Study On Relationship Between Microbial Community & Physical–Chemical Factors In Culture Environment And Disease Occurence Of Apostichopus Japonicas

In this paper, several sea cucumber(Apostichopus japonicas) culture ponds in Shaogezhuang community(for short :SGZ) and Suliu community(for short:SL)were investigated in July and August,2014, the difference between diseased ponds and non-diseased ponds was analyzed using in situ & laboratory detection, culture counting, and 16 S rRNA gene and the high-through sequencing technique. The dominant bacteria strains were isolated from the focal area of the infected sea cucumber, and their virulance were proved through artificial infection and identification.The dynamic variation of several culturable bacteria groups( heterotrophic bacteria, vibrios, nitrifying bacteria, sulphuring bacteria,nitrate-reducing bacteria, sulphurate-reducing bacteria) and several physicochemical indexes(temperature, pH, salinity, dissolved oxygen, redox potential, sulfide, organic carbon, COD, total nitrogen) were detected. The microbial community structures were analized by high-through sequencing technique, to find out the relationships between diseased ponds and non-diseased ponds. The main experimental results as follows:1 Isolation and identification of pathogenic bacteria from disease-occurred Apositchopus japonicas. infected sea cucumber collected from ponds in Shaogezhuang community and Suliu community. The dominant bacteria were isolated from the focal area of the infected sea cucumber. Artificial infection test proved that the bacterium was the causative pathogen associated with the disease. The bacterium was identified as Vibrio parahaemolyticus, Vibrio splendidus, Vibrio rotiferianus by the means of 16 S rRNA gene sequence analysis.2 Environmental factors causing skin ulcer symdrome in pond-cultured sea cucumber(Apostichopus japonicus)。The relationships between disease occurrence in the sea cucumber and environmental factors were analyzed by comparing the environmental indices in ponds containing diseased and non-diseased sea cucumber in Hongdao, Qingdao, Shandong Province. Between July 23, before any disease occurred, and August 11, 2014, we detected 4 cultivable bacterial groups(heterotrophic bacteria, vibrios, nitrifying bacteria, and sulphurate bacteria) and measured 7 physical–chemical indices(temperature, pH, salinity, dissolved oxygen,inorganic nitrogen, and chemical oxygen demand) in the pond water and detected 6bacterial groups and measured 4 physical–chemical indices(pH, redox potential,content of sulfide, and organic carbon) in the pond sediments. The number of bacterial groups in the diseased pond SGZ-1# did not differ significantly from those in the non-diseased pond SGZ-2# on the day that disease occurred, however the temperature was very high(25.94 °C) and the salinity was very low(24.47),and the concentration of the nitrite(79.56 μg/L) in pond water and the sulfide(221.1 mg/g) in the sediment were higher in the diseased pond SGZ-1#, exceeding the tolerance thresholds of the sea cucumber. The number of vibrio species in the diseased pond SL-S2# of the Suliu community was significantly higher(1.85 × 104 cfu/ml) than that in the non-diseased pond SL-N1# on the day that disease occurred, and was also higher than the number in the diseased pond SGZ-1# in the Shaogezhuang community.However, it’s contrary between SL-S2# and SL-N1#. Therefore, we infer that the disease that occurred in the sea cucumbers in Shaogezhuang was closely related to the large changes in the physical–chemical indices of the water and sediment, whereas the disease in the sea cucumbers in Suliu was closely related to the proliferation of pathogenic bacteria in the environment. Therefore, the factors associated with disease must be analyzed in individual outbreaks, including pathogenic organisms or physical–chemical factors, and the appropriate strategies should be used for disease control.3 Analysis of the microbial communities in sediment of sea cucumber(Apostichopus japonicus) cultural ponds based on high-through sequencing technique.Through analyzing the DNA of sediment microbial communities of sea cucumber in Hongdao, Qingdao, Shandong Province, using high-through sequencing and bioinformatics technique forV3 and V4 area of 16 S gene, the difference between diseased ponds and non-diseased ponds were investigated. The results revealed that Proteobacteria are absolute advantage bacteria group in both two community,abundance ratio was more than 70%.The second are bacteroidetes, abundance ratio about was 10% and the other were less than 5%. Proteobacteria in sediment of disease pond were more than in non-diseased pond at the same time. In addition, a kind of probiotics(lactic acid bacteria) was found in non-diseased ponds but did not found in diseased ponds.in Shaogezhuang comminity Lactic acid bacteria was not detected in both disease ponds and non-diseased ponds during the disease time, but was found when the diseased pond recovered to normal Suliu community.