ISOLATION AND IDENTIFICATION OF

SPONGE ASSOCIATED

CYANOBACTERIAL SYMBIONTS

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.

8

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

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

10

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

11

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

12

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.

13

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

14

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

15

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

16

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.

17

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.

18

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.

19

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.

20

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.

21

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.

22

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

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).

23

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)

24

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

25

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

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).

27

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