Evaluating Triethylamine in the Anaesthesia of Anopheles gambiae

The immobilisation of mosquitoes for injection experiments is a requirement that must be achieved for the necessary time duration to complete the process. Unfortunately, the use of cold anaesthesia in hot tropical countries is not very effective and the use of CO2 anaesthesia requires continuous exposure, which can be harmful to the experimenter. To circumvent this problem we evaluated the use of triethylamine (FlyNap) in the anaesthesia of Anopheles gambiae mosquitoes. FlyNap has been used widely in Drosophila studies, and is known to irreversibly anaesthetise mosquitoes. Very small volumes of the original FlyNap as well as different dilutions using absolute ethanol were evaluated to determine the dosage that will effectively anesthetise Anopheles gambiae female mosquitoes for the necessary duration. The results showed that the 1/10 (FlyNap/absolute ethanol) dilution worked well and could be used for the anaesthesia. Injecting live adults mosquitoes for scientific experiments is a delicate process that requires immobilising them for the required period of time. The immobilisation can be done using two main methods; chilling using cold anaesthesia (Garver & Dimopoulos 2007) or by chemical anaesthesia using CO2 (Lamacchia et al. 2013). In tropical climates cold anaesthesia is not very effective, and some mosquitoes can withstand some degree of chilling and hence may not be immobilised for long. Chemical anaesthesia, such as CO2 on the other hand, can result in the death of mosquitoes, and may be harmful to humans. A chemical anaesthesia commonly used is triethylamine. It is safe and poses no harmful effects to humans (Budavari 1989). Triethylamine formulated as FlyNap® (Carolina Biological Supply Co., NC, U.S.A.) is commonly available for anaesthetic purposes for insects. FlyNap is a mixture of 50 % triethylamine, 25 % ethanol and 25 % fragrance. It is 100 % volatile, water-soluble and can be stored in a cool, well-ventilated place. When exposed to FlyNap, insects are quickly incapacitated but remain alive and are unable to fly for several hours (Kramer et al. 1990). However, when in high doses, exposure causes death of the insect. It has been widely used in Drosophila anaesthesia (Paternostro et al. 2001; Babcock et al. 2008), and only used in lethal doses to kill mosquitoes (O’Guinn & Turell 2002; Goodman et al. 2003; Chen & Hillyer 2013). Our attempts to immobilise adult Anopheles gambiae mosquitoes using cold anaesthesia for injection experiments have proven unsuccessful, primarily due to the inability to immobilise mosquitoes long enough to complete the procedure. To circumvent this problem, this study aimed at evaluating the use of FlyNap and the dosage that will effectively anaesthetise An. gambiae long enough for experimental assays, and also result in high mosquito recovery rates. The experiment was conducted with 3and 5day-old Kisumu strain female An. gambiae mosquitoes. The mosquitoes were fed ad libitum on 10 % sucrose solution, following emergence. For each experiment, 10 mosquitoes were placed in a plastic cup and exposed to one of the following concentrations of FlyNap: 1 μl and 2 μl of stock, as well as 10 μl solutions of 1/10, 1/100, 1/1000 dilutions of FlyNap. The dilutions were prepared with absolute ethanol. The FlyNap to be tested was aliquoted onto small cotton balls and placed in the cups with the mosquitoes. To evaluate the additional effect of the ethanol in anaesthetising the mosquitoes, a control experiment using 10 μl absolute ethanol only was included for the 3and 5-day-old mosquito groups. Each cup was turned upside down to ensure that the fumes from the FlyNap did not escape and could effectively knock down the mosquitoes. The time taken for the first mosquito to be knocked down was recorded for each experiment, and the number of mosquitoes knocked down was recorded at 10-min intervals. After all mosquitoes were knocked down, they were transferred to new cups. The time taken for the first mosquito to revive was noted, and the number of revived mosquitoes was subsequently recorded at 30-min intervals. All experiments were replicated three times to determine the average time of knock *Author for correspondence. E-mail: ddesouza@noguchi.ug. edu.gh