PHYLOGENETIC AND POPULATION GENETIC STUDIES ON SOME INSECT AND PLANT ASSOCIATED NEMATODES DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Amr T. M. Saeb, M.S. * * * * * The Ohio State University 2006 Dissertation Committee: Professor Parwinder S. Grewal, Adviser Professor Sally A. Miller Professor Sophien Kamoun Professor Michael A. Ellis Approved by Adviser Plant Pathology Graduate Program Abstract: Throughout the evolutionary time, nine families of nematodes have been found to have close associations with insects. These nematodes either have a passive relationship with their insect hosts and use it as a vector to reach their primary hosts or they attack and invade their insect partners then kill, sterilize or alter their development. In this work I used the internal transcribed spacer 1 of ribosomal DNA (ITS1-rDNA) and the mitochondrial genes cytochrome oxidase subunit I (cox1) and NADH dehydrogenase subunit 4 (nd4) genes to investigate genetic diversity and phylogeny of six species of the entomopathogenic nematode Heterorhabditis. Generally, cox1 sequences showed higher levels of genetic variation, larger number of phylogenetically informative characters, more variable sites and more reliable parsimony trees compared to ITS1-rDNA and nd4. The ITS1-rDNA phylogenetic trees suggested the division of the unknown isolates into two major phylogenetic groups: the HP88 group and the Oswego group. All cox1 based phylogenetic trees agreed for the division of unknown isolates into three phylogenetic groups: KMD10 and GPS5 and the HP88 group containing the remaining 11 isolates. KMD10, GPS5 represent potentially new taxa. The cox1 analysis also suggested that HP88 is divided into two subgroups: the GPS11 group and the Oswego subgroup. Our results suggest that the HP88 group is most closely related to H. downesi K122 followed by H. marelatus Oregon, H. zealandica X1, H. megidis (UK and Jun) and H. indica EG2, respectively. Neither nd4 gene sequence- nor ND4 amino acid- based phenetic and ii phylogenetic trees were able to completely resolve the phylogenetic relationships among species of Heterorhabditis. We concluded that cox1 gene has a potential for molecular differentiation and diagnosis between closely related species within Heterorhabditis while nd4 is not an ideal target for reconstruction of phylogeny or molecular differentiation. We also advocate for more extensive sampling and deposition of representative molecular data in GenBank for all Heterorhabditis species to avoid inaccurate molecular identification. We also used the major sperm protein gene (msp) to investigate genetic diversity among 13 strains of Heterorhabditis bacteriophora. We hypothesized that H. bacteriophora strains might show genetic diversity in sequence and structure of msp genes due to large differences in their biological traits. Phenetic and phylogenetic analysis showed the presence of high genetic structuring within H. bacteriophora. Our results suggest that all the strains currently recognized as H. bacteriophora may not belong to the same species. Finally, we investigated the phylogeny and population structure of the nematode Bursaphelenchus conicaudatus and its insect vector, the yellow spotted longicorn beetle Psacothea hilaris from the Japanese islands. Phylogenetic and phenetic analyses indicated the existence of subspecies structure in B. conicaudatus and support the current subspecies groupings of P. hilaris subspecies with few exceptions. All fixation indices indicated the presence of high genetic differentiation among the local populations of both the nematode and its insect vector. The effective numbers of migrants showed definite but limited gene flow among insect and nematode local populations. iii Dedicated to my father iv ACKNOWLEDGMENTS During the course of my thesis work, there were many people who were instrumental in helping me. Without their guidance, help and patience, I would have never been able to accomplish the work of this thesis. First, I wish to thank my adviser, Parwinder S. Grewal, for intellectual support, encouragement, and enthusiasm, which made this thesis possible, and for his patience in correcting both my stylistic and scientific errors. I thank my advisory committee members, Dr. Sophien Kamoun, Dr. Sally Miller and Dr. Michael Ellis for their guidance, help, academic support and constructive suggestions that opened my eyes to much deeper insight in my research project. Also I want to thank all members of Dr. Grewal’s lab for their compassion, friendship and help especially Dr. Gunpat Jagdale. Also I want to thank all the faculty members and staff of the Department of Plant Pathology for their help, time and the highest level of education. I would like also, to express my deep respect and gratitude for all members of the Department of Entomology, where I conducted my entire research project. I do not want to forget to thank my wife, daughter and son for any inconvenience I caused them during stressful times. Finally, I present my deep gratefulness and appreciation to my home country Egypt, for giving me the opportunity to come to the USA to obtain my Ph. D. through a governmental scholarship. v VITA EDUCATION April 2002 – Present: Ph.D. Candidate, Department of Plant Pathology. The Ohio State University. February 1997 - June 2000: Master of Science, Major: Genetics. Genetic studies on some heavy metal resistance genes in bacteria. Faculty of Agriculture, Ain Shams University, Cairo, Egypt. October 1990 – June 1994: Bachelor of Science (Very Good Grade). Department of Genetics, Faculty of Agriculture, Ain Shams University, Cairo, Egypt. EMPLOYMENT June 2000 – until now: Position: Assistant Lecturer. Genetic Engineering and Biotechnology Research Institute (GEBRI), Minufiya University. Worked as a teaching member of Department of Molecular Biology and participate in the Institute scientific projects. March 1998 - June 2000: Position: Demonstrator. Genetic Engineering and Biotechnology Research Institute (GEBRI), Minufiya University. Worked as a teaching member of Genetic and Microbial Genetics Department. I also participated in the Institute’s scientific projects. October 1995 - February 1998: Position: Research Assistant. National Research Center, Dokki, Cairo, Egypt. Worked as a member of the team working on the genetic improvement of some economic microbes. I worked on heavy metal resistance in bacteria. June 1994 - September 1995: Position: Research Assistant. The Environmental Mutagensis Research Unit (EMRU), Ain Shams University, Faculty of Agriculture. Worked as a member of the research group of the scientific project dealing with genetic characterization of blue green algae. vi PUBLICATIONS Ganpati. B. Jagdale, Amr T. M. Saeb, Nethi Somasekhar, and Parwinder S. Grewal. (2006). Genetic variation and relationships between isolates and species of the entomopathogenic nematode genus Heterorhabditis deciphered through isozyme profiles. Journal of Parasitology: Vol. 92, No. 3, pp. 509–516. Amr T. M. Saeb and Parwinder S. Grewal. (2006). Intraspecific genetic variation in the major sperm protein gene of the entomopathogenic nematode Heterorhabditis bacteriophora. (Submitted). FIELDS OF STUDY Major: Plant Pathology vii TABLE OF CONTENTS Page Abstract ...........................................................................................................................ii Dedication ...................................................................................................................... iv Acknowledgments............................................................................................................v Vita..................................................................................................................................vi List of Tables....................................................................................................................x List of Figures.............................................................................................................…xiii Chapters: 1. Introduction.................................................................................................. 1 2. Inter and intra-specific genetic variation and phylogeny of Heterorhabditis: a comparison between two molecular markers………..…11 2.1 Introduction……………………………………………………..11 2.2 Materials and methods…. ……………………………………...14 2.3 Results…………………………………………………………..18 2.4 Discussion……………………………………………………....22 2.5 Summary………………………………………………………..28 2.6 Reference……………………………………………………….29 3. Investigation of the use of nd4 gene in phylogeny and molecular differentiation of entomopathognic nematodes (Rhabditida: Heterorhabditidae)…………………………………………………………50 3.1 Introduction…………………………………………………….50 3.2 Materials and methods…. ……………………………………..52 3.3 Results………………………………………………………….55 3.4 Discussion……………………………………………………...58 3.5 Summary……………………………………………………….62 viii 3.6 Reference……………………………………………………63 4. Intraspecific genetic variation in the major sperm protein gene of the entomopathogenic nematode Heterorhabditis bacteriophora…………..80 4.1 Introduction………………………………………………….80 4.2 Materials and methods…. …………………………………..83 4.3 Results……………………………………………………….87 4.4 Discussion…………………………………………………...90 4.5 Summary…………………………………………………….94 4.6 Reference…………………………………………………....95 5. Phylogeny, population structure, and gene flow in the insect tramsmitted nematode Bursaphelenchus conicaudatus and its vector Psacothea