Stomachs in your butterfly : exploring the identity and activity of proteases in the reproductive tract of female butterflies.

Reproductive traits are some of the most rapidly evolving traits in the natural world. However, research to date has focused heavily on male reproductive traits such as male genitalia, leaving female reproductive traits understudied. As a result, we know little about female reproductive adaptations or how they may have co-evolved with male reproductive traits. Butterflies provide an excellent system to study reproductive interactions and physiology from the female perspective. Female butterflies have a specialized reproductive organ called the bursa copulatrix that appears to be co-evolving with specific male traits. The bursa accepts and actively digests the complex male ejaculate, called the spermatophore. Our research focused on the proteases present in the bursa of the Cabbage White butterfly, Pieris rapae. Using a general colorimetric assay, we discovered high levels of overall protein digestion within the bursa. Using transcriptomic and proteomic approaches, we identified nine putative proteases from two main functional classes that may play a role in bursal digestion of the spermatophore. Using a zymogram approach, we conducted an in vitro protein digestion assay to measure the activity of specific bursal proteases. Finally, we investigated functionality of each protease by disrupting specific modes of protease action using targeted protease inhibitors and quantifying subsequent loss of activity. Our findings offer new insights into female bursal adaptations, and provide groundwork for future studies of co-evolution between the bursa and spermatophore