W chromosome-specific paralogs of the male-determining gene LdMasc exhibits a female- determining ability in the spongy moth, Lymantria dispar

W chromosome-specific paralogs of the male-determining gene LdMasc exhibits a female- determining ability in the spongy moth, Lymantria dispar | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article W chromosome-specific paralogs of the male-determining gene LdMasc exhibits a female- determining ability in the spongy moth, Lymantria dispar Kisuke Shoji, Kyoko Ishida, Ryota Kasahara, Hideshi Naka, Masataka G. Suzuki This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8963102/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 12 You are reading this latest preprint version Abstract Background Sex determination is a common developmental process in most organisms that exhibit sexual reproduction. Nevertheless, the modes of sex determination are highly diverse, and master sex-determining genes sometimes differ substantially even among closely related species. We explored the molecular basis underlying this striking diversity in the spongy moth, Lymantria dispar , whose master sex-determining factors exhibit continuous variation among populations. Results We previously identified the female-determining gene Fet-W and the male-determining gene LdMasc in the Japanese spongy moth ( Lymantria dispar japonica ). The number of raw RNA sequencing reads mapped to the LdMasc coding sequence region corresponding to exons 9–11 was higher in females than in males. Consistent with this finding, we identified four unigenes composed of the nucleotide sequences from LdMasc exons 9–11. Reverse-transcript polymerase chain reaction (RT-PCR) and genomic PCR using primers specific to these unigenes strongly indicated that these four unigenes originated from the W-linked gene. A nucleotide BLAST search across the entire L . d . japonica genome found at least 52 copies of nucleotide sequences nearly identical to the approximately 5.6-kb genomic sequence spanning intron 8 to exon 11 of LdMasc . Similar sequences were identified in the W chromosome contigs of Lymantria dispar dispar and Lymantria dispar asiatica , with 4 and 6 copies, respectively. We considered these sequences to be W chromosome paralogs of LdMasc and named them LdMasc-W . RT-PCR analysis demonstrated that LdMasc-W exhibits specific expression during the sex determination stage. Knockdown of LdMasc-W using embryonic RNA induced male-specific splicing of doublesex ( dsx ), a master regulatory gene for sexual development, in females. F1 hybrid females obtained by mating females of L. umbrosa , which that lacks LdMasc-W , with L. d. japonica males exhibited lethality or intersexual phenotypes. Conclusions These results strongly suggest that LdMasc-W functions to promote female determination by suppressing the expression of the male-determining gene LdMasc , similar to the female-determining gene Fem identified in other Lepidoptera species. This means that the spongy moth is a rare species possessing two W-linked female-determining genes: Fet-W and LdMasc-W . doublesex Lymantria dispar Masculinizer sex determination W chromosome Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Background Although sex determination is common among organisms that exhibit sexual reproduction between male and female individuals, the patterns of sex determination are remarkably diverse, and factors inducing sex determination vary significantly between species. Particularly in insects, differences in sex determination patterns have been observed even among closely related species [ 1 , 2 ], making them excellent models for elucidating the factors and mechanisms driving the diversification of sex determination. The spongy moth ( Lymantria dispar ; Erebidae:Lepidoptera) inhabits vast regions of the Northern Hemisphere, including Japan. Spongy moth larvae are polyphagous, feeding on over 500 deciduous tree species [ 3 ]. The species epithet disper is derived from the Latin word for separate, reflecting the moth’s striking sexual dimorphism [ 3 ]. This feature has supported a long history of research on the modes of sex determination modes in the spongy moth, which was found to possess a female-heterogametic sex chromosome system (males = ZZ, females = ZW) as early as 1934 [ 4 ]. Early studies predicted that the male-determining “M factor” was located on the Z chromosome and that the female-determining “F factor” resided on the W chromosome [ 4 ]. However, classical crossing experiments provided several lines of evidence that sex in the spongy moth is not determined solely by the presence or absence of the F factor. For example, hybrids obtained from crosses between populations in different habitats exhibit sex reversal and sex-specific lethality, resulting in a pronounced sex ratio bias [ 4 – 7 ]. Based on these findings, the M and F factors were predicted to exhibit allelic polymorphism with differential masculinizing and feminizing activity, and that sex determination occurs through their combination [ 4 – 6 ]. Under this prediction, sex-determining genes in the spongy moth would exhibit geographic divergence or subspecies differences in their sex-determining ability. Thus, the spongy moth may be a useful model species for elucidating molecular mechanisms underlying the diversification of sex-determining genes. However, the genes responsible for the F and M factors have remained unknown for nearly a century. In our previous study, we identified the gene responsible for the spongy moth F factor as Female expressed transcripts of W chromosome ( Fet-W ), and that responsible for the M factor as LdMasc , an ortholog of the Masculinizer ( Masc ) gene [ 8 ]. Masc functions as a male determination gene linked to the Z chromosome, inducing male-specific splicing of doublesex ( dsx ), which acts as a master regulatory gene for insect sex differentiation [ 9 – 11 ]. Since its identification in silkworms, Masc has been identified from at least 10 other Lepidoptera species, including Trilocha varians [ 12 ], Ostrinia furnacalis [ 13 ], the diamondback moth [ 14 ], the Mediterranean flour moth [ 15 ], and the codling moth [ 16 ], demonstrating its widespread conservation as a male-determining gene in Lepidoptera. Despite the robust conservation of Masc , its upstream regulatory factor, the female-determining gene, varies among species. In addition to Fet-W in the spongy moth, female-determining genes include Feminizer ( Fem ) in the silkworm, Fem in Polluter xylostella ( PxyFem ), and Moth-overruler-of-masculinization ( Mom ) in the pyralid moths Ephestia kuehniella and Plodia interpunctella [ 8 , 9 , 15 , 17 ]. Although no homology has been found among these genes, Fem , PxyFem , and Mom all form multicopy or tandem repeats on the W chromosome and are functionally homologous in that they provide PIWI-interacting RNA (piRNA), which leads to the degradation of Masc mRNA via the piRNA pathway [ 9 , 17 ]. In the absence of Masc protein, pre-mRNA of Bombyx mori doublesex ( Bmdsx ), which is a master regulatory gene for sexual differentiation, undergoes default splicing to produce the female isoform of Bmdsx ( BmdsxF ), thereby inducing female differentiation. [ 18 ]. Spongy moth Fet-W exhibits structural features similar to those of Fem and PxyFem , forming a cluster consisting of about 100 copies on the W chromosome [ 8 ] and encoding a sequence of approximately 70 bases complementary to the mRNA sequence of the Lymantria dispar Masc gene ( LdMasc ), which suggests that it functions as a piRNA source targeting LdMasc mRNA [ 8 ]. Although it remains unclear whether Fet-W induces the degradation of LdMasc mRNA via piRNA, it has been found to suppress LdMasc expression to induce feminization, as Fet-W knockdown increased LdMasc expression in females [ 8 ]. These findings support the notion that the sex determination mechanism in the spongy moth is largely analogous with that of the silkworm. However, several molecular discoveries have suggested that sex determination mechanisms may differ between the spongy moth and silkworm. The most notable example is the O . furnacalis Masc gene ( OsMasc ). Although OsMasc participates in male differentiation, it does not exhibit male-specific expression, in contrast to previously identified Masc genes [ 19 ]. Moreover, OsMasc is not subject to post-transcriptional repression by piRNAs in females [ 19 ]. The pyralid moth E . kuehniella possesses two copies of Masc ( EkMasc and EkMasc-B ) on the Z chromosome, which are arranged in an inverted repeat configuration [ 15 ]. Additionally, a homologous sequence of EkMasc on the W chromosome ( EkMasc W ) has been identified [ 20 ]. EkMasc W shows 94–97% homology with the region corresponding to exons 2–10 of the EkMascB cDNA sequence and is present in 17–23 copies on the W chromosome. EkMasc W is hypothesized to have arisen as a duplicate of LdMasc-B. While its function remains to be elucidated, EkMasc W produces piRNAs that are complementary to EkMasc and EkMascB [ 20 ]. As described above, female determination in the pyralid moth is controlled by piRNAs produced from another gene, E . kuehniella Mom ( EkMom ), located on the W chromosome. Visser et al. [ 20 ] hypothesized that EkMom originates from a partial sequence of Masc transposed onto the W chromosome, similar to EkMasc W . Thus, EkMasc W exhibits characteristics of a gene in the process of becoming a sex determination gene. The spongy moth shares similarities with E . kuehniella in that it possesses two copies of Masc ( LdMasc-A and LdMasc-B ) on the Z chromosome [ 8 ]. In the present study, we report that, similar to the pyralid moth carrying EkMasc W , the spongy moth carries multiple copies of nucleotide sequences linked to the W chromosome that may be designated as LdMasc-W . We examined the structural features of LdMasc-W and report its functional characteristics based on expression analyses, embryonic RNA interference (RNAi)-based knockdown experiments, and hybridization experiments between closely related species of Lymantria species. Our results showed that LdMasc-W consists of a nucleotide sequence showing extremely high homology with the genomic sequence covering introns 8–11 of LdMasc , which is structurally different from EkMasc W . Thus, LdMascW may be regarded as a W-chromosome paralog of LdMasc . LdMasc-W is present in multiple copies on the W chromosomes of three spongy moth subspecies ( Lymantria dispar dispar , Lymantria dispar asiatica , and Lymantria dispar japonica ), but with copy numbers differing among subspecies. Of the subspecies examined, L . d . japonica had the highest number (≥ 52) of LdMasc-W copies. Nevertheless, it was found that L. umbrosa , a close relative of L. d. japonica , lacks LdMasc-W . This study provides the first functional evidence that a Masc paralog linked to the W chromosome has the potential to act as a female-determining gene. Results Female-biased expression and female genome-specific presence of LdMasc Reads obtained from de novo RNA sequencing (RNA-seq) performed in our previous study [ 8 ] were mapped to the LdMasc coding sequence (CDS). A greater number of such reads aligned to the region encompassing exon 9 to the 3’ end in females than in males. Consistent with this finding, four unigenes derived in our previous study [ 8 ] were composed of sequences from exon 9 to the 3’ end of the LdMasc CDS (Fig. 1 B). The 5′-end sequences of these unigenes contained unique nucleotide sequences not present in the LdMasc gene. Genomic PCR using primers annealing to these unique regions amplified a female-specific DNA fragment (Fig. 1 C). RT-PCR using the same primers yielded female-specific amplification products (Fig. 1 D). These results strongly indicate that the nucleotide sequence spanning from exon 9 to the 3’ end of LdMasc is present on the W chromosome. Whole-genome identification of homologous sequences of LdMasc The above results suggested that nucleotide sequences homologous to LdMasc were present on the W chromosome. Therefore, we performed a nucleotide BLAST (BLASTn) search using the genomic sequence of the LdMasc gene (a 19,341-bp genomic sequence including exons 1–11) as a query sequence against the entire L . d . japonica genome. In addition to the two known LdMasc genes on the Z chromosome, we identified 52 LdMasc homologous sequences (Additional file 1: Table S1 ), all of which were found to be nucleotide sequences on contigs predicted to be derived from the W chromosome (Additional file 1: Table S1 , Fig. 2 A). Most of these sequences were approximately 5.4 kb in length, extending from a position about 13.9 kb within the LdMasc gene to its 3’ end (midway through intron 8 to the 3’ end of exon 11) (Fig. 2 A). These sequences were predicted to be nearly identical to the corresponding region of the LdMasc gene (E = 0; ≥ 99% identity). Because there were two to four homologous sequences per contig (Additional file 1: Table S1 , Fig. 2 A), these homologous sequences were also expected to constitute repetitive sequences on the W chromosome. Since all homologous sequences showed sufficient similarity to be considered derived from the LdMasc gene, we designated these homologous sequences as paralogs of LdMasc , named LdMasc-W for convenience. To validate these results, we performed a BLASTn search using the entire genomic sequences of L . d . dispar and L . d . asiatica , which are publicly available at the National Center for Biotechnology Information (NCBI). The results revealed that three nucleotide sequences showing nearly 100% homology with the approximately 13.9–17.7-kb region of the LdMasc gene were present within the contig corresponding to the W chromosome of L . d . dispar (accession no. OY755156.1) (Additional file 2: Table S2 , Fig. 2 B). In L . d . asiatica , a subspecies closer to L . d . japonica , five nucleotide sequences showing nearly 100% homology to the region from approximately 13.9 kb to near the 3’ end (~ 5.4 kb) of the LdMasc gene were found within the contig corresponding to the W chromosome (accession no. CM063456.1). We also identified one sequence showing nearly 100% homology across more than half the length of the LdMasc gene (9167–19346 bp) (Additional file 2: Table S2 , Fig. 2 B). On the other hand, the same BLASTn search against the whole-genome sequence of L. umbrosa , which is a close relative of L. d. japonica , identified a contiguous nucleotide sequence matching the entire LdMasc sequence, but failed to find a sequence corresponding to LdMasc-W (Additional file 5: Table S3 ). qPCR analysis using genomic DNA as a template again confirmed that LdMasc-W exists in approximately 6 to 65 copies in L. d. japonica females collected throughout Japan but is absent in L. umbrosa females (Fig. 2 C). For comparison, a similar analysis was performed using the predicted piRNA coding sequence of the spongy moth female-determining gene Fet-W , which was identified in our previous study [ 8 ], as a query sequence. The results revealed that 91 copies were present within a contig (accession no.: BAAIJM010000031) predicted to correspond to the W chromosome of L . d . japonica (see Additional file 6: Table S4 ). Sequences homologous to the putative Fet-W piRNA CDS were also found in the W chromosomes of L . d . dispar and L . d . asiatica , with 160 and 117 copies, respectively (Fig. 2 B, Additional file 8: Tables S6 and Additional file 9: Table S7 ). Similar analysis identified a total of 124 copies of Fet-W homologous sequences (identity = 100%) across two contigs of L. umbrosa (accession no.: BAAIJN010000033 and BAAIJN010000013) (see Additional file 9: Table S7 ). These results strongly indicate that LdMasc-W is conserved between L . dispar subspecies and that multiple copies are present on the W chromosome in all subspecies. On the other hand, our data suggests that Fet-W was highly conserved between the subspecies L. dispar and its closely related species L. umbrosa . Notably, interspecific copy number variation was greater for LdMasc-W than for Fet-W . Classification of LdMasc-W by molecular phylogenetic analysis Our analyses revealed that L . d . japonica , L . d . dispar , and L . d . asiatica possess 52, 3, and 6 copies of LdMasc-W , which we designated as LdMasc-W1 – 52 , LdMasc-WD1 – 3 , and LdMasc-WA1 – 6 , respectively. To investigate their relationships, we obtained a 5-kb genomic sequence adjacent to the 5’ end of each LdMasc-W and constructed a molecular phylogenetic tree based on their alignment. The 13 LdMasc-W genes from L . d . japonica were excluded from this analysis due to their location at the ends of contigs, which prevented the acquisition of upstream sequences exceeding 5 kb in length. Of the 39 LdMasc-W sequences subjected to phylogenetic analysis, 35 were grouped into four distinct clusters (Fig. 3 A). Cluster B consisted exclusively of LdMasc-W from L . d . japonica . The three LdMasc-W sequences from L . d . dispar ( WD1 – 3 in Fig. 3 A) were independent from those in L . d . japonica , whereas four of the six LdMasc-W genes from L . d . asiatica ( WA1 – 6 in Fig. 3 A), which is more closely related to L . d . japonica , belonged to groups A, C, and D ( WA2 , WA3 , WA5 , and WA6 in Fig. 3 A). These results suggest that the LdMasc-W genes in L . d . japonica , corresponding to LdMasc-WA2 , WA3 , WA5 , and WA6 , increased their copy numbers following the divergence of L . d . japonica from L . d . asiatica . Given the extremely low depth of the branches forming each group, we hypothesize that the copy numbers of LdMasc-W increased rapidly in each group. To verify that the 52 LdMasc-W sequences identified in L . d . japonica are indeed nucleotide sequences on the W chromosome, we performed genomic PCR using primers specifically annealed to the nucleotide sequences adjacent to the 5’ end of each LdMasc-W . As our previous molecular phylogenetic analysis results indicated that these nucleotide sequences showed homology with each other, it was impossible to design primers specific for all LdMasc-W genes. Therefore, we designed 12 primers that annealed as specifically as possible to individual LdMasc-W genes (Fig. 3 B) and used these to perform PCR with genomic DNA from both males and females as templates. The results showed that LdMasc-W21 was amplified in both males and females, while amplification products for the other LdMasc-W genes were obtained only when the female genome was used as the template (Fig. 3 C, upper panel). The same PCR analysis using genomic DNA extracted from L. umbrosa larvae as a template indicated that there were no sequences homologous to LdMasc-W in L. umbrosa genome (Fig. 3 C, lower panel), consistent with the results of BLASTn searches described above (Additional file 5: Table S3 ). Interestingly, LdMasc-W21 was also amplified in both male and female of L. umbrosa (Fig. 3 C, lower panel). These results suggest that all 52 LdMasc-W genes identified in this study, with the exception of LdMasc-W21 , are located on the W chromosome. Identification of LdMasc-W-derived transcripts and expression analysis All subsequent experiments were conducted using L . d . japonica . As described above, the four unigenes composed of the 3’ end nucleotide sequence of LdMasc were specifically expressed in females and were present only in the female genome (Fig. 1 B). Therefore, to determine whether these unigenes originate from LdMasc-W , we performed a BLASTn search using the nucleotide sequences of these four unigenes (31510, 24771, 29638, and 44942) as query sequences against the genomic sequences of all identified LdMasc-W genes. The results showed that nucleotide sequences near the 5’ ends of the four unigenes were encoded by genomic sequences of LdMasc-W2 , LdMasc-W6 , LdMasc-W8 , and LdMasc-W9 (Fig. 4 A). The nucleotide sequence near the 3’ end of unigene 44942 appeared to have originated from genomic sequences of LdMasc-W1 to LdMasc-W9 (Fig. 4 A). However, we were unable to find LdMasc-W encoding the nucleotide sequence near the 3’ end of unigene 24771. Thus, the four unigenes do not reflect all transcripts derived from LdMasc-W genes. To obtain additional transcripts from LdMasc-W , we conducted rapid amplification of cDNA ends (RACE) to identify the 5’ end using primers designed to anneal to a region common to all LdMasc-W variants (Additional file 9: Table S7 and Fig. 4 B). The resulting 5’ RACE products were TA-cloned, and the nucleotide sequences of 48 clones were determined, identifying nine distinct transcripts (see Additional file 12: Fig. S1 ). These nine transcripts were named RACE4, RACE13, RACE24, RACE35, RACE45, RACE50, RACE67, RACE69, and RACE85 (Accession no. LC889978-LC889986). Comparison of the nine nucleotide sequences with the genomic sequences of LdMasc-W genes revealed that the nine transcripts were derived from 17 LdMasc-W genes belonging to groups A, B, and D (Fig. 4 C). These results indicate that at least 17 of the 52 LdMasc-W genes identified in this study are expressed at the mRNA level. Because LdMasc-W genes were located on the W chromosome, transcripts derived from these genes should be expressed specifically in females. To verify this hypothesis, RT-PCR was performed using cDNA templates prepared from testes and ovaries with primers designed to specifically amplify each identified transcript (see Additional file 19: Table S9 ). The results revealed that four of the nine transcripts (RACE4, RACE13, RACE24, and RACE85) were expressed specifically in the ovary (see Additional file 14: Fig. S2 ). Although RACE50 was also expressed in the ovary, DNA fragments of different sizes were amplified only to a small extent in the testes (see Additional file 14: Fig. S2 ). These results indicated that the 17 LdMasc-W genes encoding these five transcripts were expressed specifically in females. To investigate whether these LdMasc-W genes also exhibit specific expression in female embryos at the sex determination stage (days 2–4 post-laying), RT-PCR analysis was performed using cDNA templates prepared from eggs of both sexes at the sex determination stage. This analysis demonstrated that transcripts with ovary-specific expression were also expressed specifically in females throughout the sex determination period (Fig. 4 D). These findings indicate that the 17 LdMasc-W genes encoding the five transcripts may possess female-specific functions at the sex determination stage. Functional analysis of LdMasc-W by embryonic RNAi Given that some LdMasc-W genes are specifically expressed in females throughout the sex determination period (Fig. 4 D) and that this gene is located on the W chromosome, we hypothesized that it is highly likely to be involved in female determination. A standard approach to evaluate the function of sex determination genes in lepidopteran insects is to examine whether functional suppression of the gene affects sexually dimorphic expression of dsx during the sex determination period [ 15 , 21 , 22 ]. Therefore, to evaluate whether LdMasc-W is involved in female determination, we examined the effects of RNAi-mediated knockdown of LdMasc-W expression during sex determination on the sexual dimorphic expression pattern of Lddsx , the ortholog of dsx in the spongy moth (Fig. 5 A). We injected small interfering RNAs (siRNAs) targeting LdMasc-W mRNA into eggs within 12 h post-laying (embryonic RNAi) to achieve LdMasc-W knockdown. Two types of siRNA targeting different nucleotide sequences within a region common to all LdMasc-W genes were used for embryonic RNAi (LdMasc-W si1 and LdMasc-W si2) (Fig. 5 B, Additional file 13: Table S8 ). Eggs injected with siRNA targeting Egfp (Egfp si) used in our previous study [ 13 ] were used as a control group. Quantification of LdMasc-W expression in embryos at 4 days post-injection (almost equivalent to 4 days post-laying) revealed that the average expression level in embryos injected with LdMasc-W si1 decreased to approximately 3.6% of that in Egfp si-injected eggs (Fig. 5 C). In contrast, the average expression level in eggs injected with LdMasc-W si2 decreased to approximately 30.4% of that in Egfp si-injected embryos (Fig. 5 C). RT-PCR analysis demonstrated that females with decreased mRNA levels of LdMasc-W via LdMasc-W si1 injection exhibited both female-type ( LddsxF ) and male-type ( LddsxM ) Lddsx expression (Fig. 5 D, E). Approximately 60% of these females showed higher LddsxM expression than LddsxF expression, but no individuals exhibited complete sex reversal (expression of only LddsxM ) (Fig. 5 E). Among females in which LdMasc-W knockdown was induced by LdMasc-W si2 injection, Lddsx expression patterns were similar to those observed in the control group (Fig. 5 D, E). These results clearly demonstrate that a drastic reduction in LdMasc-W expression induces a shift from female-type to male-type Lddsx expression in females. Induction of LddsxM expression requires expression of the male determination gene LdMasc [ 8 ]. The induced LddsxM expression observed in LdMasc-W knockdown females may have been caused by increased LdMasc expression levels in these knockdown individuals. To test this hypothesis, we quantified LdMasc expression among females in which LdMasc-W expression was apparently reduced by LdMasc-W si1 injection. The results showed significantly higher LdMasc expression levels (~ 1.5-fold higher) in LdMasc-W knockdown females, compared to females in the negative control group (Fig. 5 F). In contrast, no significant change was observed in expression levels of the female determination gene Fet-W , which suppresses LdMasc expression (Fig. 5 G). These results indicate that LdMasc-W knockdown shifts the sexually dimorphic expression of Lddsx from the female to the male mode by causing a slight increase in LdMasc expression without altering Fet-W expression levels. Thus, LdMasc-W appears to suppress LdMasc expression and induce female differentiation. Phenotypic analysis of hybrid individuals between L. d. japonica and L. umbrosa To obtain further evidence regarding whether LdMasc-W possesses female-determining ability, interspecific hybridization experiments were conducted using L. d. japonica with the highest LdMasc-W copies and its close relative L. umbrosa lacking LdMasc-W . The sexual dimorphic traits of the resulting first-generation hybrids (F1) were then examined from various perspectives. First, we created hybrid lines by crossing L. umbrosa females with L. d. japonica males (hereafter referred to as UJ-hybrid F1 line) and their reverse crosses (hereafter referred to as JU-hybrid F1 line). As control lines, laboratory lines established from eggs of L. d. japonica and L. umbrosa were subjected to the same analyses. The resulting F1 individuals were subjected to RT-PCR analysis to examine the expression pattern of Lddsx . As shown in Fig. 6 A, the expression pattern of Lddsx in embryos immediately prior to hatching in the JU-hybird F1 was normal ( LddsxM in males, LddsxF in females). On the other hand, similar RT-PCR analysis using UJ-hybrid F1 individuals demonstrated that all embryos confirmed to be genetically female (ZW individuals) expressed male-isoform of Lddsx (Fig. 6 A). Next, we quantified the expression levels of LdMasc , Fet-W , and LdMasc-W using RNA extracted from the same individual. The expression level of LdMasc in L. umbrosa and JU-hybrid F1 animals was higher in males than in females, with extremely low expression levels observed in females (Fig. 6 B). In contrast, the expression level of LdMasc in the UJ-hybrid F1 females was increased to a level equivalent to that in males (Fig. 6 B). Quantitative analysis of the expression levels of the known female determination gene Fet-W confirmed its female-specific expression in all lines (Fig. 6 B). The same analysis revealed that LdMasc-W was highly expressed only in females carrying the W chromosome derived from L. d. japonica ( L. d. japonica females and UJ-hybrid F1 females) (Fig. 6 B). These results were consistent with the fact that UJ-hybrid F1 females did not possess LdMasc-W because they inherited the W chromosome from their maternal parent, L. umbrosa . Taken together, the above findings suggest that the absence of LdMasc-W in the UJ-hybrid F1 females induced LdMasc expression at levels equivalent to males, leading to a shift in the expression pattern of Lddsx from the female-type to the male-type. The UJ-hybrid F1 eggs showed no significant difference in hatchability as compared to those in other lines (see Additional file 26: Fig. S3 ). Individuals that died during rearing were collected, and the death rate (number of dead individuals/ total number of hatched individuals) was calculated. As shown in Fig. 6 C, a significantly higher death rate was observed in the UJ-hybrid F1 individuals. When plotting the Kaplan-Meier curve, it was found that approximately 20% of individuals in the UJ-hybrid animals died within about 20 days after hatching, relatively early stages in development (Fig. 6 D). Furthermore, PCR-based molecular sexing using DNA extracted from the deceased individuals revealed that the sex ratio of the dead individuals was female-biased (Fig. 6 E). In the UJ-hybrid F1 animals, the final number of adults was 28 females and 131 males, indicating a significant male bias. These results suggest that UJ-hybrid F1 females died at a relatively early stage during the larval period. Morphological observations of sexual dimorphic traits in adults revealed no clear masculinization in the wing coloration or patterns of the UJ-hybrid F1 females, while their wing angles at rest resembled those of males (Fig. 7 A and 7 B). The antennae of the UJ-hybrid F1 females exhibited morphological characteristics similar to those of males (Fig. 7 B). The color of the scaly hairs on the legs of these females also showed the same bright coloration as the males (Fig. 7 B). The UJ-hybrid F1 females had malformed external genitalia likely due to insufficient development (Fig. 7 C). The ovaries of these females contained a number of mature eggs comparable to those of normal females, and no morphological abnormalities were observed (Fig. 7 D). However, careful observation revealed the presence of male-specific tissues at the base of the oviducts: the vas deferens, accessory glands, seminal vesicles, and ejaculatory ducts (Fig. 7 E). There was little variation among individuals in these male-like traits observed in the UJ-hybrid females. In JU-hybrid and L. umbrosa females, the oviduct, spermatheca, and seminal receptacle were present in the same region (Fig. 7 E). These results clearly demonstrated that the UJ-hybrid F1 females, which carried LdMasc and Fet-W without LdMasc-W , exhibited a morphological phenotype classified as intersex, possessing characteristics of both male and female morphology. Discussion This study revealed that multiple sequences almost identical to the approximately 5-kb genomic sequence near the 3’ end of the LdMasc gene, which is involved in male determination in the spongy moth [ 8 ], are present on the W chromosome. These homologous sequences were considered W chromosome-specific paralogs of LdMasc and designated as LdMasc-W , with varying copy numbers among L . d . dispar (3 copies), L . d . asiatica (6 copies), and L. d . japonica (52 copies) (Fig. 2 A, B). The subspecies L . d . asiatica and L . d . japonica are closely related; phylogenetic analysis revealed that LdMasc-W homologous to four of the six LdMasc-W genes had higher copy numbers in L . d . asiatica than in L . d . japonica (Fig. 3 A). Furthermore, LdMasc-W genes classified into group B were unique to L . d . japonica (Fig. 3 A). It remains unknown why LdMasc-W copy numbers increased only in L . d . japonica . The presence of false copies due to misassembly of the genome sequence can be excluded as a possible explanation because the LdMasc-W copy numbers estimated by qPCR analysis generally matched those determined through genome analysis (Fig. 2 C). The construction of chromosome-level sequence assemblies for the W chromosome in L . d . japonica will reveal the precise copy number of LdMasc-W and its location on the W chromosome in this subspecies. In the diamondback moth ( Plutella xylostella ), Masc homologous sequences are present on the W chromosome [ 15 ]. The Masc homologous sequence originates from a retrotransposon-derived mRNA that fused with the mRNA sequence of PxyMasc (a Masc ortholog of the diamondback moth) spanning exons 4–6 or exons 4–7 and was subsequently integrated into the W chromosome. This process resulted in a multicopy array, with 4–7 copies of the sequence arranged in tandem [ 15 ]. piRNAs targeting PxyMasc mRNA are produced from the region corresponding to PxyMasc exon 5 within these multicopy arrays, resulting in the degradation of PxyMasc mRNA via ping-pong amplification. Therefore, this multicopy array is predicted to function as a female determination gene in the diamondback moth and has been named PxyFem [ 15 ]. Another example of a Masc homologous sequence found on the W chromosome is EkMasc W in the Mediterranean flour moth ( Ephestia kuehniella ). EkMasc W shares approximately 94–97% homology with an EkMascB cDNA sequence encompassing exons 2–10 (~ 1.3 kb) and has 17–23 copies on the W chromosome [ 20 ]. Although this feature initially appears similar to PxyFem , EkMasc W is thought to have arisen from a duplication of LdMasc-B , rather than from a retrotransposon copy of EkMascB mRNA. Furthermore, unlike PxyFem , EkMasc W does not function as a sex-determining gene; instead, another gene ( EkMom ) is responsible for female determination [ 20 ]. Distinct from PxyFem and EkMasc W , LdMasc-W consists of a nucleotide sequence approximately 5.4 kb in length, extending from intron 8 to exon 11 of the genomic sequence of LdMasc (Fig. 2 A, B). Therefore, it is reasonable to consider that LdMasc-W is a LdMasc paralog resulting from translocation of the 3’ region of the LdMasc genomic sequence to the W chromosome, followed by an increase in its copy number due to gene duplication. LdMasc has two tandem copies on the Z chromosome [ 8 ], which suggests that it may reside within a genomic structure prone to gene duplication. Of the 52 LdMasc-W genes identified in L . d . japonica , at least 17 LdMasc-W genes were expressed specifically in females throughout the sex determination period (Fig. 4 D). RNAi-mediated knockdown of LdMasc-W caused slightly increased LdMasc mRNA levels, leading to LddsxM expression in females (Fig. 5 C–G). However, LdMasc-W knockdown resulted in only very slightly increased LdMasc mRNA levels, which may be attributable to the siRNA used in this study (LdMasc-W si1), which targeted the common region between LdMasc-W and LdMasc . In this experiment, designing siRNAs that targeted the common region of LdMasc-W and LdMasc was unavoidable due to the necessity of simultaneously knocking down the expression of at least 17 distinct mRNAs derived from LdMasc-W. LdMasc expression might have increased to a greater extent had we been able to specifically knock down LdMasc-W alone. Consistent with results obtained from embryonic RNA, a female-to-male shift in the expression pattern of Lddsx was observed in F1 hybrid female embryos obtained from the cross between L. umbrosa females and L. d. japonica males (Fig. 6 A). Note that females resulting from this crossing could carry LdMasc derived from L. d. japonica but not possess LdMasc-W . In these females, no expression of LdMasc-W was observed, whereas the expression level of LdMasc increased to levels comparable to those in males (Fig. 6 B). Furthermore, these hybrid females exhibited female-to-male sexual reversal in the morphology of their antennae, legs, and parts of their internal reproductive organs (Fig. 7 B, 7 D, and 7 E). Taken together with the above findings, it would be reasonable to conclude that LdMasc-W functions as a female determining gene that is expressed specifically in females during the sex determination period and promotes female differentiation by reducing the mRNA levels of LdMasc . The UJ-hybrid females exhibited lethality as the primary phenotype (Fig. 6 C- 6 E). This can be attributed to abnormalities in gene dosage compensation in females. It is known that abnormalities in gene dosage compensation during development cause lethality in various species, including mice, nematodes, and fruit flies [ 23 , 24 , 25 ]. In multiple Lepidoptera insects, Masc reduces gene expression levels on the Z chromosome by half in males to compensate gene dosage between two sexes [ 26 , 27 ]. It would be possible that forced expression of Masc causes unnecessary dosage compensation in the expression levels of Z-linked genes in females, resulting in female-specific lethality. In fact, forced expression of the Fem piRNA-resistant Masc ( Masc-R ) gene in the silkworm has been shown to cause female-specific lethality during the larval stage [ 11 ]. This is reminiscent of our finding that the UJ-hybrid F1 females, most of which were dead during the larval stages, expressed high level of LdMasc mRNA equivalent to that in males (Fig. 6 B- 6 E). It is highly plausible that increased expression level of LdMasc in UJ-hybrid F1 females induced inappropriate compensation for the Z-linked gene expressions, leading to female-specific lethality as the predominant phenotype. The UJ-hybrid F1 females exhibited a shift in the expression pattern of Lddsx from female to male at the pre-hatchling embryonic stage, yet the tissues showing sex reversal were limited (Fig. 7 B, 7 D, and 7 E). In Onthophagus taurus , which belongs to the family Scarabaeidae, dsx is involved in regulating sexual differentiation. However, the variety of downstream genes and the regulatory direction (activation/repression) of gene expression by dsx differ between tissues [ 28 ]. Particularly in females, tissues in which dsx regulates the expressions of genes responsible for sexual differentiations are limited. For example, many genes showing sex-specific expressions are regulated by dsx in the female horn, whereas in other tissues, sex differences are governed by factors other than dsx . Based on these findings, Ledón-Rettig et al. hypothesized that features susceptible to sexual selection or newly evolved sexual traits may require the involvement of multiple genes under the control of dsx [ 28 ]. Similarly, in the spongy moth, the degree of dependence on dsx probably differs for each sexual dimorphic trait, and thus the tissues showing sex reversal in UJ-hybrid FI females may be limited. The most noteworthy finding of this study is that LdMasc-W determines femaleness in the spongy moth through several pathways, despite the presence of the female-determining gene Fet-W in this species, as determined in our previous study [ 8 ]. As described above, RNAi-mediated knockdown of LdMasc-W increased LdMasc mRNA levels in females and induced expression of the male-type Lddsx (Fig. 5 E and 5 F). These findings suggest that LdMasc-W induces female differentiation by suppressing LdMasc expression in females. The mRNAs transcribed from LdMasc-W genes include an open reading frame (ORF) extending from exon 9 to the stop codon of LdMasc (Fig. 4 A and Additional file 12: Fig. S1 ). A comparison of the amino acid sequences encoded by these ORFs with those of the LdMasc protein revealed that amino acid sequences encoded by 43 of the 52 LdMasc-W genes were 100% identical to the corresponding amino acid sequence of the LdMasc protein (see Additional file 27: Fig. S4 ). It might be possible that the protein produced from the LdMasc-W cause a decreased expression of LdMasc mRNA. Examples of gene paralogs functioning as sex-determining genes have been discovered in several species. Dmy ( Dmrt1bY ) in medaka ( Oryzias latipes ), which adopts a heterogametic male (XX = female, XY = male) sex determination system, is a pioneering example. Dmy is located on the Y chromosome and functions as a male determination gene by inducing testis formation [ 29 ]. This gene is considered to be a Y chromosome paralog of Doublesex and mab-3 related transcription factor 1 a ( Dmrt1a ), which induces masculinization through testis development [ 30 – 32 ]. Similarly, in rainbow trout ( Oncorhynchus mykiss ), which also employs a heterogametic male sex determination system, Sexually dimorphic on the Y-chromosome ( sdY ) functions as a male determination gene [ 33 ]. The sdY gene plays a role in male determination by inducing testis formation and appears to be a Y-chromosomal paralog that originated from gene duplication of Interferon regulatory factor 9 ( Irf9 ), a gene involved in interferon regulation. The sdY gene encodes a protein homologous to the C-terminal amino acid sequence of the Irf9 protein, although the downstream cascade of sdY remains unclear. This gene is a notable example of a paralog originating from a gene unrelated to sex determination acquiring the function of a male-determining gene. The African clawed frog ( Xenopus laevis ), which employs a female heterogametic sex determination system (ZZ = male, ZW = female) like the spongy moth, has been reported as an example of a paralog functioning as a sex determination gene. The female-determining gene DM-W on the W chromosome of Xenopus laevis was identified as a W chromosome paralog of the male-determining gene DMRT1 [ 34 ]. DM-W shows extremely high homology with DMRT1 in the DNA-binding domain (DM domain), but no homology is observed in the C-terminal region. Since the C-terminal region of DMRT1 possesses transcriptional activation activity, it has been predicted that despite both proteins binding to the same target genes, they exert different transcription regulatory effects [ 35 ]. Based on these findings, a model has been proposed in which DM-W suppresses the function of DMRT1, leading to the promotion of female differentiation. As demonstrated for sdY in rainbow trout, where a protein homologous to the C-terminal amino acid sequence functions as a sex-determining gene, a protein corresponding to the C-terminal region of LdMasc produced by LdMasc-W genes may be responsible for female determination in the spongy moth. Similar to the DM-W protein that induces female differentiation by suppressing DMRT1 function, LdMasc-W protein products may induce female differentiation by inhibiting the male-determining function of the LdMasc protein. Further research is needed to elucidate the molecular mechanism underlying how LdMasc-W leads to the suppression of LdMasc function. A key remaining question is why the spongy moth possesses LdMasc-W in addition to Fet-W . We hypothesize that Fet-W alone may be insufficient to repress LdMasc expression, suggesting the necessity of an additional inhibitory mechanism. Fet-W is predicted to repress LdMasc expression by inducing the degradation of LdMasc mRNA via the piRNA pathway, similar to the Fem genes identified in other lepidopteran insects [ 8 ]. Unlike many other lepidopteran insects, the spongy moth possesses two copies of the male-determining gene LdMasc , which are tandemly arranged on the Z chromosome [ 8 ]. The expression levels of genes arranged in tandem copies are enhanced beyond those expected based on the copy number (i.e., more than doubled in this case) [ 36 ]. To counteract the increase in LdMasc copy numbers and the accompanying rise in expression levels, the spongy moth may have evolved two female determination genes. In this study, most LdMasc-W si1-injected females with nearly complete knockdown of LdMasc-W expression tended to show higher LddsxM expression than LddsxF expression, but none exhibited complete sex reversal in the expression pattern of Lddsx (Fig. 5 E, F). This finding strongly suggests that LdMasc-W functions in a complementary manner during sex determination. In summary, Fet-W alone is insufficient for female determination, and complete female determination may occur through the auxiliary female-determining function of LdMasc-W . Like the spongy moth, the Mediterranean flour moth E kuehniella possesses the female-determining gene EkMom together with a homologous sequence of EkMascB on the W chromosome, EkMasc W [ 20 ], and it possesses two copies of Masc orthologs ( EkMascA and EkMascB ) [ 15 ]. EkMasc W produces piRNAs, which may target EkMasc mRNA [ 20 ]. While the contribution of EkMasc W to female determination remains to be determined, both EkMom and EkMasc W may be required for the full achievement of female determination. Generally, when a gene playing a dominant role in sex determination (the master sex determination gene) is acquired, recombination is reduced or suppressed between homologous chromosomes, leading to the irreversible accumulation of harmful mutations [ 37 ]. Consequently, the turnover of master sex determination genes can occur at accelerated rates [ 38 – 36 ]. The presence of alternative genes is a prerequisite for the smooth execution of this process. Under these conditions, possessing two female determination genes, Fet-W and LdMasc-W , may be advantageous. Such turnover may be occurring in LdMasc-W and Fet-W . LdMasc-W copy numbers are much higher in L . d . japonica than in L . d . dispar and L . d . asiatica (Fig. 2 A, B), which may be related to a progressive shift in the function of the female-determining gene from Fet-W to LdMasc-W in L . d . japonica . To verify this hypothesis, further research is needed to clarify the function of LdMasc-W in L . d . dispar and L . d . asiatica . Conclusions Our findings demonstrated that LdMasc-W , derived from the male-determining gene LdMasc , possesses functional properties compatible with a role in female determination. Comparative analyses across three subspecies and one close relative species revealed substantial copy number variation among Fet-W , LdMasc , and LdMasc-W . Notably, LdMasc-W exhibited the highest diversity, with 0–52 copies. Similar copy number variation was observed between populations even within the same species. Phylogenetic analysis suggested that LdMasc-W copy number diversification accompanies speciation, highlighting the potential contribution of this gene to the diversification of sex determination systems. These findings would provide valuable insights into the molecular mechanisms underlying the diversification of sex determination systems. Methods Insects The Japanese spongy moth ( L . d . japonica ) used in this study was a laboratory strain primarily derived from egg masses collected in Nasu-Shiobara, Tochigi Prefecture, Japan. Another laboratory strain was established from L. umbrosa eggs collected in Kitami, Hokkaido, Japan. Species confirmations of these strains were performed by PCR-RFLP analysis of mitochondrial DNA according to the method described previously [ 42 ]. The moths were reared throughout their developmental stages in rearing chambers maintained at 25°C and 60 ± 10% humidity, under a 16-h light/8-h dark photocycle. For larval rearing, Wisteria floribunda leaves and an artificial diet were used. Post-emergence adults were allowed to mate freely within cylindrical nets (diameter, 40 cm; height, 55 cm) placed in the rearing room. Mating between L. d. japonica and L. umbrosa adults were conducted under the same condition. All eggs thus obtained were stored at 25°C in the rearing room for 1 month and then at 4°C in a refrigerator for over 100 days to break dormancy. Stereomicroscopic analysis The antennae, legs, internal and external genitalia were dissected out from adults basically within one day after emergence. Dissected genitalia was trimmed in 1×Phosphate Buffer Saline (FUJIFILM Wako) using fine-tipped forceps (No. 11252-00, FINE SCIENCE TOOLS). To prepare cuticle specimens, the external genitalia were incubated in 15% KOH at 50°C for 12 hours, followed by washing with 70% ethanol. Images of the dissected samples were acquired by a digital camera (DP73, Olympus) attached to the stereomicroscope (SZX16, Olympus). Extraction of total RNA and genomic DNA Total RNA extraction from various tissues and eggs was performed using ISOGEN reagents (Nippon Gene, Tokyo, Japan), according to the manufacturer’s instructions. Tissue homogenization was performed using a Homogenization Pestle (Scientific Specialties, Lodi, CA, USA). Genomic DNA was precipitated by adding one third the volume of ethanol to the lower layer obtained after ISOGEN treatment and centrifuging at maximum speed at 4°C.The resulting pellet was dissolved in an alkaline solution (50 mM NaOH), incubated at 95°C for 15 min, and then an equal volume of neutralizing buffer (200 mM Tris-HCl, pH 8.0) was added. The resulting genomic DNA solution was stored at 4°C. 5’ RACE analysis Poly(A) + RNA used as a template for 5’ RACE was extracted from female ovaries using Fast Track MAG mRNA Isolation Kits (Invitrogen, Carlsbad, CA, USA), following the manufacturer’s instructions. We performed 5’ RACE using the Gene Racer kit (Invitrogen), following the manufacturer’s instructions. PCR amplification of the resulting cDNA was performed using LA-Taq (Takara Bio, Kusatsu, Japan) under the following conditions: five cycles of 94°C for 2 min, 94°C for 30 s, and 72°C for 2 min; five cycles of 94°C for 30 s and 70°C for 2 min; 25 cycles of 94°C for 30 s, 68°C for 30 s, and 72°C for 2 min; followed by 72°C for 10 min. Subsequently, nested PCR was performed under the following conditions: 25 cycles of 94°C for 2 min, 94°C for 30 s, 65°C for 30 s, and 68°C for 2 min; followed by 68°C for 10 min. Primers used for 5’ RACE are listed in Additional file 13: Table S8 . RT-PCR cDNA synthesis via reverse transcription using extracted total RNA as a template was performed using the Prime Script First-Strand cDNA Synthesis Kit (Takara Bio), following the manufacturer’s instructions. We used 8 µL of total RNA for reverse transcription, with 6-mer random primers included in the kit. EmeraldAmp PCR Master Mix (Takara Bio) was used to amplify the resulting cDNA, following the manufacturer’s instructions. The nucleotide sequences of primers used for RT-PCR are shown in Additional file 19: Table S9 . qRT-PCR qRT-PCR reactions were performed using TB Green Premix Ex Taq II (Takara Bio), following the manufacturer’s instructions; cDNA was synthesized via the reactions described above. Gene expression quantification was performed as previously described [ 43 ] using the ΔΔCt method, with the Lymantria dispar elongation factor 1-α ( LdEF1-α ) gene as a reference gene. The nucleotide sequences of primers used for qRT-PCR are listed in Additional file 28: Table S10 . Estimation of gene copy numbers by qPCR The copy numbers of genes of interest were estimated by qPCR as previously described [ 44 ]. Genomic DNA prepared as described above was used as a template. The qPCR reaction was performed as described above using the same primers used for qRT-PCR (see Additional file 28: Table S10 ). The Lymantria dispar cad gene, which is an autosomal single-copy gene encoding carbamoyl-phosphate synthetase 2 aspartate transcarbamylase and dihydroorotase, was used as a reference gene [ 44 ]. Sex determination and genomic PCR using W chromosome-specific primers Molecular sexing by PCR using W chromosome-specific primers was performed as described previously [ 8 ]. Shuttle PCR was performed with LA-Taq (Takara Bio), following the manufacturer’s instructions. Individuals yielding amplification products matching the expected size were identified as female. Genomic PCR was performed under the same conditions used to amplify LdMasc-W genes (see below) using primers designed to anneal to a specific sequence in each LdMasc-W (see Additional file 29: Table S11 ). Identification of the W-chromosome-specific LdMasc paralog (LdMasc-W) To identify nucleotide sequences homologous to the LdMasc gene, a BLASTn search was performed using the 19,341-bp genomic sequence of LdMasc , encompassing exons 1–11, as a query sequence against whole-genome sequences of L . d . dispar (accession no. GCA_963576585.1), L . d . asiatica (accession no. GCA_032191425.1), and L . d . japonica (this study). The BLASTn search was performed using the BLAST interface (based on blast + 2.9.0+) integrated in GenomeMatcher 3.10 ( http://www.ige.tohoku.ac.jp/joho/portalsite/files/GenomeMatcher3.php ), with the parameters E = 0.0, ≥ 99% identity, and size ≥ 5000 bp. To determine the whole-genome sequence of L . d . japonica and L. umbrosa , genomic DNA was purified from the fat bodies of a single final-instar female larva of our laboratory strains established from L . d . japonica eggs collected in Sapporo, Hokkaido, and L . umbrosa eggs collected in Kitami, Hokkaido using the NucleoBond HMW DNA Kit (Macherey-Nagel, Düren, Germany), according to the manufacturer’s instructions. The whole-genome sequence was determined using PacBio long-read sequencing and whole-genome sequence assembly services provided by Relixa Corp. ( https://www.rhelixa.com/service/pacbio-wgs/ ). Libraries were constructed using the SMRTbell Express Template Prep Kit 2.0 (PACBIO), sequenced using the PacBio Sequel II sequencer (PacBio, Menlo Park, CA, USA), and assembled using PacBio CCS (Hifi read) software. Sequence read quality checks were performed using Nanoplot v0.11.7 software, and read assembly was conducted with Hifiasm v0.15.5-r350. All acquired data were registered in the public DNA Database of Japan (BioProject: PRJDB37639 [PSUB043264]). This analysis yielded 302 contigs (accession nos. BAAIJM010000001–BAAIJM010000302) derived from L. d. japonica genome and 1115 contigs (accession nos. BAAIJN010000001–BAAIJN010001115) from L. umbrosa genome. Contigs derived from the Z or W chromosome (see Additional file 30: Table S12 ) of L. d. japonica were identified as follows. Genomic DNA was purified from the fat bodies of one final-instar larva of each sex as described above and submitted to Relixa Corp. for whole-genome short-read sequencing ( https://www.rhelixa.com/service/wgs/ ). The obtained reads were mapped onto the 302 contigs using a data analysis service provided by Relixa Corp. Contigs showing average coverage values more than twice as high in males compared to females (longest, 44.9 Mb; total, 7) were assumed to be derived from the Z chromosome. Conversely, contigs showing average coverage values more than twice as high in females compared to males, or contigs with zero coverage in males (longest, 6.8 Mb; total, 198), were designated as W chromosome-derived contigs. Molecular Phylogenetic Analysis To clarify the phylogenetic relationships of LdMasc-W , a molecular phylogenetic analysis was conducted. BLASTn searches were performed using the nucleotide sequence of LdMasc (GenBank accession no. LC817951.1) as a query sequence against the genome sequences of L. d. japonica , L. d. asiatica , and L. d. dispar (GenBank: BAAIJM000000000.1, GCA_004115105.1, GCA_018258255.2). The obtained nucleotide sequences on the W chromosome were designated as LdMasc-W , and the upstream sequence (5 kbp) adjacent to the 5' end of each LdMasc-W was acquired. The acquired sequences were used to construct a phylogenetic tree using maximum likelihood methods with MAFFT and RAxML, employing the upstream sequence of intron 8 in LdMasc as an outgroup. Bootstrap values were set to 100. siRNA synthesis To identify nucleotide sequences unique to LdMasc-W , a BLASTn search was performed against the entire spongy moth genome sequence using a nucleotide sequence of the region shared among all LdMasc-W cDNA sequences as a query sequence. The resulting sequences were subjected to siRNA design using siDirect v2.1 ( http://sidirect2.rnai.jp/ ). siRNA synthesis was performed using the Custom Stealth RNAi siRNA Synthesis Service provided by Invitrogen ( https://www.thermofisher.com/store/v2/oligos-rna?stealth=true ). The synthesized siRNA was dissolved in injection buffer (100 mM potassium acetate, 2 mM magnesium acetate, 30 mM HEPES-KOH; pH 7.4) to a final concentration of 100 µM, as described by Fukui et al. [ 13 ]. The nucleotide sequences of siRNAs used in these experiments are listed in Additional file 31: Table S13 . siRNAs targeting Egfp were used as a control; siRNAs were designed as previously described [ 13 ]. Injection of siRNAs into eggs Egg masses obtained within 12 h after oviposition were separated into individual eggs as described previously [ 8 ]. The eggs were aligned in the same direction and fixed to the surface of a glass slide (Matsunami Glass, Osaka, Japan) using a gelatinous instant adhesive (Yamato Scientific, Hokkaido, Japan). siRNAs were injected into the eggs using glass capillary tubes (uMPm-02; Daiwa, Tokyo, Japan) connected to an electric microinjector (FemtoJet 5247; Eppendorf, Hamburg, Germany), at an injection pressure of 280–340 psi, injection time of 0.1–0.3 s, and a compensation pressure of 30 psi. The hole created by the injection was sealed with a gelatinous instant adhesive, and the eggs were stored in a controlled-temperature chamber at 25°C and 60 ± 10% humidity. Then, the eggs were individually collected into 1.5-mL microtubes and stored in a − 80°C deep freezer. Statistical analyses Some statistical analyses were performed using the Easy R (EZR) software, version 1.60 ( https://www.jichi.ac.jp/saitama-sct/SaitamaHP.files/download.html , accessed September 6, 2021). A Shapiro–Wilk test was used to evaluate the normality of the data obtained in each experiment. Since the sample size was less than 25 and the data did not show a normal distribution, the Mann–Whitney U test was used to examine the significance of differences between the two groups. Other statistical analyses were conducted with R ver. 4.4.1 [ 45 ]. Significant differences between the UJ-hybrid F1 and other lines were examined using the Kruskal-Wallis test and Dunn's post hoc test with Bonferroni correction. Declarations Declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Competing interests The authors declare no competing interests. Funding This research was funded by Grants-in-Aid for Scientific Research (B) (no. 24K01766) and a Japan Society for the Promotion of Science (JSPS) KAKENHI (A) grant (no. 25H01423) to MGS. Author Contribution Conceptualization: KS, RK and MGS. Data curation and formal analysis: KS, KI, RK and MGS. Funding acquisition: MGS. Investigation and methodology: KS, KI, RK, HN and MGS. Supervision: MGS. Writing original draft: KS and KI. All authors read and approved the final manuscript. Acknowledgement We are grateful to Mr. Kota Aoki for rearing the spongy moth larvae and teaching us how to construct the cylindrical nets used for mating adults of the spongy moth. Data Availability All data generated and analyzed during the course of this study are included in this article and its supplementary information files. For some analyses, data from publicly available repositories were used. All raw sequence data obtained by whole-genome sequence (WGS) analyses of *L. d. japonica* and *L. umbrosa* in this study have been deposited in the DDBJ Sequence Read Archive (DRA) database under the BioProject accession number PRJDB37639. The nucleotide sequences of contigs determined by the WGS analyses have been registered in DDBJ under accession numbers BAAIJM010000001- BAAIJM010000315 ( *L. d. japonica* ) and BAAIJN010000001- BAAIJM010001381( *L. umbrosa* ). The nine mRNA sequences obtained by the 5'RACE in this study have been deposited in DDBJ under accession numbers LC889978–LC889986. References Myosho T, Otake H, Masuyama H, Matsuda M, Kuroki Y, Fujiyama A, et al. Tracing the emergence of a novel sex-determining gene in medaka, Oryzias luzonensis . Genetics. 2012;191:163–70. Lee J, Fujimoto T, Yamaguchi K, Shigenobu S, Sahara K, Toyoda A, et al. W chromosome sequences of two bombycid moths provide an insight into the origin of Fem . Mol Ecol. 2024;33:e17434. Boukouvala MC, Kavallieratos NG, Skourti A, Pons X, Alonso CL, Eizaguirre M, et al. Lymantria dispar (L.) (Lepidoptera: Erebidae): current status of biology, ecology, and management in Europe with notes from North America. Insects. 2022;13:854. Goldschmidt R. “Lymantria”. Bibliographia Genetica. 1934;111:1–185. Goldschmidt R. Untersuchungen über Intersexualität. V. Z. Induct Abstl. 1930;56: 257–301. Goldschmidt R. The Material Basis of Evolution, New Haven CT: Yale University Press; 1940. Higashiura Y, Yamaguchi H, Ishihara M, Ono N, Tsukagoshi H, Yokobori S, et al. Male death resulting from hybridization between subspecies of the gypsy moth, Lymantria dispar . Heredity (Edinb). 2011;106:603–13. Moronuki Y, Kasahara R, Naka H, Suzuki MG. Identification and functional analysis of sex-determining genes in the spongy moth, Lymantria dispar (Lepidoptera: Erebidae). Insect Biochem Mol Biol. 2025;177:104219. Kiuchi T, Koga H, Kawamoto M, Shoji K, Sakai H, Arai Y, et al. A single female-specific piRNA is the primary determiner of sex in the silkworm. Nature. 2014;509:633–636. Sakai H, Sakaguchi H, Aoki F, Suzuki MG. Functional analysis of sex-determination genes by gene silencing with LNA-DNA gapmers in the silkworm, Bombyx mori . Mech Dev. 2015;137:45–52. Sakai H, Sumitani M, Chikami Y, Yahata K, Uchino K, Kiuchi T, et al. Transgenic expression of the piRNA-resistant Masculinizer gene induces female-specific lethality and partial female-to-male sex reversal in the silkworm, Bombyx mori . PLoS Genet. 2016;12:e1006203. Lee J, Kiuchi T, Kawamoto M, Shimada T, Katsuma S. Identification and functional analysis of a Masculinizer orthologue in Trilocha varians (Lepidoptera: Bombycidae). Insect Mol Biol. 2015;24:561–569. Fukui T, Kiuchi T, Shoji K, Kawamoto M, Shimada T, Katsuma S. In vivo masculinizing function of the Ostrinia furnacalis Masculinizer gene. Biophys Res Commun. 2018;503:1768–1772. Harvey-Samuel T, Norman VC, Carter R, Lovett E, Alphey L. Identification and characterization of a Masculinizer homologue in the diamondback moth, Plutella xylostella . Insect Mol Biol. 2020;29:231–240. Visser S, Voleníková A, Nguyen P, Verhulst EC, Marec F. A conserved role of the duplicated Masculinizer gene in sex determination of the Mediterranean flour moth, Ephestia kuehniella . PLoS Genet. 2021;17:e1009420. Pospíšilová K, Van't Hof AE, Yoshido A, Kružíková R, Visser S, Zrzavá M, et al. Masculinizer gene controls male sex determination in the codling moth, Cydia pomonella . Insect Biochem Mol Biol. 2023:103991. Harvey-Samuel T, Xu X., Anderson MAE, Carabajal Paladino LZ, Purusothaman D, et al. Silencing RNAs expressed from W-linked PxyMasc “retrocopies” target that gene during female sex determination in Plutella xylostella . Proc Natl Acad Sci. 2022;119:e2206025119. Yuzawa T, Matsuoka M, Sumitani M, Aoki F, Sezutsu H, Suzuki MG. Transgenic and knockout analyses of Masculinizer and doublesex illuminated the unique functions of doublesex in germ cell sexual development of the silkworm, Bombyx mori . BMC Dev Biol. 2020;20:19. Fukui T, Shoji K, Kiuchi T, Suzuki Y, Katsuma S. Masculinizer is not post-transcriptionally regulated by female-specific piRNAs during sex determination in the Asian corn borer, Ostrinia furnacalis . Insect Biochem Mol Biol. 2023;156:103946. Visser S, Yoshido A, Provazníková I, Dalíková M, Voříšková D, Chung Voleníková A, et al. A W chromosome-derived feminizing piRNA in pyralid moths demonstrates convergent evolution for primary sex determination signals in Lepidoptera. BMC Biol. 2025;23:289. Katsuma S, Sugano Y, Kiuchi T, Shimada T. Two conserved cysteine residues are required for the masculinizing activity of the silkworm Masc protein. J Biol Chem. 2015;290:26114–26124. Katsuma S, Kiuchi T, Kawamoto M, Fujimoto T, Sahara K. Unique sex determination system in the silkworm, Bombyx mori : current status and beyond. Proc Jpn Acad Ser B Phys Biol Sci. 2018;94:205–216. Belote JM, Lucchesi JC. Control of X chromosome transcription by the maleless gene in Drosophila. Nature. 1980;285:573–5. Plenefisch JD, DeLong L, Meyer BJ. Genes that implement the hermaphrodite mode of dosage compensation in Caenorhabditis elegans. Genetics. 1989;121:57–76. Takagi N, Abe K. Detrimental effects of two active X chromosomes on early mouse development. Development. 1990;109:189–201. Sugimoto TN, Kayukawa T, Shinoda T, Ishikawa Y, Tsuchida T. Misdirection of dosage compensation underlies bidirectional sex-specific death in Wolbachia -infected Ostrinia scapulalis . Insect Biochem Mol Biol. 2015;66:72–6. Arai H, Takamatsu T, Lin SR, Mizutani T, Omatsu T, Katayama Y, et al. Diverse molecular mechanisms underlying microbe-inducing male killing in the moth Homona magnanima . Appl Environ Microbiol. 2023;89:e0209522. Ledón-Rettig CC, Zattara EE, Moczek AP. Asymmetric interactions between doublesex and tissue- and sex-specific target genes mediate sexual dimorphism in beetles. Nat Commun. 2017;8:14593. Matsuda M, Shinomiya A, Kinoshita M, Suzuki A, Kobayashi T, Paul-Prasanth B, et al. DMY gene induces male development in genetically female (XX) medaka fish. Proc Natl Acad Sci. 2007;104:3865–3870. Herpin A, Schartl M. Molecular mechanisms of sex determination and evolution of the Y-chromosome: insights from the medakafish ( Oryzias latipes ). Mol Cell Endocrinol. 2009;306:51–58. Kondo M, Nanda I, Schmid M, Schartl M. Sex determination and sex chromosome evolution: insights from medaka. Sex Dev. 2009;3:88–98. Kopp A. Dmrt genes in the development and evolution of sexual dimorphism. Trends Genet. 2012;28:175–184. Yano A, Nicol B, Jouanno E, Quillet E, Fostier A, Guyomard R, Guiguen Y. The sexually dimorphic on the Y-chromosome gene (sdY) is a conserved male-specific Y-chromosome sequence in many salmonids. Evol Appl. 2013;6:486–496. Yoshimoto S, Okada E, Umemoto H, Tamura K, Uno Y, Nishida-Umehara C, et al. A W-linked DM-domain gene, DM-W, participates in primary ovary development in Xenopus laevis . Proc Natl Acad Sci. 2008;105:2469–2474. Yoshimoto S, Okada E, Oishi T, Numagami R, Umemoto H, Tamura K, et al. Expression and promoter analysis of Xenopus DMRT1 and functional characterization of the transactivation property of its protein. Dev Growth Differ. 2006;48:597–603. Loehlin DW, Carroll SB. Expression of tandem gene duplicates is often greater than twofold. Proc Natl Acad Sci. 2016;113:5988–5992. Blaser O, Neuenschwander S, Perrin N. Sex-chromosome turnovers: the hot‐potato model. Am Nat. 2014;183:140–146. Gammerdinger WJ, Conte MA, Sandkam BA, Ziegelbecker A, Koblmüller S, Kocher TD. Novel sex chromosomes in 3 cichlid fishes from Lake Tanganyika. J Hered. 2018;109:489–500. Jeffries DL, Lavanchy G, Sermier R, Sredl MJ, Miura I, Borzée A, et al. A rapid rate of sex-chromosome turnover and non-random transitions in true frogs. Nat Commun. 2018;9:4088. Vicoso B. Molecular and evolutionary dynamics of animal sex-chromosome turnover. Nat Ecol Evol. 2019;3:1632–1641. Jeffries DL, Mee JA, Peichel CL. Identification of a candidate sex determination gene in Culaea inconstans suggests convergent recruitment of an Amh duplicate in two lineages of stickleback. J Evol Biol. 2022;35:1683–1695. Arimoto M, Iwaizumi R. Identification of Japanese Lymantria species (Lepidoptera: Lymantriidae) based on PCR-RFLP analysis of mitochondorial DNA. Appl Entomol Zool. 2014;49:159–169. Yin J, Sun L, Zhang Q, Cao C. Screening and evaluation of the stability of expression of reference genes in Lymantria dispar (Lepidoptera: Erebidae) using qRT-PCR. Gene. 2020;749:144712. Belousova I, Ershov N, Pavlushin S, Ilinsky Y, Martemyanov V. Molecular sexing of Lepidoptera. J Insect Physiol. 2019;114:53–56. R Development Core Teeam. R: a language and environment for statistical computing. R Foundation for Statistical Computing; 2005. The English in this document has been checked by at least two professional editors, both native speakers of English. For a certificate, please see: http://www.textcheck.com/certificate/Sr0imE The above statement is here to inform reviewers—who may not be native speakers of English—that the English in this document has been professionally checked. If the link to the certificate above is deleted and copied into a letter then the reviewers will not see it. We STRONGLY recommend that no changes are made. Textcheck should be the last step before final formatting. Typically, authors’ changes result in errors in the English, not improvements. 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Supplementary Files MGSuzukiSupplementaryInformation.docx MGSuzukiAdditionalfile1.pptx MGSuzukiAdditionalfile2.pptx MGSuzukiAdditionalfile3TableS1.xlsx MGSuzukiAdditionalfile4TableS2.xlsx MGSuzukiAdditionalfile5TableS3.xlsx MGSuzukiAdditionalfile6TableS4.xlsx MGSuzukiAdditionalfile7TableS5.xlsx MGSuzukiAdditionalfile8TableS6.xlsx MGSuzukiAdditionalfile9TableS7.xlsx MGSuzukiAdditionalfile10.pptx MGSuzukiAdditionalfile11.pptx MGSuzukiAdditionalfile12FigureS1.pdf MGSuzukiAdditionalfile13TableS8.xlsx MGSuzukiAdditionalfile14FigureS2.pdf MGSuzukiAdditionalfile15.pptx MGSuzukiAdditionalfile16.pptx MGSuzukiAdditionalfile17.pptx MGSuzukiAdditionalfile18.pptx MGSuzukiAdditionalfile19TableS9.xlsx MGSuzukiAdditionalfile20.pptx MGSuzukiAdditionalfile21.pptx MGSuzukiAdditionalfile22.pptx MGSuzukiAdditionalfile23.pptx MGSuzukiAdditionalfile24.pptx MGSuzukiAdditionalfile25.pptx MGSuzukiAdditionalfile26FigureS3.pdf MGSuzukiAdditionalfile27FigureS4.pdf MGSuzukiAdditionalfile28TableS10.xlsx MGSuzukiAdditionalfile29TableS11.xlsx MGSuzukiAdditionalfile30TableS12.xlsx MGSuzukiAdditionalfile31TableS13.xlsx Cite Share Download PDF Status: Under Review Version 1 posted Reviews received at journal 11 Apr, 2026 Reviews received at journal 08 Apr, 2026 Reviews received at journal 01 Apr, 2026 Reviewers agreed at journal 24 Mar, 2026 Reviewers agreed at journal 23 Mar, 2026 Reviewers agreed at journal 21 Mar, 2026 Reviewers agreed at journal 21 Mar, 2026 Reviewers agreed at journal 20 Mar, 2026 Reviewers invited by journal 20 Mar, 2026 Editor assigned by journal 25 Feb, 2026 Submission checks completed at journal 25 Feb, 2026 First submitted to journal 24 Feb, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8963102","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":611425417,"identity":"9cca09e2-59e1-4a51-8b16-06c7096947d3","order_by":0,"name":"Kisuke Shoji","email":"","orcid":"","institution":"University of Tokyo","correspondingAuthor":false,"prefix":"","firstName":"Kisuke","middleName":"","lastName":"Shoji","suffix":""},{"id":611425418,"identity":"7219f631-1449-4976-bae5-d62b60d0a062","order_by":1,"name":"Kyoko Ishida","email":"","orcid":"","institution":"University of Tokyo","correspondingAuthor":false,"prefix":"","firstName":"Kyoko","middleName":"","lastName":"Ishida","suffix":""},{"id":611425419,"identity":"32577b56-3bf7-461d-81ab-c06ca4a9c25e","order_by":2,"name":"Ryota Kasahara","email":"","orcid":"","institution":"Juntendo University","correspondingAuthor":false,"prefix":"","firstName":"Ryota","middleName":"","lastName":"Kasahara","suffix":""},{"id":611425420,"identity":"ab783920-56a2-47de-a69d-eca5fc2be3d8","order_by":3,"name":"Hideshi Naka","email":"","orcid":"","institution":"Tottori University","correspondingAuthor":false,"prefix":"","firstName":"Hideshi","middleName":"","lastName":"Naka","suffix":""},{"id":611425421,"identity":"7b5f10fc-6f2b-4d17-b3da-6d36791b563d","order_by":4,"name":"Masataka G. Suzuki","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABEUlEQVRIiWNgGAWjYFACNiCuYGBsADOAQAIqzoxfyxm4FgMitTC2YdGCE8i7H0uT+DjPRnbDAbYEZh6GP3KSM5IPMPyoYWA3x6HF8EzaMcmZ29KMgVoOALUYGEtLpCUw9hxjYLZswKGlIb1Nmnfb4cQNB9gbmHn/GSTOk8gxYOBtYGA2OIBDS/9zoJY5/yFagLaAtTD+xaNFXiLtmDRvw4FEmMMSZwO1MOOzxUDiWbLljGPJxjMPsyUcnMNgbCzZ8yzhsMwxCZx+ke9PM7zxocZOtu94m+GDNwxychLHkw8+fFNjk4wrxIC2s0BiAhh1EJcIJIAYEskGOLTIAx39AVWIH6LTDpeWUTAKRsEoGHEAAC8MVNM+2jD1AAAAAElFTkSuQmCC","orcid":"","institution":"University of Tokyo","correspondingAuthor":true,"prefix":"","firstName":"Masataka","middleName":"G.","lastName":"Suzuki","suffix":""}],"badges":[],"createdAt":"2026-02-25 04:39:45","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8963102/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8963102/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":105365257,"identity":"71490700-d480-4ac7-870b-a4af141db213","added_by":"auto","created_at":"2026-03-25 08:28:56","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2175962,"visible":true,"origin":"","legend":"\u003cp\u003eFemale-biased expression and female genome-specific presence of \u003cem\u003eLdMasc\u003c/em\u003e. \u003cstrong\u003eA\u003c/strong\u003e Differences between males and females observed in the distribution of each RNA sequencing (RNA-seq) read across \u003cem\u003eLdMasc\u003c/em\u003e mRNA. The upper panel shows a schematic depiction of the structure of \u003cem\u003eLdMasc \u003c/em\u003emRNA. Boxes represent exons; numbers inside boxes are exon numbers. ATG denotes the start codon; STOP indicates the approximate position of the stop. The lower panel visualizes results obtained by mapping each read to the reference sequence (\u003cem\u003eLdMasc\u003c/em\u003e mRNA) based on information from the bam file obtained from RNA-seq. Rectangles represent individual reads. Blue and red shading indicates regions with deletions or insertions compared to the reference sequence and regions with base substitution mutations, respectively. Coverage plots were generated from the bam file obtained following RNA-seq analysis. \u003cstrong\u003eB\u003c/strong\u003e Schematic diagram of unigenes within the sequence near the 3’ end of \u003cem\u003eLdMasc\u003c/em\u003e mRNA constructed by \u003cem\u003ede novo\u003c/em\u003eRNA-seq. Red and black lines indicate regions homologous and non-homologous to the \u003cem\u003eLdMasc\u003c/em\u003e mRNA sequence, respectively. Arrows indicate the approximate positions of primers used in polymerase chain reaction (PCR) analyses. \u003cstrong\u003eC\u003c/strong\u003eResults of genomic PCR (gPCR) performed using the F1 and R1 primers shown in B. Genomic DNA prepared from male and female fat bodies was used as a template. Primers specifically amplify \u003cem\u003eLdMasc\u003c/em\u003e were used for the positive control PCR. \u003cstrong\u003eD\u003c/strong\u003e Results of reverse-transcription PCR (RT-PCR) using primers shown in B. cDNA prepared from male and female fat bodies was used as a template. PCR products were separated by electrophoresis on 1.5% agarose gel containing ethidium bromide (final concentration, 1 μg/mL).\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-8963102/v1/2c79336e3e8e4ae54f3463fa.png"},{"id":105365296,"identity":"0bf48655-52e5-45f6-a47e-a10f661eb7e8","added_by":"auto","created_at":"2026-03-25 08:29:16","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1082001,"visible":true,"origin":"","legend":"\u003cp\u003eSearch for \u003cem\u003eLdMasc\u003c/em\u003e homologous sequences across an entire genome. \u003cstrong\u003eA\u003c/strong\u003e (Right panel) Approximate positions and directions of \u003cem\u003eLdMasc\u003c/em\u003e homologous regions observed within the genome sequence of the Japanese gypsy moth (\u003cem\u003eLymantria dispar\u003c/em\u003e \u003cem\u003ejaponica\u003c/em\u003e) (see Tables S1 and S2) are shown below the schematic diagram of the \u003cem\u003eLdMasc \u003c/em\u003egene. Approximate positions of \u003cem\u003eLdMasc\u003c/em\u003ehomologous sequences found in the genomic sequence of the W chromosome in the European spongy moth (\u003cem\u003eLymantria dispar\u003c/em\u003e \u003cem\u003edispar\u003c/em\u003e) and Chinese spongy moth (\u003cem\u003eLymantria dispar\u003c/em\u003e \u003cem\u003easiatica\u003c/em\u003e) (see Additional file 2: Table S2) are similarly indicated. (Left panel) Blue lines indicate contigs containing \u003cem\u003eLdMasc\u003c/em\u003ehomologous sequences; green diamonds indicate the approximate positions of \u003cem\u003eLdMasc\u003c/em\u003ehomologous sequences; numbers to the right of each contig indicate copy numbers of \u003cem\u003eLdMasc\u003c/em\u003e homologous sequences. \u003cstrong\u003eB\u003c/strong\u003e Approximate positions and directions of \u003cem\u003eLdMasc \u003c/em\u003ehomologous regions identified in the W chromosome of the European spongy moth (\u003cem\u003eL. d. dispar\u003c/em\u003e) and Chinese spongy moth (\u003cem\u003eL. d. asiatica\u003c/em\u003e) (see Additional file 2: Table S2) are shown below the schematic diagram of the \u003cem\u003eLdMasc \u003c/em\u003egene. \u003cstrong\u003eC\u003c/strong\u003e Estimated copy numbers of \u003cem\u003eLdMasc-W\u003c/em\u003e in \u003cem\u003eL. d. japonica\u003c/em\u003e collected at indicated cities (KIT, Kitami; FK, Fukuoka; NAS, Nasu-Shjobara; TU, Toyoura; YM, Yamanashi; IW, Iwate) in Japan. \u003cem\u003eLdMasc-W\u003c/em\u003e copy numbers were estimated by qPCR (see Methods). Data represent average relative values when the single-copy gene \u003cem\u003ecad\u003c/em\u003e was set to 1.0.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-8963102/v1/dff2ab7a27bbf5ffbead14f6.png"},{"id":105365288,"identity":"4eea8387-d370-4819-ba53-5467c1f6ef85","added_by":"auto","created_at":"2026-03-25 08:29:15","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":933253,"visible":true,"origin":"","legend":"\u003cp\u003eClassification of \u003cem\u003eLdMasc-W\u003c/em\u003eby molecular phylogenetic analysis. \u003cstrong\u003eA\u003c/strong\u003e Molecular phylogenetic tree constructed using approximately 5-kb nucleotide sequences adjacent to the 5’ end of each \u003cem\u003eLdMasc-W\u003c/em\u003e. W1–W52 represent \u003cem\u003eLdMasc-W\u003c/em\u003e genes identified in the \u003cem\u003eL. d. japonica \u003c/em\u003egenome. WD1–3 and WA1–6 indicate \u003cem\u003eLdMasc-W\u003c/em\u003e genes from the W chromosomes of \u003cem\u003eL. d. dispar\u003c/em\u003e and \u003cem\u003eL. d. asiatica\u003c/em\u003e, respectively. A 5-kb genomic sequence extending from the middle of intron 8 of \u003cem\u003eLdMasc\u003c/em\u003e toward the 5’ end was used as an outgroup. Numbers on branches indicate bootstrap values. \u003cstrong\u003eB\u003c/strong\u003e(Upper panel) Schematic diagram of primer positions specifically amplifying each \u003cem\u003eLdMasc-W\u003c/em\u003e. (Lower panel) List of forward primers used in the PCR illustrated in C, and corresponding amplified \u003cem\u003eLdMasc-W\u003c/em\u003e. \u003cstrong\u003eC\u003c/strong\u003e gPCR results obtained using \u003cem\u003eLdMasc-W\u003c/em\u003e-specific primers shown in B. Genomic DNA extracted from whole-body first-instar larvae of both sexes in \u003cem\u003eL. d. japonica\u003c/em\u003e (upper panel) and \u003cem\u003eL. umbrosa\u003c/em\u003e(lower panel) was used as a template. Forward primers used for amplification are indicated at the top of the gel image. PCR products were separated on 1.5% agarose gel containing 1 μg/mL ethidium bromide.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-8963102/v1/71fa43f293aaa0371ffa64b1.png"},{"id":105365316,"identity":"b5f8080c-8fd2-43ed-86d1-f6aa042bfa91","added_by":"auto","created_at":"2026-03-25 08:29:22","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1480528,"visible":true,"origin":"","legend":"\u003cp\u003eIdentification and expression analysis of \u003cem\u003eLdMasc-W\u003c/em\u003e-derived transcripts. \u003cstrong\u003eA\u003c/strong\u003e Exon–intron structure of \u003cem\u003eLdMasc-W\u003c/em\u003e encoding the four female-specific unigenes described in Fig. 1A. (Upper panel) Schematic diagram of \u003cem\u003eLdMasc\u003c/em\u003e mRNA, with the four unigenes shown below. Numbers within boxes indicate exon numbers. Blue and green lines indicate sequences homologous to \u003cem\u003eLdMasc\u003c/em\u003e mRNA and \u003cem\u003eLdMasc-W\u003c/em\u003e, respectively. Black lines indicate sequences showing no homology to either \u003cem\u003eLdMasc\u003c/em\u003e or \u003cem\u003eLdMasc-W\u003c/em\u003e. (Lower panel) Representative example of \u003cem\u003eLdMasc-W \u003c/em\u003eencoding the four unigenes. Boxes indicate exons; straight lines indicate introns. Regions homologous to \u003cem\u003eLdMasc\u003c/em\u003e are shaded in blue; regions common between \u003cem\u003eLdMasc \u003c/em\u003eand \u003cem\u003eLdMasc-W\u003c/em\u003e are shaded in green; regions showing no homology to either \u003cem\u003eLdMasc \u003c/em\u003eor \u003cem\u003eLdMasc-W\u003c/em\u003e are shaded in black. \u003cstrong\u003eB\u003c/strong\u003eAcquisition of \u003cem\u003eLdMasc-W\u003c/em\u003e mRNA sequences through 5’ rapid amplification of cDNA ends (RACE). (Left panel) Schematic diagram of the predicted \u003cem\u003eLdMasc-W\u003c/em\u003emRNA. Arrows indicate approximate positions of the gene-specific reverse primers (GSP) used for 5’ RACE. GSP1 was used for the first PCR, and GSP2 for the nested PCR. (Right panel) Electrophoresis gel image of 5’ RACE products obtained using total RNA from ovaries. PCR products were separated on 1.5% agarose gel containing ethidium bromide at a final concentration of 1 μg/mL. \u003cstrong\u003eC\u003c/strong\u003e Molecular phylogenetic classification of transcripts identified by 5’ RACE. A phylogenetic tree was constructed using nucleotide sequences of the nine transcripts identified by 5’ RACE. \u003cem\u003eLdMasc-W\u003c/em\u003e encoding each transcript and the group to which they belong are indicated on the right side of the phylogenetic tree. Transcripts confirmed to be female-specific by RT-PCR illustrated in D are shown in red. \u003cstrong\u003eD\u003c/strong\u003eElectrophoresis gel images of RT-PCR products obtained using cDNA derived from male and female eggs at the sex determination stage (2–4 days post-laying). Primers specific the nine transcripts shown in C were used for PCR amplification. As a positive control, the same PCR was performed using primers capable of simultaneously amplifying four unigenes confirmed to be specifically expressed in females. PCR products were separated on 1.5% agarose gel containing 1 μg/mL ethidium bromide.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-8963102/v1/5c421601e265c6b77b14b6a7.png"},{"id":105365283,"identity":"edc35c03-02ae-4a99-9050-13f9ec156f40","added_by":"auto","created_at":"2026-03-25 08:29:14","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1485935,"visible":true,"origin":"","legend":"\u003cp\u003eFunctional analysis of \u003cem\u003eLdMasc-W\u003c/em\u003e by embryonic RNA interference (RNAi). \u003cstrong\u003eA\u003c/strong\u003e Schematic diagram of the sex-specific splicing pattern of \u003cem\u003eLddsx\u003c/em\u003e. Arrows indicate the approximate positions of primers used in the RT-PCR shown in D. \u003cstrong\u003eB\u003c/strong\u003e Schematic diagram of the predicted \u003cem\u003eLdMasc-W\u003c/em\u003e mRNA. Vertical bars indicate approximate positions of the siRNAs (LdMasc-W si1 and si2) targeting a region common to all \u003cem\u003eLdMasc-W\u003c/em\u003e genes used for embryonic RNAi. \u003cstrong\u003eC\u003c/strong\u003e\u003cem\u003e LdMasc-W\u003c/em\u003e mRNA levels in female eggs injected with the indicated siRNA were quantified by quantitative RT-PCR (qRT-PCR). Numbers of examined individuals: Egfp si ♀ = 4, LdMasc-W si1 ♀ = 8, LdMasc-W si2 ♀ = 8. Error bars indicate standard deviation. \u003cstrong\u003eD\u003c/strong\u003e Expression pattern of \u003cem\u003eLddsx\u003c/em\u003edetected by RT-PCR in the negative control groups (Egfp si) and female eggs with confirmed knockdown of \u003cem\u003eLdMasc-W\u003c/em\u003eexpression via injection of LdMasc-W si1 and LdMasc-W si2. Amplified products were separated by 1.5% agarose gel electrophoresis. Arrows to the right of the gel indicate the positions of \u003cem\u003eLddsxF \u003c/em\u003eand \u003cem\u003eLddsxM\u003c/em\u003e.\u003cstrong\u003e E\u003c/strong\u003e Effects of \u003cem\u003eLdMasc-W\u003c/em\u003eknockdown via LdMasc-W si1 and LdMasc-W si2 injection on sexual dimorphic expression patterns of \u003cem\u003eLddsx\u003c/em\u003e in female eggs. \u003cem\u003eLddsx\u003c/em\u003e expression changes were classified into five categories; the proportion of individuals belonging to each category is shown. \u003cstrong\u003eF\u003c/strong\u003e, \u003cstrong\u003eG\u003c/strong\u003e Expression levels of \u003cem\u003eLdMasc\u003c/em\u003e (F) and \u003cem\u003eFet-W\u003c/em\u003e (G) in the negative control group (Egfp si) and female eggs with confirmed knockdown of \u003cem\u003eLdMasc-W\u003c/em\u003eexpression were quantified by RT-qPCR. Numbers of individuals tested: Egfp si ♀ = 7, Egfp si ♂ = 3, LdMasc-W KD ♀ = 8). Error bars indicate standard deviation. ***\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01; n.s.: not significant (Mann–Whitney U test).\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-8963102/v1/5051b396603bde69e565e8fb.png"},{"id":105365259,"identity":"0229763a-c2d5-42be-8b21-018319a8db75","added_by":"auto","created_at":"2026-03-25 08:28:57","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2042563,"visible":true,"origin":"","legend":"\u003cp\u003eFunctional analysis of \u003cem\u003eLdMasc-W\u003c/em\u003e using \u003cem\u003eL. umbrosa\u003c/em\u003e and\u003cem\u003e L. d. japonica\u003c/em\u003eF1 hybrid individuals. \u003cstrong\u003eA\u003c/strong\u003e Molecular sexing by genomic PCR using the Z-chromosome (\u003cem\u003eLdMasc\u003c/em\u003e) and W-chromosome-specific primers and RT-PCR analysis of expression pattern of \u003cem\u003eLddsx\u003c/em\u003e in embryos immediately before hatching. Upper panel: Results using F1 hybrid individuals obtained from crossing between \u003cem\u003eL. d. japonica\u003c/em\u003efemales and \u003cem\u003eL. umbrosa\u003c/em\u003e males (JU-hybrid F1). Lower panel: Results using F1 hybrids produced by crossing between \u003cem\u003eL. umbrosa\u003c/em\u003e females with \u003cem\u003eL. d. japonica\u003c/em\u003e males (UJ-hybrid F1). \u003cstrong\u003eB\u003c/strong\u003e mRNA levels of \u003cem\u003eFet-W\u003c/em\u003e, \u003cem\u003eLdMasc-W\u003c/em\u003e, and \u003cem\u003eLdMasc\u003c/em\u003e were quantified by RT-qPCR in embryos immediately prior to hatching of the indicated lines. Error bars indicate standard deviation and the center line within the box indicates the median. *\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05; **\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01; ***\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001; n.s.: not significant (Mann–Whitney U test). \u003cstrong\u003eC\u003c/strong\u003e Larval death rates in the indicated lines. Significant differences between the UJ-hybrid F1 and other lines were examined using the Kruskal-Wallis test and Dunn's post hoc test (Bonferroni correction) (* \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, ** \u003cem\u003eP\u003c/em\u003e \u0026lt;0.01). \u003cstrong\u003eD\u003c/strong\u003eKaplan-Meier survival curves of UJ-hybrid F1 individuals and other lines. JU-hybrid F1, \u003cem\u003eL. d. japonica\u003c/em\u003e, and \u003cem\u003eL. umbrosa\u003c/em\u003e are shown in black as “other lines.” \u003cstrong\u003eE\u003c/strong\u003e Sex ratio of larvae that died within 20 days after hatching. The vertical axis shows the ratio of female to male dead individuals when the total number of dead individuals is set to 1.0.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-8963102/v1/57429abb4d4414baab673872.png"},{"id":105365314,"identity":"a4f44530-4d58-4d15-844d-0fdff5eb7d50","added_by":"auto","created_at":"2026-03-25 08:29:21","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":4281610,"visible":true,"origin":"","legend":"\u003cp\u003eMorphological analysis of the UJ-hybrid F1 adult females. \u003cstrong\u003eA\u003c/strong\u003e Overall dorsal view of the \u003cem\u003eL. d. japonica\u003c/em\u003e male, the \u003cem\u003eL. umbrosa\u003c/em\u003efemale, and the UJ-hybrid F1 female. \u003cstrong\u003eB \u003c/strong\u003eThe antennae, wings, and legs of the UJ-hybrid F1 female and its parents, the \u003cem\u003eL. d. japonica\u003c/em\u003e male, the \u003cem\u003eL. umbrosa\u003c/em\u003e female, and the JU-hybrid F1 female. \u003cstrong\u003eC\u003c/strong\u003e Cuticle specimens of the external genitalia in females of the indicated lines. \u003cstrong\u003eD\u003c/strong\u003e Overall view of the internal genitalia in the indicated lines. Abnormal tissues observed at the base of the ovarian tubules in the UJ-hybrid F1 females are squared by the dotted line. \u003cstrong\u003eE\u003c/strong\u003eThe abnormal tissues observed in the UJ-hybrid F1 females. For comparison, the corresponding area in the maternal parent \u003cem\u003eL. umbrosa\u003c/em\u003e and the internal genitalia of the paternal parent \u003cem\u003eL. d. japonica\u003c/em\u003e are shown. ag, accessory gland; cl, clasper; dp, dorsal chitin plate; e, egg; ed, ejaculatory duct; g, genital papilla; o, oviparous; ot, ovarian tuble; ov, oviduct; p, penis; u, uncus; sp, spermatheca; sr, seminal receptacle; sv, seminal vesicle; tes, testis; vd, vas deferens. All individuals were collected within one day after emergence and dissected immediately after collection.\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-8963102/v1/ed8ba06e01552d6354b362eb.png"},{"id":105570095,"identity":"36d6f9d1-cfb4-48df-9c64-68729bcdda9b","added_by":"auto","created_at":"2026-03-27 13:14:41","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":12518466,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8963102/v1/de5d26c7-314c-490c-b97d-a67faa2857da.pdf"},{"id":105565058,"identity":"cfb6898d-6a43-4c36-9f98-fa25e81f1ac2","added_by":"auto","created_at":"2026-03-27 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08:29:17","extension":"xlsx","order_by":30,"title":"","display":"","copyAsset":false,"role":"supplement","size":26921,"visible":true,"origin":"","legend":"","description":"","filename":"MGSuzukiAdditionalfile30TableS12.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8963102/v1/d9a7cf08f8e3f53c2145201d.xlsx"},{"id":105365301,"identity":"120078cd-1082-4de1-8429-99f9e857abda","added_by":"auto","created_at":"2026-03-25 08:29:17","extension":"xlsx","order_by":31,"title":"","display":"","copyAsset":false,"role":"supplement","size":9834,"visible":true,"origin":"","legend":"","description":"","filename":"MGSuzukiAdditionalfile31TableS13.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8963102/v1/c47c48ffcc0af7ee63d2dac0.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eW chromosome-specific paralogs of the male-determining gene \u003cem\u003eLdMasc \u003c/em\u003eexhibits a female- determining ability in the spongy moth, \u003cem\u003eLymantria dispar\u003c/em\u003e\u003c/p\u003e","fulltext":[{"header":"Background","content":"\u003cp\u003eAlthough sex determination is common among organisms that exhibit sexual reproduction between male and female individuals, the patterns of sex determination are remarkably diverse, and factors inducing sex determination vary significantly between species. Particularly in insects, differences in sex determination patterns have been observed even among closely related species [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], making them excellent models for elucidating the factors and mechanisms driving the diversification of sex determination.\u003c/p\u003e \u003cp\u003eThe spongy moth (\u003cem\u003eLymantria dispar\u003c/em\u003e; Erebidae:Lepidoptera) inhabits vast regions of the Northern Hemisphere, including Japan. Spongy moth larvae are polyphagous, feeding on over 500 deciduous tree species [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The species epithet \u003cem\u003edisper\u003c/em\u003e is derived from the Latin word for separate, reflecting the moth\u0026rsquo;s striking sexual dimorphism [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. This feature has supported a long history of research on the modes of sex determination modes in the spongy moth, which was found to possess a female-heterogametic sex chromosome system (males\u0026thinsp;=\u0026thinsp;ZZ, females\u0026thinsp;=\u0026thinsp;ZW) as early as 1934 [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Early studies predicted that the male-determining \u0026ldquo;M factor\u0026rdquo; was located on the Z chromosome and that the female-determining \u0026ldquo;F factor\u0026rdquo; resided on the W chromosome [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHowever, classical crossing experiments provided several lines of evidence that sex in the spongy moth is not determined solely by the presence or absence of the F factor. For example, hybrids obtained from crosses between populations in different habitats exhibit sex reversal and sex-specific lethality, resulting in a pronounced sex ratio bias [\u003cspan additionalcitationids=\"CR5 CR6\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Based on these findings, the M and F factors were predicted to exhibit allelic polymorphism with differential masculinizing and feminizing activity, and that sex determination occurs through their combination [\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Under this prediction, sex-determining genes in the spongy moth would exhibit geographic divergence or subspecies differences in their sex-determining ability. Thus, the spongy moth may be a useful model species for elucidating molecular mechanisms underlying the diversification of sex-determining genes. However, the genes responsible for the F and M factors have remained unknown for nearly a century.\u003c/p\u003e \u003cp\u003eIn our previous study, we identified the gene responsible for the spongy moth F factor as \u003cem\u003eFemale expressed transcripts of W chromosome\u003c/em\u003e (\u003cem\u003eFet-W\u003c/em\u003e), and that responsible for the M factor as \u003cem\u003eLdMasc\u003c/em\u003e, an ortholog of the \u003cem\u003eMasculinizer\u003c/em\u003e (\u003cem\u003eMasc\u003c/em\u003e) gene [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. \u003cem\u003eMasc\u003c/em\u003e functions as a male determination gene linked to the Z chromosome, inducing male-specific splicing of \u003cem\u003edoublesex\u003c/em\u003e (\u003cem\u003edsx\u003c/em\u003e), which acts as a master regulatory gene for insect sex differentiation [\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Since its identification in silkworms, \u003cem\u003eMasc\u003c/em\u003e has been identified from at least 10 other Lepidoptera species, including \u003cem\u003eTrilocha varians\u003c/em\u003e [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], \u003cem\u003eOstrinia furnacalis\u003c/em\u003e [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], the diamondback moth [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], the Mediterranean flour moth [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], and the codling moth [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], demonstrating its widespread conservation as a male-determining gene in Lepidoptera. Despite the robust conservation of \u003cem\u003eMasc\u003c/em\u003e, its upstream regulatory factor, the female-determining gene, varies among species. In addition to \u003cem\u003eFet-W\u003c/em\u003e in the spongy moth, female-determining genes include \u003cem\u003eFeminizer\u003c/em\u003e (\u003cem\u003eFem\u003c/em\u003e) in the silkworm, \u003cem\u003eFem\u003c/em\u003e in \u003cem\u003ePolluter xylostella\u003c/em\u003e (\u003cem\u003ePxyFem\u003c/em\u003e), and \u003cem\u003eMoth-overruler-of-masculinization\u003c/em\u003e (\u003cem\u003eMom\u003c/em\u003e) in the pyralid moths \u003cem\u003eEphestia kuehniella\u003c/em\u003e and \u003cem\u003ePlodia interpunctella\u003c/em\u003e [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Although no homology has been found among these genes, \u003cem\u003eFem\u003c/em\u003e, \u003cem\u003ePxyFem\u003c/em\u003e, and \u003cem\u003eMom\u003c/em\u003e all form multicopy or tandem repeats on the W chromosome and are functionally homologous in that they provide PIWI-interacting RNA (piRNA), which leads to the degradation of \u003cem\u003eMasc\u003c/em\u003e mRNA via the piRNA pathway [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. In the absence of Masc protein, pre-mRNA of \u003cem\u003eBombyx mori doublesex\u003c/em\u003e (\u003cem\u003eBmdsx\u003c/em\u003e), which is a master regulatory gene for sexual differentiation, undergoes default splicing to produce the female isoform of \u003cem\u003eBmdsx\u003c/em\u003e (\u003cem\u003eBmdsxF\u003c/em\u003e), thereby inducing female differentiation. [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Spongy moth \u003cem\u003eFet-W\u003c/em\u003e exhibits structural features similar to those of \u003cem\u003eFem\u003c/em\u003e and \u003cem\u003ePxyFem\u003c/em\u003e, forming a cluster consisting of about 100 copies on the W chromosome [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] and encoding a sequence of approximately 70 bases complementary to the mRNA sequence of the \u003cem\u003eLymantria dispar Masc\u003c/em\u003e gene (\u003cem\u003eLdMasc\u003c/em\u003e), which suggests that it functions as a piRNA source targeting \u003cem\u003eLdMasc\u003c/em\u003e mRNA [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Although it remains unclear whether \u003cem\u003eFet-W\u003c/em\u003e induces the degradation of \u003cem\u003eLdMasc\u003c/em\u003e mRNA via piRNA, it has been found to suppress \u003cem\u003eLdMasc\u003c/em\u003e expression to induce feminization, as \u003cem\u003eFet-W\u003c/em\u003e knockdown increased \u003cem\u003eLdMasc\u003c/em\u003e expression in females [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. These findings support the notion that the sex determination mechanism in the spongy moth is largely analogous with that of the silkworm.\u003c/p\u003e \u003cp\u003eHowever, several molecular discoveries have suggested that sex determination mechanisms may differ between the spongy moth and silkworm. The most notable example is the \u003cem\u003eO\u003c/em\u003e. \u003cem\u003efurnacalis Masc\u003c/em\u003e gene (\u003cem\u003eOsMasc\u003c/em\u003e). Although \u003cem\u003eOsMasc\u003c/em\u003e participates in male differentiation, it does not exhibit male-specific expression, in contrast to previously identified \u003cem\u003eMasc\u003c/em\u003e genes [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Moreover, \u003cem\u003eOsMasc\u003c/em\u003e is not subject to post-transcriptional repression by piRNAs in females [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. The pyralid moth \u003cem\u003eE\u003c/em\u003e. \u003cem\u003ekuehniella\u003c/em\u003e possesses two copies of \u003cem\u003eMasc\u003c/em\u003e (\u003cem\u003eEkMasc\u003c/em\u003e and \u003cem\u003eEkMasc-B\u003c/em\u003e) on the Z chromosome, which are arranged in an inverted repeat configuration [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Additionally, a homologous sequence of \u003cem\u003eEkMasc\u003c/em\u003e on the W chromosome (\u003cem\u003eEkMasc\u003c/em\u003e\u003csup\u003e\u003cem\u003eW\u003c/em\u003e\u003c/sup\u003e) has been identified [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. \u003cem\u003eEkMasc\u003c/em\u003e\u003csup\u003e\u003cem\u003eW\u003c/em\u003e\u003c/sup\u003e shows 94\u0026ndash;97% homology with the region corresponding to exons 2\u0026ndash;10 of the \u003cem\u003eEkMascB\u003c/em\u003e cDNA sequence and is present in 17\u0026ndash;23 copies on the W chromosome. \u003cem\u003eEkMasc\u003c/em\u003e\u003csup\u003e\u003cem\u003eW\u003c/em\u003e\u003c/sup\u003e is hypothesized to have arisen as a duplicate of LdMasc-B. While its function remains to be elucidated, \u003cem\u003eEkMasc\u003c/em\u003e\u003csup\u003e\u003cem\u003eW\u003c/em\u003e\u003c/sup\u003e produces piRNAs that are complementary to \u003cem\u003eEkMasc\u003c/em\u003e and \u003cem\u003eEkMascB\u003c/em\u003e [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. As described above, female determination in the pyralid moth is controlled by piRNAs produced from another gene, \u003cem\u003eE\u003c/em\u003e. \u003cem\u003ekuehniella Mom\u003c/em\u003e (\u003cem\u003eEkMom\u003c/em\u003e), located on the W chromosome. Visser et al. [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] hypothesized that \u003cem\u003eEkMom\u003c/em\u003e originates from a partial sequence of \u003cem\u003eMasc\u003c/em\u003e transposed onto the W chromosome, similar to \u003cem\u003eEkMasc\u003c/em\u003e\u003csup\u003e\u003cem\u003eW\u003c/em\u003e\u003c/sup\u003e. Thus, \u003cem\u003eEkMasc\u003c/em\u003e\u003csup\u003e\u003cem\u003eW\u003c/em\u003e\u003c/sup\u003e exhibits characteristics of a gene in the process of becoming a sex determination gene.\u003c/p\u003e \u003cp\u003eThe spongy moth shares similarities with \u003cem\u003eE\u003c/em\u003e. \u003cem\u003ekuehniella\u003c/em\u003e in that it possesses two copies of \u003cem\u003eMasc\u003c/em\u003e (\u003cem\u003eLdMasc-A\u003c/em\u003e and \u003cem\u003eLdMasc-B\u003c/em\u003e) on the Z chromosome [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. In the present study, we report that, similar to the pyralid moth carrying \u003cem\u003eEkMasc\u003c/em\u003e\u003csup\u003e\u003cem\u003eW\u003c/em\u003e\u003c/sup\u003e, the spongy moth carries multiple copies of nucleotide sequences linked to the W chromosome that may be designated as \u003cem\u003eLdMasc-W\u003c/em\u003e. We examined the structural features of \u003cem\u003eLdMasc-W\u003c/em\u003e and report its functional characteristics based on expression analyses, embryonic RNA interference (RNAi)-based knockdown experiments, and hybridization experiments between closely related species of \u003cem\u003eLymantria\u003c/em\u003e species. Our results showed that \u003cem\u003eLdMasc-W\u003c/em\u003e consists of a nucleotide sequence showing extremely high homology with the genomic sequence covering introns 8\u0026ndash;11 of \u003cem\u003eLdMasc\u003c/em\u003e, which is structurally different from \u003cem\u003eEkMasc\u003c/em\u003e\u003csup\u003e\u003cem\u003eW\u003c/em\u003e\u003c/sup\u003e. Thus, \u003cem\u003eLdMascW\u003c/em\u003e may be regarded as a W-chromosome paralog of \u003cem\u003eLdMasc\u003c/em\u003e. \u003cem\u003eLdMasc-W\u003c/em\u003e is present in multiple copies on the W chromosomes of three spongy moth subspecies (\u003cem\u003eLymantria dispar dispar\u003c/em\u003e, \u003cem\u003eLymantria dispar asiatica\u003c/em\u003e, and \u003cem\u003eLymantria dispar japonica\u003c/em\u003e), but with copy numbers differing among subspecies. Of the subspecies examined, \u003cem\u003eL\u003c/em\u003e. \u003cem\u003ed\u003c/em\u003e. \u003cem\u003ejaponica\u003c/em\u003e had the highest number (\u0026ge;\u0026thinsp;52) of \u003cem\u003eLdMasc-W\u003c/em\u003e copies. Nevertheless, it was found that \u003cem\u003eL. umbrosa\u003c/em\u003e, a close relative of \u003cem\u003eL. d. japonica\u003c/em\u003e, lacks \u003cem\u003eLdMasc-W\u003c/em\u003e. This study provides the first functional evidence that a \u003cem\u003eMasc\u003c/em\u003e paralog linked to the W chromosome has the potential to act as a female-determining gene.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eFemale-biased expression and female genome-specific presence of LdMasc\u003c/h2\u003e \u003cp\u003eReads obtained from \u003cem\u003ede novo\u003c/em\u003e RNA sequencing (RNA-seq) performed in our previous study [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] were mapped to the \u003cem\u003eLdMasc\u003c/em\u003e coding sequence (CDS). A greater number of such reads aligned to the region encompassing exon 9 to the 3\u0026rsquo; end in females than in males. Consistent with this finding, four unigenes derived in our previous study [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] were composed of sequences from exon 9 to the 3\u0026rsquo; end of the \u003cem\u003eLdMasc\u003c/em\u003e CDS (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). The 5\u0026prime;-end sequences of these unigenes contained unique nucleotide sequences not present in the \u003cem\u003eLdMasc\u003c/em\u003e gene. Genomic PCR using primers annealing to these unique regions amplified a female-specific DNA fragment (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). RT-PCR using the same primers yielded female-specific amplification products (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). These results strongly indicate that the nucleotide sequence spanning from exon 9 to the 3\u0026rsquo; end of \u003cem\u003eLdMasc\u003c/em\u003e is present on the W chromosome.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eWhole-genome identification of homologous sequences of LdMasc\u003c/h3\u003e\n\u003cp\u003eThe above results suggested that nucleotide sequences homologous to \u003cem\u003eLdMasc\u003c/em\u003e were present on the W chromosome. Therefore, we performed a nucleotide BLAST (BLASTn) search using the genomic sequence of the \u003cem\u003eLdMasc\u003c/em\u003e gene (a 19,341-bp genomic sequence including exons 1\u0026ndash;11) as a query sequence against the entire \u003cem\u003eL\u003c/em\u003e. \u003cem\u003ed\u003c/em\u003e. \u003cem\u003ejaponica\u003c/em\u003e genome. In addition to the two known \u003cem\u003eLdMasc\u003c/em\u003e genes on the Z chromosome, we identified 52 \u003cem\u003eLdMasc\u003c/em\u003e homologous sequences (Additional file 1: Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e), all of which were found to be nucleotide sequences on contigs predicted to be derived from the W chromosome (Additional file 1: Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Most of these sequences were approximately 5.4 kb in length, extending from a position about 13.9 kb within the \u003cem\u003eLdMasc\u003c/em\u003e gene to its 3\u0026rsquo; end (midway through intron 8 to the 3\u0026rsquo; end of exon 11) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). These sequences were predicted to be nearly identical to the corresponding region of the \u003cem\u003eLdMasc\u003c/em\u003e gene (E\u0026thinsp;=\u0026thinsp;0; \u0026ge; 99% identity). Because there were two to four homologous sequences per contig (Additional file 1: Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA), these homologous sequences were also expected to constitute repetitive sequences on the W chromosome. Since all homologous sequences showed sufficient similarity to be considered derived from the \u003cem\u003eLdMasc\u003c/em\u003e gene, we designated these homologous sequences as paralogs of \u003cem\u003eLdMasc\u003c/em\u003e, named \u003cem\u003eLdMasc-W\u003c/em\u003e for convenience.\u003c/p\u003e \u003cp\u003eTo validate these results, we performed a BLASTn search using the entire genomic sequences of \u003cem\u003eL\u003c/em\u003e. \u003cem\u003ed\u003c/em\u003e. \u003cem\u003edispar\u003c/em\u003e and \u003cem\u003eL\u003c/em\u003e. \u003cem\u003ed\u003c/em\u003e. \u003cem\u003easiatica\u003c/em\u003e, which are publicly available at the National Center for Biotechnology Information (NCBI). The results revealed that three nucleotide sequences showing nearly 100% homology with the approximately 13.9\u0026ndash;17.7-kb region of the \u003cem\u003eLdMasc\u003c/em\u003e gene were present within the contig corresponding to the W chromosome of \u003cem\u003eL\u003c/em\u003e. \u003cem\u003ed\u003c/em\u003e. \u003cem\u003edispar\u003c/em\u003e (accession no. OY755156.1) (Additional file 2: Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). In \u003cem\u003eL\u003c/em\u003e. \u003cem\u003ed\u003c/em\u003e. \u003cem\u003easiatica\u003c/em\u003e, a subspecies closer to \u003cem\u003eL\u003c/em\u003e. \u003cem\u003ed\u003c/em\u003e. \u003cem\u003ejaponica\u003c/em\u003e, five nucleotide sequences showing nearly 100% homology to the region from approximately 13.9 kb to near the 3\u0026rsquo; end (~\u0026thinsp;5.4 kb) of the \u003cem\u003eLdMasc\u003c/em\u003e gene were found within the contig corresponding to the W chromosome (accession no. CM063456.1). We also identified one sequence showing nearly 100% homology across more than half the length of the \u003cem\u003eLdMasc\u003c/em\u003e gene (9167\u0026ndash;19346 bp) (Additional file 2: Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). On the other hand, the same BLASTn search against the whole-genome sequence of \u003cem\u003eL. umbrosa\u003c/em\u003e, which is a close relative of \u003cem\u003eL. d. japonica\u003c/em\u003e, identified a contiguous nucleotide sequence matching the entire \u003cem\u003eLdMasc\u003c/em\u003e sequence, but failed to find a sequence corresponding to \u003cem\u003eLdMasc-W\u003c/em\u003e (Additional file 5: Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e). qPCR analysis using genomic DNA as a template again confirmed that \u003cem\u003eLdMasc-W\u003c/em\u003e exists in approximately 6 to 65 copies in \u003cem\u003eL. d. japonica\u003c/em\u003e females collected throughout Japan but is absent in \u003cem\u003eL. umbrosa\u003c/em\u003e females (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003eFor comparison, a similar analysis was performed using the predicted piRNA coding sequence of the spongy moth female-determining gene \u003cem\u003eFet-W\u003c/em\u003e, which was identified in our previous study [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], as a query sequence. The results revealed that 91 copies were present within a contig (accession no.: BAAIJM010000031) predicted to correspond to the W chromosome of \u003cem\u003eL\u003c/em\u003e. \u003cem\u003ed\u003c/em\u003e. \u003cem\u003ejaponica\u003c/em\u003e (see Additional file 6: Table \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e). Sequences homologous to the putative \u003cem\u003eFet-W\u003c/em\u003e piRNA CDS were also found in the W chromosomes of \u003cem\u003eL\u003c/em\u003e. \u003cem\u003ed\u003c/em\u003e. \u003cem\u003edispar\u003c/em\u003e and \u003cem\u003eL\u003c/em\u003e. \u003cem\u003ed\u003c/em\u003e. \u003cem\u003easiatica\u003c/em\u003e, with 160 and 117 copies, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, Additional file 8: Tables S6 and Additional file 9: Table \u003cspan refid=\"MOESM7\" class=\"InternalRef\"\u003eS7\u003c/span\u003e). Similar analysis identified a total of 124 copies of \u003cem\u003eFet-W\u003c/em\u003e homologous sequences (identity\u0026thinsp;=\u0026thinsp;100%) across two contigs of \u003cem\u003eL. umbrosa\u003c/em\u003e (accession no.: BAAIJN010000033 and BAAIJN010000013) (see Additional file 9: Table \u003cspan refid=\"MOESM7\" class=\"InternalRef\"\u003eS7\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThese results strongly indicate that \u003cem\u003eLdMasc-W\u003c/em\u003e is conserved between \u003cem\u003eL\u003c/em\u003e. \u003cem\u003edispar\u003c/em\u003e subspecies and that multiple copies are present on the W chromosome in all subspecies. On the other hand, our data suggests that \u003cem\u003eFet-W\u003c/em\u003e was highly conserved between the subspecies \u003cem\u003eL. dispar\u003c/em\u003e and its closely related species \u003cem\u003eL. umbrosa\u003c/em\u003e. Notably, interspecific copy number variation was greater for \u003cem\u003eLdMasc-W\u003c/em\u003e than for \u003cem\u003eFet-W\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eClassification of LdMasc-W by molecular phylogenetic analysis\u003c/h3\u003e\n\u003cp\u003eOur analyses revealed that \u003cem\u003eL\u003c/em\u003e. \u003cem\u003ed\u003c/em\u003e. \u003cem\u003ejaponica\u003c/em\u003e, \u003cem\u003eL\u003c/em\u003e. \u003cem\u003ed\u003c/em\u003e. \u003cem\u003edispar\u003c/em\u003e, and \u003cem\u003eL\u003c/em\u003e. \u003cem\u003ed\u003c/em\u003e. \u003cem\u003easiatica\u003c/em\u003e possess 52, 3, and 6 copies of \u003cem\u003eLdMasc-W\u003c/em\u003e, which we designated as \u003cem\u003eLdMasc-W1\u003c/em\u003e\u0026ndash;\u003cem\u003e52\u003c/em\u003e, \u003cem\u003eLdMasc-WD1\u003c/em\u003e\u0026ndash;\u003cem\u003e3\u003c/em\u003e, and \u003cem\u003eLdMasc-WA1\u003c/em\u003e\u0026ndash;\u003cem\u003e6\u003c/em\u003e, respectively. To investigate their relationships, we obtained a 5-kb genomic sequence adjacent to the 5\u0026rsquo; end of each \u003cem\u003eLdMasc-W\u003c/em\u003e and constructed a molecular phylogenetic tree based on their alignment. The 13 \u003cem\u003eLdMasc-W\u003c/em\u003e genes from \u003cem\u003eL\u003c/em\u003e. \u003cem\u003ed\u003c/em\u003e. \u003cem\u003ejaponica\u003c/em\u003e were excluded from this analysis due to their location at the ends of contigs, which prevented the acquisition of upstream sequences exceeding 5 kb in length. Of the 39 \u003cem\u003eLdMasc-W\u003c/em\u003e sequences subjected to phylogenetic analysis, 35 were grouped into four distinct clusters (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Cluster B consisted exclusively of \u003cem\u003eLdMasc-W\u003c/em\u003e from \u003cem\u003eL\u003c/em\u003e. \u003cem\u003ed\u003c/em\u003e. \u003cem\u003ejaponica\u003c/em\u003e. The three \u003cem\u003eLdMasc-W\u003c/em\u003e sequences from \u003cem\u003eL\u003c/em\u003e. \u003cem\u003ed\u003c/em\u003e. \u003cem\u003edispar\u003c/em\u003e (\u003cem\u003eWD1\u003c/em\u003e\u0026ndash;\u003cem\u003e3\u003c/em\u003e in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA) were independent from those in \u003cem\u003eL\u003c/em\u003e. \u003cem\u003ed\u003c/em\u003e. \u003cem\u003ejaponica\u003c/em\u003e, whereas four of the six \u003cem\u003eLdMasc-W\u003c/em\u003e genes from \u003cem\u003eL\u003c/em\u003e. \u003cem\u003ed\u003c/em\u003e. \u003cem\u003easiatica\u003c/em\u003e (\u003cem\u003eWA1\u003c/em\u003e\u0026ndash;\u003cem\u003e6\u003c/em\u003e in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA), which is more closely related to \u003cem\u003eL\u003c/em\u003e. \u003cem\u003ed\u003c/em\u003e. \u003cem\u003ejaponica\u003c/em\u003e, belonged to groups A, C, and D (\u003cem\u003eWA2\u003c/em\u003e, \u003cem\u003eWA3\u003c/em\u003e, \u003cem\u003eWA5\u003c/em\u003e, and \u003cem\u003eWA6\u003c/em\u003e in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). These results suggest that the \u003cem\u003eLdMasc-W\u003c/em\u003e genes in \u003cem\u003eL\u003c/em\u003e. \u003cem\u003ed\u003c/em\u003e. \u003cem\u003ejaponica\u003c/em\u003e, corresponding to \u003cem\u003eLdMasc-WA2\u003c/em\u003e, \u003cem\u003eWA3\u003c/em\u003e, \u003cem\u003eWA5\u003c/em\u003e, and \u003cem\u003eWA6\u003c/em\u003e, increased their copy numbers following the divergence of \u003cem\u003eL\u003c/em\u003e. \u003cem\u003ed\u003c/em\u003e. \u003cem\u003ejaponica\u003c/em\u003e from \u003cem\u003eL\u003c/em\u003e. \u003cem\u003ed\u003c/em\u003e. \u003cem\u003easiatica\u003c/em\u003e. Given the extremely low depth of the branches forming each group, we hypothesize that the copy numbers of \u003cem\u003eLdMasc-W\u003c/em\u003e increased rapidly in each group.\u003c/p\u003e \u003cp\u003eTo verify that the 52 \u003cem\u003eLdMasc-W\u003c/em\u003e sequences identified in \u003cem\u003eL\u003c/em\u003e. \u003cem\u003ed\u003c/em\u003e. \u003cem\u003ejaponica\u003c/em\u003e are indeed nucleotide sequences on the W chromosome, we performed genomic PCR using primers specifically annealed to the nucleotide sequences adjacent to the 5\u0026rsquo; end of each \u003cem\u003eLdMasc-W\u003c/em\u003e. As our previous molecular phylogenetic analysis results indicated that these nucleotide sequences showed homology with each other, it was impossible to design primers specific for all \u003cem\u003eLdMasc-W\u003c/em\u003e genes. Therefore, we designed 12 primers that annealed as specifically as possible to individual \u003cem\u003eLdMasc-W\u003c/em\u003e genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB) and used these to perform PCR with genomic DNA from both males and females as templates. The results showed that \u003cem\u003eLdMasc-W21\u003c/em\u003e was amplified in both males and females, while amplification products for the other \u003cem\u003eLdMasc-W\u003c/em\u003e genes were obtained only when the female genome was used as the template (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, upper panel). The same PCR analysis using genomic DNA extracted from \u003cem\u003eL. umbrosa\u003c/em\u003e larvae as a template indicated that there were no sequences homologous to \u003cem\u003eLdMasc-W\u003c/em\u003e in \u003cem\u003eL. umbrosa\u003c/em\u003e genome (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, lower panel), consistent with the results of BLASTn searches described above (Additional file 5: Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e). Interestingly, \u003cem\u003eLdMasc-W21\u003c/em\u003e was also amplified in both male and female of \u003cem\u003eL. umbrosa\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, lower panel). These results suggest that all 52 \u003cem\u003eLdMasc-W\u003c/em\u003e genes identified in this study, with the exception of \u003cem\u003eLdMasc-W21\u003c/em\u003e, are located on the W chromosome.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eIdentification of LdMasc-W-derived transcripts and expression analysis\u003c/h3\u003e\n\u003cp\u003eAll subsequent experiments were conducted using \u003cem\u003eL\u003c/em\u003e. \u003cem\u003ed\u003c/em\u003e. \u003cem\u003ejaponica\u003c/em\u003e. As described above, the four unigenes composed of the 3\u0026rsquo; end nucleotide sequence of \u003cem\u003eLdMasc\u003c/em\u003e were specifically expressed in females and were present only in the female genome (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Therefore, to determine whether these unigenes originate from \u003cem\u003eLdMasc-W\u003c/em\u003e, we performed a BLASTn search using the nucleotide sequences of these four unigenes (31510, 24771, 29638, and 44942) as query sequences against the genomic sequences of all identified \u003cem\u003eLdMasc-W\u003c/em\u003e genes. The results showed that nucleotide sequences near the 5\u0026rsquo; ends of the four unigenes were encoded by genomic sequences of \u003cem\u003eLdMasc-W2\u003c/em\u003e, \u003cem\u003eLdMasc-W6\u003c/em\u003e, \u003cem\u003eLdMasc-W8\u003c/em\u003e, and \u003cem\u003eLdMasc-W9\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). The nucleotide sequence near the 3\u0026rsquo; end of unigene 44942 appeared to have originated from genomic sequences of \u003cem\u003eLdMasc-W1\u003c/em\u003e to \u003cem\u003eLdMasc-W9\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). However, we were unable to find \u003cem\u003eLdMasc-W\u003c/em\u003e encoding the nucleotide sequence near the 3\u0026rsquo; end of unigene 24771. Thus, the four unigenes do not reflect all transcripts derived from \u003cem\u003eLdMasc-W\u003c/em\u003e genes.\u003c/p\u003e \u003cp\u003eTo obtain additional transcripts from \u003cem\u003eLdMasc-W\u003c/em\u003e, we conducted rapid amplification of cDNA ends (RACE) to identify the 5\u0026rsquo; end using primers designed to anneal to a region common to all \u003cem\u003eLdMasc-W\u003c/em\u003e variants (Additional file 9: Table \u003cspan refid=\"MOESM7\" class=\"InternalRef\"\u003eS7\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). The resulting 5\u0026rsquo; RACE products were TA-cloned, and the nucleotide sequences of 48 clones were determined, identifying nine distinct transcripts (see Additional file 12: Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). These nine transcripts were named RACE4, RACE13, RACE24, RACE35, RACE45, RACE50, RACE67, RACE69, and RACE85 (Accession no. LC889978-LC889986). Comparison of the nine nucleotide sequences with the genomic sequences of \u003cem\u003eLdMasc-W\u003c/em\u003e genes revealed that the nine transcripts were derived from 17 \u003cem\u003eLdMasc-W\u003c/em\u003e genes belonging to groups A, B, and D (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). These results indicate that at least 17 of the 52 \u003cem\u003eLdMasc-W\u003c/em\u003e genes identified in this study are expressed at the mRNA level.\u003c/p\u003e \u003cp\u003eBecause \u003cem\u003eLdMasc-W\u003c/em\u003e genes were located on the W chromosome, transcripts derived from these genes should be expressed specifically in females. To verify this hypothesis, RT-PCR was performed using cDNA templates prepared from testes and ovaries with primers designed to specifically amplify each identified transcript (see Additional file 19: Table \u003cspan refid=\"MOESM9\" class=\"InternalRef\"\u003eS9\u003c/span\u003e). The results revealed that four of the nine transcripts (RACE4, RACE13, RACE24, and RACE85) were expressed specifically in the ovary (see Additional file 14: Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). Although RACE50 was also expressed in the ovary, DNA fragments of different sizes were amplified only to a small extent in the testes (see Additional file 14: Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). These results indicated that the 17 \u003cem\u003eLdMasc-W\u003c/em\u003e genes encoding these five transcripts were expressed specifically in females. To investigate whether these \u003cem\u003eLdMasc-W\u003c/em\u003e genes also exhibit specific expression in female embryos at the sex determination stage (days 2\u0026ndash;4 post-laying), RT-PCR analysis was performed using cDNA templates prepared from eggs of both sexes at the sex determination stage. This analysis demonstrated that transcripts with ovary-specific expression were also expressed specifically in females throughout the sex determination period (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). These findings indicate that the 17 \u003cem\u003eLdMasc-W\u003c/em\u003e genes encoding the five transcripts may possess female-specific functions at the sex determination stage.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eFunctional analysis of LdMasc-W by embryonic RNAi\u003c/h3\u003e\n\u003cp\u003eGiven that some \u003cem\u003eLdMasc-W\u003c/em\u003e genes are specifically expressed in females throughout the sex determination period (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD) and that this gene is located on the W chromosome, we hypothesized that it is highly likely to be involved in female determination.\u003c/p\u003e \u003cp\u003eA standard approach to evaluate the function of sex determination genes in lepidopteran insects is to examine whether functional suppression of the gene affects sexually dimorphic expression of \u003cem\u003edsx\u003c/em\u003e during the sex determination period [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Therefore, to evaluate whether \u003cem\u003eLdMasc-W\u003c/em\u003e is involved in female determination, we examined the effects of RNAi-mediated knockdown of \u003cem\u003eLdMasc-W\u003c/em\u003e expression during sex determination on the sexual dimorphic expression pattern of \u003cem\u003eLddsx\u003c/em\u003e, the ortholog of \u003cem\u003edsx\u003c/em\u003e in the spongy moth (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). We injected small interfering RNAs (siRNAs) targeting \u003cem\u003eLdMasc-W\u003c/em\u003e mRNA into eggs within 12 h post-laying (embryonic RNAi) to achieve \u003cem\u003eLdMasc-W\u003c/em\u003e knockdown. Two types of siRNA targeting different nucleotide sequences within a region common to all \u003cem\u003eLdMasc-W\u003c/em\u003e genes were used for embryonic RNAi (LdMasc-W si1 and LdMasc-W si2) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, Additional file 13: Table \u003cspan refid=\"MOESM8\" class=\"InternalRef\"\u003eS8\u003c/span\u003e). Eggs injected with siRNA targeting \u003cem\u003eEgfp\u003c/em\u003e (Egfp si) used in our previous study [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] were used as a control group. Quantification of \u003cem\u003eLdMasc-W\u003c/em\u003e expression in embryos at 4 days post-injection (almost equivalent to 4 days post-laying) revealed that the average expression level in embryos injected with LdMasc-W si1 decreased to approximately 3.6% of that in Egfp si-injected eggs (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). In contrast, the average expression level in eggs injected with LdMasc-W si2 decreased to approximately 30.4% of that in Egfp si-injected embryos (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). RT-PCR analysis demonstrated that females with decreased mRNA levels of \u003cem\u003eLdMasc-W\u003c/em\u003e via LdMasc-W si1 injection exhibited both female-type (\u003cem\u003eLddsxF\u003c/em\u003e) and male-type (\u003cem\u003eLddsxM\u003c/em\u003e) \u003cem\u003eLddsx\u003c/em\u003e expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD, E). Approximately 60% of these females showed higher \u003cem\u003eLddsxM\u003c/em\u003e expression than \u003cem\u003eLddsxF\u003c/em\u003e expression, but no individuals exhibited complete sex reversal (expression of only \u003cem\u003eLddsxM\u003c/em\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). Among females in which \u003cem\u003eLdMasc-W\u003c/em\u003e knockdown was induced by LdMasc-W si2 injection, \u003cem\u003eLddsx\u003c/em\u003e expression patterns were similar to those observed in the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD, E). These results clearly demonstrate that a drastic reduction in \u003cem\u003eLdMasc-W\u003c/em\u003e expression induces a shift from female-type to male-type \u003cem\u003eLddsx\u003c/em\u003e expression in females.\u003c/p\u003e \u003cp\u003eInduction of \u003cem\u003eLddsxM\u003c/em\u003e expression requires expression of the male determination gene \u003cem\u003eLdMasc\u003c/em\u003e [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. The induced \u003cem\u003eLddsxM\u003c/em\u003e expression observed in \u003cem\u003eLdMasc-W\u003c/em\u003e knockdown females may have been caused by increased \u003cem\u003eLdMasc\u003c/em\u003e expression levels in these knockdown individuals. To test this hypothesis, we quantified \u003cem\u003eLdMasc\u003c/em\u003e expression among females in which \u003cem\u003eLdMasc-W\u003c/em\u003e expression was apparently reduced by LdMasc-W si1 injection. The results showed significantly higher \u003cem\u003eLdMasc\u003c/em\u003e expression levels (~\u0026thinsp;1.5-fold higher) in \u003cem\u003eLdMasc-W\u003c/em\u003e knockdown females, compared to females in the negative control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF). In contrast, no significant change was observed in expression levels of the female determination gene \u003cem\u003eFet-W\u003c/em\u003e, which suppresses \u003cem\u003eLdMasc\u003c/em\u003e expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG).\u003c/p\u003e \u003cp\u003eThese results indicate that \u003cem\u003eLdMasc-W\u003c/em\u003e knockdown shifts the sexually dimorphic expression of \u003cem\u003eLddsx\u003c/em\u003e from the female to the male mode by causing a slight increase in \u003cem\u003eLdMasc\u003c/em\u003e expression without altering \u003cem\u003eFet-W\u003c/em\u003e expression levels. Thus, \u003cem\u003eLdMasc-W\u003c/em\u003e appears to suppress \u003cem\u003eLdMasc\u003c/em\u003e expression and induce female differentiation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003ePhenotypic analysis of hybrid individuals between L. d. japonica and L. umbrosa\u003c/h2\u003e \u003cp\u003eTo obtain further evidence regarding whether \u003cem\u003eLdMasc-W\u003c/em\u003e possesses female-determining ability, interspecific hybridization experiments were conducted using L. d. japonica with the highest \u003cem\u003eLdMasc-W\u003c/em\u003e copies and its close relative \u003cem\u003eL. umbrosa\u003c/em\u003e lacking \u003cem\u003eLdMasc-W\u003c/em\u003e. The sexual dimorphic traits of the resulting first-generation hybrids (F1) were then examined from various perspectives.\u003c/p\u003e \u003cp\u003eFirst, we created hybrid lines by crossing \u003cem\u003eL. umbrosa\u003c/em\u003e females with \u003cem\u003eL. d. japonica\u003c/em\u003e males (hereafter referred to as UJ-hybrid F1 line) and their reverse crosses (hereafter referred to as JU-hybrid F1 line). As control lines, laboratory lines established from eggs of \u003cem\u003eL. d. japonica\u003c/em\u003e and \u003cem\u003eL. umbrosa\u003c/em\u003e were subjected to the same analyses. The resulting F1 individuals were subjected to RT-PCR analysis to examine the expression pattern of \u003cem\u003eLddsx\u003c/em\u003e. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, the expression pattern of \u003cem\u003eLddsx\u003c/em\u003e in embryos immediately prior to hatching in the JU-hybird F1 was normal (\u003cem\u003eLddsxM\u003c/em\u003e in males, \u003cem\u003eLddsxF\u003c/em\u003e in females). On the other hand, similar RT-PCR analysis using UJ-hybrid F1 individuals demonstrated that all embryos confirmed to be genetically female (ZW individuals) expressed male-isoform of \u003cem\u003eLddsx\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003eNext, we quantified the expression levels of \u003cem\u003eLdMasc\u003c/em\u003e, \u003cem\u003eFet-W\u003c/em\u003e, and \u003cem\u003eLdMasc-W\u003c/em\u003e using RNA extracted from the same individual. The expression level of \u003cem\u003eLdMasc\u003c/em\u003e in \u003cem\u003eL. umbrosa\u003c/em\u003e and JU-hybrid F1 animals was higher in males than in females, with extremely low expression levels observed in females (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). In contrast, the expression level of \u003cem\u003eLdMasc\u003c/em\u003e in the UJ-hybrid F1 females was increased to a level equivalent to that in males (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). Quantitative analysis of the expression levels of the known female determination gene \u003cem\u003eFet-W\u003c/em\u003e confirmed its female-specific expression in all lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). The same analysis revealed that \u003cem\u003eLdMasc-W\u003c/em\u003e was highly expressed only in females carrying the W chromosome derived from \u003cem\u003eL. d. japonica\u003c/em\u003e (\u003cem\u003eL. d. japonica\u003c/em\u003e females and UJ-hybrid F1 females) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). These results were consistent with the fact that UJ-hybrid F1 females did not possess \u003cem\u003eLdMasc-W\u003c/em\u003e because they inherited the W chromosome from their maternal parent, \u003cem\u003eL. umbrosa\u003c/em\u003e. Taken together, the above findings suggest that the absence of \u003cem\u003eLdMasc-W\u003c/em\u003e in the UJ-hybrid F1 females induced \u003cem\u003eLdMasc\u003c/em\u003e expression at levels equivalent to males, leading to a shift in the expression pattern of \u003cem\u003eLddsx\u003c/em\u003e from the female-type to the male-type.\u003c/p\u003e \u003cp\u003eThe UJ-hybrid F1 eggs showed no significant difference in hatchability as compared to those in other lines (see Additional file 26: Fig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e). Individuals that died during rearing were collected, and the death rate (number of dead individuals/ total number of hatched individuals) was calculated. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC, a significantly higher death rate was observed in the UJ-hybrid F1 individuals. When plotting the Kaplan-Meier curve, it was found that approximately 20% of individuals in the UJ-hybrid animals died within about 20 days after hatching, relatively early stages in development (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). Furthermore, PCR-based molecular sexing using DNA extracted from the deceased individuals revealed that the sex ratio of the dead individuals was female-biased (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE). In the UJ-hybrid F1 animals, the final number of adults was 28 females and 131 males, indicating a significant male bias. These results suggest that UJ-hybrid F1 females died at a relatively early stage during the larval period.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eMorphological observations of sexual dimorphic traits in adults revealed no clear masculinization in the wing coloration or patterns of the UJ-hybrid F1 females, while their wing angles at rest resembled those of males (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). The antennae of the UJ-hybrid F1 females exhibited morphological characteristics similar to those of males (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). The color of the scaly hairs on the legs of these females also showed the same bright coloration as the males (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). The UJ-hybrid F1 females had malformed external genitalia likely due to insufficient development (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC). The ovaries of these females contained a number of mature eggs comparable to those of normal females, and no morphological abnormalities were observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD). However, careful observation revealed the presence of male-specific tissues at the base of the oviducts: the vas deferens, accessory glands, seminal vesicles, and ejaculatory ducts (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE). There was little variation among individuals in these male-like traits observed in the UJ-hybrid females. In JU-hybrid and \u003cem\u003eL. umbrosa\u003c/em\u003e females, the oviduct, spermatheca, and seminal receptacle were present in the same region (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE). These results clearly demonstrated that the UJ-hybrid F1 females, which carried \u003cem\u003eLdMasc\u003c/em\u003e and \u003cem\u003eFet-W\u003c/em\u003e without \u003cem\u003eLdMasc-W\u003c/em\u003e, exhibited a morphological phenotype classified as intersex, possessing characteristics of both male and female morphology.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study revealed that multiple sequences almost identical to the approximately 5-kb genomic sequence near the 3\u0026rsquo; end of the \u003cem\u003eLdMasc\u003c/em\u003e gene, which is involved in male determination in the spongy moth [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], are present on the W chromosome. These homologous sequences were considered W chromosome-specific paralogs of \u003cem\u003eLdMasc\u003c/em\u003e and designated as \u003cem\u003eLdMasc-W\u003c/em\u003e, with varying copy numbers among \u003cem\u003eL\u003c/em\u003e. \u003cem\u003ed\u003c/em\u003e. \u003cem\u003edispar\u003c/em\u003e (3 copies), \u003cem\u003eL\u003c/em\u003e. \u003cem\u003ed\u003c/em\u003e. \u003cem\u003easiatica\u003c/em\u003e (6 copies), and \u003cem\u003eL. d\u003c/em\u003e. \u003cem\u003ejaponica\u003c/em\u003e (52 copies) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, B). The subspecies \u003cem\u003eL\u003c/em\u003e. \u003cem\u003ed\u003c/em\u003e. \u003cem\u003easiatica\u003c/em\u003e and \u003cem\u003eL\u003c/em\u003e. \u003cem\u003ed\u003c/em\u003e. \u003cem\u003ejaponica\u003c/em\u003e are closely related; phylogenetic analysis revealed that \u003cem\u003eLdMasc-W\u003c/em\u003e homologous to four of the six \u003cem\u003eLdMasc-W\u003c/em\u003e genes had higher copy numbers in \u003cem\u003eL\u003c/em\u003e. \u003cem\u003ed\u003c/em\u003e. \u003cem\u003easiatica\u003c/em\u003e than in \u003cem\u003eL\u003c/em\u003e. \u003cem\u003ed\u003c/em\u003e. \u003cem\u003ejaponica\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Furthermore, \u003cem\u003eLdMasc-W\u003c/em\u003e genes classified into group B were unique to \u003cem\u003eL\u003c/em\u003e. \u003cem\u003ed\u003c/em\u003e. \u003cem\u003ejaponica\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). It remains unknown why \u003cem\u003eLdMasc-W\u003c/em\u003e copy numbers increased only in \u003cem\u003eL\u003c/em\u003e. \u003cem\u003ed\u003c/em\u003e. \u003cem\u003ejaponica\u003c/em\u003e. The presence of false copies due to misassembly of the genome sequence can be excluded as a possible explanation because the \u003cem\u003eLdMasc-W\u003c/em\u003e copy numbers estimated by qPCR analysis generally matched those determined through genome analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). The construction of chromosome-level sequence assemblies for the W chromosome in \u003cem\u003eL\u003c/em\u003e. \u003cem\u003ed\u003c/em\u003e. \u003cem\u003ejaponica\u003c/em\u003e will reveal the precise copy number of \u003cem\u003eLdMasc-W\u003c/em\u003e and its location on the W chromosome in this subspecies.\u003c/p\u003e \u003cp\u003eIn the diamondback moth (\u003cem\u003ePlutella xylostella\u003c/em\u003e), \u003cem\u003eMasc\u003c/em\u003e homologous sequences are present on the W chromosome [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. The \u003cem\u003eMasc\u003c/em\u003e homologous sequence originates from a retrotransposon-derived mRNA that fused with the mRNA sequence of \u003cem\u003ePxyMasc\u003c/em\u003e (a \u003cem\u003eMasc\u003c/em\u003e ortholog of the diamondback moth) spanning exons 4\u0026ndash;6 or exons 4\u0026ndash;7 and was subsequently integrated into the W chromosome. This process resulted in a multicopy array, with 4\u0026ndash;7 copies of the sequence arranged in tandem [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. piRNAs targeting \u003cem\u003ePxyMasc\u003c/em\u003e mRNA are produced from the region corresponding to \u003cem\u003ePxyMasc\u003c/em\u003e exon 5 within these multicopy arrays, resulting in the degradation of \u003cem\u003ePxyMasc\u003c/em\u003e mRNA via ping-pong amplification. Therefore, this multicopy array is predicted to function as a female determination gene in the diamondback moth and has been named \u003cem\u003ePxyFem\u003c/em\u003e [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Another example of a \u003cem\u003eMasc\u003c/em\u003e homologous sequence found on the W chromosome is \u003cem\u003eEkMasc\u003c/em\u003e\u003csup\u003e\u003cem\u003eW\u003c/em\u003e\u003c/sup\u003e in the Mediterranean flour moth (\u003cem\u003eEphestia kuehniella\u003c/em\u003e). \u003cem\u003eEkMasc\u003c/em\u003e\u003csup\u003e\u003cem\u003eW\u003c/em\u003e\u003c/sup\u003e shares approximately 94\u0026ndash;97% homology with an \u003cem\u003eEkMascB\u003c/em\u003e cDNA sequence encompassing exons 2\u0026ndash;10 (~\u0026thinsp;1.3 kb) and has 17\u0026ndash;23 copies on the W chromosome [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Although this feature initially appears similar to \u003cem\u003ePxyFem\u003c/em\u003e, \u003cem\u003eEkMasc\u003c/em\u003e\u003csup\u003e\u003cem\u003eW\u003c/em\u003e\u003c/sup\u003e is thought to have arisen from a duplication of \u003cem\u003eLdMasc-B\u003c/em\u003e, rather than from a retrotransposon copy of \u003cem\u003eEkMascB\u003c/em\u003e mRNA. Furthermore, unlike \u003cem\u003ePxyFem\u003c/em\u003e, \u003cem\u003eEkMasc\u003c/em\u003e\u003csup\u003e\u003cem\u003eW\u003c/em\u003e\u003c/sup\u003e does not function as a sex-determining gene; instead, another gene (\u003cem\u003eEkMom\u003c/em\u003e) is responsible for female determination [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDistinct from \u003cem\u003ePxyFem\u003c/em\u003e and \u003cem\u003eEkMasc\u003c/em\u003e\u003csup\u003e\u003cem\u003eW\u003c/em\u003e\u003c/sup\u003e, \u003cem\u003eLdMasc-W\u003c/em\u003e consists of a nucleotide sequence approximately 5.4 kb in length, extending from intron 8 to exon 11 of the genomic sequence of \u003cem\u003eLdMasc\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, B). Therefore, it is reasonable to consider that \u003cem\u003eLdMasc-W\u003c/em\u003e is a \u003cem\u003eLdMasc\u003c/em\u003e paralog resulting from translocation of the 3\u0026rsquo; region of the \u003cem\u003eLdMasc\u003c/em\u003e genomic sequence to the W chromosome, followed by an increase in its copy number due to gene duplication. \u003cem\u003eLdMasc\u003c/em\u003e has two tandem copies on the Z chromosome [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], which suggests that it may reside within a genomic structure prone to gene duplication.\u003c/p\u003e \u003cp\u003eOf the 52 \u003cem\u003eLdMasc-W\u003c/em\u003e genes identified in \u003cem\u003eL\u003c/em\u003e. \u003cem\u003ed\u003c/em\u003e. \u003cem\u003ejaponica\u003c/em\u003e, at least 17 \u003cem\u003eLdMasc-W\u003c/em\u003e genes were expressed specifically in females throughout the sex determination period (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). RNAi-mediated knockdown of \u003cem\u003eLdMasc-W\u003c/em\u003e caused slightly increased \u003cem\u003eLdMasc\u003c/em\u003e mRNA levels, leading to \u003cem\u003eLddsxM\u003c/em\u003e expression in females (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC\u0026ndash;G). However, \u003cem\u003eLdMasc-W\u003c/em\u003e knockdown resulted in only very slightly increased \u003cem\u003eLdMasc\u003c/em\u003e mRNA levels, which may be attributable to the siRNA used in this study (LdMasc-W si1), which targeted the common region between \u003cem\u003eLdMasc-W\u003c/em\u003e and \u003cem\u003eLdMasc\u003c/em\u003e. In this experiment, designing siRNAs that targeted the common region of \u003cem\u003eLdMasc-W\u003c/em\u003e and \u003cem\u003eLdMasc\u003c/em\u003e was unavoidable due to the necessity of simultaneously knocking down the expression of at least 17 distinct mRNAs derived from LdMasc-W. \u003cem\u003eLdMasc\u003c/em\u003e expression might have increased to a greater extent had we been able to specifically knock down \u003cem\u003eLdMasc-W\u003c/em\u003e alone.\u003c/p\u003e \u003cp\u003eConsistent with results obtained from embryonic RNA, a female-to-male shift in the expression pattern of \u003cem\u003eLddsx\u003c/em\u003e was observed in F1 hybrid female embryos obtained from the cross between \u003cem\u003eL. umbrosa\u003c/em\u003e females and \u003cem\u003eL. d. japonica\u003c/em\u003e males (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). Note that females resulting from this crossing could carry \u003cem\u003eLdMasc\u003c/em\u003e derived from \u003cem\u003eL. d. japonica\u003c/em\u003e but not possess \u003cem\u003eLdMasc-W\u003c/em\u003e. In these females, no expression of \u003cem\u003eLdMasc-W\u003c/em\u003e was observed, whereas the expression level of \u003cem\u003eLdMasc\u003c/em\u003e increased to levels comparable to those in males (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). Furthermore, these hybrid females exhibited female-to-male sexual reversal in the morphology of their antennae, legs, and parts of their internal reproductive organs (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB, \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD, and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE). Taken together with the above findings, it would be reasonable to conclude that \u003cem\u003eLdMasc-W\u003c/em\u003e functions as a female determining gene that is expressed specifically in females during the sex determination period and promotes female differentiation by reducing the mRNA levels of \u003cem\u003eLdMasc\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eThe UJ-hybrid females exhibited lethality as the primary phenotype (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC-\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE). This can be attributed to abnormalities in gene dosage compensation in females. It is known that abnormalities in gene dosage compensation during development cause lethality in various species, including mice, nematodes, and fruit flies [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. In multiple Lepidoptera insects, \u003cem\u003eMasc\u003c/em\u003e reduces gene expression levels on the Z chromosome by half in males to compensate gene dosage between two sexes [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. It would be possible that forced expression of \u003cem\u003eMasc\u003c/em\u003e causes unnecessary dosage compensation in the expression levels of Z-linked genes in females, resulting in female-specific lethality. In fact, forced expression of the \u003cem\u003eFem\u003c/em\u003e piRNA-resistant \u003cem\u003eMasc\u003c/em\u003e (\u003cem\u003eMasc-R\u003c/em\u003e) gene in the silkworm has been shown to cause female-specific lethality during the larval stage [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. This is reminiscent of our finding that the UJ-hybrid F1 females, most of which were dead during the larval stages, expressed high level of \u003cem\u003eLdMasc\u003c/em\u003e mRNA equivalent to that in males (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB-\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE). It is highly plausible that increased expression level of \u003cem\u003eLdMasc\u003c/em\u003e in UJ-hybrid F1 females induced inappropriate compensation for the Z-linked gene expressions, leading to female-specific lethality as the predominant phenotype.\u003c/p\u003e \u003cp\u003eThe UJ-hybrid F1 females exhibited a shift in the expression pattern of \u003cem\u003eLddsx\u003c/em\u003e from female to male at the pre-hatchling embryonic stage, yet the tissues showing sex reversal were limited (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB, \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD, and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE). In \u003cem\u003eOnthophagus taurus\u003c/em\u003e, which belongs to the family Scarabaeidae, \u003cem\u003edsx\u003c/em\u003e is involved in regulating sexual differentiation. However, the variety of downstream genes and the regulatory direction (activation/repression) of gene expression by \u003cem\u003edsx\u003c/em\u003e differ between tissues [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Particularly in females, tissues in which \u003cem\u003edsx\u003c/em\u003e regulates the expressions of genes responsible for sexual differentiations are limited. For example, many genes showing sex-specific expressions are regulated by \u003cem\u003edsx\u003c/em\u003e in the female horn, whereas in other tissues, sex differences are governed by factors other than \u003cem\u003edsx\u003c/em\u003e. Based on these findings, Led\u0026oacute;n-Rettig et al. hypothesized that features susceptible to sexual selection or newly evolved sexual traits may require the involvement of multiple genes under the control of \u003cem\u003edsx\u003c/em\u003e [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Similarly, in the spongy moth, the degree of dependence on \u003cem\u003edsx\u003c/em\u003e probably differs for each sexual dimorphic trait, and thus the tissues showing sex reversal in UJ-hybrid FI females may be limited.\u003c/p\u003e \u003cp\u003eThe most noteworthy finding of this study is that \u003cem\u003eLdMasc-W\u003c/em\u003e determines femaleness in the spongy moth through several pathways, despite the presence of the female-determining gene \u003cem\u003eFet-W\u003c/em\u003e in this species, as determined in our previous study [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. As described above, RNAi-mediated knockdown of \u003cem\u003eLdMasc-W\u003c/em\u003e increased \u003cem\u003eLdMasc\u003c/em\u003e mRNA levels in females and induced expression of the male-type \u003cem\u003eLddsx\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF). These findings suggest that \u003cem\u003eLdMasc-W\u003c/em\u003e induces female differentiation by suppressing \u003cem\u003eLdMasc\u003c/em\u003e expression in females. The mRNAs transcribed from \u003cem\u003eLdMasc-W\u003c/em\u003e genes include an open reading frame (ORF) extending from exon 9 to the stop codon of \u003cem\u003eLdMasc\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA and Additional file 12: Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). A comparison of the amino acid sequences encoded by these ORFs with those of the LdMasc protein revealed that amino acid sequences encoded by 43 of the 52 \u003cem\u003eLdMasc-W\u003c/em\u003e genes were 100% identical to the corresponding amino acid sequence of the LdMasc protein (see Additional file 27: Fig. \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e). It might be possible that the protein produced from the \u003cem\u003eLdMasc-W\u003c/em\u003e cause a decreased expression of \u003cem\u003eLdMasc\u003c/em\u003e mRNA.\u003c/p\u003e \u003cp\u003eExamples of gene paralogs functioning as sex-determining genes have been discovered in several species. \u003cem\u003eDmy\u003c/em\u003e (\u003cem\u003eDmrt1bY\u003c/em\u003e) in medaka (\u003cem\u003eOryzias latipes\u003c/em\u003e), which adopts a heterogametic male (XX\u0026thinsp;=\u0026thinsp;female, XY\u0026thinsp;=\u0026thinsp;male) sex determination system, is a pioneering example. \u003cem\u003eDmy\u003c/em\u003e is located on the Y chromosome and functions as a male determination gene by inducing testis formation [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. This gene is considered to be a Y chromosome paralog of \u003cem\u003eDoublesex and mab-3 related transcription factor 1 a\u003c/em\u003e (\u003cem\u003eDmrt1a\u003c/em\u003e), which induces masculinization through testis development [\u003cspan additionalcitationids=\"CR31\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Similarly, in rainbow trout (\u003cem\u003eOncorhynchus mykiss\u003c/em\u003e), which also employs a heterogametic male sex determination system, \u003cem\u003eSexually dimorphic on the Y-chromosome\u003c/em\u003e (\u003cem\u003esdY\u003c/em\u003e) functions as a male determination gene [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. The \u003cem\u003esdY\u003c/em\u003e gene plays a role in male determination by inducing testis formation and appears to be a Y-chromosomal paralog that originated from gene duplication of \u003cem\u003eInterferon regulatory factor 9\u003c/em\u003e (\u003cem\u003eIrf9\u003c/em\u003e), a gene involved in interferon regulation. The \u003cem\u003esdY\u003c/em\u003e gene encodes a protein homologous to the C-terminal amino acid sequence of the Irf9 protein, although the downstream cascade of \u003cem\u003esdY\u003c/em\u003e remains unclear. This gene is a notable example of a paralog originating from a gene unrelated to sex determination acquiring the function of a male-determining gene.\u003c/p\u003e \u003cp\u003eThe African clawed frog (\u003cem\u003eXenopus laevis\u003c/em\u003e), which employs a female heterogametic sex determination system (ZZ\u0026thinsp;=\u0026thinsp;male, ZW\u0026thinsp;=\u0026thinsp;female) like the spongy moth, has been reported as an example of a paralog functioning as a sex determination gene. The female-determining gene \u003cem\u003eDM-W\u003c/em\u003e on the W chromosome of \u003cem\u003eXenopus laevis\u003c/em\u003e was identified as a W chromosome paralog of the male-determining gene \u003cem\u003eDMRT1\u003c/em\u003e [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. \u003cem\u003eDM-W\u003c/em\u003e shows extremely high homology with \u003cem\u003eDMRT1\u003c/em\u003e in the DNA-binding domain (DM domain), but no homology is observed in the C-terminal region. Since the C-terminal region of DMRT1 possesses transcriptional activation activity, it has been predicted that despite both proteins binding to the same target genes, they exert different transcription regulatory effects [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Based on these findings, a model has been proposed in which DM-W suppresses the function of DMRT1, leading to the promotion of female differentiation.\u003c/p\u003e \u003cp\u003eAs demonstrated for \u003cem\u003esdY\u003c/em\u003e in rainbow trout, where a protein homologous to the C-terminal amino acid sequence functions as a sex-determining gene, a protein corresponding to the C-terminal region of LdMasc produced by \u003cem\u003eLdMasc-W\u003c/em\u003e genes may be responsible for female determination in the spongy moth. Similar to the DM-W protein that induces female differentiation by suppressing DMRT1 function, \u003cem\u003eLdMasc-W\u003c/em\u003e protein products may induce female differentiation by inhibiting the male-determining function of the LdMasc protein. Further research is needed to elucidate the molecular mechanism underlying how \u003cem\u003eLdMasc-W\u003c/em\u003e leads to the suppression of \u003cem\u003eLdMasc\u003c/em\u003e function.\u003c/p\u003e \u003cp\u003eA key remaining question is why the spongy moth possesses \u003cem\u003eLdMasc-W\u003c/em\u003e in addition to \u003cem\u003eFet-W\u003c/em\u003e. We hypothesize that \u003cem\u003eFet-W\u003c/em\u003e alone may be insufficient to repress \u003cem\u003eLdMasc\u003c/em\u003e expression, suggesting the necessity of an additional inhibitory mechanism. \u003cem\u003eFet-W\u003c/em\u003e is predicted to repress \u003cem\u003eLdMasc\u003c/em\u003e expression by inducing the degradation of \u003cem\u003eLdMasc\u003c/em\u003e mRNA via the piRNA pathway, similar to the \u003cem\u003eFem\u003c/em\u003e genes identified in other lepidopteran insects [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Unlike many other lepidopteran insects, the spongy moth possesses two copies of the male-determining gene \u003cem\u003eLdMasc\u003c/em\u003e, which are tandemly arranged on the Z chromosome [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. The expression levels of genes arranged in tandem copies are enhanced beyond those expected based on the copy number (i.e., more than doubled in this case) [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. To counteract the increase in \u003cem\u003eLdMasc\u003c/em\u003e copy numbers and the accompanying rise in expression levels, the spongy moth may have evolved two female determination genes. In this study, most LdMasc-W si1-injected females with nearly complete knockdown of \u003cem\u003eLdMasc-W\u003c/em\u003e expression tended to show higher \u003cem\u003eLddsxM\u003c/em\u003e expression than \u003cem\u003eLddsxF\u003c/em\u003e expression, but none exhibited complete sex reversal in the expression pattern of \u003cem\u003eLddsx\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE, F). This finding strongly suggests that \u003cem\u003eLdMasc-W\u003c/em\u003e functions in a complementary manner during sex determination. In summary, \u003cem\u003eFet-W\u003c/em\u003e alone is insufficient for female determination, and complete female determination may occur through the auxiliary female-determining function of \u003cem\u003eLdMasc-W\u003c/em\u003e. Like the spongy moth, the Mediterranean flour moth \u003cem\u003eE kuehniella\u003c/em\u003e possesses the female-determining gene \u003cem\u003eEkMom\u003c/em\u003e together with a homologous sequence of \u003cem\u003eEkMascB\u003c/em\u003e on the W chromosome, \u003cem\u003eEkMasc\u003c/em\u003e\u003csup\u003e\u003cem\u003eW\u003c/em\u003e\u003c/sup\u003e [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], and it possesses two copies of \u003cem\u003eMasc\u003c/em\u003e orthologs (\u003cem\u003eEkMascA\u003c/em\u003e and \u003cem\u003eEkMascB\u003c/em\u003e) [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. \u003cem\u003eEkMasc\u003c/em\u003e\u003csup\u003e\u003cem\u003eW\u003c/em\u003e\u003c/sup\u003e produces piRNAs, which may target \u003cem\u003eEkMasc\u003c/em\u003e mRNA [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. While the contribution of \u003cem\u003eEkMasc\u003c/em\u003e\u003csup\u003e\u003cem\u003eW\u003c/em\u003e\u003c/sup\u003e to female determination remains to be determined, both \u003cem\u003eEkMom\u003c/em\u003e and \u003cem\u003eEkMasc\u003c/em\u003e\u003csup\u003e\u003cem\u003eW\u003c/em\u003e\u003c/sup\u003e may be required for the full achievement of female determination.\u003c/p\u003e \u003cp\u003eGenerally, when a gene playing a dominant role in sex determination (the master sex determination gene) is acquired, recombination is reduced or suppressed between homologous chromosomes, leading to the irreversible accumulation of harmful mutations [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Consequently, the turnover of master sex determination genes can occur at accelerated rates [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. The presence of alternative genes is a prerequisite for the smooth execution of this process. Under these conditions, possessing two female determination genes, \u003cem\u003eFet-W\u003c/em\u003e and \u003cem\u003eLdMasc-W\u003c/em\u003e, may be advantageous. Such turnover may be occurring in \u003cem\u003eLdMasc-W\u003c/em\u003e and \u003cem\u003eFet-W\u003c/em\u003e. \u003cem\u003eLdMasc-W\u003c/em\u003e copy numbers are much higher in \u003cem\u003eL\u003c/em\u003e. \u003cem\u003ed\u003c/em\u003e. \u003cem\u003ejaponica\u003c/em\u003e than in \u003cem\u003eL\u003c/em\u003e. \u003cem\u003ed\u003c/em\u003e. \u003cem\u003edispar\u003c/em\u003e and \u003cem\u003eL\u003c/em\u003e. \u003cem\u003ed\u003c/em\u003e. \u003cem\u003easiatica\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, B), which may be related to a progressive shift in the function of the female-determining gene from \u003cem\u003eFet-W\u003c/em\u003e to \u003cem\u003eLdMasc-W\u003c/em\u003e in \u003cem\u003eL\u003c/em\u003e. \u003cem\u003ed\u003c/em\u003e. \u003cem\u003ejaponica\u003c/em\u003e. To verify this hypothesis, further research is needed to clarify the function of \u003cem\u003eLdMasc-W\u003c/em\u003e in \u003cem\u003eL\u003c/em\u003e. \u003cem\u003ed\u003c/em\u003e. \u003cem\u003edispar\u003c/em\u003e and \u003cem\u003eL\u003c/em\u003e. \u003cem\u003ed\u003c/em\u003e. \u003cem\u003easiatica\u003c/em\u003e.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eOur findings demonstrated that \u003cem\u003eLdMasc-W\u003c/em\u003e, derived from the male-determining gene \u003cem\u003eLdMasc\u003c/em\u003e, possesses functional properties compatible with a role in female determination. Comparative analyses across three subspecies and one close relative species revealed substantial copy number variation among \u003cem\u003eFet-W\u003c/em\u003e, \u003cem\u003eLdMasc\u003c/em\u003e, and \u003cem\u003eLdMasc-W\u003c/em\u003e. Notably, \u003cem\u003eLdMasc-W\u003c/em\u003e exhibited the highest diversity, with 0–52 copies. Similar copy number variation was observed between populations even within the same species. Phylogenetic analysis suggested that \u003cem\u003eLdMasc-W\u003c/em\u003e copy number diversification accompanies speciation, highlighting the potential contribution of this gene to the diversification of sex determination systems. These findings would provide valuable insights into the molecular mechanisms underlying the diversification of sex determination systems.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Methods","content":"\u003ch2\u003eInsects\u003c/h2\u003e\u003cp\u003eThe Japanese spongy moth (\u003cem\u003eL\u003c/em\u003e. \u003cem\u003ed\u003c/em\u003e. \u003cem\u003ejaponica\u003c/em\u003e) used in this study was a laboratory strain primarily derived from egg masses collected in Nasu-Shiobara, Tochigi Prefecture, Japan. Another laboratory strain was established from \u003cem\u003eL. umbrosa\u003c/em\u003e eggs collected in Kitami, Hokkaido, Japan. Species confirmations of these strains were performed by PCR-RFLP analysis of mitochondrial DNA according to the method described previously [\u003cspan class=\"CitationRef\"\u003e42\u003c/span\u003e]. The moths were reared throughout their developmental stages in rearing chambers maintained at 25°C and 60 ± 10% humidity, under a 16-h light/8-h dark photocycle. For larval rearing, \u003cem\u003eWisteria floribunda\u003c/em\u003e leaves and an artificial diet were used. Post-emergence adults were allowed to mate freely within cylindrical nets (diameter, 40 cm; height, 55 cm) placed in the rearing room. Mating between \u003cem\u003eL. d. japonica\u003c/em\u003e and \u003cem\u003eL. umbrosa\u003c/em\u003e adults were conducted under the same condition. All eggs thus obtained were stored at 25°C in the rearing room for 1 month and then at 4°C in a refrigerator for over 100 days to break dormancy.\u003c/p\u003e\u003ch2\u003eStereomicroscopic analysis\u003c/h2\u003e\u003cp\u003eThe antennae, legs, internal and external genitalia were dissected out from adults basically within one day after emergence. Dissected genitalia was trimmed in 1×Phosphate Buffer Saline (FUJIFILM Wako) using fine-tipped forceps (No. 11252-00, FINE SCIENCE TOOLS). To prepare cuticle specimens, the external genitalia were incubated in 15% KOH at 50°C for 12 hours, followed by washing with 70% ethanol. Images of the dissected samples were acquired by a digital camera (DP73, Olympus) attached to the stereomicroscope (SZX16, Olympus).\u003c/p\u003e\u003ch2\u003eExtraction of total RNA and genomic DNA\u003c/h2\u003e\u003cp\u003eTotal RNA extraction from various tissues and eggs was performed using ISOGEN reagents (Nippon Gene, Tokyo, Japan), according to the manufacturer’s instructions. Tissue homogenization was performed using a Homogenization Pestle (Scientific Specialties, Lodi, CA, USA). Genomic DNA was precipitated by adding one third the volume of ethanol to the lower layer obtained after ISOGEN treatment and centrifuging at maximum speed at 4°C.The resulting pellet was dissolved in an alkaline solution (50 mM NaOH), incubated at 95°C for 15 min, and then an equal volume of neutralizing buffer (200 mM Tris-HCl, pH 8.0) was added. The resulting genomic DNA solution was stored at 4°C.\u003c/p\u003e\u003cp\u003e \u003cem\u003e5’ RACE analysis\u003c/em\u003e \u003c/p\u003e\u003cp\u003ePoly(A) + RNA used as a template for 5’ RACE was extracted from female ovaries using Fast Track MAG mRNA Isolation Kits (Invitrogen, Carlsbad, CA, USA), following the manufacturer’s instructions. We performed 5’ RACE using the Gene Racer kit (Invitrogen), following the manufacturer’s instructions. PCR amplification of the resulting cDNA was performed using LA-Taq (Takara Bio, Kusatsu, Japan) under the following conditions: five cycles of 94°C for 2 min, 94°C for 30 s, and 72°C for 2 min; five cycles of 94°C for 30 s and 70°C for 2 min; 25 cycles of 94°C for 30 s, 68°C for 30 s, and 72°C for 2 min; followed by 72°C for 10 min. Subsequently, nested PCR was performed under the following conditions: 25 cycles of 94°C for 2 min, 94°C for 30 s, 65°C for 30 s, and 68°C for 2 min; followed by 68°C for 10 min. Primers used for 5’ RACE are listed in Additional file 13: Table \u003cspan class=\"InternalRef\"\u003eS8\u003c/span\u003e.\u003c/p\u003e\u003ch2\u003eRT-PCR\u003c/h2\u003e\u003cp\u003ecDNA synthesis via reverse transcription using extracted total RNA as a template was performed using the Prime Script First-Strand cDNA Synthesis Kit (Takara Bio), following the manufacturer’s instructions. We used 8 µL of total RNA for reverse transcription, with 6-mer random primers included in the kit. EmeraldAmp PCR Master Mix (Takara Bio) was used to amplify the resulting cDNA, following the manufacturer’s instructions. The nucleotide sequences of primers used for RT-PCR are shown in Additional file 19: Table \u003cspan class=\"InternalRef\"\u003eS9\u003c/span\u003e.\u003c/p\u003e\u003ch2\u003eqRT-PCR\u003c/h2\u003e\u003cp\u003eqRT-PCR reactions were performed using TB Green Premix Ex Taq II (Takara Bio), following the manufacturer’s instructions; cDNA was synthesized via the reactions described above. Gene expression quantification was performed as previously described [\u003cspan class=\"CitationRef\"\u003e43\u003c/span\u003e] using the ΔΔCt method, with the \u003cem\u003eLymantria dispar elongation factor 1-α\u003c/em\u003e (\u003cem\u003eLdEF1-α\u003c/em\u003e) gene as a reference gene. The nucleotide sequences of primers used for qRT-PCR are listed in Additional file 28: Table \u003cspan class=\"InternalRef\"\u003eS10\u003c/span\u003e.\u003c/p\u003e\u003ch2\u003eEstimation of gene copy numbers by qPCR\u003c/h2\u003e\u003cp\u003eThe copy numbers of genes of interest were estimated by qPCR as previously described [\u003cspan class=\"CitationRef\"\u003e44\u003c/span\u003e]. Genomic DNA prepared as described above was used as a template. The qPCR reaction was performed as described above using the same primers used for qRT-PCR (see Additional file 28: Table \u003cspan class=\"InternalRef\"\u003eS10\u003c/span\u003e). The \u003cem\u003eLymantria dispar cad\u003c/em\u003e gene, which is an autosomal single-copy gene encoding carbamoyl-phosphate synthetase 2 aspartate transcarbamylase and dihydroorotase, was used as a reference gene [\u003cspan class=\"CitationRef\"\u003e44\u003c/span\u003e].\u003c/p\u003e\u003ch2\u003eSex determination and genomic PCR using W chromosome-specific primers\u003c/h2\u003e\u003cp\u003eMolecular sexing by PCR using W chromosome-specific primers was performed as described previously [\u003cspan class=\"CitationRef\"\u003e8\u003c/span\u003e]. Shuttle PCR was performed with LA-Taq (Takara Bio), following the manufacturer’s instructions. Individuals yielding amplification products matching the expected size were identified as female. Genomic PCR was performed under the same conditions used to amplify \u003cem\u003eLdMasc-W\u003c/em\u003e genes (see below) using primers designed to anneal to a specific sequence in each \u003cem\u003eLdMasc-W\u003c/em\u003e (see Additional file 29: Table \u003cspan class=\"InternalRef\"\u003eS11\u003c/span\u003e).\u003c/p\u003e\u003ch2\u003eIdentification of the W-chromosome-specific LdMasc paralog (LdMasc-W)\u003c/h2\u003e\u003cp\u003eTo identify nucleotide sequences homologous to the \u003cem\u003eLdMasc\u003c/em\u003e gene, a BLASTn search was performed using the 19,341-bp genomic sequence of \u003cem\u003eLdMasc\u003c/em\u003e, encompassing exons 1–11, as a query sequence against whole-genome sequences of \u003cem\u003eL\u003c/em\u003e. \u003cem\u003ed\u003c/em\u003e. \u003cem\u003edispar\u003c/em\u003e (accession no. GCA_963576585.1), \u003cem\u003eL\u003c/em\u003e. \u003cem\u003ed\u003c/em\u003e. \u003cem\u003easiatica\u003c/em\u003e (accession no. GCA_032191425.1), and \u003cem\u003eL\u003c/em\u003e. \u003cem\u003ed\u003c/em\u003e. \u003cem\u003ejaponica\u003c/em\u003e (this study). The BLASTn search was performed using the BLAST interface (based on blast + 2.9.0+) integrated in GenomeMatcher 3.10 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.ige.tohoku.ac.jp/joho/portalsite/files/GenomeMatcher3.php\u003c/span\u003e\u003cspan class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), with the parameters E = 0.0, \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e≥\u003c/span\u003e 99% identity, and size \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e≥\u003c/span\u003e 5000 bp. To determine the whole-genome sequence of \u003cem\u003eL\u003c/em\u003e. \u003cem\u003ed\u003c/em\u003e. \u003cem\u003ejaponica\u003c/em\u003e and \u003cem\u003eL. umbrosa\u003c/em\u003e, genomic DNA was purified from the fat bodies of a single final-instar female larva of our laboratory strains established from \u003cem\u003eL\u003c/em\u003e. \u003cem\u003ed\u003c/em\u003e. \u003cem\u003ejaponica\u003c/em\u003e eggs collected in Sapporo, Hokkaido, and \u003cem\u003eL\u003c/em\u003e. \u003cem\u003eumbrosa\u003c/em\u003e eggs collected in Kitami, Hokkaido using the NucleoBond HMW DNA Kit (Macherey-Nagel, Düren, Germany), according to the manufacturer’s instructions. The whole-genome sequence was determined using PacBio long-read sequencing and whole-genome sequence assembly services provided by Relixa Corp. (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.rhelixa.com/service/pacbio-wgs/\u003c/span\u003e\u003cspan class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Libraries were constructed using the SMRTbell Express Template Prep Kit 2.0 (PACBIO), sequenced using the PacBio Sequel II sequencer (PacBio, Menlo Park, CA, USA), and assembled using PacBio CCS (Hifi read) software. Sequence read quality checks were performed using Nanoplot v0.11.7 software, and read assembly was conducted with Hifiasm v0.15.5-r350. All acquired data were registered in the public DNA Database of Japan (BioProject: PRJDB37639 [PSUB043264]). This analysis yielded 302 contigs (accession nos. BAAIJM010000001–BAAIJM010000302) derived from \u003cem\u003eL. d. japonica\u003c/em\u003e genome and 1115 contigs (accession nos. BAAIJN010000001–BAAIJN010001115) from \u003cem\u003eL. umbrosa\u003c/em\u003e genome. Contigs derived from the Z or W chromosome (see Additional file 30: Table \u003cspan class=\"InternalRef\"\u003eS12\u003c/span\u003e) of \u003cem\u003eL. d. japonica\u003c/em\u003e were identified as follows. Genomic DNA was purified from the fat bodies of one final-instar larva of each sex as described above and submitted to Relixa Corp. for whole-genome short-read sequencing (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.rhelixa.com/service/wgs/\u003c/span\u003e\u003cspan class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The obtained reads were mapped onto the 302 contigs using a data analysis service provided by Relixa Corp. Contigs showing average coverage values more than twice as high in males compared to females (longest, 44.9 Mb; total, 7) were assumed to be derived from the Z chromosome. Conversely, contigs showing average coverage values more than twice as high in females compared to males, or contigs with zero coverage in males (longest, 6.8 Mb; total, 198), were designated as W chromosome-derived contigs.\u003c/p\u003e\u003ch2\u003eMolecular Phylogenetic Analysis\u003c/h2\u003e\u003cp\u003eTo clarify the phylogenetic relationships of \u003cem\u003eLdMasc-W\u003c/em\u003e, a molecular phylogenetic analysis was conducted. BLASTn searches were performed using the nucleotide sequence of \u003cem\u003eLdMasc\u003c/em\u003e (GenBank accession no. LC817951.1) as a query sequence against the genome sequences of \u003cem\u003eL. d. japonica\u003c/em\u003e, \u003cem\u003eL. d. asiatica\u003c/em\u003e, and \u003cem\u003eL. d. dispar\u003c/em\u003e (GenBank: BAAIJM000000000.1, GCA_004115105.1, GCA_018258255.2). The obtained nucleotide sequences on the W chromosome were designated as \u003cem\u003eLdMasc-W\u003c/em\u003e, and the upstream sequence (5 kbp) adjacent to the 5' end of each \u003cem\u003eLdMasc-W\u003c/em\u003e was acquired. The acquired sequences were used to construct a phylogenetic tree using maximum likelihood methods with MAFFT and RAxML, employing the upstream sequence of intron 8 in \u003cem\u003eLdMasc\u003c/em\u003e as an outgroup. Bootstrap values were set to 100.\u003c/p\u003e\u003ch2\u003esiRNA synthesis\u003c/h2\u003e\u003cp\u003eTo identify nucleotide sequences unique to \u003cem\u003eLdMasc-W\u003c/em\u003e, a BLASTn search was performed against the entire spongy moth genome sequence using a nucleotide sequence of the region shared among all \u003cem\u003eLdMasc-W\u003c/em\u003e cDNA sequences as a query sequence. The resulting sequences were subjected to siRNA design using siDirect v2.1 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://sidirect2.rnai.jp/\u003c/span\u003e\u003cspan class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). siRNA synthesis was performed using the Custom Stealth RNAi siRNA Synthesis Service provided by Invitrogen (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.thermofisher.com/store/v2/oligos-rna?stealth=true\u003c/span\u003e\u003cspan class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The synthesized siRNA was dissolved in injection buffer (100 mM potassium acetate, 2 mM magnesium acetate, 30 mM HEPES-KOH; pH 7.4) to a final concentration of 100 µM, as described by Fukui et al. [\u003cspan class=\"CitationRef\"\u003e13\u003c/span\u003e]. The nucleotide sequences of siRNAs used in these experiments are listed in Additional file 31: Table \u003cspan class=\"InternalRef\"\u003eS13\u003c/span\u003e. siRNAs targeting \u003cem\u003eEgfp\u003c/em\u003e were used as a control; siRNAs were designed as previously described [\u003cspan class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e\u003ch2\u003eInjection of siRNAs into eggs\u003c/h2\u003e\u003cp\u003eEgg masses obtained within 12 h after oviposition were separated into individual eggs as described previously [\u003cspan class=\"CitationRef\"\u003e8\u003c/span\u003e]. The eggs were aligned in the same direction and fixed to the surface of a glass slide (Matsunami Glass, Osaka, Japan) using a gelatinous instant adhesive (Yamato Scientific, Hokkaido, Japan). siRNAs were injected into the eggs using glass capillary tubes (uMPm-02; Daiwa, Tokyo, Japan) connected to an electric microinjector (FemtoJet 5247; Eppendorf, Hamburg, Germany), at an injection pressure of 280–340 psi, injection time of 0.1–0.3 s, and a compensation pressure of 30 psi. The hole created by the injection was sealed with a gelatinous instant adhesive, and the eggs were stored in a controlled-temperature chamber at 25°C and 60 ± 10% humidity. Then, the eggs were individually collected into 1.5-mL microtubes and stored in a − 80°C deep freezer.\u003c/p\u003e\u003ch2\u003eStatistical analyses\u003c/h2\u003e\u003cp\u003eSome statistical analyses were performed using the Easy R (EZR) software, version 1.60 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.jichi.ac.jp/saitama-sct/SaitamaHP.files/download.html\u003c/span\u003e\u003cspan class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e, accessed September 6, 2021). A Shapiro–Wilk test was used to evaluate the normality of the data obtained in each experiment. Since the sample size was less than 25 and the data did not show a normal distribution, the Mann–Whitney U test was used to examine the significance of differences between the two groups. Other statistical analyses were conducted with R ver. 4.4.1 [\u003cspan class=\"CitationRef\"\u003e45\u003c/span\u003e]. Significant differences between the UJ-hybrid F1 and other lines were examined using the Kruskal-Wallis test and Dunn's post hoc test with Bonferroni correction.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eDeclarations\u003c/h2\u003e \u003cp\u003e \u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e \u003cp\u003eNot applicable.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent for publication\u003c/strong\u003e \u003cp\u003eNot applicable.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eCompeting interests\u003c/strong\u003e \u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis research was funded by Grants-in-Aid for Scientific Research (B) (no. 24K01766) and a Japan Society for the Promotion of Science (JSPS) KAKENHI (A) grant (no. 25H01423) to MGS.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eConceptualization: KS, RK and MGS. Data curation and formal analysis: KS, KI, RK and MGS. Funding acquisition: MGS. Investigation and methodology: KS, KI, RK, HN and MGS. Supervision: MGS. Writing original draft: KS and KI. All authors read and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe are grateful to Mr. Kota Aoki for rearing the spongy moth larvae and teaching us how to construct the cylindrical nets used for mating adults of the spongy moth.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eAll data generated and analyzed during the course of this study are included in this article and its supplementary information files. For some analyses, data from publicly available repositories were used. All raw sequence data obtained by whole-genome sequence (WGS) analyses of *L. d. japonica* and *L. umbrosa* in this study have been deposited in the DDBJ Sequence Read Archive (DRA) database under the BioProject accession number PRJDB37639. The nucleotide sequences of contigs determined by the WGS analyses have been registered in DDBJ under accession numbers BAAIJM010000001- BAAIJM010000315 ( *L. d. japonica* ) and BAAIJN010000001- BAAIJM010001381( *L. umbrosa* ). The nine mRNA sequences obtained by the 5'RACE in this study have been deposited in DDBJ under accession numbers LC889978\u0026ndash;LC889986.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eMyosho T, Otake H, Masuyama H, Matsuda M, Kuroki Y, Fujiyama A, et al. Tracing the emergence of a novel sex-determining gene in medaka, \u003cem\u003eOryzias luzonensis\u003c/em\u003e. Genetics. 2012;191:163\u0026ndash;70.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLee J, Fujimoto T, Yamaguchi K, Shigenobu S, Sahara K, Toyoda A, et al. W chromosome sequences of two bombycid moths provide an insight into the origin of \u003cem\u003eFem\u003c/em\u003e. Mol Ecol. 2024;33:e17434.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBoukouvala MC, Kavallieratos NG, Skourti A, Pons X, Alonso CL, Eizaguirre M, et al. \u003cem\u003eLymantria dispar\u003c/em\u003e (L.) (Lepidoptera: Erebidae): current status of biology, ecology, and management in Europe with notes from North America. Insects. 2022;13:854.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGoldschmidt R. \u0026ldquo;Lymantria\u0026rdquo;. Bibliographia Genetica. 1934;111:1\u0026ndash;185.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGoldschmidt R. Untersuchungen \u0026uuml;ber Intersexualit\u0026auml;t. V. Z. Induct Abstl. 1930;56: 257\u0026ndash;301.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGoldschmidt R. The Material Basis of Evolution, New Haven CT: Yale University Press; 1940.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHigashiura Y, Yamaguchi H, Ishihara M, Ono N, Tsukagoshi H, Yokobori S, et al. Male death resulting from hybridization between subspecies of the gypsy moth, \u003cem\u003eLymantria dispar\u003c/em\u003e. Heredity (Edinb). 2011;106:603\u0026ndash;13.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMoronuki Y, Kasahara R, Naka H, Suzuki MG. Identification and functional analysis of sex-determining genes in the spongy moth, \u003cem\u003eLymantria dispar\u003c/em\u003e (Lepidoptera: Erebidae). Insect Biochem Mol Biol. 2025;177:104219.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKiuchi T, Koga H, Kawamoto M, Shoji K, Sakai H, Arai Y, et al. A single female-specific piRNA is the primary determiner of sex in the silkworm. Nature. 2014;509:633\u0026ndash;636.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSakai H, Sakaguchi H, Aoki F, Suzuki MG. Functional analysis of sex-determination genes by gene silencing with LNA-DNA gapmers in the silkworm, \u003cem\u003eBombyx mori\u003c/em\u003e. Mech Dev. 2015;137:45\u0026ndash;52.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSakai H, Sumitani M, Chikami Y, Yahata K, Uchino K, Kiuchi T, et al. Transgenic expression of the piRNA-resistant \u003cem\u003eMasculinizer\u003c/em\u003e gene induces female-specific lethality and partial female-to-male sex reversal in the silkworm, \u003cem\u003eBombyx mori\u003c/em\u003e. PLoS Genet. 2016;12:e1006203.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLee J, Kiuchi T, Kawamoto M, Shimada T, Katsuma S. Identification and functional analysis of a \u003cem\u003eMasculinizer\u003c/em\u003e orthologue in \u003cem\u003eTrilocha varians\u003c/em\u003e (Lepidoptera: Bombycidae). Insect Mol Biol. 2015;24:561\u0026ndash;569.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFukui T, Kiuchi T, Shoji K, Kawamoto M, Shimada T, Katsuma S. In vivo masculinizing function of the \u003cem\u003eOstrinia furnacalis Masculinizer\u003c/em\u003e gene. Biophys Res Commun. 2018;503:1768\u0026ndash;1772.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHarvey-Samuel T, Norman VC, Carter R, Lovett E, Alphey L. Identification and characterization of a \u003cem\u003eMasculinizer\u003c/em\u003e homologue in the diamondback moth, \u003cem\u003ePlutella xylostella\u003c/em\u003e. Insect Mol Biol. 2020;29:231\u0026ndash;240.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVisser S, Volen\u0026iacute;kov\u0026aacute; A, Nguyen P, Verhulst EC, Marec F. A conserved role of the duplicated \u003cem\u003eMasculinizer\u003c/em\u003e gene in sex determination of the Mediterranean flour moth, \u003cem\u003eEphestia kuehniella\u003c/em\u003e. PLoS Genet. 2021;17:e1009420.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePosp\u0026iacute;šilov\u0026aacute; K, Van't Hof AE, Yoshido A, Kruž\u0026iacute;kov\u0026aacute; R, Visser S, Zrzav\u0026aacute; M, et al. \u003cem\u003eMasculinizer\u003c/em\u003e gene controls male sex determination in the codling moth, \u003cem\u003eCydia pomonella\u003c/em\u003e. Insect Biochem Mol Biol. 2023:103991.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHarvey-Samuel T, Xu X., Anderson MAE, Carabajal Paladino LZ, Purusothaman D, et al. Silencing RNAs expressed from W-linked PxyMasc \u0026ldquo;retrocopies\u0026rdquo; target that gene during female sex determination in \u003cem\u003ePlutella xylostella\u003c/em\u003e. Proc Natl Acad Sci. 2022;119:e2206025119.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYuzawa T, Matsuoka M, Sumitani M, Aoki F, Sezutsu H, Suzuki MG. Transgenic and knockout analyses of \u003cem\u003eMasculinizer\u003c/em\u003e and \u003cem\u003edoublesex\u003c/em\u003e illuminated the unique functions of \u003cem\u003edoublesex\u003c/em\u003e in germ cell sexual development of the silkworm, \u003cem\u003eBombyx mori\u003c/em\u003e. BMC Dev Biol. 2020;20:19.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFukui T, Shoji K, Kiuchi T, Suzuki Y, Katsuma S. \u003cem\u003eMasculinizer\u003c/em\u003e is not post-transcriptionally regulated by female-specific piRNAs during sex determination in the Asian corn borer, \u003cem\u003eOstrinia furnacalis\u003c/em\u003e. Insect Biochem Mol Biol. 2023;156:103946.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVisser S, Yoshido A, Provazn\u0026iacute;kov\u0026aacute; I, Dal\u0026iacute;kov\u0026aacute; M, Voř\u0026iacute;škov\u0026aacute; D, Chung Volen\u0026iacute;kov\u0026aacute; A, et al. A W chromosome-derived feminizing piRNA in pyralid moths demonstrates convergent evolution for primary sex determination signals in Lepidoptera. BMC Biol. 2025;23:289.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKatsuma S, Sugano Y, Kiuchi T, Shimada T. Two conserved cysteine residues are required for the masculinizing activity of the silkworm Masc protein. J Biol Chem. 2015;290:26114\u0026ndash;26124.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKatsuma S, Kiuchi T, Kawamoto M, Fujimoto T, Sahara K. Unique sex determination system in the silkworm, \u003cem\u003eBombyx mori\u003c/em\u003e: current status and beyond. Proc Jpn Acad Ser B Phys Biol Sci. 2018;94:205\u0026ndash;216.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBelote JM, Lucchesi JC. Control of X chromosome transcription by the maleless gene in Drosophila. Nature. 1980;285:573\u0026ndash;5.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePlenefisch JD, DeLong L, Meyer BJ. Genes that implement the hermaphrodite mode of dosage compensation in Caenorhabditis elegans. Genetics. 1989;121:57\u0026ndash;76.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTakagi N, Abe K. Detrimental effects of two active X chromosomes on early mouse development. Development. 1990;109:189\u0026ndash;201.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSugimoto TN, Kayukawa T, Shinoda T, Ishikawa Y, Tsuchida T. Misdirection of dosage compensation underlies bidirectional sex-specific death in \u003cem\u003eWolbachia\u003c/em\u003e-infected \u003cem\u003eOstrinia scapulalis\u003c/em\u003e. Insect Biochem Mol Biol. 2015;66:72\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eArai H, Takamatsu T, Lin SR, Mizutani T, Omatsu T, Katayama Y, et al. Diverse molecular mechanisms underlying microbe-inducing male killing in the moth \u003cem\u003eHomona magnanima\u003c/em\u003e. Appl Environ Microbiol. 2023;89:e0209522.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLed\u0026oacute;n-Rettig CC, Zattara EE, Moczek AP. Asymmetric interactions between doublesex and tissue- and sex-specific target genes mediate sexual dimorphism in beetles. Nat Commun. 2017;8:14593.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMatsuda M, Shinomiya A, Kinoshita M, Suzuki A, Kobayashi T, Paul-Prasanth B, et al. DMY gene induces male development in genetically female (XX) medaka fish. Proc Natl Acad Sci. 2007;104:3865\u0026ndash;3870.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHerpin A, Schartl M. Molecular mechanisms of sex determination and evolution of the Y-chromosome: insights from the medakafish (\u003cem\u003eOryzias latipes\u003c/em\u003e). Mol Cell Endocrinol. 2009;306:51\u0026ndash;58.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKondo M, Nanda I, Schmid M, Schartl M. Sex determination and sex chromosome evolution: insights from medaka. Sex Dev. 2009;3:88\u0026ndash;98.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKopp A. Dmrt genes in the development and evolution of sexual dimorphism. Trends Genet. 2012;28:175\u0026ndash;184.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYano A, Nicol B, Jouanno E, Quillet E, Fostier A, Guyomard R, Guiguen Y. The sexually dimorphic on the Y-chromosome gene (sdY) is a conserved male-specific Y-chromosome sequence in many salmonids. Evol Appl. 2013;6:486\u0026ndash;496.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYoshimoto S, Okada E, Umemoto H, Tamura K, Uno Y, Nishida-Umehara C, et al. A W-linked DM-domain gene, DM-W, participates in primary ovary development in \u003cem\u003eXenopus laevis\u003c/em\u003e. Proc Natl Acad Sci. 2008;105:2469\u0026ndash;2474.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYoshimoto S, Okada E, Oishi T, Numagami R, Umemoto H, Tamura K, et al. Expression and promoter analysis of \u003cem\u003eXenopus\u003c/em\u003e DMRT1 and functional characterization of the transactivation property of its protein. Dev Growth Differ. 2006;48:597\u0026ndash;603.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLoehlin DW, Carroll SB. Expression of tandem gene duplicates is often greater than twofold. Proc Natl Acad Sci. 2016;113:5988\u0026ndash;5992.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBlaser O, Neuenschwander S, Perrin N. Sex-chromosome turnovers: the hot‐potato model. Am Nat. 2014;183:140\u0026ndash;146.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGammerdinger WJ, Conte MA, Sandkam BA, Ziegelbecker A, Koblm\u0026uuml;ller S, Kocher TD. Novel sex chromosomes in 3 cichlid fishes from Lake Tanganyika. J Hered. 2018;109:489\u0026ndash;500.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJeffries DL, Lavanchy G, Sermier R, Sredl MJ, Miura I, Borz\u0026eacute;e A, et al. A rapid rate of sex-chromosome turnover and non-random transitions in true frogs. Nat Commun. 2018;9:4088.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVicoso B. Molecular and evolutionary dynamics of animal sex-chromosome turnover. Nat Ecol Evol. 2019;3:1632\u0026ndash;1641.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJeffries DL, Mee JA, Peichel CL. Identification of a candidate sex determination gene in \u003cem\u003eCulaea inconstans\u003c/em\u003e suggests convergent recruitment of an Amh duplicate in two lineages of stickleback. J Evol Biol. 2022;35:1683\u0026ndash;1695.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eArimoto M, Iwaizumi R. Identification of Japanese \u003cem\u003eLymantria\u003c/em\u003e species (Lepidoptera: Lymantriidae) based on PCR-RFLP analysis of mitochondorial DNA. Appl Entomol Zool. 2014;49:159\u0026ndash;169.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYin J, Sun L, Zhang Q, Cao C. Screening and evaluation of the stability of expression of reference genes in \u003cem\u003eLymantria dispar\u003c/em\u003e (Lepidoptera: Erebidae) using qRT-PCR. Gene. 2020;749:144712.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBelousova I, Ershov N, Pavlushin S, Ilinsky Y, Martemyanov V. Molecular sexing of Lepidoptera. J Insect Physiol. 2019;114:53\u0026ndash;56.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eR Development Core Teeam. R: a language and environment for statistical computing. 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We do not accept new or revised documents on the basis of requests to \u0026lsquo;check only marked text\u0026rsquo;.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [BMC Biology](https://bmcbiol.biomedcentral.com/)","snPcode":"12915","submissionUrl":"https://submission.springernature.com/new-submission/12915/3","title":"BMC Biology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"doublesex, Lymantria dispar, Masculinizer, sex determination, W chromosome","lastPublishedDoi":"10.21203/rs.3.rs-8963102/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8963102/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eSex determination is a common developmental process in most organisms that exhibit sexual reproduction. Nevertheless, the modes of sex determination are highly diverse, and master sex-determining genes sometimes differ substantially even among closely related species. We explored the molecular basis underlying this striking diversity in the spongy moth, \u003cem\u003eLymantria dispar\u003c/em\u003e, whose master sex-determining factors exhibit continuous variation among populations.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eWe previously identified the female-determining gene \u003cem\u003eFet-W\u003c/em\u003e and the male-determining gene \u003cem\u003eLdMasc\u003c/em\u003e in the Japanese spongy moth (\u003cem\u003eLymantria dispar japonica\u003c/em\u003e). The number of raw RNA sequencing reads mapped to the \u003cem\u003eLdMasc\u003c/em\u003e coding sequence region corresponding to exons 9\u0026ndash;11 was higher in females than in males. Consistent with this finding, we identified four unigenes composed of the nucleotide sequences from \u003cem\u003eLdMasc\u003c/em\u003e exons 9\u0026ndash;11. Reverse-transcript polymerase chain reaction (RT-PCR) and genomic PCR using primers specific to these unigenes strongly indicated that these four unigenes originated from the W-linked gene. A nucleotide BLAST search across the entire \u003cem\u003eL\u003c/em\u003e. \u003cem\u003ed\u003c/em\u003e. \u003cem\u003ejaponica\u003c/em\u003e genome found at least 52 copies of nucleotide sequences nearly identical to the approximately 5.6-kb genomic sequence spanning intron 8 to exon 11 of \u003cem\u003eLdMasc\u003c/em\u003e. Similar sequences were identified in the W chromosome contigs of \u003cem\u003eLymantria dispar dispar\u003c/em\u003e and \u003cem\u003eLymantria dispar asiatica\u003c/em\u003e, with 4 and 6 copies, respectively. We considered these sequences to be W chromosome paralogs of \u003cem\u003eLdMasc\u003c/em\u003e and named them \u003cem\u003eLdMasc-W\u003c/em\u003e. RT-PCR analysis demonstrated that \u003cem\u003eLdMasc-W\u003c/em\u003e exhibits specific expression during the sex determination stage. Knockdown of \u003cem\u003eLdMasc-W\u003c/em\u003e using embryonic RNA induced male-specific splicing of \u003cem\u003edoublesex\u003c/em\u003e (\u003cem\u003edsx\u003c/em\u003e), a master regulatory gene for sexual development, in females. F1 hybrid females obtained by mating females of \u003cem\u003eL. umbrosa\u003c/em\u003e, which that lacks \u003cem\u003eLdMasc-W\u003c/em\u003e, with \u003cem\u003eL. d. japonica\u003c/em\u003e males exhibited lethality or intersexual phenotypes.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eThese results strongly suggest that \u003cem\u003eLdMasc-W\u003c/em\u003e functions to promote female determination by suppressing the expression of the male-determining gene \u003cem\u003eLdMasc\u003c/em\u003e, similar to the female-determining gene \u003cem\u003eFem\u003c/em\u003e identified in other Lepidoptera species. This means that the spongy moth is a rare species possessing two W-linked female-determining genes: \u003cem\u003eFet-W\u003c/em\u003e and \u003cem\u003eLdMasc-W\u003c/em\u003e.\u003c/p\u003e","manuscriptTitle":"W chromosome-specific paralogs of the male-determining gene LdMasc exhibits a female- determining ability in the spongy moth, Lymantria dispar","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-25 08:28:17","doi":"10.21203/rs.3.rs-8963102/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2026-04-11T12:13:25+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-08T05:44:57+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-01T22:51:32+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"159548325275294847973978913699911918240","date":"2026-03-24T13:42:58+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"199286272916953327002230922464859463873","date":"2026-03-23T06:39:36+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"111194790432648125702910155300782655813","date":"2026-03-21T11:24:45+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"18175334447378055021994303564497744470","date":"2026-03-21T10:45:41+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"287086354239363549915145628937158640851","date":"2026-03-20T21:39:19+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-03-20T16:45:12+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-02-25T19:25:07+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-02-25T07:21:24+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Biology","date":"2026-02-25T04:24:31+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [BMC Biology](https://bmcbiol.biomedcentral.com/)","snPcode":"12915","submissionUrl":"https://submission.springernature.com/new-submission/12915/3","title":"BMC Biology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"f97071c8-be07-4c94-9ef3-eaac744b4c61","owner":[],"postedDate":"March 25th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-03-25T08:28:19+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-25 08:28:17","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8963102","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8963102","identity":"rs-8963102","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}