| The mode of rice-crayfish co-culture is the main way of crayfish production at present.In order to improve the efficiency of rice and crayfish farming,fertilization should be applied to paddy fields.Fertilization could affect the water quality of rice and crayfish growing fields,and the bioturbation of Procambarus clarkii could also affect the material exchange at the soil-water interface,but the specific effect is not clear.The ammonia-N in the water body will have a peak value of 1-2 days after topdressing in the paddy field,and then the ammonia-N in the paddy field water will be in a low concentration state for a long time.Therefore,this paper studied the effects of Procambarus clarkii under different fertilization treatments on water quality and soil physical and chemical properties,and further studied the effect of short-term ammonia-N stress and recovery on the antioxidant and immune capacity and tissue structure of Procambarus clarkii.And effects of long-term ammonia-N stress on the antioxidant and immune capacity,tissue structure and intestinal flora of Procambarus clarkii,aiming to provide a reference for the healthy cultivation of Procambarus clarkii in paddy fields.1.Effects of disturbance on fertilizer release and vertical distribution of tracer particles by Procambarus clarkiiThe experimental nitrogen fertilizer,phosphorus fertilizer and potassium fertilizer were applied according to the standard of 180kg/hm~2,90kg/hm~2 and 90kg/hm~2,respectively.Six treatment groups were set up,namely,inorganic fertilizer control group,inorganic fertilizer disturbance group,organic fertilizer control group,organic fertilizer disturbance group,no fertilizer control group and no fertilizer disturbance group.Meanwhile,the tracer particle tracing method was adopted.The tracer particles were buried at 0cm and 5cm of the disturbance group and the control group,and the experiment lasted for 15 days.The results show that:The disturbance of Procambarus clarkii for 15 days will disturb the fertilizer at the depth of 0-11 cm in the soil,which increases the peak ammonia-N of the water body by51.81%after the application of inorganic fertilizer,and increases the peak value of ammonia-N in the water body by 53.02%after the application of organic fertilizer.Disturbance had a significant effect on soil total nitrogen and available phosphorus after application of inorganic fertilizer,which increased the release rate by 36.59%and 11.51%,respectively.There was a significant difference in the application of organic fertilizer to soil total nitrogen,the release rate of soil total nitrogen increased by 36.39%,and there was no significant effect on the release of available phosphorus,indicating that the application of organic fertilizer was stronger than inorganic fertilizer on the retention of phosphorus in soil.The application of organic fertilizers in rice and crayfish cultivation and the application of fertilizers below 11 cm of the soil helps to slow the release of fertilizers.2.Effects of short-term ammonia-N stress and post-exposure recovery on antioxidant and immune enzyme activities and tissue structure of Procambarus clarkiiThe Procambarus clarkii(6.33±0.73g)was exposed to different concentrations ammonia-N(0、20、40、60、80mg/L)for 48h,and then post-exposure for 48h。The contents of T-AOC,T-SOD,CAT,ACP,AKP and MDA in serum of hepatopancreas,gills,and tissue structure of hepatopancreas and gills were studied under ammonia-N stress for48h and post-exposure for 48h.The LC50 of ammonia-N to Procambarus clarkii at 24,48,72,and 96 h were 359.37,238.09,196.34,162.00 mg/L,and the safe concentration was 16.20 mg/L.After 48 h of ammonia-N stress,the T-SOD activity of the hepatopancreas in the 40,60,and 80 mg/L groups was significantly higher than that in the control group.T-AOC was significantly enhanced.After 48 hours of recovery,only hepatopancreas MDA content,gill MDA content and CAT activity,serum T-AOC did not return to normal level,other enzyme activities have recovered.Under ammonia-N stress for 48h,with the increase of ammonia-N concentration,the hepatopancreatic lumen was gradually deformed and enlarged,the number of transport vesicles increased,the respiratory epithelial cells of the gills began to fall off,and the microvascular lumen was damaged.After 48 hours of recovery,the tissue structure of the hepatopancreas basically returned to normal.Only the respiratory epithelial cells in the gills of the 40,60,and 80 mg/L groups fell off,and the microvascular lumen was damaged.In conclusion,the ammonia-N produced in the water after fertilization and fertilization of rice and crayfish would cause certain damage to the hepatopancreas,gills,and serum antioxidant immunity of Procambarus clarkii under stress for 48 hours.After48 hours of recovery,the physiological functions of hepatopancreas,gills and serum can be recovered to varying degrees,only hepatopancreas MDA content,gill MDA content and CAT activity,serum T-AOC did not return to normal level.The tissue structure of hepatopancreas was basically normal,but the gill tissue was not completely recovered.3.Effects of long-term ammonia-N stress on growth and survival,antioxidant and immunity and tissue structure of Procambarus clarkiiCurrent study explored the influence of long-term ammonia-N stress the growth and survival of Procambarus clarkii(6.18±0.68g)were exposed to different concentrations of ammonia-N(0、2、4、8、16mg/L)for 35d.The contents of T-AOC,T-SOD,CAT,ACP,AKP and MDA in serum of hepatopancreas,gills,and tissue structure of hepatopancreas and gills were studied under ammonia-N stress for 35d.Long-term ammonia-N stress for 35 days had no significant effect on the survival rate,weight gain rate,specific growth rate,meat yield and hepatopancreas index of Procambarus clarkii.After 35 days of ammonia-n stress,the T-SOD activity of gills in 4,8,and 16 mg/L groups was significantly higher than that in the control group,the serum CAT activity in the 16 mg/L group was significantly lower than that in the control group,and the serum ACP in the 4,8,and 16 mg/L groups was significantly higher than that in the control group.The activities were significantly lower than those in the control group,and there was no significant difference in the enzyme activities of each tissue in the 2mg/L group compared with the control group.After 35 d of ammonia-N stress,the tissue structure of hepatopancreas and gills in 2 and 4 mg/L groups was not damaged,while in8 mg/L and 16 mg/L groups,the lumen and transport vesicles of hepatopancreas increased,and the basement membrane in the lumen expanded into polygons.In the 8mg/L and16mg/L groups,the respiratory epithelial cells of the gills fell off,and the vacuoles formed by the damaged microvascular lumen structure became larger.In conclusion,long-term water ammonia-N(less than 2mg/L)stress for 35 days after fertilization had no significant effects on the growth and survival,antioxidant and immune capacity and tissue structure of Procambarus clarkii.4.Effects of long-term ammonia-N stress on intestinal microflora of Procambarus clarkiiThe effects of ammonia stress on intestinal microflora of Procambarus clarkii were studied by exposing(6.18±0.68)g of Procambarus clarkii to water with ammonia concentration of 0,2,4,8 and 16mg/L for 35 days.The results showed that 2,8 and16mg/L ammonia-N stress for 35 days significantly reduced the diversity of intestinal flora,but had no significant effect on the richness of intestinal flora.Ammonia-N stress affected intestinal function of Procambarus clarkii by reducing the relative abundance of Firmicutes and Actinobacteriota.Ammonia-N stress can cause significant changes in the relative abundance of Shewanella,Candidatus＿Bacilloplasma,Rhodobacter,Mycobacterium and Flavobacterium in all experimental groups,and damage the balance of intestinal flora of crayfish.It is speculated that when the ammonia-N in rice-crayfish co-culture water reaches 2mg/L,long-term culture will destroy the intestinal flora of Procambarus clarkii. |
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