Molecular mechanisms of sex determination in Lepidoptera: current status and perspectives

Abstract

Moths and butterflies (Lepidoptera) are the largest group of organisms with female heterogamety and the sex chromosome system WZ/ZZ (female/male) or exceptionally Z0/ZZ. However, the genetic basis of sex determination in Lepidoptera remained unknown for a long time until the sex-determining pathway was discovered in 2014 in the silkworm Bombyx mori. In this species, the dominant W chromosome carries a Feminizer (Fem) gene encoding a precursor of a Fem piRNA that promotes femaleness by downregulating the expression of a Z-linked gene, Masculinizer (Masc). In the W chromosome absence, Masc promotes male development and controls dosage compensation. In the 10 years since this discovery, significant progress has been made in understanding the molecular mechanisms of sex determination in Lepidoptera. Data from recent studies discussed in this review suggest a conserved role for Masc in male sex determination and dosage compensation in the clade Ditrysia, which comprises the majority of Lepidoptera. Although the primary sex-determining signals are not conserved, the presence of feminizing piRNAs of different origins in distantly related species suggests convergent evolution of a similar mechanism of female sex determination. A unique exception is zygosity-based sex determination in the butterfly Bicyclus anynana, where the primary signal is the state of the hypervariable Masc gene. In other species with a dispensable W chromosome, such as the silkmoth Samia cynthia, sex is determined by the Z:A ratio, but a molecular mechanism is not yet known. Overall, the available data suggest considerable diversity in the upstream molecular mechanisms of sex determination in Lepidoptera.

Keywords

convergent evolution / dosage compensation / feminizing piRNA / Masculinizer / moths and butterflies / sex-determining pathway

References

[1] Abe, H., Mita, K., Yasukochi, Y., Oshiki, T. and Shimada, T. (2005) Retrotransposable elements on the W chromosome of the silkworm, Bombyx mori. Cytogenetic and Genome Research, 110, 144–151.

[2] Arai, H., Herran, B., Sugimoto, T.N., Miyata, M., Sasaki, T. and Kageyama, D. (2024) Cell-based assays and comparative genomics revealed the conserved and hidden effects of Wolbachia on insect sex determination. PNAS Nexus, 3, pgae348.

[3] Arai, H., Katsuma, S., Matsuda-Imai, N., Lin, S.R., Inoue, M.N. and Kageyama, D. (2025) Prophage-encoded Hm-oscar gene recapitulates Wolbachia-induced male-killing in the tea tortrix moth Homona magnanima. eLife, 13, RP101101.

[4] Ashmore, J.S., Slippers, B., Duong, T.A. and Dittrich-Schröder, G. (2025) Understanding the genetics of sex determination in insects and its relevance to genetic pest management. Insect Molecular Biology, https://doi.org/10.1111/imb.12982.

[5] Beye, M., Hasselmann, M., Fondrk, M.K., Page, R.E. and Omholt, S.W. (2003) The gene csd is the primary signal for sexual development in the honeybee and encodes an SR-type protein. Cell, 114, 419–429.

[6] Bi, H., Li, X., Xu, X., Wang, Y., Zhou, S. and Huang, Y. (2022) Masculinizer and doublesex as key factors regulate sexual dimorphism in Ostrinia furnacalis. Cells, 11, 2161.

[7] Blackmon, H., Ross, L. and Bachtrog, D. (2017) Sex determination, sex chromosomes, and karyotype evolution in insects. Journal of Heredity, 108, 78–93.

[8] Bopp, D., Saccone, G. and Beye, M. (2014) Sex determination in insects: variations on a common theme. Sexual Development, 8, 20–28.

[9] Carabajal Paladino, L.Z., Provazníková, I., Berger, M., Bass, C., Aratchige, N.S., López, S.N. et al. (2019) Sex chromosome turnover in moths of the diverse superfamily Gelechioidea. Genome Biology and Evolution, 11, 1307–1319.

[10] Cline, T.W. and Meyer, B.J. (1996) Vive la différence: males vs females in flies vs worms. Annual Review of Genetics, 30, 637–702.

[11] Czech, B. and Hannon, G.J. (2016) One loop to rule them all: the ping-pong cycle and piRNA-guided silencing. Trends in Biochemical Sciences, 41, 324–337.

[12] Dalíková, M., Zrzavá, M., Hladová, I., Nguyen, P., Šonský, I., Flegrová, M. et al. (2017) New insights into the evolution of the W chromosome in Lepidoptera. Journal of Heredity, 108, 709–719.

[13] Deng, Z., Zhang, Y., Li, Y., Huang, K., Chen, X., Zhang, M. et al. (2021) Identification and characterization of the masculinizing function of the Helicoverpa armigera Masc gene. International Journal of Molecular Sciences, 22, 8650.

[14] Ercan, S. and Lieb, J.D. (2009) C. elegans dosage compensation: a window into mechanisms of domain-scale gene regulation. Chromosome Research, 17, 215–227.

[15] Fujii, T. and Shimada, T. (2007) Sex determination in the silkworm, Bombyx mori: A female determinant on the W chromosome and the sex-determining gene cascade. Seminars in Cell & Developmental Biology, 18, 379–388.

[16] Fukui, T., Kawamoto, M., Shoji, K., Kiuchi, T., Sugano, S., Shimada, T. et al. (2015) The endosymbiotic bacterium Wolbachia selectively kills male hosts by targeting the masculinizing gene. PLoS Pathogens, 11, e1005048.

[17] Fukui, T., Shoji, K., Kiuchi, T., Suzuki, Y. and Katsuma, S. (2023) Masculinizer is not post-transcriptionally regulated by female-specific piRNAs during sex determination in the Asian corn borer, Ostrinia furnacalis. Insect Biochemistry and Molecular Biology, 156, 103946.

[18] Geuverink, E. and Beukeboom, L.W. (2014) Phylogenetic distribution and evolutionary dynamics of the sex determination genes doublesex and transformer in insects. Sexual Development, 8, 38–49.

[19] Gopinath, G., Arunkumar, K.P., Mita, K. and Nagaraju, J. (2016) Role of Bmznf-2, a Bombyx mori CCCH zinc finger gene, in masculinisation and differential splicing of Bmtra-2. Insect Biochemistry and Molecular Biology, 75, 32–44.

[20] Han, M.J., Luo, C., Hu, H., Lin, M., Lu, K., Shen, J. et al. (2024) Multiple independent origins of the female W chromosome in moths and butterflies. Science Advances, 10, eadm9851.

[21] Harvey-Samuel, T., Norman, V.C., Carter, R., Lovett, E. and Alphey, L. (2020) Identification and characterization of a Masculinizer homologue in the diamondback moth, Plutella xylostella. Insect Molecular Biology, 29, 231–240.

[22] Harvey-Samuel, T., Xu, X., Anderson, M.A.E., Carabajal Paladino, L.Z., Purusothaman, D., Norman, V.C. et al. (2022) Silencing RNAs expressed from W-linked PxyMasc “retrocopies” target that gene during female sex determination in Plutella xylostella. Proceedings of the National Academy of Sciences USA, 119, e2206025119.

[23] Hashimoto, H. (1933) The role of the W chromosome for sex determination in the silkworm, Bombyx mori. Japanese Journal of Genetics, 8, 245–258.

[24] Hejníčková, M., Dalíková, M., Potocký, P., Tammaru, T., Trehubenko, M., Kubíčková, S. et al. (2021) Degenerated, undifferentiated, rearranged, lost: High variability of sex chromosomes in Geometridae (Lepidoptera) identified by sex chromatin. Cells, 10, 2230.

[25] Hejníčková, M., Dalíková, M., Zrzavá, M., Marec, F., Lorite, P. and Montiel, E.E. (2023) Accumulation of retrotransposons contributes to W chromosome differentiation in the willow beauty Peribatodes rhomboidaria (Lepidoptera: Geometridae). Scientific Reports, 13, 534.

[26] Hejníčková, M., Koutecký, P., Potocký, P., Provazníková, I., Voleníková, A., Dalíková, M. et al. (2019) Absence of W chromosome in Psychidae moths and implications for the theory of sex chromosome evolution in Lepidoptera. Genes, 10, 1016.

[27] Herran, B., Sugimoto, T.N., Watanabe, K., Imanishi, S., Tsuchida, T., Matsuo, T. et al. (2023) Cell-based analysis reveals that sex-determining gene signals in Ostrinia are pivotally changed by male-killing Wolbachia. PNAS Nexus, 2, pgac293.

[28] Jin, L., Walker, A.S., Fu, G., Harvey-Samuel, T., Dafa'alla, T., Miles, A. et al. (2013) Engineered female-specific lethality for control of pest Lepidoptera. ACS Synthetic Biology, 2, 160–166.

[29] Kageyama, D., Ohno, M., Sasaki, T., Yoshido, A., Konagaya, T., Jouraku, A. et al. (2017) Feminizing Wolbachia endosymbiont disrupts maternal sex chromosome inheritance in a butterfly species. Evolution Letters, 1, 232–244.

[30] Katsuma, S., Hirota, K., Matsuda-Imai, N., Fukui, T., Muro, T., Nishino, K. et al. (2022) A Wolbachia factor for male killing in lepidopteran insects. Nature Communications, 13, 6764.

[31] Katsuma, S., Kawamoto, M. and Kiuchi, T. (2014) Guardian small RNAs and sex determination. RNA Biology, 11, 1238–1242.

[32] Katsuma, S., Kiuchi, T., Kawamoto, M., Fujimoto, T. and Sahara, K. (2018) Unique sex determination system in the silkworm, Bombyx mori: current status and beyond. Proceedings of the Japan Academy, Series B, Physical and Biological Sciences, 94, 205–216.

[33] Katsuma, S., Shoji, K., Sugano, Y., Suzuki, Y. and Kiuchi, T. (2019) Masc-induced dosage compensation in silkworm cultured cells. FEBS Open Bio, 9, 1573–1579.

[34] Katsuma, S., Sugano, Y., Kiuchi, T. and Shimada, T. (2015) Two conserved cysteine residues are required for the masculinizing activity of the silkworm Masc protein. Journal of Biological Chemistry, 290, 26114–26124.

[35] Kawahara, A.Y., Plotkin, D., Espeland, M., Meusemann, K., Toussain, E.F.A., Donath, A. et al. (2019) Phylogenomics reveals the evolutionary timing and pattern of butterflies and moths. Proceedings of the National Academy of Sciences USA, 116, 22657–22663.

[36] Kawamura, N. (1988) The egg size determining gene, Esd, is a unique morphological marker on the W chromosome of Bombyx mori. Genetica, 76, 195–201.

[37] Kawaoka, S., Kadota, K., Arai, Y., Suzuki, Y., Fujii, T., Abe, H. et al. (2011) The silkworm W chromosome is a source of female-enriched piRNAs. RNA, 17, 2144–2151.

[38] Kiuchi, T., Koga, H., Kawamoto, M., Shoji, K., Sakai, H., Arai, Y. et al. (2014) A single female-specific piRNA is the primary determiner of sex in the silkworm. Nature, 509, 633–636.

[39] Kiuchi, T., Sugano, Y., Shimada, T. and Katsuma, S. (2019) Two CCCH-type zinc finger domains in the Masc protein are dispensable for masculinization and dosage compensation in Bombyx mori. Insect Biochemistry and Molecular Biology, 104, 30–38.

[40] Kristensen, N.P. and Skalski, A.W. (1999) Phylogeny and palaeontology. Handbook of Zoology, Vol. IV, Arthropoda: Insecta, Part 35, Lepidoptera, Moths and Butterflies, Vol. 1: Evolution, Systematics, and Biogeography (ed. N.P. Kristensen), pp. 7–25. Walter de Gruyter, Berlin, Germany & New York, USA.

[41] Lee, J., Fujimoto, T., Yamaguchi, K., Shigenobu, S., Sahara, K., Toyoda, A. et al. (2024) W chromosome sequences of two bombycid moths provide an insight into the origin of Fem. Molecular Ecology, 33, e17434.

[42] Lee, J., Kiuchi, T., Kawamoto, M., Shimada, T. and Katsuma, S. (2015) Identification and functional analysis of a Masculinizer orthologue in Trilocha varians (Lepidoptera: Bombycidae). Insect Molecular Biology, 24, 561–569.

[43] Lee, J., Nishiyama, T., Shigenobu, S., Yamaguchi, K., Suzuki, Y., Shimada, T. et al. (2021) The genome sequence of Samia ricini, a new model species of lepidopteran insect. Molecular Ecology Resources, 21, 327–339.

[44] Lewis, J.J., Cicconardi, F., Martin, S.H., Reed, R.D., Danko, C.G. and Montgomery, S.H. (2021) The Dryas iulia genome supports multiple gains of a W chromosome from a B chromosome in butterflies. Genome Biology and Evolution, 13, evab128.

[45] Li, X., Liu, H., Bi, H., Wang, Y., Xu, J., Zhang, S. et al. (2024) Masculinizer gene controls sexual differentiation in Hyphantria cunea. Insect Science, 31, 405–416.

[46] Ma, S., Wang, X., Fei, J., Liu, Y., Duan, J., Wang, F. et al. (2013) Genetic marking of sex using a W chromosome-linked transgene. Insect Biochemistry and Molecular Biology, 43, 1079–1086.

[47] Marec, F. and Novák, K. (1998) Absence of sex chromatin corresponds with a sex-chromosome univalent in females of Trichoptera. European Journal of Entomology, 95, 197–209.

[48] Marec, F. and Vreysen, M.J.B. (2019) Advances and challenges of using the sterile insect technique for the management of pest Lepidoptera. Insects, 10, 371.

[49] Marec, F., Neven, L.G., Robinson, A.S., Vreysen, M., Goldsmith, M.R., Nagaraju, J. et al. (2005) Development of genetic sexing strains in Lepidoptera: from traditional to transgenic approaches. Journal of Economic Entomology, 98, 248–259.

[50] Marec, F., Sahara, K. and Traut, W. (2010) Rise and fall of the W chromosome in Lepidoptera. Molecular Biology and Genetics of the Lepidoptera (eds. M.R. Goldsmith & F. Marec), pp. 49–63. CRC Press, Boca Raton, FL, USA.

[51] Martins, S., Naish, N., Walker, A.S., Morrison, N.I., Scaife, S., Fu, G. et al. (2012) Germline transformation of the diamondback moth, Plutella xylostella L., using the piggyBac transposable element. Insect Molecular Biology, 21, 414–421.

[52] Mongue, A.J., Nguyen, P., Voleníková, A. and Walters, J.R. (2017) Neo-sex chromosomes in the monarch butterfly, Danaus plexippus. G3: Genes, Genomes, Genetics, 7, 3281–3294.

[53] Moronuki, Y., Kasahara, R., Naka, H. and Suzuki, M.G. (2025) Identification and functional analysis of sex-determining genes in the spongy moth, Lymantria dispar (Lepidoptera: Erebidae). Insect Biochemistry and Molecular Biology, 177, 104219.

[54] Nguyen, P., Sýkorová, M., Šíchová, J., Kůta, V., Dalíková, M., Čapková Frydrychová, R. et al. (2013) Neo-sex chromosomes and adaptive potential in tortricid pests. Proceedings of the National Academy of Sciences USA, 110, 6931–6936.

[55] Niimi, T., Sahara, K., Oshima, H., Yasukochi, Y., Ikeo, K. and Traut, W. (2006) Molecular cloning and chromosomal localization of the Bombyx Sex-lethal gene. Genome, 49, 263–268.

[56] Ohbayashi, F., Suzuki, M.G., Mita, K., Okano, K. and Shimada, T. (2001) A homologue of the Drosophila doublesex gene is transcribed into sex-specific mRNA isoforms in the silkworm, Bombyx mori. Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology, 128, 145–158.

[57] Ohbayashi, F., Suzuki, M.G. and Shimada, T. (2002) Sex determination in Bombyx mori. Current Science, 83, 466–471.

[58] Otte, M., Netschitailo, O., Weidtkamp-Peters, S., Seidel, C.A.M. and Beye, M. (2023) Recognition of polymorphic Csd proteins determines sex in the honeybee. Science Advances, 9, eadg4239.

[59] Peloquin, J.J., Thibault, S.T., Staten, R. and Miller, T.A. (2000) Germ-line transformation of pink bollworm (Lepidoptera: Gelechiidae) mediated by the piggyBac transposable element. Insect Molecular Biology, 9, 323–333.

[60] Picq, S., Lumley, L., Šíchová, J., Laroche, J., Pouliot, E., Brunet, B. et al. (2018) Insights into the structure of the spruce budworm (Choristoneura fumiferana) genome, as revealed by molecular cytogenetic analyses and a high-density linkage map. G3: Genes, Genomes, Genetics, 8, 2539–2549.

[61] Pospíšilová, K., Van′t Hof, A.E., Yoshido, A., Kružíková, R., Visser, S., Zrzavá, M. et al. (2023) Masculinizer gene controls male sex determination in the codling moth, Cydia pomonella. Insect Biochemistry and Molecular Biology, 160, 103991.

[62] Rosin, L.F., Chen, D., Chen, Y. and Lei, E.P. (2022) Dosage compensation in Bombyx mori is achieved by partial repression of both Z chromosomes in males. Proceedings of the National Academy of Sciences USA, 119, e2113374119.

[63] Rueda-M, N., Pardo-Diaz, C., Montejo-Kovacevich, G., McMillan, W.O., Kozak, K.M., Arias, C.F. et al. (2024) Genomic evidence reveals three W-autosome fusions in Heliconius butterflies. PLoS Genetics, 20, e1011318.

[64] Saccone, G. (2022) A history of the genetic and molecular identification of genes and their functions controlling insect sex determination. Insect Biochemistry and Molecular Biology, 151, 103873.

[65] Sahara, K., Yamada, Y., Saitoh, H., Nakada, T., Asano, S., Bando, H. et al. (1997) Survival rate and egg feature of first filial (F₁) tetrapoid silkworms from tetraploid parents. Journal of Sericultural Science of Japan, 66, 341–345 (in Japanese).

[66] Sahara, K., Yoshido, A. and Traut, W. (2012) Sex chromosome evolution in moths and butterflies. Chromosome Research, 20, 83–94.

[67] Sakai, H., Oshima, H., Yuri, K., Gotoh, H., Daimon, T., Yaginuma, T. et al. (2019) Dimorphic sperm formation by Sex-lethal. Proceedings of the National Academy of Sciences USA, 116, 10412–10417.

[68] Sakai, H., Sakaguchi, H., Aoki, F. and Suzuki, M.G. (2015) Functional analysis of sex-determination genes by gene silencing with LNA–DNA gapmers in the silkworm, Bombyx mori. Mechanisms of Development, 137, 45–52.

[69] Sakai, H., Sumitani, M., Chikami, Y., Yahata, K., Uchino, K., Kiuchi, T. et al. (2016) 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 Genetics, 12, e1006203.

[70] Sawanth, S.K., Gopinath, G., Sambrani, N. and Arunkumar, K.P. (2016) The autoregulatory loop: a common mechanism of regulation of key sex determining genes in insects. Journal of Biosciences, 41, 283–294.

[71] Seiler, J. and Beye, M. (2024) Honeybees' novel complementary sex-determining system: function and origin. Trends in Genetics, 40, 969–981.

[72] Sugano, Y., Kokusho, R., Ueda, M., Fujimoto, M., Tsutsumi, N., Shimada, T. et al. (2016) Identification of a bipartite nuclear localization signal in the silkworm Masc protein. FEBS Letters, 590, 2256–2261.

[73] Šíchová, J., Nguyen, P., Dalíková, M. and Marec, F. (2013) Chromosomal evolution in tortricid moths: conserved karyotypes with diverged features. PLoS ONE, 8, e64520.

[74] Šíchová, J., Ohno, M., Dincă, V., Watanabe, M., Sahara, K. and Marec, F. (2016) Fissions, fusions, and translocations shaped the karyotype and multiple sex chromosome constitution in the northeast-Asian wood white butterfly, Leptidea amurensis. Biological Journal of the Linnean Society, 118, 457–471.

[75] Šíchová, J., Voleníková, A., Dincă, V., Nguyen, P., Vila, R., Sahara, K. et al. (2015) Dynamic karyotype evolution and unique sex determination systems in Leptidea wood white butterflies. BMC Evolutionary Biology, 15, 89.

[76] Suzuki, M.G. (2010) Sex determination: insights from the silkworm. Journal of Genetics, 89, 357–363.

[77] Suzuki, M.G., Funaguma, S., Kanda, T., Tamura, T. and Shimada, T. (2003) Analysis of the biological functions of a doublesex homologue in Bombyx mori. Development Genes and Evolution, 213, 345–354.

[78] Suzuki, M.G., Imanishi, S., Dohmae, N., Asanuma, M. and Matsumoto, S. (2010) Identification of a male-specific RNA binding protein that regulates sex-specific splicing of Bmdsx by increasing RNA binding activity of BmPSI. Molecular and Cellular Biology, 30, 5776–5786.

[79] Suzuki, M.G., Imanishi, S., Dohmae, N., Nishimura, T., Shimada, T. and Matsumoto, S. (2008) Establishment of a novel in vivo sex-specific splicing assay system to identify a trans-acting factor that negatively regulates splicing of Bombyx mori dsx female exons. Molecular and Cellular Biology, 28, 333–343.

[80] Suzuki, M.G., Kobayashi, S. and Aoki, F. (2014) Male-specific splicing of the silkworm Imp gene is maintained by an autoregulatory mechanism. Mechanisms of Development, 131, 47–56.

[81] Suzuki, M.G., Ohbayashi, F., Mita, K. and Shimada, T. (2001) The mechanism of sex-specific splicing at the doublesex gene is different between Drosophila melanogaster and Bombyx mori. Insect Biochemistry and Molecular Biology, 31, 1201–1211.

[82] Suzuki, M.G., Suzuki, K., Aoki, F. and Ajimura, M. (2012) Effect of RNAi-mediated knockdown of the Bombyx mori transformer-2 gene on the sex-specific splicing of Bmdsx pre-mRNA. The International Journal of Developmental Biology, 56, 693–699.

[83] Tamura, T., Thibert, C., Royer, C., Kanda, T., Abraham, E., Kamba, M. et al. (2000) Germline transformation of the silkworm Bombyx mori L. using a piggyBac transposon-derived vector. Nature Biotechnology, 18, 81–84.

[84] Tan, A., Fu, G., Jin, L., Guo, Q., Li, Z., Niu, B. et al. (2013) Transgene-based, female-specific lethality system for genetic sexing of the silkworm, Bombyx mori. Proceedings of the National Academy of Sciences USA, 110, 6766–6770.

[85] Tanaka, Y. (1916) Genetic studies on the silkworm. The Journal of the College of Agriculture, Tohoku Imperial University, Sapporo, Japan, 7, 129–255. http://hdl.handle.net/2115/12539

[86] Tazima, Y. (1964) The Genetics of the Silkworm. Academic Press, London, UK.

[87] Tomihara, K., Kawamoto, M., Suzuki, Y., Katsuma, S. and Kiuchi, T. (2022) Masculinizer-induced dosage compensation is achieved by transcriptional downregulation of both copies of Z-linked genes in the silkworm, Bombyx mori. Biology Letters, 18, 20220116.

[88] Traut, W. (1999) The evolution of sex chromosomes in insects: Differentiation of sex chromosomes in flies and moths. European Journal of Entomology, 96, 227–235.

[89] Traut, W. and Marec, F. (1997) Sex chromosome differentiation in some species of Lepidoptera (Insecta). Chromosome Research, 5, 283–291.

[90] Traut, W., Sahara, K. and Marec, F. (2007) Sex chromosomes and sex determination in Lepidoptera. Sexual Development, 1, 332–346.

[91] Traut, W., Vogel, H., Glockner, G., Hartmann, E. and Heckel, D.G. (2013) High-throughput sequencing of a single chromosome: a moth W chromosome. Chromosome Research, 21, 491–505.

[92] Ueno, M., Nakata, M., Kaneko, Y., Iwami, M., Takayanagi-Kiya, S. and Kiya, T. (2023) fruitless is sex-differentially spliced and is important for the courtship behavior and development of silkmoth Bombyx mori. Insect Biochemistry and Molecular Biology, 159, 103989.

[93] Van Nieukerken, E.J., Kaila, L., Kitching, I.J., Kristensen, N.P., Lees, D.C., Minet, J. et al. (2011) Order Lepidoptera Linnaeus, 1758. Animal biodiversity: An Outline of Higher-Level Classification and Survey of Taxonomic Richness (ed. Z.Q. Zhang). Zootaxa, 3148, 212–221.

[94] Van′t Hof, A.E., Whiteford, S., Yung, C.J., Yoshido, A., Zrzavá, M., de Jong, M.A. et al. (2024) Zygosity-based sex determination in a butterfly drives hypervariability of Masculinizer. Science Advances, 10, eadj6979.

[95] Visser, S., Voleníková, A., Nguyen, P., Verhulst, E.C. and Marec, F. (2021) A conserved role of the duplicated Masculinizer gene in sex determination of the Mediterranean flour moth, Ephestia kuehniella. PLoS Genetics, 17, e1009420.

[96] Vítková, M., Fuková, I., Kubíčková, S. and Marec, F. (2007) Molecular divergence of the W chromosomes in pyralid moths (Lepidoptera). Chromosome Research, 15, 917–930.

[97] Wang, Y., Chen, X., Liu, Z., Xu, J., Li, X., Bi, H. et al. (2019a) Mutation of doublesex induces sex-specific sterility of the diamondback moth Plutella xylostella. Insect Biochemistry and Molecular Biology, 112, 103180.

[98] Wang, Y.H., Chen, X.E., Yang, Y., Xu, J., Fang, G.Q., Niu, C.Y. et al. (2019b) The Masc gene product controls masculinization in the black cutworm, Agrotis ipsilon. Insect Science, 26, 1037–1044.

[99] Wei, Z., Wang, C., Zhang, X., Lv, Y., Li, Y., Gao, P. et al. (2025) CRISPR/Cas9-mediated knockout of Tektin 4-like gene (TEKT4L) causes male sterility of Cydia pomonella. Insect Biochemistry and Molecular Biology, 177, 104257.

[100] Wright, C.J., Stevens, L., Mackintosh, A., Lawniczak, M. and Blaxter, M. (2024) Comparative genomics reveals the dynamics of chromosome evolution in Lepidoptera. Nature Ecology & Evolution, 8, 777–790.

[101] Xu, J., Chen, S., Zeng, B., James, A.A., Tan, A. and Huang, Y. (2017) Bombyx mori P-element somatic inhibitor (BmPSI) is a key auxiliary factor for silkworm male sex determination. PLoS Genetics, 13, e1006576.

[102] Xu, J., Liu, W., Yang, D., Chen, S., Chen, K., Liu, Z. et al. (2020) Regulation of olfactory-based sex behaviors in the silkworm by genes in the sex-determination cascade. PLoS Genetics, 16, e1008622.

[103] Yamaguchi, J., Mizoguchi, T. and Fujiwara, H. (2011) siRNAs induce efficient RNAi response in Bombyx mori embryos. PLoS ONE, 6, e25469.

[104] Yang, X., Chen, K., Wang, Y., Yang, D. and Huang, Y. (2021a) The sex determination cascade in the silkworm. Genes, 12, 315.

[105] Yang, F., Zhang, Z., Hu, B., Yu, Y. and Tan, A. (2021b) A CCCH zinc finger gene regulates doublesex alternative splicing and male development in Bombyx mori. Insect Science, 28, 1253–1261.

[106] Yoshido, A. and Marec, F. (2023) Deviations in the Z:A ratio disrupt sexual development in the eri silkmoth, Samia cynthia ricini. Genetics, 224, iyad023.

[107] Yoshido, A., Marec, F. and Sahara, K. (2016) The fate of W chromosomes in hybrids between wild silkmoths, Samia cynthia ssp.: no role in sex determination and reproduction. Heredity, 116, 424–433.

[108] Yoshido, A., Sahara, K., Marec, F. and Matsuda, Y. (2011) Step-by-step evolution of neo-sex chromosomes in geographical populations of wild silkmoths, Samia cynthia ssp. Heredity, 106, 614–624.

[109] Yoshido, A., Šíchová, J., Kubíčková, S., Marec, F. and Sahara, K. (2013) Rapid turnover of the W chromosome in geographical populations of wild silkmoths, Samia cynthia ssp. Chromosome Research, 21, 149–164.

[110] Yoshido, A., Šíchová, J., Pospíšilová, K., Nguyen, P., Šafář, J., Provazník, J. et al. (2020) Evolution of multiple sex-chromosomes associated with dynamic genome reshuffling in Leptidea wood-white butterflies. Heredity, 125, 138–154.

[111] Zhao, Q., Li, J., Wen, M.Y., Wang, H., Wang, Y., Wang, K.X. et al. (2019) A novel splice variant of the masculinizing gene Masc with piRNA-cleavage-site defect functions in female external genital development in the silkworm, Bombyx mori. Biomolecules, 9, 318.

[112] Zheng, Z.Z., Sun, X., Zhang, B., Pu, J., Jiang, Z.Y., Li, M. et al. (2019) Alternative splicing regulation of doublesex gene by RNA-binding proteins in the silkworm Bombyx mori. RNA Biology, 16, 809–820.

[113] Zrzavá, M., Hladová, I., Dalíková, M., Šíchová, J., Õunap, E., Kubíčková, S. et al. (2018) Sex chromosomes of the iconic moth Abraxas grossulariata (Lepidoptera, Geometridae) and its congener A. sylvata. Genes, 9, 279.

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