Primary hemocyte culture of Penaeus monodon as an in vitro model for white spot syndrome virus titration, viral and immune related gene expression and cytotoxicity assays

Abstract

Immortal cell lines have not yet been reported from Penaeus monodon, which delimits the prospects of investigating the associated viral pathogens especially white spot syndrome virus (WSSV). In this context, a method of developing primary hemocyte culture from this crustacean has been standardized by employing modified double strength Leibovitz-15 (L-15) growth medium supplemented with 2% glucose, MEM vitamins (1×), tryptose phosphate broth (2.95 gl⁻¹), 20% FBS, N-phenylthiourea (0.2 mM), 0.06 μg ml⁻¹ chloramphenicol, 100 μg ml⁻¹ streptomycin and 100 IU ml⁻¹ penicillin and hemolymph drawn from shrimp grown under a bio-secured recirculating aquaculture system (RAS). In this medium the hemocytes remained viable up to 8 days. 5-Bromo-2'-deoxyuridine (BrdU) labeling assay revealed its incorporation in 22 ± 7% of cells at 24h. Susceptibility of the cells to WSSV was confirmed by immunofluorescence assay using a monoclonal antibody against 28 kDa envelope protein of WSSV. A convenient method for determining virus titer as MTT(50)/ml was standardized employing the primary hemocyte culture. Expression of viral genes and cellular immune genes were also investigated. The cell culture could be demonstrated for determining toxicity of a management chemical (benzalkonium chloride) by determining its IC(50). The primary hemocyte culture could serve as a model for WSSV titration and viral and cellular immune related gene expression and also for investigations on cytotoxicity of aquaculture drugs and chemicals.

Establishment of Shrimp Cell Lines: Perception and Orientation

“…The lack of success in spontaneous and induced cell line development subsequently paved the way for the attempts on developing fusion cell line [15,16], but, with little success. More recently, researchers have succeeded in viral gene expression [42], determinations of cytotoxicity and genotoxicity [41,43], and viral multiplication [26] employing primary cell culture systems developed from different species of penaeids.…”

“…To adjust osmolality, NaCl at a concentration ranging from 6 to 12 g/l [9,19,40,49] has also been added besides the balanced salt solutions [40,78]. Addition of vitamins [41,42], proline [49,56,57,82] and glutamine [28,82] has been proven to be the choice of supplements in the growth media. In addition, lactalbumin hydrolizate at a concentration of 0.1-1 g/l [2-4, 39, 48, 55-57], tryptose phosphate broth at 2.95 mg/ml [41,42] and TC Yeastolate at 1 g/l [56,57] have also been used as the source of peptides, amino acids and carbohydrates.…”

“…Addition of vitamins [41,42], proline [49,56,57,82] and glutamine [28,82] has been proven to be the choice of supplements in the growth media. In addition, lactalbumin hydrolizate at a concentration of 0.1-1 g/l [2-4, 39, 48, 55-57], tryptose phosphate broth at 2.95 mg/ml [41,42] and TC Yeastolate at 1 g/l [56,57] have also been used as the source of peptides, amino acids and carbohydrates. As the additional energy source 0.3-2 g/l glucose [40][41][42][55][56][57] and 0.55 g/l sodium pyruvate [19] have also been supplemented.…”

“…In addition, lactalbumin hydrolizate at a concentration of 0.1-1 g/l [2-4, 39, 48, 55-57], tryptose phosphate broth at 2.95 mg/ml [41,42] and TC Yeastolate at 1 g/l [56,57] have also been used as the source of peptides, amino acids and carbohydrates. As the additional energy source 0.3-2 g/l glucose [40][41][42][55][56][57] and 0.55 g/l sodium pyruvate [19] have also been supplemented. Buffering agents such as HEPS [28,48,82] and NaHCO 3 have been incorporated by many researchers [19,28,48,49].…”

Multifactorial interaction of growth factors on Penaeus monodon lymphoid cells and the impact of IGFs in DNA synthesis and metabolic activity in vitro

“…Well before that Braasch et al (1999) and Jose et al (2010) had reported haemocyte cultures from P. vannamei and P. monodon BrdU positive (1-2 and 22 ± 7 % respectively). Maeda et al (2003) confirmed 35 % BrdU positive cells in primary ovarian cell culture developed from Penaeus japonicus.…”