Single-Cell Peptide Profiling to Distinguish Stickleback Ecotypes with Divergent Breeding Behavior

Variation in parenting behavior is widespread across the animal kingdom, both within and between species. There are two ecotypes of the three-spined stickleback fish (<i>Gasterosteus aculeatus</i>) that exhibit dramatic differences in their paternal behavior. Males of the common ecotype are highly attentive fathers, tending to young from eggs to fry, while males of the white ecotype desert offspring as eggs. As the pituitary is a key regulator in the hypothalamic-pituitary-adrenal (HPA) axis and the hypothalamic-pituitary-gonadal (HPG) axis between the brain and body, its peptides may influence parenting behaviors. Here, we utilized matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) for high-throughput peptide analysis in single cells of pituitaries from both three-spined stickleback ecotypes. Peptide mass fingerprinting was performed using an <i>in silico</i> generated peptide library to identify detected prohormones. Differential analysis revealed POMC-derived peptides, MCH-derived peptides, and oxytocin as significantly different between the two ecotypes, with higher oxytocin levels in the common ecotype. Interestingly, these subtle chemical differences were not captured by Leiden clustering of the cellular phenotypes. These results call for further investigation of the neurochemical basis for parenting in sticklebacks.