SOLUTION TO FARMERS? EARLY IDENTIFICATION OF VIRUS CAUSING DISEASE FOR Macrobrachium rosenbergii

Macrobrachium rosenbergii is a freshwater giant prawn, with an all-male prawn breeding technology

pioneered in Malaysia by GK Aqua to commercialize the efficiency of freshwater prawn farming. Conventional farming practices results in high vertical transmission of disease as well as low productivity that could not meet supply demand and economical loss to farmers. GK Aqua came up with a solution to farmers by providing high quality post-larvae (PL) produced from specific-free pathogen (SPF) broodstock. Disease screening on brood prawns before mating procedure is an ultimate step for the prevention of the most common disease for M. rosenbergii; white-tail disease (WTD). In this process, we use pooling of RNA samples to reduce time and costs needed for identification of disease through nucleic acid extraction and PCR. We conclude that this method will help us to provide a rapid initial screening for brood prawns that will be able to produce disease-resistant PL and reduce risk of mortality to farmers.

Macrobrachium rosenbergii are commercially known throughout Southeast Asia and in Caribbean countries due to its habitat. Compared to Penaeus vannamei (white leg shrimp), freshwater giant prawn is less prone to disease and can be cultured in low density water. However, white tail disease is caused by Macrobrachium rosenbergii nodavirus (MrNV) and its co-infection, extra small virus (XSV). MrNV cause mortality in larvae and PL in which the clinical symptoms can be observed from whitish coloration on abdomen or tail muscle of PL. Adult freshwater prawn is found to be resistant towards MrNV and XSV in terms of mortality even with the presence of both viruses in their tissues. However, infected adult prawns may pass the virulence to its offspring causing 100% mortality to early life of M.rosenbergii. Therefore, it is crucial ensure brood prawn that goes into mating process are free from viruses.

In the experiment, pleopods tissues from adult M.rosenbergii will be collected for extraction, during quarantine phase prior to the mating process. The pleopods are pooled (maximum 5 each) into microcentrifuge tube and extracted using total nucleic acid extraction method. The extracted RNA samples then will be used as template for PCR method. Freshwater giant prawn is a single-stranded RNA viruses, therefore a nested PCR method is used to ensure high specificity and sensitivity of the assay. The first PCR is reverse transcriptase PCR (RT-PCR) to amplify RNA to complementary DNA (cDNA) followed by another step of nested PCR.

The results are then further analysed using 2% gel electrophoresis (Figure 1).

Figure 1. Analysis of results using gel electrophoresis.

Table 1. Results of disease screening against MrNV.

Based on the results, the initial screening of MrNV shows that one (1) out of ten (10) pooled sample is positive against MrNV at 256bp while nine (9) samples are negative at 500bp. The negative samples will then be released from quarantine to go into mating process and the sample that is tested positive will be further tested individually to identify MrNV infection in individual. In conclusion, it is emphasised that the screening for freshwater prawn against disease through nested PCR method is a crucial step to prevent transmission of WTD.