Metabolomics for in situ monitoring of attached Crassostrea gigas and Mytilus edulis: Effects of offshore wind farms on aquatic organisms.

While offshore wind power has support from countries around the world, studies show that offshore wind farms (OWFs) may affect marine organisms. Environmental metabolomics is a high-throughput method that provides a snapshot of an organism's metabolic state. To elucidate the effects of OWFs on aquatic organisms, we studied, in situ, Crassostrea gigas and Mytilus edulis attached within and outside of OWFs and their reef areas. Our results show that epinephrine, sulphaniline, and inosine 5'-monophosphate were significantly increased and L-carnitine was significantly reduced in both Crassostrea and Mytilus species from the OWFs. This may be related to immune response, oxidative stress, energy metabolism and osmotic pressure regulation of aquatic organisms. Our study shows that active selection of biological monitoring methods for risk assessment is necessary and that metabolomics of attached shellfish is useful in elucidating the metabolic pathways of aquatic organisms in OWFs.

Effects of a High-Fat Diet on Intestinal and Gonadal Metabolism in Female and Male Sea Cucumber Apostichopus japonicus.

Parental nutrient reserves are directly related to reproductive performance in sea cucumbers. This study focused on the lipid requirements of male and female sea cucumbers Apostichopus japonicus during the reproductive stage and analyzed their physiological responses to a high-fat diet (HFD). The intestinal lipid metabolites and microbiome profile changed significantly in animals fed with the HFD, as given by an upregulation of metabolites related to lipid metabolism and an increase in the predominance of Proteobacteria in the microbiome, respectively. The metabolic responses of male and female sea cucumbers to the HFD differed, which in turn could have triggered sex-related differences in the intestinal microbiome. These results suggest that the lipid content in diets can be differentially adjusted for male and female sea cucumbers to improve nutrition and promote reproduction. This data contributes to a better understanding of the reproductive biology and sex differences of sea cucumbers.

Evidence that offshore wind farms might affect marine sediment quality and microbial communities.

Offshore wind power is a typical example of clean energy production and plays a critical role in achieving carbon neutrality. Offshore wind farms can have an impact on the marine environment, especially sedimentary environments, but their influence on sediments remain largely unknown. This study, which uses the control-impact principle to define different areas, investigated the characteristics of marine sediments under the Putidao offshore wind farm in Bohai Bay, China. We used chemical and microbiological observations to evaluate sediment quality and microbial community structure. According to both the geo-accumulation index (Igeo) and contamination factor (CF) indexes, copper, chromium and zinc were the major contaminants in the offshore wind farm sediments. The pollution load index (PLI) index showed that the various sites on the wind farm were only lightly polluted compared with baseline values. Closer to the wind farm's center, the metal concentrations started to rise. The physicochemical features of the sediments could better explain changes in the microorganisms present, and screening the microbiomes showed a correlation with heavy metal levels, linking the relative abundance of microorganisms to the sediment quality index. This comprehensive study fills a knowledge gap in China and adds to our understanding of how to assess the sedimentary environments of offshore wind farms.

Transcriptome analysis of gender-biased CYP genes in gonads of the sea cucumber Apostichopus japonicus.

Gender differences in physiological characteristics are widespread in animals. Herein, differentially expressed genes (DEGs) in gonads of the sea cucumber Apostichopus japonicus were analysed by transcriptomics, and the results showed that 19,973 genes were commonly expressed in the males and females, 4186 were female-biased, and 2540 were male-biased, 4695 genes were up-regulated in the females and 3436 genes were up-regulated in the males. These DEGs were mainly associated with metabolism, including lipid metabolism, amino acid metabolism, nucleotide metabolism, energy metabolism, and cofactor and vitamin metabolism. 29 Cytochrome P450 (CYP) superfamily genes with gender differential expression were selected, and performed gene identification, phylogenetic, and functional analyses. The results indicated significant roles in multiple metabolic pathways, such as steroid hormone biosynthesis, ovarian steroidogenesis, cortisol synthesis and secretion, arachidonic acid metabolism, linoleic acid metabolism, and retinol metabolism. The findings provide insight into the molecular characteristics of physiological gender differences in sea cucumbers, and will help lay the foundation for the establishment of effective sea cucumber breeding technologies.

Plasticity of Respiratory Function Accommodates High Oxygen Demand in Breeding Sea Cucumbers.

Physiological plasticity allows animals to adjust their physiological function to abiotic and biotic variations. It has been mostly studied in the context of response to external factors and not much is known on how animals adjust their physiology to cope with variations in internal conditions. The process of reproduction implies gonadal maturation and other internal changes, bringing various challenges to the animal such as an increased demand for energy and oxygen. Here, the capacity of the sea cucumber, Apostichopus japonicus to adjust its respiratory function and physiological mechanisms during reproduction was studied using a time-lapse videography and metabolomics approach. The results showed that reproduction caused a significant increase in oxygen consumption in A. japonicus. Interestingly, breeding sea cucumbers can accommodate the high oxygen demand by accelerating respiratory rate. However, to maintain a necessary high level of respiratory activity during reproduction, sea cucumbers need consume large amounts of adenosine triphosphate (ATP). In addition, the metabolomic data suggests that oxidative stress and hormone regulation are the physiological mechanisms linking reproduction and respiratory function. Altogether, these findings suggest that plasticity of respiratory function is an effective tactic to cope with high oxygen demand during reproduction.

Metabolome responses of the sea cucumber Apostichopus japonicus to multiple environmental stresses: Heat and hypoxia.

Economically important marine organisms face severe environmental challenges, such as high temperature and low dissolved oxygen, from global climate change. Adverse environmental factors impact the survival and growth of economically important marine organisms, thereby negatively influencing the aquaculture industry. However, little is known about the responses of sea cucumbers to combined environmental co-stressors till now. In this study, ultra-performance liquid chromatography (UPLC) was utilized to obtain metabolic profiles of sea cucumbers. Changes in the concentrations of 84, 68, and 417 metabolites related to the responses of sea cucumbers to heat (26 °C), hypoxia (2 mg/L) and the combined stress, respectively, were observed and analyzed. Representative biomarkers were discussed in detail, including deltaline, fusarin C, halichondrin B and rapanone. The concentration of metabolites involved in the regulation of energy metabolism, including amino acid, carbohydrate and lipid metabolism were significantly changed, and the tricarboxylic acid (TCA)-cycle was significantly altered under heat plus hypoxia. We interpreted these changes partly as an adaptation mechanism in response to environmental stress. Based on the decreased accumulation of glutamine, we hypothesized that heat stress is the main factor that interferes with the process of glutamic acid-glutamine metabolism. The present study showed that combined environmental stressors have a more extensive impact on the metabolites of the respiratory tree in sea cucumbers than single stress. These results would facilitate further development of the sea cucumber as an echinoderm model to study mechanisms of response to adverse environments, as well as to help advance knowledge of the adaptation of marine organisms to global climate change.

Global-warming-caused changes of temperature and oxygen alter the proteomic profile of sea cucumber Apostichopus japonicus.

Multiple environmental stressors caused by global warming influence the regulation of proteins involved in various biological processes in aquatic organisms. As an important component of the marine ecosystem, sea cucumber is a suitable echinoderm species for researching stress responses. In this study, we have investigated the proteomic response of respiratory trees in the sea cucumber Apostichopus japonicus, challenged with environmental stresses by using the iTRAQ technique. Results showed that 262, 155 and 433 proteins were differentially regulated in response to heat, hypoxia, and heat plus hypoxia, respectively. Expression of key genes and proteins were measured by real-time PCR and western blot to validate the accuracy of the proteome files. Results showed that the interaction between these two stressors has an additive effect at the proteome level. Proteins involved in amino acid and carbohydrate metabolic processes were largely induced by heat while repressed by hypoxia. Exposure to multiple climate change stressors caused several proteins involved in lipid metabolic progress to be down-regulated and lipid catabolic processes were induced. Additionally, hypoxia and the combined stress induced proteins involved in iron homeostasis. ATP synthesis and gluconeogenesis were induced under heat and the combined stress, while ATP and glycogen synthesis were depressed under hypoxia. Proteins related with immune and defense response were largely induced and protein synthesis ability was depressed under all three stresses. Thus, sea cucumber may adopt different strategies to cope with varied environmental stress, and the situation in heat group is more similar with the combined treatment than hypoxia group. These proteomic changes in response to high-temperature and low-oxygen levels may provide insights into the defense strategies of sea cucumber in response to global climate changes. SIGNIFICANCE OF THE STUDY: The study focused on comparative quantitative proteomics on Apostichopus japonicus respiratory tree using iTRAQ in responses to multiple climate change stressors: heat, hypoxia and the combined stress. Data showed that A. japonicus can have an acute reaction in diverse biological pathways to both individual and interacting environmental stress, including substance metabolism, signal transduction, protein synthesis, immune response and energy production. Results indicated that sea cucumber adopted different strategies to cope with varied environmental stress, and the interaction between these two stressors has an additive effect at the proteome level. These results offer insight into the molecular regulation of A. japonicus to multiple environmental stress and reveal possible molecular events in sea cucumber under climate changes. The understanding of adaptive variation under global climate changes in aquatic organisms could be improved.

Impact of hypoxia stress on the physiological responses of sea cucumber Apostichopus japonicus: respiration, digestion, immunity and oxidative damage.

Hypoxia is one of the most frequently occurring stressors confronted by industrial cultures of sea cucumber and can cause large economic losses and resource degradation. However, its responsive mechanisms are still lacking. In this paper, the physiological responses of Apostichopus japonicus to oxygen deficiency was illustrated, including induced oxidative response and immune defense and changed digestive enzymes activities. Significantly increased activities of alpha-amylase (AMS), acid phosphatase (ACP), lactate dehydrogenase, catalase, peroxidase, succinate dehydrogenase and higher content of malondialdehyde, and decreased activities of lipase and trypsin (TRY) were observed after hypoxia exposure (dissolved oxygen [DO] 2 mg/L). Expressions of key genes showed that AMS, peptidase, ACP, alkaline phosphatase, lysozyme, heat shock protein 70 and glutathione peroxidase were increased and TRY was decreased under hypoxia. With the decline of the DO level, the decreased tendency of oxygen consumption rates was different in varied weight groups. Moreover, respiratory trees were observed degraded under long-term hypoxia stress, thus leading a negative effect of respiration. These results could help to develop a better understanding of the responsive mechanism of sea cucumber under hypoxia stress and provide a theoretical basis for the prevention of hypoxia risk.

Differential Expression of miRNAs in the Respiratory Tree of the Sea Cucumber Apostichopus japonicus Under Hypoxia Stress.

The sea cucumber, an important economic species, has encountered high mortality since 2013 in northern China because of seasonal environmental stress such as hypoxia, high temperature, and low salinity. MicroRNAs (miRNAs) are important in regulating gene expression in marine organisms in response to environmental change. In this study, high-throughput sequencing was used to investigate alterations in miRNA expression in the sea cucumber under different levels of dissolved oxygen (DO). Nine small RNA libraries were constructed from the sea cucumber respiratory trees. A total of 26 differentially expressed miRNAs, including 12 upregulated and 14 downregulated miRNAs, were observed in severe hypoxia (DO 2 mg/L) compared with mild hypoxia (DO 4 mg/L) and normoxic conditions (DO 8 mg/L). Twelve differentially expressed miRNAs were clustered in severe hypoxia. In addition, real-time PCR revealed that 14 randomly selected differentially expressed miRNAs showed significantly increased expressions in severe hypoxia and the expressions of nine miRNAs, including key miRNAs such as Aja-miR-1, Aja-miR-2008, and Aja-miR-184, were consistent with the sequencing results. Moreover, gene ontology and pathway analyses of putative target genes suggest that these miRNAs are important in redox, transport, transcription, and hydrolysis under hypoxia stress. Notably, novel-miR-1, novel-miR-2, and novel-miR-3 were specifically clustered and upregulated in severe hypoxia, which may provide new insights into novel "hypoxamiR" identification. These results will provide a basis for future studies of miRNA regulation and molecular adaptive mechanisms in sea cucumbers under hypoxia stress.

Molecular cloning of heat shock protein 10 (Hsp10) and 60 (Hsp60) cDNAs and their expression analysis under thermal stress in the sea cucumber Apostichopus japonicus.

Hsp10 and Hsp60 are important heat shock proteins (HSPs), which might be indispensable in the heat shock response and many other physiological processes. We obtained full-length cDNAs of genes hsp10 and hsp60 and classified their mRNA expression levels under thermal stress in the sea cucumber Apostichopus japonicus. Full-length hsp10 cDNA was 1528 bp containing a 5' untranslated region (UTR) of 83 bp, a 3' UTR of 1133 bp and an open reading frame (ORF) of 312 bp encoding 103 amino acid residues. Full-length hsp60 cDNA was 2560 bp containing a 118-bp 5' UTR, a 678-bp 3' UTR and a 1764-bp ORF encoding 586 amino acid residues. The deduced amino acid sequences of Hsp10 and Hsp60 shared the highest identity with sequences of the sea urchin Strongylocentrotus purpuratus, and phylogenetic trees showed that the evolution of Hsp10 and Hsp60 was almost in accord with the evolution of species. Further analysis by real-time PCR showed that the expression of hsp10 and hsp60 mRNA was highly up-regulated at 26 °C compared with other three groups (20, 22, 24 °C), and their expression in the intestine was in a time-dependent manner at 26 °C. The results suggested that hsp10 and hsp60 were involved in the heat-shock response in the sea cucumber A. japonicus.