World Aquaculture December 2019

48 DECEMBER 2019 • WORLD AQUACULTURE • WWW.WA S.ORG Shellfish Stress and Health Research at the Cawthron Institute Shellfish stress and health are two crucial factors in the success of any aquaculture operation, from the development of new species for aquaculture to growing a successful industry for existing species. Therefore, at the Cawthron Institute, we are currently working on understanding shellfish stress responses under different conditions, from hatchery to post- harvest. This work is being carried out through two current research programs that are government- funded by New Zealand’s Ministry of Business, Innovation and Employment (MBIE), the Shellfish Aquaculture Research Program, funded through the Strategic Science Investment Fund (SSIF), and the Aquatic Animals Health Program. Within the programs, we investigate all aspects of shellfish aquaculture and health in different New Zealand aquaculture species such as Pacific oyster Crassostrea gigas , geoduck Panopea zelandica and Greenshell TM mussel. Currently, health of these species is assessed by examination of histological sections of the adult animals or spat to determine the presence/absence of pathogens (Fig. 6). One of the key objectives of the research programs, however, is to develop and implement new tools to monitor health and stress of animals either in the laboratory or on farms. We are developing different assays to assess the health of adult animals by using shellfish haemolymph through flow-cytometry. Through analyses of haemolymph, we are able to determine parameters such as immune cell count and viability, relating these to the health status of the animals (Fig. 7). Additionally, we are working on developing a high-throughput enzyme- linked immunosorbent assay (ELISA) to quantify stress in the Greenshell TM mussel through the production of HSP70, as well as quantifying the expression of the HSP70 gene through Droplet Digital PCR (ddPCR). ELISA tests are able to detect the target protein (e.g. HSP70) by immune recognition by a specific antibody, allowing us to quantify the concentration of the target protein in the samples using a microplate reader. The development of both techniques (ELISA and ddPCR) to assess howmussels (adult and spat) up-regulate HSP70 will allow us to better understand how the Greenshell TM mussel responds to certain conditions and, therefore, inform appropriate changes to current industry practices. As HSP70 is a highly conserved protein among species, it is expected that the ELISA test will be also suitable to quickly assess stress in other shellfish species. The University of Otago / Cawthron oxidative stress team are also examining other species such as Pacific oysters after exposure to stressful conditions (e.g. hypoxia, emersion, temperature). Additionally, a different approach for the study of health and stress in shellfish has been developed by the team at the Auckland University of Technology (AUT) led by Professor Andrea Alfaro. In collaboration with AUT, we have been able to determine stress and health in adult mussels using a compact cell analyser to monitor haemocyte viability, ROS production and apoptosis using mussel haemolymph. Lipid content in shellfish larvae and spat has long been used as a measure of condition in the laboratory. In a novel application of fluorescent technology, we are working together with Dr. GeorgeWaldbusser of Oregon State University, who has developed an easy-to-use kit to monitor oyster fitness in the field. The kit uses handheld loupes and clip-on lenses for smartphones to take pictures of translucent spat that have been stained to determine calcification (using calcein) or lipid content (using Nile red). At Cawthron, we are currently adapting this kit to assess calcification and lipid content of Greenshell TM mussel spat, first in the laboratory and then on farms. At the same time, we are working together with researcher Alfonso Schmidt of the Malaghan Institute of Medical Research, to develop staining and image analysis techniques to assess lipid content in D-veliger larvae of the Greenshell TM mussel and species such as Pacific oysters and geoduck larvae (Fig. 8). These FIGURE 6. Histological section of Greenshell TM mussel spat after decalcification. Spat are observed under light microscopy for the presence/ absence of pathogens. Photo: Steve Webb. FIGURE 7. The author (left) together with Cawthron Aquaculture Group research assistants Joanna Copedo (middle) and Jolene Berry (right) measuring Greenshell TM mussel haemolymph chemistry. Photo: Leonardo Zamora. FIGURE 8. Greenshell TM mussel D-veliger larvae observed using confocal microscopy. Red staining (Oil Red O) shows lipid droplets, while the blue staining (DAPI) shows nuclei DNA. Scale bar 25 µm. Photo: Alfonso Schmidt.