Primary hemocyte culture of the freshwater prawn Macrobrachium rosenbergii and its susceptibility to the novel pathogen spiroplasma strain MR-1008

Primary culture of hemocyte from the giant freshwater prawn, Macrobrachium rosenbergii, was established for studies on the in vitro propagation of the novel nature pathogen spiroplasma MR-1008. In this context, a modified method of hemocyte culture from this crustacean has been standardized by employing Leibovitz-15(L-15) growth medium supplemented with 15% Fetal Bovine Serum (FBS) along with 0.1% glucose, 0.5% NaCl and antibiotics (100 U ml− 1 penicillin, 100 U ml− 1 streptomycin,1 μg ml− 1 amphotericin b) and a suitable pH of 7.20–7.40. In this medium, the hemocytes can be subcultured twice, including significant cell proliferation and viability of up to 12 days. Susceptibility of the hemocytes was investigated by challenging with spiroplasma MR-1008. Cytopathic effects (CPE) were observed as early as 48 h post-inoculation; and as the infection progressed, CPE became more apparent, with cell debris and cellular exudates present in inoculated cultures. Cell lysis was noticeable within 60 h after infection. A quantitative real-time RT-PCR was conducted to detect the immune related gene expression during the infection process at 2, 4, 12, 24, 48, and 60 h, respectively. The expression levels of lipopolysaccharide and β-1,3-glucan-binding protein (LGBP), prophenoloxidase (proPO), α-2-macroglobulin (α2M) and copper/zinc SOD (Cu/Zn-SOD) genes in hemocytes of M. rosenbergii demonstrated that these immune genes were induced by challenge with spiroplasma MR-1008. The primary hemocyte culture can be used to investigate the interaction between host and pathogen in vitro, promote enhanced understanding of spiroplasma MR-1008, and ultimately lead to prevention this crustacean disease.

► Primary hemocyte cultures of Macrobrachium rosenbergii was established.
► Hemocyte cultures retained a relatively stable system and viability to 12 days.
► Apparent CPE occurred at 48-h post-inoculation of spiroplasma MR-1008.
► Immune related genes were induced by challenge with spiroplasma MR-1008.
► It can serve as an in vitro model to assess the crustacean pathogen spiroplasma.