应用PCR-DGGE和Q-PCR分析不同体重斑点叉尾鮰皮肤、鳃和胃肠道菌群结构

Application of PCR-DGGE and Q-PCR to analyze microflora of skin,gills,and the gastrointestinal tract of Ictalurus punctatus with different weights

采用聚合酶链式反应-变性梯度凝胶电泳(PCR-DGGE)和荧光定量-聚合酶链式反应(Q-PCR)技术分析高体重(high weight,HW)和低体重(low weight,LW)斑点叉尾鮰鮰(Ietalurus punetaus)皮肤、鳃和胃肠道菌群多样性,为斑点叉尾鮰微生态研究及筛选斑点叉尾鮰源益生菌提供理论依据。结果显示,斑点叉尾鮰菌群丰富度由低到高依次为鳃、皮肤、前肠、后肠和胃。肠杆菌科(Enterobacteriaceae)和气单胞菌属(Aeromonas)是皮肤的优势菌群;肠杆菌科(Enterobacteriaceae)、气单胞菌属(Aeromonas)和肠球菌属(Enterococcus)是水体、鳃和胃的优势菌群;肠杆菌科(Enterobacteriaceae)、拟杆菌属(Bacteroidetes)、气单胞菌属(Aeromonas)和酵母菌属(Saccharomyces)是肠道的优势菌群。HW斑点叉尾鮰鳃菌群的香农多样性指数、均匀度和丰富度及前肠菌群的丰富度显著高于LW斑点叉尾鮰(P<0.05)。皮肤的黄杆菌属(Flavobacterium),胃的肠杆菌科(Enterobacteriaceae),前肠的拟杆菌属(Bacteroidetes)和双歧杆菌属(Bifidobacterium)及后肠的拟杆菌属(Bacteroidetes)、双歧杆菌属(Bifidobacterium)和酵母菌属(Saccharomyces)的拷贝数分别是101.97、107.69、106.19、103.83、106.13、103.92和104.26,均显著高于LW斑点叉尾鮰(P<0.05)。结果表明,斑点叉尾鮰皮肤、鳃、胃肠道均形成独特的菌群结构,LW和HW斑点叉尾鮰菌群结构存在明显差异,HW斑点叉尾鮰菌群多样性增加。

Ictalurus punctatus is native to the Americas and a superior freshwater fish cultured worldwide. However, acute mortalities often occur during aquaculture. In addition, the disadvantages of antibiotics have been highlighted, and a safe and effective substitute has become a hot topic. Thus, many studies have been published on the beneficial effect of probiotics, and most have targeted the microflora of the gastrointestinal tract, but few studies have investigated microflora of the gills and skin. To provide a theoretical basis to study gill, skin, stomach, foregut, and hindgut microorganisms and to screen probiotics from I. punctatus, we compared the microbial structures of the gills, skin, stomach, foregut, and hindgut from I. punctatus of different weights and explored the correlation with aquatic flora using polymerase chain reaction-denaturing gradient gel electrophoresis （PCR-DGGE） and quantitative-polymerase chain reaction （Q-PCR）. All samples possessed rich bands, but the location, number, and color of the bands were quite different among samples. The mean numbers of DGGE bands detected in the organs from low to high were gills, skin, foregut, hindgut, and stomach. The Shannon diversity index and evenness and richness of bacteria in the skin, gills, stomach, foregut, and hindgut were 3.06, 0.94, and 23.00; 2.97, 0.92, and 19.67; 3.44, 0.93, and 31.33; 3.16, 0.93, and 23.50; and 3.30, 0.95, and 26.00 in low weight （LW） I. punctatus and 3.11, 0.99, and 23.83; 3.10, 0.96, and 22.33; 3.45, 0.94, and 31.50; 3.23, 0.94, and 25.05; and 3.28, 0.95, and 25.33 in high weight （HW） I. punctatus, respectively. The Shannon diversity index, evenness, and richness of gills and richness of the foregut from HW I. punctatus were significantly higher than those of LW I. punctatus （P〈0.05）, indicating that microflora diversity was higher in HW I. punctatus than that in LW I. punctatus. Clustering and principal component analysis （PCA） distinguished LW from HW I. punctatus, and the similarity coefficients of LW and HW I. punctatus in the skin, gills, stomach, foregut, and hindgut were 0.82, 0.78, 0.78, 0.61, and 0.73, respectively, suggesting significant differences in the bacterial flora between LW and HW I. punctatus. In addition, the Shannon diversity index and evenness and richness of microflora in the aquatic environment were 3.45, 0.99, and 31.40, indicating slightly lower diversity than that of the stomach flora. The Q-PCR results showed that the dominant skin microflora were Enterobacteriaceae and Aeromonas, the dominant microflora in the water, gills, and stomach were Enterobacteriaceae, Aeromonas, and Enterococcus; and the dominant microflora in the foregut and hindgut were Enterobacteriaceae, Bacteroidetes, Aeromonas, and Saccharomyces. The numbers of Flavobacterium （10^1.97） on the skin, Enterobacteriaceae （10^7.69） in the stomach, Bacteroidetes （10^6.19） and Bifidobacterium （10^3.83） in the foregut, and Bacteroidetes （10^6.13）, Saccharomyces （10^4.26）, and Bifidobacterium （10^3.92） in the hindgut were significantly higher in HW than those in LW I. punctatus （P〈0.05）. Moreover, the total numbers of bacteria in the skin, gills, stomach, and foregut （P〈0.05） were significantly higher in HW than those in LW I. punctatus. These results reveal an increasing trend for the numbers of microflora in HW I. punctatus. The Q-PCR results were analyzed by PCA to identify the correlations between the microflora in all samples. The results demonstrated that LW and HW I. punctatus were distinguished by PCA1; the skin microflora was associated with that on gills, the foregut microflora was associated with that in the hindgut, and the stomach microflora was associated with that in the water. These results indicate the unique flora of the skin, gills, and gastrointestinal tract in I. punctatus and show that the structure and abundance of microflora are complex in LW and HW I. punctatus.