Zygosity-based sex determination in a butterfly drives hypervariability of Masculinizer.

Nature has devised many ways of producing males and females. Here, we report on a previously undescribed mechanism for Lepidoptera that functions without a female-specific gene. The number of alleles or allele heterozygosity in a single Z-linked gene (BaMasc) is the primary sex-determining switch in Bicyclus anynana butterflies. Embryos carrying a single BaMasc allele develop into WZ (or Z0) females, those carrying two distinct alleles develop into ZZ males, while (ZZ) homozygotes initiate female development, have mismatched dosage compensation, and die as embryos. Consequently, selection against homozygotes has favored the evolution of spectacular allelic diversity: 205 different coding sequences of BaMasc were detected in a sample of 246 females. The structural similarity of a hypervariable region (HVR) in BaMasc to the HVR in Apis mellifera csd suggests molecular convergence between deeply diverged insect lineages. Our discovery of this primary switch highlights the fascinating diversity of sex-determining mechanisms and underlying evolutionary drivers.

Complete male-to-female sex reversal in XY mice lacking the miR-17~92 cluster.

Mammalian sex determination is controlled by antagonistic gene cascades operating in embryonic undifferentiated gonads. The expression of the Y-linked gene SRY is sufficient to trigger the testicular pathway, whereas its absence in XX embryos leads to ovarian differentiation. Yet, the potential involvement of non-coding regulation in this process remains unclear. Here we show that the deletion of a single microRNA cluster, miR-17~92, induces complete primary male-to-female sex reversal in XY mice. Sry expression is delayed in XY knockout gonads, which develop as ovaries. Sertoli cell differentiation is reduced, delayed and unable to sustain testicular development. Pre-supporting cells in mutant gonads undergo a transient state of sex ambiguity which is subsequently resolved towards the ovarian fate. The miR-17~92 predicted target genes are upregulated, affecting the fine regulation of gene networks controlling gonad development. Thus, microRNAs emerge as key components for mammalian sex determination, controlling Sry expression timing and Sertoli cell differentiation.

Identification of gene isoforms and their switching events between male and female embryos of the parthenogenetic crustacean Daphnia magna.

The cladoceran crustacean Daphnia exhibits phenotypic plasticity, a phenomenon that leads to diverse phenotypes from one genome. Alternative usage of gene isoforms has been considered a key gene regulation mechanism for controlling different phenotypes. However, to understand the phenotypic plasticity of Daphnia, gene isoforms have not been comprehensively analyzed. Here we identified 25,654 transcripts derived from the 9710 genes expressed during environmental sex determination of Daphnia magna using the long-read RNA-Seq with PacBio Iso-Seq. We found that 14,924 transcripts were previously unidentified and 5713 genes produced two or more isoforms. By a combination of Illumina short-read RNA-Seq, we detected 824 genes that implemented switching of the highest expressed isoform between females and males. Among the 824 genes, we found isoform switching of an ortholog of CREB-regulated transcription coactivator, a major regulator of carbohydrate metabolism in animals, and a correlation of this switching event with the sexually dimorphic expression of carbohydrate metabolic genes. These results suggest that a comprehensive catalog of isoforms may lead to understanding the molecular basis for environmental sex determination of Daphnia. We also infer the applicability of the full-length isoform analyses to the elucidation of phenotypic plasticity in Daphnia.

A maternal-to-zygotic-transition gene block on the zebrafish sex chromosome.

Wild zebrafish (Danio rerio) have a ZZ/ZW chromosomal sex-determination system with the major sex locus on the right arm of chromosome-4 (Chr4R) near the largest heterochromatic block in the genome, suggesting that Chr4R transcriptomics might differ from the rest of the genome. To test this hypothesis, we conducted an RNA-seq analysis of adult ZW ovaries and ZZ testes in the Nadia strain and identified 4 regions of Chr4 with different gene expression profiles. Unique in the genome, protein-coding genes in a 41.7 Mb section (Region-2) were expressed in testis but silent in ovary. The AB lab strain, which lacks sex chromosomes, verified this result, showing that testis-biased gene expression in Region-2 depends on gonad biology, not on sex-determining mechanism. RNA-seq analyses in female and male brains and livers validated reduced transcripts from Region-2 in somatic cells, but without sex specificity. Region-2 corresponds to the heterochromatic portion of Chr4R and its content of genes and repetitive elements distinguishes it from the rest of the genome. Region-2 lacks protein-coding genes with human orthologs; has zinc finger genes expressed early in zygotic genome activation; has maternal 5S rRNA genes, maternal spliceosome genes, a concentration of tRNA genes, and a distinct set of repetitive elements. The colocalization of (1) genes silenced in ovaries but not in testes that are (2) expressed in embryos briefly at the onset of zygotic genome activation; (3) maternal-specific genes for translation machinery; (4) maternal-specific spliceosome components; and (5) adjacent genes encoding miR-430, which mediates maternal transcript degradation, suggest that this is a maternal-to-zygotic-transition gene regulatory block.

Candidate target genes of the male-specific expressed Doublesex in the termite Reticulitermes speratus.

Eusocial insects such as termites, ants, bees, and wasps exhibit a reproductive division of labor. The developmental regulation of reproductive organ (ovaries and testes) is crucial for distinguishing between reproductive and sterile castes. The development of reproductive organ in insects is regulated by sex-determination pathways. The sex determination gene Doublesex (Dsx), encoding transcription factors, plays an important role in this pathway. Therefore, clarifying the function of Dsx in the developmental regulation of sexual traits is important to understand the social evolution of eusocial insects. However, no studies have reported the function of Dsx in hemimetabolous eusocial group termites. In this study, we searched for binding sites and candidate target genes of Dsx in species with available genome information as the first step in clarifying the function of Dsx in termites. First, we focused on the Reticulitermes speratus genome and identified 101 candidate target genes of Dsx. Using a similar method, we obtained 112, 39, and 76 candidate Dsx target genes in Reticulitermes lucifugus, Coptotermes formosanus, and Macrotermes natalensis, respectively. Second, we compared the candidate Dsx target genes between species and identified 37 common genes between R. speratus and R. lucifugus. These included several genes probably involved in spermatogenesis and longevity. However, only a few common target genes were identified between R. speratus and the other two species. Finally, Dsx dsRNA injection resulted in the differential expression of several target genes, including piwi-like protein and B-box type zinc finger protein ncl-1 in R. speratus. These results provide valuable resource data for future functional analyses of Dsx in termites.

Overview of chicken embryo genes related to sex differentiation.

Sex determination in chickens at an early embryonic stage has been a longstanding challenge in poultry production due to the unique ZZ:ZW sex chromosome system and various influencing factors. This review has summarized the genes related to the sex differentiation of chicken early embryos (mainly Dmrt1, Sox9, Amh, Cyp19a1, Foxl2, Tle4z1, Jun, Hintw, Ube2i, Spin1z, Hmgcs1, Foxd1, Tox3, Ddx4, cHemgn and Serpinb11 in this article), and has found that these contributions enhance our understanding of the genetic basis of sex determination in chickens, while identifying potential gene targets for future research. This knowledge may inform and guide the development of sex screening technologies for hatching eggs and support advancements in gene-editing approaches for chicken embryos. Moreover, these insights offer hope for enhancing animal welfare and promoting conservation efforts in poultry production.

Repeated co-option of HMG-box genes for sex determination in brown algae and animals.

In many eukaryotes, genetic sex determination is not governed by XX/XY or ZW/ZZ systems but by a specialized region on the poorly studied U (female) or V (male) sex chromosomes. Previous studies have hinted at the existence of a dominant male-sex factor on the V chromosome in brown algae, a group of multicellular eukaryotes distantly related to animals and plants. The nature of this factor has remained elusive. Here, we demonstrate that an HMG-box gene acts as the male-determining factor in brown algae, mirroring the role HMG-box genes play in sex determination in animals. Over a billion-year evolutionary timeline, these lineages have independently co-opted the HMG box for male determination, representing a paradigm for evolution's ability to recurrently use the same genetic "toolkit" to accomplish similar tasks.

Dmrt1 is the only male pathway gene tested indispensable for sex determination and functional testis development in tilapia.

Sex is determined by multiple factors derived from somatic and germ cells in vertebrates. We have identified amhy, dmrt1, gsdf as male and foxl2, foxl3, cyp19a1a as female sex determination pathway genes in Nile tilapia. However, the relationship among these genes is largely unclear. Here, we found that the gonads of dmrt1;cyp19a1a double mutants developed as ovaries or underdeveloped testes with no germ cells irrespective of their genetic sex. In addition, the gonads of dmrt1;cyp19a1a;cyp19a1b triple mutants still developed as ovaries. The gonads of foxl3;cyp19a1a double mutants developed as testes, while the gonads of dmrt1;cyp19a1a;foxl3 triple mutants eventually developed as ovaries. In contrast, the gonads of amhy;cyp19a1a, gsdf;cyp19a1a, amhy;foxl2, gsdf;foxl2 double and amhy;cyp19a1a;cyp19a1b, gsdf;cyp19a1a;cyp19a1b triple mutants developed as testes with spermatogenesis via up-regulation of dmrt1 in both somatic and germ cells. The gonads of amhy;foxl3 and gsdf;foxl3 double mutants developed as ovaries but with germ cells in spermatogenesis due to up-regulation of dmrt1. Taking the respective ovary and underdeveloped testis of dmrt1;foxl3 and dmrt1;foxl2 double mutants reported previously into consideration, we demonstrated that once dmrt1 mutated, the gonad could not be rescued to functional testis by mutating any female pathway gene. The sex reversal caused by mutation of male pathway genes other than dmrt1, including its upstream amhy and downstream gsdf, could be rescued by mutating female pathway gene. Overall, our data suggested that dmrt1 is the only male pathway gene tested indispensable for sex determination and functional testis development in tilapia.

Inefficient Sox9 upregulation and absence of Rspo1 repression lead to sex reversal in the B6.XYTIR mouse gonad†.

Sry on the Y-chromosome upregulates Sox9, which in turn upregulates a set of genes such as Fgf9 to initiate testicular differentiation in the XY gonad. In the absence of Sry expression, genes such as Rspo1, Foxl2, and Runx1 support ovarian differentiation in the XX gonad. These two pathways antagonize each other to ensure the development of only one gonadal sex in normal development. In the B6.YTIR mouse, carrying the YTIR-chromosome on the B6 genetic background, Sry is expressed in a comparable manner with that in the B6.XY mouse, yet, only ovaries or ovotestes develop. We asked how testicular and ovarian differentiation pathways interact to determine the gonadal sex in the B6.YTIR mouse. Our results showed that (1) transcript levels of Sox9 were much lower than in B6.XY gonads while those of Rspo1 and Runx1 were as high as B6.XX gonads at 11.5 and 12.5 days postcoitum. (2) FOXL2-positive cells appeared in mosaic with SOX9-positive cells at 12.5 days postcoitum. (3) SOX9-positive cells formed testis cords in the central area while those disappeared to leave only FOXL2-positive cells in the poles or the entire area at 13.5 days postcoitum. (4) No difference was found at transcript levels of all genes between the left and right gonads up to 12.5 days postcoitum, although ovotestes developed much more frequently on the left than the right at 13.5 days postcoitum. These results suggest that inefficient Sox9 upregulation and the absence of Rspo1 repression prevent testicular differentiation in the B6.YTIR gonad.

Heteromorphic ZZ/ZW sex chromosomes sharing gene content with mammalian XX/XY are conserved in Madagascan chameleons of the genus Furcifer.

Chameleons are well-known lizards with unique morphology and physiology, but their sex determination has remained poorly studied. Madagascan chameleons of the genus Furcifer have cytogenetically distinct Z and W sex chromosomes and occasionally Z1Z1Z2Z2/Z1Z2W multiple neo-sex chromosomes. To identify the gene content of their sex chromosomes, we microdissected and sequenced the sex chromosomes of F. oustaleti (ZZ/ZW) and F. pardalis (Z1Z1Z2Z2/Z1Z2W). In addition, we sequenced the genomes of a male and a female of F. lateralis (ZZ/ZW) and F. pardalis and performed a comparative coverage analysis between the sexes. Despite the notable heteromorphy and distinctiveness in heterochromatin content, the Z and W sex chromosomes share approximately 90% of their gene content. This finding demonstrates poor correlation of the degree of differentiation of sex chromosomes at the cytogenetic and gene level. The test of homology based on the comparison of gene copy number variation revealed that female heterogamety with differentiated sex chromosomes remained stable in the genus Furcifer for at least 20 million years. These chameleons co-opted for the role of sex chromosomes the same genomic region as viviparous mammals, lacertids and geckos of the genus Paroedura, which makes these groups excellent model for studies of convergent and divergent evolution of sex chromosomes.

Diversity and Convergence of Sex-Determination Mechanisms in Teleost Fish.

Sexual reproduction is prevalent across diverse taxa. However, sex-determination mechanisms are so diverse that even closely related species often differ in sex-determination systems. Teleost fish is a taxonomic group with frequent turnovers of sex-determining mechanisms and thus provides us with great opportunities to investigate the molecular and evolutionary mechanisms underlying the turnover of sex-determining systems. Here, we compile recent studies on the diversity of sex-determination mechanisms in fish. We demonstrate that genes in the TGF-ß signaling pathway are frequently used for master sex-determining (MSD) genes. MSD genes arise via two main mechanisms, duplication-and-transposition and allelic mutations, with a few exceptions. We also demonstrate that temperature influences sex determination in many fish species, even those with sex chromosomes, with higher temperatures inducing differentiation into males in most cases. Finally, we review theoretical models for the turnover of sex-determining mechanisms and discuss what questions remain elusive.

The -KTS splice variant of WT1 is essential for ovarian determination in mice.

Sex determination in mammals depends on the differentiation of the supporting lineage of the gonads into Sertoli or pregranulosa cells that govern testis and ovary development, respectively. Although the Y-linked testis-determining gene Sry has been identified, the ovarian-determining factor remains unknown. In this study, we identified -KTS, a major, alternatively spliced isoform of the Wilms tumor suppressor WT1, as a key determinant of female sex determination. Loss of -KTS variants blocked gonadal differentiation in mice, whereas increased expression, as found in Frasier syndrome, induced precocious differentiation of ovaries independently of their genetic sex. In XY embryos, this antagonized Sry expression, resulting in male-to-female sex reversal. Our results identify -KTS as an ovarian-determining factor and demonstrate that its time of activation is critical in gonadal sex differentiation.

Bivalves as Emerging Model Systems to Study the Mechanisms and Evolution of Sex Determination: A Genomic Point of View.

Bivalves are a diverse group of molluscs that have recently attained a central role in plenty of biological research fields, thanks to their peculiar life history traits. Here, we propose that bivalves should be considered as emerging model systems also in sex-determination (SD) studies, since they would allow to investigate: 1) the transition between environmental and genetic SD, with respect to different reproductive backgrounds and sexual systems (from species with strict gonochorism to species with various forms of hermaphroditism); 2) the genomic evolution of sex chromosomes (SCs), considering that no heteromorphic SCs are currently known and that homomorphic SCs have been identified only in a few species of scallops; 3) the putative role of mitochondria at some level of the SD signaling pathway, in a mechanism that may resemble the cytoplasmatic male sterility of plants; 4) the evolutionary history of SD-related gene (SRG) families with respect to other animal groups. In particular, we think that this last topic may lay the foundations for expanding our understanding of bivalve SD, as our current knowledge is quite fragmented and limited to a few species. As a matter of fact, tracing the phylogenetic history and diversity of SRG families (such as the Dmrt, Sox, and Fox genes) would allow not only to perform more targeted functional experiments and genomic analyses, but also to foster the possibility of establishing a solid comparative framework.

DMRT1 is a testis-determining gene in rabbits and is also essential for female fertility.

DMRT1 is the testis-determining factor in several species of vertebrates, but its involvement in mammalian testes differentiation, where SRY is the testis-determining gene, remains ambiguous. So far, DMRT1 loss-of-function has been described in two mammalian species and induces different phenotypes: Disorders of Sex Development (46, XY DSD) in men and male infertility in mice. We thus abolished DMRT1 expression by CRISPR/Cas9 in a third species of mammal, the rabbit. First, we observed that gonads from XY DMRT1-/- rabbit fetuses differentiated like ovaries, highlighting that DMRT1 is involved in testis determination. In addition to SRY, DMRT1 is required in the supporting cells to increase the expression of the SOX9 gene, which heads the testicular genetic cascade. Second, we highlighted another function of DMRT1 in the germline since XX and XY DMRT1-/- ovaries did not undergo meiosis and folliculogenesis. XX DMRT1-/- adult females were sterile, showing that DMRT1 is also crucial for female fertility. To conclude, these phenotypes indicate an evolutionary continuum between non-mammalian vertebrates such as birds and non-rodent mammals. Furthermore, our data support the potential involvement of DMRT1 mutations in different human pathologies, such as 46, XY DSD as well as male and female infertility.

Animals that reproduce sexually have organs called gonads, the ovaries and testes, which produce eggs and sperm. These organs, which are different in males and females, originate from the same cells during the development of the embryo. As a general rule, the chromosomal sex of an embryo, which gets determined at fertilization, leads to the activation and repression of specific genes. This in turn, controls whether the cells that will form the gonads will differentiate to develop testes or ovaries. Disruption of the key genes involved in the differentiation of the gonads can lead to fertility problems, and in some cases, it can cause the gonads to develop in the 'opposite' direction, resulting in a sex reversal. Identifying these genes is therefore essential to know how to maintain or restore fertility. DMRT1 is a gene that drives the differentiation of gonadal cells into the testicular pathway in several species of animals with backbones, including species of fish, frogs and birds. However, its role in mammals ­ where testis differentiation is driven by a different gene called SRY ­ is not well understood. Indeed, when DMRT1 is disrupted in male humans it leads to disorders of sex development, while disrupting this gene in male mice causes infertility. To obtain more information about the roles of DMRT1 in mammalian species, Dujardin et al. disrupted the gene in a third species of mammal: the rabbit. Dujardin et al. observed that chromosomally-male rabbits lacking DMRT1 developed ovaries instead of testes, showing that in rabbits, both SRY and DMRT1 are both required to produce testes. Additionally, this effect is similar to what is seen in humans, suggesting that rabbits may be a better model for human gonadal differentiation than mice are. Additionally, Dujardin et al. were also able to show that in female rabbits, lack of DMRT1 led to infertility, an effect that had not been previously described in other species. The results of Dujardin et al. may lead to better models for gonadal development in humans, involving DMRT1 in the differentiation of testes. Interestingly, they also suggest the possibility that mutations in this gene may be responsible for some cases of infertility in women. Overall, these findings indicate that DMRT1 is a key fertility gene.

Functional dissection and assembly of a small, newly evolved, W chromosome-specific genomic region of the African clawed frog Xenopus laevis.

Genetic triggers for sex determination are frequently co-inherited with other linked genes that may also influence one or more sex-specific phenotypes. To better understand how sex-limited regions evolve and function, we studied a small W chromosome-specific region of the frog Xenopus laevis that contains only three genes (dm-w, scan-w, ccdc69-w) and that drives female differentiation. Using gene editing, we found that the sex-determining function of this region requires dm-w but that scan-w and ccdc69-w are not essential for viability, female development, or fertility. Analysis of mesonephros+gonad transcriptomes during sexual differentiation illustrates masculinization of the dm-w knockout transcriptome, and identifies mostly non-overlapping sets of differentially expressed genes in separate knockout lines for each of these three W-specific gene compared to wildtype sisters. Capture sequencing of almost all Xenopus species and PCR surveys indicate that the female-determining function of dm-w is present in only a subset of species that carry this gene. These findings map out a dynamic evolutionary history of a newly evolved W chromosome-specific genomic region, whose components have distinctive functions that frequently degraded during Xenopus diversification, and evidence the evolutionary consequences of recombination suppression.

Recognition of polymorphic Csd proteins determines sex in the honeybee.

Sex in honeybees, Apis mellifera, is genetically determined by heterozygous versus homo/hemizygous genotypes involving numerous alleles at the single complementary sex determination locus. The molecular mechanism of sex determination is however unknown because there are more than 4950 known possible allele combinations, but only two sexes in the species. We show how protein variants expressed from complementary sex determiner (csd) gene determine sex. In females, the amino acid differences between Csd variants at the potential-specifying domain (PSD) direct the selection of a conserved coiled-coil domain for binding and protein complexation. This recognition mechanism activates Csd proteins and, thus, the female pathway. In males, the absence of polymorphisms establishes other binding elements at PSD for binding and complexation of identical Csd proteins. This second recognition mechanism inactivates Csd proteins and commits male development via default pathway. Our results demonstrate that the recognition of different versus identical variants of a single protein is a mechanism to determine sex.

Metabolic consequences of sex reversal in two lizard species: a test of the like-genotype and like-phenotype hypotheses.

Vertebrate sex is typically determined genetically, but in many ectotherms sex can be determined by genes (genetic sex determination, GSD), temperature (temperature-dependent sex determination, TSD), or interactions between genes and temperature during development. TSD may involve GSD systems with either male or female heterogamety (XX/XY or ZZ/ZW) where temperature overrides chromosomal sex determination to cause a mismatch between genetic sex and phenotypic sex (sex reversal). In these temperature-sensitive lineages, phylogenetic investigations point to recurrent evolutionary shifts between genotypic and temperature-dependent sex determination. These evolutionary transitions in sex determination can occur rapidly if selection favours the reversed sex over the concordant phenotypic sex. To investigate the consequences of sex reversal on offspring phenotypes, we measured two energy-driven traits (metabolism and growth) and 6 month survival in two species of reptile with different patterns of temperature-induced sex reversal. Male sex reversal occurs in Bassiana duperreyi when chromosomal females (female XX) develop male phenotypes (maleSR XX), while female sex reversal occurs in Pogona vitticeps when chromosomal males (male ZZ) develop female phenotypes (femaleSR ZZ). We show metabolism in maleSR XX was like that of male XY; that is, reflective of phenotypic sex and lower than genotypic sex. In contrast, for Pogona vitticeps, femaleSR ZZ metabolism was intermediate between male ZZ and female ZW metabolic rate. For both species, our data indicate that differences in metabolism become more apparent as individuals become larger. Our findings provide some evidence for an energetic advantage from sex reversal in both species but do not exclude energetic processes as a constraint on the distribution of sex reversal in nature.

Promoter generation for the chimeric sex-determining gene dm-W in Xenopus frogs.

Many sex-determining genes (SDGs) were generated as neofunctionalized genes through duplication and/or mutation of gonadal formation-related genes. We previously identified dm-W as an SDG in the African clawed frog Xenopus laevis and found that a partial duplication of the masculinization gene dmrt1 created the neofunctionalized dm-W after allotetraploidization by interspecific hybridization. The allotetraploid Xenopus species have two dmrt1 genes, dmrt1.L and dmrt1.S. Xenopus laevis dm-W has four exons: two dmrt1.S-derived exons (exons 2 and 3) and two other exons (noncoding exon 1 and exon 4). Our recent work revealed that exon 4 originated from a DNA transposon, hAT-10. Here, to clarify when and how the noncoding exon 1 and its coexisting promoter evolved during the establishment of dm-W after allotetraploidization, we newly determined nucleotide sequences of the dm-W promoter region from two other allotetraploid species, X. largeni and X. petersii, and performed an evolutionary analysis. We found that dm-W acquired a new exon 1 and TATA-type promoter in the common ancestor of the three allotetraploid Xenopus species, resulting in the deletion of the dmrt1.S-derived TATA-less promoter. In addition, we demonstrated that the TATA box contributes to dm-W promoter activity in cultured cells. Collectively, these findings suggest that this novel TATA-type promoter was important for the establishment of dm-W as a sex-determining gene, followed by the degeneration of the preexisting promoter.

Evaluating the role of sexual antagonism in the evolution of sex chromosomes: new data from fish.

The recent increase in available molecular and genomic data for diverse taxa helps to shed new light on long-standing theories. Research into sex chromosome evolution has particularly benefited from a growing number of studies of fish, motivated by their highly diverse mechanisms of sex determination. Sexual antagonism is regularly cited as an influential force in sex chromosome emergence; however, this so far proves difficult to demonstrate. In this review, we highlight recent developments in the investigation of sexual antagonism in sex chromosome research in fish. We find strong emphasis placed on study-organism specific genomic features and patterns of recombination, rather than evidence for a comprehensive role of sexual antagonism. In this light, we discuss the alternative models of sex chromosome evolution. We conclude that fish represents a key resource for further research, provided attention is given to species-specific effects while simultaneously integrating comparative studies across taxa for a vital and comprehensive understanding of sex chromosome evolution and investigation of proposed models.

Limited evidence for extensive genetic differentiation between X and Y chromosomes in Hybognathus amarus (Cypriniformes: Leuciscidae).

Sex determination systems and genetic sex differentiation across fishes are highly diverse but are unknown for most Cypriniformes, including Rio Grande silvery minnow (Hybognathus amarus). In this study, we aimed to detect and validate sex-linked markers to infer sex determination system and to demonstrate the utility of combining several methods for sex-linked marker detection in nonmodel organisms. To identify potential sex-linked markers, Nextera-tagmented reductively amplified DNA (nextRAD) libraries were generated from 66 females, 64 males, and 60 larvae of unknown sex. These data were combined with female and male de novo genomes from Nanopore long-read sequences. We identified five potential unique male nextRAD-tags and one potential unique male contig, suggesting an XY sex determination system. We also identified two single-nucleotide polymorphisms (SNPs) in the same contig with values of FST, allele frequencies, and heterozygosity conforming with expectations of an XY system. Through PCR we validated the marker containing the sex-linked SNPs and a single nextRAD-tag sex-associated marker but it was not male specific. Instead, more copies of this locus in the male genome were suggested by enhanced amplification in males. Results are consistent with an XY system with low differentiation between sex-determining regions. Further research is needed to confirm the level of differentiation between the sex chromosomes. Nonetheless, this study highlighted the power of combining reduced representation and whole-genome sequencing for identifying sex-linked markers, especially when reduced representation sequencing does not include extensive variation between sexes, either because such variation is not present or not captured.