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

(a) Structures of wFur-encoded Oscar and wHm-t-encoded Hm-Oscar and four Wmk proteins. (b) 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. L: 100 bp DNA ladder (ExcelBand 100 bp DNA Ladder, SMOBIO Technology, Inc., Hsinchu, Taiwan). 0.1, 0.5, 1.0, and 1.5 kb markers are indicated with arrows. N: negative control (water).

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). (e) Relative expression of a Z-linked tpi gene in H. magnanima groups. Statistical significance is highlighted with different alphabets.

Hm-oscar suppressed the masculinizing function of lepidopteran Masc

(a) Relative expression levels of the male-specific BmImpM variant in transfected BmN-4 cells. The relative expression of BmImpM in the control (non-inserted pIZ/V5) and Hm-oscar/oscar co-transfected groups was normalized by setting the mean in the Hmmasc single transfected group as 100. (b) Protein structures and homologies of Hm-Oscar and Oscar proteins. (c) Relative expression levels of the BmImpM in Hm-oscar/masc transfected BmN-4 cells. The relative expression of BmImpM in each condition was normalized by setting the mean in the Hmmasc single transfected group 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, Spodoptera frugiperda; and Tv, Trilocha varians. (d) Phylogeny and homologies of the Masc protein sequences used in this study.