Biol Cell (1992) 76, 201-204 201 © Elsevier, Paris

THE CHITIN SECRETING SYSTEM FROM DEEP SEA HYDROTHERMAL VENT WORMS

Fran~oise Gaill i, Bruce Shillito l, Jean Pierre Lechaire l, Henri Chanzy 2, GErard Goffinet 3.

1 CNRS Centre de Biologic Cellulaire, 67, rue M. Gunsbourg, 94205, Ivry cedex

2 Centre de Recherches sur les Macromolecules V6g6tales, CNRS, BP 53X, 38041 Grenoble cedex

3 Institut de Zoologic, Universit6 de Liege, 22, Quai Van Beneden, B-4020 Liege (Belgique)

SUMMARY

Deep-sea hydrothermal vent worms live in tubes made of

giant f~-chitin crystallites (50 nm in diameter, several gm in length) embedded in a protein matrix. These chitin crystallites form a liquid-crystal-like structure differing from the well- known cholesteric arrangement of classical chitin-protein systems. Furthermore, and in contrast with the latter systems, the vestimentiferan chitin-protein systems are produced by goatskin-shaped glands. Rod-shaped elements in the lumen of these glands were identified by DCTEM and Au-WGA labeling and freeze fracture as chitin crystallites. The main characteristics of these "chitin secreting glands" are described.

key words: chitin, biopolymers, hydrothermal vent worms

INTRODUCTION

New types of worms, the vestimentiferans (1), have recently been discovered in extreme environments at deep-sea level

around hydrothermal vents of the Pacific ocean. Their tube is made of a chitin-protein system (2) which differs from the usual chitinous structures by several original characteristics.

In contrast with the usual natural chit.in, which is an ct form,

that of the vestimentiferan tubes is the rare g fort0 (3). The

most striking feature is that each step involved in the chitin secretion process seems to be amplified in these organisms. The lenIlth of the tube can reach more than 1,5 m in the so-

called tube worm Riftia (2). The size of the crystallites (up to 50 nm in width and several gm in length) is giant when

compared to that of the usual ~ form (3 nm in width). Using

the chitin crystallite material of another vestimentifera T e v n i a , it has been possible to demonstrate the correspondance microfibril/crystallite in the tube wall (3). The

number of chains involved in the crystallite structure may be

up to 6000 as opposed to 15 in the usual tx form (3) and the chitin secretion rate seems to be 1000 times higher than what is known in any other marine ecosystem (4). Furthermore the supramolecular organization of the chitin-protein system, which is still of the plywood type, cannot be described as cholesteric (5) but rather as "random twisted nematic" (3). Given all these characteristics one may suggest that the biogenesis mechanisms of these chitinous structures could be quite different from that of the arthropod cuticles. These results lead us to think that original chitin secreting systems may be found in such animals, and in fact, we discovered original structures opening at the worm surface. These

structures are differenciated cells associated in what we called "chitin secreting glands". We describe the main characteristics

of these glands and display data demonstrating the chitin

location in these structures.

MATERIAL AND METHODS

Several specimens of Tevnia jerichonana and Rif t ia pachyptila were collected at a depth of 2600 m during the french american HYDRONAUTE and HERO cruises with the Nautile submersible. Several animals were fixed either with

I

70 ° ethanol or in 10% salin formalin. Small pieces of vest imentum were also fixed according to classical procedures for electron microscopical studies (2). Scanning electron microscopy (SEM): SEM analysis were made on samples fixed with 10% formalin, dehydrated with

202

ethanol, critical-point-dried with ethanol and coated with gold metal. These samples were examined with a Philips 505.

Transmission electron microscope (TEM): TEM studies were

made on samples fixed in 0.4 M sodium cacodylate-buffered glutaraldehyde (3% final concentration) at pH 7.2, post-fixed with osmium tetroxide (1% final concentration) and embedded in Araldite. Serial thin sections (80 nm thick) were obtained with a Reichert Ultramicrotome. They were stained with aqueous uranyl acetate and lead citrate and examined in a Philips 201 at 80 kV accelerating voltage. Chitin affinity cytochemistry: Wheat germ agglutinin-gold complex (Au-WGA) was prepared according to Horisberger and Rosset (6). Labeling of ultrathin sections (incubation : 45 min) was carried out following the basic prodecure of Horisberger (7). Diffraction contrast tr&nsmission electron microscopy (DCTEM): DCTEM analysis were performed on unstained thin sections of .grutaraldehyde fixed specimens. These sections were observed with a Philips 400T operated at 120 kV and equipped with a low-dose attachment. The bright- field images were obtained with the help of an objective aperture of 20 I.tm selected so as to eliminate all diffracted beams with d-spacings smaller than 0,8 nm. The images and diffraction diagrams were recorded on Mitsubishi MEM trim. Freeze-fracture. Pieces of glutaraldehyde fixed Riftia vestimentum were dissected and soaked in a 30 % glycerol solution. They were frozen in Freon 22, stored in liquid

nitrogen and fractured at - 100°C (2. 10 -5 mbar) in a BAF 400 T Balzers unit. The specimens were subjected to etching at - 90°C for 15 minutes. Unidirectional shadowing was carried out with platinum and carbon and replicas obtained after a passage in a chromic acid solution. Micrographs were obtained with a Philips 201 operated at 80 kV.

RESULTS AND DISCUSSION

The chitin-secreting glands of Riftia are goatskin-shaped, 500 gm long (Fig. I). They have a long neck opening, extending to the epidermal surface. These glands are made of an epithelium surrounding a central lumen (Fig. 2). In Tevnia, this central lumen is connected with smaller ones,

the sublumens, which are borded by flat gland cells (Fig. 2). These lumens contain microfibrillar structures (Fig. 2). Even the microfibrillar pattern differs from one place to the next, these microfibrils are locally arranged in regular pattern recalling the plywood structure of the tube. Such regularity seems to be disturbed in the central lumen (Fig. 2). Locally,

gland cells exhibit microviUi surrounded by an electron-dense filamentous cell coat forming a network which spreads

throughout the glandular lumen (Fig. 2). DCTEM reveals chitin inside the glandular lumen and shows that chitin constitutes the crystalline part of the micro fibrils (Fig. 4). Affinity cytochemistry corroborates this observation: the only structures reacting with the Au-WGA complexes are the microfibrils within the lumen (Fig. 3), while the filamentous network and gland cells remain unlabeled. Freeze-fracture replicas of glands reveal the shape of the chitin fibrils (Fig. 5). When longitudinally fractured, their length may reach 3400 nm and their diameter 50 nm. These results indicate that chitin crystaUites secreted by the glands of the vestimentiferan Riftia and Tevnia are similar in shape and size to those of the tubes, and are thus not modified after their initial secretion outside the organisms. The difference concerns mainly the supramolecular organization of the crystallites. Loosely packed in the glandular lumen, they acquire in the tube a compact nematic texture (3).

LEGENDS

Fig. 1. Aspect of the chitin secreting gland in Riftia pachyptila (SEM). The arrow indicates the neck opening (scale bar: 1001am) Fig. 2. Aspect of the chitin secreting gland in Tevnia jerichonana (TEM). The central lumen (L) is connected with sub lumen (1) which are surrounded by fiat epithelial cells (C). They display rnicrofibrillar patterns (M) which exhibit local regularity. Small microvilli (m) can be seen in some areas of the cell/lumen interface (arrow). Longer ones are seen towards the central lumen where they are embedded in an electron-dense filamentous cell coat (f).(scale bar: 5 lam) Fig. 3. Au-WGA labeling in the gland of Tevniajerichonana: gold particles are exclusively located on oblique sections of chitin microfibrils (M and arrows).(scale bar: 2,5 ~tm)

Fig. 4. DCTEM (Tevnia jerichonana); microfibrils are electron dense in cross sections, indicating their crystalline nature (M and arrows).(scale bar: 2,5 ~tm)

Fig. 5. Freeze fracture (Riftia pachyptila);, chitin crystallites display different orientation. The orientations become more and more longitudinal towards the upper part (arrows).The electron light elements correspond to a shadowing effect on crystallites which are erected over the replica's surface.(scale bar: 2,5 I.tm)

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In conclusion, unlike previously studied chitinous structures, the chitin-protein complexes of vestimentiferans are first secreted by gland cells into a glandular lumen, rather than deposited directly in the extracellular compartment. The lumen content will be later extruded outside the worm and will subsequently constitute the tube wall. Vestimentiferans thus constitute an interesting tool for investigating the morphogenesis of chitinous structures. Complementary studies are in progress to elucidate the whole secretion mechanism of these biopolymers.

Acknowledgment: We thank Roger Vuong and Monique Da Conceicao for excellent technical help.

REFERENCES. '

1 Jones, M.L., The Vestimentifera, their biology, their systematic and evolutionary patterns. Oceanol. Acta, 1988, 8, 69-78. 2 Gaill, F. and Hunt, S., Tubes of deep sea hydrothermal vent worms Riftia pachyptila (Vestimentifera) and Alvinella pompejana (Annelida). Mar. Ecol. Progr. Ser., 1986, 34, 267-74. 3 Gaill, F., Persson, J., Suhiyama, J., Vuong, R. and Chanzy, H., The chitin system in the tubes secreted by deep sea hydrothermal vent worms. J. Struct. Biol., in press. 4 Gaill, F., Voss-Foucart M.F., Gerday C., Compare P., Goffinet G. (1992), chitin and protein contents in the tube of vestimentiferans from hydrothermal vents, In advances

chitin and chitosan, (Brine C.J;, Sandford P.A., Zizakis J.P. eds), London and New York, 232-236 5 Giraud-Guille, M.M. and Bouligand, Y., Chitin-protein molecular organization in arthropod. In Chitin in Nature and Technology, ed. Ch. Jeuniaux and G.W. Gooday, Plenum Publishing Corporation, New York, 1986, pp. 29-35. 6.Horisberger, M. and Rosset, J., Colloidal gold, a useful marker for transmission and scanning electron microscopy. J. Histochem. Cytochem., 1977, 25 (4), 295-305. 7 Horisberger, M., Colloidal gold: a cytochemical marker for light and fluorescent microscopy and for transmission and scanning electron microscopy. Scanning Electron Microscopy, 1981, 2, 9-31.

Received 23 November 1992