Methods to generate double - stranded RNA for in vivo RNA interference studies in giant fresh water prawn, macrobrachium rosenberg II

Abstract

Giant freshwater prawn, Macrobrachium rosenbergii (M. rosenbergii) is a highly commercial aquatic species in many countries, especially in Vietnam. This study was conducted to generate Mr-IAG double-stranded RNA (dsRNA) for application of RNA interference technique to inhibit the production of androgenic gland-specific insulin- like peptides from an androgenic gland to produce neo-female giant freshwater prawn. One step & two steps procedure for Mr-IAG dsRNA systhesis were investigated. As a result, one step procedure using T7 RiboMax Express RNAi System kit performed as the appropriate method for dsRNA synthesis in terms of RNA quality and cost effectiveness. To check the effect of Mr-IAG dsRNA as well as the optimal concentration of Mr-IAG dsRNA being necessary to inhibit the androgenic gland-specific insulin-like peptides, in vivo silencing of this gene by Mr- IAG dsRNA injection to male post larvae (PL25) were carried out with different concentrations of 1.25 µg/g, 2.5 µg/g and 5µg/g of post larvae. After 10 injections, the concentration of 1.25µg Mr-IAG dsRNA/g of post larvae seemed as the sufficient concentration to produce neo-female prawn by preventing the regeneration of appendix masculine of male prawn. However, the experiments was carried out in a short period of 21 days, hence the long-term experiments should be done for further confirmation of the effect of Mr-IAG dsRNA in neo-female prawn production. The current study using RNAi approach to control sex in giant freshwater prawn has potential application for other aquaculture species. Keywords: giant freshwater prawn, Macrobrachium rosenbergii, appendices masculinae, Mr-IAG dsRNA.