PHYSIOLOGICALLY ACTIVE FACTORS IN Ulik, CORPORA CARDIACA OF INSECTS Jennifer Jones, B. Tech. Thesis submitted for the degree of Doctor of Philosophy of the University of London and for the Diploma of Imperial College. July 1978 Imperial College of Science and Technology, Department of Zoology and Applied Entomology, Prince Consort Road, London S.W.7 2 ABSTRACT A combination of column and thin layer chromatographic techniques has been used to resolve the factors in the corpora cardiaca of Locusta migratoria migratorioides and Periplaneta americana which can affect lipid metabolism, carbohydrate metabolism and water balance. The storage and glandular lobes of the locust corpora cardiaca can be separated by dissection, unlike the glandular and storage areas of the cockroach which are in intimate contact. Pure adipokinetic hormone (AKH), isolated from locust glandular lobes has been shown to produce on injection an elevation of haemolymph lipids in locusts and haemolymph carbohydrates in cockroaches. This hormone is important in the maintenance of prolonged flight activity in the locust. The hyperglycaemic response produced in cockroaches is likely to be a pharmacological effect. By applying processes developed for the purification of AKH to cockroach corpora cardiaca, a peptide factor(s) which possesses adipokinetic, hyperglycaemic and diuretic activities has been obtained. The factor is different from AKH because AKH does not possess diuretic activity and the cockroach factor has different separation characteristics and amino acid composition. The physiological role of this factor in cockroach flight has been investigated, but its potent diuretic activity may indicate its major function. 3 The locust diuretic hormone extracted from the storage lobes is distinct from AKH as it does not possess adipokinetic or hyperglycaemic activity, and appears to be a larger molecule than AKH. Under the conditions so far employed, the material resolved from locust storage lobes which produces a hyperglycaemic response in cockroaches has the same separation characteristics as AKH but different relative adipokinetic and hyperglycaemic activities. The relative lipid mobilising and hyperglycaemic activities in de novo corpus cardiacum extracts were found to be similar to those of storage lobe extracts. The technique of in vitro culture of locust corpora cardiaca has been developed to obtain information on the synthesis and release of hormones. The biosynthetic capacity of the organ cultures was not conclusively demonstrated, but morphological studies indicated that the glandular cells remained viable during the incubation period. Electron micrographs of the storage lobes showed a degeneration of neurosecretory vesicles although a considerable quantity of paraldehyde fuchsin positive material remained in the cells. 4 ACKNOWLEDGEMENTS I wish to thank Professor T.R.E. Southwood FRS in whose department this work was carried out, and Dr. W. Nordue for his critical supervision and constant encouragement. MY thanks are also due to Dr. J.V. Stone for helpful advice and discussions. I am grateful to Dr. G.J. Goldsworthy of Hull University for providing the de novo corpora cardiaca used in this study, and to Dr. R.J. Weaver of the A.R.C. Unit of Invertebrate Chemistry and Physiology, University of Sussex for providing some of the cockroaches.• This work was carried out during the tenure of an S.R.C. Research Studentship. 5 TABLE OF CONTENTS Page INTRODUCTION 12 1. Structure of the locust cephalic neurosecretory system 13 a)Cerebral neurosecretory cells 13 b)Nerve connections within the system 15 o) The corpora cardiaca 16 2. Structure of the cockroach cephalic neurosecretory system 20 a)Cerebral neurosecretory cells 20 b)Nerve connections within the system 21 c)The corpora cardiaca 22 3. Cephalic neurosecretion and carbohydrate metabolism 23 a)General considerations of carbohydrate metabolism in insects 23 b)Involvement of the corpora cardiaea 26 c)Origins of the corpus cardiacum factors 29 d)Physiological significance of the corpus cardiacum factors 31 4. Cephalic neurosecretion and lipid metabolism 33 a)General considerations of lipid metabolism in insects 33 b)Involvement of the corpora cardiaca 35 i The adipokinetic hormone of locusts 35 ii Hypolipaemia in cockroaches and hyperlipaemia in locusts 37 iii Endogenous fat body metabolism 38 5. Cephalic neurosecretion and water balance 38 6 a) General considerations of water balance in insects 38 b) Involvement of the corpora cardiaca 39 i Factors affecting Malpighian tubule function 40 ii Factors affecting rectal function 41 c) Origins of the corpus cardiacum factors 42 6. Nature and number of active factors in the corpora cardiaca of locusts and cockroaches 43 a)Structural indications 44 b)The glandular region of the locust corpora cardiaca 46 The adipokinetic hormone 46 The hyperglycaemic factor 47 Factors affecting heart beat 48 c)The storage region of the locust corpora cardiaca 49 The hyperglycaemic factor 49 Factors affecting water balance 50 Factors affecting heart beat 51 d)The corpora cardiaca of cockroaches 51 The hyperglycaemic factor 51 Factors affecting water balance 53 Factors affecting heart beat 53 MATERIALS AND METHODS 1.Maintenance of insect colonies 55 2.Preparation of corpus cardiacum extracts 55 a)Dissection of corpora cardiaca 55 b)Methanol extracts 56 0) Saline extracts 56 7 (1) Buffer extracts 56 3. Biological assay procedures 57 a)Adipokinetic activity 57 b)Hyperglycaemic activity 57 c)Activity on flight metabolism 58 d)Diuretic activity 59 Amaranth excretion in vivo 59 Fluid secretion rate in vitro 59 e)Heart accelerating activity 60 f)Glycogen phosphorylase activation 60 4. Separation of active factors in corpus cardiacum extracts a)Filtration chromatography 61 b)Polyacrylamide gel electrophoresis 62 Anionic system 62 Cationic system 63 Gel preparation and electrophoresis . 63 Staining of gels 63 Elution of gels 64 c)Thin layer chromatography (TLC) 64 TLC of column eluates 64 TLC of gel eluates 65 Visualisation of peptide material on TLC plates 65 5. Biochemical characterisation of purified factors 65 a)Enzymic digestion 65 b)Ultra—violet absorption and fluorescence spectra 66 c)Amino acid analysis 66 6. Corpora cardiaca in organ culture 66 8 a)Culture of the corpora cardiaca 66 b)Extraction of incubation media and cultured corpora cardiaca 67 c)Morphological studies of the cultured corpora cardiaca 68 RESULTS THE CORPORA CARDIACA OF L. MIGRATORIA 1. Activities of the corpus cardiacum extracts 69 a)Effect on total haemolymph lipids 69 b)Effect on total haemolymph carbohydrates 73 c)Effect on the rate of amaranth excretion 77 d)Effect on the rate of fluid secretion by Malpighian tubules in vitro 78 e)Effect on the rate of heart beat 84 f)Effect on fat body glycogen phosphorylase levels 84 2. Purification of the corpus cardiacum factors 85 a)Purification of the glandular lobe factors 85 Glass bead column chromatography . 85 TLC of eluates 88 b)Purification of storage lobe factors 88 TLC of extracts 88 Method 1 91 Glass bead column chromatography 91 TLC of eluates 94 Method 2 94 Biogel P6 column chromatography 94 TLC of eluates 98 c) Polyacrylamide gel electrophoresis 100 Whole corpus cardiacum extracts 100 Separated lobe extracts 101 TLC of gel eluates 102 3. Activities and identity of the corpus cardiacum factors 112 a)The adipokinetic hormone 112 b)The resolved storage lobe factors 115 4. Organ culture of the corpora cardiaca 120 a)Histological appearance 120 Light microscope level 120 Electron microscope level 127 b)Adipokinetic activity in cultured corpora cardiaca and media 127 THE CORPORA CARDIACA OP P. AM,7,RICANA 1. Histological appearance of the corpora cardiaca 133 2. Activities of the corpus cardiacum extracts 136 a)Effect on total haemolymph lipids 136 b)Effect on total haemolymph carbohydrates 136 c)Effect on the rate of fluid secretion by Malpighian tubules in vitro 137 d)Effect on the rate of heart beat 139 3. Purification of the corpus cardiacum factors 139 a)Glass bead column chromatography 139 b)TLC of eluates 141 4. Activities and identity of the corpus cardiacum factors 145 a) Adipokinetic, hyperglycaemic, heart accelerating and diuretic activities 145 10 b)Flight metabolism in L. maderae 150 c)Characteristics of the purified factor 154 Enzymic digestion 154 Fluorescence and ultra—violet absorption spectra 154 Amino acid composition 155 DISCUSSION CORPUS CARDIACUM FACTORS OF L. MIGRATORIA 1.Activities of factors in glandular lobe extracts 162 2.Isolation of the adipokinetic hormone 165 3.Activities of the adipokinetic hormone 167 4.Additional glandular lobe factors 169 5.Physiological significance of the glandular lobe factors 170 6.Activities of factors in storage lobe extracts 172 7.Activities of factors in de novo corpus cardiacum extracts 175 8.Resolution of storage lobe factors and activities of the resolved factors 176 9.Physiological significance of the storage lobe factors 179 CULTIVATION OF CORPORA CARDIACA FROM L. MIGRATORIA IN VITRO 1.Morphology of the cultured glands 182 2.Activity of the cultured glands 183 CORPUS CARDIACUM FACTORS OF P. AMERICANA 1.Activities of factors in corpus cardiacum extracts 184 2.Purification and characterisation of the corpus cardiacum factor(s) 188 3.Activities of the purified corpus cardiacum factors 190 11 4. Physiological significance of the purified corpus cardiacum factor(s) 192 SUMMARY 195 REFERENCES 197 12 INTRODUCTION The cerebral neurosecretory cells and the corpora cardiaca of insects form a system analogous