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

Abstract

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 the IPV mixture was obtained after multiple rounds of crushing and centrifugation. The brain tissue primary cells with large quantities and better culture effects were selected for the IPV challenge after cultured in vitro for 5 days. The virus crude extract was filtered through a 0.22 μm filter membrane and mixed according to the volume ratio of virus crude extract to serum-free medium = 1∶9. For the experimental group, 2 mL of the mixed solution was added to each well, while the control group received 2 mL of serum-free medium per well. Samples were taken at 0, 6, 12, 24, 48, and 96 h after virus infection. RT-qPCR was used to detect the IPV load.The results showed that the cultured primary cells grew well in a 1× L-15 medium containing glutamine and serum. According to the morphology of the cells, number of axons, and other characteristics, brain tissue primary cells, primary cells from the eyestalk X organ-sinus gland complex, and thoracic ganglion tissue primary cells were divided into four types: neurosecretory cells, pseudounipolar neural cell-like, bipolar neural cell-like, and multipolar neural cell-like, respectively. In comparison, only round neural cells and bipolar-like neural cells were found in the primary cell culture of the abdominal ganglion tissue under in vitro culture. The brain tissue primary cells and X organ-sinus gland complex cells survived for 15 days, while the thoracic ganglion and abdominal ganglion tissue primary cells survived for 9 days after being cultured in vitro. After IPV infected the brain tissue primary cells, the viral load of IPV was 15.30 copies/μg RNA at 6 h post-infection (hpi) and 35.59 copies/μg RNA at 12 hpi. IPV was not detected in IPV-infected brain tissue primary cells at 24 hpi and 48 hpi. Then, IPV viral load was detected at 96 hpi, reaching 104 copies/μg RNA.In conclusion, this study successfully established a simple and convenient primary culture technology for cells from M. rosenbergii neural tissues, providing preliminary data and a platform for neuroendocrine and virus-host interactions research. An in vitro infection model of IPV was also initially established in this study. Before establishing the shrimp cell line, the cells could be used to study the mechanism of viral infection, replication, and transcription, providing primary data and a platform for studying the norovirus and neuroendocrine factors of M. rosenbergii. They could also provide critical experimental materials for further research into infection mechanisms and the development of virus and disease prevention technologies.

Keywords

• macrobrachium rosenbergii
• neural cells
• primary culture
• infectious precocity virus