Assessing the acoustic behaviour of Anopheles gambiae s.l. dsxF mutants: Implications for Vector Control

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.


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Background 22 The release of genetically modified mosquitoes which use gene-drive mechanisms to suppress 23 reproduction in natural populations of Anopheles mosquitoes is one of the scientifically most 24 promising methods for malaria transmission control. However, many scientific, regulatory and 25 ethical questions remain before transgenic mosquitoes can be utilised in the field. Mutations 26 which reduce an individual's reproductive success are likely to create strong selective pressures 27 to evolve resistance. It is thus crucial that the targeted population collapses as rapidly and as 28 completely as possible to reduce the available time for the emergence of drive-resistant 29 mutations. At a behavioural level, this means that the gene-drive carrying mutants should be at 30 least as (and ideally more) sexually attractive than the wildtype population they compete 31 against. A key element in the copulatory negotiations of Anopheles mosquitoes is their acoustic 32 courtship. We therefore analysed sound emissions and acoustic preference in a doublesex haplosufficient. We here identify significant, dose-dependent increases in the flight tones of 48 both dsxF -/and dsxF +/females when compared to dsxF +/+ control females. Flight tone 49 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 51 of control dsxF +/+ females. While flight tones from dsxF +/and dsxF -/females also elicited 52 positive phonotactic behaviour in males, this was significantly reduced compared to responses 53 to control tones. We found no evidence of phonotactic behaviour in any female genotype tested. 54 None of the male genotypes displayed any deviations from the control condition.

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A key prerequisite for copulation in anopheline mosquitoes is the phonotactic attraction of 57 males towards female flight tones within large -spatially and acoustically crowded -mating 58 swarms. Reductions in acoustic attractiveness of released mutant lines, as reported here for 59 heterozygous dsxF +/females, reduce the line's mating efficiency, and could consequently 60 reduce the efficacy of the associated population control effort. Assessments of caged 61 populations may not successfully reproduce the challenges posed by natural mating scenarios. 62 We propose to amend existing testing protocols in order to more faithfully reflect the 63 competitive conditions between a mutant line and the wildtype population it is meant to interact 64 with. This should also include novel tests of 'acoustic fitness'. In line with previous studies, Background 72 Mosquitoes represent a major global health problem, with Aedes, Anopheles and Culex species 73 acting as vectors of diseases that infect millions of people each year [1]. Malaria remains a 74 major cause of mortality and morbidity worldwide in spite of significant advances made in 75 disease control since the turn of the century [2,3]. This is in part due to the reduced efficacy 76 of current control tools such as insecticidal nets and indoor residual spraying, as well as the 77 emergence of secondary disease vectors [4,5,6]. Novel control techniques are therefore 78 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 80 haplosufficient female fertility genes, leading to a reduction in female fertility and, eventually, 81 population collapse [8,9]. The recent generation of Anopheles gambiae CRISPR/Cas9 mutants 82 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 84 mosquitoes was sufficient to lead to eventual population collapse [10]. 85 However, there are many scientific, ethical and regulatory hurdles to overcome before 86 such transgenic mosquitoes can be released in even semi-field trials [11]. It is vital that any 87 transgenic mosquitoes are subjected to rigorous testing prior to use in the field; gene transfer 88 into natural populations following release of transgenic Aedes aegypti has highlighted the 89 potential risks of release of transgenic insects [12]. On a scientific level, one important task 90 will be to maintain the gene drive's effectiveness outside of the laboratory and under more 'real 91 world' scenarios.

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A major element of this testing is the investigation of interactions with natural, non-93 mutant populations, particularly with regards to courtship behaviour. If mutant mosquitoes are 94 unable, or only less likely, to copulate with native populations then they become the less 95 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 97 habituation and mass rearing can also affect mating performance [14,15]. In this context it is 98 noteworthy that the dsxF mutants we tested were also generated from a lab-established strain 99 (G3) rather than any wildtype population [10]. Extensive testing of mutant mating fitness prior 100 to translation from laboratory mating assays is thus a key requirement for assessing a specific 101 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 103 identifying females within swarms via phonotactic responses to female flight tones and 104 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  If so, this could have substantial effects on the ability of mutants to interbreed with existing 116 mosquitoes.

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In order to address this topic, we tested the flight tones and phonotactic responses of 118 dsxF XX and XY mutants and controls. We found that whilst male (XY) mutant (dsxF -/-, 119 dsxF +/-) flight tones were not significantly different to male controls (dsxF +/+ ), female (XX) 120 mutant (dsxF -/-, dsxF +/-) flight tones had significantly higher frequencies than those of their 121 respective controls (dsxF +/+ ), with both showing an increase towards the male flight tone in a 122 seemingly dose-response fashion.

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No female showed evidence of phonotaxis to any of the acoustic stimuli we provided, 124 whilst all males showed a strong phonotactic response to tones of 400Hz (but much reduced or 125 absent responses to tones of 100Hz or 700Hz). However, a more focused phonotaxis assay 126 using the median flight tones obtained from each of the three female genotypes (dsxF +/+ , 127 dsxF +/-, dsxF -/-) found that control males responded far more strongly to the flight tones of 128 control females than to either of the mutant flight tones. Preliminary tests of dsxF -/males 129 showed a similar preference for control flight tones (Supplemental Figure 2). As such, it seems 130 likely that male mosquitoes of any genotype will demonstrate a strong preference for wildtype 131 females, with mutant females potentially reduced to a lesser attractive role.    Wing beat frequency measurements 164 A resin casing was printed using an Ultimaker 2+ 3D printer and used to house a particle 165 velocity microphone (Knowles NR-3158). The whole apparatus was held in a 166 micromanipulator placed on a vibration isolation table.

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Adult mosquitoes from each genotype were cold-sedated using ice before blue-light 168 cured glue was used to fix the tip of a tungsten wire to their thoraces, taking care not to restrict 169 or damage the wings in doing so. The tethered mosquito was mounted into the microphone 170 case and oriented such that its posterior was facing the particle velocity microphone. All 171 measurements were conducted in the same isolated room at a temperature between 21 -22°C.   Hz. These frequencies were chosen based on the prior recordings which found no female flight 209 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 211 prior to stimulus initiation. Each tone was played for 1 minute and was succeeded in turn by a 212 1-minute long silence before the next tone was played. The tones were played first from low to 213 high frequencies and subsequently from high to low, allowing mosquitoes to rest for 5 minutes 214 between forward and backward playbacks. To ensure that mosquitoes were being attracted to   By recording the flight tones of tethered female and male mosquitoes we were able to 253 calculate the median flight tones for each group (Figure 1b). All male flight tones were found 254 to be greater than all female flight tones, but we found no differences between males (Two-255 way ANOVA; p<0.001; p>0.05 respectively). The flight tones of dsxF -/females were 256 significantly different from all other groups; they were significantly higher than the other 257 female genotypes (497 ± 22.2 Hz compared to 432 ± 28.7 Hz and 380 ± 30.0 Hz for dsxF +/-258 and dsxF +/+ , respectively), and significantly lower than all male genotypes (Two-way 259 ANOVA; p<0.001; Table 1; Figure 1b). We also found a significant difference between dsxF +/-260 XX mutants and the other two female genotypes in an apparent dose response fashion (Two-261 way ANOVA; p=0.002; Figure 1b). length is far more contentious however, with conflicting reports on potential correlations (see 267 e.g. [26,27]). We measured wing lengths for each group and found significant differences 268 between the sexes (Two-way ANOVA; p<0.001; Figure 1c). Further differences were found 269 between dsxF +/and dsxF +/+ , as well as dsxF -/and dsxF +/+ , mosquitoes of both sexes (Two-270 way ANOVA; p<0.001). Individual correlation analyses for each group showed a relationship 271 between wing length and wing beat frequency only for dsxF +/+ females (Supplemental Figure   272 1). Furthermore, a linear model fit including data from all groups found no significant 273 relationship between wing length and wing beat frequency (see Supplemental Table 1). male phonotactic responses to acoustic stimuli less than 50Hz apart, these differences could 318 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 320 to be released. Updated cage trials with a starting allelic frequency of only 2.5% predicted 321 population collapse within 14 generations [31]. Generation of dsx mutants in other mosquito 322 species (such as Aedes aegypti) could not only provide a promising control method to combat 323 other mosquito populations, but also provide an ideal tool to investigate the fundamental 324 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  wildtype swarming behaviour are necessary to better understandand predictthe relevant 345 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.

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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.    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:

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Wing beat frequency ~ Sex*Genotype + Wing length 430 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.