Effects of different high temperature-pressure processing times on the sensory quality, nutrition and allergenicity of ready-to-eat clam meat.

Investigating technologies to control the allergenicity of seafood is particularly important to safeguard consumer health, but there is currently a dearth of research focused on reducing the allergenicity of clam meat. This study aimed to investigate the effects of high temperature-pressure (HTP) processing times (121 °C, 0.14 MPa; 5, 10, 15, 20 min) on the sensory quality, nutrition, and allergenicity of ready-to-eat clam meat. With the extension of HTP time, the hardness of clam meat gradually decreased, the chewiness decreased initially and then increased, and the meat became tender. HTP processing endowed clam meat with abundant esters and aldehydes. Among all the processing groups, the umami and saltiness were better at 15 min, correlating with the highest overall acceptability. Ready-to-eat clam meat contained high-protein nutritional value. Compared with raw clam meat, the tropomyosin allergenicity of clam meat treated with HTP for 15 and 20 min was significantly reduced by 51.9 % and 56.5 %, respectively (P < 0.05). However, there was no significant difference between these two groups. Appropriate HTP processing time might be an efficient condition to reduce the tropomyosin allergenicity of ready-to-eat clam meat and improve its quality, particularly for the time of 15 min. The results of this study could provide a reliable theoretical basis for the development of hypoallergenic clam foods.

Molecular Mechanisms Underlying Vibrio Tolerance in Ruditapes philippinarum Revealed by Comparative Transcriptome Profiling.

The clam Ruditapes philippinarum is an important species in the marine aquaculture industry in China. However, in recent years, the aquaculture of R. philippinarum has been negatively impacted by various bacterial pathogens. In this study, the transcriptome libraries of R. philippinarum showing different levels of resistance to challenge with Vibrio anguillarum were constructed and RNA-seq was performed using the Illumina sequencing platform. Host immune factors were identified that responded to V. anguillarum infection, including C-type lectin domain, glutathione S-transferase 9, lysozyme, methyltransferase FkbM domain, heat shock 70 kDa protein, Ras-like GTP-binding protein RHO, C1q, F-box and BTB/POZ domain protein zf-C2H2. Ten genes were selected and verified by RT-qPCR, and nine of the gene expression results were consistent with those of RNA-seq. The lectin gene in the phagosome pathway was expressed at a significantly higher level after V. anguillarum infection, which might indicate the role of lectin in the immune response to V. anguillarum. Comparing the results from R. philippinarum resistant and nonresistant to V. anguillarum increases our understanding of the resistant genes and key pathways related to Vibrio challenge in this species. The results obtained here provide a reference for future immunological research focusing on the response of R. philippinarum to V. anguillarum infection.

Expression and functional characterization of peptidoglycan recognition protein-S6 involved in antibacterial responses in the razor clam Sinonovacula constricta.

It has been recognized that peptidoglycan recognition proteins (PGRPs), structurally conserved molecules, play crucial roles in the innate immunity of invertebrate. However, few studies have been taken to explore their potential functions. In this study, a novel PGRP from the razor clam Sinonovacula constrict designated as ScPGRP-S6 was identified and characterized. The open reading frame (ORF) of ScPGRP-S6 was 666 bp in length, encoding a protein of 221 amino acid with a signal peptide (1-30) and a typical PGRP domain (39-187). The sequence alignment combined with phylogenetic analysis collectively confirmed that ScPGRP-S6 was a novel member belonging to PGRP-S family. The mRNA transcript of ScPGRP-S6 in the hepatopancreases was significantly up-regulated after peptidoglycan (PGN) stimulation, while it was moderately up-regulated after lipopolysaccharide (LPS) stimulation. The result of immunofluorescence detection demonstrated that the positive signal enhanced obviously after Vibrio parahaemolyticus challenge. Notably, the recombinant protein of ScPGRP-S6 (designed as rScPGRP-S6) exhibited high agglutination activity towards V. parahaemolyticus but weak to Staphylococcus aureus. Furthermore, rScPGRP-S6 showed strong amidase and antibacterial activity in the presence of Zn2+. Collectively, our results manifested that ScPGRP-S6 could act as a scavenger in the innate immune response of S. constricta.

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.

Quantification of the inflammatory responses to pro-and anti-inflammatory agents in Manila clam, Ruditapes philippinarum.

Inflammation is a form of innate immune response of living organisms to harmful stimuli. In marine bivalves, inflammation is a common defense mechanism. Several studies have investigated the morphological features of inflammation in bivalves, such as hemocyte infiltration. However, the molecular and biochemical responses associated with inflammation in marine bivalves remain unexplored. Here, we investigated changes in nitric oxide (NO) levels, cyclooxygenase 2 (COX-2) activity, and allograft inflammatory factor-1 (AIF-1) gene expression levels in hemolymph samples collected from Manila clam (Ruditapes philippinarum) exposed to pro- and anti-inflammatory substances. These included the pro-inflammatory agent lipopolysaccharide (LPS), and the nonsteroidal anti-inflammatory drugs (NSAIDs) ibuprofen and diclofenac, all widely used in vertebrates. Our study showed that NO levels, COX-2 activity, and AIF-1 expression increased in response to the treatments with LPS and decreased in response to the treatments with NSAIDs in a concentration-dependent manner. These results suggest that the mechanism of inflammatory responses in bivalves is very similar to that of vertebrates, and we propose that inflammatory responses can be quantified using these techniques and used to determine the physiological status of marine bivalves exposed to biotic or abiotic stresses.

Molecular Characterization of Complement Component 3 (C3) in the Pearl Oyster Pinctada fucata Improves Our Understanding of the Primitive Complement System in Bivalve.

As the central component in the complement system, complement component 3 (C3) plays essential roles in both the innate and adaptive immune responses. Here, a C3 gene (designated as pf-C3) was obtained from the pearl oyster Pinctada fucata by RT-PCR and rapid amplification of cDNA ends (RACE). The pf-C3 cDNA consists of 5,634 bp with an open reading frame (ORF) of 5,193 bp encoding a protein of 1,730 amino acids with a 19 residue signal peptide. The deduced pf-C3 protein possessed the characteristic structural features present in its homologs and contained the A2M_N_2, ANATO, A2M, A2M_comp, A2M_recep, and C345C domains, as well as the C3 convertase cleavage site, thioester motif, and conserved Cys, His, and Glu residues. Phylogenetic analysis revealed that pf-C3 is closely related to the C3s from other mollusks. Pf-C3 mRNA was expressed in all examined tissues including gill, digestive gland, adductor muscle, mantle and foot, while the highest expression was found in the digestive gland. Following the challenge with Vibrio alginolyticus, pf-C3 expression was significantly induced in hemocytes. Luciferase reporter assays indicated that pf-C3a could activate the NF-κB signal pathway in HEK293T cells. Further knockdown of pf-C3 by specific siRNA could significantly reduce the phagocytosis of V. alginolyticus by hemocytes in vitro. These results would help increase understanding of the function of C3 in the invertebrate immune system and therefore provide new insights into the roles of the primitive complement system in invertebrates.

Mitochondria are essential for antibacterial extracellular trap formation mediated by zymosan in hemocytes of Ruditapes philippinarum.

The formation of extracellular traps (ETs) is an important innate immune mechanism that serves to combat different invading pathogens. In this study, zymosan significantly induced the formation of ETs in the hemocytes of Ruditapes philippinarum, and this effect was accompanied by translocation of the mitochondria to the cell surface. Zymosan stimulation clearly induced an increase in intracellular ROS and MPO production and an overexpression of ROS-related genes (PI3K, AKT and HIF). In response to the ROS burst, the mitochondrial membrane potential decreased, and the mitochondrial permeability transition pore opened. Conversely, mitochondrial superoxide inhibitor (Mito-TEMPO) significantly inhibited the formation of ETs, suggesting that mitochondrial ROS were necessary for the formation of ETs. In addition, we found that zymosan-induced ETs showed antibacterial activities against gram-negative and gram-positive bacteria, such as Vibrio anguillarum, Vibrio harveyi, Escherichia coli and Micrococcus luteus. Taken together, these findings elucidated a new antibacterial approach for R. philippinarum and highlighted the role of mitochondria in the formation of zymosan-induced ETs.

Molecular characterization of a novel p38 MAPK cDNA from Cyclina sinensis and its potential immune-related function under the threat of Vibrio anguillarum.

The p38 mitogen-activated protein kinase (MAPK) is one important member of MAPK family and reported to serve a predominant function in regulating innate immunity after the occurrence of certain infection. In the present study, one novel p38 MAPK gene was acquired from Cyclina sinensis based on the RNA-seq analysis and designated as Csp38 MAPK. This novel gene contained a full length of 1781 bp, 1104 bp of which was deemed as open reading frames and gave rise to a peptide of 367 amino acids with a predicted molecular weight of 42.31 KDa. A conserved serine/threonine protein kinase (S_Tkc) region along with a Thr-Gly-Tyr motif was discovered in the deduced sequence. According to the phylogenetic analysis, there was a close relationship between this kinase and Meretrix petechialis p38 MAPK. As for the expression pattern, this newly-identified Csp38 MAPK was ubiquitously distributed in several tissues throughout the body but with varied abundance. After the challenge of Vibrio anguillarum, both the transcription and phosphorylation level of Csp38 MAPK in hemolymph were coordinately altered with a time-dependent manner. Besides, with the application of double strand RNA homologous to myeloid differentiation factor 88 (MyD88) of C. sinensis, the activation of Csp38 MAPK was found to obviously decrease in hemolymph after the pathogen stimulation. Hence, our experimental data presented evidence for the potential involvement of p38 MAPK in response to bacterial invaders in C. sinensis, possibly facilitating the understanding for pathogen-induced innate immunity in clams.

Two fibrinogen-related proteins (FREPs) in the razor clam (Sinonovacula constricta) with a broad recognition spectrum and bacteria agglutination activity.

Fibrinogen-related proteins (FREPs) that contain only the fibrinogen-related domain are likely involved in pathogen recognition. In this study, we identified two FREPs from the razor clam (Sinonovacula constricta), called ScFREP-1 and ScFREP-2, and investigated their roles in the immune response. Both ScFREP-1 and ScFREP-2 contained a fibrinogen-related domain at the C-terminal. ScFREP-1 and ScFREP-2 mRNAs were detected in all adult clam tissues tested, with the highest expression levels in the gill and mantle, respectively. Their expression levels were significantly upregulated after microbe infection. Recombinant ScFREPs could bind Gram-positive and Gram-negative bacteria as well as some pathogen-associated molecular patterns (PAMPs), and they could agglutinate those bacteria. These results showed that ScFREPs functioned as potential pattern recognition receptors to mediate immune response by recognizing PAMPs and agglutinating invasive microbes.

Natural hybridization between pen shell species: Pinna rudis and the critically endangered Pinna nobilis may explain parasite resistance in P. nobilis.

Recently, Pinna nobilis pen shells population in Mediterranean Sea has plummeted due to a Mass Mortality Event caused by an haplosporidian parasite. In consequence, this bivalve species has been included in the IUCN Red List as "Critically Endangered". In the current scenario, several works are in progress to protect P. nobilis from extinction, being identification of hybrids (P. nobilis x P. rudis) among survivors extremely important for the conservation of the species.Morphological characteristics and molecular analyses were used to identify putative hybrids. A total of 10 individuals of each species (P. nobilis and P. rudis) and 3 doubtful individuals were considered in this study. The putative hybrids showed shell morphology and mantle coloration intermingled exhibiting both P. nobilis and P. rudis traits. Moreover, the analyses of 1150 bp of the 28S gene showed 9 diagnostic sites between P. rudis and P. nobilis, whereas hybrids showed both parental diagnostic alleles at the diagnostic loci. Regarding the multilocus genotypes from the 8 microsatellite markers, the segregation of two Pinna species was clearly detected on the PCoA plot and the 3 hybrids showed intermediate positions.This is the first study evidencing the existence of hybrids P. nobilis x P. rudis, providing molecular methodology for a proper identification of new hybrids. Further studies testing systematically all parasite-resisting isolated P. nobilis should be undertaken to determine if the resistance is resulting from introgression of P. rudis into P. nobilis genome and identifying aspects related to resistance.

Genome-wide identification and expression profiling of TYR gene family in Ruditapes philippinarum under the challenge of Vibrio anguillarum.

Tyrosinase (EC1.14.18.1, TYR) is also called phenol oxidase, is not only involved in pigmentation but also plays an important role in modulating innate immunity in invertebrates. Tyrosinase is a copper containing metalloenzyme. The tyrosinase protein has two copper binding sites and three conserved histidines. In this study, 21 tyrosinase genes (RpTYR) were obtained from the whole genome of Ruditapes philippinarum. Their open reading frames were from 951 to 5424 aa, the range of predicted relative molecular weight from 36.72 to 203.81 kDa, and the range of isoelectric point from 4.72 to 9.88. Transcriptome analysis showed that RpTYR gene was expressed specifically in different developmental stages, adult tissues, four strains and two groups with different shell colors. Besides, the expression profiles of 21 RpTYRs were investigated against the immune response of R. philippinarum to a Vibrio challenge. The qPCR results showed that RpTYRs were involved in the immune response of R. philippinarum after Vibrio anguillarum challenge. This study provides preliminary evidence that the tyrosinases genes are involved in the immune defense and the potential immune function of R. philippinarum. Overall, these findings suggested that the expansion of TYR genes may play vital roles in larval development, the formation of shell color pattern, and immune response in R. philippinarum.

Transcriptome-wide identification and characterization of toll-like receptors response to Vibrio anguillarum infection in Manila clam (Ruditapes philippinarum).

The Manila clam (Ruditapes philippinarum), one of the major marine aquaculture species in China, is susceptible to infection with the pathogen Vibrio, which results in massive mortality and economic losses. Toll-like receptors (TLRs) are significant pattern recognition receptors (PRRs) of innate immunity that are involved in immune regulation against pathogenic invasion. Molecular characterization of Manila clam TLRs and investigations of their immune functions are essential to prevent and control Vibrio infection. In the present research, eight cDNA sequences of R. philippinarum TLRs (RpTLRs) were identified from previous transcriptome libraries and then classified into four groups, namely, P-TLR (one sequence), V-TLR (one sequence), Ls-TLR (two sequences) and sP-TLR (four sequences), based on the corresponding LRR domain arrangement of their protein structures within the typical TLR motifs. A selective pressure test firstly suggested that the molluscan P-TLR, V-TLR, Ls-TLR and sP-TLR families underwent positive selection, and different numbers of positive selection sites (PSSs) were identified in different domains of the four types of RpTLRs, as determined by PAML and analysis of website data. These findings indicated that the evolution of RpTLRs may be associated with their immune recognition and function. Furthermore, tissue-specific expression analysis showed that all RpTLRs were ubiquitously expressed in all test tissues and were dominant in hemocytes. Quantitative real-time PCR revealed that the cDNA expression of all eight RpTLRs was upregulated after injection with Vibrio anguillarum (P < 0.01) in R. philippinarum hemocytes, revealing that these RpTLRs play important roles in responding to pathogenic stimulation. In summary, these findings provide a foundation for future investigations of the molecular classification and evolutionary patterns of Toll-like receptors in invertebrates, and the innate immune responses of TLR signaling pathways in Mollusca.

The stress-immunity axis in shellfish.

It is a difficult task to describe what constitutes a 'healthy' shellfish (e.g., crustacean, bivalve). Visible defects such as discolouration, missing limbs or spines, fouling, lesions, and exoskeletal fractures can be indicative of underlying issues, senescence, or a 'stressed' animal. The absence of such symptoms is not evidence of a disease-free or a stress-free state. Now, more than ever, aquatic invertebrates must cope with acute and chronic environmental perturbations, such as, heatwaves and cold shocks, xenobiotic contaminants, intoxication events, and promiscuous pathogens expanding their host and geographic ranges. With that in mind, how does one determine the extent to which shellfish become stressed in situ (natural) or in cultured (artificial) settings to enhance disease susceptibility? Many biomarkers - predominantly biochemical and cellular measures of shellfish blood (haemolymph) - are considered to gauge immunosuppression and immunocompetence. Such measures range from immune cell (haemocyte) counts to enzymic activities and metabolite quantitation. Stressed invertebrates often reflect degraded conditions of their ecosystems, referred to as environmental indicators. We audit briefly the broad immune functions of shellfish, how they are modulated by known and emerging stressors, and discuss these concepts with respect to neuroendocrinology and immunotoxicology. We assert that chronic stress, alone or in combination with microbial, chemical and abiotic factors, increases the risk of infectious disease in shellfish, exacerbates idiopathic morbidity, and reduces the likelihood of recovery. Acute stress events can lead to immunomodulation, but these effects are largely transient. Enhancing our understanding of shellfish health and immunity is imperative for tackling losses at each stage of the aquatic food cycle and disease outbreaks in the wild.

Transcriptomic analysis reveals the immune changes associated with reproduction in the clam Meretrix petechialis.

Substantial mortality and economic losses in marine mollusk culture has drawn considerable attention in recent years. The changes in immune status and environmental stress are thought to be the main causes of shellfish summer mortality. The reproduction and immune defense are both physiologically demanding processes, therefore, the immune status of mollusk is likely to be affected by reproduction during breeding. In present study, we performed transcriptome and gene expression analyses in the clam Meretrix petechialis pre-/post-spawning. DEGs enrichment analysis revealed important immune signaling pathways and key genes changed after spawning. Further analysis showed females up-regulated genes involved in apoptosis, TLR signal pathway and heat shock, whereas males down-regulated complement-related genes after spawning. Additionally, both genders of clams up-regulated its immune response level to against Vibrio infection after spawning revealed by the changes of four immune-related DEGs. The up-regulation of two marker genes at the transcription and protein levels further confirmed that pathogen reinforced the expression differences of immune-related genes between the two groups. Our study provides a new insight into the understanding of molecular mechanisms underlying reproduction influenced immune differences in M. petechialis.

Hemocyte extracellular traps of Manila clam Ruditapes philippinarum: Production characteristics and antibacterial effects.

Extracellular traps (ETs) have been found to be an important strategy of mammals to immobilize and kill invading microorganisms. In the present study, we observed the formation of ETs in the hemocytes of marine mollusks Ruditapes philippinarum in response to challenge from bacteria Vibrio anguillarum, and examined the potential factors and signaling pathways underling this process. We detected an increase of reactive oxygen species (ROS) and myeloperoxidase (MPO) production during ETosis, accompanied by significantly up-regulated expression of ROS-related and MPO genes. The suppression of ETs structures by nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor (diphenyleneiodonium chloride, DPI) and MPO inhibitor (aminobenzoic acid hydrazide, ABAH) further confirmed the essential roles ROS and MPO played in ETosis. Furthermore, ET production could be inhibited by phosphotidylinsitol-3-kinase (PI3K) inhibitor (LY294002) and extracellular regulated protein kinase (ERK) inhibitor (U0126), suggesting the idea that both the PI3K and ERK pathways were suggested to function during ETosis. In addition, the ETosis process was accompanied by enhancement of glycolysis-related enzymatic activities, e.g., pyruvate kinase (PK) and hexokinase (HK), and over-expression of the glycolysis-related genes, e.g., PK, HK and glucose transport protein (GLUT), indicating high involvement of glycolysis in the ETosis process. Furthermore, our scanning electron microscopy (SEM) observation and antibacterial activities test successfully showed the patterns how clam ETs entrapped and killed the invading V. anguillarum. Taken together, our results revealed that ETosis with bactericidal effect increased ROS, MPO and glycolysis level and carried out in a ROS-, MPO-, PI3K-ERK-dependent manner.

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.

Molecular characterization, expression and immune functions of two C-type lectin from Venerupis philippinarum.

In the present study, two C-type lectins (designated as VpClec-3 and VpClec-4) were identified and characterized from the manila clam Venerupis philippinarum. Multiple alignment and phylogenetic relationship analysis strongly suggested that VpClec-3 and VpClec-4 belong to the C-type lectin family. In nonstimulated clams, the VpClec-3 transcript was dominantly expressed in the hepatopancreas, while the VpClec-4 transcript was mainly expressed in gill tissues. Both VpClec-3 and VpClec-4 mRNA expression was significantly upregulated following Vibrio anguillarum challenge. Recombinant VpClec-4 (rVpClec-4) was shown to bind lipopolysaccharide (LPS) and glucan in vitro, whereas recombinant VpClec-3 (rVpClec-3) only bound to glucan. In addition, rVpClec-3 and rVpClec-4 displayed broad agglutination activities towards Vibrio harveyi, Vibrio splendidus and V. anguillarum, while no agglutination activities towards Enterobacter cloacae or Aeromonas hydrophila were observed in rVpClec-3. Moreover, hemocyte phagocytosis was significantly enhanced by rVpClec-3 and rVpClec-4. All the results showed that VpClecs function as pattern recognition receptors (PRRs) with distinct recognition spectra and are potentially involved in the innate immune responses of V. philippinarum.

Immunological and oxidative stress responses of the bivalve Scrobicularia plana to distinct patterns of heatwaves.

As a consequence of global warming, extreme events, such as marine heatwaves (MHW), have been increasing in frequency and intensity with negative effects on aquatic organisms. This innovative study evaluated for the first time, the immunological and physiological response of the estuarine edible bivalve Scrobicularia plana to different heatwaves, with distinct duration and recovery periods. So, extensive immune (total haemocyte count - THC, haemocyte viability, phagocytosis rate, respiratory oxidative burst of haemocytes, total protein, protease activity, nitric oxide and bactericidal activity of plasma) and oxidative stress (lipid peroxidation - LPO, superoxide dismutase - SOD, catalase - CAT and glutathione-S-Transferase - GST) analyses were performed in an experimental study that tested the impact of heatwaves during 25 days. The survival and condition of S. plana were not affected by the exposure to the extreme events. However, our data suggested that longer heatwaves with shorter recovery periods can be more challenging for the species, since THC and phagocytic activity were most affected under the temperature increase conditions. Regarding the oxidative status, the species increased its SOD activity while MDA production slightly declined to the increase of temperature, protecting the organism from cellular damage. These results indicate that S. plana has a great capacity to adapt to environmental temperature changes, however, the expected higher frequency/duration of heatwaves with climate change trends can cause some debility of the species face to other stressors, which can compromise its success in the future.

Transcriptomic analysis of Manila clam Ruditapes philippinarum under lipopolysaccharide challenge provides molecular insights into immune response.

The Manila clam, Ruditapes philippinarum, is an economically important shellfish in marine aquaculture. A better understanding of the immune system in R. philippinarum will provide the basis for the development of strategies to mitigate the impact of infectious diseases affecting this species but can also be of relevance for other bivalves of commercial interest. In this study, the transcriptional response of the Manila clam under lipopolysaccharide (LPS) challenge was characterized using RNA sequencing. The transcriptomes of LPS challenged group of clams (LH1, LH2 and LH3), and the PBS control group (CH1, CH2 and CH3), were sequenced with the Illumina HiSeq platform. Compared with the unigene expression profile of the control group, 223 unigenes were up-regulated and 389 unigenes were down-regulated in the LPS challenged group. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses revealed that signal transduction, defense response, and immune-related pathways such as Chemokine signaling pathway, Complement and coagulation cascades, NOD-like receptor signaling pathway, and Inflammatory mediator regulation of TRP channels in sensory system were the most highly enriched pathways among the genes that were differentially expressed under LPS challenge. This study present understanding of the molecular basis underpinning response to LPS challenge and provides useful information for future work on the molecular mechanism of pathogen resistance and immunity in Manila clam.

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.