| The problem of high ammonia nitrogen often occurs in the process of aquaculture,especially in high-density aquaculture.High ammonia-N content showed toxic effects on fish or shrimp.It also reduces the growth rate or weakens immunity of animal.Moreover,the aquaculture effluent discharge will lead to environmental pollution.Therefore,research and development of healthy and ecological aquaculture model is an important guarantee for the sustainable development of aquaculture.In this study,the acute and chronic toxicity of ammonia-N to Macrobrachium rosenbergii were studied,and the mechanism of ammonia nitrogen detoxification of M.rosenbergii was analyzed by metabonomics method.The environmental effect of different culture patterns of M.rosenbergii was studied by enclosure experiment.1.Effects of short-term ammonia-N stress on the antioxidant enzymes,heat shock protein and apoptosis-related genes of M.rosenbergiiCurrent study explored the acute toxicity of ammonia-N to M.rosenbergii?3.46±0.66 g?,and the effects on oxidative stress and apoptosis in M.rosenbergii.Prawns were exposed to various concentrations of ammonia-N(0,0.5,1.88 mg·L-1)for 4 h?8h?12 h?24 h?48 h and 72 h.The activity of total superoxide dismutase?T-SOD?,catalase?CAT?,malondialdehyde?MDA?content were analyzed in muscle,haemocytes and gill of M.rosenbergii,and glutathione peroxidase?GSH-Px?in muscle was also measured.The gene expression of antioxidase?SOD,CAT,GPX?,heat shock protein?HSP60,HSP70,HSP90?and apoptosis?Caspase 2,Caspase 3,Bcl-2?in muscle and hepatopancreas were evaluated.The results showed that:?1?The half lethal concentrations of non-ionic ammonia of 24 h,48 h,72 h and 96h were 7.72,5.44,4.28 mg·L-1 and 3.78 mg·L-1 respectively.And the safe concentration was 0.378 mg·L-1.?2?The T-SOD activity was significantly increased in gill of M.rosenbergii after1.88 mg·L-1 ammonia-N expose for 8 h,and the CAT activity was also increased in gill after 1.88 mg·L-1 ammonia-N expose for 72 h.After 24 h ammonia-N expose at 0.5,1.88mg·L-1,the CAT activity was increased in haemocytes of M.rosenbergii.T-SOD activity also increased in haemocytes after 1.88 mg·L-1 ammonia-N expose for 24 h.The GSH-PX activity significantly increased in muscle after 0.5,1.88 mg·L-1 ammonia-N expose for 72 h.After 72 h ammonia-N expose,the MDA content in muscle,haemocytes and gill of M.rosenbergii in 1.88 mg·L-1 group was significantly higher than those in control and0.5 mg·L-1 group.?3?The relative mRNA expression of SOD was significantly up-regulated in muscle of M.rosenbergii after 1.88 mg·L-1 ammonia-N expose for 8 h and 24 h,respectively.And it was also significantly up-regulated in hepatopancreas after 1.88 mg·L-1 ammonia-N expose for 8 h and 48 h,respectively.The highest mRNA expression of CAT in muscle occurred at 3h in 1.88 mg·L-1 group,and it occurred at 8 h in hepatopancreas,and then both decreased.The highest mRNA expression of GPX in muscle and hepatopancreas occurred at 8 h in 1.88 mg·L-1 group.The mRNA expression of GPX hepatopancreas in was 23.86-fold and 12.21-fold in 1.88 mg·L-1 group at 8 h higher than that in the control and 0.5 mg L-1 group,respectively.?4?The relative mRNA expression of HSP60 in muscle was up—regulated at 3 h in0.5 mg·L-1 group,and that in muscle and hepatopancreas were up-regulated at 8 h,24 h and 72h in 1.88 mg·L-1 group.The relative mRNA expression of HSP70 in hepatopancreas was up—regulated at 8 h?24 h and 48 h in 1.88 mg·L-1 group,and it in muscle was up—regulated at 8 h.Meanwhile,it in hepatopancreas was up—regulated in0.5 mg·L-1 group.After 1.88 mg·L-1 ammonia-N expose,the relative mRNA expression of HSP90 in muscle was up—regulated at 8 h,12 h and 24 h,and it in hepatopancreas was up—regulated at 3 h and 72 h.After 0.5 mg·L-1 ammonia-N expose,the relative mRNA expression of HSP90 in muscle was up—regulated at 12 h,and it was also up-regulated in hepatopancreas at 72 h.?5?After 1.88 mg·L-1 ammonia-N expose,the highest mRNA expression of Caspase2 in muscle occurred at 3 h,and which in hepatopancreas at 8 h.The relative mRNA expression of Caspase 3 in muscle was up-regulated at 24 h and 48 h in 1.88 mg·L-1 group,respectively,and that in hepatopancreas was also up-regulated at 8 h and 24 h,respectively.The relative mRNA expression of BCL-2 in muscle increased significantly after the prawn treated with ammonia-N at 0.05 mg·L-1 for 3 h.However,there was no significant difference on the mRNA expression of BCL-2 in hepatopancreas after ammonia-N expose.Significant apoptosis was observed in hepatopancreatic of M.rosenbergii after ammonia-N expose at 1.88 mg·L-1 group for 72 h.2.Effects of ammonia-N stress and the post-exposure recovery on antioxidant enzymes,heat shock protein and apoptosis-related genes of M.rosenbergii juvenilesCurrent study explored the influence of ammonia-N stress and post-exposure recovery on the non-specific immune,heat shock proteins and apoptosis of M.rosenbergii.The prawns?0.16±0.06 g?were exposed to different concentrations of ammonia(0,0.43,0.64,0.85 mg·L-1 and 1.06 mg·L-1)for 48 h,and then post-exposure for 48 h.The activities of SOD,CAT,GSH-Px,MDA content were assessed in the muscle of M.rosenbergii,and the gene expression of antioxidase?SOD,CAT,GPX?,heat shock protein?HSP60,HSP70,HSP90?and apoptosis?Caspase 2,Caspase 3,Bcl-2?in muscle were also evaluated under ammonia stress for 48 h and post-exposure for 48 h.?1?The results showed that the half lethal concentrations of non-ionic ammonia of24 h,48 h,72 h and 96 h were 4.25,2.35,1.86 mg·L-1 and 1.54 mg·L-1 respectively.And the safe concentration was 0.154 mg·L-1 under the condition of?25±1??,pH 8.0±0.5.?2?The SOD activity in the muscle of M.rosenbergii significantly was raised by ammonia nitrogen at 48 h.However,compared to the control,the activities of CAT and GSH-Px in all ammonia treatments didn't change significantly at 48 h?P>0.05?.The MDA content of 0.43,0.64 and 1.06 mg·L-1 ammonia treatments were significantly higher than that of the control at 48 h?P<0.05?.After 48 h post-exposure recovery,compared to the ammonia exposure,SOD activity reduced significantly at the same ammonia treatment?P<0.05?,but they were still significantly higher than that of control group except for 0.85 mg·L-1?P<0.05?.CAT activities in 0.85 and1.06 mg·L-1 treatments reduced significantly at the same ammonia treatments after 48 h post-exposure recovery?P<0.05?,and MDA content of all treatments returned to the level of control.?3?The relative mRNA expression of CAT was significantly higher than that in the other groups after the prawn treated with ammonia-N at 1.06 mg·L-1 for 48 h?P<0.05?,and there was no significant difference between groups after 48 h post-exposure recovery.The relative mRNA expression of SOD was not significantly different after ammonia-N for 48 h,but it increased in 0.43 and 1.06 mg·L-1 groups after 48 h post-exposure recovery,which was higher than that in other groups.After ammonia-N at 0.85 mg·L-1 for 48 h,the relative mRNA expression of GPX was higher than that in the control,and there was no significant difference between groups after 48 h post-exposure recovery.?4?There was no significant difference on relative mRNA expression of HSP70 of M.rosenbergii after ammonia stress or recovery.After ammonia-N for 48 h,the relative mRNA expression HSP90 was significantly up-regulated.However,it decreased in all ammonia-N treats after 48 h post-exposure recovery,and lower than that in control.Except 0.43 mg·L-1 group,the relative mRNA expression HSP60 was also significantly up-regulated after ammonia stress for 48 h.It decreased in all ammonia-N treats after 48h post-exposure recovery,but no significant difference compares with control.?5?After ammonia-N for 48 h,the relative mRNA expression Caspase2 and Caspase3 were up-regulated compare with control.The relative mRNA expression Caspase3 decreased after 48 h post-exposure recovery,and had no significant difference with control.after 48 h post-exposure recovery,the relative mRNA expression Caspase2in 0.43 mg·L-1 group was still higher than that in control,which in the other groups returned to control level.3.Effects of long-term ammonia-N stress on growth,metabolic enzymes,antioxidant enzymes and metabolome of M.rosenbergiiCurrent study explored the influence of long-term ammonia-N stress the growth and metabolism of M.rosenbergii.Prawns?0.28±0.05 g?were exposed to different concentrations of ammonia-N(0,0.108,0.216,0.324 and 0.54 mg·L-1)for 20 d.The growth rate,survival rate and metabolism enzymes of M.rosenbergii were evaluated.The metabolome of M.rosenbergii was measured after ammonia-N treats at 0.108,0.324 and0.54 mg·L-1 for 20 d.?1?The results showed that the survival rate of M.rosenbergii significantly decreased after ammonia-N treat at 0.54 mg·L-1 compare to other groups?P<0.05?.The weight gain in 0.54 mg·L-1 decreased than that of control,but the difference was not significant between ammonia-N stress group and control group?P>0.05?.?2?The ACP,GS and GDH activities in hepatopancreas of M.rosenbergii decreased significantly after ammonia-N treats at 0.216?0.324 and 0.54 mg·L-1 compare with those in control.The AKP activity also decreased significantly after ammonia-N treats at 0.216mg·L-1.With the increase of ammonia-N concentration,the activity of GOT in hepatopancreas and muscle of M.rosenbergii increased gradually,but the difference was not significant between ammonia-N groups and control group.Ammonia-N has no significant effect on GPT activity.The urea nitrogen content in 0.216 mg·L-1 was higher than that in control group and 0.54 mg·L-1 group.?3?Ammonia-N has no significant effect on CAT activity.The highest SOD activity occurred at 0.324 mg·L-1 group,which was significantly higher than that in control group and 0.108 mg·L-1 group.The MDA content in 0.216 mg·L-11 group was the lowest,which was significantly lower than that in 0.108 mg·L-1 group.?4?Ammonia-N has significant effect on lipid,carbohydrate and protein metabolism of M.rosenbergii,such as purine metabolism,Amino sugar and nucleotide sugar metabolism,Alpha-Linolenic acid metabolism,arginine and proline metabolism,glutathione metabolism and phosphonate and phosphate metabolism,Terpenoid biosynthesis,lysine degradation and lysine biosynthesis pathways.In summary,the result of our study showed that high concentrations of ammonia nitrogen had a stress effect on M.rosenbergii,which significantly affected its survival rate and growth.In order to explore ways to reduce accumulation of ammonia nitrogen during the culture of M.rosenbergii,the environmental effect of different culture patterns of M.rosenbergii was also studied by enclosure experiment.Six treatments,with 4replicates each,were used,including prawn monoculture?MP?,prawn with aquatic plants?Lemna minor??PP?,prawn with silver carp Hypo phthalmichthys molitrix?PF?,prawn with mussels?Anodonta sp.?and H.molitrix?PMF?,prawn with Anodonta sp.and L.minor?PMP?and prawn with Anodonta sp.,L.minor and H.molitrix?PMPF?.The growth of M.rosenbergii,water quality,microbial communities in the intestine of M.rosenbergii were evaluated.4?Effects of different culture patterns on the growth of M.rosenbergii and water qualityGrowth rates of M.rosenbergii and physical,chemical and biological parameters were assessed every 10 d and on the last day of the 64 d experiment.Average weight gain in M.rosenbergii was highest in the PMPF group,although this was not statistically significant.There was also no significant difference in survival rate among treatments.Dissolved oxygen concentrations were significantly higher in the PMPF group,whereas total nitrogen and chlorophyll a concentrations were highest in the PF group.Compared to the MP group,stocking with H.molitrix?PF,PMF and PMPF?effectively reduced PO4-P.L.minor exhibited effective uptake of both N and P.In contrast to the group stocked only with H.molitrix?PF?,stocking with L.minor?PP and PMP groups?reduced phytoplankton biomass.In summary,culturing M.rosenbergii in a multitrophic PMPF system provides the potential for increased economic profits and ecological benefits.5?Effects of different culture patterns on microbial communities of intestine in M.rosenbergii and its relation to environment factorsMicrobial communities in the intestine of M.rosenbergii under six culture patterns was evaluated by high-throughput pyrosequencing technology.The relation between microbial and water environment was also analyzed.After 64 days of culturing,the microbial communities and physico-chemical factors were assessed.There were significant differences in turbidity,soluble phosphorus?PO4-P?,total nitrogen?TN?and chlorophyll-a?Chl-a?among groups,and PO4-P concentrations in MP group was significantly higher than other groups?P<0.05?.Shannon diversity index of intestinal bacteria was the highest in the MP group?4.08?,and the lowest in the PMF group?1.27?.Proteobacteria,Tenericute and Firmicutes were the major dominant phylum in intestine of M.rosenbergii in the six groups.The major dominant genus was different in six groups,which was Aeromonas in MP,PMP and PMPF groups,and Citrobacter in PP group,and Candidatus Hepatoplasma in PF and PMFgroups.The interaction analysis between intestinal bacteria and environmental factors showed that total phosphorus?TP?had significant effects on the intestinal bacterial community of M.rosenbergii?P<0.05?.Adaptation of microorganisms to the environment factors was different.The abundance of Aeromonas enteropelogenes,beneficial Enterococcus and Lactococcus garvieae were positive correlation with NO3-N and TN.Rhodobacter and Pseudomonas vranovensis were positive correlation with TP.Conclusively,culture patterns can affect the intestinal bacterial community of M.rosenbergii by affecting the nutrient concentration in the water,especially nitrogen and phosphorus. |
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