Abstract Venom is a complex trait that has convergently evolved in over 200,000 animals across the tree of life, totaling approximately ~15-30% of animal biodiversity. The prevalence of the venom phenotype demonstrates its molecular success and studying its evolution has broad applications to understanding the development of homologous tissues, the origins of novel genes, and the molecular mechanisms behind the regulation and expression of bioactive compounds. Considerably, venoms are the prototype of precision medicine: inducing a highly specific and immediate response. These attributes have fueled drug discovery efforts, leading to breakthrough venom-derived therapeutics for a wide range of conditions, from diabetes to heart disease to pain. However, the full potential of venom, in both medicine and biological research, is untapped. This unmet need arises because of the lack of robust models for genetically manipulating the development of venom glands and regulation and expression of venom bioactive peptides. The work of the Holford group was the first to characterize terebrid venom peptides as bioactive in mitigating analgesic and antitumor activity. However, without guiding principles for how venoms and venom glands develop in vivo, we, and other venom researchers, have just scratched the surface. We need model systems to revolutionize the study venom gland biology, so that we can radically transform how we generate, manipulate, and utilize venom arsenals. The cephalopod breeding program provide the tools and models necessary to tackle biological and translational questions that have remained unapproachable, such as: What drives the expression of predatory versus defensive venom components? Can we manipulate the production of specific toxins with a desired function, such as those targeting receptors involved in analgesic activity? Advancements in genetic engineering, genomics, transcriptomics, and proteomics will allow us to generate the first marine invertebrate transgenic cephalopod organisms that produce venom in specialized glands that can be investigated to explore fundamental questions about tissue development and gene regulation and expression. Specifically, we will: (1) Determine genes and proteins relevant to venom gland development, maintenance, and secretion across diverse cephalopod taxa. This objective will determine the evolutionary underpinnings between venom salivary glands and other exocrine tissues across taxa. (2) Trace the development of cephalopod salivary glands. This objective will reveal genetic pathways that can be leveraged to determine the formation and function of venom gland from diverse taxa. (3) Establish comparative cephalopod transgenic models. This objective will establish transgenic cephalopods allowing us to optimize the utility of venom glands for understanding the development of homologous tissues, the origins of novel genes, and the molecular mechanisms behind the regulation and expression of bioactive compounds. The proposed research is a new direction for the PI that is high risk-high reward, and will benefit disparate fields and industries, including developmental cellular and molecular biology and drug discovery and development. Most diseases, like Alzheimer or cancer have complex traits whose genetic characterization in model systems have been essential to finding effective therapies. Studying the evolutionary genetics in the complex trait of venom in a reliably, cultured cephalopod system will broadly impact research towards the NIH’s mission of enhancing human health.

Narrative The study of complex traits is fundamental to understanding the nature and behavior of living systems. Most diseases, like Alzheimer or cancer have complex traits whose genetic characterization in model systems have been essential to finding effective therapies. Similarly, venom is a complex trait that has evolved throughout the tree of life, and studying its evolution has broad applications to understanding the development of homologous tissues, the origins of novel genes, and the molecular mechanisms behind the regulation and expression of bioactive compounds.