Neuropeptide Diversity Encoded in Newly Sequenced Crustacean Genomes Reveals Signaling Roles during Feeding

Neuropeptides are chemically diverse signaling molecules that regulate physiology and behavior, yet many species lack neuropeptidomic characterization due to sparse genomic annotation. Here, we integrate genomic sequence information with neuropeptidomics to define the neuropeptidomes of two widely used crustacean model organisms, Callinectes sapidus and Cancer borealis. Using a curated multispecies precursor database, tBLASTn alignment, signal peptide detection, and in silico processing, we predicted more than 23,000 putative peptides across both genomes, including numerous sequences bearing hallmarks of mature neuropeptides. Mass spectrometry-based profiling provided experimental support for many predicted peptides and revealed substantial chemical diversity, including novel allatostatin B and C isoforms, an insulin-like peptide B-chain-like isoform in C. sapidus, and the first report of natalisin peptides in C. borealis. Notably, we observed an atypical precursor architecture in which a single prohormone encoded two distinct neuropeptide families, suggesting previously unrecognized modes of neuropeptide copackaging and signaling. Finally, leveraging a feeding perturbation model, we observed tissue-specific differences in the abundance of newly identified peptides in the thoracic ganglion, commissural ganglion, and pericardial organs, consistent with functional neuroendocrine roles. Together, this work expands the known repertoire of crustacean neuropeptides, provides a resource for comparative peptide biology, and establishes a genome-enabled framework for discovery of endogenous peptide signaling molecules in newly sequenced species.