Source-specific ecological and health risks of polycyclic aromatic hydrocarbons in the adjacent coastal area of the Yellow River Estuary, China.

Industrialization and urbanization have led to increasing levels of PAH pollution in highly urbanized estuaries and their adjacent coastal areas globally. This study focused on the adjacent coastal area of the Yellow River Estuary (YRE) and collected surface seawater, surface sediment, and clams Ruditapes philippinarum and Mactra veneriformis at four sites (S1 to S4) in May, August, and October 2021 to analyze the source-specific ecological and health risks and bioeffects. The findings revealed that the main sources of PAHs were traffic emission (25.2% to 28.5%), petroleum sources (23.3% to 29.5%), coal combustion (24.7% to 27.5%), and biomass combustion (19.8% to 20.7%). Further, the PMF-RQ and PMF-ILCR analyses indicated that traffic emission was the primary contributor to ecological risks in seawater and health risks in both clam species, while coal combustion was the major contributor in sediment. Taken together, it is recommended to implement control strategies for PAH pollution following the priority order: traffic > coal > petroleum > biomass, to reduce the content and risk of PAHs in the YRE.

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.

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.

Effects of dietary trans-cinnamaldehyde on growth performance, lipid metabolism, immune response and intestinal microbiota of Litopenaeus vannamei.

The purpose of this study was to evaluate the effects of dietary trans-cinnamaldehyde (TC) on growth performance, lipid metabolism, immune response and intestinal microbiota of Litopenaeus vannamei. Shrimp were randomly divided into 4 groups, with 3 replicants in each group and 70 shrimp in each replicant. The contents of TC in the four groups were 0, 0.4, 0.8 and 1.2 g kg-1, respectively. Samples were taken after 56 days, followed by a 7-day vibrio harveyi challenge experiment. The results showed that TC significantly improved the growth performance by enhancing the activity of digestive enzymes in shrimp (P < 0.05). TC also reduced the content of crude fat (P < 0.05). The addition of TC to the diet attenuated lipid deposition, as evidenced by a reduction in the content of crude fat and a decrease in plasma levels of cholesterol and triglycerides (P < 0.05). The expression of key genes for fatty acid and triglycerides synthesis were significantly down-regulated and key genes for fatty acid ß-oxidation were significantly up-regulated (P < 0.05). In addition, the immune response and antioxidant capability of shrimp were significantly enhanced by the addition of TC to the diet (P < 0.05). Meanwhile, TC could improve intestinal health by increasing the abundance of beneficial bacteria and decreasing the abundance of pathogenic bacteria, but had no significant effect on alpha diversity and beta diversity (P > 0.05). In addition, the results of histopathological sections and plasma transaminase studies showed that TC could improve the health status of hepatopancreas and was a safe nutritional supplement. After the 7-day Vibrio harveyi challenge, the cumulative mortality of shrimp decreased with increasing levels of dietary TC compared with control group (P < 0.05). These results suggested that TC could be used as a nutritional supplement for shrimp to enhance disease resistance and reduce lipid accumulation.

Process optimization for fermented siwu decoction by multi-index-response surface method and exploration of the effects of fermented siwu decoction on the growth, immune response and resistance to Vibrio harveyi of Pacific white shrimp (Litopenaeus vannamei).

The purpose of this study was to explore the optimal fermentation technology of Chinese herbal medicine formula-Siwu Decoction and the effects of fermented Siwu Decoction (FSW) on the growth performance, immune response, intestinal microflora and anti microbial ability of Litopenaeus vannamei. Response to surface methodology (RSM) was used to optimize the fermentation process of Siwu Decoction. The optimal fermentation conditions were obtained as follows: inoculation amount of mixed strains was 4.5%, fermentation time was 36 h, and the ratio of material to liquid was 20%. A total of 1260 shrimps were selected and divided into seven groups, three in parallel in each group. The dietary level of each group was as follows: Control （No additions）, USW1 (0.2% unfermented herbal medicine), USW2 (0.5% unfermented herbal medicine), USW3 (0.8% unfermented herbal medicine), FSW1 (0.2% fermented herbal medicine), FSW2 (0.5% fermented herbal medicine), FSW3 (0.8% fermented herbal medicine). The immune response and antioxidant defense ability of hemocytes and intestine were measured at 21 and 42 days of feeding and the intestinal flora and growth performance were measured at 42 days of feeding, after that, a 7-day challenge test against Vibrio harveyi was conducted. The results showed that fermented Siwu Decoction significantly improved the growth performance and body composition of Litopenaeus vannamei; significantly increased the total number of hemocytes, phagocytic activity, antibacterial activity and bacteriolytic activity of Litopenaeus vannamei, and improved the antioxidant activity of Litopenaeus vannamei; the addition of fermented Siwu Decoction significantly increased the gene expression level of hemocytes and intestinal tract of Litopenaeus vannamei, and improved the antioxidant activity of Litopenaeus vannamei. The abundance of Bacillus increased, while the abundance of Vibrio decreased. After Vibrio harveyi challenge, the cumulative mortality of FSW group was significantly lower than that of control group. Fermented Siwu Decoction may be a potential physiological enhancer in aquaculture, and can be widely used in aquaculture.

Effect of dietary Xiao-Chaihu-Decoction on growth performance, immune response, detoxification and intestinal microbiota of pacific white shrimp (Litopenaeus vannamei).

Xiao-Chaihu-Decoction (XCHD), a classical traditional Chinese medicine with diverse biological activities, is widely applied to prevent and treat many human diseases. Effects of dietary XCHD on growth performance, immune response, detoxification system, intestinal microbiota and resistance against aflatoxin B1(AFB1) of Litopenaeus vannamei was studied. Four isonitrogenous and isolipidic diets were formulated to contain 0, 1, 2, and 5 g/kg (control, XCHD1, XCHD2 and XCHD3) of XCHD, respectively. Seven hundred and eighty shrimp (1.16 ± 0.09 g) were assigned randomly to 12 tanks (400 L, three tanks each group, 65 shrimp in each tank) for 6 weeks. After sampling, 25 shrimp from each tank were selected for a 2-week AFB1 (2500 µg/kg) challenge experiment. The results indicated that the final weight, weight gain and specific growth rate in XCHD2 and XCHD3 groups were significantly increased compared to control. The protease, amylase, superoxide dismutase (SOD), glutathione s-transferase (GST), sulfotransferase (SULT) activities, total antioxidant capacity (T-AOC) and glutathione (GSH) contents in hepatopancreas were significantly increased in XCHD3 groups and the expressions of immune-related genes (Toll, Dorsal and Cru) in hepatopancreas were significantly up-regulated in XCHD2 and XCHD3 groups. High-throughput sequencing analysis revealed that the abundance of Proteobacteria decreased and the abundances of Bacteroidetes increased in XCHD2 and XCHD3 groups. Additionally, AFB1 challenge experiments showed that AFB1 caused histological damage to the hepatopancreas and significantly increased the levels of malondialdehyde (MDA) and protein carbonylation (PC) in hepatopancreas as well as the activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST). Nevertheless, XCHD could effectively alleviated the growth toxicity, immunosuppression and macromolecular damage caused by AFB1 to shrimp by inhibiting the Phase I enzyme and enhancing Phase II enzyme and antioxidant system.

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.

In vitro and in vivo immunologic potentiation of herb extracts on shrimp (Litopenaeus vannamei).

In vitro and in vivo effects of Astragalus polysaccharide (APS), chlorogenic acid (CGA) and berberine (BBR) on shrimp (Litopenaeus vannamei) were studied. In vitro test showed that the combination of APS and BBR and the combination of APS and CGA have strong immune enhancement effects and no lysosomal membrane damage on hemocyte. Then, feeding experiment was proceeded to optimize the concentrations of compound herbal extracts. Four diets containing G1-G4(0.5 g kg -1 APS + 0.5 g kg -1 BBR, 1.0 g kg -1 APS +1.0 g kg -1 BBR, 0.5 g kg -1 APS +0.5 g kg -1 CGA, 1.0 g kg -1 APS + 1.0 g kg -1 CGA) associated with the control group (common diet) were compared and determined their biomolecule damage to hepatopancreas including DNA damage, lipid peroxidation and protein carbonyl. The results indicated that G3 (0.5 g kg -1 APS +0.5 g kg -1 CGA) showed higher total hemocyte counts, phagocytic activities, antibacterial activities and bacteriolytic activities during 6 days feeding, and without biomolecule damages after 6 days post-withdrawal. Therefore, the appropriate immunostimulants formula in this study was the combination of 0.5 g kg -1 APS and 0.5 g kg-1 CGA, which was used for 6 days followed by 6 days post-withdrawal. Additionally, our study provides new support for screening composite immunostimulants formula by using primary shrimp hemocyte culture.

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.

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.

The role of Nrf2-Keap1 signaling pathway in the antioxidant defense response induced by PAHs in the calm Ruditapes philippinarum.

The NF-E2-related factor 2 (Nrf2) is a master regulator of cellular responses against environmental stresses. In this study we cloned the full-length cDNAs of the RpNrf2 encompassed 2823 bp from the clam Ruditapes philippinarum (R. philippinarum). Sequences alignment and phylogenetic analysis showed that Nrf2 was highly specific in the clams. RpNrf2 expression was detected in gill, digestive gland, mantle and adductor, which the highest transcription level was observed in gill and digestive gland. The gene expressions of RpNrf2, Kelch-like-ECH-associated Protein 1 (Keap1), Cul3-based E3 Ubiquitin Ligase (E3), Glutathione S-transferase (GST-pi), Superoxide Dismutase (SOD), Catalase (CAT) and Glutathione Peroxidase (GPx) in digestive gland was evaluated by real-time PCR after being exposed to benzo(a)pyrene (BaP) (0.25, 1and 4 µg/L) for 15 days, which showed that the expression of Nrf2 significantly increased at day 1 and day 6 after exposure (p < 0.05), and there was a negative relationship between the mRNA levels of Nrf2 and Keap1 that indicates the enhancement of Keap1 expression stimulating Nrf2 degradation. RNA interference experiments were conducted to examine the expression profiles of RpNrf2, antioxidant and detoxification genes (GST-pi, Cu/Zn-SOD, CAT and GPx) and Lipid Peroxidase (LPO) level in digestive gland exposed to BaP. The results showed that the mRNA level of Nrf2 was significantly decreased by 63.2%, and the changes of antioxidant and detoxification genes expression were consistent with the Nrf2 gene suggesting that Nrf2 is required for the induction of antioxidant and detoxification genes. Besides, the LPO levels expressed by malondialdehyde (MDA) contents were significant higher compared with the control group at 72 h post dsRNA-Nrf2 injection. In conclusion, our data demonstrated that Keap1 can sense nucleophilic or oxidative stress factors to regulate the Nrf2 signaling pathway together with E3 and Nrf2 signaling pathway plays an important role in modulating gene expression of antioxidant enzymes in bivalve mollusks.

Molecular cloning and characterization of estrogen receptor gene in the scallop Chlamys farreri: expression profiles in response to endocrine disrupting chemicals.

In order to gain insights into the mechanism of sex steroid signaling in molluscs, the full-length cDNA of estrogen receptor (ER) was isolated and characterized from Chlamys farreri for the first time. The positions of cysteine residues and other residues around them that constitute the two zinc finger motifs and the P-box are conserved. Phylogenetic analysis revealed that the CfER is an ortholog of the other mollusk ERs. Tissue distribution analysis of the CfER mRNA revealed that the expression of ER mRNA was observed in various tissues, and highest in the gonad of males and females. C. farreri were exposed for 10 days to endocrine disrupting chemicals including Benzo(a)pyrene (B(a)p) and polybrominated diphenyl ethers (BDE-47). B(a)p exposure at 0.4 and 2 µg/L caused significant increase in mRNA expression of ER and VTG, but B(a)p at 10 µg/L down-regulated CfER and VTG mRNA expression compared to control. Varying increase of ER and VTG mRNA transcripts was resulted in by BDE-47 at 0.1, 1 and 10 µg/L. These results elucidate potential roles of CfER induced by xenobiotics in C. farreri and can be helpful for investigating the mechanism of sex steroid signaling in bivalve mollusks.