Linking genomic variation in Spiroplasma endosymbionts to male production and male-killing in the pea aphid

Endosymbiotic bacteria of the genus Spiroplasma (Mollicutes) are widespread among arthropods and plants with some lineages inducing male killing in several insect hosts. In the pea aphid, Acyrthosiphon pisum, three divergent clades of Spiroplasma ixodetis have been described, but male killing has been so far reported in only one clade. Here, we aimed to assess the distribution of male-killing phenotype among the three clades and investigated links between genomic variation and male-killing phenotypes in S. ixodetis infecting pea aphids. Field collections in eastern France revealed heterogeneous infection patterns across host-plant-associated biotypes, with 17% of established aphid lines carrying S. ixodetis strains spanning three clades. Sexual reproduction of these lines was induced under laboratory conditions to assess male production as a proxy for male killing, revealing a wide range of variation from complete absence of males to normal male production. Comparative genomic analyses of Spiroplasma strains associated with reduced or normal male production and from the different clades uncovered striking divergence in genome structure, including marked differences in synteny and gene content. Considerable variation was found in the copy number of putative virulence-associated genes amongst the clades, including those encoding ribosome-inactivating proteins (RIPs), ankyrin repeat domains, and ovarian tumour (OTU) domains. Notably, none of the strains encoded Spaid, the effector responsible for male killing in S. poulsonii from Drosophila. Strains within the same clade exhibited highly similar gene repertoires yet differed in putative male killing phenotype, and no unique gene nor genomic feature could be linked to reduced male production. This study investigated the evolutionary dynamics and effects of Spiroplasma ixodetis infections on male production in the pea aphid Acyrthosiphon pisum. Our findings provide insights into the diversity and potential evolutionary trajectories of Spiroplasma–aphid associations, laying the groundwork for elucidating the genetic basis of male killing in this system. Moreover, our results highlight the challenges of pinpointing the genetic determinants of reproductive manipulation across diverse symbiont–host systems.