4 COLLECTION AND IDENTIFICATION OF MARINE SPONGES
4.1 Introduction
Sponges are the most primitive form of multicellular invertebrates
(Metazoans), belonging to the phylum, Porifera (Bergquist, 1978; Leys and
Meesch, 2006). Marine sponges are an essential and highly diverse component of
marine benthic communities, abundantly distributed from the euryhaline-estuarine
to intertidal zones and up to thousands of meters in the deep-sea (Fusetani &
Matsunaga, 1993; Hooper &Van Soest, 2002). Of the approximately 15,000 sponge
species reported (Fieseler et al., 2004), most of them occur abundantly in tropical
oceans and some populate in temperate waters, even in Polar Regions (Bruska &
Brusca, 1990; Hooper & Van Soest, 2002). Only about 1% of species inhabits the
freshwater environments (Belarbi et al., 2003). About 486 species of sponges were
identified in India so far (Thomas, 1998).
The two basic types of sponges are encrusting and free-standing. Encrusting
sponges are similar to moss because they tend to cover the surfaces of rocks. Freestanding sponges have lots of inner volume compared with their surface area. Since,
they grow into strange shapes and gigantic sizes, free standing sponges are best
known to people. The barrel sponge is a tropical sponge that can grow large enough
to fit a person inside of it. Tube sponges are well known for their diverse coloration
(Hooper and van Soest, 2002; Jonathan Bird, 2003).
4.1.1 Morphology and life cycle of sponges
Marine sponges are sessile invertebrates with a wide variety of colours,
shapes and consistencies. Neither true tissues nor organs are present, and the cells
display relatively little differentiation and tissue coordination (Simpson, 1984;
Barnes, 1987). The sponge is upright with open tubes and has a pimpled surface
and a tough and elastic texture. A sponge's body is hollow and is held in shape by
mesohyl, a jelly-like substance made mainly of collagen and reinforced by a dense
network of collagenous fibers. The mesohyl functions as an endoskeleton in most
sponges, and is the only skeleton in soft sponges that encrust hard surfaces such as
rocks. More commonly, the mesohyl is stiffened by mineral spicules or by sponging
fibers or both. Demosponges use spongin, and in many species silica spicules and
42
in some species calcium carbonate. The inner surface is covered with choanocytes,
cells with cylindrical or conical collars surrounding one flagellum per choanocyte.
The wave-like motion of the whip-like flagella drives water through the
sponge's body. All sponges have ostia, from which channels leading to the interior
through the mesohyl, and in most sponges, these are controlled by tube-like
porocytes that form closable inlet valves. Pinacocytes are plate-like cells that form
a single-layered external skin over all other parts of the mesohyl that are not
covered by choanocytes. They also have other important functions like digesting
food particles that are too large to enter the ostia (Bergquist, 1994; Ruppert et al.,
2004), while those at the base of the animal are responsible for anchoring it
(Bergquist et al., 1994). Other types of cells live and move within the mesohyl
(Bergquist et al., 1994; Ruppert et al., 2004) as detailed here:
1.
Lophocytes are amoeba-like cells that move slowly through the mesohyl
and secrete collagen fibres.
2.
Collencytes are another type of collagen-producing cells.
3.
Rhabdiferous cells secrete polysaccharides that also form part of the
mesohyl.
4.
Oocytes and spermatocytes are reproductive cells.
5.
Sclerocytes secrete the mineralized spicules ("little spines") that form the
skeletons of many sponges and in some species, provide some defense
against predators.
6.
In addition to or instead of sclerocytes, demosponges have spongocytes that
secrete a form of collagen that polymerizes into spongin, a thick fibrous
material that stiffens the mesohyl.
7.
Myocytes ("muscle cells") conduct signals and cause parts of the animal to
contract.
8.
"Grey cells" act as sponges' equivalent of an immune system.
9.
Archaeocytes (or amoebocytes) are amoeba-like cells and are totipotent. In
other words, each cell is capable of transformation into any other type of
43
cell. They also have important roles in feeding and in clearing debris that
block the ostia.
Sponges do not have nervous, digestive or circulatory systems. Instead, they
rely on maintaining a constant water flow through their bodies to obtain food and
oxygen and for waste-removal, for which the shapes of their bodies are adapted to
maximize the efficiency of the water flow. Indeed, most sponges work rather like
chimneys as they take in water at the bottom and eject it from the osculum ("little
mouth") at the top. Sponges can control the water flow by either wholly or partially
closing the osculum and ostia (the intake pores) or through varying degree of
beating of flagella, and may shut it down, if there is a lot of sand or silt in the water
(Ruppert et al., 2004).
4.1.2 Sponge associated microorganisms
Sponges are filter-feeders, having numerous tiny pores on their surface,
which allow water to enter and circulate through a series of canals where
microorganisms and organic particles are filtered out and eaten (Lee et al., 2001).
Lining these canals are special collar cells. The collar cells force water through the
sponge which brings oxygen and nutrients while removing carbon dioxide and
waste. The water brings with it bacteria and other organisms which the cells capture
and filter out. However, a few carnivorous sponges have lost these water flow
systems and the choanocytes (Hooper et al., 2002).
Sponges are well known to be the hosts for a large community of
microorganisms, which comprise a significant percentage (up to 50–60%) of the
biomass of the sponge-host (Vacelet and Donadey et al., 1977; Wang et al., 2006).
The role of these diverse microbes in sponge biology varies from source of
nutrition to mutualistic symbiosis with the sponge (Lee et al., 2001; Hentschel et
al., 2003; Hill et al., 2004).
There are two pathways through which a developing sponge acquires
bacterial symbionts. The first one is by selective absorption of specific bacteria
from the large diversity of bacteria in the surrounding water column that passes
through the sponge during filter feeding (Lee et al., 2001). The second one is by
vertical transmission of symbionts through the gametes of the sponge by inclusion
44
of the bacteria in the oocytes or larvae (Bewley and Faulkner, 1998; Schmidt, 2000;
Ruby et al., 2004; Radjasa et al., 2007). Thus, symbiotic functions that have been
attributed to microbial associates include nutrient acquisition, stabilization of
sponge skeleton, processing of metabolic waste and secondary metabolite
production (Hentschel et al., 2002).
Since sponges are simple and sessile organisms, during the course of
evolution, they have developed potent chemical defensive mechanism so as to
protect themselves from competitors, predators and infectious microorganisms
(Wang et al., 2006; Taylor et al., 2007; Remya et al., 2010). Sponges are excellent
sources of novel bioactive compounds and they yielded unusual metabolites and
bioactive compounds (Cragg et al, 2006; Newman & Hill 2006). However, in some
cases, sponge-derived compounds may be produced by symbiotic microbes rather
than by the sponges themselves (Haygood et al., 1999; Newman & Cragg et al.,
2004; Piel et al., 2006; Remya et al., 2010). Studies show that secondary
metabolites in sponges play a crucial role in their survival in the marine ecosystem
(Thakur & Müller, 2004; Newman & Cragg et al., 2004; Piel, et al. 2006). The
chemical diversity of secondary metabolites isolated from sponges includes amino
acids, nucleosides, macrolides, porphyrins, terpenoids, aliphatic cyclic peroxides
and sterols (Thakur & Müller, 2004). The biomedical and pharmaceutical
importance of these compounds are attributed to their antibacterial, anticancer,
antifungal, antiprotozoal, and antiviral activities (Osinga et al., 2001; Proksch et al.,
2002; Taylor et al., 2007).
Sponge are morphologically identified using four main criteria viz: colour,
shape, skeletal features and spicule types. Traditional taxonomy methods mainly
follow the investigation on skeletal elements. Recent investigation methods by
means of electron microscopy, chemistry and/or molecular techniques have
demonstrated that some species are actually comprised of more than one species
(Klautau et al., 1994; Solé-Cava & Boury-Esnauli, 1999). Molecular characteristics
analysis provides more precise classification criteria for species that lack
taxonomically important morphological features. By using the molecular data, one
can study all the aspects of sponge evolution (Addis & Perterson, 2005). The
phylogeny of Demospongiae was revisited recently and congruent results were
45
thereby obtained with ribosomal DNA, mitochondrial DNA and nuclear
housekeeping genes mitochondrial cytochrome c oxidase subunit.
4.2 Materials and methods
4.2.1 Sponge samplings and storage
Necessary permission was obtained from the Chief Conservator of Forests &
Chief Wildlife Warden, Govt. of Tamil Nadu, Chennai-15, for sponge-samplings.
The marine sponges were collected, with the help of fishermen, at the intertidal
pools (8-10 metre depth) of Thoothukudi-region (8° 47' N, 78° 8' E) and from
Rameshwaram-area at 4-5 m depth (9° 28' N, 79° 12' E). The sampling sites are
located in the Gulf of Mannar Biosphere region, along the Southeast coast of India
and the samplings were made during April, 2011-March, 2012. The collected
samples were immediately brought to the laboratory in cold container and stored in
the -80 °C till further process (Fig. 9).
The sponges were washed with sea water to remove all residues and blotted
in the filter paper to remove the water. The separated sponge-samples were
photographed and cut into small pieces in the size of 1-2 cm diameter and preserved
in the ethyl acetate. The small portion of the marine sponge was preserved in the
70% ethanol for the identification purpose.
Fig. 9. Phases of sponge samplings (Phase I collection was carried out in the month
of May, 2011, Phase II was carried out in the month of September 2011 and Phase
III was carried out in the month of March, 2012).
46
4.2.2 Identification of sponges
The morphological and anatomical characteristics of the collected sponges
were analysed and the species were identified through microscopic and
macroscopic comparative analyses (Jennaarruda & Carroll, 2010). The typical
characteristics taken into account includes color, size, shape and internal structure
and then the sponges were compared with the existing photographs and data
(Hooper et al., 2002).
4.2.3 Histology of skeleton and spicules preparation
Specimens for the taxonomic investigation were initially fixed in 6%
formaldehyde and later preserved in 96% ethanol. Skeletal architecture was studied
through optical microscope using the sections of 200-400 µm in thickness by
optical microscopy. Those sections were prepared by following the standard
method, as described by Alexander et al. (2010). The specimens were embedded in
epoxy resin and subsequently cut by a precise saw with a diamond wavering blade
(Leica L-1200, Germany). Spicules were prepared, as was generally accepted, by
dissolving the soft tissue of the sponge fragments in nitric acid and were examined
by optical microscopy (Nikon TE 2000-U).
4.3 Results
The identified sponge species were later authenticated/ confirmed by the
experts of Zoological Survey of India and the sponge-samples were submitted to the
sponge-repository (with unique identification numbers). Presently, 9 species of
sponges have been identified (Table 5). And the characteristic features of each
species are detailed hereunder:
47
Table. 5 List of sponges collected and identified during the study
Zoological Survey of India’s
Code
Sponge name
Cliona viridis
NZC/MBRC/S.226
Halichondria glabrata
NZC/MBRC/S.227
Mycale trincomaliensis
NZC/MBRC/S.228
Cliona quadrata
NZC/MBRC/S.229
Psammaplysilla purpurea
NZC/MBRC/S.230
Heteronema erecta
NZC/MBRC/S.231
Jaspis penetrans
NZC/MBRC/S.232
Spirastrella inconstans
NZC/MBRC/S.233
Sigmadocia petrosioides
NZC/MBRC/S.234
4.3.1 Cliona viridis
It is an excavating sponge, and found to be papillae sticking out of
calcareous substrates and covering a massive surface which has completely
overgrown and eroded the substrate; it is easily distinguished from the rather
similar yellow Cliona celata
by having a green colour. Taxonomical
classification is given here in the Table 6 & Plate 1.
Table. 6 Taxonomical classification of Cliona viridis
Kingdom
Animalia
Phylum
Porifera
Class
Demospongiae
Order
Hadromerida
Family
Clionaidae
Genus
Cliona
Species
Cliona viridis
48
Dark green color and yellowish green was observed during the naked eye
examination. It displayed overgrowth stage of Cliona viridis in massive stage, in
which the substrate has been eroded away. Excavated galleries were variable and
measured in the size of 0.3-2.5 mm in diameter. It consisted with rough, almost hard
surface (Plate-1).
4
10X
20X
Plate. 1 Morphological features and microscopic analysis of Cliona viridis
4.3.2 Halichondria Glabrata (Pallas, 1766)
It was observed to be an intertidal thickly encrusting, massive and occasionally
branching sponge, with typical volcanoe shaped oscular chimneys with green and
yellow colour. It was observed that a mild displeasing smell from the sponge body.
Firm and smooth consistency of surface was found (Table. 7 & Plate 2).
Table. 7 Taxonomical classification of Halichondria glabrata
Kingdom
Animalia
Phylum
Porifera
Class
Demospongiae
Order
Halichondrida
Family
Halichondriidae
Genus
Halichondria
Species
Halichondria glabrata
49
Colour of the sponge was observed as light pale yellowish green. With
respect to shape quite variable shapes were found. To prepare the spicules for this
sponge (Plate 2).
Plate. 2 Morphological features of Halichondria glabrata
4.3.3 Mycale trincomaliensis (Martens, 1824)
A yellowish cushion under littoral boulders and on rocks and shells in the
sublittoral was observed. A firm, compressible consistency and a fibrous interior
was found. With a hand lens a characteristic reticulate surface pattern was
discernible (Table. 8)
Table. 8 Taxonomical classification of Mycale trincomaliensis
Kingdom
Animalia
Phylum
Porifera
Class
Demospongiae
Order
Poecilosclerida
Family
Mycalina
Genus
Mycale
Species
Mycale trincomaliensis
50
Cushions of unequal thickness to massive-lobose was observed and the lobes
were present. Surface covered by small conules, raised up by the skeletal fibres and
it is giving the surface a reticulate appearance (Plate 3).
10
4X
20
Plate 3. Morphological features and microscopic analysis of Mycale trincomaliensis
4.3.4
Cliona quadrata (Grant, 1826)
A yellow boring sponge in two distinct forms were observed. Recognizable
as yellow papillae sticking out of limestone (calcareous rocks, shells, etc.); the
other was a large massive, wall-shaped sponge covered with characteristic flattened
papillae and displayed. (Table 9 & Plate 4).
Table. 9 Taxonomical classification of Cliona quadrata
Kingdom
Animalia
Phylum
Porifera
Class
Demospongiae
Order
Hadromerida
Family
Clionaidae
Genus
Cliona
Species
Cliona quadrata
51
Yellow colored body has become darker out of water and in alcohol goes
brown discolouring both the alcohol and the specimen labels. Red discoloration
surrounding ocular openings (Plate 4).
4X
10X
Plate. 4 Morphological features and microscopic analysis of Cliona quadrata
4.3.5 Psammaplysilla purpurea
Dark brown colour, fibrous body with thick hard nature was observed.
Spicules isolation was not successful (Table 10).
. Table. 10 Taxonomical classification of Cliona quadrata
Kingdom
Animalia
Phylum
Porifera
Class
Demospongiae
Order
Verongida
Family
Pseudoceratinidae
Genus
Psammaplysilla
Species
Psammaplysilla purpurea
52
20X
Plate. 5 Morphological features of Psammaplysilla purpurea
4.3.6 Heteronema erecta
A black colored excavating (or boring) sponge. Illuminating inner body with
hard surface area, Taxonomical classification of Heteronema erecta (Table 11 &
Plate 6).
Table. 11 Taxonomical classification of Heteronema erecta
Kingdom
Animalia
Phylum
Porifera
Class
Demospongiae
Order
Dictyoceratida
Family
Spongiidae
Genus
Heteronema
Species
Heteronema erecta
Plate. 6 Morphological features and Microscopic analysis of Heteronema erecta
53
4.3.7 Jaspis penetrans
Dark brown outer layer and soft light yellow colour body was observed,
encrustation with a firm consistency was also observed. Chambers irregular.
Skeleton consist of large oxeas scattered irregularly intermingled with microxeas
was observed. (Table 12 & Plate 7).
Table. 12 Taxonomical classification of Jaspis penetrans
Kingdom
Animalia
Phylum
Porifera
Class
Demospongiae
Order
Astrophorida
Family
Ancorinidae
Genus
Jaspis
Species
Jaspis penetrans
Plate. 7 Morphological features and microscopic analysis of Jaspis penetrans
4.3.8. Spirastrella inconstans (Topsent, 1888a [1887])
A brownish, hispid encrustation with a firm consistency was displayed. It has
been
reliably
identified
by
its
characteristic
microscleres
(microscopic
examination). Overgrowth on the substratum, digitate in shape. The mass found
inside the substratum never form ramifications inside; and the number of papillae
communicating to the exterior is limited in number. The incurrent and excurrent
openings was found on the same or on different papillae. Oscules 2-10 mm in
diameter; oval circular or even slit-like. Pores minute, up to 0.2 mm in diameter.
(Table. 13 & Plate 8).
54
Table. 13 Taxonomical classification of Spirastrella inconstans
Kingdom
Animalia
Phylum
Porifera
Class
Demospongiae
Order
Astrophorida
Family
Spirastrellidae
Genus
Spirastrella
Species
Spirastrella inconstans
10X
20X
Plate. 8 Morphological features and microscopic analysis of Spirastrella inconstans
4.3.9. Sigmadocia petrosioides
White colored soft body was found. Both taxonomical classification (Table
14&Plate 9).
Table. 14 Taxonomical classification of Sigmadocia petrosioides
Kingdom
Animalia
Phylum
Porifera
Class
Demospongiae
Order
Astrophorida
Family
Chalinidae
Genus
Sigmadocia
Species
Sigmadocia petrosioides
55
Plate. 9 Morphological features and microscopic analysis of Sigmadocia petrosioides
4.4 Discussion
Sponges are among the most ancestral metazoans (Medina et al., 2001) and
may hold many clues to our understanding of the evolution of early animal and
developmental processes (Martindale, 2005). They are highly diverse, abundant and
found in nearly every aquatic habitat, some freshwater and most marine, and play
numerous important ecological roles, e.g. in nutrient cycling (Lesser, 2006). Their
significant commercial importance to the pharmaceutical and biomaterials industry
is increasingly being recognized, e.g. as producers of highly potent secondary
metabolites (Faulkner 2000) useful for drug development (Munro et al., 1994).
The history of sponge-study of the India Ocean starts from 1765. A perusal
of literature reveals that 451 species of marine sponges are known to occur in India
(Pattanayak, 1999) through the works of Sollas (1884), Dendy (1887-1989),
Annandale (1914-1915), Burton (1930) and Ali (1954-56). An exhaustive survey of
the marine sponges with special reference to the Gulf of Mannar and Palk Bay has
been studied during the years 1964-67 by Thomas (1968-2006). Coral boring
sponges of the Gulf of Mannar and Palk Bay were studied by Thomas (1969). So
far about 11,000 species have been formally described globally of which 8553 are
valid species of sponges. In India marine sponges constitute 451 species. And Gulf
of Mannar and Palk Bay inhabits of 313 species belonging to 137 genera and 12
orders of demospongiae and 5 species of class Hexactinellida and 1 species of class
calcarea was recorded. (Pattanayak, 2001).
Many sponge species are extremely difficult to identify, often even by
taxonomic experts, because morphological characters for comparative morphology
are scarce and prone to homoplasies, highly variable or otherwise unsuitable for
unambiguous identification. In addition, many sponges discovered in large scale
56
biodiversity surveys remain undescribed (Hooper & Ekins 2005), partly also due to
the lack of skilled taxonomists. As a result of uncertainties in morphological
systematics, sponge species have frequently been regarded as widely distributed
(‘cosmopolitan’). However, genetic approaches, mostly using allozymes, have
clearly shown that such cosmopolitan sponge species are rare and appear to result
from over-conservative systematics,
lumping
morphologically similar
but
evolutionary distinct lineages into one widely distributed morpho-species (Klautau
et al. 1999). The question of how to describe and distinguish such genetically
distinct and reproductively isolated lineages remains, due to the difficulty of
relating those genetic differences to morphological delineation of ‘species’.
In the Palk Bay, a total of 16 specimens as belonging to 9 species of sponges
belonging to seven genera and six families were recorded from the Seagrass beds.
The genus, Spirastrella was dominant and seems to be an integral part of sea grass
ecosystems (Sivaleela et al., 2013).
Thomas (1969) identified total 20 species of boring sponges from Gulf of
Mannar and Palk Bay in south east India considered to be causing damage to the
coral reef. In his proceeding he has listed the marine sponge species that available
in Gulf of Mannar and Palk Bay region. In our present study, most of the listed
sponges were collected and identified from the same region. It impels 55 years of
evolutionary relationship and the sponge species existence.
Nowadays, molecular techniques including RFLP, RAPD and DNA
sequencing techniques are popularly used for the species identification purpose.
However, while handling marine sponges, we need a considerable caution to
perform the experiments. Marine sponges are being associated with several
microorganisms, mixed population of sponges and other marine organisms. There
could be an inaccuracy in the results due the sensitivity of the molecular techniques.
Since we use universal primers in the PCR technique it could potentially amplify
the genes from other organisms due to the mixed or contaminated DNA samples.
The presently collected/identified sponges have been used for the purpose of
extraction of their metabolites and the use of the extracted metabolites in
subsequent experiments.
57