Oyster Hemolymph Research Articles

Semi-purified Antimicrobial Proteins from Oyster Hemolymph Inhibit Pneumococcal Infection

Pneumococcal infections caused by Streptococcus pneumoniae are a leading cause of morbidity and mortality globally, particularly among children. The ability of S. pneumoniae to form enduring biofilms makes treatment inherently difficult, and options are further limited by emerging antibiotic resistance. The discovery of new antibiotics, particularly those with antibiofilm activity, is therefore increasingly important. Antimicrobial proteins and peptides (AMPs) from marine invertebrates are recognised as promising pharmacological leads. This study determined the in vitro antibacterial activity of hemolymph and unique protein fractions from an Australian oyster (Saccostrea glomerata) against multi-drug-resistant S. pneumoniae. We developed a successful method for hemolymph extraction and separation into 16 fractions by preparative HPLC. The strongest activity was observed in fraction 7: at 42 µg/mL protein, this fraction was bactericidal to S. pneumoniae and inhibited biofilm formation. Proteomic analysis showed that fraction 7 contained relatively high abundance of carbonic anhydrase, cofilin, cystatin B–like, and gelsolin-like proteins, while surrounding fractions, which showed lower or no antibacterial activity, contained these proteins in lower abundance or not at all. This work supports traditional medicinal uses of oysters and contributes to further research and development of novel hemolymph/AMP-based treatments for pneumococcal infections.

Elevated temperature and decreased salinity impacts on exogenous Vibrio parahaemolyticus infection of eastern oyster, Crassostrea virginica.

Anthropogenic carbon emissions have resulted in drastic oceanic changes, including increased acidity, increased temperature, and decreased salinity. Anthropogenic carbon emissions have resulted in drastic oceanic changes, including increased acidity, increased temperature, and decreased salinity. Few studies have directly assessed the compounded impact of alterations to oceanic conditions on oyster physiology and the relation to the presence of V. parahaemolyticus. This project investigated the relationship between projected climate scenarios and their influence on both eastern oyster, Crassostrea virginica, and the aquatic bacteria, Vibrio parahaemolyticus. Specifically, we examined whether an increase in water temperature and/or decrease in salinity would impair oyster resistance to V. parahaemolyticus, a human food and waterborne pathogen. Using a culture-dependent approach, our data revealed that the alterations in environmental conditions did not significantly impact the numbers of V. parahaemolyticus numbers within oyster hemolymph or tissues. However, we did observe a dramatic increase in the total amount of bacteria and pathogenic native Vibrio species, Vibrio aestuarianus and Vibrio harveyi. Despite detecting V. parahaemolyticus in most tissues at 7 days post-challenge, oysters were able to reduce bacterial levels below our limit of detection by 28 days of exposure. Furthermore, in our second experimental trial exploring single vs. multiple inoculation of bacteria, we observed that oysters were either able to reduce total bacterial levels to pre-treatment burdens (i.e., below our limit of detection) or die. This study demonstrates that the synergistic effects of elevated temperature and decreased salinity do not inhibit oysters from preventing the long-term colonization of exogenous V. parahaemolyticus. However, our data do show these environmental stressors impact oyster physiology and the native microbiota. This can lead to the proliferation of opportunistic pathogens, which could have impacts on oyster population numbers and ecosystem and human health.

Whole genome enrichment approach for genomic surveillance of Toxoplasma gondii

Pathogenic bacteria, viruses, fungi, and protozoa can cause food and waterborne diseases. Surveillance methods must therefore screen for these pathogens at various stages of water distribution and of food from production to consumption. Detection using nucleic acid amplification methods offer rapid identification, but such methods have limited utility for characterizing populations, variant types or virulence traits of pathogens. Whole genome sequencing (WGS) can be used to determine this information. However, pathogens must be isolated and cultured to yield sufficient DNA for WGS, which is laborious or not feasible for certain stages of parasites like oocysts of Toxoplasma gondii. We previously developed the Circular Nucleic acid Enrichment Reagent (CNER) method to make whole genome enrichment (WGE) baits for difficult-to-grow bacterial pathogens. WGE using CNERs facilitates direct sequencing of pathogens from samples without the need to isolate and grow them. Here, we made WGE-CNERs for T. gondii to demonstrate the use of the CNER method to make baits to enrich the large genomes of water and foodborne protozoan pathogens. By sequencing, we detected as few as 50 parasites spiked in an oyster hemolymph matrix. We discuss the use of WGE-CNERs for genomic surveillance of food and waterborne pathogens.

Gold Nanoparticle Paper Immunoassays for Sensing the Presence of Vibrio parahaemolyticus in Oyster Hemolymph.

Seafood contamination with Vibrio bacteria is a problem for aquaculture, especially with oysters, which are often consumed raw. Current methods for diagnosing bacterial pathogens in seafood involve lab-based assays such as polymerase chain reaction or culturing, which are time consuming and must occur in a centralized location. Detection of Vibrio in a point-of-care assay would be a significant tool for food safety control measures. We report here a paper immunoassay that can detect the presence of Vibrio parahaemolyticus (Vp) in buffer and oyster hemolymph. The test uses gold nanoparticles conjugated to polyclonal anti-Vibrio antibodies in a paper-based sandwich immunoassay. A sample is added to the strip and wicked through by capillary action. If Vp is present, it results in a visible color at the test area that can be read out by eyes or a standard mobile phone camera. The assay has a limit of detection of 6.05 × 105 cfu/mL and a cost estimate of $5 per test. Receiver operating characteristic curves with validated environmental samples showed a test sensitivity of 0.96 and a specificity of 1.00. Because the assay is inexpensive and can be used on Vp directly without the requirement for culturing, or sophisticated equipment, it has the potential to be used in fieldable settings.

Hypoxia and Anoxia Tolerance in Diploid and Triploid Eastern Oysters at High Temperature

Increasing reliance on the use of triploid oysters to support aquaculture production relies on their generally superior growth rate and meat quality over that of diploid oysters. Reports of elevated triploid mortality have generated questions about potential trade-offs between growth and tolerance to environmental stressors. These questions are particularly relevant as climate change, coastal activities, and river management impact water salinity, temperature, nutrients, pH, and oxygen levels within key estuarine oyster growing areas. In particular, the co-occurrence of warm water temperatures and low dissolved oxygen concentration (DO) events are increasingly reported in estuaries, with potentially lethal impacts on sessile, oyster resources. To investigate potential differences in DO tolerance, diploid and triploid market-sized or seed oysters were exposed to continuous normoxia (DO > 5.0 mg L–1), hypoxia (DO < 2.0 mg L–1), and anoxia (DO < 0.5 mg L–1) at 28°C and their mortalities were monitored. The hemolymph of the market-sized oysters was collected to measure cellular and biochemical changes in response to hypoxia and anoxia, whereas their valve movements were also measured. In general, about half of market-sized oysters died within about 1 wk under anoxia (LT50: 5.7–8.9 days) and within about 2 wk under hypoxia (LT50: 11.9–19.4 days) with diploid oysters tending to die faster than triploid oysters. Seed oysters took longer to die than market-sized oysters under both anoxia (LT50: 9.5–12.1 days) and hypoxia (LT50: 21.8–25.0 days) with diploid oysters (LT50: 9.5–11.8 days) dying slightly faster than triploid oysters (LT50: 11.8–12.1 days) under anoxia. Hemolymph pH decreased and plasma calcium and glutathione concentrations increased with decreasing DO, with values under anoxia being different than those under normoxia. Hemocyte density was also lower under anoxia than under either normoxia or hypoxia. Overall, few differences in physiological responses to hypoxia and anoxia were found between diploid and triploid oysters suggesting that ploidy (2N versus 3N) had limited effect on the tolerance and response of eastern oysters to low DO.

Morphological and functional characterization of the oyster Crassostrea gasar circulating hemocytes: Cell types and phagocytosis activity

Hemocytes are the circulating cells of the hemolymph of oysters and are responsible for numerous physiological functions, including immune defense. The oyster Crassostrea gasar is a native species inhabiting mangrove habitat and is of great commercial interest, cultured throughout the Brazilian coast, mainly in the north and northeast. Despite its commercial importance, little is known about its immunological aspects and defense cells, the hemocytes. This work aimed to morphologically characterize hemocytes of the oyster C. gasar and to study one of the main cellular defense response, phagocytosis, using light microscopy and flow cytometry. The results showed the presence of six hemocyte populations in C. gasar hemolymph. These comprise of large and small granulocytes, large and small hyalinocytes, blast-like cells and a rare type classified as vesicular or serous hemocytes. Hyalinocytes were highly abundant and the most heterogeneous cell population, while small granulocytes, along with vesicular hemocytes were the less abundant population. Hemocytes of C. gasar oysters demonstrated capabilities to phagocytose three different types of particles tested: zymosan A, latex particles and Escherichia coli, indicating a broad defense capacity. The zymosan A were the most engulfed particles, followed by beads, mainly phagocytized by granulocytes, the most phagocytic cells, and finally E. coli, which were the least phagocytized. This study is the first characterization of C. gasar oyster hemocytes and will support future studies that aim to understand the participation of different hemocyte types in defense responses against pathogens and/or environmental changes.

Circulating exosome miRNA, is it the novel nutrient molecule through cross-kingdom regulation mediated by food chain transmission from microalgae to bivalve?

MicroRNAs (miRNAs) can efficiently regulate gene expression at intracellular and extracellular levels. Plant-derived miRNAs are highly enriched in animal haemolymph and regulate mammalian gene expression. However, evidence for food-derived miRNAs in Mollusca species is lacking. In this study, we fed the microalga Nannochloropsis oculata to the pearl oyster Pinctada fucata martensii and detected dietary miRNAs in exosomes isolated from the haemolymph by RNA-seq. In total, 273 endogenous miRNAs were identified in all biological replicates. We identified 23 microalgae-derived miRNAs in the exosomes of pearl oyster haemolymph. Most microalgae-derived miRNAs showed high expression levels in both exosomes and microalgae and exhibited apparent variation among individuals. These food-derived miRNAs were predicted to participate in endocytosis, apoptosis, signal transduction, energy metabolism, and biomineralization by targeting multiple genes. These findings demonstrated the cross-kingdom transport of miRNAs from microalgae to bivalves and provide insights into novel nutrient transmission through the food chain.

Alterins, a new family of marine antibacterial cyclolipopeptides

Five strains of Pseudoalteromonas, isolated from oyster haemolymph, have exhibited antibacterial activity against several Gram-negative bacteria. Bioactive compounds have been identified in their cell-free supernatant and characterised as alterins, which are cyclolipopeptides comprising a heptapeptidic ring connected to a fatty acid chain. Using ultra-performance liquid chromatography–high-resolution mass spectrometry, this paper describes 37 structural analogues differing from each other by one or more amino acid residue, the length of the fatty acid chain, its hydroxylation and the presence of unsaturation.

Thermal Responses in Japanese Pearl Oysters, Pinctada fucata martensii

In order to examine the thermal responses in pearl oysters, both the mRNA expression levels of heatshock protein (Hsp) in their tissues and changes in the mineral levels of their hemolymph were analyzed using heattreated and heat-untreated pearl oysters. Pearl oysters that live in Tsukumo Bay on the Noto Peninsula were used as experimental materials. Pearl oysters kept at 25 C in artificial seawater were exposed to high temperatures (30 C or 35 C) for 1 h. After exposure, the pearl oysters were transferred into the seawater at 25 C and kept for 4 h. Thereafter, the hemolymph was collected by inserting a syringe into the adductor muscle of the heat-treated pearl oyster, and then hemocytes were separated from hemolymph by centrifugation. In addition to hemocytes, the mantles and gills were taken from heat-treated pearl oysters to analyze Hsp mRNA expression. The collected hemolymph was used to analyze its mineral concentration. The data of pearl oysters treated at a high temperature (30 C or 35 C) were compared with those of pearl oysters kept at the initial temperature (25 C) as a heat-untreated control. First, the mRNA expressions of several Hsps (Hsp20, Hsp40, Hsp70, and Hsp90) were analyzed using the hemocytes, mantles, and gills to examine the thermal responses of pearl oysters. With heat treatment, the mRNA expressions of all Hsps (Hsp20, Hsp40, Hsp70, and Hsp90) used in the present study were significantly increased. Especially, Hsp20 mRNA expression in hemocytes, mantles, and gills was remarkably increased by heat treatment. The responsiveness of Hsps mRNA expression was greatly higher at the rate of increase at 35 C than at the rate of increase at 30 C. Among the tissues used in the present study, the mRNA expression levels of Hsp20, Hsp40, Hsp70, and Hsp90 were higher in gills than in hemocytes and mantles. Second, we analyzed the mineral concentrations of hemolymph in the heat-treated pearl oysters. The calcium, magnesium, sodium, chlorine, and potassium ion levels in the hemolymph of pearl oysters kept at 35 C increased significantly as compared with those in the initial hemolymph of pearl oysters kept at 25 C. This suggests that heat treatment influences excretory organs such as gills and kidneys. The fact that the expression level of Hsps was highest in gills, which function as respiratory and International Journal of Zoological Investigations Contents available at Journals Home Page: www.ijzi.net Editor-in-Chief: Prof. Ajai Kumar Srivastav Published by: Saran Publications, Gorakhpur, India 372 excretory organs, supported the influences of the excretory system. This study is the first report to demonstrate changes in both the monovalent and divalent ions of hemolymph in response to heat treatment in Japanese pearl oysters. We plan to investigate the detailed mechanism of the above mineral regulation together with HSP expressions in the near future.

Shotgun analysis to identify differences in protein expression between granulocytes and hyalinocytes of the European flat oyster Ostrea edulis

Recovery of wild populations of the European flat oyster Ostrea edulis is important for ecosystem health and conservation of this species, because native oyster populations have dramatically declined or disappeared in most European waters. Diseases have contributed to oyster decline and are important constrains for oyster recovery. Understanding oyster immune system should contribute to design effective strategies to fight oyster diseases. Haemocytes play a pivotal role in mollusc immune responses protecting from infection. Two main types of haemocytes, granulocytes and hyalinocytes, are distinguished in O. edulis. A study aiming to explore differential functions between both haemocyte types and, thus, to enrich the knowledge of Ostrea edulis immune system, was performed by comparing the proteome of the two haemolymph cell types, using a shotgun approach through liquid chromatography (LC) coupled to mass spectrometry (MS). Cells from oyster haemolymph were differentially separated by Percoll density gradient centrifugation. Shotgun LC-MS/MS performance allowed the identification of 145 proteins in hyalinocytes and 138 in the proteome of granulocytes. After a comparative analysis, 55 proteins with main roles in defence were identified, from which 28 were representative of granulocytes and 27 of hyalinocytes, plus 11 proteins shared by both cell types. Different proteins involved in signal transduction, apoptosis, oxidative response, processes related with the cytoskeleton and structure, recognition and wound healing were identified as representatives of each haemocyte type. Important signalling pathways in the immune response such as MAPK, Ras and NF-κβ seemed to be more relevant for granulocytes, while the Wnt signalling pathway, particularly relevant for wound healing, more relevant in hyalinocytes. The differences in proteins involved in recognition and in cytoskeleton and structure suggest differential specialisation in processes of phagocytosis and internalisation of pathogens between haemocyte types. Apoptosis seemed more active in granulocytes. The differences in proteins involved in oxidative response also suggest different redox processes in each cell type.

Microbiome response differs among selected lines of Sydney rock oysters to ocean warming and acidification.

Oyster microbiomes are integral to healthy function and can be altered by climate change conditions. Genetic variation among oysters is known to influence the response of oysters to climate change and may ameliorate any adverse effects on oyster microbiome; however, this remains unstudied. Nine full-sibling selected breeding lines of the Sydney rock oyster (Saccostrea glomerata) were exposed to predicted warming (ambient = 24°C, elevated = 28°C) and ocean acidification (ambient pCO2 = 400, elevated pCO2 = 1000 µatm) for 4 weeks. The haemolymph bacterial microbiome was characterized using 16S rRNA (V3-V4) gene sequencing and varied among oyster lines in the control (ambient pCO2, 24°C) treatment. Microbiomes were also altered by climate change dependent on oyster lines. Bacterial α-diversity increased in response to elevated pCO2 in two selected lines, while bacterial β-diversity was significantly altered by combinations of elevated pCO2 and temperature in four selected lines. Climate change treatments caused shifts in the abundance of multiple amplicon sequence variants driving change in the microbiome of some selected lines. We show that oyster genetic background may influence the Sydney rock oyster haemolymph microbiome under climate change and that future assisted evolution breeding programs to enhance resilience should consider the oyster microbiome.

Screening of bacterial pathogens associated with mass summer mortality of the Pacific oyster, Crassostrea gigas, in China

The Pacific oyster (Crassostrea gigas) is an important shellfish species for aquaculture. Mass summer mortality of C. gigas has seriously hampered the development of oyster industry in recent years, while the mortality causing factors remain elusive. In order to explore the pathogenic factors associated with mass summer mortality, we performed a bacterial screening of moribund oysters collected from an oyster farm in major C. gigas aquaculture area in northern China. Fourteen strains of bacteria were isolated from hemolymph of moribund oysters, and were further identified as five Vibrio species, including Vibrio harveyi, V. alginolyticus, V. rotiferianus, V. brasiliensis, and V. owensii, based on 16S rRNA sequencing and gyrB sequencing. The virulence of bacterial strains was examined by measuring extracellular protease activity and performing experimental challenge trials. Three previously reported pathogenic Vibrio strains, V. crassostreae, V. aestuarianus and V. splendidus, were also included to better understand the virulence of the pathogens isolated in this study that can cause mortality. Experimental challenge trials revealed that V. splendidus, V. brasiliensis, V. harveyi and V. alginolyticus were all pathogenic to C. gigas, with V. alginolyticus exerting the highest level of pathogenicity. This work performed a preliminary investigation on the pathogenic factors associated with mass summer mortality of C. gigas, which should be valuable for disease surveillance.

Alterins Produced by Oyster-Associated Pseudoalteromonas Are Antibacterial Cyclolipopeptides with LPS-Binding Activity.

Discovery after discovery, host-associated microbiota reveal a growing list of positive effects on host homeostasis by contributing to host nutrition, improving hosts’ immune systems and protecting hosts against pathogens. In that context, a collection of oyster associated bacteria producing antibacterial compounds have been established to evaluate their role in non-host-derived immunity. Here, we described alterins; potent anti-Gram negative compounds produced by Pseudoalteromonas hCg-6 and hCg-42 isolated from different healthy oyster hemolymph. The strains hCg-6 and hCg-42 produce a set of at least seven antibacterial compounds, ranging from 926 to 982 Da structurally characterized as cyclolipopeptides (CLPs). Alterins share the same cationic heptapeptidic cycle connected via an amido bond to different hydrophobic hydrocarbon tails. Their MICs disclosed a potent antibacterial activity directed against Gram-negative bacteria including oyster and human pathogens that may confer a beneficial defense mechanism to the host but also represents an untapped source of new antibiotics. The alterins’ mechanisms of action have been deciphered: after binding to lipopolysaccharides (LPS), alterins provoke a membrane depolarization and permeabilization leading to bacterial lysis. As hCg-6 and hCg-42 produced a set of natural derivatives, the structure/activity relationship linked to the carbon tail is clarified. We showed that the hydrocarbon tail determines the LPS-binding properties of alterins and consequently their antibacterial activities. Its length and saturation seem to play a major role in this interaction.

IgIT-Mediated Signaling Inhibits the Antimicrobial Immune Response in Oyster Hemocytes.

The long-term evolutionary interaction between the host and symbiotic microbes determines their cooperative relationship. It is well known that the symbiotic microbes have evolved various mechanisms to either benefit or exploit the mammalian host immune system to maintain homeostasis. However, the strategies employed by the symbiotic microbes to overcome host immune responses in invertebrates are still not clear. In the current study, the hemolymph microbes in oyster Crassostrea gigas were found to be able to directly bind an oyster Ig superfamily member (IgSF) (designated as CgIgIT) to inhibit the immune responses of hemocytes. The mRNA transcripts of CgIgIT in hemocytes increased significantly after the stimulation with hemolymph microbes. CgIgIT was found to be located on the hemocyte membrane and it was able to directly bind the hemolymph microbes and polysaccharides via its three Ig domains and recruited the protein tyrosine phosphatase CgSHP2 through its ITIM. The recruited CgSHP2 inhibited the activities of CgERK, CgP38 and CgJNK proteins to reduce the productions of dual oxidase 2 (CgDuox2) and defensin 2 (CgDef2), which eventually protected the hemolymph microbes from CgDuox2/CgDef2-mediated elimination. Collectively, the results suggest that the oyster IgIT-SHP2 signaling pathway can recognize bacteria capable of residing in oyster hemolymph and inhibit innate immune responses, which contributes to the maintenance, colonization, and survival of hemolymph microbes.

Oyster hemolymph is a complex and dynamic ecosystem hosting bacteria, protists and viruses

BackgroundThe impact of the microbiota on host fitness has so far mainly been demonstrated for the bacterial microbiome. We know much less about host-associated protist and viral communities, largely due to technical issues. However, all microorganisms within a microbiome potentially interact with each other as well as with the host and the environment, therefore likely affecting the host health.ResultsWe set out to explore how environmental and host factors shape the composition and diversity of bacterial, protist and viral microbial communities in the Pacific oyster hemolymph, both in health and disease. To do so, five oyster families differing in susceptibility to the Pacific oyster mortality syndrome were reared in hatchery and transplanted into a natural environment either before or during a disease outbreak. Using metabarcoding and shotgun metagenomics, we demonstrate that hemolymph can be considered as an ecological niche hosting bacterial, protist and viral communities, each of them shaped by different factors and distinct from the corresponding communities in the surrounding seawater. Overall, we found that hemolymph microbiota is more strongly shaped by the environment than by host genetic background. Co-occurrence network analyses suggest a disruption of the microbial network after transplantation into natural environment during both non-infectious and infectious periods. Whereas we could not identify a common microbial community signature for healthy animals, OsHV-1 μVar virus dominated the hemolymph virome during the disease outbreak, without significant modifications of other microbiota components.ConclusionOur study shows that oyster hemolymph is a complex ecosystem containing diverse bacteria, protists and viruses, whose composition and dynamics are primarily determined by the environment. However, all of these are also shaped by oyster genetic backgrounds, indicating they indeed interact with the oyster host and are therefore not only of transient character. Although it seems that the three microbiome components respond independently to environmental conditions, better characterization of hemolymph-associated viruses could change this picture.

Differences in proteomic profile between two haemocyte types, granulocytes and hyalinocytes, of the flat oyster Ostrea edulis

Haemocytes play a dominant role in shellfish immunity, being considered the main defence effector cells in molluscs. These cells are known to be responsible for many functions, including chemotaxis, cellular recognition, attachment, aggregation, shell repair and nutrient transport and digestion. There are two basic cell types of bivalve haemocytes morphologically distinguishable, hyalinocytes and granulocytes; however, functional differences and specific abilities are poorly understood: granulocytes are believed to be more efficient in killing microorganisms, while hyalinocytes are thought to be more specialised in clotting and wound healing. A proteomic approach was implemented to find qualitative differences in the protein profile between granulocytes and hyalinocytes of the European flat oyster, Ostrea edulis, as a way to evaluate functional differences. Oyster haemolymph cells were differentially separated by Percoll® density gradient centrifugation. Granulocyte and hyalinocyte proteins were separated by 2D-PAGE and their protein profiles were analysed and compared with PD Quest software; the protein spots exclusive for each haemocyte type were excised from gels and analysed by MALDI-TOF/TOF with a combination of mass spectrometry (MS) and MS/MS for sequencing and protein identification. A total of 34 proteins were identified, 20 unique to granulocytes and 14 to hyalinocytes. The results suggested differences between the haemocyte types in signal transduction, apoptosis, oxidation reduction processes, cytoskeleton, phagocytosis and pathogen recognition. These results contribute to identify differential roles of each haemocyte type and to better understand the oyster immunity mechanisms, which should help to fight oyster diseases.

Optimization of paper-based nanoparticle immunoassays for direct detection of the bacterial pathogen V. parahaemolyticus in oyster hemolymph.

The detection of foodborne pathogens is critical for disease control and infection prevention, especially in seafood consumed raw or undercooked. Paper-based diagnostic tools are promising for rapid fieldable detection and provide a readout by eye due to the use of gold nanoparticle immunoprobes. Here we study different strategies to overcome these challenges in a real biological matrix, oyster hemolymph, for the detection of the pathogenic bacteria Vibrio parahaemolyticus (Vp). Nanoparticle surface chemistry, nitrocellulose speed and blocking, running steps, and antibody concentrations on the NP and nitrocellulose were all studied. Their effect on paper immunoassay signal intensity was quantified to determine optimal conditions, which enabled the detection of Vp directly from hemolymph below pathogenic concentrations.

Lethal effects of silver nanoparticles on Perkinsus marinus, a protozoan oyster parasite

Perkinsus marinus, a World Organisation for Animal Health (OIE) notifiable parasite, infects several species of oyster, including Crassostrea virginica and Crassostrea corteziensis. There is little information on possible treatments for this parasite, but the biocidal properties of silver nanoparticles (AgNP) suggest their potential use. The lethal effects of the Argovit™ formulation of AgNP was evaluated for the first time against hypnospores of P. marinus, a particularly resistant stage of the parasite that persists in the environment until favorable conditions occur for zoosporulation to be induced. Hypnospores were exposed to 1, 10 and 100 µg/mL of silver compounded in Argovit™ (corresponding to 0.009, 0.093 and 0.927 mM of Ag), to 157.47 µg/mL (0.927 mM) of silver nitrate (AgNO3) used as a positive control, and to polyvinylpyrrolidone (PVP, 1570 µg/mL) used as a vehicle control. Hypnospores in culture medium without treatment served as a negative control. Dose-dependence after 24 h of exposure to AgNP was observed. A concentration of 0.093 mM AgNP resulted in 50% mortality of P. marinus. Treatment with 0.927 mM of silver, as AgNP or AgNO3, was highly lethal, with greater than 90% mortality. Silver nanoparticles were implicated in the deformation of hypnospores. Transmission electron microscopy (TEM) revealed AgNP within the hypnospore wall and involved in the degradation of lipid droplets in the cytoplasm. AgNP were effective in a saline medium, suggesting the utility of detailed studies of the physicochemical interactions of AgNP under these conditions. These results suggest investigations of possible effect of Argovit™ formulation of AgNP against stages of the parasite like trophozoites and tomonts that develop in tissues or hemolymph of infected oysters as well as studies on its effects in the host and environment.

Spatiotemporal dynamics of Spirochaeta, the putative etiologic agent of Akoya oyster disease in pearl oysters, as determined by quantitative PCR

Since the 1990s, Akoya oyster disease (AOD) associated with reddish browning of the soft tissues and mortality of cultured pearl oyster, Pinctada fucata (Gould), has been occurring in the western part of Japan. Although transmission experiments suggested that the disease is infectious, the causative agent has not been identified. Previous studies suggested that a member of the Spirochaeta cause AOD, and Candidatus Maribrachyspira akoyae was proposed as the putative causative agent. However, difficulties in culturing the putative causal bacterium have hindered research on AOD. In this study, we developed a quantitative PCR assay to examine the spatial-temporal colonization patterns of Spirochaeta in pearl oysters. Spirochete genes were abundant in the mantle, adductor muscle, and hemolymph of oysters but scarce in the gill, crystalline style, hemocytes, heart, and digestive glands. Spirochete genes were detected in the hemolymph 2 weeks after infection, and their number increased with time and mortality. The Spirochaeta genes identified in the hemolymph of pearl oysters reared in culturing fields changed with variations in water temperature, and the number of Spirochaeta genes increased or decreased at approximately 20 °C. Hence, the bacterium was thought to grow at water temperatures above 20 °C. These results are consistent with those of infection tests and epidemiologic studies of the distribution and seasonal changes in AOD pathogens and support the hypothesis that a spirochete causes AOD.

Exploring First Interactions Between Ostreid Herpesvirus 1 (OsHV-1) and Its Host, Crassostrea gigas: Effects of Specific Antiviral Antibodies and Dextran Sulfate.

Viral entry mechanisms of herpesviruses constitute a highly complex process which implicates several viral glycoproteins and different receptors on the host cell surfaces. This initial infection stage was currently undescribed for Ostreid herpes virus 1 (OsHV-1), a herpesvirus infecting bivalves including the Pacific oyster, Crassostrea gigas. To identify OsHV-1 glyproteins implicated in the attachment of the virus to oyster cells, three viral putative membrane proteins, encoded by ORF 25, 41, and 72, were selected and polyclonal antibodies against these targets were used to explore first interactions between the virus and host cells. In addition, effects of dextran sulfate, a negative charged sulfated polysaccharide, were investigated on OsHV-1 infection. Effects of antiviral antibodies and dextran sulfate were evaluated by combining viral DNA and RNA detection in spat (in vivo trials) and in oyster hemolymph (in vitro trials). Results showed that viral protein encoded by ORF 25 appeared to be involved in interaction between OsHV-1 and host cells even if other proteins are likely implicated, such as proteins encoded by ORF 72 and ORF 41. Dextran sulfate at 30 μg/mL significantly reduced the spat mortality rate in the experimental conditions. Taken together, these results contribute to better understanding the pathogenesis of the viral infection, especially during the first stage of OsHV-1 infection, and open the way toward new approaches to control OsHV-1 infection in confined facilities.