The work described in this thesis is aimed at the study of WSSV infection in aquatic crustaceans. As mentioned above, most of the research into WSSV has been performed in shrimp species like L. vannamei and P. monodon and thus far this has not led to development of a successful treatment. The observation that C. maenas is relatively resistant to the virus opens up a new angle of study. Gathering information on this host organism and tracking its response to viral exposure opens the opportunity to identify the molecular basis for the apparent resistance of C. maenas to WSSV. Once the molecular basis is identified, a novel treatment against WSSV could be designed on this basis. For example: if C. maenas produced a miRNA with significant impact on WSSV infection then this miRNA may do the same for economically important species like L. vannamei and P.
monodon.
Shrimp species are very important in respect to the global aquaculture market, but they are not as relevant from a European perspective since the European aquaculture sector is small.
However Bateman et al. 2012 showed that WSSV can infect crustaceans in temperate regions, such as European waters, and thus there is potential for the virus to impact aquatic crustaceans in European waters [12]. Molecular knowledge of commercial European aquatic crustaceans will be useful for comparative studies and other future applications. The European lobster Homarus gammarus was selected for study in this thesis work in addition to the shore crab because it is a commercially fished species, particularly in the United Kingdom. It is furthermore, more susceptible to WSSV compared to the shore crab, providing a contrasting model for study of WSD.
C. maenas and H. gammarus do not have a large amount of available molecular information.
Therefore next generation sequencing is particularly suited for viral exposure studies in these species. Through DNA and (small)RNA sequencing experiments a large amount of information can be gathered for these species and used to form draft genomes, transcriptomes and analyses on expressed miRNAs. This basic information then provides a background against which changes through exposure to WSSV can be tracked and potentially important targets in the disease infection/resistance processes identified.
Page | 42 1.4.1 Experimental species
Two aquatic crustacean species were used in the experiments described in this thesis, the shore crab C. maenas and the European lobster H. gammarus. The shore crab was selected primarily because of its unique resistance to WSSV exposure, and the lobster both because of its important to UK fisheries and the fact that it is more susceptible to WSD compared with the shore crab.
Carcinus maenas
The European shore crab (or green crab), C. maenas, is a keystone species in the European marine environment. It has a carapace length of up to 60 mm with a width of up to 90 mm. It is an invasive species which has spread from its native waters in Europe into Australia, South Africa and the United States. C. maenas is a molluscan predator [94] and thus it can threaten local fishing industries in areas it invades. For example: in New England the shoft-shell clam (Mya arenaria) fisheries were destroyed by C. maenas [95]. C. maenas is also an important study species for biomonitoring and ecotoxicology [96, 97]. The species has been used in monitoring for heavy metal contamination [98], metal toxicity studies [99], and more recently in exposures studies with nanomaterials [100] and microplastics [101]. Despite small exceptions, C. maenas is not commercially fished or cultured.
Homarus gammarus
The European Lobster, Homarus gammarus, is a highly valued seafood commodity. It has a maximum total body length of about 60 cm, with large specimens usually 23 to 50 cm long, and a weight of 5 or 6 kg. The flavour of its meat is held in high regard by consumers, enabling this species to yield high prices on the market [102]. It is commercially fished across Europe, yielding an average of 4972.5 tonnes between 2010 and 2013 [103]. Lobster fisheries are concentrated around the United Kingdom, which accounted for 65 % of total capture (2013) [103]. H. gammarus is grown in aquaculture, but not widespread due to issues regarding cannibalistic behaviour, operating costs and density [104].
Page | 43 1.4.2 Thesis roadmap
As stated the overall aim of the project is to identify the molecular mechanism of C. maenas apparent resistance to WSSV infection. The work is centred on using bioinformatic analyses to produce a transcriptome and draft genome for C. maenas. Additionally a transcriptome for H. gammarus is generated. Lastly, an experimental infection study with WSSV in C. maenas was undertaken and the molecular responses, both mRNA and smallRNA, were analysed.
The thesis contains 6 further chapters with the following remit and content:
Chapter 2 –Research paper 1 (Published)
Molecular Mechanisms of White Spot Syndrome Virus Infection and Perspectives on Treatments
Bas Verbruggen , Lisa K. Bickley, Ronny van Aerle, Kelly S. Bateman, Grant D. Stentiford, Eduarda M. Santos and Charles R. Tyler. Viruses 2016, 8, 23
This review paper provides a critical analysis of WSSV. The review analyses the available knowledge on the WSSV infection process and includes information on the WSSV genome, current known molecular interactions between WSSV and host, and host signalling pathways important to successful infection. The review concludes with an overview on current treatment development avenues.
Chapter 3 –Research paper 2 (Published)
De novo assembly of the Carcinus maenas transcriptome and characterization of innate immune system pathways
Bas Verbruggen, Lisa K. Bickley, Eduarda M. Santos, Charles R. Tyler, Grant D. Stentiford, Kelly S. Bateman and Ronny van Aerle. BMC Genomics (2015) 16:458
This chapter describes sequencing of RNA from several C. maenas tissues and creating a transcriptome based on de novo assembly of the data. Transcripts are annotated based on sequence similarities. Functional annotation and taxonomic overviews based on significant similarities are provided. In relation to WSSV infection it was deemed important to have an initial overview of the immune system of C. maenas and therefore pathways that are likely relevant in relation to viral infection are investigated in detail.
Page | 44 Chapter 4
In this chapter a draft genome is assembled for C. maenas. Within the genome the location of genes and their introns/exons can be identified and in combination with the transcriptome the draft genome provides an excellent basis for studying WSSV in C. maenas. It also provides a significant resource for other scientists working with this species. Within the genome there are elements other than host genes that may be discovered that can have an impact on WSSV infection. For example fossilized remnants the WSSV genome could convey resistance. A genome sequence is furthermore vital for being able to identify miRNA precursors. The latter are an important class of regulatory molecules in viral infection.
Chapter 5
As for C. maenas, the genomic resources for H. gammarus are sparse. In this chapter RNA from specific tissues was isolated and sequenced for this species. The transcriptome was assembled and annotated in similar fashion as that for C. maenas. Differences in the virus relevant infection pathways were identified between H. gammarus and C maenas. In addition sequences of known WSSV receptors were compared across aquatic crustaceans.
Chapter 6
In this chapter the response of C. maenas to WSSV exposure was investigated. Individuals were injected with virus innoculum or saline solution. Over a series of timepoints, ranging from 6 hours to 28 days post injections, gill from the individuals were sampled. Isolated gill RNA was subjected to both RNA and miRNA sequencing. This sequencing data enabled identification of replicating WSSV in the samples for monitoring progression of WSSV infection. Through differential expression analysis of host transcripts and miRNAs, important elements in the infection process were identified. Pathway analysis provided additional biological context.
Chapter 7 - Discussion
The final discussion provides a critical appraisal on the key findings in this thesis and identifies both strengths and weakness in the work conducted. The findings are put into context with contemporary research into WSSV and it also provides some final conclusions on the work and suggestions for future research directions.
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