Genetic Insights into Biofilm Formation by a Pathogenic Strain of Vibrio harveyi.

The Vibrio genus includes bacteria widely distributed in aquatic habitats and the infections caused by these bacteria can affect a wide range of hosts. They are able to adhere to numerous surfaces, which can result in biofilm formation that helps maintain them in the environment. The involvement of the biofilm lifestyle in the virulence of Vibrio pathogens of aquatic organisms remains to be investigated. Vibrio harveyi ORM4 is a pathogen responsible for an outbreak in European abalone Haliotis tuberculata populations. In the present study, we used a dynamic biofilm culture technique coupled with laser scanning microscopy to characterize the biofilm formed by V. harveyi ORM4. We furthermore used RNA-seq analysis to examine the global changes in gene expression in biofilm cells compared to planktonic bacteria, and to identify biofilm- and virulence-related genes showing altered expression. A total of 1565 genes were differentially expressed, including genes associated with motility, polysaccharide synthesis, and quorum sensing. The up-regulation of 18 genes associated with the synthesis of the type III secretion system suggests that this virulence factor is induced in V. harveyi ORM4 biofilms, providing indirect evidence of a relationship between biofilm and virulence.

Microbiota of the Digestive Glands and Extrapallial Fluids of Clams Evolve Differently Over Time Depending on the Intertidal Position.

The Manila clam (Ruditapes philippinarum) is the second most exploited bivalve in the world but remains threatened by diseases and global changes. Their associated microbiota play a key role in their fitness and acclimation capacities. This study aimed at better understanding the behavior of clam digestive glands and extrapallial fluids microbiota at small, but contrasting spatial and temporal scales. Results showed that environmental variations impacted clam microbiota differently according to the considered tissue. Each clam tissue presented its own microbiota and showed different dynamics according to the intertidal position and sampling period. Extrapallial fluids microbiota was modified more rapidly than digestive glands microbiota, for clams placed on the upper and lower intertidal position, respectively. Clam tissues could be considered as different microhabitats for bacteria as they presented different responses to small-scale temporal and spatial variabilities in natural conditions. These differences underlined a more stringent environmental filter capacity of the digestive glands.

Recent advances in bivalve-microbiota interactions for disease prevention in aquaculture.

In bivalves, no clear-cut functional role of microbiota has yet been identified, although many publications suggest that they could be involved in nutrition or immunity of their host. In the context of climate change, integrative approaches at the crossroads of disciplines have been developed to explore the environment-host-pathogen-microbiota system. Here, we attempt to synthesize work on (1) the current methodologies to analyse bivalve microbiota, (2) the comparison of microbiota between species, between host compartments and their surrounding habitat, (3) how the bivalve microbiota are governed by environmental factors and host genetics and (4) how host-associated microorganisms act as a buffer against pathogens and/or promote recovery, and could thereby play a role in the prevention of disease or mortalities.

Virulence of Vibrio harveyi ORM4 towards the European abalone Haliotis tuberculata involves both quorum sensing and a type III secretion system.

Environmental Vibrio strains represent a major threat in aquaculture, but the understanding of their virulence mechanisms heavily relies on the transposition of knowledge from human-pathogen vibrios. Here, the genetic bases of the virulence of Vibrio harveyi ORM4 towards the European abalone Haliotis tuberculata were characterized. We demonstrated that luxO, encoding a major regulator of the quorum sensing system, is crucial for the virulence of this strain, and that its deletion leads to a decrease in swimming motility, biofilm formation, and exopolysaccharide production. Furthermore, the biofilm formation by V. harveyi ORM4 was increased by abalone serum, which required LuxO. The absence of LuxO in V. harveyi ORM4 yielded opposite phenotypes compared with other Vibrio species including V. campbellii (still frequently named V. harveyi). In addition, we report a full type III secretion system (T3SS) gene cluster in the V. harveyi ORM4 genome. LuxO was shown to negatively regulate the promoter activity of exsA, encoding the major regulator of the T3SS genes, and the deletion of exsA abolished the virulence of V. harveyi ORM4. These results unveil virulence mechanisms set up by this environmentally important bacterial pathogen and pave the way for a better molecular understanding of the regulation of its pathogenicity.

Implication of the Type IV Secretion System in the Pathogenicity of Vibrio tapetis, the Etiological Agent of Brown Ring Disease Affecting the Manila Clam Ruditapes philippinarum.

Vibrio tapetis is a Gram-negative bacterium that causes infections of mollusk bivalves and fish. The Brown Ring Disease (BRD) is an infection caused by V. tapetis that primarily affects the Manila clam Ruditapes philippinarum. Recent studies have shown that a type IV secretion system (T4SS) gene cluster is exclusively found in strains of V. tapetis pathogenic to clams. However, whether the T4SS is implicated or not during the infection process remains unknown. The aim of this study was to create and characterize a V. tapetis T4SS null mutant, obtained by a near-complete deletion of the virB4 gene, in order to determine the role of T4SS in the development of BRD. This study demonstrated that the T4SS is neither responsible for the loss of hemocyte adhesion capacities, nor for the decrease of the lysosomal activity during BRD. Nevertheless, we observed a 50% decrease of the BRD prevalence and a decrease of mortality dynamics with the ΔvirB4 mutant. This work demonstrates that the T4SS of V. tapetis plays an important role in the development of BRD in the Manila clam.

Potential for Genetic Improvement of Resistance to Perkinsus olseni in the Manila Clam, Ruditapes philippinarum, Using DNA Parentage Assignment and Mass Spawning.

The Manila clam Ruditapes philippinarum, a major cultured shellfish species, is threatened by infection with the microparasite Perkinsus olseni, whose prevalence increases with high water temperatures. Under the current trend of climate change, the already severe effects of this parasitic infection might rapidly increase the frequency of mass mortality events. Treating infectious diseases in bivalves is notoriously problematic, therefore selective breeding for resistance represents a key strategy for mitigating the negative impact of pathogens. A crucial step in initiating selective breeding is the estimation of genetic parameters for traits of interest, which relies on the ability to record parentage and accurate phenotypes in a large number of individuals. Here, to estimate the heritability of resistance against P. olseni, a field experiment mirroring conditions in industrial clam production was set up, a genomic tool was developed for parentage assignment, and parasite load was determined through quantitative PCR. A mixed-family cohort of potentially 1,479 clam families was produced in a hatchery by mass spawning of 53 dams and 57 sires. The progenies were seeded in a commercial clam production area in the Venice lagoon, Italy, where high prevalence of P. olseni had previously been reported. Growth and parasite load were monitored every month and, after 1 year, more than 1,000 individuals were collected for DNA samples and phenotype recording. A pooled sequencing approach was carried out using DNA samples from the hatchery broodstock and from a Venice lagoon clam population, providing candidate markers used to develop a 245-SNP panel. Parentage assignment for 246 F1 individuals showed sire and dam representation were high (75 and 85%, respectively), indicating a very limited risk of inbreeding. Moderate heritability (0.23 ± 0.11-0.35 ± 0.13) was estimated for growth traits (shell length, shell weight, total weight), while parasite load showed high heritability, estimated at 0.51 ± 0.20. No significant genetic correlations were found between growth-associated traits and parasite load. Overall, the preliminary results provided by this study show high potential for selecting clams resistant to parasite load. Breeding for resistance may help limit the negative effects of climate change on clam production, as the prevalence of the parasite is predicted to increase under a future scenario of higher temperatures. Finally, the limited genetic correlation between resistance and growth suggests that breeding programs could incorporate dual selection without negative interactions.

The marine intertidal zone shapes oyster and clam digestive bacterial microbiota.

Digestive microbiota provide a wide range of beneficial effects on host physiology and are therefore likely to play a key role in marine intertidal bivalve ability to acclimatize to the intertidal zone. This study investigated the effect of intertidal levels on the digestive bacterial microbiota of oysters (Crassostrea gigas) and clams (Ruditapes philippinarum), two bivalves with different ecological niches. Based on 16S rRNA region sequencing, digestive glands, seawater and sediments harbored specific bacterial communities, dominated by operational taxonomic units assigned to the Mycoplasmatales,Desulfobacterales and Rhodobacterales orders, respectively. Field implantation modified digestive bacterial microbiota of both bivalve species according to their intertidal position. Rhodospirillales and Legionellales abundances increased in oysters and clams from the low intertidal level, respectively. After a 14-day depuration process, these effects were still observed, especially for clams, while digestive bacterial microbiota of oysters were subjected to more short-term environmental changes. Nevertheless, 3.5 months stay on an intertidal zone was enough to leave an environmental footprint on the digestive bacterial microbiota, suggesting the existence of autochthonous bivalve bacteria. When comparing clams from the three intertidal levels, 20% of the bacterial assemblage was shared among the levels and it was dominated by an operational taxonomic unit affiliated to the Mycoplasmataceae and Spirochaetaceae families.

Transcriptomic analysis of clam extrapallial fluids reveals immunity and cytoskeleton alterations in the first week of Brown Ring Disease development.

The Brown Ring Disease is an infection caused by the bacterium Vibrio tapetis on the Manila clam Ruditapes philippinarum. The process of infection, in the extrapallial fluids (EPFs) of clams, involves alteration of immune functions, in particular on hemocytes which are the cells responsible of phagocytosis. Disorganization of the actin-cytoskeleton in infected clams is a part of what leads to this alteration. This study is the first transcriptomic approach based on collection of extrapallial fluids on living animals experimentally infected by V. tapetis. We performed differential gene expression analysis of EPFs in two experimental treatments (healthy-against infected-clams by V. tapetis), and showed the deregulation of 135 genes. In infected clams, a downregulation of transcripts implied in immune functions (lysosomal activity and complement- and lectin-dependent PRR pathways) was observed during infection. We also showed a deregulation of transcripts encoding proteins involved in the actin cytoskeleton organization such as an overexpression of ß12-Thymosin (which is an actin sequestration protein) or a downregulation of proteins that closely interact with capping proteins such as Coactosin, that counteract action of capping proteins, or Profilin. We validated these transcriptomic results by cellular physiological analyses that showed a decrease of the lysosome amounts and the disorganization of actin cytoskeleton in infected hemocytes.

Dynamics of the Pacific oyster pathobiota during mortality episodes in Europe assessed by 16S rRNA gene profiling and a new target enrichment next-generation sequencing strategy.

Infectious agents such as the bacteria Vibrio aestuarianus or Ostreid herpesvirus 1 have been repeatedly associated with dramatic disease outbreaks of Crassostrea gigas beds in Europe. Beside roles played by these pathogens, microbial infections in C. gigas may derive from the contribution of a larger number of microorganisms than previously thought, according to an emerging view supporting the polymicrobial nature of bivalve diseases. In this study, the microbial communities associated with a large number of C. gigas samples collected during recurrent mortality episodes at different European sites were investigated by real-time PCR and 16SrRNA gene-based microbial profiling. A new target enrichment next-generation sequencing protocol for selective capturing of 884 phylogenetic and virulence markers of the potential microbial pathogenic community in oyster tissue was developed allowing high taxonomic resolution analysis of the bivalve pathobiota. Comparative analysis of contrasting C. gigas samples conducted using these methods revealed that oyster experiencing mortality outbreaks displayed signs of microbiota disruption associated with the presence of previously undetected potential pathogenic microbial species mostly belonging to genus Vibrio and Arcobacter. The role of these species and their consortia should be targeted by future studies aiming to shed light on mechanisms underlying polymicrobial infections in C. gigas.

Relation between Biofilm and Virulence in Vibrio tapetis: A Transcriptomic Study.

Marine pathogenic bacteria are able to form biofilms on many surfaces, such as mollusc shells, and they can wait for the appropriate opportunity to induce their virulence. Vibrio tapetis can develop such biofilms on the inner surface of shells of the Ruditapes philippinarum clam, leading to the formation of a brown conchiolin deposit in the form of a ring, hence the name of the disease: Brown Ring Disease. The virulence of V. tapetis is presumed to be related to its capacity to form biofilms, but the link has never been clearly established at the physiological or genetic level. In the present study, we used RNA-seq analysis to identify biofilm- and virulence-related genes displaying altered expression in biofilms compared to the planktonic condition. A flow cell system was employed to grow biofilms to obtain both structural and transcriptomic views of the biofilms. We found that 3615 genes were differentially expressed, confirming that biofilm and planktonic lifestyles are very different. As expected, the differentially expressed genes included those involved in biofilm formation, such as motility- and polysaccharide synthesis-related genes. The data show that quorum sensing is probably mediated by the AI-2/LuxO system in V. tapetis biofilms. The expression of genes encoding the Type VI Secretion System and associated exported proteins are strongly induced, suggesting that V. tapetis activates this virulence factor when living in biofilm.

Rapid and efficient protocol to introduce exogenous DNA in Vibrio harveyi and Pseudoalteromonas sp.

Vibrio campbellii BAA-1116 is renowned for its bioluminescence properties, and genetic tools are available to genetically track this strain. However, many other ecologically important V. harveyi strains exist, for which only few genetic tools are available. In this study, a rapid electroporation protocol was developed to transform replicative plasmids in various environmental V. harveyi and Pseudoalteromonas strains. Moreover, a mini-Tn7 delivery system was modified to site-specifically integrate mini-transposons in the genome of V. harveyi ORM4. As a proof-of-principle, replicative plasmids carrying bioreporters were introduced by electroporation in V. harveyi ORM4 cells, and gene expression was followed at the single cell level. We could demonstrate that a flagellar gene is subjected to bimodal gene expression in V. harveyi ORM4, being highly expressed in 10% of the population in stationary phase. This study extends the possibilities to study environmental Vibrio strains and uncovers the occurrence of phenotypic heterogeneity in flagellar expression in Vibrio.

Vibrio tapetis Displays an Original Type IV Secretion System in Strains Pathogenic for Bivalve Molluscs.

The Brown Ring Disease (BRD) caused high mortality rates since 1986 in the Manila clam Venerupis philippinarum introduced and cultured in Western Europe from the 1970s. The causative agent of BRD is a Gram-Negative bacterium, Vibrio tapetis, which is also pathogenic to fish. Here we report the first assembly of the complete genome of V. tapetis CECT4600T, together with the genome sequences of 16 additional strains isolated across a broad host and geographic range. Our extensive genome dataset allowed us to describe the pathogen pan- and core genomes and to identify putative virulence factors. The V. tapetis core genome consists of 3,352 genes, including multiple potential virulence factors represented by haemolysins, transcriptional regulators, Type I restriction modification system, GGDEF domain proteins, several conjugative plasmids, and a Type IV secretion system. Future research on the coevolutionary arms race between V. tapetis virulence factors and host resistance mechanisms will improve our understanding of how pathogenicity develops in this emerging pathogen.

The One Health Concept: 10 Years Old and a Long Road Ahead.

Over the past decade, a significant increase in the circulation of infectious agents was observed. With the spread and emergence of epizootics, zoonoses, and epidemics, the risks of pandemics became more and more critical. Human and animal health has also been threatened by antimicrobial resistance, environmental pollution, and the development of multifactorial and chronic diseases. This highlighted the increasing globalization of health risks and the importance of the human-animal-ecosystem interface in the evolution and emergence of pathogens. A better knowledge of causes and consequences of certain human activities, lifestyles, and behaviors in ecosystems is crucial for a rigorous interpretation of disease dynamics and to drive public policies. As a global good, health security must be understood on a global scale and from a global and crosscutting perspective, integrating human health, animal health, plant health, ecosystems health, and biodiversity. In this study, we discuss how crucial it is to consider ecological, evolutionary, and environmental sciences in understanding the emergence and re-emergence of infectious diseases and in facing the challenges of antimicrobial resistance. We also discuss the application of the "One Health" concept to non-communicable chronic diseases linked to exposure to multiple stresses, including toxic stress, and new lifestyles. Finally, we draw up a list of barriers that need removing and the ambitions that we must nurture for the effective application of the "One Health" concept. We conclude that the success of this One Health concept now requires breaking down the interdisciplinary barriers that still separate human and veterinary medicine from ecological, evolutionary, and environmental sciences. The development of integrative approaches should be promoted by linking the study of factors underlying stress responses to their consequences on ecosystem functioning and evolution. This knowledge is required for the development of novel control strategies inspired by environmental mechanisms leading to desired equilibrium and dynamics in healthy ecosystems and must provide in the near future a framework for more integrated operational initiatives.

Ancient DNA analysis identifies marine mollusc shells as new metagenomic archives of the past.

Marine mollusc shells enclose a wealth of information on coastal organisms and their environment. Their life history traits as well as (palaeo-) environmental conditions, including temperature, food availability, salinity and pollution, can be traced through the analysis of their shell (micro-) structure and biogeochemical composition. Adding to this list, the DNA entrapped in shell carbonate biominerals potentially offers a novel and complementary proxy both for reconstructing palaeoenvironments and tracking mollusc evolutionary trajectories. Here, we assess this potential by applying DNA extraction, high-throughput shotgun DNA sequencing and metagenomic analyses to marine mollusc shells spanning the last ~7,000 years. We report successful DNA extraction from shells, including a variety of ancient specimens, and find that DNA recovery is highly dependent on their biomineral structure, carbonate layer preservation and disease state. We demonstrate positive taxonomic identification of mollusc species using a combination of mitochondrial DNA genomes, barcodes, genome-scale data and metagenomic approaches. We also find shell biominerals to contain a diversity of microbial DNA from the marine environment. Finally, we reconstruct genomic sequences of organisms closely related to the Vibrio tapetis bacteria from Manila clam shells previously diagnosed with Brown Ring Disease. Our results reveal marine mollusc shells as novel genetic archives of the past, which opens new perspectives in ancient DNA research, with the potential to reconstruct the evolutionary history of molluscs, microbial communities and pathogens in the face of environmental changes. Other future applications include conservation of endangered mollusc species and aquaculture management.

Immune priming and portal of entry effectors improve response to vibrio infection in a resistant population of the European abalone.

Since 1997, populations of the European abalone Haliotis tuberculata suffer mass mortalities attributed to the bacterium Vibrio harveyi. These mortalities occur at the spawning season, when the abalone immune system is depressed, and when temperatures exceed 17 °C, leading to favorable conditions for V. harveyi proliferation. In order to identify mechanisms of disease resistance, experimental successive infections were carried out on two geographically distinct Brittany populations: one that has suffered recurrent mortalities (Saint-Malo) and one that has not been impacted by the disease (Molène). Furthermore, abalone surviving these two successive bacterial challenges and uninfected abalone were used for several post-infection analyses. The Saint-Malo population was found to be resistant to V. harveyi infection, with a survival rate of 95% compared to 51% for Molène. While in vitro quantification of phagocytosis by flow cytometry showed strong inhibition following the first infection, no inhibition of phagocytosis was observed following the second infection for Saint-Malo, suggesting an immune priming effect. Moreover, assays of phagocytosis of GFP-labelled V. harveyi performed two months post-infection show an inhibition of phagocytosis by extracellular products of V. harveyi for uninfected abalone, while no effect was observed for previously infected abalone from Saint-Malo, suggesting that the effects of immune priming may last upwards of two months. Detection of V. harveyi by qPCR showed that a significantly greater number of abalone from the susceptible population were positive for V. harveyi in the gills, indicating that portal of entry effectors may play a role in resistance to the disease. Collectively, these results suggest a potential synergistic effect of gills and hemolymph in the resistance of H. tuberculata against V. harveyi with an important involvement of the gills, the portal of entry of the bacteria.

De novo assembly and annotation of the European abalone Haliotis tuberculata transcriptome.

The European abalone Haliotis tuberculata is a delicacy and consequently a commercially valuable gastropod species. Aquaculture production and wild populations are subjected to multiple climate-associated stressors and anthropogenic pressures, including rising sea-surface temperatures, ocean acidification and an emerging pathogenic Vibrio infection. Transcript expression data provides a valuable resource for understanding abalone responses to variation in the biotic and abiotic environment. To generate an extensive transcriptome, we performed next-generation sequencing of RNA on larvae exposed to temperature and pH variation and on haemolymph of adults from two wild populations after experimental infection with Vibrio harveyi. We obtained more than 1.5 billion raw paired-end reads, which were assembled into 328,519 contigs. Filtration and clustering produced a transcriptome of 41,099 transcripts, of which 10,626 (25.85%) were annotated with Blast hits, and 7380 of these were annotated with Gene Ontology (GO) terms in Blast2Go. A differential expression analysis comparing all samples from the two life stages identified 5690 and 10,759 transcripts with significantly higher expression in larvae and adult haemolymph respectively. This is the greatest sequencing effort yet in the Haliotis genus, and provides the first high-throughput transcriptomic resource for H. tuberculata.

Metabolic responses of clam Ruditapes philippinarum exposed to its pathogen Vibrio tapetis in relation to diet.

We investigated the effect of brown ring disease (BRD) development and algal diet on energy reserves and activity of enzymes related to energy metabolism, antioxidant system and immunity in Manila clam, Ruditapes philippinarum. We found that algal diet did not impact the metabolic response of clams exposed to Vibrio tapetis. At two days post-injection (dpi), activities of superoxide dismutase and glutathione peroxidase (GPx) decreased whereas activities of nitric oxide synthase (iNOS) and catalase increased in infected clams, although no clinical signs were visible (BRD-). At 7 dpi, activities of several antioxidant and immune-related enzymes were markedly increased in BRD-likely indicating an efficient reactive oxygen species (ROS) scavenging compared to animals which developed clinical signs of BRD (BRD+). Therefore, resistance to BRD clinical signs appearance was associated with higher detoxification of ROS and enhancement of immune response. This study provides new biochemical indicators of disease resistance and a more comprehensive view of the global antioxidant response of clam to BRD development.

Vibrio tapetis, the Causative Agent of Brown Ring Disease, Forms Biofilms with Spherical Components.

Vibrio tapetis is a marine bacterium causing Brown Ring Disease (BRD) in the Manila clam Ruditapes philippinarum. V. tapetis biofilm formation remains unexplored depite the fact that it might be linked to pathogenicity. Our objectives were to characterize the in vitro biofilm formation of V. tapetis and evaluate the effects of culture conditions. Biofilm structure and its matrix composition were examined by confocal laser scanning microscopy and scanning electron microscopy. V. tapetis was able to form biofilms on a glass substratum within 24 h. Polysaccharides and extracellular DNA of the biofilm matrixes were differently distributed depending on the V. tapetis strains. Spherical components of about 1-2 µm diameter were found at the biofilm surface. They contain DNA, proteins, and seemed to be physically linked to bacteria and of cellular nature. Transmission electron microscopy showed that the spherical components were devoid of internal compartments. Temperatures >21°C inhibit BRD whereas low salinity (2%) favor it, none of the both conditions altered V. tapetis' ability to form biofilms in vitro. We suggest therefore that biofilm formation could play a role in the persistence of the pathogen in clam than in BRD symptoms.

Development of a Taqman real-time PCR assay for rapid detection and quantification of Vibrio tapetis in extrapallial fluids of clams.

The Gram-negative bacterium Vibrio tapetis is known as the causative agent of Brown Ring Disease (BRD) in the Manila clam Venerupis (=Ruditapes) philippinarum. This bivalve is the second most important species produced in aquaculture and has a high commercial value. In spite of the development of several molecular methods, no survey has been yet achieved to rapidly quantify the bacterium in the clam. In this study, we developed a Taqman real-time PCR assay targeting virB4 gene for accurate and quantitative identification of V. tapetis strains pathogenic to clams. Sensitivity and reproducibility of the method were assessed using either filtered sea water or extrapallial fluids of clam injected with the CECT4600(T) V. tapetis strain. Quantification curves of V. tapetis strain seeded in filtered seawater (FSW) or extrapallial fluids (EF) samples were equivalent showing reliable qPCR efficacies. With this protocol, we were able to specifically detect V. tapetis strains down to 1.125 10(1) bacteria per mL of EF or FSW, taking into account the dilution factor used for appropriate template DNA preparation. This qPCR assay allowed us to monitor V. tapetis load both experimentally or naturally infected Manila clams. This technique will be particularly useful for monitoring the kinetics of massive infections by V. tapetis and for designing appropriate control measures for aquaculture purposes.

The early stages of the immune response of the European abalone Haliotis tuberculata to a Vibrio harveyi infection.

Vibrio harveyi is a marine bacterial pathogen responsible for episodic abalone mortalities in France, Japan and Australia. In the European abalone, V. harveyi invades the circulatory system in a few hours after exposure and is lethal after 2 days of infection. In this study, we investigated the responses of European abalone immune cells over the first 24 h of infection. Results revealed an initial induction of immune gene expression including Rel/NF-kB, Mpeg and Clathrin. It is rapidly followed by a significant immuno-suppression characterized by reduced cellular hemocyte parameters, immune response gene expressions and enzymatic activities. Interestingly, Ferritin was overexpressed after 24 h of infection suggesting that abalone attempt to counter V. harveyi infection using soluble effectors. Immune function alteration was positively correlated with V. harveyi concentration. This study provides the evidence that V. harveyi has a hemolytic activity and an immuno-suppressive effect in the European abalone.