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10.14457/MU.The.2004.3 24/08/2564 15:35:18 MOLECULAR PHYLOGENETIC STUDY OF CULICINE MOSQUITOES USING THE MITOCHONDRIAL CYTOCHROME OXIDASE I GENE AND THE RELATIONSHIPS WITH MOSQUITO-BORNE FLAVIVIRUSES 10.14457/MU.the.2004.3 เมื่อ 09/10/2564 02:27:14 TADA JUTHAYOTHIN A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE (ENVIRONMENTAL BIOLOGY) FACULTY OF GRADUATE STUDIES MAHIDOL UNIVERSITY 2004 ISBN 974-04-4861-5 COPYRIGHT OF MAHIDOL UNIVERSITY Thesis Entitled MOLECULAR PHYLOGENETIC STUDY OF CULICINE MOSQUITOES USING THE MITOCHONDRIAL CYTOCHROME 10.14457/MU.the.2004.3OXIDASE I GENE AND THE RELATIONSHIPS WITH เมื่อ 09/10/2564MOSQUITO-BORNE 02:27:14 FLAVIVIRUSES ……………………………………… Mr. Tada Juthayothin Candidate ……………………………………… Assoc. Prof. Pattamaporn Kittayapong, Ph.D. Major-Advisor .……………………………………… .……………………………………… Lect. Pahol Kosiyachinda Prof. Jean-Paul Gonzalez, Ph.D. M.D., Ph.D. Co-Advisor Co-Advisor …………………………………….. ………………………………………. Assoc. Prof. Rassmidara Hoonsawat, Assoc. Prof. Prayad Pokethitiyook, Ph.D. Ph.D. Dean Chair Faculty of Graduate Studies Master of Science Programme Mahidol University in Environmental Biology Faculty of Science Thesis Entitled MOLECULAR PHYLOGENETIC STUDY OF CULICINE MOSQUITOES USING THE MITOCHONDRIAL CYTOCHROME 10.14457/MU.the.2004.3OXIDASE I GENE AND THE RELATIONSHIPS WITH เมื่อ 09/10/2564MOSQUITO-BORNE 02:27:14 FLAVIVIRUSES was submitted to the Faculty of Graduate Studies, Mahidol University for the degree of Master of Science (Environmental Biology) on May 19, 2004 ……………………………………… Mr. Tada Juthayothin Candidate ……………………………………… ……………………………………… Lect. John R. Milne, Assoc. Prof. Pattamaporn Kittayapong, Ph.D. Ph.D. Member Chair ………………………………………. ……………………………………… Assoc. Prof. Vijittra Leardkamolkarn, Prof. Jean-Paul Gonzalez, Ph.D. M.D., Ph.D. Member Member ………………………………………. ……………………………………… Assoc. Prof. Rassmidara Hoonsawat, Prof. Prasert Sobhon, Ph.D. Ph.D. Dean Dean Faculty of Graduate Studies Faculty of Science Mahidol University Mahidol University ACKNOWLEDGEMENTS I would like to express my sincere gratitude and deep appreciation to Assoc. Prof. Pattamaporn Kittayapong, my major advisor for invaluable guidance, consultation, comments, suggestions and constructive criticisms. She always gave very useful information and advised me in every way that would bring me success and great development. I also would like to thank Lect. John R. Milne, Prof. Jean-Paul Gonzalez (IRD, 10.14457/MU.the.2004.3Center for Vaccine Development, Mahidol University, Thailand) and Assoc. Prof. Vijittra Leardkamolkarn (Department of Anatomy, Faculty of Science, Mahidol เมื่อUniversity, 09/10/2564 Thailand), my thesis committee, 02:27:14 who provided their valuable time to read, discuss and advise me for improving my work. Also, my thankfulness to all of the members in the Center for Vectors and Vector-borne Diseases, Faculty of Science, Mahidol University and all my friends for their kind help and friendship during my study. I would like to thank Prof. Jean-Paul Gonzalez (IRD, Center for Vaccine Development, Mahidol University, Thailand) for financial support for my study. Finally, I am thankful to my family and especially my parents who have shown their great patience, moral support, and encouragement in every way possible to enable me to succeed in my education. Tada Juthayothin May 1, 2004 Fac. of Grad. Studies, Mahidol Univ. Thesis / iv MOLECULAR PHYLOGENETIC STUDY OF CULICINE MOSQUITOES USING THE MITOCHONDRIAL CYTOCHROME OXIDASE I GENE AND THE RELATIONSHIPS WITH MOSQUITO-BORNE FLAVIVIRUSES. TADA JUTHAYOTHIN 4237529 SCEB/M M.Sc. (ENVIRONMENTAL BIOLOGY) THESIS ADVISORS: PATTAMAPORN KITTAYAPONG, Ph.D., JEAN-PAUL GONZALEZ, M.D., Ph.D., PAHOL KOSIYACHINDA, Ph.D. ABSTRACT Phylogenetic studies of mosquitoes in the Family Culicidae and in the Genus 10.14457/MU.the.2004.3Culex (subfamily Culicinae) were conducted using the mitochondrial cytochrome oxidase subunit I (COI) gene. The COI genes (642 bp) of mosquitoes were directly sequenced and 26 mosquito COI sequences were obtained from the GenBank เมื่อdatabase. 09/10/2564 Due to multiple substitution 02:27:14 and substitution bias in the sequences, which reduced the phylogenetic signal from ancestors, it was impossible to determine the relationships between mosquitoes based on nucleotide analysis. The relationships were, however, recovered from amino acid analysis in which the neighbor-joining method gave better resolution than the maximum parsimony method. This was because of the low number of informative sites in MP analysis due to little variation in COI amino acids of mosquitoes. Phylogenetic study among mosquitoes in the Family Culicidae showed that members of the Subfamily Toxorhynchitinae cluster within the clade of the Subfamily Culicinae. In the Subfamily Anophelinae clade, Anopheles mosquitoes cluster together with Chagasia bathana as sister species. Subfamily Culicinae was divided into two groups, the Aedes and Culex groups. The Aedes cluster was composed of Aedes and Armigeres mosquitoes whereas the Culex cluster was composed of Culex and Mansonia mosquitoes. The relationships among mosquitoes in the Genus Culex were investigated in three subgenera, Culex, Culiciomyia and Eumelanomyia, and the results showed that members of the Subgenus Eumelanomyia branched off first and were distantly related to the others whereas the Subgenus Culiciomyia grouped in the clade of the Subgenus Culex and was therefore considered to be closely related to Subgenus Culex. The relationships among members in the Subgenus Culex were correlated with those derived from morphological characteristics, in which members of the vishnui subgroup, i.e., Cx. vishnui and Cx. tritaeniorhynchus, grouped together. The two most similar Culex mosquitoes, Cx. gelidus and Cx. whitmorei clustered together. Mosquito-borne flavivirus phylogeny was determined using the envelope protein gene, and the nonstructural protein 3 and 5 genes. There were two distinct groups based on their vectors, Aedes and Culex mosquitoes. Comparison of mosquito phylogeny with flavivirus phylogeny revealed that coevolution between mosquitoes and flaviviruses may have occurred before Aedes and Culex separation. After separation, differences in mosquito behavior may have driven flavivirus evolution. KEY WORDS : CULICIDAE / CULEX / MOSQUITO / COI / MOLECULAR PHYLOGENY / MITOCHONDRIAL DNA / FLAVIVIRUS 258 pp. ISBN 974-04-4861-5 Fac. of Grad. Studies, Mahidol Univ. Thesis / v การศึกษาวิวัฒนาการเชิงโมเลกุลของยุงในกลุม Culicine โดยใชยีน Cytochrome Oxidase I ใน Mitochondria และความสัมพันธกับ Flavivirus ที่นําโดยยุงพาหะ (MOLECULAR PHYLOGENETIC STUDY OF CULICINE MOSQUITOES USING THE MITOCHONDRIAL CYTOCHROME OXIDASE I GENE AND THE RELATIONSHIPS WITH MOSQUITO- BORNE FLAVIVIRUSES) ธาดา จูฑะโยธิน 4237529 SCEB/M วท.ม. (ชีววิทยาสภาวะแวดลอม) คณะกรรมการควบคุมวิทยานิพนธ: ปทมาภรณ กฤตยพงษ, Ph.D., JEAN-PAUL GONZALEZ, 10.14457/MU.the.2004.3M.D., Ph.D., พหล โกสิยะจินดา, Ph.D. เมื่อ 09/10/2564 02:27:14บทคัดยอ การศึกษาวิวัฒนาการเชิงโมเลกุลของยุงในวงศ Culicidae และยุงในสกุล Culex (วงศยอย Culicinae) ได กระทําโดยใชยีน cytochrome oxidase I (COI)ใน mitochondria โดยไดมีการหาลําดับนิวคลีโอไทดจํานวน 642 นิว คลีโอไทดโดยตรงจากยุง 18 ชนิดของยีน COI รวมกับลําดับนิวคลีโอไทดของยีน COI จากยุง 26 ชนิดซึ่งไดมาจาก ฐานขอมูล GenBank ผลการวิเคราะหโดยใชลําดับนิวคลีโอไทดไมสามารถบงบอกความสัมพันธของยุงทั้งในวงศ Culicidae และสกุล Culex อันเนื่องมาจาก multiple substitution และ substitution bias ซึ่งทําใหขอมูลเชิง วิวัฒนาการจากบรรพบุรุษลดนอยลง สวนผลการวิเคราะหทางกรดอะมิโนสามารถหาความสัมพันธของยุงได โดย พบวาวิธี neighbor-joining (NJ) สามารถหาความสัมพันธไดดีกวาวิธี maximum parsimony (MP) เพราะวาขอมูลที่ ใชในการวิเคราะหโดยวิธี MP มีจํานวนนอยเนื่องจากกรดอะมิโนของยีน COI ในยุงมีความแตกตางกันนอยมาก การศึกษาสายวิวัฒนาการของยุงในวงศ Culicidae พบวายุงในวงศยอย Toxorhynchitinae อยูรวมในสาย วิวัฒนาการของวงศยอย Culicinae ซึ่งอยูในสายวิวัฒนาการของวงศยอย Anophelinae ยุงชนิด Chagasia bathana มีสายวิวัฒนาการแบบพี่นองรวมกับยุงสกุล Anopheles สวนยุงในวงศยอย Culicinae ไดแบงการวิวัฒนาการเปน 2 สายคือ สาย Aedes และสาย Culex ในกลุมของยุงสาย Aedes ประกอบดวยยุงสกุล Aedes อยูรวมกับยุงสกุล Armigeres สวนสาย Culex ประกอบดวยยุงสกุล Culex อยูรวมกับยุงสกุล Mansonia ผลการหาความสัมพันธของยุง ในสกุล Culex พบวายุงในสกุลยอย Eumelanomyia ไดแยกสายวิวัฒนาการออกมากอน สวนยุงในสกุลยอย Culiciomyia อยูในสายวิวัฒนาการรวมกับยุงในสกุลยอย Culex ซึ่งถือวาเปนสกุลยอยที่มีความสัมพันธใกลเคียงกัน สวนผลจากการหาความสัมพันธของยุงในสกุลยอย Culex พบวามีความสัมพันธสอดคลองตามลักษณะทาง สัณฐานของยุงโดย Cx. tritaeniorhynchus อยูรวมกับ Cx. vishnui และ Cx. gelidus อยูรวมกับ Cx. whitmorei สําหรับการศึกษาวิวัฒนาการของ flavivirus ที่นําโดยยุงพาหะโดยใชยีน envelope, nonstructural protein 3 และ 5 พบวาสายวิวัฒนาการไดแยกออกเปน 2 กลุมตามชนิดของพาหะคือยุง Aedes และ ยุง Culex เมื่อนําผลจากการ ศึกษาสายวิวัฒนาการของยุงมาพิจารณารวมกับสายวิวัฒนาการของ flavivirus พบวาอาจจะมีการวิวัฒนาการรวม กันระหวางยุงกับ flavivirus ที่นําโดยยุงพาหะซึ่งเกิดขึ้นกอนการแยกสายวิวัฒนาการของยุงสกุล Aedes และยุงสกุล Culex และความแตกตางทางพฤติกรรมภายหลังจากการแยกสายวิวัฒนาการของยุงทั้งสองกลุมอาจจะเปนสิ่ง กระตุนใหเกิดการวิวัฒนาการของ flavivirus ในภายหลัง 258 หนา ISBN 974-04-4861-5 CONTENTS Page ACKNOWLEDGEMENT…………….………………………………. iii ABSTRACT (ENGLISH)……………………………………………... iv ABSTRACT (THAI)………………………………………………….. v LIST OF TABLES…………………………………………………….. xiii 10.14457/MU.the.2004.3LIST OF FIGURES……………………………………………………. xv เมื่อLIST OF09/10/2564 ABBREVIATIONS…………………………………………. 02:27:14 xxiii CHAPTER 1 INTRODUCTION 1. Introduction……………………………………... 1 2. Objectives……………………………………….. 2 2 LITERATURE
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