Comparative Ultrastructural and Biochemical Studies of Four Demosponges from Gulf of Mannar, India

Marine sponge explorations at Gulf of Mannar (GoM), India in the past few years (2003-2009) revealed around forty sponge species that lead to various comprehensive studies on chemicobiological molecules including collagens and their applications. Basing on the preliminary evaluations on collagen content, four marine sponges Hyattella cribriformis, Fasciospongia cavernosa, Callyspongia fibrosa and Dysidea fragilis belong to the families Spongiidae, Thorectidae, Callyspongiidae and Dysideidae of the class Demospongiae, were chosen for macromolecular and micro-anatomical comparison. Microscopic examinations were performed to understand the form and arrangement of skeletal components in each sponge, where unique skeletal composition and spongin distribution is typical for all the sponges except D. fragilis, which is highly embedded with diversified oxeae. Biochemical analyses were performed to resolve the macromolecular content for each sponge species. Sponge D. fragilis was found to possess less protein content (57.26±3.36 mg/g freeze-dried sponge wt) when compared to other sponges and significantly, C. fibrosa possess high protein (209.55±9.53 mg/g) and other macromolecular contents, except pentose (24.12±1.21 mg/g). Different forms of collagens viz., Acid soluble (AS), salt soluble (SS) and insoluble (Ins) collagens are estimated to understand the variation in collagen make up in their respective tissues. The higher collagen content in C. fibrosa could propose to use this species as a potential model for targeting collagenous molecules towards human health benefits.


Introduction
Sponges (Phylum: Porifera) are one of the better-known, diverse multi-cellular invertebrates diverged from the common ancestor of monophyletic origin, Eumetazoa (Müller et al., 2006;Ereskovsky et al., 2010). Although phylogenetically crucial among all the sponges of Porifera, class Demospongiae has been overlooked for various anatomical and developmental studies (Brusca and Brusca, 2003). Connective tissue in Demosponges is found to be well developed and typically forms a complex and often elegant skeleton, whose rigidity varies widely among species and growth forms. The skeleton of the Demosponges was formed by gelatinous mesohyl supported with fine collagen fibers and/or a collagenous matrix (spongin) embedded with mineral spicules or oxeae. Included in the class are the bath sponges, which obviously lack spicules, but have a well-developed spongin skeleton. With respect to their body organization, cell differentiation and embryogenesis, the representatives of Porifera are characterized as model systems due to their pronounced plasticity in the determination of cell lineages (Koziol et al., 1998). These simple organisms possess numerous structural elements like collagen that are involved in cell adhesion and recognition, similar to the characteristics of more complex animals (Boute et al., 1996;Fernàndez-Busquets, 2008;Rao et al., 2011;Pallela et al., 2012).
The composition and analyses of various macromolecular and biochemical content of each sponge species is very valid in choosing potential candidate of interest, because the spicule formation is dependent on the apposition and deposition of macromolecules like protein (spongin/collagen), lipids, carbohydrates etc. (Aizenberg et al., 1996). In addition, detailed morphological studies that demosponges infer a considerable polymorphism within the species by means of their collagenous skeleton (Garrone, 1999;Garrone, 1985). Successful applications of collagens from various terrestrial sources is as vast as in the treatment of hypertension, urinary incontinence, pain associated with osteoarthritis, wound healing, tissue engineering for human organ implants etc. However, the outbreaks of contagious diseases through the highly antigenic collagens could lead to health complications like BSE and TSE (Swatschek et al., 2002). Hence, current researchers are targeting safer collagen from nature, more importantly from marine sources, as life originated from ocean. In this context, marine sponges are the ideal role models for several collagen explorations in order to meet the recent medical demands of human beings.
Despite of having an attractively simple basic organization that remains fairly constant throughout all species, they do manage to show a great variety of forms and shapes to give around 11,000 known species of sponges worldwide among which, only 8500 are considered valid (Van Soest et al., 2012). More than 300 species of sponges have been recorded from Gulf of Mannar (GoM) and Palk Bay region located on the southeast coast of India, which is considered to be one of the hotspots for marine sponges in the world (Venkataraman and Wafar, 2005). GoM possess a coastline of 315 km starting from the pilgrim town of Rameshwaram and ending at industrial town of Tuticorin. Taking their remarkable biomedical and pharmacological applications into account (Chairman et al., 2012), preliminary studies on the Demosponges from GoM were carried out in our laboratory. In the perspective of the microscopic and biochemical analyses, four marine sponges Fasciospongia cavernosa, Hyattella cribriformis, Dysidea fragilis and Callyspongia fibrosa were taken into consideration as all of them are prominent at the shallow and deep collection sites of GoM. The present paper depicts an over view of the biochemical content including the collagen of collected demosponges. This is the latest report of listing recently available sponge species in GoM area, where specific focus has been given to the macromolecular and ultrastructural differences between the experimental sponges for emphasizing the importance of these collagenated organisms, although intensive studies on collagen from Ircinia fusca were studied previously by our group. Present study may prove to be the ideal vault for further chemicobiological explorations on remaining available marine sponges in the same area; and also for the isolation, purification and analysis of therapeutically important, safer protein molecules like collagens from different sponge species of GoM.  (Montagu, 1818)] of the class Demospongiae were chosen for the present study, based on the available information on their possible ecological significance and were analyzed microscopically and biochemically. All the reagents used in the present study are of analytical grade and used without further purification.

Gross study of the sponges
Sponge specimens collected from the sea were photographed digitally (SONY) and with a CCD camera (SONY CCD IRIS, model no. SSC-M370CE, Japan) attached to a computer.

Video microscopy
Video microscopy was employed to examine the form and arrangement of skeletal components in intact

Light microscopy
Prior to the washing of each sponge species in distilled water, squashed suspension of sponge tissue was subjected to microscopic slide and observed under POLYVAR (REICHERT-JUNG) compound microscope to understand the structural array of spongin and spicules.

Electron microscopy
Scanning Electronic Microscopy was done according to the method of Bozzola and Russell (1999). Briefly, distilled water cleaned sponge tissues were dissected with thin scalpel blade and the sections were mounted over the stubs with double-sided carbon tape and were fixed with a 4% aqueous Osmium tetroxide vapours for 2 hr. A thin layer of Platinum (palladium) coat was then applied over the samples using an automated sputter coater (JEOL JFC-1600) for about 4 min. Then the samples were scanned under Scanning Electron Microscope (Model: JOEL-JSM 5600) at various magnifications.

Macromolecular analysis
Each specimen of collected sponge species was washed under running tap water for two to three times and then washed finally with 0.1M phosphate buffer (pH 7.5). For protein estimation, each experimental sponge tissue was chopped and minced using sharp dissection scissors and homogenized (10% w/v) in ice-cold 0.1M phosphate buffer (pH 7.5) using Heidolph DIAX 900 homogenizer. The homogenates were centrifuged at 500 g for 10 min and the supernatant was further recentrifuged at 5000 g for 10 min using refrigerated centrifuge (Kubota; Model: 6930). The resultant supernatant of sponge homogenates were used to estimate the total protein content by spectrophotometer (SpectraMAX Plus, Molecular Devices; Model: S/NP02512) assisted with software, SoftMax Pro ver. 5.0. following the method of Bradford (1976), using bovine serum albumin (BSA) as standard.
Total carbohydrate content was measured using the amended phenol-sulfuric acid method of Taylor (1995), whereas the total lipid content was measured by using a mixture of chloroform:methanol (1:2) according to the previous method of Brooks et al (1998).
For carbohydrate estimation, 1 g of each test sponge tissue was homogenized with distilled water and the homogenate was taken into a test tube, in which, 100 µL of 8% phenol was added. 1 mL of concentrated sulfuric acid was then added rapidly, the steam of acid being directed against the liquid surface rather than against the side of the test tube in order to obtain good mixing. The tubes were thoroughly mixed and later they were placed in a water bath for 10 to 20 minutes at room temperature to obtain yellow orange colored complex. The color was found to be stable for several hours and readings may also be taken later, accordingly. The absorbance was measured at 490 nm for hexoses and 480 nm for pentoses by using UV-Visible Spectrophotometer (Molecular Device, USA; Softmax pro 5.0). Reference standard curve constructed for the particular sugar were used to determine the amounts of carbohydrate present in each sponge species. All solutions were prepared in triplicate to minimize errors resulting from any accidental contamination.
For lipid analysis, 1 g of each experimental sponge tissue was homogenized with 10 mL distilled water and the resultant pulp was transferred to a conical flask containing 30 mL of chloroform-methanol mixture and mixed well. The extraction was left overnight at room temperature in a dark place. After this, equal volumes (20 mL) of chloroform and distilled water were added and the suspension was gently mixed by vortexing and subjected to centrifugation, to obtain three layers. The methanol layer was discarded and the lower layer of chloroform containing all the lipids was carefully collected free of interphase, by sucking out with a fine capillary tube. The organic layer was carefully evaporated by leaving the extraction in warm water (around 50℃) with a flow steam of nitrogen gas on the surface. As some lipids get polymerized or decomposed on exposure to light, heat and oxygen; the sample was covered with a dark paper to protect from light. When the solution was free of organic solvents, the total lipid content was determined and the results are expressed in terms of weight in micrograms of total lipid per gram dry tissue.

Hydroxyproline assay and collagen quantification
Different forms of collagens viz., salt soluble (SS), acid soluble (AS) and insoluble (Ins) collagens were extracted by the modified method of Prockop (1964). The content of hydroxyproline (Hyp) and total collagen was quantified according to the slightly modified methods as described earlier Jamall et al., 1981;Siddiqi and Alhomida, 2003). Briefly, the sponge tissues were cut into small pieces and homogenized in sufficient distilled water to yield 10% homogenate (w/v). Equal volume of 12 N HCl was added to each aliquot (2 mL) of homogenate and hydrolyzed at 105℃ for 18 h. Aliquots of 25 µL sponge tissue hydrolyzate, in triplicate, were added to separate vials and subjected to evaporation leading to dryness under vacuum. Sets of samples were spiked with 0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2 and 1.6 µg of standard hydroxyproline (Hyp). To each of the hydrolyzate and Hyp standard vials, 1.2 mL of 50% isopropanol was added, followed by 0.2 mL of 0.56% buffered chloramine-T solution. One ml of Ehrlich's Reagent (ER) solution was then added after an interval of 10 mins. The solutions obtained were mixed and incubated at 50℃ for 90 mins. Samples were cooled and the absorbance was measured spectrometrically at 558 nm, using water as reference and corrected for reagent blank. The absolute value of the negative intercept on the X-axis found by linear regression analysis of Hyp standards was taken to represent the Hyp content of the tissue. Quantification of total and fractionated forms (AS, SS and INS) of collagen was performed according to the standardized calculation by Neuman and Logan (1950).
The following formula was used to calculate the collagen content.
Collagen content = 7.46 [mg of Hydroxyproline per g freeze-dried sponge weight].
Where, the factor, 7.46 = ratio of the weight of collagen and weight of hydroxyproline Note: Collagen content is expressed as mg collagen / g freeze-dried sponge weight.

Statistical analyses
All the data in triplicates (n=3) was analyzed using the student's t-test to quantify macromolecules and multiple replicates of sponge tissue samples were used for microscopic observations. Statistical significance for all the biochemical analyses was defined as P < 0.05.

Results
According to our earlier explorations from the year 2006, there are a total of 40 species were collected in the Mandapam, Palk Bay and Rameswaram regions of Gulf of Mannar (GoM) ( Table 1). Depending on the abundance and past pharmacological significance, out of all the collected marine sponges, four experimental sponges Hyattella cribriformis, Fasciospongia cavernosa, Callyspongia fibrosa and Dysidea fragilis were compared to bring out their ultrastructural and biochemical analysis in the current studies.
While collecting, the sponges Hyattella cribriformis, Fasciospongia cavernosa and Dysidea fragilis were found to be comparatively more fragile and easy to be ripped off. A possible reason for the fragile nature of these sponges could be due to the lesser content of collagen or spongin in the matrix owing to less amount of strength in the skeletal network, differentiating them from Callyspongia fibrosa, where the situation is in contrary and they possess greater amount of collagen matrix. The morphotypic features and key characters along with the whole mount images of these sponges at the time of collection are presented in the Table 2. The specimen of Hyattella cribriformis is brittle and lightweight, yellowish-white or pale in color. This sponge is available at around 10 to 15 f from the seashore. Another sponge, Dysidea fragilis, is available at the depth of 15 to 20 f. Morphologically, this species appears as a mass of greenish black to yellow color. Yet another member of Demospongiae, but belonging to a different order family Callyspongiidae, Callyspongia fibrosa, is composed of finger shaped or flattened branches, surface is with strong conules and has prominent conules at growing tips. The osculae irregularly distributed, terminal, marginal, rounded or elliptical, shallow and compound. They appear to be pale yellow to brown when alive. Dermal skeleton is reticulate, meshes triangular or subdivided by tertiary fibres, which are either uni-or multispicular.

Microscopic Study Video microscopy
The four sponges showed markedly different skeletal alignment and distinctive arrangement of bundle of spicules or oxeae along with the network of spongin, when observed under video microscope (Figure 1). In these sponges, the major canals are strengthened with  Cobweb-like surface formed by huge number of spicules and sand particles, irregularly encrusting or massive and lobe-shaped. Consistency is very brittle to hold and externally appears pale pinkish to pale yellow or brownish to grayish-white.

Light Microscopy
The sponge skeletal matrix was observed under light microscope to evaluate the specular arrangement and the branching pattern of each sponge species (Figure 2). Sponge skeletal network is formed by the intervening of spongin fibers, spicules and some amorphous material to give a unique distribution of the spongin frames. These spongin frames are of different shapes due to the multiple interventions of the spongin fibers in regular as well as irregular fashion. Among the four sponges, the skeletal arrangement was almost similar for F. cavernosa and H. cribriformis, whose skeletal network was formed to be branched in multiple directions. The terminal portion of the spongin fiber in F. cavernosa (Inset) is highlighted to expose the bunch of oxeae and collagen packed by an outer cuticular layer. The junctional region of the spongin network in H. cribriformis (Inset) is covered with a membrane like material. Ramifying spongin fibers were seen in C. fibrosa, where native spongin network was observed to be thicker and internal striations of the spongin were clearly visible at high magnification (Inset). The spongin network observed in D. fragilis was unique in possessing multiple types of fibres loaded with sand grains, broken spicules and other foreign material covered by a very thin transparent layer of amorphous material, softly holding the bunch of parallely arranged detritus material (Inset) with the support of tiny collagenous material.

Scanning electron microscopy
Ultrastructural orientation of the differential skeletal make up and varied distribution of spongin in extra cellular matrix of four experimental marine sponges was analyzed by Scanning Electron Microscopy (SEM) (Figure 3-6). SEM analyses depict the branching and pattern of spongin network and the lattice distribution of silicon layers cemented around the interlacing skeletal fibers. At higher magnifications, C. fibrosa exhibited a distinct skeleton formed of thick walls of silicon based sheets layered along with the spongin and uniform sized spicules (Figure 3). The peeled off, fragmented material at the broken portions of the tissue seem to appear as collagenous layers packed at different densities. On the other hand, SEM analysis of D. fragilis depicted a number of spacious cavities called atria, formed of bundles of different variety of material composed of detritus and fragmented spicules intercalated with amorphous siliceous material rather than much fibrous protein (Figure 4).
It is clearly observed from the SEM results that carpets of amorphous material are seen much in the case of F. cavernosa ( Figure 5). The skeleton found to be majorly formed of large spongin fibers supported of the sponge tissue towards the spongin analysis, certain sponge cells appear in the section of F. cavernosa (Figure 5 C). In H. cribriformis, the skeletal network was characterized by typical spongin network rather than free spongin fibers or collagen like filaments, and the multi-junctional regions of the network were overlapped with layers of silicon and amorphous cuticular material ( Figure 6). H. cribriformis contains fibrillar extensions at the terminal fragmented portions in the skeletal network, indicating that, silicon cementing around the spongin fibers is very important in order to form the fibrillar extensions throughout the skeleton of sponge body (Figure 6 C).

Biochemical analysis
All the results for macromolecular analysis are reported  (Table 3). C. fibrosa have high protein content (209.6±9.53 mg/g freeze dried sponge wt) followed by H. cribriformis (162.72 ±8.90 mg/g) than other experimental sponge species. It could be a possible indication that the high protein content dominated the other macromolecular content like carbohydrates (pentoses and hexoses) and lipids in these two sponges. It is very clear that sponge D. fragilis has less protein content (57.26 ±3.36 mg/g) when compared to other sponges. Regarding the carbohydrate content, it is observed that C. fibrosa and H. cribriformis possess relatively high content of pentose and hexose than the other two sponges. Interestingly, although the pentose content is slightly varied in both the species of D. fragilis and F. cavernosa, the hexose content (9.6±0.5 mg/g) is similar in each species. Similarly, total lipid content of C. fibrosa and H. cribriformis is proportionately higher among the four experimental sponges (21.2 ± 1.1 and 19.5 ±0.9 mg/g, respectively).

Hydroxyproline and collagen content
Estimation of the amino acid, hydroxyproline (Hyp) is diagnostic of collagens in any organism. According to our preliminary explorations of Hyp content in all the collected sponges, six sponges along with I. fusca and S. officinalis possess high amount of Hyp than the remaining sponges and the differences between these selected sponges in their Hyp content was analyzed by spectrophotometric method. However, specific important has been given to I. fusca and S. officinalis to study separately to emphasize on the collagenous network; and four sponges considered for ultrastructural and biochemical comparison in the present study. Collagen content (based on Hyp concentration) significantly differed in all the four experimental sponges. The sponge, C. fibrosa seems to possess highest collagen content (169.19 ± 9.12 mg/g freeze-dried sponge wt) among all the four sponges (Table 3).
The solubility of collagens is a factor of extracting different forms of collagen viz., salt soluble (SS), acid soluble (AS) and Insoluble (Ins). It is clear that the AS and SS collagen content is not much varied but the content of Ins collagen differed much among the four sponges (Table 3). In all the sponges, the insoluble (Ins) form of the collagen was more than the amount of SS and AS forms of the collagen. Although the total collagen content of F. cavernosa is less than C. fibrosa and H. cribriformis, the collagen (Ins) is higher (42.8±2.1 mg/g) when compared to other sponges. However, the AS and SS collagen of C. fibrosa is higher among all the experimental sponges.

Discussion
This is the first extensive work on the skeletal architecture, isolation and characterization of collagens from Demosponges available at Gulf of Mannar (GoM) India. The GoM falls in the Indo-Pacific region, which is considered one of the world's richest in marine biological resources. It has more than 3600 species of plants and animals that make it the biologically richest coastal region in India (Kumaraguru et al., 2006).
Although the collected sponge species are localized to specific habitat locations of GoM, there is much variation in the morphology of each species due to the seasonal changes and habitat disturbances by the local trawlers. Since our studies mainly focus on the highly important protein molecules like collagen, Ircinia fusca was previously been well explored based on the remarkable collagen like fiber content . While the general structural features of sponges are visible through video microscopy, a more elaborated picture on the presence of collagen was obtained through electron microscopy. Gross and Diehl-Seifert were the first to prove the existence of collagen in marine as well as in freshwater sponges, electron microscopically (Gross et al., 1956). Furthermore, the orientation of spongin along with the fine fibrillar material embedded in extra cellular matrix of sponges is a major feature which forms the basis of differentiation for the marine sponges. Cowden (1970) has histologically proven the collagen occurrence in marine sponge tissue. Besides this, spongin skeleton formed of fine fibrils of collagen interwoven along with other connective tissue components like spicules and particulate matter could be observed through SEM.
The macromolecules are the major constituents of different forms of life and form the basis to determine the structure of these life forms, to a certain extent. The macromolecular content of different experimental sponges was estimated to reach to the sponge species with the highest amount of collagen as it is the main structural component. It is well known that sponge tissue protein content dominates other macromolecular content ranging around 40% of the total dry tissue weight (Hadas et al., 2005). Except for hexoses, C. fibrosa has shown higher collagen to relative protein, pentose and lipid content than other sponge species. Analysis of sponge biochemical components reveals that not all the organic components were resolved in the methods applied in the present research.
Collagen is the only intercellular organic framework and amounts to approximately 10% of the total organic matter in Demospongiae (Wiens et al., 1999). Genomic and complementary DNA studies showed that proteinaceous fibrous materials (collagen and spongin) contain the classic collagenous Gly-Xaa-Yaa motif (Exposito and Garrone, 1990;Boute et al., 1996), where Hydroxyproline (Hyp) occupies any one of the positions in the triplet motif other than Gly (Glycine) position. Since the ultimate goal of the project is to target the collagens, standard methods of estimating Hyp to quantify collagen from marine sponges was employed. Though it is a differential extraction for the isolation and estimation of these forms rather the total collagen, the quantification procedures are same to deduce the collagen quantity in all the sponges. As observed in the present study, collagen content is higher in Callyspongia, explanation to this may lie in the presence of high collagenous material in the heavily packaged proteinaceous sheets in the sponge tissue. Based on the present data, the potential candidate for the characterization of this collagen and collagenous nature will be verified further by various biophysical and molecular techniques.
The precipitated collagen is completely soluble in the cold acid and salt extraction buffers. However, more percentage of the precipitated collagenous material remains insoluble, which gives the Ins collagen content of the sponge tissue of all the four sponges. It is understood from the present results that the high percentage of the Ins form of the collagen in the sponges are much responsible for the tissue integrity and unique skeletal framework of the individual sponges, this fact may be responsible for the presence of high Ins collagen than AS and SS collagen forms.