Evolutionary and structural analyses uncover a role for solvent interactions in the diversification of cocoonases in butterflies

Abstract

Multi-omic approaches promise to supply the power to detect genes underlying disease and fitness-related phenotypes. Optimal use of the resulting profusion of data requires detailed investigation of individual candidate genes, a challenging proposition. Here, we combine transcriptomic and genomic data with molecular modelling of candidate enzymes to characterize the evolutionary history and function of the serine protease cocoonase. butterflies possess the unique ability to feed on pollen; recent work has identified as a candidate gene in pollen digestion. was first described in moths, where it aids in eclosure from the cocoon and is present as a single copy gene. In heliconiine butterflies it is duplicated and highly expressed in the mouthparts of adults. At least six copies of are present in and copy number varies across sub-populations. Most genes are under purifying selection, however branch-site analyses suggest genes may have evolved under episodic diversifying selection. Molecular modelling of cocoonase proteins and examination of their predicted structures revealed that the active site region of each type has a similar structure to trypsin, with the same predicted substrate specificity across types. Variation among heliconiine cocoonases instead lies in the outward-facing residues involved in solvent interaction. Thus, the neofunctionalization of duplicates appears to have resulted from the need for these serine proteases to operate in diverse biochemical environments. We suggest that may have played a buffering role in feeding during the diversification of across the neotropics by enabling these butterflies to digest protein from a range of biochemical milieux.

Cocoonase is indispensable for Lepidoptera insects breaking the sealed cocoon

“…It is worth emphasizing that these 70 cocoonase genes were found within moth and butterfly families, which indicates that the cocoonase is Lepidoptera-specific ( Fig 1 ). In addition, multiple copies of cocoonase were identified in the butterflies, consistent with recent research on Heliconius butterflies, which uncovered the role of duplicated cocoonase genes in pollen feeding [ 37 – 39 ]. While, there was single copy in most moths except Plutella xylostella , Trichoplusia ni , Plodia interpunctella and Amelois transitella , which had two copies ( S1 Fig ).…”

In silico structural and functional characterization of Antheraea mylitta cocoonase

“…11) were a trypsin-like serine protease having active site from 75 to 200 query sequence and substrate binding site from 210 to 225 query sequences in NCBI. Both the motifs belong to trypsin-like serine protease, and cocoonase-like protein has been inferred as a conserved domain (cd00190) at the positions of Detailed comparative modeling and protein structure analysis have been performed to infer functional (and perhaps adaptive) differences of heliconiine cocoonase compared with the single-copy moth cocoonase [34].…”

“…The annotation of the newly constructed sequence AmCoc (377 bp) was used to search the presence of the serine protease domain, cd00190, using SMART tool. Also, modelingbased data of 30 individual cocoonases indicated that all the cocoonase enzymes have trypsin-like specificity, and also, significant differences were noticed among the surface residues of different cocoonase types which suggest that cocoonase enzyme shows varying adaptation to different chemical environments [34].…”

“…S2), although six copies of cocoonase has been reported in Heliconius melpomene and copy number varies across H. melpomene subpopulation [16]. Also, a detailed list about the copy number variation in cocoonase genes across 18 individuals of four Heliconius melpomene (Hm) subspecies has been elaborated [34]. Nowadays, the gene editing technologies are also being used to unravel the functionality of various genes.…”

“…Gene editing technique like CRISPR/Cas9-based Bombyx mori cocoonase gene editing has been the first experimental and phenotypic evidence showing that cocoonase is a cocoon breaking determining factor [33]. Using transcriptomic and genomic data heliconiine cocoonase gene expression across additional tissues, reconstructing their phylogenetic relationships, and examining the rates of gene duplication and deletion have already been described [34]. However, there is no detailed information available about cocoonase gene from A. mylitta silkworm.…”