Emmanuelle Pales Espinosa

Lectins Associated With the Feeding Organs of the Oyster Crassostrea virginica Can Mediate Particle Selection

Despite advances in the study of particle selection in suspension-feeding bivalves, the mechanisms upon which bivalves rely to discriminate among particles have not been elucidated. We hypothesized that particle sorting in suspension-feeding bivalves could be based, in part, on a biochemical recognition mechanism mediated by lectins within the mucus that covers the feeding organs. Using Crassostrea virginica, the Eastern oyster, our investigations demonstrated that lectins from oyster mucus can specifically bind several microalgal species as well as different types of red blood cells (RBC), triggering their agglutination. Agglutination of microalgal species and RBC varied with the source of mucus (gills vs. labial palps). Hemagglutination and hemagglutination inhibition assays emphasized that mucus contains several lectins. In feeding experiments, Nitzschia closterium and Tetraselmis maculata were separately incubated with mucus before being fed to oysters. Results showed that pre-treating these microalgae with mucus significantly alters the ability of oysters to sort particles. In another experiment, oysters were fed a mixture of microspheres coated with either bovine serum albumin (BSA) or glucosamide-BSA. Results show that oysters preferentially ingest microspheres with bound carbohydrates, highlighting probable interactions between lectins and carbohydrates in the mechanisms of microalgae recognition. This study confirms the presence of lectins in mucus that covers the feeding organs of oysters and suggests a new concept with regard to particle processing by suspension-feeding bivalves: specific interactions between carbohydrates on the surface of particles and lectins within the mucus mediate the selection and rejection processes.

Microalgal Cell Surface Carbohydrates as Recognition Sites for Particle Sorting in Suspension-Feeding Bivalves

Cell surface carbohydrates play important roles in cell recognition mechanisms. Recently, we provided evidence that particle selection by suspension-feeding bivalves can be mediated by interactions between carbohydrates associated with the particle surface and lectins present in mucus covering bivalve feeding organs. In this study, we used lectins tagged with fluorescein isothiocyanate (FITC) to characterize carbohydrate moieties on the surface of microalgal species and evaluate the effect of oyster mucus on lectin binding. These analyses revealed that concanavalin A (Con A), one of six lectins tested, bound to Isochrysis sp., while Nitzschia closterium reacted with Pisum sativum agglutinin (PNA) and peanut agglutinin (PEA). The cell surface of Rhodomonas salina bound with PNA and Con A, and Tetraselmis maculata cell surface was characterized by binding with PNA, PEA, and Con A. Pre-incubation of microalgae with oyster pallial mucus significantly decreased the binding of FITC-labeled lectins, revealing that lectins present in mucus competitively blocked binding sites. This decrease was reversed by washing mucus-coated microalgae with specific carbohydrates. These results were used to design a feeding experiment to evaluate the effect of lectins on sorting of microalgae by oysters. Crassostrea virginica fed with an equal ratio of Con A-labeled Isochrysis sp. and unlabeled Isochrysis sp. produced pseudofeces that were significantly enriched in Con A-labeled Isochrysis sp. and depleted in unlabeled microalgae. Selection occurred even though two physical-chemical surface characteristics of the cells in each treatment did not differ significantly. This work confirms the involvement of carbohydrate-lectin interaction in the particle sorting mechanism in oysters, and provides insights into the carbohydrate specificity of lectins implicated in the selection of microalgal species.

Effects of temperature on hard clam (Mercenaria mercenaria) immunity and QPX (Quahog Parasite Unknown) disease development: II. Defense parameters

Quahog Parasite Unknown (QPX) is a protistan parasite affecting hard clams Mercenaria mercenaria along the Northeastern coast of the United States. The geographic distribution and occurrence of disease epizootics suggests a primary role of temperature in disease development. This study was designed to investigate the effect of temperature on constitutive and QPX-induced defense factors in M. mercenaria. Control and QPX-challenged (both experimentally and naturally) clams were maintained at 13, 21 and 27°C for 4 months. Control and experimentally-infected clams originated from a southern broodstock (Florida, no prior reports of disease outbreak) while naturally-infected clams originated from a northern broodstock (Massachusetts, enzootic area). Standard and QPX-specific cellular and humoral defense parameters were assessed after 2 and 4 months. Measured parameters included total and differential hemocyte counts, reactive oxygen species production, phagocytic activity of hemocytes, lysozyme concentration in plasma, anti-QPX activity in plasma and resistance of hemocytes to cytotoxic QPX extracellular products. Results demonstrated a strong influence of temperature on constitutive clam defense factors with significant modulation of cellular and humoral parameters of control clams maintained at 13°C compared to 21 and 27°C. Similarly, clam response to QPX challenge was also affected by temperature. Challenged clams exhibited no difference from controls at 27°C whereas different responses were observed at 21°C and 13°C compared to controls. Despite differences in infection mode (experimentally or naturally infected) and clam origin (northern and southern broodstocks), similarities were observed at 13°C and 21°C between QPX infected clams from Florida and Massachusetts. Clam response to temperature and to QPX exhibited interesting relationship with QPX disease development highlighting major influence of temperature on disease development.

Early host-pathogen interactions in marine bivalves: Evidence that the alveolate parasite Perkinsus marinus infects through the oyster mantle during rejection of pseudofeces

Parasites have developed myriad strategies to reach and infect their specific hosts. One of the most common mechanisms for non-vector transmitted parasites to reach the internal host environment is by ingestion during feeding. In this study, we investigated the mechanisms of oyster host colonization by the alveolate Perkinsus marinus and focused on how oysters process infective waterborne P. marinus cells during feeding in order to determine the portal(s) of entry of this parasite to its host. We also compared the infectivity of freely-suspended cells of P. marinus with that of cells incorporated into marine aggregates to link changes in particle processing by the feeding organs with infection success and route. Finally, we evaluated the effect of oyster secretions (mucus) covering the feeding organs on P. marinus physiology because these host factors are involved in the processing of waterborne particles. The ensemble of results shows a unique mechanism for infection by which the parasite is mostly acquired during the feeding process, but not via ingestion. Rather, infection commonly occurs during the rejection of material as pseudofeces before reaching the mouth. The pseudofeces discharge area, a specialized area of the mantle where unwanted particles are accumulated for rejection as pseudofeces, showed significantly higher parasite loads than other host tissues including other parts of the mantle. Aggregated P. marinus cells caused significantly higher disease prevalence and infection intensities when compared to freely-suspended parasite cells. Mucus covering the mantle caused a quick and significant increase in parasite replication rates suggesting rapid impact on P. marinus physiology. A new model for P. marinus acquisition in oysters is proposed.

Transcriptional changes in Manila clam (Ruditapes philippinarum) in response to Brown Ring Disease

Brown Ring Disease (BRD) is a bacterial infection affecting the economically-important clam Ruditapes philippinarum. The disease is caused by a bacterium, Vibrio tapetis, that colonizes the edge of the mantle, altering the biomineralization process and normal shell growth. Altered organic shell matrices accumulate on the inner face of the shell leading to the formation of the typical brown ring in the extrapallial space (between the mantle and the shell). Even though structural and functional changes have been described in solid (mantle) and fluid (hemolymph and extrapallial fluids) tissues from infected clams, the underlying molecular alterations and responses remain largely unknown. This study was designed to gather information on clam molecular responses to the disease and to compare focal responses at the site of the infection (mantle and extrapallial fluid) with systemic (hemolymph) responses. To do so, we designed and produced a Manila clam expression oligoarray (15K Agilent) using transcriptomic data available in public databases and used this platform to comparatively assess transcriptomic changes in mantle, hemolymph and extrapallial fluid of infected clams. Results showed significant regulation in diseased clams of molecules involved in pathogen recognition (e.g. lectins, C1q domain-containing proteins) and killing (defensin), apoptosis regulation (death-associated protein, bcl-2) and in biomineralization (shell matrix proteins, perlucin, galaxin, chitin- and calcium-binding proteins). While most changes in response to the disease were tissue-specific, systemic alterations included co-regulation in all 3 tested tissues of molecules involved in microbe recognition and killing (complement-related factors, defensin). These results provide a first glance at molecular alterations and responses caused by BRD and identify targets for future functional investigations.

Role of epicellular molecules in the selection of particles by the blue mussel, Mytilus edulis.

This study provides evidence that the suspension-feeding blue mussel, Mytilus edulis, uses biochemical cues to recognize its food. We identified lectins in mucus from the gills and labial palps, two pallial organs involved in the feeding process. These compounds were able to agglutinate rabbit and horse erythrocytes (RBC) and several species of marine microalgae representing different families. Additionally, the agglutination of RBC and microalgae was inhibited by several carbohydrates (fetuin, lipopolysaccharide (LPS), and mannose-related residues), suggesting that a suite of lectins may be present in mucus from the gills and labial palps. Results from feeding experiments, using microspheres with tailored surfaces, demonstrated that mussels preferentially ingested microspheres coated with the neoglycoproteins glucosamide-BSA and mannopyranosylphenyl-BSA but rejected in pseudofeces microspheres coated with BSA alone. The positive selection for neoglycoprotein-coated microspheres was inhibited when mussels were pre-incubated in seawater containing a solution of the same neoglycoprotein. Two surface properties of the microspheres, charge and wettability, had little effect on the observed selection process. Our results, along with our previous findings for oysters, suggest a new concept for the mechanism of particle selection in bivalves and perhaps other suspension-feeding organisms. Specifically, the selection of particles involves interactions between epiparticulate carbohydrates and lectins in the mucus produced by feeding organs.

Bivalve immunity and response to infections: Are we looking at the right place?

Significant progress has been made in the understanding of cellular and molecular mediators of immunity in invertebrates in general and bivalve mollusks in particular. Despite this information, there is a lack of understanding of factors affecting animal resistance and specific responses to infections. This in part results from limited consideration of the spatial (and to some extent temporal) heterogeneity of immune responses and very limited information on host-pathogen (and microbes in general) interactions at initial encounter/colonization sites. Of great concern is the fact that most studies on molluscan immunity focus on the circulating hemocytes and the humoral defense factors in the plasma while most relevant host-microbe interactions occur at mucosal interfaces. This paper summarizes information available on the contrasting value of information available on focal and systemic immune responses in infected bivalves, and highlights the role of mucosal immune factors in host-pathogen interactions. Available information underlines the diversity of immune effectors at molluscan mucosal interfaces and highlights the tailored immune response to pathogen stimuli. This context raises fascinating basic research questions around host-microbe crosstalk and feedback controls of these interactions and may lead to novel disease mitigation strategies and improve the assessment of resistant crops or the screening of probiotic candidates.

Pallial mucus of the oyster Crassostrea virginica regulates the expression of putative virulence genes of its pathogen Perkinsus marinus

Perkinsus marinus is a pathogen responsible for severe mortalities of the eastern oyster Crassostrea virginica along the East and Gulf coasts of the United States. When cultivated, the pathogenicity of this microorganism decreases significantly, hampering the study of its virulence factors. Recent investigations have shown a significant increase of the in vivo virulence of P. marinus exposed to oyster pallial mucus. In the current study, we investigated the effect of pallial mucus on P. marinus gene expression compared with cultures supplemented with oyster digestive extracts or with un-supplemented cultures. In parallel, parasite cells cultured under these three conditions were used to challenge oysters and to assess virulence in vivo. Perkinsus marinus mRNA sequencing was performed on an Illumina GAIIX sequencer and data were analysed using the Tuxedo RNAseq suite for mapping against the draft P. marinus genome and for differential expression analysis. Results showed that exposure of P. marinus to mucus induces significant regulation of nearly 3,600 transcripts, many of which are considered as putative virulence factors. Pallial mucus is suspected to mimic internal host conditions, thereby preparing the pathogen to overcome defense factors before invasion. This hypothesis is supported by significant regulation in several antioxidant proteins, heat shock proteins, protease inhibitors and proteasome subunits. In addition, mucus exposure induced the modulation of several genes known to affect immunity and apoptosis in vertebrates and invertebrates. Several proteases (proteolysis) and merozoite surface proteins (cell recognition) were also modulated. Overall, these results provide a baseline for targeted, in depth analysis of candidate virulence factors in P. marinus.

Early host-pathogen interactions in a marine bivalve: Crassostrea virginica pallial mucus modulates Perkinsus marinus growth and virulence.

Perkinsus marinus is an important protistan parasite of the eastern oyster Crassostrea virginica. Recent findings showed that oyster pallial organs (mantle, gills) are a major portal of entry for the parasite. Therefore, mucus covering these organs represents the first host effectors encountered by P. marinus. This study consisted of several experiments designed to investigate the effect of oyster pallial mucus on the growth, protease production and infectivity of P. marinus. In each experiment, P. marinus performance in cultures supplemented with pallial mucus (mantle, gill, or both) was compared to that of parasite cells grown in unsupplemented media or in cultures supplemented with oyster plasma or digestive extracts. P. marinus grown in media supplemented with C. virginica mantle mucus showed a significantly higher growth rate than cultures enriched with the other supplemental extracts, while cultures grown in gill mucus promoted higher protease production. Conversely, P. marinus grown in cultures supplemented with pallial mucus of the non-compatible host Crassostrea gigas (Pacific oyster) were dramatically inhibited. Challenge experiments showed a significant increase in P. marinus virulence in cultures supplemented with C. virginica pallial mucus as compared to unsupplemented cultures or to those supplemented with digestive extract or plasma. These results suggest that C. virginica mucus plays a significant role in the pathogenesis of P. marinus by enhancing the proliferation and the infectivity of this devastating parasite. The contrasting results obtained with both oyster species indicate that P. marinus host specificity may begin in the mucus.

Proteomic characterization of mucosal secretions in the eastern oyster, Crassostrea virginica

The soft body surface of marine invertebrates is covered by a layer of mucus, a slippery gel secreted by mucocytes lining epithelia. The functions of this gel are diverse including locomotion, cleansing, food particles processing and defense against physicochemical injuries and infectious agents. In oysters, mucus covering pallial organs has been demonstrated to have a major importance in the processing of food particles and in the interactions with waterborne pathogens. Given the limited information available on mucus in bivalves and the apparent wide spectra of activity of bioactive molecules present in this matrix, the characterization of these mucosal secretions has become a research priority. In this study, mucus was separately collected from the mantle, gills and labial palps of the eastern oyster (Crassostrea virginica) and analyzed by liquid chromatography and tandem mass spectrometry. Results showed the presence of a wide variety of molecules involved in host-microbe interactions, including putative adhesion molecules (e.g. c-type lectins) confirming that transcripts previously identified in epithelial cells are translated into proteins secreted in mucus. Mucus composition was different among samples collected from different organs. These results generate a reference map for C. virginica pallial mucus to better characterize the various physiological functions of mucosal secretions.