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The New Zealand Greenshell™ mussel (Perna canaliculus) supports the largest aquaculture industry in the country. However, summer mortality events and potential disease outbreaks may threaten the growth of this industry. As an approach to gauging potential threats through the seasons, a detailed histopathological examination was conducted on 256 adult cultured mussels collected from a farm between April 2018 to September 2019, which covered the austral autumn, winter, spring and summer seasons. Histological sections followed by confirmatory in situ hybridization (ISH) resulted in the identification of Perkinsus olseni at an overall prevalence of 56%. Other parasites and pathogens were identified by histology: apicomplexan parasite X (APX) (78%), copepods (Pseudomyicola spinosus or Lichomolgus uncus) (1%), Microsporidium rapuae (1%), intracellular microcolonies of bacteria (IMCs) (2%) and bacilli and cocci bacteria (4%) in gills, mantle, gonads, digestive epithelium and digestive tubules. There was a significant association between P. olseni and APX infection in mussels. This is the first report on seasonal variations of P. olseni and APX in New Zealand Greenshell™ mussel. There was a significant association between seasons and the presence of P. olseni and APX in mussels. A significant positive association between the brown material accumulation and parasites (P. olseni and APX) and between haemocytosis and P. olseni infections were recorded. A significant association between presence of parasites and health condition (healthy and unhealthy) of mussels was observed. Moreover, a significant association between digestive tubule deterioration (large lumen, with a thin epithelial wall) and P. olseni infection was noted. Therefore, this study provides information regarding the infections of potential parasites and pathogens for the first time in P. canaliculus, their seasonal variations and host-parasite interactions within a commercial farm.

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

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.

With a rising world population and pressure on ecosystems due to development, researchers need sophisticated and integrated approaches for conducting sustainability research. We outline a mixed methods practical sustainability research framework, defined as an equal-priority social–ecological methodology, whereby both community engagement and integrated biodiversity assessments are constructed to identify sustainability trade-offs. We illustrate the framework’s use for investigating the social–ecological trade-offs between preserving and removing temperate mangroves in New Zealand. We used a multistage mixed methods design with three stages to generate knowledge and identify social–ecological trade-offs. Our case study illustrates an operational approach for local mangrove management and regional coastal sustainability. Working with communities affected by ecosystem change through participatory research proved key to knowledge exchange and colearning.

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.

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. Graphical abstract

This study characterised selected peripheral blood (PB) haematological parameters, liver, serum and muscle metabolic features in three- and five-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. However, higher erythrocytes in five-year old fish, elevated thrombocyte and lymphocyte counts in three-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 were higher in males, while females had elevated levels of polyunsaturated fatty acids. Essential and non-essential amino acids in liver and serum were also elevated in females compared to males. These findings suggest differential allocation of fatty acids and amino acids to reflect requirements for gonadal, development and provisioning. Similarly, age significantly resulted in higher liver and serum abundances of some non-essential amino acids in three-year olds compared to five-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. This article is protected by copyright. All rights reserved.

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...

Meiofaunal assemblages are often overlooked, yet they are important ecological components of estuarine ecosystems. To evaluate variations in meiofaunal communities in estuarine habitats, we conduct...

Poly (I:C) is a viral pathogen-associated molecular pattern (PAMP) and a synthetic dsRNA analogue, which orchestrates antiviral responses through well conserved toll-like receptor 3 (tlr3), retinoic acid inducible gene I (RIG-1) like receptors (RLRs) and class A scavenger receptors (SR-As). This study investigated the effect of poly (I:C) in vivo on Chinook salmon (Oncorhynchus tshawytscha) haematology, innate immunity, serum and liver metabolite profiles, and lymphoid tissue cytokine transcript expression, over a 5-day period post-exposure. Poly (I:C) significantly enhanced differential monocyte and neutrophil counts, and reactive oxygen species (ROS) production in peripheral blood mononuclear cells (PBMCs). GC-MS-based metabolomics revealed that poly (I:C) significantly altered 23 liver and 13 serum metabolic features. Liver and serum metabolites involved in branched chain amino acid (BCAA) metabolism significantly increased at day 1 and returned to baseline by day 5; the citric acid cycle and ROS regulation were altered. Also, liver and serum metabolites involved in glycolysis were persistently depleted from day 1 to day 5. Liver metabolites involved in phospholipid metabolism increased at day 4 to 5 and decreased from day 2 to 4 in serum. At the molecular level, poly (I:C) upregulated antiviral ifnγ and Mx1, and anti-inflammatory il-10 in fish lymphoid tissues, which normalised to baseline by day 5. Overall, poly (I:C) induced innate and adaptive immune responses through multiple mechanisms and different levels in teleost O. tshawytscha. Findings may aid design and development of amelioration strategies against viral pathogens via metabolome reprograming. Targeted studies are recommended into identified pathways and survival biomarkers.

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.

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.

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 (1amp; 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.

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.

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.

Evaluating studies over the past almost 40 years, this review outlines the current knowledge and research gaps in the use of isolated leucocytes in salmonid immunology understanding. This contribution focuses on the techniques used to isolate salmonid immune cells and popular immunological assays. The paper also analyses the use of leucocytes to demonstrate immunomodulation following dietary manipulation, exposure to physical and chemical stressors, effects of pathogens and parasites, vaccine design and application strategies assessment. We also present findings on development of fish immune cell lines and their potential uses in aquaculture immunology. The review recovered 114 studies, where discontinuous density gradient centrifugation (DDGC) with Percoll density gradient was the most popular leucocyte isolation method. Fish head kidney (HK) and peripheral blood (PB) were the main sources of leucocytes, from rainbow trout (Oncorhynchus mykiss) and Atlantic salmon (Salmo salar). Phagocytosis and respiratory burst were the most popular immunological assays. Studies used isolated leucocytes to demonstrate that dietary manipulations enhance fish immunity, while chemical and physical stressors suppress immunity. In addition, parasites, and microbial pathogens depress fish innate immunity and induce pro-inflammatory cytokine gene transcripts production, while vaccines enhance immunity. This review found 10 developed salmonid cell lines, mainly from S. salar and O. mykiss HK tissue, which require fish euthanisation to isolate. In the face of high costs involved with density gradient reagents, the application of hypotonic lysis in conjunction with mico-volume blood methods can potentially reduce research costs, time, and using nonlethal and ethically flexible approaches. Since the targeted literature review for this study retrieved no metabolomics study of leucocytes, indicates that this approach, together with traditional technics and novel flow cytometry could help open new opportunities for in vitro studies in aquaculture immunology and vaccinology.

The antimicrobial role of itaconic acid (ITA) has been recently discovered in mammalian cells. In our previous studies, we discovered that marine molluscs biosynthesise substantial quantities of ITA when exposed to marine pathogens, but its antimicrobial function to Vibrio bacteria is currently unknown. Thus, in this study, we used an untargeted gas chromatography–mass spectrometry (GC-MS) platform to identify metabolic changes of Vibrio sp. DO1 (V. corallyliticus/neptunius-like isolate) caused by ITA exposure. Vibrio sp. DO1 was cultured in Luria-Bertani broth supplemented with 3 mM sodium acetate and with different concentrations of ITA (0, 3 and 6 mM) for 24 h. The results showed that ITA completely inhibited Vibrio sp. growth at 6 mM and partially inhibited the bacterial growth at 3 mM. A principal component analysis (PCA) revealed a clear separation between metabolite profiles of Vibrio sp. DO1 in the 3 mM ITA treatment and the control, which were different in 25 metabolites. Among the altered metabolites, the accumulation of glyoxylic acid and other metabolites in glyoxylate cycle (cis-aconitic acid, isocitric acid and fumaric acid) together with the increase of isocitrate lyase (ICL) activity in the 3 mM ITA treatment compared to the control suggest that ITA inhibited Vibrio sp. growth via disruption of central carbon metabolism.

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 β-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.

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 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. 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. 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. 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.

The culture of endemic New Zealand black-footed abalone (Haliotis iris) represents a growing aquaculture industry, which is potentially threatened by pathogens and parasites. To identify and characterise health risks, a targeted sampling event was conducted of healthy- and unhealthy-looking abalone (shell deformities, tissue damage and brown creamy substance/fluid in tissues) at a land-based farm. Histological analysis showed signs of ill health, including disrupted and swollen gills with haemocytosis, ceroid material and parasites, and muscle tissues with focal haemocytosis with parasites. For the first time in H. iris, detailed histological observations, followed by confirmatory in situ hybridisation (ISH) resulted in the identification of Perkinsus olseni (5% prevalence). Scyphidia-like ciliates (56%), Sphenophrya-like ciliates (5%), unidentified disintegrated ciliates (26%), intracellular bacterial microcolonies (IMCs) (9%), apicomplexan-like cells (1%) and bacteria (2%) were also identified across organs. There was a significant association between the presence of P. olseni and IMCs. Immunological tissue responses (haemocytosis and ceroid material) and gill pathology were evaluated semi-quantitatively and were significantly associated with P. olseni. Gross abalone appearance was also significantly associated with P. olseni and unidentified disintegrated ciliates. These findings indicate the types of pathogens and parasites found in cultured H. iris for future health assessment studies of this important aquaculture species.

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 pathways underlying responses of shrimp (Penaeus vannamei) to hypoxia and to identify 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., TCA cycle intermediates, lactic acids, 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. Biomarker analysis also indicated lactic acid as biomarker for hypoxia and model prediction. Future investigations may validate this molecule as a stress biomarker in aquaculture. This study contributes to better understanding of hypoxia in shrimp and crustaceans at the metabolic level and provides a base for future metabolomics investigations on hypoxia.

Apoptosis or programmed cell death is a fundamental process essential for an organism's development and homeostasis in the immune system of both vertebrates and invertebrates. However, little is known about apoptotic processes in marine bivalves which require further investigation to elucidate mechanisms and identify molecular effectors of cell death pathways. In this study, we characterized cellular and molecular mechanisms of apoptosis induced by lipopolysaccharide (LPS) in New Zealand Greenshell™ mussel (Perna canaliculus) haemocytes. Mussel haemocyte samples were exposed to different LPS concentrations (0, 50 and 100 μg ml−1) and incubated at 19 °C for 3 h prior to assessment of various cell health parameters via flow cytometry assays and GC/MS-based metabolomic analyses. Flow cytometry results showed slightly higher, but non-significant differences in production of reactive oxygen species between LPS-exposed and control (no LPS) samples. However, percentages of apoptotic cells determined via depolarization of the mitochondrial membrane potential and caspase-3/7 activity in LPS-exposed samples were significantly higher than in control samples, providing mechanistic information regarding initiation and progression of the apoptotic cascade. The metabolite profile of LPS-exposed haemocytes showed elevated levels of 11 metabolites compared to that of the control. These metabolites may be involved in protein and lipid degradation as a consequence of apoptosis and other immune or physiological responses. This study demonstrates that LPS could trigger apoptosis in mussel haemocytes and provides insights into apoptotic processes in mussel haemocytes. Such knowledge could be useful for understanding the immune responses of farmed bivalves to waterborne pathogens and identification of molecular biomarkers for disease management in aquaculture.