Search

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 × 10 6  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., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

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]

Abstract 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. Graphical abstract Unlabelled Image Highlights • LPS exposure triggered apoptosis in mussel haemocytes. • LPS activated caspase-3/7 activity in LPS-exposed mussel haemocytes. • LPS exposure changed inner mitochondrial membrane potential of mussel haemocytes. • Eleven metabolites were elevated in metabolite profiles of LPS-exposed mussel haemocytes. [ABSTRACT FROM AUTHOR]

Abstract Temperature fluctuations during the live storage and transport of bivalves are known to be one of the most important stressors for these markets. However, the biological mechanisms that induce these stresses and the immunological responses to stressors are not currently understood. In this study, a gas chromatography–mass spectrometry (GC–MS based) metabolomics approach was used along with flow cytometry to characterize the responses of clams during aerial exposure in two different storage conditions (ice and no ice) and storage periods (1 h and 6 h). The results showed a significant increase in haemocyte mortality and ROS production in clams exposed to room temperature (no ice) and after 6 h compared to low temperature (with ice) and 1 h, respectively. The metabolite profiles of clam haemolymph in all groups revealed changes of many metabolites between the different temperatures and sampling times. Among them, lactic acid, succinic acid, malic acid, fumaric acid and glutamic acid were identified as significantly affected by both storage condition and period. These results indicate that clam storage stresses produced biological disturbances related to energy metabolism, which was switched from aerobic to anaerobic metabolism. These findings provide insights regarding the specific metabolic consequences of bivalve live storage and transport, which may lead to improved product handling along the supply chain. In addition, this study demonstrates the effective use of metabolomics and flow cytometric approaches on bivalve haemolymph as a non-destructive shelf life assessment method. Graphical abstract Unlabelled Image Highlights • Cold storage conditions reduced oxidative stress and haemocyte mortality in clams. • Air exposure at temperatures above 16 °C disturbed the TCA cycle and aerobic metabolism. • Clams can use fatty acids as energy sources during thermal/anoxic stress. [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]