Deltamethrin induced functional mortality of Anopheles stephensi, the urban malaria vector, in relation to resistance development.

urban malaria vector, accounts for about 15% of the total malaria incidence in India1. The vector control primarily relies on the use of chemical insecticides like synthetic pyrethroids in the form of insecticide treated nets (ITNs), long-lasting insecticidal nets (LLINs) and indoor residual spraying (IRS)2. In urban areas, IRS is not feasible; hence use of ITNs and LLINs could provide an effective protection against transmission of malaria. Besides LLINs, pyrethroids are popularly used in the form of commercial mosquito repellents such as mats, liquidators and coils. Deltamethrin is one such synthetic pyrethroid receiving much attention worldwide for its use in the control of mosquito vectors especially malaria vectors due to its known higher efficacy against them3. Further, deltamethrin is one among the six pyrethroid insecticides recommended for treating mosquito nets. It acts on voltage gated sodium channel (VGSC) and by interrupting the action potential, it causes the neuron to fire spontaneously leading to eventual death of the target insects. However, selection pressure due to continuous use of synthetic insecticides can result in development of resistance4. Continuous insecticide pressure selects an insect population with (i) an altered integument permitting lesser penetration of insecticide, (ii) modifications in the structure or the expression level of detoxifying enzymes, (iii) mutations in the insecticide target sites of insects that prevent interaction with the insecticide, (iv) behavioural changes, such as avoiding contact with the insecticide5, and (v) increased cuticular thickness6. While some modifications or alterations conferring a selective advantage spread quickly in a given population under insecticide pressure, they can be associated with a high fitness cost5. In this paper, we present the results of induced leg loss leading to functional mortality of An. stephensi under deltamethrin selection pressure in the laboratory over generations. The laboratory reared strain of An. stephensi7, which is susceptible to deltamethrin, was used for the study. Female mosquitoes were allowed to feed on an artificial membrane (Parafilm ‘M’) blood feeder (defibrinated bovine blood warmed at 37°C), from the Day 3 of postemergence. Enamel bowl (22 × 18 × 5 cm) filled with dechlorinated water and lined with Whatman filter paper were used to collect the eggs. The eggs were transferred to rearing trays for hatching. The cyclic colony was maintained in the laboratory at 27 ± 2°C and 70 ± 10% RH with a photoperiod of 14 and 10 h darkness. Adult susceptibility test kits8 and deltamethrin impregnated papers with the diagnostic concentration of 0.05% were procured from the University of Sains Malaysia, Penang, Malaysia. Deltamethrin impregnated papers at different sublethal concentrations (ranged from 0.004 to 0.025%) were prepared in the laboratory as per the WHO protocol8. Adult mosquito bioassays were conducted using WHO test kits8. Three days old, 25 non-blood fed females of An. stephensi were released into the holding tube for one hour acclimatization. After acclimatization, only the healthy mosquitoes were allowed to fly from the holding tube to the exposure tube lined with deltamethrin impregnated paper. After exposure to the insecticide for one hour, the alive mosquitoes were allowed to fly back to the holding tubes and the knocked down/dead mosquitoes were gently blown to the holding tube by keeping it to downward position, and held at 27 ± 2°C and 70 ± 5% RH for 24 h. Cotton pads soaked in 10% sugar solution were provided as food source. At the end of the holding period, the tubes were shifted to a clean cloth cage where the surviving mosquitoes were allowed to get released from the tubes by opening their lids. Dead mosquitoes (true mortality) were removed and counted. The alive mosquitoes were then collected individually using a glass aspirator, observed for the number of legs present in each mosquito and released into a separate clean cloth cage (30 × 30 × 30 cm). Similar procedure was followed for the controls. The survivors were then kept for subsequent blood feeding. Utmost care was taken during handling of mosquitoes to make sure that there was no physical injury/morphological deformity to mosquitoes. Four replicates were maintained for each experiment with parallel controls. When mortality in the J Vector Borne Dis 52, September 2015, pp. 261–264