Comparative genomics and the evolution of immune genes in Drosophila

Abstract

Immune genes are among the most dynamic components of animal genomes, shaped by complex trade-offs between functional constraints, pathogen selective pressure, and environment. While Drosophila melanogaster has provided a detailed picture of innate immunity in insects, the family Drosophilidae—spanning over 60 million years and diverse ecological niches—offers a unique opportunity to study how immune systems evolve across deep evolutionary timescales. Recent large-scale sequencing efforts have now generated hundreds of high-quality drosophilid genomes, creating unprecedented opportunities for comparative analysis. However, a major limitation has been the lack of consistent gene annotations across species. In this thesis, I address this gap by generating standardized protein-coding gene annotations for 304 drosophilid species. Using a combination of comparative (CAT) and de novo (BRAKER3) annotation, guided by RNAseq and protein homology evidence, I built a consistent, high-quality gene annotation resource. I next investigated the evolutionary rates and turnover of immune-related genes relative to non-immune genes. Using models of DNA sequence evolution and trait evolution, I show that immune genes, particularly effectors and recognition proteins, evolve more rapidly than signaling genes and non-immune genes. I identified gene-level predictors of evolutionary rate, including expression level, relative solvent accessibility, gene length, and protein/genetic interactions. Finally, I used transcriptomic data from pathogen-challenged and unchallenged individuals of three non-model drosophilids to evaluate the bioinformatic recovery of known immune genes, to investigate their conservation and evolution of immune responses and discover novel immune effectors. I identified 20 candidate antimicrobial peptides that are infection-inducible, encode short, secreted proteins, and have no clear homologs in D. melanogaster, highlighting extensive lineage-specific evolution of immune effectors.