Exploratory analysis of epigenetic dynamics and small extracellular vesicles in Penaeus vannamei hemocytes under inactivated Vibrio parahaemolyticus priming

Abstract

Trained innate immunity has been linked to epigenetic regulation in vertebrates, but corresponding mechanisms in shrimp remain poorly understood. Hemocytes are the central immune cells of Penaeus vannamei, and their chromatin states may reflect regulatory responses to microbial stimulation. Extracellular vesicles (EVs) are emerging mediators of immune-related intercellular communication, although their roles in shrimp immunity remain largely unexplored. In this study, we examined histone H3K4me3 and H3K27me3 dynamics in shrimp hemocytes following priming with formalin-inactivated Vibrio parahaemolyticus AHPND (VPAHPND) using immunofluorescence-based semi-quantitative analysis across multiple time points and doses. Hemolymphderived EVs were further characterized by nano-flow cytometry at Day 7 post-priming. Semi-quantitative image analysis revealed no significant differences in H3K4me3 or H3K27me3 among groups at Day 1. At Day 2, a significant increase in H3K4me3 was detected only in the medium-dose group (7.5 x 106 CFU/mL), while H3K27me3 showed no group-level differences. By Day 4, the high-dose group (7.5 x 107CFU/mL) exhibited significantly elevated levels of both H3K4me3 and H3K27me3 compared with control and lower-dose groups. By Day 7, histone modification levels across all groups had largely returned to baseline. At Day 7, EVs were detected in shrimp hemolymph with stable size distributions but altered particle concentrations in the high-dose group. These EV observations are preliminary, based on limited sample availability and single-method characterization. Together, this study provides exploratory evidence that histone modification responses in shrimp hemocytes following bacterial priming are transient and dose-specific, accompanied by preliminary observations of hemolymph-derived EV profiles, establishing a baseline for future studies on epigenetic regulation and systemic communication in shrimp immunity.