8008772 BRADFIELD, JAMES YOUNG, IV ENDOCRINE CHARACTERIZATION OF ADULT DEVELOPMENT AND DIAPAUSE IN THE TOBACCO HORNWORM The Ohio State University PH.D. 1979 University Microfilms International300 N. Zeeb Road, Ann Arbor, M I 48106 18 Bedford Row, London WC1R 4EJ, England ENDOCRINE CHARACTERIZATION OF ADULT DEVELOPMENT AND DIAPAUSE IN THE TOBACCO HORNWORM DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By James Young Bradfield IV, B.A. ***** The Ohio State University 1979 Reading Committee: '■ Approved By William J. Collins David L. Denlinger — A ■ Adviser David J. Horn Department of Entomology VITA November 5, 1950 Born - Dallas, Texas 1973 B.A., The University of Texas at Austin 1975-1976 Teaching Associate, The Ohio State University, Columbus, Ohio. 1976-1979 Research Associate, The Ohio State University, Columbus, Ohio. PUBLICATIONS Denlinger, D. L., Campbell, J. J., and Bradfield, J. Y. 1979. Stimulatory effect of organic solvents on initiating development in diapausing pupae of the flesh fly Sarcophaqa crassipalpis and the tobacco hornworm Manduca sexta. Physiol. Entomol., in press. Streett, D. A., and Bradfield, J. Y. IV. 1978. Juvenile hormone activity in microsporidians spores. Proc. Xlth Ann. Cong. Invert. Path., pp. 199-200. FIELDS OF STUDY Major Field: Entomology Studies in endocrinology. Asst. Professor-David L. Denlinger. TABLE OF CONTENTS Page VITA .................................................. ii LIST OF TABLES .......................................... iv LIST OF FIGURES ........................................ v INTRODUCTION ............................................ 1 CHAPTER I. ENDOCRINE CONTROL OF INSECT DEVELOPMENT ........... 2 The Principal Endocrine Centers of Insect Development . 3 Endocrinology of Diapause ....................... 12 II. ENDOCRINE REQUIREMENTS FOR ADULT DEVELOPMENT IN THE TOBACCO HORNWORM ................................ 21 III. DIAPAUSE DEVELOPMENT IN THE TOBACCO HORNWORM: A ROLE FOR ECDYSONE OR JUVENILE HORMONE? ................. 36 IV. JUVENILE HORMONE TITERS IN THE LATE LARVA AND PREPUPA OF THE TOBACCO HORNWORM ......................... 53 V. CONCLUSIONS ............................. 66 APPENDIXES A. SUPPLEMENTARY EXPERIMENTS ....................... 63 B. REARING PROCEDURE FOR THE TOBACCO HORNWORM .......... 71 LIST OF REFERENCES...................................... 75 i i i LIST OF TABLES Table Page 1 Age Effect of Debraining on Prompt Initiation of Adult Development in Non-Diapause M. sexta (25°C) .... 30 2 Development of M_. sexta Abdomens Isolated at Various Times after Pupation (250C) ....................... 32 3 Developmental Response of M. sexta Pupae to Allatectomy on Day of Pupation (25°C) .................... 32 4 Diapause Duration of M. sexta Pupae Injected with a Subthreshold Dose of ?0-Hydro"xy-Ecdysone......... 43 5 Responses of Isolated Abdomens of M. sexta to Injection of 20-Hydroxy-Ecdysone ........................... 44 6 Diapause Duration of M^. sexta in Response to Allatectomy on Day of Pupation .......... 44 7 Scoring System for the M. sexta Black Mutant Larval A s s a y ..................................... 58 8 Responses of Tobacco Hornworm Wanderers and Prepuape to Topical Application and Injection of JH Analogue. .... 70 iv I LIST OF FIGURES Figure Page 1 Structures of insect metamorphic hormones.......... 7 2 Prodromes of adult development in M. sexta ......... 23 3 Developmental responses of M. sexta to debraining 1-5 days after pupation ........................... 27 4 20-hydroxy-ecdysone ED™ for diapause termination in the tobacco hornworm as a function of pupal age . 40 5 Termination of diapause in M. sexta pupae injected with 2.5 ug/gm of the JH analogue ZR-512 .......... 46 6 Dose-response plot for black mutant JH bioassay . 59 7 Juvenile hormone activity in the hemolymph of dia­ pause-destined wanderers and prepupae of M. sexta . 60 v INTRODUCTION The studies reported in this dissertation were efforts to characterize the endocrinology of adult development and diapause in the tobacco hornworm, Manduca sexta (Johansson). The research consisted of efforts along several rather distinct lines of questioning. To facilitate communication, therefore, the work is organized into separate chapters. Chapter I is an overview of insect developmental endocrinology. Chapters II, III, and IV are each presented in the form of standard research papers. In addition, one of the appendixes describes experiments which do not readily incorporate in the main body of the dissertation but which reveal potentially useful and interesting information. sexta is particularly suitable for the investigations reported here. The tobacco hornworm is fast becoming the "white rat" of insect endocrinology: many of the US laboratories active in the field today are using this insect exclusively. M. sexta thrives on an artificial diet, undergoes rapid growth, and attains a very large size. The latter attribute renders the animal especially suitable for surgery and collection of the large quantities of blood necessary for hormone analysis. Finally, one can easily generate populations destined entirely for diapause or continuous development. 1 CHAPTER I ENDOCRINE CONTROL OF INSECT DEVELOPMENT Introduction Molting and metamorphosis are features of postembryonic development common to all insects. That the control of these events is under the auspices of the neuroendocrine system - the main elements of which are the brain, prothoracic gland, and corpora allata - is a fact which was well established by work early in this century. Perhaps the most notable of the early studies are those of Fraenkel (1934), Wigglesworth (1934, 1936, 1940), Fukuda (1940, 1944), and Williams (1946, 1947, 1952). The collected efforts of the above workers and others established what is now known as the "classical scheme" of the endocrine control of molting and metamorphosis. This scheme, so well known to entomolo­ gists and ever-present in basic texts of entomology, can be summarized as follows. The coordinator of the endocrine events required for insect molting and metamorphosis is the brain. Upon receiving the appropriate stimuli, the brain literates a hormone (now known as pro- thoracicotropic hormone or PTTH) which as a profound activating effect on a second endocrine organ, the prothoracic gland. Thus activated, the prothoracic releases into the blood the molting hormone (ecdysone) 2 which, upon contact with the animal's epidermal cells, initiates the metabolic and physical process known as molting. The character of the molt, that is, whether it is larval-larval, larval-pupal, or pupal- adult, is determined by the amount of a third hormone, juvenile hormone (JH). A high level of the juvenile hormone dictates a larval molt, less a molt to the pupa, and still less an adult molt. JH is a secretory product of the corpora allata, which are paired glands in the head or neck of the insect. The activity of the corpora allata is probably regulated by the brain. A barrage of studies have added much to the classical scheme in the years since its formulation (excellent reviews by Wigglesworth, 1964, 1970; Doane, 1972; Gilbert and King, 1973; Willis, 1974). Notwith­ standing these, the classical scheme remains the essence of our concept of the control of insect development. To follow in this chapter are two sections dealing with the insect neuroendocrine system. The first will be a detailed discussion of the system and its hormonal products. The second will discuss the neuro­ endocrine system as it functions in regulating insect diapause. THE PRINCIPAL ENDOCRINE CENTERS OF INSECT DEVELOPMENT Brain That the insect brain is prerequisite for the molt was documented by Kopec (1922), who found that last instar larvae of the gypsy moth, L.ymantria dispar, failed to pupate if deprived of this organ. Subsequently, Wigglesworth, Fukuda, and Williams described the brain's essential role in the molting process: it exerts its action hormonally and by mediation of the prothoracic gland. source and chemistry of brain hormone (PTTH) Wigglesworth (1940) showed by transplantation experiments that the portion of the protocerebrum containing large neurosecretory cells (NSC's) is responsible for molt induction in Rhodnius prolixus. That the protocerebral NSC's are themselves responsible for PTTH production has been consistently suggested (Williams, 1947; Highnam, 1958; Gibbs and Riddiford, 1977; Girardie and DeReggi, 1978). The chemistry of PTTH is poorly understood. While there is general agreement that the hormone is a polypeptide (Goldsworthy and Mordue, 1974), there is consistent disagreement concerning other aspects of its character. For instance, PTTH has been reported to have molecular weights ranging from 10,000 to 30,000 dal tons in the commer­ cial silkworm Bombyx mori (Ishizaki and Ichikawa, 1967; Yamazaki and Kobayashi, 1969). Despite occasional reports of interordinal and interspecific activity (e.g., Gersch and Sturzebecher, 1970), there are probably several forms of PTTH among the insects. control of PTTH release In many insects environmental conditions are of prime importance in modulating the release of PTTH from the brain. Many different factors have been implicated, among them photoperiod (Williams, 1969), temperature (Pipa, 1976), tactile stimuli (Pipa, 1971), and moisture (Chtaki et al_., 1968). The internal means by which the release of PTTH is regulated is almost completely unknown. The classical story of