Analysing the role of semiochemicals in the oviposition substrate choices of the malaria vector mosquito Anopheles gambiae sensu lato

The search for tools that target malaria vector that resist insecticides and bite outdoors has become a research priority. Such tools will be necessary for managing residual malaria transmission and hastening the eradication of this devastating disease. This study investigated chemicals that potentially affect the oviposition substrate choices of Anopheles gambiae sensu lato (s.l.). It is foreseen that increased knowledge of the oviposition behaviour of this major malaria vectors and chemicals cues that mediate oviposition site-selection can be applied in the development of additional sampling methods and alternative interventions that to trap gravid malaria mosquitoes outdoors. To achieve a reproducible high egg-laying success of An. gambiae sensu stricto (s.s.) and An. arabiensis 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 source of blood meals. Then four bioassays were optimised for studying oviposition responses of An. gambiae s.s. in the laboratory and semi-field conditions: a WHO-tube bioassay and a wind-tunnel that detected short-range attraction in the laboratory; a two-tier choice egg-count bioassay that compared the relative proportion of eggs laid in substrates in the laboratory; and a modified BG Sentinel mosquito gravid trap that evaluated long-range attraction of gravid females to olfactory cues in the semi-field. Finally, the oviposition responses of gravid An. gambiae s.s. mosquitoes to water vapour, Bermuda grass hay infusion (hay infusion), and putative semiochemicals identified from the hay infusion and a soil infusion previously shown to elicit higher egg deposition compared to filtered Lake Victoria water (lake water) in two choice egg-count bioassays (Herrera-Varela et al. 2014), were evaluated. 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 four days. The chance of oviposition in the mosquitoes dropped when human host source of blood-meal was substituted with a rabbit (Odds ratio (OR) 0.30, 95% CI 0.14-0.66) but egg-numbers per female were not affected. All four optimised oviposition bioassays effectively showed between 15-20% shifts in oviposition substrate choices of mosquitoes with 80% statistical power and 5% significance. Using the WHO-tube bioassay, gravid An. gambiae s.s. were shown to be 2.4 times (95% CI 1.3-4.7 times) more likely to move towards high humidity in still air compared to non-gravid Preamble mosquitoes. This was more pronounced in the airflow olfactometer where the gravid mosquitoes were 10.6 times (95% CI 5.4-20.8 times) more likely to fly into a chamber with water than a dry chamber. Two-choice egg-count bioassays showed that An. gambiae s.s. were less likely to lay eggs in six-day old hay infusion (OR 0.10, 95% CI 0.03-0.33) compared to lake water. Ten putative semiochemicals were identified from the hay infusion using mass spectrometry and published electrophysiology data: 4-hepten-1-ol, 4-ethylphenol, phenylmethanol, 2-phenylethanol, indole, phenol, 3-methylindole, 3-methyl-1-butanol, 4-ethylphenol, and nonanal. Tested in two-choice egg-count bioassays, the first four listed compounds had no effect on egg deposition at the tested concentrations (between 0.01-5 parts per million) but mosquitoes were less likely to lay eggs in at least one concentration of 3-methylindole (OR 0.39, 95% CI 0.21-0.71), indole (OR 0.57, 95% CI 0.37-0.87), 3-methyl-1-butanol (OR 0.32, 95% CI 0.22-0.47), phenol (OR 0.55, 0.32-0.95), 4-methylphenol (OR 0.32, 0.18-0.57) and nonanal (OR 0.66, 95% CI 0.47-0.91) compared to lake water. In contrast to the hay infusion and hay infusion volatiles, An. gambiae s.s. were about two times more likely to lay eggs in cedrol, a sesquiterpene alcohol identified from the soil infusion, compared to lake water (OR 1.84, 95% CI 1.16-2.91). Cedrol attracted twice as many gravid mosquitoes in the semi-field also (OR 1.92, 95% CI 1.63-2.27). In the field, modified BG-Sentinel traps, electrocuting nets and OviART gravid traps with lake water and cedrol were three times more likely to trap malaria mosquitoes compared to traps with water only (OR 3.3, 95% CI 1.4-7.9). In conclusion, water vapour was shown to be a strong, non-specific pre-oviposition attractant for gravid An. gambiae s.s. in still air and moving air. It is probably the long range cue that gravid An. gambiae s.l. use to detect the presence aquatic habitats beyond the range of chemical cues. Evidence showed that An. gambiae s.s. discriminate between potential oviposition substrates and that this selective process is in-part mediated by volatile organic compounds originating from the site. Water vapour leads gravid mosquitoes to aquatic sites but semiochemicals enable the mosquitoes to discriminate and select between potential habitats. It was demonstrated that synthetic equivalents of semiochemicals found to attract gravid mosquitoes such as cedrol can be used to trap malaria mosquitoes outdoors.