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Assessing the acoustic behaviour of Anopheles gambiae s.l. dsxF mutants:
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Implications for Vector Control
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Authors: Matthew P Su1,2,3†, Marcos Georgiades1,2†, Judit Bagi1,2, Kyros Kyrou4, Andrea
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Crisanti4, Joerg T Albert1,2,*
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Affiliations
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† These authors contributed equally
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1 Ear Institute, University College London, 332 Grays Inn Road, London, WC1X 8EE, UK
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2 The Francis Crick Institute, 1 Midland Road, London, NW1 1AT, UK
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3 Division of Biological Science, Nagoya University, Nagoya, 464-8602, Japan
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4 Department of Life Sciences, Imperial College London, UK.
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* Correspondence: joerg.albert@ucl.ac.uk
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E-mails:
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MS: su.matthew.paul@h.mbox.nagoya-u.ac.jp
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MG: marcos.georgiades.18@ucl.ac.uk
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JB: j.bagi@ucl.ac.uk
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KK: kyros.kyrou14@imperial.ac.uk
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AC: a.drcrisanti@imperial.ac.uk
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JTA: joerg.albert@ucl.ac.uk
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Abstract
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Background
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The release of genetically modified mosquitoes which use gene-drive mechanisms to suppress
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reproduction in natural populations of Anopheles mosquitoes is one of the scientifically most
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promising methods for malaria transmission control. However, many scientific, regulatory and
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ethical questions remain before transgenic mosquitoes can be utilised in the field. Mutations
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which reduce an individual’s reproductive success are likely to create strong selective pressures
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to evolve resistance. It is thus crucial that the targeted population collapses as rapidly and as
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completely as possible to reduce the available time for the emergence of drive-resistant
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mutations. At a behavioural level, this means that the gene-drive carrying mutants should be at
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least as (and ideally more) sexually attractive than the wildtype population they compete
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against. A key element in the copulatory negotiations of Anopheles mosquitoes is their acoustic
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courtship. We therefore analysed sound emissions and acoustic preference in a doublesex
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mutant previously used to successfully collapse caged colonies of Anopheles gambiae s.l..
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Methods
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The flight tones produced by the beating of their wings form the signals for acoustic mating
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communication in Anopheles species. We assessed the acoustic impact of the disruption of a
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female-specific isoform of the doublesex gene (dsxF) on the wing beat frequency (WBF;
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measured as flight tone) of both males (XY) and females (XX) in homozygous dsxF- mutants
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(dsxF-/-), heterozygous dsxF- carriers (dsxF+/-) and G3 ‘wildtype’ dsxF+ controls (dsxF+/+). To
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exclude non-genetic influences, we controlled for temperature and measured wing lengths for
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all experimental animals. We used a phonotaxis assay to test the acoustic preferences of mutant
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and control mosquitoes.
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Results
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A previous study demonstrated an altered phenotype only for females homozygous for the
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disrupted dsx allele (dsxF-/-), who appear intersex. No phenotypic changes were observed for
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heterozygous carriers or males, suggesting that the female-specific dsxF allele is
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haplosufficient. We here identify significant, dose-dependent increases in the flight tones of
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both dsxF-/- and dsxF+/- females when compared to dsxF+/+ control females. Flight tone
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frequencies in all three female genotypes remained significantly lower than in males, however.
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When tested experimentally, males showed stronger phonotactic responses to the flight tones
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of control dsxF+/+ females. While flight tones from dsxF+/- and dsxF-/- females also elicited
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positive phonotactic behaviour in males, this was significantly reduced compared to responses
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to control tones. We found no evidence of phonotactic behaviour in any female genotype tested.
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None of the male genotypes displayed any deviations from the control condition.
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Conclusions
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A key prerequisite for copulation in anopheline mosquitoes is the phonotactic attraction of
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males towards female flight tones within large - spatially and acoustically crowded - mating
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swarms. Reductions in acoustic attractiveness of released mutant lines, as reported here for
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heterozygous dsxF+/- females, reduce the line’s mating efficiency, and could consequently
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reduce the efficacy of the associated population control effort. Assessments of caged
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populations may not successfully reproduce the challenges posed by natural mating scenarios.
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We propose to amend existing testing protocols in order to more faithfully reflect the
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competitive conditions between a mutant line and the wildtype population it is meant to interact
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with. This should also include novel tests of ‘acoustic fitness’. In line with previous studies,
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our findings confirm that disruption of the female-specific isoform dsxF has no effect on males;
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for some phenotypic traits, such as female flight tones, however, the effects of dsxF appear to
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be dose-dependent rather than haplosufficient.
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Keywords
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Anopheles gambiae s.l., Anopheles coluzzii, doublesex, Gene drive, Wing beat frequency,
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Flight tone, Mosquito, Acoustic communication, Hearing, Phonotaxis, Vector control
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Background
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Mosquitoes represent a major global health problem, with Aedes, Anopheles and Culex species
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acting as vectors of diseases that infect millions of people each year [1]. Malaria remains a
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major cause of mortality and morbidity worldwide in spite of significant advances made in
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disease control since the turn of the century [2, 3]. This is in part due to the reduced efficacy
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of current control tools such as insecticidal nets and indoor residual spraying, as well as the
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emergence of secondary disease vectors [4, 5, 6]. Novel control techniques are therefore
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necessary to continue the push towards disease elimination [7].
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One potential option is the utilisation of gene drive systems, which target
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haplosufficient female fertility genes, leading to a reduction in female fertility and, eventually,
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population collapse [8, 9]. The recent generation of Anopheles gambiae CRISPR/Cas9 mutants
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in which a female specific exon of the doublesex (dsxF) gene was disrupted is here of interest.
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Lab cage trials have demonstrated that the introduction of dsxF mutants into cages of wildtype
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mosquitoes was sufficient to lead to eventual population collapse [10].
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However, there are many scientific, ethical and regulatory hurdles to overcome before
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such transgenic mosquitoes can be released in even semi-field trials [11]. It is vital that any
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transgenic mosquitoes are subjected to rigorous testing prior to use in the field; gene transfer
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into natural populations following release of transgenic Aedes aegypti has highlighted the
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potential risks of release of transgenic insects [12]. On a scientific level, one important task
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will be to maintain the gene drive’s effectiveness outside of the laboratory and under more ‘real
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world’ scenarios.
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A major element of this testing is the investigation of interactions with natural, non-
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mutant populations, particularly with regards to courtship behaviour. If mutant mosquitoes are
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unable, or only less likely, to copulate with native populations then they become the less
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attractive option, which will slow down or outright frustrate the population control effort [13].
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In addition to potential direct and indirect fitness costs associated with mutations, laboratory
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habituation and mass rearing can also affect mating performance [14, 15]. In this context it is
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noteworthy that the dsxF mutants we tested were also generated from a lab-established strain
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(G3) rather than any wildtype population [10]. Extensive testing of mutant mating fitness prior
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to translation from laboratory mating assays is thus a key requirement for assessing a specific
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line’s suitability for use as part of a release program.
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The sense of hearing is a vital component of mosquito reproduction, with males
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identifying females within swarms via phonotactic responses to female flight tones and
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acoustic communication is also thought to play a role in female mate selection [16, 17, 18, 19].
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The phonotactic response is highly specific, however, with males responding only to a narrow
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range of frequencies [20]. Both male and female mosquitoes have extraordinarily sensitive and
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complex ears, but there are also significant sexual dimorphisms in auditory function and
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hearing-related behaviours [21, 22, 23].
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Chromosomally female (XX) dsxF-/- mutants display an intersex phenotype, which also
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includes an intersex morphology of their flagellar sound receivers [10]; If, and if so to what
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extent, other parts of the auditory or acoustic system are affected by the allelic disruption is
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unclear. Physiological changes that could impact the mutants’ ability to interbreed with existing
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mosquitoes, are e.g. changes in male or female flight tones – or their corresponding acoustic
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preferences. It is currently unclear if any of the dsxF mutant genotypes affects these parameters.
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If so, this could have substantial effects on the ability of mutants to interbreed with existing
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mosquitoes.
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In order to address this topic, we tested the flight tones and phonotactic responses of
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dsxF XX and XY mutants and controls. We found that whilst male (XY) mutant (dsxF-/-,
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dsxF+/-) flight tones were not significantly different to male controls (dsxF+/+), female (XX)
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mutant (dsxF-/-, dsxF+/-) flight tones had significantly higher frequencies than those of their
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respective controls (dsxF+/+), with both showing an increase towards the male flight tone in a
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seemingly dose-response fashion.
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No female showed evidence of phonotaxis to any of the acoustic stimuli we provided,
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whilst all males showed a strong phonotactic response to tones of 400Hz (but much reduced or
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absent responses to tones of 100Hz or 700Hz). However, a more focused phonotaxis assay
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using the median flight tones obtained from each of the three female genotypes (dsxF+/+,
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dsxF+/-, dsxF-/-) found that control males responded far more strongly to the flight tones of
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control females than to either of the mutant flight tones. Preliminary tests of dsxF-/- males
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showed a similar preference for control flight tones (Supplemental Figure 2). As such, it seems
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likely that male mosquitoes of any genotype will demonstrate a strong preference for wildtype
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females, with mutant females potentially reduced to a lesser attractive role.
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Methods
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Mosquito rearing
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An. gambiae G3 strain (dsxF+/+), as well as dsxF+/- and dsxF-/- mutant pupae, were reared and
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provided by the Crisanti lab at Imperial College London. Larval density was kept constant
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throughout the rearing process.
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dsxF+/+ and dsxF+/- pupae were sex separated and kept in single sex cages in incubators
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maintained at 28°C and 80% relative humidity. Light/ dark conditions included a one-hour
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ramping period of constantly increasing white light; ramp for lights-ON from zeitgeber time
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(ZT) ZT0 to ZT1; then 11 hours (ZT1-ZT12) of white light at constant intensity, followed by
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a one-hour (ZT12-13) ramping period of constantly decreasing white light, and then 11 hours
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(ZT13-ZT24) of constant darkness. All light ramps transitioned linearly between a
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Photosynthetic Photon Flux Density (PPFD) of 80 µmol/m²/s (or ~ 5929 lux) and complete
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darkness (0 µmol/m²/s or 0 lux, respectively).
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dsxF-/- pupae were not sex separated but were otherwise reared in identical conditions.
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Mosquitoes were supplied with a constant source of 10% glucose solution.
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All mosquitoes used for experiments were virgin and aged 3 – 7 days old. Mosquitoes
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were housed within temperature, humidity and light-controlled incubators for three days prior
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to all experiments, which were conducted during a time corresponding to sunset (which
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represents a time period of peak activity and swarming under natural conditions).
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Wing length measurements
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The right wings of adult mosquitoes from each genotype were removed using a pair of forceps
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whilst the mosquitoes were CO2 sedated. The wings and flagellae were then transferred to
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separate microscope slides in groups of five. Each individual sample was immediately imaged
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using a Zeiss Axioplan 2 microscope and Axiovision 4.3 software. Wing lengths were
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determined using the Axiovision 4.3 software length measurement function, calibrated to the
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nearest 0.1 mm. Three biological repeats were conducted over separate generations.
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Total sample sizes for each group: dsxF+/+ XX = 40; dsxF+/- XX = 40; dsxF-/- XX = 40;
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dsxF+/+ XY = 41; dsxF+/- XY = 40; dsxF-/- XY = 41.
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Wing beat frequency measurements
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A resin casing was printed using an Ultimaker 2+ 3D printer and used to house a particle
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velocity microphone (Knowles NR-3158). The whole apparatus was held in a
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micromanipulator placed on a vibration isolation table.
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Adult mosquitoes from each genotype were cold-sedated using ice before blue-light
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cured glue was used to fix the tip of a tungsten wire to their thoraces, taking care not to restrict
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or damage the wings in doing so. The tethered mosquito was mounted into the microphone
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case and oriented such that its posterior was facing the particle velocity microphone. All
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measurements were conducted in the same isolated room at a temperature between 21 – 22°C.
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Mosquito flight was initiated via a tarsal reflex response [24]. A small cotton ball was
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placed underneath each tethered mosquito; once the mosquito had clasped the ball, it was
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swiftly removed, with this removal stimulating flight initiation. Minimum flight length used
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was 10 seconds. The voltage timeseries waveform measured for each flying mosquito by the
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particle velocity microphone was recorded using the Spike2 software (Cambridge Electronic
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Design Ltd., UK).
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Sample sizes for each group were: dsxF+/+ XX = 30; dsxF+/- XX = 30; dsxF-/- XX = 30;
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dsxF+/+ XY = 27; dsxF+/- XY = 30; dsxF-/- XY = 30.
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Flight tone analysis algorithm
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Raw data from the Spike2 recordings were exported to Python for analysis via a custom script.
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The first and last two seconds of each flight were discarded prior to analysis. Subsequently the
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timeseries was divided into 5-second subsegments, discarding the final shorter subsegment (if
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flight length modulo 5 ≠ 0). A Fast Fourier transform (FFT) with a 200ms window was then
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applied throughout each of the subsegments. This window was shifted in 100ms increments
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(i.e. 50% overlap between successive FFTs) and applied repeatedly until the end of the flight
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segment was reached (Figure 1a).
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Limits were applied to the frequency domain of each FFT such that only frequencies
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between 200 – 1,000Hz would be extracted for analysis. For each FFT, the peak frequency was
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identified and assigned as the flight tone for the time segment over which the FFT was
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calculated. A list of peak frequencies was compiled for each of the aforementioned
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subsegments. These lists were added together and averaged, resulting in a 5-second long final
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list of average frequencies. The mean was employed in the averaging step as these values were
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normally distributed. As the list of means, in turn, tended to be non-normally distributed, the
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median was taken and assigned as the flight tone of that individual animal. The segmentation
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of the original waveform and summarization into a single 5-second long list of values served
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to moderate for potential effects of flight duration on the animals’ flight tone.
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Spectrally broad phonotaxis assay
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Female (XX) and male (XY) mosquitoes from all three genotypes were aspirated into small,
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single-sex cages in groups of 25 and kept for at least two hours in the same room used for flight
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tone experiments. All experiments were conducted at a temperature between 20 – 23°C and at
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~ ZT13 (i.e. swarming time, around the time of complete cessation of light). Throughout the
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experiment, mosquitoes were kept in constant darkness.
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A free app (TMsoft tone generator) was used to provide acoustic stimulation to caged
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mosquitoes; this stimulation consisted of three pure tones with frequencies of 100, 400 and 700
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Hz. These frequencies were chosen based on the prior recordings which found no female flight
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tones as high as 700Hz, and no male or female frequency as low as 100Hz.
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The sound source was placed next to the cage with its speaker touching the cage mesh
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prior to stimulus initiation. Each tone was played for 1 minute and was succeeded in turn by a
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1-minute long silence before the next tone was played. The tones were played first from low to
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high frequencies and subsequently from high to low, allowing mosquitoes to rest for 5 minutes
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between forward and backward playbacks. To ensure that mosquitoes were being attracted to
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the sound emitted by the sound source rather than the sound source itself, at the start of each
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experiment the sound source was placed next to the cage with its speaker touching the cage’s
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mesh with no stimulus playing. Mosquitoes that approached the sound source during either
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control or acoustic playback were counted manually using a red-light flashlight. Three
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biological repeats were conducted for each group.
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Spectrally focused phonotaxis assay
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dsxF+/+ XY mosquitoes were tested in groups of 25 as above for the broad-range assay, this
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time, however using three pure tones with frequencies equal to the recorded median flight tone
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frequencies of each of the female genotypes; 380Hz (dsxF+/+), 432Hz (dsxF+/-) and 497Hz
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(dsxF-/-). [Please note that the played dsxF+/+ control tone of 380Hz is marginally different
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from the median reported in Table 1; this is because the flight tone choice for the playback
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experiments was based on an earlier data cohort].
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Statistical analysis
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Flight tone analyses were conducted in Python. Remaining analyses were completed in Matlab
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and R. Throughout the analyses, all statistical tests used a significance level of p <0.05.
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Sample sizes for all experiments were determined via reference to published
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investigations. Within-group variation estimates were calculated when appropriate as part of
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standard statistical testing.
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Statistical tests for normality (Shapiro–Wilk Normality tests with a significance level
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of p < 0.05) were first applied to each dataset.
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Wing length measurements and flight tones were found to be normally distributed; two-
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way ANOVA tests were thus used for comparisons across the genotypes and sexes.
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For the spectrally broad phonotaxis assay, the proportion of responders to the control
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stimulus (silence) was subtracted from the proportion of responders to the stimulus tones. That
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is, to calculate the adjusted proportion of responders we calculated:
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Adjusted proportion of responders = Responders 𝑡𝑜𝑛𝑒– Responders𝑠𝑖𝑙𝑒𝑛𝑐𝑒
Total number of mosquitoes
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Where Responderstone and Responderssilence refers to the number of responders to the
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individual tones or silence respectively. One-way ANOVAs were then used to test for
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differences in responses between the stimulus tone frequencies. For the focused phonotaxis
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assay, no adjusted proportion was calculated and one-way ANOVAs were applied directly to
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the Proportion of responders.
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Results
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dsxF+/- and dsxF-/- XX mutants have different flight tones to all other XX and XY
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mosquitoes
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By recording the flight tones of tethered female and male mosquitoes we were able to
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calculate the median flight tones for each group (Figure 1b). All male flight tones were found
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to be greater than all female flight tones, but we found no differences between males (Two-
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way ANOVA; p<0.001; p>0.05 respectively). The flight tones of dsxF-/- females were
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significantly different from all other groups; they were significantly higher than the other
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female genotypes (497 ± 22.2 Hz compared to 432 ± 28.7 Hz and 380 ± 30.0 Hz for dsxF+/-
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and dsxF+/+, respectively), and significantly lower than all male genotypes (Two-way
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ANOVA; p<0.001; Table 1; Figure 1b). We also found a significant difference between dsxF+/-
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XX mutants and the other two female genotypes in an apparent dose response fashion (Two-
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way ANOVA; p=0.002; Figure 1b).
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Mosquito flight tone frequencies have been reported to show correlations with
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temperature (see e.g. [25] for Aedes), but the relation, especially for anopheline mosquitoes,
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has remained unclear. Here, temperature was tightly controlled, with all recordings being made
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between 21 and 22°C. The relationship between wing beat frequency (= flight tone) and wing
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length is far more contentious however, with conflicting reports on potential correlations (see
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e.g. [26, 27]). We measured wing lengths for each group and found significant differences
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between the sexes (Two-way ANOVA; p<0.001; Figure 1c). Further differences were found
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between dsxF+/- and dsxF+/+, as well as dsxF-/- and dsxF+/+, mosquitoes of both sexes (Two-
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way ANOVA; p<0.001). Individual correlation analyses for each group showed a relationship
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between wing length and wing beat frequency only for dsxF+/+ females (Supplemental Figure
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1). Furthermore, a linear model fit including data from all groups found no significant
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relationship between wing length and wing beat frequency (see Supplemental Table 1).
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Male, but not female, mosquitoes show positive phonotactic responses to acoustic
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stimuli mimicking female flight tones
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We tested for mosquito responses to auditory stimulation in order to investigate whether
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mutants showed altered behaviour (Figure 2a). No females from any genotype showed a
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significantly greater response to an acoustic stimulus (defined as an approach to the sound
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source) than to silence (ANOVA; p>0.05 for all comparisons; see Figure 2b top). All male
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groups tested were found to respond more strongly to tones of 400Hz, the stimulus which most
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closely mimicked wildtype female WBF, than any other stimulus type (ANOVA; p<0.05;
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Table 2; Figure 2b bottom). However, a few males also responded to the 100 and 700Hz tones.
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It seems noteworthy that the males’ flight-mediated responses to the playback tones were
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equally strong in mutants and controls, suggesting that the dsxF-/- allele does not affect male
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flight behaviour.
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We then investigated if the flight tone differences observed between females with
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different allelic combinations of dsxF (+/+, +/-, -/-) were behaviourally relevant. Specifically, we
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tested the phonotactic preferences of dsxF+/+ males to pure tones with frequencies equivalent
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to the median frequencies of females from all three genotypes (+/+ = 380 ± 30.0 Hz, +/- = 432
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± 28.7 Hz and +/- = 497 ± 22.2 Hz), at the narrow temperature range of 21 - 22 °C. Males were
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found to respond significantly more to tones similar to ‘wildtype’ dsxF+/+ female flight tones
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than to tones mimicking either of the female mutants. The ability of flight tones to induce male
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phonotaxis followed a ‘dose-dependent’ pattern with dsxF+/+ > dsxF+/- > dsxF-/- (ANOVA;
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p<0.001; Table 2; Figure 2c).
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Discussion
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Hearing plays a crucial role in mosquito copulation [28]. The phonotactic responses of
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mosquito males to the flight tones of nearby flying females (or to artificial pure tones
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mimicking such females), are an important behavioural feature for mosquito reproductive
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fitness, and reproduction [29]. As such, the ‘acoustic fitness’ of transgenic lines marked for
301
release in the field is a key requirement for the successful spread of deleterious mutations into
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wildtype populations. Here we show that the transgenic disruption of a female-specific isoform
303
of the sex-determination gene doublesex (dsxF) changes female flight tones and that mutant
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flight tones elicit substantially reduced phonotactic responses in control males. The flight tone
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changes observed were more pronounced in homozygous (dsxF-/-) than in heterozygous
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condition (dsxF-/-), indicating a dsxF+ dose-dependence of this phenotypic trait, which
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contrasts with the previously shown haplosufficiencies [10].
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Previous recordings of mosquito flight tones have implemented a variety of analytic
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techniques, but rarely implemented strict environmental controls. This is problematic given the
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significant variation for reported flight tones at different temperatures, and also the suggested
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correlations of flight tone and body size [25, 28]. Here we strictly controlled temperature, and
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also measured wing length as a proxy for body size, to control for this variability. Anopheles
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swarms form predominantly at dusk, when both light and temperature decrease rapidly [30]. It
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seems possible that during this time female Anopheles flight tones decrease rapidly in direct
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correlation to these temperature decreases; female Aedes aegypti WBF fell by around 10Hz per
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degree over similar temperature changes [25]. Given the sizeable differences we observed in
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male phonotactic responses to acoustic stimuli less than 50Hz apart, these differences could
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have a significant effect on male auditory behaviours.
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If dsxF mutants are to be released in the wild, then only heterozygous males are likely
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to be released. Updated cage trials with a starting allelic frequency of only 2.5% predicted
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population collapse within 14 generations [31]. Generation of dsx mutants in other mosquito
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species (such as Aedes aegypti) could not only provide a promising control method to combat
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other mosquito populations, but also provide an ideal tool to investigate the fundamental
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mechanisms which underly the sizeable sexual dimorphisms in mosquito auditory systems and
325
behaviours. The dsxF isoform is reported to be female-specific, it is therefore reassuring that
326
we found no differences in flight tones between male genotypes. All males not only displayed
327
typical phonotactic behaviour but furthermore retained their acoustic preference for the flight
328
tones of control (‘wildtype’) females around 400Hz (at 20-21°C). Most interestingly also, the
329
intersex phenotype of dsxF-/- females did not include the display of phonotactic behaviour,
330
possibly indicating an independence of male phonotaxis from the dsx pathway or leaving a role
331
for the male doublesex isoform (dsxM).
332
Lab-based assays in cage conditions can only partially, at best, replicate field
333
conditions. Throughout our phonotaxis experiments, we provided only a single, monofrequent
334
acoustic stimulus at any one time. This is a poor simulation of the auditory landscape of an An.
335
gambiae swarm containing many hundreds of males whose flight tones may be constantly
336
modulated [25]. The presence of multiple females within this environment may lead to
337
selection choices for individual males. This may exacerbate the phonotactic preferences we
338
discovered (see Figure 2c), with males possibly disregarding the flight tones of mutant females
339
if simultaneously presented with the sounds of wildtype ones.
340
Yet the fact that mutant males retain a strong preference for the sounds of wildtype
341
females, bodes well for the effective spread of mutant alleles into resident wildtype
342
populations. It remains to be seen though whether mutant males can successfully join the
343
natural swarms, in which Anopheles copulation occurs. Further studies of both mutant and
344
wildtype swarming behaviour are necessary to better understand – and predict – the relevant
345
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17
male-female interactions. This also holds true for a potential female choice element; although
346
we here found no differences between the male genotypes in terms of flight tones, there may
347
be other differences which influence female mate selection.
348
The argument for utilising transgenic Anopheles strains for fighting malaria grows
349
stronger with each new report of insecticide resistance or change in biting behaviour. It is
350
essential however that transgenic lines are tested thoroughly for their suitability. Not only will
351
such experimental testing improve a respective line’s chances of success, but it will also help
352
to create a more detailed profile of the specific requirements for successful release lines (e.g.
353
gene drive carriers). Given the importance of audition for all disease-transmitting mosquito
354
species, acoustic (and auditory) fitness will feature high on that list of requirements. Acoustic
355
courtship in Anopheles, finally, is inextricably linked to the mating swarm. Including swarming
356
behaviour in the pre-release testing will thus be crucial. A pipeline of testing focused on
357
mosquito acoustic mating behaviour could significantly help in boosting the efficacy of any
358
release effort. This testing could comprise a sequence of analyses, covering anatomical
359
investigation of the ear, functional tests of hearing, flight tone recordings, and phonotaxis/
360
mating assays under cage or semi-field conditions. This study utilised only a fraction of these
361
analyses and discovered ecologically relevant differences between mutant and control lines; a
362
comprehensive assessment may provide substantially more evidence which can inform the
363
decision-making process over mutant release strategies and help optimise future disease-
364
control efforts.
365
366
Acknowledgements and Funding
367
The authors would like to thank Carla Siniscalchi (Imperial College London) for providing G3
368
strain pupae. This work received funding through a pump-priming award from the BBSRC
369
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18
Vector Borne Disease (VBD) Network ANTI-VeC (AV/PP/0028/1, to J.T.A. and M.S.) and a
370
UCL Global Challenges Research Fund (GCRF) small grant (to J.T.A.) and the European
371
Research Council (ERC) under the Horizon 2020 research and innovation programme (Grant
372
agreement No 648709, to J.T.A.).
373
Author contributions
374
M.P.S., M.G., A.C. and J.T.A. contributed to the conception and design of the research. M.P.S.,
375
M.G., K.K. and J.B. performed experiments. M.P.S., M.G. and J.T.A. analysed the data.
376
M.P.S., M.G. and J.T.A. wrote the manuscript. J.T.A supervised the study. All authors read
377
and approved of the final manuscript.
378
379
Ethics approval and consent to participate
380
Not applicable.
381
Consent for publication
382
Not applicable.
383
Competing interests
384
The authors declare no competing interests.
385
Data availability
386
All data analysed in this paper are available from the authors, as well as more comprehensive
387
details on experimental or analytical methodologies.
388
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19
Figure Legends
389
Figure 1: dsxF+/-and dsxF-/- XX mutants have different wing beat frequencies (=flight tones) to all other
390
groups
391
a) Sketch of flight tone recording set-up: mosquitoes were tethered then placed at a constant distance
392
from a microphone. Temperature and humidity conditions were controlled (21-22OC; 50% RH) and
393
recordings always took place within the same two-hour window.
394
b) Calculated wing beat frequencies for each genotype – significant differences (Two-way ANOVA; *
395
p < 0.05) between groups are indicated by letter. Centre line mean; box limits, lower and upper
396
quartiles; whiskers, 5th and 95th percentiles (identical B-C).
397
Sample sizes: dsxF+/+ XX = 30; dsxF+/- XX = 30; dsxF-/- XX = 30; dsxF+/+ XY = 27; dsxF+/- XY = 30;
398
dsxF-/- XY = 30.
399
c) Wing length measurements for each genotype - significant differences (Two-way ANOVA; *
400
p < 0.05) between groups are indicated by letter.
401
Sample sizes: dsxF+/+ XX = 40; dsxF+/- XX = 40; dsxF-/- XX = 40; dsxF+/+ XY = 41; dsxF+/- XY = 40;
402
dsxF-/- XY = 41.
403
404
Figure 2: Males show a strong preference for acoustic stimuli of similar frequency to wildtype female
405
flight tones; this phonotactic response is reduced as the tone becomes increasingly different
406
a) Diagram of phonotaxis experimental set-up: Single-sex virgin cages were provided with one-minute
407
periods of stimulation in the form of three pure tones (100, 400 and 700Hz) or a one-minute period of
408
silence. The number of mosquitoes attracted to the sound source for each type of stimulus was
409
calculated.
410
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20
b) Adjusted proportion of mosquitoes responding to each stimulus type (no stimulus, 100Hz, 400Hz
411
and 700Hz respectively) for XX and XY mosquitoes from each genotype. Centre circle, median; error
412
bars represent ± SEM.
413
c) Adjusted proportion of control mosquitoes responding to each stimulus type (380Hz, 432Hz and
414
497Hz respectively) for dsxF+/+ XY mosquitoes. Centre line, median; error bars represent ± SEM.
415
416
Supplementary Figure 1: Correlations between wing length and wing beat frequency
417
a) Correlations between wing length (mm) and wing beat frequency (Hz) for all groups tested. Sample
418
sizes are the same as for wing beat frequency calculations.
419
420
Supplementary Figure 2: Phonotactic response of dsxF-/- males to phonotactic stimulation
421
a) Adjusted proportion of control mosquitoes responding to each stimulus type (380Hz, 432Hz and
422
497Hz respectively) for dsxF-/- XY mosquitoes. Centre line, median; error bars represent ± SEM.
423
424
Supplemental Information
425
Linear model fitting
426
In order to investigate the potential relationship between wing beat frequency (=flight tones)
427
and wing length (as well as other potential variables) in greater detail, we used the R package
428
‘lme4’ to fit the following equation:
429
Wing beat frequency ~ Sex*Genotype + Wing length
430
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21
We found that sex, genotype, and sex:genotype were all highly significant factors in
431
determining wing beat frequency. However, wing length was not found to significantly affect
432
wing beat frequency.
433
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22
References
434
435
1. James SL, Abate D, Abate KH, Abay SM, Abbafati C, Abbasi N, et al. Global, regional, and
436
national incidence, prevalence, and years lived with disability for 354 diseases and injuries for
437
195 countries and territories, 1990-2017: a systematic analysis for the Global Burden of Disease
438
Study 2017. Lancet. 2018;392 10159:1789-858.
439
2. O'Meara WP, Mangeni JN, Steketee R, Greenwood B. Changes in the burden of malaria in sub-
440
Saharan Africa. Lancet Infect Dis. 2010;10 8:545-55.
441
3. WHO: Malaria Fact Sheet (https://www.who.int/en/news-room/fact-sheets/detail/malaria).
442
https://www.who.int/en/news-room/fact-sheets/detail/malaria (2019).
443
4. Ranson H, N'Guessan R, Lines J, Moiroux N, Nkuni Z, Corbel V. Pyrethroid resistance in
444
African anopheline mosquitoes: what are the implications for malaria control? Trends Parasitol.
445
2011;27 2:91-8.
446
5. Lwetoijera DW, Harris C, Kiware SS, Dongus S, Devine GJ, McCall PJ, et al. Increasing role
447
of Anopheles funestus and Anopheles arabiensis in malaria transmission in the Kilombero
448
Valley, Tanzania. Malaria J. 2014;13.
449
6. Sougoufara S, Ottih EC, Tripet F. The need for new vector control approaches targeting outdoor
450
biting anopheline malaria vector communities. Parasite Vector. 2020;13 1:295.
451
7. Hemingway J, Shretta R, Wells TNC, Bell D, Djimde AA, Achee N, et al. Tools and Strategies
452
for Malaria Control and Elimination: What Do We Need to Achieve a Grand Convergence in
453
Malaria? Plos Biol. 2016;14 3.
454
8. Deredec A, Godfray HCJ, Burt A. Requirements for effective malaria control with homing
455
endonuclease genes. Proceedings of the National Academy of Sciences of the United States of
456
America. 2011;108 43:E874-E80.
457
9. Flores HA, O'Neill SL. Controlling vector-borne diseases by releasing modified mosquitoes.
458
Nat Rev Microbiol. 2018;16 8:508-18.
459
10. Kyrou K, Hammond AM, Galizi R, Kranjc N, Burt A, Beaghton AK, et al. A CRISPR-Cas9
460
gene drive targeting doublesex causes complete population suppression in caged Anopheles
461
gambiae mosquitoes. Nature Biotechnology. 2018;36 11:1062-+.
462
11. Champer J, Buchman A, Akbari OS. Cheating evolution: engineering gene drives to manipulate
463
the fate of wild populations. Nat Rev Genet. 2016;17 3:146-59.
464
12. Brossard D, Belluck P, Gould F, Wirz CD. Promises and perils of gene drives: Navigating the
465
communication of complex, post-normal science. Proceedings of the National Academy of
466
Sciences of the United States of America. 2019;116 16:7692-7.
467
13. Aldersley A, Pongsiri A, Bunmee K, Kijchalao U, Chittham W, Fansiri T, et al. Too "sexy" for
468
the field? Paired measures of laboratory and semi-field performance highlight variability in the
469
apparent mating fitness of Aedes aegypti transgenic strains. Parasite Vector. 2019;12.
470
14. Leftwich PT, Bolton M, Chapman T. Evolutionary biology and genetic techniques for insect
471
control. Evol Appl. 2016;9 1:212-30.
472
15. Soma DD, Maiga H, Mamai W, Bimbile-Somda NS, Venter N, Ali AB, et al. Does mosquito
473
mass-rearing produce an inferior mosquito? Malaria J. 2017;16.
474
16. Pennetier C, Warren B, Dabire KR, Russell IJ, Gibson G. "Singing on the Wing" as a
475
Mechanism for Species Recognition in the Malarial Mosquito Anopheles gambiae. Curr Biol.
476
2010;20 2:131-6.
477
17. Clements A. THE BIOLOGY OF MOSQUITOES VOLUME 3 TRANSMISSION OF
478
VIRUSES AND INTERACTIONS WITH BACTERIA Preface. Biology of Mosquitoes, Vol
479
3: Transmission of Viruses and Interactions with Bacteria. 2012:Viii-+.
480
18. Aldersley A, Cator LJ. Female resistance and harmonic convergence influence male mating
481
success in Aedes aegypti. Sci Rep-Uk. 2019;9.
482
19. Manoukis NC, Diabate A, Abdoulaye A, Diallo M, Dao A, Yaro AS, et al. Structure and
483
Dynamics of Male Swarms of Anopheles gambiae. J Med Entomol. 2009;46 2:227-35.
484
20. Belton P. Attraction of Male Mosquitos to Sound. J Am Mosquito Contr. 1994;10 2:297-301.
485
.CC-BY-NC 4.0 International licensepreprint (which was not certified by peer review) is the author/funder. It is made available under a The copyright holder for thisthis version posted September 6, 2020. . https://doi.org/10.1101/2020.09.06.284679doi: bioRxiv preprint
23
21. Albert Joerg T, Kozlov Andrei S. Comparative Aspects of Hearing in Vertebrates and Insects
486
with Antennal Ears. Curr Biol. 2016;26 20:R1050-R61.
487
22. Andres M, Seifert M, Spalthoff C, Warren B, Weiss L, Giraldo D, et al. Auditory Efferent
488
System Modulates Mosquito Hearing. Curr Biol. 2016;26 15:2028-36.
489
23. Su MP, Andrés M, Boyd-Gibbins N, Somers J, Albert JT. Sex and species specific hearing
490
mechanisms in mosquito flagellar ears. Nature Communications. 2018;9 1:3911.
491
24. Fraenkel G. Untersuchungen über die Koordination von Reflexen und automatisch-nervösen
492
Rhythmen bei Insekten. Zeitschrift für vergleichende Physiologie. 1932;16 2:371-93.
493
25. Villarreal SM, Winokur O, Harrington L. The Impact of Temperature and Body Size on
494
Fundamental Flight Tone Variation in the Mosquito Vector Aedes aegypti (Diptera: Culicidae):
495
Implications for Acoustic Lures. J Med Entomol. 2017;54 5:1116-21.
496
26. Cator LJ, Ng'Habi KR, Hoy RR, Harrington LC. Sizing up a mate: variation in production and
497
response to acoustic signals in Anopheles gambiae. Behavioral Ecology. 2010;21 5:1033-9.
498
27. Pantoja-Sanchez H, Gomez S, Velez V, Avila FW, Alfonso-Parra C. Precopulatory acoustic
499
interactions of the New World malaria vector Anopheles albimanus (Diptera: Culicidae).
500
Parasite Vector. 2019;12.
501
28. Andrés M, Su MP, Albert J, Cator LJ. Buzzkill: targeting the mosquito auditory system. Current
502
Opinion in Insect Science. 2020;40:11-7.
503
29. Stone CM, Tuten HC, Dobson SL. Determinants of Male Aedes aegypti and Aedes
504
polynesiensis (Diptera: Culicidae) Response to Sound: Efficacy and Considerations for Use of
505
Sound Traps in the Field. J Med Entomol. 2013;50 4:723-30.
506
30. Sawadogo SP, Costantini C, Pennetier C, Diabaté A, Gibson G, Dabiré RK. Differences in
507
timing of mating swarms in sympatric populations of Anopheles coluzzii and Anopheles
508
gambiae s.s. (formerly An. gambiae M and S molecular forms) in Burkina Faso, West Africa.
509
Parasite Vector. 2013;6 1:275.
510
31. Simoni A, Hammond AM, Beaghton AK, Galizi R, Taxiarchi C, Kyrou K, et al. A male-biased
511
sex-distorter gene drive for the human malaria vector Anopheles gambiae. Nature
512
Biotechnology. 2020.
513
514
515
.CC-BY-NC 4.0 International licensepreprint (which was not certified by peer review) is the author/funder. It is made available under a The copyright holder for thisthis version posted September 6, 2020. . https://doi.org/10.1101/2020.09.06.284679doi: bioRxiv preprint
24
Table 1: Quantification of changes to dsxF+/- XX flight tones
516
Mean values of wing lengths and flight tones for dsxF+/+, dsxF+/- and dsxF+/+ XX and XY
517
mosquitoes, with standard deviation (SD) values provided in brackets. Significant differences
518
found between dsxF-/- XX mosquitoes and any other mosquito group are starred (ANOVA on
519
ranks; *p < 0.05; ***p < 0.001).
520
521
dsxF+/+ XX
dsxF+/- XX
dsxF-/- XX
dsxF+/+ XY
dsxF+/- XY
dsxF-/- XY
Sample size,
wing length
40
40
40
41
40
41
Wing length
in mm (SD)
3.806*
(0.126)
3.894
(0.152)
3.893
(0.157)
3.604***
(0.153)
3.746***
(0.100)
3.666***
(0.109)
Sample size,
flight tone
30
30
30
27
30
30
flight tone in
Hz (SD)
388.52***
(29.97)
431.55***
(28.68)
497.18
(22.22)
590.60***
(46.10)
596.14***
(47.40)
597.71***
(47.33)
522
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25
Table 2: Quantification of phonotactic responses to acoustic stimulation
523
(Top) Median values of the number of responders to coarse phonotactic stimulation for
524
dsxF+/+, dsxF+/- and dsxF-/- XX and XY mosquitoes, with SEM values provided in brackets.
525
Significant differences found within a genotype between the response to 400Hz and 100/700Hz
526
stimulation are starred (ANOVA; *p < 0.05).
527
(Bottom) Median values of the number of responders to focused phonotactic stimulation for
528
dsxF+/+ XY mosquitoes, with SEM values provided in brackets. Significant differences found
529
between the three stimulation frequencies are starred (ANOVA; ***p < 0.001).
530
531
Sample size
(coarse)
3 cages of
25
2 cages of
25
3 cages of
25
3 cages of
25
3 cages of
25
3 cages of
25
Proportion of
responders to
control
0.02
(0.02)
0.1
(0)
0
(0)
0
(0)
0
(0)
0
(0.01)
Proportion of
responders to
100 Hz
0
(0.03)
0.09
(0.01)
0
(0.01)
0.1*
(0.03)
0.15*
(0.03)
0.1*
(0.08)
Proportion of
responders to
400 Hz
0.05
(0.02)
0.11
(0.04)
0
(0)
0.69
(0.18)
0.85
(0.07)
0.55
(0.17)
Proportion of
responders to
700 Hz
0.05
(0.03)
0.09
(0.04)
0
(0.02)
0*
(0.01)
0*
(0)
0.05*
(0.02)
Sample size
(focused)
-
-
-
6 cages of
25
-
-
Proportion of
responders to
380 Hz
-
-
-
0.75
(0.03)
-
-
Proportion of
responders to
432 Hz
-
-
-
0.54***
(0.03)
-
-
Proportion of
responders to
497 Hz
-
-
-
0.20***
(0.02)
-
-
532
533
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26
534
Supplemental table 1
535
Outputs of linear model relating wing beat frequency to sex, genotype and wing length.
536
Significant values are italicised.
537
Variable
Estimate
SE
T value
Pr(>|t|)
(Intercept)
335.44
118.73
2.825
0.00559 **
Sex (Male)
196.10
13.88
14.130
<2e-16 ***
Genotype
(Heterozygous)
39.06
14.16
2.758
0.00679 **
Genotype
(Homozygous)
108.00
14.09
7.663
6.80e-12 ***
Wing Length
13.16
31.36
0.420
0.67554
Sex (Male):
Genotype
(Heterozygous)
-44.19
18.84
-2.346
0.02072 *
Sex (Male):
Genotype
(Homozygous)
-93.51
19.16
-4.881
3.49e-06 ***
538
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