MICROSCOPY RESEARCH AND TECHNIQUE 77:296–304 (2014)

Comparative Structural Morphometry and Elemental Composition of Three Marine Sponges From Western Coast of India MAUSHMI S. KUMAR* AND BHAUMIK SHAH Department of Pharmaceutical Biotechnology, Shobhaben Pratapbhai Patel School of Pharmacy and Technology Management, SVKM’S NMIMS, Mumbai 400056, Maharashtra, India

KEY WORDS

Halichondria glabrata; Cliono lobata; Spirastrella pachyspira; ICP-AES; scanning electron microscopy

ABSTRACT Three marine sponges Halichondria glabrata, Cliono lobata, and Spirastrella pachyspira from the western coastal region of India were compared for their morphometry, biochemical, and elemental composition. One-way analysis of variance was applied for spicule morphometry results. Length, width, and length:width ratio were calculated independently. The ratio of length:width varied from 35 to 42 among the grown samples, which remained in the range of 10–22 in young sample at the beginning of studies. However, no significant change was observed in spicule width compared to length. Elemental compositions of marine sponges were determined by field emission gun-scanning electron microscope. Scanning electron microscopy data revealed that the spicules of all the three sponges were mostly composed of O (47–56%) and Si (30–40%), whereas Al (14.33%) was only detected in the spicules of C. lobata. Apart from these, K, Ni, Ca, Fe, Mg, Na, and S were additionally detected in all the three samples. Presence of heavy metals in the sponges was analyzed by inductively coupled plasma-atomic emission spectroscopy. Results showed that iron was present in a large amount in samples, followed by zinc, lead, and copper. Microsc. Res. Tech. 77:296–304, 2014. V 2014 Wiley Periodicals, Inc. C

INTRODUCTION Sponges are considered to be the simplest and oldest living multicellular organisms existing for more than 600 million years. Almost all the marine sponges operate as filter feeders collecting small particles of food from the sea water flowing through their bodies (Kelly et al., 2006; Ramel et al., 2010). They are also known as spineless animals with amazing ability to suffer damage. Some freshwater sponge species such as Ephydati fluviatilis has shown characteristic of pushing through a mesh, and given time to develop; the individual cells come together and make a new sponge (Kelly et al., 2006; Vacelet and Duport, 2004). The sponge phylum, “the pore bearers” (Porifera), has mainly three classes. The first class Calcarea have calcium carbonate spicules in the calcite form and commonly known as Calcareous sponges. The second class is Demospongiae, the most diverse class have silica spicules (mineral skeleton), and diverse forms of spongin fibre. The third class is Hexactinellida—the glass sponge, with siliceous spicules. The spicule length ranges from micrometers to centimeters, which can fuse or interlock with each other. The two classes of sponges differ in skeletal pattern, number of symmetry axes of their megascleres, which are monaxons and tetraxons in demosponges and monaxons and triaxons in hexactinellids. The diversity of skeletal study of sponges goes beyond taxonomic boundaries to include areas such as spicule chemistry and functional roles, or cellular and molecular control of the process of silica C V

2014 WILEY PERIODICALS, INC.

deposition and frameworks building (Cebrian et al., 2007). There is an urgent need to know and understand the biological diversity of potential genetic and chemical resources such as sponges, coral, gorgonians, and so forth, for the matters of ecology and conservation management. Many of these marine sponges are rapidly disappearing because of habitat destruction and overfishing. Apart from providing shelter for other species, they have been known to contain active biomolecule which are of therapeutic and pharmaceutical value. Sponges are best known for products with diverse and unique structures producers and their bioactivity. They are reported for more than 5,300 different products and every year hundreds of new compounds are being discovered (Faulkner, 2000, 2001, 2002). Most bioactive compounds from sponges belong to anti-inflammatory, antitumor, immuno- or neurosuppressive, antiviral, antimalarial, antibiotic, or antifouling class. Halichondria sp. is massive, amorphous sponge with separated inner and outer skeletons consisting of bundles of spicules arranged in Additional Supporting Information may be found in the online version of this article. *Correspondence to: Maushmi S. Kumar, Department of Pharmaceutical Biotechnology, Shobhaben Pratapbhai Patel School of Pharmacy and Technology Management, SVKM’S NMIMS, Vile Parle West, Mumbai 400056, Maharashtra, India. E-mail: [email protected] Received 9 December 2013; accepted in revised form 24 January 2014 REVIEW EDITOR: Prof. Alberto Diaspro DOI 10.1002/jemt.22343 Published online 6 February 2014 in Wiley Online Library (wileyonlinelibrary.com).

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Fig. 1. Three different species of grown marine sponges belonging to different families collected from Mumbai coastal areas in the month of March 2013. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

a random pattern. This genus produced Halichondrin B which has cell division limiting properties, and inhibits cell mitosis (Diaz et al., 1997). Spirastrella pachyspira, Levi 1958 usually grows on the corals at a depth of 4–8 ft in the Gulf of Mannar. Body is encrusting irregularly. When alive it has violet color, though pink is also noted in some parts of Indian Ocean and Red sea (Thomas, 1968) and also in our study. Oscules and pores are not visible in the living condition. In some places, the tylostyles emerging from the adjacent brushes converge and usually such an arrangement is noted in surface ridges. Cliona is a genus of Demospongia in the family Clionaidae. Species like Cliona celata are efficient excavators of calcareous materials like shells and corals (Holmes, 2000). In one of the studies, the erosion rates and erosion pattern of the Microscopy Research and Technique

TABLE 1. Results of physical morphological changes Sample December 2012 March 2013 L (mm) W (mm)

S. pachyspira (n 5 10) H. glabrata (n 5 10) C. lobata (n 5 10) S. pachyspira (n 5 10) H. glabrata (n 5 10) C. lobata (n 5 10)

13–17 14–27 24–27 4–7 1–3 8–10

45–62 37–52 27–33 5–8 4–6 8–12

tropical boring sponge Cliona albimarginata in different biogenic and non-biogenic calcareous rocks have been also investigated. The dissolution rates of the sponge on different kinds of carbonate were highly variable with reference to crystal shape and aggregation, the rock fabric and the presence of other minerals (Calcinai et al., 2007). Sponge taxonomists have

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Fig. 2. Spicule morphometry of S. pachyspira sponge (young— December 2012 and grown—March 2013) captured at 3100, where 1 unit 5 10 mm. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

focused their attention on identifying the individual sponge species on the basis of characteristic skeletal organization. Conversely, cell biologists are also interested in these primitive metazoans to understand their cellular and skeletal organization for enhanced biotechnological applications (Muller et al., 2004). Earlier, sponges have shown accumulation of elements which can be used as a biomarker to assess pollution risks and ecosystem health in the ocean (Venkateswara Rao et al., 2006). Pallela et al. (2011) have reported the first study about distinct morphological and structural variations in the spicules morphometry and aquiferous outlook of sponges (Family: Petrosiidae) available from the Gulf of Mannar. In this study, the test species were visually different and exhibited significant variations in ostia morphometry belonging to the same family. The morphometric characteristics of these marine sponges could be a possible key for the chemical and analytical speciation in

Fig. 3. Spicule morphometry of H. glabrata sponge (young— December 2012 and grown—March 2013) captured at 3100, where 1 unit 5 10 mm. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

future studies of any marine environment. The findings of chemical and biochemical nature can also help for identification and characterization of sponges in situ as well as determining whether sponges could be used as pollution indicator from the habitat sites. MATERIALS AND METHODS Sampling Sponge samples were collected, during low tide; from the inter-tidal regions by hand picking in the month of December 2012 and March 2013. The sponges were collected at the Khar Danda region (19 40 2000 N 72 490 5700 E), of the Mumbai coastal areas, India. Sponges were gently removed from the substratum, cleaned with the sea water, and were placed in plastic bags, then transferred into laboratory. The sponges were further thoroughly cleaned by mechanical removal of foreign materials followed by repeated washing with artificial sea water. All the samples were stored in deep freezer at 280 C to lessen the detrimental effects and maintain the specimen’s integrity. The Microscopy Research and Technique

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ethanol was used for final wash of spicules. The air dried spicules were mounted on microscopic glass slides and study was done for the length and width of spicules (n 5 10) of individual sponges, by a compound microscope (MOTIC-BI Advanced Series) attached to Camera. Analytical Study Presence and quantization of heavy metal like copper, lead, cadmium, zinc, and iron in sponges were analyzed using inductively coupled plasma-atomic emission spectrophotometer (ICP-AES). The Instrument Model was ARCOS from M/s. Spectro, Germany. Elemental compositions were analyzed by field emission gun-scanning electron microscopy-energy dispersive X-ray Spectroscopy (FEG-SEM-EDS). The Model used was JSM-7600F, with resolution 1.0 nm (15 kV), 1.5 nm (1 kV), accelerating Voltage: 0.1–30 kV and magnification 25–1,000,0003. The samples were coated with gold in vacuum and were mounted on aluminium stubs using double adhesive tape. They were observed in JSM-7600F SEM. EDS was performed with an Oxford X-max detector calibrated with cobalt standard at an acceleration voltage of 20 kV. Built-in standards were used for the quantification of each element (Joseph, 2003; Palella et al., 2011). Extraction of Carbohydrates Dried sponges were weighed and digested with 50 mL of 2.5 N hydrochloric acid. After boiling in water bath for 3 h it was allowed to cool at room temperature. Neutralization was performed with solid sodium carbonate (until effervescence ceased). Volume was made up to 100 mL with distilled water. After centrifugation, supernatant was taken for estimation of total carbohydrate content, using phenol–sulfuric acid method (Dubois et al., 1956; Krishnaveni et al., 1984). Fig. 4. Spicule morphometry of C. lobata sponge (young—December 2012 and grown—March 2013) captured at 3100, where 1 unit 5 10 mm. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

voucher specimens were sent to Marine Biology Regional Center, Zoological Survey of India, Chennai for identification purposes, and were identified as S. pachyspira (Class: Demospongiae; Order: Hadromerida; Family: Spirastrellidae), Halichondria glabrata (Class: Demospongiae; Order: Halichondrida; Family: Halichondriidae), and Cliona lobata (Class: Demospongiae; Order: Hadromerida; Family: Clonaidae) (Fig. 1) and are registered. Morphology of the Sponges The differential surface views of individual sponges and their tangential sections of the choanosome were observed with the help of a Photo Compound microscope. Morphometric Study of Spicules The spicules of individual sponge samples were extracted by acid digestion method (Pallela et al., 2011). The organic matter was removed with H2O2 and Microscopy Research and Technique

Extraction of Lipid Extraction was performed by most popular Folsch method. Dried sponges were kept in mixture of chloroform and methanol (2:1). After shaking for 15–20 min at room temperature, clear liquid was washed with distilled water (4 mL water for 20 mL solution). Mixture was centrifuged at lower speed 2,000 rpm and aqueous phase was discarded. Lower organic phase was evaporated on water bath to dryness. Residue was collected for estimation using phosphovanillin reagent (Folch et al., 1957; Mathew et al., 2012). Extraction of Protein Sponge species were chopped and homogenized in phosphate buffer for total protein content by Folin– Lowry method (Lowry et al., 1951; Wilson and Walker, 2000). Data Analysis Data analysis of spicules morphometry was done by applying one-way analysis of variance (ANOVA, 2013; Lowry and Richard, 2013). One-way ANOVA helped to examine individual variables for the differences among test species. When populations were significantly different, multiple comparison post hoc tests (Tukey’s HSD) were also used.

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M.S. KUMAR AND B. SHAH TABLE 2. Results for spicule morphometry for March 2013 sponge samples S. pachyspira H. glabrata

Sr. No. 1 2 3 4 5 6 7 8 9 10 Total Average Total2 Variance Std. Deviation Std. Error

L (mm)

W (mm)

L/W

L (mm)

W (mm)

L/W

L (mm)

W (mm)

L/W

0.066 0.045 0.069 0.033 0.053 0.029 0.048 0.076 0.084 0.026 0.531 0.053 0.031 0.000 0.020 0.006

0.0018 0.0014 0.0018 0.001 0.0014 0.0009 0.0012 0.0019 0.0029 0.0008 0.0151 0.0015 0.0000 0 0.0006 0.0001

36.88 32.14 38.33 33.10 38.14 32.33 40.16 40.00 29.24 33.25 353.59 35.360 12,633 14.452 3.8016 1.2022

0.0697 0.0339 0.0355 0.0385 0.0572 0.0234 0.0687 0.0640 0.0770 0.0206 0.4885 0.0488 0.0277 0.0004 0.0207 0.0065

0.0016 0.0014 0.0010 0.0009 0.0014 0.0007 0.0014 0.0014 0.0016 0.0005 0.0119 0.0011 0.0000 0 0.0003 0.0001

43.563 24.214 35.500 42.778 40.857 33.429 49.071 45.714 48.125 41.200 404.45 40.445 16872 57.131 7.5585 2.3902

0.0528 0.0778 0.0357 0.0650 0.0683 0.0519 0.0457 0.0645 0.0370 0.0478 0.5465 0.0546 0.0316 0.0001 0.0138 0.0043

0.0017 0.0019 0.0008 0.0014 0.0023 0.0012 0.0009 0.0014 0.0008 0.0011 0.0135 0.0013 0.0000 0 0.0004 0.0001

31.059 40.947 44.625 46.429 29.696 43.250 50.778 46.071 46.250 43.455 422.55 42.255 18,269 45.935 6.7776 2.1433

TABLE 3. Results of carbohydrate content in samples Conc. Amt. Amt (mg/mL) (mg/100 mL) (mg)/g sponge Collection time A490 S. pachyspira December 2012 March 2013 H. glabrata December 2012 March 2013 C. lobata December 2012 March 2013

Samples

0.081 0.398

13.0645 64.1935

1.3065 6.4194

1.3065 6.4194

0.414 0.771

66.7742 124.3548

6.6774 12.4355

6.6774 12.4355

0.535 0.554

86.2903 89.3548

8.6290 8.9355

8.6290 8.9355

TABLE 4. Results of protein content in samples Conc. Amt. Amt (mg/mL) (mg/100 mL) (mg)/g sponge A600

S. pachyspira January 2012 March 2013 H. glabrata December 2012 March 2013 C. lobata December 2012 March 2013

C. lobata

0.142 0.498

109.23 383.08

10.92 38.31

10.92 38.31

0.162 0.429

124.62 330.00

12.46 33.00

12.46 33.00

0.231 0.577

177.69 443.85

17.77 44.38

17.77 44.38

RESULTS Physical Morphology of the Sponges Samples at the collection site in the month of December 2012 were in their initial stage, younger and small. Later samples collected in the month of March 2013 were found to be in their growing stage. They were growing, attached with each other, with bigger size; however, there was no significant change in the width. Results are shown in Table 1. The three test species analyzed in this experiment were collected at the same depth and site of Khar Danda under identical environmental conditions. The external morphology of the three species varied from thick encrusting, globular structure to a more massive form and the color was light green, pink, and orange. The external surface

of S. pachyspira was pink in color, smooth, and tubular-like asconoid. H. glabrata was pale green with thin encrusting body, whereas, C. lobata appeared orange in color. Spicule Morphometry The camera images showed a significant difference in the surface view among the three species. Choanosomal comparison of spicules length, width, length:width ratio was performed and all the results are shown in the Figures 2–4 and Table 2. Results of ANOVA and Turkey HSD were analyzed. All the three test species contained similar type of oxea spicules, and did not differ in their sizes (Figs. 2–4). The L/W ratio of C. lobata (42.25) and H. glabrata (40.44) was comparatively higher than S. pachyspira (35.36) at grown stage. The L/W values for the young samples were 15.48, 10.35, and 22.54, respectively. This indicates the pattern of growth in spicules while their development. Results show that length increases by multiple folds. ANOVA results for all the varieties for spicule morphometry have shown F (14.62), F (11.26), and F (26.81) for length, width, and length:width for samples collected in December 2012. For all the samples, P value was found to be 0.05. At young stage in December 2012, the spicules structure of all the three sponges appeared similar to sigma C shape, but as they grew their structures were different. Spicule morphometry of S. pachyspira, H. glabrata, and C. lobata sponge was monoactinal monaxon with one end blunt and the other pointed (style) (Figs. 2–4). Carbohydrate, Lipid, and Protein Content Results revealed increase in carbohydrate, lipid, and protein content in all the three sponge samples in their developing stage. The lipid content increased up to 10–23-fold depending upon the variety of the samples. Results are compared in Tables 3–5. Carbohydrate content increased almost five times in S. pachyspira and protein by 3.5-fold. Microscopy Research and Technique

MICROSCOPY AND ELEMENTAL CHARACTERIZATION OF MARINE SPONGES

C. lobata December 2012 Conc. (mg/mL) W.r.t. olive oil Conc. (mg/mL) W.r.t. Cholesterol Amount (mg/g sponge) w.r.t. olive oil Amount (mg/g sponge) w.r.t. Cholesterol

Sample S. pachyspira H. glabrata C. lobata

TABLE 5. Results of lipid content in samples C. lobata S. pachyspira S. pachyspira March 2013 December 2012 March 2013

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H. glabrata December 2012

H. glabrata March 2013

118.421

1467.105

342.105

1710.526

59.211

1381.579

105.263

1304.094

304.094

1520.468

52.632

1228.070

29.605

366.776

85.526

427.632

14.803

345.395

26.316

326.023

76.023

380.117

13.158

307.018

Cu (ppm) 12.5 8.9 23.3

TABLE 6. Results of heavy metal amount in samples Zn (ppm) Fe (ppm) 87.5 51.5 109

Fig. 5. Scanning electron microscopy of S. pachyspira taken at 31,000. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Highest content was found in samples of H. glabrata with 24-fold increase in grown samples, whereas C. lobata and S. pachyspira had 12- and 5fold increase, respectively. Heavy Metal Detection Heavy metals in the sponges were analyzed by ICPAES. Results obtained from the spectroscopy are shown in following Table 6. ICP-AES result indicated presence of iron in a large amount in all the three sponge samples, followed by zinc, lead, and copper. Cadmium was not detected in other samples except C. lobata which showed its minute presence. Elemental Composition Elemental composition of sponges was found by FEG-SEM-EDS (Figs. 5–7). Elemental composition for each sample is shown in Table 7. Elemental Microscopy Research and Technique

497.9 641.8 471.5

Pb (ppm)

Cd (ppm)

17.9 8.2 67.6

ND ND 0.076

Fig. 6. Scanning electron microscopy of H. glabrata taken at 31,000. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

composition was also plotted for all the three sponges and Figures 8–10 infer the relative %. DISCUSSION The taxonomic status of all the three sponge species from the same environmental area at Khar Danda coast, Mumbai, India was studied as per their morphological and anatomical features, heavy metal presence, and elemental composition. This was the first attempt made to distinguish variations in sponge species of Khar Danda region revealing marked structural and morphological variations in the outlook as well as spicules. 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 characteristic differences in their shape and sizes of ostia

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(De Voogd et al., 2002). It is also evident in our study; that the three test species were visually different and have shown significant variations in their shape, diameter, and morphometry. The spicules have shown difference in their length and length to width ratio. The difference in architecture of ostia among the species was found in same environmental regime, which may be due to the water flow into the body or different feeding biology of species. The protein and carbohydrate 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. Analytical studies of sponges have recently become a matter of interest and secondary target energy dispersive X-ray spectrometry (EDXA/EDS) analysis of marine sponges facilitates detection of many elements, that is, O, Ca, Mg, C, H, S, P, Al, Si, K, Ca, Fe, and Ni (Araujo et al., 1999). The differences in elemental accumulation between individual sponge species are well strategized and variations probably reflect the differences in physiological conditions, in turn reflecting the passage of water

Fig. 7. Scanning electron microscopy of C. lobata taken at 31,000. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

through the aquiferous system of individual sponges (Patel et al., 1985). Depending on their structural uniqueness, different elements may be accumulated into the sponge tissues along with the ingested food particles, resulting in differential deposition on their surface, as observed in this study of three sp. ICP-AES analysis of marine sponge is also useful for detection of heavy metals, that is, Cu, Zn, Pb, Cd, Fe, As, and so forth, and help to reveal its bio-indicator role for heavy metals detection in the marine coastal area. DNA-based markers have been efficiently used in the phylogenetic analyses of sponges. Molecular biologists are working on the sponge genome to understand the molecular 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 information on the reproductive isolation of the species, which reinforced the conclusion that spicular morphology would be useful in discriminating Petrosia spp. (Bavestrello and Sara, 1992). This non-genetic approach is simple and may be useful to non-specialists as it does not require the use of traditional genetic tools. This comparison gives us the opportunity to analyze phylogenetic relationships, thus giving useful insights for the identification of sponges by non-taxonomists. Thus, sponges belonging to Porifera, though simplest in organization, exhibit diversity in morphological patterns and chemical composition. These parameters are gaining significance in taxonomic identification of sponges for ecobiotechnological exploitation of potential sponges (Hougaard et al., 1991). In the past few decades, marine sponges have shown their use for pharmaceutical, taxonomical as well as economical purposes. This study particularly has significance for in situ discrimination of sponge species by studying their morphological changes along with their spicular morphometry aspects by photographic microscopic analyses. Evaluation of sponges as a bio-indicator for heavy metal accumulation at coastal areas might also be helpful in revealing hotspot polluted zones and assist in decision-making rules for coastal management. The sponges collected from the Khar Danda region from Mumbai are undergoing a timely anthropogenic change that can make these sponges potential bio-indicators in future. Some of the species characters like carbohydrate, lipid, and protein

TABLE 7. Elemental Composition in marine sponge samples C. lobata S. pachyspira Elements O Si Al K C P H S Mg Ca Fe Na Cl Ni

H. glabrata

Wt. (%)

Atomic (%)

Wt. (%)

Atomic (%)

Wt. (%)

Atomic (%)

47.64 30.44 14.33 2.06 1.78 0.73 0.7 0.64 0.59 0.43 0.42 0.25 — —

47.47 40.4 8.14 0.93 1.23 0.38 0.46 0.32 0.24 0.17 0.12 0.15 — —

53.23 40.52 — 0.95 — — — 0.6 0.29 0.72 0.77 1.08 1.82 —

67.14 29.12 — 0.49 — — — 0.38 0.24 0.36 0.28 0.95 1.04 —

56.13 40 0.24 0.79 — — — 0.24

69.62 28.27 0.18 0.4 — — — 0.15

0.2 0.42 0.67 0.53 0.79

0.1 0.15 0.57 0.3 0.27

Microscopy Research and Technique

MICROSCOPY AND ELEMENTAL CHARACTERIZATION OF MARINE SPONGES

Fig. 8. SEM-energy dispersive X-ray spectrometry of S. pachyspira. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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analyses. This study identifies marine sponges as bioindicator of heavy metal for the Mumbai coastal areas which might be helpful in revealing hotspot polluted zones and assist in decision-making rules for coastal management. These studies encourage us to understand the marine sponge’s role as potential pollution indicator by carrying further studies. The study would take into consideration the marine water samples, their heavy metal load, and the absorbance capacity of sponge habitats in those areas. From this result, we can conclude that the sponges available at the coastal areas are undergoing timely anthropogenic changes that made these highly porous invertebrates potential bio-indicators. The morphometric characteristics of selected sponge species can be used for the chemical and analytical speciation in future studies from any marine environment. ACKNOWLEDGMENTS The authors would like to thank the Dean and staff members of School of Pharmacy and Technology Management, NMIMS for all their support and providing infrastructure for this study. REFERENCES

Fig. 9. SEM-energy dispersive X-ray spectrometry of H. glabrata. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Fig. 10. SEM-energy dispersive X-ray spectrometry of C. lobata. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

content indicate their structural composition and also the nature of bioactive compounds class which can be extracted in future for evaluation of their bioactivity. Further studies can only confirm the relation between the bioactivity and the chemical compound class and their relation to the biochemical constituents. CONCLUSION This study particularly has significance for in situ discrimination of three species of sponges by observing their morphological changes along with their spicular morphometry aspects by photographic microscopic Microscopy Research and Technique

Araujo MF, Cruz A, Humanes M, Lopes MT, da Silva JAL, da Silva JJRF. 1999. Elemental composition of Demospongiae from the eastern Atlantic coastal waters. Chem Spec Bioavail 11:25–36. Bernard S, and Mathew M, 2012. Spectrophotometric method of estimation of atorvastatin calcium using sulfo-phospho-vanillin reaction. J Appl Pharm Sci 2:150–154. Bavestrello G, Sara M. 1992. Morphological and genetic differences in ecologically distinct populations of Petrosia (Porifera; Demospongiae). Biol J Linn Soc 47:49–60. Calcinai B, Azzini F, Bavestrello G, Gaggero L, Cerrano C. 2007. Excavating rates and boring pattern of Cliona albimarginata (Porifera: Clionaidae) in different substrata. In: Porifera Research: Biodiversity, Innovation and Sustainability (ed. M. R. Cust odio, G. L^ obo-Hajdu, E. Hajdu and G. Muricy), pp. 203–210. Rio de Janeiro:  erie Livros 28, Museu Nacional. Cebrian E, Uriz M, Turon X. 2007. Sponges as biomonitors of heavy metals in spatial and temporal surveys in northwestern mediterranean: Multispecies comparison. Environ Toxicol Chem 26:2430–2439. De Voogd NJ, Van Soest RWM. 2002. Indonesian sponges of the genus Petrosia Vosmaer (Demospongiae: Haplosclerida). Zool Med Leiden 76:193–209. Diaz MC., Pomponi SA., VanSoest RWM. 1993. A systematic revision of the central West Atlantic: Halichondrida (Demospongiae, Porifera). Part III: Description of valid species. Sci. Mar. 57(4): 283– 306. Dubois M, Gilles KA, Hamilton JK, Rebers PA, Smith F. 1956. Colorimetric method for determination of sugars and related substances. Anal Chem 28:350–356. Faulkner DJ. 2000. Marine natural products. Nat Prod Rep 17:7–55. Faulkner DJ. 2001. Marine natural products. Nat Prod Rep 18:1–49. Faulkner DJ. 2002. Marine natural products. Nat Prod Rep 19:1–48. Folch J, Lees M, Stanley G. 1957. A simple method for the isolation and purification of total lipides from animal tissues. J Biol Chem 226:497–509. Goldstein J, Newbury DE, Joy DC, Lyman CE, Echlin P, Lifshin E, Sawyer L, Michael JR. 2003. Scanning Electron Microscopy and Xray Microanalysis 3rd ed. XIX, 690 p. Holmes KE. 2000. Effects of eutrphication on bioeroding sponge communities with the description of new West Indian sponges, Cliona spp. (Porifera: Hadromerida: Clionidae). Invertebrate Biol 119:125–138. Hougaard L, Christophersen C, Nielsen P, Klitgaard A, Tendal O. 1991. Biochem Systemat Ecol 19:223–235. Joseph G. 2003. Scanning electron microscopy and X-ray microanalysis springer. ISBN 978-0-306-47292-3. Retrieved March 2013, from http://books.google.com/books id5ruF9DQxCDLQC. Kelly M, Edwards A, Wilkinson M, Alvarez B, Cook M. 2006. Phylum Porifera sponges. In: The New Zealand inventory of biodiversity. Christchurch: Canterbury University Press.

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Kelly M, Edwards A, Wilkinson M, Alvarez B, Cook S, Bergquist P, Buckeridge J, Campbell H, Reiswig H, Valentine C, Vacelet J. 2009. Phylum Porifera: sponges in D. Gordon (ed.) New Zealand Inventory of Biodiversity (Volume 1), New Zealand: Canterbury University Press, pp. 23–46. Krishnaveni S, Theymoli Balasubramanian, Sadasivam S. 1984. Carbohydrate. Food Chem 15:229. Lowry, Richard. 2013. Concepts & Applications of Inferential Statistics. Retrieved March-April 2013, from http://vassarstats.net/ anova1u.html. Lowry O, Rosebrough N, Farr AL, Randall RJ. 1951. Protein measurement with folin-phenol reagent. J Biol Chem 193:265– 275. Muller WEG, Grebunjuk VA, Thakur NL, Thakur AN, Krasko A, Muller IM, Breter HJ. 2004. Oxygen-controlled bacterial growth in the sponge Suberites domuncula: Towards a molecular understanding of the symbiotic relationships between sponge and bacteria. Appl Environ Microbiol 70:2332–2341. One Way Anova (Analysis of Variance) Calculator. Retrieved January-March-April 2013, from easycalculation.com: http://easycalculation.com/statistics/one-way-anova.php.

Pallela R, Koigooraa S, Gundaa VG, Sunkarab SM, Janapalaa VR. 2011. Comparative morphometry, biochemical and elemental composition of three marine sponges (Petrosiidae) from Gulf of Mannar, India. Chem Spec Bioavail 23:16–23. Patel B, Balani MC, Patel S. 1985. Sponges ‘sentinel’ of heavy metals. Sci Tot Environ 41:143–152. Ramel G. 2010. Phyllum Porifera. Retrieved January 2013, from Earthlife: http://www.earthlife.net. Thomas PA. 1968. Two new records of silicious sponges belonging to the families Myxilliade Henstschel and Spirastrellidae Hentschel from the Indian region. J Mar Biol Assoc India 10:264– 268. Vacelet J, Duport E. 2004. Prey capture and digestion in the carnivorous sponge Asbestopluma hypogea (Porifera: Demospongiae). Zoomorphology 123:179. Venkateswara Rao J, Kavitha P, Chakra Reddy N. 2006. Petrosia testudinaria as a biomarker for metal contamination at Gulf of Mannar, southeast coast of India. Chemosphere 65:634–638. Wilson K, Walker J. 2000. Practical biochemistry: Principles and techniques. Cambridge: Cambridge University Press. p. 240. Yi Q, Wei Z, Hua L, Xingju Y, Meifang J. 2005. Cultivation of marine sponges. Chin J Oceanol Limnol 23:194–198.

Microscopy Research and Technique

Comparative structural morphometry and elemental composition of three marine sponges from western coast of India.

Three marine sponges Halichondria glabrata, Cliono lobata, and Spirastrella pachyspira from the western coastal region of India were compared for thei...
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