Establishment of hematopoietic tissue primary cell cultures from the giant freshwater prawn Macrobrachium rosenbergii.

The giant freshwater prawn Macrobrachium rosenbergii is one of the most important aquaculture species in Southeast Asia. In this study, in vitro culture of its hematopoietic tissue cells was achieved and characterized for use as a tool to study its pathogens that cause major farm losses. By transmission electron microscopy, the ultrastructure of the primary culture cells was similar to that of cells lining intact hematopoietic tissue lobes. Proliferating cell nuclear antigen (PCNA) (a marker for hematopoietic stem cell proliferation) was detected in some of the cultured cells by polymerase chain reaction (PCR) testing and flow cytometry. Using a specific staining method to detect phenoloxidase activity and using PCR to detect expression markers for semigranular and granular hemocytes (e.g., prophenoloxidase activating enzyme and prophenoloxidase) revealed that some of the primary cells were able to differentiate into mature hemocytes within 24 h. These results showed that some cells in the cultures were hematopoietic stem cells that could be used to study other interesting research topics (e.g. host pathogen interactions and development of an immortal hematopoietic stem cell line).

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

Disease is a major limiting factor in the global production of cultivated shrimp. The microsporidian parasite Enterocytozoon hepatopenaei (EHP) was formally characterized in 2009 as a rare infection of the black tiger shrimp Penaeus monodon. It remained relatively unstudied until mid-2010, after which infection with EHP became increasingly common in the Pacific whiteleg shrimp Penaeus vannamei, by then the most common shrimp species farmed in Asia. EHP infects the hepatopancreas of its host, causing hepatopancreatic microsporidiosis (HPM), a condition that has been associated with slow growth of the host in aquaculture settings. Unlike other infectious disease agents that have caused economic losses in global shrimp aquaculture, EHP has proven more challenging because too little is still known about its environmental reservoirs and modes of transmission during the industrial shrimp production process. This review summarizes our current knowledge of the EHP life cycle and the molecular strategies that it employs as an obligate intracellular parasite. It also provides an analysis of available and new methodologies for diagnosis since most of the current literature on EHP focuses on that topic. We summarize current knowledge of EHP infection and transmission dynamics and currently recommended, practical control measures that are being applied to limit its negative impact on shrimp cultivation. We also point out the major gaps in knowledge that urgently need to be bridged in order to improve control measures.

Optimisation of electroporation and lipofection protocols to derive the black tiger shrimp cell line (Penaeus monodon).

To achieve in creating permanent shrimp cell lines, cellular arrest of primary cells in the culture is needed to be firstly solved. Considering the insertion of some markers affecting cellular proliferation into primary haemocytes in order to produce the black tiger shrimp cell line and the very low percent of transduced cells previously reported in penaeid shrimps, these paved us the way to set up suitable gene delivery protocols to increase percent of transduced cells in the shrimp as our primary aim. In this study, electroporation and lipofection were used to transfer construct plasmids (pLL3.7 plasmids containing CMV promoters and pGL-IE1-126(A)-EGFP plasmids carrying WSSV IE1 promoters) into primary haemocytes. As it was difficult to distinguish between cells expressing EGFP signal and auto-fluorescence of many dead cells occurred by electroporation during the first 72 h of experiment; so, only lipofection was managed to deliver plasmids into primary cells. Surprisingly, numbers of suspected proliferative cells were derived after electroporation with no insertion of immortalising markers. These cells survived in vitro for up to 45 days with high rate of cell viability, but the number of viable cells decreased throughout the experiment. In addition, these cells expressed genes and proteins closely related to hyaline cells determined using RT-PCR and western blot. For the lipofection experiment, no green fluorescence signal was detected in any primary cell introduced with these plasmids, suggesting that plasmids were not successfully inserted into cells. Also, a number of primary haemocytes had the apoptotic cell death characteristic within 5 days after lipofection. These possibly result from using inappropriate lipofection protocol and chemical substances. In summary, finding out suitable protocols to elevate the percent of transduced cells is still necessary. Additionally, continuous shrimp cell lines would be possibly established by transforming suspected proliferative cells derived from electroporation in this study.

The cellular death pattern of primary haemocytes isolated from the black tiger shrimp (Penaeus monodon).

A key to successfully generate the penaeid shrimp cell line is to find out how primary cells died. The most suitable period to culture Penaeus monodon haemocytes was in the first 48 h of culture because cells had normal morphology, high percent of viable cells (65.29 ± 5.43%), low percent of early (11.75 ± 1.30%) and late apoptotic cells (15.47 ± 11.71%) determined by Annexin V and TUNEL including constant IAP (0.06 ± 0.01-0.07 ± 0.01) and caspase-3 expression (0.30 ± 0.06-0.39 ± 0.10) by real-time PCR throughout the experiment. Moreover, adding 50 and 250 µM of the cell permeable pan caspase inhibitor Z-VAD-FMK produced some melanised cells since the 48(th) hour, while percent of viable cells was decreased since the 24(th) hour with no difference in percent of early and late apoptotic cells compared to control at each time point. No difference of IAP and caspase-3 expression level in both Z-VAD-FMK groups was found compared to control and vehicle groups at each time point, excluding caspase-3 in 250 µM Z-VAD-FMK at the 24(th) hour was higher than control and vehicle. Supplementing sodium fluoride (NaF) induced cell membrane damage and cellular shrinkage of primary haemocytes within 2 h. Even percent of viable cells was reduced down to zero and percent of late apoptotic cells was increased by 2 h of incubation in 25 and 50 mM NaF, IAP and caspase-3 in all NaF groups was not different from control. These results indicate that a number of primary haemocytes derived in this study die through the apoptotic process.