Microbial communities associated with bacillary necrosis of Australian blue mussel (Mytilus galloprovincialis Lamarck): a study using model larval cultures by Tzu Nin Kwan BAqua (Hons) University of Tasmania Submitted in fulfilment of the requirements for the degree of Doctor of Philosophy November 2017 This thesis is dedicated to Felix Kwan who seeded in me the love for science, Agnes Chin and Lucy Chin who mentored me. All inspired me to pursue the scientist’s dream. ii Declaration This thesis contains no material which has been accepted for the award of any other degree or diploma in any tertiary institution, and to the best of my knowledge and belief, contains no material previously published or written by another person, except where due reference is made in the text of the thesis. ------------------------------------------------------ Tzu Nin Kwan (15 November 2017) Statement of Ethical Conduct The research associated with this thesis abides by the international and Australian codes on human and animal experimentation, the guidelines by the Australian Government's Office of the Gene Technology Regulator and the rulings of the Safety, Ethics and Institutional Biosafety Committees of the University. ------------------------------------------------------ Tzu Nin Kwan (15 November 2017) Statement on Authority of Access This thesis may be made available for loan and limited copying and communication in accordance with the Copyright Act 1968. iii Statement on published work The publisher of a paper comprising Chapters 2 and 3 hold the copyright for that content, and access to the material should be sought from the respective journals. The remaining unpublished content of the thesis may be made available for loan and limited copying in accordance with the Copyright Act 1968. Kwan, TN, Bowman, JP and Bolch CJS (2017) Necrotic disease in bivalve larval cultures. Journal of the Australian Society for Microbiology Inc. 28: 131-133. Published: September 2017. ------------------------------------------------------ Tzu Nin Kwan (15 November 2017) Statement of co-authorship This thesis includes work, which has been published, submitted or to be submitted for publication in a peer-review journal. The following people and institutions contributed to the publication of work undertaken as part of this thesis: Tzu Nin Kwan, Institute of Marine and Antarctic Studies, University of Tasmania (Candidate) Christopher J.S. Bolch, Institute for Marine and Antarctic Studies, University of Tasmania (Primary supervisor) John P. Bowman, School of Land and Food, University of Tasmania (Secondary supervisor) Details of the author roles: Chapter 2. Dynamic of culturable Vibrio abundance in early stage of blue mussel (Mytilus galloprovincialis Lamarck) larval rearing and associations with bacillary necrosis Chapter 3. Bacterial community dynamics associated with bacillary necrosis in Australian blue mussel (Mytilus galloprovincialis Lamarck) larval rearing induced by overfeeding iv Chapter 4. Bacterial community composition and proteolytic activity associated with bacillary necrosis in blue mussel (Mytilus galloprovincialis Lamarck) larval culture Chapter 5. Pseudoalteromonas sp. : an opportunistic pathogen of Australian blue mussel (Mytilus galloprovincialis Lamarck) larvae causing velar deformation and detachment Tzu Nin Kwan designed and carried out the experiments, collected and analysed the samples, carried out the data analysis, and wrote the manuscript. Christopher J.S. Bolch and John P. Bowman provided guidance on experimental design, data analysis and interpretation, and edited drafts of the manuscript. We the undersigned agree with the above stated work undertaken for each of the above submitted peer-reviewed and in preparation manuscripts contributing to this thesis ------------------------------------------------------ Dr. Christopher J. S. Bolch Primary supervisor Institute of Marine and Antarctic Studies, University of Tasmania ------------------------------------------------------ Prof. Craig Johnson Head, Ecology and Biodiversity Centre Institute of Marine and Antarctic Studies, University of Tasmania v Abstract Bacillary necrosis is a sporadic and rapid bacterial disease, capable of causing total collapse of larval cultures (known as larval crash) in a period of 24-48 h and is today, the most prevalent hatchery disease worldwide, affecting more than 20 bivalve species. The prevailing view is that Vibrio are primary pathogens even though this is based on limited understanding of direct causality and natural infection. As a result, this first part of the study investigated population dynamics and the role of culturable Vibrio in spontaneous outbreaks of bacillary necrosis in larval culture of Australian blue mussel (Mytilus galloprovincialis) reared under laboratory conditions. Given the importance of organic matter in regulation of Vibrio concentration in hatchery environments, the study used overfeeding of microalgae to induce bacillary necrosis. Vibrio proliferated at the beginning of overfed cultures by >20 % relative abundance. The patterns of change in Vibrio relative abundance in seawater and larvae coincided with onset of mortality (daily mortality >30 %) and progressed to a cumulative mortality > 70 % by 8 dpf. In contrast, standard fed cultures showed cumulative mortality no more than 15 %. Vibrio was not routinely detected in larvae samples except on the beginning of mortality and ranged from 2.8 to 4.7 x 102 CFU larva-1. The majority of culturable Vibrio were members of the Vibrio splendidus clade, a diverse bacterial group that is often implicated as causative agents. Interestingly, despite its increased relative abundance with mortality, challenge bioassays by immersing larvae in seawater with a series of bacterial cell concentration suggest these Vibrio isolated from the diseased cultures were unlikely pathogens. Therefore, this study in contrary to many published studies shows that Vibrio splendidus proliferation during mortality events can also result from growth of Vibrio communities normally associated with larval cultures and may not have a direct role in disease. The second part the thesis then characterised the bacterial communities from the same overfeeding experiment using molecular techniques. A total of 72 seawater and larvae samples (of which 6 associated with mass mortality) were analysed using Automated Ribosomal Intergenic Spacer Analysis (ARISA) of 16-23S rDNA intergenic spacer 1 (ITS1). Canonical analysis of principal coordinates (CAP) vi examining samples (n=72) categorised into normal, a day before (n=2), during (n=2) and a day after mortality (n=2) demonstrated systematic shifts in seawater and larval bacterial communities corresponding to the infection course of bacillary necrosis as mortality developed and subsided. Abnormal bacterial communities initiated in seawater up to 3 d prior to onset of larval mortality, and the timing of mortality coincided with the uptake of seawater communities by the larvae. This study for the first time demonstrated bacillary necrosis is potentially initiated in seawater even though the onset of bacillary necrosis remains unpredictable in both timing and scale of larval mortality. This suggests seawater is a potential important source of variation. In order to examine the link of seawater associated bacterial communities with variability in bacillary necrosis, a study was instigated that examined nineteen replicate small-scale larval cultures using 0.22 µm filtered seawater and a commercial algal food to minimize variation. Despite strict replication of larval cultures, mortality events occurred unpredictably in four of the nineteen cultures. Bacterial community analysis using Illumina MiSeq derived 16S rRNA gene taxa- abundance data showed mortality events were consistently linked with a similar group of bacteria. Linear discriminant analysis effect size (LEfSe) focusing on larval community data show sequence reads affiliated with Marinomonas sp. and Gracilibacteria were statistically more abundant in high mortality larval whilst Pseudoalteromonas distincta, Phaeobacter gallaenciensis and Vibrio splendidus were more abundant in low mortality cultures. This study also examined proteolytic activity in the larval culture seawater and for the first time showed a significant association between total proteolytic activity of rearing seawater and mortality. So far, this thesis observed that multiple bacteria can be associated with bacillary necrosis, and examinations of the interactions of some of these bacteria with mussel larvae may help progress our understanding of bacillary necrosis. The final part of this thesis investigated how bacteria isolated during outbreaks of bacillary necrosis interact with larvae in challenge assay conditions. In this study, ten bacterial colonies from the highest diluted plates (10-7) of Marine Agar (MA) were randomly isolated during a mass mortality event in laboratory larval cultures, and screened for virulence using mussel larval bioassays. A series of larval challenge vii 4 bioassays shows these isolates possessed moderate virulence (LC50 3.2±2.1 × 10 cells ml-1 at 2 day post challenge (dpc) of 6 dpf larvae in 0.22 µm filtered seawater). Interestingly, loss of virulence was observed for repeated sub-cultures. Comparison of 16S rRNA gene sequences showed that all 10 isolates were identical and represented an undescribed species of the genus Pseudoalteromonas, indicative that this bacterial strain occurred in higher concentration in the