Analysing the oviposition behaviour of malaria mosquitoes: design considerations for improving two-choice egg count experiments

Abstract

Background: Choice 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.

Methods: In 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.

Results: High 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.

Conclusion: Two-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.

Figure 1

Experimental set-up of two-choice egg-count bioassays. a makeshift huts, b cages set up in hut at icipe-Thomas Odhiambo Campus, western Kenya.

Figure 2

Illustration of the arrangement of oviposition cups and cages in two choice egg-count bioassays. The solid circles represent test cups which are arranged in the clockwise direction. Control cups (open) are positioned diagonally opposite. FL front right, BL back left, BR back right, FR front right.

Figure 3

Insemination rates of caged Anopheles gambiae s.s. and Anopheles arabiensis (Mbita strains) in standard (30 × 30 × 30 cm) and large (60 × 60 × 60 cm) cages with increasing age of the females.

Figure 4

Histogram showing the frequency distribution of egg counts from 1,443 individual Anopheles gambiae s.s. (Mbita strain) females.

Figure 5

Proportion of responses (presence of eggs) received by the test cups in two equal choice tests out of the total responses (test cups + control cups) counted per experimental round (n per round = 15–43).

Figure 6

Mean number of eggs per female laid in test and control cup and proportion of eggs laid in test cup. Analysis based on 41 simulated groups of mosquitoes.

Figure 7

Frequency distribution of the proportions of eggs laid in one cup (higher proportion) over the other in two equal choice tests for simulated groups. a all 41 groups, b groups with ≤30 individuals, c groups with >30 individuals.

Figure 8

Frequency distribution of the higher proportion (>0.5) of eggs laid in one cup by skip-ovipositing females.

Figure 9

Median proportion of eggs laid in the test cups by skip-ovipositing females (n = 4–20) in every experimental round.

Figure 10

Description of the measurable powers (black) and effect sizes (red) of tests with different sample sizes (number of mosquitoes) for two proportions at the 0.05 significance level. Solid lines sample size considerations based on power calculation for two-sample comparisons of proportions. Dashed line sample size calculation for the inference for a single proportion comparing to a known proportion (0.5) suitable for testing large groups where this baseline proportion can be confirmed.

Figure 11

Summary recommendations for implementing two-choice cage egg-count bioassays for evaluating oviposition substrate preferences of malaria vectors.