The shrimp microsporidian Enterocytozoon hepatopenaei (EHP): Biology, pathology, diagnostics and control.

Since the 1970s, much work has been devoted to the development of marine invertebrate cell culture. Despite this effort there are still no established cell lines. During the last decade, more than 40 papers on primary cell culture have been published. The phyla primarily studied are porifera, cnidaria, crustacea, mollusca, echinodermata, urochordata, and cephalochordata. A variety of methods have been investigated to improve culture conditions and prolong the lifespan of cells, including use of different pretreatments, culture media, temperature, pH, and additives; genetic modification has also been investigated. In this paper we review the vast majority of work published on marine invertebrate cell culture in the last decade (2002–2012) and emphasize new trends in biotechnological applications of cell culture.

Marine invertebrate cell culture : a decade of development

Lack of a valid shrimp cell line has been hampering the progress of research on shrimp viruses. One of the reasons identified was the absence of an appropriate medium which would satisfy the requirements of the cells in vitro. We report the first attempt to formulate an exclusive shrimp cell culture medium (SCCM) based on the haemolymph components of Penaeus monodon prepared in isosmotic seawater having 27 ‰ salinity. The SCCM is composed of 22 amino acids, 4 sugars, 6 vitamins, cholesterol, FBS, phenol red, three antibiotics, potassium dihydrogen phosphate and di-sodium hydrogen phosphate at pH 6.8–7.2. Osmolality was adjusted to 720 ± 10 mOsm kg−1 and temperature of incubation was 25 oC. The most appropriate composition was finally selected based on the extent of attachment of cells and their proliferation by visual observation. Metabolic activity of cultured cells was measured by MTT assay and compared with that in L-15 (2×), modified L-15 and Grace’s insect medium, and found better performance in SCCM especially for lymphoid cells with 107 % increase in activity and 85 ± 9 days of longevity. The cells from ovary and lymphoid organs were passaged twice using the newly designed shrimp cell dissociation “cocktail”.

https://www.dyuthi.cusat.ac.in/xmlui/bitstream/handle/purl/4063/A%20novel%20medium%20for%20the%20development%20of%20in%20vitro%20cell%20culture%20system%20from%20Penaeus%20monodon.pdf?sequence=1

A novel medium for the development of in vitro cell culture system from Penaeus monodon.

Primary cell cultures were developed from haemocytes and testis of Scylla serrata. Haemocytes were collected from live animals and cultured in double-strength L-15 medium (2× L-15) prepared in crab saline, supplemented with 5% foetal bovine serum and antibiotic–antimycotic solution (penicillin 100 U/mL, streptomycin 100 μg/mL and amphotericin B 0.25 μg/mL) with osmolality adjusted to 894 mOsm/kg. The haemocytes adhered within 2 h after seeding and showed proliferation up to 72 h. The disaggregated testis tissue fragments were seeded in 3× L-15 supplemented with non-essential amino acid mixture, lipid concentrate and antibiotic–antimycotic solution, with osmolality adjusted to 1,035 mOsm/kg with crab saline. Cells from the testis could be subcultured and maintained up to 21 d as suspension culture. Different dilutions of white spot syndrome virus (WSSV) inoculum (known virus copy number) prepared from infected Penaeus monodon were inoculated in the cultured cells, and the cytopathic effects like detachment, rounding of cells and clear areas of depleted cells were observed after 48 h in haemocyte cultures. However, WSSV-exposed testis cells did not show any obvious change until 72 h post-infection. WSSV was detected in both haemocyte and testis cultures at different time-points of infection by conventional and real-time PCR using WSSV-specific primers. The transcripts of WSSV were found to be much higher in haemocytes than in testis culture. The virus harvested from the cultured haemocytes after three passages could infect healthy P. monodon. The present study showed that mud crab haemocyte culture can support WSSV replication, and it can be used as an in vitro tool for WSSV replication.

Development of primary cell cultures from mud crab, Scylla serrata , and their potential as an in vitro model for the replication of white spot syndrome virus

Characterization of a primary cell culture from lymphoid organ of Litopenaeus vannamei and use for studies on WSSV replication

Shrimp cell lines are yet to be reported and this restricts the prospects of investigating the associated viral pathogens, especially white spot syndrome virus (WSSV). In this context, development of primary cell cultures from lymphoid organs was standardized. Poly-l-lysine-coated culture vessels enhanced growth of lymphoid cells, while the application of vertebrate growth factors did not, except insulin-like growth factor-1 (IGF-1). Susceptibility of the lymphoid cells to WSSV was confirmed by immunofluoresence assay using monoclonal antibody against the 28 kDa envelope protein of WSSV. Expression of viral and immune-related genes in WSSV-infected lymphoid cultures could be demonstrated by RT-PCR. This emphasizes the utility of lymphoid primary cell culture as a platform for research in virus-cell interaction, virus morphogenesis, up and downregulation of shrimp immune-related genes, and also for the discovery of novel drugs to combat WSSV in shrimp culture.

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Lymphoid organ cell culture system from Penaeus monodon (Fabricius) as a platform for white spot syndrome virus and shrimp immune-related gene expression.

G Poulose

Papers

Lymphoid organ cell culture system from Penaeus monodon (Fabricius) as a platform for white spot syndrome virus and shrimp immune-related gene expression.