Microbial communities associated with bacillary necrosis of Australian blue mussel (Mytilus galloprovincialis Lamarck): a study using model larval cultures

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 10\\(^2\\) 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) 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 ˜í¬¿m 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 bioassays shows these isolates possessed moderate virulence (LC50 3.2¬¨¬±2.1 ‚àöv= 10\\(^4\\) cells ml\\(^{-1}\\) at 2 day post challenge (dpc) of 6 dpf larvae in 0.22 ˜í¬¿m 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 larval culture suffering mass mortality. Histopathological examinations identified early, mid, severe, completion and post mortem stage of disease development across 3 d characterised by cleavage and detachment of velum without direct invasion of the velar tissue by bacterial cells indicative of destruction potentially mediated by toxins. However, marine-milk agar and azocasein assays detected only limited proteolytic activity of the Pseudoalteromonas isolates. Whilst more research is necessary, this study demonstrates for the first time a Pseudoalteromonas strain has been shown to negatively implicate mussel larval cultures. This thesis concludes, based on the model larval cultures that members of the Vibrio splendidus group have close links with bacillary necrosis however not necessarily as direct cause of larval mortality. Molecular techniques further show representation of Vibrio fades in the community wide changes suggestive of more complex interactions of bacteria than anticipated in shaping causality. The unpredictability and lack of association of mortality episodes with specific bacterial groups adds weight to the idea that bacillary necrosis is a condition associated with imbalance compositional changes in seawater or larval bacterial communities that can promote opportunistic interactions and influence larval health outcomes. The relevance of opportunistic pathogenesis/dysbiosis is emphasized here which adds another dimension of complexity to the current view of bacillary necrosis.