Limiting immunopathology: Interaction between carotenoids and enzymatic antioxidant defences.

The release of reactive oxygen and nitrogen species (ROS and RNS) during the inflammatory response generates damages to host tissues, referred to as immunopathology, and is an important factor in ecological immunology. The integrated antioxidant system, comprising endogenous antioxidant enzymes (e.g. superoxide dismutase SOD, and catalase CAT) and dietary antioxidants (e.g. carotenoids), helps to cope with immune-mediated oxidative stress. Crustaceans store large amounts of dietary carotenoids for yet unclear reasons. While being immunostimulants and antioxidants, the interaction of these pigments with antioxidant enzymes remains unclear. Here, we tested the interaction between dietary supplementation with carotenoids and immune challenge on immune defences and the activity of the antioxidant enzymes SOD and CAT, in the amphipod crustacean Gammarus pulex. Dietary supplementation increased the concentrations of circulating carotenoids and haemocytes in the haemolymph, while the immune response induced the consumption of circulating carotenoids and a drop of haemocyte density. Interestingly, supplemented gammarids exhibited down-regulated SOD activity but high CAT activity compared to control ones. Our study reveals specific interactions of dietary carotenoids with endogenous antioxidant enzymes, and further underlines the potential importance of carotenoids in the evolution of immunity and/or of antioxidant mechanisms in crustaceans.

Physiological and agonistic behavioural response of Procambarus clarkii to an acoustic stimulus.

This study examined the effects of an acoustic stimulus on the haemolymph and agonistic behaviour of the red swamp crayfish, Procambarus clarkii. The experiment was conducted in a tank equipped with a video recording system using six groups (three control and three test groups) of five adult crayfish (30 specimens in total). After 1 h of habituation, the behaviour of the crayfish was monitored for 2 h. During the second hour, the animals in the test groups were exposed to a linear sweep (frequency range 0.1-25 kHz; peak amplitude 148 dB(rms) re. 1 µPa at 12 kHz) acoustic stimulus for 30 min. Exposure to the noise produced significant variations in haemato-immunological parameters as well as a reduction in agonistic behaviour.

Morphology and pathology of the ectoparasitic copepod, Nicothoë astaci ('lobster louse') in the European lobster, Homarus gammarus.

Ectoparasitic copepods have been reported in a wide range of aquatic animals, including crustacean shellfish. However, with the exception of the salmon louse, Lepeophtheirus salmonis, our knowledge of such parasites in commercial species is rudimentary. The current study examines the morphology and pathology of the parasitic copepod, Nicothoë astaci (the 'lobster louse') in its host, the European lobster, Homarus gammarus. Lobsters were sampled from waters surrounding Lundy Island (Bristol Channel, UK) and all individuals collected were found to harbour female adult N. astaci in their gills, with a mean of 47·3 parasites/lobster. The majority of N. astaci were found in the basal region of pleurobranch gills. The parasite was found to attach to gill filaments via its oral sucker, maxillae and maxillipeds, and to feed on host haemolymph (blood) through a funnel-like feeding channel. It caused varying degrees of damage to the host gill, including occlusion of gill filaments and disruption to the vascular system in the central axis. Although there was evidence of extensive host response (haemocytic infiltration) to the parasite, it was displaced from the parasite attachment site and thus was observed in the central gill axis below. The region of gill filament immediately underlying the parasite feeding channel was devoid of such activity suggesting that the parasite interferes with the cellular defence and haemostatic mechanisms of the lobster in order to maintain invasion of the host.

Monitoring follow up of two areas affected by the Prestige oil four years after the spillage.

The sinking of the oil tanker Prestige in November 2002 resulted in the spill of more than 63,000 tonnes of crude oil, and polluted more than 1,000 km of coastline, especially affecting Galicia (northwestern Spain). Four years after the accident, a new biological monitoring study was undertaken of two Galician areas intensely affected by the spill, Lira and Ancoradoiro, previously evaluated in the months following the accident ( Laffon et al. 2006 ). The mussel Mytilus galloprovincialis was employed as bioindicator organism to determine both polycyclic aromatic hydrocarbons (PAH) levels and genotoxic effects. PAH were determined chromatographically in seawater samples and mussel tissues collected from November 2006 to January 2008. The results obtained showed that PAH pollution was still present in these areas, but bioaccumulation of these compounds in mussels was low, compared to reference mussels, and lower than in our previous study. DNA damage assessment was also performed in gills and hemolymph cells by means of the alkaline comet assay. DNA damage levels were higher in mussels from the exposed areas than in reference mussels. DNA damage decreased after a 7-d recovery period in the laboratory, but prolonging the recovery period up to 14 d did not contribute to less DNA damage in gill cells. Hemolymph cells were more sensitive than gill cells to the induction of DNA damage.

Effects of emersion and re-immersion on physiological and immunological variables in creel-caught and trawled Norway lobster Nephrops norvegicus.

Immune defence in creel-caught and trawled Nephrops norvegicus was investigated to assess a possible relationship between phenoloxidase (PO) activation and the total haemocyte count (THC). Capture, capture method and emersion evoked physiological and immunological responses that may have implications for the ability of N. norvegicus to survive the effects of such stressors. Haemolymph THC was always negatively related to PO activity in the trawled samples, suggesting a decreased level of the plasma serine proteinase inhibitors which reportedly regulate the ProPO system (Le Moullac et al. 1998; Fish shellfish Immunol 8:621-629). In contrast, creel-caught samples showed increased levels of both PO and THC (cf. control N. norvegicus), after a 12 h emersion period. Trawling and emersion evoked progressive and significant increases (p < 0.05) in the mean levels of haemolymph L-lactate, glucose and total ammonia. The evidence of overt activity and measured haemolymph parameters suggest that creel fishing yields N. norvegicus that are more likely to survive post-harvest treatments than those that are trawled.

Studies on the immunomodulatory effect of extract of Cyanodon dactylon in shrimp, Penaeus monodon, and its efficacy to protect the shrimp from white spot syndrome virus (WSSV).

The present study investigates the protection of shrimp Penaeus monodon against white spot syndrome virus (WSSV) using antiviral plant extract derived from Cyanodon dactylon and the modulation of the shrimp non-specific immunity. To determine the antiviral activity, the shrimp were treated by both in vitro (intramuscular injection) and in vivo (orally with feed) methods at the concentration of 2mg per animal and 2% of the plant extract incorporated with commercially available artificial pellet feed, respectively. The antiviral activity of C. dactylon plant extract was confirmed by PCR, bioassay and Western blot analysis. In the present study, anti-WSSV activity of C. dactylon plant extract by in vivo and in vitro methods showed strong antiviral activity and the immunological parameters such as proPO, O(2)(-), NO, THC and clotting time were all significantly (P<0.05) higher in the WSSV-infected shrimp treated with plant extract when compared to control groups. These results strongly indicate that in vivo and in vitro administration of C. dactylon plant extract enhances immunity of the shrimp. Based on the present data and the advantages of plant extract available at low price, we believe that oral administration of C. dactylon plant extract along with the pellet feed is a potential prophylactic agent against WSSV infection of shrimp.

Effect of sweet flag rhizome oil (Acorus calamus) on hemogram and ultrastructure of hemocytes of the tobacco armyworm, Spodoptera litura (Lepidoptera: Noctuidae).

Effect of the essential oil of Acorus calamus L. rhizomes, was studied on hemocytes of the tobacco armyworm, Spodoptera litura. The oil was administered in oral application at concentration of 500 and 1000 ppm to last instar larvae of S. litura and its effect on ultrastructure of hemocytes and hemogram was evaluated. The oil was administered in topical application at 250 microg dose to pupae to ascertain its effect on total and differential hemocyte counts. At both scanning (SEM) and transmission electron microscopic (TEM) levels, the major effect of oil treatment was observed on plasmatocytes (PLs) and granular hemocytes (GRs). SEM study revealed that the cytoplasmic projections of granular hemocytes were reduced, while the filopods of plasmatocytes remained unaffected. The vacuolization in the cytoplasm and degeneration of the organelles in both plasmatocytes and granular hemocytes was observed by TEM. However, no such deformities were observed in prohemocytes (PRs), spherulocytes (SPs), and oenocytoids (OEs). A concentration-dependent decrease has been observed in the larval body weight and hemolymph volume (HV), 24-72 h after treatment. In comparison to the controls, the maximum percentage growth inhibition (GI) was recorded to be 58.28 and 66.48, respectively at 500 and 1000 ppm after 72 h treatment. Similarly after 72 h treatment, the percentage reduction in hemolymph volume was 61.38 and 69.05, respectively at 500 and 1000 ppm. Total hemocyte count (THC), in larvae computed from five recorded hemocyte types viz. PRs, PLs, GRs, SPs and OEs, decreased only after 48-72 h of treatment. The maximum decrease in THC was recorded to be 29.15 and 49.05% at 500 and 1000 ppm, respectively, after 72 h of treatment. There was continuous decline in THC in pupae after 24-72 h treatment. DHC study revealed that both the concentrations of oil in 6th instar larvae of S. litura caused a decrease in PRs, PLs and SPs and increase in GRs and OEs after 24-72 h of treatment. Since A. calamus oil treatment causes the injury to both PLs and GRs and also affects the hemogram, it can be inferred that cellular defence reactions of S. litura are impaired.

Effect of the plant Cupressus macro-carpa (Cupressacea) on some haematolo-gical and biochemical parameters of Biomphalaria alexandrina snails.

The dry powder of the plant aereal part; Cupressus macro-carpa (Cupressacea) was tested against Biomphalaria alexandrina. LC50 & LC90 values were 59.5 & 98.8 ppm, respec-tively. Exposure of B. alexandrina to sublethal concentrations (LC0, LC10 & LC25) of C. macrocarpa for three weeks signi-ficantly decreased the number of circulating hemocytes. The magnitude of reduction was increased with increasing of the tested concentration. The main type of cell in the hemolymph of B. alexandrina was the granulocyte (71.8%), followed by large round cells or hyalinocytes (19.0%) and small round cells or undifferentiate cells (9.2%). The percentage of different hemocyte categories was changed in treated snails. In snails maintained at LC25, showed significantly higher percentages of small round cells than controls, 56.2% & 9.2% respectively. Maintainence of B. alexandrina in sublethal concentrations for three weeks significantly reduced protein & hemoglobin content in the hemolymph. Reduction in enzyme activities occurred in the hemolymph and tissues of treated snails. The enzymes were pyruvate kinase (PK), lactat dehydrogenase (LDH), hexokinase (HK) and phosphoenol pyruvate carboxy kinase (PEPCK) which are very important in metabolism of the protein and carbohydrate. The infectivity of Schistosoma mansoni miracidia was greatly reduced by exposure to the sublethal concentrations (LC0, LC10 & LC25) of Cupressus. Infection rate of B. alexandrina reached to 54.5%, 37.5% & 16.7%, respectively compared to control (81.25%). Duration of cercarial shedding and the total periodic cercarial production/snail showed significant reduction while the parasite incubation period was significantly longer (p<0.05).

More than one type of transglutaminase in invertebrates? A second type of transglutaminase is involved in shrimp coagulation.

Coagulation (clot formation) forms a physical barrier to prevent the loss of body fluid and dissemination of microbes into the haemocoel after injury or infection. Its quickness and efficiency are essential for the survival of invertebrates that rely solely on innate immunity. Transglutaminase (TG) catalyses intermolecular or intramolecular epsilon-(gamma-glutamyl) lysine bond formation, resulting in a protein polymerisation, and plays a role in blood coagulation and post-translational protein remodelling. In the present study, we cloned a TG from shrimp (Penaeus monodon) haemocyte cDNA. It was assigned as shrimp transglutaminase II (STG II). The STG II cDNA consists of a coding region of 2,274bp. The deduced protein has 757 amino acid residues with a calculated molecular mass of 85,000 Da and an isoelectric point of 5.48. RT-PCR results showed a significant level of STG II expression in haemocytes but not in hepatopancreas, in contrast to shrimp STG I (AY074924.1). The genetic distance between STG II and STG I is much larger than the distance between STG II and the TG of the kuruma shrimp (Marsupenaeus japonicus). Evidence based on tissue distribution and genetic distance suggests that no less than two types of shrimp TG exist that are encoded at different chromosomal locations. The recombinant STG II (rSTG II) incorporated a TG-specific substrate, dansylcadaverine (DCA), into clottable proteins (CP) in a calcium dependent manner. Other haemocyte- or plasma-derived TG substrate is not required for CP polymerisation but may be necessary for stable clot formation. The rSTG II catalysed clottable proteins into a long chain under transmission electron microscopy (TEM) observation. In conclusion, STG II is characterized as a haemocyte TG and is involved in coagulation.

Classification of Homarus americanus hemocytes and the use of differential hemocyte counts in lobsters infected with Aerococcus viridans var. homari (Gaffkemia).

Hemocytes of the American lobster (Homarus americanus H. Milne Edwards) were classified after examination of Wright-Giemsa stained cytocentrifuge preparations by brightfield light microscopy. Eleven hemocyte types were identified using morphologic criteria. The classification system was then used to monitor changes in the differential hemocyte count (DHC) of lobsters infected with the Gram positive coccus Aerococcus viridans var. homari, etiologic agent of gaffkemia. The appearance of less mature hemocytes in the DHCs of lobsters in the late stages of infection was similar to the 'left shift' of vertebrate inflammation. Results from this study suggest that DHCs can be used to assess and characterize inflammation in H. americanus and possibly other crustaceans.

Long-term in vitro generation of amoebocytes from the Indian horseshoe crab Tachypleus gigas (Müller).

Amoebocyte is the single type of cell circulating in the horseshoe crab hemolymph, which plays a major role in the defense system of the animal. Granules present in these cells are sensitive to nanogram quantities of bacterial endotoxins, which form the basis of the Limulus amoebocyte lysate (LAL) test. Normally, amoebocytes for the production of the LAL are collected by cardiac puncture; hence, development of the in vitro culture system for amoebocytes will reduce the variability of the lysate and help to conserve the 400 million-yr-old living fossil. In the present investigation we have attempted organ culture of gill flaps that have been shown to be the source of amoebocytes. The gill flaps were cultured at 28 degrees C on a rocker platform in a modified L-15 medium supplemented with 10% v/v horseshoe crab serum. This led to the release of amoebocytes outside the gill flaps for a period of 6-8 wk with a more or less steady number of amoebocytes during the weekly harvest. No significant difference was seen in the yield of amoebocytes from male and female horseshoe crabs. Confocal laser microscopy studies revealed significant difference in the size of amoebocytes released in vitro as compared with those obtained in vivo. Thus, we have optimized the culture conditions for the long-term generation of amoebocytes in vitro from the Indian horseshoe crab Tachypleus gigas by reducing the incidence of contamination, simulating in vivo conditions for the organ culture of gill flaps, and improvising the nutritional status using the modified L-15 medium, providing the desired osmolarity and pH.

Cell-mediated haemolytic activity of haemolymph from the Colorado potato beetle (Leptinotarsa decemlineata Say.)

Haemolytic activity was identified in cell-free haemolymph from larval and imago stages of Leptinotarsa decemlineata. The haemolytically active fraction of the haemolymph was active against human, sheep, bull, toad and mouse erythrocytes. There was no haemolysis in the presence of 0.001 M EDTA and 0.5% glutathione. The titre of haemolytic activity did not increase after injury or vaccination of the larvae with Microccocus lysodeikticus. Haemolysin, a heat-labile protein was partially purified by ammonium sulphate precipitation, gel filtration, and ion-exchange separation. SDS PAGE, electrophoresis and immunoblotting showed that the active factor was a protein with a molecular weight of approximately 55 kD. It was not bactericidal for various micro-organisms but the antibacterial activity of the lysozyme increased in the presence of haemolysin only when M. lysodeikticus were used as target cells. Spherulocytes synthesized and released the haemolytic protein in vitro. The haemolytic activity increased in the presence of lipopolysaccharide from Escherichia coli and Ca++ ions. The physiological role of the haemolysin is as yet unknown.