Differential in vivo response of soft-shell clam hemocytes against two strains of Vibrio splendidus: Changes in cell structure, numbers and adherence

Host-pathogen interaction models in aquatic species are useful tools for understanding the pathogenicity of diseases in cultured and wild populations. In this study we report the differential in vivo response of soft-shell clam (Mya arenaria) hemocytes against two strains of Vibrio splendidus. Responses were measured 24 h after injecting into the posterior adductor muscle either an endemic wild-type strain (7SHRW) or a strain associated with oyster mortalities (LGP32-GFP). Changes in hemocyte structure (percentage of rounded cells) were assessed microscopically. Changes in adherence and hemocyte numbers were analyzed by flow-cytometric cell counting. Increased percentages of rounded cells were found in response to both strains. However, values from the group infected with LGP32-GFP were significantly higher (p < 0.01) than with 7SHRW. The cell adherence was markedly diminished (p < 0.001) by LGP32-GFP whereas 7SHRW did not change it significantly. Increased numbers of hemocytes (p < 0.001) were induced by LGP32-GFP, while no significant changes were found after infection with 7SHRW. These results show the regulatory capacity of soft-shell clams hemocytes to perform specific responses against different strains of V. splendidus.     

Functional differential immune responses of Mytilus galloprovincialis to bacterial challenge

Bivalves are filter-feeders that can accumulate large numbers of bacteria, in particular Vibrio species; these can persist within bivalve tissues largely depending on their sensitivity to the hemolymph bactericidal activity. In this work, functional parameters of the hemolymph of Mytilus galloprovincialis were evaluated in response to in vivo challenge with different bacteria (Gram(−) Vibrio anguillarum and V. splendidus, Gram(+) Micrococcus lysodeikticus). Mussels were injected with heat-killed bacteria or PBS-NaCl (controls) and hemolymph sampled from 3 to 48 h post-injection (p.i.). In hemocytes, all bacteria induced significant lysosomal membrane destabilisation (LMS) from 3 h p.i. with V. splendidus > V. anguillarum > M. lysodeikticus. LMS showed recovery for both M. lysodeikticus and V. anguillarum, whereas a further time-dependent decrease was observed for V. splendidus. Bacterial challenge also induced a rapid (from 3 h p.i.) and significant increase in serum lysozyme activity; the effect was persistent with M. lysodeikticus and transient for the two Vibrio species. In order to evaluate whether in vivo challenge may affect the subsequent capacity of hemolymph to kill bacteria, the bactericidal activity was tested in an in vitro assay towards E. coli. At 48 h. p.i. hemolymph samples from V. anguillarum-injected mussels showed a significant increase in E. coli killing (+ 35% with respect to controls); a smaller effect was observed with V. splendidus-injected mussels (+ 16%), whereas M. lysodeikticus was ineffective. Moreover, hemolymph from V. anguillarum-injected mussels showed an in vitro bactericidal activity towards V. anguillarum 2-folds higher than that of controls. Changes in total hemocyte counts (THC) and in hemocyte populations were evaluated by Flow cytometry at 6 and 48 h p.i., indicating a decrease in THC followed by recovery with all bacteria. Moreover, at 6 h p.i. a general decrease in the percentage of granulocytes was observed (V. splendidus > V. anguillarum > M. lysodeikticus), followed by complete and partial recovery with M. lysodeikticus and V. anguillarum, respectively, but not with V. splendidus. The results demonstrate the existence of differential functional immune responses in M. galloprovincialis to different bacteria.     

Lysozyme gene expression and hemocyte behaviour in the Mediterranean mussel, Mytilus galloprovincialis, after injection of various bacteria or temperature stresses

The aim of the present study was to evaluate the expression of the Mytilus galloprovincialis lysozyme gene in different in vivo stress situations, including injection of bacteria Vibrio splendidus LGP32, Vibrio anguillarum or Micrococcus lysodeikticus, as well as heat shock at 30 degrees C and cold stress at 5 degrees C. Injection of V. splendidus LGP32 resulted in: (i) a general down-regulation of lysozyme gene expression, as quantified by Q-PCR; (ii) reduction in the number of circulating hemocytes; (iii) decrease in the percentage of circulating hemocytes expressing lysozyme mRNA which was now restricted to only small cells, as observed by ISH; and (iv) accumulation of hemocytes expressing lysozyme in the muscle sinus where injection took place. Injection of V. anguillarum or M. lysodeikticus induced significant up-regulation of lysozyme gene expression, but only 2-3days post-injection, with no change in the total hemocyte counts but an increased percentage of hemocytes expressing lysozyme mRNA. Neither the control injection of PBS-NaCl nor temperature stress modified the lysozyme expression pattern. Consequently, the hemocyte population appears to be capable of discriminating between stress factors, and even between 2 Vibrio species.     

Construction of a Vibrio splendidus Mutant Lacking the Metalloprotease Gene vsm by Use of a Novel Counterselectable Suicide Vector

Vibrio splendidus is a dominant culturable Vibrio in seawater, and strains related to this species are also associated with mortality in a variety of marine animals. The determinants encoding the pathogenic properties of these strains are still poorly understood; however, the recent sequencing of the genome of V. splendidus LGP32, an oyster pathogen, provides an opportunity to decipher the basis of the virulence properties by disruption of candidate genes. We developed a novel suicide vector based on the pir-dependent R6K replicative origin, which potentially can be transferred by RP4-based conjugation to any Vibrio strain and which also carries the plasmid F toxin ccdB gene under control of the P_BAD promoter. We demonstrated that this genetic system allows efficient counterselection of integrated plasmids in the presence of arabinose in both V. splendidus and Vibrio cholerae and thus permits efficient markerless allelic replacement in these species. We used this technique to construct several mutants of V. splendidus LGP32, including a derivative with a secreted metalloprotease gene, vsm, deleted. We found that this gene is essential for LGP32 extracellular product toxicity when the extracellular products are injected into oysters but is not necessary for virulence of bacteria in the oyster infection model when bacteria are injected.     

Changes in biochemical and hemocyte parameters of the Pacific oysters Crassostrea gigas fed T-Iso supplemented with lipid emulsions rich in eicosapentaenoic acid

The aim of this study was to assess the effect of dietary eicosapentaenoic acid (20:5n-3) on hemocyte parameters such as hemocyte concentration, phagocytosis, and non-stimulated reactive oxygen species (ROS) production in Pacific oysters Crassostrea gigas, as well on proximate biochemical and fatty acid compositions. One-year-old oysters (C. gigas) were fed T-Isochrysis aff. galbana (T-Iso), which is low in 20:5n-3, either alone or with supplements of a lipid emulsion rich in 20:5n-3 at 1%, 10% or 50% (dry weight of the algal ration) for up to 7 weeks. Changes in gill fatty acid composition demonstrated that the lipid emulsion was well ingested by oysters during the dietary conditioning. Biochemical analysis indicated that oysters fed supplements of 50% and, to a lesser extent, 10% lipid emulsions had a higher total lipid content compared with oysters fed other diets, suggesting a more advanced reproductive status for the oysters fed high doses of lipid emulsion. Moreover, some oysters in these two treatment groups spawned during the last three weeks of the seven-week feeding experiment. Lipid supplements had a significant influence on hemocyte concentration, phagocytic index and non-stimulated hemocyte ROS production. After 4 weeks, highest hemocyte concentrations were found in oysters fed on a supplement of 50% lipid emulsion compared with those fed on other diets but the hemocytes derived from these oysters had the lowest short-term phagocytic index. After 7 weeks of dietary conditioning, the ROS production in non-stimulated hemocytes of oysters fed 10% and 50% lipid emulsion declined. These results suggested that 20:5n-3, and perhaps its eicosanoid metabolites, affected oyster hemocyte functions; however, the reproductive status of oysters may also have interfered with the 20:5n-3 dietary effect.     

Effects of the mermithid nematode Ovomermis sinensis on the hemocytes of its host Helicoverpa armigera

Little is known about the mechanism by which mermithid nematodes avoid encapsulation responses of insect hosts. In this study, we investigated the influence of the mermithid nematode Ovomermis sinensis on host Helicoverpa armigera hemocyte number, encapsulation activity, spreading behavior and cytoskeleton. Parasitism by O. sinensis caused a significant increase in the total hemocyte counts (THC) and plasmatocyte numbers of H. armigera. However, in vivo encapsulation assays revealed that hemocyte encapsulation abilities of H. armigera were suppressed by O. sinensis. Moreover, parasitism by O. sinensis changed the spreading behavior and cytoskeletons of the host hemocytes. The results suggested that O. sinensis could actively suppress the hemocyte immune response of its host, possibly by destroying the host hemocyte cytoskeleton. This is the first report of a possible mechanism by which mermithid nematodes suppress encapsulation responses of insect hosts.     

Host-parasite relationship of the geoduck Panopea abbreviata and the green alga Coccomyxa parasitica in the Argentinean Patagonian coast

The association of the geoduck Panopea abbreviata and the green alga Coccomyxa parasitica is described. The identity of the green alga was confirmed by molecular studies; the alga was found within the hemocytes that infiltrate the connective tissue of the geoduck siphons. Cytological characteristics of hemocytes were not altered by algal infection; very often the algae were seen enveloped by a digestive vacuole within the hemocyte cytoplasm, evidencing diverse degrees of resorption. Connective cells of siphons were rarely infected by C. parasitica. The mean prevalence of C. parasitica was higher (82%) in San Matías Gulf (42°00′S, 65°05′W) than in San José Gulf (45%) (40°32′S, 64°02′W); except for spring, when the two locations showed no differences in prevalences (80%). Independently of location, season and host size, infected geoducks showed lower condition index values than uninfected ones. Regarding other bivalve species, only one specimen of the razor clam Ensis macha was found infected, and none of the oysters Ostrea puelchana and Pododesmus rudis and scallop Aequipecten tehuelchus was parasitized by the green alga.     

中华绒螯蟹血细胞原代培养条件的优化

比较了几种常见血细胞培养基(L-15、2×L-15、3×L-15、M199和RMPI-1640)对中华绒螯蟹(Eriocheir sinensis)血细胞原代培养中细胞形态以及存活率的影响,以及在筛选获得的最佳培养基中添加不同比例胎牛血清(FBS)(0%、5%、10%和15%),进一步观察了血清对中华绒螯蟹血细胞培养效果的比较。结果表明,3×L-15培养基培养效果较好,所培养的细胞形态相对完整,数量较多,培养至96 h时血细胞存活率仍大于60%;而其他4种培养基效果较差,培养12 h存活率均低于50%,且细胞形态结构变化明显。以3×L-15培养基为基础,添加不同比例胎牛血清后发现,对细胞存活有显著影响,存活率明显降低。因此,不添加血清的3×L-15培养基对中华绒螯蟹血细胞的生长较为适宜。     

Phagocytosis of the protozoan parasite, Marteilia sydneyi, by Sydney rock oyster (Saccostrea glomerata) hemocytes

QX disease is a fatal disease in Sydney rock oysters caused by the protozoan parasite Marteilia sydneyi. The current study investigates the phagocytosis of M. sydneyi by Sydney rock oyster hemocytes. It also compares the in vitro phagocytic activities of hemocytes from oysters bred for QX disease resistance (QXR) with those of wild-type oysters. After ingestion of M. sydneyi, hemocyte granules fused with phagosome membranes and the pH of phagosomes decreased. Significantly (p = <0.05) more phagosomes in QXR hemocytes showed obvious changes in pH within 40 min of phagocytosis, when compared with wild-type hemocytes. Phenoloxidase deposition was also evident in phagosomes after in vitro phagocytosis. Most importantly, ingested and melanised M. sydneyi were detected in vivo among hemocytes from infected oysters. Overall, the data suggest that Sydney rock oyster hemocytes can recognise and phagocytose M. sydneyi, and that resistance against QX disease may be associated with enhanced phagolysosomal activity in QXR oysters.     

Identification of Vibrio spp. in vibriosis Penaeus vannamei using developed monoclonal antibodies

Immunohistochemical study using monoclonal antibodies specific to various shrimp viruses and Vibrio spp. was performed in shrimp samples died from unknown cause with symptoms of black stripes on lateral sides of cephalothorax or smoky body coloration. The positive results in muscular tissue were obtained with MAb VAL57 (specific to Vibrio spp.) and in hepatopancreas tissues with MAbs VVB158 (specific to V. vulnificus) and VPC701 (specific to V. parahaemolyticus). Twelve isolates of Vibrio spp. isolated from shrimp tissues were identified with various MAbs by dot blotting, biochemical tests and 16S rRNA gene. The results revealed three groups of V. vulnificus and one group of V. shilonii. All four groups of isolated Vibrio spp. were immunologically and biochemically different. None of the V. parahaemolyticus-like bacterium was isolated. The results demonstrated that the mortality in shrimp is accompanied by the presence of Vibrio spp.     

Association of a luminous Vibrio sp., taxonomically related to Vibrio harveyi, with Clytia linearis (Thornely, 1900) (Hydrozoa, Cnidaria)

A previously unknown association between a luminous Vibrio sp., taxonomically related to the species Vibrio harveyi and a common member of the shallow/mid water communities of the Mediterranean Sea, the hydrozoan Clytia linearis is described. All the specimens of C. linearis observed under blue light excitation showed both a natural luminescence appearing as a series of fine dots due to clytin, and a clear fluorescence on the external side of the perisarc around the colonies due to the presence of luminous bacteria. Luminous bacteria were isolated from the surface of C. linearis, their phenotypic characterization as isolates was performed by several morphological, biochemical, and cultural tests, completed with 16S rDNA sequence analysis. All the isolates were referred to a Vibrio sp. taxonomically related to V. harveyi. The association of the V. harveyi-related species with C. linearis, as already suggested for another hydroid, Aglaophenia octodonta, could be explained with the activity of these bacteria of feeding on the chitinous structures present in these hydroids. Moreover, the adhesion of the luminous bacterium (here referred to as Vibrio sp. CL1) on C. linearis may contribute to the survival of this Vibrio species in the marine environment providing a suitable growth habitat.     

Transcriptomic profiling of the oyster pathogen Vibrio splendidus opens a window on the evolutionary dynamics of the small RNA repertoire in the Vibrio genus

Work in recent years has led to the recognition of the importance of small regulatory RNAs (sRNAs) in bacterial regulation networks. New high-throughput sequencing technologies are paving the way to the exploration of an expanding sRNA world in nonmodel bacteria. In the Vibrio genus, compared to the enterobacteriaceae, still a limited number of sRNAs have been characterized, mostly in Vibrio cholerae, where they have been shown to be important for virulence, as well as in Vibrio harveyi. In addition, genome-wide approaches in V. cholerae have led to the discovery of hundreds of potential new sRNAs. Vibrio splendidus is an oyster pathogen that has been recently associated with massive mortality episodes in the French oyster growing industry. Here, we report the first RNA-seq study in a Vibrio outside of the V. cholerae species. We have uncovered hundreds of candidate regulatory RNAs, be it cis-regulatory elements, antisense RNAs, and trans-encoded sRNAs. Conservation studies showed the majority of them to be specific to V. splendidus. However, several novel sRNAs, previously unidentified, are also present in V. cholerae. Finally, we identified 28 trans sRNAs that are conserved in all the Vibrio genus species for which a complete genome sequence is available, possibly forming a Vibrio “sRNA core.”     

Responses of diploid and triploid Pacific oysters Crassostrea gigas to Vibrio infection in relation to their reproductive status

Several Vibrio species are known to be pathogenic to the Pacific oyster Crassostrea gigas. Survival varies according to pathogen exposure and high mortality events usually occur in summer during gametogenesis. In order to study the effects of gametogenetic status and ploidy (a factor known to affect reproduction allocation in oysters) on vibriosis survival, we conducted two successive experiments. Our results demonstrate that a common bath challenge with pathogenic Vibriosplendidus and Vibrio aestuarianus on a mixture of mature, spawning and non-mature oysters can lead to significant mortality. Previous bath challenges, which were done using only non-mature oysters, had not produced mortality. Immunohistochemical analyses showed the affinity of Vibrio for gonadic tissues, highlighting the importance of sexual maturity for vibriosis infection processes in oysters. Mortality rate results showed poor repeatability between tanks, however, in this bath challenge. We then tested a standardized and repeatable injection protocol using two different doses of the same combination of two Vibrio species on related diploid and triploid oysters at four different times over a year. Statistical analyses of mortality kinetics over a 6-day period after injection revealed that active gametogenesis periods correspond to higher susceptibility to vibriosis and that there is a significant interaction of this seasonal effect with ploidy. However, no significant advantage of triploidy was observed. Triploid oysters even showed lower survival than diploid counterparts in winter. Results are discussed in relation to differing energy allocation patterns between diploid and triploid Pacific oysters.     

The strepsipteran endoparasite Xenos vesparum alters the immunocompetence of its host, the paper wasp Polistes dominulus

It is unexplained how strepsipteran insects manipulate the physiology of their hosts in order to undergo endoparasitic development without being entrapped by the innate immune defences of the host. Here we present pioneering work that aimed to explore for the first time several components of the cellular and humoral immune response among immature stages of the paper wasp Polistes dominulus, in both unparasitized insects and after infection by the strepsipteran endoparasite Xenos vesparum. We carried out hemocyte counts, phagocytosis assays in vitro and antibacterial response in vivo. On the whole, hemocyte load does not seem to be drastically affected by parasitization: a non-significant increase in hemocyte numbers was observed in parasitized wasps as respect to control, while the two dominant hemocyte types were present with similar proportions in both groups. On the other hand, phagocytosis was significantly reduced in hemocytes from parasitized wasps while the antibacterial response seemed to be less effective in control. These somewhat unexpected results are discussed, along with the implications of a multiple approach in immune response studies.     

Interaction of the bacteria Xenorhabdus nematophila (Enterobactericeae) and Bacillus subtilis (Bacillaceae) with the hemocytes of larval Malacosoma disstria (Insecta: Lepidoptera: Lasiocampidae)

Malacosoma disstria larvae are a pest of deciduous trees. Little is known on the interaction of bacteria with the immediate hemocytic antimicrobial responses of these insects. Incubating dead Xenorhabdus nematophila and Bacillus subtilis with a mixture of serum-free granular cells and plasmatocytes in vitro revealed differential bacterial-hemocyte adhesion and differential discharge of lysozyme and phenoloxidase but not total protein. Although active phenoloxidase adhered equally to both bacterial species, X. nematophila limited enzyme activation whereas B. subtilis enhanced activation. Serum with active phenoloxidase (as opposed to tropolone-inhibited phenoloxidase) and purified insect lysozyme increased bacterial-hemocyte adhesion of both bacterial species. An apolipophorin-III-like protein when incubated with hemocytes, limited their responses to glass slides and bacterial adhesion. However, initial binding of the protein to both bacteria increased granular cell levels with bacteria while lowering the plasmatocyte levels with adhering procaryotes. The protein also increased lysozyme and phenoloxidase activities. Although B. subtilis in vivo elicited a nodulation-based decline in total hemocyte counts and did not affect hemocyte viability, dead X. nematophila elevated hemocyte counts and damaged the hemocytes as lipopolysaccharide levels increased and X. nematophila emerged into the hemolymph. Apolipophorin-III-like protein once bound to the bacteria slowed their removal from the hemolymph.     

VP2118 has major roles in Vibrio parahaemolyticus response to oxidative stress

Background

Reactive oxygen species (ROS), including superoxide anion radical, induce chronic risk of oxidative damage to many cellular macromolecules resulting in damage to cells. Superoxide dismutases (SODs) catalyze the dismutation of superoxide to oxygen and hydrogen peroxide and are a primary defense against ROS. Vibrio parahaemolyticus, a marine bacterium that causes acute gastroenteritis following consumption of raw or undercooked seafood, can survive ROS generated by intestinal inflammatory cells. However, there is little information concerning SODs in V. parahaemolyticus. This study aims to clarify the role of V. parahaemolyticus SODs against ROS.

Methods

V. parahaemolyticus SOD gene promoter activities were measured by a GFP reporter assay. Mutants of V. parahaemolyticus SOD genes were constructed and their SOD activity and resistance to oxidative stresses were measured.

Results

Bioinformatic analysis showed that V. parahaemolyticus SODs were distinguished by their metal cofactors, FeSOD (VP2118), MnSOD (VP2860), and CuZnSOD (VPA1514). VP2118 gene promoter activity was significantly higher than the other SOD genes. In a VP2118 gene deletion mutant, SOD activity was significantly decreased and could be recovered by VP2118 gene complementation. The absence of VP2118 resulted in significantly lowered resistance to ROS generated by hydrogen peroxide, hypoxanthine–xanthine oxidase, or Paraquat. Furthermore, both the N- and C-terminal SOD domains of VP2118 were necessary for ROS resistance.

Conclusion

VP2118 is the primary V. parahaemolyticus SOD and is vital for anti-oxidative stress responses.

General significance

The V. parahaemolyticus FeSOD VP2118 may enhance ROS resistance and could promote its survival in the intestinal tract to facilitate host tissue infection.     

Hematopoietic progenitors and hemocyte lineages in the Drosophila lymph gland

The Drosophila lymph gland (LG) is a model system for studying hematopoiesis and blood cell homeostasis. Here, we investigated the patterns of division and differentiation of pro-hemocytes in normal developmental conditions and response to wasp parasitism, by combining lineage analyses and molecular markers for each of the three hemocyte types. Our results show that the embryonic LG contains primordial hematopoietic cells which actively divide to give rise to a pool of pro-hemocytes. We found no evidence for the existence of bona fide stem cells and rather suggest that Drosophila pro-hemocytes are regulated as a group of cells, rather than individual stem cells. The fate-restriction of plasmatocyte and crystal cell progenitors occurs between the end of embryogenesis and the end of the first larval instar, while Notch activity is required for the differentiation of crystal cells in third instar larvae only. Upon parasitism, lamellocyte differentiation prevents crystal cell differentiation and lowers plasmatocyte production. We also found that a new population of intermediate progenitors appears at the onset of hemocyte differentiation and accounts for the increasing number of differentiated hemocytes in the third larval instar. These findings provide a new framework to identify parameters of developmental plasticity of the Drosophila lymph gland and hemocyte homeostasis in physiological conditions and in response to immunological cues.     

Postingestive sorting of living and heat-killed Chlorella within the sea scallop, Placopecten magellanicus (Gmelin)

Suspension-feeding bivalves may enhance the energy value of their food supply by sorting particles both before and after ingestion. Previous research has indicated that the sea scallop (Placopecten magellanicus (Gmelin) (Mollusca: Bivalvia)) is capable of sorting particles within the gut both on the basis of physical properties (particle size and density) as well as chemical properties. In this study, the ability of the sea scallop to sort living from dead material solely on the basis of chemical properties was tested. The microalga Chlorella (Chlorophyta: Chlorophyceae) was chosen as the test particle because its thick cell wall remains physically intact following heat treatment, while its carbon, nitrogen, and chlorophyll a content declines. Scallops were fed a mixture of radiolabelled live and heat-killed Chlorella. We demonstrate that P. magellanicus can distinguish between living and dead algae, retaining live Chlorella cells longer than heat-killed cells. This ability to detect the subtle chemical differences between living algal material and detrital material would enhance the digestive efficiency of this species by reducing the amount of energy expended, digesting poor-quality materials. This paper presents the first study of the ability of a bivalve to distinguish between two physically identical but nutritionally different forms of the same species of microalgae.