基于线粒体COI基因的印度新曲厉螨的分子鉴定
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摘要
本研究利用线粒体COI基因对印度新曲厉螨进行分子鉴定,以期为研究该螨的生物学习性及对蜜蜂的危害提供技术支持。分别从中华蜜蜂、意大利蜜蜂工蜂体上,以及桉树花序上采集试螨,扩增印度新曲厉螨的COI基因,并将获得的COI基因序列在Genbank中Blast对比;利用Genedoc软件分析基因序列的相似性;利用MEGA 7.0计算厉螨科不同种螨之间的遗传距离,构建COI基因的系统发育树。结果表明:采自中华蜜蜂、意大利蜜蜂及桉树花序上的印度新曲厉螨样本COI基因序列完全一致,均表现出明显的A/T碱基偏向性,与同属Neocypholaelaps apicola的COI基因序列的相似性为88%。基于COI基因的印度新曲厉螨的种内的遗传距离(0.00)显著小于与厉螨科6种螨的遗传距离(0.203～0.403)且不存在重叠区域。构建的COI基因的系统发育树显示,采自印度新曲厉螨样本COI与同属的Neocypholaelaps apicola聚同一进化枝,其它不同属螨则按照属的不同聚集在不同的进化枝。本研究所用的线粒体COI基因序列的差异可应用于印度新曲厉螨的分子鉴定。
This study established a method for identifying Neocypholaelaps indica using COI gene. PCR and Sanger sequencing were used to obtain COI sequences of the samples collected from Apis cerana cerana, A. mellifera ligustica and Eucalyptus robusta, a host plant. These COI sequences were compared using Blastn in Genbank. Identities of COI gene sequences were analyzed by Genedoc, and MEGA 7.0 was used to calculate the genetic distance and construct phylogenetic trees. All COI gene sequences of N. indica samples were the same and displaying a distinct A+T bias. The sequence showed 83.3% similarity with Neocypholaelaps apicola. The intraspecific genetic distances(0.00) of N. indica samples were not only significantly lower than the interspecific genetic distances(0.203～0.403) but also showing no overlapping area between two genetic distances. Phylogenetic trees indicated that COI gene sequences can group the mites by the genera, and N. indica and N. apicola were clustered in the same branch with the bootstrap value of 88%. The COI gene used in this study could be applied in the molecular identification of N. indica from different regions and host plants.
This study established a method for identifying Neocypholaelaps indica using COI gene. PCR and Sanger sequencing were used to obtain COI sequences of the samples collected from Apis cerana cerana, A. mellifera ligustica and Eucalyptus robusta, a host plant. These COI sequences were compared using Blastn in Genbank. Identities of COI gene sequences were analyzed by Genedoc, and MEGA 7.0 was used to calculate the genetic distance and construct phylogenetic trees. All COI gene sequences of N. indica samples were the same and displaying a distinct A+T bias. The sequence showed 83.3% similarity with Neocypholaelaps apicola. The intraspecific genetic distances(0.00) of N. indica samples were not only significantly lower than the interspecific genetic distances(0.203～0.403) but also showing no overlapping area between two genetic distances. Phylogenetic trees indicated that COI gene sequences can group the mites by the genera, and N. indica and N. apicola were clustered in the same branch with the bootstrap value of 88%. The COI gene used in this study could be applied in the molecular identification of N. indica from different regions and host plants.
引文
Aliabadian M, Kaboli M, Nijman V,et al. Molecular identification of birds: Performance of distance-based DNA barcoding in three genes to delimit parapatric species [J]. PLoS ONE, 2009, 4 (1): e4119.
Ball SL, Hebert PDN, Burian SK, et al. Biological identification of mayflies (Ephemeroptera) using DNA barcodes [J]. Freshwater Science, 2005, 24 (3): 508-524.
Chen F. Study onPhylogenetic Relationships among Some Common Species of Amblyseiinae (Acari: Phytoseiidae) Based on COI Gene and ITS Gene [D]. Nanchang University, 2011. [陈芬. 基于线粒体COI基因和核糖体ITS基因的常见钝绥螨(蜱螨亚纲:植绥螨科)系统发育研究[D]. 南昌大学, 2011]
Dabert A. DNA markers in the phylogenetics of the Acari [J]. Biological Letter, 2006, 43 (2): 97-107.
Deng GF, Pan CW.Neocypholaelaps indica Evans, 1963(Acarina: Ameroseiidae) on honey bee [J]. Acta Entomologica Sinica, 1964, 13 (5): 771-772. [邓国藩, 潘錝文. 蜜蜂体上的印度新曲厉螨(Neocypholaelaps indica Evans, 1963) (Acarina: Ameroseiidae) [J]. 昆虫学报, 1964, 13 (5): 771-772]
Deng GF, Wang DQ, Gu YM. Economic Insect Fauna of China .Vol. 40. Acari: Dermanyssoideae [M]. Beijing: Science Press, 1993: 133-134. [邓国藩, 王敦清, 顾以铭. 中国经济昆虫志. 第四十册, 蜱螨亚纲, 皮刺螨总科[M]. 北京: 科学出版社, 1993: 133-134]
Folmer O, Black M, Hoeh W,et al. DNA primers for amplification of mitochondrial cytochrome C oxidase subunit I from diverse metazoan invertebrates [J]. Molecular Marine Biology Biotechnology, 1994, 3 (5): 294-299.
Ge J, Yang J, Sun WB. Phoretic mite Neocypholaelaps indica Evans infests inflorescences of Pachysandra axillari Franch and its pollinators, Apis cerana Fabricius [J]. Systematic and Applied Acarology, 2017, 22 (4): 602-604.
Hajibabaei M, Janzen DH, Burns JM,et al. From the cover: DNA barcodes distinguish species of tropical Lepidoptera [J]. Proceedings of the National Academy of Sciences of the United States of America, 2006, 103 (4): 968-71.
He LM. Molecular Identification of Pyemotes Based on Mitochondrial COI and Ribosomal ITS2 Gene [D]. Shijiazhang:Hebei Normal University, 2010. [贺丽敏. COI和ITS2基因序列在蒲螨属分子鉴定中的应用研究 [D].石家庄:河北师范大学, 2010]
Hebert PDN, Penton EH, John M,et al. Ten species in one: DNA barcoding reveals cryptic species in the neotropical skipper butterfly Astraptes fulgerator [J]. Proceedings of the National Academy of Sciences of the United States of America, 2004b, 101 (41): 14812-14817.
Hebert PDN, Stoeckle MY, Zemlak TS,et al. Identification of birds through DNA Barcodes [J]. PLoS Biology, 2004a, 2 (10): e312.
Hu F, Li YF, Sheng L,et al. Molecular detection of Callosobruchus maculatus and its allied species based on mitochondrial COI gene [J]. Plant Quarantine, 2017, 31 (3): 25-28.[胡飞, 李云飞, 盛磊, 等. 四纹豆象及其近缘种基于COI基因的分子检测技术研究 [J]. 植物检疫, 2017, 31 (3): 25-28]
Huang SX. The first discovery of main species of mites and Varroa underwoodi in China [J]. Apiculture China, 2004, 55 (2): 4-5. [黄双修. 蜜蜂外寄生螨的主要种类和恩氏瓦螨(Varroa underwoodi)在中国的首次发现 [J]. 中国养蜂, 2004, 55 (2): 4-5]
Kontschán J, Tóbiás I, Bozsik G,et al. First record of Neocypholaelaps apicola from beehives in hungary (ACARI: Mesostigmata: Ameroseiidae): Re-description and DNA barcoding [J]. Acta Zoologica Academiae Scientiarum Hungaricae, 2015, 61 (3): 237-245.
Kuang HO. Eucalyptus insects is Eucalyptus mites [J].Journal of Bee, 2003, 8: 28. [匡海鸥. 桉花虫是桉螨 [J]. 蜜蜂杂志, 2003, 8: 28]
Kumar S, Stecher G, Tamura K. MEGA7: Molecular evolutionary genetics analysis version 7.0 for bigger datasets [J]. Molecular Biology Evolution, 20165, 33 (7): 1870-1874.
Li JB, Li YX, Sun JT,et al. COI barcoding as a molecular assay for the identification of phytoseiid mites [J]. Systematic and Applied Acarology, 2012, 17 (4): 397-406.
Webster LM, Thomas RH, Mccormack GP. Molecular systematics of Acarus siro s. lat., a complex of stored food pests [J]. Molecular Phylogenetics & Evolution, 2004, 32 (3): 817-822.
Luo GC, Zhao RS. The preliminary investigations on bee mites in Taiwan [J]. Agriculture Resource of China, 1975, 9 (1): 50-56. [罗干成, 赵若素. 台湾蜂螨之生态观察[J]. 中华农业研究, 1975, 9 (1): 50-56]
Ma LM, Lin JZ. Descriptions of male and protonymph of Ameroseius longitrichus and Neocypholaelaps indica (Acari: Mesostigmata: Ameroseiidae) [J]. Entomology of Journal East China, 2006, 15 (4): 241-243. [马立名, 林坚贞. 长发美绥螨和印度新曲厉螨雄螨与前若螨描述(蜱螨亚纲:中气门目:美绥螨科)[J]. 华东昆虫学报, 2006, 15 (4): 241-243]
Ma?án P. A revision of the family Ameroseiidae (Acari, Mesostigmata), with some data on Slovak fauna [J]. ZooKeys, 2017, 704 (704): 1-228.
Moraes GJ, Narita JPZ. Description of a new species of Neocypholaelaps (Acari: Ameroseiidae) from Brazil, with a key to the world species [J]. Zootaxa, 2010, 2554 (2554): 37-44.
Narita JPZ, de Moraes GJ, Fernando LCP. Two species of Neocypholaelaps from Sri Lanka (Acari: Ameroseiidae), with description of a new species [J]. Zootaxa, 2011, 2741 (2741): 59-65.
Narita JPZ, Pédelabat M, de Moraes GJ. A new species of Neocypholaelaps Vitzthum (Acari: Ameroseiidae), with notes on the cheliceral lobes and ventral porelike stuctures of mites of this family [J]. Zootaxa, 2013, 3666 (1): 1-15.
Nicholas KB, Nicholas HBJ. Genedoc:A tool for editing and annotating multiple sequence alignments [EB/OL]. www.pscedu/biomed/genedoc. Accessed Feb 2006.
Ren GT, Guo XG. Preliminarylist of Dermanyssoidea from China [J]. Jounal Anhui Agriculture Science, 2008, 36 (36): 15915-15919, 15947. [任天广, 郭宪国. 中国皮刺螨总科名录初报 [J]. 安徽农业科学, 2008, 36 (36): 15915-15919, 15947]
Robert H. Cruickshank. Molecular markers for the phylogenetics of mites and ticks [J]. Systematic and Applied Acarology, 2002, 7 (1): 3-14.
Sakamoto H, Matsuda T, Suzuki R,et al. Molecular identification of seven species of the genus Stigmaeopsis (Acari: Tetranychidae) and preliminary attempts to establish their phylogenetic relationship [J]. Systematic and Applied Acarology, 2017, 22 (1): 91-101.
Verma S.Some observations on the life cycle of the honey bee mite, Neocepholaelaps indica Evan (Acarina: Ameroseiidae) [J]. Pest Management and Economic Zoology, 1995, 3: 65-67.
Wang K, Li YT, Zheng Y,et al. Molecular identification of Grapholita dimorpha Komai (Lepidoptera: Tortricidae) and Grapholita molesta Busck (Lepidoptera: Tortricidae) based on mitochondrial COI and COII genes [J]. Journal of Northwest A & F University (Nature Science Edition), 2016, 44 (2): 156-164. [王康, 李玉婷, 郑燕, 等. 基于线粒体COI和COII基因的沙果小食心虫与梨小食心虫的分子鉴定 [J]. 西北农林科技大学学报(自然科学版), 2016, 44 (2): 156-164]
Zhang W. Some supplement about Neocypholaelaps indica [J]. Apiculture of China, 1965, 4: 103. [张文. 关于印度新曲厉螨的一些补充 [J]. 中国养蜂, 1965, 4: 103]
Zhang Y, Guo XH, Liu GC,et al. Application of DNA barcodes to molecular systematics of Coleoptera [J]. China Journal of Applied Entomology, 2011, 48 (2): 410-416. [张媛, 郭晓华, 刘广纯, 等. DNA条形码在鞘翅目昆虫分子系统学研究中的应用 [J]. 应用昆虫学报, 2011, 48 (2): 410-416]
Zhou SH, Deng YQ, Li SY,et al. Supplementary records of Dermanyssoid mites (Acari: Parasitiformes) in Fujian province [J]. China Journal Vector Biology Control, 2012, 23 (5): 467-470. [周淑姮, 邓艳琴, 李述杨, 等. 福建省皮刺螨总科螨种记录增补 [J]. 中国媒介生物学及控制杂志, 2012, 23 (5): 467-470]
Zhu ZH, Ye H, Zhang ZY. Molecular identification of six Bactrocera species (Diptera: Tephritidae) based on mtDNA [J]. Acta Entomologica Sinica, 2005, 48 (3): 386-390. [朱振华, 叶辉, 张智英. 基于mtDNA Cytb的六种果实蝇的分子鉴定 [J]. 昆虫学报, 2005, 48 (3): 386-390]
Ball SL, Hebert PDN, Burian SK, et al. Biological identification of mayflies (Ephemeroptera) using DNA barcodes [J]. Freshwater Science, 2005, 24 (3): 508-524.
Chen F. Study onPhylogenetic Relationships among Some Common Species of Amblyseiinae (Acari: Phytoseiidae) Based on COI Gene and ITS Gene [D]. Nanchang University, 2011. [陈芬. 基于线粒体COI基因和核糖体ITS基因的常见钝绥螨(蜱螨亚纲:植绥螨科)系统发育研究[D]. 南昌大学, 2011]
Dabert A. DNA markers in the phylogenetics of the Acari [J]. Biological Letter, 2006, 43 (2): 97-107.
Deng GF, Pan CW.Neocypholaelaps indica Evans, 1963(Acarina: Ameroseiidae) on honey bee [J]. Acta Entomologica Sinica, 1964, 13 (5): 771-772. [邓国藩, 潘錝文. 蜜蜂体上的印度新曲厉螨(Neocypholaelaps indica Evans, 1963) (Acarina: Ameroseiidae) [J]. 昆虫学报, 1964, 13 (5): 771-772]
Deng GF, Wang DQ, Gu YM. Economic Insect Fauna of China .Vol. 40. Acari: Dermanyssoideae [M]. Beijing: Science Press, 1993: 133-134. [邓国藩, 王敦清, 顾以铭. 中国经济昆虫志. 第四十册, 蜱螨亚纲, 皮刺螨总科[M]. 北京: 科学出版社, 1993: 133-134]
Folmer O, Black M, Hoeh W,et al. DNA primers for amplification of mitochondrial cytochrome C oxidase subunit I from diverse metazoan invertebrates [J]. Molecular Marine Biology Biotechnology, 1994, 3 (5): 294-299.
Ge J, Yang J, Sun WB. Phoretic mite Neocypholaelaps indica Evans infests inflorescences of Pachysandra axillari Franch and its pollinators, Apis cerana Fabricius [J]. Systematic and Applied Acarology, 2017, 22 (4): 602-604.
Hajibabaei M, Janzen DH, Burns JM,et al. From the cover: DNA barcodes distinguish species of tropical Lepidoptera [J]. Proceedings of the National Academy of Sciences of the United States of America, 2006, 103 (4): 968-71.
He LM. Molecular Identification of Pyemotes Based on Mitochondrial COI and Ribosomal ITS2 Gene [D]. Shijiazhang:Hebei Normal University, 2010. [贺丽敏. COI和ITS2基因序列在蒲螨属分子鉴定中的应用研究 [D].石家庄:河北师范大学, 2010]
Hebert PDN, Penton EH, John M,et al. Ten species in one: DNA barcoding reveals cryptic species in the neotropical skipper butterfly Astraptes fulgerator [J]. Proceedings of the National Academy of Sciences of the United States of America, 2004b, 101 (41): 14812-14817.
Hebert PDN, Stoeckle MY, Zemlak TS,et al. Identification of birds through DNA Barcodes [J]. PLoS Biology, 2004a, 2 (10): e312.
Hu F, Li YF, Sheng L,et al. Molecular detection of Callosobruchus maculatus and its allied species based on mitochondrial COI gene [J]. Plant Quarantine, 2017, 31 (3): 25-28.[胡飞, 李云飞, 盛磊, 等. 四纹豆象及其近缘种基于COI基因的分子检测技术研究 [J]. 植物检疫, 2017, 31 (3): 25-28]
Huang SX. The first discovery of main species of mites and Varroa underwoodi in China [J]. Apiculture China, 2004, 55 (2): 4-5. [黄双修. 蜜蜂外寄生螨的主要种类和恩氏瓦螨(Varroa underwoodi)在中国的首次发现 [J]. 中国养蜂, 2004, 55 (2): 4-5]
Kontschán J, Tóbiás I, Bozsik G,et al. First record of Neocypholaelaps apicola from beehives in hungary (ACARI: Mesostigmata: Ameroseiidae): Re-description and DNA barcoding [J]. Acta Zoologica Academiae Scientiarum Hungaricae, 2015, 61 (3): 237-245.
Kuang HO. Eucalyptus insects is Eucalyptus mites [J].Journal of Bee, 2003, 8: 28. [匡海鸥. 桉花虫是桉螨 [J]. 蜜蜂杂志, 2003, 8: 28]
Kumar S, Stecher G, Tamura K. MEGA7: Molecular evolutionary genetics analysis version 7.0 for bigger datasets [J]. Molecular Biology Evolution, 20165, 33 (7): 1870-1874.
Li JB, Li YX, Sun JT,et al. COI barcoding as a molecular assay for the identification of phytoseiid mites [J]. Systematic and Applied Acarology, 2012, 17 (4): 397-406.
Webster LM, Thomas RH, Mccormack GP. Molecular systematics of Acarus siro s. lat., a complex of stored food pests [J]. Molecular Phylogenetics & Evolution, 2004, 32 (3): 817-822.
Luo GC, Zhao RS. The preliminary investigations on bee mites in Taiwan [J]. Agriculture Resource of China, 1975, 9 (1): 50-56. [罗干成, 赵若素. 台湾蜂螨之生态观察[J]. 中华农业研究, 1975, 9 (1): 50-56]
Ma LM, Lin JZ. Descriptions of male and protonymph of Ameroseius longitrichus and Neocypholaelaps indica (Acari: Mesostigmata: Ameroseiidae) [J]. Entomology of Journal East China, 2006, 15 (4): 241-243. [马立名, 林坚贞. 长发美绥螨和印度新曲厉螨雄螨与前若螨描述(蜱螨亚纲:中气门目:美绥螨科)[J]. 华东昆虫学报, 2006, 15 (4): 241-243]
Ma?án P. A revision of the family Ameroseiidae (Acari, Mesostigmata), with some data on Slovak fauna [J]. ZooKeys, 2017, 704 (704): 1-228.
Moraes GJ, Narita JPZ. Description of a new species of Neocypholaelaps (Acari: Ameroseiidae) from Brazil, with a key to the world species [J]. Zootaxa, 2010, 2554 (2554): 37-44.
Narita JPZ, de Moraes GJ, Fernando LCP. Two species of Neocypholaelaps from Sri Lanka (Acari: Ameroseiidae), with description of a new species [J]. Zootaxa, 2011, 2741 (2741): 59-65.
Narita JPZ, Pédelabat M, de Moraes GJ. A new species of Neocypholaelaps Vitzthum (Acari: Ameroseiidae), with notes on the cheliceral lobes and ventral porelike stuctures of mites of this family [J]. Zootaxa, 2013, 3666 (1): 1-15.
Nicholas KB, Nicholas HBJ. Genedoc:A tool for editing and annotating multiple sequence alignments [EB/OL]. www.pscedu/biomed/genedoc. Accessed Feb 2006.
Ren GT, Guo XG. Preliminarylist of Dermanyssoidea from China [J]. Jounal Anhui Agriculture Science, 2008, 36 (36): 15915-15919, 15947. [任天广, 郭宪国. 中国皮刺螨总科名录初报 [J]. 安徽农业科学, 2008, 36 (36): 15915-15919, 15947]
Robert H. Cruickshank. Molecular markers for the phylogenetics of mites and ticks [J]. Systematic and Applied Acarology, 2002, 7 (1): 3-14.
Sakamoto H, Matsuda T, Suzuki R,et al. Molecular identification of seven species of the genus Stigmaeopsis (Acari: Tetranychidae) and preliminary attempts to establish their phylogenetic relationship [J]. Systematic and Applied Acarology, 2017, 22 (1): 91-101.
Verma S.Some observations on the life cycle of the honey bee mite, Neocepholaelaps indica Evan (Acarina: Ameroseiidae) [J]. Pest Management and Economic Zoology, 1995, 3: 65-67.
Wang K, Li YT, Zheng Y,et al. Molecular identification of Grapholita dimorpha Komai (Lepidoptera: Tortricidae) and Grapholita molesta Busck (Lepidoptera: Tortricidae) based on mitochondrial COI and COII genes [J]. Journal of Northwest A & F University (Nature Science Edition), 2016, 44 (2): 156-164. [王康, 李玉婷, 郑燕, 等. 基于线粒体COI和COII基因的沙果小食心虫与梨小食心虫的分子鉴定 [J]. 西北农林科技大学学报(自然科学版), 2016, 44 (2): 156-164]
Zhang W. Some supplement about Neocypholaelaps indica [J]. Apiculture of China, 1965, 4: 103. [张文. 关于印度新曲厉螨的一些补充 [J]. 中国养蜂, 1965, 4: 103]
Zhang Y, Guo XH, Liu GC,et al. Application of DNA barcodes to molecular systematics of Coleoptera [J]. China Journal of Applied Entomology, 2011, 48 (2): 410-416. [张媛, 郭晓华, 刘广纯, 等. DNA条形码在鞘翅目昆虫分子系统学研究中的应用 [J]. 应用昆虫学报, 2011, 48 (2): 410-416]
Zhou SH, Deng YQ, Li SY,et al. Supplementary records of Dermanyssoid mites (Acari: Parasitiformes) in Fujian province [J]. China Journal Vector Biology Control, 2012, 23 (5): 467-470. [周淑姮, 邓艳琴, 李述杨, 等. 福建省皮刺螨总科螨种记录增补 [J]. 中国媒介生物学及控制杂志, 2012, 23 (5): 467-470]
Zhu ZH, Ye H, Zhang ZY. Molecular identification of six Bactrocera species (Diptera: Tephritidae) based on mtDNA [J]. Acta Entomologica Sinica, 2005, 48 (3): 386-390. [朱振华, 叶辉, 张智英. 基于mtDNA Cytb的六种果实蝇的分子鉴定 [J]. 昆虫学报, 2005, 48 (3): 386-390]