Promoting effect of raft-raised scallop culture on the formation of coastal hypoxia.

The coastal waters around the Yangma Island are an important mariculture area of raft-raised scallop and bottom-seeded sea cucumber in the North Yellow Sea, China. Large-scale hypoxia in the bottom water of this area has caused the death of a large number of sea cucumbers and heavy economic losses. To find out the formation mechanism of hypoxia, the data obtained in each August during 2015-2018 were analyzed. Compared with the non-hypoxic year (2018), the temperature, trophic index (TRIX) and dissolved organic carbon (DOC) in the bottom water were relatively higher, and the water column was stratified causing by continuous high air temperature and low wind speed meteorological conditions in the hypoxic years (2015-2017). These sites with the coexistence of thermocline and halocline, and the thickness of thermocline >2.5 m and its upper boundary >7.0 m deep, were prone to hypoxia. Spatially, the hypoxic place was highly consistent with the scallop cultivating places, and the DOC, TRIX, NH4+/NO3- and apparent oxygen utilization (AOU) at the culture sites were higher, indicating that organic matter and nutrients released by scallops may lead to local oxygen depletion. In addition, the bottom water of the culture sites had higher salinity, but lower turbidity and temperature, indicating that the slowed water exchange caused by scallop culture was a dynamic factor of hypoxia. All sites with AOU >4 mg/L at the bottom had hypoxia occurrence, even if there was no thermocline. In other words, stratification promoted the formation of hypoxia in coastal bottom water, but it was not indispensable. The raft-raised scallop culture could promote the formation of coastal hypoxia, which should arouse the attention for other coastal areas with intensive bivalve production.

Ammonia nitrogen exposure caused structural damages to gill mitochondria of clam Ruditapes philippinarum.

Ammonia nitrogen has been one of the key pollution indicators along the Chinese coastline for quite a few years. Our previous studies have proved that ammonia nitrogen is harmful for Ruditapes philippinarum clam in several aspects. Environmental concentrations of ammonia nitrogen were found to significantly decrease ATP contents and disturb ATP metabolism, in addition to reducing the potential across the mitochondrial membrane in clam gill tissues. Accordingly, mitochondrion is considered as one of the target organelles of ammonia nitrogen toxicity in clams. However, there is a lack of direct evidence to prove it. In order to reveal detail information of ammonia nitrogen toxicity on clam mitochondria and screen the related biomarker to indicate ammonia nitrogen pollution, mitochondrial parameters in gill tissues including swelling, mtDNA copy number and marker enzyme (succinic dehydrogenase, SDH) activity were measured after the clams were exposed to 0.1 mg/L and 0.5 mg/L ammonia nitrogen for 3 days and 21 days, respectively. Moreover, adverse effects of ammonia nitrogen exposure on clam mitochondrial ultra-structures, mitochondrial swelling and division were also discriminated under transmission electron microscope (TEM). Final results showed that ammonia nitrogen exposure to both concentrations significantly induced mitochondrial swelling, reduced the number of mitochondria and messed their normal structure, decreased the number of mtDNA copies and down-regulated SDH activity, all in a concentration and duration dependent manner. So, the present study helps us to better understand the structural damage of ammonia nitrogen on mitochondria in clam gill cells and provides fundamental data for ammonia nitrogen control in aquaculture.

Tissue distribution and functional characterization of mytimacin-4 in Mytilus galloprovincialis.

Antimicrobial peptides (AMPs) play fundamental roles in the innate immunity of invertebrates. Mytimacin-4 is a kind of AMP gene previously sequenced from Mytilus galloprovincialis based on an identified EST sequence in our lab. In the present study, the tissue distribution and antimicrobial activities of mytimacin-4 were further investigated. A qRT-PCR analysis revealed that mytimacin-4 transcripts were constitutively expressed in all of the tested tissues of M. galloprovincialis, with the highest expression level in the posterior adductor muscle. After challenge by Vibrio anguillarum, the expression level of mytimacin-4 gene was significantly increased at 24 h (P < 0.05) in the mantle and increased at 48 h (P < 0.05) in the posterior adductor muscle. This finding suggested that mytimacin-4 transcripts were inducible upon pathogen infection. A minimal inhibitory concentration (MIC) assay indicated that recombinant mytimacin-4 protein had potent antimicrobial activities against gram-positive and gram-negative bacteria. Among the tested microorganisms, mytimacin-4 protein exhibited strong inhibition activities against Bacillus subtilis and Vibrio parahaemolyticus with MICs of 0.315 µM and 0.62 µM, respectively. This study provides for the first time direct evidence of antimicrobial action of mytimacin-4 in M. galloprovincialis.

Digital gene expression analysis in the gills of Ruditapes philippinarum after nitrite exposure.

Due to the overload of pollutants from highly intensive anthropic activities, nitrite accumulates in offshore seawater and has been a long-lasting pollutant to the healthy aquaculture of the mollusk. In the present study, Ruditapes philippinarum was used as the target bivalve to receive nitrite exposure at environmental concentration for 1 and 7 days. Differentially expressed genes (DEGs) were detected and analyzed by a digital gene expression (DGE) approach to describe the toxicity of nitrite on the bivalve at the gene level. In the N1 group, 185 DEGs were generated and enriched in six Gene Ontology (GO) terms, including oxidoreductase activity, heme binding, tetrapyrrole binding, iron ion binding, metal binding and cation binding. The DEGs in the N1 group were also enriched in two Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, e.g., arachidonic acid metabolism and ovarian steroidogenesis. In the N7 group, 81 DEGs were generated without any GO enrichment but were enriched in five KEGG pathways, including protein processing in the endoplasmic reticulum, protein export, prion diseases, thyroid hormone synthesis and arachidonic acid metabolism. This suggested that nitrite exposure might cause adverse effects to the clams in several aspects, including oxidative damage, depressed immunity, and disorders in cell proliferation, hormone metabolism and tissue regeneration. Evaluation of oxidative stress indicated that nitrite exposure actually induced redox state imbalance by enhancing the contents of thiobarbituric acid reactive substances (TBARSs) and glutathione (GSH), and the activity of glutathione peroxidase (GSH-PX) but not superoxide dismutase (SOD). These results will provide valuable gene references for further study on the toxicology mechanism of bivalves under environmental nitrite stress.

Preparation and antioxidant properties of crayfish (Procambarus clarkii) By-products protein hydrolysates and ultra filtration fractions.

Protein isolate from crayfish by-products (CBPI) were hydrolyzed using Alcalase, neutrase, pancreatin and bromelain. Hydrolysis by Alcalase had more remarkable digesting efficiency on crayfish by-products protein than that by the other enzymes. Therefore, protein hydrolysate from Alcalase digestion (CBPHa) was selected to be fractionated by ultrafiltration according to molecular weight into three fractions F1 (MW <1kDa), F2 (MW 1-3kDa) and F3 (MW 3-10kDa). The amino acid determination revealed that CBPI had essential amino acid (EAA) close to that required for human protein synthesis. In vitro activity experiments showed that CBPHa and its fractions possessed considerable antioxidant activity. F1 exhibited the highest DPPH, superoxide radicals scavenging activities and Fe2+ chelating ability, whereas F2 showed the best hydroxyl radicals scavenging capacity and reducing power. In addition, all the fractions showed higher super oxide radical scavenging activity than the crude hydrolysates. Our findings suggest that CBPHa and their ultra filtration fractions have the potential for use in nutraceutical and functional food industries to maximize the use of crayfish processing by-products.

Gill damage and neurotoxicity of ammonia nitrogen on the clam Ruditapes philippinarum.

Ammonia nitrogen has been a potential menace to aquatic animals along the coastline of China. Presently, the toxicological effects of ammonia nitrogen were mainly concentrated on fishes, while little attention has been paid to molluscs. In this study, the clam Ruditapes philippinarum was used as the target animal to investigate the toxic effects of ammonia nitrogen. Our results showed that ammonia exposure could significantly reduce the integrity of lysosomes in a dose-dependent manner. Metabolite analysis revealed that exposure doses and duration time of ammonia nitrogen could affect the variation profiles of gill metabolites. In detail, branched chain amino acids, glutamate, choline and phosphocholine were significantly decreased after a one-day exposure. Inosine and phenylalanine were found significantly increased and ATP was decreased after a three-day exposure. The changes of metabolites implied that metabolisms of muscle element, neurotransmission and cell apoptosis of gill tissues would be affected by ammonia exposure. Such inferences were supported by the diminished muscle element, decreased concentrations of catecholamines and increased apoptosis rates, respectively. Therefore, we take advantage of metabolomics integrated with conventional biological assays to find out that ammonia exposure could cause lysosome instability, metabolic disturbance, aberrant gill structures and changes to neurotransmitters, and would result in mollusk gill dysfunction in feeding, respiration and immunity.

Metabolomics revealed more deleterious toxicity induced by the combined exposure of ammonia and nitrite on Ruditapes philippinarum compared to single exposure.

NH3-N and NO2-N always co-exist in the aquatic environment, but there is not a clear opinion on their joint toxicities to the molluscs. Presently, clams Ruditapes philippinarum were challenged by environmental concentrations of NH3-N and NO2-N, singly or in combination, and analyzed by metabolomics approaches, enzyme assays and transmission electron microscope (TEM) observation. Results showed that some same KEGG pathways with different enriched-metabolites were detected in the three exposed groups within one day, and completely different profiles of metabolites were found in the rest of the exposure period. The combined exposure induced heavier and more lasting toxicities to the clams compared with their single exposure. ACP activity and the number of secondary lysosomes were significantly increased after the combined exposure. The present study shed light on the joint-toxicity mechanism of NH3-N and NO2-N, and provided fundamental data for the toxicity research on inorganic nitrogen.

Comparative analysis of microRNA expression profiles in clam Ruditapes philippinarum after ammonia nitrogen exposure.

Ammonia nitrogen is a long-lasting pollutant along the Chinese coast. In our previous studies, the clam Ruditapes philippinarum exhibited several toxic responses to environmental concentrations of ammonia nitrogen. To elucidate the underlying mechanism of ammonia nitrogen toxicity in clams at the post-transcriptional level, microRNA (miRNA) expression profiles were investigated by high-throughput sequencing after the clams were exposed to 0.1 mg/L ammonia nitrogen for 30 days. A total of 238 miRNAs were identified, including 49 conserved miRNAs and 189 novel miRNAs. After comparative analysis, six miRNAs were significantly expressed after 1 day of exposure, with three up-regulated and three down-regulated miRNAs. In addition, 35 miRNAs were significantly expressed after 30 days of exposure, of which 16 were up-regulated and 19 were down-regulated. Furthermore, the target genes of each differentially expressed miRNA were predicted, followed by Gene Ontology (GO) category and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses. The target genes were predicted to be involved in the immune response, protein processing and transport, DNA damage repair, cellular communication, neural signaling, redox homeostasis, lipid metabolism, and biotransformation. A biological phagocytosis assay proved the speculation that ammonia nitrogen regulated the immunity of clams with the aid of a novel miRNA (novel_29). These findings support further research on miRNA levels in R. philippinarum exposed to ammonia nitrogen.

Effects of heavy metals on the expression of a zinc-inducible metallothionein-III gene and antioxidant enzyme activities in Crassostrea gigas.

Sequestration by metallothioneins and antioxidant defense are two kinds of important defense mechanisms employed by mollusks to minimize adverse effects caused by heavy metal contaminants in marine environment. In the present study, a novel metallothionein gene, CgMT-III, was cloned from Crassostrea gigas, consisting of eighteen conserved cysteine residues and encoding a MT III-like protein with two tandem ß domains. The expression level of CgMT-III transcript induced by zinc was much higher than that induced by cadmium exposure. It suggested that CgMT-III was perhaps mainly involved in homeostatic control of zinc metabolism, which was distinct from previously identified MTs in C. gigas. Among the tested antioxidant enzymes including superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), SOD and GPx showed varying up-regulations in a tissue-specific manner, while CAT activities were inhibited in both gill and hepatopancreas from C. gigas exposed to heavy metals. It can be inferred that CgMT-III was mainly involved in zinc homeostasis, and CgMT-III gene together with CAT enzyme could be potential biomarkers to indicate heavy metal, especially zinc pollution in marine organisms.

Modifications of calcium metabolism and apoptosis after ammonia nitrogen exposure imply a tumorous fate in clam Ruditapes philippinarum?

Ammonia nitrogen (NH3N) is a kind of toxic inorganic nitrogen that has been a great ecological stressor to the marine organisms for quite a few years in Chinese coastal area. Toxic mechanism of ammonia nitrogen on marine bivalve is not well elucidated, especially in calcium metabolism and apoptosis. In the present study, clams Ruditapes philippinarum were used as the experiment animals to receive NH3N exposure with environmental concentrations for 21 days. Results showed that NH3N exposure induced ROS production, decreased Ca2+ concentration, and increased caspase 3 activities in the clam gill cells. In addition, three kinds of Ca2+ exchanger genes, e. g. Na+/K+/Ca2+ exchanger 2, Na+/Ca2+ exchanger 3 and monovalent cation/H+ antiporter, exhibited significant increments in transcription to eliminate intracellular Ca2+. Besides, NH3N exposure significantly increased mRNA expression levels of key anti-apoptotic regulator Bcl-2 genes (Bcl2-1 and Bcl2-1), which would inhibit the apoptosis degree in gill cells. Taken together, increased Ca2+-extrusion and apoptosis inhibition would act cooperatively to alleviate the apoptosis degree and extend the lifespan, so that some kind of tumor might develop in oxidative damaged gill cells after NH3N exposure. Therefore, it is predicted that NH3N exposure will probably bring the clam R. philippinarum a tumorous fate, which will be a great challenge for the healthy development of molluscs aquaculture under the current pollution condition. In addition, caspase 3 activity and mRNA expression levels of Bcl2-2 and Na+/Ca2+ exchanger 3 could be used as potential clam biomarkers to indicate NH3N pollution.

Effects of ammonia nitrogen on gill mitochondria in clam Ruditapes philippinarum.

Ammonia nitrogen exposure has been found to significantly increase the early apoptosis rates of gill cells, affect the contents of ATP and disturb expressions of calcium-related genes in clam Ruditapes philippinarum. Mitochondria are the centers for energy production, initiation of apoptosis and calcium signal regulation. It is hypothesized that gill mitochondrion is a target organelle for the ammonia nitrogen. Thus, ATP metabolism together with ATP-consuming functions would be interfered by ammonia exposure. In the present study, mitochondrial transmembrane potential (MTP), ATPase activities, gill functions in clearance and respiration, and histological changes were detected to characterize the effects of ammonia to the gill mitochondria in clam R. philippinarum. Results indicated that ammonia exposure led to significant decreases in MTP, Ca2+-ATPase activity and clearance rates. However, different concentrations of ammonia nitrogen induced different variations on H+, K+-ATPase activity and respiration rates. Histological observation revealed that subacute exposure of ammonia damaged the microstructure of gill tissues. Therefore, ammonia exposure dramatically damaged the normal structure and function of mitochondria, resulting in irreversible damage in energy formation and supply. In addition, it affected Ca2+ and K+ metabolism and inhibited food intake and respiration in clam R. philippinarum.

Digital gene expression analysis in the gills of Ruditapes philippinarum exposed to short- and long-term exposures of ammonia nitrogen.

Previous study revealed severe toxic effects of ammonia nitrogen on Ruditapes philippinarum including lysosomal instability, disturbed metabolic profiles, gill tissues with damaged structure, and variation of neurotransmitter concentrations. However, the underlying molecular mechanism was not fully understood yet. In the present study, digital gene expression technology (DGE) was applied to globally screen the key genes and pathways involved in the responses to short- and long-term exposures of ammonia nitrogen. Results of DGE analysis indicated that short-term duration of ammonia exposure affected pathways in Dorso-ventral axis formation, Notch signaling, thyroid hormone signaling and protein processing in endoplasmic reticulum. The long-term exposure led to DEGs significantly enriched in gap junction, immunity, signal and hormone transduction, as well as key substance metabolism pathways. Functional research of significantly changed DEGs suggested that the immunity of R. philippinarum was weakened heavily by toxic effects of ammonia nitrogen, as well as neuro-transduction and metabolism of important substances. Taken together, the present study provides a molecular support for the previous results of the detrimental toxicity of ammonia exposure in R. philippinarum, further work will be performed to investigate the specific genes and their certain functions involved in ammonia toxicity to molluscs.

Gene expression responses in Suaeda salsa after cadmium exposure.

Coastal line is now polluted by many kinds of sewage including heavy metals discharged by intensive human activities. Cadmium is a nonessential heavy metal for organisms and can cause many kinds of adverse effect on the organisms. Suaeda salsa, a pioneer halophyte in intertidal zone of the Bohai coast, was proved to have cadmium-tolerant capacity. Given that, S. salsa was suggested as a potential coastal bio-indicator plant for cadmium contamination in the intertidal zone. Therefore, it is essential to investigate the responsive mechanism of S. salsa to cadmium since few studies focus on this subject till now. In the present study, six genes were selected to investigate the variation profiles of mRNA expression by fluorescent real-time quantitative PCR, including those involved in myo-inositol synthesis, redox reaction, salt-tolerant reaction. Results showed that cadmium exposure significantly modulate the mRNA expressions of MIPS, Nhx1, CAT2, GST, Prx Q genes. It suggested that cadmium exposure exerted an oxidative stress on S. salsa, disturbed Na(+) homeostasis across membranes and interfered with the metabolism of inositol. In addition, CAT2 gene could be used as a gene marker in S. salsa to indicate cadmium pollution.

Micromonospora rifamycinica sp. nov., a novel actinomycete from mangrove sediment.

An actinomycete strain, AM105(T), that produces rifamycin, was isolated from mangrove sediment samples collected from the South China Sea. The strain showed closest 16S rRNA gene sequence similarity to Micromonospora matsumotoense (98.0%). Chemotaxonomic characteristics of the isolate coincided with members of the genus Micromonospora. The value of DNA-DNA relatedness to M. matsumotoense (53.6%) and phenotypic differences from phylogenetically related Micromonospora species indicated that this isolate belongs to a novel species, for which the name Micromonospora rifamycinica sp. nov. is proposed. The type strain is AM105(T) (=CGMCC 4.2495(T)=DSM 44983(T)).