Effects of chronic hypercapnia and elevated temperature on the immune response of the spiny lobster, Jasus lalandii.

The West Coast rock lobster (WCRL), Jasus lalandii, inhabits highly variable environments frequented by upwelling events, episodes of hypercapnia and large temperature variations. Coupled with the predicted threat of ocean acidification and temperature change for the coming centuries, the immune response in this crustacean will most likely be affected. We therefore tested the hypothesis that chronic exposure to hypercapnia and elevated seawater temperature will alter immune function of the WCRL. The chronic effects of four combinations of two stressors (seawater pCO2 and temperature) on the total number of circulating haemocytes (THC) as well as on the lobsters' ability to clear (inactivate) an injected dose of Vibrio anguillarum from haemolymph circulation were assessed. Juvenile lobsters were held in normocapnic (pH 8.01) or hypercapnic (pH 7.34) conditions at two temperatures (15.6 and 18.9 °C) for 48 weeks (n = 30 lobster per treatment), after which a subsample of lobsters (n = 8/treatment), all at a similar moult stage, were selected from each treatment for the immune challenge. Baseline levels of haemocytes (THC ml-1) and bacteria (CFU ml-1) in their haemolymph were quantified 24 h prior to bacterial challenge. Lobsters were then challenged by injecting 4 × 104V. anguillarum per g body weight directly into the cardiac region of each lobster and circulating haemocyte and culturable bacteria were measured at 20 min post challenge. No significant differences in THC ml-1 (p < 0.05) were observed between any of the treatment groups prior to the bacterial challenge. However lobsters chronically exposed to a combination of hypercapnia and low temperature had significantly higher (p < 0.05) THCs post-challenge in comparison with lobsters chronically exposed to hypercapnia and high temperature. A significant interactive effect was recorded between temperature and pH for the post-challenge THC data (two-way ANOVA, p = 0.0025). Lobster were very efficient at rendering an injected dose of bacteria non-culturable, with more than 83% of the theoretical challenge dose (∼1.7 × 105Vibrio ml-1 haemolymph) inactivated within the first 10 min following injection. Although differences in the inactivation of V. anguillarum were observed between treatment groups, none of these differences were significant. Clearance efficiency was in the following order: Hypercapnia/low temperature > normocapnia/high temperature > normocapnia/low temperature > hypercapnia/high temperature. This study demonstrated that despite chronic exposure to combinations of reduced seawater pH and high temperature, the WCRL was still capable of rapidly rendering an injected dose of bacteria non-culturable.

The impact of holding stressors on the immune function and haemolymph biochemistry of Southern Rock Lobsters (Jasus edwardsii).

Lobsters are fished world-wide due to their status as a high value, luxury seafood. A large proportion of the product is sold via live export, with lobsters subject to a range of stressors during holding post-capture. Improving the current understanding of the immune response to these stressors assists in improving efficiency and reducing loss in the chain between capture and consumption. In this study, the immune status of four treatment groups of Southern Rock Lobster (Jasus edwardsii) were studied: controls recently landed from a fishing boat, lobsters displaying advanced shell necrosis, lobsters in an unexplained moribund state and lobsters held in a processing facility for 10 weeks in standard conditions (i.e. high density, fasted). A total of 15 immune parameters and 19 haemolymph biochemical parameters were assayed. Phenoloxidase activity was only sporadically observed in haemocyte lysate and was consistently observed at a low level in the plasma with no difference between treatments for either. Haemocyte lysate prophenoloxidase activity was detected in most individuals, with no differences found between treatments. Prophenoloxidase in the plasma showed the highest level of activity, with the shell necrosis treatment demonstrating an elevated activity level relative to the other three treatments. Cell viability was not affected in any treatment. Lobsters with shell necrosis had a reduced capacity for phagocytosis, a significantly higher total haemocyte count, fewer hyalinocytes and more granulocytes and semigranulocytes. Fasted lobsters showed an opposite shift, with significantly more hyalinocytes compared to the other treatments and very few granulocytes and semigranulocytes. The balance of a range electrolytes, minerals metabolites and enzymes were affected in shell necrosis and fasted treatments, raising them as potential markers for immunocompromised lobsters. Multivariate analysis of all assayed parameters showed that all individuals in the necrosis treatment showed a similar, distinct immune response and that the fasted treatment, along with one control and one moribund individual, showed a separate intermediate response. The remainder of the control and moribund lobsters demonstrated a distinct "non-response" in comparison. These results offer a characterisation of the physiological response to common challenges during post-capture holding of rock lobsters, demonstrating the differential response to pathogenic bacterial infection, long term fasting, non-specific moribundity and the stress of capture and transport.

Panusin represents a new family of ß-defensin-like peptides in invertebrates.

Beta_defensin have been solely found in vertebrates until ß-defensin-like peptides were described as transcript isoforms in two species of Panulirus genus. They were considered as putative antimicrobials since their biological activity have not been demonstrated. Here we purified and characterized a defensin-like peptide from the hemocytes of spiny lobster P. argus, hereafter named panusin. Structurally, panusin presents a cysteine-stabilized α/ß motif, and is prone to form homodimers. Biological activity of panusin showed broad-spectrum antimicrobial activity, characterized for being strikingly salt-resistant. Panusin did not showed hemolytic activity but was demonstrated its binding capacity to different lipid membrane models, indicating amphipathicity of ß-sheet core as driving force for its antimicrobial activity. Panusin is considered a new kind of arthropod defensin which share structural and biological features with beta-defensin from vertebrates. The presence of beta-defensin like peptides in crustacean might suggest the emergence of the evolutionary relationship of ß-defensins from vertebrates.

Behavioral Immunity Suppresses an Epizootic in Caribbean Spiny Lobsters.

Sociality has evolved in a wide range of animal taxa but infectious diseases spread rapidly in populations of aggregated individuals, potentially negating the advantages of their social interactions. To disengage from the coevolutionary struggle with pathogens, some hosts have evolved various forms of "behavioral immunity"; yet, the effectiveness of such behaviors in controlling epizootics in the wild is untested. Here we show how one form of behavioral immunity (i.e., the aversion of diseased conspecifics) practiced by Caribbean spiny lobsters (Panulirus argus) when subject to the socially transmitted PaV1 virus, appears to have prevented an epizootic over a large seascape. We capitalized on a "natural experiment" in which a die-off of sponges in the Florida Keys (USA) resulted in a loss of shelters for juvenile lobsters over a ~2500km2 region. Lobsters were thus concentrated in the few remaining shelters, presumably increasing their exposure to the contagious virus. Despite this spatial reorganization of the population, viral prevalence in lobsters remained unchanged after the sponge die-off and for years thereafter. A field experiment in which we introduced either a healthy or PaV1-infected lobster into lobster aggregations in natural dens confirmed that spiny lobsters practice behavioral immunity. Healthy lobsters vacated dens occupied by PaV1-infected lobsters despite the scarcity of alternative shelters and the higher risk of predation they faced when searching for a new den. Simulations from a spatially-explicit, individual-based model confirmed our empirical results, demonstrating the efficacy of behavioral immunity in preventing epizootics in this system.

Physiological and immunological characterization of Caribbean spiny lobsters Panulirus argus naturally infected with Panulirus argus Virus 1 (PaV1).

The present study compares 13 physiological and immunological variables between a group of healthy Panulirus argus lobsters and a group of lobsters naturally infected with Panulirus argus Virus 1 (PaV1). Viral infection was determined through histopathology and PCR. Ten of the 13 variables differed significantly between the 2 groups. Using these variables, a principal component analysis yielded 2 separate clusters: one corresponding to the healthy group and the other corresponding to the infected group. In particular, infected lobsters exhibited significantly lower levels of osmotic pressure, total hemocyte counts, plasmatic proteins, and total phenoloxidase (PO) activity in plasma, as well as significantly higher levels of cholesterol and acylglycerides. These features are consistent with metabolic wasting, hyperlipidemia, and presumed immune suppression. Infection with PaV1 appears to increase the susceptibility of lobsters to some other opportunistic pathogens, as 61.1% of infected lobsters presented infestations of ciliate epibionts (Epystilis and Zoothamniun) in the gill chamber compared with 11.5% lobsters in the healthy group. Infected lobsters also showed significantly higher levels of total PO activity in degranulated hemocytes and trypsin inhibitor activity, potentially indicating activation of immune response by the PO system during the systemic infection with PaV1.

Identification of an antagonistic probiotic combination protecting ornate spiny lobster (Panulirus ornatus) larvae against Vibrio owensii infection.

Vibrio owensii DY05 is a serious pathogen causing epizootics in the larviculture of ornate spiny lobster Panulirus ornatus. In the present study a multi-tiered probiotic screening strategy was used to identify a probiotic combination capable of protecting P. ornatus larvae (phyllosomas) from experimental V. owensii DY05 infection. From a pool of more than 500 marine bacterial isolates, 91 showed definitive in vitro antagonistic activity towards the pathogen. Antagonistic candidates were shortlisted based on phylogeny, strength of antagonistic activity, and isolate origin. Miniaturized assays used a green fluorescent protein labelled transconjugant of V. owensii DY05 to assess pathogen growth and biofilm formation in the presence of shortlisted candidates. This approach enabled rapid processing and selection of candidates to be tested in a phyllosoma infection model. When used in combination, strains Vibrio sp. PP05 and Pseudoalteromonas sp. PP107 significantly and reproducibly protected P. ornatus phyllosomas during vectored challenge with V. owensii DY05, with survival not differing significantly from unchallenged controls. The present study has shown the value of multispecies probiotic treatment and demonstrated that natural microbial communities associated with wild phyllosomas and zooplankton prey support antagonistic bacteria capable of in vivo suppression of a pathogen causing epizootics in phyllosoma culture systems.

New aspects concerning to the characterization and the relationship with the immune response in vivo of the spiny lobster Panulirus argus nitric oxide synthase.

Nitric oxide (NO) is a short-lived radical generated by nitric oxide synthases (NOS). NO is involved in a variety of functions in invertebrates, including host defense. In a previous study, we isolated and sequenced for the first time the NOS gene from hemocytes of Panulirus argus, demonstrating the inducibility of this enzyme by lipopolysaccharide (LPS) in vitro. In the present work, lobster hemocytes and gills exposed to Escherichia coli O55:B5 LPS showed an increase in both NOS activity and NOS gene expression in vivo. This response was dose and time dependent. The 3D NOS structure was predicted by comparative modeling showing the oxygenase and reductase domains. These domains contain the conserved binding motifs of NOS already found in a variety of organisms. The 3D structure prediction analysis allowed the selection of a fragment of 666bp that was cloned and subsequently expressed in E. coli BL21, in which a recombinant product of around 31KDa was obtained. Hyperimmune serum obtained from immunized rabbits was tested and employed to specifically detect the recombinant polypeptide or the endogenous NOS from lobster hemocytes by western blot and immunofluorescence. This study contributes to enlarge the existing knowledge related to NOS structure and NOS participation in the immune response in lobsters. The evaluation of an antibody capable to recognize NOS from lobsters constitutes a novel and interesting tool for the implementation of further studies on NOS functions in crustaceans.

An inducible nitric oxide synthase (NOS) is expressed in hemocytes of the spiny lobster Panulirus argus: cloning, characterization and expression analysis.

Nitric oxide (NO) is a free radical gas involved in a variety of physiological processes in invertebrates, such as neuromodulation, muscle contraction and host defense. Surprisingly, little is known about the involvement of NO synthase (NOS) in the immune system of crustaceans. This work is focused on the study of the NOS gene of the spiny lobster Panulirus argus, a crustacean with commercial interest, and its relationship with the immune response to a microbial elicitor. A NOS full-length DNA was isolated from hemocytes by reverse transcription-polymerase chain reaction (RT-PCR) using degenerated primers. The open reading frame (ORF) encodes a protein of 1200 amino acids, with an estimated molecular mass of 135.9 kDa, which contains the conserved domains and binding motifs of NOS found in a variety of organisms. NOS gene expression in lobster gills, heart, stomach, digestive gland, abdominal muscle, gut and hemocytes was studied by Real Time quantitative PCR (Real Time qPCR). The expression was higher in hemocytes, heart and gills. In addition, when lobster hemocytes were exposed in vitro to Escherichia coli O55:B5 lipopolysaccharide (LPS), an increase in the NOS activity and also in the NOS gene expression evaluated by Real Time qPCR was observed, thus demonstrating the presence of an inducible crustacean NOS by a microbial elicitor of the immune response. The information is relevant in providing basic knowledge for further studies of crustacean defense mechanisms.

Effects of mannan oligosaccharide dietary supplementation on performances of the tropical spiny lobsters juvenile (Panulirus ornatus, Fabricius 1798).

The effects of dietary mannan oligosaccharide (MOS) (Bio-Mos, Alltech, USA) on the growth, survival, physiology, bacteria and morphology of the gut and immune response to bacterial infection of tropical rock lobsters (Panulirus ornatus) juvenile were investigated. Dietary inclusion level of MOS at 0.4% was tested against the control diet (trash fish) without MOS inclusion. At the end of 56 days of rearing period, a challenged test was also conducted to evaluate the bacterial infection resistant ability of the lobsters fed the two diets. Lobster juvenile fed MOS diet attained 2.86 +/- 0.07 g of total weigh and 66.67 +/- 4.76% survival rate which were higher (P < 0.05) than the lobsters fed control diet (2.35 +/- 0.14 g total weight and 54.76 +/- 2.38% survival rate, respectively) thus providing the higher (P < 0.05) specific growth rate (SGR) and average weekly gain (AWG) of lobsters fed MOS diet. Physiological condition indicators such as wet tail muscle index (Tw/B), wet hepatosomatic index (Hiw) and dry tail muscle index (Td/B) of the lobsters fed MOS supplemented diet were higher (P < 0.05) than that of the lobsters fed the control diet. Bacteria in the gut (both total aerobic and Vibrio spp.) and gut's absorption surface indicated by the internal perimeter/external perimeter ratio were also higher (P < 0.05) when the lobsters were fed MOS diet. Lobsters fed MOS diet were in better immune condition showed by higher THC and GC, and lower bacteraemia. Survival, THC, GC were not different among the lobsters fed either MOS or control diet after 3 days of bacterial infection while bacteraemia was lower in the lobsters fed MOS diet. After 7 days of bacterial infection the lobsters fed MOS diet showed higher survival, THC, GC and lower bacteraemia than the lobsters fed the control diet. The experimental trial demonstrated the ability of MOS to improve the growth performance, survival, physiological condition, gut health and immune responses of tropical spiny lobsters juveniles.

Characterization of two isoforms of Japanese spiny lobster Panulirus japonicus defensin cDNA.

Antimicrobial peptides (AMPs) are components of the innate immune responses that form the first line of host defense against pathogens. In this study, cDNAs of two new isoforms of defensin (designated PJD1 and PJD2) from a Japanese spiny lobster Panulirus japonicus haemocytes cDNA library were cloned and sequenced. PJD1 and PJD2 consist of 656 and 673 nucleotides encoding putative proteins of 66 and 64 amino acids, respectively. The isoforms share 74.2% amino acid identity. In a phylogenetic analysis, the peptides clustered with vertebrate defensins and were closely mostly related to chicken beta-defensin. PJD1 and PJD2 were detected in all tissues examined including heart, nerves, intestine, haemocytes, gills and hepatopancreas.

Effect of environmental parameters on immune response of the Indian spiny lobster, Panulirus homarus (Linnaeus, 1758).

The high export value of the Indian spiny lobster Panulirus homarus increasingly attracts the aquaculturists for farming and fattening. However, lack of knowledge on the effect of environmental parameters on the immune system of this animal could result in high mortality, which ultimately may cause major loss to the industry. Here, we report the effect of salinity (20, 25, 35, 40, and 45 per thousand), pH (5.0, 8.0, and 9.5), dissolved oxygen (DO) (1 and 5 mg L(-1)), and ammonia-N concentration (0, 0.5, 1.5 and 3 mg L(-1)) on the immune response of P. homarus measured in the haemolymph in terms of Total Haemocyte Count (THC), phenoloxidase (PO) activity, and NBT-reduction. Our data showed significant reduction (P<0.05) in THC, and NBT-reduction at lower (20 per thousand) and higher (45 per thousand) salinities. However, PO activity showed significant disparity, showing an increasing trend from 20 to 45 per thousand. Significant reduction (P<0.05) in THC and PO activity under acidic and alkaline conditions, under hypoxic condition (1 mg L(-1)), and at the higher ammonia-N concentrations than their respective optimal conditions were observed. Thus, suggesting that extreme environmental parameters can induce modifications in the immune system of the spiny lobster P. homarus, which may enhance their susceptibility to opportunistic pathogens. The humoral parameters such as THC, PO activity, and NBT-reduction can be used as potential stress indicators for healthy management of spiny lobsters.

Allergenicity and allergens of amphipods found in nori (dried laver).

Gammaridean and caprellid amphipods, crustaceans of the order Amphipoda, inhabit laver culture platforms and, hence, are occasionally found in nori (dried laver) sheets. Amphipods mixed in nori may cause allergic reactions in sensitized patients, as is the case with other crustaceans, such as shrimp and crab, members of the order Decapoda. In this study, dried samples of amphipods (unidentified) found in nori and fresh samples of gammaridean amphipod (Gammarus sp., not accurately identified) and caprellid amphipod (Caprella equilibra) were examined for allergenicity and allergens using two species of decapods (black tiger prawn and spiny lobster) as references. When analyzed by ELISA, sera from crustacean-allergic patients reacted to extracts from amphipod samples, although less potently than to the extracts from decapods. In IgE-immunoblotting, a 37-kDa protein was found to be the major allergen in amphipods. Based on the molecular mass and the cross-reactivity with decapod tropomyosin evidenced by inhibition ELISA and inhibition immunoblotting, the 37-kDa protein was identified as amphipod tropomyosin.