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Assessing the Acoustic Behaviour of Anopheles Gambiae Sl Dsxf Mutants bioRxiv preprint doi: https://doi.org/10.1101/2020.09.06.284679; this version posted September 6, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. 1 Assessing the acoustic behaviour of Anopheles gambiae s.l. dsxF mutants: 2 Implications for Vector Control 3 Authors: Matthew P Su1,2,3†, Marcos Georgiades1,2†, Judit Bagi1,2, Kyros Kyrou4, Andrea 4 Crisanti4, Joerg T Albert1,2,* 5 Affiliations 6 † These authors contributed equally 7 1 Ear Institute, University College London, 332 Grays Inn Road, London, WC1X 8EE, UK 8 2 The Francis Crick Institute, 1 Midland Road, London, NW1 1AT, UK 9 3 Division of Biological Science, Nagoya University, Nagoya, 464-8602, Japan 10 4 Department of Life Sciences, Imperial College London, UK. 11 12 * Correspondence: [email protected] 13 14 E-mails: 15 MS: [email protected] 16 MG: [email protected] 17 JB: [email protected] 18 KK: [email protected] 19 AC: [email protected] 20 JTA: [email protected] 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.09.06.284679; this version posted September 6, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. 21 Abstract 22 Background 23 The release of genetically modified mosquitoes which use gene-drive mechanisms to suppress 24 reproduction in natural populations of Anopheles mosquitoes is one of the scientifically most 25 promising methods for malaria transmission control. However, many scientific, regulatory and 26 ethical questions remain before transgenic mosquitoes can be utilised in the field. Mutations 27 which reduce an individual’s reproductive success are likely to create strong selective pressures 28 to evolve resistance. It is thus crucial that the targeted population collapses as rapidly and as 29 completely as possible to reduce the available time for the emergence of drive-resistant 30 mutations. At a behavioural level, this means that the gene-drive carrying mutants should be at 31 least as (and ideally more) sexually attractive than the wildtype population they compete 32 against. A key element in the copulatory negotiations of Anopheles mosquitoes is their acoustic 33 courtship. We therefore analysed sound emissions and acoustic preference in a doublesex 34 mutant previously used to successfully collapse caged colonies of Anopheles gambiae s.l.. 35 Methods 36 The flight tones produced by the beating of their wings form the signals for acoustic mating 37 communication in Anopheles species. We assessed the acoustic impact of the disruption of a 38 female-specific isoform of the doublesex gene (dsxF) on the wing beat frequency (WBF; 39 measured as flight tone) of both males (XY) and females (XX) in homozygous dsxF- mutants 40 (dsxF-/-), heterozygous dsxF- carriers (dsxF+/-) and G3 ‘wildtype’ dsxF+ controls (dsxF+/+). To 41 exclude non-genetic influences, we controlled for temperature and measured wing lengths for 42 all experimental animals. We used a phonotaxis assay to test the acoustic preferences of mutant 43 and control mosquitoes. 44 Results 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.09.06.284679; this version posted September 6, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. 45 A previous study demonstrated an altered phenotype only for females homozygous for the 46 disrupted dsx allele (dsxF-/-), who appear intersex. No phenotypic changes were observed for 47 heterozygous carriers or males, suggesting that the female-specific dsxF allele is 48 haplosufficient. We here identify significant, dose-dependent increases in the flight tones of 49 both dsxF-/- and dsxF+/- females when compared to dsxF+/+ control females. Flight tone 50 frequencies in all three female genotypes remained significantly lower than in males, however. 51 When tested experimentally, males showed stronger phonotactic responses to the flight tones 52 of control dsxF+/+ females. While flight tones from dsxF+/- and dsxF-/- females also elicited 53 positive phonotactic behaviour in males, this was significantly reduced compared to responses 54 to control tones. We found no evidence of phonotactic behaviour in any female genotype tested. 55 None of the male genotypes displayed any deviations from the control condition. 56 Conclusions 57 A key prerequisite for copulation in anopheline mosquitoes is the phonotactic attraction of 58 males towards female flight tones within large - spatially and acoustically crowded - mating 59 swarms. Reductions in acoustic attractiveness of released mutant lines, as reported here for 60 heterozygous dsxF+/- females, reduce the line’s mating efficiency, and could consequently 61 reduce the efficacy of the associated population control effort. Assessments of caged 62 populations may not successfully reproduce the challenges posed by natural mating scenarios. 63 We propose to amend existing testing protocols in order to more faithfully reflect the 64 competitive conditions between a mutant line and the wildtype population it is meant to interact 65 with. This should also include novel tests of ‘acoustic fitness’. In line with previous studies, 66 our findings confirm that disruption of the female-specific isoform dsxF has no effect on males; 67 for some phenotypic traits, such as female flight tones, however, the effects of dsxF appear to 68 be dose-dependent rather than haplosufficient. 3 bioRxiv preprint doi: https://doi.org/10.1101/2020.09.06.284679; this version posted September 6, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. 69 Keywords 70 Anopheles gambiae s.l., Anopheles coluzzii, doublesex, Gene drive, Wing beat frequency, 71 Flight tone, Mosquito, Acoustic communication, Hearing, Phonotaxis, Vector control 4 bioRxiv preprint doi: https://doi.org/10.1101/2020.09.06.284679; this version posted September 6, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. 72 Background 73 Mosquitoes represent a major global health problem, with Aedes, Anopheles and Culex species 74 acting as vectors of diseases that infect millions of people each year [1]. Malaria remains a 75 major cause of mortality and morbidity worldwide in spite of significant advances made in 76 disease control since the turn of the century [2, 3]. This is in part due to the reduced efficacy 77 of current control tools such as insecticidal nets and indoor residual spraying, as well as the 78 emergence of secondary disease vectors [4, 5, 6]. Novel control techniques are therefore 79 necessary to continue the push towards disease elimination [7]. 80 One potential option is the utilisation of gene drive systems, which target 81 haplosufficient female fertility genes, leading to a reduction in female fertility and, eventually, 82 population collapse [8, 9]. The recent generation of Anopheles gambiae CRISPR/Cas9 mutants 83 in which a female specific exon of the doublesex (dsxF) gene was disrupted is here of interest. 84 Lab cage trials have demonstrated that the introduction of dsxF mutants into cages of wildtype 85 mosquitoes was sufficient to lead to eventual population collapse [10]. 86 However, there are many scientific, ethical and regulatory hurdles to overcome before 87 such transgenic mosquitoes can be released in even semi-field trials [11]. It is vital that any 88 transgenic mosquitoes are subjected to rigorous testing prior to use in the field; gene transfer 89 into natural populations following release of transgenic Aedes aegypti has highlighted the 90 potential risks of release of transgenic insects [12]. On a scientific level, one important task 91 will be to maintain the gene drive’s effectiveness outside of the laboratory and under more ‘real 92 world’ scenarios. 93 A major element of this testing is the investigation of interactions with natural, non- 94 mutant populations, particularly with regards to courtship behaviour. If mutant mosquitoes are 95 unable, or only less likely, to copulate with native populations then they become the less 5 bioRxiv preprint doi: https://doi.org/10.1101/2020.09.06.284679; this version posted September 6, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. 96 attractive option, which will slow down or outright frustrate the population control effort [13]. 97 In addition to potential direct and indirect fitness costs associated with mutations, laboratory 98 habituation and mass rearing can also affect mating performance [14, 15]. In this context it is 99 noteworthy that the dsxF mutants we tested were also generated from a lab-established strain 100 (G3) rather than any wildtype population [10]. Extensive testing of mutant mating fitness prior 101 to translation from laboratory mating assays is thus a key requirement for assessing a specific 102 line’s suitability for use as part of a release program. 103 The sense of hearing is a vital component of mosquito reproduction, with males 104 identifying females within swarms via phonotactic responses to female flight tones and 105 acoustic communication is also thought to play a role in female mate selection [16, 17, 18, 19].
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