Diverse Molecular Mechanisms Underlying Microbe-Inducing Male Killing in the Moth Homona magnanima

Abstract

Male killing (MK) is a type of reproductive manipulation induced by microbes, where sons of infected mothers are killed during development. MK is a strategy that enhances the fitness of the microbes, and the underlying mechanisms and the process of their evolution have attracted substantial attention. Homona magnanima, a moth, harbors two embryonic MK bacteria, namely, Wolbachia (Alphaproteobacteria) and Spiroplasma (Mollicutes), and a larval MK virus, Osugoroshi virus (OGV; Partitiviridae). However, whether the three distantly related male killers employ similar or different mechanisms to accomplish MK remains unknown. Here, we clarified the differential effects of the three male killers on the sex-determination cascades and development of H. magnanima males. Reverse transcription-PCR demonstrated that Wolbachia and Spiroplasma, but not OGVs, disrupted the sex-determination cascade of males by inducing female-type splice variants of doublesex (dsx), a downstream regulator of the sex-determining gene cascade. We also found that MK microbes altered host transcriptomes in different manners; Wolbachia impaired the host dosage compensation system, whereas Spiroplasma and OGVs did not. Moreover, Wolbachia and Spiroplasma, but not OGVs, triggered abnormal apoptosis in male embryos. These findings suggest that distantly related microbes employ distinct machineries to kill males of the identical host species, which would be the outcome of the convergent evolution. IMPORTANCE Many microbes induce male killing (MK) in various insect species. However, it is not well understood whether microbes adopt similar or different MK mechanisms. This gap in our knowledge is partly because different insect models have been examined for each MK microbe. Here, we compared three taxonomically distinct male killers (i.e., Wolbachia, Spiroplasma, and a partiti-like virus) that infect the same host. We provided evidence that microbes can cause MK through distinct mechanisms that differ in the expression of genes involved in sex determination, dosage compensation, and apoptosis. These results imply independent evolutionary scenarios for the acquisition of their MK ability.

Keywords: Partitiviridae; Spiroplasma; Wolbachia; endosymbionts; male killing; symbiosis.

FIG 1

Sex-determination cascade and lethal stages of male killers in H. magnanima. (a) Model of the sex-determination cascade in lepidopteran insects based on a previous report by Kiuchi et al. (30). Masc facilitates the expression of the male-type dsx splicing. Z, Z chromosome; W, W chromosome. (b) Lethal stages of male killers in H. magnanima. Both Wolbachia and Spiroplasma kill males during the mature embryonic stage. Osugoroshi viruses (OGVs) induce late MK during the larval stage. hpo, hour postoviposition.

FIG 2

Effects of male killers on doublesex (dsx) mRNA splicing in H. magnanima. (a) Nine dsx splicing variants in H. magnanima. Females had eight dsx variants (dsx-F1 to dsx-F8) encoding three DSX proteins (DSX-F1 to 3), and males showed a single variant (DSX-M). Rectangular boxes represent exons (A to J). The numbers inside the boxes indicate the numbers of base pairs. Stop codons in each variant are indicated with black or white bars. Primer binding regions are shown with a solid red color. (b) dsx-splicing patterns in hosts harboring wHm-t (W^T12 and W^T24) or wHm-c (W^C) and NSR lines. (c) dsx-splicing patterns in hosts harboring Spiroplasma (S⁺), wHm-t (W^TN10), or OGVs (L). dsx-M, male-type dsx (377 bp); dsx-F, female-type dsx (457 bp and 471 bp). β-actin was amplified as a control gene.

FIG 3

Male killers affected H. magnanima dosage compensation differently. (a to d) Normalized expression levels (TPM) and chromosomal distributions of transcripts in H. magnanima embryos. RNA-seq data (108 hpo) were used to make the following comparisons: W^T12 males versus W^T12 females (a), S⁺ males versus S⁺ females (b), L males versus L females (c), and NSR males versus NSR females (d). The chromosome number for each H. magnanima transcript-derived contig was assigned based on B. mori gene models. The boxes in the box-and-whisker diagrams represent the median and 25 to 75 percentile ranges of the expression ratios. (e to g) Quantification of Z-linked genes in H. magnanima embryos (108 hpo). The Tpi gene dose (e) was quantified to assess whether males have two Z chromosomes. Kettin (f) and Tpi (g) expression levels were normalized to that of the autosomal Ef1α gene. Error bars represent the standard error. The y axis indicates relative abundances adjusted to those of NSR females. Different letters indicate significant differences determined by the Steel-Dwass test (P < 0.05). The numbers inside the bars indicate replicates. Wol, Wolbachia wHm-t, WT12 line; Spi, Spiroplasma sHm, S⁺ line; OGV, L line; NSR, NSR line.

FIG 4

Gene expression patterns in H. magnanima harboring male killers. (a to c) Structures and expression levels of HmMasc splice variants in embryos. (a) Expression levels of HmMasc splice variants in male and female mature embryos (108 hpo). The numbers inside the bars represent base pairs (bp). Both variants had identical 5′ untranslated regions (UTRs; white) and coding sequences (blue); however, their 3′ UTRs (green, HmMasc v1; orange, HmMasc v2) differed. In the bar plot, the bar with green and orange indicate HmMasc v1 and HmMasc v2, respectively. M, male; F, female; Wol, W^T12; Spi, Spi⁺; OGV, L. (b and c) Expression dynamics of HmMasc variants in the embryos (mixture of sex-undetermined 200 to 300 embryos) of wHm-t-positive (W^T12) and negative (NSR) lines. The expression levels were quantified every 24 h from 12 to 108 hpo. Panels a to c are shown on different scales due to the different expression levels throughout embryogenesis. (d) Principal-component analysis of gene expression levels in mature embryos (108 hpo). (e) Classification of DEGs in each H. magnanima line harboring male killers. DEG expression patterns are classified into 14 clusters. The red and blue colors indicate upregulation and downregulation, respectively, between W^T12, S⁺, and L males and NSR males.

FIG 5

Male-killing Wolbachia and Spiroplasma caused abnormal apoptosis specifically in H. magnanima males. (a) Morphological characteristics of embryos (132 hpo) extracted from W^T12 and S⁺ egg masses. Male embryos were thinner and more fragile than female embryos. (b) Representative image of the midgut of a W^T12 embryo (132 hpo) stained with DAPI. Male embryos had abnormally shaped condensed nuclei (white arrows). The white arrowheads indicate heterochromatin (W chromosome). The broken white circle highlights the presumed chromatin bridge (magnification of ×1,000). (c) DNA ladders in the host lines. Genomic DNA (gDNA) and amplicons of laddered DNA (shown as “PCR”) from W^T12, S⁺, L, and NSR embryos (132 hpo). La, 100-bp DNA ladder; M, male; F, female. (d) Caspase-3 activities in different host lines for NSR, W^T12 (wHm-t-positive), and S⁺ (Spiroplasma-positive) embryos (132 hpo). The numbers inside the bars indicate replicates. Different letters indicate significant differences between groups (Steel-Dwass test, P < 0.05). (e) TUNEL assays with whole-mounted NSR, W^T12, and S⁺ male embryos (108 hpo). Green fluorescence indicates apoptosis, and blue fluorescence indicates nuclei counterstained with DAPI. The sex of the embryos was confirmed by detecting the presence or absence of heterochromatin (W chromosome).

FIG 6

Summary of the effects of male killers on H. magnanima. (a) Proposed model for explaining the effects of male killers on the host sex-related machinery. The MK Wolbachia affected sex-determination cascades and the dosage-compensation machinery, whereas Spiroplasma only affected sex determination in H. magnanima. Wolbachia possibly affects the dosage-compensation system and sex-determination cascade separately via different mechanisms or by targeting only the Masc gene and its upstream cascades, as predicted by Fukui et al. (14). In contrast, Spiroplasma utilizes a distinct but unknown mechanism that affects the sex-determination cascade. (b) Summary of the effects of male killers.