c-Jun N-terminal kinase (JNK) in Procambarus clarkii: Molecular characterization and involvement in oxidative stress-induced apoptosis during molting cycle.

The present study was conducted to characterize the full-length cDNA of c-Jun N-terminal kinase (JNK) in Procambarus clarkii (Pcjnk) and evaluate its potential function under different molt cycle. The full-length cDNA of Pcjnk covered 2937 bp with an open reading frame of 1320 bp, encoding 439 amino acids. A typical conserved TPY motif (118Thr-Pro-120Tyr) was found in Pcjnk. Quantitative real-time PCR (qRT-PCR) analysis revealed a constitutive expression of Pcjnk in the tested tissue, with the highest expression occurring in the hepatopancreas. Additionally, the present study initially revealed that relative mRNA expression of Pcjnk and apoptosis level were significantly higher in the premolt stage (D1/D2 and D3/D4 stage) as compared to other molt stages. In contrast to the levels of superoxide dismutase (SOD) and malondialdehyde (MDA), catalase (CAT) and glutathione peroxidase (GPX) level decreased significantly from the intermolt stage (C stage) to the premolt stage (D1/D2 and D3/D4 stage), then increased from the premolt stage to the postmolt stage (A and B stage). The results obtained in the present study indicated that molt could cause apoptosis induced by oxidative stress through the activation of JNK in Procambarus clarkii.

Analysis of the population structure and genetic diversity of the red swamp crayfish (Procambarus clarkii) in China using SSR markers

BACKGROUND: Procambarus clarkii produces high-quality, delicious meat that is high in protein, low in fat, and rich in calcium and phosphorus. It has become an important aquatic resource in China. Our objectives are (i) to analyze the level of genetic diversity of P. clarkii populations; (ii) to explore the genetic differentiation (Gst); and (iii) to propose appropriate strategies for the conservation. RESULTS: In this study, Shannon's index (I) and Nei's gene diversity index (H) for P. clarkii were high (I = 0.3462 and H = 0.2325 on average and I = 0.6264, H = 0.4377 at the species level) based on the SSR markers. The expected heterozygosity value of 17 microsatellite loci in 25 crayfish populations was 0.9317, the observed heterozygosity value was 0.9121, and the observed number of alleles per locus was 2.000; and the effective number of alleles per locus was 1.8075. Among the P. clarkii populations, the inbreeding coefficient within populations (Fis) was 0.2315, overall inbreeding coefficient (Fit) was 0.4438, genetic differentiation coefficient among populations (Fst) was 0.3145 and gene differentiation (Gst) was 0.4785 based on SSR analyses. The cluster analysis results obtained by unweighted pair-group method with arithmetic mean (UPGMA) analysis, principal coordinate analysis (PCoA) and STRUCTURE analysis were similar. A mantel test showed that the isolation-by-distance pattern was not significant. CONCLUSIONS: The high Gst among P. clarkii populations is attributed to genetic drift and geographic isolation. The results indicated that more P. clarkii populations should be collected when formulating conservation and aquaculture strategies.

The feeding of dietary Codonopsis pilosula polysaccharide enhances the immune responses, the expression of immune-related genes and the growth performance of red swamp crayfish (Procambarus clarkii).

Polysaccharides have many functions in aquatic animals and are widely used as immunopotentiators. However, despite the emergence of serious diseases, few studies have explored the effects of Codonopsis pilosula polysaccharide (CPP) on crustaceans. We studied the effects of CPP on the growth performance, nonspecific immunity, antioxidant activity and disease resistance of red swamp crayfish (Procambarus clarkii). Healthy crayfish (5.80 ± 0.1 g) were fed diets supplemented with 0% (control), 0.05%, 0.1%, 0.15%, 0.20%, and 0.30% CPP for 8 weeks. At the end of the 8-week feeding trial, the optimal final body weight (FBW), weight gain (WG), specific growth rate (SGR), and feed conversion ratio (FCR) were observed in the crayfish fed the diets with 0.15% and 0.20% CPP, followed by those fed the diet with 0.30% CPP and then those fed the diet with 0.10% CPP, whereas the values of these parameters were obtained with the control crayfish (P < 0.05). The crayfish fed the diets with 0.15% and 0.20% CPP exhibited a significantly higher total hemocyte count (THC) and significantly increased phenoloxidase (PO), lysozyme (LZM), hemocyte (Hc), acid phosphatase (ACP) and alkaline phosphatase (AKP) compared with those belonging to the other groups (P < 0.05). The crayfish fed the diets with 0.15% and 0.2% CPP exhibited significantly higher total superoxide dismutase (T-SOD) and glutathione peroxidase (GPx) activities, a significantly increased total antioxidant capacity (T-AOC) and a significantly lower malondialdehyde (MDA) content compared with the other groups (P < 0.05), which indicated that antioxidant capacity was significantly induced by the CPP-supplemented diets. Significantly upregulated expression of immune-related genes (anti-lipopolysaccharide factors (alf), peroxiredoxin (prx5), cathepsin B (ctsb), mitochondrial manganese superoxide dismutase (mtMnsod), cyclophilin A (cypa), glutathione peroxidase (gpx), Toll-like receptor 3 (tlr3), and heat shock protein 70 (hsp70)) was detected in the crayfish fed the diets supplemented with 0.15% and 0.20% CPP diet compared with the levels observed in the control crayfish. These results showed that dietary CPP supplementation greatly improved the growth, immunity and antioxidant capacities of crayfish, and according to the observed results, 0.15%-0.2% is the recommended optimal level of CPP dietary supplementation for crayfish.

Effects of hesperidin on the growth performance, antioxidant capacity, immune responses and disease resistance of red swamp crayfish (Procambarus clarkii).

We evaluated the effects of hesperidin on the nonspecific immunity, antioxidant capacity and growth performance of red swamp crayfish (Procambarus clarkii). A total of 900 healthy crayfish were randomly divided into six groups: the control group (fed the basal diet) and the HES25, HES50, HES75, HES100 and HES150 groups, which were fed the basal diet supplemented with 25, 50, 75, 100 and 150 mg kg-1 hesperidin, respectively. The feeding experiment lasted 8 weeks. The results indicated that compared with the control group, the crayfish groups supplemented with 50-150 mg kg-1 hesperidin had a decreased feed conversion ratio (FCR) and increased final body weight (FBW), specific growth rate (SGR) and weight gain (WG) (P < 0.05). The protein carbonyl content (PCC), reactive oxygen species (ROS) level and malondialdehyde (MDA) level in the hepatopancreas and hemocytes were significantly lower, while the total antioxidant capacity (T-AOC), glutathione peroxidase (GPx) activity, and superoxide dismutase (SOD) activity were significantly higher in the crayfish groups supplemented with 50-150 mg kg-1 hesperidin than in the control group. Supplementation with 50-150 mg kg-1 hesperidin significantly increased the activities of acid phosphatase (ACP), alkaline phosphatase (AKP), lysozyme (LZM), and phenoloxidase (PO) compared with the control group (P < 0.05); upregulated the mRNA expression of cyclophilin A (CypA), extracellular copper-zinc superoxide dismutase (ecCuZnSOD), GPxs, crustin, astacidin, Toll3 and heat shock protein 70 (HSP70) (P < 0.05); and decreased crayfish mortality following white spot syndrome virus (WSSV) infection. These findings indicate that dietary hesperidin supplementation at an optimum dose of 50-150 mg kg-1 may effectively improve nonspecific immunity, antioxidant capacity and growth performance in crayfish.

Effects of various feeding patterns of Bacillus coagulans on growth performance, antioxidant response and Nrf2-Keap1 signaling pathway in juvenile gibel carp (Carassius auratus gibelio).

The present study was conducted to evaluate the effects of various Bacillus coagulans feeding patterns on growth, antioxidant parameter and Nrf2 pathway in juvenile gibel carp. The similar size of gibel carp (initial weight: 14.33 ± 0.15 g) were subjected to three levels of B. coagulans supplementation (0, 500, and 1000 mg/kg) and two feeding modes (supplementing B. coagulans continuously or for two days of B. coagulans after 5 days of a basal diet) according to a 3 × 2 factorial design. The fish that were continuously fed 500 mg/kg B. coagulans (P2) and those fed the first basal diet for 5 days followed by 500 mg/kg or 1000 mg/kg B.coagulans for 2 days (P4 or P5) showed higher weight gain rate and specific growth rate than the other groups. Blood respiratory burst (RB), myeloperoxidase (MPO), and anti-superoxide anion free radical (AFASER) activities in the P4 group were higher than those of the control. White blood cell count (WBC), RB activity, MPO activity, and glutathione (GSH) content in the P5 group were also higher than those of the control. A similar higher trend was observed in the gene expressions of NADPH oxidase 2 (NOX2), NFE2-related factor (Nrf2), Kelch-like-ECH-associated protein(Keap1) in the P4 and NOX2, NRF2, CNC homolog 1 (Bach1), peroxiredoxin 2 (Prx2) in the P5 group compared with the control. Additionally, we observed a significantly lower level of plasma aspartate aminotransferase (AST), lower activity of alanine aminotransferase (ALT), a higher level of MPO, higher GPX activity, and increased NRF2 and Prx2 expression were all observed in the P2 treatment group compared with the control. Furthermore, the malondialdehyde (MDA) content in the P2, P3, and P4 groups was lower than that of the control. These results indicate that a diet supplemented with appropriate levels of B.coagulans could improve the growth, immune response, and antioxidant capability of gibel carp. We concluded that the pattern of two days of 500 or 1000 mg/kg B. coagulans after 5 days of a basal diet was recommended for gibel carp.

Effects of astaxanthin and emodin on the growth, stress resistance and disease resistance of yellow catfish (Pelteobagrus fulvidraco).

Yellow catfish (Pelteobagrus fulvidraco) has become a commercially important fish species in China and eastern Asia. High-density aquaculture has led to congestion and excessive stress and contributed to bacterial infection outbreaks that have caused high mortality. We investigated the effects of dietary supplementation with astaxanthin and emodin alone and in combination on the growth and stress resistance of yellow catfish. After 60 days of feeding, each group of fish (control, astaxanthin, emodin, and astaxanthin plus emodin (combination) groups) was exposed to acute crowding stress for 24 h, and a subsample of fish from the four groups was challenged with the bacterial septicemia pathogen Proteus mirabilis after the end of the crowding stress experiment. Compared with the control, the astaxanthin and emodin groups showed increases in serum total protein (TP), hepatic superoxide dismutase (SOD) activity and hepatic heat shock proteins 70 (HSP70) mRNA levels at 12 and 24 h after the initiation of crowding stress. The combination group exhibited increases in alanine aminotransferase (ALT) activity, aspartate aminotransferase (AST) activity, serum TP, hepatic SOD activity and hepatic HSP70 mRNA levels within 24 h after the initiation of crowding stress. However, decreases relative to the control were observed in the serum cortisol and glucose contents in the three treatment groups at 12 and 24 h after the initiation of crowding stress, in ALT and AST activity in the astaxanthin and emodin group at 24 h after the initiation of crowding stress, and in the serum lysozyme activity, serum alkaline phosphatase (ALP) activity, and hepatic catalase (CAT) and malondialdehyde (MDA) activity in the combination group at 24 h after the initiation of crowding stress. Additionally, the cumulative mortality after P. mirabilis infection was lower in all three treatment groups (57.00%-70.33%) than in the control (77.67%). Dietary supplementation with astaxanthin and emodin decreased the specific growth rate (SGR) and weight gain (WG) of healthy yellow catfish, although significant differences in mortality were not observed. These results indicate that dietary supplementation with 80 mg/kg astaxanthin and 150 mg/kg emodin can improve the anti-oxidative capabilities, hepatic HSP70 levels, and resistance to acute crowding stress of yellow catfish. Finally, an appropriate strategy for enhance yellow catfish stress resistance and disease resistance is proposed.

The influence of various feeding patterns of emodin on growth, non-specific immune responses, and disease resistance to Aeromonas hydrophila in juvenile Wuchang bream (Megalobrama amblycephala).

The present study was conducted to evaluate the effect of various feeding patterns of emodin on growth, non-specific immune response, and disease resistance to Aeromonas hydrophila in juvenile Wuchang bream. Healthy Megalobrama amblycephala (initial weight: 3.47 ± 0.032) were grown in a circulating water system for 8 weeks. Five groups were studied: one control group was fed with a basal diet for eight weeks (Pattern 1, P1), and three treatment groups were fed with a trial diet of 30 mg emodin kg(-1) at one-week (Pattern 2, P2), two-week (Pattern 3, P3), four-week (Pattern 4, P4) intervals. The final treatment group maintained the trial diet for the entire eight-week study duration (Pattern 5, P5). Results indicated that different feeding patterns of emodin significantly influenced the weight gain rate of Wuchang bream (P < 0.05). Fish in the P4 treatment group had significantly higher rates of weight gain (WG) than those in other treatment groups. There were no significant differences in survival rates or feed conversion ratios (FCR) between treatment groups and the control group. White blood cell count (WBC), respiratory burst activity, superoxide dismutase (SOD) activity, myeloperoxidase (MPO) activity and tumor necrosis factor-α (TNF-α) activity were shown to increase at first and then decrease from P3 condition to P5 condition. Fish under P4 treatment showed the most significant improvement of all tested parameters compared to control. Significantly higher levels (P < 0.05) of plasma aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activity were observed in P2 and P4 treatment groups when compared with the control group, while no significant differences were observed in the AST and ALT activity of fish in P2, P3, P4 and P5 treatment groups. In a bacterial challenge experiment with A. hydrophila, fish under P4 and P5 treatment showed lower cumulative mortality than the control group. The results of this study suggest that an initial 4-week feeding interval is recommended for the economic and practical culture of M. amblycephala.

[Effects of the environmental hormone cypermethrin on the reproduction of Brachionus calyciflorus].

The toxic effects of the antiandrogen cypermethrin on B. calyciflorus were investigated using the 2 d population growth rate (r) of B. calyciflorus. The effects of low-dose of cypermethrin (0.001-0.316 mg x L(-1)) on the 3 d population parameters, the 7 d resting egg production and the hatching rate of resting eggs of B. calyciflorus were also studied. The 2 d population parameters of B. calyciflorus were used to estimate the growth ability of larvae hatched from the resting eggs formed in cypermethrin. The 3 d population parameters were used to estimate the effect on the the reproduction of the offspring of B. calyciflorus, whose parent generation was preexposed in cypermethrin. The results indicated that the logarithmic concentration of cypermethrin had a linear negative correlation with the population growth rate. The half maximal effective concentration (EC50), the lowest observed effect concentration (LOEC) and no-observed effect concentration (NOEC) of cypermethrin were 14.22 mg x L(-1), 10 mg x L(-1) and 3.16 mg x L(-1), respectively. The resting egg production was decreased by 41.23% at 0.031 6 mg x L(-1) cypermethrin. The hatching rate of resting eggs was significantly decreased when formed in 0.031 6 mg x L(-1) cypermethrin. The population growth rate and mictic rate were significantly decreased for the individuals hatched from the resting eggs formed in cypermethrin. The 3 d population growth rate was significantly decreased by 15.96% compared to the control when their parent generation was pre-exposed in 0.316 mg x L(-1) cypermethrin. The results showed that the 2 d population growth rate of B. calyciflorus was less sensitive to cypermethrin. Low doses of cypermethrin reduced the resting egg production, the hatching rate of resting eggs and the population growth of hatched resting eggs, which would thus decrease the contribution of the offspring to the population growth.