Immune response in probiotic-fed New Zealand black-footed abalone (Haliotis iris) under Vibrio splendidus challenge.

Vibriosis disease is a major constraint for sustainable molluscan aquaculture. Development of strategies to enhance disease resistance during grow out would greatly reduce stock mortality and boost production yields. In this study, New Zealand black-footed abalone (Haliotis iris) were fed a commercial diet enhanced with multi-strain probiotics (Exiguobacterium JHEb1, Vibrio JH1 and Enterococcus JHLDc) for four months, then challenged with an injection of pathogenic Vibrio splendidus. Host immune responses in haemocytes were characterized using flow cytometry by measuring total haemocyte counts (THC) and viability, degree of apoptosis, and production of reactive oxygen species (ROS) 48 h post-challenge. Probiotic-fed abalone had significantly higher survival rates compared to control animals after the bacterial challenge. Infected probiotic-fed abalone also had significantly higher haemocyte viabilities, slightly lower proportions of haemocytes undergoing early apoptosis, and lower proportions of ROS-producing haemocytes compared to infected control-fed abalone. In addition, metabolite profiles of muscle tissues generated via gas chromatography-mass spectrometry (GC-MS) delivered complimentary evidence to support a perturbed ROS-regulatory system in infected abalone through changes in key metabolites associated with glutathione biosynthesis. The results of this study provide valuable information to assist in farm management practices, leading to enhance production and sustainability of the New Zealand abalone aquaculture industry.

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

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.

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 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.

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

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 (Alsever's solution at three concentrations, and specialised blood collection tubes containing lithium heparin and K2EDTA) at different temperatures and storage times. Results showed that Alsever's 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 Alsever's 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 10 s of vortexing (1000 rpm), 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 24 h (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 Alsever's solution, and samples can be prepared more efficiently.

Immune status of the spiny lobster Jasus edwardsii with tail fan necrosis.

Tail fan necrosis (TFN), a disorder commonly found in some populations of commercially fished and cultured lobsters, is thought to be initiated by injuries caused by handling and containment. The unsightly appearance of affected lobster tails significantly lowers their commercial value. Knowledge about TFN is limited. In this study we describe the morphological features of TFN and apply 6 common methods for evaluating the immune status of wild-caught Australasian red spiny lobsters Jasus edwardsii with and without TFN. The disease was more frequent in uropods than in telsons of the tail fan, and more extensive on the ventral versus the dorsal surfaces of the tail fan. Missing appendages (i.e. antenna, pereiopod or pleopod) were significantly more common and greater in number for individual lobsters affected with TFN versus those without, possibly as a result of handling in the fishery or as an indirect effect of the disease. Two immune parameters, total haemocyte count and phenoloxidase activity in the haemocyte lysate supernatant (HLS), were significantly compromised in lobsters with TFN. No differences were found in the other immune parameters, i.e. haemocyte viability, haemolymph bacterial count and the protein content of haemolymph plasma and HLS. The results are consistent with injury sustained during prior capture and handling that initiates TFN in these natural caught lobsters. These results raise some potential concerns about the fitness of lobsters in natural populations that are affected by TFN, and some potential solutions are proposed.

Innovative application of classic and newer techniques for the characterization of haemocytes in the New Zealand black-footed abalone (Haliotis iris).

Haemocytes play an important role in innate immune responses within invertebrate organisms. However, identification and quantification of different types of haemocytes can be extremely challenging, and has led to numerous inconsistencies and misinterpretations within the literature. As a step to rectify this issue, we present a comprehensive and detailed approach to characterize haemocytes using a combination of classical (cytochemical and phagocytosis assays with optical microscopy) and novel (flow cytometry with Sysmex XN-1000 and Muse(®) Cell analyser) techniques. The Sysmex XN-1000 is an innovative fluorescent flow cytometric analyser that can effectively detect, identify and count haemocytes, while the Muse(®) Cell analyser provides accurate and rapid haemocyte cell counts and viability. To illustrate this approach, we present the first report on morphological and functional features of New Zealand black-footed abalone (Haliotis iris) haemocyte cells. Two types of haemocytes were identified in this study, including type I (monocyte-like) and type II (lymphocyte-like) cells. Granular cells, which have been reported in other molluscan species, were not detected in H. iris. Cell types were categorized based on shape, size, internal structures and function. The lymphocyte-like haemocytes were the most abundant hemocytes in the haemolymph samples, and they had large nuclei and basic cytoplasms. Monocyte-like cells generally were larger cells compared to lymphocyte-like cells, and had low nucleus-cytoplasm ratios. Monocyte-like cells showed higher phagocytic activity when encountering Zymosan A particles compared to lymphocyte-like cells. The present study provides a comprehensive and accurate new approach to identify and quantify haemocyte cells for future comparative studies on the immune system of abalone and other molluscan species.