Temporal dynamics of immune response following a single-dose PACAP administration in Pacific white shrimp Litopenaeus vannamei

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a highly conserved neuropeptide with multiple biological functions including immunomodulation. While well-studied in vertebrates, its effects in invertebrates like Litopenaeus vannamei remain largely unexplored. Given the shrimp industry's global economic importance and losses due to disease, understanding PACAP's role in shrimp immunity offers a promising avenue for enhancing aquaculture sustainability and productivity. The present study evaluates the immune response to a single PACAP injection, investigating whether an individual administration is able to trigger a significant immune response, and the potential detrimental effects related to the administration route. Thus, effects of native PACAP-38 (PACAP1) and its scrambled version (PACAP5), as well as saline solution (vehicle) injection, over time were evaluated on shrimp immune responses. Samples were collected at 0-, 6-, 24-, 48-, and 72-hours posttreatment (hpt). Immune parameters such as the total number of circulating hemocytes (THC), the concentration of proteins, and hemagglutination and phenoloxidase activities, in the hemolymph were analyzed. Reverse transcription-quantitative PCR (RT-qPCR) was conducted on hemocyte, gill, and hepatopancreas samples to evaluate the differential expression of immune-related genes. Results showed that the treatments triggered an early inflammatory response marked by reduced circulating hemocytes during the first 24 hpt. PACAP-treated groups recovered baseline hemocyte levels by 48 hpt, unlike the saline control, suggesting an immunomodulatory role for PACAP. Additionally, PACAP influenced the tissue-specific expression of key immune genes such as crustin , HSP70 and superoxide dismutase . Overall, both the injection procedure and/or the administration of saline negatively impacted the shrimp's immune status, an effect that was partially mitigated by PACAP treatment. Thus, further research needs to explore new routes of administration such as dietary inclusion or immersion baths, to optimize PACAP’s immunomodulatory potential in shrimp health management. • Both injection injury and saline administration triggered inflammation. • PACAP treatment promoted a faster recovery of total hemocyte count. • PACAP modulated crustin , SOD , and HSP70 gene expression in some immune tissues. • PACAP mitigated the detrimental immune effects of saline injection.