Methods

1. Evaluating Radioprotectors to advance Sterilization Techniques applied to Aedes aegyptiMosquitoes. Stacy D. Rodriguez1, Ramaninder K. Brar2, Lisa Drake1, Hannah Drumm1, Jacob Urquidi2, & Immo A. Hansen1 Departments of Biology1 and Physics2 New Mexico State University, Las Cruces, NM 88001 D0051 A) Longevity: After evaluating sterility, we tested the effects of different proposed radio-protective agents on male mosquitoes. The males were exposed to the potential radio-protective agents for 48 hours prior to irradiation. Then longevity was measured. Introduction Sterile Insect Techniqueis a method of insect extermination in lieu of pesticides. Large amounts of sterile males are released into the environment to copulate with females. Females do not become fertilized and therefore die without reproducing - hence insect populations decline. The sterilization processis one of the challenges of using Sterile Insect Technique with mosquitoes. The fitness of sterilized males needs to be competitive with wild males; unfortunately mosquitoes are sensitive to radiation and relatively high morbidity rates after treatments have been reported (Helenski, et. al. 2006). Radioprotectors:A study in mice showed that ethanol consumption reduced the harmful effects of radiation (Monobe et. al., 2003). Only a few other potential radioprotectors have been identified for insects, including dimethyl sulphoxide(DMSO) and nordihydroguaiaretic acid (NDGA) (Helenski, et., al., 2009). In this study, we are investigating potential radioprotectors that may aid in the sterilization process of mosquitoes by evaluating the effects of different wavelengths, ethanol, and other proposed radiopotectors on longevity and reproductive fitness of X-ray sterilized mosquito males. B) Results Figure 4. Effect of potential radioprotectors on longevity post radiation A) Longevity post-irradiation after 48 hours of treatment with a potential radio-protector. Log-rank test shows no significant difference between irradiated treatments, P Value=.2452. Log-rank for trend shows no significant difference between treatments- P-Value= .0539. B) Mean longevity shows a strong trend that Lager might have radioprotective properties. Figure 2. Longer wavelengths significantly reduce male longevity. Total radiation doses for sterility employed are considerably lower (~1Gy/Steradian) than those typically used. The damage resulting from longer wavelength radiation allows for lower radiological exposure over a short time period (~5-15 min.) Above is a plot of longevity after exposure to X-radiation emanating from two different target materials (Copper and Molybdenum). The longer wavelength exposure delivered by the copper target (~1.4 Å) shows a substantially steeper death rate than that delivered by the molybdenum target (~0.7Å). • Discussion • We did achieve 100% sterility at 15 minutes of exposure to X-rays (< 1gy). Five Minute exposure resulted in 98.67% sterility. The optimal dosage, will be further evaluated in future studies. • There was no significant female fecundity difference between experimental groups that were mated with sterile and non-sterille males; however, competition experiments are necessary to evaluate the possibility of sperm selection by females. We will perform competition experiments to demonstrate the competitiveness between irradiated and unirradiated males using our sterilization technique. • Identical dosages of longer X-ray wavelengths used during irradiation significantly reduce sterile males’ longevity compared to shorter wavelengths. • Our data shows a strong trend suggesting that radioprotector-treatment is beneficial to irradiated males. We will further evaluate potential radio-protective agents in order to attain more definitive results. Methods Figure 1- A. X-Ray machine used to irradiate vials of mosquitoes consists of a collimated unfiltered beam emanating from a Molybdenum target. A brass tube houses the vial of mosquitoes. External shielding is provided by a lead tube that fits over the assembly. B. A sphere representing the total radiation coverage by an isotropic radioactive emitter. The radiation encompasses 4π of solid angle (the surface area of the Sphere defined by the radioactive source at its center). Actual dose (as noted) is typically considerably less than the total emitted. Figure 2. Male irradiation has no effect on female fecundity. Virgin females were mated with males that received different doses of ionizing radiation. . References Sterility Assessment: Mosquitoes were irradiated in small collection tubes. There were 3 different exposure times and 1 control. The experiment was done in replicates. There were 30 mosquitoes males placed in each collection tube. Post-treatment the mosquitoes were placed in cages. The mosquitoes were allowed 24 hours to recover. After recovery, the males were placed in cages with virgin females in a 1:1 ratio. The mosquitoes were allowed 72 hours for copulation. A total of 12 blood fed female mosquitoes were taken from each sample and placed into falcon tubes. The females started laying eggs 3 days post blood meal and were allowed to lay eggs for another 72 hours after the initial deposition. The eggs were harvested for fecundity and viability assays. Helinski, M.E., Parker, A.G., & Knols, B.G. (2009). Radiation Biology of Mosquitoes. Malaria Journal. (8): S6. doi:10.1186/1475-2875-8S2-S6 Monobe, M., Koike, S., Uzawa, A., & Ando, K.(2003). Effects of Beer Administration in Mice on Acute Toxicities Induced by X Rays and Carbon Ions. Journal of Radiation Research. 44, 75-80. Helinski, M.E., Parker, A.G., & Knols, G.J. (2006). Radiation-induced sterility for pupal and adult stages of the malaria mosquito Anopheles arabeinsis. Malaria Journal. 5:41: doi: 10.1.186/1475-2875-41 Figure 3. X-ray treatment is highly effective for sterilization of mosquito males. Female Virgins were mated with males that received different doses of ionizing radiation. Egg viability was then measured as an indication of sterility. This projet was supported by an Interdisciplinary Research Grant (IRG) awarded by the Office of the Vice President for Research NMSU