Cyclic Layer Deposition in Cockroach Circadian Rhythm

8/4/2019 Cyclic Layer Deposition in Cockroach Circadian Rhythm

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Pergamon 0022-1910(94)00092-l.I. Insect Physiol. Vol. 41, No. 2, 153-161,p. 1995Copyright 0 1995 Elsevier Science Ltd

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Cyclic Layer Deposition in the Cockroach(Blaberus craniifer) Endocuticle: a CircadianRhythm in Leg Pieces Cultured in vitroFRIEDRICH WEBER*Received 18 February 1994; revised 25 July 1994

Pieces from hind tibiae of freshly moulted imaginal cockr oaches were cultured in vitro. In most piecesendocuticle was deposited in vitro. This endocuticle showed either a multilamellate or a cir cadian-like str ucture. The latter type consisted of double layers each of them being composed of one lamellateand one nonlamellate layer, and thus resembled the in vivo structur e of endocuticle deposited in thecockr oach legs. Circadian -like endocuticle was deposited almost exclusively around the base of thespines. The number of double layers never exceeded the number of days in vitro; frequently, however,it was smaller. In order t o estimate the frequency of the deposition rhythm of circad ian-likeendocuticle, exogenous time marks were set by autor adiogr aphic labelling with 3H-N -acetyl-D-glucosamine. Largely independent of the temperature applied, the frequency was about one doublelayer deposited per day. Thus, it seems that in vitro deposition of endocuticle double layers is contr olledby a circadian clock. I t is supposed that the clock operating in vitro is localized in the epidermis.Cockroaches Endocuticle Growth rhythm Circadian In vitro

INTRODUCTIONPresently, the hypothesis is favoured that circadianoscillations are generated at the level of intracellularregulation, and not inside a network of interacting cells(Dunlap, 1993; Michel et al., 1993). Thus, the circadiansystem of multicellular organisms may be composed ofa multiplicity of circadian cellular clocks. In metazoansthe internal synchronization seems to be achieved by ahierarchical structure of the circadian system (Biinning,1977; Pittendrigh, 1993): main circadian pacemakers(master clocks) control frequency and phase of subor-dinate circadian clocks (slave clocks). Main circadianpacemakers are populations of coupled cells localized inthe central nervous system, in the eyes or in a centralhormonal gland. They have been identified in insects,molluscs and vertebrates (Aschoff et al., 1982; Chiba andTomioka, 1987; Underwood, 1990; Klein et al., 1991;Koumenis and Eskin, 1992). Subordinate clocks arepresumed in the nervous system, in eyes, and in hor-monal glands as well; especially in mammals they havealso been found in organs and tissues outside the centralsystems (Hardeland, 1973; Edmunds, 1988). It has to bestressed that a circadian rhythm in an organ or a tissuedoes not per se point to an autonomous expression, but

*Universitgt Miinster, Institut fiir Allgemeine Zoologie und Genetik,Schlossplatz 5, D-48149 Miinster, Germany.

only the proof that the rhythm persists under constantconditions in vitro (or in an isolated part of thebody).

Decentral circadian clocks have also been found ininsects: they are able to generate or sustain circadianrhythms. The circadian rhythm of sperm release from thetestes of the moths Anagasta kuehniella and Lymantriadispar continues under constant darkness in the iso-lated abdomen as well as in the testis-seminal ductscomplex cultured in vitro (Thorson and Riemann, 1977;Giebultowicz et al., 1988, 1989; Giebultowicz andRiemann, 1990). The prothoracic glands of the mothSamia Cynthia ricini contain a light-sensitive immediatetimer which controls the instant of gut purge in the lastlarval instar (Mizoguchi and Ishazaki, 1982, 1984).Truman (1984) described a circadian clock independentfrom the brain in Manduca sexta, which controls thephase of ecdysteroid decay on the last day of adultdevelopment. It has also been supposed that circadianrhythms of endocuticle growth, which are known fromlocusts, cockroaches, bugs and some other insects,could be controlled by a decentral circadian clock(Neville, 1975, p. 390). This hypothesis was supported byexperiments in cockroaches.

As in the other groups, in cockroaches the endocuticleof the legs is deposited in a circadian rhythm. Thestructure of this endocuticle corresponds to the two-system model of insect cuticle architecture (Neville and

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154 FRIEDRICH WEBERLuke, 1969; Neville, 1975, 1993). The endocuticle iscomposed of alternating lamellate and nonlamellatelayers. The usually considerably thicker nonlamellatelayers seem to be built up by chitin microfibrils alignedin the cuticular matrix parallel to the longitudinal axis ofthe legs. Lamellate layers consist of one or a few lamellaeresolvable under the polarizing microscope. Neville(1975) and Lukat et al. (1989) have supposed that in thecockroach lamellate layers the alignment of chitinmicrofibrils is changing helicoidally (Bouligand, 1965).During the time of postmoult endocuticle growth atevery circadian period one nonlamellate and one lamel-late layer are deposited (Wiedenmann et al., 1986).

The circadian growth rhythm continued in the legs ofthe cockroach Blaberus craniifer after several surgicaloperations (Lukat et al., 1989): (1) after extirpation ofthe optic lobes (where the main circadian pacemakersof behavioural rhythms are localized in cockroaches:Nishiitsutsuji-Uwo and Pittendrigh, 1968; Roberts,1974; Page, 1982), (2) after decapitation, (3) after extir-pation of parts of the ventral nervous cord, (4) in legpieces transplanted into the haemocoel of imaginal hostswhose endocuticle growth was already terminated. Twomore observations point to a control of endocuticlegrowth rhythm independently from the optic lobes pace-makers. (5) Once started, the circadian endocuticlegrowth rhythm in the cockroaches Leucophaea maderaeand Blaberus craniifer is insensitive to light/dark cyclesas a zeitgeber (Wiedenmann et al., 1986; Lukat et al.,1989). This is a striking difference to the light-sensitivityof the locust epidermis (Neville, 1967). (6) After rearingLeucophaea maderae in light/dark cycles of 11:ll or13 : 13 h the free-running locomotor rhythm of theadults is shortened and lengthened, respectively (Barrettand Page, 1989); however, the frequency of the endo-cuticle growth rhythm is unchanged (Page and Weber,unpublished results).

In this article evidence is given that the circadiandeposition rhythm continues locally in leg pieces takenfrom freshly moulted cockroaches and cultured in vitro(for a prepublication cf. Weber, 1985).

MATERIALS AND METH ODSCockroaches of the species Blaberus craniijk (Burm.)

(synonymous to B. fuscus) were used. The rearing con-ditions were described by Wiedenmann et al. (1986).

In the cockroach legs, the deposition of the endo-cuticle starts about 2 h after the moult and terminatesabout 3 weeks later (Runte and Weber, 1982; Wieden-mann et al., 1986). For the in vitro experiments, piecesof the hind tibiae were taken from female and maleadults l-4 h after their imaginal moult (the cuticle wasstill untanned). Endocuticle growth had not yet or onlyjust started at the moment of dissection. No differencesconcerning the in vitro deposition of endocuticle could beascertained between these cases.

The legs were disinfected by dipping into 70% alcohol(150 s). After rinsing in Ringer solution (3 x 1 min) the

tibia was put into a drop of culture medium. Piecesof 24mm length were cut off the tibia, and then splitlengthwise (the distal and proximal end of the tibiae werenot used). Muscle and fat body were not removed fromthe leg pieces. As a control a leg piece from each animalwas fixed at the moment of amputation.

As culture vessels 24 well cell culture clusters(Costar) were used, the outer wells were filled withdistilled water, the 8 inner ones with 0.6 ml culturemedium. Two half pieces were cultured in one well.To allow air exchange by diffusion the lid of the culturecluster was lifted by some mm. The culture cluster wasput into a plastic box, together with some open Petridishes filled with distilled water. The box was slowlyshaken. In most experiments the box was continuouslyperfused with a mixture of 95% 0, and 5% CO* (Mitsuiand Riddiford, 1976). In perfused cultures the variabilityof the amount of endocuticle secreted was reduced. Thecultures were incubated under continuous darkness andat constant temperature (kO.25C). If not mentionedotherwise the temperature was 27C. Usually, the pieceswere fixed after 11-14 days.

Three media were used: (1) Marks medium M20(Marks, 1973a, Gibco product), (2) S/MEM: a 5:4mixture of Schneiders Drosophila medium and mini-mum essential medium (MEM, with Hanks salts and25 mM HEPES buffer, without L-glutamine, Gibcoproducts; Howes et al., 1989) and (3) M3 after Shieldsand Sang (1977) (partly a gift from A. Diibendorfer,Zurich; Sigma chemicals and Difco yeast extract).The pH of the media was adjusted to 7.1-7.2. The mediawere used with or without a not heat-inactivated serum(M20 and S/MEM: 7.5% new born calf serum; M3: 10%fetal calf serum Myclone Plus; Gibco products).The media used did not influence endocuticle growthdifferently, and also the addition of a serum had noeffect.

No hormone was added to the culture medium in theexperiments described in this article. 167 units penicillinG, 167 ,ug streptomycin-sulfate and 0.5 pg amphotericinB (Boehringer) were added to 1 ml culture medium.Microbial growth occurred in less than 10% of thecultures. Usually, the medium was not changed duringculturing. For the histological procedures cf. Weber(1985). The sections were analysed under a polarizingmicroscope.

In order to label the deposited endocuticle H-N-acetyl-D-glucosamine (189 GBq/mmol; 1.85 MBq/mlculture medium) or a mixture of 5 tritiated aminoacids (leucine, lysine, phenylalanine, proline, tyrosine;2.59-5.18 TBq/mmol; 1.85 MBq/ml) was added to theculture medium. From two successive sections onesection was used for an autoradiography (Kodak strip-ping film AR 10 was used), and the other one forpolarizing microscopy.

The results reported here are based on 101 cultures,each containing 16 pieces. If not mentioned otherwise,from each piece one randomly selected cross section witha spine base was evaluated.


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CIRCADIAN ENDOCUTICLE DEPOSITION IN VITRO 155

RESULTSAm ount of endocuticle d eposited in vitro

As in vivo, the amount of endocuticle deposited in vitrowas usually largest in areas around the base of tibiaspines.

The hind tibia of the cockroach Blaberus craniifer isshaped like an elliptic cylinder: a cross section has theoutline of an ellipse. The two narrow sides of the tibia(the main vertex lines of the elliptic cylinder) carry alongitudinal row of 7-13 spines (apophyses). Each spinearticulates in a small oblong trough, the bottom of whichconsists of membranous cuticle. Proximally and laterallyeach trough is bounded by a basal wall. The basal wallpasses laterally into the weakly curved wall of the broadsides of the tibia (Fig. 1). In the following, the termsmembrane, basal wall and side wall (for wallof the broad sides of the tibia) are used to describeendocuticle deposition in the distinct regions of the tibiaintegument.As in vivo, the endocuticle deposited in cultured legpieces was usually thickest under the basal wall of thespine bases and in the adjacent region of the side wall(Fig. 1). Under the membrane as well as more distantfrom the basal wall the endocuticle was considerablythinner. At the lips of the leg pieces cultured in vitrooften no endocuticle was deposited.

To get a rough estimation of the amount of endo-cuticle deposited, the thickness of the endocuticle was

FIGURE 1. Endocuticle deposition starts and continues for severaldays in tibia pieces cultured in vitro in the absence of exogenousmoulting hormone. The photo shows a cross section of a tibia piecewith the base of a spine. Endocuticle deposition had not yet started atthe moment of dissection (about 1 h after imaginal moult). The piecewas fixed after 14 days in vitro. At the position marked by a line thethickness of the endocuticle (darker material) amounts to about 2/3 ofthe in uivo endocuticle deposited at this position during 14 days aftermoulting. For the endocuticle structures deposited here see the mag-nification in Fig. 2. Under the opposite basal wall distally threecircadian-like double layers and proximally many fine lamellae weredeposited, next to this basal wall only a multilamellate structure wasbuilt up (not magnified).-Culturing conditions: S/MEM withoutserum; perfusion with a mixture of 95% 0, and 5% CO,; 27.OC.-Ex exocuticle, Ep epidermis, En endocuticle, S spine, Mem membraneof the spine base, BW basal wall of the spine base, T tracheae, M

muscle, F fat body. Light microscopy.

measured at positions of the side wall next to thebasal wall (as shown in Fig. 1). From the two valuesobtained from each cross section only the larger one wasconsidered for the following calculation.

At the defined position, the mean thickness of theendocuticle deposited during 11-14 days after startingthe culture amounted to about one-third of the endo-cuticle deposited in vivo during a corresponding timeafter moulting. For example, after 12 days the meanthickness of the endocuticle was 22.5 f 9.5 pm (f SD;266 leg pieces) in vitro (in vivo: 64 f 13 pm; 17 animals).In a few cases no or only very little cuticular material wassecreted in vitro, whereas in other cases the endocuticlereached two thirds of the thickness of the endocuticledeposited in vivo (cf. Fig. 1).Multilamellate endocuticle

The endocuticle deposited in vitro consisted of twostructural types: a multilamellate type and a circadian-like type. In cross sections as well as in longitudinal(radial) sections multilamellate endocuticle showedan alternation of anisotropic and isotropic lamellae(Fig. 2). Possibly, multilamellate endocuticle was gener-ally helicoidally structured.

The number of double lamellae deposited wasfrequently larger than the number of days in vitro(up to 3 times) (a double lamella consists of an aniso-tropic and an adjacent isotropic lamella, cf. Fig. 2).The thickness of one double lamella varied between0.5 and 2.5 pm, and thus covered the range of thethickness of the double lamellae of the inner exocuticle(0.60.9 pm). Frequently, the thickness of the doublelamellae decreased during the course of deposition(Fig. 2).Circadian -like endocuticle

As endocuticle deposited in the cockroach legsin vivo, this endocuticle type can be described on thebasis of the two-system model of insect cuticle architec-ture (cf. Introduction). Circadian-like endocuticle con-sisted of alternating lamellate and nonlamellate layers(Fig. 2). Nonlamellate layers were anisotropic in longi-tudinal sections, but isotropic in cross sections. Thus,it seems that as in vivo nonlamellate layers are builtup by microfibrils aligned parallel to the longitudinalaxis of the legs. Usually, lamellate layers were con-siderably smaller than nonlamellate ones, and frequentlythey consisted of one lamella only (Fig. 4) whichappeared anisotropic in cross sections, but isotropicin longitudinal (radial) sections as in vivo. Sometimes,distal lamellate layers were relatively thick, and consistedof a few lamellae. In these cases, in cross sections as wellas in longitudinal (radial) sections anisotropic lamellaealternated with isotropic ones.

Usually, circadian-like endocuticle passed proximallyinto a multilamellate deposit (Fig. 2). In some pieces amixed type containing l-2 nonlamellate layers insidethick multilamellate deposits was detected.


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1.56 FRIEDRICH WEBER

FIGURE 2. Circadian-like and multilamellate endocuticle deposited in vitro (a magnification of the position marked inFig. 1; on the right side of the photo the membrane of the spine base). Next to the spine base 9 circadian-like double layersand proximally very fine lamellae were deposited. The first 4 lamellate and nonlamellate layers are figured in the sequence ofdeposition. A double layer consists of a lamellate layer (in this cross section with 14 lamellae appearing anisotropic and O-3lamellae appearing isotropic) and the proximal adjacent nonlamellate layer. The circadian-like structure under the membraneis less distinct. More distant from the spine base the endocuticle is multilamellate; distally rather thick, proximally very thinlamellae were deposited. A double lamella consists of an anisotropic and an adjacent isotropic lamella (+). (---) borderbetween the cuticle deposited in vivo and in vitro.-Photographed between crossed polaroids. The cross section is orientatedfor maximum brightness of birefringence. When the section is rotated by 45 relatively to this orientation all structures in the

central and left part of the figure are largely dark.

Circadian -like areasWhereas in vivo the endocuticle grows in every

region of the leg integument in a circadian rhythm,in vitro circadian-like endocuticle was detected almostexclusively in areas around the base of the spines.

The distribution of the different types of endo-cuticle around the base of the spines was analysed inan experimental series with 135 leg pieces whichwere cultured under perfusion, and showed a relativelyconsistent endocuticle growth. Both sides of thecross sectioned spine bases were evaluated separately.Depending on the position, in O-1.9% of the cases nodeposition could be detected, in 3.410.6% of the casesthe structure of the deposits was not analysable. Thefollowing percentages refer to the number of analysablecases.

Under the membrane the endocuticle was exclusivelymultilamellate in 2.6%, under the basal wall in 1 l.l%,and in the adjacent region of the side wall in 46.3% ofthe cases. It showed a circadian-like structure under themembrane in 95.7%, and under the basal wall in 84.4%of the cases. In 37.1% of the cases the circadian-likestructure crossed the border between the basal wall andthe adjacent region of the side wall (Fig. 2). However, innearly all cases the circadian-like type was replaced bythe multilamellate type within a distance of < 150 pmfrom the basal wall, only in 1.6% of the cases did thecircadian-like structure exceed this distance. The mixedtype was observed under the membrane in 1.7% of the

cases, under the basal wall in 4.5%, in the adjacentregion of the side wall in 16.5%, and outside the 150 pmdistance in 3.6% of the cases. In most cross sections theexpansion of the circadian-like endocuticle was differenton both sides of the spine bases. Obviously, the circa-dian-like areas around the spine bases were irregular inshape. Also, the numbers of circadian-like double layersdeposited on both sides were partly different.

The leg pieces used for culturing carried a longi-tudinal row of 2-3 spines. Between the correspondinglyarranged circadian-like areas only multilamellatedeposits were found. Thus, the circadian-like areas of aleg piece seemed to be isolated from each other.

As was the case in vivo, the thickness of the doublelayers varied regularly around the spine bases. Under themembrane they were considerably smaller than underthe basal wall and in the adjacent region of the sidewall. Also, like in vivo, the thickness of the double layersdecreased during the course of endocuticle growth.Usually, the double layers reached 3&60% of the thick-ness of the corresponding double layers in vivo. Insome cases, however, distal double layers were nearly asthick as in vivo (for example, about 1Opm under thebasal wall and in the adjacent region of the side wall)(cf. Fig. 2).Number of double layers deposited in vitro

The number of double layers of circadian-like endo-cuticle sometimes corresponded to the number of days


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CIRCADIAN ENDOCUTICLE DEPOSITION IN VITRO 157in uirro, but in many cases it was smaller. In a series ofexperiments leg pieces, cultured at 27 and 32C respect-ively, were fixed after 3, 6, 9 (or 10) and 12 (or 13) days.The larger number of double layers was considered whenthe number was different on the both sides of a crosssectioned spine base. After 3 and 6 days respectively, inmost pieces the number ofdouble layers corresponded tothe number of days, or it was only somewhat smaller(Fig. 2). In most pieces fixed after 9-13 days the numberof double layers was considerably smaller; however,even in two of the 4 pieces fixed after 10 days thenumber of double layers deposited and the number ofdays in culture were identical. The highest numberof double layers observed in this series was 11 (12days in vitro). On the whole, the highest number ofdouble layers observed in vitro was 12. Concerning thenumber of double layers there was no striking differencebetween pieces cultured at 27 and 32C respectively(Fig. 2). _Frequency and temperature -dependence of the endocuticlegrowth rhythm in vitro

The weak dependence of the number of double layersdeposited in vivo on the number of days (Fig. 3) couldbe interpreted as follows: (1) in contrast to in vitrogrowth (Runte and Weber, 1982), endocuticle depositionin vitro could be a discontinuous process, whichoccasionally stops and after some time starts again; (2,

l 7OCl 2O C

: 8.._P0 7-t !z *-6 5-;pE 4-= 3-

2-

3) endocuticle growth in vitro could be a continuousprocess, but (2) the frequency of the deposition ofcircadian-like endocuticle could be variable; or (3) thefrequency could be circadian, the growth, however, stopsdefinitely after a variable duration.

In order to test these possibilities, the frequency ofdouble layer formation and its temperature dependencehad to be measured. Temperature compensation isa fundamental criterion of circadian clock controlledrhythms. The growth of the endocuticle is a self-recording process which, however, lacks exogenous timemarks. It is known that chitin is added to growing cuticleby apposition (Kaznowski et al., 1986). Therefore, inorder to set time marks, 3H-N-acetyl-D-glucosaminewas added to the medium 2-3 days after starting thecultures and washed out exactly 96 h later. The pieceswere cultured at temperatures around 25 and 30Crespectively (for exact temperatures see Table 1).As expected, in the autoradiographs a distinct, heavilylabelled band covered a part of the endocuticle (Fig. 4).In the following analysis, only such cases were con-sidered, in which, after rinsing, still some (unlabelled)material was secreted (otherwise it cannot be excludedthat the secretion of endocuticle had definitely stoppedbefore the tracer was washed out).

Cross sections were prepared for autoradiographyfrom 56 pieces. In 21 pieces no endocuticle secreted afterrinsing could be detected. In these cases the labelled

Y=X

/.

. . .

.I/no d is t inc tdouble layers

0123456789 10 11 12 13number o f days In v i t ro (days a f te r mou l t ing)

FIGURE 3. Dependence of the number of circadian-like double layers deposited in vitro on the number of days in vitro(i.e. the number of days after moulting) and on culturing temperature (27 and 32C respectively). The cultures startedimmediately after moulting. They were perfused, and fixed after exactly 3, 6, 9, 10, 12 and 13 days, respectively. In vitro, thenumber of double layers corresponded sometimes with the number of days after moulting, mostly, however, it was smaller.In viva, the number of double layers corresponds rather exactly to the number of days after moulting (up to 3 weeks: as long

as endocuticle deposition continues) (Wiedenmann et al., 1986).


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158 FRIEDRICH WEBERTABLE 1. Temperature dependence of the number of circadian-likedouble layers deposited in uitro. The leg pieces were cultured in 25.0;25.5; 29.0; 30.0 and 3l.OC & 0.25C, respectively. Two or three daysafter start of the cultures H-N-acetyl-D-glucosamine was added forexactly 96 h and then washed out. Only such cases were considered,in which, after rinsing, still some (unlabelled) endocuticular materialwas deposited. A circadian-like area, which fulfilled this condition, wasdetected in 35 leg pieces. The data represent the number of areas which

showed the respective number of labelled double layersNumber of labelled

double layers 3 3.25 3.5 3.75 4 4.25 4.525.OC 4 - 3 - 125.5C 1 1 4 2 1 - -29.OC 123--3O.OC 2 - 4 - -3l.OC 1 4 1 - -

band around the spine bases covered 4 or fewer doublelayers (not shown). A circadian-like area, which fulfilledthe above condition, was detected in 35 leg pieces.In some cases it was observed that after rinsing thestructure of the endocuticle had changed from thecircadian-like to the multilamellate type. Figure 4 gives

two examples for a labelled band: in the 255C examplethe band covers about 3.5, in the 31.OC example itcovers about 4 circadian-like double layers. Table 1summarizes the data of this experiment: the numberof labelled double layers was usually 3.54; in twocases the number was a little smaller, in one case a littlelarger.From these results two conclusions can be drawn:(1) the process of deposition did not stop and startagain (otherwise the number of labelled double layersdeposited at a given temperature should be morevariable than was actually observed); (2) within thetemperature range applied the frequency was nearlytemperature-compensated. In vivo the average numberof double layers deposited during 24 h was 0.97 in26C and 0.99 in 31C (cf. Table la in Wiedenmannet al., 1986). In these in vitro experiments averagefrequencies of 0.88-0.96 were measured in the tem-perature range between 25 and 31C. The differencebetween in vivo and in vitro results is small and prob-ably caused by experimental difficulties in this in vitroanalysis.

FIGURE 4. (a, b) In order to determine the temperature dependence of the number of circadian-like double layers depositedin vitro the endocuticle was labelled by autoradiography. The photos show cross sections of tibia pieces cultured in M3 withserum and perfused with a mixture of 95% 0, and 5% CO,. The piece in Fig. 4(a) was cultured at 25.5C, the piece in Fig.4(b) at 3l.OC (+0,25C). After two days in vitro 3H-N-acetyl-D-glucosamine was added for exactly 96 h and then washedout. Fixation after altogether 13 days in vitro. From each piece two successive cross sections are shown: the one after staining(left, polarizing microscopy), the other one after autoradiography (right, light microscopy). In Fig. 4(a) the labelled band coversabout 3.5, in Fig. 4(b) it covers about 4 circadian-like double layers (cf. vertical marks left). (---) border between the cuticle

deposited in vivo and in vitro.


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CIRCADIAN ENDOCUTICLE DEPOSITION IN VITRO 159

From these results it may be concluded that thein vitro deposition of circadian-like endocuticle aroundthe base of the spines is in fact governed by a circadianclock.Incorporation of amino acids

After application of tritiated amino acids, a labelledband again appeared in the autoradiographs. In ad-dition, a diffuse labelling was also observed above theendocuticle deposited before the tracer had been added(not shown). From other investigations, too, it has beenreported that amino acids are incorporated into thecuticle not only by apposition, but also by intussuscep-tion (Kaznowski et al., 1986).

DISCUSSIONCuticle secretion in vitro-hormonal aspects

Synthesis and secretion of cuticle, chitin and N-acetyl-glucosamine-rich glycopeptides, respectively, underin vitro conditions have been reported from numerousinvestigations. Three cases can be distinguished-( 1)Synthesis and secretion have to be induced by a pulse ofan ecdysteroid in vitro. Examples refer to primary cul-tures (pieces of the integument, regenerates, imaginaldiscs) as well as cell lines (e.g. Agui et al., 1969; Marks,1973b; Diibendorfer et al., 1975; Mitsui and Riddiford,1976, 1978; Dutkowski et al., 1977; Caruelle et al., 1978;Edwards et al., 1978; Blais and Lafont, 1980; Ferkovichet al., 1981; Quennedey et al., 1983; Doctor et al., 1985;Dinan et al., 1990; Porcheron et al., 1991).+2) Thesynthesis had already been induced (or had even started)before the tissue was explanted. In such cases cuticle andchitin, respectively, were synthesized in vitro without anyhormonal supply (Miciarelli et al., 1967; Landureau,1976; Mitsui and Riddiford, 1978; Ryerse and Locke,1978; Caruelle et al., 1978; Quennedey et al., 1983;Diibendorfer and Liebig, 1992). Landureau (1976)observed that abdominal cockroach integument taken 3days before the last moult continued in vitro to secretlamellate cuticle. Also, the experiments represented hereappertain to these eases.--(3) From some insect cell linesit has been reported that the synthesis of chitin-protein-complexes, chitin and chitin-similar polysaccharides,respectively, is constitutive (Kramerov et al., 1986;Marks and Ward, 1987; Londershausen et al., 1988;Delbecque et al., 1990).-In cases (1) and (2) depositionof epicuticle as well as multilamellate procuticle has beenreported. However, integumental pieces that in vivodeposit circadian endocuticle have obviously never beencultured before, and therefore the deposition of suchcuticle in vitro has not been observed before.

The epidermis of insects has recently been recognizedas a site of ecdysteroid synthesis (Delbecque et al., 1990).It was not investigated here whether an ecdysteroidwas synthesized in the cultured Blaberus cranitjer legpieces. However, it was tested whether endocuticle depo-sition in vitro could be promoted by addition of p-ecdysone to the medium. Concentrations of lo- and

lo-lo M had no effect, higher concentrations of thehormone induced a moulting cycle (apolysis, secretionof a new epicuticle, a new exocuticle and sometimeseven some poor material structured like endocuticle)(unpublished results). Obviously, freshly moulted epider-mis of Blaberus craniifer is not refractory againstP-ecdysone.Chitin and proteins in the endocuticle deposited in vitro

The endocuticle deposited in vitro was well-structured.Thus, it seems that chitin as well as cuticle proteins weresecreted in vitro. This was confirmed by autoradiogra-phies: after application of 3H-N-acetyl-D-glucosamine(cf. Fig. 4) as well as after application of 3H-amino acids(not shown) the endocuticle deposited in vitro washeavily labelled. From dipteran species it is known thathaemolymph-born arylphorins are specifically and intactincorporated into the cuticle (cf. Kanost et al., 1990).It is not unlikely that also in cockroaches such haemo-lymph proteins are incorporated into the cuticle. Atyrosine-rich serum protein has been demonstrated inBlatta orientalis larvae (Duhamel and Kunkel, 1983).Serum proteins could be available also in the culturesdescribed in this article, as some fat body was present inthe leg pieces. On the other hand, co-cultivation of legpieces with additional fat body taken from the abdomendid not promote endocuticle growth in vitro (Weber,1985).Circadian clock controlled endocuticle deposition in vitro

The evidence is strong that double-layered endocuticledeposited in vitro is qualitatively identical with circadianendocuticle deposited in vivo in the cockroach tibia:(1) its architecture, as revealed by light microscopy, canbe described by the two-system model (Neville andLuke, 1969); (2) as in vivo the nonlamellate layers areanisotropic in longitudinal, but isotropic in cross sec-tions (relative to the axis of the tibia); (3) the frequencyof double layer formation seems to be close to one perday, and it is largely temperature-independent in thetested range from 25 to 31C. Therefore, it is reasonableto assume that the same circadian system is operatingin viva and in vitro. As some other tissues were presentin the cultured leg pieces (for example haemocytes, fatbody and muscle, cf. Fig. l), one cannot be sure thatthe clock controlling the circadian growth rhythm islocalized in the epidermis, however, it is reasonable to setup such a hypothesis. In the light of the presentlyconfirmed conception of Erwin Biinning (1977) that thecircadian oscillation is generated inside the individualcell (cf. Introduction), one has to demand that thenumerous cellular clocks inside the epidermis synchro-nize each other so that (in vivo) the whole epidermis (ofa tibia) acts as a chronometrical unity. Gap junctionsbetween adjacent epidermal cells may be a necessaryprecondition for intercellular coupling (Warner andLawrence, 1973; Caveney and Blennerhassett, 1980).Gap junctions between epidermal cells were conservedduring the in vitro culturing of cockroach leg pieces


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160 FRIEDRICH WEBER(Wieske and Weber, unpublished results). However,under the applied in vitro conditions, the ability of theepidermis to build up circadian endocuticle seemed to bemaintained almost exclusively around the base of thespines. It has been supposed that in vivo these epidermalareas act as pacemakers of intercellular synchronization(Lukat et al., 1989).

In vivo, circadian clock control of endocuticle depo-sition continues for about 3 weeks in the legs of thecockroach Blaberus craniifer (as long as postmoultendocuticle growth lasts) (Wiedenmann et al., 1986).In vitro, the circadian deposition rhythm around the baseof the spines ceased frequently after 4-8 periods. Themaximum number of periods observed in vitro was 12.Moreover, circadian clock control in vitro stoppedusually somewhat earlier than endocuticle depositionat all.Multilamellate endocuticle deposited in vitro

In vitro, for some distance from the base of thespines multilamellate endocuticle was deposited almostexclusively. Also after the transplantation of leg piecesof freshly moulted animals into the haemocoel ofhosts, circadian endocuticle was preferentially depositedaround the spine bases, whereas multilamellate endocu-title prevailed elsewhere (Lukat et al., 1989). The ques-tion arises whether multilamellate endocuticle possessesa hidden circadian structure. The quotient number ofdouble lamellae/number of double layers (determinedin adjacent regions) varied continuously from 0.95 to4.75. Therefore, it is more likely that in regions wheremultilamellate endocuticle was deposited the epidermishad relapsed into the state of differentiation beforemoulting (when the exocuticle is built up). That meansthat in multilamellate regions the endocuticle depositionseemed to be uncoupled from the circadian clock.This was probably also the case, when double layersdeposited at a position around a spine base werereplaced by multilamellate endocuticle during the courseof in vitro deposition.

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Acknowledgements-1 thank Mrs Rita Hassenriick for excellent techni-cal assistance. I thank Dr A. Dtibendorfer, University of Zurich, forthe provision of M3 medium. I am grateful to Dr M. Luff, Universityof Newcastle upon Tyne, for correcting the English, to Drs M.Luff, A. C. Neville, University of Bristol, and to G. Wiedenmann,Humboldt-Universitlt Berlin, for critically reading earlier drafts of thisarticle and for valuable comments. Financial support was provided bythe University of Miinster and by DFG (We 389/12).

Cyclic Layer Deposition in Cockroach Circadian Rhythm

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