isolation and identification of sponge associated...
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ISOLATION AND IDENTIFICATION OF
SPONGE ASSOCIATED
CYANOBACTERIAL SYMBIONTS
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30. ISOLATION AND IDENTIFICATION OF SPONGE ASSOCIATED
CYANOBACTERIAL SYMBIONTS
3.1. Introduction
Sponges are a good source of secondary metabolites among
marine invertebrates. Virtually sponges harbour prokaryotic
endobionts, and it is often assumed that, they can synthesize variety of
secondary metabolites. Unfortunately, little experimental work has
been done to test such proposals, however clear symbiotic relationship
was not established. The prokaryotic endobionts such as, heterotrophic
bacteria, fungi, actinomycetes and cyanobacteria were very well
documented in marine sponges (Table.1). Of these, some of the
heterotrophic bacteria isolated from sponges are appeared differently
from strains isolated from the surrounding seawater (Wilkinson, 1978;
Wilkonson et al., 1981). Interestingly, unique morphological types of
prokaryotes have been identified from certain sponges (Vacelet, 1975;
1981; Sanvaty, 1985; Larkum et al., 1987). The occurrences of symbiotic
cyanophytes in certain ascidians and sponges have been established
recently. But, no such studies are available on the symbiotic
cyanobacterial association in sponges from South East coast of India.
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Table 1. Works concerned with sponge associated microorganisms
Year Author (s) Aspect(s) studied
1959 Santavy Marine bacteria - invertebrate symbiosis. Thecarribean Scierosponge Ceraloporeila nichoisoni as aparadigm
1966 Vacelet et cii. Symbiosis between methane oxidizing bacteria and adeep sea carnivorous cladorhizid sponge
1971 Sara Ultrastructural aspects of the symbiosis between twospecies of the genus Aphanocapsa (Cyanophyceae) andIrcinia variabilis (Demospongiae)
1977 Vacelet and Electron microscope study of the association betweenDonadey some sponge and bacteria
1978 Wilkinson
1979* Wilkinson and Fay
1980 Wilkinson andGarrone
1981 Vacelet
1981 Wilkinson et cii.
1981 Rutzler
1982* Berthold et cii.
1985 Santavy
1985* Rutzler
1987 Wilkinson
1987* Larkum et cii
Microbial association in sponges
Nitrogen fixation in coral reef sponges with symbioticcyanobacteria
Nutrition of marine sponges. Involvement ofsymbiotic bacteria in the uptake of dissolved carbon
Algal-sponge symbioses in the coral reefs of NewCaledonia: a morphological study
Specificity of bacterial symbionts in Mediterraneanand Great Barrier Reef.sponges
An unusual blue green alga symbiotic with two newspecies of Ulosa from Carrie Bow Ray
Osciilatoria spongeiiae, the blue-green algalendosymbiont of the sponge Dysideci herbaceae
The symbiotic relationship between a blue-pigmentedbacterium and the coral reef sponge Terpios granuiosci
Association between Caribbean sponges andphotosynthetic organisms
Significance of microbial symbionts in spongeevolution and ecology
Filamentous cyanophyte, Osciiiatoria sp. in symbiosiswith sponge Dysidea herbaceae and an ascidianTrididemnum miniatuni of coral reefs
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1988 Burlando et al. Association between Calcareous Clathrina cerebrumand bacteria
1990
Santavy et al. Phenotypic study of bacteria associated withCarribean Scierosponge, Ceratoporella nicholsoni
1990
Kohlmeyer and Association of ascomycetes of the genus KoralionastesKohlmeyer. with crustaceous sponges
1990
Santavy and Colwell Comparison of bacterial communities associated withthe Caribbean Sclerosponge, Ceraloporella nicholsoni
1990* Rai Cyanobacteria in symbiosis
1992* Wilkinson Symbiotic interaction between marine sponges andalgae
1993* Unson and Faulkner Cyanobacterial symbiotic biosynthesis of chlorinatedmetabolites from Dysidea herbacea
1993* Arillo et al.
1994 Faulkner et al.
1994* Unson et al.
1994* Hinde et al.
1995 Brantley et al.
1996 Preston et a!,
1997 Schumann et al.
1998 Fuerst et al.
1998 Althoff et al.
Metabolic integration between symbioticcyanobacteria and sponges - a possible mechanism
The chemistry of some sponges and their symbionts
A brominated secondary metabolites synthesized bythe cyanobacterial symbiont Oscillatoria spongeliae of amarine sponge Dysidea herbaceae and accumulation ofthe crystalline metabolite in the sponge tissueIsolation of Oscillatoria spongeliae, the filamentouscyanobacterial symbiont of the marine sponge DysideaherbaceaeBacterial association of marine sponge Xestospongia sp.
A psychrophilic crenarchaeaon inhabits a marinesponge
Aerobic and anaerobic microorganisms in modernsponges
Archael symbionts of marine sponges
Evidence for a symbiosis between bacteria of thegenus Rhodobacter and marine sponge Halichondria
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1999 Burja et cii.
1999 Lopez et cii.
1999 Friedrich et cii.
1999 Magnino et cii.
1999 Diaz and Ward
1999 Muller
2000 Schmidt et cii,
2000 Claudia Osterhage
2002 Michio et cii.
2002 Raquel and Jadulco
2002 Ulrich Holler
2004 Taylor et cii.
2005 1 Taylor et cii
Microbial symbionts of Great Barrier reef sponges
Phyletic diversity of heterotrophic microbesassociated with Discodermia spp. (Porifera:Demospongiae)Microbial diversity in the marine sponge, Aplysinciccivernicoici
Endobionts of the coral reef sponge Theoneiici swinhoei
Perspectives on sponge cyanobacterial symbioses
16s rRNA sequences as diagnostic tools to elucidatepotential symbiotic relationship between bacteria andmarine sponge Hciuichondrici paniceciIdentification of the antifungal peptide containingsymbiont of marine sponge Theonelici swinhoei as anovel y-proteobacterium candidatus EntotheoneiiapaiciuensisIsolation, structure determination of biologicalactivity assessment of secondary metabolites frommarine derived fungi.Distribution of marine filamentous fungi associatedwith marine sponges in coral reefs of Palau andBunakeu islands, IndonesiaIsolation and structure elucidation of bioactivesecondary metabolites from marine sponge andsponge derived fungiIsolation, biological activity and secondary metaboliteinvestigations of marine derived fungi and selectedhost spongesHost specificity in marine sponges - associatedbacteria and potential implication for marinemicrobial diversityBiogeography of bacteria associated with the marinesponge Cymbcistelci concentrica
* Works related to cyanobacteria
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3.2. Materials and methods
3.2.1. Description of study area ( Fig.1)
Based on the availability of sponge samples in all the months, two
stations were selected by the present study. The study area is
comprised about 15 km. It is situated 17 km North of Kudamkulam
and 39 km from Tiruchendur in the North to Ovari in the South. The
two stations Ovari and Kuttankuzhi fall in the latitutde of
N 80 12' 57" to 80 16' 45" and longitude of F 77°47' 03" to 77054' 06".
The drainage pattern of the study area (Kuttankuzhi - Ovari) is
mainly controlled and influenced by the presence of Nambiar river.
Both the sites receive both South West monsoon (June- September)
and the North East monsoon (October-December). Non-monsoon
was noticed between January-May.
Fig. 1. Location Map of the study area
T7 455
53-36
MAAR
• Sampling stationsLiSea..'.Road
RiverDrainageBuilduplandCoastalboundaryLand
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Salinity
Salinity is maximum in the South Indian coast is found at 30 ppt.
In the southern coast the large annual variations of precipitation and
the resultant inflow of freshwater through the rivers is responsible for
the large annual variation of surface salinity. In South West monsoon
season, the Southern coast salinity is minimum value due to the heavy
rains in the winter season and flow salinity could be to the effect of
coastal currents.
Temperature
The surface water temperature increases rapidly towards North
from subtropical convergences. The surface water temperature is
minimum (120C) at the Southern coast of India and maximum about
30°C.
Geomorphology
The mountain ranges in the West and North, and the broad
undulating coastal plains in the East are the two physiographic
provinces of Tamil Nadu. The mountain range of the Western Chats
with an average altitude of 1000-1300 m is at a distance of 80 to 160 km
from the shoreline. It is a continuous range of hills with a width of 24
to 48 km from Nagercoil near Kanyakumari in the South through the
Nilgiris and the Western border of Mysore plateau up to the Tapi
valley.
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Geology
Granulitic rocks, principally of khondalites, charnockites and
granitic gneisses bordered by the sedimentary rocks exposed along the
eastern coastal plains are the principal rock types in Southern Tamil
Nadu coast. The red soils (Teri sands) observed in the area of Navaladi
region. It is situated between Kuttankuzhi and Ovari. The study area
of the coast is composed of barrier islands, near shore deltaic and shore
featured system. The whole study area is underlain with semi-
consolidated to dense calcareous sandstone.
The rivers and streams of Tamil Nadu coast are mostly dry
during the greatest part of the year and are flooded only during
monsoon. During winter dry surface airflow from land to sea in the
North East directions due to North East monsoon. In summer, there is
a complete reversal of these conditions with the moist winds blowing
from sea to land in South West direction due to South West monsoon.
3.2.2. Collection of sponge samples
Six sponge samples were collected at every month between
January to December 2002 by SCUBA diving from two sites
[Ovari (80 12'57" N, 77 047'03") and Kuttankuzhi (80 16'45" N,
77054'06" E)] located along South East coast of India in
sterilized biodegradable plastic bags and brought to the
laboratory by using ice box within 27 hours of collection.
Initially, the sponge species were identified based upon their
colour and surface ornamentation. Later, the sponge species
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were identified by the type of spicule arrangement and electron
microscopic pictures and annotated with separate identification
number (MSUSR1-3). The 'MSU' denotes Manonmaniam
Sundaranar University, 'S' denotes Shiefa and 'R' denotes
Ravikumar.
3.2.3. Isolation of sponge associated cyanobacteria
To isolate the sponge associated cyanobacterial symbionts,
sponge tissues (1 cm3) were excised from different sponge species viz.,
Coiiociathria sp (MSUSR1), Cailyspongia fibrosa(MSUSR2) and Sigmadocia
carnosa (MSUSR3) by using sterile scissors. The excised tissue was
transferred to 250 ml Erlen Meyers flask containing sterilized BG11
medium (Stainer et al., 1991). Simultaneously excised sponge tissues
(1 cm3) from each sponge samples were homogenized with sterile
BG11 medium by using electric homogenizer (REMI, Mumbai) and
inoculated into sterile BG11 medium.
Composition of BG11 Medium
Solution I (g.l-l)
NaNO3 - 300.0
K2HPO4- 8.0
Mg504.7H20 - 15.0
CaC12.2H20 - 7.2
Na2EDTA.2H20 - 0.2
Na2CO3 - 4.0
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Solution II (g.l-l)
H3B03
MnC12.4H20
NaMo04.21-120
CuSO4.5H20
Co(NO)3.6H20
- 2.86
- 1.81
- 0.39
- 0.079
- 0.0494
One ml of Solution II and 5 ml of Solution I were mixed and
made upto 1 litre for the preparation of BC 11 media. The prepared
media was further dispensed into suitable glasswares and sterilized in
an autoclave prior to inoculation. 1.5% agar was added for the
preparation of BG11 agar medium.
All the inoculated flasks were incubated in a sterile condition2s 'moIm-'S-'
for 30 days under light intensity with 12 hours light / 12
hours dark photoperiod. Triplicates were maintained for each sample.
After incubation, cyanobacterial growth in the sides, bottom of the
flasks and top of the medium were taken for identification by using
phase contrast microscope (40 x). Mixed cultures of cyanobacteria were
further purified by standard serial dilution and plating method. The
axenic culture of cyano bacterial symbionts were identified at species
level at National Facility for Marine Cyanobacteria (NFMC),
Bharathidasan University, Tiruchirapalli, Tamil Nadu, India and also
by following the method of Deshikachary (1959). Identified
cyanobacterial symbionts were deposited at NFMC and Centre for
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Marine Science and Technology, Division of Marine Microbiology and
Medicine, Tamil Nadu, India, with appropriate identification number.
The 'MSU' denotes Manonmaniam Sundaranar University, '5' denotes
Shiefa 'R' denotes Ravikumar and 'C' denotes Cyanobacteria.
3.2.4. Preparation of sponge tissues for identification
Sponge identifications are primarily based on morphology.
Some of these morphological characters vary substantially between
widely separated populations, or those living in different habitats,
whereas other features are much more consistent between individuals
irrespective of their geographic distribution. Sponge identifications
require spicule preparation to determine the diversity and geometry of
spicules in the skeleton (Hooper, 2000). Spicule preparations were
carried out by the following steps:
1. Thin sections of sponge tissue were taken in a Durham tube and
drops of concentrated nitric acid were added to the tube.
2. After vigorous reaction, another drop of acid was added. This
step was repeated several times so as to control the reaction and
production of oxide by-products.
3. After acid digestion, enough nitric acid was added to nearly fill
the tube and heated over by alcohol flame for 1 - 2 minutes.
4. Centrifugation was done at 4000 rpm for 30 seconds after
cooling.
5. Nitric acid was pipetted off leaving the spicule mass at the
bottom of the tube undisturbed.
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6. The tube was then refilled with fresh nitric acid and the spicules
were resuspended using clean, fine glass rod.
7. Again steps 3 - 5 were repeated.
8. The tube was filled firstly with demineralized water, 70%
ethanol, and then two series of 100% ethanol solutions.
Resuspending the spicules, centrifuging and decanting the
supernatant were done between each change of solution and
finally the spicules were suspended in a solution of absolute
ethanol.
9. Couple of drops of spicule solution was pipetted on to the cover
glass and spreaded out with a glass rod until all ethanol
vapourized.
10. Distribution of spicules on the cover glass was monitored under
compound microscope.
Further confirmation of sponge species were done by using SEM
pictures at Institute of Wurzburg, Germany. The procedure for the
preperation of mounts for SEM is given below (Hooper, 2000)
1. 1 - 1.5 mm thick section of sponge tissue was cut and placed in
cavity block.
2. The cavity block was covered with several drops of sodium
hypochlorite to etch the collagen from skeleton. Generally 30
seconds time is adequate for bleach.
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3. The bleach was pipetted off at the appropriate time and 70%
ethanol was added immediately and allowed to stand for
several minutes, till the bleach was completely neutralized.
4. The section was then placed on clean microscope slide and
dried.
5. Section was mounted on SEM stub with double-sided tape.
6. The specimen was then sputter -coated and viewed at 25 W.
3.3. Results
Three sponge samples from each collection sites at every month
were examined for the species of cyanobacteria associated with the
host organisms. Of these samples collected, three cyanobacterial
species viz. Phormidium angustissimum (MSUSRC1), Chroococcus minor
(MSUSRC2) and Oscillatoria amphibia (MSUSRC3) were found
associated with three sponges species viz., Sigmadocia carnosa
(MSUSR3), Callyspongia fibrosa (MSUSR2) and Colloclathria sp
(MSUSR1). The systematic position and identification parameters are
represented here under.
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3.3. 1. Identification of sponges
a) Colloclathria sp. (MSUSR1)
Taxonomic position
Phylum: Porifera Grant
Order : Poeciloscierida Topsent
Class : Demospongiae Sollas
Family : Phorbasidae de laubenfels
Genus : Colloclathria
Identification characters
Sponge composed of repent branches, 2 - 4 mm in diameter.
Texture firm and compressible. Surface uneven and hispid. Spicule size
0.3 x 0.019 mm.
b) Call ysporigiafibrosa (MSUSR2)
Identification characters
Taxonomic position
Phylum: Porifera Grant
Order : Haploscierida Topsent
Class : Demospongiae
Family : Callyspongiidae de Laubenfels
Genus : Callyspongia
Species: fibrosa
Sponge composed of massive anastomizing branches or of a
series of low tubes arising from a basal mass surface smooth or
ornamental with spines, oscules in branching forms, texture firm and
only slightly compressible, spicular oxten, 0.1 to 0.15 by 0.005 to 0.008
mm.
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Aq. I
' !
c) Sigmadocia carriosa (MSUSR3)
Taxonomic position
Phylum:
Order
Class
Family:
Genus:
Porifera Grant
Haplosclerida Topsent
Demospongiae Sollas
Adociidae de Laubenfels
Sigmadocia
Species: ccirnosa
Identification characters
The species is a proliferous mass of slender anastomizing tubes,
fused to greater or lesser extent to form a lamellar mass bearing
numerous tubular processes each terminating in a vent, surface
smooth, even, texture, soft, compressible, width of about 10 cm and
height of about 12 cm. Spicules area 0.12 to 0.14 by 0.006 to 0.008 mm
and stigmata 0.017 to 0.02 mm chord.
3.3.2. Identification of sponge associated cyanobacteria
a) Phormidium artgustissimum (MSUSRC1)
_r Taxonomic position
Class : Cyanophyceae
Order : Nostocales• 1.49, RXFamily : Oscillatoriaceae
Genus : Phormidiurn
kSpecies: augustissirnum
Identification characters
Thallus leathery, thin, pale blue green, trichome bent entangled, end
not attenuated, straight, 0.6 - 0.8 i broad, sheath colourless , cells
longer than the broad, septa not granulated, apical cell not capitate.
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b) Chroococcus minor (MSUSRC2).
Taxonomic position
Class : Cyanophyceae
Order : Chroococcales
Family: Chroococcaceae
Genus : Chroococcus
Species: minor
Identification characters
-o• e •' pP 0 -
a -a •' -, ' ,(_ .
1.p
I I.'
- p
p- - •
p . •-C. ••
'I •
.
r - -
•. •.',0? -
• •a' o-. -
Thallus slimy-gelatinous, dirty blue-green, cells spherical, 3 - 4
pm in diameter, single or in pairs, seldom 4 or 8, sheath colourless,
very thin hardly visible.
c) Oscillatoria amphibia (MSUSRC3)
Taxonomic position
Class : Cyanophyceae
Order : Nostocales
Family: Oscillatoriaceae
Genus : Oscillatoria
Species: amphibia
Identification characters
Thallus deep blue green, trichome straight, apices not attenuted,
not constricted at cross walls, 2 — 3 p broad, cells 2 — 3 times longer
than broad, 4 — 8 p long. 2 granules at the septa, end cells rounded not
capitate, calyptra absent.
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Scanning Electron Microscope Pictures showing Sponge andassociated Cyanobacterial symbionts
• •:
1b'•.
•'';is•*
:
Phormidiurn angustissimurn on Siginadocia carnosa
49'
Oscillatoric, amphibia on Colloclathria sp.
Chroo coccus minor on Callyspongia fibrosa
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3.3.3. Species composition
The frequency of occurrence of sponge associated cyanobacterial
species in Ovari site at different months reveals that, the Ph.
angustissimum (MSUSRC1) was reported from February to March, June
to November; 0. amphibia (MSUSRC3) was reported during different
months of collection except January, April, May, August, November
and December. Whereas, C. minor (MSUSRC2) was reported from
February - March, June - July and September - October (Table 2).
Table 2. Monthly occurrence of sponge associated cyanobacterial species in Ovari
CyanobacterialJan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec
species
Ph. angustissiniurn- + + - - + + + + + + -
(MSUSRCI)
C,rninor- + + - - + + - + + -
(MSUSRC2)
O.arnphibici- + + - - + + - + + - -
(MSUSRC3)
(+) Reported ; (-) Not reported
On the other hand, the frequency occurrence of Ph.
angustissimum (MSUSRC1) and 0. amphibia(MSUSRC3) in Kuttankuzhi
site was reported during January - February, June, September to
December and C. minor(MSUSRC2) was reported at different months
of collection except March, April, May and August (Table 3).
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50
45
30
25
20CD
15
10
e 5
a. 0
Table 3. Monthly occurrence of pngociated çyobacterial s pecies inKuttankuzhi
CyanobacterialJan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Decspecies
Ph. angustissimum+ + - - + - - + + + +(MSUSRC1)
C.mi nor+ + - - - + - - + + + +(MSUSRC2)
O.amphibia+ + - - - + + - + + + +(MSUSRC3)
I-t-) Keportea; -) INOt reportea
It is obvious from the present study that, three species of
cyanobacteria were reported from two collection sites. But, the
occurance of cyanobacterial species varies with the month of collection.
Moreover, the occurrence of sponge associated cyanobacteria at
different seasons indicates that, the percentage occurrence was found
higher during the South West monsoon followed by North East
monsoon in Ovari site. Whereas, the percentage of occurrence in
Kuttankuzhi site was found higher during the North East monsoon
followed by South West monsoon (Fig. 2 and 3).
Fig.2.Seasonal variation of sponge associated cyanobacterial symbionts in Ovari
Non monsoon South west North eastmonsoon monsoon
Seasons
Values are 5% level significant (5.36)
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544
..- 3o .) 3
11,
Fig.3. Seasonal variation of sponge associated cyanobacteriail symbionts in Kuttankuzhi
Non monsoon South west North eastmonsoon monsoon
Seasons
Values are 5% level significant (5.14)
Another interesting finding in the present study that, the
associations of cyanobacterial species vary with sponge species. For
instance, Ph. angustissimum (MSUSRC1) is specifically associated with
Sigmadocia carnosa (MSUSR3), 0. amphibia (MSUSRC3) is particularly
associated with the Colloclathria species (MSUSR1) and Chroococcus
minor (MSUSRC2) has specific growth on the sponge Callyspongia
fibrosa (MSUSR2) irrespective of the study sites (Tables 4 and 5).
Table 4. Cyanobacterial species diversity on different species of sponges in Ovari
Ph. angustissimum C. minor O.amphibiaName of the sponge(MSUSRC1) (MSUSRC2) (MSUSRC3)
Colloclathria sp. - - +(MSUSR1)
Call,Ispongiafibrosa - + -(MSUSR2)
Sigmadocia carnosa+ - -
(MSUSR3)
(+) Reported;(-) f'.Jot reported
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Table 5. Cyanobacterial species diversity on different species of sponges in Kuttankuzhi
Ph. angustissimum C. minor O.amphibiaName of the sponge (MSUSRC1) (MSUSRC2) (MSUSRC3)
Colloclathria sp. - - +(MSUSR1)
Callyspongiafibrosa - + -(MSUSR2)
Sigmadocia carnosa + - -(MSUSR3)
(+) Reported; (-) Not reported
ME
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3.4. biscussion
Studies in marine sponges and the microbial symbionts are
considered to be very important in both an ecological and
biotechnological view point. Marine eukaryotes and associated bacteria
should provide ideal system in which to consider microbial
biogeography as (i) bacteria in seawater should be able to disperse
among the individual of the same host yet (ii) potential for adaptation
to particular host. In the present study, the frequency in the occurrence
of cyanobacterial species showed little variation in relation to stations
and sponge species. However, the occurrence of sponge associated
cyanobacteria at different sites reveals that, the percentage occurrence
was found higher in sponges during the South West and North East
monsoon than in the non-monsoon season. Burlando (1988) found that,
the bacteria of genus Cryptophaga was found to be associated with the
sponge Cia thrina cerebrum during the cold season and disappear in the
summer. This might be a facultative relationship where the symbionts
are selected from the surrounding seawater. The present study also
found that, irrespective of the seasons, the occurrence of
Ph. angustissimum (MSUSRC1), 0. amphibia (MSUSRC3) and C. minor
(MSUSRC2) also depends upon the host tissues. This supports the
findings of Friedrich et al. (1999), where a specific association of
morphologically similar bacteria on the Aplysina cavernicoia. Moreover,
the sponge, Rhopaioeides odorabiles have specific associates with
particular bacterial strain (NW001) (Burja et al., 1999; Webster and Hill,
2001).
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It is also inferred from the present study that, the sponge
microbe association is not differed with the place of collection. Webster
et al. (2001) reported that, the occurrence of bacterial communities from
five sponge species, Kirpartrickia varialosa, Latrunculia apicalis,
Homaxinella balfourensis, Mycale ace rata and Sphaerotylus antarcitus,
collected from different Antartic sites were consistently detected
within particular species regardless of the collection sites. Similarly the
association of bacterial species with the Cymbastela concentrica sponge
was found similar over a 500 km distance (Taylor et al., 2005).
28