Comparative Animal Mucomics: Inspiration for Functional Materials from Ubiquitous and Understudied Biopolymers.

The functions of secreted animal mucuses are remarkably diverse and include lubricants, wet adhesives, protective barriers, and mineralizing agents. Although present in all animals, many open questions related to the hierarchical architectures, material properties, and genetics of mucus remain. Here, we summarize what is known about secreted mucus structure, describe the work of research groups throughout the world who are investigating various animal mucuses, and relate how these studies are revealing new mucus properties and the relationships between mucus hierarchical structure and hydrogel function. Finally, we call for a more systematic approach to studying animal mucuses so that data sets can be compared, omics-style, to address unanswered questions in the emerging field of mucomics. One major result that we anticipate from these efforts is design rules for creating new materials that are inspired by the structures and functions of animal mucuses.

Particle Selection in Suspension-Feeding Bivalves: Does One Model Fit All?

Suspension-feeding bivalves are known to discriminate among a complex mixture of particles present in their environments. The exact mechanism that allows bivalves to ingest some particles and reject others as pseudofeces has yet to be fully elucidated. Recent studies have shown that interactions between lectins found in the mucus covering oyster and mussel feeding organs and carbohydrates found on the microalga cell surface play a central role in this selection process. In this study, we evaluated whether these interactions are also involved in food selection in bivalves with other gill architectures, namely, the clam Mercenaria mercenaria and the scallop Argopecten irradians. Statistical methods were used to predict whether given microalgae would be rejected or ingested depending on their cell surface carbohydrate profiles. Eight different microalgae with previously established surface carbohydrate profiles were grown and harvested during their exponential growth phase to be used in feeding experiments. Microalgae were then used in 17 feeding experiments where different pairs of microalgae were presented to clams and scallops to evaluate selection. Decision trees that model selection were then developed for each bivalve. Results showed that microalgae rich in mannose residues were likely to be ingested in both bivalves. N-acetylglucosamine and fucose residues also seem to play a role in food particle choice in scallops and clams, respectively. Overall, this study demonstrates the role of carbohydrate-lectin interactions in particle selection in suspension-feeding bivalves displaying different gill architectures, and it highlights the importance of mannose residues as a cue for the selection of ingested particles.

Effects of salinity on hard clam (Mercenaria mercenaria) defense parameters and QPX disease dynamics.

QPX (Quahog Parasite Unknown) is a protistan parasite affecting hard clams (Mercenaria mercenaria) along the Northeast coast of the United States. The fact that QPX disease epizootics are usually observed in field sites with high salinities led to the general assumption that salinity represents an important factor for disease distribution. This study was designed to investigate the effect of salinity on QPX disease development as well as constitutive and QPX-induced defense factors in M. mercenaria. Naïve and QPX-infected (both experimentally and naturally) clams were submitted to 17 and 30 psu for 4 months. Standard and QPX-specific cellular and humoral defense parameters were assessed after 2 and 4 months. These 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 higher QPX-associated mortality in naturally infected clams maintained at high salinity compared to those held at 17 psu. Our findings also showed an increase in mortality following experimental challenge with QPX in clams submitted to 30 psu but not in those held at 17 psu. Constitutive clam defense factors and the response to QPX challenge were also affected by salinity. QPX challenge caused significant but transitory changes in hemolymph parameters that were obvious at 2 months but disappeared at 4 months. Overall, our results show that salinity modulates clam immunity and the progress of QPX disease although its impact appears secondary as compared to findings we reported earlier for temperature.

Identification, molecular characterization and expression analysis of a mucosal C-type lectin in the eastern oyster, Crassostrea virginica.

Lectins are well known to actively participate in the defense functions of vertebrates and invertebrates where they play an important role in the recognition of foreign particles. They have also been reported to be involved in other processes requiring carbohydrate-lectin interactions such as symbiosis or fertilization. In this study, we report a novel putative C-type lectin (CvML) from the eastern oyster Crassostrea virginica and we investigated its involvement in oyster physiology. The cDNA of this lectin is 610 bp long encoding for a 161-residue protein. CvML presents a signal peptide and a single carbohydrate recognition domain (CRD) which contains a YPD motif and two putative conserved sites, WID and DCM, for calcium binding. CvML transcripts were expressed in mucocytes lining the epithelium of the digestive gland and the pallial organs (mantle, gills, and labial palps) but were not detected in other tissues including hemocytes. Its expression was significantly up-regulated following starvation or bacterial bath exposure but not after injection of bacteria into oyster's adductor muscle. These results highlight the potential role of CvML in the interactions between oyster and waterborne microorganisms at the pallial interfaces with possible involvement in physiological functions such as particle capture or mucosal immunity.

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.

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.

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.

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.