II. REVIEW OF LITERATURE

The sponges or poriferans are animals of the phylum Porifera. They are primitive,

sessile, mostly marine, water dwelling filter feeders that pump water through their bodies

to filter out food particles. Sponges represent the simplest of animals, with no true tissues

(parazoa), they lack muscles, nerves, and internal organs. Their similarity to colonial

choanoflagellates shows the probable evolutionary jump from unicellular to multicellular

organisms. However, recent genomic studies suggest they are not the most ancient lineage

of animals, but may instead be secondarily simplified. There are over 5,000 modern species

of sponges known, and they can be found attached to surfaces anywhere from the intertidal

zone to as deep as 8,500 m or further. Though the fossil record of sponges dates back to the

Neoproterozoic Era, new species are still commonly discovered (Dunn et al., 2008).

2.1. Diversity of sponge-associated microorganism

Sponges (phylum Porifera) form one of the deepest radiations of the metazoa whose

evolutionary roots date back to Precambrian times. The earliest confirmed sponge fossils

were found in Precambrian rock deposits in South China that date back about 580 million

years in time (Li et al., 1998). These fossils show extraordinarily well-preserved soft

tissues, amoebocytes and even metazoan embryos. Above all, over 1000 sponge fossils

within 15 genera and 30 species have been described in Cambrian rock deposits indicating

an early radiation of this phylum. Sponges have been the focus of interest in recent years

(Fig. 1) due to their close associations with a wide variety of microorganisms and as rich

source of biologically active secondary metabolites. Though, the increasing research

interest has greatly helped in better understanding sponge-microbe interactions, still many

gaps remain unanswered. These include lack of clear information of microbial diversity,

Figure 1. Increasing research interest in marine sponge-microorganism associations. (A)

Number of publications retrieved from the ISI Web of Science database by using the

following search string: (sponge* or porifera* or demospong* or sclerospong* or

hexactinellid*) and (bacteri* or prokaryot* or microbe* or microbial or microorganism* or

cyanobacteri* or archaeon or archaea* or crenarchaeo* or fungi* or diatom* or

dinoflagellate* or zooxanthella*) not (surgery or surgical). (B) Number of sponge-derived

16S rRNA gene sequences deposited in GenBank per year. The 2006 value includes the

184 sequences submitted to GenBank from the article Taylor et al., 2007. The search string

used to recover sequences was as follows: (sponge* or porifera*) and (16S* or ssu* or

rRNA*) not (18S* or lsu* or large subunit or mitochondri* or 23S* or 5S* or 5.8S* or

28S* or crab* or alga* or mussel* or bivalv* or crustacea*). Source: Taylor et al., 2007.

factors influencing diversity in hosts, physiology of sponge associated microorganisms and

fundamental aspects of sponge symbiont ecology (Taylor et al., 2007).

Marine sponges have attracted lot of importance as source of wide variety of natural

products (Blunt et al., 2006). More novel bioactive metabolites are being reported from

sponges than from any other marine taxon, and a range of pharmacological properties have

been demonstrated (Munro, et al., 1999; Blunt et al., 2006). These bioactive compounds

are known to be beneficial to sponge in providing defense against predators

(Chanas, et al., 1997; Manz et al., 2000; Becerro et al., 2003), competitors

(Turon et al., 1996; Thacker et al., 1998; Engel and Pawlik, 2000), fouling organisms

(Sears et al., 1990; Willemsen, 1994), and microbes (Becerro et al., 1994; Newbold et al.,

1999; Thakur, et al., 2003). In some cases, the bioactive compounds appear to be produced

by associated microorganisms rather than by the sponge (Bewley and Faulkner, 1998;

Schmidt et al., 2000; Piel et al., 2004). Many types of interactions occur between sponges

and microorganisms. To a sponge, different microbes can represent food sources (Reiswig,

1971; Reiswig, 1975; Pile et al., 1996), pathogens/parasites (Lauckner, 1980; Hummel et

al., 1988; Bavestrello et al., 2000; Webster et al., 2002), or mutualistic symbionts

(Wilkinson, 1983; Wilkinson, 1992). The diversity in types of interaction is matched by the

phylogenetic diversity of microbes that occur within host sponges. Earlier studies

involving microscopy and culturing methods have showed high levels of morphological

and metabolic diversity in sponge-associated microbes (Colwell and Liston, 1962; Madri

et al., 1971; Sará, 1971; Vacelet and Donadey, 1977; Wilkinson, 1978 a, b, c). The

application of molecular tools over the past decade has greatly extended the known

diversity of microorganisms within these hosts (Preston et al., 1996; Lopez et al., 1999;

Friedrich et al., 1999; Webster et al., 2001a; Hentschel et al., 2002; Fieseler et al., 2004;

Taylor et al., 2004). Each of the three domains of life, i.e., Bacteria, Archaea, and

Eukarya, are now known to reside within sponges.

Marine sponges inhabiting both shallow- and deep-water communities are known to

occupy as much as 80% of available surfaces in some areas (Dayton, 1989). Such sustained

evolutionary and ecological success is often attributed to their intimate associations with

microbial symbionts. However, unlike many other studied host-microbe associations,

which involve only a very small number of participants [(e.g., squid-Vibrio fischeri

(Nyholm and McFall-Ngai, 2004)], amoeba-Chlamydiae (Horn and Wagner, 2004), and

Bugula-“Endobugula” symbioses (Haygood and Davidson, 1997; Lim and Haygood, 2004)

and the sponge-associated microbial communities can be highly diverse, with a range of

different microorganisms consistently associated with the same host species.

2.2. Bacterial localization

Large numbers of bacterial association has been reported for several orders of the

Demospongiae (Table 1). The sponges, Aplysina cavernicola and Ceratoporella

nicholsonii contain large numbers of bacteria that amount to 38 and 57% of the tissue

volume, respectively (Vacelet, 1975; Willenz and Hartmann, 1989). Bacterial numbers

were estimated at 6.4+4.6x108 g-1 tissue for A. aerophoba and 1.5x109 ml-1 sponge extract

for Rhopaloeides odorabile (Friedrich et al., 2001; Webster and Hill, 2001). The bacterial

distribution follows a general pattern in any given sponge. The outer, light-exposed tissue

layers are generally populated by photosynthetically active microorganisms such as

cyanobacteria and eukaryotic algae (Rützler, 1985; Wilkinson, 1992).

The inner core is populated by heterotrophic and also autotrophic bacteria. The mesohyl

contains the vast majority of microorganisms and with a few exceptions, the associated

bacteria are situated extracellularly within the mesohyl matrix. Dividing bacteria are

Table 1. List of sponges that contain variable amounts of microorganisms Hentschel et al.,

2003a).

Species

Order

Reference High density of microorganisms

Aplysina aerophoba Verongida Vacelet, 1975, Friedrich, 1998

Aplysina cavernicola Verongida Vacelet, 1975, Friedrich, 1998

Agelas oroides Agelasida Vacelet and Donadey, 1977

Plakina trilopha Homosclerophorida Vacelet and Donadey, 1977

Petrosia ficiformisb Haploslerida Vacelet and Donadey, 1977

Ircinia wistarii Dicytoceratida Wilkinson, 1978a

Jaspis stelliferra Astrophorida Wilkinson, 1978a, Fuerst et al., 1999

Theonella swinhoeic Lithistida Bewley and Faulkner, 1998

Rhopaloides odorabile Dicytoceratida Webster and Hill, 2001

Astrosclera willeyanab Agelasida Wörheide, 1998

Ceratoporella nicholsoni Agelasida Willenz and Hartman, 1989, Santavy and

Colwell, 1990

Low density of microorganisms

Pleraplysilla spinifera Dendroceratida Vacelet and Donadey, 1977

Thenea muricata Astrophorida Vacelet and Donadey, 1977

Oscarella lobularis Astrophorida Vacelet and Donadey, 1977

Grantia compressa Calcaronea Vacelet and Donadey, 1977

Acanthella acutaa Axinellida Vacelet and Donadey, 1977

Axinella polyploidesa Axinellida Vacelet and Donadey, 1977

Reniera mucosaa Haploslerida Vacelet and Donadey, 1977

Crambe spa poecilosclerida Vacelet and Donadey, 1977

Petrobiona massilianaa Calcaronea Vacelet and Donadey, 1977

Pericharax heteroaphis Clathrinida Wilkinson, 1978a

Neofibulaira irata poecilosclerida Wilkinson, 1978a

Niphates sp. Haploslerida J.Weisz and Lindquist, N (unpubl.)

a Bacteria embedded within collagen fibrils. b Bacteria contained within bacteriocytes. c Dominance of a filamentous Deltaproteobacterium, Candidatus, Entotheonella palauensis.

observed infrequently. In some sponges, such as Astrosclera willeyana and Petrosia

ficiformis, bacteria are contained within bacteriocytes (Vacelet and Donadey, 1977;

Wörheide, 1998). Bacteria are also found within vacuoles of sponge archaeocytes, where

they are lysed and digested. However, the canal system, choanocyte chambers and the

outer sponge surface are free of bacterial epigrowth in healthy sponges.

Interestingly, presence of numerous, morphologically uniform, filamentous bacteria

within the nuclei of certain sponge cells with A. aerophoba has been reported (Vacelet,

1970; Friedrich et al., 1999). These bacteria are enclosed within a vacuole in varying

number. Their presence appears to be correlated with a degeneration of the host cell,

indicating a shift towards a pathogenic interaction. Also the low occurrence of these cells

does not affect the health of the sponge. Similar associations have been reported for single

eukaryotic ciliates such as paramecium whose nuclei are infected by morphologically

similar bacteria of the genus Holospora (Hentschel et al., 2003a). However, it is not clear

whether these observations in sponges are due to infected marine ciliates that have slipped

into the sponge mesohyl.

2.3. Microscopic observations

Earlier reports establishing roles of bacteria in sponges came from microscopic studies

demonstrating the presence of large numbers of bacteria within marine sponges (Dosse,

1939; Vacelet, 1970, 1975; Vacelet and Donadey, 1977; Wilkinson, 1978c). These

investigations gave insight into the distribution and localization of bacteria within the

sponges. Large numbers of bacteria were found inside the vacuoles of archeocytes referred

to as bacteriocytes (Simpson, 1984), nucleus of sponge cells (Vacelet, 1970, 1975; Flowers

et al., 1998; Friedrich et al., 1999), and also outside of sponge cells in the mesohyl, which

is located between the cell layers that line the canal system and the choanocytes. Bacteria

can constitute as much as 60% of the mesohyl volume (Sará, 1971; Vacelet, 1975; Vacelet

and Donadey, 1977; Wilkinson, 1978 c, d; Santavy, 1985; Santavy et al., 1990). De Vos et

al. (1995) considered all marine sponges to shelter bacteria in the mesohyl and thought that

sponges with a loose mesohyl only have one or two morphological types, whereas sponges

with a dense mesohyl harbour abundant and morphological diverse types of bacteria. On

the basis of morphological studies, attempts were made to distinguish between sponge-

associated and seawater bacteria (Vacelet, 1975; Wilkinson, 1978a, b, c). Based on this

three broad categories of bacteria associated with sponges were recognized: (1) Bacteria

that were similar to those of ambient seawater and not specific to the sponge; (2) Small

numbers of intracellular bacteria that were considered to be specific to the sponge; and (3)

Large numbers within the mesohyl that appeared to be specific. In Aplysina cavernicula,

five dominant bacterial morphtypes were identified that were regarded to be specifically

associated with the mesohyl (Vacelet, 1975; Vacelet and Donadey, 1977). Of which types

C, D, and E were found in great abundance (Table 2). Subsequent studies described

morphologically similar bacteria in diverse sponges even though the abundances between

different sponge species were somewhat variable. Wilkinson, (1978c) described five

distinct bacterial morphotypes in several sponges from the Great Barrier Reef, of which

types 2 and 4 resemble Types D and E of Vacelet (1975) respectively. Subsequent studies

by Vacelet (1975) the sponges A. cavernicola and A. aerophoba were investigated for the

presence of the originally described morphotyes (Friedrich et al., 1999). Transmission

electron microscopy (TEM) revealed the presence three of the original five morphotyes.

Type C bacteria were characterized by several additional sheaths, Type D by a copious,

irregular slime layer, and Type E by a putative nuclear membrane (Table 2). It is

hypothesized that the additional outer membrane features are known to serve as shields to

Table 2. Morphological characteristics and abundances of mesohyl baceteria in Aplysina

cavernicola (Hentschel et al., 2003a).

Type

Abundance

Described in Vacelet (1975)

Diameter (μm)

Characterisitics

A

Not abundant

No

About 0.8

Precise outer border characteristic of Gram-positive cell walls

B

Not abundant

No

1.0 -1.2

Perpendicular divisions (‘spacers’) connect the outer membrane to peptidogycon

C Abundant Yes About 1.0 Presence of several additional sheaths

D Abundant Yes About 1.4 Copious, irregular slime layer

E Abundant Yes 0.8 -1.4 Enlarged periplasm/putative nuclear membrane, no peptidoglycan

O Variable Yes Variable All ‘other’ bacteria

prevent digestion by sponge archaeocytes (Wilkinson et al., 1984; Friedrich et al., 1999).

Fuerst et al. (1999) reported the occurrence of an unusual bacterial morphotype having

membrane bounded nuclear bodies in several Great Barrier Reef sponges. Because

prokaryotes generally do not possess organelles, the identification of these unusual bacteria

becomes significant. Altogether, six different subtypes were described which resembled

the general appearance of Type E according to Vacelet (1975) and Friedrich et al. (1999)

and Type 4 according to Wilkinson (1978c). Using immunogold labeling studies, the

presence of genomic DNA within the nuclear bodies was confirmed. To date, cell

compartmentalization is only known within the planctomycete division (Lindsay et al.,

1997) where, the DNA is localized within a nucleoid, termed the pirellulosome. In contrast

to free living planctomycetes, the sponge-associated bacteria do not show a polar

differentiation, which is a distinguishing feature of planctomycetes.

2.4. Culture - dependent techniques

Sponges have attracted the attention of microbiologists in recent years. Both classical

microbial techniques and cultivation approaches have been used in number of studies to

analyse the diversity of bacteria within sponges. But study of possible symbiotic

interactions between bacteria and sponges and bacterial diversity is limited by the

cultivation success. Cultivation of bacteria is always highly selective and depends on the

choice of media and culture conditions which usually allows the growth of only a small

fraction of the bacteria present within a natural sample or sponge. Thus, special skills are

required to isolate bacteria of interest. No selective cultivation methods are available to

distinguish bacteria specifically associated with sponges from others, including those

bacteria that serve as food particles. Therefore, cultivation approaches are useful in

providing general idea about the diversity of bacteria associated with sponges and their

role in these complex associations (Imhoff and Stöhr, 2003).

The microbial association in three Australian sponges has been studied and sorted

based on metabolic and physiological characteristics by Wilkinson (1978b) and Wilkinson

et al. (1981). Statistical analysis resulted in the generation of phenotypic clusters, one of

which was consistently found in all sponges examined. These include aerobic

chemoheterotrophic bacteria (Wilkinson et al., 1981), nitrogen-fixing bacteria (Shieh and

Lin, 1994), methane-oxidising bacteria (Vacelet et al., 1996), phototrophic cyanobactreia

(Wilkinson, 1978d; Simpson, 1984) and anoxygenic phototrophic bacteria (Imhoff and

Trüper, 1976). The phenotypic characteristics (Gram-negative, sticky-mucoid colonies,

presence of refractile granules, rod shaped morphology) resembled those of the Alpha-

Proteobacterium MBIC3368 that has been isolated earlier from several sponges (Lopez et

al., 1999; Hentschel et al., 2001; Webster and Hill, 2001; Olson et al., 2002; Thakur et al.,

2003). While these early studies indicated the isolation of sponge-specific microorganisms,

their respective phylogenetic identities could not be determined at that time.

A numerical taxonomic study was also performed by Santavy et al. (1990) who isolated

heterotrophic bacteria from the Caribbean sponge, Ceratoporella nicholsoni. By testing for

a large number of phenotypic attributes, the strain collection could be sorted into four

major phena. Phena 1 and 3 most closely resemebled the genus vibrio and phenon 2

showed attributes of the genus Aeromonas. Phenon 4 was composed of diverse strains

whose morphologies ranged from filaments to unusal shapes as club-, Y-, and T-shaped

cells. This phenon resembled most closely actinomycete or coryneform bacteria. However,

the ultimate conclusions regarding their phylogenetic identity could not be ascertained.

Information from cultivation studies revealed that only a minor fraction of the total

sponge-associated microbial community was amenable to cultivation on laboratory media.

Santavy et al. (1990) estimated that 3-11% of the total bacterial population of

Ceratoporella nicholsoni were culturable. Webster and Hill, (2001b) concluded that the

culturable heterotrophic bacterial community comprised only 0.1-0.23% of the total

microbial community of R. odorabile. These numbers are in the same range as estimates by

Friedrich et al. (2001), who reported only 0.15% of the total microbial community of

A. aeropoba were culturable. This general observation is consistent within the context of

natural microbial ecosystems where an estimated 1 % is currently accessible by laboratory

culture. The phenomenon and yet unsolved question of microbial ecology has suitably

been referred to as ‘the great plate count anomaly’ (Amman et al., 1995). Some facultative

anaerobic bacteria that were isolated and identified from three coral reef sponges

(Pericharax heteroraphis, Jaspis stellifera, Neofibularia irata) apparently were found

within the sponges but could not be isolated from the ambient seawater (Wilkinson,

1978a). Similarly, bacteria isolated from the Caribbean Sclerosponge, Ceratoporella

nicholsoni formed one major cluster of strains related to the genera Vibrio and Aeromonas

according to the performed numerical taxonomic analyses, whereas most bacteria from the

surrounding seawater (95%) were associated with the genera Acinetobacter, Micrococccus,

and Moraxella (Santavy and Colwell, 1990). Moreover, Wilkinson et al. (1981) described

phenotypically similar bacteria in nine sponges from the Mediterranean Sea and the Great

Barrier Reef. These isolates were reported to be sponge specific because they were not

isolated from ambient seawater. Unfortunately, the phenotypic characterization was not

comprehensive enough to identify these bacteria on the species level. Among bacteria

isolated on standard media from 40 Rhopaloeides odorabile specimens from different

regions of the Great Barrier Reef, two different bacteria were reported to be dominant

(Burja et al., 1999). By 16S rDNA sequence similarity, the most abundant isolate

(represented by strain NW001) was closely related to two bacteria isolated from an

unidentified sponge and from Aplysina aerophoba but was more distantly related to

established species of Alpha-Proteobacteria (Webster and Hill, 2001b). This bacterium

was localized within the mesohyl of the sponge, in particular surrounding the choanocyte

cells. A second abundant isolate (represented by strain NW002) was by the same criteria

found to belong to the genus Pseudoalteromonas. Unfortunately, the dominance among

cultured bacteria gives no indication concerning their relative abundance within the sponge

and, in fact, as demonstrated later by molecular genetic anyalyses of the bacterial diversity

in this sponge, Alpha-Proteobacteria were not among the dominant bacteria (Webster et

al., 2001a). A large number of aerobic chemoheterotrophic bacteria were isolated from

Halichondria panicea by using different media and culture conditions (Imhoff and Stohr,

2003).

2.5. Culture –independent techniques

Molecular genetic methods have been useful in the analysis of association between

sponges and bacteria as they help to identify sponge-associated bacteria and to analyse

their diversity. In this regard application of the 16S rDNA approach has revolutionized the

field of microbial ecology. With the use of the 16S rDNA gene as phylogenetic marker, it

has become possible not only to determine the precise phylogenetic position of

environmental bacterial populations in the evolutionary tree of life independent of their

culturability but also trace their complex ecosystems (Hentschel et al., 2003a). The

application of these techniques to environmental samples revealed a previously unseen

microbial diversity that encompasses an estimated >99% of the total microbial community

of a given habitat (Hentschel et al., 2003a). The discovery of this large pool of not yet

cultured bacteria in environmental samples is considered a milestone in environmental

microbiology.

Many cellular macromolecules can serve as evolutionary markers. In fact, phylogenetic

trees built upon elongation factors, ATPase subunits and RNA polymerases are in good

agreement with 16S rDNA gene trees (Hentschel et al., 2003a). Among the ribosomal

genes that exist ubiquitously in cells, the prokaryotic 16S and 23S (small subunit and large

subunit, respectively) and the eukaryotic 18S and 28S (Small subunit and large subunit,

respectively) genes have been very useful for revealing phylogenetic relationships. The

major advantage of rRNA’s over other macromolecules is their very high expression level

in active cells. Metabolically active bacterial cells may contain over 100,000 ribosomes

resulting in an equal number of 16S and 23S rRNAs. For bacterial phylogeny, the 16S

rRNA gene fragment of about 1500 nucleotides in length is preferable over the 23S rRNA

gene (about 3000 nucleotides) because of ease of routine sequencing. As of now, more

than 22,000 16S rRNA gene sequences have been deposited into public databases

providing an extensive reference for the evaluation of phylogenetic relationships of

bacteria. An overview of the techniques that have been developed on the basis of the 16S

rRNA gene and that have recently been applied to sponge-microbe associations is

presented below.

2.5.1. Fluorescence in- situ hybridization

To study the sponge-microbe association a innovative method known as fluorescence

in situ hybridization (FISH) which is a cultivation-independent detection of single bacterial

cells has enabled to evaluate phylogenetic identity, to visulalize their morphology and to

gain insights into the spatial arrangements of bacteria within the sponge tissue

(Hentschel et al., 2003a). This method relies on short (15-20nt) oligomeric sequences with

their 5’ end labeled to a fluorochrome dye, such as the red sulfoindocyanine (cy3) or the

green fluorescein (cy5). The FISH probe binds to the complementary target region of the

16S rRNA molecule allowing the microscopic visualization of the target cell. By virtue of

the conserved and variable regions on the 16S rRNA, probes can be designed against broad

phylogenetic groups (such as domain-specific probes), and with increasing levels of

resolution against phyla, genera, species, and even against stains. Several hundred

oligonucleotide probes have been published that are conveniently presented and updated

on the internet (http://www.microbial-ecology.de/probebase/). Thus, detection of specific

organisms requires the design of novel probes (Hentschel et al., 2003a) which can be

performed with the probe design function of the ARB program package (http://www.arb-

home.de).

Several studies have applied FISH to investigate the microbial diversity of sponges.

Schumann-Kindel et al. (1997) were the first to perform microbial diversity studies on the

Mediterranean sponges, Chondrosia reniformis and Petrosia ficiformis. Hybridization

experiments revealed that majority of bacteria belonged to the Gamma-Proteobacteria.

Delta-Proteobacteria, specifically, sulfate-reducing bacteria were also scattered throughout

the tissues of both sponges. FISH was employed by Preston et al. (1996) to confirm the

presence of the archael microorganism, Cenarchaeum symbiosum, within the tissues of the

pacific sponge, Axinella mexicana. Interestingly, about 15% of sponge-assoicated archaea

were dividing, indicating active growth of these archaea within captive sponges. The

phylogenetic profile of the microbial community of A. cavernicola and A. aerophoba was

described using previously established group-specific 16S rRNA targeted oligonucleotide

probes (Friedrich, 1998, Friedrich et al., 1999). The Delta-Proteobacteia were the most

abundant followed by the Bacteroidetes, the Gamma-Proteobacteria and the

Actinobacteria. The phylogenetic profile of R. odorabile consisted of numerous Gamma-

Proteobacteria and representatives of the Bacteroidetes. FISH also confirmed the presence

of Actinobacteria, Beta-Proteobacteria, Firmicutes, and Planctomycetales (Webster et al.,

2001a).

2.5.2. Denaturing gradient gel electrophoresis

The DGGE is higher version of molecular approach which allows the fingerprinting of

microbial communities without the need to culture the respective microorganisms

(Hentschel et al., 2003a). Following community DNA extraction, a PCR reaction is

performed. One primer in the reaction contains a GC-clamp. The PCR reaction mix is

separated into individual bands on a polyacrylamide gel with an increasing denaturing

gradient that includes urea and formamide. The GC-clamp acts as an ‘anchor’ and ensures

that each PCR product is not separated into single strands during migration into the gel. In

the ideal case, each DGGE band represents a single 16S rRNA gene sequence. Thus, each

lane represents a fingerprint of a microbial community at a given time. Individual bands

can be excised, reamplified and sequenced to obtain phylogenetic information. The

phylogenetic resolution of DGGE is somewhat limited as only partial sequences of about

500 bp in size can be separated on gradient gels.

DGGE analysis is particularly useful application for the characterization of sponge-

associated microbial communities as it not only provides insights into the overall

complexity of the microbial community, but also allows to monitor changes in community

composition of individual sponges over time. Using DGGE in combination with other

methods, Friedrich et al. (2001) have shown that the microbial community of

A. aerophoba was surprisingly resistant to experimental perturbations, such as exposure to

starvation and/or to antibiotics over a time course of 11 days. Thoms et al. (2003) have

employed DGGE to monitor microbial community changes upon long-term transplantation

of A. cavernicola to different habitats. The microbial community was also surprisingly

inert in spite of the fact that the sponges showed visual sings of stress. Importantly, DGGE

enabled researchers to make distinction between the permanent and transient fraction of the

microbial community.

2.6. 16S rDNA library construction

The construction of 16S rDNA libraries is the most labor-intensive but phylogenetically

the most informative approach (Hentschel et al., 2003a). Following community DNA

extraction and PCR amplification of the 16S rDNA genes, the products are cloned into

vectors, such as the pGEM-T-easy vector, and expressed in E.coli. Following plasmid

preparation of individual clones, the 16S rRNA genes are reamplified and sequenced.

Several computer based programs exist for the construction of phylogenetic trees. Among

the different tree construction programs, maximum likelihood and neighborhood joining

are the most frequently used. So far, only three 16S rDNA libraries have been established

from the sponges, R. odorabile, A. aerophoba and Theonella swinhoei (Webster et al.,

2001a; Hentschel et al., 2002). The results reveal a striking similarity between these

sponges. In addition, several individual eubactrerial sequences have been published from

Halichondria panicea (Althoff et al., 1998), Discodermia spp. (Lopez et al., 1999), and

Mycale hentscheli (Webb and Maas, 2002). Archaeal 16S rDNA sequences have also been

published from the sponge, Axinella mexicana (Preston et al., 1996).

2.6.1. Diversity of microorganisms from sponges

The diversity of microorganisms known from sponges was categorized in 14

recognised bacterial phyla (and one candidate phylum) consisting of both major archaeal

lineages, and assorted microbial eukaryotes (Wilkinson, 1992; Hentschel et al., 2003b;

Hentschel et al., 2006). Sequences representing the following bacterial phyla have been

recovered from 16S rDNA gene libraries and/or excised denaturing gradient gel

electrophoresis (DGGE) bands: Acidobacteria, Actinobacteria, Bacteroidetes, Chloroflexi,

Cyanobacteria, Deinococcus-Thermus, Firmicutes, Gemmatimonadetes, Nitrospira,

Planctomycetes, Proteobacteria (Alpha, Beta, Gamma and Delta proteobacteria),

Spirochaetes, and Verrucomicrobia (Althoff et al., 1998; Lopez et al., 1999; Webster et

al., 2001a; Hentschel et al., 2002; Webb and Maas, 2002; Thacker and Starnes, 2003;

Thoms et al., 2003, Taylor et al., 2004; Hill, 2004; Usher et al., 2004a; Webster et al.,

2004; Taylor et al., 2005, Gernert et al., 2005, Ridley et al., 2005b, Schirmer et al., 2005,

Steindler et al., 2005; Enticknap et al., 2006; Hentschel et al., 2006; Hill et al., 2006; Thiel

et al., 2007a). In addition, a seemingly sponge-specific candidate phylum, “Poribacteria,”

has also been reported for several sponges (Fieseler et al., 2004) (Fig. 2)

The most frequently recovered sequences in general 16S rDNA gene surveys of

sponges include those from the Acidobacteria, Actinobacteria, and Chloroflexi (Hentschel

et al., 2006). Members of several bacterial phyla, namely, the Actinobacteria,

Bacteroidetes, Cyanobacteria, Firmicutes, Planctomycetes, Proteobacteria, and

Verrucomicrobia have also been isolated in pure culture from marine sponges (Santavy

and Colwell, 1990; Burja et al., 1999; Lopez et al., 1999; Olson et al., 2000; Burja and

Hill, 2001; Hentschel et al., 2001; Webster and Hill, 2001; Webster et al., 2001; Olson et

al., 2002; Pimentel-Elardo et al., 2003; Chelossi et al., 2004; Dieckmann et al., 2005;

Figure 2. 16S rDNA-based phylogeny showing representatives of all bacterial and

archaeal phyla from which sponge-derived sequences have been obtained. Sponge-derived

sequences are shown in bold, with additional reference sequences also included. The

displayed tree is based on a maximum likelihood analysis. Bar, 10% sequence divergence.

(Taylor et al., 2007).

Kim et al., 2005; Lafi et al., 2005; Montalvo et al., 2005; Sfanos et al., 2005; Enticknap et

al., 2006; Kim and Fuerst, 2006; Lee et al., 2006; Scheuermayer et al., 2006). Sequences

from the Chlorobium (green sulfur bacteria) have not been obtained from sponges,

although positive fluorescence in situ hybridization (FISH) signals were obtained from

Rhopaloeides odorabile with a specific probe for this phylum (Webster et al., 2001a). In

contrast to marine sponges, freshwater species showed much lower bacterial diversity and

abundance. Only sequences from the Actinobacteria, Chloroflexi, and Alpha- and Beta-

Proteobacteria were recovered in a recent 16S rDNA gene library constructed from the

freshwater sponge, Spongilla lacustris (Gernert et al., 2005). Moreover, many of these

sequences were highly similar to those known previously from freshwater habitats,

suggesting that they may not represent true symbionts. With a few exceptions in the

Euryarchaeota (Webster et al., 2001c; Holmes and Blanch, 2006), archaea reported from

marine sponges are members of the phylum Crenarchaeota (Preston et al., 1996; Webster

et al., 2001c; Margot et al., 2002; Lee et al., 2003; Webster et al., 2004; Holmes and

Blanch, 2006). Lipid biomarkers also suggested the presence of both crenarchaeotes and

euryarchaeotes in a deep-water Arctic sponge, though no phylogenetic information was

provided in that study (Pape et al., 2006). The group I.1A of Crenarchaeota are extremely

prevalent in marine environments (Karner et al., 2001), and almost all sponge-derived

archaeal sequences are affiliated with this group. The best-studied sponge-associated

archaeon is the psychrophilic crenarchaeote Candidatus Cenarchaeum symbiosum, which

comprised upto 65% of prokaryotic cells within the Californian sponge, Axinella mexicana

(Preston et al., 1996; Schleper et al., 1997; Schleper et al., 1998; Hallam et al., 2006b).

Eukaryotic microbes have also been associated with sponges. Sponge-inhabiting

dinoflagellates (Sará and Liaci, 1964; Wilkinson, 1992; Hill, 1996; Hill and Wilcox, 1998;

Garson et al., 1998; Scalera-Liaci et al., 1999; Steindler et al., 2001; Webster et al., 2004;

Schönberg and Loh, 2005) and diatoms (Cox and Larkum, 1983; Gaino et al., 1994; Burja

et al., 1999; Cerrano et al., 2000; Bavestrello et al., 2000; Cerrano et al., 2004; Regoli et

al., 2004; Taylor et al., 2004; Webster et al., 2004; Totti et al., 2005) have been reported,

with the latter seemingly most prevalent in polar regions (Gaino et al., 1994; Cerrano et

al., 2000; Bavestrello et al., 2000; Regoli et al., 2004; Webster, et al., 2004; Cerrano et al.,

2004; Totti et al., 2005).

Freshwater sponges often contain endosymbiotic microalgae, primarily zoochlorellae

(Williamson, 1979; Frost and Williamson, 1980; Wilkinson, 1980; Saller, 1989; Sand-

Jensen and Pedersen, 1994; Frost et al., 1997; Bil et al., 1999). Reports of cryptomonads in

sponges were noted by Wilkinson (Wilkinson, 1992.). Marine sponge-derived fungi are

also receiving increasing attention due to their biotechnological potential (König et al.,

2006; Bugni and Ireland, 2004; Höller et al., 2000). Interestingly, of 681 fungal strains

isolated worldwide from 16 sponge species, most belonged to genera which are ubiquitous

in terrestrial habitats (e.g., Aspergillus and Penicillium) (Höller et al., 2000). It thus

remains unclear in most cases whether such fungi are consistently associated with the

source sponge, or even whether they are obligate marine species. Compelling evidence for

symbiosis of yeast with sponges of the genus Chondrilla was obtained by extensive

microscopy studies of both adult sponge tissue and reproductive structures, with strong

indications of vertical transmission of the yeast symbiont (Maldonado et al., 2005b). Little

is known about viruses in sponges, although virus-like particles were observed in cell

nuclei in Aplysina (Verongia) cavernicola (Vacelet and Gallissian, 1978). It was suggested

that these particles could be involved in sponge cell pathology. Infection of an Ircinia

strobilina- derived alphaproteobacterium by a bacteriophage isolated from seawater has

also been demonstrated (Lohr et al., 2005), although the propensity of this siphovirus to

infect the bacterium in nature is not known. Though high microbial diversity is recognized

in host-symbiont association, a given species of sponge may contain a mixture of generalist

and specialist microorganisms (Taylor et al., 2004) and the associated microbial

communities are fairly stable in both space and time (Friedrich, et al., 2001; Taylor et al.,

2004; Webster et al., 2004; Taylor et al., 2005). Thus, the several studies have revealed a

widespread existence of sponge-specific bacterial clusters, i.e., closely related groups of

bacteria which are found only in sponges (Hentschel et al., 2002).

2.6.2. Specific microorganisms associated with sponges

The notion that marine sponges might contain a specific microbiota arose some 3

decades ago from the seminal work of Vacelet (1975), Vacelet and Donadey (1977),

Wilkinson (1978a, b, c) and Wilkinson et al. (1981). Based on electron microscopy and

bacterial cultivation studies, the following three broad types of microbial association in

sponges has been proposed: (i) abundant populations of sponge-specific microbes in the

sponge mesohyl, (ii) small populations of specific bacteria occurring intracellularly, and

(iii) populations of nonspecific bacteria resembling those in the surrounding seawater

(Vacelet, 1975; Wilkinson, 1978b). One type of bacterial isolate, regarded as a single

species, was recovered from 35 taxonomically diverse sponges from several geographic

regions, but never from seawater (Wilkinson, 1984; Wilkinson et al., 1981).

Immunological experiments in which these same isolates cross-reacted with other “sponge-

specific” bacteria but not with seawater isolates were also taken as further evidence of

sponge specificity (Wilkinson, 1984). Further, these concepts were integrated into the

molecular age (Hentschel et al., 2002). They defined sponge-specific clusters as sponge-

derived groups of at least three 16S rRNA gene sequences which (i) are more similar to

each other than to sequences from other, nonsponge sources; (ii) are found in at least two

host sponge species and/or in the same host species but from different geographic

locations; and (iii) cluster together irrespective of the phylogeny inference method used

(Hentschel et al., 2002).

The hypothesis of widespread, sponge-specific microbial communities put forward by

Hentschel and colleagues (Hentschel et al., 2002) was constrained by the limited data set.

They performed phylogenetic analyses with the 190 publicly available sponge-derived 16S

rDNA gene sequences, the majority of which were from Aplysina aerophoba,

Rhopaloeides odorabile, and Theonella swinhoei. These three sponges are phylogenetically

only distantly related and were collected from the Mediterranean Sea, the Great Barrier

Reef, and Micronesia/Japan/Red Sea, respectively, yet they contained largely overlapping

microbial communities. The results of Wilkinson and others (Wilkinson et al., 1981) have

suggested that even unrelated sponges with nonoverlapping geographic ranges might share

a common core of bacterial associates. Subsequent studies have also revealed similar

observations, with reports of similar (and in some cases sponge-specific) bacteria found in

different sponge species (Thoms, et al., 2003; Hill, 2004; Fieseler et al., 2004; Lafi et al.,

2005; Montalvo et al., 2005; Schirmer et al., 2005; Hill et al., 2006; Thiel et al., 2007a).

Furthermore, both cultivation-based and molecular methods have provided evidence for

distinct microbial communities between sponges and the surrounding seawater (Wilkinson,

1978b; Santavy and Colwell, 1990; Olson and McCarthy, 2005; Taylor et al., 2005; Hill et

al., 2006).These results have indicated the uniqueness of sponge-associated microbial

communities. A total of 14 monophyletic, sponge-specific sequence clusters were

identified in the original study of Hentschel et al. (2002). These occurred in the

Acidobacteria, Actinobacteria, Bacteroidetes, Chloroflexi, Cyanobacteria, Nitrospira, and

Proteobacteria (Alpha, Delta, and Gammaproteobacteria) and, in most cases, were

strongly supported by bootstrap analyses (in all cases, the clusters were found with three

different tree construction methods). Three further clusters—each sponge specific, with the

exception of a single nonsponge sequence— were also identified in the Acidobacteria and

in a lineage of uncertain affiliation (later recognized as Gemmatimonadetes

(Hentschel et al., 2002; Zhang et al., 2003). Overall, 70% of the 190 sponge-derived

sequences available at the time fell into one of these monophyletic clusters or the other.

Interestingly, within-cluster 16S rRNA sequence similarities ranged down to as low as

77% (Hentschel et al., 2002), often considered indicative of phylum-level differences

(Hugenholtz et al., 1998). Several subsequent, mostly cultivation-independent studies have

also led to the recovery of apparently sponge-specific sequences. Approximately 50% of

16S rDNA gene sequences in a gene library obtained from the unidentified Indonesian

sponge 01IND 35 were most closely related to sequences derived from other sponges (Hill,

2004). These included members of the Acidobacteria, Nitrospira, Bacteroidetes, and

Proteobacteria, as well as several sequences in a group of uncertain affiliation. A similar

situation was reported for Discodermia dissoluta, whereby three-quarters of 160 retrieved

16S rDNA sequences were most similar to other sponge-derived sequences (Schirmer et

al., 2005). Conversely, of 21 unique sequences (each representing a unique restriction

fragment length polymorphism [RFLP] type) obtained from the Caribbean sponge,

Chondrilla nucula, only 5 retrieved other sponge-derived 16S rRNA sequences during

BLAST searches (Hill et al., 2006). Perhaps the most impressive sponge-specific cluster to

be reported so far is the candidate phylum “Poribacteria” (Fieseler et al., 2004). Fieseler

and colleagues found members of this lineage, which is moderately related to the

Planctomycetes, Verrucomicrobia, and Chlamydiae (Wiens et al., 1999), in several

sponges from geographically diverse locations, but never in adjacent seawater or sediment

samples (Fieseler et al., 2004).

2.7. Microorganisms from the sponges and their secondary metabolites.

Various microorganisms have been found in sponges. In addition a number of

biologically active compounds have been reported from marine sponges and their

associated microorganisms (Table 3). Sponges are known to be the most prolific marine

producers of novel compounds, with more than 200 new metabolites reported each year

(Taylor et al., 2007). Many more sponge-derived compounds are in different stages of

clinical and preclinical trials as anticancer or anti-inflammatory agents than compounds

from any other marine phylum (Blunt et al., 2005). The occurrence in unrelated sponges of

structurally similar compounds, particularly those which were otherwise known

exclusively from microorganisms, led to speculation that such compounds were of

microbial origin (Bewley and Faulkner, 1998; Haygood et al., 1999; Usher, et al., 2001;

Piel, 2004) (Fig. 3). The chemical synthesis of natural products can be problematic and

expensive due to their structural complexity (Aicher et al., 1992; Sipkema et al., 2005;

Butzke and Piel, 2006), but synthesis of at least some compounds by microbes helps to

obtain a sustainable, essentially unlimited supply of these compounds for testing and

subsequent drug production by cultivation of the relevant bacteria (Proksch et al., 2002;

Piel, 2004). Sponge or microbe derived compounds include a wide range of chemical

classes such as terpenoids, alkaloids, peptides, and polyketides with a wide range of

biotechnologically relevant anticancer, antibacterial, antifungal, antiviral, anti-

inflammatory and antifouling properties (Matsunaga and Fusetani, 2003; Piel, 2004;

Fusetani, 2004; Blunt et al., 2005; Keyzers and Davies-Coleman, 2005; Blunt et al., 2006;

Moore, 2006; Piel, 2006). At present anticancer drugs are attaining the attention of natural

product chemists and pharmaceutical companies, with several promising sponge-derived

compounds in clinical and preclinical cancer trials (Newman and Cragg, 2004; Blunt et al.,

2005; Simmons et al., 2005). The large numbers of novel active metabolites are reported

from sponges every year but such chemicals have not yet become successful in the

pharmaceutical industry, mainly because of supply problem (Hart et al., 2000; Proksch et

al., 2002; Thoms and Schupp, 2005). However, the nucleoside analogs Ara-A and Ara-C

have been commercialized as antiviral and anticancer agents respectively. These were not

isolated directly from sponges but are synthetic derivatives based on compounds from the

Caribbean sponge, Cryptotethia crypta (Bergmann and Feeney, 1950; Bergmann and

Feeney, 1951).

Some of the biologically active natural products are often produced in relatively small

amounts, and often by rare animals whose natural populations cannot sustain the extensive

collections required for clinical trials. Thus, alternative means are required for producing

large amounts of metabolites (anticancer compounds halichondrin B and peloruside A).

The halichondrins are a group of polyether macrolides that exhibit potent antitumor

activities (Uemura et al., 1985; Hirata and Uemura, 1986). First isolated from the Japanese

sponge, Halichondria okadai in the mid- 1980s (Hirata and Uemura, 1986), but

subsequently they were found in several other sponges from diverse geographic locations,

including Axinella spp., Phakiella carteri, Raspailia agminata, and Lissodendoryx sp.

(Hart et al., 2000). Halichondrin B (Fig. 4) was particularly sought after due to its high

cytotoxicity, and its total synthesis was reported as early as 1992 (Aicher et al., 1992).

However, due to the structural complexity of the compound, many steps were required for

its synthesis, rendering total synthesis impractical for industrial scale production. However,

the occurrence of halichondrins in many unrelated sponges suggested its microbial origin,

Figure 3. Chemical structures of jaspamide (left), from Jaspis sp. sponges, and

chondramide D (right), from the deltaproteobacterium Chondromyces crocatus. Note the

remarkable structural similarities between the compounds (Taylor et al., 2007).

Figure 4. Chemical structure of halichondrin B.

Figure 5. Chemical structure of peloruside A.

lead to looking for alternative avenues. Lissodendoryx sp., collected from the coast of

southern New Zealand, yielded the largest amounts of halichondrins and therefore became

a focus of drug supply efforts (Hart et al., 2000; Munro et al., 1999). Based on the potency

of halichondrin B and its projected demand if approved for human use, the requirement for

clinical trials was estimated to be ~10 g, with annual requirements as a commercial drug of

1 to 5 kg (Hart et al., 2000). Given that 1 metric ton of Lissodendoryx sp. sponges yielded

only 300 mg of halichondrin B and that the entire natural biomass of the sponge was

estimated to be only 289 metric tons, collection from the wild was ruled out. Aquaculture

of Lissodendoryx sp. was then investigated, with promising initial results (Munro, et al.,

1999). Nevertheless, halichondrin B may yet prove to be a success story, with a synthetic

analog, E7389, in phase I clinical trials as an anticancer compound (Simmons et al., 2005).

This simplified version of halichondrin B is more amenable to chemical synthesis but

retains the biological activities of the original compound (Choi et al., 2003).

The second example concerns the macrocyclic lactone peloruside A (West et al., 2000)

(Fig. 5) isolated from the New Zealand demosponge, Mycale hentscheli, which showed

promising anticancer properties, acting in a similar manner and potency to the widely used

cancer drug paclitaxel (Taxol) (Hood et al., 2002). With the compound currently in

preclinical trials, two avenues are being pursued in parallel to ensure a sufficient supply of

Peloruside A for subsequent clinical trials. A possibility of chemical synthesis of

Peloruside A as well as aquaculture of M. hentscheli has been reported by a New Zealand

consortium, working together with a U.S. pharmaceutical company (Jin and Taylor, 2005;

Page et al., 2005b; Handley et al., 2006). With 200 kg of sponge yielding a mere 2 g of

pure peloruside A, scaling-up is a priority, with the goal of growing >500 kg of sponge

over the coming year (Handley et al., 2006). Other compounds of pharmaceutical interest

are also produced by M. hentscheli, namely, the cytotoxic polyketide mycalamide A and

the macrolide pateamine (Perry et al., 1988; Northcote et al., 1991; Hood et al., 2001; Page

et al., 2005a). Concentrations of these metabolites in natural sponge populations vary

significantly in time and/or space (Page et al., 2005a), suggesting that complex ecological

and physical factors may be involved in their production. An improved understanding of

the ecological roles of these and other compounds could greatly benefit metabolite

harvesting programmes. Supply issues notwithstanding, the pharmacological potential of

marine sponges and other sessile invertebrates (e.g., corals, bryozoans, and ascidians) is

enormous. Although progress towards the commercial product stage has been slow, it is

highly likely that at least one of the several compounds now in clinical trials (or a synthetic

analog) will be commercialized within the next few years. A combination of improved

chemical synthesis methods with the various approaches should ensure a bright future for

this field, with sponge-derived natural products being utilized either in their natural form or

as inspiration for new, laboratory- generated compounds (e.g., via chemical proteomics)

(Piggott and Karuso, 2004). The freshwater sponges and their chemistry has received much

less attention than that of their marine counterparts. Though various lipids and a compound

with antipredator activity have been reported from freshwater sponge (Dembitsky et al.,

2003; Rezanka et al., 2006), their activity is yet to be ascertained.

Table 3. Sponges and their symbiotic microorganisms producing natural products.

Sponge

Symbiotic microorganisma

Natural productb

Reference

Aciculites orientalis

Filamentous bacteria

Theonegramide

Bewley et al., 1996b

Antarctic sponge B Pseudomonas aeruginosa

NC Jayatilake et al., 1996

Aplysina sp. B, Arthrobacter sp.

NC Hentschel et al., 2001

Aplysina sp. B, Bacillus sp.

NC Hentschel et al., 2001

Aplysina sp. B, Micrococcus sp.

NC Hentschel et al., 2001

Aplysina sp. B, Pseudoalteromonas p.

NC Hentschel et al., 2001

Aplysina sp. B, Vibrio sp.

NC Hentschel et al., 2001

Cenarchaeum symbiosum

Archeon NC Preston et al., 1996.

Dysidea herbacea

Cyanobacterium

Chlorinated etabolites

Unson and Faulkner, 1993

Dysidea herbacea

C, Oscillatoria spongeliae

Polybrominated biphenyl ethers

Flowers et al., 1998

Dysidea sp.

B, Vibrio sp.

Brominated biphenyl ethers

Elyakov et al., 1991

Halichondria okadai

B, Alteromonas sp.

Alteramide A

Shigemori et al., 1992

Halichondria okadai D, Prorocentrum lima

Okadaic acid

Kobayashi and Ishibashi, 1993

Halichondria panacea

B, Antarcticum vesiculatum

Neuroactive compounds

Perovic et al., 1998

Halichondria panicea

B, Pseudomonas insolita

NC

Müller et al., 1981

Halichondria panicea

B, Rhodobacter sp.

NC

Althoff et al., 1998

Halichondria panicea

B, Psychroserpens burtonensis

Neuroactive compounds

Perovic et al., 1998

Homophymia sp.

B, Pseudomonas sp.

Antimicrobial compounds

Bultel-Poncé et al., 1999

Hyatella sp.

B, Vibrio sp.

NC

Oclarit et al., 1994

Rhopaloeides odorabile

B, b-Proteobacteria

NC

Webster et al., 2001a

Rhopaloeides odorabile

B, g-Proteobacteria

NC

Webster et al., 2001a

Rhopaloeides odorabile

A, Actinobacteria sp.

NC

Webster et al., 2001a

Rhopaloeides odorabile

B, Cytophaga sp.

NC

Webster et al., 2001a

Rhopaloeides odorabile

Green sulfur bacteria

NC

Webster et al., 2001a

Sigmadocia symbiotica

R, Ceratodictyon spongiosum

NC

Price et al., 1984

Suberea creba

B, Pseudomonas sp.

NC

Duglas, 1994

Suberea creba

B, Pseudomonas sp.

Quinolones

Duglas, 1994

Tedania ignis

B, Micrococcus sp.

Diketopiperazines

Stierle et al., 1988

Theonella swinhoei

B, δ-Proteobacteria

NC

Schmidt et al., 2000

Theonella swinhoei

C, Aphanocapsa feldmanni

NC

Bewley et al., 1996b

Theonella swinhoei

Filamentous bacteria

Theopalauamide

Schmidt et al., 2000

Theonella swinhoei

Unicellular bacteria

Swinholide A

Bewley et al., 1996a

Unidentified sponge

A, Streptomyces sp.

Urauchimycins A and B

Imamura et al., 1993

Verongia sp.

B, Aeromonas sp.

NC

Vacelet, 1975.

Verongia sp.

B, Pseudomonas sp.

NC

Vacelet, 1975.

Xestospongia sp.

B, Micrococcus luteus

Antimicrobial compounds

Bultel-Poncé et al., 1998

aA, actinomycete; B, Bacteria; C, Cyanobacteria; D, Dinoflagellate; R, Red algae. bNC: It was not checked.