Microb. Ecol. 7:13-21 (1981)
IYIICI BI IL ECOI.OGV
Specificity of Bacterial Symbionts in Mediterranean and Great Barrier Reef Sponges* Clive R, Wilkinson, 7 , , Madeleine Nowak, i Brian Austin, 2.** and Rita R. Colwell 2 1Laboratoired'Histologieet BiologieTissulaire,Universit6ClaudeBernard,Villeurbanne69622, France;and 2Departmentof Microbiology,Universityof Maryland, CollegePark, Maryland20742, USA
Abstract. Bacteria were isolated from marine sponges from the Mediterranean and the Great Barrier Reef and characterized using numerical taxonomy techniques. A similar sponge-specific bacterial symbiont was found in 9 of 10 sponges examined from both geographic regions. This symbiont occurred in sponges of two classes and seven orders, and it probably has been associated with sponges over a long geological time scale. Another symbiont apparently specific to the sponge Verongia aerophoba was found. This sponge is yellow-orange, similar in color to the bacterial symbiont. These symbionts are two of a large mixed bacterial population present in many sponges.
Bacterial symbionts are common within many marine sponges. The granular material observed in the intercellular collagenous matrix (the mesohyl) of sponges was proved to be bacteria by Levi and Levi (7) using an electron microscope. Additional electron microscope studies have been made on the morphology of these bacteria and their relationship to sponge cells (13, 14, 17). The common conclusion drawn from these studies was that there 'are many morphologically different bacteria in the mesohyl of marine sponges. In two previous studies on bacteria isolated from temperate water sponges, no bacteria specific to the host sponges were detected; moreover, the bacteria isolated were similar to those in the ambient water (1, 10). These studies were limited in that the bacteria were characterized using several diagnostic tests only, and in one of the studies the bacterial populations in the sponge and particularly the ambient water were unrealistically low (10), During a study of four coral reef sponges, Wilkinson (16) isolated large numbers of bacteria from the sponges and the ambient water and characterized them by numerical taxonomy methods. In three of the sponges there was a common facultative anaerobic
*This paperconstitutes No. V in the seriesMicrobialAssociationsin Sponges. **Present address: Australian Instituteof Marine Science, PMB 3, Townsville,M.S.O., Queensland, 4810, Australia. ***Presentaddress: Fish DiseasesLaboratory,Weymouth,DorsetDT4 8UB, England, 0095-3628/81/0007-0013 $01.80 1981 Springer-VcrlagNew YorkInc.
14
c.R. Wilkinsonet al.
bacterial strain which was not present in the ambient water. These bacteria may not be the only sponge-specific symbionts; e.g., Eimhjellen (3) and Imhoff and T m p e r (6) have isolated phototrophic anaerobic bacteria from other marine sponges. Bacteria were isolated from sponges and the ambient water in the Mediterranean Sea to determine whether the bacteria that were specific to Great Barrier Reef sponges (16) were more widely distributed. These bacterial isolates were characterized in order to determine if ecological or metabolic groupings occurred within sponges. Numerical taxonomy methods were used to compare those bacteria isolated from Mediterranean sponges and seawater with selected strains previously isolated by Wilkinson (16) from Great Barrier Reef sponges.
Materials and Methods Sponges were collectedfrom rockyreefs adjacentto Marseiile, France[for detailssee Wilkinsonand Vacelet (20)]. Bacteria were isolatedfrom six sponges:Chondrosia reniformis, lrcinia variabilis, Petrosiaficiformis, Verongia cavernicola, V. aerophoba, and ChondriUa nucula. Two methods were used to isolate bacteria: in the fast, approximately 5 g of sponge tissue was blended in 50 ml of sterile seawater, diluted in sterile seawater, and platedby pipelling 1ml of 10- 3 and 10-4 suspensionsintosterilePetfi dishesand adding 15 ml of moltenOZR agar at 37~C(15). Ambientwater sampleswere treatedsimilarly.In the secondmethod, small blocks of sponge tissue, approximately 1 cm3, were cut fromthe interiorof the spongeusingalcohol-flamed scalpel blades and smearedover dried plates ofOZR agar. Followingincubationat 2 I~ for approximately 14 days, between 20 and 25 colonieswere isolatedrandomlyfrom the pour-platesand an additional10 colonies were picked from the smeared plates(Table 1). A set of 15 strainsrepresentingthe majorclustergroupingsof bacteria from four Great BarrierReef sponges(t6) was includedin the studyfor comparison(Table 1). In additionto characterizationtests describedpreviouslyfor coral reefsponges(16), the followingtests were used: cytochrome oxidase and indole (2); collagendigestion--a piece of membraneof pure collagenfibrils from Chondrosia reniformis [pp. 37-38 in Garrone (5)] was added to a low-nutrientbroth of 0.05% yeast extract and 0.02% casamino acids in artificial seawater and examined for 14 days for breakdown of the membrane (19); ability to grow in minimal medium plus carbon source and requirementfor addition of 0.0025% yeast extract and casaminoacids(Table2). A total of 54 characters, includingmorphology,growth characteristics, physiology, and metabolic properties, were coded for 195 bacteria from Mediterranean sponges, 32 from ambientwater, and 15 fromGreat BarrierReef sponges. These 242 strains were analyzed using simplematchingcoefficientcomparisons(SsM---positiveand negativematches)and Jaccard coefficient comparisons (Sj---positive matchesonly) and clusteredusingan unweightedaveragelinkage(12). Programs employed includedTAXAN6, UMDTAXON3, and IGPS3programpackages on the Universityof Maryland UNIVAC 1108computer.
Results Analyses obtained with the two coefficients, SsM and S j , were identical and the clusters formed with the SsM coefficient are considered here (Fig. 1). Eight clusters formed, with groups 1, 2, 4, and 8 clustering at or above 80% similarity (S) level. These four groups contained 122 strains (50.4% of the total). Three other groups (3, 6, and 7) plus a subgroup (1 a) clustered at lower levels of similarity (at or above 70%S) and contained 32 strains (13% o f the total). The interrelationships of these groups are given in Fig. 2. The largest cluster, group 1 (at or above 80%S) contained 108 strains. This was 44.6% o f all strains or 78.2% of the facultative anaerobic strains. Strains from this group were isolated exclusively from sponges and comprised the largest group within all six Mediterranean sponges (Fig. 3). This group also contained 9 of the I0 bacteria from the
Bacterial Symbionts in Sponges
15
Table 1. List of bacterial strains used in this study showing the source of strains, numbers (in parentheses), and strain numbers Mediterranean sponges
Strain Nos.
Great Barrier Reef sponges
Ra
Chondrosia reniformis
(36) R 1-R36
P
Pericharax heteroraphis
V
Ircinia variat~lis
(36) V 1-V36
J
Jaspis stellifera
F C
Petrosiaficiformis Verongia cavernicola
(30) F1-F30 (34) C 1-C34
G
Neofibalaria irata
A
Verongia aerophoba
(29) A 1-A29
I
Ircinia wistarii
N
Chondrilla nucula ambient water
(30) N1-N30
W
Strain Nos. (4) P25, P57 P85, P90 (3) J 12, J28 J69 (4) G65, G66 G72, G89 (4) I46, I60 I73, I78
(32) W 1-W32
aLetters on the left are code letters for each sponge and water.
m a j o r cluster g r o u p [Cluster f A in W i l k i n s o n (16)] f r o m three Great Barrier R e e f Sponges ( m a r k e d w i t h a triangle in Fig. I), A d j a c e n t to this g r o u p in both similarity analyses was a subcluster, group l a , which c o n t a i n e d 8 strains f r o m sponges including the one remaining o f the 10 Great Barrier R e e f strains f r o m the m a j o r cluster fA (16). All strains in these groups (1 and la) are f a c u l t a t i v e a n a e r o b e s and are c o n s i d e r e d to be marine insofar as they have an obligate r e q u i r e m e n t f o r s o d i u m ions (9). O f the 116 strains, all except 2 w e r e e x t r e m e l y m u c o i d and sticky, a characteristic o f the sponge-specific bacteria f r o m Great Barrier R e e f
Table 2. Test characters coded for in numerical classification of 242 bacterial strains from sponges and ambient water I Gram stain, 2 Motility, 3 Size: small 4 : long-thin 5 : medium 6 : short-wide 7 : large 8 Cell curvature 9 Cell pleomorphism 10 Refractile granules 11 Colony: small 12 : large 13 : mueoid 14 : spreading 1:5 : opalescent 16 : cream/buff 17 : yellow 18 : orange/pink
19 Colony: entire/irreg. 20 : smooth/rough 21 : raised/flat 22 Green diffusible 23 Brown diffusible 24 Oxidase, Kovaks 25 Oxidase, cytochrome 26 Catalase: slow 27 : rapid 28 Aerobic/facultative 29 Nitrate reduction 30 Indole 31 Voges Proskauer 32 Methyl red 33 Digestion: starch 34 : gelatin 35 : lipid 36 : agar
For details of tests, refer to Wilkinson (16).
37 Digestion: collagen 38 Na + only growth 39 K + only growth 40 Sole C: urea 41 : glucose 42 : galaetose 43 : lactose 44 : glycogen 45 : dextran 46 : sorbitot 47 : inositol 48 : manaitol 49 : glycine 50 : arginine 51 : proline 52 : creatinine 53 Min. medium growth 54 Growth requirements
16
C . R . Wilkinson et al.
22V
...- *" "" 9
.._.---'"""'"
9
""
""
IOA
~08
25C
--'-"'-
2P
-"' i
:iI :H,
.-'"
?
--'"
,.'IR
2V
IA
" ' "g1
2C 1F
~
-- ~ -- - C'" G .... ~ ~'~,~,~'"
..:::::::::: ..... "'":.......
. ...............
G .... ~ . o % L . ~ ~............. Group
4 > a ~
"~l
~N
.':::............................... ....................... .~----'?J~
"~:::;::::'-:::iii:''::'"
.............................
........
....................
-. . . .
. . . . ~. . . .
group
..........
6
4v
2~
9F
. . . . . . . . "IR 2v
~oI
:..................... {
Group
7
)70~O
3F
~,
IF
ZA IC
\ ,..................... C~
BIlCtlll, ial S t r a i n s
Group
8
>859'~.::::~A d
Fig. 1. Diagrammatic representation of the similarity matrix prepared from the S S M coefficient and unweighted average linkage clustering technique. Each of 242 bacterial strains (listed a to b on y axis) with 54 characters was compared with the strains in the same order (c to d on x axis). This order was chosen so that strains of greatest similarity were in closest proximity. The cluster groups ( s t i p p l e d ) and the similarity levels at or above which they formed are represented on the matrix. The number of strains in each cluster group is listed to the right of the matrix ( u n d e r l i n e d ) with the source of those strains. The positions of reference strains from the Great Barrier Reef are represented by a triangle on the axes. See Table 1 for code of letters.
sponges examined by Wilkinson (16). The physiological and biochemical properties of these strains are listed in Table 3, and a summary of theircharacteristics is listed in Table 4. Group 2 consists of 5 closely related aerobic strains from Chondrosia reniformis (>85%S). These strains did not have an obligate requirement for sodium, and, if the criterion for marine bacteria proposed by MacLeod (9) is followed, they may not be marine in origin. This sponge was collected near shore adjacent to numerous sewage outfalls. The properties of these strains and their summarized characteristics are given in Tables 3 and 4. Group 4 consists of 3 aerobic strains from the Great Barrier Reef sponge Ircinia wistarii [Cluster a0 in Wilkinson (16)] and 1 strain from the Mediterranean Sea. These
Bacterial Symbiontsin Sponges
17 % Similarity
t00 Group No. o f f No. Strains /
1
la
90
80
70
60
50
108
8 5___
2 3
5 I i ~lt,i/)ll/li/iltlli,,." /,"..............
4
4
2
I
II
6
~.
.....
......
~
_
13
6
8
~
,
~
. . . . . . . . . .
24
8
5
. . . . . . . . . . . . . . . . . . . . . . . .
Fig, 2. Simplified dendrogram prepared using SSMcoefficient and averagelinkage method of clustering, Groups clusteringat :>80%similarity(solidblack)and at 85%S). These strains were specific to this sponge and were not observed in any other sponge or the ambient water. They are aerobic, long, thin, curved rods which form spreading yellow, sticky colonies and have an obligate requirement for sodium. The proportion of these strains in V. aerophobarepresented in Fig. 2 may be underestimated as another 8 strains with similar colonial characteristics (dark yellow, flat, and mucoid) were isolated from the sponge but were subsequently lost becausethey requited frequent subculture or could
18
C . R . Wilkinson etal.
96 8C 70
fac aer Chondrosia
fac
aer
fac aer
Petro~ia fici forr~is
far
Irclni~. variabilis
aer
Veron~ia aerophoba
fac
aer
Ve r_on~ ~ e r n l co!~
fac
aer
Chondrilla nucula
fac
a~r
ambient water
Fig. 3. Relative proportions of facultadve anaerobic (fac) and aerobic (aer) strains expressed as percentage of total number of swains isolated from the 6 Mediterranean sponges and ambient water. Proportions of major cluster groups are represented by shading of bars.
Table 3. Summary of the physiological and biochemical tests of the major groups of bacteria isolated from Mediterranean and Great Barrier Reef (GBR) sponges and ambient water Group I Test
R
V
F
C
NO 3 - reduction Indole Voges Proskauer Acid production Starch Lipid Gelatin Collagen Agar Urea Creatinine Glucose Galactose Lactose Glycogen Dextran Sorbitol Inositol Mannitol Glycine Arginine Proline Minimal medium Growth requirement
12 4 17 0 47 68 100 92 88 86 83 100 88 100 100 92 0 0 0 0 29 82 100 92 100 100 100 100 24 55 33 36 0 0 0 0 lg 27 0 24 47 68 33 20 94 91 83 80 35 91 50 80 0 9 0 0 65 27 0 32 29 0 0 4 29 5 0 8 41 41 67 52 24 5 0 0 94 91 100 96 6 5 0 4 82 86 83 88 100 I00 100 100 0 0 0 0
A
N
GBR
Subgroup Mean la
0 100 100 90 0 90 100 20 0 10 20 40 40 I0 30 0 10 20 10 100 0 80 100 0
0 79 95 100 0 100 100 16 0 21 21 84 53 I1 53 0 5 37 5 100 5 68 100 0
0 78 100 100 0 89 100 56 0 I1 11 100 100 0 0 0 33 56 0 78 !1 78 100 0
4 78 94 95 0 81 100 33 0 19 40 83 71 5 35. 6 12 43 6 94 5 81 100 0
12 62 100 88 0 88 100 50 0 12 62 50 62 12 50 0 75 62 12 75 12 37 37 62
Group Group Group 2 4 8 100 0 0 0 0 0 0 0 0 20 100 100 40 0 100 60 100 0 100 tO0 0 100 100 0
0 0 0 0 0 100 100 0 25 50 0 0 50 0 0 0 25 0 0 0 75 100 100 0
0 0 60 0 100 100 100 0 100 40 20 1(30 100 80 80 60 40 80 100 20 0 20 0 100
Group 1 strains are listed under the sponge source. Results expressed as the percentage of strains positive for each test. (Consult Methods for letter code.)
Bacterial Symbionts in Sponges
19
Table 4. Summarized characteristics, which may be used as diagnostic traits, of the major groups formed from bacterial strains isolated from Mediterranean and Great Barrier Reef sponges and ambient water a Group 1 and la 116 sponge strains
Group 2 5 sponge strains
Chondrosia reniforrnis Group 4 f water and 3 sponge strains Group 8 5 sponge strains
Verongia aerophoba
Gram - v e , pleomorphic, variable-sized rods with refractile granules; motile; form sticky mucoid, white cream colonies with no diffusible pigments; facultative anaerobes; marine (require Na+); oxidase + r e ; do not reduce N O 3 - ; indole +ve; fermentative, form acid; > 8 0 % are VP +ve; catalase +ve (rapid); digest gelatin and most (>80%) digest lipid; use glyeine as sole C source and most (>80%) use glucose and proline; do not digest starch or agar; < 2 0 % use urea, lactose, dextran, sorbitol, mannitol, arginine as C sources. Gram - r e , large pleomorphic rods; motile; form opalescent colonies with green diffusible pigment; aerobes; nonmarine (do not require Na+); oxidase +ve; reduce N O 3 - ; indole, VP, and acid production all - v e ; oxidative; catalase + v e (rapid); use creatinine, glucose, glycogen, sorbitol, mannitol, glycine, and proline as sole C sources; do not digest starch, gelatin, lipid, agar or collagen; do not use lactose, inositol, or arginine as C sources. Gram - v e or variable, large cocci with refractile granules; nonmotile; form opalescent colonies with no diffusible pigments; aerobes; nonmarine (do not require Na +); oxidase - v e ; do not reduce N O 3 - ; indole and acid production all - v e ; catalase + r e (rapid); digest gelatin and lipid; use proline as sole C source; do not digest starch or collagen; do not use glucose, lactose, glycogen, dextran, inositol, mannitol, and glycine as C source. Gram - v e , long, thin, curved rods; motile; form yellow, mucoid, spreading colonies with no diffusible pigments; aerobes; marine (require Na+); oxidase - v e ; do not reduce N O 3 - ; indole and acid production - v e ; VP mostly +ve; catalase +ve (rapid); oxidative; digest starch, gelatin, lipid, agar; use glucose, galactose, and mannitol as sole C source; do not digest collagen or use arginine as C source.
aAbbreviations used: +ve, positive; - v e , negative; VP, Voges Proskauer.
not be isolated from among fungal contaminants. The properties of this group are represented in Tables 3 and 4. The other groups--3, 5, 6, and 7--are not considered to be significant as their similarity indices are low (Figs. I and 2) and each group consists of a mixture of aerobes and facultative anaerobes isolated from different sponges and the ambient water. These groupings have occurred because a wide range of different bacteria were examined for a limited number (54) of characters. From these analyses two groups of bacteria are believed to be sponge specific, i.e., groups 1 and la and group 8. Strains in groups 1 and la metabolize a wide range of carbohydrates and amino acids; some digest the metabolic waste products urea and creatine, and are able to digest gelatin and lipid; a few digest collagen (Table 1). Group 8 strains also metabolize a wide range of carbohydrates and sugar alcohols, and digest gelatin, starch, lipid, and agar (Table 1). There was no discernible difference in clustering of strains isolated by the direct smear technique and the pour-plate method. Strains isolated by both techniques clustered into groups 1 and 8.
Discussion The principal result of this study is that there are phenotypically similar bacterial symbionts in sponges from two distant geographical regions--the Mediterranean Sea
20
C.R. Wilkinson et al.
and the Great Barrier Reef. These symbionts clustered as group 1, a cluster of closely related strains isolated from 6 Mediterranean sponges and 3 from the Great Barrier Reef. These specific symbionts represented 40% of all the bacterial strains (551) isolated from 10 sponge species during two separate studies [this study and Wilkinson (16)] but were not found among 202 bacterial strains isolated from the ambient water in the two regions. The specific symbionts were found in two major sponge classes---the Calcarea (genus Pericharax) and seven genera of the Demospongiae comprising six orders: Choristida (Jaspis), Hadromerida (Chondrosia and Chondrilla), Poecilosclerida (Neofibularia), Haplosclerida (Petrosia), Dictyoceratida (lrcinia), and Verongida (Verongia). Considering the taxonomic and geographic distribution of the specific bacterial symbiont, it is probable that it has been associated with sponges over a considerable geological time period. Bacterial symbionts are presumably transferred from parent sponges to the next generation as numerous bacteria have been observed in the larvae of some sponges (4, 8). The symbiont was isolated from 9 of 10 species examined, and it is predicted that this symbiont will be found in most large sponge species. Another apparently specific bacterial symbiont was found in Verongia aerophoba. Group 8 strains were isolated only from V. aerophoba and not from the ambient water. It is significant that this symbiont formed yellow-orange colonies similar to the color of the sponge tissue. The significance of the other two small, closely clustered groups is not known. Strains in group 2 were only isolated from Chondrosia reniformis, but it is possible that they are not marine in origin. Strains from group 4 are not sponge specific as they were isolated from both sponge and seawater. The specific bacterial symbionts are part of a complex bacterial population present in many marine sponges. Numerous morphologically different bacteria have been observed in marine sponges (14, 17), and Vacelet (13) described five different types in Verongia aerophoba. It is not possible to correlate which morphological type corresponds to the specific symbionts, and it is conceivable that some of the observed bacteria do not grow on the low-nutrient medium used; e.g., Imhoff and Truper (6) isolated phototrophic anaerobic bacteria from some Mediterranean sponges. The function of the bacterial symbionts in the sponges is unknown. Both symbionts (groups 1 and 8) form particularly sticky mucoid colonies in culture and, provided that this is also produced in vivo, they may increase the density of the sponge intercellular matrix, thereby increasing sponge structural rigidity, The size of bacterial populations is proportional to the size and tissue density of some sponges (15). Alternatively, the mucoid material may impede phagocytosis of the bacteria by sponge amoebocytes as is the case with increased virulence in mucoid strains of Diplococcus pneumoniae which are not readily phagocytosed by blood macrophages [p. 98 in Moat (11)]. Other functions have been suggested for the bacteria, such" as digestion of material not available to the host sponge (13, 16); direct incorporation of dissolved organic matter from the seawater ( 18); and, in the case of group I symbionts, digestion and recycling of insoluble sponge collagen (19). Although the techniques employed were designed to yield ecological groupings only and the number of tests employed was inadequate to identify the bacteria, some tentative identifications, based on comparisons with the diagnostic tables of Cowan (2), are possible. Group 1 bacteria belong to the Enterobacteriaceae, possibly to a new genus. Group 2 strains are most probably a Pseudomonas sp., similar to Ps. putida. Group 4 strains are similar to those in the genera Acinetobacter, and Micrococcus and group 8
Bacterial Symbionts in Sponges
21
strains are probably Cytophaga sp., with some resemblance to Flexibacter strains. More biochemical tests and D N A base ratio tests are required before more definitive nomenclature can be attempted.
References 1. Bertrand, J. C., and J. Vacelet: L'association entre Eponges Cornees et bacteries. C. R. Acad. Sci. (Paris) [D] 273,638-641 (1971) 2. Cowan, S. T.: Cowan and Steel's Manual for the Identification of Medical Bacteria, 2nd Ed. Cambridge University Press, Cambridge (1974) 3. Eimhjellen, K. E.: Photosynthetic bacteria and carotenoids from a sea sponge Halichondrium panicea. Acta Chem. Scand. 21, 2280-2281 (1967) 4. Gallissian, M.-F., and J. Vacelet: Ultrastructure de quelques stades de I'ovogenese de spongiaires du genre Verongia (Dictyoceratida). Ann. Sci. Natl. Zool. 18,381-404 (1976) 5. Garrone, R.: Phylogenesis of connective tissue. Morphological aspects and biosynthesis of sponge intercellular matrix. In L. Robert (ed.): Frontiers in Matrix Biology, Vol. 5. S. Karger, Basel (1978) 6. Imhoff, J. F., and H. G. Tmper: Marine sponges as habitats of anaerobic phototropbic bacteria. Microb. Ecol. 3, 1-9(1976) 7. Levi, C., and P. Levi: Populations bacteriennes darts les Eponges. J. Microsc. 4, 151 (1965) 8. Levi, C., and P. Levi: Embryogenese de Chondrosia reniformis (Nardo), Demosponge ovipare, et transmission des batteries symbiotique. Ann. Sci. Natl. Zool. 18,367-380 (1976) 9. MacLeod, R. A.: On the role of inorganic ions in the physiology of marine bacteria. Adv. Microbiol. Sea 1, 95-126 (1968) 10. Madri, P. P., M. Hermel, and G. Claus: The microbial flora of the sponge Microcioniaprolifera Verrill and its ecological implications. Botan. Mar. XIV, 1-5 (1971) 11. Moat, A. G.: Microbial Physiology. John Wiley & Sons, New York (1979) 12. Sneath, P. H. A., and R. R. Sokal: Numerical Taxonomy. The Principles and Practice of Numerical Classification. Freeman and Co., San Francisco (1973) 13. Vacelet, J.: Etude en microscopie electronique de l'association entre bacteries et spongiaires du genre Verongia (Dictyoceratida). J. Microsc. Biol. Cell 23,271-288 (1975) 14. Vacelet, J., and C. Donadey: Electron microscope study of the association between some sponges and bacteria. J. Exp. Mar. Biol. Ecol. 30, 301-314 (1977) 15. Wilkinson, C. R.: Microbial associations in sponges. I. Ecology, physiology and microbial populations of coral reef sponges. Mar. Biol. 49, 161-167 (1978) 16. Wilkinson, C. R.: Microbial associations in sponges. II. Numerical analysis of sponge and water bacterial populations. Mar. Biol. 49, 169-176 (1978) 17. Wilkinson, C. R.: Microbial associations in sponges. III. Ultrastructure of the in situ associations in coral reef sponges. Mar. Biol. 49, 177-185 (1978) 18. Wilkinson, C., and R. Garrone: Nutrition of marine sponges. Involvement of symbiotic bacteria in the uptake of dissolved carbon. In D. C. Smith and Y. Tiffon (eds.): Nutrition in the Lower Metazoa, pp. 157-161. Pergamon Press, Oxford (1980) 19. Wilkinson, C. R., R. Garrone, and D. Herbage: In vitro digestion of insoluble sponge collagen by sponge symbiotic bacteria. In C. Levi and N. Boury-Esnault (eds.): Biologic des Spongiaires. Colloq. Int. C.N.R.S. No. 291,361-364 (1979) 20. Wilkinson, C. R., and J. Vacelet: Transplantation of marine sponges to different conditions of light and current. J. Exp. Mar. Biol. Ecol. 37, 91-104 (1979)
IYIICI BI IL ECOI.OGV
Specificity of Bacterial Symbionts in Mediterranean and Great Barrier Reef Sponges* Clive R, Wilkinson, 7 , , Madeleine Nowak, i Brian Austin, 2.** and Rita R. Colwell 2 1Laboratoired'Histologieet BiologieTissulaire,Universit6ClaudeBernard,Villeurbanne69622, France;and 2Departmentof Microbiology,Universityof Maryland, CollegePark, Maryland20742, USA
Abstract. Bacteria were isolated from marine sponges from the Mediterranean and the Great Barrier Reef and characterized using numerical taxonomy techniques. A similar sponge-specific bacterial symbiont was found in 9 of 10 sponges examined from both geographic regions. This symbiont occurred in sponges of two classes and seven orders, and it probably has been associated with sponges over a long geological time scale. Another symbiont apparently specific to the sponge Verongia aerophoba was found. This sponge is yellow-orange, similar in color to the bacterial symbiont. These symbionts are two of a large mixed bacterial population present in many sponges.
Bacterial symbionts are common within many marine sponges. The granular material observed in the intercellular collagenous matrix (the mesohyl) of sponges was proved to be bacteria by Levi and Levi (7) using an electron microscope. Additional electron microscope studies have been made on the morphology of these bacteria and their relationship to sponge cells (13, 14, 17). The common conclusion drawn from these studies was that there 'are many morphologically different bacteria in the mesohyl of marine sponges. In two previous studies on bacteria isolated from temperate water sponges, no bacteria specific to the host sponges were detected; moreover, the bacteria isolated were similar to those in the ambient water (1, 10). These studies were limited in that the bacteria were characterized using several diagnostic tests only, and in one of the studies the bacterial populations in the sponge and particularly the ambient water were unrealistically low (10), During a study of four coral reef sponges, Wilkinson (16) isolated large numbers of bacteria from the sponges and the ambient water and characterized them by numerical taxonomy methods. In three of the sponges there was a common facultative anaerobic
*This paperconstitutes No. V in the seriesMicrobialAssociationsin Sponges. **Present address: Australian Instituteof Marine Science, PMB 3, Townsville,M.S.O., Queensland, 4810, Australia. ***Presentaddress: Fish DiseasesLaboratory,Weymouth,DorsetDT4 8UB, England, 0095-3628/81/0007-0013 $01.80 1981 Springer-VcrlagNew YorkInc.
14
c.R. Wilkinsonet al.
bacterial strain which was not present in the ambient water. These bacteria may not be the only sponge-specific symbionts; e.g., Eimhjellen (3) and Imhoff and T m p e r (6) have isolated phototrophic anaerobic bacteria from other marine sponges. Bacteria were isolated from sponges and the ambient water in the Mediterranean Sea to determine whether the bacteria that were specific to Great Barrier Reef sponges (16) were more widely distributed. These bacterial isolates were characterized in order to determine if ecological or metabolic groupings occurred within sponges. Numerical taxonomy methods were used to compare those bacteria isolated from Mediterranean sponges and seawater with selected strains previously isolated by Wilkinson (16) from Great Barrier Reef sponges.
Materials and Methods Sponges were collectedfrom rockyreefs adjacentto Marseiile, France[for detailssee Wilkinsonand Vacelet (20)]. Bacteria were isolatedfrom six sponges:Chondrosia reniformis, lrcinia variabilis, Petrosiaficiformis, Verongia cavernicola, V. aerophoba, and ChondriUa nucula. Two methods were used to isolate bacteria: in the fast, approximately 5 g of sponge tissue was blended in 50 ml of sterile seawater, diluted in sterile seawater, and platedby pipelling 1ml of 10- 3 and 10-4 suspensionsintosterilePetfi dishesand adding 15 ml of moltenOZR agar at 37~C(15). Ambientwater sampleswere treatedsimilarly.In the secondmethod, small blocks of sponge tissue, approximately 1 cm3, were cut fromthe interiorof the spongeusingalcohol-flamed scalpel blades and smearedover dried plates ofOZR agar. Followingincubationat 2 I~ for approximately 14 days, between 20 and 25 colonieswere isolatedrandomlyfrom the pour-platesand an additional10 colonies were picked from the smeared plates(Table 1). A set of 15 strainsrepresentingthe majorclustergroupingsof bacteria from four Great BarrierReef sponges(t6) was includedin the studyfor comparison(Table 1). In additionto characterizationtests describedpreviouslyfor coral reefsponges(16), the followingtests were used: cytochrome oxidase and indole (2); collagendigestion--a piece of membraneof pure collagenfibrils from Chondrosia reniformis [pp. 37-38 in Garrone (5)] was added to a low-nutrientbroth of 0.05% yeast extract and 0.02% casamino acids in artificial seawater and examined for 14 days for breakdown of the membrane (19); ability to grow in minimal medium plus carbon source and requirementfor addition of 0.0025% yeast extract and casaminoacids(Table2). A total of 54 characters, includingmorphology,growth characteristics, physiology, and metabolic properties, were coded for 195 bacteria from Mediterranean sponges, 32 from ambientwater, and 15 fromGreat BarrierReef sponges. These 242 strains were analyzed using simplematchingcoefficientcomparisons(SsM---positiveand negativematches)and Jaccard coefficient comparisons (Sj---positive matchesonly) and clusteredusingan unweightedaveragelinkage(12). Programs employed includedTAXAN6, UMDTAXON3, and IGPS3programpackages on the Universityof Maryland UNIVAC 1108computer.
Results Analyses obtained with the two coefficients, SsM and S j , were identical and the clusters formed with the SsM coefficient are considered here (Fig. 1). Eight clusters formed, with groups 1, 2, 4, and 8 clustering at or above 80% similarity (S) level. These four groups contained 122 strains (50.4% of the total). Three other groups (3, 6, and 7) plus a subgroup (1 a) clustered at lower levels of similarity (at or above 70%S) and contained 32 strains (13% o f the total). The interrelationships of these groups are given in Fig. 2. The largest cluster, group 1 (at or above 80%S) contained 108 strains. This was 44.6% o f all strains or 78.2% of the facultative anaerobic strains. Strains from this group were isolated exclusively from sponges and comprised the largest group within all six Mediterranean sponges (Fig. 3). This group also contained 9 of the I0 bacteria from the
Bacterial Symbionts in Sponges
15
Table 1. List of bacterial strains used in this study showing the source of strains, numbers (in parentheses), and strain numbers Mediterranean sponges
Strain Nos.
Great Barrier Reef sponges
Ra
Chondrosia reniformis
(36) R 1-R36
P
Pericharax heteroraphis
V
Ircinia variat~lis
(36) V 1-V36
J
Jaspis stellifera
F C
Petrosiaficiformis Verongia cavernicola
(30) F1-F30 (34) C 1-C34
G
Neofibalaria irata
A
Verongia aerophoba
(29) A 1-A29
I
Ircinia wistarii
N
Chondrilla nucula ambient water
(30) N1-N30
W
Strain Nos. (4) P25, P57 P85, P90 (3) J 12, J28 J69 (4) G65, G66 G72, G89 (4) I46, I60 I73, I78
(32) W 1-W32
aLetters on the left are code letters for each sponge and water.
m a j o r cluster g r o u p [Cluster f A in W i l k i n s o n (16)] f r o m three Great Barrier R e e f Sponges ( m a r k e d w i t h a triangle in Fig. I), A d j a c e n t to this g r o u p in both similarity analyses was a subcluster, group l a , which c o n t a i n e d 8 strains f r o m sponges including the one remaining o f the 10 Great Barrier R e e f strains f r o m the m a j o r cluster fA (16). All strains in these groups (1 and la) are f a c u l t a t i v e a n a e r o b e s and are c o n s i d e r e d to be marine insofar as they have an obligate r e q u i r e m e n t f o r s o d i u m ions (9). O f the 116 strains, all except 2 w e r e e x t r e m e l y m u c o i d and sticky, a characteristic o f the sponge-specific bacteria f r o m Great Barrier R e e f
Table 2. Test characters coded for in numerical classification of 242 bacterial strains from sponges and ambient water I Gram stain, 2 Motility, 3 Size: small 4 : long-thin 5 : medium 6 : short-wide 7 : large 8 Cell curvature 9 Cell pleomorphism 10 Refractile granules 11 Colony: small 12 : large 13 : mueoid 14 : spreading 1:5 : opalescent 16 : cream/buff 17 : yellow 18 : orange/pink
19 Colony: entire/irreg. 20 : smooth/rough 21 : raised/flat 22 Green diffusible 23 Brown diffusible 24 Oxidase, Kovaks 25 Oxidase, cytochrome 26 Catalase: slow 27 : rapid 28 Aerobic/facultative 29 Nitrate reduction 30 Indole 31 Voges Proskauer 32 Methyl red 33 Digestion: starch 34 : gelatin 35 : lipid 36 : agar
For details of tests, refer to Wilkinson (16).
37 Digestion: collagen 38 Na + only growth 39 K + only growth 40 Sole C: urea 41 : glucose 42 : galaetose 43 : lactose 44 : glycogen 45 : dextran 46 : sorbitot 47 : inositol 48 : manaitol 49 : glycine 50 : arginine 51 : proline 52 : creatinine 53 Min. medium growth 54 Growth requirements
16
C . R . Wilkinson et al.
22V
...- *" "" 9
.._.---'"""'"
9
""
""
IOA
~08
25C
--'-"'-
2P
-"' i
:iI :H,
.-'"
?
--'"
,.'IR
2V
IA
" ' "g1
2C 1F
~
-- ~ -- - C'" G .... ~ ~'~,~,~'"
..:::::::::: ..... "'":.......
. ...............
G .... ~ . o % L . ~ ~............. Group
4 > a ~
"~l
~N
.':::............................... ....................... .~----'?J~
"~:::;::::'-:::iii:''::'"
.............................
........
....................
-. . . .
. . . . ~. . . .
group
..........
6
4v
2~
9F
. . . . . . . . "IR 2v
~oI
:..................... {
Group
7
)70~O
3F
~,
IF
ZA IC
\ ,..................... C~
BIlCtlll, ial S t r a i n s
Group
8
>859'~.::::~A d
Fig. 1. Diagrammatic representation of the similarity matrix prepared from the S S M coefficient and unweighted average linkage clustering technique. Each of 242 bacterial strains (listed a to b on y axis) with 54 characters was compared with the strains in the same order (c to d on x axis). This order was chosen so that strains of greatest similarity were in closest proximity. The cluster groups ( s t i p p l e d ) and the similarity levels at or above which they formed are represented on the matrix. The number of strains in each cluster group is listed to the right of the matrix ( u n d e r l i n e d ) with the source of those strains. The positions of reference strains from the Great Barrier Reef are represented by a triangle on the axes. See Table 1 for code of letters.
sponges examined by Wilkinson (16). The physiological and biochemical properties of these strains are listed in Table 3, and a summary of theircharacteristics is listed in Table 4. Group 2 consists of 5 closely related aerobic strains from Chondrosia reniformis (>85%S). These strains did not have an obligate requirement for sodium, and, if the criterion for marine bacteria proposed by MacLeod (9) is followed, they may not be marine in origin. This sponge was collected near shore adjacent to numerous sewage outfalls. The properties of these strains and their summarized characteristics are given in Tables 3 and 4. Group 4 consists of 3 aerobic strains from the Great Barrier Reef sponge Ircinia wistarii [Cluster a0 in Wilkinson (16)] and 1 strain from the Mediterranean Sea. These
Bacterial Symbiontsin Sponges
17 % Similarity
t00 Group No. o f f No. Strains /
1
la
90
80
70
60
50
108
8 5___
2 3
5 I i ~lt,i/)ll/li/iltlli,,." /,"..............
4
4
2
I
II
6
~.
.....
......
~
_
13
6
8
~
,
~
. . . . . . . . . .
24
8
5
. . . . . . . . . . . . . . . . . . . . . . . .
Fig, 2. Simplified dendrogram prepared using SSMcoefficient and averagelinkage method of clustering, Groups clusteringat :>80%similarity(solidblack)and at 85%S). These strains were specific to this sponge and were not observed in any other sponge or the ambient water. They are aerobic, long, thin, curved rods which form spreading yellow, sticky colonies and have an obligate requirement for sodium. The proportion of these strains in V. aerophobarepresented in Fig. 2 may be underestimated as another 8 strains with similar colonial characteristics (dark yellow, flat, and mucoid) were isolated from the sponge but were subsequently lost becausethey requited frequent subculture or could
18
C . R . Wilkinson etal.
96 8C 70
fac aer Chondrosia
fac
aer
fac aer
Petro~ia fici forr~is
far
Irclni~. variabilis
aer
Veron~ia aerophoba
fac
aer
Ve r_on~ ~ e r n l co!~
fac
aer
Chondrilla nucula
fac
a~r
ambient water
Fig. 3. Relative proportions of facultadve anaerobic (fac) and aerobic (aer) strains expressed as percentage of total number of swains isolated from the 6 Mediterranean sponges and ambient water. Proportions of major cluster groups are represented by shading of bars.
Table 3. Summary of the physiological and biochemical tests of the major groups of bacteria isolated from Mediterranean and Great Barrier Reef (GBR) sponges and ambient water Group I Test
R
V
F
C
NO 3 - reduction Indole Voges Proskauer Acid production Starch Lipid Gelatin Collagen Agar Urea Creatinine Glucose Galactose Lactose Glycogen Dextran Sorbitol Inositol Mannitol Glycine Arginine Proline Minimal medium Growth requirement
12 4 17 0 47 68 100 92 88 86 83 100 88 100 100 92 0 0 0 0 29 82 100 92 100 100 100 100 24 55 33 36 0 0 0 0 lg 27 0 24 47 68 33 20 94 91 83 80 35 91 50 80 0 9 0 0 65 27 0 32 29 0 0 4 29 5 0 8 41 41 67 52 24 5 0 0 94 91 100 96 6 5 0 4 82 86 83 88 100 I00 100 100 0 0 0 0
A
N
GBR
Subgroup Mean la
0 100 100 90 0 90 100 20 0 10 20 40 40 I0 30 0 10 20 10 100 0 80 100 0
0 79 95 100 0 100 100 16 0 21 21 84 53 I1 53 0 5 37 5 100 5 68 100 0
0 78 100 100 0 89 100 56 0 I1 11 100 100 0 0 0 33 56 0 78 !1 78 100 0
4 78 94 95 0 81 100 33 0 19 40 83 71 5 35. 6 12 43 6 94 5 81 100 0
12 62 100 88 0 88 100 50 0 12 62 50 62 12 50 0 75 62 12 75 12 37 37 62
Group Group Group 2 4 8 100 0 0 0 0 0 0 0 0 20 100 100 40 0 100 60 100 0 100 tO0 0 100 100 0
0 0 0 0 0 100 100 0 25 50 0 0 50 0 0 0 25 0 0 0 75 100 100 0
0 0 60 0 100 100 100 0 100 40 20 1(30 100 80 80 60 40 80 100 20 0 20 0 100
Group 1 strains are listed under the sponge source. Results expressed as the percentage of strains positive for each test. (Consult Methods for letter code.)
Bacterial Symbionts in Sponges
19
Table 4. Summarized characteristics, which may be used as diagnostic traits, of the major groups formed from bacterial strains isolated from Mediterranean and Great Barrier Reef sponges and ambient water a Group 1 and la 116 sponge strains
Group 2 5 sponge strains
Chondrosia reniforrnis Group 4 f water and 3 sponge strains Group 8 5 sponge strains
Verongia aerophoba
Gram - v e , pleomorphic, variable-sized rods with refractile granules; motile; form sticky mucoid, white cream colonies with no diffusible pigments; facultative anaerobes; marine (require Na+); oxidase + r e ; do not reduce N O 3 - ; indole +ve; fermentative, form acid; > 8 0 % are VP +ve; catalase +ve (rapid); digest gelatin and most (>80%) digest lipid; use glyeine as sole C source and most (>80%) use glucose and proline; do not digest starch or agar; < 2 0 % use urea, lactose, dextran, sorbitol, mannitol, arginine as C sources. Gram - r e , large pleomorphic rods; motile; form opalescent colonies with green diffusible pigment; aerobes; nonmarine (do not require Na+); oxidase +ve; reduce N O 3 - ; indole, VP, and acid production all - v e ; oxidative; catalase + v e (rapid); use creatinine, glucose, glycogen, sorbitol, mannitol, glycine, and proline as sole C sources; do not digest starch, gelatin, lipid, agar or collagen; do not use lactose, inositol, or arginine as C sources. Gram - v e or variable, large cocci with refractile granules; nonmotile; form opalescent colonies with no diffusible pigments; aerobes; nonmarine (do not require Na +); oxidase - v e ; do not reduce N O 3 - ; indole and acid production all - v e ; catalase + r e (rapid); digest gelatin and lipid; use proline as sole C source; do not digest starch or collagen; do not use glucose, lactose, glycogen, dextran, inositol, mannitol, and glycine as C source. Gram - v e , long, thin, curved rods; motile; form yellow, mucoid, spreading colonies with no diffusible pigments; aerobes; marine (require Na+); oxidase - v e ; do not reduce N O 3 - ; indole and acid production - v e ; VP mostly +ve; catalase +ve (rapid); oxidative; digest starch, gelatin, lipid, agar; use glucose, galactose, and mannitol as sole C source; do not digest collagen or use arginine as C source.
aAbbreviations used: +ve, positive; - v e , negative; VP, Voges Proskauer.
not be isolated from among fungal contaminants. The properties of this group are represented in Tables 3 and 4. The other groups--3, 5, 6, and 7--are not considered to be significant as their similarity indices are low (Figs. I and 2) and each group consists of a mixture of aerobes and facultative anaerobes isolated from different sponges and the ambient water. These groupings have occurred because a wide range of different bacteria were examined for a limited number (54) of characters. From these analyses two groups of bacteria are believed to be sponge specific, i.e., groups 1 and la and group 8. Strains in groups 1 and la metabolize a wide range of carbohydrates and amino acids; some digest the metabolic waste products urea and creatine, and are able to digest gelatin and lipid; a few digest collagen (Table 1). Group 8 strains also metabolize a wide range of carbohydrates and sugar alcohols, and digest gelatin, starch, lipid, and agar (Table 1). There was no discernible difference in clustering of strains isolated by the direct smear technique and the pour-plate method. Strains isolated by both techniques clustered into groups 1 and 8.
Discussion The principal result of this study is that there are phenotypically similar bacterial symbionts in sponges from two distant geographical regions--the Mediterranean Sea
20
C.R. Wilkinson et al.
and the Great Barrier Reef. These symbionts clustered as group 1, a cluster of closely related strains isolated from 6 Mediterranean sponges and 3 from the Great Barrier Reef. These specific symbionts represented 40% of all the bacterial strains (551) isolated from 10 sponge species during two separate studies [this study and Wilkinson (16)] but were not found among 202 bacterial strains isolated from the ambient water in the two regions. The specific symbionts were found in two major sponge classes---the Calcarea (genus Pericharax) and seven genera of the Demospongiae comprising six orders: Choristida (Jaspis), Hadromerida (Chondrosia and Chondrilla), Poecilosclerida (Neofibularia), Haplosclerida (Petrosia), Dictyoceratida (lrcinia), and Verongida (Verongia). Considering the taxonomic and geographic distribution of the specific bacterial symbiont, it is probable that it has been associated with sponges over a considerable geological time period. Bacterial symbionts are presumably transferred from parent sponges to the next generation as numerous bacteria have been observed in the larvae of some sponges (4, 8). The symbiont was isolated from 9 of 10 species examined, and it is predicted that this symbiont will be found in most large sponge species. Another apparently specific bacterial symbiont was found in Verongia aerophoba. Group 8 strains were isolated only from V. aerophoba and not from the ambient water. It is significant that this symbiont formed yellow-orange colonies similar to the color of the sponge tissue. The significance of the other two small, closely clustered groups is not known. Strains in group 2 were only isolated from Chondrosia reniformis, but it is possible that they are not marine in origin. Strains from group 4 are not sponge specific as they were isolated from both sponge and seawater. The specific bacterial symbionts are part of a complex bacterial population present in many marine sponges. Numerous morphologically different bacteria have been observed in marine sponges (14, 17), and Vacelet (13) described five different types in Verongia aerophoba. It is not possible to correlate which morphological type corresponds to the specific symbionts, and it is conceivable that some of the observed bacteria do not grow on the low-nutrient medium used; e.g., Imhoff and Truper (6) isolated phototrophic anaerobic bacteria from some Mediterranean sponges. The function of the bacterial symbionts in the sponges is unknown. Both symbionts (groups 1 and 8) form particularly sticky mucoid colonies in culture and, provided that this is also produced in vivo, they may increase the density of the sponge intercellular matrix, thereby increasing sponge structural rigidity, The size of bacterial populations is proportional to the size and tissue density of some sponges (15). Alternatively, the mucoid material may impede phagocytosis of the bacteria by sponge amoebocytes as is the case with increased virulence in mucoid strains of Diplococcus pneumoniae which are not readily phagocytosed by blood macrophages [p. 98 in Moat (11)]. Other functions have been suggested for the bacteria, such" as digestion of material not available to the host sponge (13, 16); direct incorporation of dissolved organic matter from the seawater ( 18); and, in the case of group I symbionts, digestion and recycling of insoluble sponge collagen (19). Although the techniques employed were designed to yield ecological groupings only and the number of tests employed was inadequate to identify the bacteria, some tentative identifications, based on comparisons with the diagnostic tables of Cowan (2), are possible. Group 1 bacteria belong to the Enterobacteriaceae, possibly to a new genus. Group 2 strains are most probably a Pseudomonas sp., similar to Ps. putida. Group 4 strains are similar to those in the genera Acinetobacter, and Micrococcus and group 8
Bacterial Symbionts in Sponges
21
strains are probably Cytophaga sp., with some resemblance to Flexibacter strains. More biochemical tests and D N A base ratio tests are required before more definitive nomenclature can be attempted.
References 1. Bertrand, J. C., and J. Vacelet: L'association entre Eponges Cornees et bacteries. C. R. Acad. Sci. (Paris) [D] 273,638-641 (1971) 2. Cowan, S. T.: Cowan and Steel's Manual for the Identification of Medical Bacteria, 2nd Ed. Cambridge University Press, Cambridge (1974) 3. Eimhjellen, K. E.: Photosynthetic bacteria and carotenoids from a sea sponge Halichondrium panicea. Acta Chem. Scand. 21, 2280-2281 (1967) 4. Gallissian, M.-F., and J. Vacelet: Ultrastructure de quelques stades de I'ovogenese de spongiaires du genre Verongia (Dictyoceratida). Ann. Sci. Natl. Zool. 18,381-404 (1976) 5. Garrone, R.: Phylogenesis of connective tissue. Morphological aspects and biosynthesis of sponge intercellular matrix. In L. Robert (ed.): Frontiers in Matrix Biology, Vol. 5. S. Karger, Basel (1978) 6. Imhoff, J. F., and H. G. Tmper: Marine sponges as habitats of anaerobic phototropbic bacteria. Microb. Ecol. 3, 1-9(1976) 7. Levi, C., and P. Levi: Populations bacteriennes darts les Eponges. J. Microsc. 4, 151 (1965) 8. Levi, C., and P. Levi: Embryogenese de Chondrosia reniformis (Nardo), Demosponge ovipare, et transmission des batteries symbiotique. Ann. Sci. Natl. Zool. 18,367-380 (1976) 9. MacLeod, R. A.: On the role of inorganic ions in the physiology of marine bacteria. Adv. Microbiol. Sea 1, 95-126 (1968) 10. Madri, P. P., M. Hermel, and G. Claus: The microbial flora of the sponge Microcioniaprolifera Verrill and its ecological implications. Botan. Mar. XIV, 1-5 (1971) 11. Moat, A. G.: Microbial Physiology. John Wiley & Sons, New York (1979) 12. Sneath, P. H. A., and R. R. Sokal: Numerical Taxonomy. The Principles and Practice of Numerical Classification. Freeman and Co., San Francisco (1973) 13. Vacelet, J.: Etude en microscopie electronique de l'association entre bacteries et spongiaires du genre Verongia (Dictyoceratida). J. Microsc. Biol. Cell 23,271-288 (1975) 14. Vacelet, J., and C. Donadey: Electron microscope study of the association between some sponges and bacteria. J. Exp. Mar. Biol. Ecol. 30, 301-314 (1977) 15. Wilkinson, C. R.: Microbial associations in sponges. I. Ecology, physiology and microbial populations of coral reef sponges. Mar. Biol. 49, 161-167 (1978) 16. Wilkinson, C. R.: Microbial associations in sponges. II. Numerical analysis of sponge and water bacterial populations. Mar. Biol. 49, 169-176 (1978) 17. Wilkinson, C. R.: Microbial associations in sponges. III. Ultrastructure of the in situ associations in coral reef sponges. Mar. Biol. 49, 177-185 (1978) 18. Wilkinson, C., and R. Garrone: Nutrition of marine sponges. Involvement of symbiotic bacteria in the uptake of dissolved carbon. In D. C. Smith and Y. Tiffon (eds.): Nutrition in the Lower Metazoa, pp. 157-161. Pergamon Press, Oxford (1980) 19. Wilkinson, C. R., R. Garrone, and D. Herbage: In vitro digestion of insoluble sponge collagen by sponge symbiotic bacteria. In C. Levi and N. Boury-Esnault (eds.): Biologic des Spongiaires. Colloq. Int. C.N.R.S. No. 291,361-364 (1979) 20. Wilkinson, C. R., and J. Vacelet: Transplantation of marine sponges to different conditions of light and current. J. Exp. Mar. Biol. Ecol. 37, 91-104 (1979)
