Sex Determination Pathway Research Articles

Sperm fate is promoted by the mir-44 microRNA family in the Caenorhabditis elegans hermaphrodite germline.

Posttranscriptional regulation of gene expression, typically effected by RNA-binding proteins, microRNAs (miRNAs), and translation initiation factors, is essential for normal germ cell function. Numerous miRNAs have been detected in the germline; however, the functions of specific miRNAs remain largely unknown. Functions of miRNAs have been difficult to determine as miRNAs often modestly repress target mRNAs and are suggested to sculpt or fine tune gene expression to allow for the robust expression of cell fates. In Caenorhabditis elegans hermaphrodites, cell fate decisions are made for germline sex determination during larval development when sperm are generated in a short window before the switch to oocyte production. Here, analysis of newly generated mir-44 family mutants has identified a family of miRNAs that modulate the germline sex determination pathway in C. elegans. Mutants with the loss of mir-44 and mir-45 produce fewer sperm, showing both a delay in the specification and formation of sperm as well as an early termination of sperm specification accompanied by a premature switch to oocyte production. mir-44 and mir-45 are necessary for the normal period of fog-1 expression in larval development. Through genetic analysis, we find that mir-44 and mir-45 may act upstream of fbf-1 and fem-3 to promote sperm specification. Our research indicates that the mir-44 family promotes sperm cell fate specification during larval development and identifies an additional posttranscriptional regulator of the germline sex determination pathway.

High-quality chromosome-level genomes of two tilapia species reveal their evolution of repeat sequences and sex chromosomes.

Tilapias are one of the most farmed fishes that are coined as "aquatic chicken" by the food industry. Nile tilapia and blue tilapia exhibit very recent transition of sex chromosome systems since their divergence approximately five million years ago, making them a great model for elucidating the molecular and evolutionary mechanisms of sex chromosome turnovers. Studies of their sex-determining pathways are also critical for developing genetic sex control in aquaculture. We report here the newly produced genomes of Nile tilapia and blue tilapia that integrate long-read sequencing and chromatin conformation data. The two nearly complete genomes have anchored over 97% of the sequences into linkage groups (LGs), and assembled majorities of complex repetitive regions including telomeres, centromeres and rDNA clusters. In particular, we inferred two episodes of repeat expansion at LG3 respectively in the ancestor of cichlids and that of tilapias. The consequential large heterochromatic region concentrated at one end of LG3 comprises tandem arrays of mRNA and small RNA genes, among which we have identified a candidate female determining gene Paics in blue tilapia. Paics shows female-specific patterns of single-nucleotide variants, copy numbers and expression patterns in gonads during early gonadogenesis. Our work provides a very important genomic resource for functional studies of cichlids, and suggested that unequal distribution of repeat content that impacts the local recombination rate might make some chromosomes more likely to become sex chromosomes.

A CCCH zinc finger gene regulates doublesex alternative splicing and male development in Bombyx mori.

Recent identification of a Piwi-interacting RNA (piRNA)-initiated sex determination cascade in the silkworm, Bombyx mori, provides novel insights into high diversity of insect sex determination pathways. In this system, the W-chromosome-derived Fem piRNA is the primary sex determination signal. A CCCH-type zinc finger gene Masculinizer (Masc), which is targeted by Fem piRNA-PIWI complex in female animals, is indispensable for male-specific splicing of B. mori doublesex (Bmdsx). Although many genes involved in this cascade have been identified, the regulatory mechanisms of silkworm sex determination remain to be elucidated. Here we show that another CCCH-type zinc finger gene, Bmznf-2, is a masculinization factor in B. mori. Bmznf-2 shows testis-abundant expression and loss of Bmznf-2 function via clustered regularly interspaced short palindromic repeats / single-guide RNA-mediated mutagenesis results in feminized differentiation and appearance of the female-specific splicing variants of Bmdsx transcripts in males. In contrast, there is no phenotypic consequence in mutant females. In mutant males, relative messenger RNA expression levels of female-dominant genes such as vitellogenin and sex-specific storage protein 1 are significantly elevated while several male-dominant genes are significantly down-regulated. Furthermore, male mutants show delayed developmental timing, smaller body sizes of larvae and malformation of moth wings. Our data thus reveal that Bmznf-2 plays an indispensable role in silkworm male sexual differentiation.

Direct glia-to-neuron transdifferentiation gives rise to a pair of male-specific neurons that ensure nimble male mating.

Sexually dimorphic behaviours require underlying differences in the nervous system between males and females. The extent to which nervous systems are sexually dimorphic and the cellular and molecular mechanisms that regulate these differences are only beginning to be understood. We reveal here a novel mechanism by which male-specific neurons are generated in Caenorhabditis elegans through the direct transdifferentiation of sex-shared glial cells. This glia-to-neuron cell fate switch occurs during male sexual maturation under the cell-autonomous control of the sex-determination pathway. We show that the neurons generated are cholinergic, peptidergic, and ciliated putative proprioceptors which integrate into male-specific circuits for copulation. These neurons ensure coordinated backward movement along the mate's body during mating. One step of the mating sequence regulated by these neurons is an alternative readjustment movement performed when intromission becomes difficult to achieve. Our findings reveal programmed transdifferentiation as a developmental mechanism underlying flexibility in innate behaviour.

Gtsf1 is essential for proper female sex determination and transposon silencing in the silkworm, Bombyx mori.

Sex determination pathways are astoundingly diverse in insects. For instance, the silk moth Bombyx mori uniquely use various components of the piRNA pathway to produce the Fem signal for specification of the female fate. In this study, we identified BmGTSF1 as a novel piRNA factor which participates in B. mori sex determination. We found that BmGtsf1 has a distinct expression pattern compared to Drosophila and mouse. CRISPR/Cas9 induced mutation in BmGtsf1 resulted in partial sex reversal in genotypically female animals by shifting expression of the downstream targets BmMasc and Bmdsx to the male pattern. As levels of Fem piRNAs were substantially reduced in female mutants, we concluded that BmGtsf1 plays a critical role in the biogenesis of the feminizing signal. We also demonstrated that BmGTSF1 physically interacted with BmSIWI, a protein previously reported to be involved in female sex determination, indicating BmGTSF1 function as the cofactor of BmSIWI. BmGtsf1 mutation resulted in piRNA pathway dysregulation, including piRNA biogenesis defects and transposon derepression, suggesting BmGtsf1 is also a piRNA factor in the silkworm. Furthermore, we found that BmGtsf1 mutation leads to gametogenesis defects in both male and female. Our data suggested that BmGtsf1 is a new component involved in the sex determination pathway in B. mori.

The Caenorhabditis elegans proteasome subunit RPN-12 is required for hermaphrodite germline sex determination and oocyte quality.

The proteasome is a multi-subunit complex and a major proteolytic machinery in cells. Most subunits are essential for proteasome function, and depletion of individual subunits normally results in lethality. RPN-12/Rpn12/PSMD8 is a lid subunit of the 19S regulatory particle (RP) of the 26S proteasome. Studies in Caenorhabditis elegans demonstrated that RNAi depletion of RPN-12 does not result in lethality. RPN-12 has not been well studied in higher eukaryotes. In this study, we investigate the biological significance of RPN-12 in C. elegans. We found that the null mutant rpn-12(av93) did not cause major impairment of the proteolytic activity of the proteasome. Most rpn-12(av93) hermaphrodites lack sperm leading to feminization of the germ line that can be partially rescued by mating to males. The lack of sperm phenotype can be suppressed by downregulation of TRA-1, a player in the hermaphrodite germline sex determination pathway. Also, rpn-12(av93) animals show significant nuclear accumulation of the meiotic kinase WEE-1.3, a protein predominantly localized to the perinuclear region. Interestingly, chemical inhibition of the proteasome did not cause nuclear accumulation of WEE-1.3. RPN-12 plays a previously unknown role in oogenesis and the germline sex determination pathway in C. elegans hermaphrodites.

Evolution of Sex Determination and Sex Chromosomes: A Novel Alternative Paradigm.

Sex chromosomes can differ between species as a result of evolutionary turnover, a process that can be driven by evolution of the sex determination pathway. Canonical models of sex chromosome turnover hypothesize that a new master sex determining gene causes an autosome to become a sex chromosome or an XY chromosome pair to switch to a ZW pair (or vice versa). Here, a novel paradigm for the evolution of sex determination and sex chromosomes is presented, in which there is an evolutionary transition in the master sex determiner, but the X chromosome remains unchanged. There are three documented examples of the novel paradigm, and it is hypothesized that a similar process could happen in a ZW sex chromosome system. Three other taxa are also identified where the novel paradigm may have occurred, and how it could be distinguished from canonical trajectories in these and additional taxa is also described.

An Evaluation of Differentially Spliced Genes as Markers of Sex for Forensic Entomology,.

Blow flies (Calliphoridae) are important medically and economically and are commonly used in forensics as temporal markers in death investigations. While phenotypic traits in adult flies can be sexually dimorphic, sex identification in immatures is difficult. Consequently, little is known about how sex may result in developmental disparities among sexes even though there are indications that they may be important in some instances. Since genetic mechanisms for sex are well studied in model flies and species of agricultural and medical importance, we exploit the sex-specifically spliced genes transformer (tra) and doublesex (dsx) in the sex determination pathway to optimize a sex identification assay for immatures. Using known primer sets for tra and with a novel one for dsx, we develop PCR assays for identifying sex in four forensically relevant Calliphoridae species: Lucilia sericata (Meigen), Lucilia cuprina (Wiedemann), Cochliomyia macellaria (Fabricius), and Chrysomya rufifacies (Macquart) and evaluated their performance. Band detection rates were found to range from 71 to 100%, call rates ranged from 90 to 100%, and no error was found when bands could be called. Such information is informative for purposes of testimony and in preparation for development studies. The developed assays will assist in further differentiating sexually dimorphic differences in development of the Calliphoridae and aid in more accurately estimating insect age when age predictive markers (size, development time, molecular expression) are sexually dimorphic.

Sex-determining genes distinctly regulate courtship capability and target preference via sexually dimorphic neurons.

For successful mating, a male animal must execute effective courtship behaviors toward a receptive target sex, which is female. Whether the courtship execution capability and upregulation of courtship toward females are specified through separable sex-determining genetic pathways remains uncharacterized. Here, we found that one of the two Drosophila sex-determining genes, doublesex (dsx), specifies a male-specific neuronal component that serves as an execution mechanism for courtship behavior, whereas fruitless (fru) is required for enhancement of courtship behavior toward females. The dsx-dependent courtship execution mechanism includes a specific subclass within a neuronal cluster that co-express dsx and fru. This cluster contains at least another subclass that is specified cooperatively by both dsx and fru. Although these neuronal populations can also promote aggressive behavior toward male flies, this capacity requires fru-dependent mechanisms. Our results uncover how sex-determining genes specify execution capability and female-specific enhancement of courtship behavior through separable yet cooperative neurogenetic mechanisms.

Spliceosomal Protein Gene BmSPX Regulates Reproductive Organ Development in Bombyx mori.

Sex determination and differentiation are nearly universal to all eukaryotic organisms, encompassing diverse systems and mechanisms. Here, we identified a spliceosomal protein gene BmSPX involved in sex determination of the lepidopeteran insect, Bombyx mori. In a transgenic silkworm line that overexpressed the BmSPX gene, transgenic silkworm males exhibited differences in their external genitalia compared to wild-type males, but normal internal genitalia. Additionally, transgenic silkworm females exhibited a developmental disorder of the reproductive organs. Upregulation of BmSPX significantly increased the expression levels of sex-determining genes (BmMasc and BmIMP) and reduced the female-type splice isoform of Bmdsx, which is a key switch gene downstream of the sex-determination pathway. Additionally, co-immunoprecipitation assays confirmed an interaction between the BmSPX protein and BmPSI, an upstream regulatory factor of Bmdsx. Quantitative real-time PCR showed that BmSPX over-expression upregulated the expression of the Hox gene abdominal-B (Adb-B), which is required for specification of the posterior abdomen, external genitalia, and gonads of insects, as well as the genes in the Receptor Tyrosine Kinase (RTK) signaling pathway. In conclusion, our study suggested the involvement of BmSPX, identified as a novel regulatory factor, in the sex-determination pathway and regulation of reproductive organ development in silkworms.

The fruitless gene affects female receptivity and species isolation.

Female mate rejection acts as a major selective force within species, and can serve as a reproductive barrier between species. In spite of its critical role in fitness and reproduction, surprisingly little is known about the genetic or neural basis of variation in female mate choice. Here, we identify fruitless as a gene affecting female receptivity within Drosophila melanogaster, as well as female Drosophila simulans rejection of male D. melanogaster. Of the multiple transcripts this gene produces, by far the most widely studied is the sex-specifically spliced transcript involved in the sex determination pathway. However, we find that female rejection behaviour is affected by a non-sex-specifically spliced fruitless transcript. This is the first implication of fruitless in female behaviour, and the first behavioural role identified for a fruitless non-sex-specifically spliced transcript. We found that this locus does not influence preferences via a single sensory modality, examining courtship song, antennal pheromone perception, or perception of substrate vibrations, and we conclude that fruitless influences mate choice via the integration of multiple signals or through another sensory modality.

Cellular calcium and redox regulation: the mediator of vertebrate environmental sex determination?

Many reptiles and some fish determine offspring sex by environmental cues such as incubation temperature. The mechanism by which environmental signals are captured and transduced into specific sexual phenotypes has remained unexplained for over 50 years. Indeed, environmental sex determination (ESD) has been viewed as an intractable problem because sex determination is influenced by a myriad of genes that may be subject to environmental influence. Recent demonstrations of ancient, conserved epigenetic processes in the regulatory response to environmental cues suggest that the mechanisms of ESD have a previously unsuspected level of commonality, but the proximal sensor of temperature that ultimately gives rise to one sexual phenotype or the other remains unidentified. Here, we propose that in ESD species, environmental cues are sensed by the cell through highly conserved ancestral elements of calcium and redox (CaRe) status, then transduced to activate ubiquitous signal transduction pathways, or influence epigenetic processes, ultimately to drive the differential expression of sex genes. The early evolutionary origins of CaRe regulation, and its essential role in eukaryotic cell function, gives CaRe a propensity to be independently recruited for diverse roles as a 'cellular sensor' of environmental conditions. Our synthesis provides the first cohesive mechanistic model connecting environmental signals and sex determination pathways in vertebrates, providing direction and a framework for developing targeted experimentation.

The conserved molting/circadian rhythm regulator NHR-23/NR1F1 serves as an essential co-regulator of C. elegans spermatogenesis.

In sexually reproducing metazoans, spermatogenesis is the process by which uncommitted germ cells give rise to haploid sperm. Work in model systems has revealed mechanisms controlling commitment to the sperm fate, but how this fate is subsequently executed remains less clear. While studying the well-established role of the conserved nuclear hormone receptor transcription factor, NHR-23/NR1F1, in regulating C. elegans molting, we discovered that NHR-23/NR1F1 is also constitutively expressed in developing primary spermatocytes and is a critical regulator of spermatogenesis. In this novel role, NHR-23/NR1F1 functions downstream of the canonical sex-determination pathway. Degron-mediated depletion of NHR-23/NR1F1 within hermaphrodite or male germlines causes sterility due to an absence of functional sperm, as depleted animals produce arrested primary spermatocytes rather than haploid sperm. These spermatocytes arrest in prometaphase I and fail to either progress to anaphase or attempt spermatid-residual body partitioning. They make sperm-specific membranous organelles but fail to assemble their major sperm protein into fibrous bodies. NHR-23/NR1F1 appears to function independently of the known SPE-44 gene regulatory network, revealing the existence of an NHR-23/NR1F1-mediated module that regulates the spermatogenesis program.

The X chromosome of the German cockroach, Blattella germanica, is homologous to a fly X chromosome despite 400 million years divergence

BackgroundSex chromosome evolution is a dynamic process that can proceed at varying rates across lineages. For example, different chromosomes can be sex-linked between closely related species, whereas other sex chromosomes have been conserved for > 100 million years. Cases of long-term sex chromosome conservation could be informative of factors that constrain sex chromosome evolution. Cytological similarities between the X chromosomes of the German cockroach (Blattella germanica) and most flies suggest that they may be homologous—possibly representing an extreme case of long-term conservation.ResultsTo test the hypothesis that the cockroach and fly X chromosomes are homologous, we analyzed whole-genome sequence data from cockroaches. We found evidence in both sequencing coverage and heterozygosity that a significant excess of the same genes are on both the cockroach and fly X chromosomes. We also present evidence that the candidate X-linked cockroach genes may be dosage compensated in hemizygous males. Consistent with this hypothesis, three regulators of transcription and chromatin on the fly X chromosome are conserved in the cockroach genome.ConclusionsOur results support our hypothesis that the German cockroach shares the same X chromosome as most flies. This may represent the convergent evolution of the X chromosome in the lineages leading to cockroaches and flies. Alternatively, the common ancestor of most insects may have had an X chromosome that resembled the extant cockroach and fly X. Cockroaches and flies diverged ∼ 400 million years ago, which would be the longest documented conservation of a sex chromosome. Cockroaches and flies have different mechanisms of sex determination, raising the possibility that the X chromosome was conserved despite the evolution of the sex determination pathway.

Molecular cytogenetic analysis and genetic counseling: a case report of eight 46,XX males and a literature review

Background46,XX male syndrome is a rare disorder that usually causes infertility. This study was established to identify the genetic causes of this condition in a series of 46,XX males through the combined application of cytogenetic and molecular genetic techniques.Case presentationWe identified eight azoospermic 46,XX males who underwent infertility-related consultations at our center. They all presented normal male phenotypes. In seven of the eight 46,XX males (87.5%), translocation of the SRY gene to the terminal short arm of the X chromosome was clearly involved in their condition, which illustrated that this translocation is the main mechanism of 46,XX sex reversal, in line with previous reports. However, one patient presented a homozygous DAX1 mutation (c.498G > A, p.R166R), which was not previously reported in SRY-negative XX males.ConclusionsWe proposed that this synonymous DAX1 mutation in case 8 might not be associated with the activation of the male sex-determining pathway, and the male phenotype in this case might be regulated by some unidentified genetic or environmental factors. Hence, the detection of genetic variations associated with sex reversal in critical sex-determining genes should be recommended for SRY-negative XX males. Only after comprehensive cytogenetic and molecular genetic analyses can genetic counseling be offered to 46,XX males.

Putative Mitochondrial Sex Determination in the Bivalvia: Insights From a Hybrid Transcriptome Assembly in Freshwater Mussels.

Bivalves exhibit an astonishing diversity of sexual systems, with genetic and environmental determinants of sex, and possibly the only example of mitochondrial genes influencing sex determination pathways in animals. In contrast to all other animal species in which strict maternal inheritance (SMI) of mitochondria is the rule, bivalves possess a system known as doubly uniparental inheritance (DUI) of mitochondria in which maternal and paternal mitochondria (and their corresponding female-transmitted or F mtDNA and male-transmitted or M mtDNA genomes) are transmitted within a species. Species with DUI also possess sex-associated mtDNA-encoded proteins (in addition to the typical set of 13), which have been hypothesized to play a role in sex determination. In this study, we analyzed the sex-biased transcriptome in gonads of two closely-related freshwater mussel species with different reproductive and mitochondrial transmission modes: the gonochoric, DUI species, Utterbackia peninsularis, and the hermaphroditic, SMI species, Utterbackia imbecillis. Through comparative analysis with other DUI and non-DUI bivalve transcriptomes already available, we identify common male and female-specific genes, as well as SMI and DUI-related genes, that are probably involved in sex determination and mitochondrial inheritance in this animal group. Our results contribute to the understanding of what could be the first animal sex determination system involving the mitochondrial genome.

Transcriptomics analysis revealing candidate genes and networks for sex differentiation of yesso scallop (Patinopecten yessoensis)

BackgroundThe Yesso scallop, Patinopecten (Mizuhopecten) yessoensis, is a commercially important bivalve in the coastal countries of Northeast Asia. It has complex modes of sex differentiation, but knowledge of the mechanisms underlying this sex determination and differentiation is limited.ResultsIn this study, the gonad tissues from females and males at three developmental stages were used to investigate candidate genes and networks for sex differentiation via RNA-Req. A total of 901,980,606 high quality clean reads were obtained from 18 libraries, of which 417 expressed male-specific genes and 754 expressed female-specific genes. Totally, 10,074 genes differentially expressed in females and males were identified. Weighted gene co-expression network analysis (WGCNA) revealed that turquoise and green gene modules were significantly positively correlated with male gonads, while coral1 and black modules were significantly associated with female gonads. The most important gene for sex determination and differentiation was Pydmrt 1, which was the only gene discovered that determined the male sex phenotype during early gonadal differentiation. Enrichment analyses of GO terms and KEGG pathways revealed that genes involved in metabolism, genetic and environmental information processes or pathways are sex-biased. Forty-nine genes in the five modules involved in sex differentiation or determination were identified and selected to construct a gene co-expression network and a hypothesized sex differentiation pathway.ConclusionsThe current study focused on screening genes of sex differentiation in Yesso scallop, highlighting the potential regulatory mechanisms of gonadal development in P. yessoensis. Our data suggested that WCGNA can facilitate identification of key genes for sex differentiation and determination. Using this method, a hypothesized P. yessoensis sex determination and differentiation pathway was constructed. In this pathway, Pydmrt 1 may have a leading function.

Integrated analysis of proteomics and metabolomics reveals the potential sex determination mechanism in Odontobutis potamophila

Odontobutis potamophila is a valuable species for aquaculture in China, which shows asexually dimorphic growth pattern. In this study, the integrated proteomics and metabolomics were used to analyze the sex determination mechanism. A total of 2781 significantly different regulated proteins were identified by proteomics and 2693 significantly different expressed metabolites were identified by metabolomics. Among them, 2560 proteins and 1701 metabolites were significantly up-regulated in testes, whereas 221 proteins and 992 metabolites were significantly up-regulated in ovaries. Venn diagram analysis showed 513 proteins were differentially regulated at both protein and metabolite levels. Correlation analysis of differentially-regulated proteins and metabolites were identified by Gene Ontology annotation and Kyoto Encyclopedia of Genes and Genomes pathway analysis. The results showed lipid metabolism plays an important role in sex determination. The metabolites decanoyl-CoA, leukotriene, 3-dehydrosphinganine, and arachidonate were the biomarkers in testes, whereas estrone and taurocholate were the biomarkers in ovaries. Interaction networks of the significant differentially co-regulated proteins and metabolites in the process of lipid metabolism showed arachidonic acid metabolism and steroid hormone biosynthesis were the most important pathways in sex determination. The findings of this study provide valuable information for selective breeding of O. potamophila. Significance of the studyThe male O. potamophila grows substantially larger and at a quicker rate than the female. Thus, males have greater economic value than females. However, limited research was done to analyze the sex determination mechanism of O. potamophila, which seriously hindered the development of whole-male O. potamophila breeding. In this study, four key proteins (Ctnnb1, Piwil1, Hsd17b1, and Dnali1), six most important biomarkers (decanoyl-CoA, leukotriene, 3-dehydrosphinganine, arachidonate, estrone, and taurocholate) and two key pathways (arachidonic acid metabolism and steroid hormone biosynthesis) in sex determination of O. potamophila were found by integrated application of iTRAQ and LC-MS techniques. The results give valuable information for molecular breeding of O. potamophila in aquaculture.

RNAi mediated knockdown of RpL11, RpS2, and tra-2 led to reduced reproduction of Phytoseiulus persimilis.

Phytoseiulus persimilis is one of the most important biological control agents of spider mites. Multiple studies have been conducted on factors affecting its reproduction, but limited research on related molecular mechanisms has been carried out. In this study, RNA interference of three genes, ribosomal protein L11 (RpL11), ribosomal protein S2 (RpS2), and transformer-2 (tra-2), to newly emerged females were performed through oral delivery of double-stranded RNA, and knockdown of target genes was verified using qRT-PCR analysis. When RpL11 or RpS2 was interfered, 42 and 30% P. persimilis individuals either laid no eggor had no egg hatched, whereas the remaining females had their oviposition duration reduced by 31.8 and 49.9%, fecundity reduced by 48.1 and 67.8%, and egg hatching rate reduced by 20.4 and 22.4%, respectively. In addition, offspring sex ratios were significantly male biased especially at low fecundities. When tra-2 was interfered, no significant difference in fecundity was detected, but egg hatching rate reduced by 30.6%. This study verified the possibility of RNA interference in Phytoseiidae through oral delivery, and indicated that RpL11 and RpS2 are involved in egg formation, whereas tra-2 is involved in embryo development in P. persimilis. Phytoseiid mites have different sex determination pathways compared to insects. The present study provides data and evidence at molecular biological level for future research on reproduction and sex determination of phytoseiid mites.

Mutation of doublesex induces sex-specific sterility of the diamondback moth Plutella xylostella

Doublesex (dsx)the downstream gene in the insect sex determination pathway, plays a critical role in sexual differentiation and development. The functions of dsx have been characterized in several model insect species. However, the molecular mechanism and functions of sex determination of dsx in Plutella xylostella, an agricultural pest, are still unknown. In present study, we identified a male-specific and three female-specific Pxdsx transcripts in P. xylostella. Phylogenetic analyses and multiple sequence alignment revealed that Pxdsx is highly conserved in lepidopterans. The CRISPR/Cas9 technology was used to induce mutations in the male-specific isoform, the female-specific isoform, and common regions of Pxdsx. Disruptions of Pxdsx sex-specific isoforms caused sex-specific defects in external genitals and partial sexual reversal. In addition, we found that female specific transcripts were detected in PxdsxM male mutants and male-specific transcripts were detected in PxdsxF female mutants. Mutations also caused changes in expression of several sex-biased genes and induced sex-specific sterility. This study demonstrates that Pxdsx plays a key role in sex determination of P. xylostella and suggests novel genetic control approaches for the management of P. xylostella.