Assessing the acoustic behaviour of Anopheles gambiae (s.l.) dsxF mutants: implications for vector control

Abstract

Background Release of gene-drive mutants to suppress Anopheles mosquito reproduction is a promising method of malaria control. However, many scientific, regulatory and ethical questions remain before transgenic mosquitoes can be utilised in the field. At a behavioural level, gene-drive carrying mutants should be at least as sexually attractive as the wildtype populations they compete against, with a key element of Anopheles copulation being acoustic courtship. We analysed sound emissions and acoustic preference in a doublesex mutant previously used to collapse Anopheles gambiae (s.l.) cages. Methods Anopheles rely on flight tones produced by the beating of their wings for acoustic mating communication. We assessed the impact of disrupting a female-specific isoform of the doublesex gene (dsxF) on the wing beat frequency (WBF; measured as flight tone) of males (XY) and females (XX) in homozygous dsxF− mutants (dsxF−/−), heterozygous dsxF− carriers (dsxF+/−) and G3 dsxF+ controls (dsxF+/+). To exclude non-genetic influences, we controlled for temperature and wing length. We used a phonotaxis assay to test the acoustic preferences of mutant and control mosquitoes. Results A previous study showed an altered phenotype only for dsxF−/− females, who appear intersex, suggesting that the female-specific dsxF allele is haplosufficient. We identified significant, dose-dependent increases in the WBF of both dsxF−/− and dsxF+/− females compared to dsxF+/+ females. All female WBFs remained significantly lower than male equivalents, though. Males showed stronger phonotactic responses to the WBFs of control dsxF+/+ females than to those of dsxF+/− and dsxF−/− females. We found no evidence of phonotaxis in any female genotype. No male genotypes displayed any deviations from controls. Conclusions A prerequisite for anopheline copulation is the phonotactic attraction of males towards female flight tones within mating swarms. Reductions in mutant acoustic attractiveness diminish their mating efficiency and thus the efficacy of population control efforts. Caged population assessments may not successfully reproduce natural mating scenarios. We propose to amend existing testing protocols to better reflect competition between mutants and target populations. Our findings confirm that dsxF disruption has no effect on males; for some phenotypic traits, such as female WBFs, the effects of dsxF appear dose-dependent rather than haplosufficient.

Synchronized swarming: Harmonic convergence and acoustic mating dynamics in the malaria mosquito Anopheles gambiae

“…Consistent with previous studies in free-flying An. gambiae 35,62 , and in contrast to behavior in Ae. aegypti 30,35 , our data show that females do not initiate mating interactions in response to male flight tones alone (Fig.…”

“…Artificial flight tone stimuli have been used to examine acoustic mating behavior in mosquitoes 11,26,29,30,35,39,41,62,66 as well as in midges 75 . To further dissect acoustic mating interactions during swarming, we tested swarming male flight responses to artificial female swarm tone audio playback emitted from a speaker (Fig.…”

“…Dusk lights were then turned off for the remaining 45 min of the recording to mimic sunset and the initial period of night, when male antennal fibrillae are erect to optimize detection of females 4,11,31 and circadian flight activity peaks 58,60,61 . To observe and record mating activity while minimizing light interference for the mosquitoes, the swarm cage was backlit throughout the recording with a red-light lamp 62 covered with a white paper diffuser. A white paper-lined recording booth was also placed around the swarm cage to optimize illumination.…”

“…Numerous studies have reported mosquito flight tones from single, pairs, or groups of tethered adults 26,[28][29][30][31]45,79,[82][83][84] , free-flying adults 24,32,[35][36][37]49,66,85 , or a combination of tethered and free-flying adults 10,21,25,[39][40][41][42]47,48,62,67,80 . Fewer studies have examined mating activity in swarms 6,86,87 or performed audio recordings of swarming individuals in free flight 10,35,36,39,47,49,62,66,85 , and none prior to the present study have done so simultaneously or over an entire swarm sequence. Overall, the findings presented in this study, along with other recent mosquito acoustics work 10,39,47,48,66…”

Adequacy and sufficiency evaluation of existing EFSA guidelines for the molecular characterisation, environmental risk assessment and post‐market environmental monitoring of genetically modified insects containing engineered gene drives

“…64 In line with EFSA (2013), the extent of the compositional and phenotypic analysis of GDMIs (i.e. the type and number of components and phenotypic parameters to consider [see for example James et al, 2018James et al, , 2020Aldersley et al, 2019;Su et al, 2020 for possible assessment parameters]), which are not intended for food/feed uses, is case-specific, and may vary with the nature of the insect, the genetic modification of interest, the intended outcome of the deliberate release and the level of environmental exposure. Moreover, the need for compositional and phenotypic data should be triggered by the need to test specific risk hypotheses formulated as part of the problem formulation (and which are framed by agreed protection goals).…”