RNA interference reduces PmergDNV expression and replication in an in vivo cricket model Kathy A La Fauce * , Leigh Owens School of Veterinary and Biomedical Sciences, Solander Drive, James Cook University, Townsville, Qld 4811, Australia article info Article history: Received 14 October 2008 Accepted 27 October 2008 Available online 7 November 2008 Keywords: RNA interference RNAi Acheta domesticus Penaeus merguiensis densovirus PmergDNV Gene silencing abstract RNA interference (RNAi) is an attractive anti-viral preventative because it allows interference with the expression of a viral gene in a highly sequence-specific manner. Thus, essential viral genes can be tar- geted by design, with little or no risk of undesired off-target effects. To investigate if stealth RNAis can mediate a sequence-specific anti-viral effect against PmergDNV, adult Acheta domesticus were injected with 5 lg of stealth RNAi or control stealth RNAi, targeting the capsid protein. Twenty-four hours post-injection, crickets were challenged with PmergDNV. Mortality was monitored for 14 days and real-time reverse transcriptase PCR was used to enumerate the number of copies of PmergDNV in cricket tissues. Whilst statistically not significant, trends in mortality suggest crickets injected with RNAi target- ing PmergDNV had the lowest mortality rate (11.5%) compared to crickets injected with control dsRNAi (33%) and PmergDNV alone (25%). Crickets challenged with specific dsRNAi had statistically significantly reduced PmergDNV titres by one log (3.58 Â 10 2 ) compared to crickets challenged with PmergDNV alone (3.42 Â 10 3 ). Interestingly, even the control dsRNAi was capable of reducing PmergDNV titres by one log (3.95 Â 10 2 ), but did not produce an inhibitory effect quite as strong as the targeted dsRNAi for the capsid protein of PmergDNV. The introduction of dsRNAi corresponding to the capsid protein of PmergDNV, was effective in reducing viral replication in Acheta domesticus. Administration of PmergDNV-specific dsRNAis may provide an efficient counter measure against PmergDNV in prawns. Ó 2008 Elsevier Inc. All rights reserved. 1. Introduction The worldwide production of marine prawns has substantially increased to keep pace with the worldwide demand for seafood products. However, this huge increase in cultured prawn produc- tion has not been without its setbacks. One of the most serious impediments of farming prawns is the incidence of disease. Dis- ease epizootics of economic significance are a major constraint to the industry, not only because they affect the quantity of harvest, but because disease also affects the quality and regularity of production. Most organisms have evolved protective defence mechanisms against viruses and other pathogens. Vertebrates rely on a both an ‘adaptive’ immune system that specifically targets pathogens as well as a broad-action, non-specific ‘innate’ immune response (Silverstein, 1989). On the other hand, invertebrates lack the pro- tein-based adaptive immune response found in vertebrates but are still capable of effectively fighting viral-infections (Brennan and Anderson, 2004). RNA interference (RNAi) is an evolutionary conserved gene reg- ulatory mechanism. It can be triggered by the recognition of intra- cellular long double stranded RNA, which can be transcribed from nuclear genes, replicating viruses or in the form of synthetic RNAs, resulting in sequence-specific degradation of the homologous RNA (post-transcriptional gene silencing) (Hannon, 2002; McCown et al., 2003; Hammond, 2005). Pioneering observations regarding RNA interference were reported in the nematode worm (Fire et al., 1998), plants (Waterhouse et al., 1998) and Drosophila (Ken- nerdell and Carthew, 1998). However, RNAi-related events have since been recognised in almost all eukaryotic organisms including protozoans (Bastin et al., 2001; Malhotra et al., 2002; Cottrell and Doering, 2003), fungi (Raoni and Arndt, 2003), algae (Wu-Scharf et al., 2000), nematodes (Fire et al., 1998; Caplen et al., 2001; Schott et al., 2005), plants (Fagard and Vaucheret, 2000), insects (Misquitta and Paterson, 1999; Hughes and Kaufman, 2000), fish (Nasevicuis and Ekker, 2000; Dang et al., 2008) and mammals (Wianny and Zernicka-Goetz, 2000; Caplen et al., 2001). Plants and insects encode multiple Dicer enzymes that recognize distinct precursors of small RNAs (Fire et al., 1998; Berstein et al., 2001; Lee et al., 2004; Xie et al., 2004; Tomari and Zamore, 2005). Hence, they are capable of initiating the small RNA-guided RNA interference anti-viral immunity (Hamilton and Baulcombe, 1999; Li et al., 2002) and have been recognised as an important defence against viruses in invertebrates, particularly insects (van Rij et al., 2006; Wang et al., 2006; Zambon et al., 2006). 0022-2011/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.jip.2008.10.006 * Corresponding author. Fax: +617 4779 1526. E-mail address: kathy.lafauce@jcu.edu.au (K.A. La Fauce). Journal of Invertebrate Pathology 100 (2009) 111–115 Contents lists available at ScienceDirect Journal of Invertebrate Pathology journal homepage: www.elsevier.com/locate/yjipa