家蝇发育过程中肠道可培养共生细菌的分离与鉴定

摘要/Abstract

摘要：

目的研究家蝇（Musca domestica）不同发育阶段肠道中可培养共生细菌的组成。方法在无菌条件下,对不同发育阶段家蝇虫体进行体表消毒。对卵和孵化1 d的幼虫,取5枚卵或5条幼虫,加1 ml无菌水研磨;对孵化2 d以上的幼虫、蛹和成蝇,各虫期分别取5条,解剖出肠道,加1 ml无菌水研磨。取研磨液按传统分离培养法分离和鉴定家蝇肠道的共生细菌,挑取形态有差异的单菌落,置于LB培养基中小量培养,提取菌体DNA,PCR扩增16S rDNA基因,测序后在GenBank中进行比对,鉴定到属。计数家蝇不同虫期肠道中分离的细菌菌属数的变化情况。结果通过培养分离出家蝇各阶段肠道共生细菌26属,其中,卵期未分离到共生细菌,幼虫期分离到11属,蛹期分离到19属,成蝇分离到14属。不同虫期之间存在重复,幼虫期和蛹期共有的细菌有6属,分别为变形杆菌属（Proteus）、库特氏菌属（Kurthia）、博德特氏菌属（Bordetella）、微杆菌属（Microbacterium）、产碱杆菌属（Alcaligenes）和明串球菌属（Leuconostoc）;蛹期和成蝇共有的细菌有3属,分别为鞘氨醇杆菌属（Sphingobacterium）、肠球菌属（Enterococcus）、苍白杆菌属（Ochrobactrum）;幼虫期和成蝇共有的细菌有1属,为克雷伯菌属（Klebsiella）;3个虫态共有的细菌有4属,分别为普罗威登斯菌属（Providencia）、葡萄球菌属（Staphylococcus）、香味菌属（Myroides）和假单胞菌属（Pseudomonas）。幼虫期、蛹期和成蝇革兰阳性菌的比例分别为4/11、6/19和3/14。结论在家蝇由幼虫经蛹发育到成蝇的过程中,其肠道中可培养细菌菌属数呈先上升后下降趋势,革兰阳性菌的比例逐渐下降。

关键词: 家蝇, 变态发育, 肠道, 可培养共生细菌

Abstract:

Objective To identify the intestinal cultivable bacteria in Musca domestica at different development stages. Methods Five eggs or 5 larvae at day 1 after egg hatch were collected under aseptic conditions, and were homogenized in 1 ml sterile water. At day 2 or above, 5 larvae. 5 pupae, and 5 adult worms were also collected, and their intestinal tract was isolated and homogenized in 1 ml sterile water. The intestinal symbiotic bacteria were cultured from the homogenate. Single colonies with different morphology were picked up and cultured in LB medium for small-scale production. DNA was extracted to amplify 16S rDNA by PCR. The amplification products were sequenced and blasted in GenBank to determine the bacteria genus. The numbers of genera at different developmental stages were recorded. Results A total of 26 bacterial genera were obtained, comprising 11 in larvae stage, 19 in pupae stage, and 14 in adult worms. None cultivable symbiotic bacterium was isolated from eggs. There were genus overlaps between stages. The larvae and pupae stages shared 6 genera, consisting of Proteus, Kurthia, Bordetella, Microbacterium, Alcaligenes and Leuconostoc. The pupae and adult stages shared 3 genera, consisting of Sphingobacterium, Enterococcus and Ochrobactrum. The larvae and adult stages shared 1 genus, the Klebsiella. The three stages shared 4 genera, consisting of Providencia, Staphylococcus, Myroides and Pseudomonas. The proportions of gram-positive bacteria were 4/11, 6/19 and 3/14 in the larvae, pupae and adult stages, respectively. Conclusion The genus number of cultivable bacteria from Musca domestica shows a trend of increase followed by a decrease during the development from larvae to pupae stage, accompanied with a gradual decrease of proportions of gram-positive bacteria.

Key words: Musca domestica, Metamorphotic development, Intestine, Cultivable symbiotic bacteria

中图分类号:

R384.21

刘婧, 陈丹, 庄桂芬, 黄振东, 薛志静, 张瑞玲, 张忠. 家蝇发育过程中肠道可培养共生细菌的分离与鉴定[J]. 中国寄生虫学与寄生虫病杂志, 2017, 35(2): 120-124.

Jing LIU, Dan CHEN, Gui-fen ZHUANG, Zhen-dong HUANG, Zhi-jing XUE, Rui-ling ZHANG, Zhong ZHANG. Isolation and identification of cultivable symbiotic bacteria from the intestinal tract of Musca domestica during development[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2017, 35(2): 120-124.

图/表 2

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菌属 Genus	幼虫期 Larvae	蛹期 Pupae	成蝇 Adult
变形杆菌属Proteus	+	+
普罗威登斯菌属Providencia	+	+	+
库特氏菌属Kurthia	+	+
葡萄球菌属Staphylococcus	+	+	+
香味菌属Myroides	+	+	+
博德特氏菌属Bordetella	+	+
微杆菌属Microbacterium	+	+
产碱杆菌属 Alcaligenes	+	+
克雷伯菌属 Klebsiella	+		+
明串球菌属 Leuconostoc	+	+
假单胞菌属 Pseudomonas	+	+	+
柠檬酸杆菌属 Citrobacter		+
血杆菌属 Sanguibacter		+
鞘氨醇杆菌属 Sphingobacterium		+	+
肠球菌属 Enterococcus		+	+
乳球菌属 Lactococcus		+
拉乌尔菌属 Raoultella		+
短波毛单胞菌属 Brevundimonas		+
俊片菌属 Lampropedia		+
苍白杆菌属 Ochrobactrum		+	+
肠杆菌属 Enterobacter			+
不动杆菌属 Acinetobacter			+
寡养单胞菌属 Stenotrophomonas			+
土壤杆菌 Agrobacterium			+
代尔夫特菌属 Delftia			+
漫游球菌属 Vagococcus			+
合计 Total	11	19	14