Defensins of the stable fly have developmental-specific regulation and evolve at different rates

Authors: ASGARI, DANIAL - University Of Houston; Nayduch, Dana; MEISEL, RICHARD - University Of Houston

Submitted to: Integrative and Comparative Biology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/8/2024
Publication Date: 4/18/2024
Citation: Asgari, D., Nayduch, D., Meisel, R. 2024. Defensins of the stable fly have developmental-specific regulation and evolve at different rates. Integrative & Comparative Biology. https://doi.org/10.1093/icb/icae015.
DOI: https://doi.org/10.1093/icb/icae015

Interpretive Summary: Insects produce antimicrobial peptides (AMPs) either as a response to infections or continuously during their normal activities. This study looked at how stable flies turn on and use eleven copies of genes for ancient AMPs called Defensins across their life history from larvae through adults. Stable fly larvae live in microbe-rich environments and expressed certain Defensins, while adult flies, who feed on blood which exposes flies to fewer microbe encounters, expressed different copies of the gene. Thus, specific groups of Defensin genes seem to be used by different stages of flies. Additionaly, when the genes and the proteins they code were compared to one another, differences in their sequences were found, with non-larval Defensins undergoing more changes than larval Defensins. However, while the larval Defensins had fewer changes, the changes were more extreme differences compared to other Defensins. These findings shed light on how AMPs are regulated during different developmental stages, indicating unique evolutionary pressures tied to differences in stable fly exposure to microbes during their lifetime.

Technical Abstract: Organisms produce antimicrobial peptides (AMPs) either in response to infection (induced) or continuously (constitutively) to combat microbes encountered during normal trophic activities and/or through pathogenic infections. The expression of AMPs is tightly regulated often with specificity to particular tissues or developmental stages. As a result, AMPs face varying selective pressures based on the microbes the organism’s tissue or developmental stage encounters. Here, we analyzed the evolution and developmental-specific expression of Defensins, which are ancient AMPs in insects, in the stable fly (Stomoxys calcitrans). Stable fly larvae inhibit microbe-rich environments, whereas adult flies, as blood-feeders, experience comparatively fewer encounters with diverse microbial communities. Using existing RNA-seq datasets, we identified six Defensins that were only expressed in larvae (larval Defensins) and five that were not expressed in larvae (non-larval Defensins). Each of the non-larval Defensins was expressed in at least one adult tissue sample. Half of the larval Defensins were induced by mating or feeding in adults, and all three of the induced Defensins were located downstream of canonical binding sites for an Imd transcription factor involved in the highly conserved NF-'B signaling that regulates induction of AMPs. The larval and non-larval Defensins were located in distinct genomic regions, and the protein sequences of the larval Defensins formed a monophyletic clade. There were more amino acid substitutions across non-larval Defensins, with multiple genes losing a highly conserved furin cleavage site thought to be required for the removal of the amino terminus from the mature Defensin domain. However, larval Defensins had a higher proportion of radical amino acid substitutions, altering amino acid size and polarity. Our results reveal insights into the developmental stage-specific regulation of AMPs, and they suggest different regulatory regimes impose unique selection pressures on AMPs, possibly as a result of variation in exposure to microbial communities across development.