Studies On Probiotic Mechanism Of Lactic Acid Bacteria In Litopenaeus Vannamei

Fifty-three lactic acid bacteria(LAB) strains were respectively isolated from commercial probiotics, and the intestinal tracts of shrimp(Fenneropenaeus chinensis) and fish(Acanthogobius hasta). The antibacterial and adhesive activities of these strains were examined, the results showed that Lactobacillus pentosus HC-2(HC-2) and Enterococcus faecium NRW-2(NRW-2) had higher antibacterial and adhesive activities, respectively, the identification of strains was based on the 16 S rRNA analysis. The aim of this study was to investigate and compare the probiotic effects and mechanisms of NRW-2, HC-2 and the corresponding supernatant on the shrimp(Litopenaeus vannamei).Firstly, the following four trails(three treated and one control) were carried out for four weeks: T1, shrimp fed with a commercial diet + strain HC-2(1×107 CFU g feed-1); T2, shrimp fed with a commercial diet + strain NRW-2(1×107 CFU g feed-1); T3, shrimp fed with a commercial diet + the supernatant of strain HC-2(the corresponding supernatant of 1×107 CFU g feed-1 of HC-2); and T4, shrimp fed with a commercial diet alone as a control. The results showed that the specific growth rates of shrimp fed with selected strains were higher than the control group; whereas that of the supernatant of the HC-2-containing group was lower than the control even though it was not significant. Real-time PCR was used to determine the expression levels of immune-related genes in the midgut and hepatopancreas. The results suggested that NRW-2 had higher stimulation to PEN-3α、proPO and lysozyme in the midgut at two weeks and four weeks post-feeding, respectively, while HC-2 and its corresponding supernatant had higher induction of genes in the hepatopancreas. According to the challenge tests, all of the treatment groups had significantly higher survival rate than the control group.Secondly, the effects of probiotics and the corresponding supernatant on activities of digestive enzymes in Litopenaeus vannamei were investigated. The results showed that the protease activities were significantly(P < 0.05) increased in the intestine of three treatment groups compared with the control group, but they were no significant in the hepatopancreas. The amylase and lipase activities were all increased significantly(P < 0.05) in intestine and hepatopancreas of shrimp fed with NRW-2 compares with the control group, the amylase and lipase activities were increased significantly(P < 0.05) in hepatopancreas and intestine respectively in the group of shrimp fed with HC-2, whereas the protease and amylase activities were just increased significantly in the intestine of shrimp fed with the supernatant of HC-2. This study suggested that activities of digestive enzymes in L. vannamei can be increased by NRW-2, HC-2 and the corresponding supernatant, but varied in different tissues. In addition, a microbial identification technique based on the 16 S rRNA gene sequenced with Illumina Miseq was used to thoroughly investigate the intestinal bacterial composition of L. vannamei after four dietary regimens with different probiotics or probiotic supernatant. Proteobacteria, Verrucomicrobia, and Actinobacteria were dominant in the intestines of L. vannamei, regardless of its diet. However, unique species existed in all subsets of each dietary group and the abundance of Actinobacteria was significantly increased in the intestinal bacterial community of shrimp fed with the bacteria-free supernatant of an HC-2 culture compared with the control. These results suggested that the composition and richness of bacterial species in the intestines of shrimp were influenced by dietary supplementation with probiotics or probiotic supernatant. In addition, the histology of intestines of the shrimp from four dietary groups were also described, but no obvious improvements in the intestinal histology were observed. Therefore it can be speculated that bacterial interactions in complex microbial communities may play important roles in maintaining or promoting the health of the host.Thirdly, the interaction between HC-2 and Vibrio parahaemolyticus E1(VPE1) when cocultured in Litopenaeus vannamei in vivo and in vitro was investigated. Fluorescence microscopy was used to observe the distribution of these two strains, which were labeled with DAPI and CFDA-SE, respectively, in the gut of the shrimp. Fluorescent imaging revealed that HC-2 competitively excluded VPE1 in the intestine of L. vannamei when the shrimp diet contained both these strains. The growth of both bacteria and the expression of quorum-sensing- and virulence-related genes of HC-2 or VPE1 were monitored when they were cultured alone or together in vitro. In contrast to HC-2, the population density of VPE1 decreased significantly after coculture for 8 h. A transcriptional analysis showed that the expression of the luxS gene in HC-2 increased after 8 h when it was cocultured with VPE1, suggesting that Lux S was involved in the competitive exclusion of VPE1 by HC-2. Interestingly, the aphA, opaR, and tlh genes of VPE1 were significantly upregulated in response to heat-killed HC-2 cells. This suggested that the intracellular components of HC-2 induced the expression of toxicity-related factors in VPE1 for a short time in coculture, and these may increased the risk of shrimps infected by vibrios.Lastly, the effects of the short-term addition of HC-2 on the survival rate and expression of immune-related genes of L. vannamei were investigated before or after L. vannamei infected by VPE1. The results showed that the survival rates were higher when HC-2 was added before shrimps infected than after, even though it was not significant. The transcriptional profiles of immune-related genes in the guts of L. vannamei suggest that the addition of HC-2 after shrimps infected can enhance the immunity at a certain degree, but the short-term addition of HC-2 can not significantly enhance the disease resistance of L. vannamei.