The cell cycle of epidermal cells during reprogramming in the pupa of Galleria

J. Insecf Physiol. Vol. 29, No. 10, pp. 787-794, 1983 Printed in Great Britain

Copyright

0

0022-1910/83 %3.00+ 0.00 1983 Pergamon Press Ltd

THE CELL CYCLE OF EPIDERMAL CELLS DURING REPROGRAMMING IN THE PUPA OF GALLERIA PETER WOLBEBT* and MANFRED

I(Umm~

*Zoologisches Institut der Universitat Rontgenring IO,8700 Wiirzburg, FRG tInstitut fur Humangenetik der Universitlt Koellikerstrage 2,870O Wiirzburg, FRG (Received 21 February 1983; revised 3 May 1983) Abstract-The epidermal cell cycle of the pupal mesonotum of Galleria was investigated by the determination of mitotic indices, [3H]thymidine incorporation and flow-cytophotometric analysis during the first 48 h after pupation. Immediately after the pupal ecdysis nearly all epidermal cells are arrested in G2. Thereafter only a few mitoses occur, leading to a slow increase in the number of Gl nuclei. With the onset of a mitotic wave at a pupal age of 21 h this increase becomes more rapid. On day 2, the cell population reaches a plateau in the number of Gl (resp. G2) cells, reflecting a steady state between mitotic activity and DNA synthesis. A comparison of these cell cycle changes with known data of the time course of reprogramming and ecdysteroid titre leads to the conclusion that there is no causal relationship between DNA synthesis and cellular determination in the sense of a quanta1 cell cycle, and that DNA synthesis can precede the definite rise in ecdysteroid titre. Ker Word Index: Galleria, cell cycle, reprogramming,

flow cytophotometry

A new focus of interest on cell cycle studies in the metamorphosing integument of insects arises from the linding of Besson-Lavoignet and Delachambre (1981) that epidermal cells in Tenebrio are blocked in the GZphase during most of postembryonic development. These cells appear to spend very little time in Gl, and do so only during the critical periods for moulting and metamorphosis. The present work attempts to expand these findings in order to clarify whether a GZblock is a more universal phenomenon among insects. A crucial prerequisite for such investigations is the precise knowledge of the time sequence of metamorphic events in the integument. This condition is met in the case of the pupa of Galleria, where the time course of determination leading to adult structures, and the concomitant changes of the titre of ecdysteroids have been determined in 3-h intervals (Wolbert, 1977, 1983).

INTRODUCTION

In the postembryonic development of insects, there is a close time relationship between a wave of mitoses spreading throughout the epidermis, the secretion of the morphogenetic hormones (ecdysteroids and juvenile hormones), and critical periods for moulting and cellular reprogramming in the course of metamorphosis. Very early in the history of insect endocrinology, the coincidence of these events has led to the postulate of a causal connection between the effect of hormones, cell divisions, and differentiation (Kuhn and Piepho, 1938). More recent research on this subject dealt chiefly with two questions: (1) Does the rise in the haemolymph titre of ecdysteroids trigger cell divisions in the epidermis? (2) Are DNA synthesis and mitotic division necessary prerequisites for the expression of a new developmental programme in the course of metamorphosis? (Bowers and Williams, 1964; Williams, 1965; Krishnakumaran et al., 1967 ; Madhavan and Schneiderman, 1968 ; Sehnal and Novak, 1969; Oberlander, 1969; Chase, 1970; Locke, 1970; Socha and Sehnal, 1972). Unlike any other theory, the concept of quanta1 mitoses (Holtzer et al., 1972) has stimulated research aimed at answering these questions. The results have been equivocal, and the questions remain controversial (for critical reviews see Willis, 1974; Lawrence, 1975). Several workers have concluded that DNA synthesis and mitoses are nor required for the reprogramming of epidermal cells (Kastem and Krishnakumaran, 1975; Kumaran, 1976, 1978 ; Riddiford, 1976, 1981; Riddiford and Kiely, 1981 ; Dyer et al., 1981) while others claim results supportive of, or at least consistent with, Holtzer’s theory (Wielgus and Gilbert, 1978; Wielgus et al.. 1979; Nardi and Willis, 1979: Dean et af., 1980; Besson-Lavoignet and Delachambre, 198 1).

MATERIALS AND METHODS

Animals Experiments were carried out with pupae of the greater wax moth, Galleria mellonella L. Rearing and the timing of experimental animals is described elsewhere (Wolbert, 1969). Age was determined with an accuracy off 1.5 h. All experiments were performed with a defined piece of integument, the mesothoracic tergum. Counting of mitoses Pupae of known age were fixed and embedded in paraffin wax by standard histological methods. 7.5 w sections were stained with Weigert’s iron haematoxylin and mounted in Canada balsam. Mitoses were counted for the region of the mesonotum. 787

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PETER WOLBERT and MANFRED KUBBIES

[3H]thymidine incorporation Each pupa was weighed. the mesonotum was cut out, cleaned and incubated at 25 C for 3 h on a shaker in 200 p 1of Grace’s medium containing 10 p M rH]thymidine (200 PC/ml; sp. act. 20 Ci/mmoI). The incorporation of radioactivity into single pieces was determined according to the method of Wielgus et ul. (1979) with minor modifications. DNA was dissolved by hydrolysis. 50 ill aliquots of the solution were radioassayed after the addition of 3 ml aqualuma (LKB) in a Packard scintillation counter (Prias). Incorporated radroactivity is expressed in dpm/pupa corrected by the weight of the pupa. Autoradiograph?, After incubation the epidermis of the mesothoracic tergum was carefully freed from the cuticle and fixed to a slide by fixation with Camoy’s fluid. Slides were dipped into Ilford K5 emulsion. After two weeks exposure at 4 C they were developed in Kodak D19 and fixed in Kodak rapid fixer. After dehydration the preparations were mounted in balsam. Isolatiorz of nuclei Five to ten mesonota were dissected, cleaned, and rinsed in Grace’s medium to remove adhering haemocytes. The pieces were homogenized with an Ultraturrax homogenizer in 2 ml buffer I (0.01 M Tris pH 7.5; 0.003 M CaCI,; 0.001 M MgCl,; 11.49,; Sucrose; 1p;, Triton X- 100; 20 mg/ 100 ml phenylthiourea). After rinsing the homogenizer twice with additional 2 ml of buITer the homogenate was layered for 20 min onto a cushion of buffer III (composition as buffer I but containing 20”;; sucrose) for sedimenting of cuticular pieces. The supematant was removed and centrifuged for 15 min at lOO(Yg.The pellet was washed twice with buffer 11 (same as I but without Triton). The last pellet was resuspended in 0.5 ml buffer II, mixed with 0.5 ml 20:” glycerol and kept at --20°C until flow cytophotometric analysis. Flow cytophotometr~ Nuclei were centrifuged at 5OOg for 10 min. The pellet was resuspended in a staining solution (IO5 nuclei/O.1 ml) consisting of 0.1 M Tris-buffer pH 7.4, 0.9’6 NaCI, O.l”,, Nonidet P40 and 2 pgg/ml of the fluorochrome Hoechst 33258 (Serva. Heidelberg). The samples were stained for 30 min at 37°C and cooled for measurement to 4’C. Flow measurements of Hoechst fluorescence intensity were performed with the ICP instrument manufactured by Phywe. Gottingen (now Ortho Diagnostic Instr., Westwood, MA). Following filter combinations were used: Excitation KGl, UGl. 8638 ; dichroic mirror FT 450; blocking filters KV 450, BG39. Fluorescence pulse height analysis was performed with the 2103 multichannel analyzer system of Ortho. The stored fluorescence histogram data were transmitted via asynchronous interface to a PDP 1l/23 microcomputer with dual disc drive (Digital Equipment Corporation). Data analysis: Means of fluorescence pulse height peaks were determined by non linear least square fitting of gaussian curves by the method Marquardt (Bevington. 1969). Graphical analysis of cell cycle compartments to flow histograms was by the method of Baisch et al.

(1975). Chicken erythrocytes (CRBC) were added to some samples of Galleria nuclei as internal standard (Hoehn er al., 1977). RESULTS

The mitotic wave The changes in the frequency of mitotic figures in the epidermis of the mesothoracic tergite were analyzed from pupation until 48 h after the pupal ecdysis (Fig. la). Five pupae were used for a given pupal age. The first sparse mitoses can be observed 6 h after the pupal ecdysis (the earliest pupal age under investigation). There is a IO-fold rise in the number of mitotic divisions at a pupal age of 24 h. Thirty-six h after ecdysis the mitotic activity declines rapidly to the same low level as noted at the beginning of the pupal stage. At 48 h mitotic divisions were no longer observed. During the pupal mitotic wave there are two differential cell divisions in preparation for scale formation in the Lepidoptera (Stossberg, 1938). These divisions can be distinguished by the orientation of the spindle apparatus. The spindle is vertical for mitosis I and oblique for mitosis II. In 24-h-old pupae one can observe mitosis 1 and II on the mesonotum, in 30-h-old pupae one observes exclusively mitosis II. At 27 h after pupation areas with distinctly enlarged nuclei emerge. These larger nuclei should belong to the socket and scale-forming cells as described in Ephestia kiihniella (Stossberg, 1938). They spread over the whole tergite during day 2 of pupal life. The relation between the number of these large nuclei to those of “normal” size was determined to be approximately 1 : IO. DNA synthesis In order to obtain optimal parameters for the incorporation of exogenous [3H]thymidine in the in vitro system the kinetics of the reaction was determined as a function of substrate concentration and incubation time. Pupae used for these pilot experiments were selected from day 1 after pupation without exact aging (pupal age 12 k 9 h). The relationship between the concentration of exogenous precursor in the medium and its incorporation was linear up to 8 FM. Therefore. a substrate concentration of 10 ,uM was chosen for further experimentation. Under such saturating conditions, the time course of incorporation was linear up to 8 h of incubation time. A distinct rise of incorporated radioactivity was seen after 2 h of incubation. On the basis of these data. a 3 h incubation period was chosen for all incorporation-studies of radioactivity into the DNA of mesonotal epidermal cells during the first 48 h after the pupal ecdysis. As illustrated in Fig. lb, there is a very slow rise of incorporation between 12 and 21 h. The bulk of DNA synthesis occurs in apparently biphasic fashion during the second day of pupal life. The first steep rise of radioactivity trails the onset of the mitotic wave (Fig. la) by approx 1@12 h, and the second peak succeeds the decline of mitotic figures in similar temporal distance. Autoradiographs from incubated mesothoracic terga of comparable ages reveal a high number of labelled nuclei (Fig. 2), and detailed inspection suggests that only the scale and socket forming cells are labelled. In genera1 there are con-

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Fig. 1. Cell cycle changes of epidermal cells on the pupal mesonotum of Galferiu. (a) l ----0 Changes in the proportion of G2 cells as determined by flow-cytophotometry (mean values of data from Table 1). a---0 mitotic activity. (b) [3H]thymidine incorporation (N/pupal age: IO-51 mesonota). Small horizontal bars show standard deviations. In (b) standard deviations are not shown for a pupal age of 33 h ( + 14590), 42 h ( f 11850) and 45 h ( f 14750). siderable differences in the rate of incorporation among individual pupae, as indicated by the high standard deviations in Fig. 1b.

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g. 3. Flow fluorescence histograms of nuclei from (a) 9-h-old pupae and (b) l&h-old pupae.

Cell cycle changes The changes in the relative proportions of cell cycle compartments during day 1 and day 2 after the pupal ecdysis were ascertained by flow cytophotometry at 3-h intervals. As shown in Fig. 3a, fluorescence histograms of nuclear preparations up to a pupal age of 9 h display a prominent peak at channel no. 100 and a very minor peak at channel no. 50. In relation to the chicken red blood cell standard, the relative fluorescence intensities of the two cell populations correspond to approximate DNA values of. respectively, 3.6 and 1.8 pg/nucleus (data not shown). The small accumulation of nuclei with precisely half the fluorescence of the predominant population at channel no. 100 increases with time (Fig. 3b). Since this increase coincides with that of visually determined mitoses it follows that the peak at channel no. 100 contains nuclei with a 4C DNA-content (G2phase nuclei), while the growing peak at channel no. 50 represents nuclei which have completed division (GI-phase nuclei). Table 1 summarizes the respective compartment sizes for the entire period under investigation as determined by flow cytophotometry. Immediately after the pupal ecdysis nearly all epidermal cells of the mesonotum are in G2, while Gl cells begin to appear in measurable numbers at 12 h after pupation. The relative number of these cells increases with the onset of the mitotic wave, but never exceeds 200:, of the cell population.

790

PETER WOLBERT and MANFRED KUBBIES DISCUSSION

From the data summarized in Fig. 1 and Table 1 one can derive the following interpretation of the underlying cell cycle changes: Immediately after the pupal ecdysis nearly all cells of the mesonotal epidermis are in G2. A low rate of mitotic activity prevails during the following hours, leading to a slow accumulation of cells with a 2C DNA content. As a consequence, a small Gl peak becomes visible at a pupal age of 12 h which grows during further development. DNA synthesis begins slowly at a pupal age of 12 h as revealed by [3H]thymidine incorporation, while S-phase nuclei are not seen in measurable amounts in flow histograms prior to a pupal age of 15 h. In the following hours, the situation becomes more complex. At day 2 there is a rise in mitotic activity, and also in DNA synthesis. The observed plateaus for the relative amounts of cell cycle phases are likely to reflect a kind of steady state between cell divisions and DNA synthesis. In the course of differential cell divisions for scale formation two waves of mitoses are superimposed, followed by a renewed polyploidization of socket and scale-

Table I. Flow cytophotometric analysis of cell cycle changes of mesonotal epidermal cells in the Galleriupupa during

H after pupation

the first 48 h after

Cl

pupation

5; Of cells in s

G2

3

1.4 1.5 1.7

98.6 98.5 98.3

6

1.0 1.7

99.0 98.3

9

0.7 0.9 1.8

99.3 99.1 98.2

12

1.8 1.8 5.4

98.2 98.2 94.6

15

3.8 4.2 10.5

3.0

96.2 95.8 86.5

4.8

95.3 92.5 84.0

18

4.7 7.5 11.2 5.6 6.4 7.6 8.9

4.0 3.3

_

90.4 90.3 92.4 91.1

27

IO.4 13.1

5.7 3.9

83.9 83.0

33

17.2 17.3

6.7 5.1

76.1 77.6

36+3h

19.0

5.0

76.0

39

16.5

6.5

77.0

45

21.2 17.9

3.6

75.2 82.1

21

Each line represents data deriving from one flow histogram. Dashes indicate S-phase values of equal or less than 1”; of the total population.

forming cells beginning at 27 h after pupation. This fits well with the observed peaks of t3H]thymidine incorporation at day 2. Possibly, this complex pattern is responsible for the substantial interindividual variability in radiolabelling of DNA as manifested by the high standard deviations of these measurements. The data concerning the wave of mitoses confirm and extend earlier observations in lepidopterous pupae (Krumins, 1952 for Galleria; Smolka, 1958 for Ephestia). A proportion of these mitoses should contribute to the differential cell divisions for scale and hair formation (Stossberg, 1938; Smolka, 1958). When represented in graphic form (Fig. la), the time course of exit from the G2 compartment clearly displays two successive waves both of which appear to precede, in precise temporal order, bimodal peak DNA synthesis activities (as determined by [3H]thymidine incorporation ; Fig. 1b). A corresponding biphasic pattern is not apparent from the plot of direct mitotic counts. The reasons for this discrepancy are unclear, but it is conceivable that the relatively broad mitotic wave distribution nevertheless consists of two closely overlapping subpopulations corresponding, for example, to mitoses I and II. In principle flowdata, mitotic counts and radiolabelling thus fit well together and establish a coherent temporal sequence of discrete, albeit interrelated, cell cycle events which take place after ecdysis. The described features of the cell cycle observed for the epidermal cells in the Galleria pupa are in general accord with the scheme outlined for Tenebrio (Besson-Lavoignet and Delachambre, 1981): At the beginning of the pupal stage almost all cells are blocked in G2. Such extensive blocking in G2 thus appears to be a more universal phenomenon among insects. Van Der Want and Spreij (1976) and Egberts (1979) have suggested a G2 block in imaginal discs of Calliphora. Stevens et al. (1980) were able to arrest Kc cells of Drosophila in G2 by the addition of physiological concentrations of 20-hydroxyecdysone to the medium, while Fain and Stevens (1982) found, in certain imaginal discs of Drosophila in vivo, an increasing proportion of cells in the G2/M-phase during the larval life. However, Besson-Lavoignet and Delachambre (198 I ) possibly overestimate the importance of the G2-block and release as critical phases determining moulting and metamorphosis, since in the case of the Galleria pupa only cells involved in scale formation may participate in cell cycle changes. The proportion of cells which enter the Gl compartment remains always relatively small. Given the number of epidermal cells on the mesonotum (in the range of 150.000) the results of mitotic counts show that less than 0.2:: of the cells divide synchronously even at the maximum peak of mitotic activity. Additional experiments will be needed to elucidate the dynamics of the cell cycle at day 2, and to evaluate the overall percentage of cycling cells. A newly developed BrdU-Hoechst method might be well suited for these purposes (Kubbies and Rabinovitch, 1983). Periods of cell cycle activity are intimately bound to critical phases for moulting and metamorphosis, and moulting hormones have been implicated to evoke cell divisions and DNA synthesis of epidermal cells (Kiihn and Piepho, 1938; Bowers and Williams, 1964; Williams, 1965). In the Gafleria pupa one should be

Fig. 2. Whole mount autoradiogram of mesonotal epidermis after [3H]thymidine incorporation showing labelled nuclei (e.g. arrow) in homogeneous distribution. The central, dark spot is an adhering fat body cell. In vitro incubation was from 33-36 h of pupal age.

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793

able to directly correlate the reported cell cycle Ulsamer for technical assistance. Computer software for flow studies was kindly provided by Dr P. Rabinovitch, changes with fluctuations in the ecdysteroid titre of University of Washington, Seattle. The work was supthe haemolymph. While our present work shows that from the very beginning of the pupal stage always a ported by a grant of the Deutsche Forschungsgemeinschaft. few cells of the mesonotum divide, and that detectable amounts of cells incorporating [3H]thymidine REFERENCES quite clearly occur at a pupal age of 12 h, a sharp rise Baisch H., Goehde W. and Linden W. A. (1975) Analysis of the haemolymph titre of ecdysteroids takes place of PCP-data to determine the fraction of cells in various only at 21 h after pupation (Wolbert, 1983). It therephases of the cell cycle. Radiat. Enuiron. Biophys. 12, fore appears rather unlikely that ecdysteroids are 31-39. solely responsible for recruitment, in the cell cycle, of Besson-Lavoignet M. T. and Delachambre J. (1981) The epidennal cells in the pupa of Galleria. epidermal cell cycle during the metamorphosis of Tenebrie molitor L. (Insecta: Coleoptera). Devl Biol. 83, Most reports of stimulating effects of ecdysteroids 255-265. on DNA synthesis come from work with systems like Bevington P. R. (1969) Data Reduction and Error Analysis isolated abdomen (Wielgus et al., 1979), in vitro sysfor the Physical Science, pp. 142-156. McGraw-Hill, tems of imaginal discs (Oberlander, 1969, 1972; Logan New York. et al., 1975 ; Kurushima and Othaki, 1975 ; Nardi and Bowers B. and Williams C. M. (1964) Physiology of insect Willis, 1979) or explanted embryos cultured in vitro diapause. XIII. DNA synthesis during the metamor(Bulliere and Bulliere, 1977). On the other hand, phosis of the cecropia silkworm. Biol. Bull. 126, 2055 Tenebrio epidermis does not undergo mitoses when 219. exposed to ecdysteroids in vitro (Caveney and Bulliere F. and Bulliere D. (1977) DNA synthesis and epidermal differentiation in the cockroach embryo and Blennerhasset, 1980). Krishnakumaran et al. (1967) pharate first instar larva: Moulting hormone and mitoconcluded from studies with experimentally provoked mycin. J. Insect Physiol. 23, 1475-1489. moulting in adult silkmoths that the observed stimulation of DNA synthesis is not the primary effect of Caveney S. and Blennerhasset M. G. (1980) Elevation of ionic conductance between insect epidermal cells by /Iecdysteroids. Likewise, Kumaran (1978) could not ecdysone in vitro. J. Insect Physiol. 26, 13-25. induce DNA synthesis by injecting 20-hydroxyChase A. M. (1970) Effects of antibiotics on epidermal ecdysone in Galleria larvae, and Dyer et al. (1981) metamorphosis and nucleic acid synthesis in Tenebrio concluded that there exists a temporal coincidence but molitor. J. Insect Physiol. 16, 865-884. most likely not a causal relationship between DNA Dean R. L., Bollenbacher W. E., Locke M., Smith S. L. and Gilbert L. I. (1980) Haemolymph ecdysteroid levels synthesis and ecdysteroid action on the larval epiand cellular events in the intermoult/moult sequence of dermal cells in Marx&a. Taken together this evidence Calpodes ethlius. J. Insect Physiol. 26,267-280. indicates that in certain experimental systems ecdyDyer K. A., Thornhill W. B. and Riddiford L. M. (1981) steroids may trigger DNA synthesis; however, the DNA synthesis during the change to pupal commitment initial rise of in vivo [3H]thymidine incorporation of Manduca eoidermis. Devl Biol. 84. 425-43 I. seems to be independent of ecdysteroid action. Egberts D. J. N: (1979) Late larval and prepupal DNA Our results tend also to discount the role of quanta1 synthesis in imaginal wing discs of Calliphora erythromitoses for reprogramming in the mesonotal epidermis cephala. Insect Biochem. 9,89-93. of the Galleria pupa. Determination for adult synFain M. J. and Stevens B. (1982) Alterations in the cell thesis takes place during the first 24 h after pupation. cycle of Drosophila imaginal disc cells precede metamorphosis. Devl Biol. 92, 247-258. At the time of the first sign of DNA synthesis (at a Hoehn H.. Johnston P. and Callis J. (1977) Flow cvtopupal age of 15 h) over 50% of the epidermal cells are genetics: Sources of DNA content variation among already imaginally committed (Wolbert, 1983) whereeuploid individuals. Cytogen. Cell Gene?. 19,94107. as maximal mitotic activity and DNA synthesis occurs Holtzer H., Weintraub H., Mayne R. and Mochan B. at day 2. The observed cell cycle changes are thus more (1972) The cell cycle, cell lineages and cell differentiation. likely to represent consequences than causes of the In Current Topics in Developmental Biology (Ed. by first cellular determinative event. This interpretation Moscona A. A. and Monroy A.), pp. 229-256. Academic is supported by the fact that, in other systems, chiefly Press, New York. Kastem W. H. and Krishnakumaran A. (1975) Reproa time correlation rather than direct experimental gramming in the absence of DNA synthesis in Galleria evidence invokes the quanta1 mitosis theory (Wielgus larval epidermis. Cell Dijer. 4,45-53. and Gilbert, 1978; Wielgus et al., 1979; BessonKrishnakumaran A., Berry S. J., Oberlander H. and Lavoignet and Delachambre, 1981). Moreover, Schneiderman H. A. (1967) Nucleic acid synthesis during Galleria larval epidermis cultured in vivo can synthesize insect development-II. Control of DNA synthesis in the pupal cuticle without an intervening round of DNA cecropia silkworm and other satumiid moths. J. insect 1975 ; synthesis (Kastem and Krishnakumaran, Physiol. 13, I-57. Kumaran, 1976, 1978, 1980) and inhibitors of DNA Krumins R. (1952) Die Borstenentwicklung bei der Wachsmotte Galleria mellonella L. Biol. Zbl. 71. 183synthesis cannot prevent metamorphosis in silk210. worms (Krishnakumaran et al., 1967) nor in Manduca (Riddiford, 1976, 1981; Riddiford and Kiely, 1981; Kubbies M. and Rabinovitch P. (1983) Flow cytometric analvsis of factors which influence the BrdU-Hoechst Dyer et al., 1981). This situation is thus similar to quenching effect in cultivated human fibroblasts and what we describe for the relationship between lymphocytes. Cytometry 3, 276-282. ecdysteroids and DNA synthesis: DNA synthesis can Kuhn A. and Pieoho H. (1938) Die Reaktionen der be concomitant with reprogramming, but may not be Hypodermis und der Vekonschen Driisen auf das a necessary prerequisite step. Verpuppungshormon bei Ephestia kiihniella. Z. Biol. Zbt. 58, 12-51. Acknowledgement-We

thank Gaby Dietrich and Anita

Kumaran A. K. (1976) Relationship

between DNA syn-

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and Behaviour (Ed. bv Sehnal F.. Zabza A.. Menn J. J. and Cymborowski B.), pp. 485496. Wroclaw Technical Universitv Press. Wroclaw. Sehnal F. and Novak V. J. A. (1969) Morphogenesis of the pupal integument in the waxmoth (Gallerio mellonella) andits analysis by means of juvenile hormone. Acto ent. bohemoslov 66, 1377145. Socha R. and Sehnal F. (1972) Inhibition of adult development in Tenebrio molitor by insect hormones and antibiotics. J. Insect Physiol. 18, 317-337. Smolka H. (1958) Untersuchungen an Kleinorganen im Integument der Mehlmotte. Biol. Zbl. 77,437-478. Stevens B.. Alvarez C. M., Bohman R. and O’Connor J. D. (1980) An ecdysteroid-induced alteration in the cell cycle of cultured Drosophila cells. Cell 22, 675482. Stossberg M. (1938) Die Zellvorglnge bei der Entwicklung der Fliigelschuppen von Ephestia kiihniello Z. Z. Morph. dk. Tiere 34, 175-206. Van Der Want J. J. L. and Spreij E. (1976) DNA content in imaginal disk cells of Colliphora erythrocephalo (Meigen). Nefh. J. Zoo/. 6,432-434. Wielgus J. J., Bollenbacher W. E. and Gilbert L. I. (1979) Correlations between epidermal DNA synthesis and haemolymph ecdysteroid titre during the last larval instar of the tobacco homworm Monduca sextu. J. Insect Physiol. 25,9-l 6. Wielgus J. J. and Gilbert L. I. (1978) Epidermal cell development and control of cuticle deposition during the last larval instar of Monduca sexta. J. Insect Physiol. 24,629-637. Williams C. M. (1965) DNA synthesis and hormonal control of insect metamorphosis. Science 148,670. Willis J. H. (1974) Morphogenetic action of insect hormones. A. Rev. Ent. 19,97-l 15. Wolbert P. (I 969) Die Schliipfrate der groBen Wachsmotte, Gollerio mellonella L. (Lepidoptera: Pyralidae), nach einer Rontgenbestrahlung, einer Verwundung und kombinierter Belastung im Puppenstadium. Wilhelm Roux Archiv Entw Mech 164, 134169. Wolbert P. (1977) Untersuchungen zur Imaginaldetermination an der Puppe der grol3en Wachsmotte, Gallerio mellonella L. C’erh. dt. Zool. Ges. 323. Wolbert P. (1983) Juvenilhonnon-Sensibilitat und Ecdysteroidtiter wahrend der imaginalen Determination der Epidermiszellen in der Puppe von Gallerio. Zool. Jb. Physiol. (in press).