Gut microbiome resilience of green-lipped mussels, Perna canaliculus, to starvation.

Host gut microbiomes play an important role in animal health and resilience to conditions, such as malnutrition and starvation. These host-microbiome relationships are poorly understood in the marine mussel Perna canaliculus, which experiences significant variations in food quantity and quality in coastal areas. Prolonged starvation may be a contributory factor towards incidences of mass mortalities in farmed mussel populations, resulting in highly variable production costs and unreliable market supplies. Here, we examine the gut microbiota of P. canaliculus in response to starvation and subsequent re-feeding using high-throughput amplicon sequencing of the 16S rRNA gene. Mussels showed no change in bacterial species richness when subjected to a 14-day starvation, followed by re-feeding/recovery. However, beta bacteria diversity revealed significant shifts (PERMANOVA p-value < 0.001) in community structure in the starvation group and no differences in the subsequent recovery group (compared to the control group) once they were re-fed, highlighting their recovery capability and resilience. Phylum-level community profiles revealed an elevation in dominance of Proteobacteria (ANCOM-BC p-value <0.001) and Bacteroidota (ANCOM-BC p-value = 0.04) and lower relative abundance of Cyanobacteria (ANCOM-BC p-value = 0.01) in the starvation group compared to control and recovery groups. The most abundant genus-level shifts revealed relative increases of the heterotroph Halioglobus (p-value < 0.05) and lowered abundances of the autotroph Synechococcus CC9902 in the starvation group. Furthermore, a SparCC correlation network identified co-occurrence of a cluster of genera with elevated relative abundance in the starved mussels that were positively correlated with Synechococcus CC9902. The findings from this work provide the first insights into the effect of starvation on the resilience capacity of Perna canaliculus gut microbiota, which is of central importance to understanding the effect of food variation and limitation in farmed mussels.

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.

Effect of Dietary Insect Meal and Grape Marc Inclusion on Flavor Volatile Compounds and Shell Color of Juvenile Abalone Haliotis iris.

Almost 60% of the fish meal produced globally is used in aquaculture feeds. Fish meal production relies on finite wild-marine resources and is considered as an unsustainable ingredient. Insect meal (IM) is considered a sustainable source with high levels of protein suitable for growth promotion. Grape marc (GM) is a waste byproduct of the winery industry rich in pigments with antioxidant capacity. However, the inclusion of both ingredients can affect the flavor of the meat of abalone and the color of the shell due to different nutritional profiles. The aim of this study was to evaluate the effect of the dietary inclusion of IM and GM on the flavor volatile compounds and shell color of the juvenile Haliotis iris in a 165-days feeding trial. Abalone were offered four experimental diets with different levels of IM and GM inclusion and a commercial diet (no IM or GM). Soft bodies of abalone were used to characterize volatile compounds using solid-phase microextraction gas chromatography-mass spectrometry, and color changes were analyzed in ground powder of abalone shells using color spectrophotometry 400-700 nm (visible). The results showed 18 volatile compounds significantly different among the dietary treatments. The inclusion of IM did not significantly affect the flavor volatile compounds detected, whereas the inclusion of GM reduced volatile compounds associated with lipid-peroxidation in abalone meat. The inclusion of IM and GM did not significantly affect the lightness nor the yellowness, blueness, redness, and greenness of the ground shells. The supplementation of abalone feeds with GM can help to reduce off-flavour compounds which may extend shelf-life of raw abalone meat.

Sustainable Aquafeed Formulations Containing Insect Larval Meal and Grape Marc for the New Zealand Farmed Abalone.

The aquaculture industry has been criticised for the excessive use of fish meal (FM) in feeds due to the utilisation of wild fish in the formulation and the exacerbation of overfishing marine resources. Land-based abalone aquaculture mainly uses commercial feeds (CFs) to promote faster growth, which include FM as a primary protein component. Alternative ingredients, such as insect meal (IM) and grape marc (GM) are potential candidates for FM replacement due to their suitable nutritional profile and sustainable production. This paper reports on a novel nutritional approach for the New Zealand farmed abalone, which replaces FM with IM by 10% and includes a waste by-product (GM) by 30% as a potential prebiotic source. The study was performed in two stages: (a) physico-chemical determination of diets delivered in an alginate matrix (experimental diets) and their stability in seawater compared to CF and (b) evaluation of growth and feed intake for the New Zealand black-foot abalone. There were significant differences between experimental diets and CF in terms of sinking rate, particle weight, and microscopic observations. Water stability of the experimental diets was increased by 50% in 24 and 48 hr compared to CF, producing less solid waste, and potentially reducing cleaning efforts in the farm. The inclusion of IM and GM did not compromise overall animal growth or their feed conversion ratio, however, further evaluation need to be explored in the future research. The findings revealed that the developed encapsulated feeds are a more stable food delivery method for Haliotis iris compared to the CF. Furthermore, both IM and GM can be included in feed formulations as a more sustainable strategy without compromising weight and shell gains in the abalone farming.

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.

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.

Mapping the Green-Lipped Mussel (Perna canaliculus) Microbiome: A Multi-Tissue Analysis of Bacterial and Fungal Diversity.

Poor health and mortality events of the commercially important and endemic New Zealand green-lipped mussel (Perna canaliculus) pose a threat to its industry. Despite the known importance of microbiomes to animal health and environmental resilience, the host-associated microbiome is unexplored in this species. We conducted the first baseline characterization of bacteria and fungi within key host tissues (gills, haemolymph, digestive gland, and stomach) using high-throughput amplicon sequencing of 16S rRNA gene and ITS1 region for bacteria and fungi, respectively. Tissue types displayed distinctive bacterial profiles, consistent among individuals, that were dominated by phyla which reflect (1) a fluid exchange between the circulatory system (gills and haemolymph) and surrounding aqueous environment and (2) a highly diverse digestive system (digestive gland and stomach) microbiota. Gammaproteobacteria and Campylobacterota were mostly identified in the gill tissue and haemolymph, and were also found in high abundance in seawater. Digestive gland and stomach tissues were dominated by common gut bacterial phyla, such as Firmicutes, Cyanobacteria, Proteobacteria, and Bacteroidota, which reflects the selectivity of the digestive system and food-based influences. Other major notable taxa included the family Spirochaetaceae, and genera Endozoicomonas, Psychrilyobacter, Moritella and Poseidonibacter, which were highly variable among tissue types and samples. More than 50% of fungal amplicon sequence variants (ASVs) were unclassified beyond the phylum level, which reflects the lack of studies with marine fungi. However, the majority of those identified were assigned to the phylum Ascomycota. The findings from this work provide the first insight into healthy tissue microbiomes of P. canaliculus and is of central importance to understanding the effect of environmental changes on farmed mussels at the microbial level.

Changes in metabolic profiling of whiteleg shrimp (Penaeus vannamei) under hypoxic stress.

Hypoxia is a common concern in shrimp aquaculture, affecting growth and survival. Although recent studies have revealed important insights into hypoxia in shrimp and crustaceans, knowledge gaps remain regarding this stressor at the molecular level. In the present study, a gas chromatography-mass spectrometry (GC-MS)-based metabolomics approach was employed to characterize the metabolic signatures and pathways underlying responses of Pacific white shrimp (Penaeus vannamei) to hypoxia and to identify associated candidate biomarkers. We compared metabolite profiles of shrimp haemolymph before (0 h) and after exposure to hypoxia (1 & 2 h). Dissolved oxygen levels were maintained above 85 % saturation in the control and before hypoxia, and 15 % saturation in the hypoxic stress treatment. Results showed 44 metabolites in shrimp haemolymph that were significantly different between before and after hypoxia exposure. These metabolites were energy-related metabolites (e.g., intermediates of citric acid cycle, lactic acid, alanine), fatty acids and amino acids. Pathway analysis revealed 17 pathways that were significantly affected by hypoxia. The changes in metabolites and pathways indicate a shift from aerobic to anaerobic metabolism, disturbance in amino acid metabolism, osmoregulation, oxidative damage and Warburg effect-like response caused by hypoxic stress. Among the altered metabolites, lactic acid was most different between before and after hypoxia exposure and had the highest accurate value for biomarker identification. Future investigations may validate this molecule as a stress biomarker in aquaculture. This study contributes to a better understanding of hypoxia in shrimp and crustaceans at the metabolic level and provides a base for future metabolomics investigations on hypoxia.

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.

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.

Metabolic responses of whiteleg shrimp to white spot syndrome virus (WSSV).

Outbreaks of white spot syndrome virus (WSSV) have caused serious damage to penaeid shrimp aquaculture worldwide. Despite great efforts to characterize the virus, the conditions that lead to infection and the infection mechanisms, there is still a lack of understanding regarding these complex virus-host interactions, which is needed to develop consistent and effective treatment methods for WSSV. In this study, we used a gas chromatography - mass spectrometry (GC-MS)-based metabolomics approach to compare the metabolite profiles of gills, haemolymph and hepatopancreas from whiteleg shrimp (Penaeus vannamei) exposed to WSSV and corresponding controls. The results revealed clear discriminations between metabolite profiles of WSSV-challenged shrimp and controlled shrimp in each tissue. The responses of shrimp gills to WSSV infection were characterized by increases of many fatty acids and amino acids in WSSV-challenged shrimp compared to the controls. Changes in haemolymph metabolite profiles include the increased levels of itaconic acid, energy-related metabolites, metabolites in glutathione cycle and decrease of amino acids. The WSSV challenge led to the decreases of several fatty acids and amino acids and increases of other amino acids, lactic acid and other organic compounds (levulinic acid, malonic acid and putrescine) in hepatopancreas. These alterations of shrimp metabolites suggest several immune responses of shrimp to WSSV in a tissue-specific manner, including upregulation of osmoregulation, antimicrobial activity, metabolic rate, gluconeogenesis, glutathione pathway in control of oxidative stress and shift from aerobic to anaerobic metabolism in shrimp which indicates the Warburg effect. The findings from this study provide a better understanding of molecular process of shrimp response against WSSV invasion which may be useful for development of disease management strategies.

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.

Development of a microencapsulated probiotic delivery system for New Zealand black-footed abalone (Haliotis iris).

Conventional methods of probiotics delivery to farmed aquatic animals are not efficient due to loss of probiotic's viability before the probiotics can reach their site of action. This study aims to develop a microencapsulated probiotic delivery system for black-footed abalone (Haliotis iris). An emulsion technique was used to encapsulate probiotic bacteria within chitosan-coated alginate microparticles (CALG). The efficacy of CALG microparticles in delivering probiotics to abalone was assessed using ex vivo and in vivo experiments. Microparticles (113 ± 4 µm) with encapsulation efficiency of more than 75% were developed using an internal gelation formulation approach. The ex vivo release experiments revealed the lack of probiotic discharge in the first 6 h of incubating CALG in seawater followed by a slight bacterial release within the next 20 h. The exposure of CALG microparticles to simulated gastric and intestinal media showed a significantly higher release of encapsulated bacteria in the simulated intestinal medium. The results of feeding trial revealed that the number of probiotic bacteria in probiotic-fed abalone was significantly higher than the one in the control animals. The results suggest that CALG microparticles can be used as a controlled release system for delivering viable probiotic bacteria to the gastrointestinal tract of abalone.

An integrated omics approach to investigate summer mortality of New Zealand Greenshell™ mussels.

BACKGROUND: Green-lipped mussels, commercially known as Greenshell™ mussels (Perna canaliculus Gmelin 1791), contribute > $300 million to New Zealand's aquaculture exports. However, mortalities during summer months and potential pathogenic outbreaks threaten the industry. Thermal stress mechanisms and immunological responses to pathogen infections need to be understood to develop health assessment strategies and early warning systems. METHODS: P. canaliculus were collected during a mortality event at a commercial aquaculture farm in Firth of Thames, New Zealand. Gill tissues from six healthy and six unhealthy mussels were excised and processed for metabolomic (GC-MS) and label-free proteomic (LC-MS) profiling. Univariate analyses were conducted separately on each data layer, with data being integrated via sparse multiple discriminative canonical correlation analysis. Pathway enrichment analysis was used to probe coordinated changes in functionally related metabolite sets. RESULTS: Findings revealed disruptions of the tricarboxylic acid (TCA) cycle and fatty acid metabolism in unhealthy mussels. Metabolomics analyses also indicated oxidative stress in unhealthy mussels. Proteomics analyses identified under-expression of proteins associated with cytoskeleton structure and regulation of cilia/flagellum in gill tissues of unhealthy mussels. Integrated omics revealed a positive correlation between Annexin A4 and CCDC 150 and saturated fatty acids, as well as a negative correlation between 2-aminoadipic acid and multiple cytoskeletal proteins. CONCLUSIONS: Our study demonstrates the ability of using integrative omics to reveal metabolic perturbations and protein structural changes in the gill tissues of stressed P. canaliculus and provides new insight into metabolite and protein interactions associated with incidences of summer mortality in this species.

Metabolomics investigation of summer mortality in New Zealand Greenshell™ mussels (Perna canaliculus).

Increasing water temperatures due to climate change have resulted in more frequent high mortality events of New Zealand Greenshell™ mussels (Perna canaliculus Gmelin 1791). These events have significant impacts within mussel farms which support a major shellfish industry for New Zealand. The present study investigates metabolic responses of farmed mussels during a summer mortality event in order to identify health impacts and elucidate mechanistic effects of external stressors on mussels. A gas chromatography-mass spectrometry (GC-MS)-based metabolomics approach was used to identify metabolic perturbations and flow cytometry assays were used to assess viability, oxidative stress and apoptosis of haemocytes from healthy and unhealthy mussels during a summer mortality event. The results showed significantly higher mortality and apoptosis of haemocytes in unhealthy mussels compared to healthy mussels. Reactive oxygen species (ROS) production, which is an indicator of oxidative stress was very high in both mussel groups, but no differences were observed between the two mussel groups. Metabolomics revealed alterations of many metabolites in both haemolymph and hepatopancreas (digestive gland) of unhealthy mussels compared to healthy mussels, reflecting perturbations in several molecular pathways, including energy metabolism, amino acid metabolism, protein degradation/tissue damage and oxidative stress. An increased level of itaconic acid which is an antimicrobial metabolite and biomarker of pathogen infection was observed in haemolymph, but not in hepatopancreas samples. This investigation provides the first detailed metabolic characterization of mussel immune responses to a summer mortality event and illustrates the benefits of using an integrated metabolomics and flow cytometry workflow for mussel health assessment and biomarker identification for summer mortality early detection.