Molecular investigation and genotyping of Wolbachia endobacteria in mosquito species in Sultan Marshes region

Abstract

Out of 400 mosquito specimens collected from Sultan Marshes ecosystem in Kayseri, 296 (74.0%) were identified as Culex pipiens, 63 (15.75%) as Aedes vexans, and 41 (10.25%) as Culiseta annulata. Individual DNA was extracted from adult mosquitos and PCR analysis was performed in terms of the wsp gene region. While Wolbachia positivity was detected in 119 (40.2%) female Cx. pipiens, none of the male Cx. pipiens specimens were positive. No positivity was detected in Ae. vexans and Cs. annulata samples. In the phylogenetic analyzes of the 10 isolates selected from Wolbachia positive samples, the EruWolCpip1-10 isolates were clustered in the Wolbachia B supergroup and wPip group. It was found that EruWolCpip1-9 isolates were 100% identical among themselves and these isolates genetically differed by 0.5% with EruWolCpip10 isolate. The previous isolates of WolKys1 isolated from Cx. pipiens in Kayseri region and TrERUWolLice1 isolated from Bovicola limbata in Çankırı region have 100% similarity with EruWolCpip1-9 isolates. It was also determined that the EruWolCpip1-9 isolates were 100% identical with the Cx. pipiens, Peribadotes rhomboidaria, Agriocnemis femina femina and Eurema hecabe Wolbachia isolates in the wPip group. Furthermore, while the genetic difference within the wPip group was 0.28±0.11%, the genetic difference between the Dei, Con and Ori groups which placed in the same super group with the wPip group was found as 16.9±2.3, 12.5% ±1.9% and 19.9±2.5%, respectively. The genetic difference between Haw, Riw, Mel, Aus, Mors, Uni, Pap and Alba groups in the A super group, and the wPip group in the B super group were determined as 23.5±3.0%, 17.6±2.3%, 19.1±2.5%, 22.0±2.8%, 21.0±2.7%, 22.3±2.9%, 22.0±2.8% and 20.5±2.6%, respectively. This study was carried out with the hypothesis that the genetic difference between Wolbachia isolates obtained from different arthropod species in different geographical regions and classified in the same or different super groups, and our isolates may be significant. As a result, while the genetic difference between the isolates in the same groups was found less significant, the genetic difference in different super groups was determined much more significant. These results confirmed our hypothesis

Keywords

• Genotyping
• mosquito
• Sultan Marshes ecosystem
• Turkey
• Wolbachia

Sultan Sazlığı yöresinde sivrisinek türlerinde Wolbachia endobakterisinin moleküler yöntemlerle araştırılması ve genotiplendirilmesi

Abstract

Kayseri Sultan Sazlığı ekosisteminden toplanan 400 sivrisinek örneğinin 296’sı (%74.0) Culex pipiens, 63’ü (%15.75) Aedes vexans ve 41’i (%10.25) Culiseta annulata olarak belirlenmiştir. Ergin sineklerden bireysel DNA elde edilmiş ve wsp gen bölgesi yönünden PZR analizleri gerçekleştirilmiştir. 119 (%40.2) Cx. pipiens dişisinde Wolbachia pozitifliği saptanırken, erkek Cx. pipiens örneklerinde pozitiflik belirlenmemiştir. Ae. vexans ve Cs. annulata örneklerinde de pozitiflik saptanmamıştır. Wolbachia pozitif örneklerden seçilen 10 izolatın filogenetik analizlerinde EruWolCpip1-10 izolatları Wolbachia B süper grubu ve wPip grubu içinde belirlenmiştir. EruWolCpip1-9 izolatlarının %100 identik oldukları ve EruWolCpip10 izolatıyla %0.5 genetik farklılık gösterdikleri saptanmıştır. Daha önce Kayseri’de Cx. pipiens’ten izole edilmiş WolKys1 ve Çankırı’da Bovicola limbata’dan elde edilmiş TrERUWolLice1 izolatlarının, EruWolCpip1-9 izolatlarıyla %100 benzerlik gösterdiği belirlenmiştir. EruWolCpip1-9 izolatlarının wPip grubundaki Cx. pipiens, Peribadotes rhomboidaria, Agriocnemis femina femina ve Eurema hecabe Wolbachia izolatlarıyla %100 benzer oldukları görülmüştür. wPip grubundaki genetik farklılık %0.28±0.11 belirlenirken, wPip grubu ile aynı süper gruptaki Dei, Con ve Ori grupları arasındaki genetik farklılık sırasıyla %16.9±2.3, %12.5±1.9 ve %19.9±2.5 bulunmuştur. A süper grubundaki Haw, Riw, Mel, Aus, Mors, Uni, Pap ve Alba grupları ile B süper grubundaki wPip grubu arasında genetik farklılık ise sırasıyla %23.5±3.0, %17.6±2.3, %19.1±2.5, %22.0±2.8, %21.0±2.7, %22.3±2.9, %22.0±2.8 ve %20.5±2.6 olarak saptanmıştır. Farklı coğrafyalardaki değişik artropodlarda aynı veya farklı süper gruplarda yer aldıkları belirlenmiş Wolbachia izolatları ile kendi izolatlarımız arasındaki genetik farklılığın önemli düzeyde olabileceği hipoteziyle yapılmış bu çalışma sonucunda, aynı grubun izolatları arasındaki farklılığın çok önemli olmadığı, ancak farklı süper gruplardaki izolatlar arasındaki genetik farklılığın bir hayli önemli olduğu belirlenmiştir. Bu sonuçlar hipotezimizi doğrular nitelikte bulunmuştur

Keywords

• Genotiplendirme
• sivrisinek
• Sultan Sazlığı ekosistemi
• Türkiye
• Wolbachia

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