Combined actions of bacteriophage-encoded genes in Wolbachia-induced male lethality

Abstract

Some Wolbachia endosymbionts induce male killing, whereby male offspring of infected females are killed during development; however, the origin and diversity of the underlying mechanisms remain unclear. In this study, we identified a 76 kbp prophage region specific to male-killing Wolbachia hosted by the moth Homona magnanima. The prophage encoded a homolog of the male-killing gene oscar in Ostrinia moths and the wmk gene that induces various toxicities in Drosophila melanogaster. Upon overexpressing these genes in D. melanogaster, wmk-1 and wmk-3 killed all males and most females, whereas Hm-oscar, wmk-2, and wmk-4 had no impact on insect survival. Strikingly, co-expression of tandemly arrayed wmk-3 and wmk-4 killed 90% of males and restored 70% of females, suggesting their conjugated functions for male-specific lethality. While the male-killing gene in the native host remains unknown, our findings highlight the role of bacteriophages in male-killing evolution and differences in male-killing mechanisms among insects.

Keywords: Evolutionary biology; Genetics; Microbiology; Virology.

Graphical abstract

Figure 1

Genomic comparisons of Wolbachia strains in Homona magnanima (A) An insertion, the WOwHm-t76 region, in the wHm-t genome. The wHmc_11920 of the wHm-c encodes the IS110 family of transposase and has identical sequences of wHmt_12330 of the wHm-t. wHmc_11930 of wHm-c, encoding cytoplasmic incompatibility factor A (CifA), is identical to wHmt_13170 of wHm-t. The area highlighted by (B) and surrounded by a dashed red line indicates the locus encoding pathogenesis-related genes referred to in the following section. (B) Virulence-associated genes in the WOwHm-t76 region. (C) The domain structures of wmk and Hm-oscar genes annotated by Inter-Pro. HTH: helix-turn-helix. ANK: ankyrin repeat. PP: papain-like proteinase domain. (D) Genomic loci, absent from wHm-c genomes (n = 9) but conserved in wHm-t genomes (n = 15), are highlighted in red on the wHm-t genome. Homologous genes or loci between wHm-c and wHm-t are not highlighted in red. (E) Detection of the WOwHm-t76 region (i.e., Hm-oscar gene) from Wolbachia infecting Japanese and Taiwanese H. magnanima.

Figure 2

Phylogenies and toxicities of wmk and Hm-oscar genes in the WOwHm-t76 region (A and B) Phylogenetic trees of wmk gene homologs (nucleotide) (A) and Oscar and CifB protein homologs (amino acids) (B). Phylogenetic trees were constructed based on maximum likelihood with bootstrap re-sampling of 1,000 replicates using MEGA7. Homologs of Wolbachia strains were quoted from the NCBI database, and those of wHm-a, wHm-b, and wHm-t strains were manually annotated by BLAST and HHpred searches. The wmk and Oscar homologs present in wHm-t but absent in the wHm-a, wHm-b, and wHm-c are highlighted in red (wmk-1, wmk-2, wmk-3, wmk-4, and Hm-Oscar). wmk-1, wmk-2, wmk-3, and wmk-4 in the wBol1 genome are also highlighted in red. (C) RT-PCR detection for the wmk and Hb-oscar genes. 12, 36, 60, 84, and 108 indicate hours post oviposition of Homona magnanima egg masses. Ov+: female ovary of H. magnanima (W^T12 line); Ov-: non-reverse-transcribed samples (RT minus, control) using RNA extracted from the female ovary of H. magnanima (W^T12 line); Hb: female ovary of Hypolimnas bolina; P: DNA extracted from an H. magnanima W^T12 adult female (positive control); N: negative control (water). M: DNA ladders. 1000 bp ladder for Hm-oscar, and 100 bp ladder for other genes. (D) The male ratio of adult progeny obtained from crosses between the actin–GAL4 Drosophila melanogaster line and seven UAS transgenic fly lines (wmk-1, wmk-2, wmk-3, wmk-4, cifB-like, wmk-1 and wmk-2, and wmk-3 and wmk-4; n = 11–33 independent crosses for each transgene). We counted the number of resulting offspring (females, orange; males, blue) with both actin–GAL4 and UAS (+) and siblings with UAS and Cyo (CyO) as internal controls (−). The total numbers of adult counts for each genotype and sex are shown at the bottom. Different letters indicate statistically significant differences (Steel–Dwass test, p < 0.05). Dot plots show all data points individually. Exp.: expression; (−): not expressed; (+): expressed. Female: adult female progeny; Male: adult male progeny.