内蒙古全沟硬蜱的鉴定、人工饲养及生活史观察

doi:10.12140/j.issn.1000-7423.2024.03.010

中国寄生虫学与寄生虫病杂志 ›› 2024, Vol. 42 ›› Issue (3): 345-353.doi: 10.12140/j.issn.1000-7423.2024.03.010

内蒙古全沟硬蜱的鉴定、人工饲养及生活史观察

孙连阳¹(), 崔浩¹, 董晓楠¹, 康佳美¹, 丁玉林¹^,², 习娟³, 杨洋⁴, 贺志雄⁵, 刘永宏¹^,², 赵丽¹^,²^,^*()

1 内蒙古农业大学兽医学院，呼和浩特 010000
2 农业农村部动物疾病临床诊疗技术重点实验室，内蒙古呼和浩特 010000
3 内蒙古自治区市场监督管理局综合保障中心，呼和浩特 010000
4 内蒙古自治区鄂尔多斯市检验检测中心，鄂尔多斯 017010
5 内蒙古自治区呼和浩特市回民区动物疫病防疫检疫中心，呼和浩特 010000

收稿日期:2024-03-14 修回日期:2024-05-11 出版日期:2024-06-30 发布日期:2024-07-16
通讯作者: 赵丽
作者简介:孙连阳（1999—），男，硕士研究生，从事寄生虫研究。E-mail：1317916803@qq.com
基金资助:
国家自然科学基金(32260887)

Identification, artificial rearing and observation of life cycle of Ixodes persulcatus in Inner Mongolia

SUN Lianyang¹(), CUI Hao¹, DONG Xiaonan¹, KANG Jiamei¹, DING Yulin¹^,², XI Juan³, YANG Yang⁴, HE Zhixiong⁵, LIU Yonghong¹^,², ZHAO Li¹^,²^,^*()

1 College of Veterinary Medicine, Inner Mongolia Agricultural University, Hohhot 010000, China
2 Key Laboratory of Clinical Diagnosis and Treatment Technology of Animal Diseases, Ministry of Agriculture and Rural Affairs, Hohhot 010000, Inner Mongolia, China
3 Comprehensive Security Center of Inner Mongolia Autonomous Region Market Supervision Administration, Hohhot 010000, China
4 Ordos Inspection and Testing Center, Inner Mongolia Autonomous Region, Ordos 017010, China
5 Center for Animal Epidemic Prevention and Quarantine of Huimin District, Hohhot 010000, China

Received:2024-03-14 Revised:2024-05-11 Online:2024-06-30 Published:2024-07-16
Contact: ZHAO Li
Supported by:
National Natural Science Foundation of China(32260887)

摘要/Abstract

摘要：

目的对采自内蒙古的全沟硬蜱进行形态学和分子生物学鉴定，并通过人工饲养了解全沟硬蜱的发育生活史和生物学特性。方法采集自内蒙古自治区东北部的蜱，利用3D超景深显微系统进行形态学鉴定。提取蜱DNA，PCR扩增线粒体12S rDNA和16S rDNA序列。取阳性扩增产物测序后进行BLAST比对，采用邻接法构建蜱线粒体12S rDNA和16S rDNA的系统进化树。以昆明小鼠为供血动物，在温度（25 ± 3）℃、相对湿度70%~90％条件下对蜱进行人工饲养，观察蜱生活史各阶段的时间等生物学特性。卵孵化实验随机选取10只饱血雌成蜱所产的卵各30枚，观察其孵化情况，并计算孵化率。幼蜱蜕皮实验随机选取饱血幼蜱200只，记录其蜕皮情况，并计算蜕皮率。若蜱蜕皮实验随机选取饱血若蜱100只，分为10组，每组10只，观察其蜕皮情况，并计算蜕皮率。将各阶段发育成功率统计后计算得出其综合发育率。结果形态学鉴定结果显示，采集的雌性和雄性硬蜱均符合硬蜱属的形态。PCR扩增和测序结果显示，硬蜱DNA扩增出长度为320 bp的12S rDNA序列和长度为455 bp的16S rDNA序列。BLAST序列比对分析显示，雌蜱和雄蜱线粒体12S rDNA序列与全沟硬蜱（GenBank：MF095801.1和JF758624.1）序列相似性最高，分别为99.69%和99.09%。雌蜱和雄蜱线粒体16S rDNA序列与全沟硬蜱（GenBank：MH790201.1和MH790200.1）的序列相似性最高，分别为99.75%和99.50%。进化树分析结果显示，雌蜱和雄蜱均与全沟硬蜱序列聚于同一分支上。人工饲养全沟硬蜱的生活史观察结果显示，饱血雌蜱产卵期为12~17 d，平均产卵期为14.6 d，总计产卵数约1 510~1 970枚/只，平均产卵数约1 817枚/只，日均产卵量约124枚/只；卵经21~28 d孵化为幼蜱，平均孵化期为24.8 d，孵化率为89.7%（269/300）；幼蜱吸血期为3~5 d，平均吸血期为4.5 d，饱血幼蜱经18~25 d蜕皮为若蜱，平均蜕皮期为22.7 d，蜕皮率为86.5%（173/200）；若蜱吸血期为5~8 d，平均吸血期为6.3 d，饱血若蜱经120~170 d蜕皮为成蜱，平均蜕皮期为157.2 d，蜕皮率为92.0%（92/100）；从饱血雌成蜱开始产卵，发育至下一代成蜱平均需要241.6 d，综合发育率为71.4%。结论通过形态学和分子生物学鉴定，采自内蒙古的硬蜱为全沟硬蜱。人工饲养获得全沟硬蜱从卵、幼蜱、若蜱、成蜱的生活史及其生物学特征。

关键词: 全沟硬蜱, 形态学鉴定, 分子生物学鉴定, 人工饲养, 生活史

Abstract:

Objective To identify the morphological and molecular characteristics of Ixodes persulcatus collected from Inner Mongolia and to understand their developmental life cycle and biological characteristics through artificial rearing. Methods Ticks were collected from the northeastern region of Inner Mongolia and identified morphologically using a 3D super depth microscope system. Tick DNA was extract, and mitochondrial 12S rDNA and 16S rDNA sequences was amplifed by PCR. After sequencing the positive amplification products, BLAST alignment was performed on the sequences, and the tick mitochondrial 12S rDNA and 16S rDNA genes phylogenetic trees were constructed using the neighbor joining algorithm. Kunming mice were used as blood source to artificially rear the ticks at a temperature of (25 ± 3) ℃ and a relative humidity of 70%-90%. The biological characteristics of the tick life cycle at different stages were observed. In the egg hatching experiment, 30 eggs from each of 10 fully blood-fed female adult ticks were randomly selected, their hatching was observed, and the hatching rate was calculated. A total of 200 engorged larval ticks were randomly selected for the molting experiment, and their molting status was recorded and the molting rate was calculated. 100 engorged nymphal ticks were randomly selected for the nymphal ticks molting experiment and divided into 10 groups, with 10 ticks in each group. The molting situation was observed, and the molting rate was calculated. The comprehensive development rate of each stage after calculating the success rate of development was calculated. Results The morphological identification results showed that both the collected female and male ticks conform to the morphology of Ixodes. The PCR amplification and sequencing results showed that the 12S rDNA sequence with a length of 320 bp and the 16S rDNA sequence with a length of 455 bp were amplified from tick DNA. BLAST sequence alignment analysis showed that the amplified mitochondrial 12S rDNA sequences of female and male ticks had the highest homology with the sequences of I. persulcatus (GenBank: MF095801.1 and JF758624.1), with 99.69% and 99.09%, respectively. BLAST sequence alignment analysis showed that the amplified mitochondrial 16S rDNA sequences of female and male ticks had the highest homology with the sequences of I. persulcatus (GenBank: MH790201.1 and MH790200.1), with 99.75% and 99.50%, respectively. The genetic evolution analysis results showed that both female and male ticks clustered on the same branch as the sequences of the I. persulcatus. The observed life cycle of artificially reared I. persulcatus showed that the oviposition period of engorged female adult ticks is 12-17 days, with an average oviposition period of 14.6 days. The total number of eggs laid is about 1 510-1 970 per tick, with an average oviposition of about 1 817 per tick, and a daily average oviposition of about 124 per tick. The eggs hatched into larval ticks after 21-28 days, with an average hatching period of 24.8 days and a hatching rate of 89.7% (269/300). The blood-feeding period of the larval ticks is 3-5 days, with an average blood-feeding period of 4.5 days. After 18-25 days of molting, engorged larval ticks molt as nymphal ticks, with an average molting period of 22.8 days and a molting rate of 86.5% (173/200). The blood-feeding period of nymphal ticks is 5-8 days, with an average blood-feeding period of 6.3 days. Engorged nymphal ticks molted after 120-170 days to become adult ticks, the average molting period is 157.2 days, and the molting rate is 92.0% (92/100). It took an average of 241.6 days to develop to the next generation of adult ticks from the engorged female adult ticks laying eggs, with a comprehensive development rate of 71.4%. Conclusion The ticks collected from Inner Mongolia is confirmed to be I. persulcatus through morphological and molecular biology identification. Artificial feeding experiments were conducted to obtain the biological characteristics of the life cycle of the I. persulcatus, including eggs, larval ticks, nymphal ticks, and adult ticks.

Key words: Ixodes persulcatus, Morphological identification, Molecular biological identification, Artificial feeding, Life cycle

中图分类号:

R384.4

孙连阳, 崔浩, 董晓楠, 康佳美, 丁玉林, 习娟, 杨洋, 贺志雄, 刘永宏, 赵丽. 内蒙古全沟硬蜱的鉴定、人工饲养及生活史观察[J]. 中国寄生虫学与寄生虫病杂志, 2024, 42(3): 345-353.

SUN Lianyang, CUI Hao, DONG Xiaonan, KANG Jiamei, DING Yulin, XI Juan, YANG Yang, HE Zhixiong, LIU Yonghong, ZHAO Li. Identification, artificial rearing and observation of life cycle of Ixodes persulcatus in Inner Mongolia[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2024, 42(3): 345-353.

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	(许士奇. 全沟硬蜱种群生态及蜱传病原体研究[D]. 石家庄: 河北师范大学, 2015: 2-7.)
[36]	Troughton DR, Levin ML. Life cycles of seven ixodid tick species (Acari ∶ Ixodidae) under standardized laboratory conditions[J]. J Med Entomol, 2007, 44(5): 732-740.

基因 Gene	引物序列 Primer seqence	扩增长度/bp Amplification length/bp
12S rDNA	F：AAACTAGGATTAGATACCCT	320
	R：AATGAGAGCGACGGGCGATGT
16S rDNA	F：TTAAATTGCTGTRGTATT	455
	R：CCGGTCTGAACTCASAWC

内蒙古全沟硬蜱的鉴定、人工饲养及生活史观察

Identification, artificial rearing and observation of life cycle of Ixodes persulcatus in Inner Mongolia

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