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 to the hypothalamo-neurohypophysial system of vertebrates (Scharrer,and Scharrer; 1944; Thomsen, 1952; ArvY and Gabe, 1953; HanstrOm,.1955 )• The corpora cardiaca, in addition to being the neurohaemal organ for the products of the brain
neurosecretory cell groups, may also produce intrinsic secretions.
Cerebral neurosecretion has been implicated in the control of a number of physiological processes, but the criteria for hormonal
status has been demonstrated in few instances. Some physiological
roles have been inferred principally on the basis of corpus cardiacum extract activity. Such extracts contain a multiplicity of cellular products, and investigations into the function of biologically active factors using crude whole gland extracts have frequently produced equivocal results which are difficult to
interpret.
Unless reasonably pure active factors are obtained,
investigation into the physiological significance of these factors
will be greatly impeded. The present study is concerned with
the resolution of those corpus cardiacum factors of Locusta migratoria migratorioides and Periplaneta americana thought to be
involved in the control of carbohydrate metabolism, lipid metabolism and water balance. 13
1. Structure of the locust cephalic neurosecretory system.
The major components of the locust cephalic neurosecretory
system are groups of neurosecretory cells within the brain, their
axonal pathways and the corpora cardiaca. Axons of the cerebral neurosecretory cells leave the brain in 4 pairs of nerves, the nervi corpori cardiaci (NCC) I,II,III,IV and their terminals
form the anterior storage region of the corpora cardiaca. The posterior region of the corpora cardiaca is composed of 'paren-
-chymatous' glandular cells (Highnam41961 )4,
a) Cerebral neurosecretory cells. The cerebral neurosecretory cells are in two main groups,medial
and lateral, in each half of the protocerebrum (Highnam,1965). The medial groups are closely opposed in the pars intercerebralis region, forming a triangular shaped group on the anterior face of the brain. In Schistocerca grep/aria, four cell types have been
described for the medial group using staining reaction and cell size criteria. 'A' and 'B' cells are considered to elaborate chemically different types of neurosecretory material while 'C'
and 'D' cells are considered to be non-neurosecretory (Righnam, 1966). At the electron microscope level, the 550 medial
neurosecretory cells of Locusta migratoria have been classified into three groups (Girardie and Girardie,1967). 'A' and 'B' cells are thought to correspond to two opposite phases of activity of
the same cell type, 'B' cells being the resting phase. 'C' cell
material has the same affinity for electron microscope stains as 14
'A' cell material but the cells are distinguished by 'C' type containing electron dense granules 100-200nm in diameter compared
with the 200-300nm diameter granules of 'A' cells. In the imaginal
stage 'A' cells are the most numerous (65-700) and 'C' cells
represent only 9,6 of the medial neurosecretory cells.
The lateral neurosecretory cell groups of the protocerebrum
havebeen little studied compared with the medial group. In
Locusta the lateral group contains 8-13 cells (Girardie, 1973)
and they lie superficially on the anterior face of the protocerebrum.
The lateral cell material does not show a tinctorial affinity
comparable to that of 'A' cell material either in Locusta or in
Schistocerca. In Locusta, the lateral cells contain electron
dense granules 200-300nm in diameter similar in morphological
aspect to the medial cell type (Girardie, 1973).
In addition to the medial and lateral neurosecretory cell
groups of the protocerebrum, other neurosecretory cells may occur
within the brain. Girardie (1970) has reported previously
undescribed neurosecretory cells, four in Locusta and six in
Schistocerca, situated on the outside of the pars intercerebralis
and classified as type 'D' cells. They have the same histological
characteristics as 'C' cells but contain larger electron dense
granules, 200-300nm in diameter. The presence of fushinophil
material in nerves originating in the tritocerebrum and deutero-
-cerebrum may indicate the presence of additional neurosecretory
cells in these regions of the brain (Girardie, 1973; Nason_1973). 15
b) Nerve connections within the system.
The corpora cardiaca are joined to the brain by 4 pairs of nerves which penetrate the corpora cardiaca at the junction of the storage and glandular regions,. The NCCI are the largest and most median, and the finer nerves of the NCCII, III and IV are more lateroventral.
Ventrally, the corpora cardiaca are joined to the hypocerebral ganglia by a pair of nerves and laterally to the corpora allata by the allatal nerves (NCAI) (Cazal, 1971).
The NCCI contain both neurosecretory and non-neurosecretory axon tracts from the protocerebral medial cell group, most of
which decussate in the brain before passing to the corpora cardiaca (Highnam, 1965). The NCCI also contain axons from a group of 12 cells in the tritocerebrum in Schistocerca vasa (Mason, 1973). Both lobes of Locusta corpora cardiaca receive projections from each group of medial cells (Highnam and West, 1971). Finer fibres from the NCCI travel into the posterior region of the corpora
cardiaca, but do not enter the NCAI (Mason, 1973).
Axons from the lateral cell groups run directly to the
ipsilateral anterior corpus cardiacum in the paired NCCII. Fibres
in the NCCII extend into the posterior region of the corpora cardiaca, and also enter the NCAI to end in the cortical portion of the corpora allata (Mason, 1973).
The axon tracts in the NCCIII originate from a group of cells in the tritocerebrum (Raabe, 1963) and a major branch of these
nerves connects this region of the brain with the corpora allata -16 in S. vaga (Mason, 1973).
The NCCIV are very fine nerves originating from an unknown region of the deutocerebrum (Brousse-Gaury, 1967).
c) The corpora cardiaca. The corpora cardiaca are derived from the dorsal ectodermal wall of the stomodaeum, as is the dorsal sympathetic nervous system, and have been referred to as paired pharyngeal or oesophageal ganglia (Roonwal, 1937). The same region of the stomodeal wall gives rise to the unpaired occipital or hypocerebral ganglion.
The anterior region of the locust corpora cardiaca is composed mainly of axon terminals from the cerebral neurosecretory cells, and attempts have been made during ultrastructural studies to classify the axon types present in this region on the basis of granule size, electron opacity, shape and relative proportions. The first study by Cassier and Fain-Maurel (1970) described three neurosecretory fibre types in the storage region of Locusta corpora cardiaca. Type I contains spherical electron dense granules, less than 250nm in diameter. Type II contains two sorts of granules in variable proportions: dense, well delimited spherical granules of diameter 200-250nm and 'grey grains' containing a granular substance 300nm in diameter. Type III contains three sorts of granules of comparatively large size: abundant, opaque, spherical granules 250-300nm in diameter; equally opaque oval granules
200-650nm in diameter; less opaque, grey granules 350-400nm in diameter. 17
During a second study on the corpora cardiaca of the same insect,
Cazal et al (1971) described three axon types of cerebral origin.
Type 1 axons contain 'classical' electron dense neurosecretory granules and are subdivided into catagories a, b and c; a axons contain granules 60-90nm diameter; b axons have granules of diameter 100-200nm diame4elt-and c axons contain granules 200-350nm in diameter. Type 2 axons contain electron lucent granules 280nm in diameter, containing fine flocculent material. Type 3 axon, much rarer than types 1 and 2, contains 'normal' dense granules mixed with 'structured' grains containing small dense mounds.
Cazal (1971) compared axon type la,lb and lc (Cazal et a1,1971) with type I, II and III of Cassier and Fain-Eaurel (1970), and considered types la and lb to correspond to type I, and types
II and III to correspond to type lc, but concluded that they were not entirely comparable.
In a third ultrastructural study.on the neurohaemal region of Locusta corpora cardiaca,(Goldsworthy and Taylor, unpublished observations cited in Mordue and Goldsworthy,1974), six classes of neurosecretory axon profiles have been described.
In addition to the cerebral neurosecretory axon terminals, the anterior region of the locust corpora cardiaca contain approximately18 intrinsic 'chromophobic' cells (Cazal et al,
1971; Highnam.and Goldsworthy, 1972). They are large cells, having the appearance of hypocerebral ganglion type nerve cells. 18
They are localised in the neurohaemal region accompanying the penetration of a nerve (Cazal et al, 1971) and they may contain electron dense granules (Cazal, 1971).
A third component of the locust corpora cardiaca is the glial cells. They surround the nervi corpori cardiaci and delimit a vast intercellular lacunar system.
The posterior part of the locust corpora cardiaca is glandular in appearance. It does not contain 'A' material as defined by histological staining, but does contain 'B' material, considered to be an intrinsic secretion (Highnam, 1961). Ultrastructural evidence indicates a protein synthetic activity in the glandular cells (Cazal et al, 1971). Spherical, electron dense, homogeneous, secretory granules are irregularly distributed in the cell, being most abundant in the regions distal from the cell body. These cytoplasmic projections are considered to represent a storage place of the secretory granules (Cassier and Fain-Maurel, 1970).
Cazal et al (1971) have described_two kinds of electron dense granules in the glandular cells; electron dense, homogeneous,
500nm in diameter and 'grey grains 600-800nm in diameter. Other authors consider that these cells contain only one type of secretory granule of diameter 500-600nm (Rademakere et al, 1976) that there is only one type of cell constituting the intrinsic glandular part of the locust corpora cardiaca (Cassier and Fain -
Maurel, 1970).
It is not clear if the glandular cells of the posterior region 19
of the locust corpora cardiaca can be regarded as neurosecretory cells. Anatomical, physiological and morphological criteria have le4d to this conclusion in some insects (Cazal,1948; Mormann,1965). However, the synaptic vesicles indicating a neuronal nature observed in the intrinsic cardiaca cells of Calliphora (Rormann, 1965) have not been found in Locusta (Cassier and Fain-Maurel, 1970). Cazal et al
(1971) consider the glandular region to be composed of cells with clear endocrinological characteristics, and find difficulty in considering such cells as secretory neurones.
There is clear histological evidence of an extension of the paired anterior storage region into the posterior glandular part of the corpora cardiaca in locusts (Highnam, 1961). During electron microscope studies, neurosecretory fibres containing electron dense vesicles have been found in the glandular region and are assumed to have extended from the storage region. Cassier and Fain-Maurel (1970) consider such fibres to be of type I and II axons previously described for storage region axon types. Cazal et al (1971) have
described only one type of neurosecretory axon present in the glandular region, identical with type la of the storage region (granules 60-90nm in diameter). Rademakers. et al (1976) also describe one type of axon terminal in this region containing electron dense
vesicles 95nm in diameter. Goldsworthy and Taylor (cited in Mordue
and Goldsworthy, 1974) found type 5 neurosecretory axon to be the
only class in the glandular region. The axon terminals appear to
make synaptic contact with the glandular cells (Rademakers et al, 1976).
These axon terminals may originate from cells in or near the cerebral
lateral neurosecretory cell group (Rademakers and Beenakkers, 1978). 20
2. Structure of the cockroach cephalic neurosecretory system.
The major components of the cockroach cephalic neurosecretory
system are the groups of neurosecretory cells within the brain,
their axonal pathways and the corpora cardiaca. Axons of the
cerebral neurosecretory cells leave the brain in three pairs of nerves, the NCCI, II and III, and a fourth pair not positively identified. The axons of the NCCI and II penetrate the anterior face of the corpora cardiaca, the NCCIII penetrating more dorsally (Willey,1961). These axons form the bulk of the inner core of
the gland and the intrinsic glandular cells are intermingled among the fibres,(Seshan and Levi -Montalcini, 1971).
a) Cerebral neurosecretory cells.
In the brain of P. americana are a group of approximately 100 cells along the dorsal midline of the pars intercerebralis which appear
blue-white in living specimens and stain with classical neurosecretory
stains (Gosbee et al, 1968). They contain granules up to 150nm in diameter (Bern et al, 1961; Willey and Chapman, 1962). Willey
(1961) has described four pairs of topologically distinct cell groups in the pars intercerebralis. Many cells of the most anterior groups contain cytoplasmic inclusions which are stainable with several selective neurosecretion stains.
In P americana two groups of lateral neurosecretory cells in each half of the protocerebrum with 10-40 cells in each group
have been described. These cell groups are not as laterally
situated as has been described for other insects (Willey, 1961). 21
Such cells were not described in two other studies on the same insect (Puller, 1960; Pipa, 1962).
b) Nerve connections within the system.
The corpora cardiaca are joined to the brain by three pairs of nerves (possibly four). The NCCI are the largest and most median, and the finer nerves of the NCCII and III are more lateral. The ventro —posterior lobes of the corpora cardiaca unite with the hypocerebral ganglio4 separately by means of two connectives, the same diameter as the NCCI. The ventral lobe of each half of corpus cardiacum is joined to the corpora allata by a stout extension, the
NCAI.
The NCCI leave the base of the brain on either side of the midline and enlarge to form the anterior portion of the corpora
cardiaca. The NCCI contain neurosecretory axon tracts from the protocerebral medial cell groups, most of which decussate in the
brain before passing to the corpora cardiaca. The NCCI may contain fibres from other sources (Willey, 1961).
The NCCII leave the back of the protocerebrum to join the
corpora cardiaca in close proximity to the NCCI. Axons from the lateral cell groups run directly to the ipsilateral corpora cardiaca
in the NCCII which also receives fibres from the medial neurosecretory
cell groups of the pars intercerebralis. Small branches of the NCCII extend to the end of the corpora cardiaca adjacent to the
aortal lumen. 22
The axon tracts in the NCCIII originate from an unknown source in the tritocerebrum and do not contain stainable neuro- -secretory material. The corpora cardiaca of P. americana are joined dorsally by a single dorsal comissure and the NCCIII, after leaving the lateral surface of the tritocerebrum, insert into the corpora cardiaca at the dorsal. comtisure and decussate.
c) The corpora cardiaca.
The anterio-ventral region of the cockroach corpora cardiaca is composed largely of axon terminals from the cerebral neurosecretory cells. In this part, neurosecretory material is confined to the nerve bundle, then distally 'fans out' into the cardiacum tissue (Scharrer, 1963). Neurosecretory material is more abundant in the marginal zones (Scharrer, 1952). In P. americans axon terminal profiles contain electron dense granules 150nm and 300nm in size, apparently confined to separate neurone types (Willey and Chapman, 1960; Scharrer and Kater, 1969). Cell processes considered to be of cerebral origin have been described in the corpora cardiaca of
Leucophaea maderae: they contain uniform populations of highly electron opaque granules. Small 'black' granules (90-150nm) occur in fine processes while the majority of profiles are larger containing granules 220-360nm in diameter (Scharrer, 1963).
Cell boundaries cannot be visualised satisfactorily in histological preparations of the cockroach corpora cardiaca, therefore the location of secretory products recognized by their stainability cannot be determined with certainty (Scharrer, 1963). In the posterio -
-dorsal region, some intrinsic parenchymatous glandular cells with 23
clear boundaries have been visualised in P. americana which
contain intracellular paraldehyde fuschin positive granules
(Khan and Fraser, 1962). In L. maderee intrinsic cells have
been reported to contain a spattering of electron dense granules
300nm in diameter which are morphologically indistinguishable
from those of extrinsic origin (Scharrer, 1963).
In L. maderae the pear shaped parenchymatous glandular cells
contain mixed populations of electron dense and pale granules,
200-300nm in diameter (Scharrer, 1963). The granules of low
electron opacity are presumed to derive from the more opaque
elements. The parenchymal cells are considered to exhibit
structural details of glandular and nerve cells, and consequently
termed 'neuroglandular' cells. Differences observed among the
glandular cells can be interpreted either as differences in
functional states or the existence of more than one type of
glandular cell (Scharrer, 1963).
3. Cephalic neurosecretion and carbohydrate metabolism.
a) General considerations of carbohydrate metabolism in insects.
The major sources of carbohydrate reserves in insects are flight
muscle glycogen, glycogen deposits in the fat body, and trehalose
and other sugars in the haemolymph and gastro-intestinal tract.
The fat body is the main site of glycogen deposition in the insect
body, and this reserve is mobilised as trehalose which, with the
blood trehalose, acts as a major energy supply in many insects. 24
In contrast to the haemolymph trehalose concentration, the haemolymph glucose concentration is negligible. •
Following ingestion of carbohydrates, there is no evidence
of active transport of monosaccharides through the gut wall (Treherne, 1958). They are absorbed
by passive diffusion which is facilitated by the rapid conversion
of glucose to haemolymph trehalose by the fat body. There is evidence of regulation of haemolymph trehalose'levels and in
insects, as in vertebrates, the regulation of carbohydrate metabolism may be effected in part by hormonal mechanisms. A summary of the interconversions of glucose, glycogen and trehalose
together with some possible associated regulatory mechanisms is
given in figure 1.
The two main areas for which hormonal control has been
suggested in the regulation of carbohydrate metabolism in insects are at glycogen degradation, at the step catalyzed by glycogen phosphorylase (Steele, 1963; Goldsworthy;1969, 1970) as is the
case in vertebrate carbohydrate metabolism, and at glycogen synthesis (enzyme unspecified), (Van Handel and Lea, 1965, 1970; Lea and Van Handel, 1970). In addition, it has been suggested that the degradation or removal of haemolymph trehalose may be inhibited
hormonally (Hana.oka and Takashashi, 1976), and also that the activity
of trehalose phosphate synthetase may be modified by hormones in some way as yet unknown (Wyatt, 1967). GLYCOGEN OSPHORYLASE ACTIVATED BY CYCLIC AMP INHIBITED BY GLUCOSE-6-P 741. HORMONAL CONTROL C r4, .7311
0 UDP-GLU M PYROPHOSP1 0
› c
-D ACTIVATED BY HIGH LEVELS OF GLUCOSE-6-P C
m O
TREHALOSE-6-P TREHALOSE-6-P SYNTHETASE PHOSPHATASE m ACTIVATED BY LOW LEVEL 1 OF GLUCOSE-6-P - c" n INHIBITED 4 INHIBITED BY TREHALOSEI 11 Cr) , H
Gn A1D 1VH31 NO OD 3S0 TREHALAS N3D 26
b) Involvement of the corpora cardiaca. Steele (1961) first described the effect of injected corpus cardiacum extracts in elevating haemolymph trehalose levels in
P. americana. The fat body is the source of the additional trehalose after treatment with corpus cardiacum extracts, while thoracic muscle glycogen is unaffected.
The tissue specificity of the hyperglycaemic effect has invoked comparison of the activity with that of vertebrate glucagon (Steele, 1963, 1966, 1976). Glucagon activates primarily glycogen phosphorylase in the mammalian liver, and it has been demonstrated that corpus cardiacum extracts will activate fat body glycogen phosphorylase in locusts and cockroaches (Steele, 1963;
Weins and Gilbert, 1967; Mordue and Goldsworthy, 1969; Goldsworthy,
1970). Glucagon interacts with a membrane bound adenyl cyclase
leading to an increase in the concentration of cyclic adenosine monophosphate (cANP). cAMP increases phosphorylase activity in cockroach fat body in vitro (Steele, 1964) and increases the
activity of phosphorylase extracted from the fat body of L. migratoria (Applebaum and Schlesinger, 1973). Efforts to show an activation of adenyl cyclase, and an increase in the cANP in the fat body of P. americana in response to corpus cardiacum extracts have recently been successful (Hanaoka and Takahashi, 1977). The steps involved in the activation of glycogen phosphorylase are
summarised in the diagram below. From what is known concerning
the homeostatic regulation of trehalose haemolymph levels (Wyatt,
1967), the following sequence of events following glycogen
phosphorylase activation can be envisaged. After stimulation of
27
Activation of phosphorylase in liver and proposed mechanism in
the insect fat body.
CELL MEMBRANE INACTIVE PHOSPHORYLASE B KINASE PROTEIN KINASE ADRENALIN ADENYL ACTIVE GLUCAGON CLASE PHOSPHORYLASE B KINASE CORPUS CARDIACUM EXTRACTS ? PHOSPHORYLASE PHOSPHORYLASE B A
glycogen breakdown, the fat body intracellular concentration of
glucose-6 —phosphate is increased. This overcomes the inhibition of trehalose-6 —phosphate synthetase by trehalose, and results in increased trehalose synthesis and release into the haemolymph.
Weins and Gilbert (1967) have suggested that in addition to
activating glycogen phosphorylase, the factor may also block glycolysis and activate trehalose phosphate synthetase. Increased
fatty acid oxidation results to compensate for energy demand. However, as purification of the hyperglycaemic factor has not been effected, it is not known if both activities are attributable to
the same factor. This is only one of several interpretations
which could be proposed (Wyatt, 1967). The corpus cardiacum factor 28
may inhibit the degradation or removal of haemolymph trehalose as indicated by the length of time of elevated haemolymph trehalose levels (30 hr.) following injection of corpus cardiacum extracts, compared with the relatively rapid removal of trehalose from
the haemolymph after artificial elevation by injection of trehalose
(Hanaoka and Takahashi, 1976).
The activation of flight muscle glycogen phosphorylase is unlikely to be hormonally controlled by corpus cardiacum factors. Activation of the blowfly flight muscle phosphorylase b kinase does not require adenosine triphosphate OW), nor is it dependent on cANP (Sacktor et al, 1974), and this apparent absence of a cAMP dependent kinase makes regulation of muscle glycogenblysis by corpus cardiacum hormones unlikely in this insect. However, the flight
muscle phosphorylase of Phormia regina is the only one so far fully characterised. The presence of this enzyme activity has been only noted in the muscles of the roach Blaberus discoidalis (Wyatt, 1967) and the locust L. migratoria (Goldsworthy, 1970).
Hormonal control of glycogen synthesis in insects has been
postulated, although not directly involving the corpora cardiaca.
After removal of the cerebral neurosecretory cells, glycogen has been shown to accumulate in the tissues of Calliphora erythrocephala
(Thomsen, 1952), Aedes itaeniarhynchus; (Van Handel and Lea, 1965, 1970;
Lea and Van Handal, 1970) and Locusta (Goldsworthy, 1971). This is
unlikely to be due to lack of corpus cardiacum hyperglycaemic factor
as corpus cardiacum extracts do not produce hyperglycaemia in 29 cauterised Locusta (Goldsworthy, 1971) and removal of the cerebral neurosecretory cells does not affect fat body phosphorylase levels in this species (Goldsworthy, 1970). It does not alter the rate of glycogen utilisation in the fat body of the mosquito (Lea and Van
Handel, 1970).
c) Orions of the corpus cardiacum factors.
Investigations into the origins of the hyperglycaemic activity contained in corpus cardiacum extracts have involved procedures such as ablation of the cerebral neurosecretory cells, cardiectomy, nerve section, bioassay of brain extracts and separate bioassay of storage and glandular lobe extracts in the case of the locust corpora cardiaca.
Ablation of the cerebral medial neurosecretory cells (DNC) in mosquitos causes glycogen to accumulate at the expense of lipid (Van Handel and Lea, 1970; Lea and Van Handel, 1970). Removal of
these cells in Calliphora also results in an accumulation of fat body glycogen but lipid accumulates at an increased rate (Thomsen,
1952). Ablation of the MNC in Locusta also causes glycogen to accumulate in the fat body (Goldsworthy, 1971). The mechanism of the glycogenic effect produced by MNC removal is not clear. As removal of the MNC does not impair glycogen utilisation in mosquitoes
(Van Handel and Lea, 1970), the MNC product must either suppress glycogen synthesis or stimulate a carbohydrate requiring biosynthetic reaction (Steele, 1976). However, the level of fat body glycogen
synthetase is not increased in cauterised locusts (Hill, 1972) nor is there any change in phosphorylase activity (Goldsworthy' 1971). 30
Cardiectomy or removal of the glandular lobes in L. migratoria results in decreased titres of haemolymph trehalose (Cazal, 1971) and total haemolymph carbohydrates (Jutsum and Goldsworthy, 1976), a marginal increase in haemolymph glucose (Cazal, 1971) and a fall in fat body active phosphorylase levels (Goldsworthy, 1970). Cazal (1971) found that section of the NCCI and II produced effects comparable to those of cardiectomy while Jutsum and Goldsworthy (1976) found that 1NC cautery and NCCI and II section had no effect on blood carbohydrate levels. Eleven days after glandular lobe removal, the level of haemolymph carbohydrate was found to have returned to normal (Jutsum and Goldsworthy, 1976). Section of NCCII in
P. americana is reported to cause a decrease in haemolymph trehalose levels (Gersch, 1974).
There is conflicting data on the hyperglycaemic activity of brain extracts. Slight hyperglycaemic activity has been recorded in brain extracts of Periplaneta (Ralph and McCarthy, 1964) and Carausius (Deutrieu and Gourdaux, 1967) but not in Periplaneta by
Steele (1961) or in Locusta (Mordue and Goldsworthy, 1969). In brain extracts, the active factor may be bound in a carrier protein
complex, and this could account for the low level of recorded
biological activity-(Mordue and Goldsworthy, 1969). In Locusta, hyperglycaemic activity has been detected in de novo corpora
cardiacapstructures which are regenerated at the cut ends of the
NCCI and devoid of intrinsic glandular cells (Highnam and Goldsworthy,
1972).
Bioassay of glandular lobe and storage lobe extracts of Locusta 31
corpora cardiaca shows that both parts can elevate haemolymph
carbohydrates and the level of fat body active phosphorylase, the glandular lobe extract being the more potent (Nordue and Goldsworthy, 1969). The intrinsic glandular cell factor is considered to be the
more physiologically important of the two factors (Goldsworthy and
Mordue, 1974). Although the relationship between the two hyperglycaemic factors is not clear, it has been suggested that the storage lobe
neurosecretory factor may have a tropic effect on the glandular lobe activity (Highnam, 1965; Hill, 1972).
d) Physiological significance of the corpus cardiacum factors affecting carbohydrate metabolism. The physiological role of the hyperglycaemic activity is as uncertain today as when it was first described by Steele (1961) (Steele, 1976). The relationship of haemolymph carbohydrates levels to specific physiological functions is unknown. Steele (1963) suggested that its probable function in cockroaches is to maintain an adequate
supply of haemolymph trehalose during intense activity, but the maximum response to corpus cardiacum extracts is attained only after
3-5 hours (Steele, 1963) and it is unlikely that the cockroach would
sustain intense activity for this length of time (Goldsworthy and
Mordue, 1974).
Weins and Gilbert (1965, 1967b) have related the amount of
phosphorylase activating activity in the corpora cardiaca in L.maderae to changes in the fat body glycogen content during oogenesis and suggest a possible function of the corpus cardiacum hyperglycaemic activity in the mobilisation of glycogen reserves for egg production. 32
It is known that flight causes release of neurosecretion from the storage lobes in locusts (Delphin, 1963; Highnam and Haskell, 1964), but a link between this release and mobilisation of fuels for flight has not been established. The presumed association of the hyperglycaemic factor(s) with the corpora cardiaca activity favouring fatty acid oxidation in the fat body (Weins and Gilbert, 1967) has led to speculation that the corpus cardiacum hyperglycaemic activity may be involved in providing fuel for flight in the initial stages as ketone bodies and later as trehalose (Steele, 1976).
However, there is no indication of an involvement of the corpus cardiacum hyperglycaemic factors in metabolism during flight in locusts (Jutsum and Goldsworthy, 1976 ; Goldsworthy, 1976). Little is known about the conditions governing the release of the hyperglycaemic factors from the corpora cardiaca. Normann and Duve (1969) have concluded that the brain controls the release of the hyperglycaemic factors from the corpora cardiaca in Calliphora. Goldsworthy (1970) considers that cerebral neurosecretion is not necessary for the synthesis or release of these factors. Their release from the corpora cardiaca in Periplaneta following potassium depolarisation suggests conventional nervous processes may be involved (Gersch, 1974).
Adult locusts rarely show significant elevation of haemolymph carbohydrates in response to corpus cardiacum extracts, although the same extracts can produce marked hyperglycaemia in cockroaches (Goldsworthy, 1969). Similarly, extracts of silkmoth corpora cardiaca will activate fat body phosphorylase in the cockroach but will produce no such effect in the silkmoth itself (Weins and Gilbert, 1967a). 33
It therefore appears likely that the factors from the locust and silkmoth corpora cardiaca which elicit a hyperglycaemic response in the cockroach perform a different function in these animals
(Goldsworthy, 1968; Hordue and Goldsworthy 1974).
4. Cephalic neurosecretion and lipid metabolism.
a) General considerations of lipid metabolism in insects. The major lipid component of the fat body is triglyceride, while either diglycerides or triglycerides are the major haemolymph lipids. The lipid content of the haemolymph is small compared with the fat
body, but it constitutes an important energy reserve. There are large variations in the relative proportions of haemolymph and fat body lipids, and also in the total lipid content according to age, sex, nutritional and hormonal status of the insect.
In adult P. americanaltriglycerides are the predominant
haemolymph lipid for most of the time (Nelson et al, 1967),
although in young adults diglyceride may be the major haemolynph lipid component (Downer and Steele, 1969, 1972). Adult locusts
contain predominantly diglycerides in the haemolymph (Tietz,
1963, 1967).
Required lipids are synthesised, primarily in the fat body,
from ingested lipids and carbohydrates. The conversion of carbohydrates to lipid in the fat body and its control has been investigated by Walker and Bailey (1970a,1970,1971). Intracellular 34 levels of AMP and ATP and end produot inhibition control the key
enzymes of glycolysis, which is the first stage of conversion. It appears that the synthetic systems for fatty acids are similar
to those of mammalian and avian systems. The rate of fatty acid biosynthesis is influenced by levels of fatty acids and carbohydrates
in the diet.
There is little data available concerning the degradation of lipids by the fat body. It is assumed thatp-oxidation operates in the fat body but enzyme activity has been little investigated.
Fatty acids are the preferred substrates at all ages in the fat body of S. gregaria (Walker et al, 1970). Degradation of fatty acids in the flight muscle also proceeds byj9 -oxidation (Beenakkers, 1963,
1966, 1969; Beenakkers et al, 1975).
Lipid released from the fat body can be triglyceride, diglyceride or free fatty acid depending on the insect species.
In both Locusta (Teitz, 1962, 1967) and Periplaneta (Downer and Steele, 1972; Chino and Gilbert, 1965 ), diglyceride is primarily released in vitro and replaced by lipid formed from hydrolysis of
fat body triglyceride. However, Bhakthan and Gilbert (1968) have found that free fatty acids are predominantly released from the
fat body in four genera of roach, as has Chang and Friedman (1971).
There is good evidence that lipids are transported in the haemolymph
as lipoproteins, and that glyceride release depends largely on the
addition of the glyceride on to a protein circulating in the
haemolymph (Hwangi and Goldsworthy, 1977). 35
b) Involvement of the corpora cardiaca. i. The adipokinetic hormone of locusts The work of Mayer and Candy (1969) and Beenakkers (1969)
demonstrated the existence of an adipokinetic activity in locust
corpus cardiacum extracts. Injections of corpus cardiacum extracts produce an increase in haemolymph lipid concentrations due to an increase in the diglyceride fraction (Mayer and Candy, 1969). The
fat body was shown to be the source of the additional diglycerides. It was suggested that this corpus cardiacum factor is involved in the control of lipid release from the fat body during flight, and the
presence of the active factor in the haemolymph of flown locusts was demonstrated (Mayer and Candy, 1969).
Since this work, the role of the adipokinetic hormone (AKH) in the maintenance of long term flight activity in locusts has been well established (Goldsworthy et al, 1972, 1973; Houben and
Beenakkers, 1975; Goldsworthy and Coupland, 1974; Robinson and
Goldsworthy, 1974, 1977a & b ; Spencer and Candy, 1974, 1976). In the initial stages of flight, energy is derived from trehalose
in the haemolymph (Weis-Pogh, 1952). Soon after the commencement of flight, AKH is released from the intrinsic glandular cells of the corpora cardiaca (Mayer and Candy, 1969; Goldsworthy et al, 1972a,b)
and the hormone acts in two ways. It stimulates the release of
diglycerides from the fat body into the haemolymph (Spencer and
Candy, 1974; Jutsum and Goldsworthy, 1976) and these are transported as lipoproteins to the flight muscle (Mayer and Candy, 1967). It
has been suggested that AKH acts by stimulating fat body lipase 36
activity (Spencer and Candy, 1976). The second action of AKH is to stimulate the oxidation of diglyceride in the flight muscle, possibly by increasing the flux of fatty acids into the mitochondria, the site of/a—oxidation enzymes (Robinson and Goldsworthy, 1976a, 1977). As lipid oxidation increases, so carbohydrate oxidation decreases, thereby conserving carbohydrate reserves for other metabolic activities.
Recently, the adipokinetic hormone has been isolated from the glandular lobes of the corpora cardiaca of both L. migratoria and
S. gregaria (Stone et al, 1976) and it has been suggested that the hormone may be contained in the large 600nm electron dense vesicles present in the glandular lobe cells (Mordue and Goldsworthy, 1974).
The stimulus for the release of AKH is not clear. In resting locusts, AKH is not thought to be involved in blood lipid homeostasis
(Jutsum and Goldsworthy, 1974), and the stimulus for release is likely to be connected with the flight process (Goldsworthy, 1976).
From nerve section experiments, it appears likely that a double innervation of the glandular lobe may function to control AKH release (Goldsworthy et al, 1972a). Cautery of the cerebral I1IC does not prevent AKH release (Goldsworthy et al, 1'973) but the lateral neurosecretory cell region may be, involved in the release mechanism (Goldsworthy, 1976; Rademakers do Beenakkers,1978). Haemolymph substrate concentration may also be involved in the control of
AKH release (Houben and Beenakkers, 1975; Jutsum and Goldsworthy,
1975)• 37 ii. Hypolipaemia in cockroaches and hyperlipaemia in locusts.
Injection of Locusta and Periplaneta corpus cardiacum extracts into locusts produces a hyperlipaemic response (Goldsworthy et al, 1972b). Injection of the same extracts into Periplaneta however causes a decrease in the haemolymph levels of triglyceride and diglyceride with a concommitant increase in fat body triglyceride concentration (Downer and Steele, 1969, 1972), It has been suggested that the same factor is present in the corpora cardiaca of locusts and cockroaches, the specificity of response residing at the fat body and resulting in hyperlipaemia in locusts and hypolipaemia in cockroaches (Downer, 1972). The hypolipaemic activity of the corpora cardiaca in cockroaches is thought to be involved in stimulation of dietary lipid uptake by the fat body (Downer and Steele,
1972).
The cockroach hypolipaemic factor is thought to be distinct from the cockroach hyperglycaemic factor (Downer and Steele, 1973) although no details of purification procedures have been published. The hypolipaemic activity of corpus cardiacum extracts has not been
demonstrated by Goldsworthy et al (1972b) in P. americana or in the
cockroach Gromphadorhina portentosa..
The complete lack of an adipokinetic response in P. americana
to its own corpus cardiacum extract indicates that the cockroach factor which produces hyperlipaemia in locusts performs an alternative
function in the cockroach. 38
iii.Endogenous fat body metabolism. In cockroaches, corpus cardiacum extracts appear to switch endogenous fat body metabolism in vitro to oxidation of lipid in preference to carbohydrate, in addition to reducing synthesis of lipids from glucose (Weins and Gilbert, 1965, 1967b). This activity has been associated with the hyperglycaemic activity of corpus cardiacum extracts. However, it has recently been reported that in intact cockroaches, corpus cardiacum extracts may reduce lipid oxidation
(Hoffmann and Downer, 1974).
In locusts, injections of corpus cardiacum extracts and flight are reported to increase the production of ketone bodies in the fat body (Mayer and Candy, 1969; Beenakkers, 1965). Hill et al (1972) and Bailey et al (1972) have suggested that these ketone bodies, formed during mobilisation of fat body triglyceride reserves, are either oxidised by the fat body or carried in the haemolymph for use as respiratory fuel.
5. Cephalic neurosecretion and water balance.
a) General considerations of water balance in insects.
The Malpighian tubule-hindgut complex plays a vital role in osmoregulation and excretion in insects, it being virtually impossible to separate these two functions physiologically.
The Malpighian tubules supply the hindgut with an iso-osmotic fluid containing the smaller sized haemolymph constituents more or 39 less proportional to their concentration in the haemolymph. The hindgut reabsorbs those constituents which are required and rejects others so that the composition and volume of the haemolymph remains constant or is adjusted to the requirements of the insect. The
Malpighian tubules may actively secrete substances into the primary excretory fluid, and the rectum may also add substances until finally a secondary urine is produced, either hypo- or hyper-osmotic, and eliminated from the insect. The secretion of urine by the Malphigian tubules is thought to involve the active transport of potassium ions into the distal part of the lumen, providing the driving force in urine production (Ramsay, 1958; Berridge, 1968). The movement of water, dissolved substances and ions into the lumen then occurs passively. The passage of small molecules into the lumen depends both on their concentration in the haemolymph and on the ease of crossing the cell membranes. Active transport is also involved in rectal regulation, and transport of water across the rectal wall is secondary, ions being taken up sometimes actively and water following osmotically.
As mentioned above, potassium is considered to be the'prime mover' in active transport of ions during the secretion of urine by the Malpighian tubules. It has been suggested that the apical surface of the cells have an electrogenic pump while the basal cell membrane t 4- 2+ has a Na -K activated mg dependent ATPase pump for active exchange transport (Berridge and Oschman, 1969).
b) Involvement of the corpora eardiaca.
Several studies have indicated a possible double hormonal control 40 of the water content of insects by action on two separate targets: alteration in the rate of primary urine formation by the Malpighian tubules and modulation of water reabsorption by the rectum (Cazal and
Girardie, 1968; Mordue, 1969). There is considerable variation in the literature according to the species considered and the assay technique used. This introduction therefore will be largely restricted to the data for locusts and cockroaches. i. Factors affecting Malpighian tubule function.
The storage lobes of locust corpora cardiaca are reported to contain a factor which will increase the rate of fluid secretion through the tubules in vivo (Mordue and Goldsworthy, 1969). Extracts
of glandular lobes have little effect on Malpighian tubule secretion (Mordue and Goldsworthy, 1969).
Using in vitro preparations, Cazal and Girardie (1968) have
demonstrated a factor in the storage lobes of L. migratoria which reduces the rate of excretion through the Malpighian tubules and Cazal (1971) was unable to detect the presence of a factor which
increased tubule secretion within either lobe of the corpora cardiaca of L. migratoria.
The corpora cardiaca of P. americana are reported to contain
a factor which decreases the rate of dye excretion by Malpighian tubules in vitro (Wall and Ralph, 1962) with no indication of
diuretic activity. The corpora allata of P. americana are also reported to contain an antidiuretic factor of similar activity to 41
the corpus cardiacum extracts (Wall and Ralph, 1964).
ii. Factors affecting rectal function.
It has been reported that factors which both increase and decrease water reabsorption in the rectum are present within the corpora cardiaca of L. migratoria and S. gregaria (Mordue,
1969, 1970b; Cazal and Girardie, 1968). Most investigations into hormonal control of rectal function have utilised in vitro preparations.
Corpus cardiacum extracts of S. gregaria have been reported to exert a diuretic effect upon rectal function (Mordue, 1969, 1970b) whereas corpus cardiacum extracts from L. migratoria exert an antidiuretic effect (Mordue, 1970b). The antidiuretic component is thought to be restricted to the glandular lobes (Mordue, 1970b). Cazal and Girardie
(1968) reported that both glandular and storage lobe extracts increased rectal water reabsorption in L. migratoria.
There are few reports in which cockroach corpus cardiacum extracts have been found to alter the amount of rectal water reabsorption although Wall and Ralph (1965) have reported that brain and prothoracic gland extracts increase the amount of water reabsorbed in in vitro rectal preparations.
The mode of action of the corpus cardiacum factors on the excretory system is not at all well understood. In Carausius, it has been suggested that the diuretic activity found in brain, corpora cardiaca and suboesophageal ganglion extracts (Pilcher,
1970). changes only the rate of urine production, possibly by stimulating active e transport. In Rhodnius, cGMP is thought to
be involved as 'second messenger' during stimulation of Malpighian
tubule function by the diuretic hormone from the mesothoracic
ganglion mass (Maddrell et al, 1971). It has been suggested that
in S. gregaria, the corpus cardiacum factor may elevate cellular
levels of sAMP in the Malpighian tubules which subsequently elevates
active phosphorylase and trehalase levels and thus increases the
rate of excretion (Mordue, 1970a).
Hormonal control of water absorption by the rectum is intimatly
associated with the state of hydration of the insect, but the
mechanism of water absorption modulation by the rectum is largely
unknown.
c) Origins of the corpus cardiacum factors.
Destruction of the ERG of the pars intercerebralis produces a
marked increase in the blood volume and reduction of excretion in
locusts (Highnam et al, 1965; Mordue, 1969, 1971; Cazal and
Girardie, 1968). Storage lobe extracts can elevate this low excretion
level whereas glandular lobe extracts have little effect (Mordue, 1969,
1971). These results suggest the production of a diuretic factor
by the neurosecretory cells in the brain and its transport to the
storage region of the corpora cardiaca. Girardie (1970) has suggested
that the group of four neurosecretory cells outside the main HNC
group are the source of the diuretic factor in L. migratoria. Cazal
(1971) suggests that both diuretic and antidiuretic activities of
storage lobe extracts are of cerebral origin in L. migratoria.
The antidiuretic activity of the glandular lobes, active only on 43 rectal water reabsorption, is likely to be as a result of an intrinsic secretion of the glandular cells (Cazal, 1971).
6. Nature and number of active factors in the corpora cardiaca of locusts and cockroaches.
Many vertebrate neurosecretory hormones are known to be peptides or polypeptides, and many of the activities found in the corpora cardiaca are associated with proteinaceous material. Initially, the proteinaceous nature was inferred from histological staining reactions, and subsequently confirmed by abolition of the activities following enzymic digestion of the corpus cardiacum extracts.
Many biological activities have been recorded from corpus cardiacum extracts. Before a distinction can be made between the specific and non-specific actions of the extracts (e.g. histamine is ubiquitously present in mammalian tissue extracts) and in order
to recognise which actions are of real physiological significance,
there is a requirement for the isolation and identification of the active substances present in the extracts.
The present study has attempted to clarify the involvement of
corpus cardiacum factors in lipid metabolism, carbohydrate metabolism and water balance in locusts and cockroaches. Locusts have a
principally lipid based metabolism while cockroaches primarily metabolise carbohydrates. This may be a reflection of their
different energy requirements for flight: locusts undergo long 44
migratory flights whereas cockroaches have a poorly developed flight habit and are better adapted for fast running. These different substrate requirements may be reflected in differences in the neurohormal control of substrate mobilisation, either by variation in the chemical nature of the neurohormone or in the response of the target tissue. Hypothalamic peptide hormones appear to have changed little during the course of evolution
(Barrington, 1964 ). It is therefore possible that the corpora cardiaca of different insect species will contain a family of closely related peptides, the specificity of each being determined principally at the target tissues. The classification of corpus cardiacum factors on the basis of responses produced in selected assay systems has contributed to the present confused situation concerning the neurohormal control of physiological processes in insects.
a) Structural indications.
The ultrastructural data, referred to earlier, provides an indication of the possible variety of components in the corpora cardiaca.
Attempts have been made to classify axon types and cerebral neurosecretory cells principally on the basis of shape, size and electron opacity of the granules which they contain, and also on histological staining properties. The classification of insect neurosecretory cells by their staining properties has been the subject of much controversy. Cell types A,B and C are widely used, and there is some histochemical evidence that the proteins elaborated
by these three types of cell show real differences (PrentA, 1972). 45
The stainability of a neurosecretory neurone may not be constant
throughout its length, interpreted as a chemical change in the hormone, carrier protein or membrane of the granule en route (Gabe, 1972). Granules detected at the electron microscope level are not always visualised using 'neurosecretory' staining techniques, for example the lateral neurosecretory cells of the protocerebrum of Orthoptera. These secretions must differ in some way from those of
an 'orthodox' neurosecretory neurone.
In several ultrastructural studies on the storage region of
locust corpora cardiaca, at least three and possibly as many as five axon types have been described. Attempts have been made to relate the number of extract activities to the number of axon types, and there are more activities than types. Consequently each cell
type may produce more than one active product, each factor may have more than one activity, or cells elaborating different products may not show any obvious differences in structure. The appearance of
granules may bear no relationship to the species of active peptide contained, as most of the visualised material is likely to be carrier protein (Sloper, 1957).
The neurosecretory or non-neurosecretory nature of the intrinsic glandular cells of the corpora cardiaca has been the subject of much
debate and remains unresolved. Some authors have described axon-like
processes of the glandular cells, having a denser component of neurosecretory granules in these regions and so analagous to the
axon terminal regions of the classical neurosecretory neurone 46
(Scharrer, 1963). Other authors attribute neurosecretory status to these cells on the basis of propagated action potentials (Norman, 1975) although the glandular cells and cerebral neurosecretory cell terminals are in such close juxtaposition that precision concerning the identity of the cell propagating the potential may be difficult. Neither of these criteria is considered however to be a necessary or sufficient definition of a neurosecretory neurone (Rowell, 1976). Other authors consider the intrinsic glandular cells to be non-neural on the basis of histological appearance (Cazal et al, 1971).
b) The glandular region of the locust corpora cardiaca. The adipokinetic hormone. The first insect neurosecretory hormone to be isolated, characterised and synthesised is the locust adipokinetic hormone (AKH) (Stone et al, 1976; Broomfield and Hardy, 1977). It is a blocked decapeptide, and closely resembles the structure of the only other arthropod neurohormone to be fully characterised (Fernlund, 1974), the red pigment concentrating hormone (UCH) of prawns as shown below.
AKH: PCA-Leu-Asn-Phe-Thr-Pro-Asn-Trp-Gly-Thr-NH2 1 2 3 4 5 6 7 8 9 10
RPCH: PCA-Leu-Asn-Phe-Ser-Pro-Gly-Trp-NH2
1 2 3 4 5 6 7 8
The first eight residues are homologous, except for Thr instead of Ser at residue 5 and Asn instead of Gly at residue 7. As a result 47 of structural similarity, ASH has been found to stimulate pigment concentration in prawns and shrimps, and RPCH can elicit an adipokinetic response in locusts (Mordue and Stone, 1976, 1977).
The present study has extended investigations into the pharmacological spectrum of AKH, with particular emphasis on its activity in the system of haemolymph carbohydrate elevation in cockroaches.
In addition, preliminary investigations into in vitro culture of the corpora cardiaca of L. migratoria have been carried out with a view to studying the synthetic capacity of glandular lobes maintained in vitro in terms of AKH production. Investigations into in vitro culture of insect neurosecretory systems have been concerned largely with the production of stainable material in the
MNC of cultured brains (Leloup and Gianfelici, 1966; Gianfelici,
1968a; Marks et al, 1973) and also with the in vitro transport of this material to the corpora cardiaca (Gianfelici, 1968b; Holman and Marks, 1974). However, there is no evidence to link secretory activity with morphological changes in cells (Herman, 1967;'Scharrer,
1948, 1964a,b), thus an alternative method of secretory activity assessment is required. Determination of the adipokinetic activity produced by corpora cardiaca in vitro could provide a quantitative means of assessment of secretory activity.
The hyperglycaemic factor.
The nature of the locust glandular lobe hyperglycaemic factor is unknown. It is thought to be a small peptide, similar in size to the adipokinetic hormone. It has been suggested that this factor is identical with AKH as these two activities have never been resolved from locust corpus cardiacum extracts (Nordue and Goldsworthy, 1969; see also Ode and Holwerda, 1976; Holwerda et al, 1977). Differences in the potencies of corpus cardiacum extracts from cockroaches and locusts in eliciting hyperglycaemia in cockroaches and hyperlipaemia in locusts may indicate separate identities for the adipokinetic and hyperglycaemic hormones (Goldsworthy et al, 1972b). These data however show hormonal differences between the corpora cardiaca of locusts and cockroaches and are not indicative of separate identities of the adipokinetic and hyperglycaemic factors within the corpora cardiaca of locusts or cockroaches.
The role of the hyperglycaemic activity of glandular lobe extracts
is uncertain. There appears to be little facility for the activation
of fat body glycogen phosphorylase as a means of haemolymph carbohydrate titre modulation in the locust (Goldsworthy, 1969,1970). It is therefore likely that the true physiological significance of
the glandular lobe hyperglycaemic factor lies in another direction.
Factors affecting heartbeat.
The cardioacceleratory activity of corpus cardiacum extracts has been monitored by the majority of workers using a semi—isolated heart preparation. It is generally agreed that this activity is not
due to the presence of biogenic amines in the extracts, but that it is associated with peptide material.
Nordue and Goldsworthy (1969) found that glandular lobe extracts
of the corpora cardiaca from L. migratoria caused an increase in the
beat of a semi—isolated heart preparation, and a decrease in the 49
amplitude. The cardioacceleratory activity of the corpus cardiacum extracts from S. gregaria may be restricted to the glandular region
(Highnam, 1961; Davey, 1963).
There is little evidence to support the concept of neurohormonal control of heartbeat by circulating corpus cardiacum factors in vivo.
Injections of corpus cardiacum extracts have little or no effect on heart beat in the intact animal (Mordue and Goldsworthy, 1969; Roussel and Cazal, 1969; Normann, 1972). The effect of corpus cardiacum extracts on the activity of semi—isolated hearts can however provide a useful bioassay for distinguishing between resolved factors (Mordue and Goldsworthy, 1969; Goldsworthy and Mordue, 1974).
o) The storage region of the locust corpora cardiaca.
The hyperglycaemic factor. Little is known concerning the nature of the locust storage lobe hyperglycaemic factor. The neurosecretory factor in the storage
regions of the locust corpora cardiaca and in de novo corpora cardiaca
is reported to posseseweak hyperglycaemic activity but little adipokinetic action (Highnam and Goldsworthy, 1972; Goldsworthy et al, 1973a, 1973b). It is thought that the very slight adipokinetic activity of storage lobe extracts (Goldsworthy et al, 1972b) is due to contamination with glandular lobe material (Goldsworthy and Mordue,
1974). Thus the hyperglycaemic peptide of the storage lobes, produced by the cerebral neurosecretory cells, is not thought to be identical with the glandular lobe adipokinetic hormone.
The function of the storage lobe hyperglycaemic factor in the 50 locust is as uncertain as that of the glandular lobe hyperglycaemic factor. The present study has attempted to clarify the significance of the storage lobe hyperglycaemic factor in the locust, principally
by its resolution and identification.
Factors affecting water balance. Little is known concerning the nature of the storage lobe factor
which increases the rate of amaranth clearance in vivo (Nordue and Goldsworthy, 1969) and decreases the rate of rectal water reabsorption in vitro (Mordue, 1969), or indeed if both effects are attributable
to a single factor. The activities are likely to be associated with proteinaceous material, although the presence of trypsin resistant material in extracts of corpora cardiaca from L. migratoria which
reduces Malpighian tubule secretion in vitro has been reported
(Canal, 1971).
The diuretic activity from the mesothoracic ganglionic mass of Rhodnius prolixus is associated with proteinaceous material, and is distributed between three different molecular forms, one greater than 60,000, one less than 2,000 and an insoluble particulate form
(Aston and White, 1974). The low molecular weight form is considered to qualify best as the 'true' diuretic hormone.
Goldbard et al (1970) have described two factors in the terminal
abdominal ganglion of P. americana which affect water uptake by the
rectum in vitro. One faotor of molecular weight greater than 30,000
decreases water uptake, while the second factor of approximate
molecular weight 8,000 increases water uptake. 51
The contradictory data for the diuretic and antidiuretic activities of locust corpus cardiacum extracts may be due to species difference (Mordue, 1970b),to the presence of differing amounts of antagonistic principles according to the humidity conditions under which the insects were reared (Cazal, 1971) and to the use of inadequate assay procedures (see Goldsworthy and Mordue, 1974). The present study is concerned in part with the resolution of corpus
cardiacum factors which are active in diuretic assay systems. Resolution of the factors would allow a greater depth of research into the precise involvement of the corpora cardiaca in the control
of water balance than has previously been possible. The need remains however for the developement of more specific and precise assay
procedures.
Factors affecting heartbeat. Storage lobe extracts from the corpora cardiaca of L. migratoria
increase the rate and amplitude of beating of semi-isolated heart preparations (Mordue and Goldsworthy, 1969). The nature of the active factor is unknown, but the effect of corpus cardiacum factors
on the beating of isolated hearts is likely to be a pharmacological property of a peptide hormone whose major effect is on another target tissue (Goldsworthy and Mordue, 1974).
d) The corpora cardiaca of cockroaches.
The hyperglycaemic factor.
The hyperglycaemic activity, and the heart accelerating
activity, contained in extracts of corpora cardiaca from P. americana 52
has been investigated in a series of papers by Natalizi and co-
-workers (1966,1970,1976). The hyperglycaemic activity was found to be trypsin sensitive, and resolved into two fractions following ion exchange chromatography (Natalizi and Frontali, 1966; Natalizi et al, 1970). Its chemical nature is not known in detail, but it is thought to be a peptide with a small molecular weight
Goldsworthy and Mordue, 1974). The hyperglycaemic activity has been resolved from the heart accelerating activity (Trains et al,
1976).
By means of paper chromatography, Brown (1965) resolved two substances from extracts of corpora cardiaca from P. americana which elevated haemolymph trehalose levels. The slower running substance was considered to be the more active of the two principles.
In the present study, isolation and characterisation of the corpus cardiacum hyperglycaemic factor of P. americana has been attempted. The hyperglycaemic factor is reported to be free from hypolipaemic activity (Downer and Steele, 1973) and it has also
been suggested that the cockroach hypolipaemic response and the
locust hyperlipaemic response are different responses to the same
cockroach factor (Downer, 1972). From this, it follows that the
hyperglycaemic and hyperlipaemic factors of the cockroach corpus
cardiacum are likely to be separate. Elucidation of the
pharmacological spectrum of the pure hyperglycaemic factor would
clarify the relationship between these three activities, and also
facilitate further investigation into the physiological significance
of the corpus cardiacum factor in the cockroach. 53
Factors affecting water balance.
The nature of the factors of the corpora cardiaca and the corpora allata of P. americana which inhibit the rate of dye excretion by Malpighian tubules in vitro (Wall and Ralph, 1962, 1964) are unknown. During the present study, factors resolved from extracts of corpora cardiaca from P. americana have been assayed for possible activity on excretory function in vivo and in vitro assay systems.
Factors affecting heartbeat.
At least two cardioacceleratory substances are present in the corpora cardiaca of P. americana (Unger, 1957; Gersch et al, 1960; Brown,
1965). Of the two factors resolved from extracts of the corpora cardiaca from P. americana (Gersch, 1969), one increased the heart rate but decreased the amplitude (neurohormone C) and the other increased the heartrate and the amplitude (neurohormone D). These factors have also been resolved from extracts of the brain and ventral nerve cord in P. americana (Gersch et al, 1960).
Neurohormone D is thought to be a peptide of approximate molecular weight 2,000 and is heat labile. It is thought to contain -SH groups (Gersch et al, 1960, 1963, 1964).
Brown (1965) detected three peptides in extracts of corpora cardiaca of P. americana active on semi-isolated heart preparations, and Traina et al (1976) obtained two thermostable, heterogeneous
peptide preparations possessing heart accelerating activity from
cockroach corpus cardiacum extracts. This group has reported a
total of four heart accelerating factors in the corpora cardiaca 54 of P. americana (Natalizi et al, 1970; Traina et al, 1976), two of which have similar properties to neurohormone D of Gersch et al
(1960) (Traina et al, 1974).
During the present study, factors which have been resolved from extracts of corpora cardiaca from P. americana have been assayed for heart accelerating activity as part of investigations into the pharmacological spectrum of each factor. The physiological significance of the heart accelerating factors in the intact animal however remains to be established. 55
MATERIALS AND METHODS
1.Maintenance of insect colonies. Adult L. migratoria were reared at Imperial College, London and supplemented by cultures from the Centre for Overseas Pest Research, London. The insects were maintained under crowded conditions at 28 — 1°C with a constant photoperiod of 14 hours. They were fed on a diet of fresh grass, lettuce and bran.
Adult P. americana were initially purchased from Gerrard and
Haig Ltd. (Biological Suppliers), but subsequently stocks, and a colony of Leucophea maderae were obtained from colonies reared at the A.R.C. Unit of Invertebrate Chemistry and Physiology, University of Sussex. They were maintained at 22-25°C, and fed on rat cake, bran, dog biscuits, peanuts and sliced apple.
2.Preparation of corpus cardiacum extracts. a) Dissection of the corpora cardiaca. Heads were removed from mature adult locusts. The heads were bisected longitudinally to the right of the midline. The corpora cardiaca were dissected out from the left side under saline (0.375 g/1 KC1 and 7.5 g/1 NaCl) using a binocular microscope. The glands were transferred to a small piece of glass in the dish where adhering corpora allata, fat tissue and trachea were removed. The glandular and storage regions were then separated.
The dissection of corpora cardiaca from adult cockroaches was carried out in a similar manner except that the head was bisected 56 transversly across the top of the ocelli, and the corpora cardiaca removed through the top of thehead. No attempt was made to remove the corpora allata when preparing large extracts for purification purposes.
b) Methanol extracts.
Whole corpora cardiaca, or the separated lobes, were collected into methanol in plastic microcentrifuge tubes and disrupted using an
M.S.E. 100W ultrasonic disintegrator. After centrifugation at approximatly 14,000g for 12 min., the supernatant was removed and the pellet resuspended in methanol. The pellet was extracted several times, the supernatants pooled and the methanol evaporated under nitrogen. The residue was stored in a desiccator at 40C.
c) Saline extracts.
Corpora cardiaca were collected into saline and extracted as
described above. The pooled supernatant was diluted with saline
to give the required concentration expressed as "gland pair
equivalents".
d) Buffer extracts.
Corpora cardiaca were collected into electrophoresis electrode
buffer (either Tris-glycine pH 8.3 orp-alanine-acetic acid p114.5)
diluted 1:5 with distilled water. After sonication the glands were
extracted for 1 hr. at 4°C. Following centrifugation, a few
crystals of sucrose were added to the supernatant to increase the
density for laying on top of polyacrylamide gels. 57
3. Biological assay procedures. a)Adipokinetic activity. Approximatly ten day old adult male L. migratoria were used for this assay. A 5u1 sample of haemolymph was withdrawn from the
arthrodial membrane at the base of the hind leg using an Oxford pipette sampler at time O. 20u1 of saline containing the test material were injected ventrally between the fourth and fifth abdominal segments of the experimental insect using a Temuro syringe (Shandon Ltd.). Control insects were injected with, 20u1 saline. A further 5u1 sample of haemolymph was taken after 1 hr.
The concentration of total lipid in each haemolymph sample was determined by expelling the sample into 0.5m1 concentrated
sulphuric acid and heating at 100°C for 10 min. After cooling, 50u1 were transferred to 0.5ml vanillin reagent (13mM vanillin in 11.8M phosphoric acid, Biochemica total lipid test kit). The samples were left at room temperature for 30 min. and then the developed colour read at 546nm in a Unlearn S.P. 600 spectrophotometer linked to an S.P. 45 concentration readout unit. A standard curve using a serially diluted lipid solution (1g % cholesterol) was also
prepared.
Results are expressed in units of adipokinetic activity, defined
as the amount of active principle required to release lug lipid/ul
haemolymph in 1 hr.
b)Hyperglycaemic activity. Approximatly two month old P. americana were used for this assay. 58
The procedure was the same as for the adipokinetic assay, except that the second blood sample was taken after 2 hr.
The concentration of total carbohydrates in each haemolymph sample was determined by expelling the sample into 0.5m1 10% trichloroacetic acid (T.C.A.) to deproteinise the blood. The precipitate was removed by centrifugation at 14,000g for 6 min. 50u1 of the supernatant was transferred to 0.5m1 anthrone reagent
(0.05% anthrone and 156 thiourea in 72% sulphuric acid) and the mixture heated at 100°Cfor 10 min. After cooling, the developed colour was read at 650nm (method after Roe, 1955). A standard
curve using a serially diluted solution of glucose was also
prepared.
Results are expressed in units of hyperglycaemic activity,
defined as the amount of active principle required to release lug glucose equivalentsiul haemolytph in 2 hr.
c) Activity on flight metabolism. Adult male L. maderaewere used in these investigations. They
were flown in groups of four suspended on a small roundabout
(14cm radius) as described by Goldsworthy et al,(1973a)for locusts. The temperature was not controlled during the experiments, and
the ambient temperature was approximatly 25°C.
The cockroaches were routinely flown for 10 min. as it was
found that almost all of the insects ceased flight after this time. 59
Haemolymph samples were taken immediatly before and after flight, and the total lipid and carbohydrate concentration of each sample determined. The effect of corpus cardiacum factors on changes in blood metabolites during flight and on flight duration was investigated by injecting the cockroaches with 20u1 of saline containing the test material 30 min. prior to the onset of flight. Control insects were injected with 20u1 saline.
d) Diuretic activity. Amaranth excretion rate. Adult male L. migratoria were injected with 20u1 saline containing the test material, and control insects were injected with 20u1 saline. Both test and control insects were then injected with 10u1 of 1%% amaranth solution in saline. 5u1 blood samples were taken at 5, 15 and 30 min. after injection. The samples were expelled into 0.5m1 saline and the optical density (0.D.)
read at 522nm against a blank of saline and blood from an uninjected
insect.
During preliminary experiments, it was found that large errors
in 0.D. readings were produced by the coagulation of haemolymph constituents in the saline. To overcome this, the haemolymph was expelled into 10% T.C.A. instead of saline, and the 0.D. of the
supernatant was read after centrifugation. Both fed and starved
assay insects were used.
Fluid secretion rate in vitro.
Adult male L. migratoria were used in this assay, which was 60 performed as described by Maddrell and Klunsuwan(1973)•
The alimentary tract was withdrawn with the Malpighian tubules attached. The preparation was immersed in liquid paraffin and surrounded with 200u1 of locust Ringer. Ten tubules were teased out and wound around fine glass tubes. The rate of secretion of fluid by each tubule was followed by the rate of increase in diameter of the secreted spherical drop. Readings with an eyepiece micrometer at 20 and 40 min. provided normal secretion rate values. Assay of test material was performed by
replacing the locust Ringer with 200u1 Ringer containing the test material. For control values, the preparation was surrounded with fresh Ringer. Readings were taken again at 25 and 50 min.
e)Heart accelerating activity. Adult male L. migratoria were decapitated, the abdomen opened along the mid—ventral line and the preparation pinned into a wax
dish. The heart was exposed and bathed in 100u1 locust Ringer
(Maddrell and Klunuswan, 1973). The dish was maintained at a
temperature of 30°C. The time taken for 30 beats was recorded, and the amplitude of the beat noted using an eyepiece micrometer.
For the assay of test material, the preparation was drained
of Ringer and replaced with 100u1 Ringer containing the test
material.
f)Glycogen phosphorylase activation.
Adult male L. migratoria of known age were injected with 50u1 61 of saline containing the test material. Control animals were injected with 50u1 of saline. The fat body was dissected out 1.5 hr. after the injection and homogenised in 2m1 ice cold 0.5M sodium fluoride/0.125n E.D.T.A. using a ground glass homogeniser. 0.2m1 aliquots of the supernatant obtained after centrifugation were used to measure phosphorylase activity as the release of inorganic phosphate from glucose-1-phosphate in the presence of glycogen (Sutherland and Wosilait, 1956). The assay solution comprised 30mM glucose-1-phosphate, 100mM sodium fluoride, 25mM E.D.T.A. and 0.4% glycogen in 1m1 This buffer (pH 7.0). For the measurement of total phosphorylase, 4.4mM 5 AMP were added to the solution.
The mixture was incubated at 30°C for 1 hr. then 0.2m1 TCA were added. Tissue and reagent blanks were included, and the inorganic phosphate was determined by the method of Rockstein and Herron (1951). The protein content of the enzyme extract was measured by the Rartree (1972) modification of the method of 'Lowry et al (1951).
The enzyme activity was expressed as ug inorganic phosphate liberated/ug soluble protein in enzyme extract/hr.
4. Separation of active factors in corpus cardiacum extracts. a) Filtration chromatography. Controlled pore glass beads (G-75-50, Sigma, London Chem. Co. Ltd.) were equilibrated with water and packed into a glass support 62
(Pharmacia) to form a column 2cm x 25cm. It was calibrated using blue dextran and vitamin B which gave the void and column volumes respectively.
A methanol extract of corpora cardiaca was redissolved in 1m1 distilled water, applied to the top of the column and eluted with distilled water. A flow rate of 30ml/hr. was used, and the eluate monitored for u/v absorption at 206nm using an LKB Uvicord III photometer linked to a pen recorder. 2m1 fractions were collected using a Gilson automatic fraction collector. Biogel P6 (+400 mesh, Bio—Rad Laboratories) was used under the same conditions as the
glass beads, except for a flow rate of 18m1/hr., and the collection
of 1.2m1 fractions.
Aliquots were removed from fractions eluted from the column
and diluted with saline for bioassay. The fractions found to possess activity were pooled and evaporated to dryness under reduced pressure at 50°C using a Buchii Rotavapour.
b) Polyacrylamide gel electrophoresis. Anionic system.
Stock solutions were prepared according to Davis (1964). The
polyacrylamide gels comprised a large pore gel for stacking (2.5%
acrylamide, pH 6.7) and a small pore gel for separation (7%, pH
8.9). This glycine (pH 8.3) was used as the electrode buffer and bromophenol blue was the tracker dye. 63
Cationic system. Stock solutions were prepared according to Reisfield et al, (1962). A large pore gel of 2.5% acrylamide (pH 6.8) and a small pore gel of 7% (pH 4.3) were used. The electrode buffer was p-alanine acetic acid (pH 4.5) and methyl green was the tracker dye.
Gel preparation and electrophoresis. Glass tubes (i.d. 2mm x 80mm) were filled with small pore solution to a depth of 60mm, and after polymerisation, overlayered to a depth of 10mm with large pore solution. Routinely, a buffer extract of ten pairs of corpora cardiaca was applied to each gel. Methanol extracts of corpora cardiaca, resuspended in dilute electrode buffer, were also subjected to electrophoresis. The gel tubes were placed into a Shandon disc electrophoresis apparatus, and the reservoirs filled with electrode buffer containing the tracker dye
(5ug/mi buffer). The cathode was placed in the upper reservoir and the anode in the lower for the anionic system: the electrodes were reversed for the cationic system. The gels were subjected
to a constant current of 0.5mA/gel for 20 min. then 1.5mA/gel for
30 min.
Staining of the gels. The gels were removed from the glass tubes by rimming with
a hypodermic needle and stained for 2 hr. in 0.25% Coomassie brilliant blue in acetic acid: 50% methanol 23:227. The gels were destained and stored in 7% acetic acid. They were scanned 64
at a wavelength of 560nm using a Gilford gel scanner linked to a pen recorder.
Elution of the gels.
After electrophoresis, the gels were cut into sections.
These were macerated using a ground glass homogeniser and eluted overnight with methanol. The gel was removed by centrifugation and the supernatant evaporated to dryness under nitrogen.
c) Thin layer chromatography (TLC).
TLC of column eluates.
The residue obtained after rotary evaporation of the pooled fractions was resuspended in 500u1 methanol and applied to a rapid running silica gel plate with fluorescent indicator (Rapid Plates
F254, Woelm, West Germany) which had been pre—run in methanol.
The rotary evaporation flask was washed twice with similar amounts of methanol and these were also applied to the plate.
The plate was developed in isopropanol: water: glacial acetic acid 25:10:1 (v/v) for 2.5 hr. by which time the solvent front had
moved approximatly 15cm from the sample application point. The
developed plate was air dried and viewed under u/v light. Er/v
absorbing areas were localised and immediatly scraped into methanol.
The remaining area was divided up and eluted into methanol. After
several hours, the silica gel was removed by centrifugation and
washed twice with methanol. The supernatants were pooled and
evaporated to dryness under nitrogen. Aliquots of the eluted areas 65
were subjected to bioassay and biochemical characterisation.
TLC of gel eluates.
The material obtained after the extraction of the gel sections into methanol was subjected to thin layer chromatographic separation as described above. Aliquots of the eluted areas were bioassayed for adipokinetic activity.
Visualisation of peptide material on TLC plate.
The developed plates were placed face downwards in a dessicator over a dish containing equal volumes of 2% potassium permanganate and
3N hydrochloric acid and left in the freshly generated chlorine gas for 45min. The plates were left overnight to remove all traces of chlorine, then sprayed with freshly made starch iodide (equal volumes
1% starch and 1% potassium iodide). Peptide material was visualised as a blue colouration.
5. Biochemical characterisation of the purified factors.
a) Enzymic digestion.
Inactivation experiments with proteolytic enzymes were performed on
the factors purified by column chromatography followed by TLC.
Pronase, a mixture of several proteases (Sigma NOP 5130) was
used. A 0.4% solution of pronase in freshly made 0.5% ammonium
bicarbonate was prepared. A known quantity of the purified material
was made up to 100u1 with 0.5% ammonium bicarbonate, and 20u1 of the
pronase solution were added. Suitable blanks and controls were also
set up. The samples were incubated at 40°C for 6 hr. At the end of 66 the incubation period, 0.4 ml distilled water were added to each sample and they were heated for 5 min. at 100°C to inactivate the enzyme. After cooling, the samples were centrifuged and the supernatants lyophilised. The lyophilisates were resuspended in a small quantity of distilled water and re-lyophilised to remove traces of ammonium bicarbonate. The residues were stored in a dessicator at 4°C, and assayed for biological activity.
b)Ultra-violet absorption and fluorescence spectra. The purified material was taken up in 3 ml distilled water, and the u/v absorption spectrum determined using a Perkin-Elmer double
beam spectrophotometer.
The fluorescence spectrum was read on a Unicam spectrofluorimeter
using an activation wavelength of 280nm. The u/v absorption and
fluorescence spectra of a 109M solution of tryptophan were also
determined.
c)Amino acid analysis. For quantitative amino acid analysis, a dried sample of the
purified material was hydrolysed in 5.7N hydrochloric acid for
20 hr. at 110°C in an evacuated sealed tube. The hydrolysate was lyophilised and the amino acid composition determined by the
automatic technique of Spackman et al (1958).
6. Corpora cardiaca in organ culture. a) Culture of the corpora cardiaca. Adult male L. miratoria were decapitated and the head swabbed with 67
1% iodine in 70:1() ethyl alcohol. All subsequent operations were performed in a laminar flow sterile hood. The heads were bisected longitudinally to the right of the midline and the corpora cardiaca dissected out from the left side with sterile instruments under a binocular microscope. No attempt was made to remove adhering fat tissue and trachea, and the glands were transferred using the adhering tissue to a sterile covered petri dish containing TC medium 858 (Difco Co.Ltd.), supplemented with foetal calf serum, glucose and yeast. The glands were washed three times in the sterile culture medium and then placed in plastic tissue culture cells
(Sterilin Ltd.),containing 0.2 ml culture medium, five glands to each cell. A cover slip was sealed over the top of each cell with sterile beeswax and they were placed into plastic petri dishes
(Sterilin Ltd.). Cultures were maintained at 51± 1°C for up to seven days.
b) Extraction of incubation media and cultured corpora cardiaca.
The culture medium was removed from the cell and transferred to a plastic microcentrifuge tube. It was made up to 60% methanol and the precipitated proteins removed by centrifugation. The supernatant was evaporated to dryness under nitrogen.
The cultured glands were collected into methanol and extracted as described on page 56 and the gland and media extracts were bioassayed for adipokinetic activity. Extracts were also subjected to TLC, and aliquots of the eluted areas were assayed for adipokinetio activity. 68
c) Morphological studies of the cultured corpora cardiaca.
The cultures were inspected daily using a Leitz microscope at x100
magnification.
After the incubation period, both cultured and freshly dissected
corpora cardiaca were fixed in aqueous Bouin's solution for 24 hr.
After removal of Bouin's with 70Y0 alcohol, the glands were dehydrated, cleared and embedded in ester wax. Serial sections of 110.1 were
stained with paraldehyde fuschin after permanganate oxidation (Cameron and Steele, 1959) and counterstained with Halmi's mixture
(1952).
In other experiments, cultured and freshly dissected corpora cardiaca were fixed in phosphate buffered 4% glutaraldehyde followed by post fixation in 1% osmium tetroxide (Millonig, 1962). The fixed
material was embedded in Epon, and the cut sections stained on the grids with 2% aqueous uranyl acetate followed by Reynold's lead citrate stain. The sections were examined under an AEI 306B electron
microscope. 69
RESULTS
THE CORPORA CARDIACA OF L. I1IGRATORIA
1. Activities of the corpus cardiacum extracts.
a) Effect on total haemolymph lipids.
The dose-response curve of the lipid mobilising activity of methanol extracted glandular lobes is shown in fig. 2. There is an approximately linear increase in haemolymph lipid up to about 0.01 equivalent pairs of glandular lobes. Near maximum increases (25 -
30 ug/ul) were brought about by as little as 0.02 pairs of glandular lobes. The minimum quantity required to produce a consistently measurable response was found to be 0.001 pairs of glandular lobes.
The number of arbitrarily defined units of adipokinetic activity was determined from responses to the lower doses, and methanol extracted glandular lobes were estimated to contain 2,000-3,000 adipokinetic units/pair glandular lobes.
The lipid mobilising activity of storage lobe methanol extracts was found to be some 10-20 times less than that of the glandular lobe extracts (fig. 3). The minimum amount required to produce a consistently measurable response was found to be 0.05 pairs of storage lobes, and methanol extracted storage lobes were estimated to contain 400-500 adipokinetic units/pair storage lobes.
Doses of 0.5 equivalent pairs of glandular lobe and storage
lobe methanol extracts were injected into adult male P. americana. Adipokinetic activity (units) PIG. 2.Dose-responsecurveofthelipidmobilisingactivityin L.migratoriaofa 10 30 20 0
are given.Eachgroupconsistedofatleast4animals. methanol extractofglandularlobes.Meanvaluesandstandard deviations 0.01
Dose (pairsglandular lobes) '0.02
0.03
0.04
0.05 FIG. 3. Dose—response curve of the lipid mobilising activity in L. migratoria of a methanol extract of storage lobes. Mean values and standard deviations are given. Each group consisted of at least 4 animals.
30
0 0.05 0.1 0.15 0.2 0.25 Dose (pairs storage lobes)
72
Neither was found to produce a change in the level of haemolymph
lipid substantially different from the saline injected control
(table 1).
TABLE 1
Effect of locust glandular and storage methanol extracts on total haemolymph lipids in adult male P.americana.
Material injected Total lipid concentration (ug/ul, mean -± SE)
0 2 hr
0.5 pair glandular lobes 13.6 ± 0.2 11.1 ± 0.3
0.5 pair storage lobes 14.7 -4; 1.2 11.6 ± 1.3
saline 13.8 ± 1.5 12.2 ± 1.0
At least four animals were used for each test.
Saline extracts of glandular and storage lobes, assayed at
selected doses, were found to produce responses very similar to
those produced by methanol extracts (table 2). The adipokinetic
activity therefore is equally soluble in saline and methanol.
Eight de novo corpora cardiaca were provided by Dr G.J.
Goldsworthy. Due to insufficient material, the lipid mobilising
activity of a range of doses could not be assessed. 0.5 gland
equivalents of methanol extracted de novo corpora cardiaca were
found to produce an elevation of 8.0 ± 2.4 ug/ul in locust 73 haemolymph lipids (mean value of response obtained from six animals).
TABLE 2
Comparison of the adipokinetic activity of methanol and saline
extracted glandular and storage lobes in L. migratoria.
Material injected Adipokinetic activity (units) Saline Methanol extract extract
0.002 pair glandular lobes 7.9 ± 1.2 8.3 - 1.5 0.02 pair storage lobes 4.8 -± 0.9 4.7 ±1.3 At least four animals were used for each test.
b) Effect on total haemolymph carbohydrates. Doses of 0.5 equivalent pairs of glandular lobe and storage lobe
methanol extracts were injected into adult male L. migratoria. Neither was found to produce a change in haemolymph carbohydrate
levels which was substantially different from the saline injected
control (table 3).
The dose-response curve of the hyperglycaemic response of P. americana to injections of glandular lobe methanol extracts from L. migratoria is shown in fig. 4. The response is linear up to a dose of 0.2 gland pair equivalents. The maximum increase
in blood carbohydrates 2 hr. after injection (18-25 ug/ul) was obtained only with doses in excess of 1 pair of glandular lobes. 74
TABLE 3 Effect of glandular and storage lobe methanol extracts on total
:haemolymph carbohydrates in adult male L. migratoria.
Material injected Total carbohydrate concentration (ug/u1) 0 1 hr. 0.5 pair glandular lobes 34.5 ± 1.7 38.1 ± 3.0 0.5 pair storage lobes 31.6 ± 2.2 33.0 ± 3.0
± saline 31.0 ± 2.3 32.8 3.8 At least four animals were used for each test.
The minimum quantity required to produce a consistently. . measurable response was found to be 0.05 pairs of glandular lobes. The number of arbitrarily defined units of hyperglycaemic activity was determined from responses to the lower doses, and
methanol extracted glandular lobes were estimated to contain
50-100 hyperglycaemic units / pair glandular lobes.
The hyperglycaemic activity of storage lobe methanol extracts was found to be less than half of that of the glandular
lobe extracts (fig. 5). The minimum amount required to produce
a consistently measurable response was found to be 0.1 pairs of storage lobes, and methanol extracted storage lobes were estimated to contain less than 50 hyperglycaemic units / pair
storage lobes. PIG. 4. Dose—response curve of hyperglycaemic activity in P. americana of a methanol extract of glandular lobes. Mean values and standard deviations are given. Each group consisted of at least 4 animals. 20r
0 0.05 0.1 0.15 0.2 0.25 Dose (pairs glandular lobes) PIG. 5. Dose—response curve of hyperglycaemic activity in P. americana of a methanol extract of storage lobes. Mean values and standard deviations are given. Each group consisted of at least 4 animals. 20
CD 43 0
43 rl .rI +3 0 10
0
a)E aS 0 ri QO a) Da
0 0.05 0.1 0.15 0.2 0.25 Dose (pairs storage lobes) 77
Saline extracts of glandular and storage lobes, assayed at selected doses, were found to produce responses very similar to those produced by methanol extracts (table 4). The hyperglycaemic activity therefore is equally soluble in saline and methanol.
TABLE 4 Comparison of the hyperglycaemic activity of methanol and saline extracted glandular and storage lobes in P. americana.
Material injected Hyperglycaemic activity (units) Saline Methanol extract extract 0.1 pair glandular lobes 11.5 ± 3.1 12.7 ± 2.3 0.1 pair storage lobes 9.8 ± 2.9 8.5 ± 1.7
At least four animals were used for each test.
Methanol extracted de novo corpora cardiaca, assayed at a dose of 0.5 gland equivalents, were found to produce an elevation of 3.0 ± 1.2 ug/ul in the total haemolymph carbohydrates of adult male P. americana (mean value of response obtained from six animals).
c) Effect on the rate of amaranth excretion.
During an initial series of experiments, fed insects were used as assay animals.
The injection of 1 pair of saline or methanol extracted glandular lobes into mature adult male L. migratoria did not 70
substantially alter the rate of amaranth excretion (fig. 6).
Injection of 1 pair of saline or methanol extracted storage lobes increased the rate of amaranth excretion (fig. 7). These results indicate that the diuretic activity is located mainly in the storage region and that its solubility in saline and methanol is comparable.
Fig. 8 shows the excretion of amaranth by insects which have been starved for two days. compared with fed insects. The starved animals excreted only 21.5 ± 3.7% of the injected amaranth within
30 min. compared with 62.0 ± 8.356 excreted by the fed animals in the same time. For subsequent bioassay.s, insects were starved for two days prior to the assay of test material for diuretic activity.
d) Effect on the rate of fluid production by Malpighian
tubules in vitro.
During preliminary experiments, the assay procedure utilised separate preparations for the control and assay, thus individual variations in basal secretory rates were not taken into account. The basal secretory rate however was generally found to be 1.4 nl/min.
cAMP was found to have a clear acceleratory effect on fluid secretion. 1 x 103M cAMP increased the rate by 2 nl/min.
Inclusion of 1 pair of methanol extracted storage lobes from fed 14 day old adult male L. migratoria in the incubation medium increased the rate of fluid production by 1.7 ± 0.5 nl/min. If the donor insects are starved for four days prior to removal of 79
FIG.6. The effect of glandular lobe extracts on the excretion of amaranth in L. migratoria. Extracts were assayed at a dose of 1 glandular lobe pair 80 equivalent/insect. saline extract methanol extract S S saline control
60 ♦
♦
20
0 10 20 30 min.
80
FIG.7. The effect of storage lobe extracts on the excretion of amaranth in L. migratoria. Extracts were assayed at a dose of 1 storage lobe pair equivalent/insect. 80 saline extract A methanol extract S. S saline control //
/ /
/ 60
/ / / 4, / a) / $-1 U / 1.4 / a) / / Al 40 / 43 /
ti / as / / as / 119. / A
20
0 10 20 30 min. 83.
FIG.8. The effect of feeding on the excretion of amaranth in I.migratoria. f e d
80 —a starved
6
40
20 •■••• .■••• r rr
0 10 20 30 min.
82
the storage lobes, the rate was increased by 5.0 ±1.0 nl/min. A
range of doses was assessed for activity on Malpighian tubules
in vitro (table 5). As the responses obtained were variable at
the lower doses, a dose response curve was not produced. Inclusion
of 1 pair of methanol extracted glandular lobes from both fed and
starved insects did not alter the rate of fluid production.
TABLE 5
Effect of methanol extracted storage lobes of starved adult male
L. migratoria on the rate of fluid production by Malpighian tubules
in vitro.
Dose Increase in rate of (Gland pair fluid production (nl/min.) equivalent)
0.13 2.8 ±• 0.5
0.5 2.9 ±• 0.8
2.5 5.5 ±• 1.0
Ten tubules were used for each test.
In subsequent assay procedures, the basal secretory rate
of a set of tubules was established prior to the addition of the
assay material. Fig. 9 (b) shows the mean volume of fluid produced with time by ten tubules in Ringer compared with the volume produced
by the same tubules in Ringer containing 1 pair of methanol extracted
storage lobes from locusts starved for two days. Comparisons between
the rates observed in the first and second 50 min. periods indicate
that at the dosage used, a substantial increase in fluid secretion 83
FIG.9. The effect of methanol extracts of storage lobes on fluid production by Malpighian tubules in vitro. Fluid production was initially determined in normal Ringer (e) and then in either (a) fresh Ringer (control') or (b) fresh Ringer + 1 gland equivalent of storage lobe methanol extract (A).
1.2 rcs a) 0 0 rcs o 0.8 k fat
(a) •r 1 cs-1r-1 0.4
0
6—i 0 0
0 25 50 min.
H 1 1.2 rt:s0 0 id •$-1 0.8 (b) 0 cH 0.4
0
0
0 25 '50 min. 84 is produbed. Results from the control preparation, produced at the same time (fig. 9(a)) indicate that the secretory rate of the tubules remains constant during the 100 min. assay period and is unaffected by the change in Ringer after the first 50 min. period.
e) Effect on heart beat. The cardioacceleratory activity of glandular and storage lobes was determined by inclusion of 0.5 pairs of methanol extract in the incubating medium of the isolated heart preparation. Glandular lobe extract increased the heart rate by 63.0 ± 4.5% and also decreased the amplitude of the beat. Storage lobe extract increased the heart rate by 42.0 ± 7.9% and also increased the amplitude.
Inclusion of 0.5 pairs of methanol extracted whole corpora
eardiaca increased the heart rate by 75.0'± 3.5% and also increased the amplitude.
f)Effect on fat body glycogen phosphorylase levels.
Methanol extracts of glandular and storage lobes from mature male
L. migratoria were assayed at a dose of 1 gland equivalent / insect.
Injection into two da* old adult male L. migratoria was
round to,prodi4ce little change after 1.5 hr. in either the total glycogen phosphorylase level or in the ratio of active to total
phosphorylase in the fat body-compared with the control values
(table 6). 85
TABLE 6
Effect of glandular and storage lobe methanol extracts on fat body phosphorylase activity in two day old adult male L. migratoria.
Treatment No. of Total phosphorylase Active observations specific activity phosphorylase (ug phosphate/ug % of total protein/hr) activity saline 6 0.25 ± 0.02 58.5 ±1.4 1 pair glandular lobes 12 0.18 ±0.02 60.9 ± 4.4 1 pair storage lobes 5 0.18 ± 0.03 59.8 ± 2.6
2. Purification of the corpus cardiacum factors.
a) Purification of the glandular lobe factors.
Glass bead column chromatography.
The methanol extracts of glandular lobes were redissolved in
distilled water and the aqueous extract applied to a controlled pore
glass bead column, 2 x 25 cm. The column was equilibrated and
subsequently eluted with distilled water at a flow rate of 30 ml hr - 1
Each fraction contained 2 ml effluent volume.
Fig. 10 shows a typical column profile obtained on elution of
200 glandular lobes. Two peaks of u/v absorbing material are
resolved, one at Vo (fractions 7-16) and the other at an elution
volume slightly less than V (fractions 20-30). Aqueous extracts t produce a similar profile (fig. 11). The fractions of both peaks o were pooled and water removed under reduced pressure at 50 0. 86
FIG.10. Separation of a methanol extract of the glandular lobes_from 200 L. migratoria on a G-75-50 glass bead column. 0.D. 206nm t--spooled fractions
1.111■■■•■■.11■1■:14
0 20 40 60 80 ml 87 kG.11. Separation of an aqueous extract of the glandular lobes from 170 L. migratoria on a G-75-50 glass bead column. O.D. 206nm
Illf•■■••11MilIMMIIMI=1.1.111 20 40 60 80 ml 88
The residues were bioassayed for adipokinetic and hyperglycaemic activity. The Vo peak was found to contain neither activity, whereas the Vt peak was found to contain both.
TLC of eluates.
The dry residues of the two peaks were resuspended in.methanol and subjected to TLC. No u/v absorbing areas were produced on separation of the Vo peak, whereas two u/v absorbing areas were resolved from the Vt peak of Rf's 0.67 and 0.79. These areas were eluted, as were the intervening areas between the origin and the
solvent front. All eluates were assayed for adipokinetic and
hyperglycaemic activities (fig. 12). Both activities were localised
around the area of Rf 0.67 while all other areas contained negligible
activity.
b) Purification of the storage lobe factors.
TLC of extracts.
The methanol extract of storage lobes from 70 mature adult
male L. migratoria were applied to a pre-run TLC plate. Under u/v
light, two u/v absorbing areas were visible of Rf's 0.67 and 0.79.
The origin and area Rf 0.67 were found to stain with starch iodide,
indicating the presence of peptide material.
The plate was divided up into 2 cm sections and eluted. Each
eluate was bioassayed for adipokinetic and hyperglycaemic activities
(fig. 13). These activities were localised in the area Rf 0.6-0.7.
The origin and the area Rf 0.6-0.7 were also assayed for diuretic
activity and these eluates were found to increase the rate of fluid 89
FIG.12. Hyperglycaemic and adipokinetic activity eluted from T.L.C. plate following separation of the V peak.. The hyperglycaemic and adipokinetic activities of the fractions were assayed at 0.1 and 0.05 gland pairs respectively.
2 VI
i>a 43 .1-I
420 10
0 0 cd 0 1-1 bl)
0 0.3 0.6 1
20
03 43 .11
0. 0.3 0.6 1 ORIGIN SOLVENT Rf. FRONT 90
FIG.13. Hyperglycaemic and adipokinetic activity
eluted from T.L.C. plate following separation
of a methanol extract of the storage lobes
from 70 L. migratoria. The hyperglycaemic
and adipokinetic activities were assayed at
0.1 and 0.05 gland pairs respectively.
0.3 0.6 1
0 0.3 0.6 1
ORIGIN SOLVENT Rf FRONT 91 produced by Malpighian tubules in vitro by 9.8 and 2.9 nl/min. respectively. This indicates that most of the diuretic activity of the storage lobe extracts remains at the origin.
To extend the purification procedure, storage lobe extracts were subjected to the procedure devised for AKH (Stone et al, 1976) as described for the glandular lobe extracts in the previous section.
In a subsequent purification system, the glass bead column was replaced by a column of Biogel P6.
METHOD 1 Glass bead filtration chromatography
The conditions of glass bead filtration chromatography of storage lobe extract were the same as those used for the glandular lobe
extracts.
Fig. 14 shows a typical column profile obtained on elution
of a methanol extract of storage lobes from 300 L. migratoria. Only one distinct peak of u/v absorbing material is visible, at Vo (fractions 7 - 18). The fractions of this peak were pooled, as were fractions 20 — 40. Both sets of pooled fractions were found to contain adipokinetic and hyperglycaemic activity. Aqueous extracts
were found to produce a similar profile to methanol extracts, and and fig. 15 shows the adipokineticAhyperglycaemic activity in each peak on elution of an aqueous extract of 270 fraction of the Vo storage lobes. 92
FIG.14. Separation of a methanol extract of the storage lobes from 300 L. migratoria on a G-75-50 glass bead column. 0.D. 206 nm F---wooled fractions
20 40 60 80 ml 93
FIG.15. Hyperglycaemic and adipokinetic activity in the Vo peak following elution of an aqueous extract of 100 pairs of storage lobes from a G-75-50 glass bead column.
150
100
ty i tiv f ac o
03 43
50
0 10 20 30 40 ml
0.D. 206nm A---A adipokinetic activity *---Ahyperglycaemic activity 94
TLC of eluates
The dry residues of the Vo peak and pooled fractions 20 — 40 were subjected to thin layer chromatography. No u/v absorbing areas were produced on separation of the Vo peak, but a faintly u/v absorbing spot of Rf 0.6 — 0.7 was visible on separation of fractions 20 — 40. The plates were divided up and eluted. All eluates were bioassayed for adipokinetic and hyperglycaemic activity
(figs 16 and 17). For the Vo peak and fractions 20 — 40, both. .
activities were localised in an area of Rf 0.6 — 0.7.
METHOD 2
Biogel P6 column chromatography
An alternative column material was utilised for storage lobe extracts
to obtain better separation in the lower molecular weight range and
to decrease the likelyhood of adherence of charged molecules to
the column material. Biogel P6 was selected as it is reported to
possessfew charged groups, and separates molecules in the molecular
weight range 1,000 to 6,000. Using a column 2 x 25 cm and a flow rate
were found to begin at 11 and 26 ml elution of 18 ml/hr., Vo and Vt volume respectively. The effluent was collected in 1.2 ml fractions.
The column profile obtained on elution of the methanol extract
of storage lobes from 250 L. migratoria (fig. 18) shows two major u/v
and the other after V with a pronounced absorbing peaks, one at Vo t tail. Each fraction was bioassayed for hyperglycaemic and diuretic
activity (fig. 18). The hyperglycaemic activity was found to correspond
to the tail of the peak near to Vt (fractions 26 — 35) and these
fractions, on subsequent pooling and bioassay, were also found to 95
FIG.16. Hyperglycaemic and adipokinetic activity eluted from T.L.O. plate following separation of the Vo peak. Both activities were assayed at 0.5 gland pairs.
20 4,
4,to) 4-1 01' 403 0 10 0 r4 E a) 0
bD
1:0 0.3 0.6 1
20 4,
§
4,i>a •r1 4-1 4Z 10 cd 0 erl
0 •rf .S4 0 Pi r0
0 0.3 0.6 1 ORIGIN SOLVENT Rf FRONT 96
FIG.17. Hyperglycaemic and adipokinetic activity eluted from T.I.C. plate following separation of fractions 20-40. Both activities were assayed at 0.5 gland pairs.
20 4. 91
4)
4, 03 0 10 94 (1) 03 0 1-1
0 0.3 0.6 1
20 4,rl
t>.• 4, 91 91 4' 0 W 10 0 4,
•ri 0 *1-1
0 . 0.3 0.6 1 ORIGIN SOLVENT Rf_ FRONT 97
FIG.18. Separation of a methanol extract of the storage lobes from 250 L. migratoria on a Biogel P6 column. The hyperglycaemic and diuretic activities were assayed at 0.5 and 1 gland pairs respectively. 0.D. 206 nm hyperglycaemic activity diuretic activity
20
*8-1
%OM
0 12 . 24 36 ml 98 contain adipokinetic activity. Diuretic activity was located in fractions 15 — 22. The large u/v absorbing peak at fraction 27 was found to be inactive in all assays.
TLC of eluates Fractions 25 — 27 and 29 — 35 were pooled separatly. On TLC separation, fractions 25 — 27 produced a u/v absorbing spot Rf 0.79 and fractions 29 — 35 a u/v absorbing spot of Rf 0.67. On elution and bioassay the area Rf 0.67 contained both hyperglycaemic and adipokinetic activity (fig. 19) but no diuretic activity. Area Rf 0.79 was devoid of any activity in the assay systems used.
Estimates of losses of the adipokinetic and hyperglycaemic activity during the purification procedures of storage lobe methanol extracts described above have been made. Approximatelyhalf of the original hyperglycaemic activity in the storage lobe extracts was recovered by this method: 13% lost on the Biogel P6 column and 46% on the TLC plate. The activity of the fractions was assessed at 0.2 gland pair equivalents, and this is in excess of the dose required for
maximal adipokinetic response. Thus the results for recovery of adipokinetic activity in table 7 do not give a real indication of losses incurred.
As earlier experiments have indicated that the diuretic factor
does not run under the TLC conditions employed, fractions 15 — 22 were
pooled and evaporated under reduced pressure and used for further
experimentation. 99
FIG.19. Hyperglycaemic and adipokinetic activity eluted from T.L.C. plate following separation of fractions 29-35. Both activities were assayed at 0.5 gland pairs.
^ 20 - co 43
I>) 4, n-1
4, 0 - 0 0 a) ai 0 r1
0
0.3 0.6 1
20 to 4' 4-1
0 0 10 0
0 PI •ri
0 0.3 0.6 1 ORIGIN SOLVENT Rf FRONT 100
TABLE
Purification of adipokinetic and hyperglycaemic factors from storage
lobes of locust corpora cardiaca.
(Values are for a preparation from storage lobes of 250 locusts)
FRACTION TOTAL AK RECOVERY TOTAL HG RECOVERY ACTIVITY (96) (%) (UNITS) (UNITS) methanol extract 47,088* 100 20,000 100
pooled Biogel column extract (fractions 29-35) 45,350*. 96 17,500 87 TLC fraction Rf 0.67 36,625* 78 8,125 41
Activity was assessed at 0.2 gland pair equivalents/insect
* hyperglycaemic losses cannot be correlated with adipokinetic losses
from this data as the dose used to assess adipokinetic activity was
in excess of that required for maximal response.
o) Polyacrylamide gel electrophoresis of extracts.
Whole corpus cardiacum extracts
Buffer extracts of whole corpora cardiaca were subjected to
electrophoretic separation in both anionic and cationic systems.
The electropherogram produced in an anionic system by buffer
extracts of corpora cardiaca from five twelve day old adult male
L. migratoria is shown in fig. 20. 21 protein bands are produced.
Band 1 is intensely staining and migrates almost with the ion
front. On comparison with the electropherogram obtained on 101
separation of a sample of haemolymph in the same system (fig. 25), it can be seen that bands 5, 7, 8, 13 and 15 could result from contamination with haemolymph proteins. Band 13 was invariably present in haemolymph and corpus cardiacum samples, and is probably due to a haemolymph protein. The faster migrating bands 1 - 5 of the corpora cardiaca electropherogram were not visualised in the haemolymph electropherogram.
The nine cathiodically migrating bands obtained on separation of buffer extract of corpora cardiaca from ten L. migratoria are shown in fig. 21.
Separated lobe extracts
The electropherogram produced in an anionic system by buffer extracts of the glandular lobes from ten twelve day old adult male
L. migratoria is shown in fig. 22. By comparison with the electro-..
-pherogram of whole corpora cardiaca (fig. 20), bands 6, 7, 8, 16 and
20 are no longer present.
Buffer extracts of storage lobes, separated in the same anionic
system, produce 11 bands (fig. 23) and on comparison with the electropherogram of whole corpora cardiaca, bands 1 - 6, 15, 17, and 19 are no longer present.
The main differences between the electropherograms of the glandular
and storage lobes appears to be in the faster migrating bands 1 - 15.
The origin of the gels in all cases stained deeply, indicating the
presence of proteinaceous material which had not entered the gel. 102
The electropherograms produced in an anionic system by methanol extracts of the glandular and storage lobes from ten twelve day old adult male
L. migratoria is shown in fig. 24. In both cases, very little material which could be visualised by staining with Coomassie brilliant blue was present. Glandular lobe extracts produced a faintly staining band of Rf 0.98 while storage lobe extracts produced a band of Rf 0.57.
TLC of gel eluates
Following cationic and anionic separation of 40 buffer extracted whole corpora cardiacs, the gels were cut into four segments and
eluted into methanol. The gel eluates were then subjected to TLC
(fig. 27 (a) and (b)). The plates were eluted and the eluates
bioassayed for adipokinetic activity (table 8).
In the anionic system, most of the activity is found in the
uppermost segment of the gel (in u/v absorbing areas Rf 0.64 — 0.68)
while in the cationic system, activity is found mainly in the top and
bottom segments with a little activity in the lower middle segment.
These results indicate that the activity will migrate in a cathodic
but not an anodic direction under the conditions employed. The
slowest running area in both systems was very weakly absorbing and
present in eluates from control gels, indicative of it being a
component of the electrophoretic system. The fastest running area
shown in fig. 27 (a) was visible as a blue/green spot, most intense
in the section closest to the ion front and is probably the
bromophenol blue tracker dye. Methyl green does not migrate in
this system and remains at the origin (fig. 27 (b)).
6 '7 8 • 10 11 12 1 1 1 16 1 18 1• 20 1 74 . ... Jk. . ..., .. . : , : • ORIGIN
t 560nm
FIG.20. Electropherogram of an aqueous extract of corpora cardiaca from 5 Iit migradra'In in an anionic system.
1 2 3 4 5 6 7 8 9
.. • • 1.. t .. .m ......
ORIGIN
560nm
FIG.21. Electropherogram of an aqueous extract of corpora cardiaca from 5 L. migratoria in a cationic system. + 2 _19 " ~I' " ,,' "
of-' V1
560nm1
FIG.22. Electropherogram of an aqueous extract of the glandular lobes from 10 L. migratoria in an anionic system. v ....,... ..r...-- Ir. V 1 11 :.% I .i ; '...! %,. . ■, 1 El i 4. I. ORIGIN
FIG.23. Electropherogram of an aqueous extract of the storage lobes from 10 L. migratoria in an anionic system. 107
FIG.24. Electropherogram of a methanol extract of the• separated lobes of the corpora cardiaca from 10 IzmigraLaria in an anionic system.
(a) Glandular lobes
ORIGIN
At 560nm
(b) Storage lobes
c.
ORIGIN
560nm 7 _ _ .1. .(. 7 ..• — ..: . ",:lTV 7,. " •• : ...,:,; .:1•1 • .:- • - le• .1: ..... : .. 5 ....3 e".7,..G.:.—...:11.,--#•• -.•::...... ft, ‘.: • es. 44! .. _— IN
FIG.25. Electropherogram of a haemolymph sample from I. migratoria in an anionic system. 109
FIG.26 (a). Electrophoretic separation of aqueous extracts of (i) 5 corpora cardiaca (ii)10 glandular lobes (iii)10 storage lobes from Zat mizIatoria in an anionic system.
(i) (ii) (iii) 110
FIG.26 (b). Electrophoretic separation of aqueous extracts of corpora cardiaca from 5 L. migratoria in a cationic system. 111 FIG.27. Electrophoretic separation of an aqueous extract of the corpora cardiaca from 40 L. migratoria followed by T.L.C. separation of the eluted gel segments. (a) Gel from cationic. system.
0 9, A ao 00 a
(b Gel from anionic system.
[222 - areas of adipokinetic activity 112
The losses incurred during the whole of this procedure were considerable, in the region of 70 - 80 TL.
TABLE 8
Adipokinetic activity in areas eluted from TLC plate following separation of eluted gel segments.
Area (as in fig.27) Rf from TLC Total adipokinetic activity (units)
Anionic system
A 0.80 40 B 0.69 120 C 0.653 none D 0.58 E 0.65 4,000 F 0.58
Cationic system
A 0.64 5,120
B 0.27 480
C 0.62 2,040
D 0.59 none E 0.64 5,000
3. Activities and identity of the corpus cardiacum factors.
a) The adipokinetic hormone
The factor isolated by the procedure described for the glandular
lobe extracts in results section 2a is the adipokinetic hormone
and is obtained in pure form by this method (Stone et al, 1976).
A sample of pure AKH as verified by amino acid analysis was found 113 to possess approximatly half the adipokinetic activity of methanol
extract of glandular lobes, probably on account of purification losses of appradmstely10%following column chromatography and 40% following TLC (Stone et al, 1976). Near maximal elevation in locust
haemolymph lipid was found to occur in response to injections of
10 — 15 pmol of AKH (fig. 28), and 1 — 2 pmol were the minimum
required to produce a consistently measurable adipokinetic response.
AKH was also assessed for its ability to elevate haemolymph
carbohydrate levels in P. americana (fig. 28). The pattern of
response of carbohydrate mobilisation in P. americana following
injection of pure AKH is in marked contrast to the hyperlipaemic
response in L. migratoria. A gradual increase in response is
observed up to 100 pmol AKH and some 200 — 300 pmol are necessary
to produce a maximal response. 20 — 40 pmol are necessary to
produce the smallest measurable significant hyperglycaemic response
in P. americana. The relative adipokinetic hyperglycaemic activities
of pure AKH are approximatelythe same as those for methanol extracts
of glandular lobes.
The effect of pure AKH on the heart rate and amplitude of an
isolated preparation was found to be similar to that of glandular
lobe extracts. 500 pmol of AKH caused an increase in the frequency
of 61.0 -± 2.8% and also caused a decrease in the amplitude.
Pure AKH was found to be without diuretic activity in the
in vitro Malpighian tubule assay system and had very little
effect on the fat body glycogen phosphorylase system 1.5 hr. after FIG.28. Dose-responserelationshipsforadipokinetichormonein elevating 0
levels inL.migratoria(1,---40) 20 pmol adipokinetic hormone 40 and elevatingcarbohydratelevelsinF.americana(o---q.
60
80
haemolymph lipid 100 - 10 5 • -P 02 Hyperglycaemic activity 21 115 injection into L. migratoria (table 9).
TABLE 9
Effect of pure AKH on fat body phosphorylase activity in two day
old adult male L. migratoria.
Treatment No. of Total phosphorylase Active observations specific activity phosphorylase (ug phosphate/ug % of total protein/hr) activity
Saline 6 0.25 ±0.02 58.5 ± 1.4
500 pmol AKH 4 0.22 ± 0.01 58.3 ± 3.3
TLC blank 4 0.17 ± 0.02 62.0 ± 3.9
b) The resolved storage lobe factors
A range of doses of the factor possessing hyperglycaemic and
adipokinetic activity obtained after Biogel P6 column chromatography
followed by TLC was assayed (fig. 29). Approximately twice the dose
was necessary to produce the same response as storage lobe extracts,
probably due to purification losses. Approximatly 13% of the
hyperglycaemic activity is lost after Biogel P6 column chromatography.
Losses during purification on the glass bead column were not assessed.
46% of the hyperglycaemic activity is lost after TLC. The relative
adipokihetic and hyperglycaemic activities of the purified storage
lobe factor are approximately the same as those for methanol extracts
of storage lobes.
The hyperglycaemic/adipokinetic storage lobe factor was found
to be without diuretic activity and its heart stimulating properties PIG.29. Dose—response relationships for the purified storage lobe factor in elevating haemolymph lipid levels in L. migratoria *--40 and elevating carbohydrate levels in P. americana (0----o). 20 20
)
ts 0, i n (u ty
i -1z 1-1 tiv
10 C3 ac 0
tic rl 0 ci kine
o t>) r1 ttQ ip d A
0:1
0.05 0.1 , 0.15 0.2 0.25 Dose (pairs storage lobes)
117
were found to be similar to those of storage lobe extracts.
Inclusion of 0.5 gland pair equivalents of the purified factor
in the bathing medium of an isolated heart preparation was found
to increase the rate by 52.0 ±8.6% and also to increase the
amplitude.
The diuretic factor obtained following Biogel P6 column
chromatography of methanol extracts of storage lobes was found to
be without significant adipokinetic or hyperglycaemic activities
(table 10). It also had no significant effect on the activity of
an isolated heart preparation. Assayed in both in vitro and in vivo
systems for activity on excretion, the diuretic factor was found
to increase the rate of amaranth clearance in starved mature adult
L. migratoria (fig. 30) and increase the rate of fluid production
by Malpighian tubules in vitro (fig. 31(b)).
TABLE 10
Effect of purified locust diuretic factor on haemolymph lipid levels
in L. migratoria and on haemolymph carbohydrate levels in P. americana.
Treatment I,. migratoria P. americana Total haemolymph Total haemolymph lipids (ug/ul) carbohydrates (ug/ul) 0 1hr 0 2hr 0.5 gland pair equivalent locust 12.5 ± 1.8 11.2 ± 1.3 18.8 ±1• .1 20.3 ±• 0.6 diuretic factor
saline 13.7 ± 0.9 13.1 ± 1.0 19.4 ±• 1.3 20.6 ±• 1.6
At least four animals were used for each test. % amaranth excreted FIG.30. Theeffectofthepurifiedstoragelobe 60 40 20 0
diuretic factorontheexcretionofamaranth a doseof1storagelobepairequivalent/ insect. in I l .miErEILIELa. Thefactorwasassayedat 10 118
purified factor saline control min. 20
30 119
FIG.31. The effect of the purified storage lobe diuretic factor on fluid production by Malpighian tubules in vitro. Fluid production was initially determined in normal Ringer (o) and then in either (a) fresh Ringer (controls) or (b) fresh Ringer + 1 gland equivalent of purified storage lobe diuretic factor (A).
H .1. • C.
11:5 0 0
0f-1 0.8 frs (a) 4-1
" 0.4 4-1 0
0 0
0 25 50
r-t 1.2 rcs a 0 O PI 0.8
(b ) 0 4-I 0.4 a
0 to
0 25 50 min. 120
4. Organ culture of the corpora cardiaca.
a) Histological appearance of cultured corpora cardiaca.
Light microscope level
Fig. 32 (a) to (f) show corpora cardiaca which have been maintained in vitro for three days, while fig. 32 (g) to (i) show corpora cardiaca maintained in vitro for seven days.
Fig. 32 (a) shows a pair of corpora cardiaca stained with paraldehyde fuschin (PF) and counterstained with Halmi's mixture.
There is considerable PF positive material present in the storage lobes, as is shown by figs 32 (b) and (c). In freshly dissected
glands (not shown) the PF positive material appears as small discrete
particles whereas in the cultured glands this material appears more
globular and rounded off.
The glandular region of the cultured glands appears to be
similar to that of freshly dissected glands (fig. 32 (d), (e) and
(f)), although there is some indication of involuted nuclei and
necrosis.
After seven days in culture, the PF positive material in the
storage lobes is no longer obvious as being confined to discrete
areas (fig. 32 (g)) and there appears to be general degeneration
of this region.
The glandular region has also begun to show some signs of
degeneration. The number of involuted nuclei apparently increase
(fig. 32 (h)) and the cells do not take up the stain so readily
(fig. 32 (i)). 121
FIG.32. Photomicrographs of the corpora cardiaca of Locusta migratoria following in vitro culture. (a)— (f) 3 days in vitro (a). Whole corpora cardiaca (PF). Note presence of considerable amount of stained material in storage lobes, x100. (b)and (c). Storage lobes (PF). Note presence of aggregation of PF positive material, (arrow) x400. (d). Junction of glandular and storage region, x600. (e) and (f). Glandular lobes (Haematoxylin eosin). Note some indication of necrosis (arrow). (e) x600, (f) x400.
ax — axons gl — glandular lobes sl — storage lobes
(a) 122
(b)
(C)
124
(e)
(f) 125
FIG.32 cont/d. (g) (i) 7 days in vitro (g).Storage lobes (PF). Note diffuse appearance of PF positive material, x500. (h).Glandular lobes (Haematoxylin eosin). Note indication of necrotic tissue (arrow), x400. (i).Glandular lobes (PF). x600.
(g) 126 127
Electron microscope level
Fig. 33 (d), (g) and (h) are electron micrographs of locust corpora cardiaca cultured for five days.
The storage regions (fig. 33 (g) and (h)) show 'ghosts' of neurosecretory granules and a certain amount of degeneration compared with freshly dissected storage lobe tissue (fig. 33 (e) and (f)).
The glandular region (fig. 33 (d)) of cultured corpora cardiaca in contrast does not show the same degree of degeneration and appears similar to freshly dissected glandular lobes (fig. 33 (a) to (c)) with the large electron dense granules appearing more frequently in areas away from the nucleus.
b) Adipokinetic activity in cultured corpora cardiaca and media.
Routinely five corpora cardiaca were cultured together in a culture
cell with 200u1 858 medium. Following elution of the methanol
extracted glands and media from the TLC plates, the eluates of area
Rf 0.6 - 0.7 were bioassayed for adipokinetic activity (table 11).
After three days in culture, the medium and the corpora cardiaca
contain a considerable amount of adipokinetic activity. After seven
days in culture, the quantity of adipokinetic activity has increased
but the quantity in the glands has remained approximately the same.
After seven days in culture, the aorta attached to the corpora
cardiaca could still be seen to be contracting rhythmically when
observed under the binocular microscope. 128
FIG.33. Electromicrographs of the corpora cardiaca of Locusta migratoria. (a) - (c) Freshly dissected glandular region. (a) and (b) x15,000 (c) x52,000. (d)Glandular region, 5 days in vitro, x20,000. (e)and (f) Freshly dissected storage region. (e) x40,000 (f) x52,000. (g) and (h) Storage region, 5 days in vitro, x7,000. arrow - cytoplasmic inclusion bodies bm - basement membrane er - endoplasmic reticulum f - flocculent electron dense material g - golgi body gl -glial processes gn - glial nucleus gv - golgi vesicle m - mitochondrion n - nucleus nt - neurotubules 1- breakdown of vesicular membranes? 4 woo0) .4 • ob •
• I, a • 4 ••00 0, • • • . • "gee •ittp •!' vo 411.."
• ■ • •. ,,■407.4,0
. 1_ ND
3. 00 Y:4 „ Oa, • oil _to, • 0, e 41) •4111.1,1 0 ,840
•
(q)
6z t 130
(e) 131
(f) 132
(h)
•
• * se'
• • • air 1 le • 0 e • 9 • 1 • • • • • • • •
GL • • • •
• • • •., • ,SO, • • , i • * • i $ 4 • . •0 • • ir • ' • R* •7 : 11 Q • , '• : 4* \. • i • • • j • 'kt ' 4 133
TABLE 11
Adipokinetic activity of cultured corpora cardiaca and incubation
media.
Total adipokinetic activity (units)
media glands
3 days in culture 3,875 2: 525 5,425 ± 750
7 days in culture 5,585 ± 1,075 5,150 ± 575
incubated control 465 ± 73 .10■■■••••■••• media
THE CORPORA CARDIACA OF P. AMERICANA
1. Histological appearance of the corpora cardiaca.
The corpora cardiaca of P. americana are ribbon shaped
structures. The glandular and storage regions are not anatomically
separated, although there is a predominance of glandular type cells
in the dorsal margins of the glands (fig. 34 (a) and (b)). This
necessitated the use of extracts of whole cockroach corpora cardiaca
for the purification of factors with no attempt at separation of
the glandular and storage regions.
The NCA I are a pair of stout extensions of the posterio —
—dorsal regions of the corpora cardiaca and PF positive material
can be visualised entering the corpora allata (fig. 34 (c)). Due
to this close association of the corpora cardiaca and corpora allata
in the cockroach, no attempt was made to remove the corpora allata 134 when dissecting out large numbers of corpora cardiaca for purification
purposes.
FIG. 34. Photomicrographs of the corpora cardiaca of Periplaneta americana (paraldehyde fuchsin). (a)and (b). Longitudinal section. Note presence of axons traversing the corpora cardiaca and the intimate association of stained neurosecretion and glandular cells. (a) x 300,
(b)x 500. (c). Entry of the stout NCA1 into the corpus allatum showing
the penetration of stained neurosecretion into the corpus allatum, x 300.
gl — glandular region ax — axons ca — corpus allatum st — storage region (a) 135
(b)
(c) 136
2. Activities of the corpus cardiacum extracts. a) Effect on total haemolymph lipids. The adipokinetic activity in locusts of methanol extracts of corpora cardiaca from P. americana was assessed at doses from 0.01 to 0.25 gland pair equivalents (fig. 35). The maximum response was attained only with doses in excess of 0.2 gland pair equivalents. the minimum quantity required to produce a consistently measurable response was found to be 0.01 gland pairs. Methanol extracted corpora cardiaca of P. americana were estimated to contain 500 - 600 adipokinetic units / gland pair.
Doses of 0.5 pairs of methanol extracted corpora cardiaca were injected into adult male P. americana. This did not produce a change in haemolymph lipid levels which was substantially different from the controls (table 12).
TABLE 12 Effect of methanol extracts of the corpora cardiaca from P. americana on total haemolymph lipids in adult male P. americana.
Material Total lipid concentration (ug/ul) injected 0 2hr 0.5 pair corpora cardiaca extract 12.9 ± 1.3 11.7 ± 0.5 saline 14.2 ± 2.1 14.3 ± 1.0 At least four animals were used for each test.
b) Effect on total haemolymph carbohydrates. Methanol extracts of corpora cardiaca from P. americana were injected FIG. 35. Dose-response curve of the lipid mobilising activity in L. migratoria of a methanol extract of the corpora cardiaca from P. americana. Mean values and standard deviations are given. Each group consisted of at least 4 animals.
30 ) its (un ty 20 i iv t c a tic 10 kine o dip A
0 0.05 0.1 0.15 0.2 0.25 Dose (pairs corpora cardiaca) 137a
into adult male L. migratoria at a dose of 0.5 gland pair equivalents. This did not produce a change in the level of haemolymph carbohydrate which was different from the saline injected control (table 13).
TABLE 13
Effect of methanol extracts of the corpora cardiaca from P. americana on total haemolymph carbohydrates in adult male L. migratoria.
Material Total carbohydrate concentration (ug/ul) injected 0 1hr
0.5 pair corpora cardiaca extract 33.5 ±1.7 34.9 ±2.7 saline 30.9 ± 2.1 31.8 ±3.2
At least four animals were used for each test.
The effect of methanol extracts of P. americana corpora cardiaca on the total haemolymph carbohydrate levels in adult male
P. americana is shown in fig. 36. The hyperglycaemic response is approximately linear up to a dose of 0.1 gland pairs. Near maximal responses were obtained with 0.05 gland pairs and 0.01 gland pairs were required to produce a consistently measurable response. The number of hyperglycaemic units was estimated to be 400 — 500 units
/ gland pair.
c) Effect on the rate of fluid secretion by Malpighian tubules in vitro.
Inclusion of 1 gland pair of methanol extracted corpora cardiaca FIG. 36. Dose—response curve of the hyperglycaemic activity in P. americana of a methanol extract of the corpora cardiaca from P. americana. Mean values and standard deviations are given. Each group consisted of at least 4 animals. 20
0.05 0.1 0.15 0.2 0.25 Dose (pairs corpora cardiaca) 139
from fed mature adult male P. americana in the incubation medium
of the in vitro Malpighian tubule bioassay system increased the rate
of fluid production by 4.4 nl/min.
Fig. 37 (b) shows the mean volume of fluid produced with time
by ten tubules in Ringer compared with the volume produced by the
same tubules in Ringer containing '1 gland pair equivalent of methanol
extracted corpora cardiaca from fed mature male P. americana.
Comparisons between the rates observed in the first and second
50 min. periods indicate that at the dosage used, a notable
increase in fluid secretion is produced. Results from the control
preparation processed at the same time (fig. 37 (a)) indicate that
the secretory rate of the tubules remains constant during the 100
min. assay period, and is unaffected by the change in Ringer after
the first 50 min. period.
d) Effect on heart beat.
The cardioacceleratory activity of the corpora cardiaca from
P. americana was determined by the inclusion of 0.5 gland pair
equivalents of methanol extract in the incubating medium of the
isolated heart preparation. The extract increased the heart rate
by 62.0 ± 6.6% and also increased the amplitude of the beat.
3. Purification of the corpus cardiacum factors.
a) Glass bead filtration chromatography.
The conditions of glass bead column chromatography were the same as
those used for the purification of AKH from locust glandular lobe
extracts (results section 2 a). FIG.37. Theeffectofmethanolextractscorporacardiaca (a) (b) --- r-I szt (a) freshRinger(controls)or(b)+1 from P.americanaonfluidproductionbyMalpighian tubules invitro.Fluidproductionwasinitially determined innormalRinger(e)andtheneither gland equivalentofcorporacardiacaextract(A). volume of fluid produced volume of fluid produced 1.2 0.8 0.4 0 0
140 • min min 25 25
50 50 141
Fig. 38 shows a typical column profile obtained on elution of methanol extracts of corpora cardiaca from 300 adult P. americana.
Two peaks of u/v absorbing material are visible, one at Vo (fractions
7 - 14) and the other at an elution volume less than Vt (fractions 16 — 25). The fractions of both peaks were pooled and water removed under reduced pressure. The residues were bioassayed for adipokinetic and hyperglycaemic activity. The V peak was found to contain neither activity, whereas the peak before Vt was found to contain both. Methanol extracts of the corpora cardiaca from 150 adult L. maderae were also subjected to separation on'a glass bead column under the same conditions as those used for the P. americana corpus cardiacum extracts. The resulting column profile (fig. 39) is identical to that produced by the P. americana corpus cardiacum extracts. The fractions of the two u/v absorbing peaks were pooled and water removed under reduced pressure. The residue of the Vo peak contained neither adipokinetic nor hyperglycaemic activity, while the peak before
was found to contain both. Vt
b) TLC of eluates.
The pooled fractions of the peaks obtained from both P. americana and L. maderae corpus cardiacum extracts were subjected to TLC separation under the same conditions as those used for the purification of AKH (results section 2a). In both cases, a u/v absorbing spot of
Rf 0.56 was visualised, resolved from the residue of the peak near the column volume. The whole plate of P. americana samples was assayed for hyperglycaemic, adipokinetic and diuretic activities (fig. 40).
All three activities were located in the eluate of the u/v absorbing 142
FIG.38. Separation of a methanol extract of the corpora cardiaca from 300 P. americana on a G-75-50 glass bead column. , O.D. 206nm F--Apooled fractions
0 20 40 60 80 ml 143
FIG.39. Separation of a methanol extract of the corpora cardiaca from 200 L. madera on a G-75-50 glass bead column. 0.D. 206nm fracions
0 20 40 60 80 ml
144
FIG.40. The adipokinetic, hyperglycaemic and diuretic activity elated from the TLC plate following separation of fractions 16-25. The adipokinetic, hyperglycaemic and diuretic activities were assayed at 0.1, 0.1 and 1 gland pairs respectively.
4, 20 4D 0 al Ea rl -r-I 10
O P4 V di
-P 0 1 -r-I 0.5 n-1 20 -P ti
e•-■ vi 02
QS 10 U
$-1
0 0.5 1 :10.0
4-, 0 rcj erg I> 0 0 4-1 0 0 0 0 W 41) P4 50
0 W (1.) -4 -IA 0 0 g H y •ri V. 44 0 0.5 1 ORIGIN Rf SOLVENT FRONT 145 area around Rf 0.56. The same area from the plate containing the
L.maderae samples was found to contain these three activities in amounts proportionally similar to those in the P. americana eluate.
On staining the TLC plate containing the P. americana samples • with starch iodide, only the origin stained. It is possible that the spot Rf 0.56 did not stain due to the presence of insufficient material.
4. Activities and identity of the corpus cardiacum factors. a) Adipokinetic, hyperglycaemic, heart accelerating and diuretic
activities.
A range of doses of the factor possessing adipokinetic, hyperglycaemic
and diuretic activities isolated after glass bead column chromatography
and TLC of extracts of corpora cardiaca from P. americana was assayed
(fig. 41). The relative adipokinetic and hyperglycaemic activities
of the purified factor were found to be similar to those of the
methanol extracts of corpora cardiaca. Approximatly twice the dose
of purified factor was needed to produce the same response as methanol
extracts of the corpora cardiaca, probably due to purification losses.
A range of doses of the purified factor was assayed for diuretic
activity in the in vitro Malpighian tubule assay (fig. 42). The
results indicate the factor is potent in increasing the rate of fluid
production by tubules in vitro. The results of additional assay in
the in vitro Malpighian tubule system confirmed the ability of the
purified factor to increase substantially the rate of fluid production
(fig• 43). PIG.41. Dose-response relationship for the purified factor from the corpora cardiaca of P. americana in elevating haemolymph lipid levels in L. migratoria (4,---4,) and elevating carbohydrate levels in P. americana
ty 20 i tiv ac tic ine k o Adip
0 0.05 0.1 0.15 0.2 0.25 Dose (pairs corpora cardiaca) 147
FIG.42. Dose-response relationship for the purified factor from the corpora cardiaca of P. americana in elevating the rate of fluid production by Malpighian tubules in vitro. Figures in parenthesis indicate number of tubules assayed.
(10 )
( )
6)
6) basal secretory rate
0 0.25 0.5 0.75 Dose (pairs corpora cardiaca) 148
FIG.43. The effect of the purified factor from the corpora cardiaca of P. americana on fluid production by Malpighian tubules in vitro. Fluid production was initially determined in normal Ringer (s) and then in either (a) fresh Ringer (controls) or (b) fresh Ringer + 1 gland equivalent of purified factor (A).
r. v 1.2 io a) 0 ro 0.8
(a)
cH 0.4 cH P 0
0 . 25 50 min
/1.
--- 1.2 rcs a)
rcs 0.8
(b) 7:3 0 cH 0. 4 0
0 0
0 25 50 min 149
FIG.44. The effect of the purified factor from the corpora cardiaca of P. americana on the excretion excretion of amaranth in L. migratoria. The factor was assayed at a dose of 1 gland equivalent of purified factor/insect. Ar---A purified factor saline control
60
0 40 4-0, 0 a) 4, k cd 0
20
o 10 20 30 min. 150
The injection of 1 gland pair equivalent of the purified factor into starved adult male L. migratoria produced an excretion rate which was substantially faster than the control rate (fig. 44).
Itwasincreased. by some 160%.
The effect of the purified factor on the heart rate and amplitude of an isolated heart preparation was found to be similar
to that of corpus cardiacum extracts. 0.5 gland pair equivalents of the factor caused an increase in the frequency of 44.0 I 7.410 and also caused an increase in the amplitude.
b) Flight metabolism in L. madera.
L. maderae was chosen in preference to P. americana for flight studies as it showed a greater inclination for flight.
The insects showed a tendency to fly readily for up to 10 min.
after which time they could not be induced to fly further. Insects after flight appeared sluggish. Stress during flight was indicated
by regurgitation, defaecation and vigorous wriggling movements of the
abdomen. During flight, the abdomen was held out to one side, and
the legs splayed outwards.
Changes in haemolymph metabolite levels after a 10 min. flight are shown in table 14. Lipid levels were unchanged after flight,
whereas carbohydrates were lowered by 4.8 ug/ul.
The time course of an injection of 0.2 gland pair equivalents of the purified L.maderae factor is shown in fig 45. The maximum 151
TABLE 14
The influence of flight on haemolymph metabolite levels in L. maderae.
Haemolymph metabolites % change (ugiul)
Before After 10 flight min. flight
Total carbohydrates 22.0 ±0.8 17.2 ±0.8 —22
Total lipids 11.6 ± 0.8 11.6 ±0.8 0
The figures represent the mean values from 8 animals.
response is obtained after 4 hr. falling to control level after 24 hr. The haemolymph carbohydrate levels of insects injected with saline were also increased, but much less sharply than if injected with the purified factor.
Insects were injected with 0.2 gland pair equivalents of the purified factor, and then flown after 1 hr. for 10 min. Control insects were injected with saline and flown, or with the factor and not flown. The results (fig.46) indicate that the carbohydrate mobilised under the influence of the injected factor may be utilised to some extent during flight, as the haemolymph carbohydrate levels in the factor injected flown insects fall more slowly than those in the saline injected flown insects. After 20 hr. the factor injected flown and unflown animals have elevated haemolymph carbohydrate levels, while the haemolymph carbohydrate levels of the saline injected flown
controls have returned to their normal value. FIG.45. Time course of changes in haemolymph carbohydrate levels in L. maderaefollowing injection of 0.2 gland pair equivalent of the factor purified from the corpora cardiaca.of L. madera. Each point is the mean value of 8 animals. H --* purified factor tal3 20 o saline control
•••••• .. -
W l ca
010
0
Cd
-r-I U DI Cd (ll 0 0 1 2 3 4 24 H hr. •
FIG.46. The effect of the purified factor from the corpora cardiaca of L. maderae on haemolymph carbohydrate levels during flight in L. maderae. Each point is the mean value of 8 animals.r 2.---11 injected with saline and flown 1-1 injected with factor and unflown b.0 e---o injected with factor and flown
• • 40 0
ti
a) O 30 0 a) +2
0 A, 20
PA
0 0 flight 0 • cd 10 0 0.5 1 20 hr. Injected 154
c) Characteristics of the purified factor. Enzymic digestion. Factors from the corpora cardiaca of both P. americana and L.maderae were digested by incubating with pronase. After incubation the preparations were assayed for hyperglycaemic, adipokinetic and diuretic activity (fig. 47 and 48). It can be seen that enzymic digestion of both factors abolishes both hyperglycaemic and adipokinetic activity.
The results to the diuretic activity estimations of control and digested factor were inconclusive. The diuretic activity of the undigested control was reduced to a fairly low level following the
incubation procedure, which may indicate denaturation. The comparatively
large increase in the rate of fluid production by Malpighian tubules in vitro produced by the pronase blank indicates a non-specific interference in the assay system by a component of the digestion
mixture.
Fluorescence and ultra-violet absorption spectra. The fluorescence spectrum obtained for the purified factor is shown in fig. 49a. The results are inconclusive. Amino acid analysis indicated that the amount of material present in the sample used to
obtain this spectrum (approximatly 3 nmol) would be insufficient to provide reliable data. In addition, it is possible that traces of fluorescent indicator from the TLC plate are interfering with the results obtained.
The u/v spectrum of the factor shows a maximum absorption at 155
280 nm, which coincides with that of the tryptophan sample (fig. 49b).
Amino acid composition. The preliminary data obtained on amino acid analysis of the factor obtained from extracts of corpora cardiaca from 50 P. americana following acid hydrolysis indicated that asp, ser, glu, gly, val, and phe residues may be present (fig. 50). However, insufficient material was present to give a reliable assessment.
Table 15 and fig. 51 show the results of analysis of a larger preparation from the corpora cardiaca of 300 P. americana. Several interpretations of these data are possible. If leucine is taken as unity, and all the residues are taken into account, a peptide of
17 amino acids may be present. Alternatively, there may be a mixture of two peptides, one of 6 residues and the other of 5 residues, present in proportion of 2:1. Thirdly, if valine is taken as unity, an 8 residue peptide could be present with a considerable amount of impurities. Assuming the presence of a single 17 residue molecule, it can be estimated that the corpora cardiaca from one P. americana contain 0.06 — 0.12 ug of the factor (assuming 50% purification losses). •
156
F1G.47. The hyperglycaemic, adipokinetic and diuretic activity of the factor purified from the corpora cardiaca of P. americana following pronase digestion. Activity levels are expressed as % of undigested control value.
100 H V a) o o H H P a) 0 -ri 50 N • ra W 0 k P F-f W •ri 0 0 saline pronase digested undigested 100 control control factor factor
0 0 P a) O 1> ,d 1 50
0 saline pronase digested undigested 0 control control factor factor 100 - 0 f> a) 0 .11 O 0 1-1 kPA 50 CD V Cif H Fi 4-I 0 0 4-1 •ri 0 0 1 1 saline pronase digested undigested control control factor factor 157
FIG.48. The hyperglycaemic and adipokinetic activity of the factor purified from the corpora cardiaca of I. mad era following pronase H digestion. Activity levels are expressed as o % of undigested control value. t.-0100- H 0
Pi V t>) 40 .10 Pi W 0
0 eri a) a) al a) PI . 0 1 r ■ ,-1g saline pronase digested undigested control control factor factor
H 100 — 0 P a) H 17:5 Pl% n-1 H V 0 0 H 50- .0 ..1g a) 0] cti cp N 0 PI -1-1
saline pronase digested undigested control control factor factor 158
FIG.49. The fluorescence spectrum (a) and absorption spectrum (b) of the factor purified from the corpora cardiaca of P. americana. ---- 13 nmol tryptophan standard purified factor 40 -
30 - a) 0 0 0 0 0 0 ( a ) ok 20 -
10 -
0 300 400 23M 0.3-
0.2-
(b) O 0.1. /.■••\ / • 1 / • t / • % / • %../ • • • 0 220 270 320 nm
159
FIG.50. Amino acid analysis of the factor purified from the corpora cardiaca of 40 P. americana.
•
orleu his std. + lys val phe asp? ser? g gala 570nm
440nm 160
FIG. 51. Amino acid analysis of the factor purified from the corpora cardiaca of 300 P. americana.
NH lhorleu 3 std.
gly asp glu S J phe ser val his ala lys
thr y 570nm
pro
440nm 161
TABLE 15
Amino acid composition of the purified corpus cardiacum factor possessing hyperglycaemic, adipokinetic and diuretic activities
from P. americana.
Molar proportion (leucine = 1)
found nearest integer
Aspartic acid 2.85 3
Threonine 0.47
Serine 0.72 1
Glutamic acid 2.83 3
Glycine 2.08 2
Alanine 0.79 1
Valine 1.48 2
Leucine 1.00 1
Tyrosine 0.40
Phenylalanine 1.82 2
Proline 2.20 2 162
DISCUSSION
CORPUS CARDIACUM FACTORS OF L. MIGRATORIA.
1. Activities of factors in glandular lobe extracts. The adipokinetic response of L. migratoria to injections of glandular lobe extracts obtained in this study bears a greater similarity to that obtained by Goldsworthy et al (1972) than to that obtained by Mayer and Candy (1969) in terms of the
magnitude of the response, and the amount needed to produce a consistently measurable response.It is now known that the activity
of pure AKH bears a greater resemblence to the data of Goldsworthy et al (1972) than to that of Mayer and Candy (1969). It is possible that the latter workers extracted principally the storage lobes with
some glandular lobe material. An alternative explanation is differences in diet (Goldsworthy et a1,1972). The variations in the maximal increase in haemolymph lipid (20 — 30 ug/ul) probably result from
the state of feeding and state of crowding of the assay animals, and also to slight variations in the ages of the animals.
The hyperglycaemic activity of methanol glandular lobe extracts
in P. americana is in contrast to the adipokinetic activity described
in the present study, as considerable quantities of material (0.5 —
1 gland pair) are required to produce a consistent maximum response. The maximum increase in haemolymph carbohydrates was found to be 18 — 25 ug/ul. As the mean resting haemolymph carbohydrate level in assay animals was found to be 19.1 ± 1.2 ug/ul, this represents an increase of between 90 — 100%. Saline extracts of glandular lobes 163 from L. migratoria have been reported to increase haemolymph carbohydrate levels in P. americana by 106 ± 16.3% at a dose of
1 gland pair (ffordue and Goldsworthy, 1969; Highnam and Goldsworthy, 1972). Extracts of whole corpora cardiaca from L. migratoria have been found to increase the total blood carbohydrates of P. americana from 11.6 ± 1.5 ug/ul to 23.5 ± 3.5 ug/ul (increase of 11.9 ug/ul) at a dose of 0.5 gland pairs (Goldsworthy et al, 1972b). -These data are comparable with the results of the present study.
A pair of glandular lobes from L. migratoria have been estimated to contain 2,000 — 3,000 units of adipokinetic activity and 50 — 100 units of hyperglycaemic activity. Thus the adipokinetic activity in locusts of glandular lobe extracts is some 20 times greater than the hyperglycaemic activity in cockroaches.
The inability of glandular lobe extracts to elevate active fat body phosphorylase levels in two day old adult male L. migratoria is at variance with the work of Goldsworthy (1970). The normal levels of total phosphorylase specific activity in this study are comparable with those of Goldsworthy (1970) as are the normal levels of active glycogen phosphorylase as a percentage of total activity. The different results may be due to the smaller quantities of material assayed in the present study. In order to produce a significant activation of fat body phosphorylase and haemolymph carbohydrates, doses of
1 — 1.5 gland pairs of whole corpora cardiaca were required. In the present study however, 1 gland pair of either glandular or storage lobes were assessed for activity. 164
The lack of effect of injection of glandular lobe extracts into
10 day old adult male L. migratoria on total haemolymph carbohydrate
levels is probably due to the fact that at this age, the fat body
glycogen phosphorylase levels are approaching maximum stimulation
(Goldsworthy, 1970). The elevation of haemolymph carbohydrates in
response to injections of corpus cardiacum extracts is greatest,in
6 day old insects, and maturs males never show a hyperglycaemic
response to corpus cardiacum extracts (Goldsworthy, 1969).
Glandular lobe extracts were found to increase the heart rate
and decrease the amplitude of . semi—isolated heart preparations.
These results are in agreement with those of Nordue and Goldsworthy
(1969) who resolved two fractions from extracts of whole corpora
cardiaca from L. migratoria, one of which increased heart rate and
decreased amplitude in a similar way to glandular lobe extracts.
There is little indication of a physiological role for the
heart accelerating activity of corpus cardiacum extracts in vivo.
Glucagon, the vertebrate glycogenolytic hormone, is known to have
the pharmacological effect of increasing the heart rate in a semi—
isolated heart preparation of a dog, but not in the intact animal
(Farah and Tuttle, 1960). Pharmacological activities however may be
useful in distinguishing between preparations of active factors.
The activity of corpus cardiacum extracts on heart beat are not
thought to be due to the presence of 5— hydroxytryptamine, as the
active factors have different Rf values (Mordue and Goldsworthy, 1969)
and different elution characteristics (Natalizi et a1,1970). Also, 165
there is probably an insufficient amount of 5 HT or other biological amine present in corpus cardiacum extracts to bring about the responses observed (Colhoun, 1963; Brown, 1965; Davey, 1964). The activity is likely to be due to the presence of peptide material, as the activity is abolished following enzymic digestion of corpus cardiacum extracts of P. americana (Natalizi et al, 1970).
2. Isolation of the adipokinetic hormone. The purification processes described in this study for glandular lobe extracts are essentially the same as those used by Stone et al (1976) for the isolation of the adipokinetic hormone.
The solubility of the adipokinetic and hyperglycaemic factors
in methanol is fortuitous, as a large quantity of proteinaceous
impurities is removed prior to the purification procedure. On column chromatographic separation of glandular lobe extracts, the only
area eluted from the glass bead column having adipokinetic (and hyperglycaemic) activity was the peak eluted just before the Vt
(fractions 20 — 30). The Vo peak was devoid of either activity, thus there is no indication of activity in the larger molecular weight
fraction.
On subsequent TLC and elution of the plate, both adipokinetic and hyperglycaemic activities were localised in an area Rf 0.67, which
was subsequently demonstrated by amino acid analysis to be pure AKH. The rest of the plate was also subjected to amino acid analysis, and
found to contain only inconsistent traces of peptide material. The
u/v absorbing area Rf 0.79, on subsequent mass spectroscopic analysis, 166 is thought to be cholesterol (H. Morris, personal communication).
The data obtained from polyacrylamide gel electrophoresis gives some indication of the variety of protein/peptide fractions present in extracts of corpora cardiaca from L. migratoria, although some of these are probably due to contamination with proteins from the haemolymph and hypocerebral ganglion. The top of each gel stained heavily with Coomassie blue, indicating the presence of proteins too large to enter the gel, or electrically neutral material.
Methanol extracts produced only faintly staining bands, indicating
that most of the proteins/peptides are removed by extraction into methanol. There are many problems associated with the electrophoretic separation of small peptides in polyacrylamide gels. Small molecules tend to diffuse through the gel more readily during electrophoresis,
and also visualisation of the peptides with standard stains is frequently unsatisfactory as the groups required for the staining
reaction may not be present in sufficient quantity.
The use of polyacrylamide gel electrophoresis as an analytical tool in a multicomponent extract such as those of corpora cardiaca
is limited by the degree of contaminants and artifacts. Its use as
a preparative tool as used in the present study is also limited, mainly due to the large losses incurred during elution of the resolved fractions, which are in the region of 70 — 80,4 These losses are similar to those incurred during high voltage paper electrophoresis of pure AKH (J. Stone, personal communication). The system is also
limited due to the uncharged nature of many peptide hormones, including 167
AKH. The migration of adipokinetic activity in the cationic system used in the present study may have been the result of non-specific binding of the AKH to a charged molecule or ion.
3. Activities of the adipokinetic hormone. The adipokinetic activity of AKH obtained from extracts of glandular lobes from L. migratoria was found to be essentially the same as described by Stone et al (1976). The adipokinetic response
of locusts injected with doses of up to 10 pmol AKH is very pronounced, a near maximum elevation in haemolymph lipid occuring in response to
10 - 15 pmol AKH. The smallest amount of AKH needed to produce a consistently measurable adipokinetic response is 1 - 2 pmol.
The adipokinetic activity of pure AKH is approximately half that of glandular lobe extracts, and this can be accounted for in terms of purification losses (Stone et al, 1976). Taking these losses into account, it has been estimated that there is 0.25 - 0.55 ug/ul (200 -
500 pmol) AKH in the glandular lobes from the corpora cardiaca of
each adult L. migratoria (Stone et al, 1976). This quantity of hormone was found to possess 2,000 - 3,000 units of adipokinetic activity.
Thus a unit of adipokinetic activity can be defined in terms of 0.1 - 0.2 pmol
The pattern of response of carbohydrate mobilisation in P. americana following injection of pure AKH is in marked contrast to the adipokinetic response in L. migratoria. A gradual increase
in response is observed up to 100 pmol AKH, and some 200 - 300 pmol AKH are necessary to produce a maximal response. 20 - 40 pmol are 168
required to produce the smallest measurable significant hyperglycaemic response in P. americana. Thus the relative activities of AKH are similar to those of glandular lobe extracts, the adipokinetic activity being some 20 times greater than the hyperglycaemic activity. Doses in excess of those required to induce a maximum adipokinetic response in L. migratoria (15 - 25 pmol) are barely able to bring about a significant hyperglycaemic response when injected into P. americana.
This may be the reason why previously the locust glandular lobe adipokinetic factor has been reported to be free from hyperglycaemic activity (Mordue and Goldsworthy, 1974; Goldsworthy, 1976). Other attempts to separate the adipokinetic and hyperglycaemic activities present in extracts of locust corpora cardiaca have been unsuccessful
(Grade and Holwerda, 1976; Holwerda at al, 1977) which also led to the conclusion that the two factors may be identical. Until now, the methods of extraction have not permitted the use of pure hormones.
The present study demonstrates clearly that pure AKH can cause increases in the level of carbohydrates in the haemolymph of P. americana.
Pure AKH was found to influence the beating of semi-isolated heart preparation in a similar way to glandular lobe extracts, namely to increase the frequency and decrease the amplitude of the heart beat.
This activity of purified AKH has also been observed by Goldsworthy et al (1976). It is not known if the heart accelerating activity of.
AKH is of physiological significance in the intact animal, but the pharmacological activity of other peptide hormones such as glucagon on heart preparations cautions against this assumption.
Pure AKH was found to have no effect on haemolymph carbohydrate 169 levels or on fat body active phosphorylase levels following injection into L. migratoria, probably for the same reasons as were given for the lack of effect of glandular lobe extracts (page 163). Pure AKH was also found to have no significant effect on the rate of fluid production by Malpighian tubules in vitro. As AKH was not assayed for activity on rectal water reabsorption, it is not known if AKH is distinct from the antidiuretic factor of the locust glandular lobes (Mordue, 1970).
4. Additional glandular lobe factors. After correcting for losses during purification, it has been estimated that there are 200 - 500 pmol AKH stored in the glandular lobes of each locust (Stone et al, 1976). The hyperglycaemic activity
of this quantity of hormone is 30 - 50 units. Methanolic extracts of glandular lobes contain 50 - 100 units of hyperglycaemic activity per pair of glandular lobes. Thus it is unlikely that there is an additional
factor in the glandular lobes of L. migratoria which elevates
haemolymph carbohydrates in P. americana. This does not preclude the possibility of additional glandular lobe factors which control
the elevation of active fat body phosphorylase levels during adult
development in locusts.
Structural indications that the intrinsic glandular cells of the
locust corpora cardiaca contain one type of secretory granule has been_
cited as evidence in support of the common identity of the adipokinetic and hyperglycaemic factors (Holwerdaet al, 1977). However, cells
elaborating different active material may not reflect this in their
ultrastructural appearance. In addition, the presence of neurosecretory 170
fibres containing electron dense vesicles in the glandular region (Cassie= and Fain-Maurel, 1971; Cazal et al, 1971) may contribute additional active components to homogenates of glandular lobes.
Pature work should extend investigations into the nature and number of active factors in locust glandular lobes, and also investigate the nature of the material in the large 500 - 600 nm electron dense
vesicles. As glandular lobe cell contents do not stain with conventional neurosecretory stains such as paraldehyde fuschin in locusts,the material may be different from orthodox neurosecretion
or alternatively there may be differences in the granule membrane properties.
The neurohypophysial hormones of vertebrates seem to be
synthesised as inactive precursors (Sachs et al, 1969) and the enzymes responsible for the release of oxytocin from its precursors may be packaged along with the prohormone molecules into the secretory
granules. There is also some evidence that neurohypophysial hormones may serve themselves as precursors for new hormonal factors with activities completely different from those of the parent principle (Walter, 1974). It is possible that the mechanisms of peptide neurohormone formation in insect corpora cardiaca may bear some similarities to those in the vertebrate hypothalamo-neurohypophysial
system.
5. Physiological significance of the glandular lobe factors. The role of AKH in the maintenance of prolonged flight activity 171 is now well established. This hormone mobilises diglyceride from the fat body triglyceride stores and also regulates the utilisation - of lipids during flight in locusts (Mayer and Candy, 1969; Beenakkers,
1969; Goldsworthy et al, 1972a,b, 1973; Spencer and Candy, 1974; Robinson and Goldsworthy, 1974; Jutsum and Goldsworthy, 1976). The hyperglycaemic factors of the corpora cardiaca do not appear to be involved in locust flight metabolism (Goldsworthy, 1976; Jutsum and
Goldsworthy, 1976), and this may also be true of the hyperglycaemic activity of AKH. AKH is known to exert a sparing effect on haemolymph carbohydrate levels during flight (Jutsum and Goldsworthy, 1976).
After 30 min. flight, the concentration of haemolymph carbohydrates reaches a steady state and is conserved for essential activities which may be undertaken when flight ceases. AKH facilitates the utilisation of lipid by the flight muscle, and after 30 min. the flight muscles switch from carbohydrate to lipid as the preferred substrate. No mobilisation of carbohydrate reserves in the tissues occurs as is indicated by little change in haemolymph carbohydrate concentration during the rest periods following various lengths of flight
(Goldsworthy, 1976; Jutsum and Goldsworthy, 1976). Insects other than locusts which utilise lipid for flight have not been reported to
possess an adipokinetic hormone, and it may be that the involvement of AKH in the control of flight metabolism is a special adaptation
to the 'two fuel' metabolism of mi6ratory flights in locusts
(Goldsworthy, 1976).
Rapid elevation of haemolymph carbohydrates or prolonged
maintenance of haemolymph carbohydrate levels by corpus cardiacum
factors in L. migratoria would appear to be limited by the small amount 172
of fat body glycogen (Goldsworthy, 1969) and the increase of fat
body active phosphorylase levels during adult development (Goldsworthy, 1970). The mechanisms involved in the depression
of haemolymph carbohydrate levels following cardiectomy or
glandular lobe removal (Cazall 1971; Jutsum and Goldsworthy, 1976) and the eventual return of the haemolymph carbohydrates to the normal
level (Jutsum and Goldsworthy, 1976) have yet to be elucidated.
The elevation of haemolymph carbohydrates in P. americana
following the injection of AKH is likely to be a pharmacological effect. Not only are comparatively large doses of AKH required to produce a measurable hyperglycaemic response in P. americana, but also a molecular species identical to AKH is probably not endogenous to the corpora cardiacs. of P. americana. A likely explanation of the hyperglycaemic activity of AKH is that AKH is
sufficiently structurally similar to the cockroach 's endogenous hyperglycaemic factor to be able to fit on to the receptor system
... present in the cockroach fat body, and thus produce hyperglycaemia. In an analogous way, vasopressin is sufficiently similar in
structure to oxytocin to have a low level of activity in the oxytocin assay system. Structurally similar peptides of different physiological function may therefore possess an overlap of activities in certain
assay systems.
6. Activities of factors in storage lobe extracts. The adipokinetic activity of storage lobe extracts was found
to be some 10 — 20 times less than that of the glandular lobe extracts, but only 2 — 3 times less potent than glandular lobe extracts in the 173
elevation of cockroach haemolymph carbohydrates. This agrees with other work in which glandular lobe extracts were found to be approximately 50 times more potent than storage lobe extract in terms of adipokinetic
activity (Goldsworthy et al, 1972) but only 2 - 3 times more potent in terms of hyperglycaemic activity (Mordue and Goldsworthy, 1969).
Storage lobes were estimated to contain 400 - 500 units of
adipokinetic activity / gland pair and less than 50 units of hyperglycaemic activity / gland pair. Thus the adipokinetic activity of storage lobe extract is 5 - 10 times greater than its hyperglycaemic activity. This indicates that there. is a larger proportion of hyperglycaemic activity in storage lobe extracts than can be accounted
for solely in terms of AKH contaminant from adhering glandular lobe
material, as the adipokinetic activity of AKH is some 20 times greater than its hyperglycaemic activity.
The inability of storage lobe extracts to elevate fat body active - phosphorylase levels in two day old adult male L. migratoria is at variance with the work of Goldsworthy (1970) and Mordue and
Goldsworthy (1969), possibly for the same reasons given for the lack of activity of glandular lobe extracts (page 163).
Storage lobe extracts were found to increase both the heart rate and amplitude of a semi-isolated heart preparation. These results are in agreement with those of Mordue and Goldsworthy (1969) who .
resolved two fractions from whole corpus cardiacum extracts, one of
which increased heart rate and amplitude in a similar way to storage
lobe extracts. It is not known if the cardioacceleratory activity 174 of storage lobe extracts has a physiological role in vivo.
Experiments carried out in both the in vivo and in vitro assay systems demonstrate that the corpus cardiacum factor which is active on the rate of fluid secretion by the Malpighian tubules is restricted mainly to the storage region. This agrees with the work of Mordue and Goldsworthy (1969) who, using the in vivo rate of amaranth excretion assay, found only a slight increase in the rate following injections of glandular lobe extracts but a marked increase using storage lobe extracts. Using in vitro preparations, Cazal and Girardie (1968) reported a reduction in the rate of excretion through the Malpighian tubules in response to extracts of corpora cardiaca from L. migratoria. However, the presence of a factor within the neurosecretory cells of the pars intercerebralis which increased tubule secretion was also reported.
Starvation was found to decrease the rate of amaranth excretion, as was found by Mordue (1969). Starved insects were also found to have an increased amount of diuretic activity in extracts of their storage lobes compared with extracts from fed insects.
Storage lobe extracts and subsequently resolved factors were assayed only at a dose of 1 gland pair. Mordue (1969) and Mordue and Goldsworthy (1969) found that the increase in the rate of excretion is directly proportional to the amount of corpus cardiacum extract injected up to 0.4 pairs of corpora cardiaca / 100 ul haemolymph, which is approximatly equivalent to 1 pair of corpora cardiaca / insect. Therefore at the dose tested in the present study, 175
there is sufficient material to detect activity, but the dose may
exceed that required for the production of the maximum response.
7. Activities of factors in de novo corpus cardiacum extracts. Each de novo corpora cardiaca was found to produce a blood lipid
elevation in locusts approximately equivalent to that produced by 0.002 pairs of glandular lobes or 0.04 pairs of storage lobes.
At the dosage tested (0.5 gland pairs of de novo corpora cardiaca)
the hyperglycaemic activity is almost at the limits of detectability.
An elevation of 8 ug/ul (80%) in locust haemolymph lipids and an elevation of 3 ug/ul (15%) in cockroach haemolymph carbohydrates was produced on injection of 0.5 gland pair equivalents of de novo corpora cardiaca. A response of 48 ±2% increase in locust haemolymph lipids
to 0.5 gland pairs of de novo corpus cardiacum extract was recorded by Goldsworthy et al (1973). Highnam and Goldsworthy (1972) recorded a response of 50.6 ± 5.3% increase in cockroach haemolymph carbohydrates to 1.0 gland pairs of de novo corpus cardiacum extract. Thus the data
_ produced in the present study indicate a slightly higher level of adipokinetic activity and a slightly lower level of hyperglycaemic
activity than in these two reports. However, the results are of a
similar order of magnitude.
Highnam and Goldsworthy (1972) reported that saline extracts
of de novo corpora cardiaca have a similar level of activity to extracts of the storage lobes of normal corpora cardiaca in the elevation of cockroach haemolymph carbohydrates. In the present
study, the hyperglycaemic activity of 1 pair of de novo corpora
cardiaca was found to be equivalent to that of only 0.04 pairs of 176
storage lobes.
De novo corpora cardiaca are reported to be free from glandular
cell material and contain only neurosecretory products (Goldsworthy and Highnam, 1972). It therefore appears likely that the adipokinetic and hyperglycaemic activities of de novo corpora cardiaca reported here and by Goldsworthy et al (1973) and Highnam and Goldsworthy (1972)
originate from a cerebral neurosecretory product.
8. Resolution of storage lobe factors and activities of the resolved
factors. Methanol extracts of storage lobes, on separation on a TLC plate, produce two areas of peptide material as indicated by starch — iodide
staining, one at the origin and the other at Rf 0.67. The latter spot contained adipokinetic and hyperglycaemic activities at similar relative levels to storage lobe extracts. Neither adipokinetic nor hyperglycaemic activity were detected in any other eluted areas.
_•The diuretic activity was found to be localised at the origin, indicating that the diuretic factor does not migrate in the TLC system
employed in this study.
Storage lobe extracts were subjected to the purification methods
established for the glandular lobe AKH. As with AKH, the storage lobe
adipokinetic and hyperglycaemic activities appear to be equally soluble
in water and methanol. On elution of the storage lobe extract from the glass bead column, only one area of u/v absorbing material was
eluted, the large peak at Vo. There was also a slight rise in absorbance
near to Vt. A significant amount of adipokinetic and hyperglycaemic 177
pooled fractions and pooled activities were found in both the Vo fractions 20 - 40. The Vo peak was found to contain approximately one third of the total adipokinetic and hyperglycaemic activities of
the initial extract, the rest being located in fractions 20 - 40.
peak produced on elution of an aqueous Each fraction of the Vo extract of storage lobes from the glass bead column was assayed for adipokinetic and hyperglycaemic activities, and both were found
predominantly in the tailing of the peak. Both activities in each fraction were present at an extremely low level.
Pooled fractions 20 - 40, following TLC separation, produced
a u/v absorbing area Rf 0.6 - 0.7 which contained both adipokinetic and hyperglycaemic activities in relative amounts similar to those
of storage lobe extracts. Following TLC of the pooled Vo fractions,
both activities were localised in an area Rf 0.6 - 0.7. In this case however, no u/v absorbing area was visible, probably as a result
of the small quantity of material present.
The location of adipokinetic and hyperglycaemic activity in the large molecular weight fraction on elution of storage lobe extracts
from a glass bead column may indicate non-specific binding of the factor to a species of protein present in the storage lobes but not in the glandular lobes. (There is some evidence that AKH will bind to
charged molecules or ions from the experiments involving polyacrylamide
gel electrophoresis). It could alternatively be due to a specific
association of the active factor and larger molecular weight protein,
for example a neurophysin or pro-hormone. Holwerda et al (1977), 178
starting with whole corpora cardiaca of L. migratoria, found both adipokinetic and hyperglycaemic activities in the large molecular weight fraction following column chromatography on Sephadex. They suggested this was due to unspecific binding of the factor to proteins or residual binding to natural carrier proteins.
Elution of storage lobe extracts from a Biogel P6 column produced sharper peaks compared with those obtained from the glass
bead column. In spite of the presence of fewer charged groups, the adipokinetic and hyperglycaemic activities were retained on the column
until after the column volume had been eluted. The adipokinetic and hyperglycaemic activities were found to be associated with the tailing of the large peak, fractions 25 — 27. This peak was found to be devoid of activity, and may be cholesterol as it produces a u/v absorbing area of Rf 0.79 on TLC separation.
Hyperglycaemic activity was not detected in the fractions of the
- Vo produced on the Biogel P6 column, but this may be because fractions were assayed for activity at a level lower than was used for
fractions from the glass bead column.
Each fraction eluted from the Biogel P6 column following
application of storage lobe methanol extracts was assayed for diuretic activity using the in vitro assay system, and the fractions possessing t diuretic activity were located between Voand V . This may be indicative of a molecular species larger than AKH or the storage lobe hyperglycaemic / adipokinetic factor, or it may merely reflect the .
influence of a chromatographic effect. 179
The fractions in which the diuretic activity was localised were found to be devoid of both hyperglycaemic and adipokinetic activities.
The diuretic factor was not subjected to further purification steps, and as its nature was not investigated, no conclusions can be drawn concerning its peptide nature although this is extremely likely. The diuretic activity did not coincide with a peak absorbing at 206nm , on the column profile which may have been due to the small quantity of material present or to a low absorbance at this wavelength. It is known that substances present in the corpora cardiaca such as 5 HT may have an effect on the in vitro Malpighian tubule assay system used in this study. It is unlikely however that they would be present in sufficient quantities to elicit the observed effects. The results of this study show that the diuretic factor present in storage lobe extracts of L. migratoria is distinct from the adipokinetic and hyperglycaemic factors, and may be a larger molecule.
The purified hyperglycaemic / adipokinetic storage lobe factor was assayed for its activity on heart rate and was found to have an effect similar to storage lobe extracts in increasing both the rate and amplitude. The purified diuretic factor however was found to have no effect on either the rate or the amplitude of beat in - isolated heart preparations.
9. Physiological significance of the storage lobe factors. Although the major part of the corpus cardiacum hyperglycaemic activity is in the glandular lobes, a distinct activity is thought to be present in the storage lobes (Mordue and Goldsworthy, 1969;
Goldsworthy, 1976). The presence of two factors in the corpora cardiaca 180 of locusts, one more potent than the other, may be of physiological importance or may merely represent a close similarity of structure between the factors (Mordue and Goldsworthy, 1969).
It has been concluded that the peptide produced by the intrinsic glandular cells of the locust corpora cardiaca is the more physiologically important of the two hyperglycaemic peptides (Mordue and Goldsworthy,
1969. Goldsworthy, 1970; Goldsworthy and Mordue, 1974). It now appears that the hyperglycaemic activity of glandular lobe extracts can be largely if not wholly attributed to a pharmacological activity of AKH. As has been argued previously, the hormonal control of
carbohydrate metabolism in locusts by corpus cardiacum factors, at least during flight, appears doubtful. It is possible therefore that the hyperglycaemic activity of the storage lobe factor may result merely from this factor fulfilling the structural requirements needed to elicit haemolymph carbohydrate elevation in cockroaches, in a similar way to the hyperglycaemic activity of AKH. It is unlikely
that the storage lobe hyperglycaemic factor is identical to AKE as,
although its resolution characteristics appear similar to those of
AKH, it has greater hyperglycaemic activity relative to its
adipokinetic activity than does AKH.
The slight adipokinetic activity of storage lobe extracts may be
due to contamination with glandular lobe material (Goldsworthy and Mordue, 1974). It is possible that the adipokinetic and hyperglycaemic activity of the storage lobe factor described in the present study is
a resultant of the activity of unresolved glandular lobe AKH
contaminant and the storage lobe factor providing most of the 181
observed hyperglycaemic activity. The purification procedure used in this study was.a fairly simple one, and it is possible that AKH and the storage lobe factor may co-chromatograph if they are sufficiently structurally similar. An alternative explanation is that, assuming minimal contamination with AKH, the storage lobe factor is active in both adipokinetic and hyperglycaemic assay systems. Support is lent to this suggestion by the data obtained from de novo corpus cardiacum extracts, which indicate the possible existence of a cerebral neurosecretory product possessing lower levels of adipokinetic activity relative to hyperglycaemic activity than does AKH.
The structural nature of the storage lobe factor can only be speculated upon at present. It is likely to be a small peptide.
Assuming both adipokinetic and hyperglycaemic activities of storage lobe extracts can be attributed to this peptide, it is likely to resemble RPCH rather than AKH as, on a molar basis, RPCH is more potent in elevating cockroach haemolymph carbohydrates than AKH (Nordue and Stone, 1976, 1977). RPCH does have adipokinetic activity in locusts, but is much less potent than AKH. This problem will be resolved only on the complete isolation and structure elucidation of
the storage lobe factor. An alternative purification procedure may be required if contamination with glandular lobe AKH is found to occur.
The storage lobe factor possessing adipokinetic and hyperglycaemic activities is not identical with the storage lobe diuretic factor, as
these activities are resolved on column chromatographic separation,
and also the factors produced different effects when assayed for 182
activity on semi-isolated heart preparations. The physiological role of the storage lobe factor in the locust awaits elucidation.
Cultivation of corpora cardiaca from L. migratoria in vitro.
1. Morphology of the cultured glands. The results of the present study indicate that neurosecretion present in the storage lobes at the beginning of the culture is maintained in the lobes for the duration of the culture. Investigations at the electron microscope level indicate a degeneration of the electron
dense vesicles in the storage region. This may be due to normal breakdown of the vesicles, and their not being replaced from the cell bodies due to severance of the axon tracts. Alternatively it may be due to a general degeneration of the axon terminals in the storage
region. The breakdown of the granules may explain why, at the light microscope level, the PF positive material appears as large rounded
off areas, and not as discrete granules.
No quantitative assessment concerning the amount of PF positive
material in the storage lobes was made during the incubation period.
It is unlikely that this would change a great deal over the comparatively short incubation period used in this study. Gianfelici (1968) noted accumulation of stainable material in the corpora cardiaca during
in vitro culture and interpreted this as a lack of release of the material. It could also result from a lack of normal inhibitory
feedback mechanisms which may be present in the intact animal.
Seshan and Levi -Montalcini (1971) have cultured the corpora 183
cardiaca and cerebral neurosecretory cells of P. americana in vitro for periods of 4 - 8 weeks and 1 — 3 months respectively. Holman and Marks (1973) however found degenerative changes in the neurosecretory cells of cultured L.maderae brains after 4 days in vitro with similar results for brains of Manduca sexta (Borg and Marks, 1973). In the present study, the corpora cardiaca were maintained in organ culture for up to 7 days during which time many of the glandular lobe cells remained viable as judged by morphological criteria. Numerous profiles packed with large electron dense vesicles (500 — 600nm) were seen in regions removed from the cell nucleus, and also a number of 'cytoplasmic inclusion bodies' filled with granules. These have been noted in the corpora cardiaca L.maderae especially in older animals (Scharrer, 1963). They may therefore be indicative of degenerative changes in the glandular lobe cells.
2. Activity of the cultured glands. There is no evidence to link secretory activity with morphological changes in cells (Herman, 1967; Scharrer, 1948, 1964a,b), and it is not possible to relate stainable neurosecretory material in cultured organs with specific hormones. The evidence that cultured brains continue to supply secretory products rests primarily on the assumption that the neurosecretory granules represent hormone material (Gianfelici,
1968a,b). The measurement of the secretory activity of cultured glands in terms of the production of active factors may aleviate some of these problems. In the present study, the production of adipokinetic activity was measured to assess the activity of the cultured glands. After 3 days in culture, adipokinetic activity was present in the media and glands, a higher level of activity being located in the glands. After 184
7 days in culture, there was found to be approximately the same level of activity in the glands and the media. This may indicate synthesis
and release of AKH in vitro, or alternatively it may be due to a
leeching out of the activity into the medium. This study requires extension to quantify the synthetic activity of the glandular lobe cells in vitro in terms of AKH production, and may provide a useful tool for the investigation of neurophysin or pro-hormone production.
Co-culture of the brain and corpora cardiaca may provide additional information on tropic effects within this system, and could also be used to investigate the role of the cerebral lateral neurosecretory
cell group in the release of AKH.
CORPUS CARDIAC UM FACTORS OP P. AMERICANA.
1. Activities of factors in corpus cardiacum extracts. The hyperglycaemic activity of corpus cardiac= extracts from
P. americana described in this study is essentially in agreement s with the work of Steele (1961, 1963). Steele (1963) reported that 0.002 pair of corpora cardiaca produced a 30% increase in blood
trehalose. In the present study, the lowest dose assessed for activity was 0.01 gland pairs, and this was found to produce an elevation of 6 ug/ul in haemolymph total carbohydrates which is equivalent to approximately 30% increase. However, the data expressed in percentages is not strictly comparable due to variation in the
normal values of haemolymph carbohydrates. Steele (1963) found
normal haemolymph trehalose levels in P. americana ranged from 9.1
to 13.9 ug/ul, whereas in the present study they were found to be
between 18 and 23 ugiul. Thus minor variations in the results of 185
Steele (1963) and the present study are likely to result from the differences in normal haemolymph carbohydrate levels which may in turn result from differences in diet and rearing conditions. Also in the study by Steele (1963), the cockroaches were removed from the colony 6 - 12 hr. before the beginning of each experiment and may not have fed during this period. The maximum response obtained by Steele
(1963) was an increase in haemolymph trehalose of 29.9 ug/ul. In the present study, a maximum elevation in haemolymph total carbohydrates of 18 — 25 ug/ul was obtained.
The adipokinetic activity in locusts of cockroach corpus cardiacum
extracts has not been so extensively investigated as the hyperglycaemic activity in cockroaches. Injections of 0.5 gland pairs of cockroach corpus cardiacum saline extracts into L. migratoria have been reported to increase total haemolymph lipids from 8.1 to 27.2 ug/ul in 1 hr.
(Goldsworthy et al, 1972). Injection of 0.1 gland pairs of aqueous corpus cardiacum extracts from P. americana into L. migratoria are -reported to increase haemolymph diglyceride concentration from 0.587
ug/ul to 2.858 ug/u1 (Downer, 1972). In the present study, maximum elevation of L. migratoria haemolymph lipids was obtained with 0.05 —
0.1 gland pairs of corpus cardiacum extracts from P. americana.
It has been estimated that the corpora cardiaca from one P. americana contain 500 — 600 units adipokinetic activity and 400 — 500 units of hyperglycaemic activity. Thus the adipokinetic activity of cockroach corpus cardiacum extracts is only slightly greater than its hyperglycaemic activity. These results indicate that the cockroach 186 corpora cardiaca contain hyperglycaemic activity at a level 6 - 7 times greater than that of the locust glandular lobe AKH but adipokinetic activity at a level some 4 - 5 times lower than AKH. These results are in agreement with those of Goldsworthy et al (1972), in which cockroach corpus cardiacum extracts were found to be more potent than locust corpus cardiacum extracts in elevating cockroach haemolymph carbohydrate levels but less potent in elevating locust haemolymph lipid levels.
These relative activities, different from those of AKH, indicate
that a factor similar to but not the same as AKH is present in the corpora cardiaca of P. americana. This view is supported by the fact
that although the hyperglycaemic activity of cockroach corpora cardiaca appears comparable with that of AKH at lower doses, some 10 — 20 times
more AKH than cockroach corpus cardiacum extract (on a gland pair
equivalent basis) is required to produce a maximum hyperglycaemic response in the cockroach. The adipokinetic activity of cockroach corpus cardiacum extracts is comparable with that of the locust storage
lobes at both the higher and lower doses.
Methanol extracts of corpora cardiaca from P. americana were found
to increase the rate of fluid production by locust Malpighian tubules
in vitro. This is at variance with the work of Wall and Ralph.(1962, 1964, 1965) which investigated factors influencing the rate of
excretion of indigo carmine by Malpighian tubules in vitro and rectal water reabsorption in vitro in P. americana. Corpus cardiacum, corpus
allatum and suboesophageal ganglion extracts were found to decrease
the rate of dye excretion and increase rectal water reabsorption. 187
No activity that could be clearly interpreted as diuretic was found. Penzlin (1971) however has suggested that diuretic factors are present within the corpora cardiaca of P. americana which are produced by the
neurosecretory centres in the pars intercerebralis.
Mills (1967) has suggested that a diuretic factor, active on
isolated preparations of Malpighian tubules, is released from the
terminal abdominal ganglion in P. americana. Using an in vitro rectal preparation for assay, this diuretic factor was estimated to to have a molecular weight greater than 30,000 (Goldbard et al, 1970)
and the ganglion was also reported to contain an antidiuretic factor
of molecular weight about 8,000.
In the present study, the existence of a factor in corpus cardiacum extracts of P. americana which increases the rate of fluid production by locust Malpighian tubules has been noted. The present confused situation in the literature cautions against the assumption
'that this a primary physiological function in the intact animal.
Methanol extracts of corpora cardiaca from P. americana have been found to increase the heart rate and amplitude of a semi—isolated
heart preparation in the present study. This is in agreement with
the work of Brown (1965) which reported the presence of factors in extracts of corpora cardiaca from P. americana which increased both the frequency and amplitude of contraction in a heart preparation.
Davey (1963) considers the heart accelerating factor in the corpora
eardiaca of P. americana to be confined to the intrinsic secretory
portion. The heart accelerating activity of cockroach corpus cardiacum 188 extracts is associated with proteinaceous material, as the activity is abolished with proteolytic enzymes (Davey, 1961;
Brown, 1965; Natalizi and Prontali, 1966). The heart accelerating factor(s) is not considered to be identical with the hyperglycaemic factor (Natalizi and Frontali, 1966; Traina et al, 1976).
2. Purification and characterisation of the corpus cardiacum factor(s).
Methanol extracts of corpora cardiaca from P. americana were subjected to the purification methods extablished for the locust glandular lobe AKH.
On elution of the extract from the glass bead column, two areas of u/v absorbing material were eluted, one at Voand the other before Vt (fractions 16 — 25). The Vco peak was found to be devoid of adipokinetic and hyperglycaemic activities, while pooled fractions 16 — 25 contained these activities in similar relative proportions to corpus cardiacum extracts.
Eluates of the u/v absorbing area, Rf 0.56, which was produced following TLC separation of pooled fractions 16 — 25 contained adipokinetic and hyperglycaemic activities. In addition, this area was found to contain diuretic activity when assessed in the in vitro Malpighian tubule assay system, and to increase the rate and amplitude of contraction in a semi—isolated heart preparation. Eluates of the remaining TLC plate were found to be devoid of adipokinetic, hyperglycaemic and diuretic activities.
Both the hyperglycaemic and adipokinetic activities of the 189
purified factor(s) have been shown to be associated with peptide
material bir the abolition of activity following digestion with proteolytic enzymes. This has not been conclusively demonstrated. for the diuretic activity, largely because of a reaction mixture
component which increased fluid secretion by the Malpighian tubules in vitro even in the enzyme blank assay. Also the activity of the undigested control preparations appeared to be lower than unboiled preparations, which may indicate that the diuretic activity is not
stable to incubation or boiling
Results to the fluorescence spectrum of the purified factor(s)
were inconclusive possibly due to the presence of fluorescent indicator eluted from the TLC plate along with the factor(s). The u/v absorption spectrum of the factor(s) showed a maximum absorbance
at 280nm, which indicates that tryptophan may be present.
The results obtained from amino acid analysis are open to a
'number of interpretations, and it is possible that the preparations may have contained a number of impurities. Further experimentation is required with larger quantities of material to establish if in
fact the factor is a single molecular species of 17 amino acid residues, or a mixture of smaller peptides. Traina et al (1976),
using column chromatographic techniques, have identified two heart
accelerating peptides from the corpora cardiaca of P. americana, one containing 17 amino acid residues and the other containing 12. As in the present study, these workers suggested that the peptide
preparations may be heterogeneous. These factors were reported to
be devoid of hyperglycaemic activity (Traina et al, 1976) and thus 190
differ from the factor(s) reported in the present study. The factors of Traina et al (1976) were not investigated for diuretic or adipokinetic activities.
As a 17 amino acid residue peptide may be considered rather large
to migrate in the TLC system utilised in this study, it is likely that some of the amino acids detected are due to contamination, or due to the presence of more than one peptide with similar elution
characteristics. Whichever interpretation is correct, the cockroach factor(s) does not bear much resemblence to locust glandular lobe AKH in terms of separation characteristics. The cockroach factor(s)
is eluted from the glass bead column in a smaller volume than AKH, and its Rf on TLC separation is lower. This information could. be interpreted as th. cockroach factor(s) being a larger molecule than
AKH although factors other than size influence the elution characteristics of a particular molecular species. In addition to these differences in elution characteristics, the relative adipokinetic
and hyperglycaemic activities of AKE and the cockroach factor(s) are different. AKH is extremely potent in elevating locust haemolymph lipids but has only weak hyperglycaemic activity in cockroaches. The cockroach factor(s) appears to be approximately equally potent in
the elevation of locust haemolymph lipids as in the elevation of
cockroach haemolymph carbohydrates.
3. Activities of the purified, corpus cardiacum factors(s). The activities of the purified cockroach factors(s) were found
to be approximately half of those of corpus cardiacum extracts,
probably as a result of purification losses. It is possible that 191 there is more than one species of peptide present in the purified preparation. Alternatively, the hyperglycaemic, adipokinetic, diuretic and heart accelerating activities recorded from the preparation may be attributable to a single molecular species, and these activities result from a broad pharmacological spectrum of the factor.
The cockroach factor is some 6 - 7 times more potent than AKH in the elevation of cockroach haemolymph carbohydrates and some 4 - 5 times less potent than AKH in the elevation of locust haemolumph lipids. The cockroach factor possesses only slightly more adipokinetic activity than hyperglycaemic activity, while AKH is some 20 times more potent in eliciting hyperlipaemia than hyperglycaemia. Locust AKH is thought to be a special adaptation to the 'two fuel' metabolism of locust flight (Goldsworthy, 1976), but also has a low level of activity in
the cockroach hyperglycaemic assay system. The almost equal activities of the cockroach factor in the adipokinetic and hyperglycaemic assay systems indicates a 'lower specificity' of this factor compared with
AKH, and thus the adipokinetic and hyperglycaemic activities of the cockroach factor may not reflect its primary function in the cockroach.
The heart accelerating properties of the purified cockroach factor are similar to those of the purified storage lobe factor, namely to increase both the rate and amplitude of contraction in a semi—isolated
heart preparation. The cockroach factor is not identical with the
storage lobe factor however because of different separation characteristics and different activity spectra. The storage lobe
factor does not possess diuretic activity and is also 8 — 10 times
more potent in elevating locust haemolymph lipids than in elevating 192
cockroach haemolymph carbohydrates whereas the cockroach factor possesses potent diuretic activity and has approximately equal adipokinetic and hyperglycaemic activities.
4. Physiological significance of the purified corpus cardiacum factor(s). The physiological role for the hyperglycaemic factor of the cockroach corpora cardiaca which has been put forward most often is one of mobilisation of energy reserves during periods of intense activity (Steele, 1961, 1963). The involvement of the hyperglycaemic factor in carbohydrate metabolism during flight in the cockroach however has not been extensively investigated.
Many species of cockroach have weak powers of flight and rarely fly for longer than several minutes. Instead they have developed very efficient running mechanisms. The oxygen consumption and succinic dehydrogenase activity of leg and wing muscle in P. americana hardly differ at all (Tindall and Guthrie, 1968), which indicates a low flight specialisation. Fatty acids cannot be metabolised directly by the flight muscle in cockroaches, and carbohydrates are of paramount importance in the metabolism of P. americana during flight (Polacek and Kubista, 1960). Glycogen is utilised primarily from the pterothoracal muscles, and their glycogen content decreases 36% during flight. Haemolymph trehalose is depleted by 60% after a 10 — 15 min. flight.
Injection of the purified factor into L.maderae 1 hr. before flight causes an increase in haemolymph carbohydrates which may be 193 utilised during flight with the result that haemolymph carbohydrates are depleted more slowly in the injected flown animals than in the controls. In addition, the injected insects flew for a longer period of time, up to 20 min, although the quality of flight was not assessed.
There is little indication of carbohydrate mobilisation during normal flight however, and the fact that carbohydrates are significantly depleted from the haemolymph during flight rather than being elevated as occurs with haemolymph lipids during flight in locusts may indicate that a different mechanism of mobilisation of fuel reserves is involved during flight in the cockroach.
The elevation of haemolymph carbohydrates in saline injected control cockroaches has been noted by other workers (Hanaoka and
Takahashi, 1976). It is not known if the corpus cardiacum factor is involved in the production of this effect, but the small quantity of carbohydrate mobilised during a considerably long period of time
does not suggest a mechanism of physiological importance.
Cockroaches do not show an adipokinetic response to corpus cardiacum extracts. Similarly, adult locusts do not show a
hyperglycaemic response to corpus cardiacum extracts. It has been suggested (Goldsworthy et al, 1973) that the predominance of the
adipokinetic response in locusts and the hyperglycaemic response
in cockroaches may be functionally related to differences in flight metabolism, as locusts utilise lipid during flight (Weis —Fogh, 1952)
while cockroaches oxidise carbohydrates (Sacktor, 1965). The present
study has demonstrated that the hyperglycaemic activity of locust
corpus cardiacum extracts in cockroaches is probably a pharmacological 194
effect of AKH. By the same token, it may be that the adipokinetic activity of cockroach corpus cardiacum extracts in locusts is a pharmacological effect of a factor whose primary physiological function in the cockroach is as yet unknown. It appears unlikely to be involved in the control of carbohydrate metabolism during intense activity (Goldsworthy and Mordue, 1974) and the potent activity of the cockroach factor in increasing the rate of fluid production by Malpighian tubules in vitro and also increasing the rate of amaranth clearance in locusts may indicate its major function in the cockroach. The present confused situation concerning the hormonal control of water balance in insects however indicates that
more investigation is required before this conclusion can become more
than speculative. 195
SUnIARY
1.The adipokinetic hormone (AKH), isolated from the glandular lobes of locusts, has been shown to cause elevation of haemolymph lipids on injection into L. migratoria and elevation of haemolymph carbohydrates on injection into P. americana. Larger doses (20 — 200 pmol) of AKH are required to induce hyperglycaemia in cockroaches than are required to produce hyperlipaemia in locusts (1 — 20 pmol).
2.This effect is interpreted as being a pharmacological activity of AKH because of the large doses required to elicit hyperglycaemia
and also because cockroach corpora cardiaca are not thought to contain a peptide identical with AKH.
3.It is unlikely that there is an additional factor in the glandular lobes of L. migratoria which elevates haemolymph carbohydrates in
cockroaches.
4.The adipokinetic and hyperglycaemic activities of locust storage lobe extracts may be attributable to contaminating glandular lobe AKH, but it is more likely that a second factor is present which is
structurally similar to AKH. Compared with AKH, this factor has
similar separation characteristics but greater hyperglycaemia activity relative to its adipokinetic activity. The function of
such a factor is unknown.
5.The diuretic activity of locust storage lobes has been resolved from the adipokinetic and hyperglycaemic activities. The nature of 196
the diuretic factor is unknown, but it is likely to be a peptide possibly of higher molecular weight than AKH. The diuretic factor increases the rate of fluid production by Malpighian tubules in vitro and increases amaranth clearance in vivo.
6.Using the same purification procedure as for AKH, a factor has been purified from the corpora cardiaca of P. americana which possesses
hyperglycaemic, adipokinetic, diuretic and heart accelerating activities.
Results to the amino acid analysis of this factor are open to several interpretations, possibly due to a certain heterogeneity of the preparation.
7.The factor purified from the corpora cardiaca of P. americana is not thought to be involved in the mobilisation of fat body carbohydrate
reserves during flight, and its potent diuretic activity may indicate its physiological function.
8.The in vitro culture of the corpora cardiaca from L. migratoria
was investigated, and may prove a uesful tool for study into the
synthetic activity of the glandular lobes and the release of AKH
from this region. 197
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