The Characteristic And Function Research On The Intestinal Microflora Of Shrimp

With the development of economic, people’s demand on seafood is becomingincreasingly large and shrimp culture has become one of the most important industry inaquaculture. According to fishing industry record in2010, the production of shrimpreached1.38million tons, with Litopenaeus vannamei1.22million tons, occupying84.5percent of the total shrimp production. However the intense shrimp culture inindustry also brings negative impact, for example the frequency outbreak of diseasescaused by bacteria and viruses bring great economic losses to shrimp culture industry；the abuse of medicine in curing diseases also downgrade the quality of seafood.Nowadays the best method in treating shrimp diseases is microbiology prevention. Inand out of shrimp body exists a variety of bacteria like microbiology; they are not onlybeing an important part of shrimp food chain, joining the material recycle and energyflow in culture system, but also playing an important role in maintaining ecosystembalance and improving environment quality. Like probiotic which can on the one handenhance the immunity of shrimp and on the other hand improving the cultureenvironment. The advance and basis on probiotic application is to understand theconstitute model of microbiology and screen and identify the advantage strain in shrimpgut. There are seven parts in this paper:1. Screen and identify the advantage strain in shrimp gest：in this part weisolated gest microbiology from different months and send them to16S rDNAsequencing and analysed the advantage strain of different months. The result shows thaton the month of8,9,10the major advantage strain are bacillus and vibrio. Some stainappeared in all the three month like Exiguobacterium, while some appeared only in onemonth like Bacillu sadhaeren.also, the kinds of bacteria strain goes up with the month,and this reflects the changes of gut strain with the seasons.2. By using PCR-RFLP and PCR-DGGE methods to analysis the groupcharacter of microbiology in industry culture: By using PCR-RFLP and PCR-DGGEmethods we conclude that on August the major advantage strain inshrimpgutisUnculturedbacterium,withRuegeriaspp,Vibriospp,Rhodobacterales,Streptomyces spp., Enterococcus sp. and Photobacterium damselae follows.While on Octoberthe major advantage strain in shrimp gut isUnculturedbacteriumandVibriospp.,withPhotobacteriumspp.,Neptunomonas spp.,Streptomycesspp., Ruegeria spp., Enterococcus sp and Bacillus spp follows.PCR-RFLP and PCR-DGGE all can help us in obtain some diversity of microbiology ofshrimp gut but their result may be different to some extend. However throughcombining the two result we can make a better understand of microbiology diversity,making experiment results more closer to the real state of shrimp gut and offer theorybasis on the research and application of probiotic in the future.3. Screen and examine the protect effect of strains in the inhibitory of vibrioharveyi: In this part we used cross and agar diffuse method to conform anti experimentand screened two kinds of seawater bacteria which can cause the inhibitory to thegrowth of vibrio harveyi from healthy shrimp gut. Through16sRNA sequencing, theobtained sequence are compared with sequence from GenBank by blast and establishedthe Phylogenetic dendrogram. The results showed that Jk001,Jk002have a highersimilarity with pseudoalteromonas and alpha proteobacterium respectively. Also thesafety examine results indicate that the two strains can inhibate the growth of vibrioharveyi.4. Identify3seawater bacillus and analysis their biology character: In this partwe analysed the biology character of three bacillus isolated from shrimp gut,andverified their identity by16s rRNA. Phylogenetic dendrogram analysis show that thethree strain shares the highest similarity with bacillus genara, and strain labeledKL011001has similar revolution place with Bacillus subtilis,strain labeled KL011002has similar revolution place with Bacillus pumilus, strain labeled KL011001has similarrevolution place with Bacillus licheniformis. Apart from Phylogenetic dendrogramanalysis, the difference of the three strains in using carbon in Biolog also means theybelong to the same genera but are different strains.5. The effect of three living strain in protecting Litopenaeus vannamei aganistWSSV infection: In this part we used three bacillus as major probiotic after immuneingLitopenaeus vannamei for21days with feeds embedded with only one bacteria or threebacteria, shrimp were challenge with WSSV virus through muscle injection. Afterobservation for14days, the accumulated mortality in KL011004（combination of thethree strain）、 KL011003,KL011002,KL011001immune group were50%,63.3%,73.3%,75%respectively,these results are significantly different fromcontrol group whose accumulated mortality was100%(P<0.05). Also the statisticresults shows that the accumulated mortality from KL011004immune shrimp wassignificantly lower than all the three monovalent group(P<0.05). Among themonovalent group KL011003was significantly lower than KL011002and KL011001(P<0.05), and there is no difference between KL011002and KL011001group (P>0.05).6. The expression of immune related gene of shrimp gut by feeding probiotics: On the basis of chapter5, in this part we used RT-PCR method to test the expressionlevel of caspase and trx gene during vaccination and after WSSV challenge in order tocertify the results of accumulated mortality in chapter5. The results show that incomparation with the control group, the expression level of caspase and trx invaccinated shrimp were significantly higher (P<0.05), also the gene in KL011004vaccinated group were higher than monovalent group.7. The effect of feeding feeds in which probiotics is added in slowing thepropagate rate of WSSV of Litopenaeus vannamei. This part is also based on theresults from chapter5. In this chapter we used the method of Real time PCR to examinethe copy of WSSV from gill after infection Litopenaeus vannamei in different time. Theresults show that in every examined time the WSSV copy number in gill from controlgroup is significantly higher than vaccinated group. These results indicates that theaddition of Probiotics in feeds is contribute to Litopenaeus vannamei in clearing upWSSV, enhancing the ability of Litopenaeus vannamei in resisting WSSV infection.