Transgenic expression of Nix converts genetic females into males and allows automated sex sorting in Aedes albopictus

Aedes albopictus is a major vector of arboviruses. Better understanding of its sex determination is crucial for developing mosquito control tools, especially genetic sexing strains. In Aedes aegypti, Nix is the primary gene responsible for masculinization and Nix-expressing genetic females develop into fertile, albeit flightless, males. In Ae. albopictus, Nix has also been implicated in masculinization but its role remains to be further characterized. In this work, we establish Ae. albopictus transgenic lines ectopically expressing Nix. Several are composed exclusively of genetic females, with transgenic individuals being phenotypic and functional males due to the expression of the Nix transgene. Their reproductive fitness is marginally impaired, while their flight performance is similar to controls. Overall, our results show that Nix is sufficient for full masculinization in Ae. albopictus. Moreover, the transgene construct contains a fluorescence marker allowing efficient automated sex sorting. Consequently, such strains constitute valuable sexing strains for genetic control.

Aedes albopictus can transmit the main human arboviruses including dengue, chikungunya, yellow 1 fever and Zika viruses 1-6 . Given the global expansion of this mosquito 7,8 and its increasing insecticide 2 resistance 9,10 , new and more sustainable vector control tools are urgently needed 11 . Genetic control 3 methods to decrease mosquito population, including the Sterile Insect Technique 12 and the 4 Incompatible Insect Technique 13 , have proven effective in several field trials 14-17 . These methods rely 5 on mass releases of male mosquitoes that compete with their field counterparts for mating. However, 6 in order to upscale the mass production process, an automated and more cost-efficient method for 7 separating males from females is still necessary 18,19 . Unfortunately, reliable sexing strains are not yet 8 available on an operational scale in Aedes albopictus, in part due to the limited knowledge of the sex 9 determination in this species. 10 Insects exhibit a large diversity of primary signals that activate the sex determination cascade. These 11 signals are poorly conserved between insect genera and few have been discovered so far. In 12 Drosophila melanogaster, the chromosome X:autosome ratio is the primary signal for sex 13 determination and dosage compensation. While the Y chromosome is essential for male fertility, it 14 does not determine phenotypic sex 20 . In some non-Drosophilid Diptera, the primary signal relies on a 15 male factor (M-factor) located either on the Y chromosome or in the M-locus of an autosome. Aedes 16 mosquitoes carry an M-factor located within an M-locus on the first pair of autosomes, which is called 17 Nix 21-23 . Nix was first identified in Aedes aegypti, where it consists of two exons separated by a 99-kb 18 intron, and encodes a 288-amino acid protein 21 . Stable transgenic expression of Nix in Ae. aegypti has 19 been shown to be sufficient for converting all genetic females into phenotypic fertile males, 20 confirming its role 24 . However, these phenotypic males were unable to fly, most likely because they 21 lacked myo-sex, another gene closely linked to the M-locus encoding a male-specific flight muscle 22 myosin. In Ae. albopictus, Nix is also located on chromosome I and comprises two exons with high 23 homology to Ae. aegypti Nix and a much shorter intron 25 , as well as two additional exons uncovered 24 recently 22 . Nix disruption using CRISPR/Cas9 leads to partial feminization of males, confirming the 25 role of Nix as the M-factor in this species 22 . 26 4 It remains to be shown whether Ae. albopictus Nix alone is sufficient for masculinization and if 27 genetic females transformed into phenotypic males by transgenic expression of Nix would be fertile 28 and able to fly. 29 In this work, we generated transgenic Ae. albopictus mosquitoes expressing the four main Nix 30 isoforms in genetic females and showed that all can induce partial to complete masculinization. Some 31 of these lines constitute valuable genetic sexing strains, allowing high-throughput sex sorting at the 32 neonate stage. 33

Identification of transgenic lines devoid of an M-locus 46
During characterization of Nix-expressing transgenic lines, we observed strong genetic linkage 47 between fluorescence and the male phenotype. This could be due either to transgenic fluorescent males 48 actually being masculinised genetic females, resulting in mosquito lines lacking natural males; or to 49 the insertion of the piggyBac plasmid cassette within or near the endogenous M-locus, resulting in 50 genetic linkage between the fluorescent transgene and male sex. To distinguish between these two 51 5 possibilities, we tested for the presence of endogenous and exogenous Nix by PCR (e.g. see 52 Supplementary Figure 1). In eight out of twelve lines, endogenous Nix was detected in the GFP 53 positive transgenic males. Therefore, in these lines, maleness was natural and the transgene was M-54 linked. In contrast, four of our mosquito lines were devoid of endogenous Nix, namely SM9, 1.2G, 55 2.2G and 3.1G. Interestingly, these four lines possessed the eGFP fluorescent marker, thus, they 56 expressed the shortest Nix isoforms encoded by exons 1 and 2 only (Supplementary Figure 2). In 57 some lines marked by YFP and/or DsRed fluorescence, a fraction of the males lacked the M-locus, 58 while others possessed it. However, the M-deprived males did not sire any progeny, thus they were 59 possibly sterile.  Figure 1D). These results confirmed that maleness in the SM9 line results from the 78 6 transgene's activity, which can be abolished by CRE/lox excision. The co-existence of male and 79 female tissues in the same individual also illustrates that sex determination is tissue-autonomous in Ae. 80

albopictus. 81
Characterization of Nix-expressing m/m pseudo-males 82 To evaluate whether the Nix-eGFP cassette was stable and adult pseudo-males fully viable, we first 83 determined the sex ratio in comparison to that of the parental wild strain (WT). Male ratios from SM9 84 (sex-ratio estimate ± SE = 0.56 ± 0.05), 1.2G (0.49 ± 0.05) and 3.1G (0.54 ± 0.05) transgenic strains 85 were not significantly different from that of the WT line (0.52 ± 0.10, SM9 vs. WT p-value = 0.297, 86 1.2G vs. WT p-value = 0.558, 3.1G vs. WT p-value = 0.869). 87 In Aedes mosquitoes, males and females display a significant size dimorphism, with females having a 88 larger body size 26 . We determined the body size of pseudo-males using wing length as a proxy 27 . Our 89 results showed no significant difference between the size of the wild-type and transgenic males (WT 90 male vs. SM9 pseudo-male p = 0.998), while both were significantly different from females (WT male 91 vs. WT female p-value < 0.001, SM9 pseudo-male vs. WT female p-value < 0.001, Figure 2). 92 Nix expression levels in pseudo-males vs. wild-type males were compared by RT-qPCR at the pupal 93 stage ( Figure 3A). Interestingly, pseudo-males from three different lines expressed Nix at a similar 94 level to wild-type males (SM9 males vs. WT males p-value = 0.501, 1.2G males vs. WT males p-value 95 = 0.391, 3.1G males vs. WT males p-value = 0.626). Thus, we inquired if the downstream double 96 switch genes, doublesex (dsx) and fruitless (fru), showed male-specific splicing products in pseudo-97 males. For this, we performed RT-PCR on pseudo-males of four transgenic lines. Results revealed that 98 pseudo-males displayed the same splicing pattern as WT males for both genes (Supplementary 99 Figure 5). 100

Flight ability of Nix-expressing pseudo-males 101
In contrast to what has been reported in Ae. aegypti where the lack of the M-linked gene myo-sex 102 resulted in flightless pseudo-males 24 , our Ae. albopictus pseudo-males were readily able to fly. 7 flight muscle myosins identified in Ae. aegypti, myo-sex and myo-fem, in Nix-expressing pseudo-males 105 vs. WT males by RT-qPCR. We discovered an M-linked copy of the myo-sex gene (Supplementary 106 Data 2), additional to those already known 28 , as well as an orthologue of myo-fem (Supplementary 107 Note 2). Our results showed that the collective level of myo-sex expression (all copies have 100% 108 cDNA identity) in WT females was approximately 20 fold lower compared to males (p-values < 109 0.001), and that pseudo-males expressed myo-sex at a level similar to wild type males (SM9 males vs. 110 WT males p-value = 1.000, 1.2G males vs. WT males p-value = 0.960, 3.1G males vs. WT males p-111 value = 0.843, Figure 3B). These results suggest that one or several non M-linked, endogenous myo-112 sex-like copies are efficiently upregulated in pseudo-males. Additionally, pseudo-males express myo-113 fem at similarly low levels as wild-type males (SM9 males vs. WT males p-value = 0.995, 1.2G males 114 vs. WT males p-value = 0.841, 3.1G males vs. WT males p-value = 0.614, Figure 3C). Gene 115 expression in all males tested was approximately 10,000 fold lower than in wild-type females (p-116 values < 0.001 for all combinations, Figure 3C). 117 Since we observed that genes potentially involved in flight are regulated similarly in Nix-expressing 118 pseudo-males comparing to wild-type counterparts, we tested the SM9 pseudo-males' flight ability by 119 performing a flight test as described in 29 . We observed that SM9 males had a higher escape 120 probability than WT males (p-value < 0.001, Figure 4A), suggesting that the flight capacity of SM9 121 pseudo-males was at least as high as that of WT male mosquitoes. 122

Reproductive fitness of SM9 pseudo-males 123
We compared SM9 to WT relative fertility (total number of live larvae in a given progeny) and 124 fecundity (total number of eggs laid, estimated by dividing the total number of larvae by the hatching 125 rate). We found no significant difference in SM9 hatching rate (p-value = 0.423, Figure 4B) or 126 fertility (p-value = 0.532, Figure 4C) comparing to wild-type control, thus no difference in fecundity 127 either. Then, we measured relative competitiveness between SM9 pseudo-males and wild-type males 128 by mixing equal numbers of transgenic and wild-type males with wild-type females. Similarly to Ae. aegypti 24 , we observed that pseudo-males harbour molecular characteristics of wild-155 type males. Of note, we were able to detect an undescribed M-linked homologue of myo-sex in the Ae. 9 albopictus genome, besides additional autosomal copies, characterized by a 600 nucleotide deletion 157 within the gene's non-coding region. myo-sex has been described as responsible for male flight ability 158 in Ae. aegypti and is specifically present in wild-type males, hence linked to the M-locus in this 159 species 24 . However, in Ae. albopictus, the M-linked copy does not appear to be essential for male 160 flight, as synthetic males expressing a Nix transgene but devoid of the M-linked myo-sex copy showed 161 similar total myo-sex expression level and performed better in flight tests. Moreover, we also identified 162 a strong candidate for a myo-fem orthologue, a gene described in Ae. aegypti as essential for female 163 flight 32 . In Ae. albopictus, we measured that this gene is highly expressed in wild-type females and 164 strongly repressed in wild-type males as well as transgenic pseudo-males. albopictus M-locus and that their absence might influence some aspects of the mating process that we 172 were unable to detect in our fertility and fecundity assays. 173 Besides fully masculinizing Nix transgene insertions, we also obtained a number of insertions 174 triggering no or only partial masculinization, whereas the associated fluorescence reporter genes were 175 expressed under control of the polyubiquitin promoter at comparable levels for all obtained lines. This 176 suggests that the genomic context where the Nix transgene is inserted affects its expression, and thus, 177 masculinization. There might be a dose-dependency of Nix expression during development with a 178 threshold ensuring full masculinization. However, while this might be true for GFP-bearing cassettes, 179 we failed to isolate a masculinizing line carrying a YFP or a dsRed reporter, (i.e., expressing the long 180 Nix isoforms 1 or 2). With these isoforms, we achieved partial to full apparent masculinization, but 181 these individuals were apparently infertile. Therefore, it seems that longer isoforms might not be 182 sufficient for masculinization. Alternatively, the short intron retained in our isoform 3-4 construct 183 (marked by eGFP), which we did not include in the longer isoform constructs, could carry a regulatory 184 sequence that is essential for complete masculinization and/or fertility. Finally, it is possible that lines 185 fully masculinized by the longer isoforms would have been recovered had we screened a much larger 186 sample of additional individual transgenic lines. 187 In transgenic lines where Nix transgene expression of specific isoforms is strong enough, Nix seems to 188 constitute a self-sufficient ectopic M-locus. Indeed, over >10 generations, the masculinizing lines 189 remained stable, with sex-ratios not differing significantly from the wild-type strain. This result is

Mosquito rearing 245
Aedes albopictus mosquitoes (strain BiA) were collected as larvae from a garden rainwater collector in 246 the city of Bischheim, near Strasbourg (France) in 2018 and maintained in the insectary since then at 247 25°C, 75-80% humidity, with a 14-hr/10-hr light/dark photoperiod. Larvae were reared in pans filled 248 with demineralized water and provided ground TetraMin fish food twice a day. Adult mosquitoes were 249 caged and provided with 10% sugar solution ad libitum. Females were blood-fed on anesthetised mice. 250 Eggs were laid three days later on wet kraft paper and allowed to develop for another three days before 251 being dried. 252

Molecular characterization of mosquito lines 253
Genomic DNA was extracted from pupae using NucleoSpin Tissue kit following the manufacturer's 254 instructions (Macherey Nagel, France). Total RNA was extracted using TRIzol RNA Isolation 255 Reagents (Invitrogen, ThermoFisher Scientific, France) and reverse-transcribed into cDNA using 256    Sorting is performed at the neonate stage using a COPAS device. Circled regions are the ones to be sorted by the machine. Presented graphs allow sorting of larvae based on their red and green fluorescence. Nix-expressing pseudo-males being tagged with an eGFP marker gene, the circled region sorts males. A) Sorting of 2,246 larvae from the SM9 line. B) Sorting of 1,624 larvae from the 1.2G line. C) Sorting of 3,876 larvae from the 3.1G line.