Impact of hypoxia on glucose metabolism and hypoxia signaling pathways in juvenile horseshoe crabs Tachypleus tridentatus.

Marine hypoxia poses a significant challenge in the contemporary marine environment. The horseshoe crab, an ancient benthic marine organism, is confronted with the potential threat of species extinction due to hypoxia, making it an ideal candidate for studying hypoxia tolerance mechanisms. In this experiment, juvenile Tachypleus tridentatus were subjected to a 21-day trial at DO:2 mg/L (hypoxia) and DO:6 mg/L conditions. The experimental timeline included a 14-day exposure phase followed by a 7-day recovery period. Sampling occurred on days 0, 7, 14, and 21, where the period from day 14 to day 21 corresponds to seven days of recuperation. Several enzymatic activities of important proteins throughout this investigation were evaluated, such as succinate dehydrogenase (SDH), phosphofructokinase (PFK), hexokinase (HK), lactate dehydrogenase (LDH), and pyruvate kinase (PK). Concurrently, the relative expression of hexokinase-1 (HK), hypoxia-inducible factor 1-alpha inhibitor (FIH), and hypoxia-inducible factor 1-alpha (HIF-1α), pyruvate dehydrogenase phosphatase (PDH), succinate dehydrogenase assembly factor 4 (SDH), and Glucose-6-phosphatase (G6Pase) were also investigated. These analyses aimed to elucidate alterations in the hypoxia signaling pathway and respiratory energy metabolism. It is revealed that juvenile T. tridentatus initiated the HIF pathway under hypoxic conditions, resulting in an upregulation of HIF-1α and FIH-1 gene expression, which in turn, influenced a shift in metabolic patterns. Particularly, the activity of glycolysis-related enzymes was promoted significantly, including PK, HK, PKF, LDH, and the related HK gene. In contrast, enzymes linked to aerobic respiration, PDH, and SDH, as well as the related PDH and SDH genes, displayed down-regulation, signifying a transition from aerobic to anaerobic metabolism. Additionally, the activity of gluconeogenesis-related enzymes such as PK and G6Pase gene expression were significantly elevated, indicating the activation of gluconeogenesis and glycogenolysis pathways. Consequently, juvenile T. tridentatus demonstrated an adaptive response to hypoxic conditions, marked by changes in respiratory energy metabolism modes and the activation of hypoxia signaling pathways.

Combined effects of norfloxacin and polystyrene nanoparticles on the oxidative stress and gut health of the juvenile horseshoe crab Tachypleus tridentatus.

Pollution with anthropogenic contaminants including antibiotics and nanoplastics leads to gradual deterioration of the marine environment, which threatens endangered species such as the horseshoe crab Tachypleus tridentatus. We assessed the potential toxic mechanisms of an antibiotic (norfloxacin, 0, 0.5, 5 µg/L) and polystyrene nanoparticles (104 particles/L) in T. tridentatus using biomarkers of tissue redox status, molting, and gut microbiota. Exposure to single and combined pollutants led to disturbance of redox balance during short-term (7 days) exposure indicated by elevated level of a lipid peroxidation product, malondialdehyde (MDA). After prolonged (14-21 days) exposure, compensatory upregulation of antioxidants (catalase and glutathione but not superoxide dismutase) was observed, and MDA levels returned to the baseline in most experimental exposures. Transcript levels of molting-related genes (ecdysone receptor, retinoic acid X alpha receptor and calmodulin A) and a molecular chaperone (cognate heat shock protein 70) showed weak evidence of response to polystyrene nanoparticles and norfloxacin. The gut microbiota T. tridentatus was altered by exposures to norfloxacin and polystyrene nanoparticles shown by elevated relative abundance of Bacteroidetes. At the functional level, evidence of suppression by norfloxacin and polystyrene nanoparticles was found in multiple intestinal microbiome pathways related to the genetic information processing, metabolism, organismal systems, and environmental information processing. Future studies are needed to assess the physiological and health consequences of microbiome dysbiosis caused by norfloxacin and polystyrene nanoparticles and assist the environmental risk assessment of these pollutants in the wild populations of the horseshoe crabs.

Saving the overlooked mangrove horseshoe crabs-A perspective from enhancing mangrove ecosystem conservation.

Despite being widely distributed in Asia, Carcinoscorpius rotundicauda is often overlooked and, its population status remains unclear. Moreover, it is threatened by illegal harvesting and degradation of mangrove ecosystems. Protecting its habitat is essential for population and biodiversity conservation, as mangroves provide nursery grounds and food supply for C. rotundicauda. This review discusses the biological characteristics of C. rotundicauda, including ecology, nutrition, life history, toxicology, and immunology. It also presents information about its distribution and population status. The review emphasizes the challenges faced by C. rotundicauda and proposes a conservation framework that involves the participation of local residents to facilitate conservation efforts. Collaboration between local residents and communities is proposed to protect and monitor the mangrove ecosystem. Additionally, this framework can support field research, protect C. rotundicauda juveniles and other species, and ensure the livelihood of local residents through participation in carbon trading markets and eco-industries such as eco-farming and eco-tourism.

Low-frequency noise aggravates the toxicity of cadmium in sea slug Onchdium reevesii.

Industrial development not only triggers heavy metal pollution but also introduces a less easily discernible disturbance: low-frequency noise pollution. Low-frequency noise can disrupt wildlife behavior, potentially exerting complex effects through interacting with heavy metals. Nevertheless, the cumulative impacts of low-frequency noise and cadmium (Cd) pollution on marine organisms remain largely unexplored. This study aimed to evaluate the immune defense response of sea slugs (Onchdium reevesii) exposed to Cd (1.32 mg/L) and low-frequency noise (500 Hz, 1000 Hz). Our results show that Cd exposure results in Cd2+ accumulation in the sea slug's hepatopancreas, leading to a decrease in total antioxidant capacity (TAC) and a significant increase in enzyme activities, including glutathione (GSH), lipid peroxidation (LPO), and aspartate transferase (AST). Additionally, there is a substantial upregulation in the expression of genes related to tumor protein p53 (p53), Cytochrome C (CytC), Caspase 3, and Caspase 9, as well as metallothionein (MT) and heat shock protein 70 (Hsp70) genes. Concurrently, an excessive production of reactive oxygen species (ROS) occurs in the hemocytes, resulting in apoptosis and subsequent diminished cell viability, with these effects positively correlating with the exposure duration. Furthermore, when sea slugs were exposed to both Cd and low-frequency noise, there was a decrease in the hepatopancreas's antioxidant capacity and an enhancement in hemocytes immune responses, which positively correlated with low-frequency noise frequency. The comprehensive assessment of biomarker responses highlights that low-frequency noise has the potential to amplify the deleterious effects of Cd on sea slug physiology, with this negative impact positively linked to noise frequency. Consequently, our study underscores that the combined influence of low-frequency noise and Cd pollution magnifies the effects on sea slug health. This could potentially disrupt the population stability of this species within its natural habitat, providing fresh insights into the evaluation of cumulative environmental pollution risks.

Response of moulting genes and gut microbiome to nano-plastics and copper in juvenile horseshoe crab Tachypleus tridentatus.

Nanoplastics (NPs) and heavy metals are typical marine pollutants, affecting the gut microbiota composition and molting rate of marine organisms. Currently, there is a lack of research on the toxicological effects of combined exposure to horseshoe crabs. In this study, we investigated the effects of NPs and copper on the expression of molt-related genes and gut microbiome in juvenile tri-spine horseshoe crabs Tachypleus tridentatus by exposing them to NPs (100 nm, 104 particles L-1) and/or Cu2+ (10 µgL-1) in seawater for 21 days. Compared with the control group, the relative mRNA expression of ecdysone receptor (EcR), retinoid x receptor (RXR), calmodulin-A-like isoform X1 (CaM X1), and heat shock 70 kDa protein (Hsp70) were significantly increased under the combined stress of NPs and Cu2+. There were no significant differences in the diversity and abundance indices of the gut microbial population of horseshoe crabs between the NPs and/or Cu2+ groups and the control group. According to linear discriminant analysis, Oleobacillus was the most abundant microorganism in the NPs and Cu2+ stress groups. These results indicate that exposure to either NPs stress alone or combined NPs and Cu2+ stress can promote the expression levels of juvenile molting genes. NPs exposure has a greater impact on the gut microbial community structure of juvenile horseshoe crabs compared to Cu2+ exposure. This study is helpful for predicting the growth and development of horseshoe crabs under complex environmental pollution.

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.

Polybrominated diphenyl ether-47 and food shortage impair the byssal attachment and health of marine mussels.

Polybrominated diphenyl ethers (PBDEs) are one of the most used halogenated flame retardants worldwide, and exert neurotoxicity, reproductive toxicity, endocrine interference, and carcinogenic effects on organisms. However, there are insufficient studies on the physical and immune defense at the individual level of mussels under different food conditions. To explore the defense strategy and individual health status, the thick-shelled mussels Mytilus coruscus were exposed to different BDE-47 concentrations (0, 0.1 and 10 µg/L) and nutritional conditions (feeding and starvation) for 21 days. The results showed that BDE-47 exposure and starvation significantly decreased the number of byssus threads (NBT), adhesion, and condition index (CI) of mussels, whereas increased the reactive oxygen species (ROS) production and the combined stress further declined the CI. BDE-47 exposure and starvation induced decreased adhesive capability and healthy state along with oxidative lesions in mussels. The downregulation gene expression of foot adhesion proteins (mfp-2/3/4/5/6) under starvation or combined exposure also proved the reduced adhesion of mussels. However, up-regulated mfp-1 and pre-collagens proteins (preCOL-D/P/NG) indicated mussels would adjust energy allocation to enhance the strength and extensibility of byssal threads for compensating reduced adhesion and CI. As global climate change and organic pollution have dramatically impacted the ocean, hazardous substances and the fluctuated primary productivity have frequently co-occurred, which will affect the structure of coastal biomes and fishery production.

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.

Adverse impacts of high-density microplastics on juvenile growth and behaviour of the endangered tri-spine horseshoe crab Tachypleus tridentatus.

The impacts of high-density microplastics, namely polyamine 6,6 (nylon), polymethyl methacrylate (PMMA) and polyethylene terephthalate (PET), on growth and behaviour of the endangered tri-spine horseshoe crab Tachypleus tridentatus were investigated for 100 days. Negative changes in wet weight and prosomal width of the juveniles were observed in all treatments of microplastics, but significant difference was only detected in prosomal width between control and PMMA. T. tridentatus became significantly less active upon exposure to nylon and PET. The extent of burrowing by T. tridentatus did not significantly differ among the treatments but was overall significantly reduced towards day 100. T. tridentatus exposed to PET significantly showed the lowest survival probability (30 %), compared to the other treatments (70-90 %). In conclusion, high-density microplastics compromised growth and behaviour of juvenile horseshoe crabs. Among the polymers that were tested, PET was considered more harmful and associated with higher mortality.

Effects of Microplastics on Immune Responses of the Yellow Catfish Pelteobagrus fulvidraco Under Hypoxia.

Compared with marine organisms, research on microplastics (MPs) in freshwater organisms is still less although MPs have been widely found in the freshwater ecosystem. Hypoxia is a ubiquitous issue in freshwater aquaculture, and under such scenarios, the toxic effects of MPs on typical aquaculture fish need to be clarified. In this study, we studied the effects of MPs (polystyrene) on specific growth rate (SGR), hypoxia-inducible factor-1α (HIF-1α), tumor necrosis factor-α (TNF-α), interleukin-8 (IL-8), and interferon (IFN) in the yellow catfish (Pelteobagrus fulvidraco) under hypoxic conditions. After 15 days of exposure, the SGR was not affected by MPs or hypoxia. MPs significantly increased the expressions of HIF-1α and TNF-α but inhibited the expression of IFN at high concentration MPs under normoxia. However, hypoxia significantly inhibited the expression of IL-8 and TNF-α under high MP concentration and low MP concentration, respectively. In addition, MPs had significant concentration-dependent inhibitory effects on IFN under hypoxia. Surprisingly, a positive correction between HIF-1α and TNF-α was found in fish. Although hypoxia might alleviate the effects of MPs with low concentrations, the interaction of hypoxia and MPs aggravated the negative effects of MPs on immune factors at high concentration MPs. This study provided new insight into the complex effects of hypoxia and MPs on aquatic organisms, and future studies should focus on the cellular pathways of immune cells in fish. Given that MPs could induce the immune response in fish, considerations should be paid to the impacts of MPs on freshwater aquaculture, and hypoxia should be taken into consideration when evaluating the effects of MPs.

Determination of microplastics in the edible green-lipped mussel Perna viridis using an automated mapping technique of Raman microspectroscopy.

Microplastics are prevalent in marine environments and seafood and thus can easily end up in human diets. This has raised serious concerns worldwide, particularly in Hong Kong where the seafood consumption per capita can be three times higher than the global average. This study focused on the green-lipped mussel Perna viridis, a popular seafood species which is subject to a high risk of contamination by microplastics due to its filter-feeding nature. P. viridis was collected from five mariculture sites in Hong Kong and assessed for its body load of microplastics using an automated Raman mapping approach. Microplastics were found in all sites, with an average of 1.60-14.7 particles per mussel per site, or 0.21-1.83 particles per g wet weight. Polypropylene, polyethylene, polystyrene and polyethylene terephthalate were detected among the microplastics, mainly as fragments or fibres in the size range of 40-1000 µm. It was estimated that through consumption of P. viridis, the population in Hong Kong could ingest up to 10,380 pieces of microplastics per person per year. These estimated rates were high compared to the values reported worldwide, suggesting the potential human health risk of microplastics in Hong Kong and adjacent areas.

Improved Raman spectroscopy-based approach to assess microplastics in seafood.

Microplastics represent an emerging environmental issue and have been found almost everywhere including seafood, raising a great concern about the ecological and human health risks they pose. This study addressed the common technical challenges in the assessment of microplastics in seafood by developing an improved protocol based on Raman spectroscopy and using the green-lipped mussel Perna viridis and the Japanese jack mackerel Trachurus japonicus as the test models. Our findings identified a type of stainless-steel filter membranes with minimal Raman interference, and a combination of chemicals that achieved 99-100% digestion efficiency for both organic and inorganic biomass. This combined chemical treatment reached 90-100% recovery rates for seven types of microplastics, on which the surface modification was considered negligible and did not affect the accuracy of polymer identification based on Raman spectra, which showed 94-99% similarity to corresponding untreated microplastics. The developed extraction method for microplastics was further combined with an automated Raman mapping approach, from which our results confirmed the presence of microplastics in P. viridis and T. japonicus collected from Hong Kong waters. Identified microplastics included polypropylene, polyethylene, polystyrene and poly(ethylene terephthalate), mainly in the form of fragments and fibres. Our protocol is applicable to other biological samples, and provides an improved alternative to streamline the workflow of microplastic analysis for routine monitoring purposes.

Enhanced immunity and hemocytes proliferation by three immunostimulants in tri-spine horseshoe crab Tachypleus tridentatus.

Tachypleus amebocyte lysate (TAL) is crucial in medical testing, but its industry in China has been restricted due to the decline of horseshoe crab population in recent years. Exploring methods of enhancing immunity and rapid hemocytes proliferation is urgent for the industrial horseshoe crab culture. In this study, ß-glucan (G), peptidoglycan (P), and squalene (S) were injected to horseshoe crabs at two concentrations (5 and 10 mg/kg), in order to compare their effects on total hemocyte count (THC), reactive oxygen species (ROS), and non-specific immune enzyme activities. Results showed that the THC, superoxide dismutase (SOD), catalase (CAT), and total antioxidant capacity (T-AOC) were significantly increased by three immunostimulants at different points of time; ROS was significantly increased except at two squalene groups; lysozyme (LZM) and alkaline phosphatase (AKP) activity were increased except at low dose (5 mg/kg) squalene group; malondialdehyde (MDA) activity was decreased in all treatments; and hemocyanin concentration (HC) changed little during the experiment. At the 48th hour, THC, ROS, SOD, CAT, T-AOC, LZM, and AKP activities were significantly higher in the two peptidoglycan groups than those in the control group; the low dose ß-glucan and squalene groups showed significantly higher SOD and CAT, but their THC and AKP were not significantly different from those of the control group. In general, all three immunostimulants stimulated the hemolymph parameters of horseshoe crabs, notably, peptidoglycan could significantly increase the THC and enzyme activities, suggesting that peptidoglycan can be developed as an efficient immunostimulant for horseshoe crabs.

Effects of Ocean Acidification on Molting, Oxidative Stress, and Gut Microbiota in Juvenile Horseshoe Crab Tachypleus tridentatus.

Anthropogenic elevation of atmospheric carbon dioxide (CO2) drives global-scale ocean acidification (OA), which has aroused widespread concern for marine ecosystem health. The tri-spine horseshoe crab (HSC) Tachypleus tridentatus has been facing the threat of population depletion for decades, and the effects of OA on the physiology and microbiology of its early life stage are unclear. In this study, the 1st instar HSC larvae were exposed to acidified seawater (pH 7.3, pH 8.1 as control) for 28 days to determine the effects of OA on their growth, molting, oxidative stress, and gut microbiota. Results showed that there were no significant differences in growth index and molting rate between OA group and control group, but the chitinase activity, ß-NAGase activity, and ecdysone content in OA group were significantly lower than those of the control group. Compared to the control group, reactive oxygen species (ROS) and malondialdehyde (MDA) contents in OA group were significantly increased at the end of the experiment. Superoxide dismutase (SOD), catalase (CAT), and alkaline phosphatase (AKP) activities increased first and then decreased, glutathione peroxidase (GPX) decreased first and then increased, and GST activity changed little during the experiment. According to the result of 16S rRNA sequencing of gut microbiota, microbial-mediated functions predicted by PICRUSt showed that "Hematopoietic cell lineage," "Endocytosis," "Staphylococcus aureus infection," and "Shigellosis" pathways significantly increased in OA group. The above results indicate that OA had no significant effect on growth index and molting rate but interfered with the activity of chitinolytic enzymes and ecdysone expression of juvenile horseshoe crabs, and caused oxidative stress. In addition, OA had adverse effects on the immune defense function and intestinal health. The present study reveals the potential threat of OA to T. tridentatus population and lays a foundation for the further study of the physiological adaptation mechanism of juvenile horseshoe crabs to environmental change.

Impact of Initial Feeding and Molting on Tachypleus tridentatus Gut Microbiota.

Tri-spine horseshoe crabs (HSCs) Tachypleus tridentatus have been facing the threat of population depletion for decades, and the physiology and microbiology of their early life stages are lacking. To explore what directs the change of juvenile T. tridentatus gut microbiota and how gut microbiota change, by using 16S rRNA sequencing of gut samples we detected the intestinal microbiome of juvenile HSCs and compared the impact of initial molting and initial feeding, as well as the effect of environment. Results showed that the predominant phyla in the gut microbial community of juvenile HSCs are Proteobacteria and Bacteroidetes. The richness and diversity of intestinal microbes greatly decreased after initial molting. Microbial-mediated functions predicted by PICRUSt showed that "Signal Transduction", "Cellular Processes and Signaling", "Infective Diseases" and "Digestive System" pathways significantly increased in 2nd instars. As for the effect of environment, the connection between living environment and the intestinal microbiome started to manifest after initial molting. Unexpectedly, initial feeding treatment slightly affected the intestinal microbiome of T. tridentatus in the early life stage, whereas the effect of initial molting was significant. The present study provided the first insight into the gut microbiota of T. tridentatus, and the findings led a new sight to explain what guide the change of gut microbiota.

Coastal zone use influences the spatial distribution of microplastics in Hangzhou Bay, China.

Microplastic pollution in estuarine and coastal environments has recently been characterised in several countries but few researchers have addressed the influence of different forms of coastal zone use on the distribution of microplastic. Here, microplastic particles were sampled in Hangzhou Bay, which is heavily influenced by a range of human activities, and their abundance, size, and polymer type characterised. The abundance of microplastics was 0.14 ± 0.12 items/m3 in water, 84.3 ± 56.6 items/kg dry weight of sediment, and between 0.25 ± 0.14 and 1.4 ± 0.37 items/individual in biota. These results show that Hangzhou Bay has a low level of microplastic contamination compared to other coastal systems in China, although abundance was spatially variable within the bay; relatively higher microplastic abundances were found in the southern area of the bay, which has adjacent industrial and urban land-use zones, while lower abundances were observed in the central and northern bay areas where mariculture, fisheries, and mineral and energy industries are most common. The relatively low microplastic abundance observed in the biota samples is consistent with the generally low values for the seawater and sediment samples. Pellets were the most common of four particle-shape classes (fibres, fragments, films, and pellets) in surface seawater, while fibres were most abundant in sediment and biota. Smaller-sized microplastics (<1.0 mm) were dominant in all samples. Microplastics in the surface seawater were dominated by low-density polypropylene and polyethylene particles, while rayon was dominant in the sediment and biota samples. Our results demonstrate that regional variability in anthropogenic activity and land-use are important controls on the spatial pattern of microplastic pollution in Hangzhou Bay.

Microplastics impair digestive performance but show little effects on antioxidant activity in mussels under low pH conditions.

In the marine environment, microplastic contamination and acidification may occur simultaneously, this study evaluated the effects of ocean acidification and microplastics on oxidative stress responses and digestive enzymes in mussels. The thick shell mussels Mytilus coruscus were exposed to four concentrations of polystyrene microspheres (diameter 2 µm, 0, 10, 104 and 106 particles/L) under two pH levels (7.7 and 8.1) for 14 days followed by a 7-day recovery acclimation. Throughout the experiment, we found that microplastics and ocean acidification exerted little oxidative stress to the digestive gland. Only catalase (CAT) and glutathione (GSH) showed a significant increase along with increased microplastics during the experiment, but recovered to the control levels once these stressors were removed. No significant effects of pH and microplastics on glutathione peroxidase (GPx) and superoxide dismutase (SOD) were observed. The responses of digestive enzymes to both stressors were more pronounced than antioxidant enzymes. During the experiment, pepsin (PES), trypsin (TRS), alpha-amylase (AMS) and lipase (LPS) were significantly inhibited under microplastics exposure and this inhibition was aggravated by acidification conditions. Only PES and AMS tended to recover during the recovery period. Lysozyme (LZM) increased significantly under microplastic exposure conditions, but acidification did not exacerbate this effect. Therefore, combined stress of microplastics and ocean acidification slightly impacts oxidative responses but significantly inhibits digestive enzymes in mussels.

Classification and characterization of hemocytes from two Asian horseshoe crab species Tachypleus tridentatus and Carcinoscorpius rotundicauda.

In present study, transmission electron microscopy and flow cytometry were utilized to investigate the classification, characterization and immune functions of hemocytes from horseshoe crab, Tachypleus tridentatus and Carcinoscorpius rotundicauda. Three types of hemocytes were distinguished respectively: the granular cell, the semi-granular cell and the hyaline cell by transmission electron microscopy, while three hemocyte subpopulations (Gate 1 cell, Gate 2 cell, Gate 3 cell) were classified by flow cytometry. Hyaline cell was the major cell type with the highest nuclear-cytoplasmic ratio and granular cell and semi-granular cell showed lower ratios. Immune parameters of hemocytes in horseshoe crabs were investigated by flow cytometry. Different hemocyte subpopulations respond for diverse functions. Lysosomal contents and hemocyte mortality in Gate 3 cell subpopulation were higher than that in other subpopulations, while reactive oxygen species, phagocytosis and non-specific esterase, in Gate 1 cell subpopulation, were higher than those in other subpopulations. The hemocyte types between the two species had no significant differences in staining or morphology.

Elevated pCO2 Affects Feeding Behavior and Acute Physiological Response of the Brown Crab Cancer pagurus.

Anthropogenic climate change exposes marine organisms to CO2 induced ocean acidification (OA). Marine animals may make physiological and behavioral adaptations to cope with OA. Elevated pCO2 may affect metabolism, feeding, and energy partition of marine crabs, and thereby affect their predator-prey dynamics with mussels. Therefore, we examined the effects of simulated future elevated pCO2 on feeding behavior and energy metabolism of the brown crab Cancer pagurus. Following 54 days of pre-acclimation to control CO2 levels (360 µatm) at 11°C, crabs were exposed to consecutively increased oceanic CO2 levels (2 weeks for 1200 and 2300 µatm, respectively) and subsequently returned to control CO2 level (390 µatm) for 2 weeks in order to study their potential to acclimate elevated pCO2 and recovery performance. Standard metabolic rate (SMR), specific dynamic action (SDA) and feeding behavior of the crabs were investigated during each experimental period. Compared to the initial control CO2 conditions, the SMRs of CO2 exposed crabs were not significantly increased, but increased significantly when the crabs were returned to normal CO2 levels. Conversely, SDA was significantly reduced under high CO2 and did not return to control levels during recovery. Under high CO2, crabs fed on smaller sized mussels than under control CO2; food consumption rates were reduced; foraging parameters such as searching time, time to break the prey, eating time, and handling time were all significantly longer than under control CO2, and prey profitability was significantly lower than that under control conditions. Again, a two-week recovery period was not sufficient for feeding behavior to return to control values. PCA results revealed a positive relationship between feeding/SDA and pH, but negative relationships between the length of foraging periods and pH. In conclusion, elevated pCO2 caused crab metabolic rate to increase at the expense of SDA. Elevated pCO2 affected feeding performance negatively and prolonged foraging periods. These results are discussed in the context of how elevated pCO2 may impair the competitiveness of brown crabs in benthic communities.

Effect of pH and temperature on antioxidant responses of the thick shell mussel Mytilus coruscus.

This study evaluated the combined effects of seawater pH decrease and temperature increase on the activity of antioxidant enzymes in the thick shell mussel Mytilus coruscus, an ecological and economic bivalve species widely distributed along the East China Sea. Mussels were exposed to three pH levels (8.1, 7.7 and 7.3) and two temperatures (25 °C and 30 °C) for 14 days. Activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX), glutathione (GSH), acid phosphatase (ACP), alkaline phosphatase (AKP) and glutamic-pyruvic transaminase (GPT) were measured in gills and digestive glands after 1, 3, 7 and 14 days of exposure. All enzymatic activities were significantly impacted by pH, temperature. Enzymatic activities at the high temperature were significantly higher than those at the low temperature, and the mussels exposed to pH 7.3 showed significantly higher activities than those under higher pH condition for all enzymes except ACP. There was no interaction between temperature and pH in two third of the measured activities suggesting similar mode of action for both drivers. Interaction was only consistently significant for GPX. PCA revealed positive relationships between the measured biochemical indicators in both gills and digestive glands. Overall, our results suggest that decreased pH and increased temperature induce a similar anti-oxidative response in the thick shell mussel.