SOME PHYSIOLOGICAL ASPECTS OF ADULT REPRODUCTIVE DIAPAUSE
IN THE CHRYSOMELID BEETLE,GALERUCA TANACETI (LINN.)
by
Yow Cheong, Siew, M.Sc. (1.Z.)
Thesis submitted for the Degree of Doctor of
Philosophy in the Faculty of Science, University of London
Imperial College of Science and Technology, Field Station, Silwood Park, Sunninghill, Ascot, Berkshire. May, 1963. ABSTRACT
Galeruca tanaceti Linnaeus is a short-day insect. Photoperiod
and temperature are the primary causal factors which induce, sustain and
terminate diapause. The critical photoperiod is about 13 hours at 20°C,
under controlled conditions, whereas in the field it is about 14.5 hours,
where the daily mean air temperature fluctuates around 15°C. Diapause can be sustained, terminated or reinstated depending on the photoperiod to which the beetle is subjected at any period of its adult life. The phases of diapause, ovarial maturation and oviposition are controlled by different levels of hormonal activity. Diapause is sustained by a low level of activity of the neurosecretory cells of the brain, which in turn influence a low level of activity of the neurosecretory system. Five types of neurosecretory cells, (A, A1, B, C and D) occur in the brain and the suboesophageal ganglion. These cells undergo short cycles of secretory activity throughout adult life. These cycles become shorter but more intense towards and at the phase of oviposition. The results stem from two experimental sources: (1) measurements of nuclear volumes of these cells and (2) autoradiographic investigations using 35S-DL-cystine. The corpus cardiacum/allatum complex functions in relation to the level of activity in the neurosecretory cells. Three criteria were used to determine the corpus cardiacum/allatum activity: (1) increase in volume of the glands, (2) decrease in nuclear-cytoplasmic ratios and (3) increase in nuclear volumes. To elucidate the role of the components of the neurosecretory system, cauterization and implantation experiments were carried out. These experi- ments indicate a close inter-relationship between the neuro-endocrine centres and the possibility of complex feed-back mechanisms. A comparison is made of the respiratory rates, fat and water contents in the pre-diapause, diapause and ovarial maturation phases.
ii.
TABLE OF CONTENTS Page I. Introduction
a) General Ooo 04. 0.0 ••• •o• ••• ••• ••• 1
b) Review of the literature ••• ••• ••• ••• ••• 3
II. The Life History of G. tanaceti ••• ••• ••• ••• ••• 17
III.Rearing techniques ••• ••• ••• ••• ••• ••• ••• 19
IV. Studies on the morphology of the reproductive systems with accompanying quantitative and qualitative changes at different stages of the adult life of G. tanaceti
Introduction . • • • • • ••• ..• ••• ••• ••• ••• 20
Method • ee ...... ••• ... do• • A • • ...... 20
a) Morphology of the reproductive organs ... 000 004 21 b) Quantitative and qualitative changes of the reproductive
organs at different stages of adult life history 604 23
Discussion ••• ••• SO• ••• 26
V. The neuro-endocrine complex of G. tanaceti
Introduction 004 4.0 00. 4.0 6.0 006 60.. ego 29
Method ... 000 0.0 0.0 006 600 000 000 000 30 Morphology and anatomy of the neuro-endocrine complex 31
The histology of the neurosecretory system 4104. 00. se. 35
Discussion • • . • • • • • • • • • • • • • • • •• ••• 43
VI. Studies on feeding, growth, respiration, water and fat content of G. tanaceti at different stages of its adult life history Introduction • . • • . • • • • • • • • • • • • • • • • 46
1. Feeding and growth 000 0.. 6.0 ••• 47
2. Respiration • .• • • • • • • • • • ••• 49
3. Water and fat contents • • • • • • • • • ••• 54
Discussion • • • • • • • • • • • • • • • • 0 • 59
VII.Experiments on the effects of photoperiodism and temperature on the induction and termination of reproductive diapause and on growth of G. tanaceti
Introduction 000 600 00. .00 4.9 6.6 *elk 63
Methods ••• ••• ••• ••• ••• ••• ••• ••• ••• 64
Results ••• ••• ••• ••• ••• • •• ••• ••• ••• 65
Page Rate of maturation and oviposition of G. tanaceti kept
at different pht-toperiods and temperatures ... 000 ... 68 Fecundity of G. tanaceti kept at different photoperiods and
4641 ..0 72 temperatures ...... 00 0.• 0.0 000 Ovarial maturation of G. tanaceti kept at different photo-
periods and temperatures ... 000 000 000 000 040 72 The sensitive stage of G. tanaceti in response to photoperiod-
ism and temperature 0.0 000 000 40. 000 0.0 78 Growth of reproductive organs at the photoperiod of 12 hours at 2000. e...... deo ...... 4,.. 78 Reversible effects of photoperiodism and of temperature in
G.:tanaceti 4,01. 060 0.0 004 0.0 41.. 00. 000 81
Discussion O.. .00 Ote $8. 610. 060 0.0 06. 81
VIII. Studies on the cyclical changes of the neurosecretory system during adult life history of G. tanaceti
Introduction 'OS 00. ... 0.0 04.0 see el. ... 85
Methods 004 0.6 000 066 Ott 4..6 0100 85
R esults ... 0.4 .00 000 4,00 000 1.0. 400 60* 89 1. Changes in the N.S.C. of the pars intercerebralis 89 2. Changes in the N.S.C. of the lateral groups of the brain 95 3. Changes in the N.S.C. of the suboesophageal ganglion 98 4. Qualitative and quantitative changes in the corpora
cardiaca op .00 800 000 00. Ott 06. 0.4 103 5. Qualitative and quantitative changes in the corpora
allata 0•• ••• ••• ••• ••• •O• ••• ••• 107 Discussion • •• •• • • •• • • • •• • ••• •• • • 120
IX. The dynamics of neurosecretion Introduction • • •• .• • • •• .•• • •• ••• •• • 124
Methods > 0.6 000 SOO 000 Ott .00 0.0 .00 125
Results 000 O.. .0. 600 660 64,0 600 Ott 127 a) Demonstration of short range secretory cycles in the N.S.C. by the use of 3 S-DL-cystine in auto- radiographic study ...... eel e.. *of 127 b) Experimental demonstration of a possible "neuro-
endocrine momentum" 00. $00 0010 Ott 4100 132
Discussion ... 000 000 .0. 000 00. .00 000 135 iv. Page X. Experimental investigations into the role of the neurosecretory system in controlling reproductive diapause, ovarial maturation and oviposition in G. tanaceti
Introduction .• • • •• •• • • a • • • • • • • II • • • • • • 141 Methods •• • ... •• • •• • ... •• • • •• •• • • • . 141 Results ... • • • ... •• • •• • •• • ...... 144 a) Cauterization experiments ... •• • •• • •• • •• • 144 b) Implantation experiments ... •• • •• • ... •• • 150 Discussion . •• • •• • •• • ••• •• • ••• ... •• • 152
XI. General discussion • • • • • ... •• • •• • ... •• • • • • 156
Summary .. • • • III • • • • • • • • • • a • • • • • • • • 0 • • • • 168
Acknowledgements ... •• • •• • ••• •• • ... •• • ... • • • 171
Bibliography .. • •• • • • • • • •• • •• • ••. •• • ...... 172 1. I. INTRODUCTION a) General
The phenomenon of diapause was first demonstrated by Duclaux (1869).
He observed that the egg batches of Bombyx mori invariably failed to hatch at room temperatures whereas, similar batches which had been chilled previously for 40 days in an ice box, hatched successfully when returned to the higher temperature.
This phenomenon of diapause in insects has recently stimulated much research as is evident from the reviews by Bonnemaison (1945), Andrewartha
(1952), Lees (1955, 1956), Church (1955), Hinton (1957), Willer (1957a),
Danilevskii (1961), Harvey (1962), de Wilde (1962) and van der Kloot (1960,
1962).
The problem of arrested growth has become appreciated as a highly
complex mechanism for the preservation of the species in regions where
seasonal climatic conditions are unfavourable for continuous growth and for reproduction. It has long been recognised that the environment ultimately controls diapause. During the last 25 years, however, with the classical
work of Kogure (1933) on Bombyx mori, investigations into the effects of photoperiodism and temperature have shown that these are the primary causal
factors, inducing, sustaining and terminating diapause.
From the physiological standpoint, these external factors form only
part of the problem. The other aspect of the problem revolves mainly around
the physiological and bio-chemical control of growth.
It is now known from the classical research of Wigglesworth, Fukuda
and Williams that the cessation of growth and the accompanying metabolic
adjustments are under hormonal control. The neurosecretory system which
has been under active investigations during the last 15 years, contains 2. the main endocrine centres. The neurosecretory system is thought to provide the link, coordinating the internal physiology of the insect with the external environment. Indeed, it is this link, as Lees (1955) suggests, that permits the mechanism underlying diapause to function as a timing device, synchronizing the periods of dormancy and active growth with the seasonal changes in the environment.
There has not been any attempt so far by workers of diapause to investigate the physiology of imaginal diapause as outlined in the above scheme. de Wilde and his associates (1958, 1959, 1960) partially approached the problem in their study on the role of the corpus allatum as an endocrine
centre, controlling imaginal diapause.
The principal theme in this study is to consider the role of the neurosecretory system in the control of induction and termination of diapause,
in response to the effects of photoperiodism and temperature.
The Chrysomelid beetle, Galeruca tanaceti L., passes through well-
defined physiological phases in its adult life. Because of this, it offers
possibilities of an integrated investigation into the external factors
controlling diapause and the internal physiology of the insect.
I have divided the adult life history into four physiological phases,
namely: the pre-diapause, diapause, ovarial maturation and oviposition
phases, because of certain distinctive physiological conditions.observed in
the beetle in the course of its adult life.
This work has been divided into seven main sections, namely:-
1. the study on the growth of the reproductive organs through the natural
adult life history;
2. the effects of photoperiodism and temperature on the incidence of
diapause, ovarial maturation and oviposition; 3. 3. the metabolic adjustments during diapause and ovarial maturation phases;
4. morphological, histological and cytological studies of the neurosecretory
system;
5. the occurrence of cytological and morphometrical changes in the neuro-
secretory system in the different physiological phases;
6. the dynamics of neurosecretion;
7. the functional significance of the neurosecretory system in the overall
control of diapause, ovarial maturation and oviposition; and finally
8. a synthesis of the experimental observations in the discussion.
b) Review of, the literature
A comprehensive survey of the literature is beyond the scope of this
thesis, because of the vast amount of data available on the various aspects
of the phenomenon of diapause. A few of the more important recent reviews
by eminent workers hare been mentioned in the general introduction. There-
fore,I will attempt to confine myself to reviewing only those aspects which
appear relevant to the present investigation.
The term diapause was first used by Henneguy (1904, cited by Lees
1955, Andrewartha, 1952) to denote the condition of arrested growth in
either the developing or in the adult insect. Following Henneguy, many
authors have used the term indiscriminately sometimes, including in it
torpor caused by cold or draught. This has created unnecessary confusion.
Andrewartha (1952) in emphasizing the physiological implications of diapausi,
introduced the term "physiogenesis" as analogous with the term "morphogenesis"
in its morphological aspect when considering diapause. He considered
diapause "as a stage in physiogenesis which must be completed as a 4.
prerequisite for the resumption of morphogenesis". Way (1962) in stressing
the temperature dependence of physiogenesis said," characteristically the
diapausing insect can resist cold, has a low metabolic rate and, unlike
the quiescent insect, is not immediately reactivated by favourable climate,
but remains in diapause until a temperature-dependent process of physiological
development has been completed". This definition is not wholly acceptable
in some cases of larval and imaginal diapause as it neglects a probable
photoperiod-dependent process in the physiogenesis of diapause. The larvae rc of Dendrolimus pint will enter diapause within 30 days at 19-20°C. when
the photoperiod is reduced to 9 hours per day. Diapause, however, can be
terminated at any time if the larvae are exposed to continuous illumination
after 14 days. (Gayspitz 1949, 1953). It would appear that larval develop-
ment in D. pini is directly controlled by photoperiod. A photoperiod-
dependent process seems operative in physiogenesis in imaginal diapause and
is found in the present study on G. tanaceti. G. tanaceti remains in
diapause at a photoperiod of 16 hours at 20°C. but diapause can be terminated
at the same temperature when the photoperiod is reduced to 14 hours or
below within 21 days. Diapause can be reinstated within 30 days when
ovipositing females are subjected to a photoperiod of 16 hours.
In different species the occurrence of diapause has been found to
be highly variable and may take place at any stage of the life cycle. It's
induction and termination can be controlled by different external stimuli
in the different species. To this nay be added examples of diapause in
both parasites and hosts, in which there is, as a rule, perfect synchron-
ization of their emergence. Schoonhoven (1960, 1962) obtained evidence
that the activation of the parasite, Eucarcelia rutilla in the host, 5. Bupalus piniarius was brought about by the presence of the prothoracic glands hormone in the host, through parabiosis experiments. These facts illustrate the difficulty of defining diapause in the ecological sense.
A more satisfactory definition may be formulated with an endocrinological bias when there is more knowledge in this field.
Kogure (1933) was the first author to analyse the effects of photo- periodism and temperature on the incidence of diapause in the eggs of Bombyx mori. His discoveries did not stimulate further investigations until 1948 onwards, when important leads were made by Danilevskii (1948, 1949, 1951),
Gayspitz (1949, 1953), Dickson (1949), Way and Hopkins (1950) and Lees
(1953a, 1953b).
The production of diapausing eggs in the bivoltine and tetravoltine races of Bombyx mori has been found to be determined by the effects of long day lengths and high temperatures; whereas short day lengths and low temperatures induced the production of non-diapausing eggs.
Danilevskii (1948) found that in the nymphalid butterfly Araschfita levana the effects of short day at moderate temperature induced the larvae to develop into diapausing pupae but long days and high temperatures resulted in non-diapausing pupae. This work has been confirmed by Miler (1955) who found that the diapausing pupae in the spring yielded the levana form, whereas the non-diapausing pupae produced exclusively the prorsa form.
This is an example of seasonal polymorphism which is linked with diapause through the agency of photoperiodism.
In Grapholitha molesta, Dickson (1949) found that the effects of photoperiodism in controlling diapause was only operative within a narrow temperature range of about 20-25°C. Larvae bred in the absence of light did not enter diapause. As the photoperiod was increased to more than 3 6. hours per day so did the incidence of diapausing larvae. The percentage of diapausing larvae reached 100 at about 12 hour photoperiod. As the day length was increased beyond 13 hours, the incidence of diapause decreased again. With higher or lower temperatures there was no incidence of diapause, irrespective of the day lengths. G. molesta provides an interesting example of a temperature-dependent-photoperiodic reaction on diapause.
The induction of diapause in the pupae of Diataraxia oleracea was found to be influenced by temperature and photoperiod during the larval stage (Way and Hopkins, 1950). Low temperatures and short photoperiods tended to induce diapause, while high temperatures and long photoperiods tended to avert it. Similar results have been obtained in Metatetranychus ulmi (Lees,1953a). In these two species, temperature has been regarded as a "token" stimulus in the same sense as photoperiod (Lees,1955). In
D. oleracea the photoperiod was operAtive between the beginning of the
"moulting sleep", prior to ecdysis, to the last instar. The third and fifth day of the last instar were also sensitive. A single diapause- preventing-photoperiod during the "moulting sleep" was probably sufficient to prevent diapause. This introduces the concept that the sensitive stage is not necessarily the responsive stage. de. Wilde (1962) reviewed the literature and listed 11 species studied so far, which show that the responsive stage is quite remote in time to the sensitive stage. The extreme case is that of Bombyx mori in which the photoperiodic response is delayed by one generation.
There have been relatively few studies on marginal diapause. The species studied include: Dytiscus, marginalis (Joly, 1945), Nomadacris septemfasciata (Norris, 1959, 1962), Leptinotarsa decemlineata (de Wilde, 7. 1954), Coccinella septempunctata (Hodek and Cerkasov, 1960a, 1961),
Semiadalia 11-notata (Hodek and Cerkasov, 1960b), Sitona cylindricollis
(Davey, 1956), Stenocranus mihutus (Willer, 1957b) and Ceuthorrhynchus pleurostioma (Ankersmit, 1960). In all the examples cited above except in D. maroinalis, S. 11-notata and S. cylindricollis, for which no experimental data are available, the primary causal factor in controlling the incidence of diapause has been shown to be photoperiodism, whereas the
effect of temperature, modified the photoperiodic reaction of the insect.
It was the general opinion that the critical photoperiod at a particular temperature, limited the incidence of diapause. Among the species cited, the autumn race of C. pleurosticupa and S. minutus are short-day species.
Danilevskii (1961) in his comprehensive review of the photoperiodism
and seasonal development in insects listed some 120 species which responded
to photoperiodism, studied so far in which 88 are "long-day" and 6 are
"short-day" diapausing species. de Wilde (1962), however, suggested that
many of the insects with autumnal reproductive activities might belong to
the short-day type.
Many insects, just prior to entry into diapause, feed actively and
build up large reserves of fat and glycogen, thus enabling them to survive
through the unfavourable conditions during diapause. This build up of reserves varies in different insects depending on the intensity i.e.
duration of diapause. In Scoliopterix libatrix fat may constitute up to
56% of dry weight, (Sacharov, 1930) and up to 60% in hibernating females of
Culex pipiens. (Buxton, 1935). However, in others, fat reserves are not so
abundant, as they have been found to feed periodically. Examples of this
category are seen in Coccinella septemfasciata, Semiadalia 11-notata (Hodek
and Cerkasov, 196Cal b, 1961), Sitona cylindricollis (Davey, 1956) and 8. Leptinotarsa decemlineata (Busnel, 1939). Waloff (1949) found in Ephestia elutella that fat constituted 47.5% of dry weight in November end this fat content was reduced to 40.8% in May after diapause; whereas the water content remained fairly constant throughout diapause. As a contrast,
Gilpinia polytoma may be cited as an example, in which less than 2% of the energy stores are modified during the period of prepupal diapause. The adult sawfly does not feed, but if the prepupa overwinters four or five times the fecundity is little affected (Prebble, 1941).
Accompanying the process of fat deposition, some authors have found a steady decline in the ratio of water to dry matter. For example, in the codling moth Cydia pomonella, the water content falls from 72% in June to
58% in August (Ushatinskaya, 1952, cited by Lees, 1955). This deficiency of water is considered a potent factor in retarding growth and hydration is considered a normal requirement of the developing orthopteran egg,
Locustana pardalina (Matthee, 1951) and Melangplus diffexentialis (Slifer,
1938, 1946, 1948, 1950). In insect imaginal diapause, however, the water content does not normally decrease during diapause, as seen in
L. decemlineata (Busnel, 1939), Anatolica eremite (Edelman 1951, cited by
Lees), Sitona cylindricollis (Davey, 1956) and Semiadalia 11-notate (Hodek and 'berkasov, 1960).
Another feature of diapausing insects is their marked lower level of metabolism, reflected in lower respiratory rates. Thus in Anatolica eremita, the oxygen consumption declines progressively after September, when the beetle ceases to feed, and is at its lowest in January. In January, the rate of oxygen uptake is about one-tenth of that when feeding resumes after diapause in April (Edelman, 1951). Bodine and his co-workers
(Bodine 1934, 1941; Bodine and Boell, 1934) observed that oxygen uptake 9.
in the egg of Melanonlus differentialis is almost completely insensitive to respiratory poisons, such as cyanide and carbon monoxide during diapause,
but highly sensitive before and after diapause. They concluded that mitosis,
growth and differentiation are associated with a functional cytochrome system.
In Hyalophora cecropia, the rate of oxygen consumption of diapausing pupae
at 25°C., averages 16.3 ± 9.03 mm 3/gm. live weight/hour, is only 1.4% that
of mature larvae and 5% of pupae prior to adult moult (Schneiderman and
Williams,1953). Williams and his co-workers have contributed much towards
the understanding of oxidative metabolism in diapausing insects. It is
found that within hours after pupation in the diapausing pupae of, the
Cecropia silkworm, the activity and absorption bands of cytochromes b and c
become undetectable, whereas cytochromes a and a3 also decline sharply
(Shappiro and Williams,1957a, 1957b). In contrast, in the non-diapausing
pupae, the cytochrome system declines very little. The cytochrome decline
has-been considered as a direct result of a sudden drop in the titre of the
thoracic glands hormone (ecdysone), (Shappiro 1956, cited by Harvey 1962).
These studies show that the low metabolism of the diapausing pupae is
associated with the pr sence of low level of cytochromes 83 and b5 and the
absence of c, save in the musculature. This system appears to be incompatible
with growth and is resistant to cyanide and carbon monoxide. This aspect
of respiration has been reviewed by Lees (1955), Wigglesworth (1954) and
recently by Harvey (1962).
Imaginal diapause is associated invariably with the failure of
development of the reproductive organs, particularly the ovaries. This
has been confirmed in all the species studied so far, with the possible
exception of Aoelastica alni (cited by Hodek and C'erkasom,1960). There is 10.
variability, however, in males in the extent to which the testes are developed at the onset and during diapause. In Sitona cylindricollis, no meiotic divisions have been seen in the dormant testes, whereas in
Semiadalia 11-notata free-living spermatozoa are stored in the vesiculce
seminales and copulation and fertilization take place during diapause
(Hodek and Cerkasov, 1960). In Coccinella septelpunct‘ta there is continuous
spermatogenesis during the diapausing phase.
Wigglesworth (1934) was the first to suggest that diapause in insects
may be due to a temporary failure in the secretion of a growth hormone.
Following the location of the neurosecretory cells in the brain of Rhodnius
(HanstrOm, 1938), Wigglesworth (1940) extirpated the region containing the
neurosecretory cells from Rhodnius larvae at about the "critical" period
and transplanted them into the abdomen of larvae decapitated soon after
feeding. These headless larvae moulted. On this evidence, he tentatively
concluded that the growth hormone came from the neurosecretory cells.
Sellier (1949), working on the diapausing 9th instar larvae of Gryllus
campestris, found that diapause could be suppressed by transplanting the
brain from another, earlier instar non-diapausing larva.
Fukuda (1940a,b) was the first worker to discover the functional
rale of the prothoracic glands and their control of moulting and development
of the pupae of the silkworm Bombyx mori by ligaturing experiments. The
relationship between the brain and the prothoracic glands in H. cecropia
was established experimentally by Williams (1947). When he implanted an
activated brain i.e. one which had been chilled previously, into the
isolated abdomen of a diapausing pupa, it did not induce development,
whereas it did cause metamorphosis of the anterior half from which the 11. brain had been removed. But an isolated diapausing abdomen could be
induced to metamorphose if it was provided with an active brain plus prothoracic glands. Williams subsequently (1948b, 1952) established that the source of the activation of the prothoracic glands came from the neurosecretory cells of the brain. Thus in H. cecropia pupal diapause is due to the inactivity of the neurosecretory cells of the brain and the prothoracic glands. Chilling activates the brain which in turn activates
the prothoracic glands. This work has been confirmed in Cephus cinctus
(Church, 1955). The mechanism by which ecdysome regulates moulting remains
a challenge (Harvey, 1962).
Van der Kloot (1955) from electro-physiological experiments found
that the brain of the Cecropia silkworm during diapause is electrically
silent. There is also a rapid decline in cholnesterase in the brain.
During diapause the cholinergic action of the brain increases steadily.
Chilling speeds up the increase. When the accumulated cholinesterase is
at a high level of concentration, the brain becomes electrically active,
causing the neurosecretory cells to release the brain hormone.
The humoral basis of voltinism in Bombyx mori has been studied in-
dependently by Fukuda and Hasegawa. Their investigations point to the
suboesophageal ganglion as the neuro-endocrine centre which produces a
diapause hormone which is circulated in the blood and is absorbed by the
eggs. These eggs subsequently become diapausing eggs. (Fukuda, 1951 a,b,
1952; Hasegawa, 1952). Whereas, when the adults are subjected to low
temperatures and short photoperiods, the suboesophageal ganglion is inhibited
and does not produce this diapause hormone, thus non diapause eggs are laid.
Fukuda's experiments implicate the brain as a controlling centre of the 12. suboesophageal ganglion, inhibiting or stimulating it via the nerve
commissures to produce the hormone. Recently Morohoshi (1959) has provided
evidence that the corpus allatum hormone could counteract the suboesophageal
ganglion hormone in B. morl.
So far, the humoral mechanism of imaginal diapause has not been
completely worked out in any insect. The striking feature of imaginal
diapause is the failure in development of the ovaries. On this basis, the
endocrine centres controlling development and maturation of the gonads may
well throw some light on this problem. Wigglesworth (1936) observed that
the corpora allata were necessary for the production of ripe eggs in
Rhodnius. Since then, many authors have confirmed his findings, e.g. in
Leucophaea maderae (Scharrer, 1946a, b). aiiscus marginalis (joly, 1945)
Calliphora erythrocephala (Thomsen, 1940, 1952), Melanoplus differentialis
(Pfeiffer, 1939, 1945), Oncopeltus fasciatus (Johansson, 1958), and recently(
in Dermestes maculatus (Ladduwahetty, 1962). On the other hand, egg
maturation proceeds without interruption in allatectomizedC,arausius morosus,
unless the glands are extirpated at an early nymphal stage (Pflugfelder, 1937a
b, 1939). Similarly, the corporaallata are apparently not necessary for
egg production in certain Lepidoptera, e.g. Bombyx mori (Fukuda, 1944).
But in these cases, the developing pupae have fully mature eggs and
probably there is still a high level of the corpus allatum hormone in the
body (see Williams, 1956a).
These facts led de Wilde et al. (1958, 1959) and de Wilde (1960)
to investigate into the role of the corpus allatum in relation to the
imaginal diapause in LeFtinotarso decemlineata. Extirpation and implant-
ation of corpora allata suggested that diapause in this species occurred 13.
as a consequence of the inactivity of the corpora allata.
Thomsen (1952) demonstrated that the medial neurosecretory cells of
the pars intercerebralls of Calliphora exerted an overall controlling
influence on ovarial development. The influence of the neurosecretory cells
appears to be twofold; primarily, they activate the corpus allatum and,
secondly, their secretions promote ovarial development independently of the
corpus allatum.
In Schistocerca cregaria there is evidence of a positive control of
ovarial development by the neurosecretory cells of the pars intercerebrali+s
(Highnam,1961; 1962 a, b, c). His results were derived from the histological
examination of the neurosecretory system and were substantiated by cauter-
ization and implantation experiments. In Schistocerca Highnam has been able,
to correlate activity of the neurosecretory cells in terms of the amount of stainatthe material in the cells. His hypothesis is that the neurosecretory
system is "active" when it contains small amounts of stainable material and
that it is less active when the material accumulates within it. This
hypothesis has been substantiated by autoradiographic studies in S. oreciaria
(Highnam, 1962c).
In Aedes aeoypti, ovarial development is shown to depend on the
release of a hormone from the brain (Gillett, 1958). Similar results have
been obtained in Dermestes maculatus in which ovarial development is totally
inhibited in young females deprived of their medial neurosecretory cells,
whereas in allatectomized females, the eggs develop up to the stage of yolk
deposition (Ladduwahetty, 1962).
In Iphita limbata Sal, the medial neurosecretory cells have been
shown to play a major rele in inducing oviposition (Nayar., 1958). Trans- 14. plantation of medial neurosecretory cells from a mature female to an
immature one stimulated oviposition almost immediately.
However, in Caralaaius morosus and Clitumnus extradentatus (Dupont-
Raabe,1952, 1954) and in Oncopeltus fasciatus (Johansson,1958) the medial neurosecretory cells do not influence ovarial development but control
fecundity.
There is now growing evidence that these neurosecretory cells also
control protein metabolism (Thomsen, 1952; Thomsen and Miller, 1959, 1960;
Highnam, 1962c). The results of Strangkways-Dixon(1960, 1961) in Calliphora
suggest that the medial neurosecretory cells control protein ingestion,
whereas, the corpora allata control carbohydrate ingestion. It would seem
that the process of ovarial development under the control of the neuro-
secretory cells is mediated in protein metabolism.
The studies on reproduction in Leucophaea maderae and Diploptera
punctata indicate that a highly complex humoral mechanism is involved in
the regulation of developing eggs in the brood sac (Engelman 1957, 1958, 1959,
1960). During "pregnancy" the ovaries are quiescent as a result of inactivity
of the corpora allata which in turn are restrained by'the brain via nervous
pathways. The suboesophageal ganglion in his experiments, however, showed
a stimulating effect on the corpora allata. The corpora allata could be
activated during "pregnancy" by the removal of ootheca from the brood sac.
This activation, however, did not take place if the eggs were implanted into
the body cavity immediately after the removal of the ootheca. This indicates
that the developing eggs in the brood sac release a substance which causes
the brain to inhibit the corpora allata. Engelman's experiments further
indicate that the genital apparatus transmits nervous impulses to:the 15. brain for the control of the corpora allata.
The literature on neurosecretion has been reviewed by Scharrer and
Scharrer (1954), Bodenstein (1954), Van der Kloot (1960, 1962), Welsh (1959)
and the Proceedings of the three International Symposia on Neurosecretion
(1953, 1957, 1961).
The criterion for distinguishing neurosecretory cells from other
neurones has been generally adopted on the basis of Scharrers' (1954)
definition, " neurosecretory cells are nerve cells which show
cytological evidence of secrdtion". Johansson (1958) and Ladduwahetty (1962)
have found secretory inclusions in the cells which are probably motor neurones.
To avoid confusion, Van der Kloot (1060) suggests that the term "neuro-
endocrine" cells should be used to designate the neurosecretory cells which
elaborate and release hormones. Berns (1962) pointed out that a variety
of inclusions present in the neurones, may mimic neurosecretory material,
such as neuromelanin, stored metabolites and pigmented materials of unknown
significance. He defines a neurosecretory cell as one in which "...the
signs of secretory activity can be related to the production of chemical
agents with measurable physiologic effects, agents definable as hormones...".
A distinctive feature of neurosecretion is the axonal transport of
neurosecretory material to the organs of storage and release via the nervi
corporis cardiaci I and II. The organ of storage of secretion from the
brain, is the corpus cardiacum as seen in Leucophaea maderae (Scharrer, 1951,
1952), Iphitalimbata (Nayar, 1956), Schistocerca grecoria (Highnam, 1961),
Blaberus craniifer (Hodgson and Geldiay, 1959) and Carabus nemoralis (Klug,
1958/1959). However, in some insects, the corpus allatum appears to have
taken over the storage function as in Dermestes maculatus (Ladduwahetty, 1962) 16.
The two staining techniques which have been most widely used in work on neurosecretion are Gomori's (1941) chrome-haematoxylin/Phloxine method (CHP,) and a modified Gomori's (1950) Aldehyde/Fuchsin (Paraldehyde
Fuchsin) method (PF.).
Using either or both these staining techniques, two main types of neurosecretory cells, designated as A and B-cells have been described by a number of workers. Those cells that stain blue-black with CHP. and purple with PE are designated as A cells. The B cells are phloxinophil and stain red with CHP.and red or bluish green with PR (Scharrer, 1955 on Leucophaea, suboesophageal ganglion; M. Thomsen, 1954 on several species of Hymenoptera;
Nayar, 1955 on Iphita limbata, on nerve ring; Johansson, 1958 on Onco_ptltus fasciatus brain, suboesophageal,first thoracic and last abdominal ganglia;
Fraser, 1959a Lucilia caesar, larval brain, 1959b L. caesar last abdominal ganglia; Highnam, 1961 on Schistocerca preciaria on brain and Ladduwahetty,
1962 on Dermestes maculatus, brain). According to Delphin (1963) who has made a thorough survey of the staining reactions of the A and B cells reported by the above workers on different species, concludes that these cells are similar if not identical to one another. In addition, various authors have described additional celltypes. In these cells designated as
C and D or III and IV there is as yet no uniformity in classification.
Thus the C cells present in Oncopeltus described by Johansson (1958) do not necessarily correspond to the C cells described by Ladduwahetty (1962) in Dermestes. It seems that standardization of staining techniques is much to be desired in comparative histological investigation of this nature. 17.
II. The Life History of G. tanaceti
From the beginning of September to the end of November, G. tanaceti lays batches of diapausing eggs which overwinter. The eggs measure up to
1.4 mm.in length and are laid in batches of about 60. The eggs are held together, protected and cemented to the plant by a creamy fluid secreted by the spermathecal gland. This secretion turns black and hardens within hours.
The larvae hatch out in April to May of the following Spring. The newly hatched larvae measure between 1.5 to 2 mm. in length, feed voraciously for the next 3 - 4 weeks mainly on the leaves of Stellaria graminea L. although a few have been found feeding on Achillea millefolium L. The full-grown larvae which measure up to 2 cm. in length, then either burrow into the ground to pupate or build a "cocoon" in the dry vegetation beneath the food plants. The period of pupation is from 12 - 16 days.
The adults emerge about the first week of June, in a pre-diapause phase which lasts between 8 - 16 days'during which they feed actively.
Following this pre-diapause phase, the adults remain in reproductive diapause, feeding periodically but spending most of the time in the ground or beneath the tussock grass. Females, on dissection right through the pre- and diapause phases, reveal completely undifferentiated ovarioles which are about 1 mm. long. The germarium and spermathecal gland remain small. No sperm bundles have been seen in the vesiculae seminales at this stage.
Development of ovarioles begins from about the middle of August and thaturation of the terminal oocytesis completed by mid-September. Copulation takes place just before the formation of fully mature. oocytes. The 18. females oviposit successive batches of eggs from mid-September to the end of November. The interval between the successive ovipositions varies between 7 - 10 days. The adults die in December.
The adult beetles are seldom seen throughout their life Iistory except during the short period of oviposition, when the females move up the stems and leaves of the tussock grass or up the inflorescence of
Achilles and oviposit their eggs. 19.
III. Rearing techniques.
The best period for collecting G. tanaceti in the field was in the larval stage, during April to May. The larvae were kept in Watkin and
Donkaster cages containing Stellaria graminea L.. About 40 - 50 larvae of the same age were reared per cage kept in the insectary.
When the larvae pupated, they were carefullyremoved from their pupation "cocoons" by a pair of fine forceps and kept in petri dishes, with moistened filter paper at about 20°C. in the C.T.room.
The adults, on emergence, were removed and transferred to separate
W. & D. cages containing Stellaria, their exact date of emergence being noted. These were kept in the insectary. Usually no more than 35 adults were placed in one cage. At the pe,k of adult emergence, about 60 such cages were used. The food plants had to be replenished every 5 - 7 days.
A stock of beetles in sustained diapause was kept in the C.T. rooms.
These were placed at controlled conditions, at a photoperiod of 16 hours at 20°C. 20.
IV. Studies on the morphology of the reproductive systems with accompanying quantitative and qualitative changes at different stages of the adult life in G. tanaceti
Introduction
The sustained reproductive diapause throughout the summer, followed by a long maturation period of about 30 days, finally leading to oviposition of successive batches of eggs, form the interesting features of the adult life of G. tanaceti.
This study, therefore, attempts to evaluate the quantitative and qualitative changes of the reproductive organs at different stages of its
adult life under natural conditions. It forms, in fact, the framework upon
which other physiological investigations are based.
As investigations into the physiological changes in the insect male reproductive organs have been neglected, it seemed desirable to examine the reproductive condition of the males while the females were in the phase of
diapause.
Only a short description is given of the morphology of the reproductive
organs. A more detailed account of them has been given by Donia (1958).
Method,
a) Morphological studies.
Normal techniques were employed in morphological studies. Beetles
were dissected either under Ringer's solution or in Bouin's fluid.
b) Quantitative and qualitative studies.
1. Females. 7 - 8 females were dissected at definite time intervals
which represebt the main physiological phases of adult life. (See rearing 21. techniques, page 19 ). Morphometric measurements were made on the components of the ovarioles and on the width of the spermathecal gland.
The spermatheca was then squashed and stained with aceto-carmine and examined for the presence of spermatozoa. The mean lengths of ovarioles, germaria, oocyte I and oocyte II were calculated from at least 60 ovarioles at each stage.
2. Males. Morphometric measurements were made on the testicular lobes and on the width of the accessory glands. The testes were squashed in aceto-carmine to ascertain if spermatogenesis proceeded normally during the phase of diapause. They esiculae semir4ales were also examined for the presence or absence of stored spermatozoa. The male reproductive organs were examined at stages comparable to those of the females. a) Morphology of the reproductive organs
1) Female Fig. 1.
Each ovary consists of 40 - 42 meroistic telotrophic ovarioles. The bursa copulatrix is small and is present inside the long sac-like vagina.
The spermatheca lies on the dorsal wall of the vagina. The spermathecal duct, according to Donia (1958), is distinctive of G. tanaceti in the
Chrysomelidae, in that it is very short, 0.33 mm long and is straight without any coiling. It opens anteriorly into the bursa copulatrix.
2) Male Fig. 1.
A pair of bilobed testes, enclosed in a common testicular sheath is situated dorsally in the middle of the abdominal cavity, the right testis being more'anterior. Two vase efferentia arise ventrally from each testicle lobe uniting immediately to form the vas deferens. The two vase deferentia run posteriorly and unite to form the ejaculatory duct. Just 22
A
3 mm. B
3mm,
Fig. 1. A. Dorsal aspect of 2. reproductive organs of G. tanaceti. B. Spermatheca and gland. C. Dorsal aspect of &reproductive organs. aed., aedeagus; a.g., accessory gland; cor., cornu; c.sh., common testicular sheath; ej.d., ejaculatory duct; m.l. muscular layer; nod., nodulus; sp.d., spermathecal duct; sp.g., spermathecal gland; sp.m., spermathecal muscles; t., testicular lobe; v. vagina; v.d., vas deferens; v.s. vesicula seminalis. 23. prior to their junction, each vas deferens becomes slightly dilated, forming
a small vesicula seminalis. A pair of accessory glands arises posteriorly
at the junction between each of the vas deferens and the vesicula seminalis
and runs anteriorly into the abdomen.
b) Quantitative and qualitative changes of the reproductia organs at different stages of adult life history
i) Females
No measurements were taken of the lengths of fully mature ovarioles
because there was no practical method of determining their values accurately.
This was equally true of measuring the lengths of the spermathecal glands,
but it was found that the width of the spermathecal gland provided a good
basis for assessing its relative activity. Aceto-carmine squashes of the
spermathecae gave a reliable method of determining the presence of bundles
of spermatozoa, the sperm heads being stained red with aceto-carmine.
The results of these observations are tabulated in Table 1.
During the phases of pre-diapause and diapause, there was no differ-
entiation of oocytes in the ovarioles of G. tanaceti. The spermathecae On
microscopical examination revealed the absence of spermatozoa, suggesting
that no copulation had taken place during the phase of diapause. The
spermathecal gland remained developed.
On the termination of diapause, the ovarian maturation period commenced
and extended over a period of 30 days, during which there was a progressive
but gradual differentiation of oocytes. Growth, however, was localized in
the terminal oocytes, oocyte I. In a fully mature ovariole just prior to
ovulation of oocyte I, onlylthree oocytes could be seen in the vitellarium.
In the post ovulation period, there was immediate differentiation of oocyte
IV from the germarium. Growth meanwhile took place in oocyte II which
TAhlP 1. Qualitative and quantitative changes in female reproductive organs of G. tanaceti. Each stage based on 7 - 8 dissections
Physiological Age Length of Ovariole Length of Length of Length of Spermatheca Width of phase ovariole condition oocyte I oocyte II germaria full empty sp. gl.
Pre-diapause 0 day 0.76 mm Undifferentiated 0.34 mm 0 8 110 p 1 " 4.79 mm 0.34 " 0 8 110 p 8 days 0.80 mm 0.35" 0 7 130 p
Diapause 16 days 0.82 mm II - 0.35" 0 7 130 p 24 " 0.83 mm I/ - - 0.36 " 0 7 130 p 40 " 0.86 mm n 0.37 " 0 7 140 p 56 1.06 mm oocyte 0.3E " 0 7 160 p formation Differentiation Maturation 68 1.46 mm Oocyte I 0.33 mm - 0.4C " 6 1 220 p 82 11 1.90 mm Oocytes I & II 0.75 mm 0.30mm 0.47 " 7 0 240 p 96 It *Oocytes I II III *1.28 mm 0.35" 0.52" 7 0 330 p IV (1.07 mm)
Oviposition Just *Oocytes I II III *1.35 mm 0.38 " 0.52 " 7 0 350 p before IV 290 p
Post- 1 day Oocytes I II III 0.93 mm 0.38 ' 0.50 " 7 0 280 p oviposition 3 days Oocytes I II III 1.14 mm 0.40' 0.51 " 7 0 300 p 5 n *Oocytes I II III *1.25 mm 0.40' 0.52" 7 0 320 p IV (1.15 mm)
* Ovulation of Oocyte I. 25. now became oocyte I in the vitellarium. This meant that the terminal oocytes developed at the expense of all the nutr,ition available to the adjacent ova.
The spermathecal gland during this period became enlarged progressively and was largest just before oviposition. Post-mortem of beetles just after oviposition revealed relatively smaller spermathecal glands. It was evident that this gland provided the secretion that held and protected the eggs when they were laid in batches.
During the post oviposition period, which lasted 7 to 10 days, a similar sequence of events took place in egg maturation and egg different- iation. The maturation of the terminal oocytes during this period appeared to be more rapid than those in the first phase of maturation. This differential speed in development of terminal oocytes and its maturation seemed to be paralleled by the differential levels of endocrine secretory activity. This problem will be discussed in greater detail (see chapters
VIII and IX).
ii) Males
Belling's iron aceto-carmine technique provided a rapid method of
examining squashed testes for presence of meiotic divisions in spermato- genesis. The experiments showed up meiotic division figures which were
especially common around the periphery of the testicular lobes throughout the phase of diapause.
Examination of the vesiculae seminales revealed the presence of
bundles of spermatozoa, only during the post-diapause period. In the post- diapause phase there was also increased activity in the accessory glands.
They were twice as wide as those in the phase of diapause. 26.
There was an apparent increase in the size of the testes during diapause. This might account for the fact that spermatogenesis evidently took place during this period, whereas no fully mature spermatozoa were released into the vesiculae seminales. The validity of this explanation has yet to be established experimentally. Copulation took place about 75 days after the imaginal moult, a period when the females had actively developing oocytes. Repeated copulations had been observed to take place in the laboratory.
The observations of these experiments are tabulated below:-
Table 2. Changes in male reproductive organs of G. tanaceti based on 7 to 8 dissections at different stages of the adult life.
••••••••110. Physiological Age Mean length of Condition of Width of Vesiculae phase each testicular testes accessory seminales lobe gland 1 day 1.14 mm. All stages 122 p empty of meiosis 8 days 1.26 " 117 p
Diapause 16 11 1.55 " 164 p
24 11 1.40 " H 135 p
40 " 1.60 " “ 141 p
56 11 1.98 " 11 293 i Full of sperm - Maturation atozoa 68 11 1.74" 330 p
Discussion
The reproductive activity in G. tanaceti has been shown to occur in autumn and early winter. The induction and termination of reproductive diapause has been shown to be under the direct influence of photoperiodism and temperature. (See chapter VII). 27.
Diapausing G. tanaceti have completely undifferentiated ovarioles.
A similar condition has been found in Coccinella septeml=mnc+Ate (Hodek and
Cerkasov, 1961). In ytiscus marginalis (Joly, 1945) and in Leptinotarsa decemlineata (de Wilde, 1954), however, during the phase of reproductive diapause, ovulation continues but the immature ova soon degenerate and are resorbed by the ovarian wall.
In G. tanaceti, the males, however, during diapause, have more or less mature testes, which is in agreement with Semiadalia 11-notata (Hodek and qCerkasov, 1960b) These authors suggest that spermatogenesis as a process requires less expenditure of energy than egg development. In G. tanaceti there are no bundles of spermatozoa stored in the vesiculr.e seminales during the phase of diapause. In S. 11-notata on the other hand, the vesiculae seminales are filled with free-living spermatozoa during the hibernation period. Copulation,in fact, takes place in S. 11-notata in the hibernation quarters before the emergence of females in the spring.
Development of ovarioles and the spermathecal glands seems to proceed simultaneously in G. tanaceti. This is not surprising as the spermathecal gland has been shown to be implicated in successful oviposition of batches of eggs.
In G. tanaceti, growth has been shown to be confined wholly to the terminal oocytes during ovarian development. This mechanism of extreme differential oocyte development may prove to be common to insects which lay batches of eggs. This brings to mind an analogous situation in Leucophaea maderae and Diploptera purctata in which during "pregnancy" the ovaries are inactive and are controlled by the activity of the corpora allata which in turn are restrained by the brain. There is a further restraining feed-back 28. mechanism from the ootheca to the corpora allata via the brain (Engelman,
1957, 1959, 1960). It seems to me that the hypothesis put forward by
Engelman (1957, 1959) that the corpora allata secrete the gonadotropic hormones cyclically in Leucophaea and Diploptera and thus repress further egg development during "pregnancy" does not explain fully the observations made in G. tanaceti. This aspect will be dealt with later (See chapter
VIII). 29. V. The neuro-endocrine complex of G. tanaceti
The stomatogastric nervous system and the neurosecretory system are best considered as one neuro-endocrine complex. These two apparently distinct systems are in fact connected intimately by nervous pathways via the brain and the corpus cardiacum and the corpus cardiacum/allumtum via the suboesophageal ganglion. The corpus allatum-suboesophageal nerve has been traced in only a few insects so far, e.g. in the Ephemeroptera by Arvy
and Gabe (1953a), Oncopeltus fasciatus (Johansson, 1958), Periplaneta americana.
(Harker, 1960c; Willey, 1961), Locusta mioratoria (Staa1,1961),
Schistocerca grecoria (Delphin, 1963) and in G. tanaceti. It is probable that
the existence of this fine nerve has been overlooked in most insects.
Knowledge of insect endocrinology has been derived mainly from studies
on the neurosecretory system which has been centred mainly around the-inter-
cerebralis-cardiacum-allatum" system (Scharrer and Scharrer, 1944, 1954;
Hanstrec, 1953). There is, as yet, hardly any study on the functional
integration of the neurosecretory system and the stomatogastric nervous
system in spite of their intimate relationships. This may prove to have its
shortcomings. Day (1943) observed that the ovaries of Lucille sericata
failed to develop after the severance of the recurrent nerve. More recently,
however, Clarke and Langley (1962) have shown that there was a complete
cessation of growth and moulting in Locusta mioratoria L. when the frontal
connections were severed or the frontal ganglion removed at any time during
the first 90% of the stadium.
In the present study of G. tanaceti, a morphological and anatomical
description is given of the neuro-endocrine complex. The histology of the
neurosecretory system is treated in greater detail. Four staining 30. techniques which are specific for neurosecretory cells have been employed for the confirmation of the distinct neurosecretory cell types. However, two staining techniques have been used more extensively, namely, the paraldehyde fuchsin/light green technique after Halmi (1952) and the Alcian blue/phloxine technique after Delphin (1963).
The term "neurosecretory system" has been retained in this study to ascribe to the parts of the neuro-endocrine complex which elicit neuro- secretion phenomena.
Method
Insects were dissected in 70% ethyl alcohol for morphological and anatomical studies.
The neurosecretory system attached with the oesophagous were dissected out under Bouin's fluid and fixed in it for 18-24 hours for histological studies. Dehydration was achieved through the alcohol series and the tissue was cleared in cedar wood oil. Embedding was in either paraffin wax with ceresin (m.pt. 55°C, 3 changes of 10 minutes each) or paraffin wax (m.pt.
58°C, 3 changes of 20 minutes each). Serial sections were cut at 4 u.
The sections were stained with:-
a) paraldehyde fuchsin/light green, modified from Halmi's techneque (1952), potassium permanganate oxidation followed by Lugol's iodine, (henceforth referred to as PF); kr) Alcian blue/Phloxine,potassium permanganate oxidation, after Delphin
(1963), henceforth referred to as ABP);
c) Chrome haematoxylin/Phloxine after Gomori's technique (1941) henceforth referred to as CHP);
d) Heidenhaies Azan/aniline blue after Pantin (1948) (henceforth referred to as Azan). 31.
Morphology and anatomy of the neuro-endocrine complex.
The neuro-endocrine complex of G. tanaceti is depicted in Figs 2 & 3.
The corpus cardiacum is roughly spindle shaped, its distal end being joined to a compact spheroidal corpus allatum. There is a thin anterior projection of the corpus cardiacum which runs anteriorly just out- side the dorsal aorta to the region of the frontal ganglion. This corpus cardiacum projection has been seen in Hydrous piceus (Coleoptera) (Cazal,
1948). The dorsal aorta running on the dorsal aspect of the oesophagous dilates in the region of the corpus cardiaca and thus, is in close
association with the glands which therefore are considered "neuro-haemal" organs in the sense of Carlisle and Knowles (1953).
Two nerves, the nervi corpus cardiaci I and II (N.C.C. I & II)
leave the posterior aspect of the protocerebrum, connect the brain to the
corpus cardiacum. N.C.C. I is a relatively large nerve composed of axons
from the medial group of neurosecretory cells of the pars intercerebralis
of the protocerebrum. N.C.C.II is a fine nerve, composed of axons from the
lateral group of neurosecretory cells of the lateral lobe of the protocerebrum.
This fine nerve leaves the brain laterally to N.C.C. I and runs posteriorly,
slightly dorso-medially over N.C.C. I thus entering the corpus• cardiacum
medially to the large nerve. These two pairs of nerves innervating the
corpora cardiaca occur in all pterygote insects (Cazal, 1948).
A third pair of nerves, N.C.C. III leaves the lateral aspect of the
tritocerebrum and enters the antero-lateral aspect of the corpus cardiacum
independently of N.C.C. I and II. Its origin has not been determined in
G. tanaceti and no neurosecretory cells have been found in the tritocerebrum.
The nerve is probably homologous with N.C.C. III of Carausius morosus 32. whose origin has been traced to one or two neurosecretory cells of the tritocerebrum (Dupont-Raabe 1954, 1956). The N.C.C. III has also been found in Oncopeltus fasciatus (Johansson, 1958) but he was unable to locate its origin.
Just anterior to the brain and lying on the dorsal wall of the gut is the relatively large frontal ganglion. The recurrent nerve from the frontal ganglion runs back along the mid-dorsal line of the oesophagous, expanding slightly to form the hypocerebral ganglion which lies between the two corpora cardiaca.. Two pairs of nerves, oesophageal 1 and 2 leave the hypocerebral ganglion. The oesophageal 1 runs laterally into the corpus cardiacum. The oesophageal 2 runs obliquely backwards, almost immediately bifurcates; one branch innervating the dorsal wall of the oesophagous and the other branch runs laterally along the gut to the crop where it expands very slightly forming the ingluvial ganglion.
A fine nerve arising from the maxillary nerve of the suboesophageal ganglion connects the suboesophageal ganglion to the corpus cardiacumi allatum. This fine nerve expands to form a small ganglion from which two nerves arise, one entering the corpus cardiacum and the other into the corpus allatum.
Four main groups of neurosecretory cells of the protocerebrum can be seen as bluish white specks under illumination. Two groups of these neuro- secretory cells lie dorso-medially in the pars intercerebralis of the brain.
The other two groups of neurosecretory cells lie on the dorsal aspect in the lateral protocerebral lobes of the brain. Tht: fifth group of neuro- secretory cells lie mid-ventrally in the suboesophageal ganglion. 33
Fig 2
Fig. 2. Dorsal aspect of the neuro-endocrine complex of G. tanaceti. c.al., corpus allatum; c.c., corpus cardiacum; fr.g., frontal ganglion; hy.g., hypocerebral ganglion; N.C.C. I,II, III, nervic corporis cardiaci I, II, III; oe. 1,2, oesophageal nerve 1, 2; re.n., recurrent nerve; soe, al. n., suboesophageal-allatum nerve. FF, 9 3
NCC II IsICC III re n by
Fig. 3. Lateral aspect of the neuro-endocrine complex of G. tanaceti. Abbreviations as in Fig. 2. c.c.p., anterior projection of corpus cardiacum; d.aor., dorsal aorta; i.g., ingluvial ganglion. 35.
The histology of the neurosecretory system.
The brain is invested by a thin connective tissue sheath consisting of two layers: the outer non cellular neural lamella and an inner neural sheath. The neural sheath is composed of a continuous layer of syncytial sheath cells having small nuclei. The cortical zone comprises neuro- secretory cells, gliel cells, globuli cells and ordinary motor neurones.
The neuropile which comprises the bulk of the medulla zone is made up principally of axons.
Five distinct cell types have been established in G. tanaceti on the basis of their tinctorial affinities in employing the different stains.
A summary of their characteristics is given in Table 3.
Medial grc-ips of neurosecretory cells of the pars intercerebralis
The two medial groups of neurosecretory cells (henceforth referred to as N.S.G.), lie close together in the dorso-medial aspect of the pars
intercerebralis of the protocerebrum. Each group of cells is lodged in
each hemisphere of the brain. Their axons run anteriorly, then sweep down-
wards and decussate. In serial sections, cut sagitally, there is no distinct
demarkation of the two groups of cells which for convenience have been
considered as one group. There are 42 - 46 A-cells containing varying
amounts of granular inclusions at different stages of the life cycle (see
chapter VIII for details). There are 10 - 13 B-cells, distributed between
the A-cells. The B-cells,using the PF. technique, stain up differently
at different stages and because of their location and numbers remain constant,
they are considered to exist in two physiological phases, designated as B1
and B cells. II Table 3. The neurosecretory cell types of G. tanaceti„ their occurrence, numbers and stianingreactins
,1 A-cells A -cells BI-cells B -cells C-cells D-cells I II CHP Blue inclusion Blue inclusion Red inclusion Red inclusion Blue cyto-plasm Blue granules with red inclusion PF Purple Purple Yellowish-brown Green Green cytoplasm Green with red inclusion APB Blue Blue Red Red Blue-green cyto- Blue plasm with red inclusion Azan Yellowish red Yellowish red Blue Blue Occurrence *M.G.P.I. 9.) L.G.P.I. *i) M.G.P.I. *i) M.G.P.I. *i) M.G.P.I. * L.G.P.I. ii) S.G. ii) S.G. ii) S.G. No.of cells 42 - 46 i) 2 - 3 i) 10 - 13 i) 10 - 13 2 - 5 6-13 ii) 2 - 3 ii) 8+ ii) 8+ 2 medial VG ° B and B cells are E emarks I II taken as o e. different physiological stages of B-cells. 8 phloxinophil B.-cells. *M.G.P.I. medial groups of pars intercerebralis L.G.P.I. - lateral groups of pars intercerebralis S.G. suboesophageal ganglion. 37.
The C-cells, the largest of the N.S.C. occur in varying numbers
from two to five. They tend to lie close to the lateral periphery of the
main group of N.S.C.
Lateral group of neurosecretory cells of the protocerebrum
The lateral group of N.S.C., one in each cerebral hemisphere, is
located dorso-laterally in the protocerebral lobe just outside the pars
intercerebrals of the brain.
cells. (The Each lateral group of N.S.C. is composed of 2 - 3AI staining affinity is similar to A-cells of the medial group using PF, CHP
and ABP, but the histological picture of the cytoplasmic inclusions is
slightly different (cf. Deiphin, 1963)) and 6 - 13 D-cells. These D-cells
are generally smaller than the other N.S.C.
Suboesophageal ganglion neurosecretory cells Firs 4 & 5.
The suboesophageal ganglion contains 2 - 3 A cells (comparable to those of the lateral group of A cells in the brain), and 8 B cells
distributed in 3 groups. These B-cells particularly the posterior group
of 2 cells,are strongly phloxinophil and only show up using the CHP or
ABP.techniques. They are therefore not necessarily identical to the
B-cells of the brain. In Periplaneta americana the B-cells of the sub-
oesophageal ganglion, however, are not Pnloxinophil and can only be
demonstrated with PF.(Fuller, 1960). Similarly in Schistocerca gregaria,
the B-type cells of the ventral ganglia are never phloxinophil and there-
fore not identical with the B-cells of the brain (Delphin, 1963). Standard-
itation of techniques may explain the nature of this apparent diversity of
B tell types, in different insects. 38
Fig.4
VO.
BR- Cell
20p C Cell
D Cell
Fig. 4. Neuroseoretory Cell types of G. tanaceti. a. BI cell in 0 day old .9; b. BI cell in during oviposition; c. BI cell ! in phase of post-oviposition; d. BII cell in diapausing,y; e. BII cell in maturing 9; f. C cell in diapausing y; g. C cell in maturing y; h. D cell; i. AI cell of suboesophageal ganglion in ovipositing va., vacuoles. 39. Neurosecretory pathways
The pathways traversed by the axons of the A-cells are very distinct owing to the presence of stained secretory material carried along them.
This material is granular at certain stages, giving the impression that the secretions from the neurosecretory cells are discharged continuously; whereas at other stages especially during the maturation and oviposition phases, the secretion along the axons are continuous. The pathways traversed by the B and C-cells are less defined; their secretions are probably less in amount and therefore are maskted by the A-cell secretions. Because their axons run along those of the A-cells initially, it seems reasonable to assume that they continue their entire pathways together. The axons from the two medial groups of N.S.C. of the pars intercesebralis pass anteriorly into the neuropile. They then curve posteriorly and decussate, entering the N.C.C.I.
Thus each corpus cardiacum is innervated by axons from the opposing medial group of N.S.C. This condition seems typical in all insects studied so far (Van der Kloot, 1960).
The axons from the lateral group of N.S.C., however, run directly into the N.C.C. II without decussating. Their pathways are never as distinct as those of the medial group of cells.
The neurosecretory pathways of the suboesophageal ganglion have not been traced successfully. There is, however, evidence that the axons of the A cells and the 4 B-cells run into the neuropile, beyond which their I direction is unknown. The axons of the two large B cells of the posterior
group traverse backwards along the ventral nerve cord. This observation
supports the finding of the axonal routes of N.S.C. of the suboesophageal
ganglion of S. precaria (Delphin, 1963). 40
Fig 5
D Cell C Cell A i Ceh A Cell NCC II B Cell NCC I
B. ont4--
AiCell nu
Fig. 5. A. Arrangement of Neurosecretory Cell groups and their pathways of the brain in G. tanaceti ( schematic ). B. Arrangement of Neurosecretory Cell groups of the suboesophageal ganglion. 41. Some of the axons of N.C.C.I and II which innervate the corpus cardiacum traverse their entire lengths into the corpus allatum. These axons may be considered to constitute the cardiaco-allatum nerve which is very distinct in some insects as in Hydrous piceus (Cazal, 1948).
The corpus cardiacum
The corpus cardiacum is of nervous origin (Hanstrtm, 1940) but neurones are uncommon in the functional organ. It consists of two types of cells. The first type of cells forming an ill-defined cortex appear to be of secretory cells. These cells are, on the whole, larger than those of the medulla and often have globules of secretory product in the cytoplasm.
This secretion stains bluish-green with P.F. These cells no doubt correspond to the "chromophile cells" described by Cazal (1948).
The second cell type appears to be non-secretory and are scattered in
the matrix of the corpus cardiacum. Some are located between the more well defined secretory cells of the periphery. They correspond to the
"chromophobe cells" of Cazal (1948). A third cell type has been described by Johansson (1958) in Oncopeltus as "chromophobe glia-like cells". These
cells are probably similar to the second type of cells of G. tanaceti.
The axons from the N.C.C. I and II intermingle in the anterior portion
of the corpus cardiacum, which has taken the functional role for storage of
the neurosecretory materials from the brain. Droplets of stainable secretory material lie within or outside the axons between the chromophile and
chromophobe cells. Some secretory granules have been observed intracellularly
suggesting that there is probably an intimate relationship between the axons
of the neurosecretory cells and the secretory cells of the corpus cardiacum.
This problem may be solved by electron-microscopy. 42.
The connective membrane of the corpus cardiacum folds in, thus partitioning the gland into distinct lobules, a condition similarly described by Cazal (1948).
The corpus allatum.
The corpus allatum is a compact body invested by a thin connective membrane which is continuous with that of the corpus cardiacum.
Cell outlines are discerned with difficulty, especially in the in- active diapausing gland. In Iphita the gland is syncytial (Nayar, 1956)
but not in Rhodnius (Wigglesworth, 1934).
In the inactive gland, the nuclei are packed closely together being distributed quite homogenously in the gland. In the active gland, increase in volume has been shown solely due to the increase of the cell volume.
Some cells, especially those of the periphery, increase by several times
expaning outwards, thus distributing the nuclei heterogenously. In these active glands,vacuoles are seen inter- and intracellularly.
Contrary to the condition observed in Dermestes maculatus (Ladduwa-
hetty, 1962), no neurosecretion from the brain has been observed in the gland. No mitotic activity of the corpus allatum has been discerned in
G. tanaceti. In older females, some nuclei enlarge as much as 40 times the original size and in these,pycnosis is evident. Mendes (1948) describes four types of cells in the corpus allatum of Melanoplus differentialis. In
G. tanaceti during the oviposition phase, cells corresponding to Mendes' description could be seen. I believe that while his classification is convenient for description, there exists in G. tanaceti only one type of cells and the different sizes represent different levels of activity of the
individual cells. 43.
Discussion
The intimate ar.a;omical relationship between the stomatogastric nervous system and the neurosecretory system is well known in all orders of insects (Cazal, 1948). The role of the frontal ganglion in L. miqratoria as a mediator for ensuring continuous secretion of neurosecretory material, via the stimulation of stretch receptors by the movements of the pharynx has been suggested by Clarke and Langely (1962). The two oesophageal nerves which pass from the corpus cardiacum and the hypocerebral ganglion to innervate the oesophagous have been found to contain neurosecretory matter presumably from the N.S.C. of the brain (E. Thomson, 1954). For these reasons it seems plausible to integrate the stomatogastric nervous system and the neurosecretory system as one neuro-endocrine complex. This will override confusion over terms like the "retro-cerebral" organs (= corpus cardiacum/allatum) as part of the stomatogastric system while some authors in describing the neurosecretory system include the corpus cardiacum- allatum complex.
The general anatomical features of the "neuro-endocrine" complex are comparable to that in beetles described by Cazal (1948). The discovery of the corpus allatum-suboesophageal nerve is of particular interest. This,
I believe, is the first record of the existence of this nerve in the
Coleoptera.
Scharrer's (1954) definition of N.S.C. are nerve cells which "show cytological evidence of secretory activity", seems inadequate. This has been pointed out by Van der Kloot (1960) and Bern (1962). Bern maintains that "...:if the signs of secretory activity can be related to the production of chemical agents definable as hormones, then it can be concluded that such 44. calls are definitely neurosecretory". With the present knowledge of insect endocrinology, it is difficult to satisfy the requirements set forth in his definition of N.S.C.
In this study I. have included only those neurones which 1) show cytological evidence of secretion; 2) are capable of discharging their secretions along their axons into the corpus cardiacum and 3) show cyclical morphometric changes as neurosecretory cells. In the case of B and C cells from which secretions along the axons have not been detected, probably because they are masked by those from the A cells, however, their products have stained up in the corpus cardiacum.
The most prominent group of N.S.C. are the medial cells located in the pars intercerebralis. Their existence in two groups is confirmed by the decussation of their axonalpathways. Neurosecretory cells designated as A, BI, BI, and C are found in these two groups. These two groups consist of 42 - 46 A-cells, 10-13 B- cells and 2 - 5 C-cells.
The lateral N.S.C. groups in G. tanaceti are comparable to those described in Hyalophora cecropia (Williams, 1948), Iphita limbata (Nayar,
1955), Dermestes maculatus (Ladduwahetty, 1962), Calliphora erythrocephala
.(Thomson, 1952) and in Adelphocoris lineolatus (Ewen, 1962). However, the distribution of cell types found in this group of N.S.C. seem to vary in the
few insects which have been studied. Thus, in Iphita, the lateral cells are phloxinophil and presumably the B-cell type. In Dermestes, this group contains 5 N.S.C., four of which are B-cells while the fifth is a large
C-cell. In A. lineolatus 3 B-cells are located in each lateral group. In
G. tanaceti there are present in this group 2 - 3 AI cells and a highly variable number of D-cells, depending on the physiological state of this 45. insect. Their number is generally high in the maturation and oviposition phases, suggesting that they have a special role to play in these processes.
(cf. Arvy, 3ounhio1 and Gabe, 1953; Arvy and Gabe, 1953a, Formigoni, 1956;
Kobayashi, 1957 and Lhovte, 1952). It is interesting to note that these cells stain green with PF ond thus resemble B-cells but with ABP they stain blue and therefore are not similar to the characteristic phloxinophil
B-cells of the medial group, hence D-cells. The A-cells in this group have been designated AI type because of their persistently different cytoplasmic
appearance from those of the typical A-cells of the medial group.
The 2 or 3 N.S.C. of the suboesophageal ganglion are designated as
A/ because of their tinctorial affinity with the AI cells of the lateral
groups of the brain. No B cells have been discerned using the PF technique.
But with CHP and ABP, 8 phloxinophil cells have been located, distributed
in 3 groups in the suboesophageal ganglion. This suggests that the B-cells
of the suboesophageal ganglion are not identical to those of the brain,
confirming Fuller's (1960) and Delphin's (1963) studies.
There is still some controversy over the validity of the distinct
cell types. Some authors, namely, Lhoste (1952), H. Thomson (1954), Nayar
(1955), de Lerma (1956) and Brandenberg (1956) believe that the A and B
cells (let alone the C and D-cells) belong to the same category but are
seen in two phases of a secretory cycle.
In G. tanaceti the B-cells of the pars intercerebralis have been
found to be capable of transformation; these cells are thought to be in-
volved in the control of diapause and will be discussed more fully in a later
chapter. The five cell types, however, have been found to be persistently
distinet from one another at all stages. Their distribution is also
distinctive. 46.
VI. Studies on feeding, growth, respiration, water and fat content of G. tanaceti at different stages of its adult life history,
Introduction
From the foregoing chapter, it is evident that G. tanaceti enters into diapause soon after the imaginal moult. There is a cessation of ovarial development during this phase. Termination of this phase occurs about the middle of August, after a relatively short period of about nine weeks of facultative diapause.
It has long been recognised that diapause in insects is invariably accompanied by a fall in level of metabolism and therefore a decline in locomotory and feeding activity. There is also commonly a pre-diapause phase during which the insect feeds ravenously and builds up large reserves of fat and glycogen within the body. This process is usually followed by a decrease of water content. The metabolic adjustments of insects in diapause have been reviewed by Lees (1955, 1956) and more recently by
Harvey (1962).
It is now generally accepted that imaginal diapause is caused by the failing of corpora allata to secrete the gonadotropic hormone. This gonado- tropic hormone has been shown to be implicated in oxidative metabolism by
Thomsen (1949), Thomsen and Hamburger (1955), who found a marked decrease in oxygen uptake following allatectomy in Calliphora erythrocephala. This conclusion was supported by Sgges;e•, (1960) who worked on Leucophaea maderae and by de Wilde and Stegwee (1958) on Leptinotarsa decamlineata,.
In this study, growth, respiration, water and fat contents formed a physiological basis of comparison between beetles in diapause and those in the phase of ovarian maturation. The biochemical aspects of respiration .are beyond the scope of this thesis. 47.
The work is divided into three subsections; 1) feeding and growth,
2) respiration and 3) water and fat contents. An attempt is made to
synthesize the various findings in these experiments in the discussion. It
is considered that the aspects in this section have been treated in a preliminary manner.
1. Feeding and growth
Method
Male and female beetles kept in the insectary were dissected at
definite time intervals, (see chapter IV), throughout the adult life. The
contents of the gut were noted so as to determine whether they had been
feeding.
16 males and 16 females emerged on 1st July were marked individually
with Britfix colour dope. The weights of the beetles were taken every four
days to follow the growth of the internal organs. In addition, the weight
cycle of individual beetles was followed.
Result
Dissections to expose the gut contents revealed that both males and
females probably fed intermittently throughout diapause. The beetles, which
had fed previously, contained varying amounts of food in the large crop.
During the phase of ovarian development, the females fed more regularly and
the crops were normally filled with green plant matter (Stellaria leaves).
The results of these findings are summarized in Table 4.
The weight of the insect provides a good index of internal growth.
Fig. 6 depicts the mean weight of 16 males and 16 females taken at four
day intervals since their emergence on the 1st July. The weight ratios
calculated from the mean weights of successive values to the initial mean Table 4. Examination of the gut of 7-- 8 males and females of G. tanaceti of different ages
Physiological Alimentary Alimentary Alimentary Alimentary phase Age Sex :canal (full) canal (empty) Sex canal (full) canal (empty) Phase
1 day yy 0 7 de 2 6 Pre-diapause 11 8 days " 7 0 7 0
16 " II 5 2 II 4 3 Diapause 24 " It 3 4 4 3 Diapause 40 " II 4 3 3 4 56 " II 5 2 It 2 5
68 " 7 0 11 5 2 Maturation
It It 5 2 Copulation Maturation 82 " 7 0 96 It 5 2
Just 4 3 before Oviposition Just 5 2 after 1 day 3 4 Post- 3 days II 7 0 oviposition 5 II 6 1 49.
weights show that there was hardly any growth of the females throughout the period of diapause. Although there were fluctuations in weights of
individual beetles in this period, the weight ratios showed that there was, nevertheless, no appreciable loss in weight in the group as a whole. These
observations further suggest that the beetles must feed to maintain their normal weights in the diapause period.
There was an initial gain in weight in both males and females up to
12 days after emergence. This was no doubt largely associated with initial
intake of food.
The period of ovarian maturation corresponded closely with the
increase in weight of females. Loss in weight in the oviposition period was
due to laying of the batches of eggs. The weight cycle of one typical
female superimposed over the mean weight cycle in fig. 6 illustrates this
point.
2. Respiration
Method
Warburg respirometers were used for the determination of oxygen up-
take and carbon dioxide output. The respirometers were immersed in a
water bath which was thermostatically controlled at 25°C.. A detailed
account of the technique is given by Umbreit et al. (1947). The technique
for estimating carbon dioxide output was by the "Direct-method" (p.16-18).
0.2 mis of 10% NaOH was used to absorb the CO2 in the 02 uptake determinations
The limitations of the "Warburg" techniques are fully discussed by Umbreit
(1947, p. 11-12).
Determinations of the respiratory rates were performed on whole
insects. Eight males and eight females of known ages, kept in the insectary, F iq. 6 1—...--Oviposit ion phase 190 Pre-Iiapouse -41--Oiapause phase--II—Ovarian maturation phase ' phase
180
170
160
150
140 t; 99 batch ^.• 130 batch E E120
110 e- 100 -c
'6— 90
60
70 "yr 60
50
40 0
25 3 12 2.0
July August September October November
Fig. 6. Fluctuations in weight of cb' and 9T of G. tanaceti in the different physiological phases throughout adult life since imaginal emergence on 1st July. Mean weights are based. on 16 o1 and 16 w. Note weight cycle of typical 9 illustrating fluctuations in weight after laying batches of eggs. 51. were used throughout the experiments. The respiratory rates were determined at definite intervals corresponding to the periods given in the previous section, except in the phase of oviposition.
Two beetles per respirometer with four replicates were used in each set of experiments. Some experiments had to be repeated to obtain more consistent results. Readings of manometers were taken at 15 minute intervals for a period of five hours. The weights of the beetles were taken at the end of the experiments. A thermo-barometer was used throughout the experiments to correct any varying temperature:or atmospheric pressures in the course of the experiments. The flask constant for each respirometer was calculated from the formula given by Umbreit et al. (1947). The corrected manometer readings were then converted to volumes of gas consumed or evolved, standardizing the values at temperatures of 25°C and 760 mm. atmospheric pressure. The calculated volumes of 0 uptake were then plotted on a graph and the mean of 2 the four replicates was calculated and the results were then expressed in cubic millemetres of 0 consumed or CO evolved by one gramme live weight per hour. 2 2 The CO output was found to be erratic, unlike the 0 uptake. The 2 2 difficulty was overcome by calculating the amounts of CO2 produced at intervals of 1 or li hours. This method gave consistent results. The mean value of CO output was derived from at least 12 calculations for each set 2 of experiments.
The respiratory quotient (R.Q.) which would serve to indicate the general nature of metabolism was the ratio of CO produced per unit weight 2 per unit time divided by the corresponding 02 uptake.
Results
The results obtained are summarized in Table 5.
Tablc4 Oxygen uptake and carbon dioxide output of G. tanaceti throughout its adult life . Measurements were made with Warburg respirometers, based on 6 74Vana899;250C760 mm. pressure 99 031 0 uptake CO output 0 uptake CO output Physiological 2 2 phase Age mm3/gr./hr. mm3/gr./hr. R.Q. mm3hr./hr. mm3/gr./hr. R.Q.
1 day 645 503 0.78 896 762 0.85 Pre-diapause 8 days 696 626 0.90 903 831 0.92 16 days 690 573 0.83 955 888 0.95 24 " 461 401 0.87 923 877 0.95 Diapause 40 " 297 264 0.87 429 356 0.83 56 422 397 0.94 533 442 0.83 68 " 523 481 0.92 548 499 0.91 Maturation 82 " 719 654 0.91 942 867 0.92 96 " 954 878 0.92 53.
The males had a higher respiratory rate than the females at any comparable stage during the adult life. The highest respiratory rates of the females during diapause were at 8 and 16 days after the imaginal moult.
This period coincided with the period of active feeding. The R.Q. of
females was 0.90 at the age of 8 days, suggesting that oxidation was
probably one of proteins. The lowest R.Q. was 0.78 in 1 day old females.
At this period, the beetles had not begun to feed and stored fat seemed to be the main source of energy. The R.Q. throughout diapause did not normally
fall below 0.87. This value implied the combustion of a mixture of a varying
amount of fat, protein and carbohydrate. This rather high R.Q. during the
phase of diapause was not surprising in view of the fact that the insects
fed throughout this period. During the phase of ovarian maturation
G. tanaceti resumed active feeding and the R.Q. increased to about 0.92.
This suggests the oxidation of mainly protein and carbohydrate. There was
a higher level of metabolism as shown by the higher respiratory rates.
In males, a high R.Q. was maintained up to 24 days, probably
reflecting a longer active feeding period. The rather high throughout
diapause again suggested the combustion of ingested food.
The curves of 0 uptake in both males and females were roughly 2 U-shaped. U-shaped curves are considered characteristic of pupal develop-
ment generally. These results are comparable to those recorded in Anatolica
eremita (Edelman, 1951). In G. tanaceti, however, the 02 uptake during
diapause was higher than that in A. eremita implying that Anatolica is a
species with a more intense diapause. This insect does not feed during
the phase of diapause. 54. 3. Water and Eat contents
Method
Groups of 3 - 5 beetles of both sexes were weighed and then dried in an oven regulated at 110°C for 6 hours. The dehydrated beetles were cooled in a desiccator. They were weighed again to determine the dry weight and the water content. The dehydrated beetles were powdered, placed in a thimble and the ether soluble fat was extracted for 12 - 24 hours in a micro soxhlet extracting apparatus. After complete fat extraction, the ether-fat solution was concentrated by distilation, transferred to a weighing bottle and allowed to evaporate in a dust-free fumigant cupboard.
At least 3 replicates were carried out of each experiment. The water and fat contents were determined at definite times of the adult life.
Results a) Females The results of the experiments are summarized into table 6.
The results given in table 6 are the aggregate of the three replicates carried out in each stage. The results of the replicates gave fairly consistent values. In cases when one of the replicates Gave a significantly large discrepancy, the experiment was repeated.
The salient features of the experiments show that the water content of the females was high up to 8 days (78.8%) but tended to fluctuate at lower value (about 71.1%) during the diapausing phase, and showed no increase even after termination of diapause. These results are comparable to the studies made on a few species of beetles having an imaginal diapause, e.g.
Leptinotarsa decemlineata (Busnel, 1939), Anatolica eremite (Edelman, 1951)
Sitona qlindricollis (Davey, 1956) and Semiadalia 11-notata (Hodek and
Cerkasov, 1960). Table 6. W. Weights, water and fat contents of Galeruca tanaceti of different ages
Physiological Age Live wt.(mg) Dry wt. (mg) Percentage Fat wt.(mg.) Percentage Fat wt. per phase water fat dry wt. in mg, -c 1 day 630.44 137.61 78.2 16.92 12.3 1.54 Prs-diapause 8 days 682.69 144.89 78.8 17.45 12.1 1.75 16 " 631.96 194.50 69.2 24.14 12.4 2.41 24 " 577.91 173.24 . 70.0 18.62 10.7 2.07 Dipause 40 " 787.06 226.22 71.3 22.38 .9.9 2.03 56 si 890.83 238.36 73.2 26.45 11.1 2.05. 68 " 1415.79 373.84 73.6 36.15 9,7 2.78 maturation 82 " 1869.23 542.44 71.0 46.22 8.5 4.20 96 " 2309.47 608.44 73.7 49.47 8.1 3.81 Just after oviposition 1309.29 374.68 71.4 20.82 5.6 1.74 56.
The percentage fat/dry weight remained just over 12% during the pre-diapause phase, but tended to decrease gradually during the phases of diapause and ovarian maturation. The fat/dry weight percentage fell to
5.6 just after oviposition. The last column in the table shows the mean weight of fat per female. The results suggest that fat was being built up to the 16th day (1.54 mg. in 1 day old and 2.41 mg. in 16 days old
But the amount of fat present was fairly constant throughout diapause.
This suggests that fat was not utilized at all for energy during this phase,
Or, alternatively, it was replenished as soon as it was used up. During the period of ovarial development, the fat content increased up to 4.0 mg. per beetle but fell to 1.74 mg. in beetles which had oviposited. This was no doubt due to the fat contained in the ripe eggs.
The results of percentage fat/dry weight suggest that fat was being utilized during the phase of diapause and apparently contradicts the results of fat content per female. On closer examination, the two sets of results are in no way contradictory. Table 1 on morphometric measure- ments of the reproductive organs at different stages of diapause show that there is slight growth of the gonads and hence the increase in dry weight. Therefore, it was tentatively concluded that there was some detectable growth in the female reproductive organs (or other organs) although there was no oocyte differentiation during diapause. The trends, however, shown in these experiments, complement each other. b) Males (Table 7).
The water co ent of males paralleled that of the females, being high (79.0%) on the 8th day, and falling to about 72% during the subsequent stages. The significance of this is not known. The percentage Table 7. 05. Weights, water and fat contents of males of G. tanaceti of different ages
Physiological Age Live wt.(mg) Dry wt.(mg) Percentage Fat wt.(mg) Percentage Fat wt. per phase water fat dry wt. in mg.
lday 567.83 124.49 78.1 12.03 9.7 1.00 Prc-diapause 8days 580.10 117.61 79.7 10.03 8.5 1.00 16 491.69 135.13 72.5 12.47 9.2 1.25 24 549.20 161.79 70.5 16.68 10.3 1.52 40 " 582.83 163.66 71.9 14.87 9.1 1.45 Diapause 56 " 552.70 154.89 72.0 12.89 8.3 1.2S
Maturation 64 it 748.40 202.79 72.9 16.44 8.1 1.24
oo F ig.7 3^ o I 70
2 o[ so.V O 8 5
--4, io • • u.1:1 0. 40
O► E 30 E R , _ zo 0 b g 1 0 •
0
0-7 1000
900
600 O 1
600 E cV E 500 0' o—o 99 O 400
300 cy
Mt alter I 8 I6 24 40 56 68 82 96 days oviposihon PreA6oposse-1 Reproductive diapause Ovarian maturation phase phase phase
Fig. 7. Trends in water and fat content; in oxygen consumption and R.Q. of G. tanaceti in different physiological phases. Note weight of fat per 9 increases during phase of ovarian maturation and decreases after oviposition. Absolute fat content does not decrease "dfiring diapause. 59. fat weight/dry weight fluctuated between 8.0% to 10.0%. The amount of fat per male was found to increase from 1.0 mg. (1 day old d') to 1.52 mg. per male at the end of the pre-diapause phase. Then, the values tended to fall again in subsequent stages. The significance of this is not known.
As in the females, fat was considered either not utilized or, if utilized, soon replaced, during the diapause phase.
Discussion
The results from the foregoing experiments suggest that G. tanaceti passes through four physiological pbases during its adult life. The pre- diapause phase which lasts from 0 - 8 <16 days in the females is a period of relatively active feeding, high level of metabolic activity, high water content and a gradual building up of fat deposition (from 1.54 mg. per at 1 day old to 2.41 mg. per y at 16 days old). It is noteworthy that some of the B type N.S.C. of the pars intercerebralis are in the B1 phase in the 0 to 8 days old females, whereas, during diapause, these cells occur
phase. The presence of B cells also coincides with the periods in the BII I of higher metabolic activity as is evident in their occurrence during the ovarian maturation and oviposition phase. (See chapter VIII).
During the phase of diapause, dissections of beetles have revealed the presence of food in the gut, suggesting that they have fed. Diapausing insects which have been found to feed during the dormant period are not uncommon, e.g. Sitona cylindricollis (Davey, 1956), Trogoderma qranarium
(Burges, 1960), Dendrolimus pini (Gayspitz, 1949) and Anax imperator
(Corbet, 1956).
The respiratory rates of both males and females in G. tanaceti in diapause, decreased to a minimal "maintenance level" (Harvey, 1962). 60.
Slnma (1960a,b) showed that during the phase of diapause of the sawfly,
Cephalcia abietes, a decrease of 0 consumption is accompanied by a 2 corresponding decrease of cytochIome cxidase and succinic dehydrogenase activity. This confirmed the earlier observations of Agrell (1949),
3odine et al.(1952), Ludwig and Barsa (1955) that the highest activity of succinic dehydrogenase in insects is associated with a period of locomotory activity and growth (cited by Sl6ma, 1960a).
Harvey (1962) pointed out that during the entire life cycle, the intensity of oxidative metabolism is closely adjusted to the demands for
available energy. These demands can be resolved into three components, those associated with growth, maintenance and effector activities. He
added that respiration of the insect at any time may be viewed as a resultant of these components. The respiration in G. tanaceti may now be equated as follows. During the pre-diapause phase, a high respiratory rate was observed as a result of a high level of effector activities
(locomotory and feeding) and a low maintenance level, while in the diapause phase, the low respiration meets the demand of a lower level of effector
activities and maintenance level. During the phase of ovarian maturation, the respiration must necessarily be higher to meet in addition the demands
for growth. Feeding has been shown to be significant in raising the 02 uptake in Trocioderma granarium (Burges, 1960) and in Samia cynthia (Jones
and Wilson, 1959).
The 0 uptake was continuous in G. tanaceti throughout its life. 2 However, there was some evidence of cyclic CO2 release. This discontinuous
CO release seems to be a common feature in diapausing insects. Some 2 earlier authors e.g. Dreyer (1932) and Agrell (1951a) have found extremely 61. low R.Q. valuee and have attributed wrongly, either to the incomplete combOttion of fat or to the conversion of an "oxygen-poor" into an "oxygen- rich" substrate. However, the observations of Punt (1950) and Schneiderman and Williams (1953) have shown that although 02 uptake is continuous, the evolution of CO can be intermittent. Punt (1957) cites examples of 2 discontinuous CO2 output in some adult beetles. The cycles of CO2 release in bursts can vary from one per minute to 25 per hour (Hamilton, 1959) and half-hourly bursts to one per 24 hours or more (Schneiderman and Williams,
1955).
In G. tanaceti the CO output was found to be erratic when manometric 2 readings were made at 15 minutes intervals but when the readings were taken at 1 hour to I* hours intervals, they gave consistent R.Q. This suggests that CO was released in bursts between 1 to 1* hours. However, it was 2 realized that the "Direct Method" employed here was a crude method of determination of CO2 evolution. As the experiment proceeded, the 02 concentration in the respirometer declined and this may have had side effects on the true nature of the CO output. The periodic brief opening 2 of the spiracles to release bursts of CO2 during diapause is considered as a mechanism of water conservation (Buck, 1962).
The percentage of fat body/dry weight did not rise substantially prior to entry into diapause in G. tanaceti. Whearas, in some instances of intense diapause as in Scoliopterix libatrix fat constitutes 56% of dry weight (Sacharov, 1930). In hibernating females of Culex pipiens fat forms up to 60% of dry weight (Buxton, 1935).
It has been found in several insects that allatectormy causes enlargement of the fat body, viz. : Melanoplus differentialis (Pfeiffer, 1945) 62.
Calliphora erythrocephala (Thomsen, 1952), Lucilia sericata and Sarcophaqa
securifera (Day, 1943), Oncopeltus fasciatus (Johansson, 1958), Dermestes maculatus (Ladduwahetty, 1962) and in G. tanaceti. From these observations,
it could be surmised that the presence of the corpus allatum hormone
(gonadotropic or metabolic hormone) somehow controls the excessive deposition
of fat and its absence leads to fat accumulation. In G. tanaceti, during
the phase of diapause, the corpus allatum has been found to function at a
very low level and one would expect this deficiency of the c.a. activity to
lead to fat depositions. This was in fact found to be partly true. Fat
was being gradually built up and was maintained at a certain level through-
out diapause. Unfortunately, these results in G. tanaceti cannot be
expressed with confidence since small samples were used in these experiments.
However, it seems reasonable to suggest that in G. tanaceti the corpus
allatum or some other unknown source of hormone functions at a level of
activity which is sufficiently high to control excessive accumulation of
fat. This equilibrium of fat deposition is no doubt compensated by the
small amounts of food ingested to provide the raw materials for the
manufacture of fat.
Some insects, before entering diapause, feed actively and build up
a huge deposit of fat and glycogen within their body. This accumulation
of fat, I suggest, may be a consequence of the failure of activity of the
corpora allata. 63.
VII. Experiments on the effects of photoperiodism and temperature on the induction and termtnation of reproductive diapause and on growth of G. tanaceti
Introduction
Most authorities to-day accept the significance of neuro-endocrine mechanisms in the regulation of diapause. Whereas opinions differ as to the pecise role of the endocrine system in the chain of events which leads to the onset or termination of diapause. The N.S.C. of the brain, while retaining.a sensory function may well provide the link between the external environment and the endocrine system. The classical work of Williams
(1946, 1947, 1948, 1950) on the pupal diapause of Hyalophora cecropia has established this link experimentally and found that chilling of the brain for a certain period, up to six weeks, activates the N.S.C. of the brain.
The N.S.C. in turn activate the prothoracic gland which promotes metamorphosis and growth. Van der Kloot (1955, 1958) provides both physiological and biochemical results to confirm Williams' experiments.
The determination of voltinism in Bomby's mori by Fukuda (1951-1953) implicated the action of the brain and the suboesophageal ganglion as a link mediating the external and internal environments.
The external environment, therefore, ultimately determines the onset or termination of diapause. Photoperiodism and temperature are now regarded as external stimuli by most authorities in initiating, sustaining and finally terminating larval, pupal and imaginal diapause.
In a majority of insects studied, namely Leptinotarsa decemlineata
(de Wilde, 1955) Antheraea pernyi (Tanaka, 1944) and Harrisina brillians
(Smth and Langston, 1953), photoperiodism is considered as the primary causal factor. The effect of temperature has been found to modify the 64. photoperiodic effect on diapause as evident in the studies on Diataraxia oleracea (Way and Hopkins, 1950), Bombyx mori (Kogure, 1933),
Metatetram,chus ulmi (Lees, 1953a, 1953b), Barathra brassicae (Matsumoto et a1.1953;,0tuka and Santa, 1955) andAbraxas miranda (Masa.ki, 1958)
The aim in this study was to investigate the effects of photoperiodism and temperature on the induction and termination of reproductive diapause in
G. tanaceti. The role of the food plant, which may contribute to a more subtle and intricate balance of the total natural environment of the insect, has not been considered.
In these experiments an attempt was made to study simultaneously the growth rates and fecundity of G. tanaceti kept at different photoperiods.
The sensitive stage of the insect was also considered in regard to its response to photoperiod and temperature.
As a control, to follow their growth, insects subjected to a photo- period of 12 hours at 20°C were dissected at regular intervals from 8 to 30 days, for examination of the reproductive organs. Qualitative and
quantitative measurements were made on the male and female reproductive
organs. This will provide a basis for comparison of growth of the re- productive organs under experimental conditions with those under normal
conditions.
Methods
a) Each cage contained a minimum of 12 72 and 12 4661 which were subjected
to fixed photoperiods varying between 18 hours to 8 hours at 24 hour cycles
at a temperature of 20°C., since adult emergence. Specially constructed
light-proof, well-ventilated cabinets with time switches were used in some 65. of these experiments. The intensity of lighting varied from 80 lux to
240 lux (inside the cage).
Three further sets of experiments kept at photoperiods of 16 hours,
12 hours and 8 hours were set up at 15°C.. A time switch was set regulating a 16 hours of lighting in this C.T. room. The dark phases of the two shorter photoperiods were accomplished by covering and uncovering the cages with black polythene sheets at more or less fixed times.
As a control at 20°C. 13 females and 12 males were kept initially at a photoperiod of 18 hours for two weeks, followed by a decrease of 2 hours every two subsequent weeks to a final photoperiod of 8 hours.
Individual beetles were marked with Britfix colour dope for identific- ation. From 24 days after the start of the experiments when the beetles had shown a response in some photoperiod regimes each individual beetle was weighed every four days. A record was kept of the total number of batches of eggs laid by all the beetles. By comparing successive weights of individuals, it was possible to know which particular beetle had oviposited.
Each set of experiments lasted 84 days (except in the experiments for photo- periods of 18 hours and 16 hours at 20°C where the diapausing beetles had
to be sacrificed for further experiments). b) 3 - 4 males and females were dissected at regular intervals from 8 days to 30 days at the photoperiod of 12 hours at 20°C. Qualitative and
quantitative measurements were taken of the reproductive organs. (Same
method as in chapter IV).
Results
In interpreting the results, the experimental beetles were classified
into three categories;- 1) diapausing beetles or those in which weights 66. remained more ox less constant throughout the experimental period. Post- mortem dissections confirmed the absence of ovarial differentiation;
2) non-diapausing females or those in which weights increased steadily and were accompanied by abdominal distension and absence of oviposition and, finally 3) ovipositing females. Only ovipositing females were considered as those in which reproductive diapause had successfully terminated.
The results are summarized in Tables8, 9 and 10.
It was found that all the experimental females kept at 18 hour photo- period at 2000. had undifferentiated ovarioles. At a photoperiod of 16 hours, no beetles oviposited but 25% of them had differentiated ovarioles,
although there was no yolk formation in the terminal oocytes even after 72
days. At photoperiods below 14 hours, most of the beetles had terminated reproduction diapause but there was varying percentages of ovipositing females, depending on the photoperiods (see table 8). The critical photoperiod at o . 20 C. lq G. tanaceti was between 12 to 14 hours. However, at photoperiod
of 10 hours the incidence of diapause increased to 83.3%
All the beetles at 15°C at photoperiods of 16 hours, 12 hours and
8 hours' had terminated their reproductive diapause. At photoperiod of 16
hours, 63.6% oviposited with an average of 1.1 egg batches per female.
It was found that although the ovarioles differentiated oocytes readily, and
the maturation of eggs proceeded normally, full fecundity was not achieved.
Their abdomens were consequently filled with mature eggs and dissections
revealed symptoms of egg resorption in varying numbers of eggs in the
vitellarium at the end of the experiments. o Beetles kept at photoperiods of 12 hours and 8 hours at 15 C,
produced larger percentages of ovipositing females than those kept at the • Table 8. The incidence of diapause, non-diapause and oviposition of G. tannceti subjected at different constant photoperiods at 20°C with accompanying fecundity. Results based on 12 - 13 9 at - - each phOtoPeriod
Photoperiod Percentage Percentage Percentage Total No. Av.No. of Av. No. of Percentage diapause at non- non-diapause of batches eggs/ eggs/batch survival end of Expt. diapause Ovipositing laid ovip. 9 expt. period
18 hr 100 0 - - - - 90.2 16 " 75 25 0 - 83.3 14 " 8.3 91.7 90.7 20 2.2 48.4 75 12 " 0 100 83.3 37 3.7 52.4 66.7 10 " 0 100 16.7 3 1.5 41.3 75 8 " 0 100 63.6 18 2.6 53.3 23.3
Tabble 9. The incidence of diapause, non-diapause and oviposition of G. tanaceti subje;ted at different constant photoperiods at 15°C with accompanying fecundity. Results based on 12 - 13 5,) at each photoperiod
Photoperiod Percentage Percentage Percentage Total No. Av. No. of Av. No. of Pezcentvge diapause at non- non-diapause of batches eggs,/ eggs/batch survival end of Expt. diapause Ovipositing laid ovip. 9 Expt. period
.110.1.1.111•110.....01110 16 hr 0 100 63.6 8 1.1 32.'3 63.6 12 " 0 100 100 37 3.7 48.1 81.9 8 fl 0 100 90.9 27 2.7 42.7 51.8 ••••••••••1•.11111. ,•••••••1
Table 10. Subjected to decreasing photoperiod at 20°C (2 weeks at each photoperiod frDp 18 hours to 8 hours) ..11•1111••••••••• ••••• 0 100 100 37 2.8 52.1 69.2 -2 68. same photoperiod at 20°C. The mean number of eggs per batch laid at o o o 15 C, however, was lower than those at 20 C.. This suggests that at 15 C. there was probably less ingestion of food and therefore less egg growth.
The production of eggs varied directly to the amount of food ingested has been established in Oncopeltus fasciatus by Johansson (1954, 1958).
Fig. 8 depicts the incidence of diapause in the beetles kept at different photoperiods at 20°C. and at 15°C..
All the beetles, i.e. 100%, subjected to decreasing photoperiods from 18 to 16 - 14 - 12 - 10 to 8 hours, two weeks at each photoperiod, terminated diapause successfully. The beetles commenced laying eggs (mean
52.1 eggs per batch), shortly after being transferred to the 12 hour photo- period but ovarial development and egg maturation had proceeded at the photoperiod of 14 hours.
The results of these experiments indicated and confirmed that
G. tanaceti is a "short-day" species, and a low temperature (i.e. 15°C) has a compensatory effect of short day-lengths.
Rate of maturation and oviposition of G. tanaceti kept at different photo- periods and temperatures
By following the increase in weight of individual beetles at four day intervals, it was possible to identify which beetle had terminated diapause. The time taken to determine diapause and of subsequent oviposition
Was thus available. The results are summarized in table 11, 12 and 13.
Fig. 11 depicts the time taken for development of ovarioles and
subsequent oviposition in individual beetles kept at different photoperiods
at 20°C. and 15°C. 69
A. Fiq. 8
100 NO MD
80 0
• 60
U "1:1J 171 .0 40
I • 20 U
0 I8Hr I6Hr. I4 Hr. 12 Hr. 10Hr 8Hr.
B. 20°C. 100
8ci. 80
60
s , 205 - 40 15 2 10 20 U 5 r, "4). 11. 0 0 Ib• Hr. 14 Hr. kwir PHOTO PE RIODS
•
Fig. 8.A. The effect of photoperiod at 20°C. and 15°C. on the incidence of diapause in G. tanaceti. (dotted line represents hypothetical photoperiodic reaction curve at 15°C.) B. The effect of decreasing photoperiods, 2 hours of photoperiod per 2 weeks, from 18 hours to 8 hours at 20°C. on the incidence of diapause. Note oviposition commences at 12 hour photoperiod. Table 11. The effect of photoperiod and a time factor involved in the teraination of reproductive diapause and on the subsequent oviposition of G. tanaceti. Temperature 20°C. Days IN Photo- 120' period 24 28 32 36 40 44 48 52 56 60 64 68 72 76 80 84 18 hr 0 0 0 0 0 0 0 0 0 0 0 0 0 0 16 " 0 0 0 0 0 8.3 8.3 8.3 8.3 8.3 8.3 25 0.).4.t(0 0 14 " 25 50 58.3 66.7 75 83.3 E3.3 91.7 91.7 91.7 91.7 91.7 91.7 91.7 91.7 91.7 r-4 M •Cr4 12 " 68.3 58.3 66.7 75 83.3 83.3 83.3 83,3 90.9 90.9 90.9 100 100 100 100 100 0. 44 $4 RI .r4 10 " 16.7 16.7 16.7 16.7 16.7 33.3 33.3 33.3 41.7 80 80 100 100 100 100 foo -0 8 It 8.3 33.3 36.4 54.5 63.6 72.7 90.9 100 100 130 1)0 100 100 100 100 - - - - - ,_ ------.. - - 1 - 4 1 2 1 - 1 - 2 3 1 - 1 4 2 5 7 2 2 - 3 1
1 "• -^ 1 ''' ''' .' •" 1 - - - 1 2 4 1 5 2 1 1 1
18 " ------44 J: 0 4-) 16 " - - - - • fp 0 -0 14 " - 61 - - 61.3 60 51.5 54 54.5 58 - 49 25 25.3 O 0 01 O. 12 " 70 - 52 54.7 55 56 59 59 55.2 55.5 52.7 62.5 36.5 - 24.7 16 • W O 0) 10 " - 48 - - 49 27 -
< 0 8 I t - - 63 59.5 61.7 63 52.2 41 50 48 26
Table 12. The effect of photoperiod and a time factor involved in the termination of reproductive diapause and in the subsequent oviposition of G. tanaceti Temp. 15°C Days Photo period 24 28 32 36 40 44 48 52 56 60 64 68 72 76 80 84 cn 0 0 a I 16 hr 9.1 27.3 63.6 63.6 90.9 100 100 100 100 100 100 100 100 100 100 100 ei .r-I RS m E -,-, E F-i *0 a12 " 63.6 81.8 100 100 100 100 100 100 100 100 100 100 100 100 100 100 0 0 U) Lk -1-) in 0 g 8 n 15.4 30.8 38.5 46.2 61.5 69.2 76.9 76.9 84.6 90.9 100 100 100 100 100 100 . R. Ca -1-) 0. i ts, A-, a) 16 " lim V. .... * - 1 - 1 2 - - 3 1 - Ili 44 >4)1" -1:1 0 LH X0=12 " - - - 5 2 3 5 2 -2 3 6 7 1 o 0w •r-1 .4-1 O .0 co > 8" O. Ia. •1M. ••1 - 2 1 1 2 4 3 2 6 5 Z-0 - ° o 16 " - - - - - 42 - 32 45 - - cr, 23.3 24 Co 4-1 (13 FA 0 .0 - - .. ••• - 61 48.5 60.3 53 0 12 " 54 35 48.6 - 40.7 43.9 22 •o k Z 0 8" - - .. - - 49 41 49 32 62 42.3 33.5 - 39.3 40 24
Table 13. The effect of decreasing photoperiod and a time factpr involved in the termination of reproductive diapause and in the subsequent oviposition of G. tanaceti Temp. 200c. 8 hr/d Photoperiodl 16 hr/d 1 14 hr/d 1 12 h /d 1 10 hr/d Days 24 28 32 36 40 44 48 52 56 60 64 68 72 76 80 84 % of females terminated diapause 0 0 7.7 46.2 53.8 92.3 100 100 10C 100 100 100 100 100 100 100 No. of batches of eggs laid by YIZ 4 4 6 9 7 2 1 2 2 Av. No. of eggs per batch 69.3 57.8 51.5 39.1 57.1 55.5 54 50 42 - 72.
Fecundity of G. tanaceti kept atdifferent photopertods and temperatures
During each period of weighing, the number cif batches of eggs faid were noted, and the number of eggs per batch was' counted. The results are shown in tables 11, 12 and 13.
Beetles showing the highest-featndity were those kept at the photo- o period of 12 hours at 20 C.. The average number of egg batches laid by an ovipositing female was 3.7 with an average of 52.4 eggs per batch (cf. beetles under natural conditions laid an average of 5 egg batches, average number of eggs per batch was 61.9): Beetles with a comparable fecundity were those kept at photoperiod of 12 hours at 15°C.. The average number of egg batches laid was 3.7 per female but the mean number of eggs per batch was 46.1. Although the beetles subjected to a decreasing photoperiod had a relatively lower fecundity than those kept at the 12 hour photoperiod, the results were not conclusive. These experiments lasted 84 days. In the experiment on the effects of decreasing photoperiods, the oviposition period was confined to 36 days instead of the 60 days in those beetles kept at 12 hour photoperiod.
The total egg production could be taken as an index of the effective- ness of the photoperiod regime and of temperature in the termination of diapause in this species.
Ovarial maturation of G. tanaceti kept at different photoperiods and temperatures.
Growth in G. tanaceti has been shown to be associated with the development and maturation of the ovaries and this in turn has been correlated with increase in weight.
The means of weights of all beetles kept at different photoperiods 73. were calculated. These were taken 24 days after the beginning of the experiments (in some, after 28 days) and these were treated as the basic initial value. The means of weights obtained subsequently, at four day intervals, were expressed as ratios of the initial means. These calculated ratios of weights indicated the rate of growth of the internal reproductive organs. These growth rates are depicted in figs. 9 & 10.
There was practically no growth in beetles kept at photoperiods of
18 hours and 16 hours at 20°C..
The general trend of growth in beetles kept at photoperiods of 14 hours and below at 20°C. was one of steady increase until the middle of the oviposition period, when growth generally tended to be on the decline. In contrast, beetles kept at 10 hour photoperiod at 20°C., increase in weight was gradual until the end of the experiment. At this photoperiod, although the ovarial cycle was normal, ovulation and hence oviposition was retarded, probably as a result of a low level of endocrine activity. Dissections of the beetles, at the end of the experiment, showed signs of oocyte degeneration and resorption. This condition is comparable to that in the diapausing
adults of tiscus maroinalis (Joly, 1945) and Leptinotarsa decemlineata
(de Wilde, 1954).
The general trends in maturation of beetles kept at 15 C at the photo-
period of 12 hours and of 8 hours were comparable to those at 20°C..
Beetles subjected to decreasing photoperiod (decrease of 2 hours in
2 weeks) showed a steady indrease in growth rate until the end of the photo-
period of 12 hours. At the 10 hour photoperiod there seemed a decline in
growth. It appears that the photoperiod of 10 hours at 20°C. is incompatible
with growth in this species. The maximum growth reflected by the maximum 74
F g .9 z0 A. 8hr/day
1-5 10
10 5
I I I a 0
2.0 B. 10hr/day
1.5 10
1-0 -o o
20 C. 12hr/ day - -0- a.) 1.5 _c
1.0 5 CM cr
0 0 w°
25_ D. 14hr/ day 0 cc 2-0 0 E 1— 1.5 0Z C.) 1.0 $ Li -10 1 .5 _ E. lbhriday
1.0
05
F. 18hriday
1-0
0-5
P • 1 24 28 32 36 40 44 48 52 56 60 64 68 72 76 80 84 DAYS
Fig. 9. Weight ratios of w of G. tanaceti kept at different photoperiods at 2000. Dotted lines represent theoretical weight ratios, taking into account the weight of eggs laid. The figure also illustrates the distribution of oviposition at different photo-regimes. 75
F iq 10 2.5 _ A. 8hr/ day - Ay------20
1.5 i10
10 5
a 0
2-0E B. 12 hr/day -°-- —II— —Z,---71:-147.-1-_ 4,12:22...7..v 110 i-5 _.-'':----- ;,------"- 1.0 c--- S
I I I I I I I I I 0
a 2.5 C. I 6hr/day
20
0 1.5
0 1.0
f • ' 0 o
_C ber 0'C) Num
D. Decreasing pnotoperiods at 20°C.
--- 16 hr. 411r.
25
2.0
I.5 110
1.0 S . . I I I 24 28 32 36 40 44 48 52 56 60 64 68 72 76 80 8 4 DAYS
Fig. 10. Weight ratios of 99 of G. tanaceti kept at photoperiods of 16 hours, 12 hours and 8 hours at 15''C. and at decreasing photoperiods at 200C. Dotted lines represent theoretical weight ratios, taking into account the weight of eggs laid. The figure also illustrates the distribution of oviposition at different photo-regimes. Fig. II
80 H80_ B. 0 0 0 0
70 70 • 8 60 8 0 0 60 8 S. 0 O• • 0. 0 0 O• 0 8$ 50 8 • 50 8- 8 • 00 v-) 0§. 0 • O• >--Y • 00 o >- • oo < • 0 40 a ° S a 40 • • • • • • 2* 30 30 • • o
20 20
o egipositim_ 9 10 O ovipositima • maturing 9 • maturing 9
Ibnr. I2hr. 8hr. 18 hr. I I6h r. 14 hr. 12 hr. IOhr. 8hr.
PHOTOPERIODS PHOTOPERIODS
Fig. 11. Rate of..:oVarial maturation and oviposition of individual .n of G. tanaceti, subjected to different photoperiods. A. at 15°C. and B. at .20°C..
17 Fig. 12
I6
IS
14 a 13
45 12
A II 0 AO
9
Reproductive Ovarian Ovi position diapaust maturation
20 mean air temperature .,,....,...... —.11 ------.. 15 rr r . ... • ."N- .--...," ,-, ...,. 4-1 5 „.• r.....----..../ i --- .------,
I mean ground temperature I I o i 1 . . s . 1 June July August September October November
Fig. 12. The main physiological phase of adult life history of G. tanaceti, controlled by hours of day-length and temperature, under natural conditions. (Hours of day-length is derived from difference of hours between sunrise and sunset. Data, 1962). 78. number of eggs laid occurred when the beetles were being subjected to the photoperiod of 12 hours. Thus, there exists a close relationship between growth rates and the effects of photoperiodism and temperature to the extent which diapause is terminated on the one hand and fecundity on the other, via successful ovipositions.
The sensitive stage of G. tanaceti in response to photoperiodism and temperature
According to de Wilde (1962) who reviewed the effects of photoperiod- ism on insects and mites, all stages, excepting the pupae of the insect life cycle, may be sensitive to photoperiodism.
Some preliminary experiments were carried out on G. tanaceti to determine the sensitive stage. Eggs were kept in the refrigerator and were therefore subjected to darkness most of the time. When diapause development was completed, (terminology of Andrewartha, 1952) the larvae hatched out and were divided into two sets. One set of larvae was bred throughout the larval period at a photoperiod of 12 hours at 20°C. and the other set was bred at a photoperiod of 16 hours and 20°C.. The adults which emerged from these two sets responded normally to photoperiodism.
From this preliminary investigation, it was concluded that the sensitive and responsive stage are the same, namely, in the imagos of the same generation in G. tanaceti. This result is comparable to those found in Leptinotarsa decemlineata (de Wilde, 1954) and in Coccinella septemrunctata(Hodek, 1958).
Growth of reproductive organs at the photoperiod of 12 hours at 20°C.
Beetles which were subjected to a photoperiod of 12 hours at 20°C. were dissected at definite intervals before oviposition (30 days). The
Table 14. Qualitative and morphometric changes of reproductive organ; of G. tanaceti kept at 99 12 hr/d. 20°C.
Age Length of Ovariole Length of Length of Length Df Spermatheca Width of sp. ovariole condition oocyte I oocyte II germarim gland (mm) (mm) (mm) (mm) (mm) 8 days 0.83 Undifferentiated - . 0.33 All empty 150 p it it 12 " 0.85 - 0.34 180 u Beginnings of li - 0,40 190 p 16 " 0.88 oocyte I it 20 " 1.12 Differentiation 0.36 - 0.50 200 p oocyte I 24 ,, 1.34 Oocyte I & II 0.54 0.19 0.52 Full of sperm* 230 p il 30 " 1.86 Ovulation 1.19 0.43 0.53 260 p oocyte I, II & III
* Spermatozoa bundles
Table 15. Qualitative and morphometric changes in the male reproductive organs of G. tanaceti kept at 12 hour photoperiod at 20°C.
Age Mean length of Condition of Width of Vesiculae seminales testicular lobe testes acesscry glands
8 days 1.40 mm. All stages of meiosis 123 p Empty 12 " 1.43 " 126 p ft to 11 16 " 1.51 " 137 p 20 " 1.52 " 175 p It 24 " 1.68 n 254 p Full of spermatozoa 30 " 1.72 11 298 p 81. qualitative and morphometric changes of the male and female reproductive organs are tabulated in tables 14 and 15. Measurements were taken from
3 - 4 specimens in each stage.
The results show that there was an initial period lasting about 16 days when the beetle was still in diapause. By 30 days, however, the dissected beetles revealed fully mature eggs, some of which had been ovulated. This period of development and maturation of ovarioles appeared to take place at a faster rate than under normal conditions; period of maturation lasts about 30 days (see table 14). In males, the period of diapause appeared to be extended to over 20 days at a photoperiod of 12 hours at 20C (see table 15).
Reversible effects of photopexiodism and of temperature in G. tanaceti
G. tanaceti had shown sustained reproductive diapause at photoperiods of 18 hours and 16 hours at 20°C.. Such diapausing beetles, irrespective of the duration in these regimes, could be induced to oviposit in less than 30 days when transferred to a photoperiod of 12 hours (see chapter X).
Conversely, if ovipositing or maturing beetles, in which diapause had o terminated, were transferred to a photoperiod of 16 hours at 20 C., further oviposition ceased in between 20 - 30 days (see chapter IX).
These findings in G. tanaceti are comparable to the result seen in
Dendrolimus pini (Gayspitz, 1949, 1953). In some cases, however, it has been shown that a few photoperiodic daily cycles may be decisive as in
Diataraxia oleracea (Way and Hopkins, 1950).
Discussion
G. tanaceti evidently belongs to the "short-day" species with a 82. facultative reproductive diapause. Up to now there have been very fev studies: viz; the bivoltine race of Bombyx mori (Kogure, 1933), some races of
Antheraea pernvi (Danilevskii and Gayspitz, 1948), Abraxas miranda (Masaki,
1958), Chorizaqrotis auxiliarus (Jacobson and Blakeley, 1959) Stenocranus minutus (Willer, 1957b, 1958) and the autumn race of the weevil
C'uthorrhynchus pleurostiqma (Ankersmit, 1960).
G. tanaceti is also fairly unique in having two diapause stages in its life cycle, namely, in the egg stage and in the imaginal stage.
Danilevskii (1961) cites two other species both Lepidopterous, as having two diapause phates in the life cycle, namely Operoptera brumata and Exapte conqelatella, which have a summer diapause in the pupal stage and a winter diapause in the egg-stage.
Evidence from the foregoing experiments has indicated that oviposition in maturing females must be regarded as the criterion of successful terminatio of reproductive diapause. This interpretation may seem at first sight to differentiate the process of ego maturation from the process of oviposition.
They may well be two distinct physiological processes. I have some some evidence from morphometric analyses of the neuro-endocrine system which are substantiated by autoradiographic studies that these two processes are controlled by different levels of hormones; a higher level of hormonal activity stimulating oviposition, while a lower level maintaining continual ovarial develbpment.
The effects of photoperiodism and temperature are considered es the primary causal factors in controlling diapause in G. tanaceti. From the experimental evidence available it appears that photoperiodism is the primary causal factor and the effect of temperature modifies it. 83.
Investigations on "short-day" insects have shown that high temperatures may promote diapause as in B. mori (Kogure, 1933) and A. miranda (Masaki,
1958). The results obtained in G. tanaceti in this respect are in agreement with these two species.
As a contrast, experiments with "long-day" insects have shown that high temperatures tend to avert diapause as in D. oleracea (Way and Hopkins,
1950), M. ulmi (lees, 1953), Pieris brassicae (David and Gardiner, 1952) and in Acronycta rumicis (Danilevskii, 1961).
Unfortunately, there is insufficient data on the effects of photo- periodism at 15°O. on G. tanaceti to establish an empirical relationship between the effects of photoperiodism and of temperature as an overall influencing factor.
The effects of gradual changes in daylength have not been thoroughly investigated in the past. In Anax imperator there is evidence that diapausing nymphs metamorphose promptly when subjected to a gradually increasing photo- period (Corbet, 1956). On the other hand, in Acronycta rumicis, shortening of daylength in itself is not a factor which regulates diapause. Here the effect is wholly determined by the critical photoperiod in the course of the shortening of daylengths (Tsvetaeva, cited by Danilevskii. 1961). It has been shown that G. tanaceti subjected to a decrease of photoperiods from
18 hours to 8 hours, decreasing by 2 hours every two weeks, 100% of the females successfully terminateddiapause, whereas, at none of the constant photoperiods to which G. tanaceti was subjected, had this 100% positive effect. This aspect of the dynamics of daylengths in diapause has yet to be studied more fully.
The r6le of light and dark phases of unnatural cycles have been 84. shown to control diapause. Their effect depends on the absolute lengths of both these phases. Studies of this aspect have been carried out in a in few species, e.g. /Metatetranychus ulmi light and dark phases appear to be antagonistic to each other; long light periods favouring uninterrupted development and long dark periods favouring diapause (Lees, 1953). The significance of the natural 24 hour cycles of light and dark phases in controlling diapause is much to be desired. This may lead to some under- standing of photoperiodic reaction in relation to diurnal rhythms of physiological and effector activities.
The actual site of perception of photoperiodism has not been definitely located, although there is sufficient evidence that the head and thorax provide such effective areas, arising mainly from the work of
Gayspitz (1957), (cited by de Wilde, 1962) on Dendrolimus, Acrenye%a and
Pieris and Lees (1960b) on Met:pure viciae. In G. tanaceti the neuro-
endocrine system has been shown to be implicated in the control of diapause,
ovarial maturation and oviposition. (see chapters VIII to X). 85.
VII. Studies on the cyclical changes of the neurosecretory system during adult life history of G. tanaceti
Introduction
Since the discovery by Wigglesworth (1936) that the secretion of the corpus allatum will induce egg development in Rhodnius, many authors have confirmed his finding. de Wilde et al (1959) have evidence that diapause in Leptinotarsa decemlineata occurs as a result of an "endocrine deficiency syndrome" of the corpora allata.
The studies of Williams (1947, 1943, 1952) on H. cecropia establish that the neurosecretory cells of the brain as the source, producing a hormone which activates the prothoracic gland to produce ecdysone
(Butenandt and Karlson, 1954). Ecdysone initiates growth and moulting.
Thomsen (1952) has evidence in Calliphora erythrocephala that the medial N.S.C. of the brain exert an overall control over the endocrine centre. In Dermestes maculatus, the medial N.S.C. have been shown to control the normal functioning of the corpus allatum and oviposition (Ladduwahetty,
1962), and in Carabus nemoralis, the medial N.S.C. are thought to control the activity of the corpus allatuth (Klug 1958/59). Similarly, the studies of Highnam (1961, 1962) and Gillett (1958) have shown that the N.S.C. of the brain control ovarial development.
The N.S.C. of the suboesophageal ganglion produce a diapause hormone which determines voltinism in Bombyx mori (Fukuda, 1951, 1952 Hasegawa, 1952;
This resume makes it compelling that a comparative histo-physiological study of the neurosecretory system of G. tanaceti is necessary. ivletpzb
The technique for the rearing of G. tanaceti has been described 86. on p. 19 In this series of experiments, the same males and females which had been used for morphometric studies on their reproductive organs
(chapter IV), were used in the present study oh the neurosecretory system.
The staining technique used was PF., counterstained with Erhlich's haematoxylin and light green. All serial sections were cut at 4 p. a. Determination of nuclear volume
The nuclear volumes were calculated on the assumption that the nuclei were perfect spheroids. The general formula for the volume was then:.
V a2b where a and b were two diameters. Measure- 3 --- 8 ments of the diameters of the nuclei were taken in sections where they were largest. This meant that the calculated values are not to be taken as absolute volumes but rather a close approximation of their size. As the nuclear volumes served as a basis of comparison of the nuclear sizes of the different stages,this technique has proved to work very satisfactorily.
Morphometric studies of the corpora cardiaca and allata
Three values were determined as a basis of comparison fox the morphometrical changes of the corpus cardiacum and corpus allatum, each taken in turn of the different stages of the beetle's life. The three values were:- 1) the volume of the gland, 2) the number of nuclei and
3) the nuclear-cytoplasmic ratio or the nuclear density.
Determination of volume
Outlines of successive sections of the glands were drawn under
10 x 45 magnification, with a camera lucida. The area of the outline draw- ing was measured with a planimeter graduated in centimetres. The area was 2 then converted into 01.1 by a conversion graph. This graph was constructed 2 from areas of known values in which have corresponding values in Cm. 2; 87. with the planimeter from the camera lucida drawings. The volume of the section was calculated by multiplying the area by 4 ( = thickness of the section). The total volume of the gland was the summation of these calculated volumes of the sections. Five calculated volumes using the above method were determined to test the accuracy of subsequent modified methods.
In diapausing and maturing females, outline drawings of the glands were made of alternate sections. The areas of the skipped sections were taken as intermediate between consecutive sections. This method was found to be satisfactory.
In ovipositina females, the glands were very much larger and volumes of every fourth section were calculated. The total volume was derived by multiplying all the calculated volumes of sections by four. This again gave results which were very near to those calculated by the first method.
These modified methods were used subsequently with confidence.
Determination of nuclear-cytoplasmic ratio
An accurate determination of the density of nuclei met with certain difficulties. These difficulties resulted from the crowding of nuclei and the irregularity of nuclei distribution. I have modified Scharrer's (1958) technique, by directly counting the number of nuclei in three representative sections selected in approximately the same location, i.e. the largest section in the middle of the series plus the fourth section from the middle onetbher side. The total area of the three sections and the sum of nuclei counted in the area permitted the calculation of the number of nuclei per 2 mmm?. . In addition, the nuclear density were calculated from the second and the penultimate sections of the series. This was done because 88. observations indicated that the peripheral cells of the glands were generally larger.
Determination of nuclear number
This index was necessary to determine whether volumetric increase in the corpora cardiaca and allata was due to a rise in cytoplasmic content or whether this was accompanied by an increase in the number of nuclei. The following general formula by Engelman (1957) and of Scharrer and Von Harnack
(1958) was used:-
N =N xV 1 Ax (T + 2r)
where N = total number of nuclei in each gland;
N number of nuclei counted in.A; 1 A = total area of five sections selected for nuclear counts;
V = volume of each gland;
T = thickness of section;
2r= average nuclear diameter.
This formula could be satisfactorily used in diapausing females, when the average diameter was less than 8 p. In ovipositing females, where the nuclear diameter was greater than 8 p, part of the formula was modified as follows:- T was taken always as 4 a) but where 2r was greater than 8 p but less than 12 p, the fraction (T + 2r) was substitutedby (4 + 12) b) if 2r was greater than 12 p but less than 16 p, the fraction
(T + 2r) was substituted by (4 + 16).
Otherwise Engelman's formula gave a higher estimate. 89.o.
The calculated values using the above modified methods were tested against the actual counts of nuclei in detailed maps of successive sections and
showed no more than 5% error in five test cases.
Analyses of varying amounts of stainable material in the neurosecretory systems at different stages of life history
The A-cells of the brain using PF. exhibited varying amounts of
stainable inclusions in the cytoplasm of the cell. The A-cells were divided into five arbitrary categories designated as stages I, II, III, IV
and V. A-cells in stage I had little neurosecretory material, whereas cells
in stage V were packed with aggregates of neurosecretion. These arbitrary
stages are depicted in figure 13 .
Relative quantities of A-cell secretion discharged along the axom
at the time of fixation were represented by arithmetical symbols from -, +
H- and +++.
Relative quantities of different secretions of all types stored in
the corpora cardiaca at various stages were represented by the same symbols
as above.
An index of A-cell sizes was calculated on those cells which were
either ellipsoidal, flask-shaped or spherical. In 17 beetles, when all
the A-cells were examined it was found that only about 10% were irregular
in shape. In the others, the cell volume was calculated with the formula:- 2 V = 4 yr a b where a and b were the two diameters; 3 8 -b was the diameter of the cell along the axoftalaxis (excluding the axon):,
It must be stressed that these calculated values served only as-a compatative
index of the cell sizes, rather than their absolute volumes. 89. liesults
1. Changes in the N.S.C. of the pars intercerebralis a. A-cells
The percentage distribution of the stages of the 42 - 46 A-cells in the pars intercerebralis of all female beetles at known ages were first examined and the results are summarized below:-
Table 16. Percentage distribution of A-cells in the pars intercerebralis of G. tanaceti 99; each stage based on 4 - 5 beetles
Physiological Stage Stage Stage Stage Stage Phase Age I II III IV V 0 day 17.2% 33.1% 45.0% 4.7% Pre-diapause 1 " 2.7% 15.6% 29.9% 47.6% 4.2% 8 days 26.1% 44.9% 29.0%
16 tl 10.8% 32.4% 46.1% 10.8%
11 Diapause 24 .11.• 11.8% 14.3% 41,0% 32.9% 56 It 9.3% 18.5% 13.0% 27.8% 31.5% 68 10.0% 13.6% 8.2% 30.9% 37.3% olaturation 82 It - 28.9% 39.5% 31.6% 96 6.1% 12.2% 30.5% 38.2% 13.0%
Just be- 14.9% 41.6% 31.2% 12.3% IMO Oviposition fore Just 14.9% 17,2% 31.6% 35.6% 0.6% after
1 day 2.7% 8.7% 13.4 38.3% 36.9% Post- oviposition 3 days 9.1% 47.3% 41.8% 1.8% 5 " 5.6% 33.3% 45.1% 16.0%
During the phases of diapause and ovarial maturation, most of the
A-cells were in stages III, IV and V. But during the short oviposition
phase, most of the cells were in stages I, II and III. Then at varying
periods during the post-oviposition phase, there was a gradual but distinct 90. shift of distribution of the cells containing more secretion to cells generally containing intermediate amounts of secretion. Thus, 1 day after oviposition, 38.3% of the cells were in stage IV and 36.9% in stages V. Two days later, none of the cells belonged to stage V and 47.3% were in stage II and 41.8% in stage III. This gradual shifting in frequencies of cells containing varying amounts of secretion at different time intervals, suggests that the arbitary stages probably represent phases of a secretory cycle.
The nuclear volumes of all the measurable A-cells havebeen determined
in the different stages of the life cycle. The nuclear volumes have been taken as an index of assessment of the relative activity of the N.S.C., the
larger the nuclei, the more active the cells (cf. Lea and Thomsen, 1962).
The results are presented in table 17.
In 0 and 1 day Old females, the stages IV and V of A-cells tended to
have larger nuclei than the other stages. Whereas, at all other periods,
stage I and II cells tended to have larger nuclei than those containing
more secretory material in them. It would seem, therefore, that cells
generally containing fewer inclusions in them, were more active than those having more inclusions, except in those just after the imaginal moult.
Here, it could be considered that the A-cells were in their initial secretory
cycles, and therefore the more active the cells, the more secretion they
had synthesized in them.
Nuclear measurements of all A-cells in their designated stages have
been plotted in fig. 14. The salient features show that : 1) although
the cells with the same histological appearance (hence the same stage),
their nuclear volumes varied i.e. they appeared to be in different levels
of activity at the same age, 2) there appeared to be long cycles of 91. secretory activity within the same stage in different periods of the same
physiological phase and 3) these long range cycles of activity gradually
intensified towards and at the phase of oviposition.
A-"cell volumes" The calculated "cell volumes" summarized below, show a positive
relationship with the nuclear volumes, i.e. large cells tended to have large nuclei. 3 Table 18. Means of A-"cell volumes" in p of the pars intercerebralis of G. tanaceti 4, based on all measurable cells of 4 - 5 beetles per stage
Physiological Stage Stage Stage Stage Stage phase Age I II III IV V 0 day - 1:98.8 1734.5 1722.4 1934.1 Pre-diapause 1 " 1060.6 1386.8 1662.8 1948.8 1914.4 8 days - 1451i2 1917.6 2184.9 16 days - 1449.5 1523.2 1538.2 1570.0 Diapause 24 " - 1813.2 1463.0 1463.8 1314.5 56 " 2104.9 1954.1 2043.2 1814.5 1595.1
68 1I 4090.3 4059.4 3554.7 2942.1 2454.8
II I Maturation 82 mm0 4211.9 2660.8 3099.8 2417.7 96 I 4595.9 3336.1 3479.3 3470.4 3088.0 Just. be- 3340.0 2902.3 2843.5 2464.6 Oviposition fore Justafter 6924.9 4213.4 3833.5 3539.7 1 day 5804.0 5135.5 4142.2 3525.2 2893.8 Post- oviposition 3 days 3942.6 4181.2 3496.7 2749.4 5 " 4013.0 3948.3 3315.9 2997.6 3 Table 17. Mean nuclear volumes p of A-cells of pars intercerebralis of feaale G. tanaceti at different stages of adult life based on all "measurable" nuclei of 4 - 5 beetles per stage.
Physiological Age Stage 1 Stage II Stage III Stage IV Stage V _phase in des ± 0 341.2 = 15.6 331.1 7.9 333.0 - 6.0 391.7 ± 7.4 ± Pre-diapause 1 317.4 326.4 - 7.1 343.3 - 4.3 366.4 4.0 416.0 ± 10.5 ± 8 386.6 = 18.3 383.0 ± 7.3 381.7 7.4 ± ± 16 422.7 ± 15.3 330.2 7.8 359.5 5.9 348.0 - 10.2 Diaoause 24 350.1 ± 17.2 296.2 - 9.6 266.6 ± 3.1 263.6 4.2 + 56 582.1 432.0 - 18.9 342.0 - 13.5 232.8 ± 12.2 262.3 - 10.3 68 445.0 ± 22.6 431.6 ± 12.6 411.7 ± 25.2 352.7 - 6.6 342.1 j- 7.4 Maturation 82 616.1 337.3 305.8 ± 11.1 322.3 ± 9.2 356.7- 8.8 96 522.4 477.4 ± 30.2 494.1 ± 20.1 447.3 ± 13.2 387.5 ± 19.0 Just before 491.3 ± 15.8 489.9 ± 9.9 412.2 - 3.2 418.8 - 13.4 ± Oviposition Just after 599.3 9.06 563.3 ± 16.3 528.3 - 13.1 512.3 - 9.7 551.0 564.2 - 27.0 441.1 - 13.5 427.9± 11.2 412.2 - 6.9 Post- 1 day 576.1 - 28.8 oviposition 3 days 495.0 ± 12.2 496.5 - 5.9 478.9 ± 5.8 448.2 5 days 625.8 514.7 - 10.0 490.4 - 7.4 464.8 ± 9.1 93
Fig. 13
sta9c I JI IH Iv I
b) sta9eT
s tage I
20.
Fig. 13. Secretory stages of A-cells of pars intercerebralis of the brain in G. tanaceti. a. phase of diapause, b. phase of ovarian maturation, and c, phase of oviposition.
94
80 - stage I . 700
600
500
400 • • • 300 •
stage it 700 — • • • • 600 ■ ■ r
.—, 500 I r j•-• 400 r F c 300 r 200 700- stagell. 600 I. • 0 500 ▪ • L 400' r 11. S L • Oc 300 1 Lt.) 200 _J stage EL 600 z 500
400 V 300 200 600 500 stage11. L. I. 400 - I •
300 200 I I I I 0 I 8 _LB. IA. 1 3 SI DAYS ,_Pre-thopause D i apause ion ma t urotion___Ov 'position Post oviposihon phase phase phase phase
Fig. 14. Distribution of nuclear volumes of all A-cells, stages I, II, III, IV and V of G. tanaceti throughout adult life. Note cyclical fluctuations of nuclear volumes of same cell stages throughout life history. 95. b) B-cells of the medial groups of pars intercerebralis
The 3-cells of the pars intercerebralis apparently existed in two physiological phases, B1 and B . Cells in 3 phase stained yellowish II brown and in B phase, they stained green with PF.. However, they both II stained up red with CHP. or ABP. The total number of B-cells was found to be constant 10 - 13. Cells in B phase appeared on imaginal emergence
(beetles less than 24 hours old) but by 8 days their number decreased and was followed by a reciprocal increase in numberof BI, cells. By 16 days all 3-cells were in B phase and occurred predominantly throughout diapause. II By 68 days, when diapause was terminated, most of the 3-cells occurred in D phase with a reciprocally smaller number of cells in BII phase. Just I before oviposition, the frequency of 3I cells was 11.7 and dropped abruptly to 4.3 just after oviposition. It would seem that they are implicated in oviposition. Their nuclear volumes also increased towards the oviposition phase as the A-cells, suggesting a higher level of activity in them during this phase.
The number of C-cells varied from 2 - 5 throughout the different phases and in some beetles they were completely unidentified at some stages.
However, they appeared in their apparently full numbers during the oviposition phase. Their functional significance is not known. The results obtained on B and C cells are tabulated in Table 19.
2. Changes in the N.S.C. of the lateral group of the brain
It has been said before that two types of N.S.C. were located in the lateral groups. Observations made on the histology of these cells revealed that there were no apparent cycles of secretion within them in contrast to the A-cells of the medial group. However, their numbers 3 , Table 19. Cyclical changes in frequencies and nuclear volumes (in p ) of BI, BII and C-cells of the pars intercerebralis of G. tanaceti ?9, based on 4-5 females per stage
Physiological f. Nuclear vol. f. Nuclear vol. f. Nuclear vol. B phase Age 8, BI-cells II BII-cells C C-cells 0 day 10.0 323.8 4.3 537.8 Pre-diapause 1 II 8.0 364.5 3.5 342.6 4.3 538.2 8 days 4.5 380.4 7.5 300.8 1.0 476.0 16 I. - 12.0 324.0 2.3 443.5 Diapause 24 1. - - 13.0 344.6 4.0 616.3 56 2.7 388.9 10.0 362.3 0.3 516.7 68 " 10.7 356.2 1.3 359.3 3.3 524.9 maturation 82 12.0 386.2 3.7 542.1 96 9.7 424.3 3.0 366.2 2.3 487.9
Oviposition Just before 11.7 352.7 1.3 393.5 5.0 475.6 Just after 4.3 428.6 6.3 494.1 4.0 601.7 1 day 7.5 425.7 4.3 531.1 2.3 646.8 Post- 0.5 oviposition 3 days 12.5 393.7 649.8 4.5 546.8 5 n 9.' 368.8 1.0 607.3 4.3 516.5 Table 20. Cyclical changes in frequencies and nuclear volumes of AI and D cells of each lateral group of N.S.C. of brain in G. tanaceti at different stages of adult life, each stage based on 4-5 y_y_
D-cells DTcells Physiological AI cells AI-cells 3 3 phase Ace frecuency nuclear volume „u frequency Nuclear volume p 0 day 2.6 345.4 6.5 314.1 Pre-diapause 1 " 2.5 356.4 7.3 339.2 8 days 1.6 350.1 7.8 337.4 16 1.9 340.9 8.9 315.4 Diapause 24 11 1.7 310.3 7.7 312.7 56 1.3 316.9 10.2 330.7 68 ), 2.7 363.3 10.8 354.5 Ovarial q 1.7 397.2 10.5 384.6 maturation 82 96 1.7 424.2 11.5 360.3
100 11 2.7 415.6 10.1 382.8 Oviposition 100 1.2 417.3 9.8 376.0
101 1.0 472.1 7.0 512.9 Post- SI 2.3 496.1 11.0 492.6 oviposition 103 105 1.4 581.3 11.6 461.4 98. fluctuated at different stages of the life cycle. Nuclear volumes of these cells revealed certain trends which might be correlated with activity of these cells (Table 20).
The mean frequencies of A/-cells varied from 1.0 to 2.7. In fact,
-cells in the lateral groups was 2 - 3 cells. But the normal number of AI at all stages, since some of these cells contained little or no secretion, they failed to stain up. The cells depleted of secretion suggest that they were probably non-functional at that stage. The same applied to the D-cells.
Their number varied from 6 - 13, probably as not all the cells were functional at any one phase. Their mean frequencies, however, showed a steady increase from the diapause phase to the oviposition phase. There was also a steady increase in nuclear volumes towards the oviposition phase.
This coincided with increased activity of the N.S.C. of the medial groups.
3. Changes in the N.S.C. of the_suboesophageal ganglion
Only 2 - 3 AI cells comparable to the histology of AI-cells of the lateral groups of the brain, were discerned in the suboesophageal ganglion using the PE technique. After using the CHP. and ABP. techniques, 8 phloxinophil cells, i.e. B-cells, have also been found distributed in three groups. The functional significance of these phloxinophil cells is problem- atical. The nuclear volumes of the AI-cells were relatively large during the pre-diapause phase, denoting greater secretory activity. During diapause, their nuclear volumes decreased, being lowest just after the 3 termination of diapause when the mean volume was 303.2 p . Coinciding with 3 ovarian maturation, the mean volumes increased steadily to 659.4 xi just after oviposition. This high level of secretory activity of the AI-cells was maintained with minor fluctuations throughout the oviposition phase. 99.
Table 21. Cyclical changes in frequencies and nuclear volumes of the A 1 of the suboesophageal ganglion of G. tanaceti,n based on 4 - 5 beetles per stage,
Physiological Mean nuclear 3 stage Age Frequencies volumes, p
0 day 2.0 488.6 Pro-diapause 1 " 1.5 477.2 8 days 0
16 " 0.8 329.4 Diapause 24 " 1.0 350.4 56 2.0 312.0
68 2.0 303.2 Maturation 82 2.0 393.6 96 2.0 496.2
Just before 2.0 546.3 Oviposition Just after 2.0 659.4
1 day 2.0 867.2 Post- 3 days 2.0 632.5 oviposition 5 days 2.0 844.6
Unfortunately it has not been possible to trace the entire axonal I=7,thways of these cells. The corpus allatum-suboesophageal nerve exists in
G. tanaceti and it is tempting to suggest that these cells may have a stim- ulatory effect on the activity of the corpus allatum. Engelman (1957) has
experimental evidence that the suboesophageal ganglion stimulates the
activity of the corpus allatum via nervous impulses. Harker's (1960c)
observations on Periplaneta americana, on the other hand, show that the di rection of theaxonal transport of neurosecretory material is from the
corpus catdiacum to the suboesophageal ganglion. The subtle relationships
100.
between ,he various endocrine centres has yet to be resolved.
In considering the quantitative amounts of stainable neurosecretory
materials of the system at any stage, it seems desirable. to take into
account the amount of secretion, particularly that of the A-cells, along
the axons of the N.S.C. of the pars-intercerebralis. The same symbols
were used to denote the relative amounts of secretions of other cell-types
stored in the corpus cardiacum. The observed results are summarized below:-
Table 22. The quantitative amounts of stainable neurosecretory material present in the neurosecretory system of females of G. tanaceti at different stages of adult life; each stage based on examination of 4 - 5 specimens
Physiological A-cell secretion Storage in corpus cardiacum phase Age along the axons A-cell Bicell BII and D C-cells
0 day -H- 4/0
Pte-diapause 1 "
8 days 40,
16 Ott 24 Di apause 40 11 56 I I +++
68 -1-1-+ +++ +++ 00 Maturation 82 -H- 4-14- 44+
96 I I -H-I-
Just before -H- Oviposition Just after 4-1-
1 N + ++ ++ + 0/. Post - 3 'i 4- + ++ + - oviposition 5 )4 + + ++ + + ? 101.
There were small amounts of A-cell secretion transported along the axons throughout diapause. At the stage of termination of diapause and at the initial stages of ovarial development, the amounts of stainable
uiaterial was at a maximum (3+) along the axons. Then, towards the phase of oviposition, the quantity of stainable material declined and was maintained at a relatively low value (+).
The quantity of A-cell secretion stored in the corpus cardiacum was
fairly substantial at day-0. The amount increased by day-16 to 3+ and this level was maintained throughout diapause until day-56, when the quantity
began to diminish. The phase of ovarial maturation corresponded with build up of secretion along the axons following the release of a vast
quantity of secretion from the medial groups of N.S.C.. Simultaneously,
the amount of secretion began to accumulate in the corpus cardiacum. At
the phase of oviposition, the amount of A-cell secretion stored in the
corpus cardiacum was generally small, although the N.S.C. were at a high
level of secretory activity. These observations suggest a differential rate of rdlease of secretion from the cells, transportation along the axons
and discharge from the corpus cardiacum, at different phases.
The B-cells secretion stored in the corpus cardiacum followed a
similar pattern depending on the activity of either the cells in the B1
phase or the B phase. Secretion from the "chromophile cells" of the II corpus cardiacum was discerned in the gland. at certain stages
but because its tiAorial affinity was similar to the BII secretion,
the amount of B secretion from the brain is doubtful. Further, the D-cells II of the lateral groups of N.S.C. stained up similarly to the BII cells
with PF. and hence the presence of their secretion stored in the corpus
F ig. 15
Pre-d lapause_i D apause_iOvarian maturation—it Oviposition phase phase 900— phase phase
AI Cells SG 800
700 e6. -13 BriCetls 600 C AI Cells
C Cells E soo A Cells
D Cells O
400 BI Ceils
300
200*-
O a 16 24 56 68 82 96 J.B. J.A. 1 3 3 DAYS
Fig. 15. Cyclical changes in mean nuclear volumes of all cell types at different physiological phases of G. tanaceti throughout adult life. Note nuclear volumes tend to increase towards the phase of oviposition. 3 103. cardiacL.m could not be measured accurately. During the phases of maturation and oviposition when there were relatively few cells in the B11 phase and generally a larger number of D-cells, the secretion in the corpus cardiacum was considered to be mainly of D-cells.
4. Qualitative and quantitative changes in the corpora cardiaca
Three quantitative values formed the basis of comparison of the changes which occurred in the corpora cardiaca throughout the adult life of the beetles. The results of this investigation are set forth in Tables
23 & 24.
The volume of the corpus cardiacum was smallest at the imaginal moult but increased to twice the size by the 16th day. This volume was maintained more or less throughout diapause. During post-diapayse, the
volume of the gland increased steadily and was greatest just before ovi- position reaching 500% of the original volume. Just after oviposition the gland decreased considerably in volume. There appears, therefore, a correlation between glandular activity of the corpus cardiacum and
oviposition. In the phase of post-oviposition, there was a tendency for
the volume to increase again.
The increase in size has been attributed mainly to increase in
cytoplasmic content of the gland ( see fig. 16). The calculated nuclear number of the gland fluctuated in different beetles between 318 to 752
during the "active phases". This suggests that there is high variability
of nuclear numbers in beetles even at the same physiological phase.
Therefore increase in volume of the gland cannot be attributed to an
increase in number of cells. The amounts of neurosecretory material from Table 23. Quantitative changes in the corpus cardiacum of G. tanaceti females at different stages of the adult life, based on 6 - 10 glands per stage.
Total Mean nuclear Physiological Mean volume 3\ Nuclear density phase Age (100 thousand,u ) No. of Nu/an' nuclear No. diamater 0 day 4.39 6,783 372 7.6 ,u Pre-diapause 1 " 6.26 6,651 551 7.6 p
16 days 9.08 5,597 622 7.4 p 24 6.03 5,302 422 7.3 p Diapause 40 9.06 4,556 51! 7.2 Al 56 8.61 3,670 403 7.7 p 68 14.01 2,996 531 7.7 p Maturation 82 ft 17.50 2,389 521 7.5 p 96 it 15.42 2,257 433 7.3 p Just before 21.81 2,476 462 8.7 p Oviposition Just after 14.48 3,050 436 8.2P 1 day 17.40 2,592 439 8.2 p Post- 3 days 17.69 3,072 467 8.4 AI oviposition 5 days 22.57 3,166 642 8.6 p Table 24. Quantitative changes in the corpus cardiacum of G. tInaceti w, based on 6 - 10 glands per stage.
Physiological Range in vol. of c.c. Range in nuclear- Range in number phase Age (in 100 thousand u3) cytoplasmic ratio of nuclei immil•••••••••••• 0 day 4.30 - 4.46 6,271 - 7,355 346 - 410 Pre-diapause 1 s, 4.86 - 7.88 6,165 - 7,207 419 - 724 16 days 6.70 -11.60 4,833 - 6,084 510 •- 701 Diapause 24 " 5.54 - 7.21 4,978 - 5,449 377 •474 40 " 7.18 -11.13 4,314 - 4,936 443 - 615 56 " 7.34 -11.85 3,063 - 4,098 353 - 454 68 " 11.37 -16.51 2,636 - 3,565 375 - 667 Maturation 82 " 15.13 -19.84 2,172 - 2,714 441 - 579 96 " 13.46 -20.82 1,892 - 2,616 318 567 Just before 17.85 -24.75 2,228 - 2,874 427 - 499 Oviposition Just after 11.65 -17.77 2,468 - 3,333 385 - 504
1 day 14.00 -22.27 2,192 - 3,090 397 - 488 Post- 3 days 12.06 -20.18 3,072 - 3,252 376 - 519 oviposition 5 " 20.34 -27.25 2,779 - 3,791 571 - 752
0 (31 106 FigA6 41(27161
24 7000
22
E E 20 b000 • _ ••
0 0 ;' o 16 - woo E C C
0 4P- 0
w 12 •4000 ° E E >• 10 0
8- ?.` 3000 •• 'vs .4 z
4 2000
7O0
2400
300
# n 0 I I 24 40 56 61 82 96 J B. JA. I 3 • 5 DAYS Pre•cliapause4—.— i a pause F_Ovarian maturation 4— Oviposition .1 L Post•roiposi lion —i phase phase phase phase
Fig. 16. Graphic representation of morphological changes in the corpus cardiacum of W at different physiological phases of G. tanaceti throughout 1 adult life. Note increase in volume of the gland is, inversely related to the nuclear-cytoplasmic ratio. For details see text. 107. the braid stc'ed L1 the corpu'i cardiacum might contribute some change in volume but this would be negligible. The secretory "chromophile" cells enlarged correspondingly with the size of the gland. There was also increase in their nuclear volumes (table 23) at the phases of ovarial maturation and oviposition. During these phases, they contained within them large granules 7 x 5 u stained green with PF.. The matrix of the gland also contained large vacuoles.
5. Qualitative and quantitative changes in the corpora allata
The sequence of changes in the corpus allatum during adult life was no less spectacular as compared to the corpus cardiacum. The results are summarized in tables 25 and 26 and in fig. 17. There was the initial small increase in volume during the pre-diapause phase. The small volume of the gland was maintained during diapause. The nuclei were packed closely, being distributed homogenously in the matrix of the gland. The inactive phase was characterized by a low cytoplasmic content, absence of vacuoles 3 and small nuclei (volume about 150 p ) of a uniform size.
At the termination of diapause, the gland increased steadily in volume. This was accompanied by a marked increased in the cytoplasmic content and in nuclear volume. During this phase, the nuclei differentiated into two types; about 50% of them, mostly the peripheral cells, increased in size far faster than those of the central region of the gland (see table
25). The peripheral cells, no doubt, formed the main secretory cells.
The gland assumed a characteristic heterogenous distribution of nuclei, due to the differential 4growthdrate of the individual cells and the presence of vacuoles. Table 25. Cyclical changes in the corpus allatum of C. tanaceti ??, based on 6 - 10 glands per stage .1•1•11.0•1•01•••••m00....•••••••••••• .•11.. Physiological Mean vol. of c.a. Nuclear cytoplasmic Number of Ratio of nuclear phase Age (100 thousand.0) ratio (no. of nuclei/ nuclei density of mm2) pheral/mid recilon 0 day 0.98 20,750 307 0.89 Pre-diapause 1 day 1.33 19,222 336 0.92
16 days 0.78 23,185 245 0.84 24 " 0.86 21,471 248 0.83 Diapause 40 " 0.94 20,231 263 0.77 56 " 1.08 18,500 25_ 0.£1
68 " 1.25 14,367 238 0.93 Maturation 82" 2.60 9,880 269 0.86 96H 6.55 4,802 376 0.84
Just before 21.96 2,538 408 0.69 Oviposition Just after 11.56 3,386 323 0.59
1 day 11.12 4,080 311 0.77 Post- 3 days 20.33 2,712 312 0.79 oviposition 5 ty 13.13 3,451 271 0.79
0 CO • Table 26, Cyclical changes in the corpus allatum of G. tanaceti based on 6 - 10 glands per stage Physiological Range in vol. 04 c.a. Range in nuclear-:range in nuclear Range no. phase Age (100 thousand u ) cytoplasmic ratio cytoplasmic ratio of nuclei (mid region) (periphEral region) 0 day 0.83 - 1.42 18,315 - 22,921 16,522 - 21,983 223 - 397 Pre-diapause 1 " 1,08 - 1.69 16,703 - 22,099 15,294 - 22,540 244 - 466 16 days 0.60 - 1.19 22,794 - 26,471 18,209 - 24,889 195 - 377 24 " 0.72 - 1.18 21,038 - 26,789 16,471 - 22,034 217 - 312 Diapause 40 " 0.72 - 1.16 20,859 - 24,875 16,86 - 18,904 223 -• '04 56 0.78 - 1.21 16,832 - 22,723 16,186 - 18,321 212 - 295 68 " 0.93 - 1.41 7,433 - 22,250 9,369 - 17,027 208 - 260 Maturation 82 " 1.78 - 4.91 8,356 - 13,316 6,901 - 11,923 190 - 408 96 " 5.91 - 11.84 3,710 - 6,953 3,118 - 5,652 278 - 467 Just before 16.60 - 29.88 2,427 - 3,453 1,833 - 2,361 363 •- 502 Oviposition Just after 8.09 - 21.14 3,061 - 4,528 1,299 - 3,771 209 - 405 1 day 7.77 - 14.47 3,752 - 5,038 2,500 - 4,521 247 - 407 Post- 3 days 15.18 - 25.98 2.710 - 3,278 1,922 - 2,775 269 - 421 oviposition 5 10.19 - 15.87 2,270 - 5,957 2,540 - 3972 171 - 362 110
Fiq.17
30 27000
25000
20 •A N 23000
21000
to 19000
-.140001 d n usa 120003
ho 10' t
0 I
10 8 . 10000
o 6 6000°
E E O 4 • 6000
2 4000 7.7
0 2000
500
X400 0
c aoo 00 • 4200 E ; 100
0 1 16 24 40 56 68 • 82 96 I 3 5 DAYS .Pre D a pause IHOyarian maturation_o_Ovipositioru L Post-oYiposition Phase phase phase phase
Fig. 17. Graphic representation of morphological changes in the corpus allatum of 9 at different physiological phases of G. tanaceti. Note increase in volume of the gland is inversely related to the nuclear-cyto- plasmic ratio. For details see text.
III Fig. I8
500
400 I . . . . " . ; .
• r r T1 . • • E 300 • . .73 ir O 1.- 1 • i. 7 r ii. t
200 & ?.. [IL " 1 . 1
t P " P • Ii •Ib I •• • s• bi= rI - F i 111. r . • : . 100
It I 1 _t 1
posit ion B. phase
4000
F—Ovarian matured ion—I phase
3000
Giant 111.106
2000 i___.Rcproductive dicipause--1 phase
. c 1000-
700 E 600 0 > 500 I P"gi4T"el
400
300
200
100
1111 11 0 I 16 24 40 68 82 96 B. 4A. I 3 5 DAYS
Fig. 18.A.Cyclical changes in nuclear volume of 'chromophile' cells of the corpus cardiacum at different physiological phases of G. tanaceti. B. Changes in nuclear volumes of corpus allatum of 9; at different physiologi;a1 phases. Note the appearance of giant nuclei (volume greater than 1500p1 during phase of oviposition. The two curves after 68 days illustrate the size limits of all nuclei of the gland. 112
Fig. 19
Fig. 19. a. Section of corpus allatum of diapausings? in G. tanaceti. b. Corpus allatum during phase of ovarian maturation. c. Corpus allatum during phase of oviposition. d. Corpus cardiacum during phase of diapause. e. Corpus cardiacum during phase of ovarian maturation. f. Anterior part of corpus cardiacum during phase of oviposition. ax., axons; chb.c., chromophobe cell; chp.c., chromophile cell; chp.s., chromophile cell secretion; o.n., giant nucleus; n.s.m. neurosecretory material from the brain; va., vacuoles. I I 3 fig.20
Fig. 20. Medial group of Neurosecretory Cells in pars intercerebralis of 99 of G. tanaceti. a . In 1 day old ci?. b. 8 days old 9. c. 24 days old 2. d. 68 days old e. Just before oviposition. f. Just after oviposition. All photomicrogra phs about same magnification. A. A-cell; BI., BI cell; BII., BII cell. 1 1 4 tig.21 'i•••• • • jr **s
• • \-
• 4 ..- . 'fr.::•,-.,-•4, •.• ...,, - '..,-,----,,,;,.„.-„,_ .---:*-=-i:-.A,p...i,, t-•-. • .:-.... -747.-,..p."''....•.-rtzvez,-.....". ,.._4- ...... z.k . ". '.,-.:,....u.....4.*". -• " .'.P'. 4 ":•,1.1t..,. '' .4 t i. ....' • • •• • . • . t /.• 1 •••••• .•
•
,
..•
•
Fig. 21. Axons containing varying amounts of stainable neurosecretory material from the medial group of N.S.C. of n at different physiological phases in G. tanaceti. a. 1 day old 9.. b. 24 days old c. 68 days old f. 68 days old ?. d. Ovipositing e. Non-ovipositing mature 9. I15 a.
4 C.O..
fr7 c, '0*
e. f.
)Fig. 22. Corpus cardiacum and corpus allatum ofd at different physiological phases in G. tanaceti. a. 0 day old Q. Note substantial neurosecretory material from brain; small c.a.. b. Corpus cardiacum of 68 days old 2. Note large amount of neuro- secretory material from the brain. c. Corpus cardiacum of ovipositing q. Note large droplets of chromophile cell secretion. d. Corpus cardiacum of 'post-ovipositing 9. Little neurosecretory material from the brain. e. Corpus allatum of ovipositing 9. Note giant nuclei and vacuoles. f. Corpus allatum' of older ovipositing 9. Note giant nucleus in pycnosis...... ,•?...,., .,),„., \ 7-4-.•foil'ile,- ;. , ,t••;011-•1-.; • , ,.: ..../c:f'-.i:."...:,,Is-'6;,c -,-,...-Zx-v,ve?•Mt.r.: -y PI . _ 1.,„,?•&,..kcsl,o . ;..1-,-. 7 . ..* it. •,...'N '7.4'.. 1,VIC, ^ .144 :F...*:c. '4. • 0 4' t • 4 , .• ez ,,, , 1,47,
•
•t:1 "Z.W.. C-
•
. "7-,••77- .• •
4 rit'isiA7t
Fig. 23. Neurosecretory Cells of suboesophageal ganglion of G. tanaceti.. a. 2A1 cells of pre-oviposition 9 . b. 2A1 cells of 5 days post-oviposition 9. c. 2A1- cells of 3 days post-oviposition d. 2B cells_of_posterior group, their axons traversing backwards. e. 4 B cells of medial group of maturing 9. f. 2 13 cells of posterior group of ovipositing q, 117
•
-
Fig. 24. a. N.S.C. of medial group of ipositing 9 of G. tanaceti, subjected to 16 hours photoperiod at 20°C. after 30 days. b. As in a. Note large C cell. c. D cells of lateral group in non-ovipositina mature 9. d. N.S.C. of medial group in 3 days post-ovipositing 2. e. N.S.C. of medial group in mature 9 having N.S.C. of the suboesophageal ganglion `cauterized. Note N.S.C. similar to those of diapausing f. Axons of non-ovipositing mature 9. Note large amounts of stainable neurosecretory material similar to that in maturing N. 118 fig. 25 4,11PTIP•F„- •.' _ff., • ti
f.
Fig. 25. a. D cells of lateral group in ovipositing 9 of G. tanaceti. b. Corpus cardiacumAllatum of 1 day post-oviposition 9. Note practically no neurosecretory material from brain. c. N.S.C. of medial group in 9 just after oviposition. d. N.S.C. of medial group in y 3 days after oviposition. d. N.S.C. of medial group in 9 3 days after oviposition. e. 2 B cells of anterior group of suboesophageal ganglion in 3 days after oviposition. f. Corpus allatum of ovipositing 119.
During Liie phase or oviposition, the gland increased to a maximum
volume (more than 20 times that of the diapausing gland). There was a
marked corresponding increase in cytoplasmic content and the incidence of
giant nuclei comparable to those described by de Lerma (1932), Palm (1947),
Kaiser (1954) and by Scharrerand Von Harnack (1958).
Just after oviposition, the corpus Ealatum decreased significantly
suggesting an unknown role for the gland in the process of oviposition.
Throughout the phase of oviposition, however, the corpus allatum maintained
a high level of secretory activity as was evident from their volumes,
nuclear-cytoplasmic ratios, persistence of giant nuclei and the occurrence
of vacuoles.
The increase in size of the gland was attributed solely to the
increase in nuclear-cytoplasmic content. During the phase of ovarial
maturation, there appeared to be a slight increase in nuclear numbers.
However, no mitotic figures had been seen at any stage and the variability
in nuclear number among the individual beetles made it unlikely to be an
important factor. During the phase of post-oviposition, some nuclei
showed symptoms of pycnosis. These nuclei assumed an irregular shape and
contained very diffuse chromatin material and indeed towards the end of the
life cycle, the number of nuclei decreased.
The higher level of activity of the corpus allatum as witnessed by
four indices, correlated with the process of oocyte differentiation, oocyte
maturation and oviposition. These results are in agreement with the related seasonal changes in corpus allatum/to the reproductive phase in Dytiscus
marginalis (Joly, 1945) and the general seasonal activity in Carabus
nemoralis (Klug, 1958/59). 120.
It was observed that the peripheral cells of the corpus allatum were generally larger than those of the middle region of the gland (see table 25 - ratio of nuclear density of peripheral region/mid region).
Just after oviposition, this ratio was lowest 0.59, implying that the cyto- plasmic contents of these peripheral cells were 6C2,11arger than those of the middle region.
These histo-physiological data suggest that during oviposition, the stored secretion of the corpus allatum could contribute to the large size of the gland, since after oviposition the gland decreased significantly in volume. It appears that autoradiographic studies may elucidate the dynamics of secretory activity of the corpus allatum.
Discussion
The pre-diapause phase has been separated from the diapause phase, because of some physiological differences (see chapter VI). There is now evidence from a study of the neurosecretory system that this pre-diapause phase is different from the diapause phase proper. Of particular interest is the occurence of B-cells in the B physiological phase. The nuclear I volumes have been considered as an index of secretory activity of the N.S.C..
During the pre-diapause phase, which lasted between 8-16 days after the imaginal moult, measurements of nuclear volumes of all the N.S.C. have shown that they are larger than the same cells of the phase of diapause.
It is thus considered that the neurosecretory system is more active in the pre-diapaUse phase. There seems to be a direct correlation in the higher level of activity of the N.S.C. with higher metabolic activity.
The phase of diapause is characterized by the apparent absence of
B-cells in their B phase. The activity of the corpus cardiacum/allatum I 121. is at its lowest. There is, however, a substantial amount of storage in
A-cell secretion in the corpus cardiacum. As the secretory activity of the A-cells are lowest during this phase, it appears that the discharge of the stored secretory material from the corpus cardiacum is at a slow rate.
This, in fact, has been substantiated in autoradiographic studies (see chapter IX).
During the phases of ovarial maturation, there is renewed activity in the N.S.C. of the brain and of the suboesophageal ganglion, while the B-cells revert to their B physiological phase. This is accompanied by an increase I in the activity of the corpus cardiacum/allatum. The secretory activity
cells of the suboesophageal ganglion parallels that of the of the 2AI activity of the corpus allatum. A possible source of hormonal stimulation
may come from the suboesophageal ganglion via the corpus allatum-suboeso-
phageal nerve. If Engelman's (1957, 1958, 1960) hypothesis is correct,
in that the suboesophageal ganglion stimulates the activity of the corpus
allatum, then, in G. tanaceti, the N.S.C. of the brain stimulate the
suboesophageal ganglion which in turn releases its tropic effect on to the
corpus allatum. This hypothesis cannot be ruled out.
The activity of the N.S.C. is considered to be stimulated by short
photoperiod lying between 15 and 206C..
During the phase of oviposition, the activity of the neurosecretory
system attains its maximum level. It is also striking that this higher
level of neuro-endocrine activity is maintained throughout the subsequent
oviposition periods. Unlike the conditions in Leucophaea maderae (Scharrer
and Von Harnack, 1958, Engelman, 1957), there is no quiescence of the
corpora allata after ovulation. On this ground, the complex feed-back 122. mechanism of the ootheca in the control of the corpus allatum activity propounded by Engelman (see page 27 ) does not satisfy the observations made in G. tanaceti.
These observations imply that diapause, ovarial maturation and oviposition are controlled by different levels of hormonal activity of the neurosecretory system. During the phase of diapause, the neurosecretory system functions, but probably at a low level and is capable of controlling metabolism at the "maintenance level". But this level is insufficient for ovarial development. This is contrary to what has been seen in some instances of diapause, as in Hyalophora cecropia 1947, 1948), in which the N.S.C. are thought to cease their function at this phase.
In Cephus cinctus there is evidence that the neurohormone from the N.S.C. of the brain has to reach a certain level to be capable of activating the prothoracic glands (Church, 1955).
The phase of ovarial development demands a higher level of endocrine activity for differentiation, development and maturation of oocytes. During the phase of oviposition, the neuro-endocrine system functions at a high level probably to ensure the development of oocytes and the process of oviposition.
The role of the corpus cardiacum has not been fully understood.
The gland extracts have been shown to stimulate contractions of gut muscles and malphigian tubules (Cameron, 1953), to stimulate other centres, e.g. pericardial cells, to produce a pharmacological active agent (Davey, 1961
1962); to stimulate followed by depression of nervous activity (Ozbas and
Hodgson, 1958; Milburn et al, 1960).
It has been known that copulatory reflexes in mantids and cock- 123. roaches are controlled by efferent-motor activity in the phallic nerves.
Roeder (1935) has shown that the decapitation of these male insects by the females preceding copulation elicits very intensive copulatory reflexes from the headless males. The inhibition of these reflexes normally is controlled by the suboesophageal ganglion. If a corpus cardiacum extract is applied to an intact coackroach nerve, bursts of nerve impulses develop in the phallic motor nerves mimicking the effects produced by decapitation.
The corpus cardiacum is thought to inhibit the activity of the inhibitory centre in the suboesophageal ganglion (Milburn et al., 1960).
The corpus cardiacum in G. tanaceti decreased in volume just after
each oviposition, suggesting a role of the c.c. in oviposition. It is tempting to speculate here that the corpus cardiacum may release its secretion to inhibit an inhibitory centre, thus eliciting well-conditioned
ovipository reflexes which is necessary for the successful laying of a
batch of eggs over fairly long time intervals.
There is some evidence from the measurements of nuclear volumes of
the N.S.C. throughout the definite intervals of the life-history of G. tanaceti
that these cells undergo cycles of activity. These cycles of activity have
been shown to fluctuate in intensity during any one major physiological phase.
The secretory cycles of activity tend to intensify during the phase of
ovarial maturation and more so towards and during the phase of oviposition.
In order to understand these apparent cycles of activities of the N.S.C. an
attempt will be made to discuss the dynamics of neurosecretion from evidence
obtained in autoradiographic studies in the following chapter. 124.
IX. The dynamics of neurosecretion
There is evidence from the preceding study that the neurosecretory cells undergo cycles of activity during any one major physiological phase.
These cycles of activity are more intensified towards the phase of ovi-
position. These observations led me to the tentative conclusion that
diapause, ovarial development and oviposition are controlled by different
levels of hormonal activities. It was therefore necessary to ascertain and
substantiate this conclusion by a more direct proof. This was achieved by 35 the use of radioactively labelled amino acid, S-cystine in an auto- radiographic study.
Sloper (Sloper, 1958; Sloper et al., 1960) has shown that this method
can give consistent measures of neurosecretory activity in the rat
hypothalamo-hypophysial neurosecretory system. Highnam (1962c) also using
35S-cystine, is able to compare the activity of the neurosecretory system
of females reared with mature males against females reared without males in
Schistocerca gregaria.
In the present study attempts were made to elucidate and determine 35 the following processes by the application of S-cystine:-
a) to compare the rates of secretory activities of the N.S.C. of the pars
intercerebralis +hroughout the three main physiological phases, i.e. in
the diapause, maturation and oviposition phases;
b) to see whether the N.S.C. undergo cyclical activities;
c) to determine the period of storage of neurosecretion in the corpus
cardiacum, and finally,
d) to ascertain the relationship between the size of the nucleus and the
intensity of secretory activity of the N.S.C.. 125.
This study includes the formulation of a hypothesis which attempts to explain the dynamics of neurosecretion, namely a concept here named the
"neuro-endocrine momentum".
An attempt is made in the discussion to include the interpretation of the terms "activity" or "inactivity" of the neurosecretory system based on histological observations.
Methods a) Autoradiographic technique 35 9.3 ma. of 5-D1,-cystine having a specific activity of 0.119 mc./ing. on 3rd August, 1962, was obtained from the Radiochemical Centre, Amersham.
The radioactive amino acid was dissolved in 0.1 ml. of 0.8 N 1431. and 2.6 mis.
of Ringer's solution. 10 microlitres of radioactive solution was injected by means of a microsyringe into each diapausing female. Each beetle there- 35 fore received 4 pc. of S..
It was found that injection of 5 p1. of the radioactive solution was
a more suitable volume for the mature and ovipositing females which had much
distended abdomens. Therefore, the original solution was halved. This was
achieved by freeze-drying the solution and half of the original volume of
distilled water was added to dissolve the amino acid. Each beetle, therefore, 35 received 4 pc. of S..
30 beetles were used to represent each physiological phase. Samples
of 3 beetles were dissected and fixed at intervals varying between 20 minutes
to seven days after injection. The neuro-endocrine system was dissected
out, fixed and sectioned.
A modified technique by Pelc and Howard (1952) was used after dewaxing.
The slides were brought to water followed by potassium permanganate 126. oxidation and then stained with PF., fixed in 95% alcohol for 10 minutes and then brought down through the alcohol to water. The slides were washed for at least one hour in distilled water with 5 minute changes.
Kodak AR 10 stripping film was then layered onto the slides. The slides were then arranged in a light-proof slide box, kept in a sealed polythene bag filled with silica gel and exposed at about 4°C.froim14 to 70 days until the reduced silver grains were of a reasonable density for counting.
The autoradiograms were then developed in Kodak D-19 B developer and fixed in Amphix rapid fixer. Number of silver grains per N.S.C. (A-cells) were counted in sections having the largest area under oil immersion. The diamaters of the nuclei were measured and the volumes of the nuclei, thus, can be calculated. At least 20 of the N.S.C. were considered in each 35 insect. Uptake of S. by B-cells was similarly determined in the unstained areas surrounded by the stained A-cells of the pars intercerebralis. A 2 mean of 4 - 6 readings of a standard area, 225 p of the ordinary neurones of the brain was taken for a background count.
The relative activity of the N.S.C. was calculated from the number of silver grains in the largest section of each cell over the number of silver grains in the standard area i.e. the background. The mean of the A-cells largest areas during the phase of diapause was slightly smaller than the 2 standard background area (225 p ); means of A-cells area in maturing
females was approximately the same and in ovipositing females mean A-cells area was larger than the standard background area. b) Demonstration of a possible neuro-endocrine "momentum" in G. tanaceti.
Beetles which had oviposited at least once were transferred from the insectdry into the laboratory at a photoperiod of 16 hours at 20°C— 127.
This condition has been found to be unfavourable for oocyte maturation and oviposition.
Likewise, beetles with well developed ovarioles but which had not previously oviposited, were transferred to a photoperiod of 16 hours at 20°C..
As a control, maturing females were transferred from the insectary and kept at a photoperiod of 12 hours per day at 20°C.. This environment had been shown to be favourable for oviposition. At the end of 30 days, the number of batches of eggs and the number of beetles surviving until the end of the experiment were noted.
Results
In diapausing females, 20 minutes after injection, at three weeks period of exposure, gave a reasonable reduced grain count. In maturing and ovipositing females, however, a longer time exposure was needed as at this time presumably the growing oocytes took up a great amount of the 3r available .5.S-D L-cystine. In these instances the time exposure was extended to 1 - 2 months to obtain the comparable reduced silver grains density.
The results are summarized in Table 27.
When these experiments were performed it was unknown how long each secretory cycle lasted in the different physiological phases. Therefore, these experiments must be regarded as preliminary. However, the trends and consistencies of the results rendered them useful in elucidating and confirming several points.
All the A-cells of the pars intercerebralis of the brain in diapausing females completed a cycle of activity between 42 - 7 2 hours. This 35 necessarily assumes that uptake and discharge of S. by the N.S.C. was Table 27. Distribution of radioactivity in i, A and B-cells, t 120 hours after 'injection of 35S-D L-cystine into the haemolymph of G. tanaceti females representing three physiological phases
Physiological No. of Time after Ratio of No. of Ratio of No. of Ratio of No. of condition of specimens injection silver graird: per silver grains per silver grains per females A-cell/225 u2 B-cells/225 p2 A ce11/225 ,u2 3 20 min 1.13 0.83 0.80 3 3 hr 1.64 1.04 1.16 3 9 hr 1.94 1.09 1.28 Diapausing 3 18 hr 2.36 1.38 1.52 3 24 hr 2.74 1.83 1.58 3 42 hr 1.63 1.68 0.95 3 72 hr 1.14 0.91 0.86 3 120 hr 0.95 0.80 0.74 3 20 min 1.63 1.25 0.94 3 1 hr 1.68 1.23 3 3 hr 1.99 1.53 1.95 Maturing 2 9 hr 3.22 2,03 3 18 hr 1.51 1.21 1.14 2 24 hr 1.23 1.06
3 1 hr 2.41 1.40 3 3 hr 4.50 2.85 Ovipositing 3 9 hr 3.56 2.81 2 18 hr 2.08 1.21 129. directly correlated with the synthesis of neurosecretory material and their subsequent release. In maturing females, a complete cycle of neurosecretory activity lasted between 18 - 24 hours and in ovipositing females, the overall cycle was completed between 13 - 14 hours. The ratio of the number of silver grains in the N.S.C. to that in the background cells gave an index of the intensity of the cycle. Fig. 26 depicts these results. 35 The uptake and discharge of S. is depicted by normal distribution curves in the three sets of experiments, suggesting that the sythesis of neurosecretion and its subsequent discharge was a gradual and a continuous process.
The B-cells seemed to follow the same cycles of activity as the
A-cells but with a lower uptake of cystine. This did not necessarily indicate that these cells were functioning at a lower level of activity.
The uptake of cystine must be directly proportional to the relative requirements of the different N.S.C. for their synthesis of neurosecretion.
It has yet to be demonstrated biochemically in insects that cystine is an essential precursor to neurosecretory material particularly in the B-cells.
The 2A cells of the suboesophageal ganglion were stained lightly I and were therefore not identified in all the specimens. In diapausing and in some maturing females when identified, they were seen to follow similar cycles of activity as their counterparts in the brain (see table '''7) but with a lower uptake of cystine.
The relationship between nuclear volumes and activity has been adequately confirmed in these experiments. Smaller nuclear volumes in the diapausing females have been shown to be less active than the larger nuclei of the ovipositing females. But at any stage in any specimen, the nuclear volumes of the N.S.C. varied from cell to cell. This means that the Ce ll- bac kground ratio 20 10 30 2.5 I.5 Fig. 26.Graphicrepresentation ofshortrangesecretorycycles ofAand 99 ofG.tanaceti representing thethreemainphysiological phaseswere used. --s--m-- = cells ofparsintercerebralis bytheuseof --,--o-- = Acellsofovipositing 92.;--o--o--= B cellsofovipositing I
3
= A cellsof maturedW;--o--a-- =Bcellsofmaturing A cells ofdiapausing Hours otter 9
injection of 18
W;
24
35 S-DL-Cystine 35 S-DL-Cystine inautoradiography. = Bcells ofdiapausing 42 Fig. 26
92; fl; 72 0 Fiq.27 A. B.
•040_ 0 330 0 'as
= 0 I U [0
0- IVA g 1.2 u
alb
214
0 0 1. 12 V
3 9 18 24 42 72 120 I 3 9 18 24
Hours after injection of 35S-DL-Cystine
35 Fig. 27. Histogram of relative S. activity in A cells, along axons and corpus cardiacum from * to 120 hours after injection of 35S-DL-Cystine in G tanaceti. A. Diapausing 99. 3. Ovipositing 99. Note in diapausing 7 S. is accumulated in corpus cardiacum up to 120 hours, whereas in ovipositing ?9, most of 35.E. is discharged by 24 hours. For details see text. 132. 42 - 46 A-ceils of the pars intercerebralis functioned at different levels of activity at any one stage.
The ratio of the number of silver grains in the anterior storage lobe of the corpus cardiacum to the number of silver grains in its cortex was calculated. The greater number of silver grains in the anterior lobe 35 was attributed to the accumulation of S. from the brain. Further counts were made of the number of reduced silver grains on the axons over the same area in the background cells. This ratio can provide some information in the rate of discharge of secretion along the axons.
The results are depicted in Fig. 27. The ratio of silver grains along the axons to the background showed that there was continuous discharge 35 of S. during the entire cycle of activity. The rate of discharge appeared to be greater at the period of maximum uptake of cystine i.e. 24 hours 35 after injection with S. in the diapausing females, and after 3 hours in ovipositing females. In diapausing females, there was a continuous build- ing up of silver grains in the corpus cardiacum up to 120 hours after the 35 injection of S. This implied that most of the neurosecretion discharged during the 42 - 72 hour cycle was stored in the corpus cardiacum for a period up to 120 hours. In ovipositing females, however, there was a building up of neurosecretion in the corpus cardiacum up to 18 hours but by 24 hours most of it had been discharged. b) Experimental demonstration of a possible "neuro-endocrine momentum"
The results of this set of experiments are summarized in Table 28.
The average number of eggs per batch was about the same under all three treatments. "Maturing females" subjected to a photoperiod of 16 o, hours at 20 laid a total of 13 batches of eggs at the end of 30 days. Table 28. Experiment to demonstrate a possible "neuro-endocrine momentum" by subjecting G. tanaceti having two different levels of neuro-endocrine activity to an unfavourable environment.
Previous No. of insects Treatment Duration No. of Average No. Average No. Percentage physiological of experiment batches of of ergs per of batch per survival conditions eggs laid batch female
Maturing 16 hr/d females 12q9 20°C. 30 days 13 53.t 1.1 66.7
Ovipositing 16 hr/d females ln? 126" 20°C. 30 days 21 56.5 1.8 41.7
Control Maturing 12 hr/d females 1o9? 1000' 20°C. 30 days 23 54.1 2.3 90.0 134.
They ceased to oviposit at the end of 21 days. "Ovipositing females" subjected to the same unfavourable environment laid a total of 21 batches of eggs and again, towards the end of the experiment, there was no sign that the surviving females were capable of further oviposition. Post- mortem dissections revealed undeveloped eggs in the ovarioles.
This concept of a "neuro-endocrine momentum" can help to explain these results. Ovipositing females with their higher level of neuro- endocrine activity take a longer period to adjust their neurosecretory system under the influence of the unfavourable environment. Consequently, their neurosecretory system produced diminishing levels of hormones but sufficient to mature and oviposit a second batch of ova. Maturing females, however, started with a lower level of neuro-endocrine activity and thus their diminishing hormonal levels were too low for maturation and oviposition of the second batch of eggs.
Histological observations on the neurosecretory system of these experimental beetles, using the ABP. technique, revealed two interesting features: 1) the activity of the N.S.C. reverted to a condition similar to that of maturing females and 2) the A-cells contained within them phloxinophil granules. These cells are comparable to the "castration cells" of the suboesophageal ganglion of L. madexae (Scheirer, 1955) and some Hymenopterous species in which Thomsen (1954) found both red and blue granules using the CHP. technique. The existence of this mosaic type cell is attributed to an altered metabolism consequent to transferring the beetle from a "favourable" to an "unfavourable" environment (cf. Scharrer
(3, von Harnack, 1960, 1961). 135.
Discussion
Evidence from this preliminary autoradiographic study supports the preceding findings that the N.S.C. undergo cycles of secretory activity throughout the adult life. If the measure of uptake and discharge of
353 DL-cystine by the N.S.C. is indicative of the normal cycles of secretory activity, it is concluded that these are short cycles. In diapausing females, their neurosecretory cycles last 42 - 72 hours and are at a low level of activity.In ovipositing females, the neurosecretory cycles are even shorter, i.e. about 13 hours per cycle and the activity is functioning at yet a higher level than in the maturing females. If the amount of uptake 35 of S. can be directly correlated with the amount of synthesis of neuro- secretion within the A-cells, these results can be numerically expressed, i.e. the ovipositing females possess N.S.C. which produce about 10 times as much hormone as diapausing females during the same period, and twice as much as the maturing females. The same trends appear in the AI and B cells and probably in C and D-cells.
The neurosecretory cycles have been shown to fluctuate in intensity of secretion during the diapause period. These cycles tend to increase their intensity during the phase of ovarial maturation and probably reach the "peak" levels during oviposition. This high level of activity is maintained throughout the phase of oviposition. This gradual building up in intensity in the neurosecretory cycles suggests a momentum effect subsequent to each cycle. This has, therefore, tempted me to introduce a new concept, namely, a "neuro-endocrine momentum" which appears to me would explain these cycles of secretory activities. This "neuro-endocrine momentum" effect could on the other hand be argued that it would "drive" 136. the neurosecretory system to such a high intensity of activity that the insect may die of sheer exhaustion. To counteract this, once a "peak" level of activity has been attained, there must be a restraining source.
Engelman (1957, 1958, 1959, 1960) has evidence that in Leucophaea and
Diploptera, the reproductive organs are such a source which controls the
activity of the corpus allatum via the brain. The reproductive organs of
G. tanaceti or an unknown neuro-endocrine centre may well restrain the
activity of the neurosecretory system. Such feedback mechanisms are not uncommon in insect physiology. Hodgson (1962) postulates the hypothesis that the corpus cardiacum as a source in the feed-back mechanism which acts upon the central nervous system with the result that the insect becomes
quiescent after a period of hyper-activity.
There is also evidence from the autoradiographic studies that during
the phase of lower neuro-endocrine activity, the rate of transportation of
neurosecretion along the axons is slower than when the neuro-endocrine
system is active (see fig. 27). Thus, the ratio of silver grains in the
axons to the background is higher in diapausing females than in ovipositing 35 females during the same period after injection of S-D L-cystine. Moreover, 35 the build up of S. in the corpus cardiacum is higher in the ovipositing
females than in diapausing females during comparable times and thus further
substantiates this argument. There is some further evidence that the
neurosecretion stored in the corpus cardiacum is discharged at differential 35 rates; thus, in ovipositing females, most of the 54 accumulated during
the first 18 hours, is discharged by 24 hours, while in diapausing females, 35 the S. is accumulated continuously up to 120 hours after injection of
35S. To conclude, it appears that when the neurosecretory system is 137. functioning at a high level, there is more production of hormones. These hormonesare continuously released and transported at a high rate along the axons into the corpus cardiacum where they are temporarily accumulated and then discharged to meet the requirements of the insect. Thus, in the phase of oviposition, a high level of hormone is needed for oocyte differentiation and maturation and probably also for the process of oviposition (cf.Nayar,
1958).
Many workers have attempted to interpret histological observations of the neurosecretory system in terms of "activity" and "inactivity". The presence of many inclusions in the A-cells has been considered "active" as compared to cells having few and small inclusions,. (Dupont-Raabe, 1952; Arvy and Gabe, 1952, 1953; Formigoni, 1956; Herlant-Meewis and Faquet,1956).
However, Highnam (1961) pointed out that "the amount of material contained in a N.S.C. at any time depends both upon its rate of synthesis and its rate of discharge. " He maintains that in a neurosecretory system contain- ing small amounts of material, the material is discharged as soon as it is formed and therefore, the system as a whole is "active". He was able to substantiate his hypothesis by the use of radioactively labelled cystine which was taken up more rapidly in the neurosecretory system havino few inclusions than in systems packed with secretions. Ladduwahetty (1962) prefers to use the term "release" as an alternative to "active". She attributes the neurosecretory system to be in a state of "high release" when the A-cells contain few aggregate of secretion and the axons and the endocrine organs containing large amounts of material.
In G. taneceti, the criterion for assessing "activity" by histological observations of the N.S.C. is untenable as the insects at any stage of the 138. life cycle have N.S.C. which contain varying amounts of inclusions and varying nuclear volumes. It appears, therefore, that not all the 42 - 46
A-cells of the medial groups function at the same level of activity at any
one stage. This argument is substantiated by the autoradiographic studies, 35 since there is an appreciable build up of S. along the axons one hour 35 after the injection of S-D L-cystine, which is long before the mean peak
of uptake of cystine by all the N.S.C.. This suggests that some N.S.C. are
more 'active' than others.
It seems reasonable to assume that a N.S.C. at its initial cycle of
activity contains none or little secretion. In the course of its subsequent
cycles of activity, if the rate of synthesis is greater than the rate of
discharge, more secretions will be accumulated until the cell is packed
with inclusions. This suggestion seems justifiable in the phase of
diapause and to a lesser extent in the phase of ovarial maturation in which
there is low release of neurosecretion. Indeed, histological observations
support this view, as most of the cells are packed with inclusions during
these two phases (see table 13).
During the phase of ovarial maturation, the production of neuro-
secretory material is stepped up. The rate of synthesis is probably still
greater than the rate of release, since the cells are packed with secretion.
Then, when the cells are packed with inclusions, there is a rapid release
of secretion, as vast amounts of secretion can be sden along the axons of
maturing females. This process of rapid release of secretion reduces the
cells to stages II or III.
During the phase of oviposition, the cells become more active,
producing more secretion but they can generally be designated to stages 139.
II, III and IV. This probably means that there is a continuous release of neurosecretion from the cells and the rate of release is equal to the rate of synthesis; comparable condition is seen in Schistocerca oreaaria reared with mature males during the phase of ovarial development (Highnam,
1961, 1962).
The hypothesis of the dynamics of neurosecretion in G. tanaceti is set forth in figure 28 which illustrates the mechanism of the three main physiological phases. Fig. 28 a) Rate of synthesis > rate of discharge (predominantly in phase of diapause)
rate of discharge (predominantly in phase of ovarian maturation) rapid discharge
1 1 stage I
c) continuous discharge of neurosecretion
rate of synthesis = rate of discharge
( predominantly in phase of oviposition
28. Hypothesis illustrating dynamics of neurosecretion and discharge in A cells during phases of diapause, ovarian maturation and oviposition in G. tanaceti. 141.
X. Experimental investigations into the role of the neurosecre:ory system in controlling reproductive diapause, ovarial maturation and oviposition in G. tanaceti
Introduction
The preceding studies have demonstrated that spectacular changes occur in the neurosecretory system from the phase of diapause to the ultimate phase of oviposition. It was evident primarily that final experimental proof was needed to ascertain the individual role played by the components of the neurosecretory system in controlling diapause, ovarial maturation and, finally, oviposition. Secondly, it was desirable to
investigate the relationship between the various components.
Owing to the lack of knowledge in insect endocrinology and research
faciaitiesavailable at this level, rather crude methods have to be employed
in these preliminary investigations. This study is divided into two sections:-
The first part is devoted to cauterization of the various components of the
neurosecretory system and to study their overall effects particularly on
the reproductive system. The second part is devoted to implantation
experiments mainly to supplement the first part of the work.
Methods
a) Cauterization experiments
1. Cauterization of medial or lateral groups of N.S.C.
An A.C. microcautery, built by Mr. J.W. Siddorn of Silwood Park,
was used for these experiments. The effective temperature for cautery was
between 700-900°C.. Prior to all operations, the bench was sterilized with
"Dettol" and all operating instruments were sterilized over a methylated
spirit-lamp. 142.
Females representing the three main physiological phases were used in these cauterization experiments. A small depression was made in the plasticine operating platform to accommodate the thorax and abdomen of the insect, with the head resting on the rim of the depression. A thin plasticine pad was then placed over the insect holding it in position.
Filter paper was placed under the insect's head to absorb any regurgitated food or haemolymph. The operation was performed under a binocular micro- scope, 10 x 6.3 with a blue filter on the spot light. A small window was cut open in the dorsum of the head capsule by two pairs of watchmaker's
No. 4 forceps. The brain was then carefully exposed, avoiding injury to the dorsalaorta and the large lateral tracheal trunks. Filter paper was used to absorb some of the haemolymph around the brain to facilitate cautery.
The N.S.C. appeared quite distinctly with a light bluish tinge, under illumination. The hot cautery was then introduced gently to destroy all the N.S.C. of the two medial groups or the lateral groups. In some cases, when the original cuticle of the head capsule was destroyed during the a operation, a piece of cuticle from the elytron of/sterilized dead insect was substituted and the wound was sealed with paraffin wax.
2. Cauterization of corpora cardiaca-allata
The same procedure outlined above was employed. The corpora cardiaca/allata were approached by cutting two small windows on the sides of the head behind the compound eyes. The large lateral tracheal trunk was displaced carefully by a thin seeker, thus exposing the glands which were completely extirpated or cauterized.
3. Cauterization of N.S.C. of suboesophaqeal ganglion
Experiment was carried out as before but with the ventral side of 143. the beetle uppermost. The antenna were stretched forward and pinned down by a thin plasticine pad. In this way, the cervical membrane was fully extended. A sharp needle was needed to cut open the cervical membrane, exposing the suboesophageal ganglion. The N.S.C. lying in the ventral aspect of the ganglion could be seen distinctly and were cauterized. In this operation no paraffin wax was used to seal the wound. The insect, on release from the operating platform, retracted its head and the wound was telescoped back into the thorax.
4. Controls
Insects used for controls had their head capsule similarly cut open but no injury was inflicted on the components of the neurosecretory system.
The wounds were then sealed with paraffin wax.
The females were then placed in previously sterilized Petri dishes to recover. They were then transferred to a photoperiod of 12 hours at
20C.or to the insectary. At the end of the experiment the surviving females were dissected and morphometric measurements were then taken of the reproductive organs. Where possible, the remaining components of the neurosecretory system were examined. b) Implantation experiments.
The components of the neurosecretory system were divided into
1) the brain, 2) the suboesophageal ganglion and 3) the corpora cardiaca/ allata. Individual or combinations of the components were implanted into the recipient diapausing females, The components of the neurosecretory were dissected in vivo from donors, usually ovipositing females. The recipient females were placed dorso-laterally on a depression made in the plasticine operating platform. An incision was made by fine scissors 144. on the ventro-lateral aspect of the 4th to 6th abdominal segments. The individual components or their combinations were then inserted into the abdomen by a pair of fine forceps. The wound was sealed with paraffin wax.
The recipients were then kept in previously sterilized Petri dishes for a few hours to recover. They were transferred to cages contain- ing Stellaria and were kept at a 16 hour photoperiod at 20°C. for 25 days.
The surviving females were dissected and morphometric measurements were taken of the gonads,
Controls
Implantation of components of the neurosecretory system from diapausing males were performed as above, with diapausing recipient females.
Results a) Cauterization experiments
1. Diapausing females
These beetles had been kept previously at a photoperiod of 16 hours at 20°C.. Their imaginal age varied from 15 to 35 days old. The operated beetles were transferred to a photoperiod of 12 hours at 20°C..
The results of this first series of experiments are summarized in
Table 29.
The criterion for deciding whether these operated beetles had terminated reproductive diapause was based on morphometric measurements of the reproductive organs. Dissections also revealed the presence of food
in the gut, thus ruling out the possibility that their feeding habit had
been affected by the operation.
The morphometric measurements of the reproductive organs are
tabulated in Table 30. Table 29. Cautery of various components of the neurosecretory system in diapausing females of G. tanaceti and its effects in determining the physiologicEl phase when subjected to 12 hr. at 20°C.
Part of neuro- No. of Treatment No. of No. )f dia- No. of non- No. of ovi- secretory system insects survivors pausing W diapausing 2y positing 99
cauterized -11.01•••••••.•• A. M.G. N.S.C. 16 99 20 days at 5 1 * 1 0 12 hr/da.c, at 20°C. Controls 4 ?? If 2 0 2 0
B. L.G. N.S.C. 17 y9 6 6 0 0 Controls 4 w 3 0 0 ,, C S.G. N.S.C. 16 yy 6 6 o Controls 4 yy ft 4 0 2 2
D. C.C. and C.A. 16 In 5 5 0 0 Controls 4 99 3 0 3
* N.S.C. not completely cauterized. M.G. = Medial groups L.G. = Lateral groups S.G. = Suboesophageal ganglion C.C. = corpus cardiacum C.A. = Corpus allatum Table 30. Morphometric measurements of the reproductive organs and condition of spermathecal glands in operated females of G. tanaceti based on all surviving females
-•••m••••••••• ••••0•11.... Part of N.S. Sp. gland Sp. gland Ovarial condition Ovariole Oocyte I Germarium cauterized Empty Full Diameter in ii Diff. Und. mean length (mm} (mm) (innil A. M.G. N.S.C. 4 1 150 4 0.86 0.36 *1 1.13 0.32 0.55 Controls 0 2 250 2 0 1.83 0.70 0.53
B. L.G. N.S.C. 6 0 140 0 6 0.82 0.34 Controls 1 2 230 3 0 1.52 0.58 0.56
C. S.G. N.S.C. 6 0 120 0 6 0.82 0.35 Controls 0 4 250 4 0 2.33 1.07 0.55
D. C.C. & C.a. 5 0 120 0 5 0.86 0.36 Controls 0 3 230 3 0 1.50 0.56 0.54
N.S.C. = Not completely cauterized. N.S. = Neurosecretory system 147.
The results show quite clearly that cautery of any component of the neurosecretory system dompletelt impeded termination of diapause. There was also the striking absence of copulation taking place, although mature males were present in the cages.
2.Mat allaaltralsa In the second series of experiments maturing females from the insectary were used, following the procedure as given previously. The operated females were kept in the insectary under natural conditions for 20 days, a period long enough for oviposition to occur in the controls. The surviving females were dissected and measurements were taken of the reproductive organs. In the maturing females, however, it was difficult to be conclusive whether further ovarial development had proceeded since the operations. But it was found that the length of the germarium during the phase of ovarial maturation varied from 0.40 mm.to 0.52 mm.(see chapter I). The maturing females selected for this series of experiments were approximately 80 days old, i.e. the oocytes I had not yet ovulated and therefore the germarium measured about
0.47 mm.in length.
The results of this series of experiments are set out in Table 31.
The results again show quite clearly that cautery of any component of the neurosecretory system completely prevented further oocyte development and maturation.
3. Ovipositinq females
In the third series of experiments, an attempt was made to study the role of the N.S.C. of the suboesophageal ganglion in controlling oviposition.
14 ovipositing females had their N.S.C. of suboesophageal ganglion
cauterized on 20.viii.and were kept in the insectary until 8.1x. Five survived the experiment but none ovipositied. From Table 31. Morphometric measurements of the reproductive organs and condition of oocytes in operated maturing females of G. tanaceti based on all surviving females
Part of N.S. No. of No. of Sp. gland Sp. gl. Ovariel Germarium No. of egg cauterized beetles survivors Full Empty diameter .in condition length(Tn mm) batches laid No-further de- A. M.G. N.S.C. 14 99 8 6 2 210 velopment. Egg 0.47 resorption 11 B. L.G. N.S.C. 14 99 7 6 1 230 0.46 0 11 C. S.G. N.S.C. 14 99 10 8 2 230 0.47 0 0 D. C-C.. & C.A. 14 99 5 5 0 210 0.45 Controls 12 99 11 11 0 290 Corpora latea 0.52 >12 batches in all 149. the results, it appeared that the N.S.C. of the suboesophageal ganglion could either a) control the secretory activity of the brain and corpora cardiaca/allata, to function at a level suitable for oviposition or b) contribute a hormone necessary for oviposition. In order to test the two alternatives, two further sets of cauterization experiments followed by implantations were performed.
The N.S.C. of the suboesophageal ganglion were cauterized in 24 ovi- positing females. In 12 of the females the brain and the corpora cardiaca/ allata complex from ovipositing females were implanted into them. In this set of experiments, six survived the double operation after 18 days, but none oviposited. In the remaining 12 females, the suboesophageal ganglion from ovipositing females were implanted into them: After 18 days, four survived the double operation and all four oviposited but only one batch of eggs per female during this period. It appeared that the suboesophageal ganglion produced a hormone necessary for oviposition.
Of the cauterized diapausing females, only 35% survived until inspection-
Cauterization of any of the groups of N.S.C. produced atrophied corpora cardiaca/allata. The operated beetles appeared to contain more fat bodies than normal unoperated beetles of the same physiological phase. The percentage survival of maturing females was over 50%. The dependence of one component on the other components of the neurosecretory system, for a fully functional state, was again quite apparent.
In those surviving beetles which had the N.S.C. of their suboesophageal ganglion cauterized, a histological examination of the N.S.C. of the brain was made, using the ABP. technique. The N.S.C. of the brain revealed from their histology and nuclear sizes, in the "maturing females" (20 days after 150. operation) that they probably functioned at a low level of activity comparable to those of the diapausing females. In ovipositing females, however, three days after the cautery of the N.S.C. of the suboesophageal ganglion, revealed that their N.S.C. in the brain were comparable to those of normal ovipositing females. Whereas, the N.S.C. of the brain of the cauterized ovipositing females, 20 days after the operation, were comparable to those of diapausing females. b. Implantation experiments
In this series of implantation experiments, each recipient diapausing female only received one component or components of the neurosecretory system from one ovipositing or maturing female. Thus the effects of the implantations were not very marked, but the response seen in the reproductive organs was quite cleai-cut in most cases.
The results of the implantations experiments are tabulated in Table
32.
Implantations of the whole neurosecretory system, brain only, corpora cardiacaMlata, corpora cardiaca/allata and suboesophageal ganglion terminated diapause in all the recipient diapausing females. However, im- plantations of the suboesophageal ganglion only, the recipients sustained diapause in six out of the seven survivors. From this result, it appeared that the suboesophageal ganglion hormone did not initiate ovarial differentiation. Implantation of the brain and suboesophageal ganglion terminated diapause in five out of the six survivors. This set of results were hence inconclusive.. Nevertheless, it is tempting to suggest that the brain and the suboesophageal ganglion are "antagonistic" in their influence Table 32. Measurements of the reproductive organs of diapausing females after implantation of various parts of the neurosecretory system from maturing or ovipositing donors after 25 days. Data based on 12 recipients for each set of experiments Ovarial Part/s No. of Sp. gl. Sp. gland condition Ovariole Oocyte I Germarium implanted survivors Empty Full width in,u Diff. Und. length (in mm) (in mm) (in mm)
A. Whole 4 4 0 250 4 0 1.05 0.41 0.49 complex B Brain only 8 8 0 280 8 0 1.11 0.42 0.50 C. S.G. only 7 2 5 210 1 1.05 0.28 0.51 1 6 0.95 - 0.45 D. C. C. & C.A. 5 4 1 260 5 0 1.08 0.28 0.49 E. Brain & S.G. 6 5 1 250 5 1.05 0.34 0.49 1 0.99 - 0.47 F, C.C. & C.A. + 6 6 0 260 6 0 1.08 0.36 0.50 S.G. G. Controls 6 6 0 180 0 6 0.91 0.38 152. on each other, whereas the suboesophageal ganglion has a "tropic" effect on the corpora cardiaca/allata. From the above series of experiments, differentiation of the ovarioles into oocyte I and slight enlargement of the spermathecal gland and germarium, it was considered that diapause had terminated. (cf. to controls). It is, of course, noteworthy that in those recipients which had terminated diapause, successful copulation had taken place.
Discussion
The neurosecretory system clearly functions as a unit. If a component of it is destroyed, the remaining system ceases to function in a normal physiological state and is thus unable to respond to the stimuli of the environment. This suggests a delicate relationship and interdependence of one neuro-endocrine centre on the others, involving probably complex feed- back mechanisms. The nature of these feed-back mechanisms is not fully understood.
When G. tanaceti is deprived of the N.S.C. of the medial groups, ovarial development ceases, thus confirming the earlier findings of many workers. The deprivation of medial N.S.C. resulting in cessation of ovarial development, is probably the consequence of two processes. The first is that these cells stimulate the activity of the corpus allatum to produce the gonadotropiAlormone, thus resulting in ovarial development. The absence obviously restrains the activity of the gland. The second process is that the neurosecretion from these cells can directly stimulate ovarial develop- ment and conversely its absence will not. Implantations of "active" brain alone has been shown sufficient to terminate diapause. This also suggests that a certain level of hormones from the brain, overriding the normal level 153. of hormones in diapausing females is sufficient to, initiate the "break" of diapause. The results also suggest that the brain can discharge its neurosecretion without the intervention of the corpora cardiaca. This is in agreement with the findings of Williams (1948b), Sellier (1949), Nayar (1958), and Ladduwahetty (1962).
Cautery of the lateral groups of N.S.C. in G. tanaceti completely retards egg development. The lateral groups of N.S.C. have been found to belong to AI and D-cells, quite distinct from those of the medial groups. It is likely that the lateral groups of N.S.C. collaborate with those of the medial groups to produce a single or multiple hormones, capable of inducing ovarial development or stimulating the activity of the corpus allatum. This suggestion is in agreement with Williams' (1948b) hypothesis that the medial and lateral cells collaborate to produce a single developmental factor in
Hyalophora cecropia, In G. tanaceti, cautery of either the medial N.S.C. or the lateral N.S.C. produces atrophied corpora cardiaca/allata. These results confirm that the activity of the corpora cardiaca/alleta are directly under the tropic influence of the brain factor (cf. Thomsen, 1952; Johansson, 1958;
Ladduwahetty, 1962).
Thomsen (1952), however, observes that cautery of the lateral cells only partially retards egg development in Calliphora erythrocephala and she attributes the medial N.S.C. as the main neuro-endocrine centre.
The role of the N.S.C, of the suboesophageal ganglion is still problematical. In G. tanaceti, their extirpation after 20 days influences a low level of activity in the N.S.C. of the brain, although the beetles are subjected to a photoperiod of 12 hours at 20°C.. Their extirpation in ovipositing females after 3 days, however, has been shown in no way to retard 154. the activity of the N.S.C. of the brain, whereas 20 days after operation, the N.S.C. of the brain reverted to the diapausing condition. This suggests that if a feed-back mechanism is involved, it is operative only after several cycles of secretory activity. Implantation of "active" suboesophageal ganglia, into diapausing females did not terminate diapause. On the other hand, extirpation of N.S.C. of the ganglion has been demonstrated to sustain diapause. It means that the hormone produced by the suboesophageal ganglion does not initiate oocyte differentiation, and yet its presence is necessary for the N.S.C. of the brain to function in response to the stimuli of the environment. This suggests a feed-back mechanism from the suboesophageal ganglion to ensure a continual activation of the brain factor in its response to the environment. Further cauterization and implantation experiments appear to clotd the issue. Ovipositing females will not oviposit even containing mature oocytes if deprived of the N.S.C. of this ganglion and on implantation of "active" ganglion from ovipositing donors, the operated females will lay eggs. This suggests at least a dual function of the ganglion,
Extirpation of the corpora cardiaca/allata sustain diapause, whereas implantation of "active" corpora cardiaca/allata terminate diapause. These results are in agreement with similar experiments performed in Leptinotarsa decemlineata (de Wilde and Stegwee, 1958; de Wilde et al., 1959) and in
Dytiscus marginalis (Jolt', 1945), both species having an imaginal diapause.
There is evidence from the foregoing experiments that the neurosecretion from the brain is effective in stimulating oocyte differentiation and yet in the preceding cauterization experiments, no oocyte differentiation occurred.
This meant that the N.S.C. of the brain were incapacitated to produce the effective hormonal level for ovariole differentiation. This suggests that 155. there exists a feed--•back mechanism from the corpora cardieca/allata on the brain, probably via the cardiaco.allatum nerve.
In the implantation experiments, the overall growth of the reproductive organs of the diapausing recipients, was small but not surprising. Each recipient received the component or components of the neurosecretory system from only one donor. Previous work on the dynamics of the N.S.C. have shown that they perform short cycles of secretion and thus are capable of producing small amounts of neurosecretion during the period when they were physiologically active in the abdomen of the recipient. Also, the neuro- secretion stored in the corpora cardiaca in ovipositing females has been shown to be of short duration (less than 24 hours) and thus in the implant- ation of these glands they contained probably small amounts of hormones from the brain.
The cauterization of various components of the neurosecretory system of maturing females, resulted in a cessation of ovarial development. This supports a previous observation that a certain level of hormonalactivity has to be maintained for continual ovarial development and oocyte maturation.
These series of experiments have thrown some light on the nature of the problem of the inter-relationship of the various neuro-endocrine centres, rather than providing clear-cut answers to the problem. Future work on the nature of feed-back mechanisms is envisaged. 156.
XI General discussion
The detailed aspects of the investigations in this thesis have
already been discussed in their respective sections. This final chapter is,
therefore, devoted to a general synthesis of the salient features of reproductive diapause in G. tanaceti. The theme will revolve around the
neurosecretory system which is considered as the main neuro-endocrine centre
mediating the external environment and the internal physiology of"the" beetle.
The N.S.C. of the brain are believed to serve as connecting links
between the nervous and the endocrine systems (E. Scharrer, 1952), thereby
translating nervous stimuli into hormonal effects (Scharrer and Brown, 1962).
This concept has been generally accepted at the last three Symposia on Neuro-
secretion and Comparative Endocrinology (Takewaki, 1962; Heller and Clarke,
1962; and Gorbman and Barrington, 1962). Lees (1960) demonstated in Mei:pure
viciae that the site of photoreception lies in the -brain and suggested that
the N.S.C. are the probable locality.
The effects of photoperiodism and temperature have been shown to be
the primary causal factors controlling the incidence of diapause in G. tanaceti„
Short day lengths and intermediate temperatures tend to terminate diapause,
the reaction is thus comparable to that in Bombyx mori (Kogure, 1933),
Antheraea nernyi, Dasychira nudibunda (Gayspitz, 1953), and the autumnal race
of Ceuthorrhynchus pleurostiama (Ankersmit, 1960).
The mode of action of photoperiodism on the site of photoreception is
still far from fully understood. Whether the rhythm of the light phase
followed by the dark phase of daily photoperiodic cycles, provides the stimuli
for similar rhythm of secretory activity in the N.S.C. awaits further studies.
So far, only two studies point to this sort of mechanism. Klug (1958/59) 157. secured evidence in Carabus nemoraIis correlating diurnal activity cycles with secretory cycles of the N.S.C.. Thus, between noon and 6 p.m. he found
15 cells packed with secretion and between 9 p.m. and 4 a.m. all the cells were empty. The studies of Harker (1956, 1958 and 1960a, b, c) on the diurnal rhythm of activity in Periplaneta americana implicate the close association of cyclical activity of the neurosecretory system with the rhythm of daily photoperiodism. In G. tanaceti, however, there appears to be no direct correlation of the cycles of secretory activity of the N.S.C. with the rhythm of light and dark phases.
The striking feature of imaginal diapause in G. tanaceti is the virtual cessation of ovarial differentiation and development. This is due primarily to the failure of the N.S.C. of the brain to produce a sufficiently high concentratidn of hbrmones necessary for the initiation of oocyte differentiation and development. The low activity of the N.S.C. in turn restrains the corpus allatum from producing the gonadotropic hormone necessary for egg maturation. This hormonalcontrol of ovarial development has been documented in many insects viz., Rhodnius prolixus (Wigglesworth,
1936), Calliphora erythrocephala (Thomsen, 1942, 1952), Culex pipiens form molestus (Clements, 1956), Leucophaea maderae (Scharrer 1946a,b), Dermestes maculatus (Ladduwahetty, 1962) and in Schistocerca oredaria (Highnam, 1961,
1962) and in G. tanaceti,
The phase of diapause in Dytiscus maroinalis (Jolt', 1945) and
Leptinotarsa decemlineata (de Wilde, 1960) has been directly attributed to the failure of the activity of the corpus allatum. Implantation of several
"active" corpora allata has induced oviposition in these two species.
The phase of diapause in G. tanaceti is controlled by long photoperiods 158. at intermediate temperatures and during this time the neurosecretory system shows a low level of activity. It is striking in G. tanaceti that during this phase, the B-cells of the pars intercerebralis occur predominantly
in the B phase. The B cells secretion appears analogous in its action II II to the "diapausing hormone" produced by the suboesophageal ganglion N.S.C. in Bombyx mori (Fukuda 1951 to 1953). Whereas, during the phases of pre- diapause, ovarial maturation and oviposition, these cells occur in the B1 phase, co-inciding with the phases of higher metabolic activity. It is tempting to suggest that the B1 phase cells may stimulate the activity of
the corpus allatum. This low level of hormonal activity, particularly that
of the corpus allatum, appears to be the direct cause of a low level of metabolism. This is inferred from the experimental evidence of Thomsen (1949),
Thomsen and Hamburger (1955) in Calliphora erythrocephala that allatectomy reduced oxygen uptake by 24%, while implantation of 3 "active" additional
corpora allata increased oxygen uptake by 19%. Comparable results have been
obtained in Pyrrhocoris apterus (Novak et Al.,1960) and in Leptinotarsa
decemlineata (de Wilde and Stegweet 1958; de Wilde$ 1960).
Thomsen (1952) suggests that the N.S.C. and corpus cardiacum produce
hormones which do not affect the ovaries directly but via protein metabolism.
Removal of the corpora cardiaca in Periplaneta americana has been found to
cause disturbances in protein metabolism (Bodenstein, 1954). The production
of proteinase by the intestinal cells in Calliphora erythrocephala has recently been correlated with hormone production by the N.S.C. (Thomsen and
Willer, 1960). In Schistocerca preperia blood protein concentration has
been correlated with the activity of the neurosecretory system; the more
active the neurosecretory system, the higher the concentration of protein 159. in the blood (Highnam, 1962c).
The work of Strangwayp —Dixon (1959,.1961) in Calliphora indicates that the M.N.S.C. controls protein ingestion while the secretion of the corpus allatum controls ingestion of carbohydrate. Whether the neuro- secrecory system controls the physiology of feeding in G. tatlacet hes yet to be resolved. However, G. tanac9tj feeds during the phase of diapause.
The endocrine control of feeding in G. tanaceti which is a phytophagous insect, may be quite different from that in Calliphora.
There is no substantial building up of fat reserves in G. tanaceti before the phase of diapause. The respiratory quotients calculated at different times during diapause suggest that fat combustion is not the source of energy and that the "maintenance metabolic level" is maintained by occasional feeding. The gradual increase of weight in fat per beetle in the period of diapause has been attributed to the low level of activity of the corpus allatum. This is based on the evidence that allatectomy invariably is followed by an increase of fat deposition vize in Melanoplus differentialis
(Pfeiffer, 1945), Calliphora erythrocephaZa (Thomsen, 1942, 1952), Lucilia sericata and Sarcophaca securifera (Davy, 1943), Oncopeltus fasciatus
(Johansson, 1958), Dermestes maculatus (Ladduwahetty1 1962) and in G. tanaceti.
Under natural conditions, G. tanaceti terminates diapause at about the first week of August in a gradually decreasing photoperiod of about
14.5 hours. The N.S.C. in response to the stimuli of this decreasing photo- period (at thecritical\threshold) are at a higher level of activity.
Accompanying this higher level of activity of the N.S.C., the corpora
cardiacum and allatum also have a higher level of activity as is evident
from their increase in volumes, which correspond with increase of their 160. cytoplasmic content and of their nuclear volumes. Because of this higher level of endocrine activity, the ovarioles differentiate and development of oocytes proceeds, provided a certain hormonal level is maintained. The terminal oocytes I, however, develop at the expense of the available nutrition, while oocytes II and III remain small. This differential rate of oocyte development implies probably a complex humoral mechanism whose nature remains to be resolved ,
The phase of ovarial maturation is also accompanied by a higher level of metabolism, probably as a result of a higher level of activity of the neurosecretory system. The fat content also increases during this phase but in the form of yolk which is deposited in the maturing oocytes.
During the phase of oviposition, the neurosecretory system probably functions at its peak. The A-cells of the brain of ovipositing females have 35 been estimated by autoradiography using S-D L-cystine, to be about 10 times as active as those of diapausing females and twice as active as those of maturing females. The corpus allatum increases to about 20 times its original
volume. At this phase, "giant nuclei" occur in the corpus allatum, indicat-
ing the high level of activity of the gland. These giant nuclei have been described in Gryllotalpa (de Lerma, 1932; Palm, 1947) in Leucophaea maderae
(Scharrer and von Harnack, 1958) and in Melanoplus differentialis (Mendes,
1948). In Gryllotalpa de Lerma (1932) thought that these giant nuclei were
formed by the fusion of several nuclei. There is no evidence of this in
G. tanaceti. The occurrence of giant nuclei coincides with period of high
level of activity of the corpus allatum in the above species.
Just before oviposition, the volumes of the corpus cardiacum/allatum
probably reach their maximum sizes. Within minutes of ovipositions, there 161. is the remarkable decrease in volumes of these glands. It has been suggested (see page 123) that the corpus cardiacum secretion may control the, process of oviposition. The decrease in volume of the corpus allatum has been attributed to a sudden discharge of its secretion to ensure a rapid maturation of the terminal oocytes. The first phase of ovarial maturation lasts 25 - 30 days, whereas the period of terminal oocytes maturation between successive ovipositions, last 7 - 10 days. The differential rates of oocyte maturation are probably controlled by the different levels of hormonal activity in these two phases.
The results obtained in autoradiographic studies have supported previous observations, based on changes in nuclear volumes of the N.S.C., that these cells undergo cycles of secretory activity. The autoradiographic studies have further demonstrated that these cycles of activity are of short duration, thus in ovipositing females, a cycle of activity is completed in about 13 hours. Comparable results have been obtained in Chaoborus larvae (Fuller,
1960). However, Fuller used a more drastic treatment namely, he inserted a hot wire into the second abdominal ganglion of Chaoborus. The larvae were fixed and examined at intervals after stimulation. There was a gradual fell in amounts of secretion in the N.S.C., so that after 1 hour, all the cells of the pars intercerebralis were empty, followed by a refilling of secretion in the cells by 13 hours. The studies of Klug (1958/59) in Carabus nemoralis and Harker (1956, 1958, 1960) in Periplaneta americana on diurnal rhythm of activity in these two species, lend support of these short range secretory cycles of the N.S.C., Further Lea and Thomsen (1962) using dark-field illumination, were able to follow cyclical changes in living medial N.S.C. in Calliphora which were fed on sugar followed by meat. In Calliphore 162. the cycle of secretory activity in newly emerged females is completed in 4 days. These findings are significant in that they have thrown some light on the dynamics of neurosecretion.
Some authors have not managed to find secretory changes, particularly in B-cells correlated with physiological processes and, therefore, have considered them to be inactive (Ewen, 1962; Klug, 19509 and Mitsuhashi and
Fukaya, 1960). This view appears to be no longer tenable. Other authors have implied in their analyses, in correlating the activity of the neuro- secretory system with reproduction or metamorphosis, that the N.S.C. undergo one long cycle of activity culminating in oviposition or ecdysis (Dupont-
Raabe, 1951, 1952; Arvy, Bounhiol and Gabe, 1953; Arvy and Gabe, 1953;
Nayar, 1953; Highnam, 1961, 1962 and Ladduwahetty, 1962). This concept may not hold for all insects, as there are apparently cycles of shorter range.
The N.S.C. have been demonstrated in G. tanaceti to function at a low level of intensity during the phase of diapause. At the termination of diapause, the activity intensifies tending to function at a higher level during the process of oviposition. This tendency towards a gradual building up of intensity of secretory activity has led me to introduce a new concept namely the "neuro-endocrine momentum" to explain the dynamics of neuro- secretion. This concept helps to explain that once the neurosecretory system has attained a high level of activity in response to a favourable
environment, it will take some time to attune itself to an unfavourable environment (see page 132). It is also noteworthy that when G. tanaceti is subjected to a photoperiod of 12 hours at 20°C.(a photoperiod which leads to successful termination of diapause) it takes 20 days to terminate diapause.
The concept of "neuro-endocrine momentum" appears to explain this result 163. quite satisfactorily, namely, that this photo-regime stimulates the N.S,C..
The N.S.C. undergo cycles of activity gathering momentum at each cycle, until at 20 days when the level of activity is sufficiently high, the oocytes differentiate and develop.
In G. tanaceti the neurosecretory system is considered to exert an overall control on reproductive diapause, ovarial development and oviposition.
These distinct physiological phases are controlled by different hormonal levels. Experiments involving cauterizations of various components of the the neurosecretory system indicate that/neuro -endocrine Complex functions as a whole unit, with complex feed-back mechanisms which are not fully understood.
The N.S.C. of the brain of G. tanaceti, for theoretical considerations, are presumed to exert an overall controlling influence on the other neuro- endocrine centres. This central control by the N.S.C. of the brain has been suggested in metamorphosis of Rhodnius (Wigglesworth, 1940); pupal growth in Hyalophora ceCropia (Williams, 1948, 1952); in the stimulation of the suboesophageal ganglion to produce the diapause hormone in Bombyx mori
(Fukuda, 1952) and in reproduction in Call,iphpra erythrosepba (Thomsen 1952),
In contrast, however, the N.S.C. of the brain do not appear to play a dominant rele in reproduction of some insects, viz Carau:ius morosus,
Clitumnus extradentatus (Dupont-Raabe, 1952), Oncopeltus fasciatus (Johansson,
1958) and in Rhodnius prolixus (Wigglesworth, 1936).
The corpora cardiaca/allata in G. tanaceti depend for their function on either nervous stimuli or an allatropic hormone from the N.S.C. of the brain. When the N.S.C. are cauterized in either diapausing or maturing females, the corpora cardiacabllata atrophied. Similar results have been observed in Calliphora (Thomsen, 1952), Oncopeltus (Johansson, 1958) and in 164.
Dermestes (Ladduwahetty, 1962). When the corpora cardiacabliata of
G. tanaceti are extirpated, the N.S.C. of the brain reverted to an inactive condition in maturing females. This suggests that there is a feed-back mechanism from the corpora cardiaca/allata to the brain N.S.C. and that these two centres mutually stimulate each other. This feed-back mechanism of the corpus allatum on the M. N.S.C. of Calliphora has been experimentally demonstrated (Lea and Thomsen, 1962). The close inter-relationship of the
N.S.C. of the brain and the corpus allatum has been shown in Carabus nemoralis in its diurnal activity by measurements of nuclear volumes (Klug, 1958/59).
The N.S.C. of the suboesophageal ganglion in G. tanaceti from cauterization experiments followed by histological examinations of the N.S.C. of the brain, suggests yet another feed-back mechanism to the brain. This could be brought about in two ways) the first is directly to the brain via the circum oesophageal commissures and the second way is via the corpus allatum suboesophageal nerve to the corpus cardiacum/allatum, in turn to the brain. This mechanism has yet to be resolved. The cautery of the N.S.C. of the suboesophageal ganglion in ovipositing females containing mature eggs inhibits oviposition. On implantation of a suboesophageal ganglion from an ovipositing female donor, immediately leads to successful oviposition. This result indicates that the suboesophageal ganglion produces a hormone which is involved in oviposition. The corpus cardiacum secretion has been suggested (see page 123) to cause ovipository reflexes during oviposition
This experiment could therefore be viewed that the hormone from the sub- oesophageal ganglion stimulates the corpus cardiacum to release the factor leading to successful oviposition. This complex mechanism is probable, in view of the existence of the fine corpus allatum►suboesophageal nerve, 165. which connects the suboesophageal ganglion to the corpus cardiacum/allatum.
In G. tanaceti, reproduction occurs during autumn and early winter.
Why a species should evolve a summer reproductive diapause when the food supply is abundant remains problevatical. The classical concept of the phenomenon of diapause is thought of as a physiological mechanism which enables the insect to survive under a period of unfavourable condition.
Richards and Waloff (1961) suggest that summer diapause may well be an effective device to avoid peak incidence of predation and of parasitism.
The interesting feature of ovarial development in G. tanaceti is that growth has been shown to be confined wholly to the terminal oocytes. There appears to be a selective value in an autumn species having this interesting reproductive mechanism, especially in G. tanaceti, in which the adults do not survive the winter. It appears advantageous to the beetle to devote all the available nutrition to ensure the production of one batch of eggs over a short period, rather than developing all the oocytes at the same rate over a relatively longer period, wheh the temperature may become too adverse for normal metabolism in autumn.
The diversity of the different cell types reported by various workers necessitates standardized staining techniques before their apparent distinctiveness can be accepted. However, in the evolution of the insects, it is not difficult to conceive that different neuro-endocrine centres have evolved into different neurosecretory systems which produce different hormones to meet the diversified environmental stimuli to which they axe subjected. There is, on the other hand, no valid reason to accept that all insect species should have the same types of N.S.C. distributed in the same pattern. 166.
Studies on the neurosecretory system have been centred mainly around the "intercerebralis-cardiacum-allatum" system (Hanstrft, 1953; Scharrer
and Scharrer, 1944, 1954). This system in insects has been analogised with
the hypothalammhypophyseal system of chordates by Hanstrtim (1941) and by
Scharrer and Scharrer (1944). In both, there is an epithelial ectodermal rudiment (the adenohypophyokisor the corpus allatum) which may be homologized hypothetically with a cephalic nephridium. Thia flaes during growth with a nervous rudiment (the neurohypophysis or the corpus cardiacum) to form a secretory complex. Both neurohypophysis and corpus cardiacum are innervated
from N.S.C. (in the hypothalamus and in the pars intercerebralis of the
protocerebrum respectively (cited by Wigglesworth, 1954). The intercerebralis-
cardiacum-allatum system has been extended to analogize with the sinus gland/
X-organ of the crustacea (Hanstrtfm, 1949).
The study of the intercerebralis-cardiacum-allatum system in relation
to different physiological processes may be an unrealistic approach to the
problem in view of the facts that a) Fukuda (1951 - 1953) has shown that the
suboesophageal ganglion produces a hormone which controls voltinism in
Bombyx mori; b) Ewen (1962) has some evidence that the B-cells of the
thoracic-abdominal ganglia in Adelphocoris lineolatus may probably be concerned
with the final stages of egg formation or perhaps with oviposition, and
c) Deiphin (1963) has experimental evidence in Schistocerce greciaria that
the hormonal activity of the last abdominal ganglion appears to play a
special role in ovarial maturation.
It is suggested here,that the whole central nervous system and its
allied endocrine glands have to be examined in correlation with maJor
physiological processes before one may appraise their functional significance. 167.
Electron microscopy, coupled with experiments, may resolve the function and nature of the feed-back.mechanisms of the neuro-endocrine centres. 168.
Summary
1. G. tanaceti Linn. has an imaginal reproductive diapause in which there
is a complete cessation of ovarial differentiation and development.
In the males, however, spermatogenesis proceeds normally, although no
spermatozoa are stored in the vesiculae seminales; the accessory glands
remain undeveloped.
2. Photoperiod and temperature are the primary causal factors • inducing,
sustaining and finally, terminating diapause.
3. G. tanaceti is a short day insect, the critical photoperiod is about 13
hours at 20°C.; it is higher at 15°C. and under natural conditions in
the field, where the daily mean air temperature fluctuates around 15°C.
the critical photoperiod is about 14.5 hours.
4. Diapause can be sustained, terminated or reinstated, depending on the
photoperiod to which a female is subjected at any period of its adult
life.
5. The adult life history has been divided into four main physiological
phases; pre-diapause, diapause, maturation and oviposition. The major
part of the study has been on the changes in the neurosecretory system
throughout these phases.
6. Using four different staining techniques, 5 basic neurosecretory cells
designated as types A, AI, B, C and D. have been located in the brain
and in the suboesophageal ganglion. Their locality and distribution
have been mapped. The most prominent groups of neurosecretory cells form
the medial groups in the pars intercerebralis of the brain. 169.
7. The B-cells are thought to exist in two physiological phases, B1 and B IT. The occurrence of the B type of cells coincides with the phases of I higher endocrine activity in the pre-diapause, ovarial maturation and
oviposition phases, whereas the BI, cell types occur predominantly during
diapause.
8. All the neurosecretory cells undergo cycles of activity as has been
observed by measurements of their nuclear volumes, throughout adult life.
These cycles of secretory activity tend to intensify towards the phase of
oviposition.
9. These observations were substantiated by the use of a radioactively 35 labelled amino acid S-D L-cystine in autoradiographic studies. The 35 uptake and discharge of S.by the A- and B-cells of the pars inter-
cerebralis suggest that the N.S.C. undergo short cycles of activity. In
diapausing females, the N.C.S. complete a cycle of activity in 42 - 72
hours in maturing females in less than 24 hours, and in ovipositing
females in about 13 hours. of cystine 10. On the assumption that the rate of uptake/indicates the incorporation
and synthesis of neurosecretion, the neurosecretory cells of ovipositing
females are empirically expressed as 10 times more active than those of
the diapausing ones and twice as active as those of maturing females.
11. In relation to this increase in the level of activity of the neuro-
secretory cells, the corpus cardiacumbllatum follow a similar pattern
in increase of activity. This is evident from the following: (1) the
glands increase in volume, (2) there is decrease in nuclear-cytoplasmic
ratio and (3) increase in nuclear volumes of the secretory cells of the
glands. 170.
12. It is concluded that the phases of diapause, ovarial development and
oviposition are controlled by different levels of endocrinal syndromes.
13. Cautery of any components of the neurosecretory system, leads to a low
functional level of activity of the remaining neurosecretory system,
irrespective of the stimuli of the environment.
14. Implantation experiments suggest that probably several hormones are
involved in maintaining a favourable syndrome.
15. Respiratory rates, fat and water contents have been compared in the pre-
diapause, diapause and ovarial maturation phases.
16. Morphometric measurements of the female reproductive organs have been
made throughout adult life. Oocyte development is confined only to the
terminal oocytes. 171.
Acknowledgements
I wish to record my thanks to Professor 0.W. Richards, F.R.S. for providing me with research facilities in his Department.
I am very grateful to my Supervisor of Studies, Dr. N. Waloff, for her continued interest, constructive criticisms and for translating some
Russian papers.
Thanks are due to Dr. K.C. Highnam, who introduced me to the technique of autoradiography.
I wish to thank Mr. J.W. Siddorn and Mr. D. J. Cross for help in photography, Dr. F. Delphin for introducing me to the technique of Alcian
BlueAhloxine and Miss J.C.Marlow for her help with autoradiography.
Finally, my appreciations are due to Mr. M. J. Way, Dr. C.T.Lewis,
Dr. F. Call, Dr. H.H. El Shatoury for their interesting discussions; to Mr.
R.G. Davies, Mrs. M. van Emden for translating several German papers and to
Mrs. G.T. Sarney for typing the manuscript.
This research was carried out during the tenure of a Commonwealth
Scholarship awarded by the Government of the United Kingdom. 172. BIBLIOGRAPHY
AGRELL, I. 1951a. A contribution to the histolysis-histogenesis problem in insect metamorphosis. Acta physiol. scand., 23, 179-186. AGRELL, I. 1951b. The diapause problem. Anne biol., 55, 287-295. ANDREWARTHA, H.G. 1952. Diapause in Relation to the Ecology of insects. Biol. Rev., 27, 50-107. ANKERSMITo. G.W. 1960. On the Influence of Photoperiod and Temperature on the life cycle of some Univoltine insects. In HrdY, I. (Ed.), The Ontogeny of Insects. Prague, pp. 277-282. ARVY, L. and GABE, M. 1953a. Donnees histophysiologiques sur la neuro- secretion chez les ParOpteres (Ephemeropteres et Odonates). Z. Zellforsch., 38, 591-610. ARVY, L. and GABE, M. 1953b. Donnees histophysiologiques sur les glandes endocrine dephaliques de Brachyotera risi Morton (Plecoptere). Arch. Zool. exp. gen., 22, 105-122. ARVY, L., BOUNHIOL, J.-J. and GABE, M. 1953. Deroulement de la neurosecretion protocerebrale chez Bombyx mori L. au cours du developpement post- embryonnaire. C.R. Acad. Sci., Paris, 236, 627-629. BERN, H.A. 1962. The Properties of Neurosecretory cells. In Takewaki, K. (Ed.), Progress in Comparative Endocrinology. pp. 117-132. BODENSTEIN, D. 1954. Endocrine mechanisms in the life of insects. Recent Progr. Hormone Res., 10, 157-179. BODINE, J.H. 1934. The Effect of cyanide on the oxygen consumption of normal and blocked embryonic cells (Orthoptera). J. cell. comp. Physiol., 4, 397-404. BODINE, J.H. 1941. The cell - some aspects of its functional ontogeny. Amer. Nat., 75, 97-106. BODINE, J.H. and BOELL, E.J. 1934. Respiratory mechanism of normally developing and blocked embryonic cells (Orthoptera). J. cell. comp. Physiol., 5, 97-113. BONHAG, P.F. 1958. Ovarian Structure and Vitellogenesis in insects. Annu.Rev. Ent., 3, 137-160 BONNEMAISON, L. 1945. Arrets de dbveloppement et diapauses. Ann. Epiphyt., 11, 19-56. 173.
BRANDENBURG, J. 1956. Das endokrine System des Kopfes von Andrena vaqa Pz. (Ins. Hymenopt.) and Wirkung der Stylopisation (Stylops, Ins. to Strepsipt.). Z. Morph. Okol. Tiere, 45, 343-364. BUCK, J. 1962. Some Physiological Aspects of Insect Respiration. Annu. Rev. Ent., 7, 27-56. BURGES, H.D. 1960. Studies on the Dermestid Beetle, Troqoderma qranarium Everts. - IV. Feeding, growth and respiration with particular reference to diapause larvae. J. Ins. Physiol. 5, 317-334. BUSNEL, R.G. 1939. Etudes physiologiques sur le Leptinotarsa decemlineata Say. Theses Fac. Sci. Univ. Paris, no. 55, 207 pp. BUTENANDT, A. and KARLSON, P. 1954. Uber die Isolierung eines Metamorphose- Hormons der Insekton in kristallisierter Form. Z. Naturf., 9b, 389-391. BUXTON, P.A. 1935. Changes in the composition of adult Culex pipiens during hibernation. Parasitology, 27, 263-265. CAMERON, M.L. 1953. Secretion of an orthodiphenol in the corpus cardiacum of the insect. Nature, Lond., 122, 349-350. ,V CARSLILE, D.B. and KNOWLES, F.G.W. 1953. Neurohemal organs in Crustaceans. Natures Lond., 172, 404-405. CARSLILE, D.B. and KNOWLES, F.G.W. 1959. Endocrine Control in Crustaceans. Cambridge University Press. 120 pp. CAZAL, P. 1948. Les glandes endocrines retro-cerebrates des insectes (etude morphologique). Bull. biol. Suppl., 32, 1-227. CHURCH, N.S. 1955. Hormones and the termination and reinduction of diapause in Cephus cinctus Nort. (Hymenoptera:Cephidae). Caned. J. Res., D., 33, 339-369. CLARKE, K.U. and LANGLEY, P.A. 1962. Factors concerned in the interaction of growth and molting in Locusts miqratoria L. Conf. European Endocrinologists, London. 1962, pp. 25-26. CLEMENTS, A.N. 1956. Hormonal control of ovary development in mosquitoes. J. exp. Biols, 33, 211-233. CORBET, P.S. 1956. Environmental factors influencing the induction and termination of diapause in the Emperor Dragonfly Anax imperator Leach (Odonata e Aeshnidae). J. exo. Biol., 33, 1-14. 174. DANILEVSKII, A.S. 1948. Photoperiodic reactions of insects in conditions of artificial illumination. [In Russian;1 C.R. Acad. Sci. U.R.S.S. (N.S.), 60, No. 3, 481-484. DANILEVSKII, A.S. 1949. The dependence of the geographical distribution of insects on the ecological peculiarities of their life cycles. [In Russian] Ent. Oboz. 30, 194-207. DANILEVSKII, A.S. 1951. On the conditions favouring a several year diapause in Lepidoptera. [In Russian.' Ent. Oboz., 31, 386-392. DANILEVSKII, A.S. 1961 Photoperiodism and seasonal development of insects. [In Russian.] Leningrad University Press 241 pp. DANILEVSKII, A.S. and GAYSPITZ, K.F. 1948. The influence of the daily periodicity of illumination on the seasonal rhythm of insects. 1In Russian] C.R. Acad. Sci. U.R.S.S. 52, no. 2, 337-340. DAVEY, K.G. 1956. The Physiology of dormancy in the Sweetclover weevil (Sitona cylindricollis (Fehr.)). Canad. J. Res., D, 34, 86-98. DAVEY, K.G. 1961. The mode of action of the heart accelerating factor from the corpus cardiacum of insects. Gen. Comp. Endocrinol. 1, 241 DAVEY, K.G. 1962. Changes in the pericardial cells of Periplaneta americana induced by exposure to homogenates of the corpus cardiacum. 3111.1art. J. micr. Sci., 103, 349-358. DAVID, N.A.L. and GARDINER, B.O.C. 1952. Laboratory breeding of Pieris brassicae L. and Apantiles glomeratus L. Proc. R. ent. Soc. Lond. (A), 27, 54-56. DAY, M.F. 1943. The Function of the corpus allatum in Muscoid Diptera. Biol. Bull., Wood's Hole, 84, 127-140. DELPHIN, F. 1963. Studies on neurosecretion in Schistocerca gregaria Forskal (Orthoptera: Acrididae). Univ. London Ph.D. Thesis 242 pp. DICKSON, R.C. 1949. Factors governing the induction of diapause in the oriental fruit moth Laspeyresia molesta. Ann. ent* Soc. Amer., 42, 511537. DONIA, A.R. 1958. Reproduction and reproductive organs in some Chrysomelidae (Coleoptera). Univ. London Ph.D. Thesis. 215 pp. DREYER, W.A. 1932. The effect of hibernation and seasonal variation of temperature on the respiratory exchange of Formica ulkei Emery. Physiol. Zo81., 5, 301-331. 175. DUCLAUX, M.E. 1869. De l'influence du froid de l'hiver sur le developpement de l'embryon de du ver 1 soie et sur l'oclosion de la graine. C.A. Acad. Sci., Paris, 69, 1021-1022. DUPONT-RAABE, M. 1951. Etude morphologique et cytologique du cerveau de quelques phasmides. Bull. Soc. zool. Fr., 76, 386-397. DUPONT-RAABE, M. 1952. Contribution a l'etude du IrOle endocrine du cerveau et notamment de la pars intercerebralis chazlesphasmides. Arch. Zool. exp. 4n., 89, 128-138. DUPONT-RAABE, M. 1954. Le role endocrine du cerveau dans la regulation des Phenomenes d'adaptation chromatique et de la ponte chez les Phasmides. Pubbl. Staz. zool. Napoli, 24 suppl., 63-66. DUPONT-RAABE, M. 1956. Quelques donnees relatives aux phenomenes de neuro- secretion chez lesp,hasmides. Ann. Sci. nat. Zool., 11 ser., 18, 293-303. ENGELMAN, F. 1957. DieSteuerung der Ovarfunktion bei der ovoviviparen Scharbe Leucophaea maderae (Fabr.). J. Ins. Physiol., 1, 257-278. ENGELMAN, F. 1958. The Stimulation of the corpora allata in Diploptera punctata (Blattaria). Anat. Rec., 132, 432-433. ENGELMAN, F. 1959. The Control of reproduction in Diploptera punctata (Blattaria)d Biol. Bull.,Wood's Hole, 116, 406-419. ENGELMAN, F. 1960. Mechanisms controlling reproduction in two viviparous cockroaches (Blatteria). Ann. N.Y. Acad.. Sci., 89, 516-536. EWEN, A.B. 1962. Histophysiology of the Neurosecretory System and Retro- cerebral Endocrine Glands of the Alfalfa Plant Bug, Adelphocoris lineolatus (Goeze) (Hemiptera s Minidae). J. Morph„ 111,255-274. FORMIGONI, A. 1956. Neurosecretion et organes endocrines chez Apis mellifica L. Ann. Sci. nat. Zool., 11 ser., la, 283-291. FRASER, A. 1959a. Neurosecretion in the Brain of the Larva of the Sheep Blowfly Lucilia caesar. Quart. J. micr. Sci., 100, 377-394. FRASER, A. 1959b. Neurosecretory cells in the abdominal ganglia of larva of Lucilia caesar L. (Diptera). Quart. J. micr. Sci., 100, 395-399. FUKUDA, S. 1940a. Induction of pupation in silkworms by transplanting the prothoracic gland. Proc. imp. Acad. Japan, 16, 414-416. FUKUDA, S. 1940b. Hormonal control of moulting and pupation in the silkworm. Proc. imp. Acad. Japan, 16, 417-420. 176.
FUKUDA, S. 1944. The hormonal mechanism of larval moulting and metamorphosis in the silkworm. J. Fac. Sci. Tokyo Univ., sect. 4, 6, 477-532. FUKUDA, S. 1951a. Alteration of voltinism in the silkworm by decapitating the pupa. Zool. Maq. Tokyo, 60, 119-120. FUKUDA, S. 1951b. Factors determining the production of non-diapausing eggs in the silkworm. Proc. imp. Acad. Japan, 27, 582-587. FUKUDA, S. 1952. Function of the pupal brain and suboesophageal ganglion in the production of non-diapause and diapause eggs in the silkworm. Annot. zool. lap., la, 149-155. FUKUDA S. 1962. Hormorif control of Diapause in the Silkworm. In Takewaki, K. (Ed.), Progress in Comparative Endocrinology, pp. 337-340. FULLER, H.B. 1960. Morphologische und experimentelle Untersuchungen fiber die neurosekretoAschen Verhbltnisse in Zentralnerven-system von Blattiden und Culiciden. Zool. Jb. Allq. Zool., 69, 223-250. GAYSPITZ, K.F. 1949. Light as a factor regulating the cycle of development of the pine Lasiocampid Dendrolinus pini L. [In Russian.] C.:A. Acad. Sci. U.R.S.S., 68, 781-784. GAYSPITZ, K.F. 1953. Reactions of monovoltine butterflies to prolongation of day length. [In Russian.]_ Ent. Oboz., 33, 17-31. GILLETT, J.D. 1958. Induced ovarian development in decapitated mosquitoes by transfusion of haemolymph. J. exp. Biol., 35, 685-693. GOMORI, G. 1941. Observations with differential stains on human islets of Langerhans. Amer. J. Path., 17, 395-406. GORBMAN, A. (Ed.)'1959. Symposium Comparative Endocrinology. Proc. Columbia Univ. SympA on Comparative Endocrinology, held at Cold Spring Harbor, New. York. John Wiley & Sons, Inc.,New York. HALMI, N.S. 1952. Differentiation of two types of basophils in the Adenohypophysis of the rat and mouse. Stain Tech., 27, 61-64. HAMILTON, A.G. 1959. The infra-red gas analyser as a means of measuring the —carbon dioxide output of individual insects. Nature, Land., 184, 3670.369. HANSTR8M, B. 1938. Zwei Probleme betreffs der hormonalen Lokalisation im Insektenkopf. Acta Univ. Lund. N.F., Avd. 2, 39, 1-17. HANSTROM,/elinige Parallelen im Bau und in der Herkunft der in kretorischen Organe der Arthropoden und der Vertebraten. Univ.. Lund., N.F., Avd. 2, 37,1-19. 177.
11 HANSTROM, B. 1949. Three Principal incretory organs in the animal kingdom. Bull. biol., Suppl., 33, 182-209. HANSTRCM B. 1953. Neurosecretory pathways in the head of crustaceans, insects and vertebrates. Nature, Lond., 171, 72-73. HARKEk, J.E. 1956. Factors controlling the diurnal rhythm of activity in Periplaneta americana L. J. exp. Biol., 33, 224-234. HARKER, J.E. 1958. Diurnal rhythms in the animal kingdom. Biol. Rev.,33, 1-L. HARKER, J.E. 1960a. The effect of perturbations on the environmental cycle of the diurnal rhythm of activity of Periplaneta americana L. J. exp. Biol., 32, 154-163. HARKER, J.E. 1960b. Internal factors controlling the suboesophageal ganglion neurosecretory cycle in Periplaneta americana L. J. exp. Biol., 37, 164-170. HARKER, J.E. 1960c. Endocrine and Nervous Factors in insect Circadian rhythms. Cold. Spr. Harb. Symp. quant. Biol., 25, 279-287. HARVEY, W.R. 1962. Metabolic Aspects of Insect Diapause. Annu. Rev. Ent., 7, 57-80. HASEGAWA, K. 1952. Studies on the voltinism in the silkworm Bombyx mori L., with special reference to the organs concerning determination of voltinism. J. Fac. Agric. Tottori Univ., 1, 83-124. HERLANT-MEEWIS, H. and PAQUET, L. 1956. Neuro4cretion et mue chez Carausius morosus Brdt. Ann. Sci. nat. Zool. 11 ser., 18, 163-168. HIGHNAM, K.C. 1961. The histology of the neurosecretory system of the adult female desert locust Schistocerca gregaria. Quart. J. micr. Sci., 102, 27-38. HIGHNAM, K.C. 1962a. Neurosecretory control of ovarian development in Schistocerca oregano. Quart. J. micr. Sci., 103, 57-72. HIGHNAM, K.C. 1962b. Variations in neurosecretory activity during oocyte development in Schistocerca preclaria. J,Endocrin., 24, 1-2. HIGHNAM, K.C. 1962c. Neurosecretory control of ovarian development in the Desert Locust. Proc. 3rd Int. Symp. Neurosecretion, Bristol, 1961. pp. 379-390 178. HINTON,H.E. 1957. Some Aspects of Diapause. Sci. Progr., 45, 307-320. HODEK, I. 1958. Influence of temperature, relative humidity and photoperiod- icity on the speed Of development of Coccinella septempunctata L Acta Soc. ent. 6echoslay., 55, 121-141. HODEK, I. and CERKASOV, J. 1960a. Prevention and artificial induction of the imaginal diapause in Coccinella 7-punctata L. Nature, Lond., 187, 345. HODEK, I. and CERKASOV, J. 1960b. Changes in the physiological state of hibernating imagoes of the lady-bird Semiadalia 11-notata Schneid.. In Hrdy; I (Ed.), The Ontogeny of Insects. Prague, pp. 249-252. HODEK, I and CERKASOV, J. 1961. Prevention and artificial induction of imaginal diapause in Coccinella septempunctata L. (Col.: Coccinellidae). Ent. exp. appl., 4, 179-190. HODGSON, E.S. 1962. Neurosecretion and Behaviour in Arthropods. In Takewaki, K. (Ed.), Progress in Comparative Endocrinology. pp. 180 - 187. HODGSON, E.S. and GELDIAY, S. 1959. Experimentally induced release of neurosecretory materials from roach corpora cardiaca. Biol. Bull., Wood's Hole, 117, 275-283. HRDY, I. (Ed.) 1960. The Ontogeny of Insects. Acta symposia de evolutione insectorum, Praha, 1959. JACOBSON, L.A. and BLAKELEY, 0.E. 1959. Development and behaviour of the army cutworm in the laboratory. Ann. ent. Soc. Amer., 52, 100-105. JOHANSSON, A.S. 1954. Corpus alb:turn and egg production in starved-milkweed bugs. Nature Lond., 174, 89. JOHANSSON, A.S. 1958. Relation of nutrition to endocrine-reproductive functions in the milkweed bug Oncopeltus fasciatus (Dallas) (Heteroptera Lygaeidae). Nytt. Mag. Zool. Oslo, 7, 1-132. JOLY, P. 1945. La function ovarienne et son contrOle humotal chez les dytiscides. Arch. ,Zool. exp. gen., 84, 47-164. JONES* B.M. and WILSON, R.S. 1959. Studies on the action of phenylthiourea on the respiratory metabolism and spinning behaviour of the cynthia silkworm. Biol. Bull., Wood's Hole, 117, 482-491. 179. KAISER, P. 1954. Cyclic changes in the corpora allata and their relation to the function of the ovaries in Hydrophilus. Ann.Acad.bras.Sci., 26, 283-288. KLUG, H. 1958/59. Histo-physiologische Untersuchungen Uber die Aktiviat- speriodik bei Carabiden. Ass.Z.Humboldt-Univ.Berlin (Math.- nat.R.), 8, 405-434. KOBAYASHI, M. 1957. Sanshi Shikenjo akoku, 15, 181-273. (cited by van der Kloot 1960 and 1962). KOGURE, M. 1933. The influence of light and temperature on certain characters of the silkworm, Bombyx marl. J.Dep.Agric.Kyushu Univ., 4, 1-93 LADDUVAHETTY, A.M. 1962. The reproductive cycle and neuroendocrine relations in Dermestes maculatus (Coleoptera : Dermestidae). Univ.London Ph.D. Thesis. 151 pp.
LEA, A.O. and THOMSON, F. 1962. Cycles in the synthetic activity of the
medial neurosecretory cells of Calliphora erythrocephala and their regulation. Proc.3rd.Int.Symp.Neurosecretion, Bristol, 1961. pp. 345-347. LEES, A.D. 1953a. Environmental factors controlling the evocation and termination of diapause in the fruit tree red spider mite, Metatetranychus ulmi Koch. (Acarina Tetranychidae). Ann. eppl. Biol., 40, 449-486. LEES, A.D. 1953b. The significance of the light and dark phases in the photo- periodic control of diapause in Metatctranychus ulmi Koch. Ann. appl. Biol., 40, 487-497. LEES, A.D. 1955. The Physiology of Diapause in Arthropods. Cambridge University Press. 151 pp. LEES, A.D. 1956. The physiology and biochemistry of diapause. Annu. Rev. En 1, 1-16. LEES, A.D. 1960. Some aspects of animal photoperiodism. Cold Spr. Harb. Symp. quant. Biol., 25, 261-268. LERMA,de B. 1932. Osservazioni sui corpora dilate del Gryllotalpa. Arch.zool.ital., 17, 417-433. 180.
LERMA, de B. 1956. Corpora cardiaca et neurosecretion protocerebrale chez le Coleopthre, Hydrous piceus L. Ann. Sci. nat. Zool., 11 ser., 18, 235-249.
LHOSTE, J. 1952. Donnees histo-physiologiques sur les cellules neurosecret- rices cephaliques et le complexe retrocerebral de Forficula auricularia L. Arch. Zool. exp. gen., 89, 169-183. MASAKI, S. 1958. The response of a "short day" insect to certain external factors: the induction of diapause in Abraxas miranda Butler (Lepidoptera, Geometridae). Jap. J. appl. Ent. Zool., Tokyo, 2, 285-294. MATSUMOTO, S., SANTA, H. and OTUKA, M. 1953. Studies on the diapause in the cabbage army-worm Barathra brassicae L. 2. The effect of temperature and photoperiod on the induction of diapause. Oyo-Kontyu, 9, 45-51. MATTHtE, J.J. 1951. The structure and physiology of the egg of Locustana pardalina Walk. Bull. Dep. Agric. S. Afr. Sci., No.316, 83 pp• MENDES, M.V. 1948. Histology of the corpora allata of Melanoplus different-
iales (Orthoptera Saltatoria). Biol. Bull., Vood's Hole, 94, 194-207.
MILBURN, N., WEIANT, E.A. and ROEDER, K.D. 1960. The release of efferent nerve activity in the roach, Periplaneta americana by extracts of the corpus cardiacum. Biol. Bull., Wood's Hole, 118, 111-119. MITSUHASHI, J. and FUKAYA, M. 1960. The hormonal control of larval diapause in the rice stem borer, Chilo suppressalis. III. Histological studies on the neurosecretory cells of the brain and the secretory cells of the corpora allata during diapause and post
diapause. Jap. J. appl. Ent. Zool., Tokyo, 4, 127-134.
MOROHOSHI, S . 1959. Hormonal studies on the diapause and non-diapause eggs of the silkworm Bombyx mori. J.Ins. Physiol., 3, 28-40. MULLER, H.J. 1955. Die Saisonformembildung von Araschnia levana, ein photo- periodisch gesteuerter Diapause-Effekt. Naturwissenschaften, 42, 134-135. 181.
MULLER, H.J. 1957a. Die Wirkung exogener Faktoren auf die zyklische Formen- bildung der Insekten, insbesondere der Gattung Euscelis (Hon. Auchenorrhyncha). Zool. Jb. Abt. I. Syst. (Ock.), Geogr. Biol., 85, 317-430. MULLER, H.J. 1957b. Uber die Diapause von Stenocranus minutus Fabr. (Hom. Auchenorrhyncha). Beitr. Ent., 7, 203-226. MULLER, H.J. 1958. Uber den Einfluss der Photoperiode auf Diapause and Korpergrosse der Delphatide, Stenocranus minutus Fabr. (Homoptera, Auchenorrhyncha). Zool. Anz., 160, 294-312. NAYAR, K.K. 1953. Neurosecretion ih Iphita limbata Stal. Curr. Sci., 22, 149.
NAYAR, K.K. 1955. Studies on the neurosecretory system of Iphita limbata Stal. I. Distribution and structure of the neurosecretory cells of the nerve ring. Biol. Bull., Wood‘ Hole, 108, 296-307. NAYAR, K.K. 1956. The structure of the corpus allatum of Iphita limbata (Hemiptera). Quart. J. micr. Sci., 97, 83-88. NAYAR, K.K. 1958. Studies on the neurosecretory system of Iphita limbata Stal. V. Probable endocrine basis of oviposition in the female insect. Proc. Indian Acad. Sci., (B), 47, 233-251. NORRIS, M.J. 1959. The influence of day-length on imaginal diapause in the red locust, Nomadacris septemfasciata (Serv.). Ent. exp. appl., 2, 154-168. NORRIS, M.J. 1962. Diapause induced by photoperiod in a tropical locust, Nomadacris septemfasciata (Serv.). Ann. appl. Biol., 50, 600- 603. NOVAK, V.J.A., SLAMA, K. and WENIG, K. 1960. Influence of implantation of corpus allatum on the oxygen consumption of Pyrrhocoris apterus. In Hrdl: I. (Ed.), The Ontogeny of Insects. Prague, pp. 147- 151. OTUKA, M. and SANTA, H. 1955. Studies on the diapause in the cabbage army- worm, Barathra brassicae L. 3. The effect of the rhythm of light and darkness in tilF induction of diapause. Bull. Nat. 182.
Inst. Agr. Sci. (Tokyo), C, No.5. (NBgy3 Gijutsu Kenedo Okoku). OZBAS, S. and HODGSON, E.S. 1958. Action of insect neurosecretion upon central nervous system in vitro and upon behaviour. Proc. net. Acad. Sci., 44, 825-830. PALM, N.B. 1947. Notes on the structure of the corpora allata in Gryllotalpa. Forh. K. fisiogr. SUllsk.ImInd., 17, 130-140. PANTIN, C.F.A. 1946. Notes on Microscopical Technique for Zoologists. -I 4 e 4 I. ( , Cambridge University Press. 1st Ed. 79 pp. PELC, S.R. and HOWARD, A. 1952. Techniques of Autoradiography and the Application of the Stripping-Filth Method to Problems of Nuclear Metabolism. Brit. Med. Bull., 8, 132-135. PFEIFFER, I.Sti. 1939. Experimental study of the function of the corpora allata in the grasshopper, Melanoplus differentialis. J. exp. Zool., 82, 439-461. PFEIFFER, I.W. 1945. Effect of the corpora allata on the metabolism of adult female grasshoppers. J. exp. Zool., 99, 183-233. PFLUGFELDER, O. 1937a. Bau, Entwicklung and funktion der Corpora allata and cardiaca von Dixippus morosus Br. Z. wiss. Zool., 149, 477-512. PFLUGFELDER, O. 1937b. Untersuchungen 'Fiber die Funktion der Corpora allata der Insekten. Verb. dtsch. zool. Ges., 39, 121-129. PFLUGFELDER, O. 1939. Wechselwirkungen von DrUsen innerer Sekretion bei D.ixippus morosus Br. Z. wiss. Zool., 152, 384-408. PREBBLE, M.L. 1941. The diapause and related phenomena in Gilpinia polytoma (Hartig). IV. The influence of food and diapause upon reprod- uctive capacity. Canad. J. Res,, D, 19, 417-436. PROCEEDINGS OF THE FIRST INTERNATIONALSYMPOSIUM ON NEUROSECRETION. Held at Naples, 1953. (Published 1954). PROCEEDINGS OF THE SECOND INTERNATIONAL SYMPOSIUM ON NEUROSECRETION. Held at Lund, 1957. (Published 1958). 183.
PROCEEDINGS OF THE THIRD INTERNATIONAL SYMPOSIUM ON NEUROSECRETION. Held at Bristol, 1961. (Published 1962). PUNT, A. 1950. The respiration of insects. Physiol. comp., 2, 59-74. PUNT, A., PARSER, W.J. and KUCHLEIN, J. 1957. Oxygen uptake in insects with cyclic carbon dioxide release. Biol. Bull., Wood's Hole, 112, 108-119. RICHARDS, O.V. and WALOFF, N. 1961. A study of a natural population of Phytodecta olivacea (Forster) (Coleoptera, Chrysomeloidea). Phil. Trans. Series B, 244, 205-257.
ROEDER, K.D. 1935. An experimental analysis of the sexual behaviour of the praying mantis (Mantis religiose L.). Biol. Bull., Wood's Hole, 69, 203-220. SACHAROV, N. L. 1930. Studies in cold resistance in insects. Ecology, 11, 505-517. SAGESSER, H. 1960. Uber die Wirkung der Corpora allata auf den Sauerstoff- verbrauch bei der Schabe Leucophaea maderae (Fabr.). J. Ins. Physiol., 5, 264-285. SCHARRER, B. 1946a. The role of corpora allata in the development of Leucophaea maderae (Orthoptera). Endocrinology, 38, 35-45. SCHARRER, B. 1946b. The relationship between corpora allata and reproductive organS in adult LeUcophaea maderae (Orthoptera). Endocrinology, 38, 46-55. SCHARRER, B. 1951. The storage of neurosecretory material in the corpus cardiacum. Anat. Rec., 111, 554-555. SCHARRER, B. 1952. Neurosecretion. XI. The effects of nerve section on the intercerebralis-cardiacum-allatum system of the insect Leucoph- aea maderae. Biol. Bull., Wood's Hole, 102, 261-272. SCHARRER, B. 1955. "Castration cells" in the central nervous system of an insect Leucophaea maderae (Blattaria). Trans. N.Y. Acad. Sci., Ser.II, 17, 520-525. SCHARRER, B. 1959. The role of neurosecretion in neuroendocrine integration. In Gorbman, A. (Ed.), Symposium Comparative Endocrinology, pp. 134-148. 184.
SCHARRER, B. and HARNACK, von M. 1958. Histophysiological studies on the corpus allatum of Leucophaea maderae. I. Normal life cycle in male and female adults. Biol. Bull., Wood's Hole, 115, 508-520. SCHARRER, B. and HARNACK, von M. 1960. Castration effects in the insect Leucophaea maderae. Anat. Rec., 138, 382. SCHARRER, B. and HARNACK, von M. 1961. Histophysiological studies on the corpus allatum of Leucophaea maderae. III. The effect of castration. Biol. Bull., Wood's Hole, 121, 193-208. SCHARRER, B. and SCHARRER, E. 1944. Neurosecretion. VI. A comparison between the intercerebralis-cardiacum-allatum system of insects and the Hypothalamo-Hypophy seal system of the vertebrates. Biol. Bull. Wood's Hole, 87, 242-251. SCHARRER, E. 1952. The general significance of the neurosecretory cells. Scientia, 46, 172-183. SCHARRER, E. and BROWN, S. 1962. Neurosecretion in Lumbricus terrestris. Gen. Comp. Endocrinol., 2, 1-3. SCHARRER, E. and SCHARRER, B. 1954. Hormones produced by neurosecretory cells. Recent Progr. Hormone Res., 10, 183-240. SCHNEIDERMAN, H.A. and WILLIAMS, C.M. 1953. The physiology of insect diapause. VII. The respiratory metabolism of the Cecropia silkworm during diapause and metamorphosis. Biol. Bull., Wood's Hole, 105, 320-334. SCHNEIDERMAN, H.A. and WILLIAMS, C.M. 1954. The physiology of insect diapause. IX. The cytochrome oxidase system in relation to the diapause and development of the Cecropia silkworm. Biol. Bull., Wood's Hole, 106, 238-252. SCHNEIDERMAN, H.A. and WILLIAMS, C.M. 1955. An experimental analysis of the discontinuous respiration of the Cecropia silkworm. Biol. Bull. Wood's Hole, 109, 123-143. SCHOONHOVEN, L.M. 1960. Diapause in Bupalus pini (Lep, Geometridae) in relation to host-parasite synchronization. In Hrdlr, I. (Ed.), The Ontogeny of Insects. Prague, pp. 261-264. 185.
SCHOONHOVEN, L.M. 1962. Synchronization of a parasite/host system, with special reference to diapause. Ann. appl. Biol., 50, 617-621. SELLIER, R. 1949. Diapause larvaire et macropt4risme chez Gryllus campestris (Orth.). C.R. Acad. Sci., Paris, 228, 2055-2056. SHAPPIRIO, D.G. and WILLIAMS, C.M. 1957a. The cytochrome system of the Cecropia silkworm. I. Spectroscopic studies of individual
tissues. Proc. roy. Soc. B., 147, 218-232. SHAPPIRIO, D.G. and WILLIAMS, C.M. 1957b. The cytochrome system of the Cecropia silkworm. II. Spectrophotometric studies of oxidative enzymes in the wing epithelium. Proc. roy. Soc. B., 147, 233- 246. SLAMA, K. 1960a. Metabolism during diapause and development in sawfly metamorphosis. In firdy., I. (Ed.), The Ontogeny of Insects. Prague, pp. 195-201. SLAMA, K. 1960b. Physiology of Sawfly Metamorphosis. I. Continuous Respiration in Piapausing Prepupae and Pupae. J. Ins. Physiol., 5, 341-348. SLIFER, E.H. 1938. The formation and structure of a special water-absorbing area in the membranes covering the grasshopper egg. Quart. J. micr. Sci., 22, 437-458. SLIFER, E.H. 1946. The effects of xylol and other solvents on diapause in the grasshopper egg; together with a possible explanation for the action of these agents. J. exp. Zool., 102, 333-356. SLIFER, E.B. 1948. Isolation of a wax-like material from the shell of the grasshopper egg. Disc. Faraday Soc., No.3, 182-187. SLIFER, E.H. 1950. A microscopical study of the hydropyle and hydropyle cells in the developing egg of the grasshopper, Melanoplus differentialis. J. Morph., 87, 239-273. SLOPER, J.C. 1958. The application of newer histochemical and isotope techniques for the localization of protein-bound cystine or cysteine to the study of hypothalmic neurosecretion in normal and pathological conditions. In Bargman, W., Hanstr3m, B. and Scharrer, B. and E. (Eds.), Proc.IInd.Int.Symp.Neurosecretion. Lund, 1957. pp. 20-25. 186.
SLOPER, J.C., ARNOTT, D.J. and KING, B.C. 1960. Sulphur metabolism in the pituitary and hypothalmus of the rat: a. study of radioisotope uptake after the injection of 35S-DL-cystine, methionine and sodium sulphate. J. Endocrin., 20, 9-23. SMITH, 0.J. and LANGSTON, R.L. 1953. Continuous laboratory propagation of Western grape loaf Skeletonizer and parasites by prevention of diapause. J. econ. Ent., 46, 477-484 STAAL, G.B. 1961. Studies on the physiology of phase induction in Locusta migratoria (migratoroides R. & F.). Laboratoriurn voor Entomol- ogle, lAageningen, Nederland. Publikatie Fonds Landhouw Export Bureau, 1916 - 1918, No.40, 1-125. STRANGMS-DIXON, J. 1960. Hormones, selective feeding and reproduction in Calliphora. In Hrdi, I. (Ed.), The Ontogeny of Insects. Prague, pp. 137-139. STRANGWAYS-DIXON, J. 1961. The relationship between nutrition, hormones and reproduction in the blowfly Calliphora erythrocephala Meig. I. Selective feeding in relation to the reproductive cycle, the corpus allatum volume and fertilization. J. exp. Biol., 38, 225-236. TAKE6AKI, K. (Ed.) 1962. Progress in Comparative Endocrinology. Proc.3rd.Int. Symp.comp.Endocrinol., Held at Oiso, Japan, 1961. 383 pp. TANAKA, Y. 1944. Effect of daylength on hibernation of the Chinese oak silkworm (In Japanese). Aerie. Hort. (N6gy6 oyobi Engel), 19, no.9. THOMSEN, E. 1942. An experimental and anatomical study of the corpus allatum in the blowfly Calliphora erythrocepnala Meig. Vidensk. Medd. dansk. naturh. Foren. Kbh., 106, 319-405. THOMSEN, E. 1949. Influence of corpus allatum on oxygen consumption of adult Calliphora erythrocephala Meig. J. exp. Biol., 26, 137-149. THOMSEN, E. 1952. Functional significance of the neurosecretory brain cells and the corpus cardiacum in the female blowfly Calliphora erythrocephala Meig. J. exp. Biol., 29, 137-172. 187.
THOMSEN, E. 1954. Studies on the transport of neurosecretory material in Calliphora erythrocephala by means of ligaturing experiments. J. exp. Biol., 31, 322-330.' THOMSEN, E. and HAMBURGER, K. 1955. Oxygen consumption of castrated females of the blowfly, Calliphora erythrocephala Meig. J. exp. Biol., 32, 692-699. THOMSEN, E. and MOLLER, I. 1959. Neurosecretion and intestinal proteinase activity in an insect, Calliphora erythrocephala Meig. Nature, Lond., 183, 1401-1402. THOMSEN, E. and MOLLER, I. 1960. Further studies on the function of the neurosecretory brain cells of the adult Calliphora females. In Hrq, I. (Ed.), The Ontogeny of Insects. Prague, pp. 121-126. THOMSEN, M. 1954. Neurosecretion in some Hymenoptera. Biol. Skr., 7 (5),1-24. UMBRIET, W.k., BURRIS, R.H. and STAUFFER, J.F. 1947. Manometric Techniques and Related Methods for the Study of Tissue Metabolism. Burgess Publishing Co., Minneapolis, Minn., 1945. 203 pp. VAN DER KLOOT, V.G. 1955. The control of neurosecretion and diapause by physiological changes in the brain of the Cecropia silkworm. Biol. Bull., hood's Hole, 109, 276-294. VAN DER KLOOT, W.G. 1958. The resting potentials of neurons in the Cecropia brain during diapause and development. Proc.lOth.Int.Congr. Ent., Montreal, Cue., 1956, 2, 79. VAN DER KLOOT, W.G. 1960. Neurosecretion in insects. Annu. Rev. Ent., 5, 35-52. VAN DER KLOOT, W.G. 1962. Comparative Endocrinology of the Invertebrates. Annu. Rev. Physiol., 24, 491-516. II1ALOFF, N. 1949. Observations on larvae of Ephestia elutella HUbner (Lep. Phycitidae) during diapause. Trans. P. Ent. Soc. Lond., 100, 147-159. WAY, M.J: 1962. Definition of diapause. Ann. appl. Biol., 50, 595-596. M.J. and HOPKINS, B.A. 1950. The influence of photoperiod and temperature on the induction of diapause in Diataraxia oleracea L. (Lepidop- tera). J. exp. Biol., 27, 365-376. 188.
WELSH, J.H. 1959. Neuroendocrine substances. In Gorbman, A. (Ed.), Symposium Comparative Endocrinology, pp. 121-133. WELSH, J.H. 1962. Introductory notes to NeuroendocrinePthenomena. In Takewaki, K. (Ed.), Progress in Comparative Endocrinology, pp. 115-117. WIGGLESWORTH, V.B. 1934. The physiology of ecdysis in Rhodnius prolixus (Hemiptera). II. Factors controlling moulting and "Metamorph— osis". Quart. J. micr. Sci., 77, 191-222. WIGGLESWORTH, V.B. 1936. The function of the corpus allatum in the growth and reproduction of Rhodnius prolixus (Hemiptera). Quart. J. micr. Sci., 79, 91-121. WIGGLESWORTH, V.B. 1940. The determination of characters at metamorphosis in Rhodnius prolixus (Hemiptera). J.exp. Biol., 17, 201-222. WIGGLESWORTH, V.B. 1948. The functions of the corpus allatum in Rhodnius prolixus (Hemiptera). J. exp. Biol., 25, 1-14. WIGGLESWORTH, V.B. 1952. Hormone balance and control of Metamorphosis in Rhodnius prolixus (Hemiptera). J. exp. Biol., 29, 620-631. WIGGLESWORTH, V.B. 1954. The Physiology of Insect Metamorphosis. Cambridge University Press. 152 pp. WIGGLESWORTH, V.B. 1957. The action of growth hormones in insects. Symp. Soc. exp. Biol., 11, 204-227. VIGGLESINORTH, V.B. 1962. Hormones in relation to metamorphosis. In Takewaki, K. (Ed.), Progress in Comparative Endocrinology. pp. 316-321. WILLEY, R.B. 1961. The morphology of the stomadeal nervous system in Periplaneta americana (L.) and other Blattaria. J.Morph., 108, 219-261. WILDE, de J. 1954. Aspects of diapause in adult insects, with special regard to the Colorado beetle, Leptinotarsa decemlineata Say. Arch. n6erl. "tool., 11, 375-385. WILDE, de J. 1955. The significance of the photoperiod for the occurrence of diapause in the adult Leptinotarsa decemlineata Say. Proc.lst. Int.Photobiol.Congr., Amsterdam, 1954. pp. 96-101. 189.
WILDE, de J. 1960. Diapause in the Colorado beetle (Leptinotarsa decemlineata Say.) as an endocrine deficiency syndrome of the corpus allata. In Hrdl, I. (Ed.), The Ontogeny of Insects. Prague, pp.226-230.. WILDE, de J. 1962. Photoperiodism in Insects and Mites. Annu. Rev. Ent., 7, 1-26. WILDE, de J. and STEGWEE, D. 1958. Two major effects of the corpus allatum in the adult Colorado beetle (Leptinotarsa decemlineata Say.). Arch. neerl. Zool., 13, 277-289. WILDE, de 3., DUINTJER, C.S. and MOOK, L. 1959. Physiology of diapause in the adult Colorado beetle Leptinotarsa decemlineata Say. I. The Photoperiod as a controlling factor. J. Ins. Physiol., 3, 75-85.
WILLIAMS, C.M. 1946. Physiology of insect diapause. I. The role of the brain in the production and termination of pupal dormancy in the giant silkworm Platysamia cecropia. Biol. Bull.t Woods Hole, 90, 234-243. WILLIAMS, C.M. 1947. Physiology of insect diapause. II. Interaction between pupal brain and prothoracic glands in the metamorphosis of the giant silkworm, Platysamia cecropia. Biol. Bull., Wood's Hole, 22, 89-98. WILLIAMS, C.M. 1948a. Physiology of insect diapause. III. The prothoracic glands in the Cecropia silkworm with special reference to their significances in embryonic and postembryonic development. Biol. Bull., Vloods Hole, 94, 60-65. WILLIAMS, C.M. 1948b. Extrtnsic control of morphogenesis as illustrated in the metamorphosis of insects. Grolp,th Symposium, 12, 61-74. WILLIAMS, C.M. 1952. Physiology of insect diapause. IV. They brain and prothoracic glands as an endocrine system in the Cecropia silkworm. Biol. Bull., wod's Hole, 103, 120-138.
WILLIAMS, C.M. 1956a. The juvenile hormone of insects. Nature, Lond., 178, 212-213. 190.
WILLIAMS, C.M. 1956b. Physiology of insect diapause. X. An endocrine mechanism for the influence of temperature on the diapausing pupa of the Cecropia silkworm. Biol. Bull., Woods Hole, 110, 201-218.