Stable primary embryonic cells of Artemia are suitable for tracing the process of V. anguillarum and V. parahaemolyticus infection

The cell line is an important experimental tool to investigate the mechanism of host-pathogen interactions and to diagnose and cure diseases. However, their application in aquatic invertebrates is limited because they lack immortalized cell lines. The brine shrimp Artemia produces encysted diapause gastrula embryos under stress conditions. The biological advantages of Artemia embryos, such as quick switching on of the cell cycle upon short-term hydration and easy control of microbial contamination via decapsulation, makes them a candidate material for cell culture in vitro. This study identified five subpopulations of embryonic cells at the gastrula stage based on morphological characteristics. After 3–4 h of hydration, embryonic cells were derived from the cysts and successfully cultured for 10 days in 1.2 × L-15 medium containing 15% fetal bovine serum, with a cell viability of more than 90%. Using this primary cell culture system, Vibrio anguillarum and V. parahaemolyticus strains were screened as possible infectious Vibrio strains using laser confocal microscopy observations. The pathological features of viable Vibrio-challenged embryonic cells were further studied by comparing formaldehyde-inactivated and autoclaved Vibrio and polystyrene beads using SEM observation. The results confirmed that both Vibrio strains could challenge embryonic cells and cause some cytopathological features, such as cell aggregation and concaves. Our study provides the first evidence that in vitro cell culture of Artemia embryos can be used to demonstrate the characteristics of Vibrio infection. The primary cell culture system established in this study will facilitate research on the mechanism of host-pathogen interactions in aquatic animals.