Kayseri yöresinde Culex pipiens biyotipleri ve Culex torrentium’un Real time PCR ile araştırılması ve moleküler karakterizasyonu
Öz
Bu çalışmada, Haziran-Ağustos 2014 tarihleri arasında Kayseri yöresinde çeşitli bölgelerden toplanmış sivrisinek örneklerinde Culex pipiens kompleks biyotipleri ve Cx. torrentium moleküler olarak araştırılmıştır. Saha çalışmaları boyunca toplam 1052 dişi sivrisinek örneklemesi yapılmış ve bunların 315’i (%29.9) morfolojik teşhis analizleriyle Cx. pipiens kompleks ve/veya Cx. torrentium olarak teşhis edilerek ayrılmıştır. Ayrılan örneklere ait genomik DNA izolatlarının ilk basamak Real time PCR analizlerinde tamamının Cx. pipiens kompleks nesillerine ait oldukları saptanmış, Cx. torrentium pozitifliği belirlenmemiştir. İkinci basamak Real time PCR analizlerinde Cx. pipiens komplekste belirlenen izolatların, 311’inin Cx. pipiens form pipiens nesillerine ait oldukları tespit edilmiş, Cx. pipiens form molestus pozitifliği görülmemiştir. Kalan 4 örnek, analizlerde her iki biyotip yönünden de amplifikasyon göstermemiştir. Real time PCR sonuçlarının konfirmasyonu ve hibrit nesillerin araştırılması amacıyla tüm örneklere ait izolatların ACE-2 ve CQ11 microsatellite DNA analizleri gerçekleştirilmiştir. Analizlerde Cx. pipiens form pipiens olarak belirlenen tüm izolatların ilgili DNA markerları ile pipiens biyotipine özgü bant profilleri sergilediği görülmüş ve izolatların konfirmasyonu sağlanmıştır. Real time PCR analizlerinde biyoformlar yönünden amplifikasyon göstermeyen 4 izolatın microsatellite DNA analizleriyle Cx. pipiens form pipiens ve Cx. pipiens form molestus hibritleri oldukları tespit edilmiştir. Cx. pipiens form pipiens ve hibrit nesillere ait birer izolatın mitokondrial cytochrome oxidase I (mt-COI) gen bölgesine göre barkodlaması yapılmış ve filogenetik analizleri gerçekleştirilmiştir. Her iki izolatın sekans analiz sonuçlarına göre yeni bir haplotip oldukları belirlenmiş ve haplotip bazında Cx. pipiens komplekse ait GenBank’ta kayıtlı homolog nesillerle filogenetik ilişkileri ortaya konmuştur
Anahtar Kelimeler
Culex pipiens biyotipleri Culex torrentium Kayseri moleküler karakterizasyon Real time PCR
Kaynakça
- Aldemı̇r A, Boşgelmez A (2006): Population dynamics of adults (Diptera:Culicidae) in Gölbaşı District, Ankara. Turk J Zool, 30, 9-17. stages of mosquitoes barcoding: Complementing morphological
- Cranston PS (1987): Keys to the adults, male hypsopygia, fourth-instar larvae and pupae of the British mosquitoes (Culicidae) with notes on their ecology and medical importance. Freshw Rev, 152.
- Cywinska A, Hunter FF, Hebert PD (2006): Identifying Canadian mosquito species through DNA barcodes. Med Vet Entomol, 20, 413-424.
- Darriba D, Taboada GL, Doallo R ve ark. (2012): jModelTest 2: More models, new heuristics and parallel computing. Nat Methods, 9, 772.
- Darsie RF Jr, Samanidou-Voyadjoglou A (1997): Keys for the identification of the mosquitoes of Greece. J Am Mosq Control, 13, 247-254.
- Di Luca M, Toma L, Boccolini D ve ark. (2016): Ecological distribution and CQ11 genetic structure of Culex pipiens complex (Diptera: Culicidae) in Italy. PLoS One, 11, e0146476.
- Duzlu O, Yildirim A, Inci A ve ark. (2016): Molecular investigation of Francisella-like endosymbiont in ticks and Francisella tularensis in ixodid ticks and mosquitoes in Turkey. Vector Borne Zoonotic Dis, 16, 26-32.
- Ergunay K, Gunay F, Erisoz Kasap O ve ark. (2014): Serological, molecular and entomological surveillance demonstrates widespread circulation of West Nile virus in Turkey. PLoS Negl Trop Dis, 8, e3028.
- Farajollahi A, Fonseca DM, Kramer LD ve ark. (2011): "Bird biting" mosquitoes and human disease: a review of the role of Culex pipiens complex mosquitoes in epidemiology. Infect Genet Evol, 11, 1577-85.
- Fonseca DM, Keyghobadi N, Malcolm CA ve ark. (2004): Emerging vectors in the Culex pipiens complex. Science, 303, 1535-38.
- Gomes B, Kioulos E, Papa A ve ark. (2013): Distribution and hybridization of Culex pipiens forms in Greece during the West Nile virus outbreak of 2010. Infect Genet Evol, 16, 218-225.
- Gomes B, Sousa CA, Novo MT ve ark. (2009): Asymmetric introgression between sympatric molestus and pipiens forms of Culex pipiens (Diptera: Culicidae) in the Comporta region, Portugal. BMC Dev Biol, 9, 262.
- Gunay F, Alten B, Simsek F ve ark. (2015): Barcoding Turkish Culex mosquitoes to facilitate arbovirus vector incrimination studies reveals hidden diversity and new potential vectors. Acta Trop, 143, 112-120.
- Harbach RE (1985): Pictorial keys to the genera of mosquitoes, subgenera of Culex and the species of Culex (Culex) occurring in southwestern Asia and Egypt, with a note on the subgeneric placement of Culex deserticola (Diptera: Culicidae). Mosquito Systematics, 17, 83-107.
- Hebert PDN, Ratnasingham S, deWaard JR (2003): Barcoding animal life: Cytochrome c oxidase subunit 1 divergences among closely related species. Philos Trans R Soc Lond B Biol Sci, 270, 96-99.
- Hubalek Z (2008): Mosquito-borne viruses in Europe. Parasitol Res, 103, 29-43.
- Huelsenbeck JP, Ronquist F (2001): MRBAYES: Bayesian inference of phylogenetic trees. BMC Bioinformatics, 17, 754-755.
- Inci A, Yildirim A, Njabo KY ve ark. (2012): Detection and molecular characterization of avian Plasmodium from mosquitoes in central Turkey. Vet. Parasitol, 188, 179-184.
- Kearse M, Moir R, Wilson A ve ark. (2012): Geneious basic: An integrated and extendable desktop software platform for the organization and analysis of sequence data. BMC Bioinformatics, 28, 1647-49.
- Knight KL, Stone A (1977): A catalog of the mosquitoes of the world (Diptera: Culicidae). Entomological Society of America (2d ed.), Washington, USA.
- Korkmaz S, Yıldırım A, Düzlü Ö ve ark. (2016): Kayseri yöresinden toplanmış Culex pipiens komplekse ait sivrisinek (Diptera: identifikasyonu.Türkiye Parazitol Derg, 40, 199-204.
- Kumar NP, Rajavel AR, Natarajan R ve ark. (2007): DNA barcodes can distinguish species of Indian mosquitoes (Diptera: Culicidae). J Med Entomol, 44, 1-7.
- Laurito M, Oliveira TM, Almiron WR ve ark. (2013): COI barcode versus morphological identification of Culex (Culex) (Diptera: Culicidae) species: A case study using samples from Argentina and Brazil. Mem Inst Oswaldo Cruz, 1, 110-122.
- Librado P, Rozas J (2009):DnaSP v5: A software for comprehensive analysis of DNA polymorphism data. Bioinformatics, 25, 1451-52.
- Malcolm CA, Bourguet D, Ascolillo A ve ark. (1998): A sex-linked acetylcholinesterase-mediated insecticide resistance in Culex pipiens. Insect Mol Biol, 7, 107-120. insensitive
- Öter K (2007): İstanbul’da Görülen Sivrisinek Türlerinin Tespiti. İstanbul Üniversitesi Sağlık Bilimleri Enstitüsü, Parazitoloji Anabilim Dalı, Doktora Tezi, İstanbul.
- Reusken CB, de Vries A, Buijs J ve ark. (2010): First evidence for presence of Culex pipiens biotype molestus in the Netherlands, and of hybrid biotype pipiens and molestus in northern Europe. J Vector Ecol, 35, 210-212.
- Rudolf M, Czajka C, Borstler J ve ark. (2013): First nationwide surveillance of Culex pipiens complex and Culex torrentium mosquitoes demonstrated the presence of Culex pipiens biotype pipiens/molestus hybrids in Germany. PLoS ONE, 8, 71832.
- Schaffner E, Angel G, Geoffroy B ve ark. (2011): The mosquitoes of Europe. CD-ROM.
- Shaikevich EV, Vinogradova EB, Bouattour A ve ark. (2016): Genetic diversity of Culex pipiens mosquitoes in distinct populations from Europe: Contribution of Cx. quinquefasciatus in Mediterranean populations. Parasit Vectors, 9, 47.
- Smith JL, Fonseca DM (2004): Rapid assays for identification of members of the Culex (Culex) pipiens complex, their hybrids, and other sibling species (Diptera: culicidae). Am J Trop Med Hyg, 70, 339-345.
- Vinogradova EB, Shaikevich EV, Ivanitsky AV (2007): A study of the distribution of the Culex pipiens complex (Insecta: Diptera: methods of identification). Comp Cytogenet, 1, 129-138.
- Wang G, Li C, Guo X ve ark. (2012): Identifying the main mosquito species in China based on DNA barcoding. PLoS One, 7, e47051.
- Ward RA (1992): Third supplement to “A catalog of the mosquitoes of the World” (Diptera: Culicidae). Mosquito Systematics, 24, 177-230.
- Weitzel T, Braun K, Collado A ve ark. (2011): Distribution and frequency of Culex pipiens and Culex torrentium (Culicidae) in Europe and diagnostic allozyme markers. J Eur Mosq Control Assoc, 22-37.
- Werblow A, Klimpel S, Bolius S ve ark. (2014): Population structure and distribution patterns of the sibling mosquito species Culex pipiens and Culex torrentium (Diptera: Culicidae) reveal different evolutionary paths. PLoS One, 9, e102158.
- Yıldırım A, İnci A, Düzlü O ve ark. (2013): Detection and molecular characterization of the Wolbachia endobacteria in the Culex pipiens (Diptera: Culicidae) specimens collected from Kayseri province of Turkey. Vet J Ankara Univ, 60, 189-194. Geliş tarihi: 15.03.2017 / Kabul tarihi: 28.07.2017
Investigation of Culex pipiens biotypes and Culex torrentium by Real time PCR in Kayseri region andmolecular characterization of the isolates
Öz
The biotypes of Culex pipiens complex and Cx. torrentium were molecularly investigated in the mosquito specimens collected from different regions of Kayseri between June and August 2015 in this study. A total of 1052 female mosquito specimens were sampled during the field surveys and 315 (29.9%) out of these were morphologically identified and reserved as Cx. pipiens complex and/or Cx. torrentium. The first step Real time PCR analyses of genomic DNA isolates from these reserved specimens revealed that all the specimens were belong to Culex pipiens complex and there was no Cx. torrentium positivity. Among the isolates determined in Culex pipiens complex, 311 were determined as belong to lineages of Cx. pipiens form pipiens according to second step Real time PCR analyses and there was no positivity for Cx. pipiens form molestus. The remaining 4 isolates showed no amplification for both two biotypes. ACE-2 and CQ11 microsatellite DNA analyses were carried out on the isolates from all specimens in order to confirm the real time PCR results and investigate the hybrid lineages. All the isolates determined as Cx. pipiens form pipiens showed specific banding pattern for pipiens biotype with the related DNA markers in the analyses result in the confirmation of the isolates. The 4 isolates that did not show amplification for both biotypes in Real time PCR assays were designated as the hybrids of Cx. pipiens form pipiens and Cx. pipiens form molestus with the microsatellite DNA analyses. DNA barcoding and phylogenetic analyses were performed on one isolate belong to each Cx. pipiens form pipiens pipiens and hybrids based on mitochondrial cytochrome oxidase I (mt-COI) gene region. Both isolates were determined as new haplotypes according to the sequence analyses and their phylogenetic relations based on haplotypes with the available homolog linages belong to Cx. pipiens complex in GenBank were revealed
Anahtar Kelimeler
Culex pipiens biotypes Culex torrentium Kayseri molecular characterization Real time PCR
Kaynakça
- Aldemı̇r A, Boşgelmez A (2006): Population dynamics of adults (Diptera:Culicidae) in Gölbaşı District, Ankara. Turk J Zool, 30, 9-17. stages of mosquitoes barcoding: Complementing morphological
- Cranston PS (1987): Keys to the adults, male hypsopygia, fourth-instar larvae and pupae of the British mosquitoes (Culicidae) with notes on their ecology and medical importance. Freshw Rev, 152.
- Cywinska A, Hunter FF, Hebert PD (2006): Identifying Canadian mosquito species through DNA barcodes. Med Vet Entomol, 20, 413-424.
- Darriba D, Taboada GL, Doallo R ve ark. (2012): jModelTest 2: More models, new heuristics and parallel computing. Nat Methods, 9, 772.
- Darsie RF Jr, Samanidou-Voyadjoglou A (1997): Keys for the identification of the mosquitoes of Greece. J Am Mosq Control, 13, 247-254.
- Di Luca M, Toma L, Boccolini D ve ark. (2016): Ecological distribution and CQ11 genetic structure of Culex pipiens complex (Diptera: Culicidae) in Italy. PLoS One, 11, e0146476.
- Duzlu O, Yildirim A, Inci A ve ark. (2016): Molecular investigation of Francisella-like endosymbiont in ticks and Francisella tularensis in ixodid ticks and mosquitoes in Turkey. Vector Borne Zoonotic Dis, 16, 26-32.
- Ergunay K, Gunay F, Erisoz Kasap O ve ark. (2014): Serological, molecular and entomological surveillance demonstrates widespread circulation of West Nile virus in Turkey. PLoS Negl Trop Dis, 8, e3028.
- Farajollahi A, Fonseca DM, Kramer LD ve ark. (2011): "Bird biting" mosquitoes and human disease: a review of the role of Culex pipiens complex mosquitoes in epidemiology. Infect Genet Evol, 11, 1577-85.
- Fonseca DM, Keyghobadi N, Malcolm CA ve ark. (2004): Emerging vectors in the Culex pipiens complex. Science, 303, 1535-38.
- Gomes B, Kioulos E, Papa A ve ark. (2013): Distribution and hybridization of Culex pipiens forms in Greece during the West Nile virus outbreak of 2010. Infect Genet Evol, 16, 218-225.
- Gomes B, Sousa CA, Novo MT ve ark. (2009): Asymmetric introgression between sympatric molestus and pipiens forms of Culex pipiens (Diptera: Culicidae) in the Comporta region, Portugal. BMC Dev Biol, 9, 262.
- Gunay F, Alten B, Simsek F ve ark. (2015): Barcoding Turkish Culex mosquitoes to facilitate arbovirus vector incrimination studies reveals hidden diversity and new potential vectors. Acta Trop, 143, 112-120.
- Harbach RE (1985): Pictorial keys to the genera of mosquitoes, subgenera of Culex and the species of Culex (Culex) occurring in southwestern Asia and Egypt, with a note on the subgeneric placement of Culex deserticola (Diptera: Culicidae). Mosquito Systematics, 17, 83-107.
- Hebert PDN, Ratnasingham S, deWaard JR (2003): Barcoding animal life: Cytochrome c oxidase subunit 1 divergences among closely related species. Philos Trans R Soc Lond B Biol Sci, 270, 96-99.
- Hubalek Z (2008): Mosquito-borne viruses in Europe. Parasitol Res, 103, 29-43.
- Huelsenbeck JP, Ronquist F (2001): MRBAYES: Bayesian inference of phylogenetic trees. BMC Bioinformatics, 17, 754-755.
- Inci A, Yildirim A, Njabo KY ve ark. (2012): Detection and molecular characterization of avian Plasmodium from mosquitoes in central Turkey. Vet. Parasitol, 188, 179-184.
- Kearse M, Moir R, Wilson A ve ark. (2012): Geneious basic: An integrated and extendable desktop software platform for the organization and analysis of sequence data. BMC Bioinformatics, 28, 1647-49.
- Knight KL, Stone A (1977): A catalog of the mosquitoes of the world (Diptera: Culicidae). Entomological Society of America (2d ed.), Washington, USA.
- Korkmaz S, Yıldırım A, Düzlü Ö ve ark. (2016): Kayseri yöresinden toplanmış Culex pipiens komplekse ait sivrisinek (Diptera: identifikasyonu.Türkiye Parazitol Derg, 40, 199-204.
- Kumar NP, Rajavel AR, Natarajan R ve ark. (2007): DNA barcodes can distinguish species of Indian mosquitoes (Diptera: Culicidae). J Med Entomol, 44, 1-7.
- Laurito M, Oliveira TM, Almiron WR ve ark. (2013): COI barcode versus morphological identification of Culex (Culex) (Diptera: Culicidae) species: A case study using samples from Argentina and Brazil. Mem Inst Oswaldo Cruz, 1, 110-122.
- Librado P, Rozas J (2009):DnaSP v5: A software for comprehensive analysis of DNA polymorphism data. Bioinformatics, 25, 1451-52.
- Malcolm CA, Bourguet D, Ascolillo A ve ark. (1998): A sex-linked acetylcholinesterase-mediated insecticide resistance in Culex pipiens. Insect Mol Biol, 7, 107-120. insensitive
- Öter K (2007): İstanbul’da Görülen Sivrisinek Türlerinin Tespiti. İstanbul Üniversitesi Sağlık Bilimleri Enstitüsü, Parazitoloji Anabilim Dalı, Doktora Tezi, İstanbul.
- Reusken CB, de Vries A, Buijs J ve ark. (2010): First evidence for presence of Culex pipiens biotype molestus in the Netherlands, and of hybrid biotype pipiens and molestus in northern Europe. J Vector Ecol, 35, 210-212.
- Rudolf M, Czajka C, Borstler J ve ark. (2013): First nationwide surveillance of Culex pipiens complex and Culex torrentium mosquitoes demonstrated the presence of Culex pipiens biotype pipiens/molestus hybrids in Germany. PLoS ONE, 8, 71832.
- Schaffner E, Angel G, Geoffroy B ve ark. (2011): The mosquitoes of Europe. CD-ROM.
- Shaikevich EV, Vinogradova EB, Bouattour A ve ark. (2016): Genetic diversity of Culex pipiens mosquitoes in distinct populations from Europe: Contribution of Cx. quinquefasciatus in Mediterranean populations. Parasit Vectors, 9, 47.
- Smith JL, Fonseca DM (2004): Rapid assays for identification of members of the Culex (Culex) pipiens complex, their hybrids, and other sibling species (Diptera: culicidae). Am J Trop Med Hyg, 70, 339-345.
- Vinogradova EB, Shaikevich EV, Ivanitsky AV (2007): A study of the distribution of the Culex pipiens complex (Insecta: Diptera: methods of identification). Comp Cytogenet, 1, 129-138.
- Wang G, Li C, Guo X ve ark. (2012): Identifying the main mosquito species in China based on DNA barcoding. PLoS One, 7, e47051.
- Ward RA (1992): Third supplement to “A catalog of the mosquitoes of the World” (Diptera: Culicidae). Mosquito Systematics, 24, 177-230.
- Weitzel T, Braun K, Collado A ve ark. (2011): Distribution and frequency of Culex pipiens and Culex torrentium (Culicidae) in Europe and diagnostic allozyme markers. J Eur Mosq Control Assoc, 22-37.
- Werblow A, Klimpel S, Bolius S ve ark. (2014): Population structure and distribution patterns of the sibling mosquito species Culex pipiens and Culex torrentium (Diptera: Culicidae) reveal different evolutionary paths. PLoS One, 9, e102158.
- Yıldırım A, İnci A, Düzlü O ve ark. (2013): Detection and molecular characterization of the Wolbachia endobacteria in the Culex pipiens (Diptera: Culicidae) specimens collected from Kayseri province of Turkey. Vet J Ankara Univ, 60, 189-194. Geliş tarihi: 15.03.2017 / Kabul tarihi: 28.07.2017
Ayrıntılar
| Birincil Dil | İngilizce |
|---|---|
| Konular | Veteriner Cerrahi |
| Diğer ID | JA47MA99JF |
| Bölüm | Araştırma Makalesi |
| Yazarlar | |
| Yayımlanma Tarihi | 1 Eylül 2018 |
| Yayımlandığı Sayı | Yıl 2018 |