Two NF-κB subunits are associated with antimicrobial immunity in Hyriopsis cumingii.

The NF-κB pathway activated by bacteria and viruses produces a series of antimicrobial peptides that participate in the innate immune response. In this study, two NF-κB subunits were cloned and identified from Hyriopsis cumingii (named Hcp65 and Hcp105) using RT-PCR and RACE. The predicted Hcp65 protein possessed a N-terminal Rel homology domain (RHD) and an Ig-like/plexins/transcription factors domain (IPT); the Hcp105 contained an RHD, an IPT domain, 6 ankyrin (ANK) domain and a death domain. Quantitative reverse transcription PCR (qRT-PCR) showed that Hcp65 and Hcp105 were constitutively expressed in the detected tissues, and were significantly up-regulated in hemocytes, hepatopancreas and gill of mussels challenged with lipopolysaccharide (LPS), peptidoglycan (PGN) and polyinosinic-polycytidylic acid (poly I: C). The dsRNA-mediated silencing of Hcp65 and Hcp105 caused significant reduction of immune genes such as lysozyme (HcLyso), theromacin (Hcther), whey acid protein (HcWAP), LPS-binding protein/bactericidal permeability protein (HcLBP/BPI) 1 and 2. In addition, subcellular localization experiments showed that Hcp65 and Hcp105 proteins were expressed in both the nucleus and cytoplasm of HEK-293T cells, and Hcp50 proteins (mature peptide of Hcp105) were mainly localized in the nucleus. The recombinant Hcp65 and Hcp50 protein could form homodimer and heterodimer and bind κB site in vitro. These results provide useful information for understanding the role of NF-κB in mollusks.

RNAi-mediated knock-down of the dopamine beta-hydroxylase gene changes growth of razor clams.

Dopamine beta-hydroxylase (DßH) plays an essential role in the synthesis of catecholamines (CA) in neuroendocrine networks. In the razor clam, Sinonovacula constricta a novel gene for DßH (ScDßH-α) was identified that belongs to the copper type II ascorbate-dependent monooxygenase family. Expression analysis revealed ScDßH-α gene transcripts were abundant in the liver and expressed throughout development. Knock-down of ScDßH-α in adult clams using siRNA caused a reduction in the growth rate compared to control clams. Reduced growth was associated with strong down-regulation of gene transcripts for the growth-related factors, platelet derived growth factors A (PDGF-A) (P < 0.001) 24 h after ScDßH-α knock-down, vascular endothelial growth factor (VEGF1) (P < 0.001) and platelet derived growth factor B (PDGF-B-2) (P < 0.001) 24 h and 48 h after ScDßH-α knock-down and transforming growth factor beta (TGF-ß1) (P < 0.001) 48 h and 72 h after ScDßH-α knock-down. Taken together the results suggest that the novel ScDßH-α gene through its role in CA synthesis is involved in growth regulation in the razor clam and possibly other bivalves.

Involvement of a novel Ca2+-independent C-type lectin from Sinonovacula constricta in food recognition and innate immunity.

Bivalve lectins perform a crucial function in recognition of foreign particles, such as microalgae and pathogenic bacteria. In this study, a novel C-type lectin form Sinonovacula constricta (ScCL) was characterized. The full-length cDNA of ScCL was 1645 bp, encoding a predicted polypeptide of 273 amino acids with one typical carbohydrate-recognition domain. ScCL has the highest similarity and closest phylogenetic relationship with the C-type lectin from Solen grandis. Real-time PCR analysis showed that ScCL was expressed in all tested tissues, with the highest expression in the foot and the lowest expression in hemocytes. Agglutination activity of ScCL was Ca2+-independent. ScCL showed the strongest agglutination on Chlorella vulgaris, the modest agglutination on Platymonas subcordiformis, Nannochloropsis sp., and Thalassiosira pseudonana, the weakest agglutination on Chaetoceros sp., and no agglutination on Isochrysis zhanjiangensis. Meanwhile, agglutination tests and western blot analysis revealed that the recombinant ScCL protein could agglutinate Staphylococcus aureus and Vibrio harveyi, but could not agglutinate Vibrio anguillarum, Bacillus cereus, or Vibrio parahaemolyticus. Furthermore, ScCL had a high binding activity with LPS and mannose, a low binding activity with LTA, and no binding activity with PGN. The expression of ScCL in the gill of S. constricta fed with C. vulgaris and T. pseudonana was significantly increased at 1 and/or 3 h. After injection with S. aureus, the expression of ScCL in the gill was significantly increased at 3, 6, and 24 h. These results indicated that ScCL was involved in food particle recognition and immunity of S. constricta.

Galectin-mediated immune recognition: Opsonic roles with contrasting outcomes in selected shrimp and bivalve mollusk species.

Galectins are a structurally conserved family of ß-galactoside-binding lectins characterized by a unique sequence motif in the carbohydrate recognition domain, and of wide taxonomic distribution, from fungi to mammals. Their biological functions, initially described as key to embryogenesis and early development via recognition of endogenous ("self") carbohydrate moieties, are currently understood as also encompassing tissue repair, cancer metastasis, angiogenesis, adipogenesis, and regulation of immune homeostasis. More recently, however, numerous studies have contributed to establish a new paradigm by revealing that galectins can also bind to exogenous ("non-self") glycans on the surface of potentially pathogenic virus, bacteria, and eukaryotic parasites, and function both as pathogen recognition receptors (PRRs) and effector factors in innate immunity. Our studies on a galectin from the kuruma shrimp Marsupenaeus japonicus (MjGal), revealed that it functions as a typical PRR. Expression of MjGal is upregulated by infectious challenge, and can recognize both Gram (+) and Gram (-) bacteria. MjGal also recognizes carbohydrates on the shrimp hemocyte surface, and can cross-link microbial pathogens to the hemocytes, promoting their phagocytosis and clearance from circulation. Therefore, MjGal contributes to the shrimp's immune defense against infectious challenge both as a PRR and effector factor. Our studies on galectins from the bivalve mollusks, however, have shown that although they can function in immune defense as MjGal, protistan parasites take advantage of the recognition roles of the host galectins, for successful attachment and host infection. We identified in the eastern oyster Crassostrea virginica two galectins (CvGal1 and CvGal2) that not only recognize a large variety of bacterial species, but also the protozoan parasite Perkinsus marinus. Like the shrimp MjGal, both oyster galectins function as opsonins, and promote parasite adhesion and phagocytosis. However, P. marinus survives intrahemocytic oxidative killing and proliferates, eventually causing systemic infection and death of the oyster host. In the softshell clam Mya arenaria we identified a galectin (MaGal1) that displays carbohydrate specificity and recognition properties for sympatric Perkinsus species (P. marinus and P. chesapeaki), that are different from CvGal1 and CvGal2. Our results suggest that although galectins from bivalves can function as PRRs, Perkinsus parasites have co-evolved with their hosts to subvert the galectins' immune functions for host infection by acquisition of carbohydrate-based mimicry.

Larval Geoduck (Panopea generosa) Proteomic Response to Ciliates.

The innate immune response is active in invertebrate larvae from early development. Induction of immune response pathways may occur as part of the natural progression of larval development, but an up-regulation of pathways can also occur in response to a pathogen. Here, we took advantage of a protozoan ciliate infestation of a larval geoduck clam culture in a commercial hatchery to investigate the molecular underpinnings of the innate immune response of the larvae to the pathogen. Larval proteomes were analyzed on days 4-10 post-fertilization; ciliates were present on days 8 and 10 post-fertilization. Through comparisons with larval cultures that did not encounter ciliates, proteins implicated in the response to ciliate presence were identified using mass spectrometry-based proteomics. Ciliate response proteins included many associated with ribosomal synthesis and protein translation, suggesting the importance of protein synthesis during the larval immune response. There was also an increased abundance of proteins typically associated with the stress and immune responses during ciliate exposure, such as heat shock proteins, glutathione metabolism, and the reactive oxygen species response. These findings provide a basic understanding of the bivalve molecular response to a mortality-inducing ciliate and improved characterization of the ontogenetic development of the innate immune response.

Molecular characterization and expression analysis of fibrinogen related protein (FREP) genes of Manila clam (Ruditapes philippinarum) after lipopolysaccharides challenge.

Ruditapes philippinarum has high economic value and is distributed all over the world. Fibrinogen associated protein (FREP) is a type of pattern recognition receptor, participates in the innate immune response to eliminate pathogens after the invasion of pathogenic microorganisms. In this study, three FREP genes (FREP-1, FREP-2, and FREP-3) were identified and characterized from R. philippinarum. The protein sequence of FREPs were highly conserved with those homologous in vertebrates, and FBG domain possessed significantly high structural conservation in polypeptide binding site and Ca2+ binding site. The tissues expression analysis of FREPs in three shell color strains and two population of R. philippinarum were examined, with the highest expression level in gill and hepatopancreas. Besides, FREP genes were demonstrated to be induced by lipopolysaccharides injection, the significantly changes were observed after LPS injected. Our results suggest the involvement of FREPs in response to LPS injection, and it might exert a significant function on the innate immune defense of the Manila clam.

A sialic acid-binding lectin with bactericidal and opsonic activities from Ruditapes philippinarum.

In the present study, a sialic acid-binding lectin was cloned and characterized from Manila clam Ruditapes philippinarum (designed as RpSabl). The open reading frame of RpSabl encoded a polypeptide of 162 amino acids with a calculated molecular mass of 17.7 kDa. Analysis of the conserved domain suggested that RpSabl was a new member of the sialic acid-binding lectins family. In non-stimulated clams, RpSabl transcripts were constitutively expressed in all five tested tissues, especially in hepatopancreas. After Vibrio anguillarum challenge, the expression of RpSabl mRNA in hepatopancreas was significantly up-regulated at 3 h (3.8-fold, P < 0.05), 6 h (4.9-fold, P < 0.05), 12 h (12.3-fold, P < 0.01) and 24 h (9.7-fold, P < 0.01), while RpSabl transcripts in hemocytes was only significantly up-regulated at 6 h (8.5-Fold, P < 0.01). RNAi-mediated knockdown of RpSabl transcripts affected the survival rates of Manila clam against V. anguillarum, perhaps mainly due to the inhibited expression of antibacterial effectors (e.g. lysozyme and defensin). Moreover, recombinant protein of RpSabl (rRpSabl) possessed binding activities towards lipopolysaccharides (LPS), peptidoglycan (PGN) and glucan in vitro. Coinciding with the Pathogen-associated molecular patterns (PAMPs) binding assay, rRpSabl displayed broad bacterial-agglutination properties towards Vibrio harveyi, Vibrio splendidus, V. anguillarum, Enterobacter cloacae and Aeromonas hydrophila. Meanwhile, the phagocytosis and encapsulation ability of hemocytes could be significantly enhanced by rRpSabl incubation. All these results showed that RpSabl could function as a versatile molecule involved in the innate immune responses of R. philippinarum.

A peptidoglycan recognition protein involved in immune recognition and immune defenses in Ruditapes philippinarum.

Peptidoglycan recognition proteins (PGRPs) are important pattern recognition receptors in the innate immune system of invertebrates. In the study, a short PGRP (designed as RpPGRP) was identified and characterized from the manila clam Ruditapes philippinarum. The open reading frame of RpPGRP encoded a polypeptide of 249-amino acids with a calculated molecular mass of 27.2 kDa and an isoelectric point of 6.62. Multiple alignments and phylogenetic analysis strongly suggested that RpPGRP was a new member of the PGRP superfamily. In non-stimulated clams, RpPGRP exhibited different tissue expression pattern, and highly expressed in hepatopancreas and hemocytes. Expression of RpPGRP transcripts was significantly up-regulated in hemocytes of clams post Vibrio anguillarum or Micrococcus luteus challenge. The recombinant RpPGRP (rRpPGRP) exhibited high affinity to PGN, LPS and zymosan in a concentration-dependent manner. With a broad spectrum of bacterial binding activities, rRpPGRP exhibited strong agglutination activity to Escherichia coli, Vibrio splendidus, V. anguillarum and M. luteus. Furthermore, rRpPGRP exhibited Zn2+-dependent amidase activity and catalyzed the degradation of insoluble PGN. Especially, rRpPGRP exhibited significant antibacterial activity against E. coli and M. luteus. Moreover, the biofilm formation of E. coli could be inhibited after rRpPGRP incubation in the presence of Zn2+. This inhibitory effect of rRpPGRP might attribute to its amide bactericidal activity. Taken together, rRpPGRP played important roles in PGRP-mediated immune defense mechanisms, especially by recognizing antigens and eliminating bacteria.

A preliminary attempt to explore the potential functions of a tetraspanin gene (MmTSPAN) in the innate immunity of hard clam Meretrix meretrix: Sequence features and expression profiles.

Tetraspanins belong to the transmembrane 4 superfamily (TM4SF), and play crucial roles in immune responses. In the present study, a novel tetraspanin gene (designated MmTSPAN) was cloned and characterized from the hard clam Meretrix meretrix. The complete cDNA sequence of MmTSPAN contained an open reading frame (ORF) of 816 bp, which encoded a protein of 271 amino acids. MmTSPAN exhibited highly similarity with previously identified tetraspanins from other species. It contained four transmembrane domains (12-35 aa, 69-92 aa, 99-123 aa and 238-261 aa), characteristic CCG motif and four conservative cysteine residues. The mRNA transcripts of MmTSPAN were ubiquitously detectable in all the tested tissues, with the highest expression level in hepatopancreas. Temporal transcriptional levels in the hepatopancreas revealed significant up-regulation of MmTSPAN by Vibrio splendidus stimulation, with a 3.14-fold increase at 6 h compared to the control, and reaching 32.98-fold at 24 h. These results provide useful information for further study of the function of tetraspanin in the innate immune system of M. meretrix, and may offer a new therapeutic target for diseases of M. meretrix.

Novel Ca2+-independent C-type lectin involved in immune defense of the razor clam Sinonovacula constricta.

C-type lectins (CTLs) are important pattern recognition molecules that participate in bacterial binding and agglutination by specific recognition of carbohydrates from pathogens. In this study, a full-length cDNA of CTL was cloned from Sinonovacula constricta (designated ScCTL-2). ScCTL-2 has a length of 981 bp, a 5'-untranslated region (UTR) of 47 bp, a short 3'-UTR of 37 bp, and an open reading frame (ORF) of 894 bp, which encodes a polypeptide of 298 amino acid residues. The deduced amino acid of ScCTL-2 possesses a conserved carbohydrate-recognition domain (CRD) similar to that of C31-E171. Spatial distribution analysis demonstrated that ScCTL-2 was constitutively expressed in all tested tissues, with dominant expression in foot and siphon and weak expression in hepatopancreas. The mRNA expression level of ScCTL-2 in gills and hepatopancreas was significantly upregulated at 6 and 12 h after challenge with the pathogen Vibrio parahaemolyticus. The recombinant ScCTL-2 showed specific binding and agglutinate capacities to all examined Gram-negative bacterial species, namely, Escherichia coli, Vibro anguillarum, and V. parahaemolyticus in a Ca2+-independent manner. However, these binding activities were not detected in Gram-positive Micrococcus luteus. Our results indicated that ScCTL-2 could be a novel pattern recognition receptor that can specifically recognize Gram-negative microorganisms in the innate immunity of S. constricta.

Identification and characterisation of a novel small galectin in razor clam (Sinonovacula constricta) with multiple innate immune functions.

Galectins are lectins possessing an evolutionarily conserved carbohydrate recognition domain (CRD) with affinity for ß-galactoside. The key role played by innate immunity in invertebrates has recently become apparent. Herein, a full-length galectin (ScGal) was identified in razor clam (Sinonovacula constricta). The 528 bp open reading frame encodes a polypeptide of 176 amino acids with a single CRD and no signal peptide. ScGal mRNA transcripts were mainly expressed in hemolymph and gill, and were significantly up-regulated following bacterial challenge. Recombinant rScGal protein binds to and aggregates various bacteria, and has affinity for peptidoglycan, lipoteichoic acid and d-galactose. The protein also stimulates hemocytes to phagocytose invading bacterial pathogens. ScGal is an important immune factor in innate immunity, and a small protein with multiple important functions.

From the raw bar to the bench: Bivalves as models for human health.

Bivalves, from raw oysters to steamed clams, are popular choices among seafood lovers and once limited to the coastal areas. The rapid growth of the aquaculture industry and improvement in the preservation and transport of seafood have enabled them to be readily available anywhere in the world. Over the years, oysters, mussels, scallops, and clams have been the focus of research for improving the production, managing resources, and investigating basic biological and ecological questions. During this decade, an impressive amount of information using high-throughput genomic, transcriptomic and proteomic technologies has been produced in various classes of the Mollusca group, and it is anticipated that basic and applied research will significantly benefit from this resource. One aspect that is also taking momentum is the use of bivalves as a model system for human health. In this review, we highlight some of the aspects of the biology of bivalves that have direct implications in human health including the shell formation, stem cells and cell differentiation, the ability to fight opportunistic and specific pathogens in the absence of adaptive immunity, as source of alternative drugs, mucosal immunity and, microbiome turnover, toxicology, and cancer research. There is still a long way to go; however, the next time you order a dozen oysters at your favorite raw bar, think about a tasty model organism that will not only please your palate but also help unlock multiple aspects of molluscan biology and improve human health.

Molecular cloning and expression analysis of C-type lectin (RpCTL) in Manila clam Ruditapes philippinarum after lipopolysaccharide challenge.

The Manila clam, Ruditapes philippinarum, is one of the most commercially important marine bivalves. C-type lectins (CTLs) are pattern recognition receptors (PRRs) that play important roles in the identification and elimination of pathogens by the innate immune system. In this study, a new CTL (RpCTL) was identified in the Manila clam, R. philippinarum. The full-length RpCTL cDNA is 802 bp, with an open reading frame of 591 bp, encoding 196 amino acids, including an N-terminal signal peptide and a carbohydrate recognition domain (CRD). RpCTL contains conserved CRD disulfide bonds involving four cysteine residues (Cys30-Cys104, Cys124, and Cys132), and the EPN (Glu94-Pro95-Asn96) and WND (Trp119-Asn120-Asp121) motifs. Quantitative reverse transcription (RT)-PCR detected RpCTL transcripts mainly in the gill, siphon, and hepatopancreas in three shell-color strains (zebra, white, and white-zebra strains) and two unselected populations of R. philippinarum, and the gene was highly expressed in the hepatopancreas after lipopolysaccharide treatment. Antimicrobial activity assays of recombinant RpCTL against both Gram-positive and Gram-negative bacteria showed that RpCTL inhibits microorganismal growth. In a survival test, RpCTL inhibited and killed Vibrio anguillarum in R. philippinarum. These results suggest that RpCTL participates in the pathogen identification process of R. philippinarum as a PRR and in its immune defense system.

Molecular characterization, expression, and antibacterial activity of a c-type lysozyme isolated from the manila clam, Ruditapes philippinarum.

Lysozyme is an antibacterial enzyme that is widely distributed in nature and plays an important role in the prevention of bacterial infections. In this study, a c-type lysozyme (designated as "RpCLYZ") was cloned and characterized from the manila clam, Ruditapes philippinarum. The full-length cDNA was 619 bp with an open reading frame (ORF) of 447 bp, and encoded a polypeptide of 148 amino acids with a calculated molecular mass of 17.0 kDa and an isoelectric point of 4.83. RpCLYZ was found to share high sequence similarity with c-type lysozymes from other invertebrates. The mRNA transcript of RpCLYZ was universally expressed in a wide range of tissues, especially in gills and mantle. Challenge with Vibrio anguillarum, significantly induced mRNA expression of RpCLYZ, which reached a maximum level 48 h after bacterial challenge. Recombinant RpCLYZ (rRpCLYZ) exhibited antibacterial activities against both Gram-positive and Gram-negative bacteria. Additionally, the optimal pH and temperature for rRpCLYZ activity were determined to be 4.5 and 20 °C, respectively. These results suggest that RpCLYZ participates in innate immune responses against bacterial invasion.

A defensin-like antimicrobial peptide from the manila clam Ruditapes philippinarum: Investigation of the antibacterial activities and mode of action.

Defensins are small cysteine-rich cationic proteins that are ubiquitously present in both vertebrates and invertebrates and constitute the front line of host innate immunity. In the present study, a defensin-like antimicrobial peptide (designed as RpdefB) was identified and characterized from the manila clam Ruditapes philippinarum. The open reading frame of RpdefB encoded a 70-amino acid polypeptide with a calculated molecular mass of 7.5 kDa and isoelectric point of 8.16. Multiple alignments and phylogenetic analysis strongly suggested that RpdefB was a new member of the defensin family. In non-stimulated clams, RpdefB transcripts were constitutively expressed in all five tested tissues, especially in the hepatopancreas. After Vibrio anguillarum challenge, expression of RpdefB mRNA in hemocytes was significantly up-regulated at 6 h, 12 h and 72 h. The synthetic peptide RpdefB showed high antibacterial activity against the Gram-negative bacterium Vibrio splendidus. Moreover, membrane integrity analysis revealed that RpdefB increased the membrane permeability of Escherichia coli and then resulted in cell death. Overall, our results suggested that RpdefB played an important role in the elimination of invading bacterium, perhaps through membrane-disruptive activity.

Molecular characteristics, expression, and antimicrobial activities of i-type lysozyme from the razor clam Sinonovacula constricta.

Lysozyme is a key component of the innate immune system, which plays a pivotal role in early defense against pathogen infection. In this study, an i-type lysozyme homology was identified from the razor clam Sinonovacula constricta (designated as ScLYZ) through RACE approaches. The full-length cDNA of ScLYZ was 768 bp and encoded a polypeptide of 140 amino acid residues. SMART analysis revealed that ScLYZ processed a signal peptide (1-18 aa) and a destabilase domain from 25 to 133 aa. Two catalytic residues (Glu36 and Asp47) and two specific motifs ["CL(E/L/R/H)C(I/M)C" and "MDVGSLSCG(P/Y) (F/Y)QIK"] of the i-type lysozyme were highly conserved in the ScLYZ sequence. Multiple sequence alignments and phylogenetic analysis indicated that ScLYZ could be a new member of the i-type lysozyme subfamily. Tissue distribution analysis revealed that ScLYZ was constitutively expressed in all examined tissues, and the highest expression was found in the hepatopancreas. After the razor clams were challenged by Vibrio parahaemolyticus, the mRNA levels of ScLYZ increased in the gill and hepatopancreas. Moreover, the recombinant protein was expressed in Escherichia coli, and the refolded ScLYZ showed highly antimicrobial activities against V. parahaemolyticus and Vibrio splendidus. The minimal inhibitory concentration toward V. parahaemolyticus was 8.2 µmol/mL. All our results supported that ScLYZ was involved in the innate immune defense of razor clam by inhibiting the growth of invasive pathogens.

Two macrophage migration inhibitory factors (MIFs) from the clam Ruditapes philippinarum: Molecular characterization, localization and enzymatic activities.

Macrophage migration inhibitory factor (MIF) is an evolutionarily ancient cytokine-like factor and plays a critical role in both innate and adaptive immunity. In the present study, two MIFs (designed as RpMIF-1 and RpMIF-2, respectively) were identified and characterized from the clam Ruditapes philippinarum by rapid amplification of cDNA ends (RACE) approaches. The full-length cDNA of RpMIF-1 and RpMFI-2 consisted of 531 and 722 nucleotides, encoding a polypeptide of 113 and 114 amino acid residues, respectively. Multiple alignments and phylogenetic analysis revealed that both RpMIF-1 and RpMIF-2 belonged to the MIF family. The conserved catalytic-site Pro2 for tautomerase activity was identified in the deduced amino acid sequences of RpMIFs. Both RpMIF-1 and RpMIF-2 transcripts were constitutively expressed in examined tissues of R. philippinarum with dominant expression in hepatopancreas, gills and hemocytes. Immunolocalization analysis showed that RpMIF-1 and RpMIF-2 proteins were expressed in examined tissues with the exception of adductor muscle and foot. After Vibrio anguillarum and Micrococcus luteus challenge, the mRNA expression of RpMIFs was significantly modulated in hemocytes, gills and hepatopancreas. Recombinant RpMIF-1 and RpMIF-2 proteins possessed significant tautomerase activity and oxidoreductase activity, indicating that these two proteins was perhaps involved in inflammatory responses. In summary, our results suggested that RpMIF-1 and RpMIF-2 played an important role in the innate immunity of R. philippinarum.

Immune state correlates with histopathological level and reveals molluscan health in populations of Modiolus kurilensis by integral health index (IHI).

Quantitative analysis of the histopathological and immune parameters of bivalve Modiolus kurilensis collected from water areas with different level of ecotoxicological stress was performed. Significant differences between samples from polluted and non-polluted sites were revealed for total haemocyte count; percentage of agranulocytes; size and internal complexity of agranulocytes and granulocytes; phagocytic activity; percentage of NBT-positive cells; hemolytic activity and plasma protein concentration; percentage of the optical density of haemolymph major polypeptide bands at 55 kDa, 78 kDa, and 124 kDa; concretion coverage area in the kidney tubules; thickness of the tubular basement membrane; nephrocyte shape; and karyopyknosis of the kidneys; and hypervacuolisation; necrosis; karyopyknosis; haemocyte infiltration; fibrosis; and invasion of the digestive gland. Analysis of the global histopathological condition index based on the weighted indices also revealed that both the digestive gland and kidneys showed significantly greater histopathological changes in the bivalves collected from polluted water. Bivalve histopathology is an established tool in aquatic toxicology. However, it reflects a morphological picture of change, which, as a rule, can be clearly recorded only at the later stages of pathology, and in some cases, indicates an adaptation to stressors within the physiological norm. In this respect, a promising and highly sensitive biomarker of the functional state of bivalves, in terms of norm and pathology as well as their habitat, is the evaluation of immune status in combination with morphological changes. However, the use of different methods and scales of assessment and the diagnosis of biomarkers, characterised by different profiles of the stress response, makes it difficult to compare the results of different studies. We propose a reliable and powerful system for assessing the physiological state of bivalve molluscs, expressed in the integral health index (IHI) and based on the standardisation of the numerical values for all parameters that have significant differences between animals collected from impacted and non-impacted water areas. In our study, IHI calculated in three variants (for histopathological parameters, for immunological parameters, and in combination) showed the most significant differences in each of the cases, but the strongest difference (-4.07) was in calculating the total IHI, which included both the immune and histopathological parameters (p = 0.00005).

Two I84 family protease inhibitors from Chinese razor clams Sinonovacula constricta expressed in response to environmental challenges.

Protease inhibitors play critical roles in numerous biological processes including host defense in all multicellular organisms. Eighty three evolutionary families of protease inhibitors are currently accommodated in the MEROPS database and the I84 family currently consists of 3 novel serine protease inhibitors from the eastern oyster Crassostrea virginica. In this study, we identified 2 new I84 family members from the Chinese razor clam Sinonovacula constricta, scSI-1 and scSI-2, using cDNA cloning and sequencing. The scSI-1 cDNA consisted of 494 bp with a 282 bp ORF encoding a 93-amino acid polypeptide that was predicted to have a 19-amino acid signal peptide and a 74-residue mature protein with a calculated molecular mass of 8248.5 Da. The scSI-2 cDNA was 490 bp long with a 273 bp ORF encoding a 90-amino acid polypeptide that was predicted to have an 18-amino acid signal peptide and a 72-residue nature protein with a calculated molecular mass of 7528.4 Da. ScSI-1 and scSI-2 shared high sequence similarity with the 3 known members of I84 family and both expressed primarily in the clam digestive glands. Protease inhibitory activity in the clam plasma also exhibited the signature kinetic characteristics of the I84 members from the oyster. In addition, levels of scSI-1 and scSI-2 gene expression in digestive glands and the protease inhibitory activity in plasma elevated significantly in clams challenged by bacterial injections and Vibrio harveyi was more effective than Staphylococcus epidermidis in inducing the gene expression and plasma protease inhibitory activity. Moreover, drastic changes of salinity and temperature also caused significant changes in the gene expression and plasma activity. These results indicated that scSI-1 and scSI-2 represented 2 new members of the I84 family and they likely play a role in clam host defense against infections and in reactions against physiochemical stressors.

A potential microRNA regulation of immune-related genes in invertebrate haemocytes.

Bivalve mollusks have been employed as sentinel organisms in environmental health programs due to their sedentary lifestyle, filter-feeding behavior and their ability to accumulate pathogens or toxin molecules inside tissues. Endocrine disrupting chemicals (EDCs) can be up taken and bioaccumulated, and due to sensibility of mollusks to these EDCs, being able to cause immune alterations. Recently, microRNAs (miRNAs) were shown to be involved in modulation and buffering developmental processes against the effects of environmental alterations and pathogenic microorganisms. Moreover, it is suggested that this miRNAs are incorporated into the estrogen-controlled immune network, regulating mechanism of immune gene expression at the posttranscriptional level, modulating immune responses as phagocytosis, redox reaction and apoptosis in bivalve haemocytes. Thus, miRNAs can be used as biomarkers that specifically elucidate immunotoxic effects caused by exogenous biotic or abiotic factors, and can act as useful tools in integrated monitoring environmental health programs. In this review, we aim to describe the investigations that have been carried out on miRNAs in bivalve mollusks, especially those associated with immune responses against infectious agents and xenobiotic exposure.