Assessing the acoustic behaviour ofAnopheles gambiaes.l.dsxFmutants: Implications for Vector Control

Abstract

Background: The release of genetically modified mosquitoes which use gene-drive mechanisms to suppress reproduction in natural populations of Anopheles mosquitoes is one of the scientifically most promising methods for malaria\ transmission control. However, many scientific, regulatory and ethical questions remain before transgenic mosquitoes can be utilised in the field. Mutations which reduce an individual's reproductive success are likely to create strong selective pressures to evolve resistance. It is thus crucial that the targeted population collapses as rapidly and as completely as possible to reduce the available time for the emergence of drive-resistant mutations. At a behavioural level, this means that the gene-drive carrying mutants should be at least as (and ideally more) sexually attractive than the wildtype population they compete against. A key element in the copulatory negotiations of Anopheles mosquitoes is their acoustic courtship. We therefore analysed sound emissions and acoustic preference in a doublesex mutant previously used to successfully collapse caged colonies of Anopheles gambiae s.l.. Methods: The flight tones produced by the beating of their wings form the signals for acoustic mating communication in Anopheles species. We assessed the acoustic impact of the disruption of a female-specific isoform of the doublesex gene (dsxF) on the wing beat frequency (WBF; measured as flight tone) of both males (XY) and females (XX) in homozygous dsxF- mutants (dsxF-/-), heterozygous dsxF- carriers (dsxF+/-) and G3 'wildtype' dsxF+ controls (dsxF+/+). To exclude non-genetic influences, we controlled for temperature and measured wing lengths for all experimental animals. We used a phonotaxis assay to test the acoustic preferences of mutant and control mosquitoes. Results: A previous study demonstrated an altered phenotype only for females homozygous for the disrupted dsx allele (dsxF-/-), who appear intersex. No phenotypic changes were observed for heterozygous carriers or males, suggesting that the female-specific dsxF allele is haplosufficient. We here identify significant, dose-dependent increases in the flight tones of both dsxF-/- and dsxF+/- females when compared to dsxF+/+ control females. Flight tone frequencies in all three female genotypes remained significantly lower than in males, however. When tested experimentally, males showed stronger phonotactic responses to the flight tones of control dsxF+/+ females. While flight tones from dsxF+/- and dsxF-/- females also elicited positive phonotactic behaviour in males, this was significantly reduced compared to responses to control tones. We found no evidence of phonotactic behaviour in any female genotype tested. None of the male genotypes displayed any deviations from the control condition. Conclusions: A key prerequisite for copulation in anopheline mosquitoes is the phonotactic attraction of males towards female flight tones within large - spatially and acoustically crowded - mating swarms. Reductions in acoustic attractiveness of released mutant lines, as reported here for heterozygous dsxF+/- females, reduce the line's mating efficiency, and could consequently reduce the efficacy of the associated population control effort. Assessments of caged populations may not successfully reproduce the challenges posed by natural mating scenarios. We propose to amend existing testing protocols in order to more faithfully reflect the competitive conditions between a mutant line and the wildtype population it is meant to interact with. This should also include novel tests of 'acoustic fitness'. In line with previous studies, our findings confirm that disruption of the female-specific isoform dsxF has no effect on males; for some phenotypic traits, such as female flight tones, however, the effects of dsxF appear to be dose-dependent rather than haplosufficient.

Hitting the right note at the right time: Circadian control of audibility in Anopheles mosquito mating swarms is mediated by flight tones

“…S8) but also faster and thus more difficult to catch. Whatever the ultimate reason, the observed flight tone changes are bound to affect the mutual audibility in a hearing system, where tonal changes of ~50 Hz can reduce responses by >60% ( 29 ).…”

“…The population-genetic consequences of such interindividual variance in mating compatibility could be substantial. While the molecular bases for mosquito flight tone frequencies are still largely unknown, recent work has linked first genetic pathways and also demonstrated the behavioral impact of mutant phenotypes ( 29 ).…”

“…Previously reported differences in harmonic convergence event counts, which have been proposed as an acoustic interaction between males and females (21), are either directly explained by this intrinsic noise or indicative of underlying differences in the baseline flight tones. For example, the finding that (i) sons of pairs that showed more harmonic convergence before mating had greater mating success and that (ii) these offspring themselves showed more harmonic convergence before mating (32) simply indicates that a 3:2 ratio of flight tones provides audibility and, thus, mating benefits and (iii) that flight tones are heritable traits [also confirmed in (29)]. Attempts to use the short-lived harmonic convergence events as an indicator of genetic fitness have been largely inconclusive, but harmonic convergence has been found to be predictive of successful mating attempts (33), which is expected as the phonotactic chase that precedes copulation is an acoustically guided male behavior, which will benefit from the male flying at 1.5 times the female flight wingbeat frequency.…”

Serotonin modulation in the male Aedes aegypti ear influences hearing

“…Male attraction to the sound of flying, conspecific females is a clear, highly reproducible behavior (Andrés et al, 2020). Indeed, Frontiers in Physiology frontiersin.org Frontiers in Physiology frontiersin.org male attraction to female flight sounds is so strong that males can even be attracted to speakers playing pure tones of relevant frequencies; this frequency range is typically relatively narrow, with changes in pure tone frequency of >50 Hz leading to significant reductions in phonotactic response (Su et al, 2020). Male phonotaxis occurs predominantly within swarms, which form at specific times of the day and whose formation may be linked to serotonin expression levels (Anstey et al, 2009;Somers et al, 2022).…”

“…aegypti flight performance (Ngai et al, 2019). This is highly relevant in the context of mosquito acoustic communication, as flight plays a key role in the hearingmediated mating behavior of mosquitoes, with even relatively small changes in the frequency of flight sounds resulting in significant changes in the male responses (Andrés et al, 2020;Su et al, 2020). However, it remains unknown whether manipulation of the serotonergic system in Ae.…”