Moths and butterflies (Lepidoptera) have sex chromosome systems with female heterogamety, and 2 models, W-dominance and Z-counting, have been proposed to determine sex. The W-dominant mechanism is well known in Bombyx mori. However, little is known about the Z-counting mechanism in Z0/ZZ species. We investigated whether ploidy changes affect sexual development and gene expression in the eri silkmoth, Samia cynthia ricini (2n = 27♀/28♂, Z0♀/ZZ♂). Tetraploid males (4n = 56, ZZZZ) and females (4n = 54, ZZ) were induced by heat and cold shock, and then, triploid embryos were produced by crosses between diploids and tetraploids. Two karyotypes (3n = 42, ZZZ and 3n = 41, ZZ) were identified in triploid embryos. Triploid embryos with 3 Z chromosomes showed male-specific splicing of the S. cynthia doublesex (Scdsx) gene, whereas 2-Z triploid embryos showed both male- and female-specific splicing. From larva to adult, 3-Z triploids showed a normal male phenotype, except for defects in spermatogenesis. However, abnormal gonads were observed in 2-Z triploids, which showed both male- and female-specific Scdsx transcripts not only in the gonads but also in somatic tissues. Two-Z triploids were thus obviously intersexes, suggesting that sexual development in S. c. ricini depends on the Z:A ratio and not only on the Z number. Moreover, mRNA-seq analyses in embryos showed that relative levels of gene expression are similar between samples with different doses of Z chromosomes and autosome sets. Our results provide the first evidence that ploidy changes disrupt sexual development but have no effect on the general mode of dosage compensation in Lepidoptera.

Biology Centre of the Czech Academy of Sciences Institute of Entomology Branišovská 31 370 05 České Budějovice Czech Republic

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