Obtaining Nix-expressing transgenic Ae. albopictus lines
Because additional exons were unknown when this project began, we first built a transgenesis plasmid comprising only Nix exon 1, intron 1 and exon 2 under the control of the endogenous Nix promoter (~2 kb preceding the ATG start codon, see Supplementary Note 1). We included an OpIE2-eGFP fluorescence marker for identification of transgenic individuals (Fig. 1c). About 300 Ae. albopictus embryos were injected with this plasmid. Among the surviving G0 adults, we obtained 24 males showing transient expression (TE) of the fluorescent marker and few females, some of which showing deformed genitalia at the pupal stage. After crossing en masse TE males with wild-types females, we recovered 29 eGFP positive G1 pupae, including 22 males and 7 females. We further outcrossed the 22 positive males en masse with wild-type females. In the G2 generation, we obtained 395 males expressing GFP while 53 were negative, and 185 females expressing eGFP while 151 were negative. Of these, 12 randomly selected eGFP males were outcrossed individually with WT females, creating the lines named herein SM1 to SM12. Two crosses, (SM8 and SM10), did not produce eggs and were not further investigated. In all the other progenies but SM4, 100% of the G3 males expressed eGFP. GFP positive females were also observed, although these were rare or absent in most lines. Interestingly, in SM9 57% of eGFP positive females (larger body size and female antennae) showed an intersex phenotype with deformed genitalia (Supplementary Fig. 1). Transgenic lines were amplified over several generations and in five of them (SM1, SM3, SM7, SM9, and SM12), 100% of the males were GFP positive.
Isoform naming follows work published in ref. 22. Grey boxes are Nix exons, with darker grey being the translated parts. White triangles are piggyBac 5′ and 3′ inverted terminal repeat (ITR) sequences, orange boxes represent an AttP landing site included for potential future purposes, purple boxes represent loxP recombination sites, white arrows are promoters, pink polygons are SV40 polyA sequences, green, red and yellow boxes are eGFP, DsRed and YFP gene sequences, respectively. Drawing not to scale. Plasmids carry a Nix isoforms 3–4 and a GFP marker gene under the control of the OpIE2 promoter b Nix isoforms 3–4 and a GFP marker gene under the control of the polyubiquitin promoter, c Nix isoform 1 and a DsRed marker gene under the control of the polyubiquitin promoter, and d Nix isoform 2 and a YFP marker gene under the control of the polyubiquitin promoter. Detailed plasmid sequences can be found under Addgene references #173505, #173665, #173666, #173667.
The second injection mix comprised three different transgenesis plasmids to express the newly reported Nix isoforms22, labelled with different fluorescence markers (Fig. 1a, b and d with DsRed, YFP and eGFP respectively) under the control of the Ae. aegypti poly-ubiquitin promoter (PUb). We injected about 700 embryos with this mix, from which approximately 100 TE individuals were obtained. Similar to our previous observations, we obtained mostly male G0 TE individuals that were split in five pools of males and outcrossed en masse with wild-type females. Strikingly, in the G1 generation, 70–100% of the transgenic individuals of each pool were males. Some of the transgenic females in at least three pools were deformed or partially masculinised. Most transgenic G1 individuals expressed more than one fluorescent marker, indicating that they carried multiple piggyBac insertions with different transgenes. In each of five G1 pools, 8 transgenic males were randomly selected and outcrossed individually with WT females in order to generate lines carrying a single fluorescent marker. From these 40 individual founder males, we retained 7 independent lines in which 100% of the males were fluorescent (namely 1.2R, 1.2G, 2.2G, 3.1YR, 3.1G, 4.4Y, 5.1GR) for further analysis.
Identification of transgenic lines devoid of an M-locus
During characterization of Nix-expressing transgenic lines, we observed strong genetic linkage between fluorescence and the male phenotype. This could be due either to transgenic fluorescent males actually being masculinised genetic females, resulting in mosquito lines lacking natural males; or to the insertion of the piggyBac plasmid cassette within or near the endogenous M-locus, resulting in genetic linkage between the fluorescent transgene and male sex. To distinguish between these two possibilities, we tested lines with 100% fluorescent males for the presence of endogenous and exogenous Nix by PCR (e.g. see Supplementary Fig. 2). In eight out of twelve lines, endogenous Nix was detected in the GFP positive transgenic males. Therefore, in these lines, maleness was natural and at least one copy of the transgene was M-linked. In contrast, four of our mosquito lines were devoid of endogenous Nix, namely SM9, 1.2G, 2.2G and 3.1G. Interestingly, these four lines possessed the GFP fluorescent marker, thus, they expressed the shortest Nix isoforms encoded by exons 1 and 2 only. In some lines marked by YFP and/or DsRed fluorescence, a fraction of the males lacked the M-locus, while others possessed it. However, the M-deprived males did not sire any progeny, thus they were possibly sterile.
Single males from the four M-free GFP lines (hereafter termed pseudo-males) were backcrossed to wild-type females for several generations to eliminate additional non-fully masculinizing transgene insertions. Following this step, we obtained three lines (SM9, 1.2G and 3.1G) where only pseudo-males showed eGFP fluorescence, without residual fluorescent females or intersex individuals. Further tests and analyses were performed on the SM9 line, and some experiments were replicated on the other two lines.
Confirmation of the role of Nix transgenes in masculinization
To further confirm the role of Nix transgenes in masculinization, we injected SM9 embryos with a plasmid expressing CRE recombinase in order to excise the Nix–eGFP transgenic cassette, which is flanked by lox recombination sites (Fig. 1). At the pupal stage, efficiently injected individuals showed a striking loss of eGFP expression at the injection site (posterior pole), indicative of lox cassette excision. While these pupae should have developed into phenotypic males (due to the eGFP-marked Nix transgene cassette), they showed female genitalia, hence demasculinization of their posterior pole (Fig. 2a–c, Supplementary Fig. 3). Adult mosquitoes hatching from these pupae showed antero-posterior gynandromorphism, having male heads and female genitalia. Strikingly, a single individual, which arose from an embryo accidentally injected in the anterior pole rather than in the posterior, showed the opposite gynandromorphic phenotype, with a female head and male genitalia (Fig. 2d). These results confirmed that maleness in the SM9 line results from the transgene’s activity, which can be abolished by CRE/lox excision. The co-existence of male and female tissues in the same individual also illustrates that sex determination is tissue-autonomous in Ae. albopictus.
a Representative transgenic male pupa and male adult from the SM9 line. b Representative non-transgenic female pupa and female adult from the SM9 line. c Transgenic SM9 male pupa and adult injected as embryos in the posterior pole with CRE-recombinase that excised the Nix-eGFP cassette in the injected region. These individuals show a male anterior body with female genitalia. d Transgenic SM9 male pupa and adult injected with CRE-recombinase in the anterior pole of the embryo. Note the female anterior body and male genitalia. Scale bars in the bottom right corner of each picture represent 1 mm.
Characterization of Nix-expressing pseudo-males
To evaluate whether the Nix-eGFP cassette was stable and adult pseudo-males fully viable, we first determined the sex ratio in comparison to that of the parental wild-type (WT) line (Fig. 3a, Supplementary Table 2). Male ratios from SM9 (sex-ratio estimate ± SE = 0.56 ± 0.05), 1.2G (0.49 ± 0.05) and 3.1G (0.54 ± 0.05) transgenic strains were not significantly different from that of the WT line (0.52 ± 0.09, SM9 vs. WT p-value = 0.165, 1.2G vs. WT p-value = 0.528, 3.1G vs. WT p-value = 0.760).
a Sex ratio comparison between the WT line and the SM9, 1.2G and 3.1G transgenic lines. Sex ratio of the WT line was counted manually on N = 3 independent batches of pupae, while sex ratios of transgenic lines were counted on N = 3 independent batches of neonate larvae using COPAS. Grey dots in rectangles represent the total numbers of males and females (right y-axis). Grey dots in the middle part represent the sex ratio of each replicate. Black dots are the estimate values with vertical lines being 95% confidence intervals. Sex ratios were compared using linear generalised mixed-effect model. None of the sex ratios were significantly different from that of the WT line: SM9 vs. WT p-value = 0.165, 1.2G vs. WT p-value = 0.528, 3.1G vs. WT p-value = 0.760. b Wing length comparison between N = 38 wild-type males, N = 39 wild-type females and N = 48 Nix-expressing SM9 pseudo-males represented as violin plots with jitter grey data points. Wing length was measured on ImageJ software from pictures of dissected right wings taken under a binocular microscope. Comparisons were performed using linear model: WT male vs. WT female p-value < 0.001, SM9 male vs. WT female p-value < 0.001, WT male vs. SM9 male p-value = 0.998.
In Aedes mosquitoes, males and females display a significant size dimorphism, with females having a larger body size26. We determined the body size of pseudo-males using wing length as a proxy27. Our results showed no significant difference between the size of the wild-type and transgenic males (WT male vs. SM9 pseudo-male p = 0.998), while both were significantly different from females (WT male vs. WT female p-value < 0.001, SM9 pseudo-male vs. WT female p-value < 0.001, Fig. 3b, Supplementary Data 2).
Nix expression levels in pseudo-males vs. wild-type males were compared by RT-qPCR at the pupal stage (Fig. 4a, Supplementary Data 3). Interestingly, pseudo-males from three different lines expressed Nix at a similar level to wild-type males (SM9 males vs. WT males p-value = 0.501, 1.2G males vs. WT males p-value = 0.391, 3.1G males vs. WT males p-value = 0.626). Thus, we inquired if the downstream double switch genes, doublesex (dsx) and fruitless (fru), showed male-specific splicing products in pseudo-males. For this, we performed RT-PCR on pseudo-males of four transgenic lines. Results revealed that pseudo-males displayed the same splicing pattern as WT males for both genes (Supplementary Fig. 4).
RT–qPCR results are represented by −ΔCT, which reflects the relative expression level of each gene in a given treatment, CT values being inversely proportional to the expression levels. AalRpS7 was used as endogenous reference gene. Grey dots represent each data point. Black dots represent the mean value of the N = 3 biological replicates, vertical lines represent 95% confidence intervals. On each panel, distinct letters represent significant difference in an ANOVA followed by a pairwise Tukey test (p-value < 0.001). a Nix relative expression. b Relative comparison of myo-sex orthologues total expression levels. c Relative expression of the candidate orthologue of the Ae. aegypti myo-fem gene, LOC109402113. d Relative expression of the candidate orthologue LOC115254984, which is annotated as a putative pseudo-gene. This primer pair could also amplify LOC115254986 due to high sequence similarity but this other pseudo-gene seems not to be expressed.
Expression of myo-sex orthologues in Ae. albopictus pseudo-males
Contrarily to Ae. aegypti where the lack of the M-linked gene myo-sex resulted in flightless pseudo-males24, our Ae. albopictus pseudo-males were readily able to fly. Consequently, we wondered whether an essential orthologue of myo-sex was present in the Ae. albopictus’s M-locus. Using the current version of the Ae. albopictus genome (Vectorbase release 52, 20 May 2021), we found two homologous copies of Ae. aegypti myo-sex located on two distinct unplaced scaffolds (SWKY01000423 and SWKY01000369), annotated under the identifier references LOC109430926 and LOC109412105, respectively24,28. We designed primer pairs specific to each of these two genes, exploiting differences in their non-coding regions. Interestingly, one of these PCR markers suggested the presence of a male-specific copy of myo-sex, characterized by a 664 bp deletion in its non-coding sequence (Supplementary Note 2). These results do not align with the currently available genomic data, and suggest that a third, M-linked copy of the myo-sex gene exists or that the genome assembly concerning one of the above-mentioned copies is erroneous possibly due to high similarity between them. Hence, we compared the expression levels of the Ae. albopictus orthologues of myo-sex in Nix-expressing pseudo-males lacking the M-linked copy vs. WT males by RT-qPCR. All myo-sex copies being 100% identical at the cDNA level, RT-qPCR reflects global expression of all copies combined. Our results showed that pseudo-males expressed myo-sex at a level similar to wild-type males (SM9 males vs. WT males p-value = 1.000, 1.2G males vs. WT males p-value = 0.960, 3.1G males vs. WT males p-value = 0.843), and that this level was approximately 20 fold higher compared to WT females (p-values < 0.001 for all combinations, Fig. 4b, Supplementary Data 3). These results suggest that one or several non M-linked, endogenous myo-sex-like copies are efficiently upregulated in pseudo-males.
Expression of myo-fem orthologue in Ae. albopictus pseudo-males
Another sex-specifically expressed flight gene is myo-fem, described in Ae. aegypti as essential for female flight29. In Ae. albopictus, this gene has several potential orthologues. We compared their expression by RT-PCR between WT males and females and identified a putative myo-fem orthologue (LOC109402113) based on its strong female-specific expression pattern. RT-qPCR results revealed that pseudo-males express myo-fem at similarly low levels as wild-type males (SM9 males vs. WT males p-value = 0.995, 1.2G males vs. WT males p-value = 0.841, 3.1G males vs. WT males p-value = 0.614, Fig. 4c, Supplementary Data 3). Gene expression in all males tested was approximately 10,000 fold lower than in wild-type females (p-values < 0.001 for all combinations). Notably, another potential orthologue located on the same genomic scaffold as LOC109402113 and annotated as a pseudo-gene with detectable RNA expression, LOC115254984, displayed a very similar expression profile (Fig. 4d, Supplementary Data 3).
Flight ability and reproductive fitness of SM9 pseudo-males
Since we observed that genes potentially involved in flight are regulated similarly in Nix-expressing pseudo-males comparing to wild-type counterparts, we tested the SM9 pseudo-males’ flight ability by performing a flight test as described in ref. 30. We observed that SM9 males had a higher escape probability than WT males (p-value < 0.001, Fig. 5a, Supplementary Table 3), suggesting that the flight capacity of SM9 pseudo-males was at least as high as that of WT male mosquitoes.
a Percentage of males that successfully escaped the flight test device. Grey dots in rectangles represent the total numbers of males that remained inside the flight tunnel and that escaped (right y-axis). Black dots are the estimate values with vertical lines being 95% confidence intervals. N = 3 replicates with an average of 82 ± 13 males were performed. To test the effect of the lines on the flight test success, we used linear generalised mixed-effect model and Bernoulli distribution assumptions with “replicate” as random effect. p-value < 0.001. b Hatching rate measured by dividing the number of progeny by the number of eggs on N = 3 egg batches. Dried eggs were counted, submerged, placed in a vacuum chamber for 30 mn and allowed to hatch for 24 h before counting larvae. Grey dots in rectangles show the total numbers of eggs that hatched or did not hatch (right y-axis). Black dots are the estimate values with vertical lines being 95% confidence intervals. Hatching rate was compared by linear generalised mixed-effect model: p-value = 0.423. c Fertility measured by the number of progeny sired by 30 males crossed with 60 females. Black dots represent the mean value of the N = 3 biological replicates, vertical lines represent 95% confidence intervals. The effect of line on fertility was tested using a linear model: p-value = 0.532. d To estimate SM9 male competitiveness, N = 5 competition assays were performed, crossing 30 WT males and 30 SM9 males to 30 females. In their progeny, the percentage of SM9 pseudo-males was measured by COPAS and compared to the expected percentage (dashed line) by linear generalised mixed-effect model: 10.2 ± 0.4 % of transgenic progeny vs. 27.9% of expected value (p-value < 0.001). Grey dots in rectangles are representative of the total numbers of transgenic SM9 pseudo-males and non-transgenic individuals in the progeny (right y-axis). Black dots are the estimate values with vertical lines being 95% confidence intervals. In all panels, grey dots in the middle part of the graph represent pooled replicates.
We compared SM9 to WT relative fertility (total number of live larvae in a given progeny) and fecundity (total number of eggs laid, estimated by dividing the total number of larvae by the hatching rate). We found no significant difference in SM9 hatching rate (p-value = 0.423, Fig. 5b, Supplementary Table 4) or fertility (p-value = 0.532, Fig. 5c, Supplementary Table 5) comparing to wild-type control, thus no difference in fecundity either. Then, we measured relative competitiveness between SM9 pseudo-males and wild-type males by mixing equal numbers of transgenic and wild-type males with wild-type females. If males from both lines were equally competitive, we would expect 50% of the females to be inseminated by a WT male (producing WT progeny), and 50% being inseminated by a transgenic pseudo-male. SM9 males producing 55.8% of GFP-positive sons, and their fecundity and fertility being similar to that of WT, we would therefore expect 27.9% of the total progeny from the competition assay to be GFP-positive. In this experiment, we observed an estimated mean of 10.2 ± 0.4% of GFP-positive progeny indicating a reduced competitiveness (p-value < 0.001, Fig. 5d, Supplementary Table 6). The same competitiveness assay was performed between 1.2G pseudo-males and WT males, and between 3.1 G pseudo-males and WT males (Supplementary Table 6). Both gave a similar result (1.2G pseudo-males vs. WT males 12.5 ± 0.3% of transgenic progeny, p-value < 0.001, 3.1G pseudo-males vs. WT males 9.6 ± 0.4% of transgenic progeny, p-value < 0.001).
Automated sex sorting of transgenic pseudo-males
The transgenesis plasmids carrying a fluorescent marker under strong promoters, neonate larvae can be sorted according to their fluorescence, which, in this case, is sex specific. Similarly to what has been developed in Anopheles mosquitoes31,32, sex sorting can be automated using a COPAS device (Union Biometrica) which allows separation of particles based on fluorescence levels. Using COPAS on the SM9 line, we were able to separate green fluorescent males from non-fluorescent females (Fig. 6a). However, due to the weak activity of the OpIE2 promoter driving eGFP at the neonate stage, the fluorescence was not always strong enough to get well separated positive vs. negative clouds by COPAS. Lines 3.1G and 1.2G express higher levels of GFP due to the high activity of the PUb promoter at the neonate stage and provide clearer sex separation (Fig. 6b, c). In all cases, batches of several thousands of neonate larvae could be repeatedly sex separated using COPAS at a speed of approximately 2400 larvae per minute. Visual screening at the pupal stage confirmed perfect sex separation.
Sorting is performed at the neonate stage using a COPAS device. Presented graphs show the fluorescence profile of a representative sample of each line as log(Green) = f(log(Red)). Fluorescence clusters are detected automatically using the ‘kmeans’ function from the R package ‘stat’. Nix-expressing pseudo-males being tagged with an eGFP marker gene, the top green cluster is composed of males, while females are in the bottom black cluster. a 3728 larvae from the SM9 line carrying an OpIE2-GFP marker. Here automated clustering detected the lowest male larvae as females, which depicts the difficulty of separating males from females at the neonate stage using an OpIE2-GFP marker. b 6235 larvae from the 1.2G line carrying a polyubiquitin-GFP marker. c 1624 larvae from the 3.1G line carrying a polyubiquitin-GFP marker.
