豪猪血蜱的形态学和分子生物学鉴定

Morphological and molecular biological identification of Haemaphysalis hystricis

为豪猪血蜱的鉴定提供准确依据,从湖南省怀化地区猪獾体表采集蜱,运用体视显微镜超景深系统观察其形态学特征;提取蜱体壁总DNA,经PCR扩增测序获得其COX1、ITS2和16S rDNA基因序列,与GenBank数据库中序列比对,并构建系统发育树,进行同源性分析。结果显示,该蜱种雌蜱呈南瓜子形,前窄后宽;假头基为矩形,基突后缘短薄,末端刺宽圆;孔区圆形;须肢圆短,第3节背面有1对刺;盾板两侧缘呈深弧状弯曲,刻点散布均匀;颈沟短而深;缘垛较宽但不明显;无外距,内距粗短呈锥状;生殖孔为圆形,前端与基节Ⅱ中部平行,后端止于基节Ⅲ前缘;气门板呈逗点形;肛门圆形,肛沟位于肛后呈酒杯状。雄蜱除体型较小、体色较浅、盾板覆盖全背、颈沟短而浅、缘垛明显形状狭长、生殖孔与基节Ⅱ平行饱满呈唇形、肛门平行于第1缘垛呈圆形突出状、肛沟呈马蹄状等特征外,其他结构与雌蜱的相似。该蜱种COX1、ITS2和16SrDNA基因序列大小分别为749、1 656和440 bp。经测序比对,该蜱种COX1、16SrDNA基因序列与GenBank中已有豪猪血蜱COX1、16S rDNA基因序列的相似度分别可达99.21%、99.32%以上;该蜱种ITS2基因序列在GenBank中尚未有记录,其仅与长角血蜱ITS2基因序列(KX450306)的相似度较高为91.32%。通过COX1、ITS2和16S rDNA基因系统发育树分析,该蜱种与其他血蜱种聚于同一属分支下。结果表明,从猪獾体表采集的蜱种为豪猪血蜱,结合形态结构及分子标记特征较易鉴定该蜱种。

This experiment was conducted to provide an accurate basis for the identification of Haemaphysalis hystricis.Ticks were collected from the body surface of Arctonyx collaris in Huaihua,Hunan Province.The morphological features were observed by the super-depth-of-field system of stereomicroscope.Extracted total DNA of perisome of the ticks was used to amplify the gene sequences of cytochrome oxidase subunit I(COX1),nuclear second internal transcribed spacer(ITS2),and mitochondrial 16 S ribosomal DNA(16 S r DNA).These gene sequences were compared with the existing sequences in Gen Bank.The phylogenetic trees were constructed for homology analysis.The results showed that the female ticks were pumpkinshaped,anterior margin nearly narrow,posterior margin broad;basis capituli subrectangular,posterior margin of cornua thinly short with broadly rounded apex;porose area round;palp short rounded,posterodorsal spurs on segment 3;lateral grooves of scutum deeply curved,punctations uniform in distribution,cervical groovers short and very deep;festoons broad and indistinct;no external spurs,internal spurs moderately short and blunt,awl-shaped;genital opening round,anterior margin parallelling the medially of coxae Ⅱ,posterior margin finished to the anterior border of coxae Ⅲ;peritreme comma-shaped;anus round,anal groove behind the anus,goblet-shaped.Male ticks were moderately small,colour moderately shallow,scutum covering dorsal;cervical groovers short,very shallow;festoon obviously narrow,distinct;genital opening parallelling the medially of coxae Ⅱ,moderately round,lip-shaped;anus parallelling the first festoon,round,slightly convex;anal groove horseshoe-shaped.Other structures of the males were closer to those of the females.The size of the amplified COX1,ITS2,and 16 S r DNA gene sequences of the ticks in this study was found to be approximately 749,1 656 and 440 bp,respectively.After sequencing and contrasting,the similarity between the COX1 and 16 S r DNA gene sequences of the ticks in this study and the COX1 and 16 S r DNA gene sequences of the existing H.hystricis in Gen Bank was more than99.21% and 99.32%,respectively.There was no homogeneous ITS2 gene sequence of the ticks in this study in Gen Bank.The ITS2 gene sequence of the ticks in this study was 91.32% similar to ITS2 gene sequence of H.longicornis(KX450306).Through analysis of COX1,ITS2 and 16 S r DNA gene,the ticks in this study and other Haemaphysalis sp.recorded in Gen Bank clustered under the same genus branch in phylogenetic trees.The results suggested that the ticks collected from the body surface of A.collaris were H.hystricis,which were easy to distinguish and identify by significant morphological features and molecular marker characteristics.