Mosquitoes
Experiments were conducted using a laboratory population of An. gambiae s.s. reared as follows: larvae were fed on Tetramin® fish food and maintained at temperature of 27 ± 1°C. Adult mosquitoes were kept inside mosquito cages measuring 30 cm × 30 cm × 30 cm in a separate room, where temperatures were maintained at 27°C and relative humidity at 70–90%. The adults were fed on 10% glucose solution delivered through Whatman® filter paper. The insectary has12:12 LD photoperiod. Three to 8 days old nulliparous females, starved for 6–8 hours were selected for the experiments.
The semi-field system
Experiments were conducted within a semi-field enclosure, also referred to as the screen house, available at the Ifakara Health Institute (IHI), Tanzania [17]. The semi field system had three compartments each approximately 200 square metres, and one of which was used for this study. The experimental chamber was devoid of vegetation.
Mosquito collection
A counter flow geometry trap (the MMX® model) made by the American Biophysics Corporation [18, 19] was used to comparatively evaluate mosquito responses towards the test compounds. This trap consists of an oval shaped plastic casing (the collection container) enclosing an extended inner tubing where the bait is inserted (the attractant plume tube). It has two fans blowing air in opposite directions. The smaller fan (the attractant plume fan) located directly on top of the attractant plume tube blows air out. Simultaneously, the larger fan (the exhaust fan), which is located near the top of the trap, sucks air upwards through the trap, thereby creating a counter current suction mechanism. Attracted mosquitoes trace the path of the expelled air current, which carries the volatiles from the bait. When the insects reach near the lower end of the trap, they are sucked into the collection container by the more powerful current of the exhaust fan. At the end of the experiment, the collection tube is closed using a plastic seal after which the trap is disconnected from the 12-volt battery that powers it.
Test compounds
Two standard mosquito repellents with comparable efficacy but different modes of action were used. The repellents were: 1) an inhibitor, N, N diethyl-3-methylbenzamide (deet) and 2) a spatial repellent, para-methane-3, 8, diol (PMD). A synthetic odour blend recently developed and tested at the IHI (Patent pending), was used as the test medium instead of a human volunteer. In summary, this blend consists of carboxylic acids, ammonia and carbon dioxide.
Experimental procedures
Competitive binary assays were performed to assess the relative changes in attractiveness of the synthetic odour blend when a repellent was added to it. Two MMX® traps were set up 20 metres apart inside the screen house. One of the traps (control trap) was baited with the synthetic odour blend delivered using nylon strips inserted into the attractant plume tube of the MMX® trap (Figure 1). The other trap (treatment trap) was baited with the same blend plus an additional nylon strip containing a particular concentration of either PMD or deet. Deet was dissolved in ethanol and tested at concentrations of 0.001%, 0.01%, 0.1%, 1%, 10% and 100%. PMD, which is solid at room temperature, was dissolved in distilled water and tested at concentrations of 0.025% 0.25%, 2.5%, 25% and 50%. To prepare the 50% w/w solution of PMD, 500 mg of the solid was melted in a water bath and mixed with 500 ml of distilled water. A blank strip was added to the batch of strips used in the control trap so that the number of strips in both traps was equal. The use of the nylon strips in dispensing odours has been described in detail elsewhere (Okumu et al., unpublished). Carbon dioxide was added to both traps at a constant rate of 500 ml/min.
Two hundred female mosquitoes were released at the centre of the screen house, 10 m equidistant from the traps. The number of mosquitoes trapped in either of the two traps was considered an estimate of attractiveness of the baits. Four to six replicates each lasting 6 hours were conducted for each concentration of PMD and deet, the location of the treatment and control traps being rotated between replicates. We conducted two experiments per night; the first one starting at 19.00 Hrs and ending at 01.00 Hrs and the second running between 01.10 Hrs and 07.10 Hrs.
Statistical analyses
Data was analysed using SPSS version 15 (SPSS Inc., Chicago). Bait attractiveness was measured on the basis of relative mosquito catches in treatment versus control traps. The preference by mosquitoes to fly to either the treatment trap (the trap containing the repellent plus the synthetic blend) or the control trap (the trap containing only the synthetic blend) was coded as 1 and 0 respectively, and then weighted by the number of mosquitoes caught per trap per replicate (i.e. relative mosquito response). The proportion of mosquitoes caught in the treatment trap (Pt) was computed for each repellent at the different test concentrations, taking the total number of mosquitoes collected in both traps as the denominator.
Data was fitted onto a binary logistic regression and Pt was estimated as a function of the categorical variables, trap location (x1) and phase of the night (x2). A stepwise backward-conditional method was used to assess the significance of the independent variables prior to inclusion in the regression models. The intercept obtained was exponentiated to determine the odds for the treatment compared to the control. The odds were then used to estimate the probability that the mosquitoes would be trapped preferentially in the trap baited with the synthetic blend plus the repellent (i.e. estimated probability = Odds/(1+Odds)). Finally, a linear regression analysis was performed on Pt and respective repellent concentrations of either Deet or PMD to determine if there was a dose-response relationship between them.