Microbiological Quality of Oysters and Mussels Along Its Market Supply Chain.

Oysters and mussels are known vectors of foodborne pathogens because of their immobile and filter-feeding nature leading to the accumulation of biological particles in their tissues. Accumulated bacteria which comes from the culture environment and unsanitary handling can cause food poisoning if these shellfish are consumed raw or partially processed. This study determined the incidence of bacterial pathogen contamination along the different channels of the oyster and mussel supply chain through a time-distribution simulation analysis. First, the route of the fresh bivalve products from a local farm to its market was established through interviews. From the data gathered, a simulation experiment was conducted following the observed time-temperature conditions and the actual bulk packaging material used by the traders. The presence of target pathogens Escherichia coli, Salmonella spp., Vibrio parahaemolyticus, and Vibrio cholerae were detected using standard conventional culture techniques. Initial E. coli counts in both mussels and oysters were higher than the safety limit of 330 MPN in 100 g tissue. Interestingly, E. coli counts in mussels decreased after 6 h and maintained low numbers after more than 24 h postharvest. Counts in oysters however increased to 1000 MPN in 100 g tissue. V. parahaemolyticus in mussels and oysters showed a gradual increase in counts with increasing holding time albeit in numbers that are lower than the safety limit of 1000 cfu g-1 tissue. Qualitative detection of Salmonella and V. cholerae showed the presence of both pathogens in all the sampling points. All four pathogens were also detected in the culture waters and in the sediment. Results of the study showed that the culture environment and the handling practices contribute greatly to the pathogen contamination in oysters and mussels.

A lanthanide-based high-sensitivity fluorescence method for the on-site rapid detection of thermostable direct hemolysin of Vibrio parahaemolyticus.

Vibrio parahaemolyticus is a common foodborne pathogen in seafood, which often causes seafood borne bacterial gastroenteritis or food poisoning. Thermostable direct hemolysin (TDH) is considered to be one of the main virulence factors involved in this pathogen. The most clinical V. parahaemolyticus isolates produce TDH. Therefore, high sensitivity and specificity detection of TDH are of great significance for food safety and early diagnosis of diseases caused by V. parahaemolyticus. In this study, we developed a rapid, sensitive immunochromatographic test paper assay for the quantitative detection of TDH in seafood samples using time-resolved fluorescence techniques. First, we completed the preparation of fluorescent detection antibodies by coupling lanthanide fluorescent nanospheres with homemade high-affinity polyclonal antibodies based on the principle of the double-antibody sandwich. The lanthanide fluorescent nanospheres used in this study are characterized by a large stokes shift and a long fluorescence lifetime, which effectively reduces background noise and improves detection sensitivity. In addition, the method can be completed within 15 min for the detection of TDH, has a detection limit below 50 ng/mL and good linearity in the range of 50-5000 ng/mL. Moreover, it has good specificity and no cross-reactivity with Vibrio vulnificus hemolysin (VVH), Clostridium perfringens α toxin (CPA) or C. perfringens ε toxin (ETX). Finally, the sensitivity of this method was unchanged when the three simulated samples of Patinopecten yessoensis, Ruditapes philippinarum, and Scapharca broughtonii tested, indicating that the method is not affected by samples in a complex matrix. In conclusion, this study establishes a practical new method for on-site rapid detection of TDH, which is easy to operate, fast response, easy to carry and can be implemented under the field conditions without expensive equipment and professional person.

Thermal tolerances of Popenaias popeii (Texas hornshell) and its host fish from the Rio Grande Basin, Texas.

Freshwater mussels are particularly sensitive to hydrologic changes, including streamflow and temperature, resulting in global decline. The Devils River in south-central Texas harbors the endangered freshwater mussel Popenaias popeii (Unionidae; Texas hornshell). There is concern that water withdrawals from the underlying aquifer may be negatively impacting this species. To assess this risk, we evaluated upper thermal tolerances (LT05 and LT50) of larvae (glochidia) and juveniles from two sites. After being acclimated to 27 °C, glochidia were subjected to five experimental temperatures (30, 32, 34, 36, and 38 °C) and non-acclimated control (20 °C) for 12-h and 24-h while juveniles were subjected to three experimental temperatures (30, 32, and 36 °C) and non-acclimated control (20 °C) for 96-h. We overlaid tolerance estimates against in situ water temperature and discharge data to evaluate thermal exceedances. Additionally, we reviewed upper thermal tolerances of P. popeii's presumed host fish (Carpiodes carpio, Cyprinellas lutrensis, and Moxostoma congestum) and their congeners. Stream temperatures only occasionally exceeded mussel LT05/50 and fish CLMax/LTMax, likely due to the Devils River's large spring input, highlighting the importance of protecting spring flows. We provide a practical framework for assessing hydrological needs of aquatic ectotherms, including the parasite-host relationship, which can be used to optimize environmental management.

Hologenome analysis reveals independent evolution to chemosymbiosis by deep-sea bivalves.

BACKGROUND: Bivalves have independently evolved a variety of symbiotic relationships with chemosynthetic bacteria. These relationships range from endo- to extracellular interactions, making them ideal for studies on symbiosis-related evolution. It is still unclear whether there are universal patterns to symbiosis across bivalves. Here, we investigate the hologenome of an extracellular symbiotic thyasirid clam that represents the early stages of symbiosis evolution. RESULTS: We present a hologenome of Conchocele bisecta (Bivalvia: Thyasiridae) collected from deep-sea hydrothermal vents with extracellular symbionts, along with related ultrastructural evidence and expression data. Based on ultrastructural and sequencing evidence, only one dominant Thioglobaceae bacteria was densely aggregated in the large bacterial chambers of C. bisecta, and the bacterial genome shows nutritional complementarity and immune interactions with the host. Overall, gene family expansions may contribute to the symbiosis-related phenotypic variations in different bivalves. For instance, convergent expansions of gaseous substrate transport families in the endosymbiotic bivalves are absent in C. bisecta. Compared to endosymbiotic relatives, the thyasirid genome exhibits large-scale expansion in phagocytosis, which may facilitate symbiont digestion and account for extracellular symbiotic phenotypes. We also reveal that distinct immune system evolution, including expansion in lipopolysaccharide scavenging and contraction of IAP (inhibitor of apoptosis protein), may contribute to the different manners of bacterial virulence resistance in C. bisecta. CONCLUSIONS: Thus, bivalves employ different pathways to adapt to the long-term co-existence with their bacterial symbionts, further highlighting the contribution of stochastic evolution to the independent gain of a symbiotic lifestyle in the lineage.

Physiological and molecular responses to hypoxia stress in Manila clam Ruditapes philippinarum.

Hypoxia has become one of the major environmental problems in the aquaculture industry. As one of the most commercially important bivalves, Manila clam Ruditapes philippinarum may be suffering substantial mortality attributable to hypoxia. The physiological and molecular responses to hypoxia stress in Manila clam were evaluated at two levels of low dissolved oxygen: 0.5 mg/L (DO 0.5 mg/L) and 2.0 mg/L (DO 2.0 mg/L). With the prolongation of hypoxia stress, the mortality rate was 100% at 156 h under DO 0.5 mg/L. In contrast, 50% of clams survived after 240 h of stress at DO 2.0 mg/L. After the hypoxia stress, some severe structural damages were observed in gill, axe foot, hepatopancreas tissues, such as cell rupture and mitochondrial vacuolization. For the hypoxia-stressed clams, the significant rise and decline of enzyme activity (LDH and T-AOC) was observed in gills, in contrast to the reduction of glycogen content. Furthermore, the expression levels of genes related to energy metabolism (SDH, PK, Na+/K+-ATPase, NF-κB and HIF-1α) was significantly affected by the hypoxia stress. It is therefore suggested that the short-term survival of clams under hypoxia may be dependent on stress protection by antioxidants, energy allocation, and tissue energy reserves (such as glycogen stores). Despite this, the prolongation of hypoxia stress at DO 2.0 mg/L may cause the irreversible damages of cellular structures in clam tissues, eventually leading to the death of clams. We therefore support the hypothesis that the extent of hypoxia impacts on marine bivalves may be underestimated in the coastal areas.

A sticky carbohydrate meets a mussel adhesive: Catechol-conjugated levan for hemostatic and wound healing applications.

The stickiest natural polysaccharide, levan, plays a role in metalloproteinase activation, which is an important step involved in the healing of injured tissue. However, levan is easily diluted, washed away, and loses adhesion in wet environments, which limits its biomedical applications. Herein, we demonstrate a strategy for fabricating a levan-based adhesive hydrogel for hemostatic and wound healing applications by conjugating catechol to levan. Prepared hydrogels exhibit significantly improved water solubilities, and adhesion strengths to hydrated porcine skin of up to 42.17 ± 0.24 kPa which is more than three-times that of fibrin glue adhesive. The hydrogels also promote rapid blood clotting and significantly faster healing of rat-skin incisions compared to nontreated samples. In addition, levan-catechol exhibited an immune response close to that of the negative control, which is ascribable to its significantly lower endotoxin level compared to native levan. Overall, levan-catechol hydrogels are promising materials for hemostatic and wound healing applications.

Anomaly Detection in Biological Early Warning Systems Using Unsupervised Machine Learning.

The use of bivalve mollusks as bioindicators in automated monitoring systems can provide real-time detection of emergency situations associated with the pollution of aquatic environments. The behavioral reactions of Unio pictorum (Linnaeus, 1758) were employed in the development of a comprehensive automated monitoring system for aquatic environments by the authors. The study used experimental data obtained by an automated system from the Chernaya River in the Sevastopol region of the Crimean Peninsula. Four traditional unsupervised machine learning techniques were implemented to detect emergency signals in the activity of bivalves: elliptic envelope, isolation forest (iForest), one-class support vector machine (SVM), and local outlier factor (LOF). The results showed that the use of the elliptic envelope, iForest, and LOF methods with proper hyperparameter tuning can detect anomalies in mollusk activity data without false alarms, with an F1 score of 1. A comparison of anomaly detection times revealed that the iForest method is the most efficient. These findings demonstrate the potential of using bivalve mollusks as bioindicators in automated monitoring systems for the early detection of pollution in aquatic environments.

Tissue-Specific Toxicokinetics of Aqueous Radium-226 in an Estuarine Mussel, Geukensia demissa.

Radiological contamination of coastal habitats poses potential risk for native fauna, but the bioavailability of aqueous radium (Ra) and other dissolved metals to marine bivalves remains unclear. This study was the first to examine the tissue-specific disposition of aqueous 226Ra in a coastal mussel, specifically the Atlantic ribbed mussel Geukensia demissa. Most organ groups reached steady-state concentrations within 7 days during experimental exposure, with an average uptake rate constant of 0.0013 mL g-1 d-1. When moved to Ra-free synthetic seawater, mussels rapidly eliminated aqueous 226Ra (average elimination rate constant 1.56 d-1). The biological half-life for aqueous 226Ra ranged from 8.9 h for the gills and labial palps to 15.4 h for the muscle. Although previous field studies have demonstrated notable 226Ra accumulation in the soft tissues of marine mussels and that, for freshwater mussels, tissue-incorporated 226Ra derives primarily from the aqueous phase, our tissue-specific bioconcentration factors (BCFs) were on the order of (8.3 ± 1.5) × 10-4 indicating low accumulation potential of aqueous 226Ra in estuarine mussels. This suggests marine and estuarine mussels obtain 226Ra from an alternate route, such as particulate-sorbed Ra ingested during filter-feeding or from a contaminated food source.

MS-CLAM: Mixed supervision for the classification and localization of tumors in Whole Slide Images.

Given the size of digitized Whole Slide Images (WSIs), it is generally laborious and time-consuming for pathologists to exhaustively delineate objects within them, especially with datasets containing hundreds of slides to annotate. Most of the time, only slide-level labels are available, giving rise to the development of weakly-supervised models. However, it is often difficult to obtain from such models accurate object localization, e.g., patches with tumor cells in a tumor detection task, as they are mainly designed for slide-level classification. Using the attention-based deep Multiple Instance Learning (MIL) model as our base weakly-supervised model, we propose to use mixed supervision - i.e., the use of both slide-level and patch-level labels - to improve both the classification and the localization performances of the original model, using only a limited amount of patch-level labeled slides. In addition, we propose an attention loss term to regularize the attention between key instances, and a paired batch method to create balanced batches for the model. First, we show that the changes made to the model already improve its performance and interpretability in the weakly-supervised setting. Furthermore, when using only between 12 and 62% of the total available patch-level annotations, we can reach performance close to fully-supervised models on the tumor classification datasets DigestPath2019 and Camelyon16.

Are clam-seagrass interactions affected by heatwaves during emersion?

The increased frequency of heatwaves expected in the context of global warming will affect socio-ecological systems such as shellfish beds at intertidal seagrass meadows. A mesocosm experiment was performed to assess the effects of a simulated atmospheric heatwave during low tide on the bioturbation indicators and growth of the commercial juvenile native Ruditapes decussatus and the introduced clam R. philippinarum, and on their interactions with the seagrass Zostera noltei. Under the heatwave, heat dissipation at 5 cm depth was significantly greater in the sediments below Z. noltei than below bare sand, the photosynthetic efficiency (Fv/Fm) of Z. noltei decreased and the clams tended to grow less. Furthermore, after the heatwave clams below bare sand tended to burrow deeper than those below Z. noltei, indicating that seagrass provided a refuge for clams. Ruditapes philippinarum grew less, and did not burrow as deeply as R. decussatus, which may imply greater vulnerability to desiccation and heat at low tide. The particle displacement coefficient (PDC) of R. philippinarum indicated lower bioturbation values in Z. noltei than in bare sand and was a suitable bioturbation indicator for juvenile Ruditapes spp. clams. In Z. noltei coexisting with R. philippinarum, the Fv/Fm values were higher than without clams after a recovery period, which may be linked to the assimilation of phosphate excreted by the clams and suggests a facilitative interaction. No such interaction was observed with R. deccusatus, probably because of its deeper burrowing depth. The findings suggest reciprocal facilitative interactions between R. philippinarum and Z. noltei and the potential contribution of Z. noltei to the sustainability of clams under global warming scenarios, which may support management actions aimed at enhancing the coexistence between shellfishing activities and seagrass conservation.

Methodological advances in the detection of biotoxins and pathogens affecting production and consumption of bivalve molluscs in a changing environment.

The production, harvesting and safe consumption of bivalve molluscs can be disrupted by biological hazards that can be divided into three categories: (1) biotoxins produced by naturally occurring phytoplankton that are bioaccumulated by bivalves during filter-feeding, (2) human pathogens also bioaccumulated by bivalves and (3) bivalve pathogens responsible for disease outbreaks. Environmental changes caused by human activities, such as climate change, can further aggravate these challenges. Early detection and accurate quantification of these hazards are key to implementing measures to mitigate their impact on production and safeguard consumers. This review summarises the methods currently used and the technological advances in the detection of biological hazards affecting bivalves, for the screening of known hazards and discovery of new ones.

Understanding the aggregation, consumption, distribution and accumulation of nanoparticles of polyvinyl chloride and polymethyl methacrylate in Ruditapes philippinarum.

Plastic products have become an integral part of our life. A widespread usage, high stability, uncontrolled disposal and slow degradation of plastics in the environment led to the generation and accumulation of nanoparticles of polymers (NPs) in the marine environment. However, little is known about the aggregation, consumption and distribution of NPs from common polymers such as polyvinyl chloride (NP-PVC) and polymethyl methacrylate (NP-PMMA) inside marine animal physiologies. In the current study, two types of polymers (PVC and PMMA) × four exposure concentrations (1, 5, 15 and 25 mg/L) × four times (4, 8, 12 and 24 h) exposure studies were conducted to understand the consumption and distribution of luminescent NP-PVC (98.6 ± 17.6 nm) and NP-PMMA (111.9 ± 37.1 nm) in R. philippinarum. Under laboratory conditions, NP-PVC showed a higher aggregation rate than NP-PMMA in seawater within a period of 24 h. Aggregations of NPs increased with an increase in initial NP concentrations, leading to significant settling of nanoparticles within 24 h exposure. Such aggregation and settling of particles enhanced the consumption of NPs by benthic filter-feeding R. philippinarum at all exposure concentrations during 4 h exposure. More interestingly, NP-PVC and NP-PMMA were observed in large amounts in both liver and gills (22.6 % - 29.1 %) of the clams. Furthermore, NP-PVC was detected in most organs of R. philippinarum as compared to NP-PMMA. This study demonstrates that different polymers distribute and accumulate differently in the same biological model under laboratory exposure conditions based on their chemical nature.

Norgestrel causes digestive gland injury in the clam Mactra veneriformis: An integrated histological, transcriptomics, and metabolomics study.

The potential adverse effects of progestins on aquatic organisms, especially non-target species, are of increasing concern worldwide. However, the effect and mechanism of progestin toxicity on aquatic invertebrates remain largely unexplored. In the present study, clams Mactra veneriformis were exposed to norgestrel (NGT, 0, 10, and 1000 ng/L), the dominant progestin detected in the aquatic environment, for 21 days. NGT accumulation, histology, transcriptome, and metabolome were assessed in the digestive gland. The bioconcentration factor (BCF) was 386 and 268 in the 10 ng/L NGT group and 1000 ng/L NGT group, respectively, indicating efficient accumulation of NGT in the clams. Histological analysis showed that NGT led to the swelling of epithelial cells and blurring of the basement membrane in the digestive gland. Differentially-expressed genes and KEGG pathway enrichment analysis using a transcriptomic approach suggested that NGT primarily disturbed the detoxification system, antioxidant defense, carbohydrate and amino acid metabolism, and steroid hormone metabolism, which was consistent with the metabolites analyzed using a metabolomic approach. Furthermore, we speculated that the oxidative stress caused by NGT resulted in histological damage to the digestive gland. This study showed that NGT caused adverse effects in the clams and sheds light on the mechanisms of progestin interference in aquatic invertebrates.

Hepatitis A virus detection by RT-qPCR in shellfish samples from three Moroccan Atlantic coastal areas: Dakhla, Oualidia, and Moulay Bousselham.

This comparative study assessed hepatitis A virus (HAV) contamination in shellfish harvesting areas in Morocco, and the correlations between viral contamination and rainfall. To this aim, HAV contamination was evaluated in 156 shellfish samples collected at three Moroccan coastal areas (52 samples/area): Dakhla (class A), Oualidia (class B), and Moulay Bousselham (class C). Samples were collected monthly between March 2018 and March 2019, and included oysters from different farms at the Oualidia and Dakhla coastal areas, and wild mussels at the Moulay Bousselham lagoon. HAV was detected by RT-qPCR in 24/156 (15.38%) samples: 16/145 (11.03%) mussel (Mytillus galloprovincialis) and 8/156 (5.13%) oyster (Crassostea gigas) samples. The 16 mussel-positive samples (16/52) were collected at Moulay Bousselham, and the eight oyster-positive samples (8/52) were collected at Oualidia, and none at Dakhla (class A). The highest HAV contamination rate was recorded at Moulay Bousselham lagoon (class C) (30.77%), while it did not exceed 8% at Oualidia (class A). A nonsignificant, positive correlation between HAV-positive samples and mean rainfall was observed. The important HAV presence in bivalve mollusks from the Oualidia and Moulay Bousselham lagoons indicate the need of viral surveillance of bivalve mollusks and of improving wastewater quality to enhance shellfish safety.

Parasites and their impact on thick-shelled river mussels Unio crassus from two populations in Luxembourg.

The thick-shelled river mussel Unio crassus Philipsson, 1788 is a species native to many European habitats, with declining populations. The impact of parasite communities on health status of this species is poorly understood. In this study, parasites of 30 U. crassus specimens from the Our and Sauer Rivers in Luxembourg were identified morphologically and, in some cases, using molecular genetic methods. The findings were correlated to selected parameters (total length, visceral weight, shell lesions, gonadal stage). The 2 populations did not differ in shell length, visceral weight, number of males and females, gonadal scoring, shell lesions, and the occurrence of glochidia. The prevalence and infestation intensities of detected Trichodina sp., Conchophthirus sp., and freshwater mite larvae did not differ between the 2 populations, whereas the prevalence and infestation intensities of mite eggs, nymphs, and adults were significantly higher in the Sauer River. Rhipidocotyle campanula and European bitterling Rhodeus amarus larvae were only present in the Sauer. Histopathology revealed the destruction of the gonads by R. campanula and tissue damage by the mites. The only significant correlation of the selected parameters was a positive correlation between R. amarus occurrence and total length as well as a negative correlation between R. amarus occurrence and gonadal stage. In the Sauer River, 2 mussels were found to be hermaphrodites.

The Inhibitory Effects of RNA-Interference-Mediated Guanylate Cyclase Knockdown on Larval Metamorphosis and Early Progeny Growth of Razor Clam.

Guanylate cyclase (GC, cGMPase) is a key enzyme in organisms, catalyzing the synthesis of cGMP from GTP, thus making cGMP work. cGMP plays a vital role in the regulation of cell and biological growth as a second messenger in signaling pathways. In this study, we screened and identified cGMPase from the razor clam Sinonovacula constricta, which encoded 1257 amino acids and was widely expressed in different tissues, especially the gill and liver. We also screened one double-stranded RNA (dsRNA), cGMPase, which was used to knockdown cGMPase at three larval metamorphosis development stages: trochophores-veliger larve, veliger larve-umbo larve, and umbo larve-creeping larvae. We showed that interference at these stages significantly inhibited larval metamorphosis and survival rates. cGMPase knockdown resulted in an average metamorphosis rate of 60% and an average mortality rate of 50% when compared with control clams. After 50 days, shell length and body weight were inhibited to 53% and 66%, respectively. Thus, cGMPase appeared to regulate metamorphosis development and growth in S. constricta. By examining the role of the key gene in the metamorphosis development of S. constricta larvae and the growth and development period, we can provide some data reference for studying the growth and development mechanism of shellfish, and the results provided basic information for the breeding of S. constricta.

Patterns of dreissenid mussel invasions in western US lakes within an integrated gravity model framework.

Freshwater invasive species, such as the quagga mussel (Dreissena rostriformis bugensis), are causing over $1 billion USD annually in damages to water infrastructure, recreation, and the environment. Once established, quagga and other dreissenid mussels are extremely difficult to eradicate. Preventing the spread of these invasives is critical and of high management concern. Invasive dreissenid establishment is predicated upon both successful dispersal from a source and suitable habitat in the uninfested waterbody to which they are transported. Recreational boaters have become predominant dispersal vectors making it possible to forecast the risk of invasion of waterbodies for more targeted management and prevention. We developed an integrated mussel dispersal model that couples a constrained gravity model and habitat suitability model to forecast future invasions. The model simulates boater movement between lakes, the likelihood of boats transporting mussels, and the likelihood that those mussels survive in the environmental conditions of the new lake. Model output was most sensitive to changes in boater threshold, then buffer zones, while not as sensitive to changes in habitat suitability. From an initial infested source pool of 11 among 402 Western inland US lakes, we forecast additional lakes infested in several possible simulation scenarios. Constraining movement reduced connectivity between waterbodies with amplifying effects at different distance levels. This model can be used to determine waterbodies most at risk for dreissenid mussel invasion and to highlight the importance of multifactor integrated models in environmental management.

High temperatures enhance the strength of multiple predator effects in a typical crab-clam system.

Although marine heatwaves pose urgent threats to marine life, our understanding of how these events influence interactions between key species in marine ecosystems is still inadequate. Herein, we examined the behavioral mechanisms by which heat regulates multiple predator effects in different foraging systems that include Asian paddle crabs (Charybdis japonica) and swimming crabs (Portunus trituberculatus) by quantifying their predation and competition at two temperatures. Our results show that non-independent multiple predator effects occurred in the conspecific treatment of Asian paddle crabs and in the interspecific treatment, whereas independent multiple predator effects occurred in the conspecific treatment of swimming crabs. Asymmetrical behavior responses of these crabs to competition and heat triggered divergences in multiple predator effects. High temperatures increased the strength of multiple predator effects but did not alter their types. The reason is that heat negatively impacts predation by enhancing aggressive interactions, outweighing its direct positive effects on predation.

Molecular Cloning and Characteristics of a Lectin from the Bivalve Glycymeris yessoensis.

C-type lectins (CTLs) are a family of carbohydrate-binding proteins that mediate multiple biological events, including adhesion between cells, the turnover of serum glycoproteins, and innate immune system reactions to prospective invaders. Here, we describe the cDNA cloning of lectin from the bivalve Glycymeris yessoensis (GYL), which encodes 161 amino acids and the C-type carbohydrate recognition domain (CRD) with EPN and WND motifs. The deduced amino acid sequence showed similarity to other CTLs. GYL is a glycoprotein containing two N-glycosylation sites per subunit. N-glycans are made up of xylose, mannose, D-glucosamine, 3-O-methylated galactose, D-quinovoses, and 3-O-methylated 6-deoxy-D-glucose. The potential CRD tertiary structure of the GYL adopted CTL-typical long-form double-loop structure and included three disulfide bridges at the bases of the loops. Additionally, when confirming the GYL sequence, eight isoforms of this lectin were identified. This fact indicates the presence of a multigene family of GYL-like C-type lectins in the bivalve G. yessoensis. Using the glycan microarray approach, natural carbohydrate ligands were established, and the glycotope for GYL was reconstructed as "Galß1-4GlcNAcß obligatory containing an additional fragment", like a sulfate group or a methyl group of fucose or N-acetylgalactosamine residues.