method of detecting bacteria in bivalve gills

8/7/2019 Method of detecting Bacteria in Bivalve gills

1/4

Notes 1373SAMUELSSON, G., AND G. OQUIST. 1980. Effects ofcopper chloride on photosynthetic electron trans-port and chlorophyll-protein complexes of Spi-nacia oleracea. P lant Cell Physiol. 21: 445-454.SELLNER, K. G., L. LYONS, E. S. PERRY, AND D. B.

HEIMARK. 1982. Assessing physiological stressin Thalassiosira jluviatilis (Bacillariophyta) andDunaliella tertiolecta (Chlorophyta) with DCMU-enhanced fluorescence. J. Phycol. 18: 142-148.SHARP, J. H., M. J. PERRY, E. H. RENGER, AND R. W.EPPLEY. 1980. Phytoplankton rate processes inthe oligotrophic waters of the central North Pa-cific. J. Plankton Res. 2: 335-353.SHELDON, R. W. 1984. Phytoplankton growth ratesin the tropical ocean. Limnol. Oceanogr. 29: 1342-1346.SHIOI, Y., H. TAMAI, AND T. SASA. 1978. Inhibitionof photosystem II in the green alga Ankistrodesmusfalcatus by copper. Physiol. Plant. 44: 434-438.SHULENBERGER,E., AND J. L. REID. 198 1. The Pacificshallow oxygen maximum, deep chlorophyll max-imum, and primary productivity, reconsidered.Deep-Sea Res. 28: 90 l-9 19.STEEMANN NIELSEN, E., L. I&IMP-NIELSEN, AND S.WIUM-ANDERSEN. 1969. The effect ofdeleteriousconcentrations of copper on the photosynthesis ofChlorella pyrenoidosa. Physiol. Plant. 22: 112 l-1133.AND S. WIUM-ANDERSEN. 1970. Copper ionsaspoison in the sea and freshwater. Mar. Biol. 6:93-97.STRICKLAND, J. D., AND T. R. PARSONS. 1972. A prac-tical handbook for seawater analysis, 2nd ed. Bull.Fish. Res. Bd. Can. 167.

TUSSEN, S. B. 1979. Diurnal oxygen rhythm and pri-mary production in the mixed layer of the AtlanticOcean at 2ON. Neth. J. Sea Res. 13: 79-94.TRIPATHY, B. C., AND P. MOHANTY. 1980. Zinc-in-hibited electron transport of photosynthesis in iso-lated barley chloroplasts. Plant Physiol. 66: 1174-1178.VENRICK, E. L., J. R. BEERS, AND J. H. HEINBOKEL.1977. Possible consequences of containing mi-croplankton for physiological rate measurements.J. Exp. Mar. Biol. Ecol. 26: 55-76.VINCENT, W. F. 1980. Mechanisms of rapid photo-synthetic adaptation in natural phytoplanktoncommunities. 2. Changes in photochemical ca-pacity as measured by DCMU-induced chloro-phyll fluorescence. J. Phycol. 16: 568-577.-, P. J. NEALE, AND P. J. RICHERSON. 1984. Pho-toinhibition: Algal responses to bright light duringdiel stratification and mixing in a tropical alpinelake. J. Phycol. 20: 201-2 11.WELSCHMEYER, N. A., AND C. J. LORENZEN. 1985.Chlorophyll budgets: Zooplankton grazing andphytoplankton growth in a temperate fjord andthe Central Pacific Gyres. Limnol. Oceanogr. 30:1-21.WOOD, A. M. 1983. Available copper ligands and theapparent bioavailability of copper to natural phy-toplankton assemblages. Sci. Total Environ. 28:5 l-64.

Submitted: 11 April 1985Accepted: 10 April 1986

Limnol. Oceanogr.. 31(6), 1986, 1373-13760 1986, by the American Society of Limnology and Oceanography, Inc.

A simple method to detect bacterial associations in bivalve gillsAbstract-Recent discoveries have shown thatendosymbiotic bacteria may be widespread amongbivalve species in different habitats. A simplemethod is described which allows the detectionof bacterial-gill association with an ordinarylight microscope.

The discovery of a rich fauna in the vi-cinity of hydrothermal vents raised severalimportant questions about food chain re-lationships at great depths. A symbiotic as-sociation was first shown between chemo-lithotrophic sulfur-oxidizing bacteria andthe gutless worm Riftia pachyptila Jones,one of the most abundant species of this

vent fauna (Cavanaugh et al. 198 1; Felbeck198 1; Southward et al. 198 1). Later, twolarge species of bivalve Mytilidae were foundto carry similar bacteria in their gill tissue(Cavanaugh 1983; Fiala-Medioni 1984; LePennec and Hily 1984; Le Pennec and Prieur1984). These chemolithotrophic bacteria arethought to constitute a major source of theenergy and nutrition of their hosts (cf. Jan-nasch 1985). Symbiotic bacteria and pos-sible chemosynthetic capabilities have alsobeen described for animals living in shallowsulfide-rich environments. These includegroups such as oligochaetes (Phallodrilusleukodermatus and Phallodrilus planus:


2/4


3/4

Notes 137510. Stain for 10 min in freshly preparedGiemsa solution: 4 ml of Giemsa (Merck,Art. 9204); 4 ml of phosphate buffer 0.2 M;pH 6.8; 92 ml of distilled water.11. Wash in water for 5 min and air-dry.The preparation can be viewed immedi-ately with the light microscope. For highmagnification (X 100 optics), immersion oilis placed directly on the dried droplet with-out need of a cover glass. If wanted, the slidecan be made permanent: keep 10 min inxylol and mount in Euparal (or another res-in).This procedure yields preparations of dis-rupted cells at the periphery of the dropsites. Giemsa stains DNA of nuclei and bac-teria. Results with S. subtruncata show ineach droplet of gill cell suspension manyassemblages of bacteria-shaped organelles.We have used a minimum of 10 animals(each gill gave one droplet of cell suspen-sion). We also tested mantle and foot tissuefrom S. subtruncata, and gill tissues fromother bivalves (Mytilus edulis, Mytilus gal-loprovincialis, Ostrea edulis, Crassostrea gi-gas, Ruditapes philippinarum, Venus galli-na); bacteria-shaped organelles were notobserved. The results with S. subtruncataare shown in Fig. 1. Large numbers of bac-teria-shaped organelles were confirmed byTEM to be Gram-negative bacteria (Bouvyet al. 1986) which had been released fromtheir packed assemblages (Fig. 1A). Two dif-ferent types of bacteria are present, one rod-shaped (about 3 x 0.6 pm) (Fig. 1B) and theother shorter and wider (about 2 x 1 pm)(Fig. 1C).

It is also possible to use the Gram methoddirectly with fresh tissue. The procedureused is as above except that step 5 is omittedand after air-drying, the preparation isGram-stained. Gram-negative bacteria werefound in S. subtruncata (Bouvy et al. 1986).The method can be useful in a systematicsearch for bacterial associations among bi-valves and could also be used to detect sur-face bacteria as well as bacteria in other tis-sues of other species. However, it should beconsidered as a first step to detect bacteria-shaped organelles; further TEM techniquesshould be used to confirm the characteris-tics of procaryotic cells. After that, enzy-matic assays, chemical analyses, carbon

dioxide fixing, nitrogen fixing, etc. are nec-essary to determine metabolic pathways andactual symbiotic relationships.C. Thiriot- Qui&reuxStation Zoologique06230 Villefranche-sur-MerFrance

J. SayerLaboratoire Arago66650 Banyuls-sur-MerFrance

ReferencesBERG, C. J., AND P. ALATOLO. 1984. Potential che-mosynthesis in molluscan mariculture. Aquacul-ture 39: 165-179.Bouw, M., AND OTHERS. 1986. Sur la presence debatteries dans la branchie dun Mollusque Bivalvelittoral Spisula subtruncata (Da Costa). C.R. Acad.Sci. Paris. 303: 257-262.CAVANAUGH, C. M. 1983. Symbiotic chemoauto-trophic bacteria in marine invertebrates from sul-phide habitats. Nature 302: 58-61.-, S. L. GARDINER, M. L. JONES, H. W. JANNASCH,AND J. B. WATERBURY. 198 1. Prokaryotic cells

in the hydrothermal vent tube worm Riftia pa-chyptila Jones: Possible chemoautotrophic sym-bionts. Science 213: 340-342.DANDO, P. R., E. C. SOUTHWARD, N. B. SOUTHWARD,N. B. TERWILLIGER, AND R. C. TERWILLIGER. 1985.Sulphur-oxidizing bacteria and haemoglob in in gillsof the bivalve mollusc Myrtea spinifera. Mar. Ecol.Progr. Ser. 23: 85-98.FELBECK, H. 198 1. Chemoautotrophic potential ofthe hydrothermal vent tube worm Riftia pachyp-tilu Jones (Vestimentifera). Science 213: 336-338.-. 1983. Sulfide oxidation and carbon fixationby the gutless clam Solemya reidi: An animal-bacteria symbiosis. J. Comp. Physiol. 152: 3-l 1.-, G. LIEBEZEIT, R. DAWSON, AND 0. GIERE. 1983.CO, fixation in tissues of marine oligochaetes(Phallodrilus leukodermatus and P. planus) con-taining symbiotic chemoautotrophic bacteria. Mar.Biol. 75: 187-189.FIALA-MEDIONI, A. 1984. Mise en evidence par mi-croscopie electronique a transmission de labon-dance de batteries symbiotiques dans la branchiede Mollusques Bivalves de sources hydrother-males profondes. C.R. Acad. Sci. Paris 298(3): 487-492.FISHER, R. M., AND S. C. HAND. 1984. Chemoau-totrophic symbionts in the bivalve Lucinaflori-dana from sea grass beds. Biol. Bull. 167: 445-459.GIERE, 0. 198 1. The gutless marine oligochaete Phal-lodrillus leukodermatus. Structural studies on anaberrant tubificid associated with bacteria. Mar.Ecol. Progr. Ser. 5: 353-357.


4/4

1376 NotesJANNASCH, H. W. 1985. The chemosynthetic supportof life and the microbial diversity at deep-sea hy-drothermal vents. Proc. R. Sot. Lond. Ser. B 225:277-297.LE PENNEC, M., AND A. HILY. 1984. Anatomie, struc-ture et ultrastructure de la branchie dun Mytilidaedes sites hydrothermaux du Pacifique oriental.Oceanol. Acta 7: 5 17-524.-, AND D. PRIEUR. 1984. Observations sur lanutrition dun Mytilidae dun site hydrothermalactif de la dorsale du Pacifique oriental. C.R. Acad.Sci. Paris 298(3): 493-498.OTT, J., G. RIEGER, R. RIEGER, AND F. ENDERES. 1982.New mouthless interstitial worms from the sulfidesystem: Symbiosis with prokaryotes. Mar. Ecol.(Pubbl. Sta. Zool. Napoli 1) 3: 313-333.

POWELL, M. A., AND G. N. SOMERO. 1985. Sulfideoxidation occurs in the animal tissue of the gutlessclam, Solemya reidi. Biol. Bull. 169: 164-l 8 1.SOUTHWARD, A. J., AND OTHERS. 198 1. Bacterial sym-bionts and low 12V3C ratios in tissues of Pogo-nophoru indicate unusual nutrition and metabo-lism. Nature 293: 616-620.VETTER, R. D. 1985. Elemental sulfur in the gills ofthree species of clams containing chemoauto-trophic symbiotic bacteria: Possible inorganic en-ergy storage compound. Mar. Biol. 88: 33-42.

Submitted: 11 February 1986Accepted: 2 July 1986

Limnol. Oceanogr., 3 l(6). 1986, 1376-13830 1986, by the American Society of Limnology and Oceanography, Inc.

A simple fiber-optic microprobe for high resolution lightmeasurements: Application in marine sediment lAbstract -A fiber-optic microprobe is de-

scribed which is inexpensive and simple to buildand use. It consists of an 80-pm optical fiberwhich at the end is tapered down to a roundedsensing tip of 20-30-km diameter. The detectoris a hybrid photodiode/amplifier. The probe hasa sensitivity of 0.0 1 PEinst m-2 s-l and a spectralrange of 300-1,100 nm. Spectral light gradientswere measured in fine-grained San Francisco Baysediment that had an undisturbed diatom coatingon the surface. The photic zone of the mud wasonly 0.4 mm deep. Measured in situ spectrashowed extinction maxima at 430-520,620-630,670, and 825-850 nm due to absorption by chlo-rophyll a, carotenoids, phycocyanin, and bacte-rio-chlorophyll a. Maximum light penetration inthe visible range was found in both the violet andthe red at 5 400 and 2 700 nm.

Most of our knowledge about light dis-tribution and microalgal photosynthesis inaquatic ecosystems is based on studies inthe water column. In many lakes and coastalmarine environments, however, benthicmicroalgae contribute significantly to pri-mary production (e.g. Hunding 197 1; Gar-

I Supported by a research fellowshiptional Research Council to B.B.J. from the Na-

gas 197 1; Cadee and Hegeman 1974; Hart-wig 1978). The benthic systems differ fromthe plankton in certain respects which makethe study of microbenthic photosynthesisand its regulation by light experimentallydifficult: the photic zone is very narrow insediments, light scattering is strong, andmany benthic microalgae migrate verticallyin response to changing environmental con-ditions such as light.Earlier techniques for measuring light insediments have not been able to resolve thespectral light gradients with a high spatialresolution. Light penetration has been stud-ied by covering a large light sensor with mil-limeter-thin layers of sediment or benthicmicroalgae (e.g. Hoffmann 1949; Taylor1964; Taylor and Gebelein 1966; Jorgensenet al. 1979; Haardt and Nielsen 1980) or byinserting a small light probe into the sedi-ment (Fenchel and Straarup 1971). Only inthe leaves of higher plants have light mea-surements recently been made on a verysmall scale by the use of fiber-optics (Vo-gelmann and BjSrn 1984).Since the development of microelectrodetechniques for the measurement of chemicalgradients and of photosynthesis at I lOO-

method of detecting bacteria in bivalve gills

Documents