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First report on chitinous holdfast
in sponges (Porifera)
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Research
Hermann Ehrlich1, Oksana V. Kaluzhnaya3, Mikhail V. Tsurkan4,
Alexander Ereskovsky5, Konstantin R. Tabachnick6, Micha Ilan7,
Allison Stelling8, Roberta Galli8, Olga V. Petrova9, Serguei V. Nekipelov9,
Victor N. Sivkov9, Denis Vyalikh10, René Born11, Thomas Behm1,
Andre Ehrlich2, Lubov I. Chernogor3, Sergei Belikov3, Dorte Janussen12,
Vasilii V. Bazhenov1 and Gert Wörheide13,14,15
1
Cite this article: Ehrlich H, Kaluzhnaya OV,
Tsurkan MV, Ereskovsky A, Tabachnick KR, Ilan
M, Stelling A, Galli R, Petrova OV, Nekipelov SV,
Sivkov VN, Vyalikh D, Born R, Behm T, Ehrlich
A, Chernogor LI, Belikov S, Janussen D,
Bazhenov VV, Wörheide G. 2013 First report
on chitinous holdfast in sponges (Porifera).
Proc R Soc B 280: 20130339.
http://dx.doi.org/10.1098/rspb.2013.0339
Received: 11 February 2013
Accepted: 18 April 2013
Subject Areas:
biochemistry, evolution, structural biology
Keywords:
chitin, Porifera, holdfast, endemic sponges
Authors for correspondence:
Hermann Ehrlich
e-mail: [email protected]
Gert Wörheide
e-mail: [email protected]
Institute of Experimental Physics, and 2Institute of Mineralogy, TU Bergakademie Freiberg,
09599 Freiberg, Germany
3
Limnological Institute SB RAS, 664033 Irkutsk, Russia
4
Max Bergmann Centre for Biomaterials, Leibniz Institute of Polymer Research, 01062 Dresden, Germany
5
Mediterranean Institute of Biodiversity and Ecology, CNRS, IRD, Aix-Marseille University,
13007 Marseille, France
6
P.P. Shirshov Institute of Oceanology, Russian Academy of Sciences, Moscow, Russia
7
Department of Zoology, Tel Aviv University, Tel Aviv 69978, Israel
8
Carl-Gustav-Carus Klinikum, TU Dresden, 01307 Dresden, Germany
9
Department of Mathematics, Komi SC UrD RAS, Syktyvkar, Russia
10
Institute of Solid State Physics, TU Dresden, 01062 Dresden, Germany
11
Institute of Materials Science, TU Dresden, 01062 Dresden, Germany
12
Forschungsinstitut und Naturmuseum Senckenberg, 60325 Frankfurt am Main, Germany
13
Department of Earth and Environmental Sciences, Palaeontology & Geobiology, and 14GeoBio-Center,
Ludwig-Maximilians-Universität München, Richard-Wagner-Strasse 10, 80333 München, Germany
15
Bayerische Staatssammlung für Paläontologie und Geologie, Richard-Wagner-Strasse 10,
80333 München, Germany
A holdfast is a root- or basal plate-like structure of principal importance that
anchors aquatic sessile organisms, including sponges, to hard substrates. There
is to date little information about the nature and origin of sponges’ holdfasts in
both marine and freshwater environments. This work, to our knowledge,
demonstrates for the first time that chitin is an important structural component
within holdfasts of the endemic freshwater demosponge Lubomirskia baicalensis.
Using a variety of techniques (near-edge X-ray absorption fine structure,
Raman, electrospray ionization mas spectrometry, Morgan–Elson assay and Calcofluor White staining), we show that chitin from the sponge holdfast is much
closer to a-chitin than to b-chitin. Most of the three-dimensional fibrous skeleton
of this sponge consists of spicule-containing proteinaceous spongin. Intriguingly,
the chitinous holdfast is not spongin-based, and is ontogenetically the oldest part
of the sponge body. Sequencing revealed the presence of four previously undescribed genes encoding chitin synthases in the L. baicalensis sponge. This
discovery of chitin within freshwater sponge holdfasts highlights the novel and
specific functions of this biopolymer within these ancient sessile invertebrates.
1. Introduction
Electronic supplementary material is available
at http://dx.doi.org/10.1098/rspb.2013.0339 or
via http://rspb.royalsocietypublishing.org.
Environmental characteristics are known to influence the gross morphology of many
benthic organisms including sponges (Porifera; [1,2]). This variability in shape is the
consequence of both strategic (during a long-term evolutionary response to environmental pressures) and tactical (in response to local environmental pressures)
processes. At local scales, morphology correlates with wave action, current flow
rate and sedimentation for both a number of sponge species and entire sponge
assemblages [3–6]. Wave action and current flow rate have direct influences not
only on sponge morphology, but also on the strength of attachment to substrata
& 2013 The Author(s) Published by the Royal Society. All rights reserved.
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2. Material and methods
(a) Sponge samples
Specimens of L. baicalensis were collected in Lake Baikal near Bolshie
Koty Settlement (518540 1200 N, 1058060 0200 E) from 15–25 m depths
(water temperature 3–48C) by SCUBA during 2009–2012.
The samples collected were placed immediately in containers
with Baikal Lake water and ice, and transported to the
Limnological Institute SB RAS (Irkutsk) for 1.2 h at a constant
water temperature (3–48C).
(b) Isolation of chitin-based holdfast
The specimens of L. baicalensis were initially carefully inspected for
the intactness of their skeletons, and the presence of macroalgae or
invertebrates, using a stereomicroscope. Neither contaminants
nor damage were observed for the collected species. The isolation
of the chitin-based holdfast was performed according to the
alkali-based treatment steps as described in the electronic
supplementary material in details.
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Proc R Soc B 280: 20130339
According to observations by transmission electron
microscopy and scanning electron microscopy, fibrous
material is found within the basal plates of young sponges,
and within the holdfasts of adult ones. Intriguingly, to our
best knowledge, to date no reports exist containing detailed
bioanalytical investigations confirming that the fibrous
material observed is really collagen-like spongin. However,
from a methodological point of view, verification of the presence of spongin within skeletal formations is very simple.
This arises from the excellent solubility of spongin in alkaline
solutions. This property of spongin is well known, and was
first described by Kunike [21]. Our preliminary investigations
attempted to isolate peptides from the spongin-based skeletons
of different demosponges. These studies show that the alkali
solution hydrolyses spongin. Obtained is a hydrolysate of
amino acids with no residual peptides visible on SDS-PAGE
gels after staining with Coomassie and the very sensitive
silver stain. These results agree well with those reported previously [22] about the strong insolubility of spongin, which
aimed to appropriate peptides for proteomics research.
Our recent findings of chitin within skeletons of both
marine demosponges from the Order Verongida [12,13] as
well as of hexactinellids [23] relied on the fact that spongin,
in contrast to chitin, is soluble in a 2.5 M NaOH solution.
Therefore, we used this simple test in the present study. We
decided to use the endemic freshwater sponge Lubomirskia
baicalensis (Pallas, 1773) for these investigations (figure 1a), as
the grey or brownish coloured holdfast of this sponge is particularly visible after it has been detached from stones or
other rocky substrates (figures 1b and 2a; electronic supplementary material, figure S1). Initial experiments showed
dissolution of the holdfast-containing skeletal fragments after
2–4 h in 2.5 M NaOH at 378C. The presence of residual fibrous
matter could also be seen which strongly resembled the shape
of the sponge holdfast (figure 2; electronic supplementary
material, figures S3 and S4). Isolation of the holdfast in the
form of an alkali-resistant fibrous material still containing siliceous spicules motivated us to carry out, to our knowledge, the
first ever detailed analytical, biochemical and genetic investigations to identify chitin as a possible candidate as the main
structural component of the sponge holdfast.
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by its holdfast. For example, wave action is known to affect
sponge morphological types, as many of the more delicately
branching species are destroyed by drag [4,5]. Therefore, the
limits of morphological adaptation for any particular sponge
species may reduce species richness at sites of high wave exposure
flow. Wave action removes delicate sponge forms such as the pedunculate and arborescent shapes. Encrusting and robust species
are more suited to this environment [7]. However, the biomechanical basis for such morphological changes has rarely been
documented [5]. The holdfasts of sponges that live in muddy
substrates often have complex tangles of root-like growths, however the holdfasts of organisms that live on smooth surfaces
(such as the surface of a boulder) have the base of the holdfast
literally glued to the surface. For example, the globular demosponge Cinachyra subterranea, van Soest & Sass [8] has been
found on vertical walls and the floors of caves flooded with
water at marine salinity levels [8]. Attachment to the rock is
accomplished not by a root of spicules, but by a smooth, flat,
disk-like holdfast. Some sponges, e.g. species of Tentorium,
may show adaptation to both soft bottom and hard substrate [9].
Detailed analysis of the literature with regard to the
nature and origin of the poriferan holdfast suggests that it
is a complex structure that is initially developed by the
larvae. When a sponge begins its life cycle, it is a microscale
free-swimming larva in the water column. The tiny sponge
must settle down on a substrate and establish a niche for
itself to survive. After settlement, the larvae metamorphoses
and begin to transform their organization into the adult body
plan. During this process, the outermost layer of cells (the
pinacoderm) covers the metamorphosing sponge, as well as
the adult body [10]. The basal pinacoderm (basopinacocytes)
secretes a mixture of spongin (collagen-like protein) and complex carbohydrates ( probably in the form of a fibrillar
spongin –polysaccharide complex) that allows the animal to
attach to a substrate [11]. The spongin attachment plaques
can be seen as the precursor of the sponge holdfast: it is
secreted by basopinacocytes, and the protein– carbohydratebased glue secreted by these cells holds the sponge in place.
According to the traditional point of view, spongin is the
basic component of the sponges’ organic skeleton. Taxonomically, spongin is a character of the class Demospongiae,
which comprises the highest number of known species (currently more than 8000). Only the representatives of marine
demosponge Order Verongida possess skeletons that consist
mostly (up to 70%) of chitin, and not spongin [12,13].
It was proposed [14] that spongin sticks the animal to its substratum [15], links its skeletal spicules together, and is also
present within the coat of sponge gemmules [16]. Although the
spongin matrix has been defined as an exoskeleton [16], spongins
exhibit different morphological aspects among demosponges
and vary according to the tissues. It is currently not known if
all spongin assemblies are equivalent [11,17], or whether or
not they are entirely made of short-chain sponge collagens [14].
Thus, according to traditional point of view the reticulate
skeleton in most demosponges arises from the basal spongin
plate [18] and their spicules (if present) are cemented by varied
amounts of spongin in the form of bundles and networks.
Formation of the spongin attachment plaques (¼ anchoring
layer, basal layer and spongin lamella) as the possible precursor
of the sponge holdfast has been investigated during aggregate
differentiation in demosponge cells [15] and for settlement
and metamorphosis of the parenchymella larvae [19,20] for
freshwater sponges, including the metamorphed larva stage.
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(a)
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(b)
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Proc R Soc B 280: 20130339
Figure 1. (a) Underwater image of the endemic Baikal Lake sponge Lubomirskia baicalensis shows that this branched 50 cm tall demosponge is attached to the
rocky substrate. The bright green colour is due to a symbiotic algae (Zoochlorella) that lives in the external tissue layer of the sponge. (b) The sponges are attached
to the hard substrate via plate-like holdfast (arrows) that morphologically differ from fibrous spongin-based and silica spicules-containing skeleton. (Online version
in colour.)
(a)
(b)
(c)
(d)
Figure 2. The holdfast of L. baicalensis (a, arrow) became brownish during drying in air. The microstructure of the holdfast is quite visible using light microscopy
(b – d ). The holdfast after 12 h of alkali treatment still shows light pigmentation and contains both spicules (b) and residual microparticles from the rocky substrate
to that the sponge was attached (c). The alkali-resistant fibrous network within the holdfast becomes visible in the light microscope after 7 days of insertion in 2.5 M
NaOH solution at 378C. Scale bars, 100 mm. (Online version in colour.)
(c) Analytical methods
Analytical methods like Raman spectroscopy, near-edge X-ray
absorption fine structure (NEXAFS) spectroscopy, Calcofluor
White (CFW) staining, as well as electrospray ionization
mass spectrometry (ESI-MS) and estimation of N-acetyl-D-glucosamine (NAG) contents are represented in the electronic
supplementary material.
(d) Chitin synthase gene detection from the genome of
Lubomirskia baicalensis
Specimens of L. baicalensis to be used for RNA isolation were frozen
in liquid nitrogen; those to be used for DNA isolation were stored in
70 per cent ethanol at 48C. Total genomic DNA from sponge tissue
was extracted using the PureLink Genomic DNA kit (Invitrogen).
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3. Results
(b) Identification of chitin within holdfast of
Lubomirskia baicalensis
(a) Structural peculiarities of the Lubomirskia
baicalensis holdfast
Habitat conditions of Baikal sponges differ considerably from
those of other freshwater sponges owing to hydrological and
hydrochemical peculiarities of Lake Baikal, such as great
depths, long ice periods, low water temperatures in summer
(10–128C) in the upper layers, high oxygen content and low
concentrations of organic matter [24]. Lubomirskia baicalensis
(figure 1) has a branched shape and an encrusting base with
erect (30–60 cm up to 1 m high) dichotomous branches with
rounded apices. The diameter of branches varies from 1 to
4 cm ranging from cylindrical to flattened shapes. The colour
of live specimens is brilliant green, which is due to the
symbionts inhabiting the external layer of sponges [25].
Ectosomal skeleton consists of spicule tufts from primary spongin fibres. The spicule skeleton consists of megascleres oxeas,
uniformly spined (145–233 9–18 mm; [26]). Lubomirskia
baicalensis is common on rocks, boulders and wood along the
entire shoreline at a depth from 3–4 m to more than 50 m.
Sponges could be easily mechanically detached from the
hard substrates such that the holdfast remained strongly
attached to the sponge body (figures 1b and 2a; see also electronic supplementary material, figure S1). Initially, we used
insertion of selected L. baicalensis specimens into 2.5 M
NaOH at 378C to examine the chemical stability of the
sponge skeletons to alkali treatment, because it is a well
known fact that spongin can be easily dissolved in solutions
that are up to 5 per cent alkali even at room temperature [21].
This stands in contrast to chitin, which is resistant to similar
alkali treatment at temperatures of up to 508C [12,13,23,27].
The light microscopy image (figure 2b) of L. baicalensis holdfast after 12 h incubation in alkaline solution shows the presence
of residual siliceous spicules as well as brownish pigmented
organic matter with some mineral microparticles (figure 2c)
that are resistant to the treatment. This organic material remains
undissolved in 2.5 M NaOH, even after incubation over 7 days
at 378C, and shows very intense characteristic fluorescence of
chitin after specific CFW staining (see the electronic supplementary material, figure S2). Siliceous spicules, however, are
Recently, NEXAFS technique has been successfully applied to
determine key differences between electronic properties
related to the light adsorption by polysaccharides and proteins, even within diverse biominerals [28–30]. We used
NEXAFS spectroscopy to explore site-specific electronic properties of L. baicalensis cleaned holdfast samples (measured on
500 500 mm areas) in order to gain insight into the nature of
the organic components. NEXAFS experiments, performed at
the carbon K edge, provided evidence that the contribution
of carbon is mainly owing to the organic part of the holdfast
(figure 3). Moreover, the carbon K-edge spectrum of this
sponge holdfast showed all the typical absorption features
of chitin and not those of spongin (figure 3), or collagen,
as was the case for spicules of the hexactinellid Hyalonema
sieboldi in previous studies by our team [30]. Both chitin
and collagen spectra exhibited a strong peak at approximately 288 eV that is associated with the C 1s ! p*
resonance involving acetamido (2NH(C ¼ O)CH3) group—
and pepty (–NH –C(O) –) group—character orbitals. Careful
inspection of the spectra indicates, however, that energies
of this peak are different for chitin (approx. 288.5 eV), spongin
(approx. 288.1 eV) and collagen (approx. 288.2 eV). It has been
shown [31,32] that the observed 0.3 eV shift manifests upon the
conversion of carboxyl bonds in lone amino acids into amide
bonds in peptide chains. While the 0.3 eV shift is rather
small, it has been well documented in the studies cited
above. It appears to provide a ‘sensible’ base for ‘in situ’ identification of polysaccharides and proteins—including naturally
occurring biocomposites, such as sponge holdfast—without
the need of preliminary disruption or extraction. In these analyses, we also show that the C¼O-character absorption peak of
the holdfast chitin is distinguishable from a strong cellulose
peak reported at 289.5 eV (see figure 3; [33]).
The chitin molecule consists of NAG (GlcNAc) residues,
including the acetamide group at the C-2 position of glucosamine, the secondary hydroxyl group at C-3 and the primary
hydroxyl group at C-6 positions [34]. Therefore, estimation of
GlcNAc is the crucial step for chitin identification in organic
matrices of unknown origin.
4
Proc R Soc B 280: 20130339
not more visible after this treatment in contrast to some mineral
particles. We observed that the alkali-resistant mineral microparticles of the substrate origin are still tightly bound into
chitinous fibres (figure 2c; electronic supplementary material,
figure S2). Because of this strong incrustation of the chitinous
fibrillar network with the mineral phase, we assume that
chitin and not spongin, which was dissolved during insertion
of the holdfast into alkaline solution, may be responsible for
attachment of the sponge to the rocky substrate.
The treatment of alkali-resistant matrix of the holdfast
with 3 M hydrochloric acid (HCl) leads to disappearance of
the mineral particles, however the fibrous matrix remains
stable. In our previously published work, we provided
strong evidence that chitin is also resistant to dissolution in
HCl [28]. This property can be also effectively used for isolation of chitin that contains calcium carbonate-based
minerals as residual material. To confirm this discovery of
the presence of chitin within the holdfast of L. baicalensis,
we used a multitude of sensitive bioanalytical methods as
presented below.
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Total RNA was isolated from fresh or deep-frozen sponge specimens
using a Trizol Reagent kit (Sigma). cDNA was synthesized using
a Reverta kit (AmpliSens, Russia). Comparison of the known
chitin synthase mRNA sequences of freshwater sponge Spongilla
lacustris (HQ668146; HQ668147) and marine sponge Amphimedon
queenslandica (XP_003385441) revealed highly conserved regions
which have been chosen for designing several degenerate primers
(see the electronic supplementary material). PCR products obtained
with the primer pair of ChsFW_L1 (50 -GGACATGTTGGATTCTG
ATCCCC-30 ) and ChsFW_R4 (50 -CTCCGTGGATCAGGCAGC
TGAACTC-30 ) was subsequently cloned and sequenced (see the
electronic supplementary material).
Chitin synthase (CHS) genes were identified by comparison with the CHS sequences registered in GenBank using
‘BLAST-X’ tools at the National Center for Biotechnology
Information (NCBI) web site (http://www.ncbi.nlm.nih.gov).
The amino acid sequence encoded by the obtained CHS
cDNA was deduced using the software EDITSEQ (DNAStar Inc,
USA). The sequences obtained in this study were submitted
to GenBank and can be retrieved under the accession nos
JX875071 – JX875074.
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288.52 eV
289.3 eV
TEY (arb. units)
0.6
spongin skeleton
chitin
holdfast
285.0 eV
cellulose(plant)
cellulose(bact.)
0.4
0.2
284
286
288
290
E (eV)
292
294
Figure 3. Detailed NEXAFS spectra taken at the C 1s threshold for L. baicalensis
cleaned holdfast, spongin-based skeleton, chitin standard (Fluka) and two different celluloses of the plant and bacterial origin, respectively. Careful analysis
reveals an energy shift approximately 0.3 eV of the C 1s ! p* acetamido
(–NH(C¼O)CH3) group peak in holdfast chitin relative to pepty (–NH–
C(O)–) group position in collagen-like spongin. Furthermore, the spectra of
the holdfast differ from those obtained for cellulose samples. (Online version
in colour.)
Mass spectroscopy is one of the most sensitive methods
for analysis of D-glucosamine, which is the only product of
chitin acid hydrolysis. The obtained ESI-MS spectrum of
the hydrolyzed L. baicalensis holdfast sample is very similar
to the spectra of a D-glucosamine standard (see the electronic
supplementary material, figure S5), and consists of three
main signals with m/z ¼ 162.18, 180.02 and 359.61. The signals at m/z ¼ 180.02 clearly shows the presence of dGlcN
molecules in the sample and corresponds to a [M þ Hþ]
species of dGlcN (calculated molecular weight of 179.1).
The signal at m/z ¼ 162.06 corresponds to [M 2 H2O þ Hþ]
dGlcN ion (calculated: 162.1) which is the loss of one water
molecule [35,36]. The weak signal at m/z ¼ 359.13 corresponds to [2 M þ Hþ] species which is the proton-bound
dGlcN non covalent dimer [36]. The sample at m/z ¼ 201 corresponds to [M 2 H2O þ Kþ] and [(GlcN)2 þ Kþ] adducts
with a potassium ion which is common for natural samples.
Interestingly, the sample can be completely hydrolyzed at
608C, but is stable in 6 M HCl at room temperature for
at least 24 h.
To quantify chitin in our samples, we measured the
amount of N-acethylglucosamine released by chitinases
using a Morgan–Elson colorimetric assay [37], which is the
most reliable method for the identification of alkali-insoluble
chitin owing to its specificity [38]. We detected 775.3 + 0.3 mg
N-acetyl-glucosamine per mg of L. baicalensis holdfast.
The results of Raman spectroscopy of the cleaned
L. baicalensis holdfast are represented in the electronic supplementary material, figure S6. The Raman spectra for achitin and L. baicalensis cleaned fibrous holdfast fibre material
were nearly identical, also in agreement with published reports
on chitin identification using Raman spectroscopy [39].
In summary, the analytical investigations described above
clearly show the presence of chitin within the holdfast of
L. baicalensis. This observation raises the question of the
presence of the corresponding CHS genes, which have thus
far not been described. We have, therefore, carried out the
corresponding genetic analysis, as described below.
4. Discussion
The presence of chitin in both evolutionary older marine and
evolutionary younger freshwater sponges [42] suggests that
the CHS genes found in this study represent shared ancestral
character states of sponges, and maybe even of a possible
common ancestor within the metazoan lineage (see the electronic supplementary material, figure S7). Thus, we suggest
that the chemistry, structure, morphology and biomechanical
properties of poriferan holdfasts were crucial throughout the
evolutionary history of sponges. Since dislodgment is
mostly fatal for adult sponges, the role of the holdfast is a critical
one. For sponges, holdfast morphology and sediment cohesiveness are important determinants of the maximum tensile
force they are able to withstand under specific environmental
conditions.
There are no doubts that fixation is very important for all
sedimentary animals, especially those which have little
opportunity to move or to re-build these body parts. Intriguingly, to our best knowledge, there are no reports regarding
the chitinous origin of the holdfast of other aquatic invertebrates with exception of hydrorhiza of the hydroid
Myriothela cocksi [43]. Hydrorhiza is a rootstock by which a
hydroid is attached to other objects. The adhesion of the
hydrorhiza to the substratum is affected only by the perisarc
layer covering the flattened extremities of the adhesive tentacles. This perisarc is about 6 mm thick and is composed of
true chitin [43].
The comparably smaller sponge-class Calcarea is characterized by skeletal spicules of calcite, and holdfasts within
this taxon are less expressive, than those found among members of the class Demospongiae, which are notably more
diverse, reach large sizes and inhabit more different solid
substrata (for review see [8]). Searching for specific types of
fixation including some hypothetical adhesive substances
for fixation in different classes of Porifera is a challenging
task for future studies.
Proc R Soc B 280: 20130339
0
We have isolated and characterized four new CHS gene
fragments from the freshwater sponge L. baicalensis: CHS_LB01
(1088 bp), CHS_LB02 (924 bp), CHS_LB03 (1077 bp) and
CHS_LB04 (1232 bp). The first three sequences were identified
from sponge RNA, while CHS_LB04—from sponge DNA.
These sequences included the intron in position 935–1073
(139 bp). The sizes of the deduced hypothetical protein
fragments of Baikalian sponge CHSs were 308–364 aa. A comparison of CHS amino acid sequences yielded an identity of
74.5–92.5 per cent. BLAST-X analyses indicated that the protein sequences of CHS from Baikalian sponge were most similar
to CHS of S. lacustris, AEI55440 (73–98%), A. queenslandica,
XP_003385441 (52–54%), Hydra magnipapillata, XP_002162504
(39–43%), Nematostella vectensis, XP_001633545 (43–46%) and
Branchiostoma floridae, XP_002592717 (35–39%). CLUSTALX alignment of L. baicalensis CHS predicted proteins with those from
the closest relatives revealed the conserved domain specific
for all types of CHSs. This domain includes catalytically critical sequences GEDR and QRRRW [40] in the all amino acid
sequences (figure 4).
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(c) Characterization of chitin synthase genes in
Lubomirskia baicalensis
288.1 eV
0.8
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Proc R Soc B 280: 20130339
Figure 4. Alignment of the C-terminal end of L. baicalensis CHS predicted proteins (L_baic_01, L_baic_02, L_baic_03 and L_baic_04) with predicted proteins from
Spongilla lacustris (S_lac_5605, GenBank accession no. AEI55440), Amphimedon queenslandica (Amph_queen, GenBank accession no. XP_003385441), Hydra magnipapillata
(Hydra_magn, GenBank accession no. XP_002162504), Nematostella vectensis (Nemat_vect, GenBank accession no. XP_001633545) and Branchiostoma floridae (Bran_flori,
GenBank accession no. XP_002592717). The alignment was performed using the CLUSTALX v. 2.0.10 program [41]. Amino acids that are conserved among eight to nine (white
symbols on black background) and five to seven (black symbols on grey background) sequences are highlighted. The conservative domains (GEDR and QRRRW) of CHS are
marked with asterisks (*).
Within the phylum Porifera, different attachment
methods have evolved over time. For example, the settled
larvae of Halichondria moorei undergoing metamorphosis
were found to possess a complex glycocalyx lining the cells
on their upper surface [44]. This structure, which has been
referred to as the sponge larval coat, was present on neither
adult sponges, nor on unsettled larvae. It was suggested
that this sponge’s mechanism for larval attachment bears
some similarity to the adhesion of many cultured cells to
their substrates. This hypothesis is supported by the absence
in sponge larvae of specialized cement glands, which are
known to be involved in substrate attachment in other
marine invertebrates [44].
Our finding of the presence of chitin within holdfast of
L. baicalensis suggests the existence of corresponding chitinproducing cells. Intriguingly, these cells are definitively not
located within chitin-containing gemmules. In numerous freshwater sponges, gemmules consist of a rough coating (mainly
proteinaceous spongin and silicious gemmuloscleres) and
are filled with archaeocytic totipotent cells and many undifferentiated and polynucleated cells [45]. It was found that newly
formed gemmular cells are first surrounded by a squamous cell
layer which later degenerates and becomes surrounded in turn
by a layer of columnar cells. The columnar cells secrete both
internal and external chitinous membranes. Jeuniaux [46]
reported that 1–3% of the dry weight of the gemmules’ coating
is chitin. However, while the production of dormant, encapsulated gemmules by freshwater sponges and a few marine
species is a well-documented biological phenomenon [47],
there are to date no reports about gemmule production by any
species of the endemic family Lubomirskiidae, freshwater
sponges in the Baikal Lake, which is monophyletic with the
globally distributed Spongillidae [48]. Thus, an obvious object
of investigation with respect to identification of chitin is the
poorly investigated larva of L. baicalensis.
One of the most intriguing questions that shall be
addressed by further investigations is to decipher the role of
the holdfast in sponge attachment, and how holdfasts maintain
their adhesive prosperities underwater and in highly saline as
well as in freshwater conditions. Such information may have a
high potential for use in biomimetic fields, where it could
be exploited for the design of biocompatible adhesives with
tuneable physico-chemical properties.
Financial support by DFG ( projects EH 394/1–1) is gratefully
acknowledged. Furthermore, this study was funded by the Erasmus
Mundus DAAD Programme 2011, RFBR no. 11–04–00323-a, no. 12–
02–00088-a, G-RISC (German-Russian Interdisciplinary Science
Center) grant 2012 and the CryPhysConcept Programme. We cordially thank Prof. Eike Brunner for use of the facilities at Institute
of Bioanalytical Chemistry, TU Dresden.
Downloaded from http://rspb.royalsocietypublishing.org/ on June 18, 2017
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