RNAi treatment of broodstock to reduce disease impacts in farmed prawns Melony Sellars Jeff Cowley, Min Rao, Brian Murphy, Dean Musson Aquaculture Program - CSIRO Agriculture and food flagship

Knowledge to-date for prawns & RNAi RNAi antivirals reduce viral loading of Penaeid prawns through tail muscle injection of all commercially relevant viruses in juveniles under laboratory conditions of broodstock Black Tiger females from GAV under commercially comparable conditions Antivirals and the administration process do not adversely impact the ability of female Black Tiger prawns to spawn Injection is the only reliable administration method at present

The challenge for our industry GAV is endemic in Black Tiger prawns indigenous to coastal waters and it’s not uncommon for wild broodstock used in hatcheries to be infected with GAV at high prevalence GAV is transmitted vertically from broodstock to progeny very effectively GAV infections carried into ponds with seedsock are a primary contributor to ‘disease-related’ production losses during grow-out

Potential solutions to this challenge Source broodstock from elsewhere Expensive to collect broodstock from remote locations Potential for introducing new pathogens (FRDC Project 2013/036) Viability of local commercial broodstock suppliers Undertake PCR-based screening selection for GAV-free or GAV-low broodstock Expensive, labour intensive and requires laboratory support Not practical when infection prevalence and loads are high Stimulate the RNAi pathway to reduce or clear GAV infections by injecting broodstock with GAV-specific dsRNA Grow-out of progeny from cleared parents and assessment of progeny performance – now underway Registration requirements - Australian Pesticides and Veterinary Medicines Authority (APVMA) Establish virus-free or disease-resistant breeding populations

Potential solutions to this challenge Source broodstock from elsewhere Expensive to collect broodstock from remote locations Potential for introducing new pathogens (FRDC Project 2013/036) Viability of local commercial broodstock suppliers Undertake PCR-based screening selection for GAV-free or GAV-low broodstock Expensive, labour intensive and requires laboratory support Not practical when infection prevalence and loads are high Stimulate the RNAi pathway to reduce or clear GAV infections by injecting broodstock with GAV-specific dsRNA Grow-out of progeny from cleared parents and assessment of progeny performance – now underway Registration requirements - Australian Pesticides and Veterinary Medicines Authority (APVMA) Establish virus-free or disease-resistant breeding populations

Project goals Demonstrate dsRNA antivirals can abrogate or reduce vertical transmission of GAV to progeny Quantify improvements in health, survival and growth performance of progeny that are spawned from GAV infected parents that receive the antiviral compared to progeny from GAV infected parents that do not receive the antiviral Deliver proof-of-concept that antivirals can be up-scaled for use in commercial hatcheries and result in progeny with lowered viral infection loads that realise commercial benefits in prawn health, growth and survival

Experimental workflow 2016-2017 Reproductive performance data Growth and survival performance data GAV load broodstock GAV load broodstock GAV load broodstock GAV load female spawner GAV load emrbyos GAV load PL15 GAV load PL60 GAV load PL100 GAV load PL140 GAV load PL180 11July 29July 15Aug 20Aug 20Sept Feb/Mar Destination GAV antivirals that reduce vertical transmission Ablate & inject antiviral or saline Spawning 10 treatment families 10 control families Source broodstock from Qld EC GAV PCR screen Inject antiviral or saline Harvest ponds once marketable size Stock 2 treatment, 2 control ponds

Thankyou This research is supported by the Australian Prawn Farmers Association, Fisheries Research and Development Corporation, Science and Industry Endowment Fund and CSIRO