Dhakad, P.; Newman, D.; Obbard, D.

Background Drosophila melanogaster has been a valuable model for dissecting the molecular architecture of innate immunity. However, the family Drosophilidae encompasses over 4000 species, spanning deep evolutionary divergences and diverse ecologies. Here, we use immune challenge with the gram-negative pathogen Providencia rettgeri to investigate the conservation and evolution of immune responses in three non-model drosophilid species, Hirtodrosophila cameraria, H. confusa, and Scaptodrosophila deflexa, each of which diverged from D. melanogaster over 45 million years ago. Results We find that all three species retain a core set of immune signaling and recognition genes, but exhibit substantial variation in effector gene content and inducibility. In particular, Scaptodrosophila deflexa lacks orthologs of multiple antimicrobial peptides (AMPs) known from D. melanogaster, including DptA, AttA, and AttC, and shows little transcriptional response to bacterial-challenge with Providencia rettgeri. In contrast, both of the Hirtodrosophila species exhibit substantial transcriptional responses, including strong induction of canonical Imd pathway genes. Microbiome profiling of our samples revealed higher Providencia abundance in H. cameraria, and high levels of the defensive symbiont Spiroplasma in S. deflexa, potentially explaining differences in infection outcome. Our combined annotation and expression analysis of these species also allowed us to identify 20 novel AMP-like candidates, many with structural features like known AMPs. Conclusions Our study demonstrates the feasibility of functional immune analyses in non-model Drosophila species and reveals striking lineage-specific differences in immune gene repertoire and expression. These findings highlight the importance of non-model, wild-derived taxa for uncovering novel immune effectors and understanding evolutionary forces shaping insect immunity.