Interactive effects of elevated temperature and Photobacterium swingsii infection on the survival and immune response of marine mussels (Perna canaliculus): A summer mortality scenario.

The New Zealand Greenshell™ mussel (Perna canaliculus) is an economically important aquaculture species. Prolonged increases in seawater temperature above mussel thermotolerance ranges pose a significant threat to mussel survival and health, potentially increasing susceptibility to bacterial infections. Using challenge experiments, this study examined the combined effects of increased seawater temperature and bacterial (Photobacterium swingsii) infection on animal survival, haemocyte and biochemical responses of adult mussels. Mussels maintained at three temperatures (16, 20 and 24 °C) for seven days were either not injected (control), injected with sterile marine broth (injection control) or P. swingsii (challenged with medium and high doses) and monitored daily for five days. Haemolymph and tissue samples were collected at 24, 48, 72, 96, 120 h post-challenge and analysed to quantify bacterial colonies, haemocyte responses and biochemical responses. Mussels infected with P. swingsii exhibited mortalities at 20 and 24 °C, likely due to a compromised immune system, but no mortalities were observed when temperature was the only stressor. Bacterial colony counts in haemolymph decreased over time, suggesting bacterial clearance followed by the activation of immune signalling pathways. Total haemocyte counts and viability data supports haemocyte defence functions being stimulated in the presence of high pathogen loads at 24 °C. In the gill tissue, oxidative stress responses, measured as total antioxidant capacity and malondialdehyde (MDA) levels, were higher in infected mussels (compared to the controls) after 24h and 120h post-challenge at the lowest (16 °C) and highest temperatures (24 °C), indicating the presence of oxidative stress due to temperature and pathogen stressors. Overall, this work confirms that Photobacterium swingsii is pathogenic to P. canaliculus and indicates that mussels may be more vulnerable to bacterial pathogens under conditions of elevated temperature, such as those predicted under future climate change scenarios.

Quantification of Photobacterium swingsii and characterisation of disease progression in the New Zealand Greenshell™ mussel, Perna canaliculus.

Greenshell™ mussels (Perna canaliculus) are endemic to New Zealand and support the largest aquaculture industry in the country. Photobacterium swingsii was isolated and identified from moribund P. canaliculus mussels following a mass mortality event. In this study, a challenge experiment was used to characterise, detect, and quantify P. swingsii in adult P. canaliculus following pathogen exposure via injection into the adductor muscle. A positive control (heat-killed P. swingsii injection) was included to account for the effects of injection and inactive bacterial exposure. Survival of control and infected mussels remained 100% during 72-hour monitoring period. Haemolymph was sampled for bacterial colony counts and haemocyte flow cytometry analyses; histology sections were obtained and processed for histopathological assessments; and adductor muscle, gill, digestive gland were sampled for quantitative polymerase chain reaction (PCR) analyses, all conducted at 12, 24, 48 h post-challenge (hpc). The most profound effects of bacterial injection on mussels were seen at 48 hpc, where mussel mortality, haemocyte counts and haemolymph bacterial colony forming were the highest. The quantification of P. swingsii via qPCR 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 P. canaliculus mussels and provides histopathological insight into the tissue injury caused by the action of injection into the adductor muscle. The multi-technique methods used in this study can be applied for use in early surveillance programs of bacterial infection on mussel farms.

Histopathological investigation of four populations of abalone (Haliotis iris) exhibiting divergent growth performance.

The black-foot abalone (paua), Haliotis iris, is a unique and valuable species to New Zealand with cultural importance for Maori. Abalone are marine gastropods that can display a high level of phenotypic variation, including slow-growing or 'stunted' variants. This investigation focused on identifying factors that are associated with growth performance, with particular interest in the slow-growing variants. Tissue alterations in H. iris were examined using histopathological techniques, in relation to growth performance, contrasting populations classified by commercial harvesters as 'stunted' (i.e., slow-growing) and 'non-stunted' (i.e., fast-growing) from four sites around the Chatham Islands (New Zealand). Ten adults and 10 sub-adults were collected from each of the four sites and prepared for histological assessment of condition, tissue alterations, presence of food and presence of parasites. The gut epithelium connective tissue, digestive gland, gill lamellae and right kidney tissues all displayed signs of structural differences between the slow-growing and fast-growing populations. Overall, several factors appear to be correlated to growth performance. The individuals from slow-growing populations were observed to have more degraded macroalgal fragments in the midgut, increased numbers of ceroid granules in multiple tissues, as well as increased prevalence of birefringent mineral crystals and haplosporidian-like parasites in the right kidney. The histopathological approaches presented here complement anecdotal field observations of reduced seaweed availability and increased sand incursion at slow-growing sites, while providing an insight into the health of individual abalone and sub-populations. The approaches described here will ultimately help elucidate the drivers behind variable growth performance which, in turn, supports fisheries management decisions and future surveillance programs.

The effect of simulated marine heatwaves on green-lipped mussels, Perna canaliculus: A near-natural experimental approach.

Marine heatwaves (MHW) are projected for the foreseeable future, affecting aquaculture species, such as the New Zealand green-lipped mussel (Perna canaliculus). Thermal stress alters mussel physiology highlighting the adaptive capacity that allows survival in the face of heatwaves. Within this study, adult mussels were subjected to three different seawater temperature regimes: 1) low (sustained 18 °C), 2) medium MHW (18-24 °C, using a +1 °C per week ramp) and 3) high MHW (18-24 °C, using a +2 °C per week ramp). Sampling was performed over 11 weeks to establish the effects of temperature on P. canaliculus survival, condition, specific immune response parameters, and the haemolymph metabolome. A transient 25.5-26.5 °C exposure resulted in 61 % mortality, with surviving animals showing a metabolic adjustment within aerobic energy production, enabling the activation of molecular defence mechanisms. Utilisation of immune functions were seen within the cytology results where temperature stress affected the percentage of superoxide-positive haemocytes and haemocyte counts. From the metabolomics results an increase in antioxidant metabolites were seen in the high MHW survivors, possibly to counteract molecular damage. In the high MHW exposure group, mussels utilised anaerobic metabolism in conjunction with aerobic metabolism to produce energy, to uphold biological functions and survival. The effect of exposure time was mainly seen on very long-, and long chain fatty acids, with increases observed at weeks seven and eight. These changes were likely due to the membrane storage functions of fatty acids, with decreases at week eleven attributed to energy metabolism functions. This study supports the use of integrated analytical tools to investigate the response of marine organisms to heatwaves. Indeed, specific metabolic pathways and cellular markers are now highlighted for future investigations aimed at targeted measures. This research contributes to a larger program aimed to identify resilient mussel traits and support aquaculture management.

Histopathological changes in the greenshell mussel, Perna canaliculus, in response to chronic thermal stress.

Climate change associated temperature challenges pose a serious threat to the marine environment. Elevations in average sea surface temperatures are occurring and increasing frequency of marine heatwaves resulting in mortalities of organisms are being reported. In recent years, marine farmers have reported summer mass mortality events of the New Zealand Greenshell mussel, Perna canaliculus, during the summer months; however, the etiological agents have yet to be determined. To elucidate the role of thermal stress, adult P. canaliculus were exposed to three chronic temperature treatments: a benign control of 17 °C and stressful elevations of 21 °C and 24 °C. Eight mussels per treatment were collected each month throughout a 14-month challenge period to identify and investigate histopathological differences among P. canaliculus populations exposed to the three temperatures. Histopathology revealed several significant deleterious alterations to tissues associated with temperature and exposure time. Increasing temperature and progression of time resulted in 1) an increase in the number of focal lipofuscin-ceroid aggregations, 2) an increase in focal hemocytosis, 3) an increase in the thickness of the sub-epithelial layer of the intestinal tract and 4) a decreased energy reserve cell (glycogen) coverage in the mantle. Prolonged exposure, irrespective of temperature, impacted gametogenesis, which was effectively arrested. Furthermore, increased levels of the heat shock protein 70 kDa (HSP 70) were seen in gill and gonad from thermally challenged mussels. The occurrence of the parasite Perkinsus olseni at month 5 in the 24 °C treatment, and month 7 at 21 °C was unexpected and may have exacerbated the fore-mentioned tissue conditions. Prolonged exposure to stable thermal conditions therefore appears to impact P. canaliculus, tissues with implications for broodstock captivity. Mussels experiencing elevated, temperatures of 21 and 24 °C demonstrated more rapid pathological signs. This research provides further insight into the complex host-pathogen-environment interactions for P. canaliculus in response to prolonged elevated temperature.

Metabolite Changes of Perna canaliculus Following a Laboratory Marine Heatwave Exposure: Insights from Metabolomic Analyses.

Temperature is considered to be a major abiotic factor influencing aquatic life. Marine heatwaves are emerging as threats to sustainable shellfish aquaculture, affecting the farming of New Zealand's green-lipped mussel [Perna canaliculus (Gmelin, 1791)]. In this study, P. canaliculus were gradually exposed to high-temperature stress, mimicking a five-day marine heatwave event, to better understand the effects of heat stress on the metabolome of mussels. Following liquid chromatography-tandem mass spectrometry analyses of haemolymph samples, key sugar-based metabolites supported energy production via the glycolysis pathway and TCA cycle by 24 h and 48 h of heat stress. Anaerobic metabolism also fulfilled the role of energy production. Antioxidant molecules acted within thermally stressed mussels to mitigate oxidative stress. Purine metabolism supported tissue protection and energy replenishment. Pyrimidine metabolism supported the protection of nucleic acids and protein synthesis. Amino acids ensured balanced intracellular osmolality at 24 h and ammonia detoxification at 48 h. Altogether, this work provides evidence that P. canaliculus has the potential to adapt to heat stress up to 24 °C by regulating its energy metabolism, balancing nucleotide production, and implementing oxidative stress mechanisms over time. The data reported herein can also be used to evaluate the risks of heatwaves and improve mitigation strategies for aquaculture.

Acute thermal stress and endotoxin exposure modulate metabolism and immunity in marine mussels (Perna canaliculus).

Mass mortalities of New Zealand Green-lipped mussels (Perna canaliculus) are thought to be associated with increased water temperatures and immune challenges from opportunistic pathogens. However, the combined effects of acute thermal stress and immune stimulation on mussels are poorly understood. To investigate these responses, adult mussels were exposed to different temperatures (26 °C [thermal stress] vs 15 °C [ambient]) and a bacterial-derived endotoxin injection (with vs without) to mimic a pathogen infection. Various immunological and metabolic parameters were measured over two days via enzyme staining reactions, flow cytometry, and metabolomic profiling. None of the treatments impacted total and differential haemocyte counts, haemocyte viability or production of reactive oxygen species. Acid phosphatase and phenoloxidase activities were detected only within granulocytes (not in hyalinocytes), although their relative expressions also were not affected. Conversely, metabolite profiling exposed impacts of thermal stress and endotoxin exposure at a metabolic level, indicative of physiological changes in energy expenditure and partitioning. At the higher water temperature, free fatty acid and amino acid constituents increased and decreased, respectively, which supports an elevated energy demand and higher metabolic rate due to thermal stress. Ultimately, energy production is being sustained via multiple routes including the glycolysis pathway, TCA cycle, and ß-oxidation. Additionally, branched-chain amino acids, the urea cycle and the glutathione pathway were affected by the higher temperature. The metabolic response of mussels exposed to endotoxin exposure resulted in increased metabolite response largely linked to protein and lipid degradation. After 5 days of exposure, survival data confirmed a severe physiological impact of the higher temperature through incidences of mortality. However, the thermal challenge in combination with the specific endotoxin treatment applied did not lead to a synergistic effect on mortality. These findings provide new insights into the relationship between thermal stress and immunity to better understand the immune defence system in mussels.

Pathogenicity and virulence of bacterial strains associated with summer mortality in marine mussels (Perna canaliculus).

The occurrence of pathogenic bacteria has emerged as a plausible key component of summer mortalities in mussels. In the current research, four bacterial isolates retrieved from moribund Greenshell࣪ mussels, Perna canaliculus, from a previous summer mortality event, were tentatively identified as Vibrio and Photobacterium species using morpho-biochemical characterization and MALDI-TOF MS and confirmed as V. celticus, P. swingsii, P. rosenbergii, and P. proteolyticum using whole genome sequencing. These isolates were utilized in a laboratory challenge where mussels were injected with cell concentrations ranging from 105 to 109 CFU/mussel. Of the investigated isolates, P. swingsii induced the highest mortality. Additionally, results from quantitative polymerase chain reaction analysis, focusing on known virulence genes were detected in all isolates grown under laboratory conditions. Photobacterium rosenbergii and P. swingsii showed the highest expression levels of these virulence determinants. These results indicate that Photobacterium spp. could be a significant pathogen of P. canaliculus, with possible importance during summer mortality events. By implementing screening methods to detect and monitor Photobacterium concentrations in farmed mussel populations, a better understanding of the host-pathogen relationship can be obtained, aiding the development of a resilient industry in a changing environment.

Effects of seawater temperature and acute Vibriosp. challenge on the haemolymph immune and metabolic responses of adult mussels (Perna canaliculus).

The New Zealand Greenshell™ mussel (Perna canaliculus) is an endemic bivalve species with cultural importance, that is harvested recreationally and commercially. However, production is currently hampered by increasing incidences of summer mortality in farmed and wild populations. While the causative factors for these mortality events are still unknown, it is believed that increasing seawater temperatures and pathogen loads are potentially at play. To improve our understanding of these processes, challenge experiments were conducted to investigate the combined effects of increased seawater temperature and Vibrio infection on the immune and metabolic responses of adult mussels. Biomarkers that measure the physiological response of mussels to multiple-stressors can be utilised to study resilience in a changing environment, and support efforts to strengthen biosecurity management. Mussels acclimated to two temperatures (16 °C and 24 °C) were injected with either autoclaved, filtered seawater (control) or Vibriosp. DO1 (infected). Then, haemolymph was sampled 24 h post-injection and analysed to quantify haemocyte immune responses (via flow-cytometry), antioxidant capacity (measured electrochemically) and metabolic responses (via gas chromatography-mass spectrometry) to bacterial infection. Both seawater temperature and injection type significantly influenced the immune and metabolite status of mussels. A lack of interaction effects between temperature and injection type indicated that the effects of Vibrio sp. 24 h post-infection were similar between seawater temperatures. Infected mussels had a higher proportion of dead haemocytes and lower overall haemocyte counts than uninfected controls. The proportion of haemocytes showing evidence of apoptosis was higher in mussels held at 24 °C compared with those held at 16 °C. The proportion of haemocytes producing reactive oxygen species did not differ between temperatures or injection treatments. Mussels held at 24 °C exhibited elevated levels of metabolites linked to the glycolysis pathway to support energy production. The saccharopin-lysine pathway metabolites were also increased in these mussels, indicating the role of lysine metabolism. A decrease in metabolic activity (decreases in BCAAs, GABA, urea cycle metabolites, oxidative stress metabolites) was largely seen in mussels injected with Vibrio sp. Itaconate increased as seen in previous studies, suggesting that antimicrobial activity may have been activated in infected mussels. This study highlights the complex nature of immune and metabolic responses in mussels exposed to multiple stressors and gives an insight into Vibrio sp. infection mechanisms at different seawater temperatures.

Metabolite profiling of abalone (Haliotis iris) energy metabolism: a Chatham Islands case study.

INTRODUCTION: The Chatham Islands has some of the most prized black-footed abalone (Haliotis iris) beds in New Zealand. This well-managed fishery includes restrictions on catch and size limits, selective fishing methods, and shellfish management. However, recent declines in biomass and growth parameters have prompted omics research to characterise the biological responses of abalone, potentially contributing towards animal management strategies. OBJECTIVES: The aim of this study was to characterise the metabolite profiles of slow and fast growing, juvenile and adult abalone, relating to metabolites supporting energy metabolism. METHODS: A gas chromatography-mass spectrometry metabolite profiling, applying methyl chloroformate alkylation, was performed on juvenile and adult abalone samples collected from Point Durham and Wharekauri sites, Chatham Islands, New Zealand. RESULTS: The results obtained from haemolymph and muscle samples indicated that abalone from the fast-growing area, Wharekauri, fuelled metabolic functions via carbohydrate sources, providing energy for fatty acid and amino acid synthesis. Conversely, higher amino acid levels were largely utilised to promote growth in this population. The metabolism of juvenile abalone favoured anabolism, where metabolites were diverted from glycolysis and the tricarboxylic acid cycle, and used for the production of nucleotides, amino acids and fatty acids. CONCLUSIONS: This research provides unique physiological insights towards abalone populations supporting the use of metabolomics as a tool to investigate metabolic processes related to growth. This work sets the stage for future work aimed at developing biomarkers for growth and health monitoring to support a growing and more sustainably abalone fishery.

Evaluation of immune stimulatory products for whiteleg shrimp (Penaeus vannamei) by a metabolomics approach.

The use of probiotics, prebiotics and dietary fiber has become a common practice in shrimp aquaculture as alternatives to antibiotic treatment. However, not much is known about the metabolic mechanisms underlying the effects of probiotics and immunostimulant used in shrimp aquaculture. In this study, a gas chromatography-mass spectrometry (GC-MS) based metabolomics approach was used to characterize metabolite profiles of haemolymph and gills of whiteleg shrimp (Penaeus vannamei) exposed to four treatments (cellulose fiber, probiotics with Vibrio alginolyticus, a combination of cellulose fiber and V. alginolyticus and a control treatment). The cellulose fiber was administrated as a feed additive (100 mg⋅Kg-1 feed), while the probiotics was applied in the water (105 UFC⋅mL-1 culture water). The results showed significant differences in haemolymph metabolite profiles of immune stimulated treatments compared to the control and among treatments. The combination of cellulose fiber and probiotics resulted in greater differences in metabolic profiles, suggesting a better immune stimulation with this approach. The changes in haemolymph metabolome of treated shrimp reflected several biochemical pathway modifications, including changes in amino acid and fatty acid metabolism, disturbances in energy metabolism and antimicrobial activity and stress responses. For gill tissues, significant differences were only found in lactic acid between the probiotic group and the control. Among the altered metabolites, the increases of itaconic acid in haemolymph, and lactic acid in both haemolymph and gill tissues of immune-stimulated suggest the potential use of these metabolites as biomarkers for health assessment in aquaculture.

Physiological responses of juvenile New Zealand geoduck (Panopea zelandica) following emersion and recovery.

The New Zealand geoduck clam is a unique seafood delicacy, with animals selling for up to $US 220-330/kg. Stress accumulated during transport of juveniles to grow-out sites represent a bottleneck in the aquaculture process. In this study, the physiological responses of juvenile geoducks following emersion (3- and 8-h), and recovery (1- and 5-days) were investigated. An integrated approach of flow cytometry, osmolality and metabolomics, along with behavioural assessments was used. Both cellular and chemical haemolymph parameters and metabolite profiles were recorded for P. zelandica juveniles and are reported herein for the first time. An increase in haemolymph osmolality was experienced with an increase in emersion period, with significant differences seen between the 3- and 8-h emersion groups after 5 days of recovery. Viability measures of haemocytes varied insignificantly between experimental groups, creating baseline ranges. The proportion of haemocytes undergoing respiratory burst activity did not appear to be affected by emersion and re-immersion. Haemocyte mitochondrial membrane potential was highest following 1-day of recovery, likely linked to metabolic readjustment, and increased glycolysis, taking place following emersion. Metabolomics analyses suggest that protein, lipid and carbohydrate metabolite classes assist with energy production in geoducks. Activation of anaerobic metabolic pathways, with a high dependence on succinate, were prominent in the 8-h exposure group, with metabolic recovery still taking place following 5-days of immersion, mainly due to proteins restoring energy reserves. Elucidating the physiological responses of juvenile geoduck subjected to transport stress can aid cultivation methods already underway to develop a novel, high value aquaculture industry.

The Effects of Live Transport on Metabolism and Stress Responses of Abalone (Haliotis iris).

The New Zealand abalone industry relies mostly on the export of processed products to distant Asian markets, notably China. Over the past five years, live export of high quality abalone from New Zealand has proven successful. However, transport of live animals is associated with multiple stressors that affect survival and meat quality at the end of the transport phase. Better understanding of transport-derived stress is needed to improve transport conditions and recovery at destination to ensure high product quality and safety throughout the supply chain. To this end, we applied an untargeted GC-MS-based metabolomics approach to examine the changes in metabolite profiles of abalone after a 2-day transport event and subsequent water re-immersion for 2 days. The results revealed alterations of many metabolites in the haemolymph and muscle of post-transported abalone. Decreased concentrations of many amino acids suggest high energy demands for metabolism and stress responses of transported abalone, while increases of other amino acids may indicate active osmoregulation and/or protein degradation due to oxidative stress and apoptosis. The accumulation of citric acid cycle intermediates and anaerobic end-products are suggestive of hypoxia stress and a shift from aerobic to anaerobic metabolism (resulting from aerial exposure). Interestingly, some features in the metabolite profile of reimmersed abalone resembled those of pre-transported individuals, suggesting progressive recovery after reimmersion in water. Evidence of recovery was observed in the reduction of some stress biomarkers (e.g., lactic acid, succinic acid) following reimmersion. This study revealed insights into the metabolic responses to transport stress in abalone and highlights the importance of reimmersion practices in the supply chain of live animal exports.

Beyond relaxed: magnesium chloride anaesthesia alters the circulatory metabolome of a marine mollusc (Perna canaliculus).

BACKGROUND: The New Zealand Green-lipped mussel industry is well-established providing vastly to aquaculture exports. To assess mussel health and reproduction status, visual examination of organs and/or collection of haemolymph is commonly applied. Anesthetics, such as magnesium chloride (MgCl2) can be utilized to prevent muscle contraction and keep shells open during sampling. The specific effects of muscle relaxing agents on baseline metabolism in invertebrates is unknown, but it is evident that molecular, cellular and physiological parameters are altered with these chemical applications. To this end, metabolomics approaches can help elucidate the effects of relaxing agents for better assessment of their use as a research tool. METHODS: Adult Green-lipped mussels were anaesthetized for 3 h in a MgCl2 bath, whereafter haemolymph samples were collected and analyzed via gas chromatography-mass spectrometry applying methyl chloroformate alkylation derivatization. RESULTS: Anesthetized mussels were characterized as non-responsive to manual manipulation, with open valves, and limited siphoning function. Metabolite profiling revealed significant increases in the abundances of most metabolites with an array of metabolic activities affected, resulting in an energy imbalance driven by anaerobic metabolism with altered amino acids acting as neurotransmitters and osmolytes. CONCLUSION: This research is the first to use a metabolomics approach to identify the metabolic consequences of this commonly used bivalve relaxing technique. Ultimately the use of MgCl2 anesthetization as a sampling strategy should be carefully evaluated and managed when performing metabolomics-related research.

Effect of anticoagulants on farmed giant kokopu, Galaxias argenteus (Gmelin 1789) haematological parameters and erythrocyte fragility.

We sought to determine a compatible anticoagulant for routine haematological and physiological assessments with giant kokopu (Galaxias argenteus), an endemic New Zealand fish. We observed that blood treated with lithium heparin (LH) rapidly coagulated and haemolysed, making it unsuitable for G. argenteus. Dipotassium ethylenediaminetetraacetic acid (K2 EDTA) and trisodium citrate (citrate) effectively prevented blood coagulation. K2 EDTA-treated erythrocytes exhibited the least mean haemolysis and mean corpuscular fragility. Further studies into prolonged storage effects of citrate and K2 EDTA are recommended to find a compatible anticoagulant for use with G. argenteus blood.

Haematological and metabolic profiles associated with age and sex in giant kokopu (Galaxias argenteus) (Gmelin 1789) broodstock.

This study characterized selected peripheral blood (PB) haematological parameters, liver, serum and muscle metabolic features in 3- and 5-year-old male and female giant kokopu (Galaxias argenteus) broodstock reared indoor at 16°C. Sex and age did not affect PB total cell count and haematocrit values. Nonetheless, higher erythrocytes in 5-year-old fish, elevated thrombocyte and lymphocyte counts in 3-year-old fish indicate age-specific cellular regulation. Higher thrombocyte counts in female fish suggest sex-specific regulation. At a metabolic level, liver abundance for long chain saturated fatty acids (FAs) was higher in males, whereas females had elevated levels of polyunsaturated FAs. Essential and non-essential amino acids (AAs) in liver and serum were also elevated in females compared to males. These findings suggest differential allocation of FAs and AAs to reflect requirements for gonadal, development and provisioning. Similarly, age significantly resulted in higher liver and serum abundances of some non-essential AAs in 3-year-olds compared to 5-year-old fish, suggesting higher metabolism in younger fish. Overall, results enhance our understanding of sex- and age-based differences in fish haematology, muscle, liver, and serum metabolite profiles in healthy G. argenteus. Future studies should carefully consider potential age- and sex-specific differences in metabolic responses.

Metabolic and immune responses of Chinook salmon (Oncorhynchus tshawytscha) smolts to a short-term poly (I:C) challenge.

Polyinosinic:polycytidylic acid [poly (I:C)] was administered in vivo to Chinook salmon (Oncorhynchus tshawytscha) post-smolts to determine the immune responses on haematological and cellular functional parameters, including spleen (SP), head kidney (HK) and red blood cell (RBC) cytokine expression, as well as serum metabolomics. Poly (I:C) in vivo (24 h exposure) did not affect fish haematological parameters, leucocyte phagocytic activity and phagocytic index, reactive oxygen species and nitric oxide production. Gas chromatography-mass spectrometry-based metabolomics revealed that poly (I:C) significantly altered the serum biochemistry profile of 25 metabolites. Metabolites involved in the branched-chain amino acid/glutathione and transsulphuration pathways and phospholipid metabolism accumulated in poly (I:C)-treated fish, whereas those involved in the glycolytic and energy metabolism pathways were downregulated. At cytokine transcript level, poly (I:C) induced a significant upregulation of antiviral ifnγ in HK and Mx1 protein in HK, SP and RBCs. This study provides evidence for poly (I:C)-induced, immune-related biomarkers at metabolic and molecular levels in farmed O. tshawytscha in vivo. These findings provide insights into short-term effects of poly (I:C) at haematological, innate and adaptive immunity and metabolic levels, setting the stage for future studies.

Flow cytometric characterization of hemocytes of the flat oyster, Ostrea chilensis.

The flat oyster, Ostrea chilensis, native to New Zealand (NZ) and Chile is considered an important ecological, cultural and fisheries resource. Currently, commercial landings of this species in NZ are restricted due to low population numbers caused by ongoing mortalities resulting from the presence of the haplosporidian parasite, Bonamia exitiosa. More recently, the arrival of B. ostreae in NZ led to major mortalities in farmed stocks. To understand how diseases caused by Bonamia spp. affect this oyster species, a more complete understanding of its biology, physiology and immune system is needed. The present study characterized, for the first time, hemocytes of adult O. chilensis, from the Foveaux Strait, NZ, using flow cytometry (FCM) and histology. Based on the internal complexity of the hemocytes, two main circulating hemocyte populations were identified: granulocytes and hyalinocytes (accounting for ~30% and ~70% of the total circulating hemocyte population, respectively). These were further divided into two sub-populations of each cell type using FCM. A third sub-population of granulocytes was identified using histology. Using FCM, functional and metabolic characteristics were investigated for the two main hemocyte types. Granulocytes showed higher phagocytic capabilities, lysosomal content, neutral lipid content and reactive oxygen species production compared to hyalinocytes, indicating their important role in cellular immune defence in this species. Methods of hemocyte sampling and storage were also investigated and flow cytometric protocols were detailed and verified to allow effective future investigations into the health status of this important species.

In vitro immune response of chinook salmon (Oncorhynchus tshawytscha) peripheral blood mononuclear cells stimulated by bacterial lipopolysaccharide.

We investigated cellular functional and targeted immune cytokine responses of farmed Chinook salmon (Oncorhynchus tshawytscha) peripheral blood mononuclear cells (PBMCs) in vitro to LPS from Escherichia coli (E. coli) serotypes O111: B4 and O55: B5, and a phorbol ester phorbol 12-myristate 13-acetate (PMA). Bacterial LPS and PMA significantly (p < 0.05) induced reactive oxygen species (ROS) production in O. tshawytscha PBMCs, and enhanced by interferon (IFN)-inducible cytokine production. Cellular phagocytosis was significantly enhanced with PMA and E. coli serotype O111: B4 LPS after 1 and 2 h respectively. At the molecular level, LPS and PMA significantly (p < 0.05) upregulated pro-inflammatory cytokine gene transcripts for IFNγ, TNF-α, and anti-inflammatory IL-10, 24 h post-stimulation. This response is postulated to be mediated via the MyD88 and TRIF pathways in TLR4, or synergistic TLR1 and TLR2 receptors. This is the first report of LPS induced immune related in vitro responses in farmed O. tshawytscha PBMCs.