STUDIES ON NEUROSECRETION IN SCHISTOCERCA GREGARIA FORS& (ORTHOPTERA ACRIDIDAE). by Frederick Delphin, M.Sc. (Rangoon). Thesis submitted for the Degree of Doctor of Philosophy in the Faculty of Science, University of London. Imperial College of Science and Technology, Field Station, Silwood Park, Sunninghill, Ascot, Berkshire. February 1963. 0 STUDIES ON NEUROSECRETION IN SCHISTOCERCA GREGARIA FOPSKAL (ORTHOPTERA : ACRIDIDAE). by Frederick Delphin, Dipl.Journ. (Rangoon). Thesis submitted for the Diploma of Imperial College. Imperial College of Science and Technology, Field Station, Silwood Park, Sunninghill, Ascot, Berkshire. February 1963. ABSTRACT. The literature on neurosecretion is reviewed, and a key to the cell types drawn up. Two new differential staining techniques for staining neurosecretion are reported, and older techniques re-examined critically, The histology of the neurosecretory cells of the ventral ganglia of Schisto- cerca aregaria has been investigated in detail. Four main types (A, B, C and D) occur in the ventral ganglia. Such cells are absent in the frontal, hypocerebral and ingluvial ganglia. Axonal transport of neurosecretion from the brain to the corpora cardiaca, backwards and forwards along the ventral connectives, and along the peripheral nerves, has been demonstrated experimentally, but not along the circum-oesophageal connectives. The anatomy of the nervous system is described. The distribution of the neuro- secretory cells supports the anatanical findings that the third thoracic ganglion is formed by the fusion of the metathoracic and first three abdominal ganglia. Autoradiographic investigations of secretion give results which are not in conflict with the histological inferences. Evidence is obtained that neurosecretory cells of the ventral ganglia undergo changes correlated with ovarian maturation, though 'castrat- ion' cells are absent. Oviposition is under nervous control. The A2 neurosecretory cells of the third thoracic and first four abdominal ganglia appear to be important in flight and water metabolism. They have been shown to discharge their secretions under experimental conditions of dehydration, and when the metabolic rate is increased as a result of a high level of flight activity. TABLE OF CONTENTS. Page I. INTRODUCTION 1 (a) General 1 (b) Historical Review 3 II. TECHNIQUES 17 (a) Rearing Techniques 17 (b) Histological Techniques 17 A. Paraldehyde-Fuchsin Technique 21 B. Chrome Haematoxylin-Phloxine Technique 26 C. Pararosaniline Chloride as a Neurosecretory Stain 30 D. Alcian Blue-Phloxine as a Selective Stain for NSS 33 III. ANATOMY OF NERVOUS SYSTEM 36 (a) General 36 (b) Structure of the Ganglia 39 IV. NEUROSECRETION 51 (a) Classification of Neurosecretory Cells 51 (b) Staining Characteristics of the Types of Neurosecretory Cells in the Ventral Ganglia of Schistocerca gregaria 71 (c) Distribution of Neurosecretory Cells in the Ventral Ganglia of Schistocerca gregaria 93 (d) Discussion 107 Page V. NEUROSECRETION IN RELATION TO PHYSIOLOGICAL ACTIVITIES 117 (a) Secretory Activity of the Neurosecretory Cells of the Ventral Ganglia during Maturation and Oviposition 117 (b) Role of the Last Abdominal Ganglion in Female Maturation and Oviposition 131 (c) Effect of Ovariectomy on the Histological Picture of Neurosecretory Cells 143 (d) Transport of Neurosecretory Material as studied by Ligation Experiments 144 (e) Secretory Dynamics of A3 Cells as studied by Autoradicgraphy 168 (f) Release of Neurosecretory Material induced by Sustained Flight and Fatigue 174 (g) Neurosecretion in Relation to Water Metabolism 185 VI. GENERAL DISCUSSION 193 VII. SUMMARY 214 VIII. ACKNOWLEDGMENTS 217 IX. BIBLIOGRAPHY 219 I. INTRODUCTION. (a) General. Students of neurosecretion in insects have in the past devoted their attention mainly to the pars intercerebralis and corpus allatum/corpus cardiacum complex, e.g., Viigglesworth (1940) on Rhodnius prolixus Stgl, Nayar (1953, 1955, 1956) on Iphita limbata Stgl, E.Thomsen (1952, 1954) on Calliphora erythrocephala Meig., B.Scharrer (1941, 1955, 1956) on Leucophaea maderae (Fabricius), Highnam (1961, 1962) on Schistocerca gregaria (Forskgl), Johansson (1957, 1958) on Oncopeltus fasciatus Dail.; to name a few. More recently a number of workers have described neurosecretory cells in the ganglia of the ventral nerve cord: Fraser (1959) on Lucilia Caesar L., Geldiay (1959) on Blaberus craniifer, Johansson (1958) in Oncopeltus fasciatus Dallas, Hiller (1960) in Periplaneta americana and Chaoborus, and Clements (1956) in Culex pipiens. As far back as 1940 Day had described what he thought were neurosecretory cells in the ventral ganglia of the moth Eacles imperialis Dru., while Kobayashi (1957) working on Bombyx mori L. found more neurosecretory cells in the ventral ganglia than in the brain: 30 in the brain as against 80 in the suboesophageal and 1,100 in the thoracic and abdominal ganglia. The presence of neurosecretory cells in the ventral ganglia of these few investigated insects, combined with the fact that little was known of their structure and distribution and nothing of their function prompted me to study the neurosecretory cells of the ventral ganglia of S.gregaria in some detail. The primary objectives of this study are therefore:- 1. to describe the different types of neurosecretory cells revealed in the 2. ventral ganglia by means of various differential staining techniques, and to examine critically some of the techniques; 2. to see what changes (if any) take place in the neurosecretory cells during various stages of the life cycle, and if possible, to correlate these findings with the development of sexual maturity in the female; 3. to explore the extent to which other physiological processes (e.g. flight, water balance) are correlated with changes in the ventral neurosecretory system; and 4. to determine what happens to the secretions and where and how they are conducted in the body. The work falls into three main sections: a. Morphological and Histological; b. Experimental; and c. General discussion of the findings. 3. (b) Historical Review. Research in insect endocrinology has proceeded along three main lines: histological, cyto— and biochemical, and experimental. 1. Histological. The first evidence of the presence of hormones of neuroendocrine origin was obtained in 1922 when Kopec removed the brains of freshly moulted last instar larvae of the moth Lymantria dispar and found that the larvae failed to pupate, but it was not until 1935 that Weyer, working on the brain of the honey bee, Apis mellifera L., showed that the neurosecretory cells of the pars intercerebralis might be the source of the hormone. In 1937 Scharrcr, working on the brain of Bombus, and a year later Hanstrbm, working on the brain of Rhodnius, found similar cells in the pars intercerebralis. Of special interest at about this time was the work of Schrader (1938) who published photographs of sections of the brain of Ephestia kiihniella; he stated that he failed to find any neurosecretory cells there, but from the photographs it appears that they are present but that he failed to recognize them. Two years after Schrader, in 1940, Day described cells, undoubtedly of a neurosecretor•; nature, in the suboesophageal ganglion of Lepidoptera, but he, like Schrader, was wary of pronouncing them neurosecretory, probably because his was the first report of the existence of neurosecretory cells outside the brain, and because the stains available at that time were not specific for revealing neurosecretion. The staining methods in neurosecretory work at the time employed four general histological stains: Heidenhain's Haematoxylin, Heidenhain's 4. Azan, and the trichrome stains of Masson and Mallory. Then in 1949 Bergmann employed Gomori's (1941) Chrome—haematoxylin/Phloxine method (henceforth called CHP) for selective staining of the neurosecretory cells of mammals, and Stutinsky (1952) subsequently applied the method to insects. In 1952 Halmi modified Gomorits (1950) Aldehyde/Fuchsin (Paraldehyde/Fuchsin, hence— forth called PF) for the differential staining of the pancreas, and Gabe (1953) further modified it and adapted it for staining neurosecretory cells of insects. These two staining techniques greatly facilitated the study of neurosecretion. Employing one or the other or both of these differential staining methods together with one or more of the four general histological stains mentioned earlier, various workers have gone on to describe a variety of different types of neurosecretory cells in insects belonging to widely separated orders. Quite accidentally in 1954 Scharrer first used the symbols A and B to denote the two main types of neurosecretory cells in the suboesophageal ganglion of Leucopha,:a maderae. Scharrer, however, used only one stain, PF, the main purpose of her paper being to describe the unique "castration cells" hitherto found nowhere else in insects, and the criteria she gave for differentiating the A—type from the B—type cell were not quite complete. The credit for the first successful attempt to bring some order into the classification of neurosecretory cells, therefore, must go to Nayar (1955) who used CHP and PF as well as Heidenhain's Haematoxylin and Azan in his work on the brain of Iphita limbata, although he unfortunately failed to get satisfactory results with PF, a fact which may be due to the sample of basic Jr- rl fuchsin he used, as discussed on page2O-25of this work. Nayarls classific— 5. ation has since been accepted and followed by a number of workers.