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Multi-omics Approaches to Investigate Responses of New Zealand Green-lipped™ Mussels to Environmental Stress and Pathogen Loads

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Thesis(38.58 MB)

Date

2023

Authors

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Alfaro, Andrea
Venter, Leonie

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Doctor of Philosophy

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Auckland University of Technology

Abstract

GreenshellTM mussels are important ecosystem engineers, with an established position in the aquaculture sector of New Zealand. Significant industry losses have been experienced, in terms of mass mortalities, on mussel farms in recent years. The exact cause of these mortalities is unknown, however association with increased water temperatures experienced during summer months have been deemed as a likely cause. Literature suggests that summer mortality involves intrinsic (immune dysfunction, gametogenesis, and spawning) and extrinsic (frequency and duration of heatwaves) factors, causing a physiological tipping point resulting in mussel death. Pathogen loads seem to proliferate during the summer months resulting in disease outbreaks. Novel research is required to study the impact of these mortalities and to mitigate disease outbreaks to assure health and sustainability for this industry. In this study, a multi-disciplinary approach, along with physiological evaluations, were employed to investigate the investigate the interplay of selected bacterial pathogens Photobacterium spp. and Vibrio spp. and temperature changes threatening the health status of adult, green-lipped mussels, Perna canaliculus. This thesis consists of two literature reviews (Chapter 2 and Chapter 3) and six experimental chapters (Chapter 4 -Chapter 9). Chapter 2 evaluates the immune status of GreenshellTM mussels and Chapter 3 explores physiological biomarkers relating to Vibrio sp. infections in mussels. Building upon the insights gained from extensive literature reviews, the focus now shifts to the experimental chapters, aiming to illuminate potential intersections and broader implications within the research framework. The first experimental chapter of this thesis (Chapter 4) assesses the effect of magnesium chloride (MgCl2) on adult GreenshellTM mussels’ physiology and metabolic response. MgCl2 significantly impacted the haemolymph metabolome in anesthetised mussels, indicating major physiological dysregulation. In Chapter 5, four bacterial isolates retrieved from moribund P. canaliculus, from a previous summer mortality event, were identified as V. celticus, P. swingsii, P. rosenbergii and P. proteolyticum using whole genome sequencing. Additionally, mussels injected with P. swingsii showed high mortality, along with expression of virulence genes (hsp60, zm, vcpA, toxR, ompU, mshA, chi, lip, and plp), suggesting pathogenesis of this bacterium to GreenshellTM mussels. Bacterial progression utilising P. swingsii was further investigated to better understand the mussel immune response and bacterial effect on mussel mortalities (Chapter 6). This study showed that the most profound effects of bacterial infection on mussels were seen at 48 hours post challenge (hpc) where mussel mortality, haemocyte counts and haemolymph colony forming units were the highest. The quantification of P. swingsii via targeted PCR showed highest levels of bacterial DNA at 12 hpc in the adductor muscle, gill, and digestive gland. Histopathological observations suggested a non-specific inflammatory response in all mussels associated with a general stress response. This study highlights the physiological effects of P. swingsii infection in GreenshellTM mussels and provides histopathological insight into the tissue injury caused by the action of injection into the adductor muscle. In Chapter 7, the impact of temperature stress on the metabolome of mussels was investigated utilising metabolomics. Mussels were exposed to two temperatures, 16 °C and 24 °C for five-days, creating a controlled laboratory marine heatwave environment. The metabolite changes in the presence of temperature stress provided insights into several pathways involved in defence and repair mechanisms, which reallocated energy away from organismal growth towards maintenance. Since all mussels in this experiment survived, this suggests that P. canaliculus has the potential to adapt to heat stress up to 24°C, by regulating their energy metabolism, balancing nucleotide production, and implementing oxidative stress mechanisms overtime. In the next chapter (Chapter 8), an experiment was performed to investigate the effects of thermal stress, bacteria, and combined stressors on selected immunological parameters and the survival of mussels. The total haemocyte count, viability, bacterial counts, total antioxidant capacity, and lipid peroxidation were used as indicators to measure an immune response of infected mussels to different temperatures. Water temperature at 24°C significantly affected immune functions and led to oxidative stress and reduction of immunosurveillance in the P. swingsii infected mussels. The combination of temperature-pathogen stress affected the survival of mussels with highest mortality at 24°C in the presence of bacteria. This chapter demonstrated that mussels have lower tolerance to the combined effects of high temperature stress and pathogen infection. Chapter 9 investigated the effect of bacterial coinfection, on mussel size, bacterial clearance efficiency and metabolic response, considering juvenile and adult mussels. This study showed greater mortality in juvenile mussels within the bacterial coinfection group, suggesting that susceptibility of small mussels to bacterial infections are greater than in adults. Large decreases in energy metabolites were detected in mussels when exposed to multiple bacterial pathogens. Potentially due to high energy expenditure and metabolite functions to support immunity and protein synthesis during this pathogen interactions. Collectively, these findings demonstrated a rigorous exploration of complex biological processes within New Zealand Greenshell™ mussels (P. canaliculus) and deepened the understanding of their general health, disease progression, transmission mechanism and host-pathogen interactions. These findings can be used to help assess P. swingsii transmission risk within and among GreenshellTM mussels’ populations and facilitate appropriate management and restoration strategies for both wild and cultured mussel species.

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http://hdl.handle.net/10292/16730

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