Manila Clam Ruditapes Philippinarum Research Articles

Metabolic adaptation of the clam Ruditapes philippinarum during air exposure and the positive effects of sodium nitroprusside pretreatment.

The Manila clam (Ruditapes philippinarum), as one of the shellfish living in the intertidal zone, is known for its strong ability to withstand air exposure. Sodium nitroprusside (SNP), a donor of nitric oxide (NO), has been shown to be useful for antioxidant and immune regulation in aquatic animals. In this study, an untargeted metabolomics (LC-MS/MS) technique was employed for the first time in Manila clam to analyze the metabolic and histological impacts after air exposure and the positive effects of SNP pretreatment. During air exposure, a significant increase in taurine, L-glutamate, and several polyunsaturated fatty acids in clams was detected, which indicates that clams may experience inflammatory reactions, oxidative stress, and an increase in blood ammonia content. When clams were exposed to SNP for 6h, arginine, spermine, L-glutamic acid, and glutathione content were all upregulated, indicating that the SNP exposure induced NO production and improved antioxidant capacity in clams. When the clams were exposed to air after SNP pretreatment, there were no significant differences in the levels of taurine, L-glutamate, or aliphatic acids between the experimental and control groups. Gill tissue was more severely damaged in clams directly exposed to air than in those that experienced air exposure after SNP pretreatment, especially in clams exposed to air for a long time (72h). Both metabolomics and tissue section structure indicated that SNP pretreatment decreased the stress responses caused by air exposure in R. philippinarum. These findings provided fresh insights and a theoretical foundation for understanding the tolerance to air exposure and physiological functions of SNP (or NO) in R. philippinarum.

Environmental influence on summer survival of Manila Clam Ruditapes philippinarum: A case study in an aquaculture bay

The aquaculture of Manila clam (Ruditapes philippinarum) is under threat due to its high mortality in summer. To investigate the environmental influence on mortality of the species, we have conducted field surveys in the aquaculture area in Laizhou Bay, during the summers of 2019 and 2020. Environmental and biological data were collected. The results of data analysis have shown that in 2020, large-scale mortality events were not recorded and spatial variations of environmental variables were no significant within the survey area. However, in August 2019, significant variations of environmental variables were detected in the middle tidal area. This area was charactered with relatively high-water temperature (with maximum temperature of 31.49 °C and mean value of 26.17 ± 1.62 °C), elevated hydrogen sulfide concentrations (with a maximum concentration of 24.72 μmol/L and a mean concentration of 7.89 ± 4.14 μmol/L), and low dissolved oxygen concentrations (with a minimum concentration of 3.35 mg/L and a mean concentration of 6.27 ± 0.42 mg/L). Correspondingly, high mortality of the clam was recorded in the area, the abundance of live Manila clams has decreased by 80%. No significant abnormalities of environmental variables and clam growth were observed in other regions in 2019. Glycogen content and condition index of the clam were analyzed to investigate the relationship between environmental stress and the health of the clam. Compared to the condition index, glycogen content is a more sensitive indicator of the health status of the clam because changes in glycogen content appeared earlier than changes in condition index and mortality. A principal component analysis further indicated that the high mortality of the clam corresponds with a few environmental variables, including elevated temperature, hypoxia and the concentration of hydrogen sulfide. The simultaneous presence of these multiple environmental stressors could have triggered alterations in the physiological responses of the clam.

Embryonic development, survival and growth of triploid Zebra Clam II strain of the Manila clam Ruditapes philippinarum

Triploid breeding with large individuals, high yield, and excellent disease resistance is widely used in aquaculture. However, the functional roles of triploid in the Zebra Calm II strain of Manila clam Ruditapes philippinarum has not been highlighted. In this study, the Zebra Clam II strain of the Manila clams were treated with cytochalasin B (CB) at a final concentration of 0.5 mg/L at 26 °C for 17 min to induce triploidity. The results showed that triploid induction rate of first-hatched D-larvae was 64.73 % and the triploid proportion of 180-day-old larvae was 72.5 %. Furthermore, the hatching rate in triploid (60.54 ± 2.19 %) was significantly lower than that in the control group (92.47 ± 0.58 %) (p < 0.05). Moreover, the development time of D-larvae in the experimental group was delayed by 145 min, and the survival rate of 15-day-old larvae in experimental group (46.08 ± 4.49 %) was significantly lower than that in the control group (62.57 ± 2.68 %) (p < 0.05), but have no significantly changes in the survival rate of triploids in larval to adult stages (p > 0.05). However, the daily relative growth rate of experimental group was significantly higher than that the control group at all daily ages except 360 days (p < 0.05). Moreover, at 480 days of larvae, the growth of the shell length and shell width in the experimental group was 19.2 % and 22.3 % higher than that in the control group, respectively. Altogether, the results in this study provide a scientific basis for the triploid breeding of Zebra Clam Ⅱ, which has great potential to improve the quality and efficiency of clam farming.

Sex-specific responses of Ruditapes philippinarum to ocean acidification following gonadal maturation

Ocean acidification (OA) can seriously affect marine bivalves at different levels of biological organization, generating widespread consequences on progeny recruitment and population maintenance. Yet, few effort has been devoted to elucidating whether female and male bivalves respond differentially to OA in their reproductive seasons. Here, we estimated differences in physiological responses of female and male Manila clams (Ruditapes philippinarum) to OA during gonadal maturation. In comparison to OA-stressed male clams, females significantly depressed activities in enzymes related to energy metabolism (NKA, T-ATP), antioxidant defence (SOD and MDA), and non-specific immune function (ACP), and downregulated expression of AMPK that plays a key role in cellular metabolism, indicating that sex did significantly affect responses of R. philippinarum to OA. Such sex-based differences can be likely couched in energetic terms, given the much more energetically expensive cost of egg production than that of sperms. These results indicate that sex-specific responses to OA during reproductive seasons do exist in marine bivalves, and therefore accounting for such sex specificity is of paramount importance when projecting population sustainability and formulating conservation strategies in an acidifying ocean.

Exploring the newly emerging effects of native seagrasses on survival and growth of non-native juvenile clams

The Manila clam (Ruditapes philippinarum) has been introduced into the Atlantic European coast for commercial exploitation. In this region, the population dynamics of this clam species may be determined by the interaction with the native dwarf eelgrass (Zostera noltei). We performed an experiment in the tidal flats of an estuary located in northern Spain to analyse the interaction between Z. noltei and juvenile Manila clams. Based on existing knowledge, it was hypothesised that seagrasses act as a nursery protecting juveniles from predation, which is an important cause of natural mortality, and that seagrass density influences the survival, growth and condition of clams. The results indicate that the effects of seagrasses on juvenile clams depends on shoot density, which mainly determine the relative prevalence of positive versus negative interactions. Dense seagrass meadows protect juvenile clams from predation, likely decreasing their visibility and reducing the efficiency of predators. However, in these dense meadows, a decline in the condition of clams was also observed, although not in shell growth. This decline can be due to the fact that food supply in dense meadows is slow and does not fulfil the replenishment rates required by filter-feeding organisms or because competition for space with the seagrass causes stress. Clams are larger in populations where predator access is prevented, possibly indicating selective predation on larger organisms when this interaction is allowed. The knowledge gained from this experiment is of great interest for analysing possible future trends in clam populations and the derived social implications given its high economic value.

Genetic and Haplotype Diversity of Manila Clam Ruditapes philippinarum in Different Regions of China Based on Three Molecular Markers.

China has the largest production yield of Manila clam Ruditapes philippinarum in the world. Most of the clam seeds for aquaculture are mainly derived from artificial breeding in southern China, likely resulting in the loss of genetic variation and inbreeding depression. To understand the genetic and haplotype diversity of R. philippinarum, 14 clam populations sampled from different regions of China were analyzed by three molecular markers, including COI, 16SrRNA and ITS. Based on the results of the COI and ITS genes, the 14 populations showed a moderate to high level of genetic diversity, with an average haplotype diversity of 0.9242 and nucleotide diversity of 0.05248. AMOVA showed that there was significant genetic differentiation among all populations (mean FST of the total population was 0.4534). Pairwise FST analysis showed that genetic differentiation reached significant levels between Laizhou and other populations. Two Laizhou populations showed great divergence from other populations, forming an independent branch in the phylogenetic tree. The shared haplotypes Hap_2 and Hap_4 of COI appeared most frequently in most clam populations. In contrast, 16SrRNA analysis of the clam populations revealed the dominated haplotype Hap_2, accounting for 70% of the total number of individuals. The haplotype diversity of the Laizhou population (Laizhou shell-wide (KK) and Laizhou dock (LZMT)) was relatively higher than other populations, showing multiple unique haplotypes (e.g., Hap_40, Hap_41 and Hap_42). These findings of genetic and haplotype diversity of clam populations provide guiding information for genetic resource conservation and genetic improvement of the commercially important R. philippinarum.

Transcriptomics and metabolomics reveal the molecular and metabolic adaptation to heat stress in Manila clam Ruditapes philippinarum

Temperature is an important environmental factor affecting metabolism, growth and life activities of marine bivalves. To reveal the molecular and metabolic responses to heat stress, comparative transcriptomics and metabolomics were performed in the commercially important bivalve, Manila clam Ruditapes philippinarum. Comparative transcriptomics revealed a total of 3980 differential genes (DEGs) mainly involved in energy metabolism, protein processing, glycerolphospholipid metabolism, inoxidizability and anti-apoptosis. Comparative metabolomic analysis identified 293 differential metabolites (DEMs) in the heat-stressed clams, including Acetyl-CoA, Beta-D-Glucose, phosphatidylcholine and free amino acids. For the heat-stress clams, they showed the significantly increased enzyme activities of AKP and ACP, as well as antioxidant-related enzyme activities (SOD and CAT) and oxidative damage substance (MDA). The changes in gene expression of glycolysis-related genes (GLUT1, GLK, HK1 and GPI6) and metabolites (Beta-D-Glucose and Acetyl-CoA) reflect the adjusted energy metabolism in the heat-stressed clams. The protein processing in endoplasmic reticulum may play the important roles in the stability of cell membrane structure during heat adaptation. The increased contents of glycerophospholipid (PC) may be essential to maintain the stability and permeability of cell membrane in the heat-stressed clams. Molecular chaperones (HSP70 and HSP90) may participate in the degradation of misfolded proteins to maintain cell homeostasis during the heat stress. To defense the heat stress, clams also initiate the protection and defense mechanisms by activating antioxidant system, innate immune system and anti-apoptotic system. These findings will shed new lights on molecular and metabolic adaptation to heat stress in the intertidal mollusks in a warming climate at the global scale.

Comparative evaluation of most probable number and direct plating methods for enumeration of Escherichia coli in Ruditapes philippinarum, and effect on classification of production and relaying areas for live bivalve molluscs

A precise and accurate method for enumeration of Escherichia coli is critical to classify production and relaying areas for live bivalve molluscs. Paired comparison of most probable number and direct plating count of E. coli was performed on a total of 918 samples of Manila clams (Ruditapes philippinarum) collected from official monitoring and from a regional shellfish monitoring program conducted from 2011 to 2017 in Emilia Romagna region, Italy. The records of E. coli enumeration resulting from both MPN reference method and alternative TBX count method were compared with McNemar's test using three cut-off values, namely 230, 700 and 4.600 MPN/100g, based on the requirements laid down in Regulation (EU) 2019/627. The relative trueness was calculated. Significant differences were observed between the two methods for each investigated cut-off: these incongruities concerned a total of 129 (14%), 131 (14%) and 56 (6%) samples observed as negative with TBX but positive by MPN (respectively using 230, 700 and 4.600 E. coli as cut-off), as well as, quite the opposite, a total of 20 (2,2%), 8 (0,9%) and 5 (0,5%) samples observed negative by MPN but positive with TBX method. A negative bias is reported between the two methods. Applying the outcomes of the two different methods to a real scenario, the Veterinary Competent Authority could classify production and relaying areas in the same area of classification only for 69% of samples. This high degree of disagreement between MPN and direct plating outcomes clearly shows the different and not negligible impact of the two methods on the specific E. coli requirements for the classification of production and relaying areas for R. philippinarum. Analytical methods, either official or validated, if used officially by Veterinary Authority must lead to the same classification of production and relaying areas for live bivalve molluscs.

Transcriptional profiling of pigmentation-regulated long non-coding RNAs in the mantle of Manila clam (Ruditapes philippinarum)

Long non-coding RNAs (lncRNAs) play important roles in a variety of biological processes and have received extensive attention in recent years. However, the expression and roles of lncRNAs in the Manila clam (Ruditapes philippinarum) are still poorly understood. In this study, we identified lncRNAs in the mantle of clams with different shell colors (orange (O), zebra (Z), white (W), and white zebra (WZ)). Using poly(A)-independent and strand-specific RNA-seq, we obtained 183,818,4888 clean reads and 32,541 lncRNA transcripts. In total, 1988 lncRNAs were identified as differentially expressed lncRNAs in comparisons between six pairs of groups. Fourteen mRNAs and their target genes were found to be related to the shell color pathway of melanin and porphyrin metabolism. Protein kinase C, calmodulin, melanogenesis associated transcription factor, peroxidase-like protein, tyrosine decarboxylase, myeloperoxidase, putativetyrosinase-like protein tyr-3, dopamine beta-hydroxylase, 5-aminolevulinate synthase, tyrosine aminotransferase, uroporphyrinogen-III synthase, hephaestin, delta-aminolevulinic acid dehydratase and ferrochelatase and their corresponding 77 cis-acting lncRNAs are thought to play important roles in the melanin synthesis pathway and porphyrin metabolic pathway. This study identified the catalog of lncRNAs expressed in the mantle of Manila clam, and the results provide insights into understanding the molecular regulation of shell color and genetic breeding in R. philippinarum.

Impaired reproduction, energy reserves and dysbiosis: The overlooked consequences of heatwaves in a bivalve mollusc

Extreme events like Marine Heatwaves (MHWs) are becoming more intense, severe, and frequent, threatening benthic communities, specifically bivalves. However, the consequences of non-lethal MHWs on animals are still poorly understood. Here, we exposed the Manila clam Ruditapes philippinarum to non-lethal MHW for 30 days and provided an integrative view of its effects. Our result indicated that albeit non-lethal, MHW reduced clam's energy reserves (by reducing their hepato-somatic index), triggered antioxidant defenses (particularly in males), impaired reproduction (via the production of smaller oocytes in females), triggered dysbiosis in the digestive gland microbiota and altered animals' behaviour (by impacting their burying capacity) and filtration rate. Such effects were seen also at RNA-seq (i.e. many down-regulated genes belonged to reproduction) and metabolome level. Interestingly, negative effects were more pronounced in males than in females. Our results show that MHWs influence animal physiology at multiple levels, likely impacting its fitness and its ecosystem services.

Kinetics of uptake and depuration of synthetic pyrethroid insecticides in manila clam (Ruditapes philippinarum).

Synthetic pyrethroid insecticides (SPIs) are frequently detected in water bodies and sediments, and they show high toxicity to aquatic organisms, but their toxicity kinetics remain unknown. In this work, the kinetics of uptake and depuration of three SPIs, fenpropathrin (FP), cypermethrin (CM) and deltamethrin (DM) were evaluated in manila clams (Ruditapes philippinarum) for the first time through a bioconcentration-semi-static test. Clams were exposed to three SPIs of different concentrations (2 ng/mL and 20 ng/mL) for 4 days, followed by a 10-day depuration stage. The results indicated that adult manila clams could absorb SPIs rapidly, and the bioconcentration factor (BCF) values of SPIs were different at high and low concentrations of contaminants. The depuration rate constants (k2) of SPIs in adult manila clams ranged from 0.024 h-1 to 0.037 h-1. The bioaccumulation factors ranged from 319.41 to 574.38. And the half-lives (t1/2) were in the range of 18.49 to 29.22 h. These results showed that manila clams have a high bioconcentration capacity, and SPIs have a high cumulative risk for bivalves. Moreover, after 10 days of elimination, SPIs can still be detected in manila clams at all concentrations, indicating that the complete elimination of SPIs required a longer time.

CO2-Induced Ocean Acidification Alters the Burrowing Behavior of Manila Clam Ruditapes philippinarum by Reversing GABAA Receptor Function.

Biological burrowing behavior is an important driver shaping ecosystems that is being threatened by CO2-induced ocean acidification; however, the effects of ocean acidification on burrowing behavior and its neurological mechanism remain unclear. This study showed that elevated pCO2 significantly affected the burrowing behaviors of the Manila clam Ruditapes philippinarum, such as increased foot contraction, burrowing time, and intrabottom movement and decreased burrowing depth. Delving deeper into the mechanism, exposure to elevated pCO2 significantly decreased extracellular pH and increased [HCO3-]. Moreover, an indicator GABAA receptor, a neuroinhibitor for movement, was found to be closely associated with behavioral changes. In situ hybridization confirmed that the GABAA receptor was widely distributed in ganglia and foot muscles, and elevated pCO2 significantly increased the mRNA level and GABA concentration. However, the increase in GABAA receptor and its ligand did not suppress the foot movement, but rather sent "excitatory" signals for foot contraction. The destabilization of acid-base homeostasis was demonstrated to induce an increase in the reversal potential for GABAA receptor and an alteration in GABAA receptor function under elevated pCO2. This study revealed that elevated pCO2 affects the burrowing behavior of Manila clams by altering GABAA receptor function from inhibitory to excitatory.

Effects of Manila clam Ruditapes philippinarum culture on the macrobenthic community

Macrobenthic bioturbation affects the environment, and variances in habitat, such as decreased dissolved oxygen concentration and increased ammonia concentration, affects the macrobenthic community. The relationship between macrobenthos and habitat factors may be a mutually causal relationship. The bottom-sowing culture for Manila clam Ruditapes philippinarum in many coastal countries plays an important role in coastal fisheries, and the relationship between Manila clam and other macrobenthos affects the ecological stability of the bottom-sowing culture zone. It is necessary to explore the relationship between them to manage the waters used for the bottom-sowing culture for Manila clam. In this study , from June to October 2021, the field investigation on macrobenthos including Manila clam and their habitat factors, such as particle size, nutrient content, redox potential, and organic matter content, in 21 experimental communities enclosed by bolting-silk net in Shuangdao Bay, Weihai, Shandong, China was conducted, during which macrobenthos functional groups were determined by feed sources and motor behavior. The results showed that Manila clam biomass was 7.215±0.984 g/m2 (calculated by dry soft tissue weight), and it was positively correlated with the biomass of macrobebthos functional groups B1, G1 and P1; water content in sediment; sulfide content in interstitial water; the Shanon-Wiener diversity index; Pielou’s evenness index and the W statistic of the ABC (Abundance-Biomass Comparison) curve (p&amp;lt; 0.05). Moreover, it was negatively correlated with sediment particulate size and HCl-NO3 content in sediment (p&amp;lt; 0.05). The action of Manila clam to habitats was the dominant role of the interaction between clam and habitat. Thus, the bottom-sowing culture for Manila clam does not decrease the macrobenthic community stability, and the invasion of other macrobenthos into the bottom-sowing culture zones for Manila clam may be accidental or inevitable. The results of our study suggest that the management of the bottom-sowing culture for Manila clam should be conducted from ecosystem level; i.e., fishing and aquaculture in the same waters are regarded as two components of an ecosystem; manage them together, rather than treat them separately.

In vitro study of deltamethrin-induced extracellular traps in hemocytes of Ruditapes philippinarum

Deltamethrin (DLM), a broad-spectrum pesticide, has been proven to have toxic effects on aquatic organisms. Here, we detected the formation of extracellular traps (ETosis) formation in Manila clam (Ruditapes philippinarum) hemocytes stimulated by three concentrations of DLM (0.01, 0.1 and 1 μg/mL) in vitro, and explored the underlying mechanisms induced by this pesticide. Extracellular DNA structure observation and quantitative results indicated that DLM exposure could obviously induce hemocytes ETosis, especially under high concentration of DLM induction. Moreover, DLM increased the levels of myeloperoxidase (MPO) and reactive oxygen species (ROS) in a dose-dependent manner, and enhanced the mRNA expression of several ROS-related genes. DPI (NADPH oxidase inhibitor) and ABAH (MPO inhibitor) could substantially inhibit DLM-induced extracellular traps (ETs), suggesting that the induced ETs release was caused by the induction of the ROS burst and MPO production. In addition, three concentrations of DLM-induced ETs were also accompanied by mitochondrial dysfunction, such as increasing the production of mitochondrial ROS, leading to a decrease in mitochondrial membrane potential (MMP) and activation of mitochondrial permeability transition pore (MPTP). Taken together, these results will shed new light on the immunotoxicity of DLM in clams and perhaps lays the foundation for health assessment in bivalves.

The combined effects of polyethylene microplastics and benzoanthracene on Manila clam Ruditapes philippinarum

Microplastic (MP) toxicity has recently been explored in various marine species. Along with the toxicity of plastics polymer itself, additional substances or pollutants that are absorbed onto it may also be harmful. In the present study, we investigated the combined impacts of polyethylene microplastics (PE MPs) and an organic pollutant (Benzo(a)anthracene, BaA) on Manila clam Ruditapes philippinarum during a one-week exposure. Two PE MPs concentrations (26 μg L−1 and 260 μg L−1) and one BaA concentration (3 μg L−1) were tested. The clams were exposed to BaA and PE MPs either alone or in combination. BaA and PE MPs were incubated before the combined exposure. The biological effects of PE MPs and BaA on the clams were evaluated by considering several assays such as feeding rate, anti-oxidant enzyme activities, and the expression levels of stress-related genes. The feeding rate significantly decreased in individual PE MPs and individual BaA groups while it remained unchanged in combined groups. Superoxide dismutase (SOD) was the most affected among the biochemical parameters. Malondialdehyde (MDA), and glutathione peroxidase (GPx) activities were slightly affected, whereas no changes were observed in glutathione s-transferase (GST) activities. CYP1A1, CYP3A4, and HSP70 gene expressions displayed slightly significant changes. Considering all stressor groups, high PE MPs exposure (260 μg L−1 PE MPs) more effectively altered the biological parameters in the clams compared to individual low PE MPs and BaA exposure, and their combination. The results also indicated the negligible vector role of PE MPs to transport BaA into the clam tissues.

Population Genetics of Manila Clam (Ruditapes philippinarum) in China Inferred from Microsatellite Markers.

The Manila clam (Ruditapes philippinarum) is one of the most commercially important bivalves along the coast of China. With the continuous expansion of clam farming scale, it may lead to some serious problems, including loss of genetic variation, inbreeding depression, and reduced effective population size (Ne). In the present study, eleven microsatellite markers were used to investigate the genetic diversity and differentiation among 13 clam populations along the coast of China. As a result, 150 alleles were detected according to the genotyping results of eleven microsatellite loci. The observed heterozygosity (Ho) was estimated to be ranging from 0.437 to 0.678, while the expected heterozygosity (He) was calculated to be varying from 0.587 to 0.700. Fst values between populations ranged from 0.0046-0.1983. In particular, the Laizhou population had the highest genetic variability, which was significantly different from the others (all Fst values > 0.1). For all the clam populations, there was no significant linear regression between genetic and geographic distance, indicating that these populations do not follow a pattern of isolation by distance (IBD). Genetic structure was estimated according to NJ, principal coordinates (PCoA), and structure-based clustering. Estimates of effective population size range from dozens to thousands among different populations, based on linkage-disequilibrium and molecular coancestry methods. The results reveal the genetic diversity of clams and verify the hypothesis that clam population differentiation may be influenced by the mode of southern breeding and northern culture, providing guiding information for natural resource conservation and genetic breeding of clams.

New insights into the oxidative damage and antioxidant defense mechanism in Manila clams (Ruditapes philippinarum) exposed to 8:2 polyfluoroalkyl phosphate diester stress

8:2 perfluoroalkyl phosphate diester (8:2 diPAP) is the main precursor of perfluoroalkyl carboxylic acids, and it has been detected in a wide range of environments. In this study, conventional biochemical and histopathological analyses and transcriptome methods were used to investigate the accumulation and oxidative stress of 8:2 diPAP in Manila clams (Ruditapes philippinarum) as well as the clam's defense mechanisms for the first time. The hepatopancreas was the main target organ for 8:2 diPAP accumulation; the concentration reached 484.0 ± 15.5 ng/g after 7 days of exposure to 10 μg/L of 8:2 diPAP, which was 2–100 times higher than that found in other organs. 8:2 diPAP accumulation resulted in significant lipid peroxidation, and the change in malondialdehyde content was highly correlated with 8:2 diPAP accumulation (r > 0.8). The antioxidant enzymes catalase and peroxidase were significantly activated at 7 days of exposure. Although the levels subsequently returned to normal, this restoration was unable to prevent damage. Histopathological analysis showed that 8:2 diPAP exposure resulted in inflammatory damage to the hepatopancreas, which failed to resolve during the recovery period. Transcriptomic analyses showed that the expression of differentially expressed genes had different degrees of positive/negative correlation with antioxidant indicators, and they were significantly enriched in cell death regulatory pathways such as autophagy, apoptosis, and necrosis. The core factor expression results indicated that 8:2 diPAP exposure induced activation of the organismal autophagy factor followed by a shift towards apoptosis. In addition, pathways related to amino acid metabolism and energy metabolism were involved in determining the cell fate of Manila clams. Overall, these results indicated that 8:2 diPAP induced peroxidation of membrane lipids, disturbed physiological processes, and ultimately initiated programmed cell death in Manila clams. The findings of this study provide new insights into the mechanism of toxicity of 8:2 diPAP exposure in marine bivalves.

Changes in biochemical metabolites in manila clam after a temporary culture with high-quality microalgal feed mixed with the dinoflagellate species Karlodinium veneficum and K. zhouanum

Phytoplankton composition is an important factor affecting the growth and physiological biochemical characteristics of filter-feeding bivalves. With the increasing trend in dinoflagellate biomass and blooms in mariculture areas, how the physio-biochemical traits and seafood quality of the mariculture organism are affected by the dinoflagellates, especially those at nonfatal levels, is not well understood. Different densities of two Karlodinium species, namely K. veneficum (KV) and K. zhouanum (KZ), mixed with high quality microalgal food Isochrysis galbana was applied in feeding manila clam Ruditapes philippinarum in a 14-day temporary culture, to comparatively study how the critical biochemical metabolites such as glycogen, free amino acids (FAAs), fatty acids (FAs), volatile organic compounds (VOCs) in the clam were affected. The survival rate of the clam showed dinoflagellate density and species specificity. The high-density KV group inhibited survival to 32% lower than that of the pure I. galbana control, respectively, while KZ at low concentrations did not significantly affect the survival compared with the control. In the high-density KV group, the glycogen and FAA contents decreased (p < 0.05), indicating that energy and protein metabolism were significantly affected. Amount of carnosine (49.91 ± 14.64 to 84.74 ± 8.59 μg/g of muscle wet weight) was detected in all the dinoflagellate-mixed groups, while it was not present in the field samples or in the pure I. galbana control, showing that carnosine participated in the anti-stress activities when the clam was exposed to the dinoflagellates. The global composition of FAs did not significantly vary among the groups. However, contents of the endogenous C18 PUFA precursors linoleic acid and α-linolenic acid significantly decreased in the high-density KV group compared to all the other groups, indicating that high density of KV affected the metabolisms of fatty acids. From the results of the changed VOC composition, oxidation of fatty acids and degradation of free amino acids might occur in the clams exposed to dinoflagellates. The increased VOCs, such as aldehydes, and decreased 1-octen-3-ol probably produced a more fishy taste and reduced food flavor quality when the clam was exposed to the dinoflagellates. This present study demonstrated that the biochemical metabolism and seafood qulity of the clam were affected. However, KZ with moderate density in the feed seemed to be beneficial in aquaculture for increasing the content of carnosine, a high-valued substance with multiple bioactivities.

ITRAQ-based proteomic analysis reveals proteins associated with cold stress response in two different populations of Manila clam (Ruditapes philippinarum).

Water temperature is one of the key environmental factors for marine ectotherms and a change in temperature beyond and organism's capacity limits can cause a series of changes to physiological state and damage to the organism. Understanding how organisms adapt to complex environments is a central goal of evolutionary biology and ecology. Ruditapes philippinarum is an ecologically and scientifically important marine bivalve species. To uncover the molecular mechanisms of acclimation of R. philippinarum to low-temperature stress, iTRAQ-based quantitative proteomics was conducted to compare the proteomes of the north and south populations of R. philippinarum under low-temperature stress. The results showed a total of 6355 and 6352 proteins were identified in two populations, respectively. Among these, 94 and 83 were differentially abundant proteins (DAPs), and most of DAPs were related to oxidation-process, protein binding, or an integral component of membrane. According to the results of KEGG pathway enrichment analysis, most of DAPs in both populations are involved in immune-related pathways, while other population-specific significant abundance proteins of south population and north population were enriched in biosynthesis of amino acids (Enolase, Glutamine synthetase) and unsaturated fatty acids pathways (3-ketoacyl-CoA thiolase, Stearoyl-CoA desaturase), respectively, indicating that two population of clams may have different cold-stress regulation mechanisms. Our study provides new insights into different cold stress tolerance mechanisms in northern and southern populations of R. philippinarum using iTRAQ-based proteomics. This work contributes to a better understanding of molecular basis on cold stress response and adaptations, which shed lights on evolutionary biology and general ecophysiology of R. philippinarum.

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

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