Camp. Biochem. Phwiol. Vol. 105A, No. 4, pp. 679687, 1993 0300-9629/93 $6.00 + 0.00

Printed in Great Britain 0 1993 Pergamon Press Ltd

PHYSIOLOGY OF LARVAL DIAPAUSE IN THE WAX MOTH, GALLERIA MELLONELLA:

AN ULTRASTRUCTURAL ANALYSIS

P. GASSIER* and B. CYMBOROWSKI~$

*Universiti Pierre et Marie Curie, Laboratoire d’fivolution des ctres Organists, 105, boulevard Raspail, 75006 Paris VI, France; tDepartment of Invertebrate Physiology, Warsaw

University, 93 Zwirki i Wigury, 02-089 Warszawa, Poland (Fax 0048-2222-5342)

(Received 8 October 1992; accepted I1 November 1992)

Abstract-l. The wax moth, Galleriu mellonella enters larval diapause when l-day-old last instar larvae are transferred from an optimal rearing temperature of 30 to 18°C. In the haemolymph of diapausing larvae very low ecdysteroid titres exist which are due to synthetic inactivity of the prothoracic glands and not due to a reduction in prothoracic gland cell numbers. Ultrastructural analysis of the prothoracic glands taken from diapause-committed S-day-old last instar larvae, show all the signs of inactivity as far as the development of the endoplasmic reticulum and mitochondrial system are concerned. Whereas in the same age last instar non-diapausing larvae, the glands are extremely active as judged by their ultrastructure.

2. Paraldehyde-fuchsin staining and ultrastructural studies have revealed that in diapause-committed S-day-old last instar larvae of Galleriu mellonella, neurosecretory ceils of the protocerebrum accumulate the neurosecretory vesicles in their perikaria. The protocerebral cells of control (non-diapausing) larvae of the same age contain only a small amount of neurosecretory vesicles.

3. The corpora allata of diapause-committed larvae seems to be inactive despite the high titre ofjuvenile hormone found in their haemolymph. The endoplasmic reticulum and mitochondrial system, organellae involved in the juvenile hormone biosynthesis, are reduced.

4. The possible mechanism controlling larval diapause of Galleria mellonella is discussed.

INTRODUCTION

Development of the wax moth, Galleria meIlonelIa

is strongly affected by a low temperature of 18°C. When l-day-old last instar larvae were transferred from an optimal rearing temperature of 30°C to 18”C, their further development was completely blocked at the stage of the post-spinning larva (Smietanko et al., 1989; Mikolajczyk and Cymborowski, 1993). This facultative arrested development, being the consequence of changes in the neuroendocrine sys- tem, can be defined as diapause (Mikolajczyk and Cymborowski, 1993; Muszyiiska-Pytel et al., 1992). Ecdysteroid titre in the haemolymph of these larvae is maintained at a low level which is due to the nervous and humoral inhibition of the prothoracic glands (PG). The inhibitory nervous input probably comes from the brain and is transmitted to the PG via

When diapausing larvae were transferred from 18°C to diapause-termination conditions of 30°C development was initiated and pupation was syn- chronous within 5-7 days as a result of rhythmic production and liberation of ecdysteroids into their haemolymph (Cymborowski et al., 1989, 1991).

In this paper we have investigated the cellular events of the endocrine centres and resultant hor- monal changes, which is a necessary prelude to elucidating the factors and mechanisms involved in the neurohormonal control of diapause in Galleriu

mellonella larvae. Specifically, the following questions were addressed. (1) Is steroidogenic refractoriness a result of atrophy of the prothoracic glands, or (2) is it a consequence of a reduction in the activity of the PTTH-stimulating system? (3) The cellular basis of JH-activating and producing systems during larval dianause were also investigated.

the suboesophageal ganglion and paired cervical _ nerve (Muszyiiska-Pytel et al., 1992).

In diapausing larvae there is a high titre MATERIALS AND METHODS

of juvenile hormone (JH) in the haemolymph Stock maintenance c

(Smietanko er al., 1989). It is, therefore, probable that as a consequence of the action of JH, the PG

The wax moth (Galleria mellonella) larvae were

remain refractory to stimulation by prothoracico- reared in constant darkness at 30°C on a semi-

tropic hormone (PTTH). artificial diet prepared as described by Sehnal (1966). Larvae were kept at 30°C up to the first day of the

$To whom all correspondence should be addressed.

last (7th) instar. Thereafter they were transferred into the diapause-inducing temperature of 18°C and

679

680 P. CASSIEW and B. CYMB~ROWSKI

used for experiments after different times spent at this temperature. Larvae reared continuously at 30°C served as controls.

Quantification of ecdysteroids

Ecdysteroid titre determinations were performed on control and diapause-destinated larvae. The haemolymph samples were collected daily beginning from day 5 of the last instar of Galleria mellonella larvae reared at 30’C or transferred to 18°C. Samples (20 ill) of haemolymph were extracted by two treat- ments with 100% methanol followed by centrifu- gation (15 min, 500 rpm). Supernatants were pooled, evaporated to dryness under nitrogen at 40°C and then diluted in the appropriate buffer for an enzyme immunoassay (EIA) for ecdysteroids according to Porcheron et al. (1989). Acetyleholinesterase was used as a label. In this assay there is a good cross- reactivity between ecdysone and 20-hydroxyecdysone (Porcheron et al., 1989), hence 20-hydroxyecdysone was used as a standard and the results were expressed in picograms of 20-hydroxyecdysone equivalents per microlitre of haemolymph.

~orF~oiog~ca~ and cytological analysis

The paraldehyde-fuchsin histological technique of Dogra and Tandam (1964) was used for neuro- secretory cell examination. The brains of diapausing and non-diapausing last instar larvae were dissected out into Ringer’s solution, fixed in Bouin’s fluid and embedded in paraffin. The whole-mount preparations or Sprn sections were stained with paraldehyde- fuchsin prepared from basic fuchsin (Allied Chemical Co., C.I.425.00). The number of neurosecretory cells, their position in the brain, and the degree of accumu- lation of the neurosecretory material was recorded according to previous works (Cymborowski, 1970, 1973; Muszynska-Pytel, 1986).

The prothorac~c gIands of control and diapausing Iarvae were also fixed in Bouin’s fluid to preserve the size and shape of the PG cells and their morphology was examined under the dissection microscope.

Transmission electron microscop~l

For electron microscopy, the brain, prothoracic glands and corpora allata of diapausing and non- diapausing larvae of the last instar were removed under stereoscopic microscope. The tissues were fixed for 20 min in cold (4°C) fixative (3% glutaralde- hyde in 0.1 M cacodylate buffer with 8% sucrose) and rinsed in the same buffer. After 1% osmium postfixation and dehydratation, the tissues were embedded in Epon 812. Semi-thin sections (0.5-l pm) were stained with 1% tholuidine blue in 1% sodium borate solution. Ultra-thin sections collected on copper grids were stained with uranyl acetate and contrast enhanced by lead citrate (Reynolds. 1963).

RESULTS

Eedysteroid titres in Contras and drayage-committed Iast instar Galieria larvae

Last instar Galieria meiionella larvae reared con- tinuously at 30°C showed a high peak (approx. 1000 pg of 20-hydroxyecdysone equivalents/p I of haemolymph) of ecdysteroids on day 8 in the second half of the instar. This was followed by a rapid decrease in ecdysteroid titre at the time of pupation (Fig. 1).

In the larvae which were transferred to 18°C on the first day of the last instar and reared at this temperature, haemoIymph ecdysteroid titre remained very low. There was only one small peak of ecdy- steroid titre (about 27 pg of 20-hydroxyecdysone equivalents/g1 of haemolymph) occurring on day 8 after transfer to 18°C. This ecdysteroid peak was insufficient to stimulate development and morpho- genie processes leading to metamorphosis and the larvae did not pupate.

Morphological and cytological investigations of the prothoracic glands of control and diapausing Galleria larvae

The prothoracic glands of control last instar larvae kept at 30”~ were significantly bigger than the PG’s of diapausing insects {Fig. 2). In control larvae there are approx. 55 polyploid ceils per gland, whereas after three months at 18°C only 18-20 cells per gland were found.

1100 r-

900 - 3OQC Pupation

r g 700 - /r

/ ~

I

I

.F

““I

z J 300

? x1 * -1

c 100 -

0 ot z J

Dow of 7th instar

Fig. I. Ecdysteroid titres in control 3O’C reared insects (A) and diapanse-committed 18;‘C kept last instar Gul(eriu meilon&t larvae. Samples of haemolymph were assayed by EIA. Each point represents mean (*SD) of 4-5 separate determinations. Data are presented as pg of 20- hydroxyecdysone (20E) equivalents (eqts) per ~1 of haemo- lymph. Asterisks indicate the time when the brain, prothoracic glands and corpora allata were taken for EM

study.

Larval diapause in Galleria mellonellu 681

Fig. 2. Morpholo~cal changes of prothoracic gland cells in diapausing (A) and non-diapausing (B) the last instar Gafleria mellonefia larvae. After three months spent in diapause there is a noticabie reduction in number and size of the PG ceils. For better preservation of the structure the glands were fixed in

Bouin’s fluid.

In order to check whether the low titre of ecdy- steroids in the haemolymph of these larvae was due to reduced numbers of prothoracic gland cells or to inactivity, ultrastructural analysis was performed. It was found that after 8 days spent at 18°C none of the prothoracic gland cells showed any sign of secretory activity. The cells consisted of irregularly shaped giant nuclei filling nearly the whole cell body. The chromatin was in the form of compact masses (Fig. 3A). The nuclei were surrounded by a thin layer of cytoplasm containing only minute fragments of endoplasmic reticulum with free lying ribosomes. Only small numbers of mitochondria could be seen in the cytoplasm and the cells had a smooth plasmic membrane surface.

However, prothoracic gland cells taken at the peak ecdysteroid haemolymph titre on day 8 from control larvae reared at 30°C (see Fig. I), were much better developed than PG cells of diapausing larvae. The nuclei were lobulated with greater amounts of more homogenous chromatin compared to the nuclei of diapausing larvae. The endoplasm was thick with well-developed rough endoplasmic reticulum cister- nae and with numerous mitochondria (Fig. 38). A thick layer of radial channels appeared on the cell surface. These peripheral channels of the PG cells have been considered to be distinct signs of secretory activity (Mala et al., 1974).

&Ference in the amount and distribution of‘ neuro- secretion in the neurosecretory cells of ~rotocerebru~~ of diapa~ing and non-dia~ausing Galleria larvae

The largest group of peptidergic neurosecreto~ cells in the brain of Gaiterin mellonella is located in its protocerebrum (Fig. 4). Using the method of total preparation staining of Dogra and Tandam (1964) they could be divided into medial cells, situated in pars intercerebralis and lateral cells, located in pars Iateralis.

The percentage of neurosecretory cells filled with

neurosecretory vesicles in relation to their total number was taken as the index of accumulation of neurosecretion in the brain protocerebrum of dia- pausing and non-diapausing larvae. It was found that after three months spent at 18°C there was a high (46%) degree of accumulation of neurosecretion both in the medial and lateral groups of protocerebrum cells (Fig. 4a). In the brain of non-diapausing larvae taken on day 8 of the last instar the percentage of neurosecretory cells filled with neurosecretion did not exceed 15% of the total number of protocerebral cells. Cells with different degrees of accumulation of neurosecretion can be seen in Fig. 4b.

The most conspicuous differences between neuro- secretory cells of brain protocerebrum of diapause- committed and non-diapausing larvae are those observed at the ultrastructural level (Fig. 5). Firstly, the perikarya of neurosecretory cells of diapause- committed larvae exhibited a high accumulation of secretory granules compared with non-diapausing cells (compare Figs 5A and C). The accumulation of secretory material occurs mainly around the Golgi areas (Fig. SB). Secretory vesicles of different sizes and electron densities are observed in the same cell. The cytoplasm of these cells is very compact with barely distinguishable mitochondria.

The neurosecretory cells of non-diapausing 3O”C- maintained larvae contained rather small amounts of secretory material (Fig. 5C). Furthermore, the secretory vesicles were of different sizes and electron densities. The cytoplasm frequently contained lytic laminated, or partially laminated, bodies (Fig. 5D). This type of ultrastructures indicates an active trans- port and turn-over of synthesized material, and poss- ibly the existance of a crinophagic processes.

Corpora allata of diapausing and non -diapausing Galleria larvae

Since in diapausing 18’C-maintained Galleria mellonella larvae there is a high juvenile hormone titre

682 P. GASSIER and B. CYM~ROWSKI

in the haemolymph (Smietanko et af., 1989) it was diapausing larvae were also taken on day 8 of the decide’ d to investigate the ultrastructure of these instar, at the peak of ecdysteroid titres in t glands in comparison with non-diapausing larvae haemolymph (see Fig. 1). kept a .t 30°C. The tissues for electron microscopy It was surprising to find that despite the high study of diapause-committed larvae were taken on titre in the haemol~ph of diapause-com~tted day 8 after moulting to the last instar and trans- instar larvae, their corpora allata were inac ferred from 30 to 18°C. The corpora allata of non- judged on an ultrastructural basis. The corpora al

last heir

JH last :tive lata

Fig. 3. Transmission electron microscopy studies of the prothoracic glands of S-day-old last larval instar of Gulleriu me~lonell~. (A) In diapause-committ~ 18°C kept larvae, glandular cells contain a sinuous outlined polyploid nucleus (N) provided with numerous nucleoli (n) and clumps of heter~hromatin (arrows). The reduced cytoplasm shows only some ribosomes and scarce mitochondria (M). The peripheral cytoplasmic infolding-formed labyrinth, in the vicinity of the basal laminae (BL) is reduced. (B) In non-diapausing larvae well-developed cytoplasmic areas contain numerous mitochondria (M) and short cisternae of rough endoplasmic reticulum (*). The nucleus swelled and the peripheral labyrinth is apparent

(arrow). BL, basal lamina; n, nucleoli; Tr, tracheole. G x 10,000.

Larval diapause in Galleria melhnelia 683

Fig. 4. Neurosecretory cells of Galieria meZ~onel~a brain stained with paraldehyd~fuchsin. (a} Neuro- secretory cells of protocerebrum of diapausing larvae which spent three months at 18°C. (b) Neuro- secretory cells of the same region of control 30°C kept 8-day-old the last instar. The higher degree of accumulation of secretory material is observed in the brain of diapausing larvae in comparison with

non-diapausing insects.

cells had large fragmented nuclei with patches of well-condensed chromatin (Fig. 6A). The cytoplasm was thin with only small numbers of mitochondria and a smooth endoplasmic membrane surfaces, organellae normaily involves in the biosynthesis of the juvenile hormone (Cassier, 1990).

On the contrary, the corpora allata of non- diapausing larvae were much better developed. The cytoplasm was very thick with rough endoplasmic reticulum and numerous mitochondria of different shapes (Fig. 6B). The surface of the corpora allata ceils undulated and contained many groups of secretory vesicles of varying sizes.

DISCUSSION

The low ecdysteroid titre in the haemolymph of diapause-destinated last instar Gaffer~a meffo~effa

larvae results from inhibition of ecdysone production by their prothoracic giands. This can be caused by a substantial reduction in the number of the pro- thoracic gland cells of diapausing larvae. However, this is not obvious since it was shown by Sehnal et al.

(1988) that extirpation of more than 90% of the prothoracic glands has no significant effect on the ecdysteroid haemolymph titre of last instar Galleriu

meffoneiia larvae. The cells remaining after extirpa- tion undergo hypertrophy and in vitro can produce amounts of ecdysteroids comparable with whole glands from intact insects.

The fact that only a few days after transfer of l-day-old last instar larvae from 30 to 18°C the ecdysteroid titre drops to low levels (See Fig. 1) supports earlier suggestions of nervous inhibition of the PG at lowered temperatures (Mikolajczyk and Cymborowski, 1993). The ecdysteroid titre in haemo- lymph of diapausing larvae significantly increased after severance of the nerve cord behind the brain or

suboesophageal ganglion (Muszyriska-Pytel et al.,

1993). The ultrastructure of the PG cells of diapause-

committed last instar larvae revealed signs of their synthetic inacti~ty, which supports previous findings (McDaniel et al., 1976; Zimowska et al., 1985; Sehnal et al., 1986). The lack of radial channels on the cell surface is considered to be an obvious sign of secretarial inactivity of prothoracic glands (Mala et

al., 1974). The most obvious sign of inactivity con- cerns the reduction of the mitochondrial system and rough endoplasmic reticulum normally involved in ecdysteroids biosynthesis.

The synthetic inactivity may occur as a reaction to low synthesis of PTTH in the brain (Williams, 1947; Kono, 1973) or inhibition of the PTTH release from the cerebral neuroendocrine complex (McDanieI and Berry, 1967; Williams, 1969; Boven, 1984). On the other hand the ecdysone production may be sup- pressed by the mechanism in which the PG them- selves become refractory to PTTH stimulation during diapause (Agui, 1975; Browning, 1981; Ciancio et al.,

1986). Our results from histological and ultra- structural analyses support the idea of inhibition of PTTH release rather than low PTTH synthetic activity of the larval brain during diapause. In the protocerebrum neurosecretory cells among which there are PTTH producing cells (Muszynska-Pytel, 1986, 1987a), a high accumulation of secretory gran- ules is observed compared to non-diapausing larvae. The varying sizes of neurosecretory vesicles may be due to differences in their contents (peptidergic or aminergic) as has been suggested by Warton and Dutkowski, 1977, 1978). This accumulation of neuro- secretory vesicles suggests a slow-release process.

Another possibility is that the high titre of juvenile hormone found in haemolymph of 18”C- maintained larvae (Smietanko et al., 1989) might

684 P. GASSIER and B. CYMBOROWSW

direct

(Gym 1980;

1985) (Bollc

tly or indirectly inhibit the prothoracic glands We were surprised to find that in diap lborowski and Stolarz, 1979; Safranek et al., committed Galleria larvae the corpora allata

Hiruma and Agui, 1982; Zimowska et al., synthetic inactivity rather than activity based c 8. JH can inhibit release of PTTH from the brain high juvenile hormone titre in the haemolymph mbacher, 1988), and can also inhibit synthesis of CA inactivity in these larvae might be due to teroids in the PG and their response to PTTH bition of release of allatotropin from neurosect

trai et al.. 1989). cells (high degree of accumulation of sect

Fig. 5. Transmission electron microscopy studies of the protocerebral neurosecretory cells of 8day-old last larval instar of Galleria mellonella. (A) Diapause-committed 18” kept larvae. The perikarya of the neurosecretory cell is filled with neurosecretory vesicles showing various electron densities. Ax, begining of axon; GC, glial cell; N, nucleus; N, nucleoli. G x 9200. (B) Details of A showing the activity of the Golgi apparatus (G) and coexistance of various electron-dense neurosecretory vesicles. Arrows. rough endoplasmic reticulum. G x 19.500. (C) Neurosecretory cells of non-diapausing larvae. The perikarya of neurosecretory cells contain only a reduced number of neurosecretory vesicles strictly located in the Golgi area. N, nucleus. G x 13,400. (D) Details of Fig. SC. Neurosecretory vesicles are frequently associates with lamellar or dense lytic bodies (arrow) indicative of a crinophagic process.

M. mitochondria. G x 27,000.

show m the I. The

inhi- metory -etory

Larval diapause in Galieria mellonella 685

Fig. 6. Transmission electron microscopy studies of the corpora allata of I-day-old last larval instar of Galleria mellonella. (A) Diapause-committed 18°C kept larvae. The gland shows a high nucleocytoplasmic ratio. The reduced cytoplasmic areas contain only ribosomes, scarce mitochondria (M). G, Golgi apparatus; N, nucleus. G x 9200. (B) Non-diapausing larvae. Hypertrophy and cytoplasmic areas containing numerous mitochondria (M) and well-developed rough endoplasmic reticulum can be seen.

NF, neurosecretory fibres; N, nucleus; n, nucleoli; Tr, tracheoie; BL, basal laminae. G x 9200.

material) located in the pars inter~erebralis of the brain (Mus2y~ska-~tel, 1987b). This discrepancy can be explained by the possibility that in diapausing larvae the juvenile hormone degrading system may be inhibited.

The high activity of the corpora allata of non- diapausing larvae which is reflected by increased amounts of different mitochondria and rough endo- plasmic reticulum cisternae in their cytoplasm was

found on day 8 of the last instar. Fain-laurel and Cassier (1969) associate the various mitochondrial types observed in the corpora allata in Locusta

migratoriu with the state of glandular activity and the nature of secretory products. This activity correlates well with the second peak of JH found in prepupae of Galleriu larvae (Sedlak, 1985; Rembold and Sehnal, 1987). The presence of neurosecretory vesicles at the surface of corpora allata cells could be

686 P. CA~%ER and B. CYMB~ROWSKI

related to the release of PTTH as in Manduca sexta

(Agui et al., 1980).

Acknowledgemenrs-This work was supported in part by grant KBN 14-SOI-GR-44 and by the Universiti Pierre et Marie Curie. Paris VI. The authors thank Mr Tomasz Szczuka for technical assistance, Miss Karen Birmingham from University of Bath for language corrections- and Professor D. S. Saunders, University of Edinburgh U.K. for critical appraisal of the ma~u~ript.

REFERENCES

Agui N. (1975) Activation of the prothoracic glands by brains in vi&o. J. Insect Physiol. 21, 903-913.- _

Agui N., Bollenbacher W. E., Granger N. A. and Gilbert L. I. (1980) Corpus allatum is release site for insect prothoracicotropic hormone. Nature 285, 669-670.

Bollenbacher W. E. (1988) The interendocrine regulation of larval-pupal development in the tobacco hornworm, hfanduea sexta: a model. J. insecr Physiol. 43, 941-947.

Boven M. F.. Saunders D. S.. Bollenbacher W. E. and Gilbert L. I: (1984) in vitro reprogramming of the photo- periodic clock in an insect brain-retrocerebral complex. Proc. natn. Acad. Sci. 81, 5881-5884.

Browning T. 0. (1981) Ecdysteroids and diapause in pupae of Heliotis puncligera. J. Insect Physiol. 27, 7 157 19.

Cassier P. (1990) Morphology, histology and ultrastructure of JH producing glands in insects. in Morphogenetic Hormones of Arthropods (Edited by Gupta A. P.), pp. 83-94. Rutgers University Press, London.

Ciancio M. J.. Watson R. D. and Bollenbacher W. E. (1986) Competency of Manduca sexta prothoracic glands to synthesize ecdysone during development. Mol. Cell. Endocr. 44, 171-178.

Cymbotowski B. (1970) Investigation on the neuro- hormonal factors controlling circadian rhythm of loco- motor activity in the house cricket Acheta domesticus L. II. Daily hist~hemi~l changes in the neuro~retory ceils of the pars intercerebralis and subesophageal ganglion. 2001. Pal. 20, 127-149.

Cymborowski B., Muszynska-Pytel M.. Porcheron P. and Cassier P. (1991) Haemolymph ecdysteroid titres controlled by a circadian clock mechanism in larvae of the wax moth, Galleria mellonella. J. Insect Physiol. 31, 35-40.

Cymborowski B. (1973) Les variations diurnes de l’activite des cellules neuros&cretrices du cerveau du grillon (Acheta

- domesticus L.). Acrida 2, 299-306. Cvmborowski B.. Smietanko A. and Delbecaue J. P. (1989)

?Circadian modulation of ecdysteroid titer in G&let& meilonella larvae. Comp. Biochem. Physiol. 94A, 43 I-438.

Cymborowski B. and Stolarz G. (1979) The role of juvenile hormone during larval pupal transformation of Sp~optera littoralis: Switchover in the sensitivity of the prothoracic glands to juvenile hormone. J. Insect Physiol. 25, 939-942.

Dogra G. S. and Tandam B. K. (1964) Adaptation of certain histological techniques for in situ demonstration of the neuro-endocrine system of insects and other animals. Q. J. Microsc. Sri., 105, 455466.

Fain-Maurel M.-A. and Cassier P. (1969) Pleomorphisme mitochondrial dans les corpora allata de Locusra migrafo- ria migracorioides (R. et F.) au tours de la vie imaginale. Z. ZeNforsch. 102, 543-553.

Hiruma K. and Agui N. (1982) Larval-pupal transform- ation of the prothoracic glands of Mamesfra brassicae. J. Insecr Physiol. 28, 89-95.

Kono Y. (1973) Light and electron microscopic studies on the neurosecretory control of diapause incidence in Pieris rapae crucivora. J. Insect Ph_~siol. 19, 255-272.

Mala J., Novak V. J. A., Blazsek J. and Blazs A. (1974) The effects of juvenile hormone on the prothoracic glands of Galleria mellonella L. 1. Mornhoionv of the gland in the course of the postembryonic’develipment. A%a biol. Hung. 25, 85-95.

McDaniel C. N. and Berry S. J. (1967) Activation of the prothoracic glands of Anrherea polyphemus. Nature 214, 1032-1033.

McDaniel C. N., Johnson E., Saum 1. and Berry S. J. (1976) Ultrastructure of active and inhibits prothoracie glands. J. Insect Physiol 22, 473-481.

Mikolajczyk P. and Cymborowski B. (1993) Influence of lowered temperature on developmental rhythms of the wax moth Galleria mellonella: putative role of ecdy- steroids. Comp. Biochem. Physiol. (in press).

Muszytiska-Pytel M. (1986) Changes in the brain neuro- secretory cells of the wax moth, Galleria mellonella L. (Lepidoptera), during larval-pupal development. Folia Biol. (Krakoaw) 34, 435-448.

Muszynska-Pytel M. (1987a) Some aspects of activity regulation of Galleria mellonella PTTH cells. Arch. Insecr Biochem. Physiol. 5, 2 I l-224.

Muszynska-Pytel M. (1987b) Allatotropic activity of the median neurosecretory cells of Galleria mellonelZa (Lepidoptera) larval brain. Experienria 43, 9088909.

Muszy~ska-Pytel M., Trzcinska R., Aubry M., Pszcz$kowski M.. and Cvmborowski B. (1992) Reeu- lation of prothoracic glands activity in diapausing la<ae of the wax moth, Gaileria mellonella L. (Lepidoptera). Molec. Biol. 23, 33-41.

Porcheron P., Moriniere M., Grassi J. and Pradelles P. (1989) Development of an enzyme immunoassay for ecdysteroids using acetylcholinesterase as label. Insect Biochem. 19, 117-122.

Reynolds E. S. (1963) The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J. Cell Biol. 17, 208212.

Rembold H. and Sehnal F. (1987) Juvenile hormones and their titer regulation in Galleria mellonella. Inserr B~o~hem. 17, 997-1001.

Safranek L., Cymborowski B. and Williams C. M. (1980) Effects of juvenile hormone on ~dysone-dependent development in the tobacco homworm, Manduca sexfa. Biol. Bull. 158, 2488256.

Sakurai S., Okuda M. and Ohtaki T. (1989) Juvenile hormone inhibits ecdysone secretion and responsiveness to prothoracicotropic hormone in prothoracic glands of Bombyx mori. Gen. camp. Endocr. 75, 222-230.

Sedlak B. J. (1985) Structure of endocrine glands. in Comprehensive Insect Physiology Biochemistry and Phar- macology (Edited by Kerkut G. A. and Gilbert L. I.), pp. 25-60. Pergamon Press, Oxford.

Sehnal F. (1966) Kritisches Studium der Bionomie und biometric der in verscheidenen ~~ns~dinungen gezuchteten Wachsmotte, Galleria mellonella L. Z. w&s. Zool. 174, 5382.

Sehnal F., Delbecque J. P., Maroy P. and Maia J. (1986) Ecdysteroid titres during larval life and metamorphosis of Galleria mellonella. Insect Biochem. 16, 157-162.

Sehnal F., Fonagy A., Akai H. and Kallenborn H. G. (1988) Prothoracic glands and ecdysteroid titre in Galleria mellonella larvae. J. Insect Physiol. 43, 609614.

Smietanko A., Wisniewski J. R. and Cymborowski B. (1989) Effect of low rearing temperature on development of Gallerio mellonella larvae and their sensitivity to juvenilizing treatment. Comp. Biochem. Physiol. 92A, 1633-1639.

Wartoti S. S. and Dutkowski A. B. (1977) Ultrastructure of the neurosecretory cells of pars intercerebralis of Galleria meZlo~lla (Lepidoptera) after no~d~neline adminis- tration. Gen. camp. Endocr. 33, I79-186.

Warton S. and Dutkowski A. B. (1978) Ultrastructurai

Larval diapause in Galleria mellonella 687

analysis of the action of reserpine on the brain neuro- Williams C. M. (1969) Photo~riodism and the endocrine endocrine system of the wax moth, ~al~er~a mellonel~a L., aspects of insect diapause. Symp. Sot. exp. Biol. 23, Lepidoptera. CeII 7%~. ffes. 192, 143-155. 285-300.

Williams C. M. (1947) Physiology of insect diapause. II. Zimowska G., Handler A. M. and Cymborowski IX (1985) Interaction between the pupal brain and prothoracic Cellular events in the prothoracic glands and ecdysteroid gland in the metamorphosis of the giant silkmoth, titres during the last-larval instar of Spodoptera littoralis. Platysamia cecropia. &of. Bull. 93, 89-97. J. Insect Physiol. 31, 331-340.