BACKGROUND: The primary signals of sex determination in insects are diverse and evolve erratically. This also appears to be the case with moths and butterflies. In the silkworm Bombyx mori, female development is triggered by a W chromosome-derived Feminizer (Fem) piRNA that degrades the mRNA of the Z-linked Masculinizer (Masc) gene, which controls male development. We investigated whether this mechanism is conserved in another group of Lepidoptera. RESULTS: We identified a putative feminizing piRNA and many partial copies of the EkMasc gene on the W chromosome of Ephestia kuehniella. The piRNA is generated by a repetitive W-linked sequence named E. kuehniella Moth-overruler-of-masculinization (EkMom). EkMom piRNA shows high similarity to a region of Z-linked EkMasc and is expressed at the onset of female development, but has no relationship to the B. mori Fem piRNA. We then mapped small RNA-seq data from embryos of the related Plodia interpunctella to the PiMasc gene and identified a single small RNA, a PiMom piRNA, able to target PiMasc and with high sequence identity to the EkMom piRNA. Both the PiMom and EkMom repeats are present in high copy number and form a single cluster on the W chromosome. In both species, the Mom piRNA is responsible for Masc mRNA cleavage, clearly demonstrating that the Mom piRNA triggers female development. CONCLUSIONS: Our study provides multiple lines of evidence that Mom piRNA is the primary sex-determining signal in two pyralid moths and highlights a possible pathway for the origin of feminizing piRNAs in Lepidoptera. The similarity in female sex determination between the phylogenetically distant species suggests convergent evolution of feminizing piRNAs in Lepidoptera.

Biology Centre of the Czech Academy of Sciences Institute of Entomology 370 05 České Budějovice Czech Republic

Department of Ecology and Evolutionary Biology University of Kansas Lawrence KS 66045 USA

Faculty of Medicine 2 Charles University Prague and University Hospital Motol 150 06 Prague 5 Czech Republic

Faculty of Science University of South Bohemia 370 05 České Budějovice Czech Republic

Groningen Institute for Evolutionary Life Sciences University of Groningen 9747 AG Groningen The Netherlands

Laboratory of Entomology Wageningen University and Research 6700 AA Wageningen The Netherlands

Present Address European Molecular Biology Laboratory 69117 Heidelberg Germany

Zobrazit více v PubMed

Nöthiger R, Steinmann-Zwicky M. A single principle for sex determination in insects. Cold Spring Harb Symp Quant Biol. 1985;50:615–21. 10.1101/sqb.1985.050.01.074. PubMed

Wilkins AS. Moving up the hierarchy: a hypothesis on the evolution of a genetic sex determination pathway. BioEssays. 1995;17:71–7. 10.1002/bies.950170113. PubMed

Sánchez L. Sex-determining mechanisms in insects. Int J Dev Biol. 2008;52:837–56. 10.1387/ijdb.072396ls. PubMed

Baker BS, Wolfner MF. A molecular analysis of PubMed

Saccone G. A history of the genetic and molecular identification of genes and their functions controlling insect sex determination. Insect Biochem Mol Biol. 2022;151:103873. 10.1016/j.ibmb.2022.103873. PubMed

Salz HK, Erickson JW. Sex determination in PubMed PMC

Beye M, Hasselmann M, Fondrk MK, Page RE, Omholt SW. The gene PubMed

Hall AB, Basu S, Jiang X, Qi Y, Timoshevskiy VA, Biedler JK, et al. A male-determining factor in the mosquito PubMed PMC

Criscione F, Qi Y, Tu Z. GUY1 confers complete female lethality and is a strong candidate for a male-determining factor in Anopheles stephensi. eLife. 2016;5:e19281. 10.7554/eLife.19281.001. PubMed PMC

Krzywinska E, Dennison NJ, Lycett GJ, Krzywinski J. A maleness gene in the malaria mosquito PubMed

Sharma A, Heinze SD, Wu Y, Kohlbrenner T, Morilla I, Brunner C, et al. Male sex in houseflies is determined by PubMed

Zou Y, Geuverink E, Beukeboom LW, Verhulst EC, van de Zande L. A chimeric gene paternally instructs female sex determination in the haplodiploid wasp PubMed

Liu P, Yang W, Kong L, Zhao S, Xie Z, Zhao Y, et al. A DBHS family member regulates male determination in the filariasis vector PubMed PMC

Meccariello A, Salvemini M, Primo P, Hall B, Koskinioti P, Dalíková M, et al. PubMed

Traut W, Sahara K, Marec F. Sex chromosomes and sex determination in Lepidoptera. Sex Dev. 2007;1:332–46. 10.1159/000111765. PubMed

Sahara K, Yoshido A, Traut W. Sex chromosome evolution in moths and butterflies. Chromosoma Res. 2012;20:83–94. 10.1007/s10577-011-9262-z. PubMed

Hejníčková M, Koutecký P, Potocký P, Provazníková I, Voleníková A, Dalíková M, et al. Absence of W chromosome in Psychidae moths and implications for the theory of sex chromosome evolution in Lepidoptera. Genes. 2019;10(12):1016. 10.3390/genes10121016. PubMed PMC

Yoshido A, Šíchová J, Pospíšilová K, Nguyen P, Šafář J, Provazník J, et al. Evolution of multiple sex-chromosomes associated with dynamic genome reshuffling in PubMed PMC

Hashimoto H. The role of the W chromosome for sex determination in the silkworm. Bombyx mori Jap J Genet. 1933;8:245–7. 10.1266/jjg.8.245.

Tazima Y. The genetics of the silkworm. London: Academic Press; 1964.

Abe H, Mita K, Yasukochi Y, Oshiki T, Shimada T. Retrotransposable elements on the W chromosome of the silkworm, PubMed

Katsuma S, Kiuchi T, Kawamoto M, Fujimoto T, Sahara K. Unique sex determination system in the silkworm, PubMed PMC

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;509:633–6. 10.1038/nature13315. PubMed

Sakai H, Sakaguchi H, Aoki F, Suzuki MG. Functional analysis of sex-determination genes by gene silencing with LNA–DNA gapmers in the silkworm, PubMed

Kaneda T, Matsuda-Imai N, Kosako H, Shoji K, Suzuki MG, Suzuki Y, et al. The masc–PSI complex directly induces male-type PubMed PMC

Tomihara K, Kawamoto M, Suzuki Y, Katsuma S, Kiuchi T. Masculinizer-induced dosage compensation is achieved by transcriptional downregulation of both copies of Z-linked genes in the silkworm. PubMed PMC

Katsuma S, Kawamoto M, Kiuchi T. Guardian small RNAs and sex determination. RNA Biol. 2014;11:1238–42. 10.1080/15476286.2014.996060. PubMed PMC

Czech B, Hannon GJ. One loop to rule them all: the ping-pong cycle and piRNA-guided silencing. Trends Biochem Sci. 2016;41:324–37. 10.1016/j.tibs.2015.12.008. PubMed PMC

Kristensen NP, Skalski AW. Phylogeny and palaeontology. Handbook of Zoology, Vol. IV, Arthropoda: Insecta, Part 35, Lepidoptera, Moths and Butterflies, Vol. 1: Evolution, Systematics, and Biogeography. Edited by: Kristensen NP. Berlin & New York: Walter de Gruyter, 1999. p. 7–25. 10.1515/9783110804744.

Fukui T, Kawamoto M, Shoji K, Kiuchi T, Sugano S, Shimada T, et al. The endosymbiotic bacterium PubMed PMC

Lee J, Kiuchi T, Kawamoto M, Shimada T, Katsuma S. Identification and functional analysis of a PubMed

Wang YH, Chen XE, Yang Y, Xu J, Fang GQ, Niu CY, et al. The PubMed

Harvey-Samuel T, Norman VC, Carter R, Lovett E, Alphey L. Identification and characterization of a PubMed PMC

Deng Z, Zhang Y, Li Y, Huang K, Chen X, Zhang M, et al. Identification and characterization of the masculinizing function of the PubMed PMC

Visser S, Voleníková A, Nguyen P, Verhulst EC, Marec F. A conserved role of the duplicated PubMed PMC

Pospíšilová K, Van ’t Hof AE, Yoshido A, Kružíková R, Visser S, Zrzavá M, Bobryshava K, Dalíková M, Marec F. PubMed

Li X, Liu H, Bi H, Wang Y, Xu J, Zhang S, et al. PubMed

Moronuki Y, Kasahara R, Naka H, Suzuki MG. Identification and functional analysis of sex-determining genes in the spongy moth, PubMed

Van’t Hof AE, Whiteford S, Yung CJ, Yoshido A, Zrzavá M, de Jong MA, et al. Zygosity-based sex determination in a butterfly drives hypervariability of PubMed PMC

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 PubMed

Fukui T, Shoji K, Kiuchi T, Suzuki Y, Katsuma S. PubMed

Yoshido A, Marec F, Sahara K. The fate of W chromosomes in hybrids between wild silkmoths, PubMed PMC

Yoshido A, Marec F. Deviations in the Z:A ratio disrupt sexual development in the eri silkmoth, PubMed

Dalíková M, Zrzavá M, Hladová I, Nguyen P, Šonský I, Flegrová M, et al. New insights into the evolution of the W chromosome in Lepidoptera. J Hered. 2017;108:709–19. 10.1093/jhered/esx063. PubMed

Hejníčková M, Dalíková M, Potocký P, Tammaru T, Trehubenko M, Kubíčková S, et al. Degenerated, undifferentiated, rearranged, lost: high variability of sex chromosomes in Geometridae (Lepidoptera) identified by sex chromatin. Cells. 2021;10:2230. 10.3390/cells10092230. PubMed PMC

Harvey-Samuel T, Xu X, Anderson MAE, Carabajal Paladino LZ, Purusothaman D, Norman VC, et al. Silencing RNAs expressed from W-linked PubMed PMC

Robinson R. Lepidoptera Genetics. 1st ed. Oxford: Pergamon Press; 1971.

Traut W, Vogel H, Glockner G, Hartmann E, Heckel DG. High-throughput sequencing of a single chromosome: a moth W chromosome. Chromosom Res. 2013;21:491–505. 10.1007/s10577-013-9376-6. PubMed

Dalíková M, Zrzavá M, Kubíčková S, Marec F. W-enriched satellite sequence in the Indian meal moth, PubMed

Vítková M, Fuková I, Kubíčková S, Marec F. Molecular divergence of the W chromosomes in pyralid moths (Lepidoptera). Chromosoma Res. 2007;15:917–30. 10.1007/s10577-007-1173-7. PubMed

Marec F. Synaptonemal complexes in insects. Int J Insect Morphol Embryol. 1996;25:205–33. 10.1016/0020-7322(96)00009-8.

Zrzavá M, Hladová I, Dalíková M, Šíchová J, Õunap E, Kubíčková S, et al. Sex chromosomes of the iconic moth PubMed PMC

Gotter AL, Levine JD, Reppert SM. Sex-linked PubMed

Van’t Hof AE, Nguyen P, Dalíková M, Edmonds N, Marec F, Saccheri IJ. Linkage map of the peppered moth, PubMed PMC

Smith DA, Gordon IJ, Traut W, Herren J, Collins S, Martins DJ, et al. A neo-W chromosome in a tropical butterfly links colour pattern, male-killing, and speciation. Proc Biol Sci. 2016;283:20160821. 10.1098/rspb.2016.0821. PubMed PMC

Mongue AJ, Nguyen P, Voleníková A, Walters JR. Neo-sex chromosomes in the monarch butterfly, PubMed PMC

Deng Z, Zhang Y, Zhang M, Huang J, Li C, Ni X, et al. Characterization of the first W-specific protein-coding gene for sex identification in PubMed PMC

Charlesworth B, Charlesworth D. The degeneration of Y chromosomes. Philos Trans R Soc Lond B Biol Sci. 2000;355:1563–72. 10.1098/rstb.2000.0717. PubMed PMC

Skaletsky H, Kuroda-Kawaguchi T, Minx PJ, Cordum HS, Hillier L, Brown LG, Repping S, Pyntikova T, Ali J, Bieri T, Chinwalla A, Delehaunty A, Delehaunty K, Du H, Ginger G, Fulton L, Fulton R, Graves T, Hou SF, Latrielle P, Leonard S, Mardis E, Maupin R, McPherson J, Miner T, Nash W, Nguyen C, Ozersky P, Pepin K, Rock S, Rohlfing T, Scott K, Schultz B, Strong C, Tin-Wollam A, Yang SP, Waterston RH, Wilson RK, Rozen S, Page DC. The male-specific region of the human Y chromosome is a mosaic of discrete sequence classes. Nature. 2003;423:825–37. 10.1038/nature01722. PubMed

Bachtrog D. A dynamic view of sex chromosome evolution. Curr Opin Genet Dev. 2006;16:578–85. 10.1016/j.gde.2006.10.007. PubMed

Hori T, Asakawa S, Itoh Y, Shimizu N, Mizuno S. PubMed PMC

Backström N, Ceplitis H, Berlin S, Ellegren H. Gene conversion drives the evolution of PubMed

Chen JM, Cooper DN, Chuzhanova N, Férec C, Patrinos GP. Gene conversion: mechanisms, evolution and human disease. Nat Rev Genet. 2007;8:762–75. 10.1038/nrg2193. PubMed

Bachtrog D. Y-chromosome evolution: emerging insights into processes of Y-chromosome degeneration. Nat Rev Genet. 2013;14:113–24. 10.1038/nrg3366. PubMed PMC

Hamm DC, Harrison MM. Regulatory principles governing the maternal-to-zygotic transition: insights from PubMed PMC

Zhang M, Xu P, Pang H, Chen T, Zhang G. Expression analysis of mRNA decay of maternal genes during PubMed PMC

Wang W, Yoshikawa M, Han BW, Izumi N, Tomari Y, Weng Z, et al. The initial uridine of primary piRNAs does not create the tenth adenine that is the hallmark of secondary piRNAs. Mol Cell. 2014;56:708–16. 10.1016/j.molcel.2014.10.016. PubMed PMC

Klattenhoff C, Theurkauf W. Biogenesis and germline functions of piRNAs. Development. 2008;135:3–9. 10.1242/dev.006486. PubMed

Ghildiyal M, Zamore PD. Small silencing RNAs: an expanding universe. Nat Rev Genet. 2009;10:94–108. 10.1038/nrg2504. PubMed PMC

Kawaoka S, Kadota K, Arai Y, Suzuki Y, Fujii T, Abe H, et al. The silkworm W chromosome is a source of female-enriched piRNAs. RNA. 2011;17:2144–51. 10.1261/rna.027565.111. PubMed PMC

Yoshido A, Marec F, Sahara K. Resolution of sex chromosome constitution by genomic PubMed

Yoshido A, Yamada Y, Sahara K. The W chromosome detection in several lepidopteran species by genomic in situ hybridization (GISH). J Insect Biotechnol Sericol. 2006;75:147–51. 10.11416/jibs.75.147.

Sakai H, Sumitani M, Chikami Y, Yahata K, Uchino K, Kiuchi T, et al. Transgenic expression of the piRNA-resistant PubMed PMC

Marec F. Genetic control of pest Lepidoptera: induction of sex-linked recessive lethal mutations in

Kearse M, Moir R, Wilson A, Stones-Havas S, Cheung M, Sturrock S, et al. Geneious basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics. 2012;28:1647–9. 10.1093/bioinformatics/bts199. PubMed PMC

R Core Team. R: a language and environment for statistical computing. Vienna: R Foundation for Statistical Computing; 2018.

Wickham H. Ggplot2: elegant graphics for data analysis. New York: Springer-Verlag; 2016.

Untergasser A, Cutcutache I, Koressaar T, Ye J, Faircloth BC, Remm M, et al. Primer3—new capabilities and interfaces. Nucleic Acids Res. 2012;40:e115. 10.1093/nar/gks596. PubMed PMC

Challis RJ, Kumar S, Dasmahapatra KK, Jiggins CD, Blaxter M. Lepbase: the Lepidopteran genome database. bioRxiv. 2016. 10.1101/056994.

Katoh K, Misawa K, Kuma K, Miyata T. MAFFT: a novel method for rapid multiple sequence alignment based on fast Fourier transform. Nucleic Acids Res. 2002;30:3059–66. 10.1093/nar/gkf436. PubMed PMC

Katoh K, Standley DM. MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol Biol Evol. 2013;30:772–80. 10.1093/molbev/mst010. PubMed PMC

Huelsenbeck JP, Ronquist F. MRBAYES: Bayesian inference of phylogenetic trees. Bioinformatics. 2001;17:754–5. 10.1093/bioinformatics/17.8.754. PubMed

Buntrock L, Marec F, Krueger S, Traut W. Organ growth without cell division: somatic polyploidy in a moth, PubMed

Mediouni J, Fuková I, Frydrychová R, Dhouibi MH, Marec F. Karyotype, sex chromatin and sex chromosome differentiation in the carob moth,

Šíchová J, Nguyen P, Dalíková M, Marec F. Chromosomal evolution in tortricid moths: conserved karyotypes with diverged features. PLoS One. 2013;8:e64520. 10.1371/journal.pone.0064520. PubMed PMC

Ferguson KB, Visser S, Dalíková M, Provazníková I, Urbaneja A, Pérez-Hedo M, et al. Jekyll or hyde? The genome (and more) of PubMed PMC

Kato A, Albert PS, Vega JM, Birchler JA. Sensitive fluorescence in situ hybridization signal detection in maize using directly labeled probes produced by high concentration DNA polymerase nick translation. Biotech Histochem. 2006;81:71–8. 10.1080/10520290600643677. PubMed

Traut W, Sahara K, Otto TD, Marec F. Molecular differentiation of sex chromosomes probed by comparative genomic hybridization. Chromosoma. 1999;108:173–80. 10.1007/s004120050366. PubMed

Carabajal Paladino LZ, Nguyen P, Šíchová J, Marec F. Mapping of single-copy genes by TSA-FISH in the codling moth, PubMed PMC

Frydrychová R, Marec F. Repeated losses of TTAGG telomere repeats in evolution of beetles (Coleoptera). Genetica. 2002;115:179–87. 10.1023/A:1020175912128. PubMed

Watanabe T, Tomizawa S, Mitsuya K, Totoki Y, Yamamoto Y, Kuramochi-Miyagawa S, et al. Role for piRNAs and noncoding RNA in de novo DNA methylation of the imprinted mouse PubMed PMC

Suzuki MG, Suzuki K, Aoki F, Ajimura M. Effect of RNAi-mediated knockdown of the PubMed

Ephestia kuehniella small RNAseq. BioProject. 2024. https://www.ncbi.nlm.nih.gov/bioproject/?term=PRJNA1107079. Accessed 15 August 2025.