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Horseshoe crabs are marine chelicerates which have existed for about 450 million years. Their long coexistence with microbial pathogens presumably is due to their efficient immune system. Herein, the first large-scale transcriptome and microRNA (miRNA) dataset of embryonic stages of the horseshoe crab Tachypleus tridentatus were presented. It provides a general overview of global gene expression changes during horseshoe crab’s early development. This study focuses on the stages 18–20 of the embryonic development of T. tridentatus, which are the first three major embryonic molt stages and involve the formation of several vital organs. We identified 138,260 unigenes, including 34 immune-related gene families such as pattern recognition receptors, signaling and transduction pathway genes, antimicrobial peptides, and coagulation cascade genes. Additionally, 1522 miRNAs (1046 known miRNAs and 476 novel miRNAs) were predicted to 29,584 target genes. Numerous mRNA-miRNA interactions were also identified. Results of Quantitative PCR proved that the immune genes such as Spaetzle, ankyrin-3, peptidoglycan-recognition protein, coagulogen, TNF-receptor-associated factor, and CD-9 antigen were negatively regulated by miR-5735, miR-200, miR-145, miR-199, miR-144, and miR-322, respectively. Futhermore, we confirmed the target regulatory relationship between miR-200 and ankyrin-3 by dual luciferase reporter gene assay. To better understand the complex developmental gene regulatory networks in T. tridentatus, the time-course gene expression data analysis is the first step. Our results provide a valuable resource for analyzing the functions of gene repertoires in embryonic developmental stages （Stage 18 to Stage 20）of horseshoe crab.

To reveal the differences in the microbial composition of skin mucus and the habitat of Chinese sturgeon (Acipenser sinensis) in different physiological states, we compared the community structure of the skin mucus flora of healthy and sub-healthy Chinese sturgeons and explored the correlation between the structure of the bacterial community and physiological state of Chinese sturgeons. Skin mucus and aquaculture water of healthy and sub-healthy Chinese sturgeons were collected, and the bacterial flora composition was analyzed using high-throughput sequencing technology. The results showed that microbial diversity and richness were significantly higher in the sub-healthy group than in the healthy group (P < 0.05). Statistical analysis of the sequences showed that many unique operational taxonomic units (OTUs) were present in the mucus samples compared with the aquaculture water, and the number of shared OTUs with the aquaculture water changed according to the health status. The principal component and principal coordinate analyses between different groups showed a strong separation tendency, and the relative distance between samples within groups was small. There were significant differences in the composition and dominant species of skin mucus and aquaculture water communities of Chinese sturgeons at the phylum and genus levels. The dominant phyla in the water were Proteobacteria (31.84%) and Firmicutes (24.37%); the dominant phyla in the healthy group were Proteobacteria (55.23%) and Bacteroidetes (21.00%); and the percentage of Proteobacteria (40.23%) in the sub-healthy group, but the proportion of Acidobacteria (18.29%), Gemmatimonadetes (10.08%), Actinobacteria, and Verrucomicrobia increased. The dominant flora in water samples were Sphaerochaeta (10.56%) and Cloacibacillus (7.95%). The dominant flora in the healthy group were Ralstonia (19.83%) and Mucilaginibacter (8.11%). The dominant flora in the sub-healthy group, compared to the healthy group, consisted mainly of Sphingomonas (16.71%) and WPS-1_genera_incertae_sedis (5.05%). Functional prediction revealed that the functional abundance of flora in the sub-healthy group showed a decreasing trend compared with that in the healthy group, including environmental adaptation, biological metabolism, and signal transduction. Similarity and difference analysis of the mucus microbiome of Chinese sturgeons in different physiological states and aquaculture water showed that it changed with the physiological state, and the genus Sphingomonas was the dominant genus of mucus flora in the sub-healthy group, which can be used as a health marker. By comparing the microbial communities in the skin mucus of healthy and sub-healthy Chinese sturgeons, different microbial community structures were observed in different health states, which provides a new perspective for their healthy culture and monitoring.

Exposing marine organisms to contemporary contaminants, such as perfluorooctanoic acid (PFOA) and nano-titanium dioxide (nano-TiO2), can induce multifaceted physiological consequences. Our investigation centered on the responses of the mussel, Mytilus coruscus, to these agents. We discerned pronounced disruptions in gill filament connections, pivotal structures for aquatic respiration, suggesting compromised oxygen uptake capabilities. Concurrently, the respiratory rate exhibited a marked decline, indicating a respiratory distress. Furthermore, the mussels' clearance rate, a metric of their filtration efficacy, diminished, suggesting the potential for bioaccumulation of deleterious substances. Notably, the co-exposure of PFOA and nano-TiO2 exhibits interactive effects on the physiological performance of the mussels. The mussels' digestive performance waned in the face of heightened PFOA and nano-TiO2 concentrations, possibly hampering nutrient assimilation and energy accrual. This was mirrored in the noticeable contraction of their energy budget, suggesting long-term growth repercussions. Additionally, the dysregulation of the gut microbiota and the reduction in its diversity further confirm alterations in intestinal homeostasis, subsequently impacting its physiological functions and health. Collectively, these findings underscore the perils posed by escalated PFOA and nano-TiO2 levels to marine mussels, accentuating the need for a deeper understanding of nanoparticle-pollutant synergies in marine ecosystems.

The Chinese sturgeon (Acipenser sinensis), a critically endangered migratory fish native to the Yangtze River estuary, is experiencing alarming population declines. Understanding the physiological and biochemical profiles of this species is paramount for its conservation. However, due to limited sample availability, blood biochemical parameters have remained understudied. In this study, we examined blood chemistry in artificially cultured Chinese sturgeon ranging from 2 to 15 years of age. Our results revealed age-related trends: total protein (TP), albumin (ALB), globulin (GLO), total cholesterol (CHOL), high-density lipoprotein (HDL), low-density lipoprotein (LDL), estrogen (E2), testosterone (T), testosterone undecanoate (11-KT), and red blood cell count (RBC) increased with age, while glucose (GLU), uric acid (UA), and serum creatinine (CREA) decreased. Levels of C-reactive protein (CRP) declined from 3 to 7 years but rose from 8 to 15 years. Blood parameters showed stabilization with age, indicating enhanced resilience and immunity. Significant alterations in parameters at ages 2–3 and 14–15 suggest critical developmental stages. These findings are crucial for understanding sturgeon growth, development, migration, and reproduction, underscoring the necessity for targeted conservation efforts during pivotal life stages.

Objectives Mortality and associated risk factors in young and elderly haemodialysis patients with end-stage kidney disease (ESKD) have not been well examined in China. Therefore, we aimed to assess the all-cause mortality and risk factors associated with all-cause mortality between young and elderly haemodialysis patients in China.Design A population-based multicentre retrospective cohort study.Setting Using the Dialysis Initiation based on Fuzzy mathematics Equation study data, patients with ESKD undergoing maintenance haemodialysis from 24 centres in China from 1 January 2008 to 30 September 2015.Participants 1601 enrolled patients with ESKD were categorised into young group (18–44 years old) and elderly (≥60 years old) group.Outcome measures The primary outcome was all-cause mortality. We estimated overall survival using a log-rank test. Cox proportional hazard regression analysis was implemented to identify risk factors and HR associated all-cause mortality.Results During a mean follow-up of 48.17±25.59 months, of the 1601 subjects, 319 (19.92%) patients death, including 64 (9.97%) in young group and 255 (26.59%) in elderly group, respectively. The cumulative survival in elderly group was lower than young group (Log Rank tests=63.31, p

IntroductionAs one of the megadiverse countries, the effectiveness of wildlife protection in China is of great significance to global biodiversity conservation. With continued evolution and revisions, China’s Wildlife Protection Law has listed over 140 marine species; however, it is still inclined toward terrestrial animals.MethodsTo narrow the gap between compliance and enforcement, we collected 1,309 effective responses from various coastal cities of China through an anonymous online questionnaire survey, to investigate their exposure, understanding and attitudes toward Wildlife Protection Law for marine species (mWPL).ResultsMost respondents demonstrated an overall good understanding about the context, necessity and effectiveness of mWPL. The fisher communities were found to be more aware of the dissemination and implementation of mWPL. However, they understood less of the penal system, and exhibited negative attitudes toward the necessity and punishment of the legislation, probably due to the conflicts between resource utilizations and legislative interventions. The participants also indicated that seahorses, horseshoe crabs and corals were commonly subjected to illegal exploitations.DiscussionWhile most respondents suggested greater fines, tighter laws and better public enforcement, we advocate the exploration of bottom-up options such as community engagement and environmental education to improve compliance and implementation of mWPL for the benefit of marine wildlife conservation in China.

Juvenile tri-spine horseshoe crabs (Tachypleus tridentatus) were exposed to determine the effects of single and combined stresses of polystyrene nanoplastics (nano-PS) and heavy metal (Cu2+) on antioxidant enzyme parameters. The juveniles were exposed to a 21-day 100-nm polystyrene concentration (104 particles l-1) and a concentration of Cu2+ (10 µg l-1) followed by a recovery period of 7 days. The in vivo antioxidant activity for whole horseshoe crab was analyzed. The results revealed that all antioxidant parameters, i.e., superoxide dismutase (SOD), catalase (CAT), glutathione (GSH), malondialdehyde (MDA), and lipid peroxidation (LPO), showed both increased and decreased levels in different experimental groups of horseshoe crabs having different experimental conditions compared to the control group at three time points, i.e., on days 7, 14, and 21. Similarly during the recovery period, SOD, CAT, and MDA showed decreased levels in all experimental groups, while GSH and LPO showed increased levels in all experimental groups of horseshoe crabs under the influence of different experimental conditions of nanoplastics and heavy metals compared to the control group on day 28. These results showed that the exposure of nano-PS and Cu2+ had precise effects on juvenile horseshoe crabs. Integrated biomarker responses showed that nano-PS and Cu2+ had adverse effects on juvenile horseshoe crabs. By principal component analysis, the potentially toxic effects of nano-PS and Cu2+ on horseshoe crabs were obtained.

As global climate change has dramatically impacted the ocean, severe temperature elevation and a decline in primary productivity has frequently occurred, which has affected the structure of coastal biomes. In this study, the sex-specific responses to temperature change and food availability in mussels were determined in terms of digestive performance. The thick-shelled mussels Mytilus coruscus (male and female) were exposed to different temperature and nutritional conditions for 30 days. The results showed that the digestive enzymes of mussels were significantly affected by temperature, food, sex, and their interactions. High temperature (30°C) and starvation significantly decreased amylase, lysozyme, and pepsase activities of female mussels, while trypsin and trehalase did not change significantly at the experimental end. The activity of amylase, trypsin, and trehalase was significantly reduced in males at high temperature (30°C) under starvation treatment, but high temperature (30°C) elevated pepsase. Unsurprisingly, starvation caused the reduction of lysozyme and pepsase under 25°C in males. Amylase, lipase, and trehalase were higher in female mussels compared with males, while the enzymatic activities of lysozyme, pepsase, and trypsin were higher in male mussels than females. Principal component analysis showed that different enzyme activity indexes were separated in male and female mussels, indicating that male and female mussels exhibited significantly different digestive abilities under temperature and food condition change. The study clarified sex-specific response difference in mussel digestive enzymes under warming and starvation and provided guidance for the development of mussel aquaculture (high temperature management and feeding strategy) under changing marine environments.

Seasonality has a significant effect on the physiology of fish, especially the effect of water temperature changes. In the present study, the growth, innate immune parameters and liver function indices of two rescued wild adult Chinese sturgeons under captive conditions were monitored for 1 year. The results showed that the total annual weight loss rate of the male was −4.58% and the total weight gain rate of the female was 24.12%, in which the weight of both individuals registered highly significant differences in summer, fall and winter (p < 0.01). The male Chinese sturgeon also exhibited negative specific growth rates (−0.1 to −0.8%) during spring to fall, whereas positive specific growth rates, ranging from 0.03 to 0.11%, were recorded in the female. Seasonality also affected the innate immune parameters of the two Chinese sturgeons, in which leukocytes had been increasing since spring and C-reactive protein (CRP) content was significantly higher (p < 0.05) in summer than fall in both individuals. The CRP level of the male Chinese sturgeon showed a significant increase from fall to winter (p < 0.05), suggesting that it may have contracted infection or inflammation during this study period. With the analysis of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), transaminase (AST:ALT) ratio, alkaline phosphatase, albumin to globulin ratio and triglycerides, it was found that the liver function of the captive Chinese sturgeons was adversely affected along seasonal changes, with the highest degree of liver impairment in winter. In combining observations from growth performance and changes in innate immune and liver function parameters, the present findings deduced that the male Chinese sturgeon under study was more susceptible to seasonal changes than the female. For better indoor culture of adult Chinese sturgeons, monitoring of hematological parameters to detect early signs of inflammation and liver function abnormality should be conducted with routine veterinary care during prolonged captivity.

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, β-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.

As an endangered animal species, the Chinese sturgeon (Acipenser sinensis) has been artificially cultured during recent years. In the present experiment, the performance of a common formulated feed and a mixed natural feed on the growth and blood physiology of Chinese sturgeon were compared during one year of indoor culture. In terms of specific growth rate, the formulated diet group showed a higher value (4.2%) compared with the natural food group (−1.8%) during one year of indoor culture. Alanine aminotransferase activities of the sturgeons in formulated diet group was significantly higher than that in the natural food group throughout whole experiment period (p < 0.05). The total protein albumin, albumin and globulin contents of the natural food group were significantly higher than that of the formulated diet group in February and May (p < 0.05). The C-reactive protein contents of the formulated diet group were significantly higher than that of natural food group in May and August (p < 0.05). The activities of total cholesterol, high-density cholesterol, and low-density cholesterol were significantly higher in the natural food group than those in the formulated diet group (p < 0.05). The activities of triglycerides were significantly higher in the natural food group than those in the formulated diet group in February and May (p < 0.05). Almost half of the biochemical indicators examined show significant seasonal fluctuations, indicating that seasons have a substantial impact on the Chinese sturgeon. Overall, these results indicate that natural food exerted a positive impact on physiological indicators compared to the formulated diet.

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.

Marine hypoxia caused by nutrient enrichment in coastal waters has become a global problem for decades, especially diel-cycling hypoxia that occurs frequently in the summer season. On the contrary, sudden rainstorms, and freshwater discharge make salinity in estuarine and coastal ecosystems variable, which often occurs with hypoxia. We found mass mortality of the Hong Kong oyster Crassostrea hongkongensis in the field where hypoxia and salinity fluctuation co-occur in the summer season during the past several years. To investigate the effects of diel-cycling hypoxia and salinity changes on the hemocyte immune function of C. hongkongensis, oysters were exposed to a combined effect of two dissolved oxygen (DO) concentrations (24 h normal oxygen 6 mg/L, 12 h normal oxygen 6 mg/L, and 12 h hypoxia 2 mg/L) and three salinities (10, 25, and 35‰) for 14 days. Subsequently, all treatments were restored to constant normal oxygen (6 mg/L) and salinity under 25‰ for 3 days to study the recovery of hemocyte immune function from the combined stress. Hemocyte parameters were analyzed by flow cytometry, including hemocyte mortality (HM), total hemocyte count (THC), phagocytosis (PHA), esterase (EST) activity, reactive oxygen species (ROS), lysosomal content (LYSO), and mitochondrial number (MN). The experimental results showed that diel-cycling hypoxia and salinity changes have obvious interactive effects on various immune parameters. In detail, diel-cycling hypoxia and decreases in salinity led to increased HM, and low salinity caused heavier impacts. In addition, low salinity, and diel-cycling hypoxia also led to decreases in LYSO, EST, and THC, while the decrease of PHA only occurs in the early stage. On the contrary, ROS production increased significantly under low salinity and hypoxic conditions. After 3-day recovery, THC, PHA, EST, LYSO, and MN were basically restored to normal, while HM and ROS were still significantly affected by diel-cycling hypoxia and salinity change, indicating that the combined stress of diel-cycling hypoxia and salinity changes had latent effects on the immune function of C. hongkongensis. Our results highlight that diel-cycling hypoxia and salinity change may impair the health and survival of the Hong Kong oyster C. hongkongensis and may be the key factors for the mass mortality of this oyster in the field.

Marine microplastics pollution is a major environmental concern in marine ecosystems worldwide, yet the biological impacts of microplastics on the coastal biota are not yet fully understood. We investigated the impact of suspended microplastics on the energy budget of the mussels Mytilus coruscus using the Cellular Energy Allocation (CEA) approach. The mussels were exposed to control conditions (no microplastics) or to one of the three concentrations of 2 μm polystyrene microspheres (10, 104, and 106 particles/L) for 14 days, followed by 7 days of recovery. Exposure to high concentrations of microplastics (104 or 106 particles/L) increased cellular energy demand (measured as the activity of the mitochondrial electron transport system, ETS) and depleted cellular energy stores (carbohydrates, lipids, and proteins) in the mussels whereas exposure to 10 particles/L had no effect. Carbohydrate levels decreased already after 7 days of microplastics exposure and were restored after 7 days of recovery. In contrast, the tissue levels of lipids and proteins declined more slowly (after 14 days of exposure) and did not fully recover after 7 days following the removal of microplastics. Therefore, the total energy content and the CEA declined after 7–14 days of exposure to high microplastics concentrations, and remained suppressed during 7 days of subsequent recovery. These findings demonstrate a negative impact of microplastics on energy metabolism at the cellular level that cannot be restored during a short time recovery. Given a close link of CEA with the organismal energy balance, suppression of CEA by microplastics exposure suggests that bioenergetics disturbances might lead to decreases in growth and productivity of mussels’ populations in environments with heavy microplastics loads.

Gut microbiota play a very important role in the health of the host, such as protecting from pathogens and maintaining homeostasis. However, environmental stressors, such as ocean acidification, hypoxia, and warming can affect microbial communities by causing alteration in their structure and relative abundance and by destroying their network. The study aimed to evaluate the combined effects of low pH, low dissolved oxygen (DO) levels, and warming on gut microbiota of the mussel Mytilus coruscus. Mussels were exposed to two pH levels (8.1, 7.7), two DO levels (6, 2 mg L−1), and two temperature levels (20, 30°C) for a total of eight treatments for 30 days. The experiment results showed that ocean acidification, hypoxia, and warming affected the community structure, species richness, and diversity of gut microbiota. The most abundant phyla noted were Proteobacteria, Bacteroidetes, and Firmicutes. Principal coordinate analysis (PCoA) revealed that ocean acidification, hypoxia, and warming change microbial community structure. Low pH, low DO, and increased temperature can cause shifting of microbial communities toward pathogen dominated microbial communities. Linear discriminant analysis effect size (LEfSe) showed that the significantly enriched biomarkers in each group are significantly different at the genus level. Phylogenetic investigation of communities by reconstruction of unobserved states (PICRUSt) analysis revealed that the gut microbiome of the mussels is associated with many important functions, such as amino acid transport and metabolism, transcription, energy production and conservation, cell wall, membrane and envelope biogenesis, and other functions. This study highlights the complexity of interaction among pH, DO, and temperature in marine organisms and their effects on the gut microbiota and health of marine mussels.

The Qingtian paddy field carp (Cyprinus carpio var qingtianensis) is a local carp cultivated in the rice field of Qingtian county, Zhejiang province, China. Its high tolerance to hypoxia makes it an ideal organism for studying the molecular regulation mechanism during hypoxia process as well as reoxygenation following hypoxia in fish. In this study, we counted the differentially expressed genes (DEGs) altered during hypoxic exposure and reoxygenation process. The results indicated that 2236 genes (1506 up-regulated genes and 730 down-regulated genes) were differentially expressed between the control and hypoxic groups. The results from Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis indicated that 1152 of 2236 genes were enriched, and those genes participated in energy metabolism, reactive oxygen species (ROS) elimination, acceleration of cell apoptosis, inhibition of growth, and other processes. We found activation of the pentose phosphate pathway in hypoxia treatment, suggesting that carbohydrates not only provide energy for metabolism but also provide NADPH for protecting the body from oxidative damage and ribosomes for promoting RNA synthesis. During reoxygenation, 4509 genes (1865 up-regulated genes and 2644 down-regulated genes) were differentially expressed. The results of KEGG enrichment analysis indicated that 2392 of 4509 genes were enriched, and participated in pyruvate and lactic acid metabolism, synthesis of amino acids and lipids, inhibition of cell apoptosis, regulation of cell growth and differentiation, and other processes. These differentially expressed genes effectively alleviate the body acidosis and promote the normal growth and development of the body. Through the analysis of KEGG pathway enrichment, we observed that the physiological regulation of Qingtian paddy field carp during the processes of hypoxia and reoxygenation is not a simple and reversible process. This work first reported the adaptive mechanism of hypoxia and the recovery mechanism of reoxygenation after hypoxia in common carp, and also provided new insights for the physiological regulation of fish under hypoxia treatment.

This review provides the updated information and analysis of research development on horseshoe crabs over the past 30 years and raises some suggestions for future studies on horseshoe crabs. Horseshoe crab is a unique marine species and attracts many scholars in various research fields. However, to date, the development of horseshoe crab research has not been analyzed and reported from a bibliometric perspective. Bibliometric analysis is a unique tool for evaluating the development of a specific research area by analyzing relevant publications and researchers from all over the world. In this study, Web of Science, Google Scholar, and PubMed were used to collect relevant data on horseshoe crabs. VOSviewer software was used to visualize cooperation and major keywords in horseshoe crab research. From 1989 to 2019, scholars emphasized the physiological characteristics, medical value, and ecological conservation of horseshoe crabs. Some topics in this field have gradually developed because of some reasons explained in this review. Some important or recent studies have been discussed, and some potential research topics for future research have been suggested.

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.

In order to investigate the ecotoxicological effects of nano-ZnO particles and seawater acidification on marine bivalves, the thick shell mussels, Mytilus coruscus were subjected to joint treatments with different nano-ZnO concentrations (0 [control], 2.5 [medium] and 10 mg L-1 [high]) under two pH levels (7.7 [low]and 8.1 [control]) for 14 days. The results showed that respiration rate (RR), absorption efficiency (AE), clearance rate (CR), O:N ratio and scope for growth (SFG) were significantly reduced with nano-ZnO concentration increase, but ammonium excretion rate (ER) was increased. Low pH significantly reduced CR, RR, SFG, and O:N ratio of the mussels especially under high nano-ZnO conditions, and significantly increased ER. Principal component analysis (PCA) showed consistent relationships among most tested parameters, especially among SFG, RR, O:N ratio and CR under the normal pH and 0 nano-ZnO conditions. Therefore, seawater acidification and nano-ZnO interactively impact the ecophysiological responses of mussels and cause more severe effects when they appear concurrently.

Next-generation sequencing was used to analyze the effects of toxic microcystin-LR (MC-LR) on silver carp (Hypophthalmichthys molitrix). Silver carps were intraperitoneally injected with MC-LR, and RNA-seq and miRNA-seq in the liver were analyzed at 0.25, 0.5, and 1 h. The expression of glutathione S-transferase (GST), which acts as a marker gene for MC-LR, was tested to determine the earliest time point at which GST transcription was initiated in the liver tissues of the MC-LR-treated silver carps. Hepatic RNA-seq/miRNA-seq analysis and data integration analysis were conducted with reference to the identified time point. Quantitative PCR (qPCR) was performed to detect the expression of the following genes at the three time points: heme oxygenase 1 (HO-1), interleukin-10 receptor 1 (IL-10R1), apolipoprotein A-I (apoA-I), and heme binding protein 2 (HBP2). Results showed that the liver GST expression was remarkably decreased at 0.25 h (P < 0.05). RNA-seq at this time point revealed that the liver tissue contained 97,505 unigenes, including 184 significantly different unigenes and 75 unknown genes. Gene Ontology (GO) term enrichment analysis suggested that 35 of the 145 enriched GO terms were significantly enriched and mainly related to the immune system regulation network. KEGG pathway enrichment analysis showed that 18 of the 189 pathways were significantly enriched, and the most significant was a ribosome pathway containing 77 differentially expressed genes. miRNA-seq analysis indicated that the longest miRNA had 22 nucleotides (nt), followed by 21 and 23 nt. A total of 286 known miRNAs, 332 known miRNA precursor sequences, and 438 new miRNAs were predicted. A total of 1,048,575 mRNA–miRNA interaction sites were obtained, and 21,252 and 21,241 target genes were respectively predicted in known and new miRNAs. qPCR revealed that HO-1, IL-10R1, apoA-I, and HBP2 were significantly differentially expressed and might play important roles in the toxicity and liver detoxification of MC-LR in fish. These results were consistent with those of high-throughput sequencing, thereby verifying the accuracy of our sequencing data. RNA-seq and miRNA-seq analyses of silver carp liver injected with MC-LR provided valuable and new insights into the toxic effects of MC-LR and the antitoxic mechanisms of MC-LR in fish.The RNA/miRNA data are available from the NCBI database Registration No. : SRP075165.

With the release of large amounts of CO2, ocean acidification is intensifying and affecting aquatic organisms. In addition, salinity also plays an important role for marine organisms and fluctuates greatly in estuarine and coastal ecosystem, where ocean acidification frequently occurs. In present study, flow cytometry was used to investigate immune parameters of haemocytes in the thick shell mussel Mytilus coruscus exposed to different salinities (15, 25, and 35‰) and two pH levels (7.3 and 8.1). A 7-day in vivo and a 5-h in vitro experiments were performed. In both experiments, low pH had significant effects on all tested immune parameters. When exposed to decreased pH, total haemocyte count (THC), phagocytosis (Pha), esterase (Est), and lysosomal content (Lyso) were significantly decreased, whereas haemocyte mortality (HM) and reactive oxygen species (ROS) were increased. High salinity had no significant effects on the immune parameters of haemocytes as compared with low salinity. However, an interaction between pH and salinity was observed in both experiments for most tested haemocyte parameters. This study showed that high salinity, low salinity and low pH have negative and interactive effects on haemocytes of mussels. As a consequence, it can be expected that the combined effect of low pH and changed salinity will have more severe effects on mussel health than predicted by single exposure.

The rising anthropogenic atmospheric CO2 results in the reduction of seawater pH, namely ocean acidification (OA). In East China Sea, the largest coastal hypoxic zone was observed in the world. This region is also strongly impacted by ocean acidification as receiving much nutrient from Changjiang and Qiantangjiang, and organisms can experience great short-term natural variability of DO and pH in this area. In order to evaluate the defense responses of marine mussels under this scenario, the thick shell mussel Mytilus coruscus were exposed to three pH/pCO2 levels (7.3/2800 μatm, 7.7/1020 μatm, 8.1/376 μatm) at two dissolved oxygen concentrations (DO, 2.0, 6.0 mg L−1) for 72 h. Results showed that byssus thread parameters, such as the number, diameter, attachment strength and plaque area were reduced by low DO, and shell-closing strength was significantly weaker under both hypoxia and low pH conditions. Expression patterns of genes related to mussel byssus protein (MBP) were affected by hypoxia. Generally, hypoxia reduced MBP1 and MBP7 expressions, but increased MBP13 expression. In conclusion, both hypoxia and low pH induced negative effects on mussel defense responses, with hypoxia being the main driver of change. In addition, significant interactive effects between pH and DO were observed on shell-closing strength. Therefore, the adverse effects induced by hypoxia on the defense of mussels may be aggravated by low pH in the natural environments.