Abstract
BackgroundChoice egg-count bioassays are a popular tool for analysing oviposition substrate preferences of gravid mosquitoes. This study aimed at improving the design of two-choice experiments for measuring oviposition substrates preferences of the malaria vector Anopheles gambiae senso lato, a mosquito that lays single eggs.MethodsIn order to achieve high egg-laying success of female An. gambiae sensu stricto (s.s.) and Anopheles arabiensis mosquitoes in experiments, four factors were evaluated: (1) the time provided for mating; (2) the impact of cage size, mosquito age and female body size on insemination; (3) the peak oviposition time; and, (4) the host sources of blood meal. Choice bioassays, with one mosquito released in each cage containing two oviposition cups both with the same oviposition substrate (100 ml water), were used to measure and adjust for egg-laying characteristics of the species. Based on these characteristics an improved design for the egg-count bioassay is proposed.ResultsHigh oviposition rates [84%, 95% confidence interval (CI) 77–89%] were achieved when 300 male and 300 blood-fed female An. gambiae s.s. were held together in a cage for 4 days. The chances for oviposition dropped (odds ratio 0.30; 95% CI 0.14–0.66) when human host source of blood meal was substituted with a rabbit but egg numbers per female were not affected. The number of eggs laid by individual mosquitoes was overdispersed (median = 52, eggs, interquartile range 1–214) and the numbers of eggs laid differed widely between replicates, leading to a highly heterogeneous variance between groups and/or rounds of experiments. Moreover, one-third of mosquitoes laid eggs unequally in both cups with similar substrates giving the illusion of choice. Sample size estimations illustrate that it takes 165 individual mosquitoes to power bioassays sufficiently (power = 0.8, p = 0.05) to detect a 15% shift in comparative preferences of two treatments.ConclusionTwo-choice egg count bioassays with Anopheles are best done with a two-tier design that (1) implements a parallel series of experiments with mosquitoes given a choice of two identical substrates choices and, (2) uses a single mosquito in each test cage rather than groups of mosquitoes to assess the preference of a test or control solution. This approach, with sufficient replication, lowers the risk detecting pseudopreferences.
Highlights
Choice egg-count bioassays are a popular tool for analysing oviposition substrate preferences of gravid mosquitoes
Including males in holding cages after blood meals increases the proportion of ovipositing females The odds of a female An. gambiae s.s. laying eggs were nine times greater if, after a blood meal, she was held with males than without them (OR = 9.0, 95% confidence interval (CI) 7.9–9.5, p < 0.01)
Whilst the total number of eggs laid by females held with males [2,904 eggs] was three times as high as the total number laid by females kept separated from males after bloodmeals [994 eggs], the mean number of eggs laid per female was similar in mixed-sex cages [66 eggs, 95 % CI 44–99 eggs] and female only cages (54 eggs, 95% CI 36–82) highlighting the benefit of observing individual rather than groups of mosquitoes
Summary
Choice egg-count bioassays are a popular tool for analysing oviposition substrate preferences of gravid mosquitoes. Like all vector control interventions, these too have limitations and when used in isolation could fall short in areas with: (1) strains selected for physiological resistance to insecticides [7,8,9,10]; (2) secondary vector species that live and bite outdoors [11, 12]; and, (3) cryptic vector sub-groups that bite in the early evening and/or bite outdoors [13] These and a complex of other factors, including increasing drug resistance and high costs of interventions, make malaria resurgence a grim reality [14]. New strategies with novel tools that combine with LLINs and IRS to target these elusive groups of vectors in addition to the major vectors could prevent the resurgence of disease and hasten malaria elimination
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