Gravid Anopheles gambiae sensu stricto avoid ovipositing in Bermuda grass hay infusion and it's volatiles in two choice egg-count bioassays

Abstract

Background: A number of mosquito species in the Culex and Aedes genera prefer to lay eggs in Bermuda grass (Cynodon dactylon) hay infusions compared to water alone. These mosquitoes are attracted to volatile compounds from the hay infusions making the infusions effective baits in gravid traps used for monitoring vectors of arboviral and filarial pathogens. Since Bermuda grass is abundant and widespread, it is plausible to explore infusions made from it as a potential low cost bait for outdoor monitoring of the elusive malaria vector Anopheles gambiae s.s.

Methods: This study investigated preferential egg laying of individual An. gambiae s.s. in hay infusion or in tap water treated with volatiles detected in hay infusion headspace compared to tap water alone, using two-choice egg-count bioassays. Infusions were prepared by mixing 90 g of dried Bermuda grass (hay) with 24 L of unchlorinated tap water in a bucket, and leaving it for 3 days at ambient temperature and humidity. The volatiles in the headspace of the hay infusion were sampled with Tenax TA traps for 20 h and analysed using gas chromatography coupled to mass spectrometry.

Results: In total, 18 volatiles were detected in the infusion headspace. Nine of the detected compounds and nonanal were selected for bioassays. Eight of the selected compounds have previously been suggested to attract/stimulate egg laying in An. gambiae s.s. Gravid females were significantly (p < 0.05) less likely to lay eggs in hay infusion dilutions of 25, 50 and 100 % and in tap water containing any of six compounds (3-methylbutanol, phenol, 4-methylphenol, nonanal, indole, and 3-methylindole) compared to tap water alone. The oviposition response to 10 % hay infusion or any one of the remaining four volatiles (4-hepten-1-ol, phenylmethanol, 2-phenylethanol, or 4-ethylphenol) did not differ from that in tap water.

Conclusions: Anopheles gambiae s.s. prefers to lay eggs in tap water rather than Bermuda grass hay infusion. This avoidance of the hay infusion appears to be mediated by volatile organic compounds from the infusion. It is, therefore, unlikely that Bermuda grass hay infusion as formulated and used in gravid traps for Culex and Aedes mosquitoes will be suitable baits for monitoring gravid An. gambiae s.s.

Keywords: Anopheles gambiae; Bermuda grass; Egg-count cage bioassays; GC–MS; Hay infusions; Oviposition behaviour; Volatile detection.

Fig. 1

Egg-laying responses of Anopheles gambiae s.s. tested individually to Bermuda grass hay infusion and key organic volatiles of the infusion and the mean number of eggs laid per individual female tested. The bar chart shows the mean proportion of eggs laid in control and test substrates in choice egg-count bioassays, error bars show the 95 % confidence intervals (95 % CI). The odds ratios, including their 95 % CI and P values present the output of the statistical analysis based on generalized linear models. The experiments with tap water in both the control and test cup serve as reference based on the underlying assumption that gravid females lay an approximately equal proportion of eggs (1:1) in either test or control cup if both contain the same choice. The analysis aims to detect a statistically significant deviation from the reference distribution

Fig. 2

Relative amount detected in the headspace of Bermuda grass hay infusions of the volatiles evaluated in cage bioassays. Average relative amount (normalized against standard) and 95 % confidence interval. The compounds are listed in each plot in order of volatility (retention time based on GC analysis on a DB-5 column). a The four compounds detected in highest amount. b The compounds previously suggested to mediate positive oviposition responses