Unique physiological and regulatory activity drives divergent toxin and non-toxin gene expression in rattlesnake accessory venom glands.

Understanding the mechanisms by which organs and tissues evolve new physiological functions is central to understanding the evolution of novelty. This is particularly interesting in the context of related tissues that evolve specialized, yet complementary, functions. Snake venom glands are an attractive system to test hypotheses related to the evolution and specialization of novel physiological function, as these modified salivary glands have evolved over ∼60 MY to synthesize and store venom. Front-fanged venomous snakes (elapids and viperids) possess two types of venom glands: the main and accessory glands. The larger main gland produces greater quantities of venom toxins and has been studied extensively, while the smaller accessory gland has received less attention. Here, we explore gene expression differences between main and accessory venom glands across three rattlesnake species (Crotalus cerberus, C. oreganus concolor and C. viridis). Our findings indicate that accessory glands express most venom genes at significantly lower levels than the main gland, with a few exceptions that may represent biologically relevant contributions of accessory glands to venom. The two glands also exhibit distinct trans-regulatory environments that we link to key differences in their underlying physiology and secretory roles. Our results further suggest that two signaling pathways that regulate venom, the unfolded protein response (UPR) and extracellular signal-regulated kinase (ERK), show significantly lower activation in the accessory gland. These findings provide insight into the physiological and functional diversification of snake venom systems, highlighting how distinct glandular systems have evolved contrasting and complementary roles driven by distinct physiological and regulatory mechanisms.