment replicated seven times. Scores were analyzed using ANOVA and Tukey HSD tests. Blood-feeding results post-irradiation are visualized in Figure 1. In our controls (0 gray), we observed that 25–50% of the mosquitoes took a full or partial blood meal, but fed percentages were inversely proportional to doses applied. At 65 gray and above, less than 10% of treated mosquitoes fed on blood (F= 6.7484, df = 6, 48, P < 0.0001). Above 85 gray, feeding effectively ceased (F= 4.8956, df = 6, 48, P < 0.0007). In the higher irradiation doses (65 gray and above), we observed the mosquitoes hovering over the blood sausage but not landing. In our control (0 gray) treatments, we observed that ca. 50% took a blood meal, but as the amount of radiation increased the number of blood meals decreased: ≥65 gray there were no fully engorged mosquitoes; less than 10% of treated Ae. aegypti at and above 65 gray acquired even partial blood meals. This demonstrates that irradiating males for the purpose of sterilization also reduces the tendency for females to blood-feed when given an opportunity, thereby reducing the risk from an unintentionally released irradiated female that could be synergized to other control programs, such as IIT. Mosquitoes are medically important vectors currently being evaluated for SIT testing programs (Benedict and Robinson 2003), but there are some concerns with SIT for mosquito control due to the unintentional release of females. When comparing the SIT methods used against agricultural pests to methods used for mosquitoes, we can observe some differences (Alphey et al. 2010). For example, when SIT is used for mosquito control, it is enacted for public health purposes, such as stopping the spread of disease to humans or reducing the presence of a potential vector (Ponlawat and Harrington 2005). However, in this study, we have documented that the proportion of females that blood-feed is significantly lower in irradiated vs non-irradiated females. Although we can confidently demonstrate that irradiated females do not blood-feed as frequently, the impact of releasing irradiated females unintentionally with their male cohorts for control purposes of mosquito-borne diseases and its risk for public health need to be further addressed.

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