| Chinese mitten crab,Eriocheir sinensis,is commonly known as river crab or hairy crab.It is one of the most important economic species of freshwater aquaculture and distributed widely in China.The Chinese mitten crab is famous for its delicious,nutritious and economic value at home and abroad.In recent years,as the price of Chinese mitten crabs has steadily increased and the market demand has been increasing,farmers have begun to compete to expand the scale of farming and increase the degree of intensification,but at the same time,it also brings huge challenge to the breeding industry of Chinese mitten crab.A series of problems such as abuse of the pesticides and antibiotic,environmental degradation,and host immune fatigue have brought great losses to the breeding industry of Chinese mitten crab.In order to promote the concept of healthy cultivation,we investigated the effect of dietary different level of L-tryptophan and its metabolites-melatonin for the glucose metabolism,immune performance and gut microbial diversity.This study will establish a theoretical basis for the healthy cultivation of the Chinese mitten crab.?1?After 30 days of feeding with diet which added 4 levels of L-tryptophan?L-tryptophan account for the diet were 0.36%,0.47%,0.73%and 1.05%,named 1,2,3and 4,respectively?,the blood glucose metabolism related indexes,expression of the main rate-limiting enzyme in tissues during glycolysis and the antioxidant and immune indexes in serum and hepatopancreas were measured.The results showed that adding different levels of L-tryptophan to feed had no significant effect on serum glucose level,its value was approximately 4.62 mmol/L.With the increasement of L-tryptophan in diets,the content of glycogen in the hepatopancreas was on the rise.Among them,the content of glycogen in the fourth group was significantly higher than that in the first group?P<0.05?.In addition,L-tryptophan had no significant effect on the activity of glycogen phosphorylase?a and b??P>0.05?.Pyruvate kinase?PK?and glycogen synthase kinase 3?GSK3?mRNA relative expression by RT-PCR was detected in ten different tissues?eyestalk,thoracic ganglion,cerebral ganglion,intestinal tract,hepatopancreas,stomach,heart,hemolymph,muscle and gill?,respectively.The results showed that PK and GSK3 were expressed in tissues but the relative expression among different tissues were significantly different?P<0.05?.The relative expression of PK mRNA was the highest in the heart and the lowest in the eyestalk?P<0.05?.The relative expression of GSK3 mRNA was the highest in the thoracic ganglion and the lowest in the eyestalk?P<0.05?.In addition,we analyzed the relative expression of PK and GSK3 mRNA in four tissues of crabs which were fed with different L-tryptophan dietary.From the results,we could see that there was no significant difference in the relative expression of PK mRNA in heart and cerebral ganglion?P>0.05?.But the relative expression of PK mRNA in thoracic ganglion and muscle were improved significantly with the increasement of L-tryptophan in diets?P<0.05?.Similarly,there was no significant difference in the relative expression of GSK3 mRNA in cerebral ganglion?P>0.05?,but the relative expression of GSK3mRNA were up-regulated significantly with the increasement of L-tryptophan in diets in thoracic ganglion,heart and gill?P<0.05?.The different L-tryptophan dietary had no significant effects on the activity of T-AOC,T-SOD and ACP in the serum and T-AOC in the hepatopancreas of the Chinese mitten crab?P>0.05?,but the activity of CAT and AKP in serum were on the rise.The activity of CAT and AKP in serum of the forth group were higher than those in the first group?P<0.05?.Besides,crabs which were fed with experimental diets had higher activity of the T-SOD in the hepatopancreas when compare with controls?P<0.05?.The activity of CAT in the hepatopancreas of the third and forth group higher than those in the first and second group significantly?P<0.05?.The value of CAT in the first group was the lowest.?2?Through high-throughput sequencing technology,the 16S rRNA V3-V4 region was amplified in the gut microbiota of the Chinese mitten crabs after feeding with the four diets.The high-throughput sequencing of the Illumina platform found that adjunction of the L-tryptophan can significantly increase the richness and diversity of intestinal microbiota.Besides,the dominant phylum in the intestine of the Chinese mitten crab were Tenericutes,Proteobacteria,Firmicutes,Chloroflexi and Actinobacteria.The L-tryptophan in diets increased the richness of Proteobacteria,Firmicutes and Actinobacteria in the intestine significantly.These bacterias are all dominant bacteria and have a certain role in promoting the immunity of Chinese mitten crab.?3?We investigated the effect of melatonin?MT?on the hemolymph glucose metabolism and the capacity of antioxidant and immune in serum and hepatopancreas.First of all,we explored the regulation of eyestalk on the hemolymph glucose level.It was found that eyestalk ablation,especially bilateral,caused a significant decrease in the hemolymph glucose level by removing the unilateral or bilateral eyestalk of the Chinese mitten crab?P<0.05?.Injection of melatonin induced hyperglycemia in a dose-dependent manner both in intact and ablated crabs.A dose of 10-6 mole/crab melatonin increased the level of hemolymph glucose level and differed significantly from the other doses(10-12,10-10,10-9,10-8 and 10-7 mole/crab)?P<0.05?.CHH?crustacean hyperglycemic hormone?mRNA relative expression by RT-PCR was detected in all ten different tissue samples?eyestalk,thoracic ganglion,cerebral ganglion,intestinal tract,hepatopancreas,stomach,heart,hemolymph,muscle and gill?from intact crabs,and had tissue-specific characteristics.Based on the relative expression of CHH mRNA in the above tissues,eyestalk,thoracic ganglion,intestinal tract and hemolymph were selected to estimate the effect of melatonin on the relative expression of CHH mRNA.Bilateral eyestalk ablation caused a significant increase in the relative expression of CHH mRNA in the thoracic ganglion,intestinal tract and hemolymph compared with the controls?P<0.05?.In addition,injection of melatonin into intact or ablated crabs elevated the CHH mRNA level in the eyestalk,thoracic ganglion and intestinal tract tissues compared with controls?P<0.05?.Administration of melatonin resulted in a significant decrease in total carbohydrates and glycogen levels with an increase in phosphorylase activity levels in the hepatopancreas and muscle in intact and ablated crabs?P<0.05?.The ablation of the bilateral eyestalk had no significant effects on the activity of T-AOC,T-SOD and ACP in the serum of the Chinese mitten crab?P>0.05?,but the activity of the CAT and AKP in the serum decreased significantly when compared with controls?P<0.05?.And not only that,the crabs which were removed the eyestalk had the lower activity of T-SOD and CAT in the hepatopancreas?P<0.05?.On the one hand,for the intact crabs,the injection of melatonin improved the activity of T-SOD,CAT,ACP and AKP in the serum?P<0.05?,but no significant effects on T-AOC were found in the serum?P>0.05?.On the other hand,for the ablated crabs,the injection of melatonin increased the activity of T-AOC,T-SOD,CAT,ACP and AKP in the serum and the activity of T-SOD and CAT in the hepatopancreas?P<0.05?.However,whenever eyestalk was ablated or not,the T-AOC did not change obviously in the hepatopancreas of crabs which injected with melatonin?P>0.05?.The results show that the effect of L-tryptophan in the diets on the blood glucose metabolism was not significant,but it can improve the antioxidant and immune capacity.In addition,appropriate L-tryptophan was added to diets can enrich the superior bacteria in the intestinal tract of Chinese mitten crab which is contribute to defend against the disease.The metabolites-melatonin can induce hyperglycemic effect in Chinese mitten crab,but this effect is probably not mediated via eyestalk factors such as CHH.Besides,melatonin can also improve the body's antioxidant capacity properly. |
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