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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. [Display omitted] • GC−MS metabolomics revealed shrimp responses to immune stimulatory products. • More metabolic responses to immune stimulators in haemolymph than in gills. • The combination of cellulose fibers and probiotics yielded better immune stimulation. • Cellulose fibers could be used as a prebiotic for shrimp. [ABSTRACT FROM AUTHOR]

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. Image 1 • Unknown summer mortality causes perturbations in mussel metabolism. • Metabolomics allows classification of mussels from different affected levels. • Accumulation of itaconic acid may indicate the involvement of pathogen infections. • Itaconic acid has potential to be a biomarker for pathogen infection. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • Immune responses of Perna canaliculus infected with Vibrio sp. varied by host sex. • Infection with Vibrio sp. led to changes of metabolite profiles of mussels. • Metabolic responses of Perna canaliculus infected with Vibrio sp. did not vary by host sex. • Itaconic acid is a potential biomarker for pathogen infection in marine bivalves. Abstract Massive mortalities due to pathogens are routinely reported in bivalve cultivation that have significant economic consequences for the global aquaculture industry. However, host-pathogen interactions and infection mechanisms that mediate these interactions are poorly understood. In addition, gender-specific immunological responses have been reported for some species, but the reasons for such differences have not been elucidated. In this study, we used a GC/MS-based metabolomics platform and flow cytometry approach to characterize metabolic and immunological responses in haemolymph of male and female mussels ( Perna canaliculus ) experimentally infected with Vibrio sp. Sex-based differences in immunological responses were identified, with male mussels displaying higher mortality, oxidative stress and apoptosis after pathogen exposure. However, central metabolic processes appeared to be similar between sexes at 24 h post injection with Vibrio sp. DO1. Significant alterations in relative levels of 37 metabolites were detected between infected and uninfected mussels. These metabolites are involved in major perturbations on the host’s innate immune system. In addition, there were alterations of seven metabolites in profiles of mussels sampled on the second day and mussels that survived six days after exposure. These metabolites include itaconic acid, isoleucine, phenylalanine, creatinine, malonic acid, glutaric acid and hydroxyproline. Among these, itaconic acid has the potential to be an important biomarker for Vibrio sp. DO1 infection. These findings provide new insights on the mechanistic relationship between a bivalve host and a pathogenic bacterium and highlight the need to consider host sex as a biological variable in future immunological studies. [ABSTRACT FROM AUTHOR]

Plastic biodegradation by insects has made significant progress, opening up new avenues for the treatment of plastic waste. Wax moth larvae, for example, have attracted the attention of the scientific community because they are known to chew, ingest, and biodegrade natural polymer bee waxes. Despite this, we know very little about how these insects perform on manufactured plastics or how manufactured plastics affect insect metabolism. As a result, we studied the metabolism of greater wax moths (Galleria mellonella) fed on molasses-supplemented polylactic acid plastic (PLA) blocks. An analysis of the central carbon metabolism (CCM) metabolites was performed using liquid chromatography triple quadrupole mass spectrometry (LC-QQQ-MS), while an analysis of untargeted metabolites and lipids was conducted using liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QToF-MS). In total, 169 targeted CCM metabolites, 222 untargeted polar metabolites, and 196 untargeted nonpolar lipids were identified within the insect samples. In contrast, compared to control larvae, PLA-fed larvae displayed significantly different levels of 97 CCM metabolites, 75 polar metabolites, and 57 lipids. Purine and pyrimidine metabolisms were affected by PLA feeding, as well as amino acid metabolism, carbohydrates, cofactors, vitamins, and related metabolisms. Additionally, PLA exposure disrupted insect energy metabolism and oxidative stress, among other metabolic disturbances. The larvae fed PLA have lower levels of several lipids, suggesting a reduction in lipid reserves, and ceramide levels are likely to have changed due to apoptosis and inflammation. The study indicates that G. mellonella larvae could ingest PLA but this process causes some metabolic stress for the host. Future studies of the molecular pathways of this biodegradation process might help to provide strategies for stress reduction that would speed up insect digestion of plastic. • PLA disrupted wax moth energy metabolism and oxidative stress. • PLA-fed wax moth larvae displayed altered purine and pyrimidine metabolisms. • PLA-fed wax moth larvae were lower in several lipid reserves. [ABSTRACT FROM AUTHOR]

[Display omitted] • Hypoxia led to the change of 44 metabolites and 17 pathways in shrimp haemolymph. • Hypoxic stress caused disturbances in energy metabolism, osmoregulation and oxidative damage. • Warburg effect-like response was observed in shrimp responded to hypoxia exposure. • Lactic acid could be a reliable biomarker for hypoxia and other stress conditions. 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. [ABSTRACT FROM AUTHOR]

[Display omitted] • Shrimp responded to WSSV in a tissue specific manner. • WSSV caused metabolic changes in gills, haemolymph and hepatopancreas of shrimp. • WSSV challenge led to changes in osmoregulation and energy pathways in shrimp. • Shrimp can produce itaconic acid as an antimicrobial compound. • WSSV induced Warburg effect in shrimp. 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. [ABSTRACT FROM AUTHOR]

Abstract New Zealand Chinook salmon (Oncorhynchus tshawytscha) industry has great potential for growth and expansion. While production is relatively free of health problems, there is limited literature on haematology, and immunological tools to safeguard against possible future health threats. The current study aim was to characterise New Zealand farmed O. tshawytscha peripheral blood cellular composition, develop a micro-volume method to isolate peripheral blood mononuclear cells (PBMCs) and validate a microcapillary flow cytometry assay kit for PBMC cell count and viability assessment. We used light microscopy to characterise peripheral blood and PBMC cellular composition in combination with a flow cytometer Sysmex XT 2000i Haematology Analyser. ImageJ version 1.52 was used for cell size characterisation of freshly stained blood. The stability of PBMCs stained with the Muse® Cell Count and Viability Assay Kit and the Trypan blue assay stains were studied at 4 °C and 21 °C for 60 min; while the Muse® Cell Count and Viability Assay Kit was validated against the Trypan blue assay haemocytometer chamber to assess PBMC count and viability. Findings showed that O. tshawytscha smolt yearlings had total blood cell counts in the range of 1.9–2.7 × 106 μL−1. Differential cell counts revealed five cell types, comprising 97.18% erythrocytes, 2.03% lymphocytes, 0.67% thrombocytes, 0.09% monocytes, and unquantifiable neutrophils. Using micro-volumes of blood and Lymphoprep™, we successfully isolated fish PBMCs. Significantly, stained PBMCs remained stable for up to 45 min at 4 °C and 21 °C; while validation of the Muse® protocol showed that this microfluidic instrument delivered more accurate and precise viability results than the haemocytometer. The Muse® protocol is rapid, easy to use, has quick calibration steps, and is suitable for field use to facilitate onsite sample processing. These findings pave the way for future assessments of fish health and in vitro immunological studies in O. tshawytscha. Graphical abstract Image 1 Highlights • Salmon smolts peripheral blood total cell count was estimated at 2.3 × 106 μL−1. • Differential cell counts revealed five cell types dominated by 97.18% erythrocytes. • Fish PBMCs successfully isolated from a micro blood volume (<300 μL). • Isolated PBMCs were majorly composed of lymphocytes and monocytes. • Muse® Cell Analyser delivered accurate and precise results as the haemocytometer. [ABSTRACT FROM AUTHOR]