Studies Of Effects On Litopenaeus Vannamei Bacterial Ecology Caused By Probiotics Application And Mechanism Of Interaction Between Microalgae And Probiotics

Litopenaeus vannamei is becoming the most valuable aquaculture animal in this world, and the most proportion of the product come from China. The data show that the product of L. vannamei is about 1.5769 million tons in the 2014, the total value over ten billion RMB. L. vannamei was introduced into China from 1988, until now, the history of culture was over 27 years. Following the culture technique improved continuly, shrimp culture density also became higher, this process resulted in more and more shrimp diseases break out during the culture. In order to control the shrimp disease and improve water quality, probiotics was successful used into the shrimp farming process in the past decades. Because of the former research usually based on the microbe culture methods, and this kind of methods was difficult to illustrate the mechanism of probiotic effects and variation of bacterial community structure, the deficiency about the basic data of microbe community then hamper the probiotic’s development and application.Bacillus subtilis FY99-01 and Nitrobacter winogradskyi Y3-2 which isolated and cultured in the state laboratory of agricultural microbiology was choosed as probiotics in this study, and an outdoor culture experiment was conducted at Beihai city, Guangxi province. In this experiment, denaturing gradient gel electrophoresis（DGGE） and high-throughput sequencing tecniuqes were used to analyse the effects on total bacteria and vibiro ecolgy of shrimp culture water and intestinal tract during the different culture period, which cased by application of probiots.Algae is a key role in aquaculture system, complicated relationship between algae and bacteria may be impact the effects of probiotic. Algae and nitrifying bacteria utlize the same substrate, especially, they determine the form of nitrogen, even the direction of nitrogen cycle, however, few previous studies foucus on this topic, so, a series models of bacteria-algae interaction were constructed in this study try to demonstrate the effect on bacterial nitrification cased by algae in aquaculture ponds.The mainly findings in this study as follows:（1） B. subtilis FY99-01 was applied in shrimp ponds can obviously improve the water quality, exectly, reduced the p H value significantly in prophase（0 30 days）, reduced the concentration of soluble orthophosphate（SOP） in middle and later period（30 84 days）, decreased the concentration of nitrite in later period（60 84 days）. Meanwhile, B. subtilis FY99-01 was used also can improve the shrimp growth rate, product, feed conversation ratio and survival ratio. The effects of N. winogradskyi Y3-2 was weaker than the B. subtilis FY99-01, N. winogradskyi Y3-2 was applied can not improve water quality and shrimp growth rate during the experiment, especially, nitrite concentration was not reduced.（2） The dominated bacterial species of culture water was different in different culture period. In prophase, the dominated microbes belong to β-proteobacteria and Verrucomicrobiae; In middle stage, the dominated microbes belong to α-proteobacteria, Actinobacteria, Verrucomicrobiae, Cyanobacteria and Bacilli; In later stage, the dominated bacterial species and ratio list as follows: α-proteobacteria（30.46 ± 7.70%）, γ-proteobacteria（12.72 ± 4.11%）, Chloraplast（7.16 ± 3.22%）, Actinobacteria（7.10 ± 3.55%）, Flavobacteria（6.32 ± 2.34%）, Cyanobacteria（5.52 ± 8.87%）, β-proteobacteri（4.30 ± 1.42%）, Sphingobacteria（4.03 ± 1.39%）, Planctomycetacia（3.30 ± 0.46%）, δ-proteobacteria（2.57 ± 0.60%）, Verrucomicrobiae（1.55 ± 0.74%） etc..（3） The dominated bacterial speices at class level of Litopenaeus vannamei intestinal tract as follows: γ-proteobacteria（60.77 ± 16.51%）, α-proteobacteria（15.45 ± 8.98%）, Sphingobacteria（4.01 ± 2.16%）, Actinobacteria（3.83 ± 3.75%）, Flavobacteria（3.80 ± 0.69%）, Planctomycetacia（1.00 ± 0.60%） �'-proteobacteriaetc. Vibiro was the most important population in L. vannamei intestinal tract, it most abundance species were Vibrio shilonii, V. hepatarius and an uncultured Vibrio sp.（Gene number: AY374396.1）. Interestingly, Planctomycetacia was found dominated in shrimp intestinal tract, as we know, anaerobic ammonium oxidation bacteria（Anammox） come from this cluster, Anammox is very important for nitrogen cycle in nature.（4） At the aspects of shrimp culture water microbial ecolgy, application of B. subtilis FY99-01 and N. winogradskyi Y3-2 in different culture period can impact shrimp culture water bacterial community structure differently, and the impact of B. subtilis FY99-01 was bigger than the nitrifying bacteria. UPGAMA of DGGE showed that the effect caused by B. subtilis FY99-01 was bigger in prophase and middle stage than the later stage. Sequences data from DGGE gel showed that proportion of a human pathogen WP01（Klebsiella pneumoniae MP3₁A,JN644624.1） and WP03（Uncultured organism SBXY₇96,JN429468.1） decreased caused by B. subtilis FY99-01 application in prophase, reversely, the proportion of B. subtilis increased. N. winogradskyi Y3-2 also decreased the ratio of WP01. In middle stage, WM08（Cryobacterium psychrotolerans,JN626519.1）, WM09（Leptolyngbya fragilis,EF420112.1） and WM11（Uncultured Verrucomicrobiales bacterium,FJ516959.1） appeared in groups which used B. subtilis FY99-01, at the same time, a human pathogen WM10（Acinetobacter radioresistens SMA4,JQ618289.1） disappeared. Application of N. winogradskyi Y3-2 made the WM08 and WM09 appeared, and decreased the ratio of WM10. In later stage, the abundance of WA01, WA03 and WA07 which belong to Verrucomicrobiae decreased or even disappeared caused by B. subtilis FY99-01 application;Two species of bacteria named WA03 and WA11 which belong to Verrucomicrobiae and one denitrifying bacteria WA04 which belong to Bacilli were decreased by N. winogradskyi Y3-2 application. In the later stage, results based on high-throughput sequencing showed that application of B. subtilis FY99-01 can decrease the abundance of α-proteobacteria significantly（P < 0.05）, increase the abundance of Flavobacteria, Cyanobacteria, Bacilli and Chloroplast; The effects caused by N. winogradskyi Y3-2 was weaker, it was found can only increase the abundance of Chloroplast. At genus level, B. subtilis FY99-01 can increase the abundance of Bacillus spp.（P < 0.05）. In a word, the application of probiotic optimizing the bacterial community structure in shrimp culture water.（5） The probiotics used in the shrimp culture water also effect the bacterial community sturucter of shrimp intestinal tract, B. subtilis FY99-01 and N. winogradskyi Y3-2 increase the bacterial diversity of shrimp intestinal tract. The application of B. subtilis FY99-01 decreased the abundance of Vibrio spp.（especilly shrimp pathogen V. parahaemolyticus）、Rhodobacter spp., and increased the abundance of Pseudomonas spp.、Corynebacterium spp.. The application of N. winogradskyi Y3-2 also reduce tha ratio of Vibiro spp., nevertheless, increased the abundance of Pseudomonas spp. and Chitinophaga spp.. The above results domanstrated that the probiotics which used in this study were safely and advantage to the shrimp health.（6） Algae growth inhibited the bacterial nitrification significantly, light exposure also inbibit the bacterial nitrification, and algae growth and light exposure co-effect could enhance the inhibition. Algae mainly inhibited nitrifying bacterial growth, and light mainly inhibit nitrifying bacterial activity. The model results demonstrated that algae growth and light exposure in wild shrimp culture ponds can result in the nitrifying bacteria ineffect to decrease the anmmonia and nitrite.（7） The mainly mechanism of M. aeruginosa FACHB-915 inhibited the nitrifying bacteria’s activity and growth through compete for utilize ammonia. Microcystin-LR had no effect on the bacterial nitrification.