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Immunoelectrophoretic Techniques Used for Systematic Investigation

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Immunoelectrophoretic Techniques Used for Systematic Investigation AN ABSTRACT OF THE THESIS OF Lloyd James Lockwood for the Ph.D. in Genetics-Physiology presented on/4gazeiNz3 Title: Immunoelectrophoretic Techniques Used for Systematic Investigation Redacted for Privacy Abstract Approved: - / W. P. Stephen Immunotaxonomic studies have largely been based on serology in which either one of two parametershas been used for establishing relationships:(1) the number of precipitin arcs formed on Ouehterlony plates, or (2) the total amount of precipitin resulting from reactions with a standard antiserum. Recognizing the restrictions of both methods, electrophoresis and immunology, it seems desirable to develop and evaluate techniques incorporating these primary tools as a means of determining protein homologies. Immunoelectrophoretic analysis (IEA) has special applicability for it provides a means of more objectively evaluating homologies at the generic and family levels. With the prime purpose of evaluating and develop- ing techniques, two hypotheses are posed:(1) that homologous pairs of proteins are more common in related species and less common in more distantly related species, and (2) that proteins from distantly related organisms with common Rf values, when separated on acrylamide gels, can more likely be considered to be homologous if they exhibit the same antigenic properties. The purpose of this study was to determine the extent to which IEA can be used to establish isology and possibly homology among soluble insect proteins. Insect species whose relationships are well known have been used to determine whether IEA reflects on the propinquity of the species tested. The contention and conclusion of this investi- gation was that the techniques presented are of highly significant value in establishing phyletic relationships in closely and distantly related organisms. Immunoelectrophoretic Techniques Used for Systematic Investigation by Lloyd James Lockwood A Thesis submitted to Oregon State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy June 1974 APPROVED: Redacted for Privacy Arofessor of Entomology in charge of major Redacted for Privacy dead of Genf ics Institute Redacted for Privacy Dean of Graduate School Date thesis is presented ii-/`/7, /973 Typed by Jane Teller for Lloyd James Lockwood TABLE OF CONTENTS I. Introduction 1 II. Materials and Methods 15 Biological Material 15 Preparation of Antigens 18 Preparation of Antisera 20 Collection of Antisera 23 Animal Treatment 23 Blood Collection 23 Blood Treatment 25 Disc Electrophoresis 26 Double Immunodifussion 31 III. Results and Discussion 37 Preparation of Antigens 37 Preparation of Antisera 38 Collection of Antisera 43 Disc Electrophoresis 44 Immunoelectrophoretic Analysis 48 Orthoptera 57 Cockroach Intrasubfamily Cross Reactions 62 Cockroach Intersubfamily Cross Reactions 68 Cockroach Intrafamily Cross Reactions . 74 Apinae and Bombinae 83 Interordinal Comparison 92 IV. Conclusion 97 Bibliography 101 LIST OF TABLES I. Classification of cockroaches 17 II. Injection Schedule I for the production of antisera 21 III. Injection Schedule II for the production of antisera 22 IV. Soluble protein concentration extracted in 40% sucrose and determined by a modified Lowry Test of thoracic muscle extracts used as antigens 39 V. Number of arcs produced by IEA using undiluted sera after Schedule I was administered 41 VI. Number of antigens reacting in IEA with homologous antisera 42 VII. Protein concentration of antisera 45 VIII. Homologous and heterologous reactions which formed arcs G 1-gl and G 1 2-g3a . 51 IX. Homologous and heterologous reactions which formed the arcs G 3-g4s, G 3-g6s and G 3-g61s 52 X. Homologous and heterologous reactions which formed arcs G 1 + 2-g3a and G 3-g6a 53 XI. Homologous and heterologous reactions which formed arcs G la and G 1,2 -gi, 2, 3 54 XII. Homologous and heterologous reactions which formed arcs G 1-gla and G 2-g3, 5a . 55 XIII. Number of arcs formed in homologous and heterologous reactions of cockroaches . 60 XIV. Classification of cockroaches (Rehn, 1951) 63 XV. Heterologous reactions within the subfamily Blaberinae, tribe Blaberini 64 XVI. Heterologous reactions between species of the subfamilies Epilamprinae and Blaberinae 70 XVII. Heterologous reactions between the subfamilies Blaberinae and Blattinae 75 XVIII. Heterologous reactions between the subfamilies Blattinae and Epilamprinae . 79 XIX. Number of arcs formed in homologous and heterologous reactions of the bees 84 LIST OF FIGURES 1. Rabbit ear illustrating good vascularization 24 2. Stained protein bands of L. maderae illustrating interfaces of four gel concentrations and grid numbering systems 28 3. Polyacrylamide gel cutter 29 4. Inserting acrylamide gel into cutter 29 5. Cutting acrylamide gel 30 6. Gel cut into equal halves 30 7. Agar covered gel in a petri dish 32 8. B. californicus x anti-B. californicus primary development 34 9. B. californicus x anti-B. californicus secondary development 34 10. B. californicus x anti-B. californicus 10-day development 34 11. B. orientalis x anti-P. americana heavy precipitin barrier 36 12. Composite drawing of precipitin arcs of P. americana x anti-P. americana 47 13. P. americana x anti-P. americana stained bisected gel and precipitin arcs 47 14. B. appositus x anti-B. californicus stained bisected gel, grid and precipitin arcs 49 15. Graphic representation of arcs G 1, 2-g3a and G 1-gla 51 16. Graphic representation of arys G 3-g4s, G 3-g6s, and G 3-g6 s 52 17. Graphic representation of arcs G 1+ 2-g3a and G 3-g6a 53 18. Graphic representation of arcs G la and G 1 + 2-gl, 2, 3 54 19. Graphic representation of arc G 1-gla and G 2-g3, 5a 55 20. A. mellifera x anti-A. mellifera, Set I, Slide 1 58 21. A. mellifera x anti-A. mellifera, Set I, Slide 2 58 22. A. mellifera x anti-A. mellifera, Set I, Slide 3 58 23. A. mellifera x anti-A. mellifera, Set II, Slide 1 59 24. A. melliferax anti-A. mellifera, Set I, Slide 2 59 25. B. giganteus x anti-B. giganteus 61 26. B. craniifer x anti-B. craniifer 61 27. L. maderae x anti-L. maderae 61 28. A. marmorata x anti-B. craniifer 65 29. A. mormorata x anti-B. giganteus 65 30. E. posticus x anti-B. craniifer 65 31. E. posticus x anti-B. gigantues 66 32. B. fumigata x anti-B. craniifer 66 33. B. fumigata x anti-B. giganteus 66 34. B. craniifer x anti-B. giganteus 67 35. B. discoidalis x anti-B. giganteus 67 36. B. orientalis x anti-P. americana 69 37. B. giganteus x anti-L. maderae 71 38. L. maderae x anti-B. giganteus 71 39. B. craniifer x anti-L. maderae 71 40. L. maderae x anti-B. craniifer 72 41. A. marmorata x anti-L. maderae 72 42. E. posticus x anti-L. maderae 72 43. B. discoidalis x anti-L. maderae 73 44. B. fumigata x anti-L. maderae 73 45. B. giganteus x anti-P. americana 76 46. B. craniifer x anti-P. americana 76 47. B. discoidalis x anti-P. americana 77 48. A. marmorata x anti-P. americana 77 49. B. fumigata x anti-P. americana 78 50. P. americana x anti-B. giganteus 78 51. E. posticus x anti-P. americana 78 52. P. americana x anti-L. maderae 80 53. L. maderae x anti-P. americana 80 54. B. orientalis x anti-L. maderae 80 55. A. mellifera x anti-A. mellifera 85 56. B. appositus x anti-B. californicus 87 57. B. appositus x anti-B. californicus: whole gel 87 58. B. appositus x anti-B. californicus: bisected gel 88 59. B. californicus x anti-A. mallifera 88 60. A. mellifera x anti-B. californicus 89 61. B. vosnesenskii x anti-A. mellifera 89 62. B. appositus x anti-A. mellifera 90 63. B. occidentalis x anti-A. mellifera 90 64. B. craniifer x anti-A. mellifera 93 65. L. maderae x anti-A. mellifera 95 66. L. maderae x anti-B. californicus 95 67. B. appositus x tni-L. maderae 96 68. P. americana x anti-A. mellifera 96 IMMUNOELECTROPHORETIC TECHNIQUES USED FOR SYSTEMATIC INVESTIGATION I. INTRODUCTION Taxonomy historically has been based on com- parative morphology, and relationships established using phenotypic characters have resulted in widely accepted animal phylogenies. The high phenotypic correlation that exists among closely related species has resulted in very stable taxonomic treatment. Among more distantly related organisms, however, where the degree of phenotypic correlation is low, decisions as to the relationships and their concomitant taxonomic treat- ment are often based on highly subjective criteria. Especially in the absence of good fossil records the choice of phenotypic characters as "primitive" criteria suffers from this lack of objectivity. It has thus been the objective of many workers during the past half century to seek other more objective means of evaluating inter-organism relation- ships. Several of the more contemporary biological disciplines, such as genetics, cytology, behavior and ecology, have led to changes or clarification of 2 inter-taxa relationships. One of the most intriguing areas for exploration has been comparative biochemistry. Since the general acceptance of Mendelian genetics it has been postulated that a knowledge of the chemical structure of individual chromosomes would provide the ultimate tool in the determination of phyletic relationships. The premise holds that the establishment of base sequences would permit the establishment of structural homology among species. Fitch (1966), then with Margoliash (1967, 1969), determined the amino acid sequences of cytochrome-C from 20 eukaryotic species ranging from fungi to man. On the basis of this information they constructed a phylogenic tree to show the relationships among these species. Their results are in good agreement with zoological opinion. The ultimate definitive factor would appear to be the sequence of the principal specific purines and pyrimidines in DNA.
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