Large-scale field sampling reveals Spiroplasma infection in Lymantria dispar japonica and Lymantria umbrosa in Hokkaido, Japan

The spongy moth ( Lymantria dispar ) exhibits significant sex ratio distortion owing to interspecific hybridization with its closely related species, Lymantria umbrosa , in Hokkaido. This study investigated the involvement of sex-manipulating endosymbiotic bacteria by screening 268 egg masses collected from multiple sites in Hokkaido for infection with Wolbachia and Spiroplasma . PCR analysis amplified Spiroplasma 16S rRNA genes from 40 egg masses, and phylogenetic analyses revealed that the identified Spiroplasma belonged to the Spiroplasma ixodetis clade. Vertical transmission was confirmed in a subset of infected lineages, but no significant sex ratio distortion was observed in L. umbrosa -derived infected egg masses. Although one egg mass exclusively produced female adults, nuclear DNA polymorphism analysis and mitochondrial DNA haplotyping revealed that this lineage originated from backcrosses between L. dispar japonica females and L. umbrosa males. All-female broods were reproduced in experimentally generated Spiroplasma -negative backcrosses, indicating that the observed sex-ratio bias could be attributed to genetic effects of interspecific hybridization rather than to endosymbiont infection. These findings provide the first evidence of Spiroplasma infection in L. dispar japonica and L. umbrosa populations in Hokkaido, and indicate that infection with this bacterium does not necessarily induce male killing.