云南边境地区埃蠓属一新种形态学和分子生物学鉴定
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摘要
目的采用形态学和分子生物学方法对在中国-老挝-越南三国交界地区采集蠓虫样本中的1种埃蠓进行系统分类鉴定。方法 2018年5月在云南省江城县中老越三国交界地区采集蠓虫样本,提取蠓虫基因组DNA,分别采用ITS-1基因和COⅠ基因特异引物进行PCR扩增、序列测定。采用Clustal X2.1软件、DNAStar软件和Mega 7.0进行序列分析;同时对蠓虫进行压片,在体视显微镜下观察形态学特征,并计算AR、HR、PR、CR和TR1、TR2、TR3等数值。结果江城采集到的1只雌性蠓虫具有两复眼相连、无毛、复眼间有一额鬃;触角15节,触须5节,第3节中部膨大有1小圆形感觉器窝;唇片鬃左右各2根,大颚端部有齿7枚,第5齿钝小。翅有2个发达径室,径2室和径1室长度之比值2.069,除翅基淡斑外,有9个淡斑,中2脉(M2)中部两侧淡斑相连,翅沿中脉有5个暗斑,除端部小暗斑外,其余4块均覆及径脉,径中横脉处暗斑扩延至翅后缘。中脉基部泡状膨大,被暗斑覆盖,第2径室(2R2)端部1/2和基部1/6被浅色暗斑覆盖。后足跗节1~3节各有1个端刺。腹部有2个球形受精囊,略不等大,有颈,另有1退化小囊等形态学特征。遗传进化分析结果显示:在ITS-1基因构建系统进化树上,江城县采集的蠓虫与环纹埃蠓形成一个进化分支,核苷酸同源性在92%~93%之间,而与其他蠓虫核苷酸同源性均低于81%;在COⅠ基因构建的系统进化树上,江城采集的蠓虫位于独立进化分支内,与其他蠓虫核苷酸同源性均低于84%。结论形态学和分子生物学鉴定结果均提示江城采集的这只蠓虫为埃蠓属1新种,丰富了我国蠓虫种类资源。
Objective To systematically classify and identify one species of Allohelea Kieffer,1917 collected from the border area of China-Laos-Vietnam border area,Yunnan province,using morphological and molecular biological methods.Methods Specimens of midges were collected in Jiangcheng county,Yunnan province in May 2018.DNA was extracted from midge,PCR amplification and sequencing were performed by using ITS-1 gene and COⅠgene specific primers,respectively.The sequence analysis was performed by using Clustal X2.1,DNAStar and Mega 7.0 bioinformatics softwares.Meanwhile,midge body was subject to Clear remainder,dehydration and mounts the body on the slide.Identification of midge based on morphologic characters under the Stereomicroscope,and the numerical values of AR,HR,PR,CR and TR1,TR2,TR3 were calculated.Results A female midge collected from Jiangcheng county had morphology characteristics such as:two connected compound eyes,hairless,and a frontal bristle between the compound eyes,15 segment antennae,5 segment tentacles with a small rounded sensory fossa in the middle of segment3,2 bristles on each sides of the lip,7 teeth at the end of the maxilla of which the fifth is blunt and small.There were two developed radial cells in the wing with the ratio of the length of them was 2.069.Except for the clear patches on the base of the wing,there were nine clear patches on the wing.The clear patches on both sides of the middle veins 2(M2)were connected.There were 5 gray patches along the middle vein of wing.Except for the small gray patches at the end,the other 4 gray patches covered the middle vein,and the gray patches on the cross veins extend to the rear edge of wing.At the base of middle vein,there was enlarged bubble with gray patches cover.Theend 1/2 and base 1/6 of the 2 nd radial cell(2 R2) were covered by light gray patches. There was an end spine on each of the 1-3 segments of the hind tarsal segment. The abdomen had two slightly unequal and spherical spermathecas with Necks,and one degenerated sac. The phylogenetic analysis based on ITS-1 gene sequences showed that midges collected in Jiangcheng county and Allohelea annulata Yu,2005 formed an evolutionary branch and nucleotide homology was 92%-93%,while the nucleotide homology with other midges was less than 81%. On the tree based on CO Ⅰgene,the midges collected from Jiangcheng county formed an independent evolutionary branche and the nucleotide homology with other midges was less than 84%. Conclusion The results of morphological and molecular biological identification suggested that this midge collected at Jiangcheng is a new species of Allohelea,which enriches the species resources of midges in China.
Objective To systematically classify and identify one species of Allohelea Kieffer,1917 collected from the border area of China-Laos-Vietnam border area,Yunnan province,using morphological and molecular biological methods.Methods Specimens of midges were collected in Jiangcheng county,Yunnan province in May 2018.DNA was extracted from midge,PCR amplification and sequencing were performed by using ITS-1 gene and COⅠgene specific primers,respectively.The sequence analysis was performed by using Clustal X2.1,DNAStar and Mega 7.0 bioinformatics softwares.Meanwhile,midge body was subject to Clear remainder,dehydration and mounts the body on the slide.Identification of midge based on morphologic characters under the Stereomicroscope,and the numerical values of AR,HR,PR,CR and TR1,TR2,TR3 were calculated.Results A female midge collected from Jiangcheng county had morphology characteristics such as:two connected compound eyes,hairless,and a frontal bristle between the compound eyes,15 segment antennae,5 segment tentacles with a small rounded sensory fossa in the middle of segment3,2 bristles on each sides of the lip,7 teeth at the end of the maxilla of which the fifth is blunt and small.There were two developed radial cells in the wing with the ratio of the length of them was 2.069.Except for the clear patches on the base of the wing,there were nine clear patches on the wing.The clear patches on both sides of the middle veins 2(M2)were connected.There were 5 gray patches along the middle vein of wing.Except for the small gray patches at the end,the other 4 gray patches covered the middle vein,and the gray patches on the cross veins extend to the rear edge of wing.At the base of middle vein,there was enlarged bubble with gray patches cover.Theend 1/2 and base 1/6 of the 2 nd radial cell(2 R2) were covered by light gray patches. There was an end spine on each of the 1-3 segments of the hind tarsal segment. The abdomen had two slightly unequal and spherical spermathecas with Necks,and one degenerated sac. The phylogenetic analysis based on ITS-1 gene sequences showed that midges collected in Jiangcheng county and Allohelea annulata Yu,2005 formed an evolutionary branch and nucleotide homology was 92%-93%,while the nucleotide homology with other midges was less than 81%. On the tree based on CO Ⅰgene,the midges collected from Jiangcheng county formed an independent evolutionary branche and the nucleotide homology with other midges was less than 84%. Conclusion The results of morphological and molecular biological identification suggested that this midge collected at Jiangcheng is a new species of Allohelea,which enriches the species resources of midges in China.
引文
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[2] Mellor PS,Boorman J,BaylisM. Culicoides biting midges:their role as arbovirus vectors[J]. Ann Rev Entomol,2000(45):307-340.
[3]虞以新.中国蠓科昆虫(昆虫纲双翅目共2卷)[M].北京:军事医学科学出版社,2006:1324.
[4]孙虹,虞以新,柯明剑.中国埃蠓属一新种(双翅目:蠓亚科)[J].中国媒介生物学及控制杂志,2008,19(1):35-36.
[5]虞以新,严格.福建省埃蠓属二新种[J].寄生虫与医学昆虫学报,2004,11(1):36-39.
[6]刘金华,严格,虞以新.中国海南省埃蠓二新种[J].动物分类学报,1996,21(3):358-360.
[7]王静林,李顺祥,叶少飞,等.云南省华宁县荒川库蠓形态和遗传多态性研究[J].中国国境卫生检疫杂志,2018,41(1):16-20.
[8] Sossah C,Baldet T,Delecolle JC,et al. Molecular detection of Culicoides spp. and Culicoides imicola,the principal vector of bluetongue(BT)and African horse sickness(AHS)in Africa and Europe[J]. Vet Res,2004(35):325-337.
[9] Dallas JF,Cruickshank RH,Linton YM,et al. Phylogenetic status and matrilineal structure of the biting midge,Culicoides imicola,in Portugal,Rhodes and Israel[J]. Medical and Veterinary Entomology,2003(17):379-387.
[10] Romero-Alvarez D,Escobar LE. Oropouche fever,an emergent disease from the Americas[J]. Microbes Infect,2018,20(3):135-146.
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