HAIRPIN-RNA EXPRESSION CASSETTE BY TWO-STEP CLONING SYSTEM AND ITS POTENTIAL AS AN IMMUNE STIMULANT IN SHRIMP

Vanvimon Saksmerprome*, Patai Charoonart, Boonsirm Withyachumnarnkul

National Center for Genetic Engineering and Biotechnology, Thailand Science Park, Pathumthani and Centex Shrimp, Mahidol University, Bangkok Thailand
E-mail: vanvimon.sak@biotec.or.th We demonstrate an improved method for delivering RNAi-based immunity to large shrimp populations. Long sequences of double-stranded RNA (dsRNA) have recently been used to enhance viral resistance, through an RNA interference (RNAi) mechanism, in shrimp aquaculture. Since dsRNA-mediated knockdown efficiency of viral genes appears to be dose dependent, a large-scale production of dsRNA is necessary for antiviral/therapeutic applications of RNAi for shrimp farm operations. A new design of hairpin-RNA expression vector, followed by transformation into RNase-deficient E.coli HT115, offers a quick preparation of a large amount of long dsRNA (normally >300 nt). The hairpin RNA consists of a forward strand and a 100-base shortened reverse strand, and the unpaired 100-base region on the forward strand serves as a loop. This strategy reduces 3-step to 2-step cloning of hairpin construct into DNA expression cassette, thus bypassing difficulty in joining a small loop piece into a large carrier vector. A total RNA of ~4 mg is generally obtained from 100 mL bacterial culture.

The bacterially expressed hairpin RNA specific to RNA-dependent RNA polymerase (RdRp) gene of yellowhead virus (YHV) can induce antiviral immunity in Penaeus vannamei shrimp. Viral protection by exogenous dsRNA demonstrates the potential of dsRNA produced by 2-step-cloning hairpin cassette as an immune stimulant in shrimp (Figure 1). We investigate further if the putative viral promoters can be regulated by shrimp transcriptional activators. The hairpin specific to YHV is cloned into the viral promoter-contained plasmids, followed by intramuscular injection of the constructs into shrimp. We examine protection against YHV and level of viral mRNA after plasmid injection. Successful hairpin expression driven by such promoters demonstrates the potential of DNA-based vaccine for effective antiviral immunity in shrimp.

CHALLENGES AND OPPORTUNITIES OF OFFSHORE AQUACULTURE IN THE PACIFIC NORTHWEST

Langdon*, C.

Coastal Oregon Marine Experiment Station and Dept. Fisheries and Wildlife,
Hatfield Marine Science Center,
Oregon State University,
2030 S. Marine Science Drive,
Newport, Oregon 97365 Projected high rates of global and US population growth coupled with declines in many wild fisheries will put increasing pressure on aquaculture to meet future consumer demand for seafood. The US currently imports about 80% of its seafood, resulting in a large annual trade deficit of about $10 billion and a dependence on other countries to supply affordable, high-quality seafood in the future. Offshore aquaculture has been identified by NOAA and other organizations as a possible means of providing the US with a sustainable domestic source of seafood.

The Pacific Northwest offers some unique opportunities for offshore aquaculture, such as 1) clean, cold seawater that allows production of valuable cold-water species, 2) high wave and wind energy that could provide offshore facilities with sources of power and 3) a strong fishing community that could participate in farming initiatives.

In 2008, a forum was held at the Hatfield Marine Science Center, Oregon State University, Oregon, US, to discuss the potential of offshore aquaculture in the Pacific Northwest. The forum was attended by a wide range of participants, including scientists, administrators, fishing industry representatives, non-profit organizations and the media. Details of participants, the program and findings are given on the forums web site: Error! Hyperlink reference not valid. .

The forum made two overarching recommendations: 1) education and outreach should be undertaken to discuss the challenges and opportunities of offshore aquaculture for local communities and 2) demonstration projects should be established to determine technical, biological, economic and environmental aspects of offshore aquaculture. These recommendations and other findings of the forum will be presented.