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Home , Alvinella pompejana, Annelid, Riftia pachyptila

MARINE ECOLOGY PROGRESS SERIES Vol. 34: 267-274, 1986 - Published December 19 Mar. Ecol. Prog. Ser.

Tubes of (Vestimentif era) and (Annelida)

' CNRS Centre de Biologie Cellulaire, 67 Rue Maurice Gunsbourg, 94200 Ivry sur Seine, France Department of Biological Sciences, University of Lancaster, Bailrigg. Lancaster LA1 4YQ. England

ABSTRACT: The aim of this study was to compare the structure and chemistry of the dwelling tubes of 2 living close to deep sea hydrothermal vents at 12"48'N, 103'56'W and 2600 m depth and collected during April 1984. The Riftia pachyptila tube is formed of a proteoglycan/ complex whereas the Alvinella pompejana tube is made from an unusually stable glycoprotein matrix containing a high level of elemental . The A. pompejana tube is physically and chemically more stable and encloses within the tube wall material.

INTRODUCTION the submersible Cyana in April 1984 during the Biocy- arise cruise (12"48'N, 103O56'W). Tubes were pre- The Pompeii Alvinella pompejana, a poly- served in alcohol, or fixed in formol-saline, or simply chaetous , and Riftia pachyptila, previously rinsed and air-dried. considered as pogonophoran but now placed in the Some pieces of tubes were post-fixed with osmium putative Vestimentifera (Jones 1985), are tetroxide (1 O/O final concentration) and embedded in found at a depth of 2600 m around deep sea hydrother- Durcupan. Thin sections were stained with aqueous mal vents. R. pachyptila where the vent water uranyl acetate and lead citrate and examined using a (anoxic, rich in hydrogen sulphide, temperatures up to Phillips EM 201 TEM at the Centre de Biologie 15°C) mixes with surrounding seawater (oxygenated, Cellulaire, CNRS, Ivry (France). no hydrogen sulphide, 2°C) (Arp & Childress 1981) Scanning electron microscope (SEM) observations while A. pompejana forms incrustations on the white were made on fixed samples, dehydrated with ethanol, smokers closer to the hot spring, by secreting - critical-point dried and sputter coated with gold. ised organic tubes and inhabiting zones of active mix- Samples were examined using a Phillips 505 SEM at ing of hot, reducing, acidic, metal-rich, fluid with cold, the Centre de Biologie Cellulaire Ivry (France). well-oxygenated seawater (Desbruyeres et al. 1983). Neutral hexose was determined using the orcinol- Here, within a few decimeters, the temperature sulphuric acid method (Barker et al. 1963). changes from 200 "C to 18 "C. Amino acid analyses were performed on unfixed Both species live in the tubes they secrete. These samples hydrolysed in 3 M methanesulfonic acid for tubes have been analyzed in a morphological and 48 h at 105OC, in vacuo. Hydrolysates were brought to biochemical way to see if the original location of these starting pH (2.0) with sodum hydroxide and analysed species may have an influence on their tube charac- using an LKB 4101 analyser. teristics. Elemental compositions were determined either using a Phillips PW1400 X-ray fluorescence spectro- meter in the Department of Environmental Sciences at MATERIALS AND METHODS Lancaster or using the Lancaster Biological Sciences Department Jeol JSM5OA scanning electron micro- Tubes from both worms, AlvineLla pompejana and scope X-ray microprobe analyser with Kevex detection Rftia pachyptila, were collected at 2600 m depth by and Link Systems data processing. Probe spots of 1 cm2

@l Inter-Research/Printed in F. R. Germany 268 Mar. Ecol. Prog. Ser. 34: 267-274, 1986

were applied at X l00 magnification. Samples were jana tube is tortuous and smaller (10 cm length, 1.5 cm either analysed whole after a brief wash in distilled in diameter, 1 mm thickness). Both tube walls are con- water and drying or as powdered ash produced by centrically multilayered (Fig. 1B & 2B). The A. pompe- heating at 500°C in air for 24 h. jana tube is fibrous (Fig. 1B) and appears to be made of Chitin contents were determined gravimetrically successive fibril layers disposed as in plywood. In each after removal of protein at 105OC in l M potassium layer, fibrils are parallel but their orientation varies hydroxide for 24 h (Hunt & Nixon 1981). Elemental from one layer to the successive one in a discontinuous sulphur was extracted from dry material by repeated way (Fig. ID). The fibril orientation varies through the extraction with either spectroscopic grade carbon di- whole tube thickness, giving an irregular pattern of sulphide or n-hexane and the content determined fibril section rows (Fig. lC, D). Sinusoidal patterns are gravimetrically. The identity and purity of the sulphur observed where irregular series of nested arcs may be extracted was established by X-ray microprobe analy- recognized in semi-thin sections (Fig. 1C). Such sis and (in the Chemistry Department at Lancaster) by patterns are not detected in the tube of R. pachyptrla. A mass spectrometry on a Hewlett-Packard 5995 GUMS, fibrillar aspect does appear in the R. pachyptila tube at with direct entry probe. high magnification, where opaque fibrillar layers Thermal transitions were measured on l mm X alternate with electron-light ones (Fig. 2D). 5 mm specimens, of established orientation relative to Filamentous bacteria associate with the inner sur- tube geometry, held lightly in a 1 mm gap between face of the Alvinella pompejana tube (Fig. lA, D). optical quality quartz plates which formed a con- Numbers of these bacteria (50 pm long, 2.5 pm diame- taining 50 % glycerol in distilled water. This cell was ter) vary from region to region of the same tube, rod- supported in a water bath containing a heater, ther- shape bacteria often being present also. Numerous mostat and digital thermocouple thermometer. Ther- bacteria are enclosed inside the tube of A. pompejana mal transitions were observed, using a cathetometer, (Fig. 1C & 2A, C). In contrast, only a few non-fila- through the glass wall of the bath. mentous (45 nm diameter) bacteria occur in the Riftia Swehng and shrinkage estimations of chemical sta- pachyptila tube outer surface (Fig. 2B). bhty were made on samples, similar to those used for Structurally dissimilar, the tubes of the 2 worms are thermal transition measurements, held in slots in also compositionally unalike. Inorganic content of the shallow baths on the stage of a travelling microscope. Riftia pachyptila tube is low (3.0 % ash) but that of The range and molecular weights of solubilizable Alvinella pompejana high (29 % ash). Extraction of A. protein in the tubes was determined, after solubiliza- pompejana tube with carbon disulphide yielded 12 to tion in 2 % SDS pH 6.8 Tridglycine treatment buffer, 25 % of free elemental sulfur. Some elemental sulfur by slab SDS polyacrylamide gel electrophoresis (SDS- has been found in accumulations associated PAGE). Samples (l mg) were triturated with a small with another hydrothermal species, Paral- volume of solubilization buffer, heated at 100°C for vinella sp. (Juniper et al. in press). 15 min, centrifuged and applied to 10 O/O gels, in an This elemental sulfur in the AlvineUa pompejana LKB slab gel-electrophoresis apparatus, using 20 mA tube may come from the bacteria it encloses, as bac- cm-' for 4 h in pH 8.3, 2 % SDS-Tridglycine running teria have been implicated as a source of elemental buffer. Staining was with Kenacid Blue. Molecular sulfur in clam (Vetter 1985). Epibiotic bacteria weights were estimated by comparison with mobilities have been described at the dorsal surface of A. pompe- of protein standards of known molecular weight. jana (Gaill et al. 1984) and on the tube inner surface (Desbruyeres et al. 1985); this is the first time they have been recorded within wall material. We know neither RESULTS AND DISCUSSION the origin of these latter bacteria, nor their metabolism type. One hypothesis is that as the tube inner surface is The cylindrical, virtually straight, tubes of kftia covered by a high density of bacteria, newly secreted pachyptila can achieve 2 m or more length (5 cm tube material from the will capture bacteria, diameter, 2 mm wall thickness). The Nvinella pompe- enclosing then in the wall. Such a process will not

Fig. 1. Alvinella pompejana tube. (A) Scanning electron micrograph (SEM) of filamentous bacteria from the inner surface of the tube (~6000).(B) The tube is made of superimposed layers (transverse section, SEM X 100). (C) Oblique semi-thin sections of the tube; 3 layers of bacteria (b) are enclosed between the fibril layers (r),fibril sections display sinusoidal patterns, which look like irregular inversed nested arcs (a) (~150).(D) Filamentous bacteria (b) associated with the inner surface of the tube. A fibrillar structure is detected inside the tube (t). Fibnl sections are parallel in each row (r) which are parallel with the tube surface (S) (semi-thin section, x600). (E)The tube is made of successive layers of parallel fibrils (1, 2, 3). Fibril orientation varies from one layer to the successive one In a discontinuous way (SEM x 1700) m B-' - ,'?r 270 Mar. Ecol. Prog. Ser 34: 267-274, 1986

occur in i&ftia pachyptila where the bacteria are quantities of granular particulate material. X-ray endosymbiotic (Cavanaugh et al.1981). tomography suggests that some of this is crystalhe The Riftia pachyptila tube contains no free sulfur. iron and ; hydrogen detected dur- Elements identified by X-ray fluorescence and X-ray ing dilute mineral acid treatment supports this view. microprobe are given in Table 1. Microscopically, R. The Riftia pachyptila tube contains chitin (20 % by pachyptila tubes seem relatively uncontaminated by gravimetric analysis after boiling in 1M potassium detritus, while Alvinella pompejana tubes incorporate hydroxide for 24 h) and both tubes contain neutral

Fig. 2. Alvinella pompejana and kftia pachypljla tubes. (A) Ultrastructural aspect of the bacteria enclosed in A. pompejana tube (X2000). (B) Ultrastructural aspect of the multllayer structure of R. pachyptila tube (~2000).(C) Aspect of the tube matrix of A. pompejana: a row of nested areas is seen in the lower left of the micrograph, where the tube has a filamentous aspect, and smalI bactena are present in the upper part (~4000).(D) High magnif~cationof (C) (X16000) Gaill & Hunt: Tubes of hydrothermal vent worms 271

Table 1. kftia pachyptila and Alvlnella pompajana whole typical of mollusc shell matrix protein (Degens et al. and ashed tube elemental compositions determined by X-ray 1967). In A. pompejana an elevated glycine and high fluorescence analysis (A) and X-ray microprobe analysis (B). & Values are counts as a percentage of total counts serine content resembles beta fibroins (Lucas Rudall 1968). Notable in R. pachyptila is the high cystelne Element Rifha pachyptila Alvinella pompejana content (10 %). The serine (25 O/O) and glycine (20 %) Whole Ash Whole Ash contents in A. pornpejana suggest a limited range of A B A B slmilar . Unfortunately, SDS-PAGE of tube matenal treated with solubilization buffer ylelded a F Tr. 0 0 Tr. 0 0 very small trace of protein In the extracts, witnessing Na T 10 2 1 1 8 the stability of the material. In contrast, SDS-PAGE of M g Tr. 11 23 1 5 6 R. pachyptila tube solubilisates suggests a multi-pro- Al 1 0 1 1 13 tein system (Fig. 3). There are numerous proteins evi- Si 1 0 4 3 1 1 P Tr. - 12 11 18 40 dent, spanning the molecular weight range 120 000 to S 77 51 36 46 56 10 12 000, but the 2 domlnant species occur at molecular C1 9 21 Tr. 7 Tr. Tr. weights 16 500 and 12 000, respectively. K 5 1 Tr. 4 Tr. Tr. Both tube materials display considerable stabhty. Ca 5 5 18 20 14 22 Ti Tr 0 0 Tr 0 0 This is both physical and chemical. Thermal transi- Fe 1 Tr. 3 1 3 5 tions are small in water between 20 and 100°C. The Cu Tr. 0 0 Tr. 0 0 Alvinella pompejana tube extends by 7 %; a sharp Zn Tr. 0 0 4 1 4 shrinkage at 60°C compensates exactly for a 3 % expansion between from 20 to 60°C, and a final exten-

hexose, 7.5 % for Alvlnella pompejana and 3.2 O/O for R. sion occurs sharply at 80°C. The fiftia pachyptila tube pachyptila. Both tubes contain protein and it is likely shrinks overall by only 3 O/O over the temperature range that this comprises the major balance of organic but there are several transitions; slight shrinkage material. Table 2 gives amino acld analyses of the between 20 and 40°C is compensated for by swelhng whole tubes, companng these with other worm and po- up to 60°C, and a small sharp shrinkage up to 80°C gonophore tubes. Both tubes have compositions typical precedes a slight expansion with a sudden 2.5 '10 of structural proteins but of different types. In R. shrinkage at 90°C. Such complex behaviour may pachyptila tube a high aspartate-glycine pattern is reflect the composite nature of the polymer. The small

Table 2. Riftia pachyptia, Alvinella pompejana and other worm tube amino acld compositions expressed as the number of individual resldues per l000 total residues

Amino acid R. pachyphla A. pornpejana Serpulid"' Sabellariidb,d Eunicide Pogonophorani -- p -- Aspa~c 111 67 167 7 6 6 2 113 Threonine 49 39 7 9 40 3 9 50 Serine 75 250 52 192 43 87 Glutamic 73 5 3 104 54 20 68 Proline 58 14 4 2 3 9 81 63 Glycine 144 206 104 263 380 128 Alanine 7 8 118 7 6 8 3 46 5 7 98 tr 9 5 13 4 2 Valine 7 2 63 45 4 3 42 53 Methionine 2 15 7 8 0 12 Isoleucine 3 2 21 40 2 2 19 3 8 Leucine 39 27 4 9 4 6 35 47 Tyrosine 53 4 7 17 20 0 5 6 Phenylalanine 2 9 64 23 0 34 Omithine - - 8 0 Lysine 62 17 18 32 33 3 9 Hishdine 24 16 7 14 11 23 Arginine 5 9 20 19 4 2 175 86

" Average of 3 species, Hydroidas norvegicus, Eupomatus sp., Pomatoceros lamark~i Average of 4 species, Sabellana floridensis, S. ka~paraensis,Phragmatopoma lapidosa, P. nloerchj C,d Mitterer 1971, Bubel et al. 1983 P Hyalinoecia tubicola; new data ' S~boglinumsp. (Foucart et al. 1965) 272 Mar. Ecol. Prog. Ser 34: 267-274, 1986

material but subsequent treatment with 1.0 M potassium hydroxide causes 50 % thickening, swelling and delamination, again wlth loss of 5 '10 protein. The phylogenetic position of the Pogonophora remains disputed; the presence of an adult polymeric and schizocoelic larval development indicates protosome affinity while cuticular structure, setae, opisthosomal and photoreceptor cells point to annelid relations (Hickman et al. 1984). Jones (1985) places IZlftia in a new phylum, the Vestimenti- fera. Hence, a comparison of pogonophore tube com- position relative to polychaete annelid worm tubes is justified on phylogenetic and functional grounds. Unfortunately, we have insufficient data to compare R. pachyptila compositionally with pogonophoran Fig. 3. Riftia pachyptda tube. Solubilized extract of the species from non-deep sea hydrothermal sites. More median layers SDS-PAGE electrophoresed. Dens~tometer data are available for the varied tube types from scan and molecular weights indicated in kilodaltons X 10' polychaete worms (Mitterer 1971). Polychaete families which secrete tubes fall into 3 main types. Chaeto- changes suggest a very stable structure; important for pteridae, and secrete flexible A. pompejana which Lives at higher temperatures than membranous or homy uncalcified tubes; often incor- R. pachyptila. porating mud or sand. Serpul.idae produce calcified The chemical stability of the tubes is also marked. tubes. Fragile tubes of mucus or cementing material, They show little bulk swelling or dissolution in mild incorporating sand or clay, are produced by , reagents, e.g. mercaptoethanol, guanidinium hydro- Maldaniidae, Sabellaridae, Amphictenidae, Amphare- chloride or detergents, but the fiftia pachyptila tube tidae, and Nereidae. Other families pro- does swell rapidly and partially dissolve in anhydrous duce tubes of mucus or no tube at all (Defretin 1971). formic acid. This solution contains the bulk of the A distinct difference between the R2ftia pachyptila chitin-proteoglycan and some protein. The residue and Alvinella pompejana tubes is that the former con- shows slight solubility in mercaptoethanol and more so tains chitin while the latter does not. This is in accor- in formamide. As already mentioned, proteins from the dance with known properties of pogonophorans com- R. pachyptila tube can be solubilized for SDS-PAGE. pared to other polychaete tubes. The only analysis The Al~nellapompejana tube is much more stable available for a pogonophoran tube is from Siboglinum with no appreciable solubility in formic or dichlorace- sp. (Foucart et al. 1965) where the chitin content was tic acids. A 7 % swelling in 1.0 M potassium hydroxide 33 %, rather more than R. pachyptila with 20 '10,but solubilizes 5 '10 protein. The elemental sulfur may completely contrasting with polychaete tubes where assist stability since after its removal 3 O/oprotein is chitin is absent. extracted by water and then 1.5 % by 4 M guanidinium The striking feature of polychaete tubes is their lack hydrochloride. Hydrochloric acid (12 M) for 30 s of morphological and chemical uniformity. Mor- removes iron and zinc without solubilizing organic phologically, the Riftia pachyptila tube (viewed as an Table 3. Riftia pachyptda, Alvinella pompejana and other worm tube amino acid compositions broken down into specific types or groups. Acidic: Asp + Glu; Basic: Lys + His + Arg; Polar: Acidic + basic + Ser + Thr + Tyr; Apolar: Pro + Ala + Val + Ile + Leu + Phe. Hydrophobic index calculated from data in Levitt (1976). Units as in Table 2

R. pachyptlla A. pompejana Serpulid Sabellariid Eunicid Pogonophora: (+ SD) (+ SD) Siboglinum sp.

Acidic 184 120 272 (f 71) 130 (_+32) 82 181 Basic 145 53 43 (f l) 89 (& 14) 219 148 Polar 506 509 463 (+ 79) 470 (f 20) 384 522 Apolar 280 272 317 (2 55) 255 (f 21) 224 292 Polar: apolar 1.8 1.8 1.5 (t 0.06) 1.9 (k0.13) 1.7 1.8 Hydrophobic index -233 1.2 -223 (f 121) -184 (294) -505 -202 Glycine 114 206 104 (k 9) 263 (2 36) 380 128 Tyrosine 53 4 7 17 (f 4) 20 (t 18) 0 5 6 Cysteine 98 tr 9 (k 10) 5 (t2) 13 42 Gaill & Hunt: Tubes of hydrothermal vent worms 273

annelid type) is of the membranous, horny group while The plywood architecture of the Alvinella pornpe- that of Alvinella pompejana is of the fragile , jana tube and the nested arcs series observed in ob- incorporating exogenous . Table 3 compares lique sections indicate a liquid crystalline-like organi- amino acid compositions of the R. pachyptila and A. zation (Bouligand 1971) whose type has to be analysed. pompejana tubes with those of other and Liquid crystalline-like organization has also been pogonophorans. Such an analysis demonstrates the noted in some pogonophoran tubes where chitin is inconsistency of worm-tube protein types. Acidic involved in the structure (Gupta & Little 1975). amino acid contents vary widely, never falling below The physical and chemical stabihties of both Riftia 8 % of total but rising in some serpulids to 30 % of total pachyptila and Alvinella pompejana tubes point to amino acids. Likewise basic amino acid levels show proteins which are strongly self-interactive either via wide variation. A more consistent feature is the ratio of multiple weak interactions or through covalent bridg- polar to apolar amino acids which indicates a signifi- ing. The former may be implicated partly in R. pachy- cantly polar protein character. A, pornpejana is the ptila tube stabilization since the combined effects of only worm tube to show a positive hydrophobic index, formic acid (non-hydrolytically) and formamide cause suggesting that non-aqueous stabilizing interactions appreciable solubilization. For A. pompejana tube no are favoured. Perhaps this also helps to explain the reagent causes significant solubilization; formic acid intimate association of elemental sulfur with the struc- fails as a chaotropic agent and only alternate extremes ture. of pH bring about hydrophilic swelling. This suggests The most striking feature of the Riftia pachyptila that ionic interactions, probably with inorganic compo- tube analysis is its close correspondence to that of the nents, have some stabilizing influence but that cova- pogonophoran Siboglinum sp. This coupled with the lency is a major factor. There is insufficient cysteine in high chitin content speaks for a close affinity between A. pompejana protein to explain stability through di- the 2 species. R. pachyptila tube protein is also not sulfide bridging. In R. pachyptila tube a high cysteine unlike some of the other worm tube proteins. The content might point to disulfide as being an important po1ar:apolar ratio is high and the hydrophobic index structural determinant. However, this cannot account very negahve, indicating a structure llkely to be for residual stability after formic acid treatment since strongly hydrated and with hydrogen-bonded and disulfide breaking agents produce very little solubili- other polar interactions favoured. The glycine content zation. is high in Alvinella pompejana and lower in R. Both dwelling tube types have high chemical stabil- pachyptila. High glycine contents in invertebrate ity, an aspect accentuated in Alvinella pompejana. s!rLc!urn! pi~teiiis,coupied wiih iow acidic amino acid This may be related to location, as this worm lives contents, suggest extended sheet conformations, not close to the chimney in a region of high temperature usually associated with calcification. The reverse situ- and exotic chemistry. ation points to more elaborate folding and also to Fossilized worm tubes have been found in areas of calcification potential. ancient extinct hydrothermal vent activity (Haymon et Physical and chemical stability is common in al. 1984, Oudin & Constantinou 1984, Banks 1985). extracellular structural materials produced by inverte- Such tubes are coated with concentric layers of brates (Vincent 1982). Stability can be achieved in amorphous silica intercalated with thin sulfide several ways. Simple polymers of repetitive monomer laminae. Probably the silica is formed by replacement structure tend to linear conformations; these in turn of organic material during fossilization, but the tend to paracrystalline assemblies with stabllization hypothesis of biogenic origin for sulfide laminae is commonly involving hydrogen-bonded and Van der corroborated by the presence of bacterial growth layers Wads interchain regular interactions (Hunt & Oates and elemental sulfur inside the tube in Alvinella pom- 1978). Globular proteins with high helical content also pejana tubes. These results may give some indication tend to self-assemble as sheets or fibres (Cohen 1966). about tube type, allowing fossilized vestimentiferan Such materials can be further stabilized by inter- and tubes to be distinguished from polychaetous ones. intrachain covalent cross-links of which diverse types are now known. Elemental sulfur should be also con- Acknowledgements. The authors gratefully acknowledge the sidered with protein composition as a factor affecting chief scientist of the Biocyarise cruise, Daniel Desbruyeres, the hydrophobic character and stability of the Riftia and M. C. Laine for technical assistance. Dr. R. Macdonald and Mr. J. Bowman of the Department of Environmental pompejana tube. Sciences and Dr. S. Breuer of Chemistry Department at Lan- X-ray diffraction studies of the tubes (unpubl.) indi- caster University are thanked for help with X-ray fluor- cate a paracrystalline chitin component of the ves- escence and mass spectral analyses respectively. timentiferan tube; that of the polychaete shows no orientation at medium to high diffraction angles. 274 Mar. Ecol. Prog. Ser 34: 267-274, 1986

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This article was submitted to the editor; it was accepted for printing on October 3, 1986