Mytilus Coruscus Research Articles

Regulation of innate immunity in marine mussel Mytilus coruscus: MicroRNA Mc-novel_miR_196 targets McTLR-like1 molecule to inhibit inflammatory response and apoptosis.

Toll-like receptors (TLRs) are crucial players in immune recognition and regulation, with aberrant activation leading to autoimmune, chronic inflammatory, and infectious diseases. MicroRNAs (miRNAs) have been shown to regulate gene expression at transcriptional and post-transcriptional levels. While miRNA-mediated regulation of TLR signaling has been studied in mammals, the underlying mechanisms of TLR-miRNA interactions in molluscs remain unclear. In a previous study, one of the TLR genes potentially targeted by miRNAs was identified and named McTLR-like1. McTLR-like1 was later found to be targeted by miRNA Mc-novel_miR_196 through bioinformatic prediction. In this study, we aim to experimentally determine the interaction between McTLR-like1 and Mc-novel_miR_196, as well as their functional role in the innate immune response of molluscs. The results showed that the expression of Mc-novel_miR_196 was suppressed, while the expression of McTLR-like1 was enhanced in M. coruscus hemocytes treated with lipopolysaccharide (LPS). Moreover, in vitro assays demonstrated that Mc-novel_miR_196 directly targets the 5' UTR of McTLR-like1 and leads to the down-regulation of proinflammatory cytokines in hemocytes. In addition, co-transfection experiments confirmed that Mc-novel_miR_196 inhibits McTLR-like1 and inhibits the expression of proinflammatory cytokines. The Tunel assay also showed that Mc-novel_miR_196 inhibited apoptosis in hemocytes induced by LPS. Our findings suggest that microRNA Mc-novel_miR_196 acts as a regulator of innate immunity in M. coruscus by targeting McTLR-like1 and inhibiting inflammatory response and apoptosis. These results provide further insights into the complex molecular mechanisms underlying TLR signaling in molluscs.

Assessing the Impact of Microplastic Filaments Contaminated with PAHs on Mytilus coruscus Larvae through Surface Contact.

In recent years, microplastics have been of great concern in environmental and health research. In field surgeries and laboratory investigations, research interests were focused on the retention of microplastics inside of animals by ingestion and the series of negative effects after that. However, such large plastic debris and filaments are hardly eaten by small animals, like zooplankton, planktonic larvae, etc. In this study, the surface contact between plastic filaments contaminated with polycyclic aromatic hydrocarbons (PAHs) and mussel pediveliger larvae has been investigated to figure out the effects of "non-digestive tract route of exposure" on subject animals. In a 1600 mL artificial seawater medium, high mortalities of mussel larvae were recorded after being exposed to two PAHs-contaminated (benzo[α]pyrene (BaP) and phenanthrene (Phe)) filaments for 5 days, 68.63% for BaP and 56.45% for Phe on average. We suggest that the surface contact was the dominant pathway to transfer PAHs from contaminated filaments to larvae and that the risk of contaminated plastic ropes transferring hydrophobic organic compounds (HOCs) to larvae in mussel aquaculture should be taken seriously.

Nano-TiO2 aggravates the adverse effect of pentachlorophenol on antioxidant and immune response in anti-predatory mussels

Nano-TiO2 can act as a vector to organic compounds, such as pentachlorophenol (PCP) posing a potential threat to the marine ecosystems. Studies showed that nano pollutant toxicity can be modulated by abiotic factors, but little is known about the potential influence of biotic stressors (such as predators) on the physiological responses to pollutants in marine organisms. We explored the effects of n-TiO2 and PCP on the mussel Mytilus coruscus in the presence of its natural predator, the swimming crab Portunus trituberculatus. Exposure to n-TiO2, PCP, and predation risk showed interactive effects on antioxidant and immune parameters of the mussels. Elevated activities of catalase (CAT), glutathione peroxidase (GPX), acid phosphatase (ACP) and alkaline phosphatase (AKP), suppressed activity of superoxide dismutase (SOD), lower levels of glutathione (GSH) and increased malondialdehyde (MDA) levels indicated dysregulation of the antioxidant system and immune stress induced by single PCP or n-TiO2 exposure. Integrated biomarker (IBR) response values showed the effect of PCP was concentration dependent. Of the two used n-TiO2 sizes (25 and 100 nm), larger particles induced higher antioxidant and immune disturbances indicating higher toxicity possibly due to higher bioavailability. Compared to single PCP exposure, the combination of n-TiO2 and PCP enhanced the imbalance of SOD/CAT and GSH/GPX and led to elevated oxidative lesions and activation of immune-related enzymes. Overall, the combined impacts of pollutants and biotic stress exhibited a greater magnitude of adverse effects on antioxidant defense and immune parameters in mussels. The toxicological effects of PCP were exacerbated in the presence of n-TiO2, and the deleterious impact of these stressors was further amplified under predator-induced risk after prolonged (28 days) exposure. However, the underlying physiological regulatory mechanisms governing the interplay of these stressors and predatory cues on mussels remain elusive, warranting further investigation.

Population Genetic Analysis of the Wild Hard-shelled Mussel, Mytilus Unguiculatus (Valenciennes 1858) in South Korea Using a Microsatellite Multiplex Assay

The Korean or hard-shelled mussel, Mytilus unguiculatus, previously known as Mytilus coruscus, is one of the most economically and ecologically important bivalves in South Korea. However, the population size of this species has drastically reduced owing to overharvesting and habitat shrinkage. Because its genetic information is poorly documented, we contributed, in this study, the genetic diversity and structural analyses of 246 adult samples of M. unguiculatus from seven populations along the coastal areas of the mainland and islands of South Korea using a microsatellite multiplex assay. Genetic diversity analyzed from eleven polymorphic microsatellite loci was consistently moderate (0.50–0.57) in all populations. No recent bottleneck was found, indicating that the number of the studied populations did not decrease to an extent that resulted in a reduction of genetic diversity. Additional tests did not reveal any genetic structure across them, possibly resulting from constant gene flow, strong dispersal of planktonic larvae, and genetic admixture between wild populations. These results suggest that M. unguiculatus populations along the coastal areas of South Korea should be managed as a single unit. Our study provides crucial information for future genetic monitoring, conservation management, and population restoration plan in preparation for the rapid decline in bivalve resources.

Protective effect of mussel polysaccharide on cyclophosphamide-induced intestinal oxidative stress injury via Nrf2-Keap1 signaling pathway.

The hard-shelled mussel (Mytilus coruscus) has been used as a traditional Chinese medicine and health food in China for centuries. Polysaccharides from mussel has been reported to have multiple biological functions, however, it remains unclear whether mussel polysaccharide (MP) exerts protective effects in intestinal functions, and the underlying mechanisms of action remain unclear. The aim of this study was to investigate the protective effects and mechanism of MP on intestinal oxidative injury in mice. In this study, 40 male BALB/C mice were used, with 30 utilized to produce an animal model of intestinal oxidative injury with intraperitoneal injection of cyclophosphamide (Cy) for four consecutive days. The protective effects of two different doses of MP (300 and 600 mg/kg) were assessed by investigating the change in body weight, visceral index, and observing colon histomorphology. Moreover, the underlying molecular mechanisms were investigated by measuring the antioxidant enzymes and related signaling molecules through ELISA, real-time PCR, and western blot methods. The results showed that MP pretreatment effectively protected the intestinal from Cy-induced injury: improved the colon tissue morphology and villus structure, increased superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) activities, and reduced malondialdehyde (MDA) content in serum and colon tissues. Meanwhile, MP also significantly increased the expression levels of SOD, GSH-Px, heme oxygenase-1 (HO-1), and nuclear factor E2-related factor 2 (Nrf2) mRNA in colon tissues. Further, western blot results showed that the expression of Nrf2 protein was significantly upregulated while kelch-like ECH-associated protein 1 (Keap1) was significantly downregulated by MP in the colonic tissues. This study indicates that MP can ameliorate Cy-induced oxidative stress injury in mice, and Nrf2-Keap1 signaling pathway may mediate these protective effects.

Effects of polystyrene microplastics on dechlorane plus bioaccumulation in the thick-shell mussel

The combined pollution of microplastics (MPs) and persistent organic pollutants (POPs) have attracted increasing attention from the international community in recent years. MPs can affect the toxicity, bioenrichment rate and bioavailability of POPs through adsorption and other interactions. Dechlorane Plus (DP) is a chlorinated flame retardant and a typical. DPs used mainly in various polymer materials, such as electrical wire and cable coating. The pollutions of MPs and DPs (syn and anti isomers, syn-DP [s-DP] and anti-DP [a-DP]) are ubiquitously present in the environment. However, the effect of MPs on the bioaccumulation of DP has not been reported. In this study, thick-shell mussels (Mytilus coruscus) were exposed to DPs (10 ng/L, DP10), DPs and polystyrene microplastics (PS) (10 ng/L DP +10 μg/L PS, DP10/PS10; 10 ng/LDP+20 μg/L PS, DP10/PS20) for 28 days to investigate the effect of MPs on DPs enrichment. Thick-shell mussels accumulated DPs in the adductor muscle, gill, and gonad showed an increasing trend with time, but the concentration of DPs in the visceral mass does not show a time-dependent manner. The concentration of DPs in the gonads and gills was significantly affected by the concentration of PS (p < 0.05), but there was no significant correlation between the concentration of DPs and the concentration of PS in the adductor muscle and visceral mass (p > 0.05). Moreover, DPs showed selective enrichment of syn-DP in thick-shell mussel tissues, and there was no significant correlation between this result and PS concentration (p > 0.05). These findings provide a new entry point for studying the interaction between microplastics and persistent organic pollutants in marine organisms.

Effect of cadmium in the gonads of mussel (Mytilus coruscus): an ionomics and proteomics study.

The mussel Mytilus coruscus is an important and very popular seafood in China and widespread along the eastern coast of China. In this study, we investigated the molecular response of mussel gonads to cadmium accumulation at two concentrations (80 and 200µg/L) for 30days using ionomics and proteomics techniques. The shrinkage of the cells and moderate hemocytic infiltration were observed in the Cd-treated groups. The strontium, selenium (Se), and zinc contents were significantly altered, and the relationships between iron, copper, Se, manganese, calcium, sodium, and magnesium were also significantly altered. Label-free quantitative proteomics analysis revealed a total of 227 differentially expressed proteins. These proteins were associated with multiple biological processes, including the tricarboxylic acid cycle, structural reorganization of cells, biosynthesis of amino acids, inflammatory response of cells, and tumorigenesis. Nonetheless, our ionomics and proteomics analysis revealed that mussels could partly alleviate the adverse effects of Cd by altering the metal contents and correlations between minerals, thereby enhancing the biosynthesis of some amino acids and activity of antioxidant enzymes. Overall, this study provides an insight into the mechanism underlying Cd toxicity in mussel gonads from a metal and protein perspective.

Outer membrane vesicles induce the mussel plantigrade settlement via regulation of c-di-GMP

Despite the importance of outer membrane vesicles (OMVs) in benthic animal settlement, the underlying molecular mechanism remains elusive. Here, the impact of OMVs and OMVs synthesis-related tolB gene in Mytilus coruscus plantigrade settlement was tested. The OMVs were extracted from Pseudoalteromonas marina through density gradient centrifugation, and a tolB knockout strain, achieved by homologous recombination, was utilized for the investigation. Our results demonstrated that OMVs could significantly enhance M. coruscus plantigrades settlement. Deleting the tolB resulted in downregulation of c-di-GMP, accompanied by a reduction of OMV production, a decline in bacterial motility and increasing biofilm-forming ability. Enzyme treatment resulted in a 61.11% reduction in OMV-inducing activity and a 94.87% reduction in LPS content. Thus, OMVs regulate mussel settlement via LPS, and c-di-GMP is responsible for the OMV-inducing capacity. These findings provide new insights into the interactions between bacteria and mussels.

Two-Component System Response Regulator ompR Regulates Mussel Settlement through Exopolysaccharides.

The outer membrane protein (OMP) is a kind of biofilm matrix component that widely exists in Gram-negative bacteria. However, the mechanism of OMP involved in the settlement of molluscs is still unclear. In this study, the mussel Mytilus coruscus was selected as a model to explore the function of ompR, a two-component system response regulator, on Pseudoalteromonas marina biofilm-forming capacity and the mussel settlement. The motility of the ΔompR strain was increased, the biofilm-forming capacity was decreased, and the inducing activity of the ΔompR biofilms in plantigrades decreased significantly (p < 0.05). The extracellular α-polysaccharide and β-polysaccharide of the ΔompR strain decreased by 57.27% and 62.63%, respectively. The inactivation of the ompR gene decreased the ompW gene expression and had no impact on envZ expression or c-di-GMP levels. Adding recombinant OmpW protein caused the recovery of biofilm-inducing activities, accompanied by the upregulation of exopolysaccharides. The findings deepen the understanding of the regulatory mechanism of bacterial two-component systems and the settlement of benthic animals.

Chlamydomonas reinhardtii-derived mytichitin-CB/hispidalin chimera efficiently inhibits the growth of bacteria by disrupting their membrane integrity

The antimicrobial peptides Mytichitin-CB and Hispidalin were isolated from Mytilus coruscus and Benincasa hispida, respectively. Our previous studies showed that Mytichitin-CB, when expressed in Chlamydomonas reinhardtii, inhibits the bacterial growth with moderate efficiency. Naturally isolated or Pichia pastoris-expressed Hispidalin also exerts moderate efficiency for inhibiting the growth of bacteria. Here, we expressed a chimeric peptide composed of Mytichitin-CB and Hispidalin that we named as MCH in C. reinhardtii. MCH was stably expressed and its yield accounted for 0.18% of total soluble proteins of the host cells. C. reinhardtii-derived MCH inhibited the growth of both Gram-positive and Gram-negative bacteria by disrupting their cell membrane integrity. Its minimal inhibitory concentration (MIC) were ranged between 20 and 40 μg/mL, demonstrating that MCH is more potent for inhibiting the bacterial growth than individual microalgae-expressed Mytichitin-CB and yeast-expressed Hispidalin. Of more importance, MCH was temperature- and pH-tolerant, protease-resistant and exhibited low hemolytic activity against rat erythrocytes and low cytotoxicity to human embryonic kidney 293 (HEK 293) and Madin-Darby bovine kidney (MDBK) cells. Therefore, we conclude that C. reinhardtii-derived chimeric peptide MCH has a great potential for being developed to replace traditional antibiotics in the future.

Dietary exposure to nTiO2 reduces byssus performance of mussels under ocean warming

Nano‑titanium dioxide (nTiO2) is a widely used nanomaterial posing potential ecological risk for marine ecosystems that might be enhanced by elevated temperatures such as expected during climate change. nTiO2 may affect benthic filter feeders like mussels through waterborne exposures and via food chain due to the adsorption on/in algae. Mussel byssus are proteinaceous fibers secreted by byssal glands of the mussels for attachment. Byssus production and mechanical properties are sensitive to environmental stressors but the combined effects of warming and nTiO2 on byssus performance of mussels are unclear hampering our understanding of the predation and dislodgement risk of mussels under the multiple stressor scenarios. We explored the effects of a short-term (14-day) single and combined exposures to warming (28 °C) and 100 μg L−1 nTiO2 (including food co-exposure) on the byssus performance of the thick shell mussel Mytilus coruscus. The mechanical strength (measured as the breaking force) of the byssal threads was impaired by warming and nTiO2 (including food co-exposure), but the number and length of the byssal threads were increased. The mRNA expression levels of mussel foot proteins (mfp-3, mfp-5) and pre-collagens (preCOL-D, preCOL-P, preCOL-NG) were up-regulated to varying degrees, with the strongest effects induced by warming. This indicates that the physiological and molecular mechanisms of byssus secretion are plastic. However, downregulation of the mRNA expression of preCOL-D and preCOL-P under the combined warming and nTiO2 exposures indicate the limits of these plasticity mechanisms and suggest that the attachment ability and survival of the mussels may be impaired if the pollution or temperature conditions further deteriorate.

High Fischer ratio oligopeptides from hard-shelled mussel: Preparation and hepatoprotective effect against acetaminophen-induced liver injury in mice

High Fischer ratio oligopeptides (HFOPs) have a variety of physiological activities, but there are few literatures reporting the hepatoprotective effects of HFOPs against acetaminophen (APAP)-induced liver injury. The objectives of this study were to establish the preparation process of HFOPs from hard-shelled mussel (Mytilus coruscus) (HFOPs-HM) and investigate the hepatoprotective effects and potential mechanisms of HFOPs-HM against APAP-induced liver injury in mice. Therefore, trypsin and papain were selected as the endo and exogenous proteases for preparing HFOPs-HM according to the degree of hydrolysis (DH) of hard-shelled mussel hydrolysates, and activated carbon of XHJ-200 displayed the best effect to remove aromatic amino acids according to the Fischer ratio (OD220/OD280) and its parameters were optimized as adsorption time of 3.0 h and solid-liquid ratio of 1:10. The HFOPs-HM's Fischer ratio and average molecular weight were 26.03 (>20) and 878.54 Da, respectively. Furthermore, a mice model of liver injury induced by APAP was established. HFOPs-HM could significantly improve liver index and histopathological features, decrease the serum alanine aminotransferase (ALT) and aspartate transaminase (AST) activities, increase the glutathione content and superoxide dismutase (SOD) and catalase (CAT) activities. Further research demonstrated that HFOPs-HM could alleviate liver oxidative stress in liver tissue via activating the Nrf2 pathway to initiate the intracellular antioxidant enzyme system. In addition, HFOPs-HM also suppressed liver inflammation by downregulating the secretion of TNF-α, IL-1β, and IL-6. Thus, HFOPs-HM could serve as an auxiliary functional dietary molecule applied in function products to alleviate drug-induced liver injury.

Combined toxic effects of nanoplastics and norfloxacin on mussel: Leveraging biochemical parameters and gut microbiota

Antibiotics and nanoplastics (NPs) are among the two most concerned and studied marine emerging contaminants in recent years. Given the large number of different types of antibiotics and NPs, there is a need to apply efficient tools to evaluate their combined toxic effects. Using the thick-shelled mussel (Mytilus coruscus) as a marine ecotoxicological model, we applied a battery of fast enzymatic activity assays and 16S rRNA sequencing to investigate the biochemical and gut microbial response of mussels exposed to antibiotic norfloxacin (NOR) and NPs (80 nm polystyrene beads) alone and in combination at environmentally relevant concentrations. After 15 days of exposure, NPs alone significantly inhibited superoxide dismutase (SOD) and amylase (AMS) activities, while catalase (CAT) was affected by both NOR and NPs. The changes in lysozyme (LZM) and lipase (LPS) were increased over time during the treatments. Co-exposure to NPs and NOR significantly affected glutathione (GSH) and trypsin (Typ), which might be explained by the increased bioavailable NOR carried by NPs. The richness and diversity of the gut microbiota of mussels were both decreased by exposures to NOR and NPs, and the top functions of gut microbiota that were affected by the exposures were predicted. The data fast generated by enzymatic test and 16S sequencing allowed further variance and correlation analysis to understand the plausible driving factors and toxicity mechanisms. Despite the toxic effects of only one type of antibiotics and NPs being evaluated, the validated assays on mussels are readily applicable to other antibiotics, NPs, and their mixture.

Trophic niche and potential carbon source of three reef-associated fishes of Zhongjieshan Islands.

To clarify the trophic relationship of important rock fishes, we analyzed trophic niche of three typical rockfish species (Oplegnathus fasciatus, Sebastiscus marmoratus and Conger myriaster) in the Zhongjieshan Islands in summer 2020, based on the carbon and nitrogen stable isotope techniques. We calculated the contributions of major carbon sources [macroalgae, phytoplankton, suspended particulate organic matter (POM) and substrate organic matter (SOM)]. The results showed that: 1) the δ13C values of the three species ranged from -21.44‰ to -15.21‰, with an average value of (-16.85±1.12)‰, while the δ15N values ranged from 8.32‰ to 10.96‰, with an average value of (9.69±0.66)‰. There were significant differences in carbon and nitrogen stable isotopes among the three species. 2) There was small niche overlap between O. fasciatus and S. marmoratus, indicating that the interspecific competition was not intense. There was no overlap between C. myriaster and the first two, indicating feeding differentiation. 3) The total ecotone area, corrected core ecotone area, and food source diversity of C. myriaster were the highest, indicating that it had a more generalized diet and richer food sources. 4) With Mytilus coruscus as a baseline organism, the trophic level of C. myriaster was the highest (3.38), followed by S. marmoratus (3.09), and the trophic level of O. fasciatus was the smallest (3.00). 5) Results of the stable isotope mixture model (SIAR) showed that POM was the main carbon source of the three species, contributing 57.4%, 57.9%, and 92.0% of the total, respectively. In addition, the contribution rate of SOM was also high for O. fasciatus and S. marmoratus, which was 21.5% and 33.9%, respectively. This study could provide basic information and reference for understanding trophic structure and marine food web in Zhongjiashan Islands.

Morphological change and differential proteomics analysis of gill in Mytilus coruscus under starvation.

Mytilus coruscus is a dominant shellfish in the Yangtze estuary and its adjacent sea area. Food deprivation often occurs during their growth due to fluctuations in algal abundance caused by seasonal freshwater flushing and high-density aquaculture mode. To investigate the coping strategies of M. coruscus to starvation stress, electron microscopy and differential proteomic analysis were performed on the critical feeding organ gill of the mussels after 9days of starvation. The electron microscopy results showed that the cilia of the mussel gills were dissolved, and the gaps between gill filaments widened under starvation. Differential proteomic analysis revealed that phagocytosis-related proteins such as ATPeV1E, ATPeV1C, LAMP1_2 and CTSL were significantly upregulated, and the phagocytosis pathway was significantly enriched (p < 0.05). In addition, the corin content in gill and myeloperoxidase level as well as the number of dead cells in blood were both significantly increased (p < 0.05). What's more, proteomic data suggested that immune maintenance, cellular transport and metabolism related pathways were significantly enriched, which illustrated an immune and metabolism responses under starvation. This study reveals for the first time that phagocytosis functions as an essential strategy for M. coruscus to cope with starvation, which provides new scientific knowledge and a theoretical basis for understanding the adaptation mechanisms of mussel to starvation and for rational optimization of mussel culture patterns.

Mussel settlement mediated by bacterial VgrG proteins via extracellular outer membrane vesicles

T6SS plays an important role in biofilm formation, but how bacterial T6SS regulates biofilm formation and its effect on marine invertebrate settlement is still unclear. Here, the role of T6SS spike protein VgrG and VgrG protein synthesis gene on biofilm formation and Mytilus coruscus settlement was examined. Deletion of the core gene vgrG of T6SS resulted in a decrease in the growth capability of Leisingera aquaemixtae and inhibition of the ability of biofilm formation. The OMV content and inducing activity of ΔvgrG biofilms were decreased respectively by 20.72% and 59.70% compared with the wild-type biofilms. OMVs induced the settlement of M. coruscus, while VgrG proteins did not induce mussel settlement directly. Addition of VgrG proteins resulted in the upregulation of OMVs in the ΔvgrG biofilms. Adding OMVs caused no increasing inducing activity of ΔvgrG biofilms. Interestingly, the inducing activity of ΔvgrG biofilms both by adding VgrG proteins alone, and by adding VgrG proteins and OMVs in combination increased significantly, and restored to the inducing capability of wild-type biofilms. Thus, bacterial T6SS spike protein VgrG could mediate mussel settlement by increasing the OMV content of biofilms. The present findings shed new light on the interaction between T6SS and marine invertebrate settlement.

Exploring the Role of a Novel Interleukin-17 Homolog from Invertebrate Marine Mussel Mytilus coruscus in Innate Immune Response: Is Negative Regulation by Mc-Novel_miR_145 the Key?

Interleukin-17 (IL-17) represents a class of proinflammatory cytokines involved in chronic inflammatory and degenerative disorders. Prior to this study, it was predicted that an IL-17 homolog could be targeted by Mc-novel_miR_145 to participate in the immune response of Mytilus coruscus. This study employed a variety of molecular and cell biology research methods to explore the association between Mc-novel_miR_145 and IL-17 homolog and their immunomodulatory effects. The bioinformatics prediction confirmed the affiliation of the IL-17 homolog with the mussel IL-17 family, followed by quantitative real-time PCR assays (qPCR) to demonstrate that McIL-17-3 was highly expressed in immune-associated tissues and responded to bacterial challenges. Results from luciferase reporter assays confirmed the potential of McIL-17-3 to activate downstream NF-κb and its targeting by Mc-novel_miR_145 in HEK293 cells. The study also produced McIL-17-3 antiserum and found that Mc-novel_miR_145 negatively regulates McIL-17-3 via western blotting and qPCR assays. Furthermore, flow cytometry analysis indicated that Mc-novel_miR_145 negatively regulated McIL-17-3 to alleviate LPS-induced apoptosis. Collectively, the current results showed that McIL-17-3 played an important role in molluscan immune defense against bacterial attack. Furthermore, McIL-17-3 was negatively regulated by Mc-novel_miR_145 to participate in LPS-induced apoptosis. Our findings provide new insights into noncoding RNA regulation in invertebrate models.

Investigation of physiological energetic response of the thick shell mussel, Mytilus coruscus, to microplastics and low salinity: Potential countermeasures to multi-environmental changes

Microplastics (MPs) contamination and salinity fluctuation are two common phenomena on the East China Sea coast. The purpose of this experiment was to evaluate the combined effects of MPs and salinity on the energy budget of the thick shell mussel Mytilus coruscus. Juvenile mussels were exposed to six combined treatments with three MPs levels (0, 10 and 1000 items L−1) and two salinity levels (15, 25) for 28 d. The clearance rate, food absorption efficiency, respiration rate, ammonium excretion rate, and O:N ratio were significantly reduced, and the fecal organic dry weight ratio was significantly increased at salinity 15, inducing a reduction in the scope for growth. Under salinity 15, 1000 items L−1 MPs had significant negative effects on growth at Day 28. However, under salinity 25, the results were opposite in that the level of 1000 items L−1 MPs brought an obvious increment in scope for growth at Day 28. This meant that there were interactions between salinity and MPs in scope for growth at Day 28. These results suggest that the physiological energetics of juvenile M. coruscus can acclimate to MPs contamination with little physiological effect under normal salinity. This experiment has revealed that although marine MPs contamination is of concern, marine organisms likely have a significant capacity to adapt to it.

An Interleukin-17 Isoform from Thick Shell Mussel Mytilus coruscus Serves as a Mediator of Inflammatory Response

The inflammatory cytokine interleukin-17 (IL17) plays an important role in innate immunity by binding to its receptors (IL17Rs) to activate immune defense signals. To date, information on members of the IL17 family is still very limited in molluscan species. Here, a novel member of the IL17 family was identified and characterized from thick shell mussel Mytilus coruscus, and this gene was designated as McIL17-1 by predicting structural domains and phylogenetic analysis. McIL17-1 transcripts existed in all examined tissues with high expression levels in gills, hemocytes and digestive glands. After the stimuli of different pathogen associated molecular patterns (PAMPs) for 72 h, transcriptional expression of McIL17-1 was significantly upregulated, except for poly I:C stimulation. Cytoplasm localization of McIL17-1 was shown in HEK293T cells by fluorescence microscopy. Further, in vivo and in vitro assays were performed to evaluate the potential function of McIL17-1 played in immune response. McIL17-1 was either knocked down or overexpressed in vivo through RNA inference (RNAi) and recombinant protein injection, respectively. With the infection of living Vibrio alginolyticus, a high mortality rate was exhibited in the McIL17-1 overexpressed group compared to the control group, while a lower mortality rate was observed in the McIL17-1 knocked down group than control group. In vitro, the flow cytometric analysis showed that the apoptosis rate of McIL17-1 inhibited hemocytes was significantly lower than that of the control group after lipopolysaccharide stimulation. These results collectively suggested that the newly identified IL17 isoform is involved in the inflammatory response to bacterial infection in M. coruscus.

Effects of L-arginine on Nitric Oxide Synthesis and Larval Metamorphosis of Mytilus coruscus.

To investigate the regulatory functions of L-arginine and nitric oxide (NO) on Mytilus coruscus metamorphosis, M. coruscus larvae were exposed to an inhibitor of nitric oxide synthase (NOS), aminoguanidine hemisulfate (AGH), and a substrate for NO synthesis, L-arginine. We observed that NO levels showed a significant increase, and this trend continued with L-arginine treatment. When NOS activity was inhibited, the larvae could not synthesize NO, and metamorphosis was not inhibited even in the presence of L-arginine. On transfecting pediveliger larvae with NOS siRNA followed by L-arginine exposure, we found that the larvae did not produce NO and that the larval metamorphosis rate was significantly increased, suggesting that L-arginine regulates M. coruscus larval metamorphosis by promoting NO synthesis. Our findings improve our understanding of the effects of marine environmental factors on larval metamorphosis of mollusks.