Two Complete Genomes of Male-Killing Wolbachia Infecting Ostrinia Moth Species Illuminate Their Evolutionary Dynamics and Association with Hosts

Abstract

Wolbachia is an extremely widespread intracellular symbiont which causes reproductive manipulation on various arthropod hosts. Male progenies are killed in Wolbachia-infected lineages of the Japanese Ostrinia moth population. While the mechanism of male killing and the evolutionary interaction between host and symbiont are significant concerns for this system, the absence of Wolbachia genomic information has limited approaches to these issues. We determined the complete genome sequences of wFur and wSca, the male-killing Wolbachia of Ostrinia furnacalis and Ostrinia scapulalis. The two genomes shared an extremely high degree of homology, with over 95% of the predicted protein sequences being identical. A comparison of these two genomes revealed nearly minimal genome evolution, with a strong emphasis on the frequent genome rearrangements and the rapid evolution of ankyrin repeat-containing proteins. Additionally, we determined the mitochondrial genomes of both species' infected lineages and performed phylogenetic analyses to deduce the evolutionary dynamics of Wolbachia infection in the Ostrinia clade. According to the inferred phylogenetic relationship, two possible scenarios were proposed: (1) Wolbachia infection was established in the Ostrinia clade prior to the speciation of related species such as O. furnacalis and O. scapulalis, or (2) Wolbachia infection in these species was introgressively transferred from a currently unidentified relative. Simultaneously, the relatively high homology of mitochondrial genomes suggested recent Wolbachia introgression between infected Ostrinia species. The findings of this study collectively shed light on the host-symbiont interaction from an evolutionary standpoint.

Keywords: Bacterial endosymbiont; Genome rearrangement; Horizontal transmission; Introgression; Lepidoptera; Mitochondrial genome.

Fig. 1

Characterization of male killing in wSca-infected O. scapulalis. a Brood sex ratios in the wSca-infected matriline. The female:male ratio of each mating is shown. Tetracycline treatment was conducted to remove wSca from a subpopulation of the second generation. Sexing was conducted based on the morphology of the pupal abdominal tips. b An adult female moth infected with wSca. Bar, 10 mm. c The sex ratios of wSca-infected larvae at 4 and 14 dph. The number indicates the sample size of each group. d The body length of wSca-infected and uninfected larvae at 4 dph. Data presented are mean ± standard error of the mean. Dot plots show all data points individually. An asterisk denotes statistical significance (P < 0.001; N.S., not significant, P > 0.1; Wilcoxon rank-sum test). e Splicing patterns of Osdsx in uninfected, wSca-infected, and infection-cured embryos at 96 hpo. The results of technical duplicates of the RT-PCR assay are presented for each sample. The numbers indicate individual samples (biological duplicates for each condition). Adult female and male moths were used as positive controls. The letters F and M indicate female- and male-type splicing variants, respectively. AF: adult female; AM: adult male; NT: no template

Fig. 2

Circular map of the wFur and wSca genomes. Circles arranged in order from outer to inner indicate the following: the locations of annotated coding sequences (CDS) on the positive (outermost) and the negative (second outermost) strands, the position of annotated RNAs, GC content, and GC skew. The GC content and GC skew are calculated in 10 kb windows and expressed as deviations from an average of the whole sequence

Fig. 3

Phylogenetic relationship of Supergroup B Wolbachia genomes. The maximum likelihood tree constructed from concatenated protein sequences of 339 single-copy orthologs is shown. wMel and wRi (Supergroup A) were used as outgroups. The strain names and their hosts are labeled. If no suitable strain name is available, it is denoted by “NA”. Clades composed of almost the same strains are collapsed, and the number of contained strains is labeled. Branch support calculated using 1000 replicates of ultrafast bootstrap is shown on the nodes. The sequences determined in this study are highlighted in red and bold, while strains associated with other lepidopterans are in blue

Fig. 4

Synteny conservation between the wFur and wSca genomes. Dots and lines represent the alignments generated by nucmer program. Forward matches are shown in red, while reverse matches are shown in blue

Fig. 5

Phylogenetic relationship of mitochondrial genomes of Ostrinia and allied moth species. The maximum likelihood tree constructed from concatenated sequences of 13 proteins with partitions for each protein is shown. Two pyralid species (Lista haraldusalis and Ephestia kuehniella) were used as outgroups. Branch support calculated using 1000 replicates of ultrafast bootstrap is shown on the nodes. The sequence determined in this study are highlighted in red and bold

Fig. 6

A proposed model for evolutionary history of Wolbachia infection in Ostrinia moths. Each circle represents an assumed individual species in evolutionary time scale. Wolbachia-infected subpopulations are depicted in a dark color. Black arrowheads indicate when the divergence of mitochondrial haplotypes currently found in infected and uninfected Clade III Ostrinia moths began. a The establishment of Wolbachia infection can be traced back to the beginning of mitochondrial divergence at the maximum, or b more recently by assuming introgressive transfer from an unidentified (possibly extinct) relative