New technologies to enhance resistance to oyster herpes virus

Abstract

Aquaculture is the fastest growing farmed food sector in the world and is a key part of global food security, encompassing the farming of fishes, molluscans and crustaceans. The Pacific oyster Crassostrea gigas is a globally important species, farmed commercially across the world. The major threat facing sustainable growth of Pacific oyster aquaculture is the spread of disease, chiefly the pathogenic oyster herpes virus microvariant (OsHV-1 uVar). Biosecurity has so far proven the best way to reduce the impact of OsHV-1, but has not been totally successful as outbreaks have continued to occur where oysters are grown in the open seas.

Vaccination against OsHV-1 is not currently possible as oysters lack a sophisticated adaptive immune system. The cost of OsHV-1 outbreaks is large, both in terms of food and economic losses, and is the focus of research and development worldwide. For example, selective breeding programmes have been used to develop strains of oysters that are more resistant to OsHV-1 through the realisation of genetic improvement. The basis of genetic resistance to OsHV-1 is unclear, although the potential for improvement is promising. However, the technology available for studying OsHV-1 and implementing practical gains in disease resistance are severely lacking compared to terrestrial livestock and to other aquaculture species, particularly finfish.

Transgenesis and genome editing are powerful tools that have been used to study gene function in a wide range of species and have resulted in development of improved food crops and animals. However, the application of transgenesis and particularly genome editing in marine invertebrates is well behind that of other commercially important animals. Difficulties arise due to lack of basic methods for key processes, such as use of cell cultures, and limited understanding of OsHV-1 pathogenesis. However, aspects of Pacific oyster biology make them well suited to improve these technologies. In particular, oysters can be spawned in controlled environments and produce a vast number of embryos that develop rapidly.

Additionally, the economic importance from being one of the top aquaculture molluscs globally makes them the ideal species through which application of findings has a direct route to impact.

To improve upon the protocols available for producing primary cell cultures, I developed a new large explant method using tissues from adult and juvenile Pacific oysters. Primary cultures were established from heart, mantle, muscle, gill, gonad tissue, as well as hemocytes.

This method had benefits over existing approaches as cultures were maintained for longer (up to 10 weeks) and displayed novel cell morphologies. This new method is logistically simpler than previous methods so that cultures can be established more easily and with fewer specialised reagents. Additionally, this method led to the discovery of entire tissues and large tissue fragments that remained active in the tissue culture system for up to 10 weeks after being excised from the donor animal. Hearts continued to beat rhythmically, mantle tissues continued to move and produce mucus, and gill tissues continue to actively move media surrounding them. This new whole-organ system, as well as the incremental improvements in primary cell culture, could have major benefits for studying marine invertebrate cell culture and potentially for the development of an immortalised marine mollusc cell line.

To further develop the tissue explant system for studying OsHV-1, I made tissue explants taken from juvenile oysters and exposed them to infectious OsHV-1 in a controlled laboratory environment. Using qPCR, histology and electron microscopy I collected strong evidence to support the conclusion that OsHV-1 can replicate in heart, mantle, and gill tissue explants. To further validate the use of tissue explants as a model for OsHV-1 infection, tissue explants were taken from oysters originating from two different oyster producers in the UK, which show different responses to OsHV-1 in laboratory challenges.

The viral load of the media and within tissues was quantified using qPCR and showed that the source of the oyster influenced the outcome of the tissue explant infection. This work offers a new model for OsHV-1 infection which can be more tightly controlled than existing models, offering the potential to disentangle the complexities of OsHV-1 pathogenesis. This also demonstrates another application for the whole tissue explant method described earlier.

To improve the methods available for transgenesis in Pacific oysters, I used an iterative approach to compare existing approaches and test new approaches. This is the first work to directly compare different oyster transgenesis methods, which have been limited to cross-species comparisons. By using high throughput methods such as electroporation, lipofection and lentivirus mediated transfection oyster embryos and gametes can be transfected at a scale that is unmatched in either finfish or terrestrial livestock. Using electroporation of a plasmid containing the GFP protein an oyster embryo that was clearly healthy and swimming with a clear and distinct fluorescence was detected. Furthermore, lipofection and lentivirus mediated transfection were attempted in Pacific oysters for the first time. The transfection efficiency using electroporation was very low, and no clear signs of fluorescence was detected using any of the other approaches used. This work highlights the difficulties that exist for molluscan genome engineering but provides the most comprehensive and detailed attempts yet.

To apply the methods developed for transgenesis to genome editing, I used the electroporation approach to introduce Cas9 ribonucleoprotein designed based on previous successful research that used microinjection. This work also tested a different electroporation system which has some key advantages over the system used for chapter 5. DNA sequencing did not detect clear evidence of genome editing.

Overall, my research has demonstrated significant development in applying genetic engineering and tissue culture methods to the Pacific oyster, with potential for application in marine molluscs broadly. I have also demonstrated for the first time that functional oyster tissues can be maintained in a cell culture system and provide a useful tool for studying OsHV-1.