Exploring the mechanism of nonylphenol-induced ovarian developmental delay of manila clams, Ruditapes philippinarum: Applying RNAi to toxicological analysis.

Nonylphenol (NP) contamination in the coastal environment of China poses ecological risks to aquatic organisms. However, the endocrine disruptive impacts of NP on bivalves, particularly on ovarian development, remain poorly understood. In this study, Manila clams Ruditapes philippinarum at the developing stage of gonad were exposed to 1.0 µg/L NP for 21 days. Utilizing RNA interference (RNAi) to suppress ER gene expression, we observed a delay in ovarian development as evidenced by histological observations under both NP and NPRi (NP with ER-RNAi) treatment, with Vtg elevation exclusive to the NP group. Comprehensive analyses encompassing transcriptomics, real-time quantitative PCR, and steroid hormone measurement revealed significant alterations in aldosterone synthesis, estrogen signaling, and thyroid hormone synthesis. These pathways showed similar perturbations in both NP and NPRi groups compared to controls. Notably, the NPRi group exhibited distinct enrichment in PPAR and insulin signaling pathways, may implicating these in ER function suppression. Steroid hormone biosynthesis was notably reduced in both treatments, pointing to a profound impact on hormone synthesis. The contrast between in vivo and in vitro findings suggests that NP's detrimental effects on ovarian development may primarily involve neuroendocrine regulation of steroidogenesis. This investigation highlights the complex dynamics of NP-induced endocrine disruption in bivalves, emphasizing the pivotal role of ER and associated pathways.

Effects of Benzo[a]pyrene on Food Metabolism and Reproductive Endocrine and Ovarian Development in Female Scallop Chlamys farreri at Different Reproductive Stages.

Benzo[a]pyrene (B[a]P), a polycyclic aromatic hydrocarbon (PAH) with the most carcinogenic effects of all the PAHs, has multiple toxic effects on marine bivalves. We investigated the interference mechanism of B[a]P on food metabolism (sugars, proteins, and sugars), and on reproductive endocrine and ovarian development in female scallops (Chlamys farreri). Scallops were exposed to different concentrations of B[a]P concentrations of 0, 0.38, 3.8, and 38 µg/L throughout gonadal development. Total cholesterol and triglyceride contents in the digestive glands were increased, and their synthesis genes were upregulated. The plasma glucose contents decreased with the inhibition of glycogen synthesis genes and the induction of glycolysis genes in the digestive gland. The results showed that B[a]P had endocrine-disrupting effects on scallops, that it negatively affected genes related to ovarian cell proliferation, sex differentiation, and egg development, and that it caused damage to ovarian tissue. Our findings supplement the information on B[a]P disruption in gonadal development of marine bivalves. Environ Toxicol Chem 2024;43:748-761. © 2023 SETAC.

Effects of dietary dihydromyricetin on growth performance, antioxidant capacity, immune response and intestinal microbiota of shrimp (Litopenaeus vannamei).

A 56-day culture trial was conducted to evaluate the effects of dietary dihydromyricetin (DMY) on growth performance, antioxidant capacity, immune response and intestinal microbiota of shrimp (Litopenaeus vannamei). 840 healthy shrimp (1.60 ± 0.21 g) in total were fed with four different levels of DMY diets at 0 (Control), 100 (D1), 200 (D2), and 300 (D3) mg/kg, respectively. Samples were collected after the culture trial, and then, a 7-day challenge experiment against Vibrio parahaemolyticus was conducted. The results demonstrated that DMY significantly enhanced the activity of protease, amylase and lipase as well as the expression of lipid and protein transport-related genes (P < 0.05). The results of plasma lipid parameters indicated that DMY reduced lipid deposition, manifested by significantly (P < 0.05) decreased plasma total cholesterol (T-CHO), triglyceride (TG) and low-density lipoprotein cholesterol (LDL-C). The expression of genes involved in fatty acid ß-oxidation and triglyceride catabolism was significantly up-regulated (P < 0.05), and genes involved in triglyceride synthesis were significantly down-regulated in DMY groups when compared to control group (P < 0.05). Moreover, dietary DMY also significantly (P < 0.05) increased the total antioxidant capacity (T-AOC), antioxidant enzymes activity and glutathione (GSH) content of shrimp, and a significant increase of total hemocytes count (THC), phagocytic rate (PR), antibacterial activity (AA) and bacteriolytic activity (BA) was observed in DMY groups (P < 0.05). The addition of DMY to the diet significantly augmented immune response by up-regulating the expression of genes related to toll-like receptors (Toll) signaling pathway, immune deficiency (IMD) signaling pathway and intestinal mucin. Furthermore, dietary DMY could modulate the composition and abundance of intestinal microbiota. In conclusion, DMY showed promising potential as a functional feed additive for shrimp to improve the growth performance and physiological health.

Insights into mechanism of DNA damage and repair-apoptosis in digestive gland of female scallop Chlamys farreri under benzo[a]pyrene exposure during reproductive stage.

As one of the most carcinogenic persistent organic pollutants (POPs), benzo[a]pyrene (B [a]P) brings high toxicity to marine bivalves. Digestive gland is the most important metabolism-related organ of aquatic animals. This study conducted the digestive gland transcriptome of Chlamys farreri under B[a]P treatment at reproductive stages. And the reproductive-stage dependence metabolism-DNA repair-apoptosis process of scallops under 0, 0.04, 0.4 and 4 µg/L B[a]P was studied by qRT-PCR. The results demonstrated that the detoxification metabolism was disturbed after ovulation except for CYP3A4. In antioxidant system, antioxidant enzyme CAT and GPX, and GGT1 (one of the non-enzymatic antioxidants synthesis gene) continuously served the function of antioxidant defense. Three types of DNA repair were activated under B[a]P stress, however, DNA strand breaks were still serious. B[a]P exposure weakened death receptor pathway as well as enhanced mitochondrial pathway, surprisingly suppressing apoptosis in scallops. In addition, ten indicators were screened by Spearman correlation analysis. This study will provide sound theoretical basis for bivalve toxicology and contribute to the biomonitoring of marine POPs pollution.

Characterization and removal mechanism of a novel enrofloxacin-degrading microorganism, Microbacterium proteolyticum GJEE142 capable of simultaneous removal of enrofloxacin, nitrogen and phosphorus.

In the study, a novel ENR-degrading microorganism, Microbacterium proteolyticum GJEE142 was isolated from aquaculture wastewater for the first time. The ENR removal of strain GJEE142 was reliant upon the provision of limited additional carbon source, and was adaptative to low temperature (13 â„ƒ) and high salinity (50‰). The ENR removal process, to which intracellular enzymes made more contributions, was implemented in three proposed pathways. During the removal process, oxidative stress response of strain GJEE142 was activated and the bacterial toxicity of ENR was decreased. Strain GJEE142 could also achieve the synchronous removal of ammonium, nitrite, nitrate and phosphorus with the nitrogen removal pathways of nitrate → nitrite → ammonium → glutamine → glutamate → glutamate metabolism and nitrate → nitrite → gaseous nitrogen. The phosphorus removal was implemented under complete aerobic conditions with the assistance of polyphosphate kinase and exopolyphosphatase. Genomic analysis provided corresponding genetic insights for deciphering removal mechanisms of ENR, nitrogen and phosphorus. ENR, nitrogen and phosphorus in both actual aquaculture wastewater and domestic wastewater could be desirably removed. Desirable adaptation, excellent performance and wide distribution will make strain GJEE142 the hopeful strain in wastewater treatment.

Comparison and quantification of estrogen receptor-mediated responsiveness to endocrine disruptors in bivalves by using complementary model and a novel yeast assay approach.

Endocrine disrupting chemicals (EDCs) in estuaries and coastal habitats have been widely detected over the world and caused global concern. Bivalves have been shown to be vulnerable to endocrine disruption. However, estrogen receptors (ERs) sensitivity to steroids and EDCs has long been considered to be restricted to vertebrates. In the present study, a computational simulation docking model was applied to qualitatively predict the binding behavior of two bivalve ERs to estradiol and compared the docking activity with zebra fish ERa. A novel reconstituted yeast system was constructed by using transcriptional activator GAL-4 consists of ER-expressing plasmid and ERE (estrogen responsive element)-containing plasmid. The assays showed that bivalve ER specifically activate transcription in response to tested steroids and EDCs, but the activation ability is weaker compared to zebra fish ERa. The results corroborate the presence of an active ER in bivalve molluscs and provide a promising tool for screening of marine environmental pollutants active in disturbing ERs of bivalves, as well as understanding the underlying mechanism across taxonomic groups and phyla.

Transcriptomic response to GnRH down regulation by RNA interference in clam Ruditapes philippinarum, suggest possible role in reproductive function.

Gonadotropin-releasing hormone (GnRH) plays a key role in the control of the reproductive axis in vertebrates, however, little is known about its function in reproductive endocrine regulation in molluscs. In the present study, RNA-seq was used to construct transcriptomes of Ruditapes philippinarum testis and ovaries of control and GnRH suppressed individuals using RNA interference. GnRH suppression caused 112 and 169 enriched KEGG pathways in testis and ovary, with 92 pathways in common in both comparisons. The most enriched KEGG pathways occurred in the "Oxidative phosphorylation", "Dorso-ventral axis formation", "Thyroid hormone synthesis" and "Oxytocin signaling pathway" etc. A total of 1838 genes in testis and 358 genes in ovaries were detected differentially expressed in GnRH suppressed clams. Among the differentially expressed genes, a suit of genes related to regulation of steroid hormones synthesis and gonadal development, were found in both ovary and testis with RNAi of GnRH. These results suggest that GnRH may play an important role in reproductive function in bivalves. This study provides a preliminary basis for studying the function and regulatory mechanism of GnRH in bivalves.

Bioaccumulation, Detoxification, and Biological Macromolecular Damage of Benzo[a]pyrene in Exposure in Tissues and Subcellular Fractions of Scallop Chlamys farreri.

Because of the persistence and high toxicity of benzo[a]pyrene (B[a]P), the bioaccumulation and detoxification mechanisms of B[a]P have been studied extensively at the tissue level; but the data at the subcellular level in bivalves have not been reported. The present study was conducted to investigate the effects of B[a]P exposure on bioaccumulation, detoxification, and biomacromolecular damage in gills, digestive glands, and their subcellular fractions of the scallop Chlamys farreri. The subcellular fraction contains cytoplasm, mitochondria, microsome, nucleus, cell membrane, and overall organelle. The results demonstrated that B[a]P accumulation showed a clear time-dose effect. Based on the time-dependent accumulation of B[a]P in subcellular fractions, we speculated that the intracellular migration order of B[a]P was cell membrane, organelle, and nucleus in turn. Considering the difference of B[a]P accumulation may be related to B[a]P metabolism, we have further confirmed that the activities of B[a]P metabolizing enzymes in scallop tissues and subcellular fractions were significantly tempted by B[a]P (p < 0.05), including 7-ethoxyresorufin O-deethylase (increased), glutathione-S-transferase (GST; decreased), and superoxide dismutase (increased). First, GST was detected in bivalve cytoplasm and microsome. Second, B[a]P exposure also caused biomacromolecules damage. The results demonstrated that mitochondria and microsome were more vulnerable to lipid peroxidation than cell membrane and nucleus. Taken together, the present study fills some of the gaps in our knowledge of the bioaccumulation and detoxification mechanisms of C. farreri exposed to B[a]P in subcellular fractions and deeply explores the transportation and the main metabolic and damage sites of polycyclic aromatic hydrocarbons (PAHs) in cells, which helped us to comprehensively understand the toxic mechanism of PAHs on bivalves. Environ Toxicol Chem 2022;41:2353-2364. © 2022 SETAC.

Impact of P-Chloroaniline on Oxidative Stress and Biomacromolecules Damage in the Clam Ruditapes philippinarums: A Simulate Toxicity Test of Spill Incident.

As a hazardous chemical, p-chloroaniline (PCA) shows intensive adsorption and accumulation after entering the aquatic ecosystem, which can be enriched in organisms and cause damage. With the objective of achieving an integrated and mechanistic view of the toxic effects of PCA in the marine sentinel organism Ruditapes philippinarum, Manila clams were exposed to different concentration of PCA (0.5, 2 and 5 mg/L) for 15 days. Focusing on the gills, first targeting the toxic and digestive gland, the metabolic detoxification organ, we detected dose- and time-related changes inantioxidase activities and biomacromolecular damages in treated clams. Glutathione S-transferase (GST) activity and glutathione (GSH) contents were significantly induced, and superoxide dismutase (SOD) activity increased at the beginning of exposure and then decreased. The malondialdehyde (MDA) and protein methylation (PC) contents which represent lipid peroxidation and carbonylation of proteins, increased first with exposure time and then decreased in the digestive gland. DNA strand break levels were consistently higher than those in the control group. The digestive gland showed more sensitivity to the stress of PCA than the gills. GST and MDA in the gill and GST, GSH, SOD, DNA strand break level in the digestive gland showed significant correlation with PCA exposure, which indicated that these parameters can be used as sensitive biomarkers to indicate toxic effects from chloraniline leakage.

Reproductive toxicity induced by benzo[a]pyrene exposure: first exploration highlighting the multi-stage molecular mechanism in female scallop Chlamys farreri.

Reproductive toxicity induced by benzo[a]pyrene (B[a]P) exposure has received great ecotoxicological concerns. However, huge gaps on the molecular mechanism still exist in bivalves. In this study, reproduction-related indicators were investigated in female scallops Chlamys farreri during life cycle of proliferative, growth, mature, and spawn stages, under gradient concentrations of B[a]P at 0, 0.04, 0.4, and 4 µg/L. Meanwhile, a multi-stage ovarian transcriptome analysis under 4 µg/L B[a]P exposure was also conducted to elucidate the potential molecular mechanisms. The results indicated that life-cycle exposure to 0.4 and 4 µg/L B[a]P significantly decreased GSI and sex steroid levels. Even 0.04 µg/L B[a]P could play the adverse role in DNA integrity at the mature and spawn stages. Ovarian histological sections showed that B[a]P inhibited the maturation and release of oocytes. Through the functional enrichment analysis of differentially expressed genes (DEGs) from transcriptome data, 18 genes involved in endocrine disruption effects, DNA damage and repair, and oogenesis were selected and further determined by qRT-PCR. The downregulation of genes involved in steroidogenic and estrogen signaling pathways indicated that B[a]P could cause endocrine disruption through both receptor-dependent and receptor-independent pathways. The variations of gene expressions involved in DNA single-strand break and repair implied the presence of toxic mechanisms similar with vertebrates. Additionally, the changes of gene expressions of cell cycle, apoptosis, and cell adhesion suggested that exposure to B[a]P possibly caused the reproductive toxicity effects by affecting oogenesis. Taken together, this study was a pioneer in combining genome-wide transcriptomic analysis with its corresponding reproductive indicators (GSI, sex steroid levels, DNA single-strand break, and histological sections) to explore the bivalves' toxic mechanisms under B[a]P exposure. Meanwhile, some genes involved in estrogen signaling pathway and DNA damage were firstly analyzed in bivalves, and the expression data might be useful in establishing new hypotheses and discovering new biomarkers for marine biomonitoring.

Possible roles of gonadotropin-releasing hormone (GnRH) and melatonin in the control of gonadal development of clam Ruditapes philippinarum.

Gonadotropin-releasing Hormone (GnRH) is a key reproductive endocrine regulator, and melatonin is considered as a potent candidate in the regulation of photoperiod-related reproductive endocrinology. Nevertheless, their function during gonadal development of molluscs has not been uncovered yet. In the present study, RNAi of GnRH and melatonin injection were conducted on marine bivalve manila clam Ruditapes philippinarum. Tissue section showed that gonadal development was significantly inhibited in male clams injected with GnRH dsRNA for 21 days. For GnRH RNAi treatment group, the expression levels of steroid synthetic enzyme genes 3ß-hydroxysteroid dehydrogenase (3ß-HSD), 17ß-hydroxysteroid dehydrogenase (17ß-HSD), cytochrome P450 (CYP3A) and melatonin receptor homolog (MTNR) gene were significantly down-regulated in female clams while significantly up-regulated in male clams. In melatonin injection group, the expression of GnRH was significantly inhibited and the expression of 3ß-HSD, 17ß-HSD, CYP3A and MTNR genes also increased which was in line with the GnRH dsRNA injection group in male clams. These results suggest that melatonin may affect GnRH expression and both have effects on gonadal development of bivalves. This study provides evidence for understanding the effects of melatonin and GnRH on reproductive endocrinology and gonadal development in bivalve molluscs.

Simultaneous aerobic removal of phosphorus and nitrogen by a novel salt-tolerant phosphate-accumulating organism and the application potential in treatment of domestic sewage and aquaculture sewage.

Phosphorus (P) and nitrogen (N) pollution are the worldwide challenging problem. In the present study, a new salt-tolerant phosphate-accumulating organism (PAO) was isolated and identified as Bacillus subtilis GHSP10. Strain GHSP10 did not produce hemolysin and showed high susceptibility to antibiotics. The favorable phosphorus removal C/N ratios, P/N ratios, temperature, salinities, pH values and shaking speeds of strain GHSP10 were 10-20, 0.1-0.2, 28 °C, 0-3%, 7.5-8.5 and 100-250 r/min. Besides, strain GHSP10 could conduct heterotrophic nitrification-aerobic denitrification and the maximal removal efficiencies of ammonium, nitrite and nitrate were 99.52%, 81.10% and 95.84% respectively. Moreover, the phosphorus removal process of strain GHSP10 was achieved under entirely aerobic conditions, and glycogen and poly-ß-hydroxybutyrate could provide energy source for the phosphorus removal process of strain GHSP10. The amplification of ppk, hao, napA, narG, nirK genes as well as the expression of polyphosphate kinase helped to reveal the removal pathways of phosphorus and nitrogen, providing theoretical support for the phosphorus removal, nitrification and aerobic denitrification abilities of strain GHSP10. Furthermore, efficient removal of phosphorus and nitrogen from both domestic sewage and aquaculture sewage could be accomplished by strain GHSP10. This study may provide a hopeful candidate strain for simultaneous removal of phosphorus and nitrogen pollution from both freshwater sewage and saline sewage.

Heterotrophic nitrification-aerobic denitrification characteristics and antibiotic resistance of two bacterial consortia from Marinomonas and Halomonas with effective nitrogen removal in mariculture wastewater.

Heterotrophic nitrification-aerobic denitrification (HNAD) characteristics and antibiotic resistance of two bacterial consortia, Marinomonas communis & Halomonas titanicae (MCH) and Marinomonas aquimarina & Halomonas titanicae (MAH), and their single isolates (MC, MA, and H) were determinated in this study. When cultured in sole and mixed N-source media (NH4+-N and/or NO2--N of 10 mg/L), MCH and MAH exhibited greater efficiency and stability of inorganic-N removal than single isolates, and these strains preferred to remove NH4+-N by simultaneous HNAD in mixed N-source media. Meanwhile, 45%-70% of NH4+-N and/or NO2--N was mainly converted to organic nitrogen (15%-25%) and gaseous nitrogen (30%-40%) by these strains, and more inorganic-N was transformed to intracellular-N by MCH and MAH via assimilation instead of gaseous-N production by denitrification. Both isolates and their consortia had the maximal NH4+-N or NO2--N removal efficiency above 95% under the optimum conditions including temperature of 20-30 °C, C/N ratios of 15-20, and sucrose as carbon source. Interestingly, bacterial consortia performed greater nitrogen removal than single isolates under the low temperature of 10 °C or C/N ratios of 2-5. In real mariculture wastewater, MCH and MAH also showed higher NH4+-N removal efficiency (65%-68%) and more stable cell quantity (4.2-5.2 × 108 CFU/mL) than single strains, due to the interspecific coexistence detected by bacterial quantitation with indirect immunoassay. Additionally, these isolates and consortia had stronger resistances to polypeptides, tetracyclines, sulfonamides, furanes, and macrolides than other antibiotics. These findings will be conducive to the applications of HNAD bacteria of Marinomonas and Halomonas on reducing nitrogen pollution in mariculture or other saline environments.

Effects of biogenic amines on the immune response and immunoregulation mechanism in hemocytes of Litopenaeus vannamei in vitro.

The effects of three biogenic amines (DA, NE and 5-HT) on the immune signaling pathway and immune response of hemocytes in shrimp were investigated through in vitro experiments. The results showed that the G protein effectors (AC, PLC), the second messengers (cAMP, DAG), Calmodulin (CaM) and protein kinases (PKA, PKC) of DA and NE groups shared a similar trend in which all intracellular signaling factors increased significantly and reached the maximum at 3 h. The concentrations of AC, cAMP and PKA in 5-HT groups decreased significantly compared with the control group, while the concentrations of PLC, CaM, DAG and PKC in 5-HT groups increased markedly. The immune parameters such as total hemocyte count (THC), cell viability, antibacterial activity and bacteriolytic activity, as well as prophenoloxidase (proPO) activity in three biogenic amines groups decreased significantly, while the phenoloxidase (PO) activity increased significantly. The phagocytic activity in DA and NE groups decreased significantly, while that in 5-HT groups increased markedly and reached the highest level at 1 h. Among these three biogenic amines, DA showed the strongest effect on the immune activity of the hemocytes, whereas 5-HT had the least effect. In addition, we speculated that DA and NE might regulate phagocytosis by activating intracellular AC-cAMP-PKA pathway while 5-HT might inhibit intracellular AC-cAMP-PKA pathway. Moreover, the activation of proPO system might be related to PLC-DAG-PKC and PLC-CaM pathway.

Temporal transcriptome analysis in female scallop Chlamys farreri: First molecular insights into the disturbing mechanism on lipid metabolism of reproductive-stage dependence under benzo[a]pyrene exposure.

Polycyclic aromatic hydrocarbons (PAHs) are one of the most widespread persistent organic pollutants (POPs) in marine environment. Benzo[a]pyrene (B[a]P), the most toxic carcinogen of PAHs, is widely studied as a representative that interferes with lipid metabolism. However, the underlying molecular mechanisms of lipid metabolism by B[a]P interference towards bivalve, one of the marine-pollution bio-indicators have not been elucidated yet, especially during gonadal development which is closely associated with lipids. In this study, female scallops Chlamys farreri were cultured with natural and 4 µg/L B[a]P exposed seawater, respectively, and a multi-stage (proliferative, growth, mature, and spawn stage) ovarian transcriptome profiling was performed to decipher the reproductive stage-dependence disturbing mechanisms on lipid metabolism caused by B[a]P in bivalves. The results revealed the potential molecular mechanism of B[a]P-induced triglycerides (TGs) accumulation, which probably resulted from the collaboration of promoting synthesis and inhibiting metabolization of TGs, notably, this mechanism also occurred at spawn stage. Correspondingly, B[a]P and TGs contents measured in ovary offered direct biochemical evidences for the interference effects and stage-dependent accumulation patterns of B[a]P. Moreover, the gene expressions of fatty acids synthesis related enzymes were down-regulated cooperatively, illustrating the molecular compensatory mechanism that reduced susceptibility from oxidative damage. And these results further emphasized the important role of prostaglandins (PGs) in immune response mediated by arachidonic acid metabolism. In addition, this study explored the underlying molecular mechanism affected by B[a]P on sterol metabolism, which possibly posed a threat to normal reproductive functions in bivalves. Taken together, our findings filled the gap of the stage-dependent interference molecular mechanisms on lipid metabolism behind bivalves, and provided a new perspective for investigating the adaptive mechanisms of bivalves under POPs stress.

Biomonitoring of polycyclic aromatic hydrocarbons (PAHs) from Manila clam Ruditapes philippinarum in Laizhou, Rushan and Jiaozhou, bays of China, and investigation of its relationship with human carcinogenic risk.

This study examined the marine environment and seafood safety using chemical monitoring and multiple biomarkers. Samples were collected from three bays on the Shandong Peninsula in China, Laizhou, Rushan and Jiaozhou, in March, May, August, and October of 2018 and 2019. The polycyclic aromatic hydrocarbon (PAH) concentrations in sediments and tissue samples from the clam Ruditapes philippinarum and multiple biomarkers were measured. All the sampling sites were found to be medium-PAH-contaminated areas (100-1000 ng/g d.w.). According to the correlation analysis, ethoxyresorufin-o-deethylase (EROD) and superoxide dismutase (SOD) activity in the clam's digestive gland were sensitive to PAHs (p < .05), but the incremental lifetime cancer risk (ILCR) was lower than the priority risk level (10-4) at most sampling sites. EROD, SOD and acetylcholinesterase activity exhibited significant correlations with the ILCR values (p < .01), suggesting that they may serve as good indicators for assessing safe seafood consumption levels for human beings.

Phosphorus and nitrogen removal by a novel phosphate-accumulating organism, Arthrobacter sp. HHEP5 capable of heterotrophic nitrification-aerobic denitrification: Safety assessment, removal characterization, mechanism exploration and wastewater treatment.

A novel phosphate-accumulating organism (PAO), Arthrobacter sp. HHEP5 was isolated from mariculture effluents. It produced no hemolysin and was susceptible to most antibiotics. It had removal efficiencies of above 99% for 1-10 mg/L phosphorus at 18-28 °C, pH 5.5-8.5, salinities 0-3%, C/N ratios 5-20, P/N ratios 0.1-0.2 and 20-260 rpm. It exhibited simultaneous aerobic phosphorus removal, nitrification and denitrification with the highest ammonium, nitrite, nitrate removal efficiencies of 99.87%, 100%, 99.37%. Phosphorus removal was accomplished by assimilating phosphate with the existence of polyphosphate kinase completely under aerobic condition. Genes involved in nitrogen removal were amplified. 99% of phosphorus and 95% of nitrogen in both mariculture and domestic wastewater were removed by HHEP5. This study provided sound methods for future screening of PAOs and new perspectives for renewed cognition of phosphorus removal process. Wide adaptation and remarkably aerobic phosphorus, nitrogen removal performances would make HHEP5 a promising candidate in wastewater treatment.

Benzo[a]pyrene exposure induced reproductive endocrine-disrupting effects via the steroidogenic pathway and estrogen signaling pathway in female scallop Chlamys farreri.

Benzo[a]pyrene (B[a]P), as one of the typical polycyclic aromatic hydrocarbons and environmental contaminants, may cause endocrine disrupting effects and reproductive impairments in bivalves. However, the molecular mechanisms are still not fully understood. In this study, three reproductive stages (proliferative stage, growing stage and mature stage) of female scallops Chlamys farreri were exposed to B[a]P at 0, 0.38 and 3.8 µg/L. The present study determined the adverse effects of B[a]P on gonadosomatic index, circulating hormone concentrations, endocrine-associated gene expression and ovarian histology. Significant decrease in sex hormones including progesterone (P), testosterone (T) and 17ß-estradiol (E2), was observed in B[a]P-treated C. farreri at growing stage and mature stage. These effects were associated with down-regulated expression of steroidogenic enzymes, including 3ß-HSD, CYP17 and 17ß-HSD, which were regulated by the upstream adenylate cyclase (Adcy) - protein kinase (PKA) signaling pathway. Ovarian transcript levels of estrogen receptor (ER) and caveolin-1 (cav-1) were decreased in B[a]P-treated C. farreri. Vitellogenin (Vtg), an estrogen-mediated gene involved in ovarian development, was down-regulated by B[a]P. Furthermore, ovarian histology was investigated to clarify the impairment of B[a]P on ovaries at growing stage and mature stage. Overall, the present results elucidated the anti-estrogenic mechanisms along the steroidogenic pathway and estrogen signaling pathway for the stage-dependent endocrine-disrupting effects of B[a]P. This finding provides important information regarding to the underlying molecular mechanisms of B[a]P-induced endocrine disruption in different reproductive stages of bivalves. In addition, the adverse effects should be taken into concertation during protection of bivalves germplasm resources and comprehensive evaluation of ecological risks.

Effects of feeding level and C/N ratio on water quality, growth performance, immune and antioxidant status of Litopenaeus vannamei in zero -water exchange bioflocs-based outdoor soil culture ponds.

Two trials were conducted as follows: the first trial was a 90-day experiment to determine the effects of reducing feeding level on shrimp status; the second trial (90 days) is based on the first trial to explore the suitable C/N ratio of biological flocs for shrimp culture in outdoor soil ponds. Results showed that the BFV levels increased gradually and then tended to be stable in the treatment groups. Concentrations of TAN and NO2-N were maintained low level in each treatment pond during experimental period. The final body weight, biology body length and yield of the shrimp in each trial with no significantly different (P > 0.05) while food coefficient and THC of the shrimp in 70% feeding level and C/N12 treatment were slightly lower than in the 100% feeding level and C/N16 treatment respectively (p < 0.05). The antibacterial activity and bacteriolytic activity in C/N16 treatment group were higher than in C/N12 (p < 0.05), while there were no significant difference between the two feeding levels (70%,100%) (P > 0.05). The shrimp in 70% feeding level and C/N12 treatment had the higher T-AOC in both the plasma and the hepatopancreas when compared with 100% feeding level and C/N16 treatment group (p < 0.05). The SOD activity of plasma in 70% treatment group was higher than in 100% (p < 0.05), while it was no significant difference between the two C/N ratios (12,16) in both the plasma and the hepatopancreas (P > 0.05). The effects of two feeding levels and C/N ratios on the GSH level and the ratio of GSH/GSSG in the plasma and the hepatopancreas of shrimp showed no significant difference (P > 0.05). The results showed that 70% feeding level and C/N12 ratio could provide adequate nutrition for shrimp to maintain a normal physical health status with the presence of bioflocs.

Evidence that dopamine is involved in neuroendocrine regulation, gill intracellular signaling pathways and ion regulation in Litopenaeus vannamei.

The transport of ions and ammonia in gills may be regulated by neuroendocrine factors. In order to explore the mechanism of dopamine (DA) regulation, we investigated hemolymph neuroendocrine hormones, gill intracellular signaling pathways, ion and ammonia transporters, hemolymph osmolality and ammonia concentration in Litopenaeus vannamei after injection of 10-7 and 10-6 mol DA per shrimp. The data showed a significant increase in crustacean hyperglycemic hormone (CHH) concentration at 1-12 h and a transient significant decrease in corticotrophin-releasing hormone (CRH), adrenocorticotropic hormone (ACTH) and cortisol concentration under DA stimulation. The up-regulation of guanylyl cyclase (GC) mRNA, cyclic guanosine monophosphate (cGMP) and protein kinase G (PKG) concentration, together with the down-regulation of DA receptor D4 mRNA and up-regulation of cyclic adenosine monophosphate (cAMP), protein kinase A (PKA), diacylglycerol (DAG) and protein kinase C (PKC) concentration suggested the activation of complicated intracellular signaling pathways. The expression of cAMP response element-binding protein (CREB), FXYD2 and 14-3-3 protein mRNA was significantly increased by PKA regulation. The increase in Na+/K+-ATPase (NKA) activity and the stabilization of V-type H+-ATPase (V-rATPase) activity were accompanied by an up-regulation of K+ channel, Na+/K+/2Cl- cotransporter (NKCC), Rh protein and vesicle associated membrane protein (VAMP) mRNA, resulting in an increase in hemolymph osmolality and a decrease in hemolymph ammonia concentration. These results suggest that DA stimulates the secretion of CHH and inhibits the release of cortisol, which activates intracellular signaling factors to facilitate ion and ammonia transport across the gills, and may not affect intracellular acidification.