Analysis of the genomes of a male-killing Spiroplasma and its co-infecting Rickettsia reveals a case of concerted genome expansion

Maternally inherited symbionts are central to arthropod biology, functioning both as mutualistic partners and as reproductive parasites. Genomic analyses provide critical insight into these interactions. Here, we sequenced, assembled and examined the genomes of Spiroplasma and Rickettsia co-infecting the lacewing Mallada desjardinsi , with the aim of elucidating the male-killing phenotype of Spiroplasma and predicting potential phenotypes for Rickettsia . In Spiroplasma , we identified a set of candidate effector genes. However, Spaid-like genes, where present, lacked the functional domains previously demonstrated to be important for male-killing. The Rickettsia genome contained two cifA/cifB gene pairs, consistent with the capacity to induce cytoplasmic incompatibility (CI). Unexpectedly, both symbiont genomes were markedly expanded relative to congenerics. We describe this pattern – that contrasts with the classical trajectory of genome reduction in symbionts – as secondary genome expansion. Expansion was driven by extensive proliferation of mobile elements: Rickettsia harboured an exceptionally high number of insertion sequences, while Spiroplasma accumulated both insertion sequences and prophage regions. Collectively, our findings indicate that the canonical Spaid-mediated male-killing mechanism is not conserved in the Spiroplasma of M. desjardinsi , while Rickettsia may induce CI. Moreover, the parallel genome expansions observed suggest that secondary expansion events may be influenced by host-associated factors rather than occurring stochastically.