Amino Acid Frequency in the Proteome Is Not Associated With Realised Thermal Limit nor Dietary Niche Breadth in 35 Lepidoptera Species.

Amino acids are the building blocks of proteins that perform essential physiological functions. Theory suggests that the proteome composition-the amino acid frequencies across all proteins in a genome-is associated with an organism's optimal growth temperature, offering insights into species' temperature limits. This hypothesis, however, is largely based on prokaryotic models and has not been thoroughly tested in complex multicellular eukaryotes, where many amino acids must be acquired through diet. Here, we integrated multiple databases and analysed amino acid frequencies in the proteomes of orthologous and non-orthologous genes from 35 Lepidoptera species to test for correlations with maximum observed temperatures and diet breadth. Using a robust phylogenetic comparative approach, we found no evidence that proteome composition correlates with temperature or diet breadth, which are important ecological traits for Lepidopterans and affect their interactions with other species. These results suggest that, unlike in simpler organisms, animal proteome composition is shaped more strongly by intrinsic biophysical and energetic constraints than by ecological factors such as temperature exposure or dietary specialisation. Our study bridges evolutionary genetics with ecological physiology across a diverse group of insects and highlights the importance of publishing well-designed null results. These findings also emphasise the limitations of using proteome composition as a proxy for ecological adaptation in multicellular species, while opening avenues of future research to further explore the complex interplay between genetics, physiology, and environment in shaping biodiversity.