Transient expressions of the Hm-oscar gene resulted in female-biased sex ratios

Male ratio of adult progeny obtained from cRNA-injected groups (wmk-1, wmk-2, T2A-brigged wmk-1 and wmk-2, wmk-3, wmk-4, T2A-brigged wmk-3 and wmk-4, GFP, and Hm-oscar; n = 5–15 independent replicates using different egg masses), the non-injected NSR line, and the MK wHm-t-positive WT12 line. The total numbers of adult females and males are shown at the bottom. Different letters indicate significant differences (Steel–Dwass test, P < 0.05). The dot plots show all data points individually.

Hm-Oscar induced lethality in male embryos with the female-type sex determination

(a) Sex ratio of the hatched larvae and unhatched embryos in the Hm-oscar-expressed, wHm-t-infected, and non-infected/expressed groups. Females and males were discriminated based on the presence or absence of W chromatin. The number of individuals is indicated in brackets. (b) Splicing patterns of the downstream sex-determining gene dsx of H. magnanima embryos (5 days post oviposition). Abbreviations: HmOsc, Hm-oscar injected group; GFP, GFP-injected group; WT12, wHm-t-infected line; NSR, non-infected/injected line. M and F indicate W chromatin-negative (ZZ: male genotype) and W chromatin-positive (ZW: female genotype) mature embryos, respectively. dsx-F and dsx-M represent female and male-specific splicing variants, respectively.

Hm-oscar-overexpressed male embryos showed higher levels of Z-linked gene expression

(a-d) Normalized expression levels (TPM) and chromosomal distributions of transcripts in H. magnanima embryos. RNA-seq data of embryos (108 hpo) were used to make the following comparisons: Hm-oscar-injected males versus Hm-oscar injected females (a), GFP-injected males versus GFP-injected females (b), WT12 males versus WT12 females (c), and NSR males versus NSR females (d). The chromosome number for each H. magnanima transcript-derived contig was assigned based on Bombyx mori gene models. The X axis represents the chromosome number of B. mori (shown as chr01 to chr28), and chr01 and chr15 (highlighted in orange) correspond to the Z chromosome of H. magnanima (Arai et al., 2023a).

Hm-Oscar suppressed the masculinizing function of lepidopteran Masc

Relative expression levels of the male-specific BmImpM variant in transfected BmN-4 cells. The expression of BmImpM was normalized using the housekeeping gene rp49. The expression levels of BmImpM in the Masc and Hm-Oscar/Oscar co-transfected cells were normalized by setting each Masc-transfected cell as 100. The dot plots show all data points individually. Each experimental condition was replicated three times. Abbreviations: Hm, Homona magnanima; Bm, Bombyx mori; Of, Ostrinia furnacalis; Pm, Papilio machaon; Sf, Spodoptra frugiperda; and Tv, Trilocha varians.