Metabolic diversity in epibiotic microflora associated with the Pompeii worms Alvinella pomPejana and A. caudata (Polychaetae: Annelida) from deep-sea ...
Marine Biology
Marine Biology 106, 361-367 (1990)
@ Springer-Verlag 1990
Metabolic diversity in epibiotic microflora associated with the Pompeii worms Alvinella pomPejana and A. caudata (Polychaetae: Annelida) from deep-sea hydrothermal vents D. Prieur, S. Chamroux, P. Durand, G. Erauso, Ph. Fera, C. Jeanthon, L. Le Borgne, G. M~vel and P. Vincent UPR 4601 CNRS and University P. & M. Curie, Station Biologique, BP 74, F-29682 Roscoff C6dex, France Date of final manuscript acceptance: April 20, 1990. Communicated by J. M. P6r6s, Marseille
Abstract. Specimens of alvinellid polychaetes (Alvinella pompejana Desbruy6res and Laubier, 1980 and A. caudata Desbruy6res and Laubier, 1986) and their tubes were sampled from deep-sea hydrothermal vents at 13°N from the manned submersible "Nautile" during the "Hydronaut" cruise (October to November 1987) on the East Pacific Rise. Samples were subjected to bacterial analysis aboard the mother ship "Nadir" to detect bacteria involved in the nitrogen and sulphur cycles, in non-specific heterotrophic processes, and displaying resistance to selected heavy metals. Cultures were incubated at different temperatures under atmospheric and "in situ" (250 atm) pressures. Bacterial growth was observed in enrichment cultures for most metabolic types screened. Heavy-metalresistant bacteria were also detected in many samples. No filamentous bacterial form was observed in the cultures. The results demonstrate a high metabolic diversity in episymbiotic flora, indicating that the worm (A. pompejana or A. caudata), its tube and its epiflora represent a complex micro-ecosystem.
Introduction The food web in deep-sea hydrothermal vent ecosystems is fuelled by chemoautotrophy, mainly based on sulphuroxidizing bacteria (Felbeck and Somero 1982, Tuttle et al. 1983). These bacteria are either free-living (Ruby et al. 1981) and attached to rock surfaces (Jannasch and Wirsen 1981), or associated with invertebrate species (Cavanaugh et al. 1981, Fiala-M6dioni 1984, Gaill et al. 1984 a). Although some of the free-living bacteria are ingested by invertebrates such as Bathymodiolus thermophilus (Le Pennec and Prieur 1984), bacterial primary production is more efficiently effected within invertebrate tissues. Endosymbiotic bacteria have been found associated with the Vestimentifera (Cavanaugh et al. 1981) and Bivalvia (Fiala-M6dioni 1984), but not with the Alvinellidae (Desbruybres et al. 1983). While the endosymbiotic bacteria
living in vestimentiferan or bivalve tissues display an extremely weak morphological diversity, and seem in the main to be unique, the epibiotic bacteria of the polychaetes Alvinella pompejana and A. caudata are morphologically extraordinarely diverse (Gaill et al. 1987). Alvinellapompejana (Desbruybres and Laubier, 1980) and A. caudata (Desbruy6res and Laubier, 1986) secrete their tubes on the walls of active sulphide chimneys, where the temperature ranges from 20 ° to 40 °C (Desbruy6res et al. 1982). Both species have a dense epibiotic microflora, composed of rod-shaped, spiral-curved and prosthecate bacteria scattered on the surface of the worm integument, and clump-like bacteria (rods, cocci, filaments) in the intersegmentary spaces (Gaill et al. 1984a). In addition, Alvinella pompejana and A. caudata have specific populations of episymbiotic bacteria (Gaill et al. 1987). The epidermis of A. pompejana has expansions located at the level of the dorsal intersegmentary parts which bear filamentous, sheathed bacteria (0.3 to I #m diam, 1 to 200 #m long) while A. caudata has modified posterior parapods, the digital lobes of which bear larger bacterial filaments (2.5 #m diam, 600 #m long). Both species live in tubes, the inner wall of which is covered by various bacteria, some filamentous (Desbruybres et al. 1983). The above studies were in contrast with previous work, which indicated that polychaetous annelids usually lacked associated bacteria (Sieburth 1975). The role of this epibiotic microflora has been investigated by several authors. Although Alvinella pompejana and A. caudata have a functional digestive tract and are able to ingest particles including bacterial cells (Desbruy~res et al. 1983), atrophic role has been suggested for their epibiotic micro flora, based on the analyses of stable carbon and nitrogen isotopes in the worm tissues (Desbruy6res et al. 1983). Detection of enzymes specifically involved in CO2-fixation (Tuttle 1985) indicated that at least some of these bacteria were autotrophic, probably sulphur-oxidizing. In situ experiments using radiolabelled bicarbonate have demonstrated that antibiotics decrease uptake of labelled compounds by Alvinella spp., suggesting a
362
D. Prieur et al. : Metabolic diversity in epibiotic microflora of Pompeii worms
trophic role of the epibiotic bacteria, complementary to that of planktonic bacteria in the digestive tract (AlayseDanet et al. 1986). However, bicarbonate uptake in those experiments was restricted to a few coccoid bacteria, suggesting metabolic diversity (autotrophy and heterotrophy) of bacteria species or physiological plasticity in response to environmental conditions. The name "Pompeii w o r m " was first given to Atvinella pompejana (Desbruy6res and Laubier, 1980) since it is subjected to mineral particles precipitated when hot vent-waters enriched with mineral solutions discharge into cold, deep seawater. These minerals include high amounts of zinc, iron and hydrogen sulphide, and high concentrations of heavy metals (Baross et al. 1982). Laubier et al. (1983) noted that part of the outer tissues of A. pompejana and some associated filamentous bacteria contained sulphur granules. Gaill et al. (1984 b) examined A. pompejana with an electron probe, and found high concentrations of zinc, arsenic and sulphur in different epidermal areas of the worm. The detection of metallothionein-like proteins in A.pompejana tissues (Cosson-Mannevy etal. 1986) led these authors, Alayse-Danet et al. (1986) and Prieur and Jeanthon (1987) to propose a detoxification role for this microflora. The above authors suggested two roles for the epibiotic microflora of the Pompeii worms: a t r o p h i c role that would implicate a dominant autotrophic microflora, and a detoxifying role through intracellular accumulation or extracellular transformation of toxic compounds. To test these hypotheses, we carried out a detailed survey of the worm's microflora which involved screening bacterial metabolic types that could be involved in these processes.
Materials and methods Samples were obtained from deep-sea hydrothermal vents located on the East Pacific Rise at 13°N (Desbruy6res et al. 1982) during the cruise "Hydronaut", from October to November 1987. Specimens of Alvinellapompejana and A. caudata, tubes of A. pompejana, and unidentified tubes were collected by the submersible "Nautile" from three actives sites, "Totem", "Parigo", "Pogonord" (Alayse-Danet 1988) and brought to the surface in an insulated box. Specimens of Paralvinella grasslei (Desbruy+res and Laubier, 1982), that do not bear epibiotic bacteria, were also sampled as controls. Aboard the mother ship "Nadir", specimens were washed three times in 0.2/~mfiltered seawater. Then the dorsal integument of A. pompejana, the rear part of A. caudata, and small pieces of A. pompejana tubes and unidentified tubes were carefully dissected with sterile tools. The fragments were rinsed once more in 0.2 #m-filtered seawater, and homogenized with a "Polytron" grinder, washed with sterile seawater, and flame-sterilized. One whole small specimen of P. grasslei was also processed and homogenized in the same way. Portions of the homogenate were inoculated into liquid culture media, or diluted and plated on media suitable for the bacterial metabolism to be detected. Sulphur-oxidizing bacteria were cultivated in liquid media with thiosulphate or thiocyanate as electron donors (Adair and Gundersen 1969, Tuttle and Jannasch 1972) and in sulphide gradients containing Na2S (Nelson and Jannasch 1983). Sulphate-reducing bacteria were cultivated in a medium containing lactate and acetate, according to the method of Abd el Malek and Rizk (1958) and Postgate (1966). Bacteria involved in the nitrogen cycle were cultivated in suitable culture media for nitrifying, nitrate-respiring, denitrifying, aerobic and anaerobic nitrogen-fixing bacteria, ac-
cording to Lewis and Pramer (1958), Chamroux (1972), Postgate (1972) and Mevel (1986), respectively. Non-specific heterotrophic bacteria were cultivated on agar plates containing 2216E medium (Oppenheimer and Zobell 1952) and incubated under aerobic and anaerobic conditions. TCBS (thiosulphate :citrate : bile : sucrose) medium (Kobayashi et al. 1963) was used for the enrichment of vibrios. Two additional media containing amino acids (cas-amino acids Difco, 0.5 g 1 1, Chamroux personal communication) or carbohydrates as carbon sources were also used. Manganese-oxidizing heterotrophic bacteria were enriched on agar plates according to Nealson (1978). Heavy-metal-resistant heterotrophic bacteria were cultivated on 2216E medium, supplemented with silver (10 mg 1-1), copper (90 mg l-t), cadmium (80 mg 1-1), zinc (100 mg 1-1) or arsenate (1770 mg 1-1), according to the tests by Jeanthon and Prieur (1990). Incubation temperatures were selected corresponding to earlier measurements of temperature in Alvinella spp. habitats (Desbruy6res et al. 1982). Liquid cultures and agar plates were incubated at 20° and 40 °C, under atmospheric pressure. In addition, selected cultures in liquid media were incubated at 20° and 80 °C under "in situ" hydrostatic pressure (250 arm). Cultures displaying bacterial growth were processed for isolation of pure bacterial strains for further qualitative studies.
Results Sulphur bacteria (Table 1) Cultures of sulphur-oxidizing bacteria using thiosulphate as electron donor, were obtained from the body wall of Alvinella pompejana, and A. caudata and unidentified tubes after incubation at 20 °C under both atmospheric and "in situ" pressure. N o bacterial growth was observed at 40 °C. Only few cultures displayed bacterial growth in media containing thiocyanate or in sulphur gradients. Sulphate-reducing bacteria were cultivated under atmospheric pressure from tubes of Alvinella pompejana and unidentified tubes and from Paralvinella grasslei, at 20 ° and 40 °C, but seemed to be absent from the body wall of A. pompejana and A. caudata. However, three samples incubated at 80 °C under "in situ" pressure, contained sulphate reducers.
Nitrogen bacteria (Table 2) The first step in nitrification (oxidation of N H , to NO2) was detected only in one sample of Alvinella pompejana, incubated at 20 ° and 40 °C under atmospheric pressure. The second step (oxidation of N O / t o NO3) was detected in two samples of unidentified tubes, incubated at 20 ° and 40 °C under atmospheric pressure. Nitrate respiration (with production of nitrites) was present in almost all samples incubated at 20 ° and 40°C under atmospheric and "in situ" pressures. Denitrification, with the production of nitrogen gas, also occurred in these samples incubated at 40 °C at atmospheric pressure, but was only observed at 20 °C in one sample incubated at "in situ" pressure. Nitrate respiration was also observed in one sample of an A. pompejana tube, incubated at 80 °C under "in situ" pressure. Nitrogen fixation was detected under anaerobic conditions for A. pompejana and A. caudata homogenates incubated at 20 °C.
D. Prieur et al. : Metabolic diversity in epibiotic microflora of Pompeii worms
363
Table 1. Alvinella pompejana, A. caudata and Paralvinella grasslei. Presence of sulphur bacteria in cultures from Alvinellidae and their tubes (East Pacific Rise, 13 °N) at active sites "Totem" (A), and "Parigo" (B). + : bacterial growth observed; - : no bacterial growth observed; w: weak bacterial growth; nt: not tested. The designation numbers of the samples taken are shown in parentheses
Site Pressure (atm) T(°C) Thiosulphate oxidation Thiocyanate oxidation Na 2 S oxidation Sulphate reduction
A. pompejana
Tube,
A. caudata
(2) (2) (10)(29)
A. pompejana (30)
(1) (1) (23)(7)
B 250 80 nt +
B 1 20 + w nt . .
B 1 20 + nt
B 1 40
A B 250 250 20 80
w nt
nt
nt +
B 1 40 nt .
A 1 20 nt nt + .
Unidentified tubes
P. grasslei
(3) ( 3 ) ( 2 0 ) ( 2 0 ) ( 1 1 )
(22)
A A 250 250 20 80 + -
B 1 20 +
B i 40 -
B 1 20 +
B 1 40 -
A 250 20 +
A 1 40 -
nt
nt +
nt +
nt +
nt +
nt -
+
(23)
nt +
Table 2. Alvinella pompejana, A. caudata and Paralvinella grasslei. Presence of nitrogen bacteria in cultures from Alvinellidae and their tubes (East Pacific Rise, 13 °N). Further details as in legend to Table 1
Site Pressure (atm) T(°C) NH 4 oxidation NO 2 oxidation NO 3 respiration Denitrification Aerobic N2 fixation Anaerobic N 2 fixation
A. pompejana
Tube,
A. caudata
Unidentified tubes
P. grasslei
(2) (2) (10)(29)
A. pornpejana (30)
(1) (1) (7) (23)
(3) ( 3 ) ( 2 0 ) ( 2 0 )
(22)
B 1 20
B 1 40
A A 250 250 20 80
B i 20
B 1 40
B 1 20
B 1 40
A 1 40
+ -
+ +
+
-
+ + -
+ + +
+ +
+ nt
+
-
+ + -
B 1 20 +
B 1 40 +
B B 250 250 20 80
+ -
+ +
+ +
-
+
-
+
-
B 250 80 + -
Table 3. Alvinella pompejana, A. caudata and Paralvinella grasslei. Presence of non-specific heterotrophic bacteria in cultures from Alvinellidae and their tubes (East Pacific Rise, 13 °N). Site C: active site "Pogonord". TCBS: Kobayashi et al. (1963) medium. Further details as in legend to Table 1
A. pompejana
A. caudata
Tube,
A. pompejana
Site Pressure (atm) T (°C) Aerobic 2216E Anaerobic 2216E TCBS Amino acids Carbohydrates
Unidentified tubes
P. grasslei
(34)
(28)
(29)
(30)
(30)
(23)
(35)
(14)
(23)
(11)
(30)
(22)
C 1 20 + + nt nt +
B 1 20 + + + +
B 1 40 --
B 1 20 + + + + +
B 1 40 + + nt nt nt
A 1 20 + + +
C 1 20 + + nt nt nt
A 1 40 + nt nt + +
A 250 80 nt nt nt nt +
B 1 40 + nt nt + nt
B 1 40 nt nt + nt nt
A 1 40 -+ +
N o n - s p e c i f i c h e t e r o t r o p h i c b a c t e r i a ( T a b l e 3) E x c e p t f o r Paralvinella grasslei, a l m o s t all s a m p l e s c o n tained non-specific heterotrophic, aerobic or facultative anaerobic bacteria growing on 2216E medium. Cultures w e r e o b t a i n e d a f t e r i n c u b a t i o n a t 20 ° a n d 4 0 ° C f r o m Alvinella caudata a n d A . p o m p e j a n a t u b e s a n d f r o m u n i d e n t i f i e d t u b e s ; n o c u l t u r e w a s o b t a i n e d f r o m A. pompejana a t 4 0 ° C . I d e n t i c a l r e s u l t s w e r e a c h i e v e d u s i n g a m i n o acids or c a r b o h y d r a t e s as the sole c a r b o n source; in this case, positive responses were also recorded for P. grasslei s a m p l e s . O n e s a m p l e o f A. eaudata d i s p l a y e d
bacterial growth on the carbohydrate medium after incub a t i o n a t 80 ° C u n d e r " i n s i t u " p r e s s u r e . Vibrio-like b a c teria were only observed on TCBS medium from one s a m p l e o f a n A. pompejana t u b e , a f t e r i n c u b a t i o n a t 40 °C.
H e a v y - m e t a l - r e s i s t a n t b a c t e r i a ( T a b l e 4) H e t e r o t r o p h i c b a c t e r i a r e s i s t a n t t o silver, c o p p e r , c a d m i u m , z i n c a n d a r s e n a t e w e r e c u l t i v a t e d f r o m all t h e s a m p l e s o f Alvinella pompejana, A. caudata, a n d A. pompe-
364
D. Prieur et al. : Metabolic diversity in epibiotic microflora of Pompeii worms
Table 4. AIvinella pomejana, A. caudata and Paralvinella grasslei. Presence of heavy-metal-resistant and manganese-oxidising bacteria in cultures from Alvinellidae and their tubes (East Pacific Rise, 13 °N) Site C: active site "Pogonord". Further details as in legend to Table 1
A. pompejana
Site Pressure (atm) T (°C) Silver Copper Cadmium Zinc Arsenic Mn oxidation
A. caudata
Unidentified tubes
P. grasslei
(29) (34) (10)
Tube, A. pornpejana (30)
(35)
(14)
(20)
(11)
(22)
B 1 20 + + + + + +
B 1 20 + + + + + +
C 1 20 + + + + + +
A 1 40 . + +
B 1 40 + + + . + -
B 1 40 -
A 1 40 -
+
+ -
C 1 20 + + + + + +
A 1 40 -
Table 5. Alvinella pompejana and A. caudata. Ratios of colony-
forming units growing on 2216E medium and metal-supplemented media for Alvinellidae and their tubes (East Pacific Rise, 13 °N). Site C: active site "Pogonord". Further details as in legend to Table 1
A. pompejana Tube, A.pompe(29) (34) jana (35) Site Silver Copper Cadmium Zinc Arsenic
A. caudata Unidentifled tubes (30) (20)
B
C
C
B
B
55.5 11.1 7.7 12.2 38.8
31.8 28.3 0.8 23.8 32.7
54.3 56.1 9.6 52.1 42.4
44.3 55.6 6.1 84.5 15.4
0.08 0.04 0.4 nd 5.2
jana tubes incubated at 20 °C. N o culture was obtained from A. pompejana samples incubated at 40 °C. At this teInper.ature, on!y arsenate-resistant bacteria were cultivated from A, caudata, from unidentified tubes, and f r o m Paralvinella grasslei. In this latter species, no strain resistant to silver, copper, cadmium or zinc was detected. The relative abundance of heavy-metal-resistant bacteria was estimated by dividing the numbers of colony-forming units ( C F U ) growing on metal-amended media by the numbers of C F U growing of 2216E medium (Table 5). Cadmium-resistant bacteria were least represented, with 0.4 to 9.6% of C F U growing on 2216E medium. Excepted for unidentified tubes, silver, copper, zinc, and arsenate-resistant bacteria always accounted for > 10%, and often > 50% of the culturable heterotrophic microflora. H a l f of the culturable heterotrophic microflora from the body wall of A. caudata seemed resistant to the metals tested (except cadmium). Manganese-oxidizing bacteria were cultivated at 20°C from A. pompejana, tubes of A. pompejana and A. caudata, and at 40 °C from A. caudata and one sample of unidentified tubes. The ratio of manganese-oxidizing bacteria and heterotrophic bacteria growing on 2216E medium fluctuated between 24 and 67%.
Discussion and conclusions
The genera Alvinella and Paralvinella (polychaetous annelids) belong to the subfamily of Alvinellidae created by
.
.
Desbruy6res and Laubier (1980) within the family of Ampharetidae. These two genera have been described from deep-sea hydrothermal vents of the East Pacific Rise (Desbruy6res and Laubier 1980, 1982, 1986). The two Alvinella species, A. pompejana and A. caudata (Desbruy6res and Laubier 1986), build U-shaped multilayered proteinaceous tubes on the surface of zincsulphide diffusers and, in a few cases, on the walls of black smokers (Desbruy6res et al. 1985). Desbruy6res etal. (1983) reported that the tubes inhabited by the worms penetrate sufficiently deep into the walls of the chimneys as to come m contact with the hot fluids. The m a x i m u m temperature to which the Pompeii worms are exposed is not known, but they live in a temperature gradient between 20 ° and 40 °C, and are the most thermophilic invertebrates found in the vent biotopes (Desbruy6res et al. 1983, Gaill et al. 1987). They are exposed to a mixture of seawater and vent fluids and to a constant discharge of sulphide particles precipitating from the smoker fluids (Desbruy6res et al. 1983). The two Alvinella species bear a dense epibiotic microflora, composed of coccal, spiral-curved and rodshaped cells, and sheathed and unsheathed filaments, the latter constituting 60 to 80% of the epibiotic bacterial biomass (Desbruy~res et al. 1985, Gaill et al. 1987). A trophic or/and a detoxifying role of these microflora has been suggested, and the present study screened bacterial metabolisms that could be involved in such process(es). Paralvinella grasslei, a polychaete f r o m a neighbouring habitat but with no associated microflora was used as a control. Although dominant in term of biomass, the filamentous bacteria were not cultivated in thiosulphate-containing liquid medium nor in sulphide gradients• This does not necessarily mean that the filaments observed in earlier studies are not sulphide-oxidizing bacteria. Few marine filamentous sulphide-oxidizing bacteria have been successfully cultivated, and large cells similar to those associated with Alvinella spp., have never been obtained in earlier culture (Nelson personal communication). Certainly, culture assays from coastal vents where similar filamentous bacteria occur (Jacq et al. 1989) would be useful for improve culturing methods. However, although limited to a fraction of the microflora, the results obtained here confirm the metabolic
D. Prieur et al. : Metabolic diversity in epibiotic microflora of Pompeii worms
diversity of the epibiotic bacteria of Alvinella spp., as previously suggested by Alayse-Danet et al. (1986). Both heterotrophic and autotrophic bacteria were cultivated, confirming the occurrence of these two metabolic types within the microflora. Non-specific heterotrophic bacteria, both aerobic and facultative anaerobic, were cultivated from almost all the samples studied. However, presumptive vibrios were found only in tube samples, in agreement with previous data of Prieur and Jeanthon (1987), who found very few fermentative strains associated with A. pompejana samples. At this stage, it is not possible to determine the complete and exact role of the heterotrophic component of the microflora, and further biochemical and nutritional characterizations of the numerous strains obtained in pure culture are needed. One specimen of Paralvinella grasslei was included in the sample analysed. This species does not have an epibiotic microflora and, in contrast to Alvinella spp. specimens, few cultures, even of heterotrophic bacteria, were obtained from this species. These results agree with previous SEM observations, and this would mean that collection by a submersible does not induce significant contamination. The heterotrophic microflora appeared to be adapted to the high concentrations of heavy metals to which the worms are exposed. Recent experiments by Jeanthon and Prieur (1990) indicated that 71% of the strains studied displayed multi-resistance to the metals tested. However, in order to play a role in detoxification, as suggested by several authors, these bacteria may have the ability to concentrate metals. Such ability has been previously noted for aquatic and marine bacteria (see Belliveau et al. 1987 for review), e.g. the concentration of chromium by epiphytic bacteria of the crab Helice crassa (Johnson et al. 1981). Experiments on metal concentration by selected isolates are under progress in our laboratory. Manganese has been found to be toxic to several bacterial species (Hajj and Makemson 1976); in vent fluids, concentrations up to 206/~M1-1 have been measured (Baross et al. 1982). Enrichment cultures indicated that 24 to 67% of the heterotrophic microflora cultivated during the cruise "Hydronaut" was able to oxidize manganese. By this process, heterotrophic microflora could transform this metal into an oxidized form, probably less toxic to the worm. Sulphur-oxidizing bacteria were found in almost all samples; this is not surprising considering the primordial role of sulphide oxidation in the hydrothermal ecosystem (Jannasch and Wirsen 1979). Autotrophic, but mainly mixotrophic, strains were isolated in pure culture, this result being also in agreement with those of Ruby et al. (1981). By oxidizing sulphide, sulphur-oxidizing bacteria could certainly detoxify the microenvironment of the worm. However, a part of the associated microflora is able to produce hydrogen sulphide. In a previous study, Prieur and Jeanthon (1987) noted that most of the heterotrophic bacteria associated with Alvinella pompejana could produce hydrogen sulphide from cysteine. In the present study, sulphate reducers, which also produce hydrogen sulphide, were found in Alvinella spp. and tube samples, suggesting some metabolic exchanges between
365
the two components (sulphur-oxidizing and sulphatereducing) of the sulphur bacteria. Bacteria involved in the nitrogen cycle have rarely been reported in previous studies of deep-sea vents (Lilley et al. 1983). The results obtained here appear rather unbalanced. While nitrate-respiring and denitrifying bacteria occurred in almost all the samples, nitrifying bacteria were cultivated from only three samples. Preliminary assays on denitrification activity performed in our laboratory indicated rapid nitrogen production. Since nitrogen fixation was rather low (three positive samples), these results could indicate a possible loss of nitrogen to the worm ecosystem. However, other preliminary tests showed that 45% of the denitrifying bacteria were capable of assimilative reduction of nitrate and, therefore, of active participation in the synthesis of organic nitrogen. The results presented clearly indicate that the microflora associated with Alvinella pompejana and its tube, and with A. caudata and unidentified tubes was certainly more complex than previously suspected. Both aerobic and anaerobic metabolic processes, usually confined to different redox conditions, were detected. Two explanations are suggested: (1) because of the high density of bacteria living on the worm and tube surface, microanaerobic zones could exist within an oxygenated environment, as previously reported for several types of biofilms (Sieburth 1987). (2) The metabolic processes could arise from exposure to successive contrasting environmental conditions arising from vent activity and consequent fluctuations in the hydrothermal fluid temperatures. Recent measurements of "in situ" temperature (Desbruy~res et al. unpublished data) revealed fluctuations in the range of 10 to 20 C ° within a few seconds. The fact that true denitrification (with nitrogen production) was more frequent at 40°C than at 20°C, and that sulphate reduction in A. pompejana and its tube occurred only at 80 °C, favours the second hypothesis. The occurrence of bacteria growing at 80 °C cultivated from samples of A. pompejana and its tube also confirmed that the worms are periodically exposed to increases in temperature of such amplitude that they are forced to leave their tubes (Desbruy6res personal communication). Growth of sulphate-reducing bacteria at 80 °C is also in agreement with Baross and Deming (1985) and Desbruy~res et al. (1985), who reported that Alvinella spp. tubes were cemented to smoker walls by sulphide deposits. However, although the data obtained confirm the occurrence of autotrophic and heterotrophic metabolisms within the epibiotic microflora of the two species of Alvinella, quantitative data are needed to really understand the role of the different components of the microflora. The recent report of similar bacterial associations on an annelid (Halicryptus spinulosus) from coastal environments, (Oeschger and Schmaljohann 1988) could be of great value in facilitating (because of its accessibility) experiments and cultures, prior to further deep-sea vent studies. Acknowledgements. The authors thank A.-M. Alayse-Danet for inviting three of them to participate in the cruise "Hydronaut"
366
D. Prieur et al. : Metabolic diversity in epibiotic microflora of Pompeii worms
organized by IFREMER. Thanks also to Dr. D. Desbruy+res and Dr. E Gaill for their critical review of the manuscript, and to C. Faidy and A. Marhic, for their technical assistance. This work was supported by IFREMER (Grant 88.2.470219), CNRS (GDR "Ecoprophyce") and PNEHO.
Literature cited Abd E1 Malek, Y., Rizk, S. G. (1958). Counting of sulphate reducing bacteria in mixed bacterial populations. Nature, Lond. 182: p. 538 Adair, F. W, Gundersen, K. (1969). Chemoautotrophic sulfur bacteria in the marine environment: isolation, cultivation and distribution. Can. J. Microbiol. 15:345-353 Alayse-Danet, A. M. (1988). Rapport prrliminaire des recherches effectures pendant la campagne "Hydronaut". Ifremer/DERO/ EP (Drpartement Etudes et Recherche Ocraniques/Environnement Profond), Brest, France Alayse-Danet, A. M., Gaill, F., Desbruy~res, D. (1986). In situ bicarbonate uptake by bacteria-Alvinella associations. Mar. Ecol. 7:233-240 Belliveau, B. H., Starodub, M. E., Cotter, C., Trevors, J. T. (1987). Metal resistance and accumulation in bacteria. Biotech. Adv. 5: 101-127 Baross, J. A., Deming, J. W (1985). The role of bacteria in the ecology of black smoker environments. Bull. biol. Soc. Wash. 6: 355-371 Baross, J. A., Lilley, M. D., Gordon, L. I. (1982). Is the CH4, H 2 and CO venting from submarine hydrothermal systems produced by thermophilic bacteria? Nature, Lond. 298:366-368 Cavanaugh, C. M:, Gardiner, S. L., Jones, M. L., Jannasch, H. W, Waterbury, J. B. (1981). Prokaryotic cells in the hydrothermal vent tube worm Riftia pachyptila Jones: possible chemoautotrophic symbionts. Science, N.Y. 213:340-342 Chamroux, S. (1972). Etude des bactrries marines rrduisant le nitrate. 1: Isolement. Annls Inst. Pasteur, Paris 122:475-481 Cosson-Mannevy, M. A., Cosson, R., Gaill, E (1986). Mise en 6vidence de protrines de type mrtallothion6ine chez deux invertrbrrs des sources hydrothermales, le pogonophore vestimentifrre Riftia pachyptila et l'annrlide polychrte Alvinella pompejana. C. r. hebd. Sranc. Acad. Sci., Paris 302:347-352 Desbruyrres, D., Crassous, P., Crassle, J., Khripounoff, A., Reyss, D., Rio, M., Van Praet, M. (1982). Donn6es 6cologiques sur un nouveau site d'hydrothermalisme actif de la ride du Pacifique oriental. C. r. hebd. Sranc. Acad. Sci., Paris 295:489-494 Desbruyrres, D., Gaill, E, Laubier, L., Fouquet, Y. (1985). Polychaetous annelids from hydrothermal vent ecosystem: an ecological review. Bull. biol. Soc. Wash. 6:103-116 Desbruy+res, D., Gaill, F., Laubier, L., Prieur, D., Rau, G. H. (1983). Unusual nutrition of the "Pompeii worm" Alvinella pompejana (polychaetous annelid) from a hydrothermal vent environment: SEM, TEM, 13C and lSN evidence. Mar. Biol. 76:201-205 Desbruyrres, D., Laubier, L. (1980). Alvinella pompejana gen. sp. nov., Ampharetidae aberrant des sources hydrothermales de la ride Est-Pacifique. Oceanol. Acta 3:267-274 Desbruyrres, D., Laubier, L. (1982). Paralvinella grasslei, new genus, new species of Alvinellidae (Polychaeta: Ampharaetidae) from the Galapagos rift geothermal vents. Proc. biol. Soc. Wash. 95:484-494 Desbruyrres, D., Laubier, L. (1986). Les Alvinellidae, une famille nouvelle d'ann~lides polych~tes infrodres aux sources hydrothermales sous marines: systrmatique, biologic et 6cologie. Can. J. Zool. 64:2227-2245 Felbeck, H., Somero, G. N. (1982). Primary production in deep-sea hydrothermal vent organisms: role of sulfide-oxidizing bacteria. Trends biochem. Sciences 7:201-204 Fiala-Mrdioni, A. (1984). Mise en +vidence par microscopic 61ectronique /t transmission de l'abondance de bactrries symbio-
tiques dans la branchie de mollusques bivalves de sources hydrothermales profondes. C. r. hebd. Sranc. Acad. Sci., Paris 298:487-492 Gaill, F., Desbruy+res, D., Prieur, D. (1987). Bacterial communities associated with "Pompeii worms" from the East Pacific Rise hydrothermal vents: SEM, TEM observations, Microb. Ecol. 13:129-139 Gaill, E, Desbruy~res, D., Prieur, D., Gourret, J. P. (1984a). Mise en ~vidence de communaut~s bact~riennes 6pibiontes du "ver de Pompri" (Alvinella pompejana). C. r. hebd. Sranc. Acad. Sci., Paris 298:553-558 Gaill, E, Halpern, S., Quintana, C., Desbruyrres, D. (1984b). Prrsence intracellulaire d'arsenic et de zinc associ6s au soufre chez un Polychrte des sources hydrothermales. C. r. hebd. Sranc. Acad. Sci., Paris 298:331-335 Hajj, H., Makemson, J. (1976). Determination of growth of Sphaerotilus discophorus in the presence of manganese. Appl. envirl Microbiol. 32:699-702 Jacq, E., Prieur, D., Nichols, P., White, D. C., Porter, T., Geesey, G. G. (1989). Microscopic examination and fatty acid characterization of filamentous bacteria colonizing substrata around subtidal hydrothermal vents. Archs Microbiol. 152:64-71 Jannasch, H. W., Wirsen, C. O. (1979). Chemosynthetic primary production at East Pacific sea floor spreading centers. BioSci. 29:592-598 Jannasch, H. W, Wirsen, C. O. (1981). Morphological survey of microbial mats near deep-sea thermal vents. Appl. envirl Microbiol. 41:428-538 Jeanthon, C., Prieur, D. (1990). Resistance to heavy metals of heterotrophic bacteria isolated from the deep-sea hydrothermal vent polychaete Alvinellapompejana. Prog. Oceanogr. (in press) Johnson, I., Flower, N., Loutit, M. W (1981). Contribution of periphytic bacteria to the concentration of chromium in the crab Helice crassa. Microb. Ecol. 7:245-252 Kobayashi, T., Enomoto, R., Sakazaki, R., Kuwuhara, S. (1963). A new selective isolation medium for pathogenic Vibrios: TCBS agar. Jap. J. Bact. 18:387-391 Laubier, L., Desbruy~res, D., Chassard-Bouchaud, C. (1983). Microanalytical evidence of sulfur accumulation in a polychaete from deep-sea hydrothermal vents. Mar. Biol. Lett 4:113-116 Le Pennec, M., Prieur, D. (1984). Observations sur la nutrition d'un Mytilidae d'un site d'hydrothermalisme actif de la dorsale du Pacifique oriental. C. r. hebd. Sranc. Acad. Sci., Paris 298: 493 -498 Lewis, R. E, Pramer, D. (1958): Isolation of Nitrosomonas in pure culture. J. Bact. 76:524-528 Lilley, M. D., Baross, J. A., Gordon, L. I. (1983). Reduced gases and bacteria in hydrothermal fluids: the Galapagos spreading center and 21°N East Pacific Rise. In: Rona, P. A., Brstrom, K., Laubier, L., Smith, K. L., Jr. (eds.) Hydrothermal processes at sea floor spreading centers. Plenum Press, New York, p. 411419 Mevel, G. (1986). Contribution /t l'~tude de la nitrification dans l'eau et le srdiment d'~cosystrmes littoraux et estuariens. Etude in situ et exprrimentale. Thrse doctorat d'Etat. Universit6 de Brest Nealson, K. H. (1978). The isolation and characterization of marine bacteria which catalyse manganese oxidation. In: Krumbein, W E. (ed.) Environmental biogeochemistry and geomicrobiology. Vol. 3. Ann Arbor Science Publishers Inc., Ann Arbor, Michigan, p. 847-858 Nelson, D. C., Jannasch, H. W (1983). Chemoautotrophic growth of a marine Beggiatoa in sulfide-gradient cultures. Archs Microbiol. 136:262 269 Oeschger, R., Schmaljohann, R. (1988). Association of various type of epibacteria with Halicryptus spinulosus (Priapulidae). Mar. Ecol. Prog. Set. 48:285-293 Oppenheimer, C. H., Zobell, C. E. (1952). The growth and viability of sixty-three species of marine bacteria as influenced by hydrostatic pressure. J. mar. Res. 11:10-18
D. Prieur et al. : Metabolic diversity in epibiotic microflora of Pompeii worms Postgate, J. R. (1966). Media for sulphur bacteria. Lab. Pract. 15: 1239-1244 Postgate, J. R. (1972). The acetylene reduction test for nitrogen fixation. In: Norriss, J. R., Ribbons, P. W. (eds.) Methods in microbiology. Vol. 6B. Academic Press, London, p. 343-356 Prieur, D., Jeanthon, C. (1987). Preliminary study of heterotrophic bacteria isolated from two deep-sea hydrothermal vent invertebrates: Alvinella pompejana (polychaete) and Bathymodiolus thermophilus (bivalve). Symbiosis 4:87-98 Ruby, E. G., Wirsen, C. O., Jannasch, H. W. (1981). Chemolithotrophic sulfur-oxidizing bacteria from the Galapagos rift hydrothermal vents. Appl. envirl Microbiol. 42: 317324 Sieburth, J. McN. (1975). Microbial seascapes. University Park Press, Baltimore, Md
367
Sieburth, J. McN. (1987). Contrary habitats for redox-specific processes: methanogenesis in oxic waters and oxidation in anoxic waters. In: Sleigh, M. A. (ed.) Microbes in the sea. Ellis Horwood Ltd., Chichester, p. 11-31 Tuttle, J. H. (1985). The role of sulfur-oxidizing bacteria at deep-sea hydrothermal vents. Bull. biol. Soc. Wash. 6:335-343 Tuttle, J. H., Jannasch, H. W. (1972). Occurrence and type of Thiobacillus-like bacteria in the sea. Limnol. Oceanogr. 17: 532-543 Tuttle, J. H., Wirsen, C. O., Jannasch, H. W. (1983). Microbial activities in the emitted hydrothermal waters of the Gal/tpagos Rift vents. Mar. Biol. 73:293-299
