我国不同地区熊蜂短膜虫的感染情况及亲缘关系

doi:10.3864/j.issn.0578-1752.2019.06.014

摘要/Abstract

摘要：

【目的】探明熊蜂肠道寄生虫——短膜虫（Crithidia bombi）的流行规律以及不同蜂种、不同地区间寄生的熊蜂短膜虫的亲缘关系。【方法】调查内蒙古、甘肃、青海、四川4个省（自治区）的25种1 007只熊蜂的短膜虫感染情况,对不同蜂种、不同地区间熊蜂短膜虫的感染率进行卡方检验（SPSS 22.0）,并对熊蜂短膜虫的内转录间隔区ITS序列进行PCR扩增、克隆、测序。利用特异的ITS引物扩增待检测的熊蜂肠道总DNA,之后进行凝胶电泳检测,通过是否扩增出675 bp的熊蜂短膜虫ITS基因片段来判断待检测的蜂群是否感染了熊蜂短膜虫,再进一步测序,分析不同蜂种、不同地区寄生的熊蜂短膜虫之间的亲缘关系。【结果】在调查的所有样品中,有20种262只熊蜂被短膜虫感染,感染率为26.0%。感染率最高的两种熊蜂是白背熊蜂（Bombus festivus）和火红熊蜂（B. pyrosoma）,而西伯熊蜂（B. sibiricus）的短膜虫感染率最低;青海省熊蜂的短膜虫感染率最高,而内蒙古熊蜂的短膜虫感染率最低;雄蜂的感染率极显著高于工蜂和蜂王。另外,除了甘肃省的黑尾熊蜂（B. melanurus）和猛熊蜂（B. difficillimus）,四川省的疏熊蜂（B. remotus）、兴熊蜂（B. impetuosus）和弗里熊蜂（B. friseanus）所感染的短膜虫与其他短膜虫亲缘关系较远之外,其余大部分的熊蜂短膜虫的亲缘关系较近。【结论】熊蜂短膜虫对我国熊蜂的感染广泛,且不同蜂种、不同地理环境和不同级型间熊蜂短膜虫的感染率均有差别。熊蜂经过长期群体进化演变,不同地区形成相对固定的优势种类,因此各地区所捕捉到的熊蜂种类不同。对我国不同地区熊蜂短膜虫感染情况的统计分析侧重于样本量≥30的种类,这些种类是各地区的优势种类,另外一些熊蜂虽未能采集足够的样本,但根据已采集的样本量分析得出的感染率较高。熊蜂短膜虫存在寄主专一性,且对熊蜂宿主的感染具有适应性。我国部分地区熊蜂感染的短膜虫的亲缘关系总体来说较强,但也受到地理位置及其寄主熊蜂蜂种的影响。

关键词: 熊蜂, 熊蜂短膜虫, 内转录间隔区, 感染率, 亲缘关系

Abstract:

【Objective】The objective of this study is to clarify the prevalence of Crithidia bombi, an intestinal parasite of bumblebees, and its genetic relationship among different species in different regions.【Method】In this study, the infection status of 1 007 bumblebees from 25 species in four provinces (autonomous region) including Inner Mongolia, Gansu, Qinghai, and Sichuan was investigated, and the infection rate of C. bombi in different species and regions was analyzed by chi-square test (SPSS 22.0). PCR amplification, cloning and sequencing were carried out based on the ITS sequences of C. bombi. The specific ITS primers were used to amplify the total DNA in the intestines of bumblebees, and gel electrophoresis was performed. The infection status of bumblebee colony was determined by whether 675 bp of ITS gene fragment of C. bombi was amplified or not, and further sequencing was carried out to analyze the genetic relationship of C. bombi in different species and regions. 【Result】Out of all the samples investigated, 262 bumblebees of 20 species were infected by C. bombi, with an infection rate of 26.0%. Among them, Bombus festivus and B. pyrosoma had the highest infection rates, and B. sibiricus had the lowest infection rate. The infection rate of bumblebee was the highest in Qinghai Province and lowest in Inner Mongolia. The infection rate of male bee was significantly higher than that of worker bee and queen bee. The genetic relationship of most C. bombi was relatively close, except for B. melanurus and B. difficillimus in Gansu Province, and B. remotus, B. impetuosus, B. friseanus in Sichuan Province.【Conclusion】The infection of C. bombi is widespread in China, and the infection rate is different among different species, regions and castes of bumblebee. After a long-term of population evolution, the dominant species of bumblebee are relatively fixed in different regions, so the bumblebee species collected in different regions are different. The statistical analysis of the infection of C. bombi in different regions of China focused on the sample size ≥30, which are the dominant species in each region. Some bumblebees have not collected enough samples, but the infection rate is high according to the sample that we already get. There is host specificity in C. bombi, and C. bombi are adaptive to the infection of the host. In some regions of China, the genetic relationship between the C. bombi is relatively strong on the whole, but it is also affected by the geographical location and the species of bumblebee.

Key words: bumblebee (Bombus spp.), Crithidia bombi, internal transcribed space (ITS), infection rate, genetic relationship

唐裕杰,王刘豪,李凯,李继莲. 我国不同地区熊蜂短膜虫的感染情况及亲缘关系[J]. 中国农业科学, 2019, 52(6): 1110-1118.

TANG YuJie,WANG LiuHao,LI Kai,LI JiLian. Infection Status and Genetic Relationship of Crithidia bombi in Different Regions of China[J]. Scientia Agricultura Sinica, 2019, 52(6): 1110-1118.

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图/表 7

表1

各地区所调查的熊蜂的种类和数量"

省（自治区） Province (autonomous region)	纬度 Latitude	经度 Longitude	海拔 Elevation (m)	蜂种 Species	采集数 Collection number	感染数 Infected number
内蒙古 Inner Mongolia	48°41.448′	122°45.290′	419	密林熊蜂B. patagiatus	41	5
	48°41.448′	122°45.290′	419	明亮熊蜂B. lucorum	4	3
	49°31.497′	117°16.951′	712	西伯熊蜂B. sibiricus	50	0
	49°28.953′	119°40.439′	602	盗熊蜂B. filchnerae	2	2
	49°28.953′	119°40.439′	602	藓状熊蜂B. muscorum	1	0
甘肃 Gansu	36°34.728′	102°58.077′	2096	黑尾熊蜂B. melanurus	7	1
	36°34.728′	102°58.077′	2096	西伯熊蜂B. sibiricus	1	1
	36°49.855′	102°39.003′	2210	火红熊蜂B. pyrosoma	19	13
	36°49.855′	102°39.003′	2210	明亮熊蜂B. lucorum	2	1
	36°49.855′	102°39.003′	2210	盗熊蜂B. filchnerae	3	1
	34°25.522′	102°17.784′	3454	稳纹熊蜂B. waltoni	7	0
	34°25.522′	102°17.784′	3454	拉熊蜂B. laesus	1	0
	34°42.456′	102°30.892′	3288	密林熊蜂B. patagiatus	52	12
	34°42.456′	102°30.892′	3288	兴熊蜂B. impetuosus	2	0
	38°09.402′	100°11.705′	2912	红束熊蜂B. rufofasciatus	103	15
	38°09.402′	100°11.705′	2912	苏氏熊蜂B. sushkini	3	2
	38°09.402′	100°11.705′	2912	伪猛熊蜂B. personatus	2	0
	38°09.402′	100°11.705′	2912	克什米尔熊蜂B. kashmirensis	2	1
	38°09.402′	100°11.705′	2912	越熊蜂B. supremus	3	1
	37°56.221′	100°57.951′	3418	猛熊蜂B. difficillimus	4	1
	37°59.971′	100°45.020′	3214	拉达克熊蜂B. ladakhensis	6	0
	37°59.971′	100°45.020′	3214	昆仑熊蜂B. keriensis	7	1
	38°13.043′	100°56.944′	2835	小雅熊蜂B. lepidus	3	0
青海 Qinghai	37°10.689′	102°03.091′	2596	明亮熊蜂B. lucorum	10	9
	37°10.689′	102°03.091′	2596	火红熊蜂B. pyrosoma	11	7
	37°10.689′	102°03.091′	2596	西氏熊蜂B. sichelii	5	4
	37°10.689′	102°03.091′	2596	小雅熊蜂B. lepidus	9	7
	37°10.689′	102°03.091′	2596	密林熊蜂B. patagiatus	1	0
	37°10.689′	102°03.091′	2596	兴熊蜂B. impetuosus	3	0
	37°41.408′	100°34.319′	3461	稳纹熊蜂B. waltoni	10	0
	37°41.408′	100°34.319′	3461	拉达克熊蜂B. ladakhensis	3	0
	37°41.408′	100°34.319′	3461	伪猛熊蜂B. personatus	2	0
	37°41.408′	100°34.319′	3461	红束熊蜂B. rufofasciatus	1	0
	37°41.408′	100°34.319′	3461	克什米尔熊蜂B. kashmirensis	1	0
四川 Sichuan	28°19.672′	103°07.999′	2062	兴熊蜂B. impetuosus	80	12
	28°19.672′	103°07.999′	2062	小雅熊蜂B. lepidus	12	2
	28°19.672′	103°07.999′	2062	三条熊蜂B. trifasciatus	8	5
	28°19.672′	103°07.999′	2062	白背熊蜂B. festivus	40	28
	28°19.672′	103°07.999′	2062	常熊蜂B. avanus	10	10
	30°02.905′	101°58.049′	2833	疏熊蜂B. remotus	159	32
	28°32.822′	102°10.638′	1785	弗里熊蜂B. friseanus	317	86
合计Total					1007	262

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蜂种 Species	样本数（只） Sample number	感染率 Infection rate (%)	卡方值 χ²value	P值 P value
白背熊蜂B. festivus	40	70.0A	36.368	1.63E-09
火红熊蜂B. pyrosoma	30	66.7A	23.695	0.000001
弗里熊蜂B. friseanus	317	27.1B	0.057	0.812
疏熊蜂B. remotus	159	20.1BC	2.931	0.087
密林熊蜂B. patagiatus	94	18.1BC	3.178	0.075
红束熊蜂B. rufofasciatus	104	14.4C	7.274	0.007
兴熊蜂B. impetuosus	85	14.1C	6.323	0.012
西伯熊蜂B. sibiricus	51	2.0D	15.457	0.000084

省（自治区）Province (autonomous region)	样本数（只） Sample number	感染率 Infection rate (%)	卡方值 χ²value	P值 P value
青海 Qinghai	56	48.2A	12.668	0.000372
四川 Sichuan	626	28.0B	0.457	0.499
甘肃 Gansu	227	22.0B	1.945	0.163
内蒙古Inner Mongolia	98	10.2C	12.655	0.000375

级型 Caste	样本数（只）Sample number	感染率 Infection rate (%)	卡方值 χ²value	P值 P value
雄蜂 Male	76	44.7A	12.467	0.000414
工蜂 Worker	929	24.5B	0.556	0.456
蜂王 Queen	2	0C	0.703	0.402