Primary Culture of Macrobrachium rosenbergii Neural Cells and its Application in Virus Research

Macrobrachium rosenbergii is one of the most popular species in aquaculture. However, the M. rosenbergii farming industry has been facing an ongoing iron prawn syndrome (IPS) crisis since 2010, resulting in substantial economic losses to the farming industry. Infectious precocity virus (IPV) is a novel virus of Flaviviridae found in recent years that can cause sexual precocity and associated slow growth in healthy M. rosenbergii. It is believed to have a specific correlation with IPS. There are very few cell lines of crustaceans that can be used for studying the response of cells to pathogens. Even though the primary culture technology of blood and muscle cells in M. rosenbergii has gradually matured, there have been few studies on the primary culture of neural cells. Quantitative results of different tissues of IPV-positive M. rosenbergii have indicated that nerve-rich tissues, such as eyestalk, brain, and thoracic ganglion tissues, have a higher viral load, which explains why IPV has neurotropic tissue characteristics. To provide an in vitro cell platform for studying the virus-host interactions of IPV, a simple, stable, and feasible primary culture method was established for the nervous tissue primary cells of M. rosenbergii.In this study, healthy prawns with a body length of about 10–12 cm, healthy appendages, and vitality were selected for the experiments. The body surface of M. rosenbergii was first disinfected with 75% alcohol. On the clean bench, the nervous tissues, including the brain, X organ-sinus gland complex from the eyestalk, thoracic ganglion, and abdominal ganglion tissues, were isolated and washed in PBS buffer containing 100 U/mL penicillin and streptomycin, 100 U/mL amphotericin, and 80 U/mL gentamicin, 2–3 times. The tissues were then placed in 5 mL of 0.5% papain solution. After digestion at 25 ℃ for 5 min, 1× L-15 medium containing 15% FBS, 140 mmol/L D-glucose, and antibiotics (100 U/mL penicillin and streptomycin, 100 U/mL amphotericin, and 80 U/mL gentamicin) were added to terminate digestion. Next, the cells were seeded onto a 24-well plate and allowed to settle in darkness at 25 ℃ for 45 min. After adhesion, cells were transferred to a cell incubator and cultured in the dark at 28 ℃. The nervous tissues' primary cells at different time points were observed under an inverted microscope, and the morphological changes were recorded through imaging. The compound eye tissue of IPV-positive M. rosenbergii was placed in SM buffer, and