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
References
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- Baldo L, Prendini L, Corthals A ve ark. (2007): Wolbachia are present in southern African scorpions and cluster with supergroup F. Curr Microbiol, 55, 367-373.
- Bandi C, Anderson TJ, Genchi C ve ark. (1998): Phylogeny of Wolbachia in filarial nematodes. Proc R Soc, 265, 2407-2413.
- Barr AR (1980): Cytoplasmic incompatibility in natural populations of a mosquito, Culex pipiens. L. Nature, 283, 71-72. 5. Behbahani A (2012): Wolbachia infection and mitochondrial DNA comparisons among Culex mosquitoes in South West Iran. Pak J Biol Sci, 15, 54-57.
- Berticat C, Rousset F, Raymond M ve ark. (2002): High Wolbachia density in insecticide-resistant mosquitoes. Proc Biol Sci, 269, 1413-16.
- Bourtzis K, Dobson SL, Braig HR ve ark. (1998): Rescuing Wolbachia have been overlooked. Nature, 391, 852-853.
- Bourtzis K, Dobson SL, Xi Z ve ark. (2014): Harnessing mosquito-Wolbachia symbiosis for vector and disease control. Acta Trop, 132, 150-163.
- Dobson SL, Bourtzis K, Braig HR ve ark. (1999): Wolbachia infections are distributed throughout insect somatic and germ line tissues. Insect Biochem Mol Biol, 29, 153-160.
- Doudoumis V, Alam U, Aksoy E ve ark. (2013): Tsetse- Wolbachia symbiosis: Comes of age and has great potential for pest and disease control. J Invertebr Pathol, 112, 94-103.
- Duron O, Labbe P, Berticat C ve ark. (2006): High Wolbachia density correlates with cost of infection for insecticide resistant Culex pipiens mosquitoes. Evolution, 60, 303-314.
- Duron O, Raymond M, Weill M (2011): Many compatible Wolbachia strains coexist within natural populations of Culex pipiens mosquito. Heredity, 106, 986-993.
- Echaubard P, Duron O, Agnew P ve ark. (2010): Rapid evolution of Wolbachia density in insecticide resistant Culex pipiens. Heredity, 104, 15-19.
- Felsenstein J (1985): Confidence intervals on phylogenies: An approach using the bootstrap. Evolution, 39, 783-791.
- Glick J (1992): Illustrated key to the female Anopheles of southwestern Asia and Egypt (Diptera: Culicidae). Mosquito Systematics, 24, 125-153.
- Goward CR, Scawen MD, Murphy JP ve ark. (1993): Molecular evolution of bacterial cell-surface proteins. Trends Biochem Sci, 18, 136-140.
- Guz N, Kocak E, Akpınar E ve ark. (2012): Wolbachia infection in Trissolcus species (Hymenoptera: Scelionidae). Eur J Entomol, 109, 169-174.
- Harbach RE (1985): Pictorial keys to the genera of mosquitoes, subgenera of Culex and the species of Culex (Culex) occuring in southwestern Asia and Eygpt, with a note on the subgeneric placement of Culex deserticola (Diptera: Culicidae). Mosquito Systematics, 17, 83-107.
- Hurst GDD, Jiggins FM, von der Schulenburg JHG ve ark. (1999): Male-killing Wolbachia in two species of insect. Proc R Soc Lond B Biol Sci, 266, 735-740.
- Inci A, Dik B, Mumcuoglu YK ve ark. (2014): Detection and molecular characterization of the Wolbachia endobacteria in chewing lice species collected from the angora goats in Central Anatolia Region of Turkey. 5th International Conference on Phthiraptera, Utah, United States. 2-7 August, p.76.
- Iturbe-Ormaexte I, Walker T, O’Neill SL (2011): Wolbachia and the biological control of mosquito-borne disease. EMBO Rep, 12, 508-518.
- Johnson KN (2015): The impact of Wolbachia on virus infection in mosquitoes. Viruses, 7, 5705-17.
- Kasap H, Kasap M (1983): Türkiye Anophelinae türleri. Türk Hij Den Biyol Derg, 40, 39-52.
- 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. Bioinformatics, 28, 1647-49.
- Kittayapong P, Baisley KJ, Baimai V ve ark. (2000): Distribution and diversity of Wolbachia infections in southeast Asian mosquitoes (Diptera: Culicidae). J Med Entomol, 37, 340-345.
- Lo N, Paraskevopoulos C, Bourtzis K ve ark. (2007): Taxonomic status of the intracellular bacterium Wolbachia pipientis. Int J Syst Evol Microbiol, 57, 654-657.
- Noda H, Koizumi Y, Zhang Q ve ark. (2001): Infection density of Wolbachia and incompatibility level in two planthopper species, Laodelphax striatellus and Sogatella furcifera. Insect Biochem Mol Biol, 31, 727-737.
- O’Neill SL, Giordano R, Colbert AM ve ark. (1992): 16srRNA endosymbionts associated with cytoplasmic incompatibility in insects. Proc Natl Acad Sci USA, 89, 2699-02. bacterial
- Osei-Poku J (2012): The evolution and genetics of vector competence in mosquito disease vectors. PhD thesis, Clare College, University of Cambridge, Cambridge: 211.
- Pidiyar VJ, Jangid K, Patole MS ve ark. (2003): Detection and phylogenetic affiliation of Wolbachia sp. from Indian mosquitoes Culex quinquefasciatus and Aedes albopictus. Curr Sci, 84, 1136-39.
- Postiglione M, Tabanli S, Ramsdale CD (1973): The anopheles of Turkey. Rivista di Parassitologia, 34, 127-159.
- Rasgon JL, Scott TW (2003): Wolbachia and cytoplasmic ıncompatibility in the california Culex pipiens mosquito species complex: Parameter estimates and ınfection dynamics in natural populations. Genetics, 165, 2029-38.
- Rasgon JL, Scott TW (2004): An initial survey for Wolbachia (Rickettsiales: Rickettsiaceae) infections in selected California mosquitoes (Diptera: Culicidae). J Med Entomol, 41, 255-257.
- Ravikumar H, Ramachandraswamy N, Sampathkumar S ve ark. (2010): A preliminary survey for Wolbachia and bacteriophage WO infections in Indian mosquitoes (Diptera: Culicidae). Trop Biomed, 27, 384-393.
- Ricci I, Cancrini G, Gabrielli S ve ark. (2002): Searching for Wolbachia (Rickettsiales: Rickettsiaceae) in mosquitoes (Diptera: Culicidae): Large polymerase chain reaction survey and new identifications. J Med Entomol, 39, 562- 567.
- Rowley SM, Raven RJ, McGraw EA (2004): Wolbachia pipientis in Australian spiders. Curr Microbiol, 49, 208- 214.
- Saitou N, Nei M (1987): The neighbor-joining method: A new method for reconstructing phylogenetic trees. Mol Biol Evol, 4, 406-425.
- Schaffner E, Angel G, Geoffroy B ve ark. (2001): The mosquitoes of Europe (CD-Rom). Institut de Resherche Pour le Développement, Montpellier, France.
- Sungpradit S, Nuchprayoon S (2010): Wolbachia of arthropods applications. Chula Med J, 54, 605-621. nematodes: Biology and
- Sunish IP, Rajendran R, Paramasivan R ve ark. (2011): Wolbachia endobacteria in a natural population of Culex quinquefasciatus from filariasis endemic villages of South India and its phylogenetic implication. Trop Biomed, 28, 569-576.
- Tamura K, Stecher G, Peterson D ve ark. (2013): MEGA6: Molecular evolutionary genetics analysis version 6.0. Mol Biol Evol, 30, 2725-29.
- Vandekerckhove TT, Watteyne S, Willems A ve ark. (1999): Phylogenetic analysis of the 16S rDNA of the cytoplasmic bacterium Wolbachia from then ovel host Folsomia candida (Hexapoda, Collembola) and its implications for Wolbachial taxonomy. FEMS Microbiol Lett, 180, 279-286.
- Walker T, Moreira LA (2011): Can Wolbachia be used to control malaria? Mem Inst Oswaldo Cruz, 106, 212-217.
- Walker T, Klasson L, Sebaihia M ve ark. (2007): Ankyrin repeat domain-encoding genes in the wPip strain of Wolbachia from the Culex pipiens group. BMC Biol, 5, 39.
- Werren JH (1997): Biology of Wolbachia. Annu Rev Entomol, 42, 587-609.
- Werren JH, Windsor DM (2000): Wolbachia infection frequencies in insects: Evidence of a global equilibrium? Proc R Soc Lond B, 267, 1277-85.
- Wiwatanaratanabutr I (2013): Geographic distribution of wolbachial infections in mosquitoes from Thailand. J Invertebr Pathol, 114, 337-340.
- Yen JH, Barr AR (1971): New hypothesis of the cause of cytoplasmic incompatibility in Culex pipiens. L. Nature, 232, 657-658.
- Yildirim A, Inci A, Duzlu 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.
- Zabalou S, Apostolaki A, Livadaras I ve ark. (2009): Incompatible insect technique: incompatible males from a Ceratitiscapitata (Diptera: Tephritidae) genetic sexing strain. Entomol Exp Appl, 132, 232-240.
- Zhou W, Rousset F, O’Neill SL (1998): Phylogeny and PCR-based classification of Wolbachia strains using wsp gene sequences. Proc R Soc Lond Ser B, 265, 509-515. Geliş tarihi: 02.02.2017 / Kabul tarihi: 28.07.2017
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
References
- Almeida F, Moura AS, Cardoso AF ve ark. (2011): Effects of Wolbachia on fitness of Culex quinquefasciatus (Diptera: Culicidae). Infect Genet Evol, 11, 2138-43.
- Baldo L, Prendini L, Corthals A ve ark. (2007): Wolbachia are present in southern African scorpions and cluster with supergroup F. Curr Microbiol, 55, 367-373.
- Bandi C, Anderson TJ, Genchi C ve ark. (1998): Phylogeny of Wolbachia in filarial nematodes. Proc R Soc, 265, 2407-2413.
- Barr AR (1980): Cytoplasmic incompatibility in natural populations of a mosquito, Culex pipiens. L. Nature, 283, 71-72. 5. Behbahani A (2012): Wolbachia infection and mitochondrial DNA comparisons among Culex mosquitoes in South West Iran. Pak J Biol Sci, 15, 54-57.
- Berticat C, Rousset F, Raymond M ve ark. (2002): High Wolbachia density in insecticide-resistant mosquitoes. Proc Biol Sci, 269, 1413-16.
- Bourtzis K, Dobson SL, Braig HR ve ark. (1998): Rescuing Wolbachia have been overlooked. Nature, 391, 852-853.
- Bourtzis K, Dobson SL, Xi Z ve ark. (2014): Harnessing mosquito-Wolbachia symbiosis for vector and disease control. Acta Trop, 132, 150-163.
- Dobson SL, Bourtzis K, Braig HR ve ark. (1999): Wolbachia infections are distributed throughout insect somatic and germ line tissues. Insect Biochem Mol Biol, 29, 153-160.
- Doudoumis V, Alam U, Aksoy E ve ark. (2013): Tsetse- Wolbachia symbiosis: Comes of age and has great potential for pest and disease control. J Invertebr Pathol, 112, 94-103.
- Duron O, Labbe P, Berticat C ve ark. (2006): High Wolbachia density correlates with cost of infection for insecticide resistant Culex pipiens mosquitoes. Evolution, 60, 303-314.
- Duron O, Raymond M, Weill M (2011): Many compatible Wolbachia strains coexist within natural populations of Culex pipiens mosquito. Heredity, 106, 986-993.
- Echaubard P, Duron O, Agnew P ve ark. (2010): Rapid evolution of Wolbachia density in insecticide resistant Culex pipiens. Heredity, 104, 15-19.
- Felsenstein J (1985): Confidence intervals on phylogenies: An approach using the bootstrap. Evolution, 39, 783-791.
- Glick J (1992): Illustrated key to the female Anopheles of southwestern Asia and Egypt (Diptera: Culicidae). Mosquito Systematics, 24, 125-153.
- Goward CR, Scawen MD, Murphy JP ve ark. (1993): Molecular evolution of bacterial cell-surface proteins. Trends Biochem Sci, 18, 136-140.
- Guz N, Kocak E, Akpınar E ve ark. (2012): Wolbachia infection in Trissolcus species (Hymenoptera: Scelionidae). Eur J Entomol, 109, 169-174.
- Harbach RE (1985): Pictorial keys to the genera of mosquitoes, subgenera of Culex and the species of Culex (Culex) occuring in southwestern Asia and Eygpt, with a note on the subgeneric placement of Culex deserticola (Diptera: Culicidae). Mosquito Systematics, 17, 83-107.
- Hurst GDD, Jiggins FM, von der Schulenburg JHG ve ark. (1999): Male-killing Wolbachia in two species of insect. Proc R Soc Lond B Biol Sci, 266, 735-740.
- Inci A, Dik B, Mumcuoglu YK ve ark. (2014): Detection and molecular characterization of the Wolbachia endobacteria in chewing lice species collected from the angora goats in Central Anatolia Region of Turkey. 5th International Conference on Phthiraptera, Utah, United States. 2-7 August, p.76.
- Iturbe-Ormaexte I, Walker T, O’Neill SL (2011): Wolbachia and the biological control of mosquito-borne disease. EMBO Rep, 12, 508-518.
- Johnson KN (2015): The impact of Wolbachia on virus infection in mosquitoes. Viruses, 7, 5705-17.
- Kasap H, Kasap M (1983): Türkiye Anophelinae türleri. Türk Hij Den Biyol Derg, 40, 39-52.
- 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. Bioinformatics, 28, 1647-49.
- Kittayapong P, Baisley KJ, Baimai V ve ark. (2000): Distribution and diversity of Wolbachia infections in southeast Asian mosquitoes (Diptera: Culicidae). J Med Entomol, 37, 340-345.
- Lo N, Paraskevopoulos C, Bourtzis K ve ark. (2007): Taxonomic status of the intracellular bacterium Wolbachia pipientis. Int J Syst Evol Microbiol, 57, 654-657.
- Noda H, Koizumi Y, Zhang Q ve ark. (2001): Infection density of Wolbachia and incompatibility level in two planthopper species, Laodelphax striatellus and Sogatella furcifera. Insect Biochem Mol Biol, 31, 727-737.
- O’Neill SL, Giordano R, Colbert AM ve ark. (1992): 16srRNA endosymbionts associated with cytoplasmic incompatibility in insects. Proc Natl Acad Sci USA, 89, 2699-02. bacterial
- Osei-Poku J (2012): The evolution and genetics of vector competence in mosquito disease vectors. PhD thesis, Clare College, University of Cambridge, Cambridge: 211.
- Pidiyar VJ, Jangid K, Patole MS ve ark. (2003): Detection and phylogenetic affiliation of Wolbachia sp. from Indian mosquitoes Culex quinquefasciatus and Aedes albopictus. Curr Sci, 84, 1136-39.
- Postiglione M, Tabanli S, Ramsdale CD (1973): The anopheles of Turkey. Rivista di Parassitologia, 34, 127-159.
- Rasgon JL, Scott TW (2003): Wolbachia and cytoplasmic ıncompatibility in the california Culex pipiens mosquito species complex: Parameter estimates and ınfection dynamics in natural populations. Genetics, 165, 2029-38.
- Rasgon JL, Scott TW (2004): An initial survey for Wolbachia (Rickettsiales: Rickettsiaceae) infections in selected California mosquitoes (Diptera: Culicidae). J Med Entomol, 41, 255-257.
- Ravikumar H, Ramachandraswamy N, Sampathkumar S ve ark. (2010): A preliminary survey for Wolbachia and bacteriophage WO infections in Indian mosquitoes (Diptera: Culicidae). Trop Biomed, 27, 384-393.
- Ricci I, Cancrini G, Gabrielli S ve ark. (2002): Searching for Wolbachia (Rickettsiales: Rickettsiaceae) in mosquitoes (Diptera: Culicidae): Large polymerase chain reaction survey and new identifications. J Med Entomol, 39, 562- 567.
- Rowley SM, Raven RJ, McGraw EA (2004): Wolbachia pipientis in Australian spiders. Curr Microbiol, 49, 208- 214.
- Saitou N, Nei M (1987): The neighbor-joining method: A new method for reconstructing phylogenetic trees. Mol Biol Evol, 4, 406-425.
- Schaffner E, Angel G, Geoffroy B ve ark. (2001): The mosquitoes of Europe (CD-Rom). Institut de Resherche Pour le Développement, Montpellier, France.
- Sungpradit S, Nuchprayoon S (2010): Wolbachia of arthropods applications. Chula Med J, 54, 605-621. nematodes: Biology and
- Sunish IP, Rajendran R, Paramasivan R ve ark. (2011): Wolbachia endobacteria in a natural population of Culex quinquefasciatus from filariasis endemic villages of South India and its phylogenetic implication. Trop Biomed, 28, 569-576.
- Tamura K, Stecher G, Peterson D ve ark. (2013): MEGA6: Molecular evolutionary genetics analysis version 6.0. Mol Biol Evol, 30, 2725-29.
- Vandekerckhove TT, Watteyne S, Willems A ve ark. (1999): Phylogenetic analysis of the 16S rDNA of the cytoplasmic bacterium Wolbachia from then ovel host Folsomia candida (Hexapoda, Collembola) and its implications for Wolbachial taxonomy. FEMS Microbiol Lett, 180, 279-286.
- Walker T, Moreira LA (2011): Can Wolbachia be used to control malaria? Mem Inst Oswaldo Cruz, 106, 212-217.
- Walker T, Klasson L, Sebaihia M ve ark. (2007): Ankyrin repeat domain-encoding genes in the wPip strain of Wolbachia from the Culex pipiens group. BMC Biol, 5, 39.
- Werren JH (1997): Biology of Wolbachia. Annu Rev Entomol, 42, 587-609.
- Werren JH, Windsor DM (2000): Wolbachia infection frequencies in insects: Evidence of a global equilibrium? Proc R Soc Lond B, 267, 1277-85.
- Wiwatanaratanabutr I (2013): Geographic distribution of wolbachial infections in mosquitoes from Thailand. J Invertebr Pathol, 114, 337-340.
- Yen JH, Barr AR (1971): New hypothesis of the cause of cytoplasmic incompatibility in Culex pipiens. L. Nature, 232, 657-658.
- Yildirim A, Inci A, Duzlu 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.
- Zabalou S, Apostolaki A, Livadaras I ve ark. (2009): Incompatible insect technique: incompatible males from a Ceratitiscapitata (Diptera: Tephritidae) genetic sexing strain. Entomol Exp Appl, 132, 232-240.
- Zhou W, Rousset F, O’Neill SL (1998): Phylogeny and PCR-based classification of Wolbachia strains using wsp gene sequences. Proc R Soc Lond Ser B, 265, 509-515. Geliş tarihi: 02.02.2017 / Kabul tarihi: 28.07.2017
Details
| Primary Language | English |
|---|---|
| Subjects | Veterinary Surgery |
| Other ID | JA57UZ62RP |
| Journal Section | Research Article |
| Authors | |
| Publication Date | September 1, 2018 |
| Published in Issue | Year 2018 |