Comparative morphometry, biochemical and elemental composition of three marine sponges (Petrosiidae) from Gulf of Mannar, India. Chemical Speciation and Bioavailability 23, 16–23

Comparative morphometry biochemical and elemental

composition of three marine sponges (Petrosiidae) from

Gulf of Mannar India

Ramjee Pallelaac Srikanth Koigooraa Venu Gopal GundaaMadhavendra Sakunthala Sunkarab and Venkateswara Rao Janapalaa

aToxicology Unit Biology Division Indian Institute of Chemical Technology Hyderabad - 500 607 IndiabElectron Microscopy Division Indian Institute of Chemical Technology Hyderabad - 500 607 IndiacMarine Bioprocess Research Center Pukyong National University Busan - 608 737 Republic of Korea

E-mail jvraoiictgmailcom

ABSTRACT

Three marine sponges Neopetrosia similis Xestospongia testudinaria and Petrosia nigricans from the Gulfof Mannar Southeast coast of India were compared based on their morphometric parameters and theirbiochemical and elemental composition These sponges showed differences in spicule protein (spongin)ratio of 5 1 11 1 and 13 1 respectively Xestospongia testudinaria possessed the longest oxeae 305times than P nigricans and 147 times than N similis Spectral analysis revealed that the spicules of thesesponges are mostly composed of O (450) and Si (29ndash45) whereas Al and Ca (4ndash5) were additionallydetected only in the spicules of P nigricans In contrast the percentage elemental composition inpinacodermal sections was significantly different as O and Si were the maximum (23ndash50) followedby Cl and Al as moderate (1ndash17) and Na S Fe and Ca in minor quantities (02ndash6) Cluster analysisand traditional taxonomic arrangements show that X testudinaria and N similis have a close relationshipwhereas P nigricans was hypothesized to be their sister group The present findings could be the key foridentifying sponges in situ as well as determining whether sponges could be used to assess pollution inthe sea

Keywords marine sponges petrosiidae spicules elemental composition Gulf of Mannar

INTRODUCTION

Sponges (Porifera) are the most ancient metazoans with

ubiquitous benthic distribution They are distributed along

all latitudes from intertidal to deep-sea Many sponge

species produce toxic substances enabling their survival in

competitive marine environments Some of the metabolites

have beneficial pharmaceutical effects for humans with anti-

inflammatory cytotoxic antitumor antibiotic and anti-viral

activities (Scheuer 1978 ndash 1983 Faulkner 1984 Uemura

et al 1985 Kim et al 1998 Gunasekera et al 1990)

Recently several researchers have tried to develop in vitro

cultivation systems for sponges (Demospongiae) to

produce compounds of medicinalypharmaceutical value in

biotechnological processes without disturbing the

ecosystem Most of these workers collect the specific

sponge species for microbial and sponge cell cultures

The taxonomical identification of specimens during the

collection period is a complex task More complete infor-

mation on the anatomy and morphometry of sponges may

be helpful to identify the collected sponges up to genus

level

The majority of sponges are divided into three classes

viz Calcarea (sponges with spicules composed of calcium

carbonate) Hexactinellida (the glass sponges with siliceous

spicules) and Demospongiae (having opaline or anhydrous

siliceous spicules andyor proteinaceous fibres) according to

the composition of their skeleton (Bergquist 2001 Hooper

et al 2002) Demosponges possess leuconoid structure

with folded choanoderm and continuous pinacoderm

Formation of the mesohyl is more diverse in the

Demospongiae which are characteristically thickened with

spicules of both mega and microscleres with one to four

rays and dispersed collagenous fibres (spongin) both or

neither (Harrison and de Vos 1991) Sponge taxonomists

have focused their attention on understanding the character-

istic skeletal organization in order to identify individual

species On the other hand cell biologists are also investi-

wwwchemspecbiocouk

doi 103184095422911X12966340771966

16 Chemical Speciation and Bioavailability (2011) 23(1)

gating these primitive metazoans to understand their orga-

nization on the cellular and skeletal level for more enhanced

biotechnological applications (Muller et al 2004) Besides

the intrinsic components demosponges can incorporate

sediments and foreign substances into their spicules

skeleton (Araujo et al 1999) Previous studies have

demonstrated that accumulation of elements in marine

sponges can be used as a biomarker to assess pollution

risks and ecosystem health in the ocean (Venkateswara Rao

et al 2006 2007 2008) Therefore species identification

based on morphological and elemental composition would

further facilitate sorting of species for use in applied

biotechnology

Numerous biologically active molecules have been

isolated from sponges belonging to the family Petrosiidae

(Kim et al 1998 Giner et al 1999 Aoki et al 2002

Venkateswarlu et al 1993 Choi et al 2004 Blunt et al

2006 Park et al 2007) The sponges included in this

family have thus been valuable to chemists and have also

found their way into biotechnological applications

However many genera have been included in this family

and characterizing the discriminating features between the

species is essential to identify specific genera Hence here

qualitative and quantitative morphological variations and

elemental compositions of three sponges belonging to

Petrosiidae collected at Gulf of Mannar India are

compared According to the World Sponge Database

these three sponges are identified as Xestospongia testudi-

naria Lamarck 1815 Neopetrosia similis Ridely and Dendy

1886 and Petrosia nigricans Lindgren 1887

(Desqueyroux-Faundez and Valentine 2002) Previously

they were considered as three different species under

Petrosia genera ie P testudinaria P similis and P

nigricans respectively The structural dissimilarities

between these three taxa were analysed using advanced

microscopy The present findings on the morphological and

elemental variations between the taxa are essential for in

situ identification and form a basis for in vitro cellyfragment

cultures for producing bioactive secondary metabolites

(Gunda and Janapala 2009)

METHODOLOGY

Sampling

Sponge samples were collected during low tide from the

shallow sub-tidal regions between 15 and 25 feet by

snorkelling and skin-diving The sponges were collected

at the Mandapam region (Lat 9 10 0 to 9 50 0 N Long 78

10 0 to 79 07 0 E) of the Gulf of Mannar Biosphere Reserve

India (Figure 1) Sponges were gently removed from the

substratum without any tissue damage and were placed in

plastic bags underwater then transferred into large

containers of aerated seawater for transport to the labora-

tory Before analysis the sponges were thoroughly cleaned

by mechanical removal of foreign materials followed by

repeated washing with artificial sea water (Millero 1996)

The voucher specimens were submitted to the National

Institute of Oceanography (NIO) Goa for depository

purposes and were identified as Xestospongia testudinaria

Neopetrosia similis and Petrosia nigricans (Class

Demospongiae Order Haplosclerida Family Petrosiidae)

at Vizhinjam Research Centre of Central Marine Fisheries

Research Institute (ICAR) Vizhinjam

Thiruvananthapuram India Based on our earlier expedi-

tions (2000 to 2007) it was noticed that only these three

genera of Petrosiidae family exist at the Mandapam coast of

the Gulf of Mannar India

Ramjee Pallela Srikanth Koigoora Venu Gopal Gunda Madhavendra Sakunthala Sunkara and Venkateswara Rao Janapala 17

Figure 1 Location Map of sponge sampling site (Mandapam region Gulf of Mannar Southeast Coast of India)

Morphology and architecture of the sponges

The differential surface views of individual sponges and

their tangential sections of the choanosome were observed

with the help of a video microscope (High scope Compact

Micro vision system Model No KH-2200 MD2) and

digital photographs were obtained

Morphometry of spicules

Specified pieces of individual species consisting of two

primary layers of cells (pinacoderm and choanoderm) and

an inner cellular region (mesohyl) were (a minimum of

three replicates each) digested with concentrated HNO3 and

allowed to stand for 2 ndash 3 h until all the pieces were

dissolved and then heated gently over a Bunsen flame

until the liquid was clear The aliquots were centrifuged

at 5000 g for 10 min and the precipitates were re-centrifuged

after every wash with distilled water for three times

Additional organic matter was removed with H2O2 and

finally washed with absolute ethanol and air dried The air-

dried spicules on microscopic glass slides were mounted

with a DPX mount and then covered with a glass cover slip

to study the length and width of spicules (n frac14 500) of

individual sponges by a compound microscope

(POLYVAR Reichert- Jung light microscope) attached to

Ethovision-version 23 (Noldus Information Technology

The Netherlands) through a CCD camera (Sony CCD

IRIS Model No SSC-M370CE) The magnification was

calibrated with the aid of ocular and stage micrometers

(ERMA Tokyo Japan)

Analytical methods

The elemental composition of pinacoderm (Al Ca Cl Fe

Na O S and Si) and spicular membrane (silicalemma) in

each test sponge (n frac14 5) was analysed by HITACHI S-520

scanning electron microscope equipped with energy disper-

sive X-ray analysis (EDXA) Briefly the processed samples

were mounted on aluminium stubs using double adhesive

tape coated with gold in HITACHI HUS-SGB Vacuum and

observed in Hitachi S-520 SEM Then EDXA was carried

out with an Oxford Link ISIS-300 detector calibrated with

cobalt standard at an acceleration voltage of 20 kV Built-in

standards were used for the quantification of each element

For quantifying the total protein and carbohydrates 1 g of

each individual sponge species was chopped into small

pieces and homogenized in sufficient volumes of phosphate

buffer by the Heidolph DIAX 900 homogenizer The total

carbohydrate content was assayed by the phenolndash sulfuric

acid method (Taylor 1995) whereas the total protein was

estimated by Bradfordrsquos method (Bradford 1976)

Data analysis

The mean value and standard error (+ SE) of the length

and width of spicules percentage amount of spicules per

gram (dry weight) total protein content and elemental

composition in silicalemma and pinacoderm were calcu-

lated for each variable of the independent species The

suitability of the data was evaluated to examine the indi-

vidual variables for the differences among test species by

one-way analysis of variance (ANOVA) When populations

were significantly different multiple comparison post-hoc

tests (Tukeyrsquos HSD) were performed to see which popula-

tions differed from one another

Phylogenetic relationships among the three sponges of

lsquoPetrosiidaersquo family were analysed using STATISTICA

software (STATSOFT Inc Version 60) The percentage

ratios of individual parameters such as pentose hexose

spicule length width and lengthywidth and silicon ratio of

spicule versus pinacoderm were taken in multiple replicates

as the input to analyse the phylogenetic relationships among

the test sponges For each parameter cluster analysis (CA)

was used to elucidate the closest potential of the measured

cluster between the three genera where CA was represented

as hierarchical tree plots and a dendrogram was prepared

based on the single linkage (nearest neighbour) method

RESULTS

Morphology and architecture of the sponges

The three test species analysed in the present experiments

were collected at the same depth and locality under identical

environmental conditions The current study is mainly

based on the distinct morphological and biochemical differ-

ences between these sponges (Table 1) The external

morphology of each species varies from thick encrusting

globular to a more massive form and the colour patterns are

from a brown-beige colour to blackish blue often asso-

ciated with well-lit environments The external surface of X

testudinaria is smooth and compact which appears to be

covered by a fine ectosomal layer Small bundles of slender

oxea are grouped at nodes to give granular surface in N

similis However the architecture of P nigricans was

totally different from the other species by the zig-zag

brushy arrangement of oxeae

It is evident from the video microscopic images that a

significant difference in the surface view was found

between the three species The images indicated that the

differences in the architecture of walls surrounded by ostia

were quite distinctive between the sponges When

compared to the other two sponges X testudinaria is

hispid to touch with clear openings of ostia surrounded

by precise arrangement of spicules (oxeae) bounded with

spongin In addition a number of large and slender oxeae

crowded as rounded meshes were present where dense

interstitial reticulation of free spicules offered the sponge

a stony texture (Figure 2)

Morphometry of spicules

All the test species possessed similar type of oxea spicules

(85 ndash 95) but differed in their sizes (Figure 3) The mean

lengths (L) and widths (W) of oxeae among the three

species were statistically different (L One way ANOVA

Ffrac122597 frac14 46365 Tukeyrsquos HSD P50001 and W

Ffrac122597 frac14 32245 Tukeyrsquos HSD P50001 respectively)

It is evident from the Table 1 that the length and width

ratios did not differ significantly (P5005) The LyW ratio

of X testudinaria was comparatively high (3703) while

18 Elemental composition of three marine sponges in Gulf of Mannar

compared to other two genera P nigricans and N similis

(3061 and 3585 respectively)

Analytical methods

The amount of protein (mostly referred to as spongin)

required for binding the spicules to form unique skeletal

architecture is significantly different in all the species

Comparatively the amount of protein estimated in N

similis was 35-fold higher than P nigricans and 16-fold

higher than X testudinaria The spicule protein ratio

(mg g 1) between the species was significantly different

at P5001 at 13 1 11 1 and 5 1 in P nigricans X

testudinaria and N similis respectively However the

pentose and hexose (carbohydrate) contents were signifi-

cantly higher (415 times) in P nigricans than in the other

two sponges

The composition of the inorganic envelope (silicalemma)

of spicules and uptake of the elements in the pinacosomal

sections of each test sponges was analysed by SEM

equipped with an EDXA which indicated that the silica-

lemma of spicules in all test species was composed almost

exclusively of O (55 ndash 61) and Si (36ndash 49) however Al

and Ca (4 ndash 5) were additionally quantified in P nigri-

cans The mean percentage composition of O in spicules

did not differ significantly (ANOVA analysis

Ffrac12212 frac14 0033 P frac14 0968) However the percentage accu-

Ramjee Pallela Srikanth Koigoora Venu Gopal Gunda Madhavendra Sakunthala Sunkara and Venkateswara Rao Janapala 19

Figure 2 Video micrographs of surface view and choanosomal sections of three marine species a Petrosia nigricans b Neopetrosia similis

c Xestospongia testudinaria 1 surface view (406) 2 section of choanosomal layer (406) and 3 at 220X

Figure 3 Micro morphological variations (by light microscopy

and scanning electron microscopy) in the spicules (oxeae) of three

different Petrosiidae sponges

mulation of Si in X testudinaria was significantly different

in P nigricans at P5005 (Ffrac12212 frac14 2708 P frac14 0107) It

is evident from the results that the relatively high composi-

tion of O was noticed in the spicular envelopes of P

nigricans N similis and X testudinaria (167 123 and

113 respectively) compared with that of Si (Figure 4)

The elemental analysis of surface pinacosome of the

three genera indicated the accumulation of elements such

as Al Ca Cl Fe Na O S and Si in different proportions

The order and range of percentage accumulation of the

elements in these genera were oxygen (28 ndash 50) and silicon

(23 ndash 35) as maximum chlorine (8 ndash 16) and aluminium (1 ndash

17) as moderate and sodium (3 ndash 6) sulfur (06 ndash 14) iron

(02 ndash 21) and calcium (03 ndash 27) in minor quantities One

way ANOVA (p5005) and mean values were compared

using Tukeyrsquos HSD pair wise comparison test which

indicated that O and Na are not significant among P

nigricans and N similis While comparing N similis and

X testudinaria all the elements are significantly different

except S (Figure 5)

The majority of chosen parameters (hexose pentose

spicule length and the ratios of spicule lengthywidth and

silicon of silicalemmaypinacoderm) used to study the

phylogenetic relationship through dendrogram analysis

between the sponges revealed that X testudinaria and N

similis are more closely related as compared to P nigricans

(Figure 6) It is apparent from the results that morphometric

biochemical and elemental parameters could also be used as

markers to assess the taxonomic similarities and dissimila-

rities between the sponges

DISCUSSION

This is the first study to show distinct morphological and

structural variations in the spicule morphometry and

aquiferous outlook of sponges (Family Petrosiidae) avail-

able from the Gulf of Mannar It is evident that the test

species were visually different and exhibited significant

variations in their ostia (number shape and diameter) and

morphometry of oxeae The protein (spongin) and carbo-

hydrate components differed between the sponges These

components are responsible for binding the spicules to

build up a specialized shape of ostia that influence the

water flow into the sponge body It is well known that

each genus has its own architecture as demonstrated by the

different morphology of ostia This has been demonstrated

and documented between the seven species collected at

SW Sulawesi reefs (Eastern Indonesia) which have char-

acteristic differences in their shape and sizes of ostia (de

Voogd and van Soest 2002)

The striking difference in percentage spicules by dry

weight in X testudinaria was relatively higher (amp 2 fold)

than P nigricans and 16 fold than N similis Also X

testudinaria possessed 305 times longer spicules than P

nigricans and 147 times that of N similis The growth of

the spicule is mainly based on specialized cells called

sclerocytes and axial filament which is formed by silica-

teins responsible for elongation of spicules and their

width is enhanced by the apposition of the silica

(Garrone 1978 Simpson 1984) In addition Aizenberg

et al (1996) concluded that intra-crystalline macromole-

cules (protein carbohydrates etc) play an important role

by their direct or indirect apposition and deposition to

form canalicular tracts of the sponge skeleton

EDXA of inorganic envelope spicular silicalemma and

the pinacodermal surface of the three sponges exhibited

significant changes in elemental composition The present

results indicated that some of the elements are limited to

the surface but many more might have been accumulated

in body wall The EDXA analysis is restricted only to

surface elemental composition and not to the tissue

accumulation which may be a key to identify the different

genera of Petrosiidae family It is evident from our earlier

ICP-MS analysis that more number of metals of higher

amount were quantified in P testudinaria (X testudinaria)

(Venkateswara Rao et al 2006) The elemental variation

between individual sponges between the species is well

documented (Patel et al 1985) and these variations in

elemental deposition may reflect the anatomical differences

of the individual sponges (Garrone et al 1981 Simpson

1984)

DNA based markers have been efficiently used in the

phylogenetic analyses of sponges Molecular biologists are

working on the sponge genome to understand the mole-

cular mechanism of evolution of metazoan genes and

diseases but knowledge of their biological and ecological

characteristics is so far very limited (Yi et al 2005)

Further analyses using specific loci have provided informa-

tion on the reproductive isolation of the species which

reinforced the conclusion that spicular morphology would

20 Elemental composition of three marine sponges in Gulf of Mannar

Figure 4 The percentage elemental composition of inorganic

envelope (silicalemma) in three sponge species (Family Petrosii-

dae) under sympatric conditions at Gulf of Mannar Statistical

analysis was determined using ANOVA (P5005) and means were

compared using Tukeyrsquos HSD pair-wise comparisons test Statis-

tical significance as compared to P nigricans ns no significant

difference (P5005)

Ramjee Pallela Srikanth Koigoora Venu Gopal Gunda Madhavendra Sakunthala Sunkara and Venkateswara Rao Janapala 21

Figure 5 Percent elemental uptake in the pinacosomal layer of each sponge species (Family Petrosiidae) under sympatric conditions at Gulf

of Mannar Statistical analysis was determined using ANOVA (P5005) and means were compared using Tukeyrsquos HSD pairwise comparisons

test Statistical significance as compared to P nigricans (a) Statistical significance as compared to N similis (b) ns no significant

difference (P5005)

Figure 6 Dendrogram as obtained from average linkage cluster analysis (STATISTICA Ver 60) using the parameters of morphometry

biochemical and elemental analysis to assess the phylogenetic relationship between the three sponges of the Petrosiidae family

be useful in discriminating Petrosia spp (Bavestrello and

Sara 1992) In the present work we have analysed the

phylogenetic relationship based on the relative morpho-

metry biochemical and elemental composition which is a

new sequential approach to assess the linkage evidence

among the sponges The current non-genetic approach is

simple and may be useful to non-specialists as it does not

require the use of traditional genetic tools The present

comparison gives us the opportunity to analyse phyloge-

netic relationships thus giving useful insights for the

identification of sponges by non-taxonomists

As the demand for pharmacologically potent natural

products is constantly increasing attempts for the biotech-

nological production of sponge tissues are being made

Several studies suggest secondary metabolite production

by sponge symbionts (Molinski 1993 Oclarit et al 1994

Bewley et al 1996 Hentschel et al 2001) whereas

others indicate their production by sponge cells (Uriz

et al 1996 ab Garson et al 1998 Turon et al 2000)

Sponges possess strong regenerative capacities (Simpson

1984) and pieces of live sponge tissue are able to grow

and regenerate into healthy sponges This potency has

been used on a broad range of sponge species for the

cultivation of sponge tissue samples in both half-open

systems and open sea aquaculture (Osinga et al 1999)

CONCLUSION

Marine demosponges have been widely utilized for their

economical pharmaceutical as well as taxonomical

purposes in the past few decades The present observations

are particularly remarkable for ascertaining in situ discri-

mination of three members of Petrosiidae by observing

their morphological features along with their spicular and

anatomical aspects by advanced microscopic analyses

From our present and previous results we can conclude

that the sponges available at the Gulf of Mannar are

undergoing a timely anthropogenic disturbance that made

these organisms potential bio-indicators In their turn the

morphometric characteristics of these findings could be a

possible key for the chemical and analytical speciation of

future species available in any marine environment

ACKNOWLEDGMENTS

The authors thank the Director IICT for providing the

facilities and constant encouragement throughout the study

and also to Task-force project (CMM 004) of Council of

Scientific and Industrial Research (CSIR) which is the

funding source for the present work We are also grateful

to Mr T Sathish Bioengineering and Environmental

Centre IICT for calculating phylogenetic relationship

among the sponges The authors RP SK and GVG

thank CSIR for providing Senior Research Fellowships

REFERENCES

Aoki S Naka Y Itoh T Furukawa T Rachmat R Akiyama

S and Kobayashi M (2002) Lembehsterols A and B novel

sulfated sterols inhibiting thymidine phosphorylase from the

marine sponge Petrosia strongylata Chem Pharm Bull

50(6) 827 ndash 830Aizenberg J Ilan M Weiner S and Addadi L (1996) Intra-

crystalline macromolecules are involved in the morphogenesis

of calcitic sponge spicules Conn Tiss Res 34(4) 255 ndash 261

Araujo MF Cruz A Humanes M Lopes MT da Silva JAL

and da Silva JJRF (1999) Elemental composition of Demos-

pongiae from the eastern Atlantic coastal waters Chem Spec

Bioavail 11(1) 25 ndash 36Bavestrello G and Sara M (1992) Morphological and genetic

differences in ecologically distinct populations of Petrosia

(Porifera Demospongiae) Biol J Linn Soc 47(1) 49ndash 60

Bergquist PR (2001) Porifera (sponges) encyclopedia of life

sciences John Wiley and Sons Ltd New York

Bewley CA Holland ND and Faulkner DJ (1996) Two classes

of metabolites from Theonella swinhoei are localized in

distinct populations of bacterial symbionts Cell Mol Life

Sci 52(7) 716 ndash722Blunt JW Copp BR Munro MHG Northcote PT and

Prinsep MR (2006) Marine natural products Nat Prod

Rep 23(1) 26 ndash 78Bradford MM (1976) A rapid and sensitive method for the

quantitation of microgram quantities of protein utilizing the

principle of protein-dye binding Anal Biochem 72(1 ndash 2)

248 ndash 254Choi HJ Bae SJ Kim ND Jung JH and Choi YH (2004)

Induction of apoptosis by dideoxypetrosynol A a polyacety-

lene from the sponge Petrosia sp in human skin melanoma

cells Int J Mol Med 14(6) 1091ndash 1096de Voogd NJ and van Soest RWM (2002) Indonesian sponges

of the genus Petrosia vosmaer (Demospongiae Haploscler-

ida) Zoologische Mededelingen (Leiden) 76(16) 193 ndash 209

Desqueyroux-Faundez R and Valentine C (2002) Family Petro-

siidae van Soest 1980 In Hooper JNA and van Soest

RWM (eds) Systema Porifera A guide to the classification

of sponges pp 906 ndash917 Kluwer AcademicyPlenum Publish-

ers New YorkFaulkner DJ (1984) Marine natural products metabolites of

marine invertebrates Nat Prod Rep 1 551 ndash598Garrone R (1978) Phylogenesis of connective tissue Morpholo-

gical aspects and biosynthesis of sponge intercellular matrix

In Robert L (ed) Frontiers of matrix biology Vol 5 pp 1 ndash

250 Karger Press BaselGarrone R Simpson TL and Pottu-Boumendil J (1981) Ultra-

structure and deposition of silica in sponges In Simpson TL

and Volcani BE (eds) Silicon and siliceous structures in

biological systems pp 495 ndash 525 Springer-Verlag New York

Garson MJ Flowers AE Webb RI Charan RD and McCaf-

frey EJ (1998) A spongeydinoflagellate association in the

haplosclerid demosponge Haliclona sp cellular origin of

cytotoxic alkaloids by Percoll density gradient fractionation

Cell Tiss Res 293(2) 365 ndash373Giner JL Gunashekar SP and Pomponi SA (1999) Sterols of

the marine sponge Petrosia weinbergi implications for the

absolute configurations of the antiviral orthoesterols and

weinbersterols Steroids 64(12) 820 ndash824Gunasekera SP Gunasekera M Longley RE and Schulte AK

(1990) Discodermolide a new bioactive polyhydroxylated

lactone from the marine sponge Discodermia dissolute J

Org Chem 55(16) 4912ndash 4915

22 Elemental composition of three marine sponges in Gulf of Mannar

Gunda VG and Janapala VR (2009) Effects of dissolved oxygen

levels on survival and growth in vitro of Haliclona pigmenti-

fera (Demospongiae) Cell Tiss Res 337(3) 527 ndash 535

Hooper JNA van Soest RWM and Debrenne F (2002)

Phylum Porifera Grant 1836 In Hooper JNA and van

Soest RWM (eds) Systema Porifera A Guide to the

classification of sponges pp 9ndash 13 Kluwer Academic

Plenum Publishers New York

Harrison FW and de Vos L (1991) Porifera Vol 2 In Harrison

FW and Westfall JA (eds) Microscopical anatomy of

invertebrates pp 29 ndash 89 Wiley-Liss New York

Hentschel U Schmid M Wagner M Fieseler L Gernert C

and Hacker J (2001) Isolation and phylogenetic analysis of

bacteria with antimicrobial activities from the Mediterranean

sponges Aplysina aerophoba and Aplysina cavernicola FEMS

Microbiol Ecol 35(3) 305 ndash 312

Kim JS Im KS Jung JH Kim YL Kim J Shim CJ and

Lee CO (1998) New bioactive polyacetylenes from the

marine sponge Petrosia sp Tetrahedron 54(13) 3151ndash 3158

Millero FJ (1996) Chemical Oceanography 2nd edn p 469

CRC Press Boca Raton

Molinski TF (1993) Marine pyridoacridine alkaloids structure

synthesis and biological chemistry Chem Rev 93(5) 1825ndash

1838

Muller WEG Grebenjuk VA Pennec GL Schroder HC

Brummer F Hentschel U Muller IM and Breter HJ

(2004) Sustainable production of bioactive compounds by

sponges-cell culture and gene cluster approach A review

Mar Biotechnol 6(2) 105 ndash 117

Oclarit JM Okada H Ohta S Kaminura K Yamaoka Y

Iisuka T Miyashiro S and Ikegami S (1994) Anti-bacillus

substance in the marine sponge Hyatella species produced by

an associated Vibrio species bacterium Microbios 78(314)

7 ndash 16

Osinga R Tramper J and Wijffels RH (1999) Cultivation of

marine sponges Mar Biotechnol 1(6) 509 ndash532

Park C Jung JH Kim ND and Choi YH (2007) Inhibition of

cyclooxygenase-2 and telomerase activities in human leukemia

cells by dideoxypetrosynol A a polyacetylene from the marine

sponge Petrosia sp Int J Oncol 30(1) 291 ndash 298

Patel B Balani MC and Patel S (1985) Sponges lsquosentinelrsquo of

heavy metals Sci Tot Environ 41(2) 143 ndash 152

Scheuer PJ (1978-1983) Marine natural products Chemical and

biological perspectives Vol (1 ndash 5) Academic Press Inc New

York

Simpson TL (1984) The cell biology of sponges pp 1 ndash 662

Springer-Verlag New York

Taylor KACC (1995) A modification of the phenolysulfuric

acid assay for total carbohydrates giving more comparable

absorbances Appl Biochem Biotechnol 53(3) 207ndash 214

Turon X Becerro MA and Uriz MJ (2000) Distribution of

brominated compounds within the sponge Aplysina aero-

phoba coupling of X-ray microanalysis with cryofixation

techniques Cell Tiss Res 301(2) 311 ndash 322

Uemura D Takahashi K Yamamoto T Katayama C Tanaka

J Okumura Y and Hirata Y (1985) Norhalichondrin A an

antitumor polyether macrolide from a marine sponge J Am

Chem Soc 107(16) 4796ndash4798

Uriz MJ Becerro MA Tur JM and Turon X (1996a)

Location of toxicity within the Mediterranean sponge

Crambe crambe (Demospongiae Poecilosclerida) Mar

Biol 124(4) 583 ndash 590

Uriz MJ Turon X Galera J and Tur JM (1996b) New light on

the cell location of avarol within the sponge Dysidea avara

(Dendroceratida) Cell Tiss Res 285(3) 519 ndash 527

Venkateswara Rao J Kavitha P Chakra Reddy N and Gnanesh-

war Rao T (2006) Petrosia testudinaria as a biomarker for

metal contamination at Gulf of Mannar southeast coast of

India Chemosphere 65(4) 634 ndash 638

Venkateswara Rao J Kavitha P Srikanth K Usman PK and

Gnaneshwar Rao T (2007) Environmental contamination

using accumulation of metals in marine sponge Sigmadocia

fibulata inhabiting the coastal waters of Gulf of Mannar India

Toxicol Environ Chem 89(3) 487 ndash 498

Venkateswara Rao J Srikanth K Ramjee P and Gnaneshwar

Rao T (2009) The use of marine sponge Haliclona tenuir-

amosa as bioindicator to monitor heavy metal pollution in the

coasts of Gulf of Mannar India Environ Mon Assess

156(1 ndash 4) 451ndash 459

Venkateswarlu Y Reddy VRM Srinivas KNVS and Venka-

teswara Rao J (1993) A new isoquinoline from a sponge

Petrosia sp Ind J Chem 32(B) 704

Yi Q Wei Z Hua L Xingju Y and Meifang J (2005)

Cultivation of marine sponges Chin J Oceanol Limnol

23(2) 194 ndash 198

Ramjee Pallela Srikanth Koigoora Venu Gopal Gunda Madhavendra Sakunthala Sunkara and Venkateswara Rao Janapala 23