健康和溶藻弧菌感染条件下斜带石斑鱼消化道菌群结构的研究
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摘要
本文以斜带石斑鱼仔稚鱼、不同生长速度的两批幼鱼及溶藻弧菌感染后的斜带石斑鱼为研究对象,利用纯培养方法结合PCR/DGGE方法对石斑鱼消化道菌群结构进行研究,主要结果如下:
1、斜带石斑鱼仔稚鱼消化道菌群结构的研究以斜带石斑鱼孵化后2 d(未开口)仔鱼、8 d(开口仔鱼)、22 d仔鱼、40 d稚鱼为研究对象,分析由孵化到稚鱼的不同生长阶段消化道菌群结构。结果表明,仔稚鱼消化道可培养细菌总数随着生长日龄的增加而逐渐递增,由4.0×102 CFU/尾增加到1.4×107 CFU/尾;2 d仔鱼分离到乳酸菌,未分离到弧菌;8 d、22 d仔鱼及40 d幼鱼均未分离到乳酸菌,22 d仔鱼、40 d幼鱼分离到弧菌;开口前后可培养细菌种类变化较大,种类由多到少。2 d、8 d、22 d仔鱼和40 d幼鱼可培养细菌种类分别为6种、3种、5种、10种;2 d、8 d及22 d仔鱼与进水口细菌种类有一定的相似,8 d仔鱼及22 d仔鱼与其饵料细菌种类有一定程度的相似。
DGGE结果显示,2 d、8 d和22 d仔鱼消化道菌群DGGE图谱相似,仔鱼和40 d稚鱼消化道菌群DGGE图谱存在明显差异;仔鱼与养殖水体菌群DGGE图谱具有一定的相似性;22 d仔鱼与后期饵料轮虫菌群图谱相似性较高,8 d仔鱼与早期饵料牡蛎受精卵的相似性较低。
2、不同生长速度斜带石斑鱼幼鱼消化道菌群结构的研究
利用纯培养方法结合PCR/DGGE方法,分析不同生长速度70 d幼鱼消化道菌群结构。纯培养试验结果表明,生长速度慢的幼鱼消化道细菌总数(5.4×106 CFU/g)少于生长速度快的幼鱼消化道细菌总数(9.0×106 CFU/g),但生长速度慢的幼鱼消化道弧菌数量(6.0×105 CFU/g)明显多于生长速度快的幼鱼(3.4×105 CFU/g),生长速度慢的幼鱼消化道乳酸菌数量(1.5×105 CFU/g)也多于生长速度快的幼鱼消化道乳酸菌数量(6.0×104 CFU/g)。水样中分离到的细菌总数(5.4×105 CFU/ml)、弧菌数(2.2×103 CFU/ml)均少于幼鱼消化道;水样中未分离到乳酸菌。生长速度慢的幼鱼消化道中四种弧菌占细菌总数的12.3%,生长速度快的幼鱼消化道中仅两种弧菌,占细菌总数的比例降低到3.6%;生长速度慢的幼鱼消化道中假单胞菌所占细菌比例12.0%,生长速度快的幼鱼消化道中假单胞菌占细菌总数降低到5.6%。
仅在生长速度快的幼鱼消化道分离到短小芽孢杆菌、克劳氏芽孢杆菌和嗜冷杆菌且数量占绝对优势。抑菌试验表明,短小芽孢杆菌、克劳氏芽孢杆菌和嗜冷杆菌对弧菌具有抑制生长作用。提示短小芽孢杆菌、克劳氏芽孢杆菌和嗜冷杆菌可能与石斑鱼的生长和健康有关。系统进化树结果表明,两组幼鱼消化道的17种细菌隶属于γ-变形杆菌纲,β-变形杆菌纲和芽孢杆菌纲。
DGGE结果显示,生长速度慢的幼鱼消化道菌群多样性明显低于生长速度快的幼鱼消化道。割胶测序结果显示,生长速度快的幼鱼消化道优势菌隶属于不动杆菌属细菌(Acinetobacter baumannii)、结核杆菌(Mycobacterium tuberculosis)、脱硫肠球菌属细菌(Desulfotomaculum reducens)和Petrotoga mobilis;生长速度慢的幼鱼优势菌均隶属于不动杆菌属(Acinetobacter)。
3、溶藻弧菌感染后斜带石斑鱼消化道菌群结构的研究
溶藻弧菌感染后斜带石斑鱼消化道菌群分析结果显示,对照组胃(1.2×105 CFU/g )、前肠(5.1×106 CFU/g)和后肠(2.8×105 CFU/g)和实验组的胃(3.0×105 CFU/g)、前肠(3.1×106 CFU/g)、后肠(3.1×106 CFU/g)的细菌总数量相近;对照组石斑鱼胃和后肠细菌种类明显多于实验组石斑鱼胃和后肠,但对照组前肠细菌种类少于实验组前肠细菌种类。
DGGE结果显示,对照组和实验组细菌多样性具有明显差异。斜带石斑鱼胃菌群结构在灌胃溶藻弧菌后第1 d到第3 d细菌多样性变化不大,但第4 d细菌种类明显减少,第5 d细菌种类则呈回升趋势;前肠细菌多样性分析显示,实验组第1 d和对照组有一定的相似性,实验组第3 d和第4 d相似性较高,细菌多样性明显减少;后肠细菌多样性分析显示,灌胃溶藻弧菌第1 d时,细菌种类较多,第4 d时,细菌多样性降到最低,对照组和实验组第1 d和第5 d相似性较高,实验组第3 d和第4 d相似性较高。割胶测序结果显示,Escherichia coli、Petrotoga mobilis和Vibrio furnissii三种菌在对照组和实验组5 d内的胃、前肠、后肠均检测到,实验组第4 d细菌可检出细菌种类很少,而Shewanella sp.和Vibrio fischeri泳带很强,为第4 d的优势菌。
In the present study, traditional culture-based techniques and denaturing gradient gel electrophoresis (DGGE) tequnique were used to study the gut microbiota of larvae, juvenile and two groups of young grouper Epinephelus cioides with different grow rate. In addition, the gut microbiota of E. cioides infected artificially with Vibrio alginolyticus was investigated. The general results are presented as follows:
1. The aim of the study was to investigate the gut microbiota of the different stages from the eggs hatch to juvenile grouper. Larvae and juvenile samples were collected at day 2, 8, 22 and day 40 post hatch. The results showed that the total cultivable bacterial number increased from the larvae stage to juvenile stage, from 4.0×102 CFU to 1.4×107 CFU per fish. Lactic acid bacteria were only found in the 2 d larvae grouper and no Vibrio sp. observed. Vibrio was found in the 22 d larvae and 40 d juvenile grouper. Six bacterial species were found in the gut of 2 d larvae, but only three bacterial species were found in the gut of 8 d larvae. The cultivable bacterial species are similar between the larvae and inlet water, while those in 8 d and 22 d larvae are similar with there diets.
DGGE results showed that the diversity of gut microbiota of 2 d, 8 d and 22 d of larvael grouper was similar. However, the DGGE profiles of the larvae and juvenile grouper showed an apparent difference. The DGGE profile of 22 d larvae grouper was similar to that of rotifer. There was a low similarity of the DGGE profile between 8 d larvae and its diets (fertilized eggs of the Pacific oyster Crassostrea gigas).
2. The gut microbiota of two groups of juvenile grouper Epinephelus coioides, slow growing and fast growing grouper at 70 days post–hatch, were investigated by using traditional culture-based techniques and PCR/DGGE technique. Traditional culture-based techniques showed that the total bacterial number in the slow growing grouper ( 5.4×106 CFU/g ) was lower than those in the gut of fast growing grouper ( 9.0×106 CFU/g ). However the number of Vibrio and Lactic acid bacteria in the slow growing grouper ( 6.0×105 CFU/g and 1.5×105 CFU/g respectively ) were higher than those in the fast growing fish ( 3.4×105 CFU/g and 6.0×104 CFU/g ). The number of total bacteria ( 5.4×105 CFU/ml ), Vibrio ( 2.2×103 CFU/ml ) in the inlet water were lower than those in the gut of juvenile grouper, and no lactic acid bacteria observed in the inlet water. Four Vibrio species were isolated and comprised 12.3% of the total gut bacteria in slow growing grouper, whereas only two Vibrio species were isolated and comprised 3.6% of the total bacteria in fast growing grouper. Pseudomonas comprised 12.0% in the slow growing grouper, wheras only 5.6% in the fast growing grouper.
Bacillus pumilus, Bacillus clausii and Psychrobacter sp. were only isolated and dominated in the gut of the fast growing fish. The three species showed antagonistic effect on pathogenic Vibrio, this may cause the lower number and less species of Vibrio in the gut of fast growing grouper and suggesting fast growing fish might harbor a favorable microbiota. The bacteria in the gut of grouper could be classified into three groups belonging toγ-proteobacteria,β-proteobacteria and Bacilli class.
The results of DGGE analyses showed that the bacterial diversity in the gut of slow growing grouper was obvious lower than that in the fast growing grouper. Sequencing results showed that the fast growing grouper was dominated by Acinetobacter baumannii、Mycobacterium tuberculosis、Desulfotomaculum reducens and Petrotoga mobilis, however the slow growing grouper gut was dominated by Acinetobacter.
3. The results of DGGE analyses showed the bacterial diversity in the gut microbiota of grouper after administered intragastrically with Vibrio alginolyticus . Sequencing results showed that the stomach、fore gut and hind gut samples were collected at day 1 to day 5 after administered intragastrically with Vibrio alginolyticus. The results showed that the total cultivable bacteria in the stomach, fore- and hind-gut of the experiment group were similar with the control group. The bacterial species in the stomach and hind gut of the experiment group were obvious less than the control group, however the species in the fore hand of experiment group were more than the control group.
DGGE results showed that the bacterial diversity were obvious differences between the experiment group and control group. The microbiota of stomach was relative stable in the first three days after administered intragastrically with V. alginolyticus, whereas many bacteria disappeared in the fourth day, but appeared again in the fifth day. The fore gut bacterila diversity of the experiment group was no change in the first day compared to control group . The bacterial diversity was similar in the third and fourth day , with the lowest species. The hind gut bacterial diversity of the experiment group was much more in the first day , howere it reduced to the lowest in the fourth day. Sequencing results showed that Escherichia coli、Petrotoga mobilis and Vibrio furnissii were isolated from the stomch, fore and hind gut of both control group and experiment group . There were fewer bacterial species in the gut of experiment group in the fourth day, however, Shewanella sp. and Vibrio fischeri dominated.
1、斜带石斑鱼仔稚鱼消化道菌群结构的研究以斜带石斑鱼孵化后2 d(未开口)仔鱼、8 d(开口仔鱼)、22 d仔鱼、40 d稚鱼为研究对象,分析由孵化到稚鱼的不同生长阶段消化道菌群结构。结果表明,仔稚鱼消化道可培养细菌总数随着生长日龄的增加而逐渐递增,由4.0×102 CFU/尾增加到1.4×107 CFU/尾;2 d仔鱼分离到乳酸菌,未分离到弧菌;8 d、22 d仔鱼及40 d幼鱼均未分离到乳酸菌,22 d仔鱼、40 d幼鱼分离到弧菌;开口前后可培养细菌种类变化较大,种类由多到少。2 d、8 d、22 d仔鱼和40 d幼鱼可培养细菌种类分别为6种、3种、5种、10种;2 d、8 d及22 d仔鱼与进水口细菌种类有一定的相似,8 d仔鱼及22 d仔鱼与其饵料细菌种类有一定程度的相似。
DGGE结果显示,2 d、8 d和22 d仔鱼消化道菌群DGGE图谱相似,仔鱼和40 d稚鱼消化道菌群DGGE图谱存在明显差异;仔鱼与养殖水体菌群DGGE图谱具有一定的相似性;22 d仔鱼与后期饵料轮虫菌群图谱相似性较高,8 d仔鱼与早期饵料牡蛎受精卵的相似性较低。
2、不同生长速度斜带石斑鱼幼鱼消化道菌群结构的研究
利用纯培养方法结合PCR/DGGE方法,分析不同生长速度70 d幼鱼消化道菌群结构。纯培养试验结果表明,生长速度慢的幼鱼消化道细菌总数(5.4×106 CFU/g)少于生长速度快的幼鱼消化道细菌总数(9.0×106 CFU/g),但生长速度慢的幼鱼消化道弧菌数量(6.0×105 CFU/g)明显多于生长速度快的幼鱼(3.4×105 CFU/g),生长速度慢的幼鱼消化道乳酸菌数量(1.5×105 CFU/g)也多于生长速度快的幼鱼消化道乳酸菌数量(6.0×104 CFU/g)。水样中分离到的细菌总数(5.4×105 CFU/ml)、弧菌数(2.2×103 CFU/ml)均少于幼鱼消化道;水样中未分离到乳酸菌。生长速度慢的幼鱼消化道中四种弧菌占细菌总数的12.3%,生长速度快的幼鱼消化道中仅两种弧菌,占细菌总数的比例降低到3.6%;生长速度慢的幼鱼消化道中假单胞菌所占细菌比例12.0%,生长速度快的幼鱼消化道中假单胞菌占细菌总数降低到5.6%。
仅在生长速度快的幼鱼消化道分离到短小芽孢杆菌、克劳氏芽孢杆菌和嗜冷杆菌且数量占绝对优势。抑菌试验表明,短小芽孢杆菌、克劳氏芽孢杆菌和嗜冷杆菌对弧菌具有抑制生长作用。提示短小芽孢杆菌、克劳氏芽孢杆菌和嗜冷杆菌可能与石斑鱼的生长和健康有关。系统进化树结果表明,两组幼鱼消化道的17种细菌隶属于γ-变形杆菌纲,β-变形杆菌纲和芽孢杆菌纲。
DGGE结果显示,生长速度慢的幼鱼消化道菌群多样性明显低于生长速度快的幼鱼消化道。割胶测序结果显示,生长速度快的幼鱼消化道优势菌隶属于不动杆菌属细菌(Acinetobacter baumannii)、结核杆菌(Mycobacterium tuberculosis)、脱硫肠球菌属细菌(Desulfotomaculum reducens)和Petrotoga mobilis;生长速度慢的幼鱼优势菌均隶属于不动杆菌属(Acinetobacter)。
3、溶藻弧菌感染后斜带石斑鱼消化道菌群结构的研究
溶藻弧菌感染后斜带石斑鱼消化道菌群分析结果显示,对照组胃(1.2×105 CFU/g )、前肠(5.1×106 CFU/g)和后肠(2.8×105 CFU/g)和实验组的胃(3.0×105 CFU/g)、前肠(3.1×106 CFU/g)、后肠(3.1×106 CFU/g)的细菌总数量相近;对照组石斑鱼胃和后肠细菌种类明显多于实验组石斑鱼胃和后肠,但对照组前肠细菌种类少于实验组前肠细菌种类。
DGGE结果显示,对照组和实验组细菌多样性具有明显差异。斜带石斑鱼胃菌群结构在灌胃溶藻弧菌后第1 d到第3 d细菌多样性变化不大,但第4 d细菌种类明显减少,第5 d细菌种类则呈回升趋势;前肠细菌多样性分析显示,实验组第1 d和对照组有一定的相似性,实验组第3 d和第4 d相似性较高,细菌多样性明显减少;后肠细菌多样性分析显示,灌胃溶藻弧菌第1 d时,细菌种类较多,第4 d时,细菌多样性降到最低,对照组和实验组第1 d和第5 d相似性较高,实验组第3 d和第4 d相似性较高。割胶测序结果显示,Escherichia coli、Petrotoga mobilis和Vibrio furnissii三种菌在对照组和实验组5 d内的胃、前肠、后肠均检测到,实验组第4 d细菌可检出细菌种类很少,而Shewanella sp.和Vibrio fischeri泳带很强,为第4 d的优势菌。
In the present study, traditional culture-based techniques and denaturing gradient gel electrophoresis (DGGE) tequnique were used to study the gut microbiota of larvae, juvenile and two groups of young grouper Epinephelus cioides with different grow rate. In addition, the gut microbiota of E. cioides infected artificially with Vibrio alginolyticus was investigated. The general results are presented as follows:
1. The aim of the study was to investigate the gut microbiota of the different stages from the eggs hatch to juvenile grouper. Larvae and juvenile samples were collected at day 2, 8, 22 and day 40 post hatch. The results showed that the total cultivable bacterial number increased from the larvae stage to juvenile stage, from 4.0×102 CFU to 1.4×107 CFU per fish. Lactic acid bacteria were only found in the 2 d larvae grouper and no Vibrio sp. observed. Vibrio was found in the 22 d larvae and 40 d juvenile grouper. Six bacterial species were found in the gut of 2 d larvae, but only three bacterial species were found in the gut of 8 d larvae. The cultivable bacterial species are similar between the larvae and inlet water, while those in 8 d and 22 d larvae are similar with there diets.
DGGE results showed that the diversity of gut microbiota of 2 d, 8 d and 22 d of larvael grouper was similar. However, the DGGE profiles of the larvae and juvenile grouper showed an apparent difference. The DGGE profile of 22 d larvae grouper was similar to that of rotifer. There was a low similarity of the DGGE profile between 8 d larvae and its diets (fertilized eggs of the Pacific oyster Crassostrea gigas).
2. The gut microbiota of two groups of juvenile grouper Epinephelus coioides, slow growing and fast growing grouper at 70 days post–hatch, were investigated by using traditional culture-based techniques and PCR/DGGE technique. Traditional culture-based techniques showed that the total bacterial number in the slow growing grouper ( 5.4×106 CFU/g ) was lower than those in the gut of fast growing grouper ( 9.0×106 CFU/g ). However the number of Vibrio and Lactic acid bacteria in the slow growing grouper ( 6.0×105 CFU/g and 1.5×105 CFU/g respectively ) were higher than those in the fast growing fish ( 3.4×105 CFU/g and 6.0×104 CFU/g ). The number of total bacteria ( 5.4×105 CFU/ml ), Vibrio ( 2.2×103 CFU/ml ) in the inlet water were lower than those in the gut of juvenile grouper, and no lactic acid bacteria observed in the inlet water. Four Vibrio species were isolated and comprised 12.3% of the total gut bacteria in slow growing grouper, whereas only two Vibrio species were isolated and comprised 3.6% of the total bacteria in fast growing grouper. Pseudomonas comprised 12.0% in the slow growing grouper, wheras only 5.6% in the fast growing grouper.
Bacillus pumilus, Bacillus clausii and Psychrobacter sp. were only isolated and dominated in the gut of the fast growing fish. The three species showed antagonistic effect on pathogenic Vibrio, this may cause the lower number and less species of Vibrio in the gut of fast growing grouper and suggesting fast growing fish might harbor a favorable microbiota. The bacteria in the gut of grouper could be classified into three groups belonging toγ-proteobacteria,β-proteobacteria and Bacilli class.
The results of DGGE analyses showed that the bacterial diversity in the gut of slow growing grouper was obvious lower than that in the fast growing grouper. Sequencing results showed that the fast growing grouper was dominated by Acinetobacter baumannii、Mycobacterium tuberculosis、Desulfotomaculum reducens and Petrotoga mobilis, however the slow growing grouper gut was dominated by Acinetobacter.
3. The results of DGGE analyses showed the bacterial diversity in the gut microbiota of grouper after administered intragastrically with Vibrio alginolyticus . Sequencing results showed that the stomach、fore gut and hind gut samples were collected at day 1 to day 5 after administered intragastrically with Vibrio alginolyticus. The results showed that the total cultivable bacteria in the stomach, fore- and hind-gut of the experiment group were similar with the control group. The bacterial species in the stomach and hind gut of the experiment group were obvious less than the control group, however the species in the fore hand of experiment group were more than the control group.
DGGE results showed that the bacterial diversity were obvious differences between the experiment group and control group. The microbiota of stomach was relative stable in the first three days after administered intragastrically with V. alginolyticus, whereas many bacteria disappeared in the fourth day, but appeared again in the fifth day. The fore gut bacterila diversity of the experiment group was no change in the first day compared to control group . The bacterial diversity was similar in the third and fourth day , with the lowest species. The hind gut bacterial diversity of the experiment group was much more in the first day , howere it reduced to the lowest in the fourth day. Sequencing results showed that Escherichia coli、Petrotoga mobilis and Vibrio furnissii were isolated from the stomch, fore and hind gut of both control group and experiment group . There were fewer bacterial species in the gut of experiment group in the fourth day, however, Shewanella sp. and Vibrio fischeri dominated.
引文
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