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.