TISSUE & CELL 1986 18 (2) 297-304 (~ 1986 Longman Group Ltd

HARTMUT GREVEN* and WERNER PETERS1

LOCALIZATION OF CHITIN IN THE CUTICLE OF TARDIGRADA USING WHEAT GERM AGGLUTININ- GOLD CONJUGATE AS A SPECIFIC ELECTRON- DENSE MARKER

Key words: Tardigrada, cuticle, chitin, wheat germ agglutinin

ABSTRACT. Wheat germ agglutinin, a lectin with binding sites specific for N-acetyl- glucosamine residues, was coupled with colloidal gold as an electron-dense marker and used for the electron microscopical localization of chitin in the cuticles of Echiniscus testudo (Heterotardigrada, Echiniscoidea), Macrobiotus hufelandi (Eutardigrada, Parachela) and Milnesium tardigradum (Eutardigrada, Apochela). In all species investigated the cuticular layer next to the epidermis, the procuticle, was labelled, and in Milnesium tardigradum additionally the so-called intracuticle. In Echiniscus testudo only parts of the dorsal intracuticle showed some additional labelling. The other cuticular layers (ventral intracuticle in Echiniscus testudo and epicuticles of all species investigated) remained unlabelled. Specificity of binding was controlled by competitive inhibition with triacetyl chitotriose. The results are discussed with regard to a chitin-containing procuticle and the occurrence of the intracuticle in Tardigrada.

Introduction

Reports about the occurrence of chitin in Tardigrada have for long been contradic- tory. Marcus (1929) assumed that chitin is absent, whereas Cu6not (1932) considered the cuticle of Tardigrada to be chitinous or chitinoid (Cudnot, 1949; see Richards, 1951). More recently Baceetfi and Rosati (1971) have shown in an ultrastructural study of the cuticle of a eutardigrade that the cuticle layer next to the epidermis is degraded by chitinase. Bussers and Jeuniaux (1973) degraded major parts of the cuticle of Macrobiotus sp., Echiniscus merokensis and Milnesiurn tardigradum with a highly purified chitinase and used this specific reaction to prove the occurrence of chitin in the cuticle of Tardgrada. However,

*Zoologisches Institut der Universit~it Miinster, Hiif- terstr. 1, D-44 Miinster (Wesff.), Federal Republic of Germany.

tInstitut fiir Zoologie II der Universit~it Diisseldoff, Universitatsstr. 1, D-4000 Dtisseldorf, Federal Republic of Germany. Received 28 October 1985. Revised 18 November 1985.

20

Bussers and Jeuniaux (1973) could not localize it in the complex multilayered cuti- cle of Tardigrada at the light microscopical level, as the cuticle is only up to 2/~m thick.

In spite of the negative results obtained by Crowe et al. (1971) for the determination of chitin in the cuticle of Macrobiotus areolatus, it is now generally accepted that the cuticle of Tardigrada contains chitin (Greven, 1980, 1984; Ramazzotti and Maucci, 1983).

In order to confirm and extend previous findings this ultrastructural study attempts to localize chitin in the cuticle of three representatives of Tardigrada with wheat germ agglutinin (WGA). This lectin is highly specific for N-acetyl-glucosamine. It has a binding site which is able to bind a sequence of 3/3-(1,4)-linked residues of this sugar (Allen et al., 1973; Goldstein et al., 1975) and therefore has a strong affinity to oligo- and polymers of N-aeetyl-glucos- amine, especially chitin. The lectin coupled with colloidal gold as an electron-dense marker is a useful tool for the ultra- structural localization of chitin (Peters and

297

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Latka, 1985). The specifity of binding of the WGA-goldconjuga te can be controlled by competitive inhibition.

In addition to the localization of chitin in the cuticle layers it was expected to gain some information about the so-called intra- cuticle which has been described in several Tardigrada (Baccetti and Rosati, 1971; Greven, 1975, 1982, 1984).

Materials and Methods

Specimens of Echin i scus t es tudo (Heterotar- digrada), M a c r o b i o t u s h u f e l a n d i and MiIne- s i u m t a r d i g r a d u m (both Eutardigrada) were collected from mosses growing on a roof near the Zoological Institute in Miinster.

They were fixed for several hours in 2-5% glutaraldehyde buffered with 0.1M cacodylate, pH 7.2, treated with 50mM ammonium chloride for 45min to block aldehyde groups (Ruth, 1983), dehydrated in a graded series of ethanol and embedded in the water-soluble resin Lowicryl K4M (Ruth et al. , 1981); polymerization took place at 4°C.

Gold sol with grains of 17 nm in diameter was obtained by reducing tetrachloroauric acid with sodium citrate according to Frens (1973), Horisberger (1979) and Peters et al. (1983). The relatively large size of grains was chosen to enable location also at lower magnifications. Wheat germ agglutinin (WGA) was obtained from Serva, Heidel- berg. Its small size (M=40,000 dalton) prevented direct coupling With colloidal gold; W G A had to be coupled with bovine serum albumin (BSA) (Sigma, Munich) by glutaraldehyde (Horisberger and Russet, 1977) before it could be used for labelling

GREVEN AND PETERS

the gold sol at pH 7.4. The labelling condi- tions were described previously in detail by Peters et al. (1983). The labelled gold sol was stabilized with polyethylene glycol (M=20,000; Serva, Heidelberg) and stored at 4°C. Before use the gold content in the probes was checked by measuring the optic- al density at 520nm (Goodman et al . , 1981) and diluted appropriately for labelling.

Ultrathin sections of Lowicryl-embedded material were obtained with a diamond knife and mounted on grids which had been coated with Formvar and stabilized with carbon. Sections were equilibrated with phosphate-buffered saline containing 1 mM CaCla and 1 ram MgCI2 for 5-10 min before they were incubated with WGA-BSA-go ld conjugate diluted 1:50; with the same phosphate-buffered saline. After 10 rain incubation at room temperature the grids were washed twice in double distilled water, stained with uranyl acetate and lead citrate and examined in a Zeiss EM 9-$2 electron microscope. The specifity of binding was controlled by competitive inhibition with 0-3M N-acetyl-glucosamine or with 5 10 mM triacetyl chitotriose. WGA-BSA-go ld sol and the resl~ective sugar were mixed for 10 rain before the sections were transferred to this medium. After several rinses with buffer and distilled water the sections were examined with the electron microscope.

Results

The cuticle of Tardigrada consists of several layers. The following main layers can be distinguished: (1) the epicuticle, which can be divided into sublayers, may be very different in the various species and even

Fig. 1. Echiniscus testudo, dorsal cuticle. The cuticle next to the epidermis (ep) is labelled with WGA-BAE-gold conjugate, x19,000.

Fig. 2. Echiniscus testudo. Transition from dorsal (d) to ventral (v) cuticle. Note the unlabelled rather thick ventral intracuticle (i). The dorsal intracuticle is either labelled or nut. × 18,000.

Fig. 3. Echiniscus testudo. Dorsal cuticle with labelled intracuticle (i). x29,000.

Fig. 4. Echiniscus testudo. Ventral cuticle with unlabelled intracuticle (i). x30,00fl.

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300

vary on the dorsal and ventral sides of the same organism; (2) the intracuticle; and (3) the procuticle. An intracuticle and a 'wax- layer' which is localized between intra- and procuticle have not been identified in all Tardigrada examined until now (Greven, 1984).

Echiniscus testudo Of all species investigated this has the most complex cuticle. The fibrous layer next to the epidermis, which is called procuticle, shows WGA-BSA-gold conjugate labelling particularly its apical zone (Figs 1-3). The electron-dense ventral intracuticle localized above the procuticle is completely unlabel- led (Fig. 4), but the frequently rather narrow dorsal intracuticle shows occasional labelling (Figs 2, 3). The thick dorsal epi- cuticle with its lacunae and the ventral epicuticle with its characteristic rods and striations are entirely free of label (Figs 1-4).

Milnesium tardigradum This species is the only eutardigrade with a distinct intracuticle between the epi- and procuticle and a discontinuous 'wax layer' which is underlying the intracuticle. Both layers, however, are rarely apparent after glutaraldehyde fixation alone (Fig. 5; see also Baccetti and Rosati, 1971). In Milne- sium tardigradum pro- and intracuticle show heavy labelling which may also be apparent on the 'wax-layer'. The epicuticle is devoid of any label (Fig. 5).

Macrobiotus hufelandi The only cuticular layers which are not labelled heavily are the electron-dense epi- cuticle and its sublayers (Fig. 6).

Different parts of the epidermis in the

GREVEN AND PETERS

three species investigated, such as the basal lamina and vesicles, are also labelled with gold particles.

After competitive inhibition with 10raM triacetyl chitotriose, single gold particles in cuticular layers may be interpreted as un- specific background (Fig. 7).

A schematic diagram showing the dis- tribution of gold labelling in the cuticle layers of the three species examined is shown in Fig. 8.

Discussion

The results show that the cuticle of Eutar- digrada and Heterotardigrada contains con- siderable numbers of W G A binding sites. Binding of WGA-BSA-gold conjugate is not inhibited by 0-3 M N-acetyl-glucosamine or 5 mM triacetyl chitotriose. However, in- cubation with 10mM triacetyl chitotriose causes complete inhibition. This suggests the presence of chitin rather than glycopro- reins, the latter having a lower binding affinity to W G A than chitin. These findings largely agree with the results of Baccetti and R0sati (1971) and Bussers and Jeuniaux (1973) who were able to degrade parts of cuticle with chitinase. In Macrobiotus hufe- landi and in Milnesium tardigradurn only the epicuticle is free of chitin. Echiniscus testudo, however, shows an additional layer between the epi-and procuticle, tentatively being named intracuticle by Greven (1975, 1984), which appears chitin-free.

The occurrence of chitin in the cuticle of Tardigrada has been used for speculations about their phylogenetic relations (Crowe et al., 1971; Baccetti and Rosati, 1971; Bus- sers and Jeuniaux, 1973; Jeuniaux, 1975). However, chitin occurs widespread in the animal kingdom--(Jeuniaux, 1963, 1971;

Fig. 5. Milnesium tardigradum. The layers between epicuticle and epidermis are labelled. The position of the intracuticle is marked by arrows, x33,000.

Fig. 6 Macrobiotus hufelandi. The cuticle layers beneath the epicuticle are labelled, x 20,000.

Fig. 7. Macrobiotus hufelandi. Competitive inhibition with triacetyl chitotriose nearly completely abolishes the label, x39,000.

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302

e

i

p

GREVEN AND PETERS

N b c

Fig. 8. Schematic representation of the cuticle layers of the three species of Tardigrada investigated. Dotted areas indicate the cuticular layers containing chitin. (a) Echiniscus testudo, dorsal cuticle; (b) same species, ventral cuticle; (c) Milnesium tardigradura; (d) Macrobiotus hufelandi (modified after Greven, 1984). e, epicuticle; i, intracuticle; p, procuticle.

Peters, 1968), and it is likely that the ability to synthesize chitin is an ancestral feature in metazoa; agreement in such features (sym- plesiomorphism) cannot be used to reveal phylogenetic relationships (Hennig, 1950, 1966, 1982).

Tardigrada and Arthropoda have cuticles which consist of several layers with different morphological appearance and chemical composition. In both taxa chitin is present only in the procuticle (endocuticle), but not in the epicuticle (see Richards, 1951; Neville, 1975), Tardigrada, however, have their own characteristic cuticular layers (i.e. tripartite convoluted layer, intracuticle, etc.; see Greven, 1982). The chemical composition of these layers and perhaps other features might help to elucidate possible phylogenetic rela- tionships between Tardigrada and Arthropo- da.

A special but poorly understood feature of the tardigrade cuticle is the presence of an intracuticle. Baccetti and Rosati (1971) introduced this name for a distinct layer between pro-and epicuticle in a species of Eutardigrada which is very much like Mil- nesiurn tardigradum (Greven, 1975) and suggested that it is composed of untanned glycoproteins and lipids but devoid of chi- tin. The intracuticle in Milnesium tardigra- dum was named on the basis of its identical structure and location (Greven, 1972; Dewel and Dewel, 1979). Although different in structure a similarly positioned distinct

layer was described in species of the genus Echiniscus. Some Eutardigrada, however, appear to lack such an intracuticle (Greven, 1972, 1975, 1982; Kristensen, 1982). Others show a more or less distinct, occasionally electron-dense zone beneath the epicuticle which was tentatively called intracuticle (Greven, 1972, 1975; Shaw, 1974; Greven and Robenek, 1983).

The same applies to a similarly positioned electron-dense layer in the cuticle of some marine Heterotardigrada which was named either intracuticle (Greven, 1975, 1984; Greven and Grohe, 1975) or procuticle 2 by Kristensen (1982) and HaUas and Kristen- sen (1982). In Macrobiotus hufelandi the occurrence of an intracuticle is doubtful (Greven, 1972; Shaw, 1974; Greven and Robenek, 1983). Our results suggest that contrary to the findings of Baccetti and Rosati (1971)a chitin-free intracuticle does no t exist either in Macrobiotus hufelandi or in Milnesium tardigradum. The competitive inhibition by triacetyl chitotriose (see above) indicates that gold labelling of the intracuticle in Milnesiurn tardigradum is related to chitin and not to glycoproteins, which are suggested to be components of the intracuticle and its inner ' tanned flake' by cytochemical tests of Baccetti and Rosati (1971). Electron-dense or otherwise dis- stinct cuticular layers beneath the epicuticle of Tardigrada may be chemically modified parts of the chitin-containing procuticle.

CHITIN IN THE CUTICLE OF TARDIGRADA

Their darkening could be an effect of tan- ning processes (see Crowe et al., 1971; Baccetti and Rosati, 1971).

Although the ultrastructure of the distinct layer between epi-and procuticle in Milne-. sium tardigradum and Echiniscus testudo may depend on the fixative used, conven- tional transmission electron microscopy shows that it is structurally different in these species. In Milnesium tardigradum it consists of a rather homogeneous layer with more or less distinct fibers (Baccetti and Rosati, 1971; Greven, 1972; Dewel and Dewel, 1979). In the dorsal cuticle of Echi- niscus testudo it is a thin layer penetrated by vertical structures. In the ventral region it is a more electron-dense zone with occasional lamellar structures (Greven and Robenek, 1983). In both species this layer shows a similar arrangement of wavy microfibers when freeze-fractured (Greven and

303

Robenek, 1983), which are, for instance, not visible in Macrobiotus hufelandi. Never- theless, the layers seem to be, at least partly, of different chemical composition.

Cytochemical studies of further tardi- grade cuticles should lead to a better under- standing of the nature and the distribution of the so-called intracuticles. These layers are not only of interest for their permeabil- ity (Greven and Greven, 1986), but also may provide additional taxonomic criteria for the Tardigrada.

Acknowledgements The authors are very much obliged to Mrs Inge Latka for excellent technical assistance and to Dr U. J. Santore for critically reading the English version of the manu- script.

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