Tim Young

Differential expression of novel metabolic and immunological biomarkers in oysters challenged with a virulent strain of OsHV-1

&NA; Early lifestages of the Pacific oyster (Crassostrea gigas) are highly susceptible to infection by OsHV‐1 &mgr;Var, but little information exists regarding metabolic or pathophysiological responses of larval hosts. Using a metabolomics approach, we identified a range of metabolic and immunological responses in oyster larvae exposed to OsHV‐1 &mgr;Var; some of which have not previously been reported in molluscs. Multivariate analyses of entire metabolite profiles were able to separate infected from non‐infected larvae. Correlation analysis revealed the presence of major perturbations in the underlying biochemical networks and secondary pathway analysis of functionally‐related metabolites identified a number of prospective pathways differentially regulated in virus‐exposed larvae. These results provide new insights into the pathogenic mechanisms of OsHV‐1 infection in oyster larvae, which may be applied to develop disease mitigation strategies and/or as new phenotypic information for selective breeding programmes aiming to enhance viral resistance. Graphical abstract Figure. No caption available. HighlightsHerpesvirus‐induced metabolic responses were investigated in oyster larvae by GC‐MS.Host metabolism changes are suggestive of Irg‐1‐like activation.Energy and lipid metabolism was substantially disturbed during infection.Activation of immunoresponsive gene 1 and the Warburg effect is hypothesised.Metabolomics is a powerful approach to study disease in early oyster life stage.

Identification of candidate biomarkers for quality assessment of hatchery-reared mussel larvae via GC/MS-based metabolomics

To ensure environmental and economic sustainability of future aquaculture growth, large-scale hatchery production of mollusc larvae is required. However, variation in larval quality currently limits potential maximum yields. Identification of biomarkers which reflect the immediate physiological condition of larvae during hatchery production could help monitor and determine causes of variation. Metabolomics is well-suited to this task due to its capacity for providing an instantaneous snapshot of the physiology of an organism through analysis of its metabolite profile. As a test, we applied GC/MS-based metabolomics for this purpose. Using a variety of univariate and multivariate feature selection methods, we identified four metabolite–metabolite ratios involving levels of succinate, glycine, alanine, pyroglutamate and myristic acid as candidate biomarkers for assessing mussel larval quality. These metabolites are known to have roles in energy metabolism, osmotic regulation, immune function and cell–cell communication. We anticipate that further investigation of these metabolites and their associated biochemical pathways will yield a more complete understanding of the factors responsible for larval production variability.

Metabolic profiling of mussel larvae: effect of handling and culture conditions

Gas chromatography mass spectroscopy was applied to characterize the metabolic profiles of hatchery-reared mussel (Perna canaliculus) larvae before and after a prolonged handling and water exchange process, and to investigate the effect of culture conditions. A decrease in succinate and an increase in alanine were observed after the water exchange, which indicated alterations in energy production and osmotic balance. However, these variations were subtle and it is unlikely that the water exchange practice had any lasting negative effects on larval physiology and performance. Multivariate pattern recognition tools (hierarchical clustering, principal component analysis and projection to latent squares discriminant analysis) were used to assess metabolite variations in larvae reared in low-density static and high-density flow through systems and to construct a culture condition classification model. Twelve metabolites contributed most towards the model, which indicated differences in energy, protein and lipid metabolism. The clear group separations were not represented by observable variations in morphological traits. This suggests that growth performance is metabolically buffered through an adaptive physiological mechanism to provide similar developmental characteristics under these conditions.

Putative involvement of adrenergic receptors in regulation of mussel (Perna canaliculus) larval settlement

Abstract Settlement responses were investigated for mussel (Perna canaliculus) larvae after exposure to catecholamines and their precursor metabolites. Settlement and mortality assays were conducted in Petri plates with chemical treatments (L-phenylalanine, L-tyrosine, L-DOPA, dopamine hydrochloride and epinephrine at various concentrations) and controls. The proteinogenic amino acids L-phenylalanine and L-tyrosine were both effective inducers (~65%) of larval settlement at 10−5 mol l−1 compared with controls (4%). Exposure of larvae to L-DOPA, dopamine and epinephrine resulted in maximum settlement induction (50, 60 and 51%, respectively) at 10−5 mol l−1. Larval mortalities were low (comparable to controls) across all concentrations of L-phenylalanine and L-tyrosine treatments, whereas high mortalities (>60%) were observed for L-DOPA, dopamine and epinephrine at concentrations ≥ 10−4 mol l−1. Our results indicate that P. canaliculus larval settlement is under endogenous regulation by a catecholaminergic mechanism. Furthermore, the inductive effects of all tested metabolites in the epinephrine biosynthesis pathway point to a putative involvement of adrenergic-type receptors in the regulation of larval settlement in this mussel species.

Metabolic and immunological responses of male and female new Zealand Greenshell™ mussels (Perna canaliculus) infected with Vibrio sp.

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 hosts 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.

Neurophysiological control of swimming behaviour, attachment and metamorphosis in black-footed abalone (Haliotis iris) larvae

Experiments were conducted to test the effect of a range of chemicals on larval responses in swimming behaviour, attachment and metamorphosis of the black-footed abalone (Haliotis iris). The effect of antibiotics on larval survival was first tested within negative (filtered seawater) and positive (GABA at 10−5, 10−4 and 10−3 mol L−1) control assays over 3 days. This experiment corroborated the effectiveness of using antibiotics to improve survival of larvae without obvious synergistic interactions with the GABA inducer or confounding effects of potential bacterial interactions. Chemical treatments (acetylcholine, potassium chloride, dopamine and glutamine) were then tested at various concentrations for their ability to modulate swimming behaviour and induce larval attachment and metamorphosis over 14 days. Generally, larval state shifted from swimming to attached, and from attached to metamorphosed, in the control and treatments over time. However, the peak percentage of attached and metamorphosed larvae varied in time among chemicals and concentrations. While overall percent metamorphosis was minimally enhanced after 14 days of exposure to some chemical treatments at certain concentrations, all treatments displayed significant capacities to down-regulate larval swimming and induce early attachment and metamorphosis. Mortality was recorded throughout the duration of the experiment, and was generally low (<20%) across controls and most treatments for exposures of less than 12 days. Interpretations of specific results from this study are used to elucidate neurophysiological control of larval activities for this abalone species. Comparisons with other marine invertebrates highlight the specificities of chemical cues and endogenous regulatory mechanisms across relatively closely related taxa.

Investigation of early mussel (Perna canaliculus) development using histology, SEM imaging, immunochemistry and confocal microscopy

ABSTRACT A comprehensive study, incorporating histology, light microscopy, scanning electron microscopy, immunochemistry and confocal microscopy, was performed to investigate embryogenesis and larval development of the New Zealand Greenshell™ mussel, Perna canaliculus. Detailed observations with this multi-technique approach revealed a gastrula stage at 18 hours post-fertilization, with the appearance of a blastopore, apical sense organ and enclosing vegetal pole. Early D-stage larvae showed limited alimentary organogenesis and clear initiation of a developing nervous system. Shell morphology of D-larvae was characterized by a flat, hinged, pitted–punctate prodissoconch I shell, followed closely by commarginal growth lines within the prodissoconch II shell. Early umbo larvae had a protruding functioning velum, and well-developed posterior adductor and velar retractor muscles. Significant progression in neuronal development occurred just before the umbo stage with noticeable paired cerebral, pedal and visceral ganglia. Shell morphology was characterized by further prodissoconch II secretion with a more rounded umbonate appearance. During the transition through the pediveliger stage, rapid development of the gill rudiment, eye spot and functioning foot was observed with ongoing neuronal development. The first appearance of the dissoconch shell layer took place during this transition, at which point the nervous system was highly distinct with innervations extending throughout muscle regions and between ganglia. This study provides the first comprehensive documentation of the developmental stages of P. canaliculus larvae from fertilization to settlement. The study highlights the advantages of using a combination of techniques to understand larval development and provides crucial information to identify larval performance during larval rearing.

Practical Fertilization Procedure and Embryonic Development of the New Zealand Geoduck Clam (Panopea Zelandica)

Cultivation of the geoduck Panopea zelandica (Quoy & Gaimard, 1835) requires knowledge on embryonic development to produce spat in hatcheries. This study investigated the development of P. zelandica embryos at 15°C and 35 ppt and the optimal sperm:egg ratios for fertilization under hatchery conditions. Panopea zelandica broodstock were induced to spawn by serotonin injection. Sperm and eggs were collected and fertilization was conducted at sperm:egg ratios of: 50:1, 100:1, 500:1, 1000:1 and 10,000:1 over 40 min. The optimal sperm:egg ratio was Panopea zelandica eggs (~80 μm diameter) developed the first and second polar bodies within 15–20 and 50–55 min post-fertilization, respectively. The blastula appeared at ~8 hpf, including the X R and X L cells and the presumptive shell field depression. Gastrulation occurred at 12–18 hpf with organic material apparent at the shell field depression. The mid-stage trochophore, which appeared at around 35 hpf had an apical plate with an apical tuft. The shell field spread to form the periostracum, which expanded and folded into right and left segments covering the late trochophore. The early D-stage veliger appeared at 45 hpf with the soft body being enclosed by two valves and the appearance of the velum. These observations will serve as the basis for future analyses of P. zelandica embryogenesis and for optimization of commercial production of D-veliger larvae.

Multi-strain probiotics enhance immune responsiveness and alters metabolic profiles in the New Zealand black-footed abalone (Haliotis iris)

Abstract We assessed whether dietary administration of a multi‐strain probiotic (Exiguobacterium JHEb1, Vibrio JH1 and Enterococcus JHLDc) lead to enhanced immune responsiveness in juvenile New Zealand black‐footed abalone (Haliotis iris). Two groups of abalone were fed (1% body weight per day) over a four‐month period with different diets. The control diet consisted of a standard commercial pellet feed (AbMax 16), whereas the treatment diet was additionally enriched with the probiotic mix. At the end of the experiment, probiotic‐fed animals showed improved growth compared with control‐fed abalone in length (32.3% vs 22.3%), width (31.9% vs 20.7%) and wet weight (109.6% vs 72.8%), respectively. Haemolymph sampling was conducted at the beginning of the experiment and after 2 and 4 months. Haemolymph samples were analysed for total haemocyte count (THC) and viability, presence of apoptotic cells and production of Reactive Oxygen Species (ROS). Compared with control abalone, probiotic‐fed abalone had significantly higher THC (1.9 × 106 vs 5.6 × 105 cells), higher viability (90.8% vs 75.6%), higher percentage of ROS‐positive cells (19.4% vs 0.5%) and higher numbers of non‐apoptotic cells (88.0% vs 78.0%), respectively. These results indicate that the probiotic‐enriched diet enhanced the immunostimulatory mechanisms, with a simultaneous low‐level up‐regulation of ROS production as a priming mechanism of the antibacterial defence system. Metabolomics‐based profiling of foot muscle tissue additionally revealed that probiotic‐fed abalone differentially expressed 17 unique metabolites, including amino acids, fatty acids and TCA cycle related compounds. These data suggest that the probiotic‐supplemented diet can also alter central carbon metabolic processes, which may improve the survival, as well as the growth of abalone. HighlightsA multi‐strain probiotic enhanced immune responsiveness in juvenile abalone.Probiotic‐fed abalone had higher haemocyte cell counts and viability.Probiotic‐fed abalone had higher numbers of ROS‐positive and non‐apoptotic cells.Metabolomics profiling resulted in differential expression of metabolites in foot.Probiotics diet can alter central carbon metabolism to improve growth and survival.

Effect of antiaggregants on the in vitro viability, cell count and stability of abalone (Haliotis iris) haemocytes

ABSTRACT The ability to successfully prepare and preserve haemocyte cells for microscopy and flow cytometry is critical for the investigation of animal immune systems. In this study, we observed the total cell count, in vitro viability and stability of New Zealand black‐footed abalone (Haliotis iris) haemocytes with different antiaggregants and handling protocols. Haemocyte stability was evaluated by direct observation of haemocytes under the microscope and calculating the aggregation index. Haemocyte counts and viability were measured via flow cytometry and tested for the effect of different antiaggregants (Alsevers solution at three concentrations, and specialised blood collection tubes containing lithium heparin and K2EDTA) at different temperatures and storage times. Results showed that Alsevers solution is an effective antiaggregant at haemolymph:antiaggregant dilution ratios of 1:1, 1:2 and 1:3. Lithium heparin was ineffective as an antiaggregant, whereas K2EDTA was similarly as effective as Alsevers solution. The influence of different mixing techniques (vortex, pipetting and flipping) were subsequently tested using the K2EDTA Microtainer® tubes, revealing that proper mixing should be performed immediately. High cell viability can be achieved by mixing samples by either 10s of vortexing (1000rpm), 10 times pipetting or 20 times flipping. The in vitro storage of abalone haemocytes in AS and K2EDTA as antiaggregants at ambient room temperature was highly effective for up to 24h (75–85% viability; 0.05–0.15 aggregation index) and is recommended for haemocyte studies in H. iris. Utilization of K2EDTA Microtainer® tubes were advantageous since they are more cost effective compared to Alsevers solution, and samples can be prepared more efficiently. HIGHLIGHTSAlsevers solution is an effective antiaggregant at haemolymph:AS 1:1, 1:2 & 1:3.High haemocyte viability was achieved by mixing 10s of vortexing (1000rpm).High haemocyte viability was achieved by mixing 10 times pipetting.High haemocyte viability was achieved by mixing 20 times flipping.K2EDTA Microtainer® tubes are cost effective to store abalone haemocytes.