Triangle Sail Mussel Research Articles

The First High-Quality Genome Assembly of Freshwater Pearl Mussel Sinohyriopsis cumingii: New Insights into Pearl Biomineralization.

China leads the world in freshwater pearl production, an industry in which the triangle sail mussel (Sinohyriopsis cumingii) plays a pivotal role. In this paper, we report a high-quality chromosome-level genome assembly of S. cumingii with a size of 2.90 Gb-the largest yet reported among bivalves-and 89.92% anchorage onto 19 linkage groups. The assembled genome has 37,696 protein-coding genes and 50.86% repeat elements. A comparative genomic analysis revealed expansions of 752 gene families, mostly associated with biomineralization, and 237 genes under strong positive selection. Notably, the fibrillin gene family exhibited gene family expansion and positive selection simultaneously, and it also exhibited multiple high expressions after mantle implantation by transcriptome analysis. Furthermore, RNA silencing and an in vitro calcium carbonate crystallization assay highlighted the pivotal role played by one fibrillin gene in calcium carbonate deposition and aragonite transformation. This study provides a valuable genomic resource and offers new insights into the mechanism of pearl biomineralization.

Low-complexity domain-containing protein (LCDP), induced accumulation of calcium carbonate individual crystals to irregular polycrystals in the triangle sail mussel Hyriopsis cumingii

The nacre layer is composed of sheet-like aragonite crystals, with often loosely arranged polycrystal aragonite sheets which may induce poor mechanical properties in shells. In this study, a full-length low-complexity domain-containing protein (LCDP) cDNA from the triangle sail mussel Hyriopsis cumingii was generated and its role in shell formation investigated. The full-length cDNA was 1058 bp; it had an open reading frame (ORF) of 714 bp encoding 237 amino acids and contained a 20-amino acid signal peptide at the N-terminus and two low-complexity domains. H. cumingii LCDP was not homologous with other species. Tissue expression analyses showed that LCDP was specifically expressed in the mantle. In shell repair assays, significantly higher LCDP expression was observed in the shell repair group from days 12–21 (p < 0.01). After LCDP silencing, aragonite flake shapes in pearl layers became irregular with disordered deposition, while calcium carbonate (CaCO3) crystal surfaces in prismatic layers became rougher and organic matrices between crystals appeared skeletonized, indicating the importance of biomineralization. Our in vitro CaCO3 crystallization assays showed that LCDP induced single crystals to polycrystals, probably via loose arrangement between aragonite flakes. These results provide new insights on freshwater mollusk biomineralization and a theoretical basis for improved pearl quality.

Hc-transgelin is a novel matrix protein gene involved in the shell biomineralization of triangle sail mussel (Hyriopsis cumingii)

In triangle sail mussel (Hyriopsis cumingii), shell biomineralization is a complicated process that involves multiple gene products. Shell matrix proteins are involved in the formation of the organic framework and play an important role in the regulation of calcium carbonate deposition. In this study, A new shell matrix protein gene Hc-transgelin was identified in H. cumingii. The full-length cDNA of Hc-transgelin was 1200 bp, including a 501 bp open reading frame, which encoded 166 amino acids. Hc-transgelin is rich in lysine, it accounts for 11.40% of the protein. The predicted transgelin protein contained a conserved calmodulin homologous domain. A tissue-specific expression assay indicated that Hc-transgelin exhibited significantly highest expression in the mantle. Furthermore, Hc-transgelin in situ hybridization detected positive signals at the edge of the mantle outer fold, where nacre and prismatic layer biomineralization occur. An RNA interference assay showed that the shape of aragonite flakes in nacre changed and their growth was inhibited, and cracks appeared in the prismatic layer organic sheath when the expression of Hc-transgelin was suppressed. In a shell repair assay, a higher expression of Hc-transgelin appeared from day 12 to day 25 when the nacre accumulated quickly. These findings indicate that Hc-transgelin may be involved in the formation of aragonite flakes in the nacre and play a role in the formation of the organic sheath in the prismatic layer of the shell. This study provides new insights into the role of the Hc-transgelin gene and also contributes to the molecular understanding of mollusk shell formation.

Genomic selection for improvement of growth traits in triangle sail mussel (Hyriopsis cumingii)

The triangle sail mussel, Hyriopsis cumingii, is the most important aquaculture species for freshwater pearl production in China. However, one major problem of freshwater pearl mussel industry is the low profit despite high pearl yield, and the focus of current research has now switched from yield to the improvement of pearl quality. It is essential to enhance the quality of pearls by improving the growth performance of mussels and, to this end, genomic selection (GS) has been proved as a useful method for selective breeding. In the present study, 764 mussels (496 for reference population, 268 for selection candidates) were genotyped by sequencing (GBS) and a total of 97,090 single nucleotide polymorphisms (SNPs) were identified. Based on genome-wide SNPs, the heritability was estimated to be moderate to high for the following five growth traits: shell length, shell height, shell width, shell weight, and body weight (0.36 ± 0.11 to 0.51 ± 0.10). Then, the effects of five GS models (i.e., GBLUP, BayesA, BayesB, BayesCπ, and BayesLASSO) and different SNP numbers (selected using three SNP screening strategies) on the predictive ability (PA) of the genomic estimated breeding values (GEBVs) were compared. The GBLUP model containing 10,000 SNPs selected by GWAS strategy was established and was used to calculate GEBVs in selection candidates. The individuals with high GEBVs were then selected as broodstock for the fast-growth strain (FS), and the rest for control strains (CS). At last, encouraging results were detected that the FS performed significantly better than CS in terms of growth, with different traits increasing by 5.74–28.77% and 11.93%–54.58% at the WY and CM sites, respectively. These results may assist in the future large-scale commercial application of GS in H. cumingii and provide a cost-efficient GS strategy for other aquaculture species as well.

β-arrestin interacts with TRAF6 to negatively regulate the NF-κB pathway in triangle sail mussel Hyriopsis cumingii

As members of arrestins family, β-arrestins are widely expressed in monocytes, macrophages, neutrophils and other immune cells. They can regulate the immune response of bodies through various ways. In the present study, a β-arrestin homolog named Hcβ-arrestin was cloned and identified from Hyriopsis cumingii. Predicted Hcβ-arrestin protein contained a conserved arrestin domain, which could be further divided into arrestin-N (39-192aa) and arrestin-C (211-365aa). Amino acid sequence alignment showed that it had the highest identity with Mytilus galloprovincialis and Mytilus edulis counterpart, which was 89.02% and 87.68%, respectively. Furthermore, real-time quantitative PCR analysis showed that the Hcβ-arrestin gene was widely expressed in the detected tissues and with the highest expression in hepatopancreas. The transcription of Hcβ-arrestin in hepatopancreas and gill of mussels was significantly up-regulated after stimulation with peptidoglycan, lipopolysaccharide (LPS) and polyinosinic polycytidylic acid. Knockdown of Hcβ-arrestin gene significantly increased the expression of some antibacterial effector genes, such as lysozyme, LPS-binding protein/bactericidal permeability increasing protein and theromacin in hepatopancreas and gills of LPS stimulated mussels, but only had little effect on TLR pathway genes. In addition, GST pull-down assay confirmed that Hcβ-arrestin can bind to HcTRAF6 protein in vitro. Dual luciferase reporter assay showed that the co-expression of HcTRAF6 and Hcβ-arrestin inhibited the activation of NF-κB reporter by HcTRAF6. These findings indicated that Hcβ-arrestins could interact with HcTRAF6 to negatively regulate the NF-κB pathway in H. cumingii.

Effective treatment of aquaculture wastewater with mussel/microalgae/bacteria complex ecosystem: a pilot study

The discharge of aquaculture wastewater increased significantly in China. Especially, high content of nitrogen and phosphorus in wastewater could destroy the receiving water environment. To reduce the pollution of aquaculture wastewater, farmed triangle sail mussel (Hyriopsis cumingii) was proposed to be cultivated in the river. This was the first time that bacteria (Bacillus subtilis and Bacillus licheniformis) and microalgae (Chlorella vulgaris) were also used and complemented ecosystem functions. The pollutants in wastewater were assimilated by Chlorella vulgaris biomass, which was then removed through continuous filter-feeding of Hyriopsis cumingii. While, Bacillus subtilis and Bacillus licheniformis enhanced the digestive enzyme activities of mussel. It demonstrated that approximately 4 mussels/m3 was the optimal breeding density. Under such condition, orthogonal experiment indicated that the dose of Bacillus subtilis, Bacillus licheniformis, and Chlorella vulgaris should be 0.5, 1, and 2 mL respectively. Compared with mussel, mussel/microalgae, mussel/bacteria system, treatment ability of the mussel/microalgae/bacteria system in batch experiment was better, and 94.67% of NH3-N, 92.89% of TP and 77.78% of COD were reduced after reaction for 6 days. Finally, 90 thousand mussels per hectare of water were cultivated in Kulv river in China, and the field experiment showed that water quality was significantly improved. After about 35 days of operation, NH3-N, TN, TP and COD concentration were maintained around 0.3, 0.8, 0.3, and 30 mg/L respectively. Therefore, the mussel/microalgae /bacteria system in this study showed a sustainable and efficient characteristic of aquaculture wastewater bioremediation.

Combined effects of ration levels and temperature on immune responses of the triangle sail mussel Hyriopsis cumingii

Nowadays, the cultivation mode of Hyriopsis cumingii urgently needs transformation and upgrading, and realizing sustainable aquaculture lacks the selection of suitable algae and the exploration of culture conditions. This experiment is to evaluate combined effects of ration levels (low ration, medium ration and high ration) and temperature (13.0 ± 0.2°C, 23.0 ± 0.2°C, and 33.0 ± 0.2°C) on immune responses of H. cumingii. Activities of immune enzymes (alkaline phosphatase, superoxide dismutase, catalase and lysozyme) and expression of immune-related genes (interleukin-17 and inhibitor of apoptosis protein) in mussels were analysed during 21 days exposure period. Results suggested that the diatom Navicula pelliculosa was suitable algae for H. cumingii, the optimal ration level and temperature of H. cumingii were the medium ration and 23℃. Semi-starvation, excessive dietary, low- or high-temperature stimulated immune activities, and when beyond the immune regulation range, most immune enzyme activities decreased. Acute stress also stimulated expression of HcIL-17 and IAP, and long-term stress of discomfort temperature and ration levels led to immune dysfunction and affected expression of these genes. The study has practical significance in realizing the sustainable aquaculture of H. cumingii and provides a broader perspective regarding the water ecosystem.

Characterization of a novel toll-like receptor and activation NF-κB signal pathway in triangle sail mussel Hyriopsis cumingii.

Toll-like receptor (TLR) family plays an important role in innate immunity for detection of and defense against microbial pathogens. In this study, a novel toll-like receptor (HcTLRn) was characterized from freshwater pearl mussel H. cumingii. The complete sequence of HcTLRn was 3725 bp, and the open reading frame (ORF) encoded 718 amino acid residues. Predicted HcTLRn protein possessed seven atypical leucine-rich repeat (LRR) domains, two typical LRR subfamily domains, a C-terminal domain LRR, a transmembrane domain and an intracellular Toll/interleukin-1 (IL-1) receptor domain. Transcripts of HcTLRn were constitutive expressed in the tissues of healthy mussels and were markedly induced in hepatopancreas and gills after lipopolysaccharide (LPS), peptidoglycan (PGN) and polyinosinic polycytidylic acid (ploy I: C) stimulation. Knockdown of HcTLRn in vivo significantly decreased the mRNA levels of TLR pathway transcription factors p65 and p105 as well as antimicrobial peptides (AMPs) including lysozyme (HcLys), theromacin (HcTher), whey acidic protein (HcWAP), LPS-binding protein/bactericidal permeability increasing protein (HcLBP/BPI) 1 and 2 after mussels challenged by LPS. In situ hybridization results showed that HcTLRn mRNA was significantly increased in hemocytes after LPS, PGN and poly I:C stimulation. HcTLRn protein was mainly expressed in hepatopancreas and gills and was significantly increased after LPS stimulation. Moreover, recombinant extracellular domain of HcTLRn (HcTLRn-ECD) proteins could bind to a variety of bacterial and pathogen-associated molecular patterns such as LPS, PGN, and poly I:C in vitro. Subcellular localization results showed that HcTLRn was mainly distributed near the cell membrane and in cytoplasm. Over-expression of HcTLRn activated the NF-κB luciferase reporter in HEK293T cells. Collectively, these results suggested that HcTLRn was a TLR family member that might play an important role in activation of NF-κB signal pathway in Mollusca.

Dorsal regulates the expression of two phage lysozymes acquired via horizontal gene transfer in triangle sail mussel Hyriopsis cumingii

Dorsal is a Rel/NF-κB transcription factor, which forms a key part of the Toll pathway. Lysozyme is a ubiquitous enzyme that degrades bacterial cell walls. In this study, a Dorsal homolog was cloned and characterized from triangle sail mussel Hyriopsis cumingii, namely, HcDorsal. Dorsal consisted of 3041 bp, including a 1938 bp open reading frame encoding a 645 amino acid protein. The deduced HcDorsal protein contained a Rel homology domain and an Ig-like, plexin, transcription factor domain. Analysis of expression patterns showed that HcDorsal was highly expressed in the hepatopancreas of H. cumingii. The expression level of HcDorsal continuously increased after Vibrio parahaemolyticus stimulation. When HcDorsal was knocked down by siRNA interference, two phage lysozyme genes (HcLyso1 and HcLyso2) obtained by horizontal gene transfer were significantly downregulated in hemocytes of mussels. Furthermore, knockdown of HcLyso1 and HcLyso2 could weaken V. parahaemolyticus clearance ability. Recombinant HcLyso1 and HcLyso2 proteins accelerated the bacterial clearance in vivo in mussels and evidently inhibited the growth of V. parahaemolyticus. These results suggested that HcDorsal could be activated after V. parahaemolyticus stimulation and then modulate the immune response through the transcriptional regulation of HcLyso1 and HcLyso2, thereby playing a protective role in mussels.

Expression and functional characterization of the CUB domain-containing protein from the triangle sail mussel (Hyriopsis cumingii) in response to pathogenic infection.

The complement C1r/C1s, Uegf, and Bmp1 (CUB) domains, which are most exclusively found in extracellular and plasma membrane-related proteins, are involved in various biological processes. In this study, a CUB domain-containing protein (designed as HcCDCP) was cloned and characterized from freshwater pearl mussel (Hyriopsis cumingii). The 2280bp complete cDNA of the HcCDCP contained a 1002bp open reading frame, which encoded a protein with 333 amino acids. The predicted HcCDCP protein contained a typical CUB domain and a transmembrane region. The tissue distribution analysis indicated that the HcCDCP was detected in all tissues, and the highest expression was found in hepatopancreas followed by gills. After infection with bacteria (i.e., Staphylococcus aureus and Vibrio parahaemolyticus), virus (white spot syndrome virus) and virus analogs (poly[I:C]), the mRNA level of the HcCDCP was significantly upregulated, suggesting that the HcCDCP might be involved in host immune defense response. The RNA interference revealed that the silencing of the HcCDCP could evidently inhibit the expression levels of lysozyme and tumor necrosis factor. Moreover, the recombinant protein of the CUB domain (rCUB) possessed binding capacity to eight different kinds of bacteria. The polysaccharide binding assay showed that the rCUB specifically bound to lipopolysaccharide, peptidoglycan, and D-mannose. This study provided valuable information for exploring the biological roles of CDCPs in the host defense system of mollusks.

Growth, calcium content, proximate composition, and fatty acid composition of triangle sail mussel (Hyriopsis cumingii) fed five different microalgal diets

Sustainable cultivation of triangle sail mussel Hyriopsis cumingii lacks a suitable, optimal diet. To determine the optimum microalgal diet H. cumingii, we tested five microalgal diets (Cyclotella sp., Chlorella pyrenoidosa, Scenedesmus dimorphus, Monoraphidium contortum, and a mix of these four microalgae) using single factor experiments. We measured body growth, pearl growth, proximate composition, fatty acid composition, and calcium (Ca) content in the mantle and foot of H. cumingii. Overall, diet had significant effects on the growth of H. cumingii (P < .05), and the group fed Cyclotella sp. showed the greatest (P < .05) increases in weight, shell length, height, and width, and pearl weight. The Ca content of the mantle and foot also differed significantly between treatments (P < .05); the highest content in the foot was observed in mussels fed Cyclotella sp., while the highest content in the mantle was found in mussels fed S. dimorphus and M. contortum. The crude protein contents of the foot and mantle were highest in mussels fed Cyclotella sp., and both were positively correlated with growth; the moisture content of the foot was negatively correlated with growth. The mussels fed the mixed microalgae had the highest polyunsaturated fatty acids (PUFAs) proportion (P < .05), while the group fed Cyclotella sp. showed the lowest PUFAs and the highest saturated fatty acids (SFAs) and monounsaturated fatty acids (MUFAs) proportions (P < .05). The proportions of SFAs and MUFAs were positively correlated with growth, and the proportion of PUFAs was negatively correlated with growth. Principal component analysis showed that the fatty acid composition differed among diet groups. In summary, mussels showed different growth, Ca content, and proximate and fatty acid compositions when fed different microalgal diets. Cyclotella sp. was the optimum microalgal diet, followed by S. dimorphus. Based on growth rate and tissue compositions of H. cumingii obtained here, a suitable diet for this species should include these microalgae.

Antioxidant responses of triangle sail mussel Hyriopsis cumingii exposed to toxic Microcystis aeruginosa and thermal stress

Harmful algal blooms (HABs) and thermal stress as climate changes become more common in global water ecosystem, especially under eutrophic habitats. Here our study examined the combined impacts of bloom forming cyanobacteria Microcystis aeruginosa and thermal stress on the antioxidant responses of the ecologically important species triangle sail mussel Hyriopsis cumingii. The differential responses of a series of enzymes, e.g. superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and glutathione-S-transferase (GST), as well as signal metabolites including reactive oxygen species (ROS), malondialdehyde (MDA) and glutathione (GSH) involved in antioxidant defense mechanisms were analyzed during 14 d exposure to toxic cyanobacterium M. aeruginosa and 7 d depuration period. The activities of SOD and GPx as well as the content of ROS and MDA in H. cumingii increased, while CAT activity reduced due to M. aeruginosa exposure. Thermal stress resulted in decrease of CAT, the accumulation of GSH and the enhance of GST and SOD. Meanwhile, the interactive effects among M. aeruginosa, thermal stress and time were also observed on most parameters except for GST activity. The total amount of microcystins (MC) in sail mussels increased with concentrations of exposed M. aeruginosa, independently of the presence or absence of thermal stress. Although around 50% of MC in mussels dropped in the depuration period, most parameters showed alterations because of cyanobacteria exposure and thermal stress. Overall, these findings suggested that toxic cyanobacteria or thermal stress induces oxidative stress and severely affects the enzymes activities and intermediates level associated with antioxidant defense mechanisms in sail mussels respectively. More importantly, the toxic impacts on sail mussels could be intensified by their combination.

Antioxidant responses of triangle sail mussel Hyriopsis cumingii exposed to harmful algae Microcystis aeruginosa and high pH.

In lakes and reservoirs, harmful algal blooms and high pH have been deemed to be two important stressors related to eutrophication, especially in the case of CO2 depletion caused by dense blooms. However, the effects of these stressors on the economically important shellfish that inhabit these waters are still not well-understood. This study evaluated the combined effects of the harmful algae Microcystis aeruginosa (0%, 50%, and 100% of total dietary dry weight) and high pH (8.0, 8.5 and 9.0) on the antioxidant responses of the triangle sail mussel H.cumingii. The mussels were exposed to algae and high pH for 14 d, followed by a 7-day depuration period. Reactive oxygen species (ROS) in the mussel hemolymph, antioxidant and detoxifying enzymes, such as glutathione-S-transferase (GST), glutathione (GSH), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and malondialdehyde (MDA) in the digestive glands were analyzed during the experimental period. GST, SOD and GPx activity levels and the content of GSH increased following exposure to toxic M.aeruginosa, whereas CAT activity was inhibited. pH showed no significant effects on the immune defense mechanisms and detoxification processes. However, a high pH could cause increased ROS and MDA levels, resulting in oxidative injury. After a 7-day depuration period, exposure to toxic M.aeruginosa or high pH resulted in latent effects for most of the examined parameters. The treatment group exposed to the highest pH (9.0) displayed an increased oxidation state compared with the other pH treatments (8.0 and 8.5) for the same concentrations of toxic M.aeruginosa. The trends observed for ROS, MDA, GPx, GST, SOD and GSH levels indicated that a high density of toxic algae could result in severe and continuous effects on mussel health.

The Suppressor of Cytokine Signaling-6 Gene and Its Expression during Wound Healing and Infection in the Triangle Sail Mussel Hyriopsis cumingii

Suppressors of cytokine signaling (SOCS) family members can inhibit multiple biological functions of cytokines, hormones, and growth factors. In this study, a SOCS6 gene homolog from triangle sail mussel Hyriopsis cumingii (HcSOCS6) was cloned and characterized. The full-length cDNA of HcSOCS6 was 1,823 bp and encoded 458 amino acid residues. No introns were found in the genomic sequence. The deduced HcSOCS6 protein possesses highly conserved functional domains, including the Src homology 2 domain and the SOCS box. Phylogenetic analysis showed that HcSOCS6 was clustered with other molluscan genes. Transcripts of HcSOCS6 were constitutively expressed in all examined tissues, at high levels in the hepatopancreas and gills and low levels in hemocytes. Transcription of HcSOCS6 was significantly upregulated in hemocytes and the hepatopancreas after injecting with lipopolysaccharide, but it was only upregulated in hemocytes after Aeromonas hydrophila stimulation. The expression of HcSOCS6 in mantle significantly increased during wound healing, especially on the third day. These results suggested that HcSOCS6 was involved in the immune response and wound healing in the triangle sail mussel.

Combined effects of toxic Microcystis aeruginosa and hypoxia on the digestive enzyme activities of the triangle sail mussel Hyriopsis cumingii

Nowadays, eutrophication is a very popular environmental problem in numerous waters around the world. The main reason of eutrophication is the enrichment of the nutrient, which results in the excessive growth of phytoplankton and some of them are toxic and harmful. Fortunately, some studies have shown that some bivalves can filter the overgrown phytoplankton in water, which may alleviate water eutrophication. However, the physiological effects of toxic cyanobacteria on filter feeding animal have not been clarified very well. In this experiment, digestive enzyme activities in Hyriopsis cumingii exposed to different concentrations of the toxic Microcystis aeruginosa (0, 5 * 105 and 5 *106 cell ml-1) at two dissolved oxygen (DO) levels (6 and 2 mg l-1) for 14 days were investigated. Toxic M. aeruginosa significantly affected all digestive enzyme activities throughout the experiment. At high toxic M. aeruginosa concentration, the activities of cellulase, amylase and lipase in digestive gland and stomach were significantly increased (P<0.05). However, hypoxia reduced the activities of cellulase, amylase and lipase in digestive gland and stomach. Conflicting effects were observed between toxic M. aeruginosa and DO in most digestive enzyme activities during the exposure period. Therefore, it is not conducive for the digestion and absorption of M. aeruginosa in H. cumingii under hypoxic conditions. H. cumingii is tolerant to toxic M. aeruginosa and may remove toxic cyanobacteria from waters under normal DO conditions.

Integrated transcriptome analysis of immunological responses in the pearl sac of the triangle sail mussel (Hyriopsis cumingii) after mantle implantation

For pearl culture of bivalve Hyriopsis cumingii, implantation of the sabio may cause nucleus discharge and increased host death rates. We performed a transcriptome analysis of the pearl sac of H. cumingii for 30 days after mantle implantation; 293863 unigenes were obtained, and 27176 unigenes were identified using nr, nt, KO, Swiss-Prot, Pfam, GO, and KOG databases. We detected 4878 differentially expressed genes (DEGs) through pairwise comparisons. We speculated that the physical condition of the recipient mussels returned to normal in about one month; the period was divided into six vital phases (0, 2 h–6 h, 12 h–24 h, 48 h to 7 days, 14 days and 30 days) on the basis of the overall similarities in DEGs. We compared the DEGs between time points and identified key immune-related genes. Our findings provide information on the immunological reactions induced by implantation in pearl mussels.

HcCUB-Lec, a newly identified C-type lectin that contains a distinct CUB domain and participates in the immune defense of the triangle sail mussel Hyriopsis cumingii

As pattern recognition receptors (PRRs), C-type lectins (CTLs) play crucial roles in recognizing and eliminating pathogens in innate immunity. In this study, a novel CTL (HcCUB-Lec) was identified from the triangle sail mussel Hyriopsis cumingii. The full-length of HcCUB-Lec cDNA was 1558 bp with an open reading frame of 1281 bp that encodes a putative protein of 426 amino acid residues, including an N-terminal signal peptide, a complement Uegf Bmp1 (CUB) domain, a single carbohydrate recognition domain (CRD), and a transmembrane domain. Quantitative real-time PCR analysis revealed that HcCUB-Lec transcript was distributed in all examined tissues with the highest levels in hepatopancreas and was significantly upregulated in gills and hepatopancreas after immune challenge with Staphyloccocus aureus and Vibrio parahaemolyticus. When HcCUB-Lec was silenced by RNAi, the expression levels of three antimicrobial peptides, including whey acidic protein (HcWAP), defensin (HcDef), and lysozyme (HcLyso), were dramatically decreased in gills. The recombinant HcCUB-Lec and its individual CUB and CRD domains can bind with Gram-positive bacteria (S. aureus and Bacillus subtilis), Gram-negative bacteria (V. parahaemolyticus and Aeromonas hydrophila), and polysaccharides (lipopolysaccharide and peptidoglycan). Moreover, rHcCUB-Lec and its domains could also agglutinate S. aureus and V. parahaemolyticus in the presence of Ca2+ and can clear V. parahaemolyticus in H. cumingii. Results of this study suggest that HcCUB-Lec acts as an antimicrobial PRR that participates in the innate immune responses of H. cumingii.

Cloning and Analysis of Gene Expression of Two Toll Receptors in Freshwater Pearl Mussel Hyriopsis cumingii

Toll receptors are involved in innate immunity of invertebrates. In this study, we identify and characterize two Toll genes (named HcToll4 and HcToll5) from triangle sail mussel Hyriopsis cumingii. HcToll4 has complete cDNA sequence of 3,162 bp and encodes a protein of 909 amino acids. HcToll5 cDNA is 4,501 bp in length and encodes a protein of 924 amino acids. Both deduced HcToll4 and HcToll5 protein contain signal peptide, extracellular leucine rich repeats (LRRs), and intracellular Toll/interleukin-1 (IL-1) receptor domains. Quantitative real-time PCR analysis revealed that HcToll4 and HcToll5 were largely distributed in the hepatopancreas and could be detected in the gills and mantle. HcToll4 and HcToll5 expression could respond to Staphylococcus aureus, Vibrio parahaemolyticus, White spot syndrome virus (WSSV) or Poly I:C challenge. RNA interference by siRNA results showed that HcToll4 and HcToll5 were involved in the regulation of theromacin (HcThe) and whey acidic protein (HcWAP) expression. Based on these results, HcToll4 and HcToll5 might play pivotal function in H. cumingii innate immune response.

Three newly identified galectin homologues from triangle sail mussel (Hyriopsis cumingii) function as potential pattern-recognition receptors

Galactoside-binding lectins, also known as galectins, play crucial roles in innate immune response in invertebrates. In this study, three cDNA sequences from Hyriopsis cumingii were identified and collectively called HcGalec genes. Each of the three deduced HcGalec proteins contained a galactose-binding lectin domain or a GLECT domain. All the three HcGalec genes are mainly present in the hepatopancreas and gills, and their expression is induced at 24 h after bacterial challenge. Three recombinant HcGalec proteins can bind and agglutinate (Ca2+-dependent) various microorganisms, including Gram-positive and Gram-negative bacteria. These proteins can attach to mannan and peptidoglycan. Meanwhile, the expression of the three HcGalec genes in the gills were significantly down-regulated after dsRNA interference (HcGalec1-RNAi, HcGalec2-RNAi, and HcGalec3-RNAi) and Vibrio parahaemolyticus injection. The expression levels of some antimicrobial peptides, including lysozyme 1 and lysozyme 2, were also markedly decreased after dsRNA interference. Overall, these results suggested that these three HcGalec proteins may function as potential receptors participating in the innate immune responses of H. cumingii against bacterial infection.

A galectin contributes to the innate immune recognition and elimination of pathogens in the freshwater mussel Hyriopsis cumingii

Galectins are members of the lectin superfamily. They function as pattern recognition receptors in the innate immune system of vertebrates and invertebrates. A galectin homolog from the triangle sail mussel Hyriopsis cumingii (HcGal2) was cloned and characterized. HcGal2 mRNA was expressed in all tissues examined, displaying particular enrichment in mantle tissue. Interestingly, rHcGAL2 protein was only detected in the mantle, hemocytes, and gills, suggesting that post-transcriptional regulation may occur. HcGal2 expression was induced in the mantle, liver, and hemocytes after exposure to lipopolysaccharides, Gram-negative bacteria (Aeromonas hydrophila), and Gram-positive bacteria (Staphylococcus aureus). The transcript significant upregulated was also detected after implantation in the mantle, pearl sac, liver, and hemocytes. Recombinant HcGAL2 protein (rHcGAL2) agglutinated Gram-positive and Gram-negative bacteria. In addition, rHcGAL2 promoted phagocytosis by hemocytes in vivo. Our data suggest that HcGal2 functioned as a pattern recognition receptor in against the pathogenic microbes and contributed to the “non-self” recognition and elimination in H. cumingii.