Horizontal gene transfer rivals gene duplication as a source of anti-parasitoid immune innovation in the Drosophilidae

Abstract

Macroparasites are among the most important agents of natural selection in their host populations, but anti-macroparasite immunity is poorly understood. Vinegar flies (Drosophilidae) and the parasitoid wasps that infect them are emerging models to study how animals defend against macroparasite attack. The canonical anti-parasitoid immune mechanism in insects is melanotic encapsulation, which involves cell-mediated encapsulation coupled with prophenoloxidase (PPO)-mediated melanization of parasitoid embryos. Recently, we discovered the horizontal transfer (HGT) of a bacterially-derived humoral anti-parasitoid effector, Cytolethal distending toxin B (CdtB), across insects, including four drosophilid lineages. Here, we assessed the prevalence of these two anti-parasitoid immune mechanisms in 406 drosophilid and four outgroup species. We found that melanotic encapsulation was relatively uncommon among species with known anti-parasitoid immune responses. While PPO duplications were found in 88 species, the most salient PPO gene underlying melanotic encapsulation, PPO3, was restricted to Drosophila melanogaster and its close relatives. PPO genes were present in lower copy number per genome in the Drosophilidae than in outgroup lineages and evolved slowly. We found cdtB in the genomes of 93 species and estimated at least 11 independent cdtB gains across the Drosophilidae as early as ∼44 mya and as recently as ∼6 mya. cdtB acquisition was subsequently associated with higher net diversification rates in some clades. We conclude that humoral immune effectors may play a more important role than previously appreciated in anti-parasitoid immunity in insects and that an immune innovation arising repeatedly from HGT is potentially associated with the evolutionary success of these animals. Significance Statement Insects have evolved robust immune strategies to overcome parasitoid wasp challenge. The prevailing mechanism involves encapsulation of immature parasitoids using melanin-producing blood cells dependent on duplicated prophenoloxidase (PPO) genes. Another anti-parasitoid response evolved through the horizontal gene transfer (HGT) of a toxin-encoding gene (cdtB) from endosymbionts to the genomes of insects. We show the number of species encoding PPO duplications and species encoding cdtB were similar in the Drosophilidae. HGT, but not PPO duplication, was associated with higher rates of diversification within some fly lineages, indicating that the HGT events or an associated trait may confer a fitness advantage. These results suggest that anti-parasitoid immune mechanisms that act independently of melanotic encapsulation responses are effective and pervasive in the Drosophilidae.