University of Alberta
The cytology and physiology of coordinated behaviour in the freshwater sponge,
Ephydatia muelleri
by
Glen Ronald Douglas Elliott
A thesis submitted to the Faculty of Graduate Studies and Research
in partial fulfillment of the requirements for the degree of
Doctor of Philosophy
In
Physiology, Development and Cell Biology
Department of Biological Sciences
Edmonton, Alberta
Spring, 2009
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"Education: the path from cocky ignorance to miserable
uncertainty." -- Mark Twain
Abstract
Sponges are an ancient group of animals and therefore hold important clues about
how multicellularity and early coordination mechanisms arose. Recent molecular and
physiological studies suggest they have cell signalling systems similar to those found in
higher metazoans indicating that sponges are more integrated than previously
appreciated. I used a small freshwater sponge with modern imaging and physiological
approaches to study mechanisms of coordination in this phylum.
In response to mechanical stimuli, Ephydatia muelleri carries out a series of
highly coordinated peristaltic-like contractions. The sponge body has three distinct
regions: the ectosome, choanosome, and osculum, which inflate and contract in sequence
to expel water from the aquiferous system. Epithelial cells (myocytes), in particular
those in the ectosome, contain large tracts of actin that are presumed to control lowering
and raising of the apical surface of the sponge and in the contractions of the canals.
Some cells lining the canals and all cells lining the osculum were found to have a pair of
short cilia that may be sensory and used to detect water flow.
Cells in the mesohyl arrest crawling as a wave of contraction passes, suggesting
an extracellular signal may pass between cells. I tested the role of glutamate, y-aminobutyric acid (GABA) and nitric oxide in propagation of contractions. Glutamate induced
contractions in a dose-dependent manner, and its action was blocked by AP3 and delayed
by kynurenic acid. GAB A did not induce contractions, but caused twitches of the
choanosome. Nitric oxide synthase was localized, using antibodies and a NADPHdiaphorase assay, to the mesenchyme cells surrounding canals, in the osculum and in
choanocytes; this finding was functionally confirmed by a cGMP assay.
I dedicate this thesis especially to my family and friends who made this
possible for me to keep my sanity.
If sponges are seen as an elementary hydrostatic skeleton in which tubes of water
are lined by contractile cells, these results suggest that control over a hydrostatic skeleton
evolved prior to the origin of nerves and true muscle. Due to their circumpolar
distribution and ease of collection and culturing, freshwater sponges represent a practical
model system for investigating physiological signalling systems involved in the
coordination and propagation of contractions within the sponge body.
Table of Contents
Chapter 1 : General Introduction
1
1.1 The origin and evolution of muscular and nervous systems
1
1.2 Behaviour in sponges
11
1.3 Anatomy of a sponge
13
1.4 A brief history of studies on sponge behaviour
16
1.5 Evidence and possible mechanisms for a coordinated contractile system in cellular
sponges
20
1.6 The aims of the present research
23
1.6 Literature cited
25
Chapter 2 : Coordinated contractions effectively expel water from the aquiferous system
of a freshwater sponge
36
2.1 Introduction
36
2.2 Materials and methods
39
2.2.1 Sponge collecting and culturing
39
2.2.2 Digital video time-lapse microscopy and image analysis
40
2.2.3 Fixation for fluorescence and confocal microscopy
41
2.2.4 Fixation for scanning electron microscopy
42
2.3 Results
42
2.3.1 Description of the juvenile sponge
42
2.3.2 Description of the inflation-contraction behaviour
45
2.3.3 Response to the addition of inedible ink particles
48
2.3.4 The kinetics of the inflation-contraction cycle
51
2.3.5 Kinetics of twitches, ripples and local contractile events
69
2.3.6 The contractile apparatus of the sponge
72
2.4 Discussion
72
2.4.1 Rates of contraction
80
2.4.2 Coordination of effectors
81
2.4.3 Evidence for effector tissue and signal propagation
83
2.4.4 Comparison with other contractile systems
85
2.5 Supplementary movies
87
2.6 Literature cited
90
Chapter 3 : Morphology of the juvenile sponge, Ephydatia muelleri: A phylotypic body
plan
98
3.1 Introduction
3.2 Materials and methods
98
101
3.2.1 Sponge collecting and culturing
101
3.2.2 Fixation for scanning electron and light microscopy
102
3.2.3 Fixation for fluorescence and confocal microscopy
103
3.2.4 Pharmacological manipulations
104
3.3 Results
104
3.3.1 Morphology of the gemmule
107
3.3.2 General anatomy of the juvenile sponge
110
3.3.3 Subdermal cavity and choanosome
113
3.3.4 Apical pinacoderm
121
3.3.5 Porocytes
126
3.3.6 Osculum
3.4 Discussion
131
131
3.4.1 The sponge body plan
134
3.4.2 Epithelia
136
3.4.3 Choanosome - flow control and feeding
137
3.4.4 Osculum
139
3.5 Conclusion
139
3.6 Literature cited
141
Chapter 4 : Evidence for Glutamate, GABA and NO in coordinating behaviour in the
sponge, Ephydatia muelleri (Demospongiae, Spongillidae)
149
4.1 Introduction
149
4.2 Materials and methods
152
4.2.1 Collection and culturing of sponges
152
4.2.2 High performance liquid chromatography (HPLC-MS)
153
4.2.3 Digital time-lapse and data acquisition
155
4.2.4 Test substance application
155
4.2.5 Fluorescence microscopy
156
4.2.6 NADPH-diaphorase histochemical detection of nitric oxide synthase
157
4.2.7 cGMP assay for nitric oxide reaction
158
4.3 Results
4.3.1 Description of the freshwater sponge
158
158
4.3.2 Evidence of neurotransmitter molecules in sponge tissues: HPLC-MS analysis.
159
4.3.3 Evidence for a metabotropic glutamate signalling system
160
4.3.4 Evidence for function of glutamate receptors
169
4.3.5 Role of calcium in contractions
175
4.3.6 Evidence for a GABA signalling system
178
4.3.7 Evidence for nitric oxide signalling system
178
4.4 Discussion
188
4.4.1 Neuroactive amino acids in sponge tissue
188
4.4.2 Evidence for metabotropic glutamate signalling system
191
4.4.3 Evidence for a metabotropic GABA signalling system
194
4.4.4 A potential role for nitric oxide signalling
196
4.4.5 Evolution of ligand based coordination pathways
197
4.5 Conclusions
198
4.6 Literature cited
200
Chapter 5 : General Discussion
5.1 Overview
208
208
5.2.1 Behaviour in cellular sponges
209
5.2.2 A note on methodology for behavioural assays
212
5.3 Sponge morphology
213
5.3.1 Cytology of contractile cells
214
5.3.2 Ciliated and sieve cells
218
5.3.3 Do sponges have an epithelium?
220
5.3.4 Morphology of a 'primitive synapse'
221
5.4 Sponge Physiology
224
5.4.1 Physiology of peristalsis in sponges
224
5.4.2 Mechanism of signal propagation and contraction
225
5.4.2.1 Metabotropic glutamate signalling pathway
229
5.4.2.2 Metabotropic GABA signalling pathway
230
5.4.2.3 Nitric oxide signalling pathway
233
5.4.2.4 Other signalling molecules
234
5.4.2.5 Calcium dynamics
235
5.5 Concluding remarks
236
5.6 Literature cited
238
Appendix 1 : Field observations of freshwater sponges in lakes surrounding Bamfield
Marine Sciences Center
249
List of Tables
Table 1-1. History and major milestones in sponge research
18
Table 2-1. Duration of the phases in the inflation-contraction cycle in response to
different stimuli
52
Table 2-2. Rates of contraction in different regions of Ephydatia muelleri
53
Table 4-1. Amino acid levels for tissue from Ephydatia muelleri and Spongilla lacustris
by HPLC-MS analysis
163
Table 4-2. Kinetics of the excurrent and incurrent canal contractions
168
List of Figures
Figure 1 -1. Proposed hypotheses in the progression of the steps involved in the evolution
of nerve and muscle
4
Figure 1-2. Metazoan phylogeny of the major clades based on recent molecular and
character evidence
7
Figure 1-3. Current phylogeny of poriferan classes based on current knowledge of
molecular phylogenetic relationships
10
Figure 1-4. The anatomy of a sponge
15
Figure 2-1. Scanning electron micrograph fracture and schematic diagram illustrating the
principal features of Ephydatia muelleri: the apical pinacoderm, sub-dermal cavity,
choanosome, and basal pinacoderm
44
Figure 2-2. The response of Ephydatia muelleri to mechanical agitation
47
Figure 2-3. Uptake and clearing of inedible ink by a 7-day-old sponge
50
Figure 2-4. The duration of the inflation-contraction cycle depends on the resting
diameter of the largest excurrent canals
57
Figure 2-5. Stereomicrographs showing the sponges, and enlargements of the regions of
the principal canals as measured in Figure 2-4
59
Figure 2-6. Contraction of incurrent and excurrent canals in a sandwich preparation.... 61
Figure 2-7. Analysis of the kinetics during the inflation-contraction cycle showing waves
of contraction that propagate along and across incurrent and excurrent canals
64
Figure 2-8. Stereomicrograph images and changes in diameter of the tip and base of the
osculum during a contraction
66
Figure 2-9. Closure of a field of porocytes in the apical pinacoderm correlates with
contraction of the choanosome after stimulus by agitation
68
Figure 2-10. Rapid contractions ('twitches') occur simultaneously in distinct regions of
the choanosome after uptake of inedible ink
71
Figure 2-11. Actin distribution and morphology of apical endopinacocytes
74
Figure 2-12. Morphology of the aquiferous canals and choanocyte chambers studied by
scanning electron microscopy of sponges preserved when relaxed and after stimulus
when contracted
76
Figure 2-13. Summary diagram illustrating the temporal coordination of contractions by
the aquiferous canals, apical pinacoderm, ostia, osculum, during a single inflationcontraction event in Ephydatia muelleri
77
Figure 3-1. General morphology of the adult and gemmulated sponges by light and
scanning electron microscopy
106
Figure 3-2. The morphology of the gemmule from Ephdatia muelleri viewed by
scanning electron microscopy
109
Figure 3-3. General anatomy of the freshwater juvenile sponge by light and scanning
electron microscopy
112
Figure 3-4. Description of the choanosome - subdermal cavity, aquiferous canal system,
and mesohyl - viewed by thick section and scanning electron microscopy
115
Figure 3-5. The choanocyte chamber and apopyle viewed by scanning electron and
epifluorescence microscopy
118
Figure 3-6. Ciliated cells in the excurrent canal system viewed by scanning electron
microscopy
120
Figure 3-7. The apical pinacoderm 'tent' viewed by light, scanning electron and
epifluorescence microscopy
123
Figure 3-8. Disassembly by Cytochalasin B and subsequent recovery of labelled actin
viewed by epifluorescence microscopy
125
Figure 3-9. Porocytes in the apical pinacoderm seen by scanning electron,
epifluorescence and light microscopy
128
Figure 3-10. The basal pinacoderm viewed by scanning electron and epifluorescence
microscopy
130
Figure 3-11. The osculum viewed by scanning electron and confocal microscopy
133
Figure 4-1. HPLC-MS chromatographs of amino acids in tissue of Ephydatia muelleri
and Spongilla lacustris
162
Figure 4-2. Response of Ephydatia muelleri to 80 uM L-glutamate
165
Figure 4-3. Dose-dependence of contractions of Ephydatia muelleri treated with Lglutamate
167
Figure 4-4. Effect of the allosteric inhibitor of glutamate receptors, AP3 on L-glutamate
triggered contractions
172
Figure 4-5. Effect of the competitive inhibitor of metabotropic glutamate receptors,
Kynurenic acid on glutamate triggered contractions
174
Figure 4-6. Response of Ephydatia muelleri to 80 uM L-glutamate and agitation in Ca2+free medium and Ca2+Mg2+-free medium
177
Figure 4-7. Contraction of the choanosome and excurrent canals of Ephydatia muelleri in
response to 250 uM GABA
180
Figure 4-8. Localization of nitric oxide synthase in Ephydatia muelleri by NADPHdiaphorase staining
182
Figure 4-9. Localization of nitric oxide synthase in Ephydatia muelleri by neuronal nitric
oxide synthase and universal nitric oxide synthase antibodies
185
Figure 4-10. Nitric oxide synthase activity shown by cGMP assay in Ephydatia muelleri
187
Figure 5-1. Theoretical diagram showing the body organization of the contractile and
coordination systems in a thin versus thick walled sponge
217
Figure 5-2. Diagrammatic drawings of possible synaptic patterns found in the freshwater
sponge primitive synapse based on the summary of data from the Cnidaria
222
Figure 5-3. Proposed pathways of signal propagation in freshwater sponge contractions.
227
Figure 5-4. Possible pathways for signal propagation to occur in the freshwater sponge.
232
Chapter 1 : General Introduction
1.1 The origin and evolution of muscular and nervous systems.
The differentiation of muscle and of nerves was a major step in the evolution of true
metazoans, but how they arose, and in what ancestor this occurred, is still unclear.
Recorded observations of sponge contractions date back as far as Aristotle (348-322 BC)
(see Aristotle, 1498), but it was not until the mid-18th Century that the first hypotheses to
account for the origin of coordination and contraction in animals were proposed. Two
ideas prevailed: the neuromuscular system was proposed to have either evolved from a
single epithelial cell type that differentiated into two cell types (muscle and nerve) or
from two independent cell types, one epithelial and the other mesodermally derived.
Kleinberg (1872) proposed that both neurons and muscle cells arose from a single
neuromuscular cell as seen in cnidarians, diploblastic animals that have an epithelial cell
with a contractile basal process. His contemporaries, the Hertwig brothers (Hertwig and
Hertwig, 1878) disagreed with the interpretation that these cells were a precursor to
neurons because cnidarians have separate neuronal sensory and motor cells. The Hertwig
brothers nevertheless agreed with Kleinberg that muscular and nervous systems arose
through differentiation of a single precursor cell that had both a coordinating and
contractile function (Parker, 1910).
The opposing view, that these two types of cell had a simultaneous independent
origin, and that the union of the two was a derived feature, was held by Claus (1878) and
Chung (1880). As evidence they gave the independent differentiation of nerve and
muscle tissue from separate tissue layers, and their secondary union during the ontogeny
1
of vertebrates. This theory, unlike that of the Hertwigs and Kleinberg, gains support
from highly derived metazoans, vertebrates, rather than from the basal metazoans.
Parker (1910) also proposed an independent origin for muscle and nerve tissue,
but based his ideas on evidence from the Porifera, the earliest evolving metazoans.
Parker (1919) postulated that early in the evolution of the Metazoa, independent effectors
such as muscle cells existed prior to the development of nerves. Sensory cells originated
from some type of flagellated epithelial cell with connections to epitheliomyocytes within
the outer epithelium layer. The sensory cell and the epitheliocyte differentiated into a
third cell, a neuron, which connected to the muscle cells to form the three-cell
neuromuscular system (sensory cell, neuron, and muscle cell). He considered sponge
myocytes, as independent effectors, to be the first muscular organs to arise, around which
the first neural tissue and sense cells developed giving rise to the condition found today
in other basal metazoans. He postulated that to this receptor-effector system an adjuster
and central organ (motor nerve and ganglion) was added, giving the complete
neuromuscular mechanism found in the higher metazoans. However, Horridge (1966)
suggests that Parker's interpretation is open to objection on the grounds that conduction
systems must be developed before the independent effectors and sensory cells can
activate areas large enough to give a significant response.
In the mid-late 20th Century, these ideas were revisited when non-neural (neuroid)
conduction was found in many phyla (Figure 1-1). Neuroid conduction is the
transmission of electrical signals through epithelial sheets, as seen in hydrozoans,
ctenophores, tunicates and even the skin of amphibians (Mackie, 1970; Anderson, 1980;
Mackie, 2004). Both Horridge (1968) and Mackie (1970; 1990) envisioned that electrical
2
Figure 1-1. Proposed hypotheses in the progression of the steps involved in the
evolution of nerve and muscle. Mackie (1970) proposed that the steps in the evolution of
nerve and muscle were: A) a primordial myoepithelium; B) movement of protomyocytes
(contractile bases of epithelium) from an epithelium towards the interior; C) protoneurons
evolve to convey excitation to the myocytes from the exterior, where all cells were
electrically coupled indicated by circular arrows. Current flows from the depolarized
regions of membrane to groups of electrically interconnected cells forming effective
conducting units; D) Neurosensory cells and interneurons evolve that connect to one
another and to the myocytes by chemically transmitting, polarized junctions. Dashed
lines (by the author) indicate low resistance pathways through which action potentials can
flow. Pavans de Ceccatty (1974) proposed that sponges show the primitive step followed
by the Cnidarians and finally the Ctenophores in the evolution of nerves and muscle, E)
Sponges have an epithelial surface that is composed of flat exopinacocytes with cell
bodies extending deeper into the connective tissue. The contractile cells are connected to
both the exopinacocytes and themselves. All cells have secretory vesicles; F) The
myoepithelium of the Cnidaria is a muscle sheet composed of the contractile bases
forming above the basal membrane, and G) The epithelium, nerves and muscle of the
Ctenophores consist of individual cells comprising an epithelium with sensory cells, and
muscle cells in a layer below the basal membrane, which is connected by neurons from
the sensory cells.
3
B
»* F
Figure 1-1
synapses likely evolved before chemical synapses, based largely on the condition seen in
hydromedusa and ctenophore epithelia. Little was known about the situation in Porifera,
and the phylogenetic paradigm at that time was that hydrozoans were the more basal
group of cnidarians.
A new understanding of metazoan phylogenies, molecular expression of
mesodermal genes and transcription factors has revitalized the debate of the evolution of
mesoderm and true muscle in basal metazoans (Figure 1-2). Current phylogenetic studies
now concur that anthozoans (corals and sea anemones) are the ancestral cnidarian group
(Collins et al., 2006; Burton, 2008). It is not clear that the molecules for electrical
synapses (pannexins) are even present in their genome (e.g., Magie and Martindale,
2008), so if there are no gap junctions in anthozoans, but they are in hydrozoans and in
other metazoans, then electrical synapses must have evolved independently multiple
times in metazoans. Regarding mesoderm, the current paradigm is that unlike bilaterally
symmetrical animals, cnidarians are diploblastic, lacking a middle layer or mesoderm that
is embryologically derived. However, in the jellyfish Podocoryne carnea, Spring et al.
(2002) have shown that the entocodon, a mesoderm-like tissue that develops during
medusa development, is homologous with the third germ layer, the mesoderm of
bilaterians (Figure 1-1). They found that this tissue gives rise to both the striated and
smooth muscle of the developing medusa, and expresses Brachyury, Mef2 and Snail
genes that are involved in muscle cell differentiation in other animals. Transcription
factor Mef2 may be involved in the expression of muscle-specific genes such as
tropomyosin and myosin heavy chain II (Spring et al., 2002). Spring and colleagues
(2002) also suggest that the common ancestor of the cnidarians and bilaterians
5
Figure 1-2. Metazoan phylogeny of the major clades based on recent molecular and
character evidence modified from Burton (2008) (Bridge et al., 2001; Medina et al.,
2001; Halanych, 2004; Martindale et al., 2004; Burton, 2008).
6
Lophotrocozoa
Ecdysozoa
Deuterostomia
Acoelomorpha
Scyphozoa
Cubozoa
Hydrozoa
Stauromedusae
Ceriantharia
Zoanthinaria
Antipatharia
Scleractina
Corallimorpha
Boloceroididae
Non-Boloceroididae
Ctenophora
Placozoa
Porifera
Figure 1-2.
7
not only had genes that function in muscle cell differentiation, but that they may have
already employed the genes to develop and differentiate a contractile system similar to
those found in the true triploblasts. Seipel and Schmid (2005) suggest that the striated
and smooth muscle cells may have evolved directly and independently from non-muscle
cells based on comparisons with cnidarians homologs (myosin heavy chain II) to
bilaterian mesoderm and myogenic regulators, which are expressed in polyp stages. They
argue that diplopblasty evolved secondarily in cnidarians; thus, a tri-layered ancestor
gave rise to striated and smooth muscle in the higher metazoans. These ideas are
provocative and although the concept of a triploblastic ancestor to cnidarians is not
entirely accepted it is agreed that there is a homology of the entocodon (middle layer in
cnidarians) and mesoderm within the metazoan (Burton, 2008).
Current phylogenies suggest the Porifera is the most basal animal group having
arisen before the placozoans, ctenophores, cnidarians and bilaterians (protostomes and
deuterostomes). Relationships among sponge groups are still controversial. Most
molecular analyses suggest sponges are paraphyletic (Kruse et al., 1998; Zrzavy et al.,
1998; Borchiellini et al., 2001; Medina et al., 2001; Peterson and Butterfield, 2005;
Figure 1-3), but one study suggests they are monophyletic (Philippe et al., 2009).
Hexactinellids (glass sponges) and demosponges form a clade of siliceous sponges
(Silicea) and calcareous sponges and homoscleromorphs are more closely related to other
metazoans (Wang and Lavrov, 2007). Homoscleromorphs have been interpreted as
demosponges that have retained an ancestral mitochondrial genome and features such as
a presence of true epithelium, acrosomes, and cross-striated rootlets of the flagella base
8
Figure 1-3. Current phylogeny of poriferan classes based on current knowledge of
molecular phylogenetic relationships based on phylogeny by Erpenbeck and Worheide
(2007). Dashed lines indicate branches of particular uncertain molecular hypotheses.
9
(remaining former
poecilosclerid, hadromerid
and halichondrid families)
Poecilosclerida s.s.
Tethyidae + Hemiasterellidae
Halichondriidae + Suberitidae
Clionaidae + Spirastrellidae
Agelasida + Axinellidae
Spirophorida
Astrophorida
Spongillina
marine Haplosclerida
Halisarcida
Chondrosida
Verongida
Dendroceratida
Dictyoceratida
- < ! ! ^ J Amphidiscophora
- < ^ J Hexasterophora
O
to
3
o
i/i
•a
o
D
5*
a>
to
x
01
n
»-*
5'
to_
51
- < d j Calcinea
- < ^ J Calcaronea
CD
n
SL
e
0)
Homoscleromorpha
Eumetazoa
Choanoflagellata
Figure 1-3.
10
that are lost in most sponges (Gaino et al., 1987; Boute et al., 1996; Ereskovsky and
Boury-Esnault, 2002). Nevertheless, all of these hypotheses imply that a sponge-like
animal (with canal-like gut) must have given rise to all other metazoans. Much more
must be known about sponges at the cellular, molecular, and physiological levels before
one can contemplate whether sponges could be derived from a triploblastic ancestor.
Until now, sponge cell biological studies are quite scant (Simpson, 1984) and molecular
biology studies are only just beginning; the longest history is of behavioural studies.
1.2 Behaviour in sponges
Sponges can to contract (Schmidt, 1866; Weissenfels, 1990; Nickel, 2004), react to
mechanical (damage or tension), electrical and chemical stimuli (Parker, 1910; McNair,
1923; Emson, 1966; Prosser, 1967; Pavans de Ceccatty, 1979; Nickel, 2004), and move
(McNair, 1923; Jones, 1957; Kilian and Wintermann-Kilian, 1979; Fishelson, 1981;
Bond and Harris, 1988; Bond, 1992). Responsive behaviour has been documented in all
classes of Porifera (Demospongiae, Calcarea, and Hexactinellida) in adults and larvae
(Elliott et al., 2004; see review Leys and Meech, 2006; Elliott and Leys, 2007). The
coordination of this behaviour has been discussed over the last 100 years in terms of
trying to understand whether or not sponges possess a nervous system. One difficulty in
generalizing about coordination in Porifera is that two classes of sponges are cellular
while one class, the Hexactinellida, is syncytial.
The Hexactinellida (glass sponges) have a unique type of behavioural response.
Upon mechanical stimulation, sedimentation, or electrical stimulation, electrical impulses
travel through their syncytial tissues causing the flagella to stop beating (Leys and
11
Mackie, 1997; Leys et al., 1999). The bulk of the glass sponge tissue consists of one
giant multinucleate cell termed the trabecular reticulum that is draped over spicules in 2-5
um thin strands (Leys, 1999). The trabecular reticulum is continuous with the dermal
membrane, the atrial membrane and the flagellated chambers (Mackie and Singla, 1983;
Leys, 1999; Leys, 2003). Leys and colleagues (1997; 1999) confirmed the long-term
suspicions of Bidder (1923), Mackie (1979), Lawn et al. (1981), and Mackie et al. (1983)
that the propagated feeding current arrests were caused by electrical events. They
showed that despite the absence of nerves, action potentials were conducted at 0.26
cm-sec"1 through the tissue. Although this rate of propagation is at the low end of neuroid
conduction velocities, the electrical impulses are all-or-none events that have absolute
and relative refractory periods of 30 s and 150 s, respectively (Leys and Mackie, 1997;
Leysetal., 1999).
In contrast to the Hexactinellida, demosponges and calcareous sponges are
cellular and as far as is known, lack gap junctions or equivalent electrical junctions
between cells, and consequently they behave quite differently. These sponges have
waves of contractions, which are thought to travel through specific muscle-like cells
called myocytes (Bagby, 1965; Reiswig, 1971; Simpson, 1984). Other contractile
cellular events have been reported in cellular sponges, such as the healing behaviour of
Leucosolenia complicata (Jones, 1957), in which the excised part of a cut tube comes
together to form a new membrane thus regenerating the tube shape of the sponge.
Not only do adult and juvenile demosponges exhibit contractile behaviour, but the
larvae of some demosponge and calcareous sponges also show dramatic behavioural
responses. Sponge larval behaviour consists of phototaxis (Warburton, 1966; Bergquist
12
and Sinclair, 1968; Bergquist et al., 1970; Wapstra and van Soest, 1987; Woollacott,
1990; 1993; Maldonado and Young, 1996; 1999; Leys and Degnan, 2001; Elliott et al.,
2004), geotaxis (Warburton, 1966), chemotaxis (Avila and Carballo, 2006), and rheotaxis
(Maldonado and Young, 1999), but since the focus of this thesis is on behaviour in adult
sponges, I will not be discussing the mechanisms of larval behaviour here.
1.3 Anatomy of a sponge
The sponge body is organized into three functional regions: the ectosome, choanosome,
and osculum all of which are centered around an empty gemmule husk (Figure 1-4). The
ectosome consists of the apical pinacoderm, a three layered tent-like structure forming
the outer layer of the sponge that is draped the choanosome and spicule skeleton, and
subdermal cavity, a common reservoir for water to enter into the sponge choanosome.
The choanosome consists of the aquiferous system including incurrent and excurrent
canals, choanocyte chambers, mesohyl and basal pinacoderm. Within the choanosome,
the most striking feature is the branching nature of the aquiferous canal system with the
choanocyte chambers budding off the excurrent canals. Water enters the sponge through
the ostia in the apical pinacoderm into the subdermal cavity and enters the choanosome
via prosopyles, which lead to incurrent canals and choanocyte chambers. Filtered water
flows from the choanocyte chambers via apopyles into the excurrent canals and is vented
out of the osculum. Finally, the osculum, which protrudes to the distal end of the animal,
is the common vent for filtered water to exit the sponge excurrent canal system.
13
Figure 1-4. The anatomy of a sponge. A) Side view light micrograph image of a
sponge showing the osculum (osc), apical pinacoderm (apd), gemmule (g), spicule tract
(sp) and choanosome (cho). Scale bar, 1 mm. B) Top view light micrograph image of a
sponge showing the location of the incurrent canals (in), horizontal branching excurrent
canals (ex), osculum (osc) and gemmule (g). The excurrent canals consisted of
peripheral canals (p), two main canals (m) which converge on a central canal (c) at the
base of the osculum. Scale bar, 1 mm. Fracture (C) and schematic diagram (D)
illustrating the principal features of Ephydatia muelleri: the apical pinacoderm (apd), subdermal cavity (sdc), choanocytes (ch), and basal pinacoderm (bpd). The apical
pinacoderm consists of an inner layer of endopinacocytes (enp) and outer layer of
exopinacocytes (exp); porocytes (p), which form the ostia (os), are sandwiched between
the two layers. The choanosome contains incurrent (in) and excurrent (ex) aquiferous
canals, choanocyte chambers (cc) and spicule tracts (sp) that support the apical
pinacoderm. A thin collagenous middle region (mesohyl, me) houses mobile cells.
Prosopyles (pp), the entrance to chambers are formed by perforate 'sieve'-like cells.
Apopyles (ap) vent water from chambers. Water flow is indicated by dotted blue line.
Scale bar, 20 um.
14
ma .
WWg< v•^*»
Figure 1-4.
15
1.4 A brief history of studies on sponge behaviour.
Although Aristotle observed and recorded contractions occurring in sponges, Grant
(1825) was the first to notice and accurately describe the water currents that exited
through the osculum, which he called fecal orifices. He saw mysterious water currents
that came from the sponge, and carried out the first experiments to observe the response
of sponge tissues to corrosive acids and red hot wires, neither of which caused the tissue
to contract or orifices to close. He erroneously concluded that there was a complete lack
of irritability or contractibility and that the observations made by Ellis and Knight in
1765 were attributed by "some optical deception a little assisted by the imagination"
(Grant, 1825). Nevertheless, it was these experiments that made him decide that sponges
were animals, and he defined Porifera as "simple, aquatic, soft, animals, without
perceptible nervous or muscular filaments or organs of sense, with fibrous internal
skeletons sometimes supported with siliceous and sometimes calcareous spicula, the body
permeated with soft gelatinous flesh, covered externally with minute absorbent pores and
terminates in large open vents" (Grant, 1825).
Over the last century scientists have experimented on sponges often using similar
techniques, but always on different animals, and mostly observing different structures
respond (ostia in some cases, oscula in others, canals in yet others) (Table 1-1). These
studies showed that sponges respond to mechanical, chemical and electrical stimulation.
Over time researchers have homed in on the freshwater sponge, as a practical model to
study coordinated contractions because they are easily collected and culturing requires no
flow, they can be manipulated experimentally on a microscope, and they have transparent
tissues. Specific findings of importance were McNair's (1923) observation that
16
responses were different when the animal was stimulated at the tip or base of the
osculum, and that rates of contraction were slower on the inside than on the outside of the
osculum - but in either case, these rates were pretty fast at up to 370 utn-s"1. Prosser's
(1962; 1967) use of neurostimulants demonstrated the likely presence of receptors for a
great number of pharmacological substances, and showed that the sponge could detect
differences between these (i.e., they were not all perceived as toxins). The development
of sandwich preparations (Wintermann, 1951) provided a hint that contractions were part
of a more 'global' behaviour, and also gave the first observations that cells in the
mesohyl stop crawling as waves of contraction passed by.
Notably much of the early work was limited by microscopy techniques at the
time. For example, cinefilm was used to film contractions, but no computerized imaging
and software analysis tools were available to dissect patterns in the activities observed.
The perfusion systems available were large, and as such would have required massive
amounts of drugs required to test responsiveness of the animal. Recording changes to 20
urn diameter ostia at the same time would have been extremely difficult if not impossible,
hence oscula were usually observed. Finally, it was not until the 1960s and use of
transmission electron microscopy that the surface of Calcarea and Demospongiae were
determined to be cellular. Hence all previous physiological experiments must have
worked within the framework that responses could have travelled through syncytia. It
has only been since the 1980s that it has been known that glass sponges (Hexactinellida)
are the only group of sponge for which this is true (Mackie and Singla, 1983). Finally, all
of this work was carried out under the phylogenetic framework of the time which
suggested that sponges were a unique group of animals, separate from all other
17
Table 1-1. History and major milestones of sponge research.
Date
Author
Species
Milestone
322 BC
Aristotle
unknown
Sponges contract to damaging stimuli.
1740-60
Linneaus
many
Decided sponges are plants based on their
lack of sentience.
1765
Ellis & Knight
unknown
Observed openings (ostia) contract and dilate.
1825
Grant
unknown
Sponges move water, respond to acid and
heat by closing ostia.
1910
Parker
Stylotella heliophila
Observed the contraction of the osculum and
observed no transmission of the oscular
contractions to other oscula.
1923
McNair
Ephydatia fluviatilis
First to suggest that the sponge contraction
was a propagated phenomenon.
1937
Bidder
Leuconia
Grantia
Clathrina
Haliclona pygmaea
Sycon
Stylotella
Described musculo-skeletal cells and muscle
bands in several species.
1951
Wintermann
Ephydatia fluviatilis
Pioneered a method for growing sponges in
the space under coverslips.
1952
Kilian
Ephydatia fluviatilis
Thought that local arrests in feeding current
were caused by contraction of the ostia, the
osculum and the endopinacoderm, the latter
by causing the flagellated chambers to
become compressed and mechanically
impede flagellar beating.
1960
Pavans de
Ceccatty
Tethya lyncurium
Showed that whole sponges were capable of
contracting up to one third of their expanded
volume, and that the wave of contraction
spread throughout the sponge body from local
sites on the dermal membrane.
1962
Prosser et al.
Ephydatia fluviatilis
Spongilla lacustris
First to characterize the ionic requirements
for contraction to occur in the osculum.
1966
Emson
Cliona celata
Concluded that the mechanical stretching of
myocytes within the oscular membrane
sphincter propagated the contraction along
the osculum.
18
Date
Author
Species
Milestone
1967
Loewenstein
Haliclona microciona
Showed increased transfer of ions between
dissociated cells in culture - suggests
electrical coupling possible.
1967
Prosser
Ephydatia fluviatilis
Spongilla lacustris
Microciona
Found no evidence for propagation from one
part of the osculum to another, or evidence of
electrical excitability in several marine and
freshwater species.
1969
Pavans de
Ceccatty
Euspongia
Hippospongia
Using frame by frame analysis of time-lapse
cinematographic film he observed propagated
waves of contractions in the oscular
membranes. The contractions came in two
forms: one type involved a slow, general
contraction in which the oscular crown
constricted symmetrically like a sphincter,
and the other type concerned locally
propagated wave-like responses.
1971
Reiswig
Marine demosponges
Verongia
Found that oscular contractions were
frequently synchronized in the same
individual.
1971
Pavans de
Ceccatty
Euspongia officinalis
First use of biogenic amines to alter
spontaneous contractions.
1979
Mackie
Review and new work
on Rhabdocalyptus
dawsoni
Suggests that since propagated waves of
contraction occur independent of general
contractions, there must be more than one
mechanism involved; reported rapid arrests of
flow in hexactinellid sponges.
1981
De Vos & Van
De Vyver
Ephydatia fluviatilis
First description of spontaneous contractions
in the freshwater sponge.
1991
Fishelson
Tethya
Moving of entire colonies.
1984
Weissenfels
Ephydatia fluviatilis
Characterized the 'spontaneous' or
endogenous contractions that propagate
across the sponge body
1990
Weissenfels
Ephydatia fluviatilis
Characterized the repetitive nature of
contractions of the whole body.
1997
Leys & Mackie
Rhabdocalyptus
dawsoni
Recorded first action potential in a sponge.
2003
Carpaneto et al.
Axinella polypoides
First to patch-clamp record from isolated
demosponge cells.
2004
Nickel
Tethya wilhelma
Documented rhythmic and stimulated whole
body contractions.
19
metazoans - under today's hypothesis of paraphyly I understand that metazoan features
should be reflected in the sponge body plan. Today's experiments have obvious technical
and conceptual advantages, and it is with those that the mechanisms of coordination of
contractile events in demosponges are being re-examined (Meech, 2008; Kosik, 2009).
1.5 Evidence and possible mechanisms for a coordinated contractile system in
cellular sponges.
Most coordinating systems require two components: a messenger for propagating a signal
over distance, and an effector or a means of responding to the signal. It is well established
that sponges lack muscle, yet have contractile cells. Myocytes in both marine and
freshwater sponges form an epithelium that surrounds the osculum, ostia, and aquiferous
canal system and this epithelium has been shown to contract in many species (Parker,
1910; McNair, 1923; Bagby, 1965; Emson, 1966; Fishelson, 1981).
In sponges, three possible coordination systems have been hypothesized:
mechanical, electrical and chemical transmission of a stimulus (Jones, 1962). Although
both messenger and effector systems are poorly known in sponges, the rate of the
propagated events observed suggests certain hypotheses. First, the rates of propagation of
contractile waves in both freshwater and marine sponges are too slow for electrical
conduction (20-30 urn-sec"1) (Parker, 1910; McNair, 1923; Mackie, 1979; Weissenfels,
1990). However, local electrically conducted events cannot be completely ruled out since
Reiswig (1979) found that when Phorbas amaranthus was stimulated, the flaps would
contract in less than one second, which is surprisingly rapid compared to any other events
reported from other demosponges. Also, Lowenstein (1967) found that ion flow could be
20
measured between two dissociated sponge cells approximately 20 minutes after they were
placed in contact. However, this work has never been corroborated, and all further
attempts to establish evidence of gap junctions or other junctions that would allow
electrical coupling between cells in cellular sponges have been unsuccessful (Green and
Bergquist, 1979; Pavans de Ceccatty, 1979). Finally, to date there is no evidence for
molecules that might be in a proto-gap junction (pannexins or hemichannel) from the
trace files of the sponge genome.
Mechanical transmission of a signal between cells is possible, as local responses
to a tactile stimulus cause local contractions. Because these do not propagate throughout
the whole sponge body successive stretching of the membrane may be the propagated
trigger, however transmission of the signal is more likely by way of a chemical
messenger molecule released by mechanical stretching. There is now substantial
evidence for chemical signalling within the Porifera from studies showing
immunolocalization, cloning and functional studies with nitric oxide (Giovine et al.,
2001), cAMP (Simpson and Rodan, 1976; Gaino and Magnino, 1996), a metabotropic
glutamate receptor (Perovic et al., 1999), GABA (Emson, 1966; Perovic et al., 1999),
abscisic acid (Zocchi et al., 2001; Zocchi et al., 2003), cyclic ADP-ribose (Zocchi et al.,
2001; Zocchi et al., 2003), serotonin (Lentz, 1966; Weyrer et al., 1999); acetylcholine
(Emson, 1966; Pavans de Ceccatty, 1971; Thiney, 1972; Pavans de Ceccatty, 1976),
epinephrine (adrenalin) (Lentz, 1966; Pavans de Ceccatty, 1971; Thiney, 1972; Pavans de
Ceccatty, 1976), norepinephrine (Thiney, 1972; Emson, 1966), eserin (Lentz, 1966;
Thiney, 1972; Pavans de Ceccatty, 1976), and tryptamine (Emson, 1966). Amino acids,
calcium, ATP, or NO could also be involved in signal propagation (Prosser et al., 1962;
21
Emson, 1966; Giovine et al., 2001). The release of intracellular calcium across the cell
membrane may coordinate the contractile behaviour of cells (Pavans de Ceccatty, 1974;
Lorenz et al., 1996; Leys and Meech, 2006). The rate of propagation of the contractile
waves in sponges from 1-30 seconds is similar to that of calcium waves during cell-cell
communication between astrocytes, which are gap junction coupled in culture; however,
it is important to note that calcium waves could travel more slowly in whole organisms
and most measured rates are from artificial preparations.
Three molecules seem to be the most likely candidates for signalling in cellular
sponges: glutamate (L-Glu), y-aminobutyric acid (GABA), and nitric oxide (NO).
Glutamate is broadly distributed, evolutionarily conserved in intercellular signalling
systems in organisms with and without a nervous system, and it is an excitatory
neurotransmitter in the vertebrate central nervous system and astrocytes, and in many
invertebrates. GABA is an amino acid whose structure is conserved from bacteria
through metazoans and whose function in neural transmission is mainly inhibitory. In
animals without a nervous system, GABA has been localized in cells involved in body
contractions, swimming and feeding behaviour. Nitric oxide is a ubiquitous signalling
molecule in diverse physiological processes such as stress response, vascular tone
regulation, immune defense and nervous transmission (Bredt and Snyder, 1994). And so
it is on these three molecules that I have concentrated efforts. Future work could
investigate a host of others, as discussed in Chapter 5.
22
1.6 The aims of the present research
The aims of this thesis were: a) to determine whether contractions in sponges reflected a
coordinated event, which would be a singular difference in how such contractions were
viewed by earlier physiologists, b) to revisit in detail the morphology of the freshwater
sponge so as to better understand the means by which contractions were effected and the
conduit for signal propagation, and c) to assess the roles of three different molecules in
sponge contractions.
In Chapter 2,1 describe the response of the sponge to mechanical stimuli. This
response is stereotypical and travels in a peristaltic-like wave down the canals of the
sponge, first inflating and then contracting the canals so as to expel water from the whole
aquiferous system. In this chapter, I also describe other faster events such as twitches
and the diaphragm-like contraction of the surface tissue or apical pinacoderm. The
inflation-contraction cycle was spatially and temporally coordinated resulting in a
reproducible, orchestrated series of contractions passing through specific body regions of
the sponge in order. One of the most interesting observations in this section is that cells
of the mesohyl arrest crawling as a wave of contraction passes that suggests that an
extracellular signal may pass between cells.
In Chapter 3,1 present a very detailed morphological description of the juvenile
sponge using light, scanning electron and immunofluorescence microscopy. Three distinct
regions are easily identified, the ectosome, choanosome, and osculum, whose interactions
were coordinated to carry out contractions of the whole animal. The ectosome contains
the apical pinacoderm - two epithelia sandwiching around a thin collagenous mesohyl and the subdermal cavity, a common incurrent reservoir of water that was to be filtered.
23
Within the apical pinacoderm a vast network of condensed actin tracts were visible that
run the entire length of the sponge. The choanosome houses the aquiferous system,
which includes canals (incurrent and excurrent), choanocyte chambers, and basal
pinacoderm. The osculum vents water from the choanosome. Interestingly, all
endopinacocytes in the osculum, and a few within the excurrent canals possess a pair of
short presumed sensory cilia that may be used to detect water flow.
In the fourth chapter, I investigate the roles of extracellular signalling molecules
in the coordination of the peristaltic contractions. This work consists of measurements of
free amino acids in the sponge tissue by precipitation using HPLC-MS (High
performance liquid chromatography with mass spectroscopy) and testing whether some
of these signalling systems (L-Glu, GAB A, NO) were active in the freshwater sponge.
Twelve active amino acids were identified in sponge tissue lysate including both
glutamate and GABA. These molecules were tested to observe what if any function they
play in the peristaltic contraction of the aquiferous canals. Glutamate induced a dosedependent stimulation of the inflation contraction cycle that was blocked by both an
allosteric and competitive inhibitor to the metabotropic glutamate receptor signalling
system. GABA is an inhibitory signal that did not induce inflation-contraction cycles,
but was found to have a role in triggering twitches of the incurrent canals. Nitric oxide
synthase was located in cells of the apical pinacoderm, choanocytes and aquiferous canal
system and an active signalling system was shown by accumulation of cGMP, a
secondary signalling molecule in the nitric oxide pathway.
In Chapter 5,1 summarize the above work and reflect on the findings in the
context of the evolution of Poriferan and metazoan signalling molecules and mechanisms.
24
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35
Chapter 2 : Coordinated contractions effectively expel water from the
aquiferous system of a freshwater sponge
2.1 Introduction
Sponges (phylum Porifera) have a fossil record of over 600 million years (for example,
see Conway-Morris, 1993). Despite their ancient origin, they possess a vast repertoire of
genes that encode regulatory signalling molecules, many of which are homologous to
those in higher animals (Morris, 1993; Muller, 2003; Adell et al., 2007). Recent studies
also indicate that sponge embryogenesis is characterized by a spatio-temporal pattern of
gene expression that defines different regions or layers of the developing larva (Larroux
et al., 2006). Other research has shown that aspects of the immune response (Wiens et
al., 2004), respiration, maintenance of homeostasis (Zocchi et al., 2001), and even control
of body shape as a result of changes to the stiffness of the extracellular matrix (Wilkie et
al., 2004), have many features in common with equivalent physiological processes in
higher animals. These examples illustrate the sponge's ability to regulate its
developmental and physiological functions; however, coordinated movements of the
whole animal in response to external stimuli - the quintessential feature of Eumetazoa are not well known.
In contrast to its molecular and physiological complexity, the sponge is a
structurally simple animal. The sponge body is composed of at least 8 types of cells
arranged around an extensive aquiferous canal system built for filter feeding (Simpson,
1984). It is often suggested that sponges lack conventional epithelia, with typical cellcell junctions and a basement membrane which would create sealed internal
A version of this chapter has been published as Elliott, GRD and Leys, SP. 2007. Coordinated
contractions effectively expel water from the aquiferous system of a freshwater sponge.
Journal of Experimental Biology 210:3736-3748.
26
compartments (Tyler, 2003). However, sealing junctions, though not often dense or beltform, are present in sponge epithelia (Woollacott and Pinto, 1995; Gonobobleva and
Ereskovsky, 2004). Homoscleromorph sponges have a clear basement membrane
containing type IV collagen, a diagnostic feature of basal laminae (Boute et al., 1996;
Boury-Esnault et al., 2003), and complexes of extracellular matrix underlying the
epithelium in other demosponges have recently been found to contain a type of collagen
that is functionally equivalent to Type-IV of basement membranes (Aouacheria et al.,
2006). Although sponges lack typical organs and nervous tissue, they do have contractile
cells called myocytes (actinocytes of Boury-Esnault and Rutzler, 1997) that structurally
and pharmacologically resemble primitive smooth muscle cells allowing certain
contractile behaviour to occur (Parker, 1910; Prosser et al., 1962; Bagby, 1965; Prosser,
1967). The extent of coordination of this behaviour is the question addressed here in this
study.
As a filter-feeder, the main problem encountered by a sponge is likely intake of
unwanted material into the aquiferous system. Like other filter feeders, sponges have
developed mechanisms to control the feeding current - but these differ in the two
physiologically distinct types of sponges. Glass sponges (Class Hexactinellida) form
syncytial tissues during early embryogenesis, and this tissue allows them to arrest their
feeding current by propagating action potentials (Lawn et al., 1981; Lawn, 1982; Mackie
et al., 1983; Leys and Mackie, 1997; Leys et al., 1999). These animals apparently lack
any contractile tissues. In contrast, cellular sponges (Classes Calcarea & Demospongiae)
control their feeding current by contracting centralized sphincters or cells that line the
aquiferous canal system (Leys and Meech, 2006). The slow rate of contractions recorded
37
to date suggests there is no electrical coupling between cells as suggested by Mackie
(1979) and reiterated by Nickel (2004). So far ultrastructural studies have not identified
gap junctions in cellular sponges (Green and Bergquist, 1979; Garrone et al., 1980;
Lethias et al., 1983), but since proteins immunoreactive to anti-connexin antibodies were
found in penatulaceans (the most basal anthozoans) (Anctil and Carette, 1994), innexinor connexin-like molecules may yet surface from the current sponge genome project
(Joint Genome Institute, 2005-7). Nevertheless, in the presumed absence of such
junctions cellular sponges must possess another mechanism for coordinating effective
responses to stimuli.
Over a century of research has explored the intricacies of sponge responsiveness,
but because each study has observed different structures (ostia, oscula, or choanosome) in
different animals, the events have been thought to be localized and decremental (not
propagated); cellular sponges have not been considered capable of the coordinated
behaviours of higher animals (Jones, 1962; Mackie, 1979; Pavans de Ceccatty, 1979).
However, a fresh look at the activities of species in two demosponge genera, Tethya and
Ephydatia, suggests cellular sponges are able to propagate contractions both
endogenously and in response to external stimuli. Tethya is an opaque ball-shaped
sponge that contracts rhythmically, shrinking to one third of its normal size in 21 minutes
(Pavans de Ceccatty et al., 1960; Reiswig, 1971); similar contractions can be triggered by
natural stimuli (touch of a crustacean) (Nickel, 2004) and by chemical stimuli (Parker,
1910; Emson, 1966; Ellwanger and Nickel, 2006). In juveniles of Ephydatia, a
transparent encrusting sponge, waves of contraction travel through the canals and
chambers, taking up to one hour to entirely encompass the entire sponge (de Vos and Van
38
de Vyver, 1981; Weissenfels, 1984; 1990).
The objective of this study was to determine whether responses to stimuli
amount to a coordinated event; that is, do the various parts of the sponge - the
pinacoderm, ostia, osculum, and aquiferous canals - function together to propagate
contractions in a directional manner throughout the sponge's body. This study presents
the first characterization of the inflation (dilation)-contraction behaviour of Ephydatia
muelleri. Due to its small size and transparency, and the simplicity of its body design,
Ephydatia muelleri offers a practical model for future physiological studies.
2.2 Materials and methods
2.2.1 Sponge collecting and culturing
Gemmules (reduction bodies) and pieces of the freshwater sponge Ephydatia muelleri
(Lieberkuhn, 1955) were scraped from sunken trees or submerged rocks in Frederick
Lake, B.C. (48° 47' 51.7559"; 125° 2' 58.5600") at a depth of 0-3 m and stored in
unfiltered lake water at 4°C in the dark until use (Ricciardi and Reiswig, 1993). Bags
with sponge pieces were aerated monthly, and gemmules stored in this way were viable
for at least one year. The gemmules were removed from the spicule skeleton by gently
rubbing sponge fragments between 2 pieces of wet corduroy. Loose gemmules were
washed in cold distilled water (4°C) to remove debris, sterilized with a 1% hydrogen
peroxide (H2O2) solution for 5 minutes, and rinsed with cold distilled water to remove
excess H2O2.
Using sterile pipettes, gemmules were transferred to Petri dishes containing
Strekal's growth medium (0.9 mM MgS0 4 -7H 2 0, 0.5 mM CaC0 3 , 0.1 mM
39
Na 2 Si0 3 -9H 2 0, 0.1 mM KC1) (Strekal and McDiffett, 1974) or M-media (0.5 raM
MgS0 4 -7H 2 0, ImM CaCl2-2H20, 0.5 mM NaHC0 3 , 0.05 mM KCl, 0.25 mM
Na 2 Si0 3 -9H 2 0) (Funayama et al., 2005). For whole-mount preparations, single
gemmules were placed on an ethanol-washed, flamed glass or plastic 22 mm2 coverslip in
Petri dishes. For sandwich preparations, one 18 mm2 cover slip was mounted with dental
wax (Hygenic Corporation, USA) at the corners on a cover slip-bottom culture dish
(Willco Wells B. V., United Kingdom) that had been sterilized in 30% H 2 0 2 and rinsed
with 100% ethanol prior to use. Two gemmules were placed at the edge of the raised
coverslip, and dishes were left undisturbed at room temperature (21°C) in the dark. The
growth medium was replaced every 48 hours.
2.2.2 Digital video time-lapse microscopy and image analysis
Time-lapse imaging was carried out using either an inverted compound microscope
(Zeiss Axioskop) or a stereomicroscope (Olympus SZX-12). Images were captured with
digital cameras (Ql-Cam monochrome with color filter, Retiga monochrome and SONY
CCD), which were interchangeable on both microscopes. Image capture and analysis
was carried out using Northern Eclipse version 7 (Empix Imaging Inc., Mississauga)
from both live video feed and digitally taped material. Stimulation of the juvenile
sponges consisted of exposing sponges to water-soluble black calligraphy ink (Sumi
black ink, Delta Art Supplies, Edmonton) at a concentration of 1 drop (25 uL) of 100X
diluted ink in 1 ml of culture water (final dilution 4000X) or vigorous shaking (2-4 Hz) of
the culture medium over the sponge in the Petri dish for 1 minute (hereafter called
agitation as published elsewhere by de Vos and Van de Vyver, 1981). Images were
40
captured by Northern Eclipse every 5, 10, or 20 s, as indicated for each experiment. The
use of water jets, pin pricking or damage to sponge tissue did not solicit an inflationcontraction cycle; these stimuli only generated local contractions of tissue.
Changes in diameter of the canals for every first, fifth, tenth or twentieth image
of the aquiferous canals, ostia, osculum, and apical pinacoderm were measured in
triplicate using Northern Eclipse, and data were logged to MS Excel 2003. In whole
preparations, measurements of the aquiferous canals were taken at the center (diameter of
217.73 ±14.93 \im), middle (diameter of 107.38 ±3.75 urn), and peripheral canals
(diameter of 40.16 ±1.39 urn). In sandwich preparations (e.g., Figure 2-6), the inner
diameter of the canals was measured at 2 locations (100 and 300 \im apart) along a single
canal. For area measurements (Figure 2-7), images of ink-fed sponges were converted to
greyscale with Adobe Photoshop, two regions of 1450 urn by 1350 um (those occupied
by canals) were thresholded from 0 to 130 and the black area (that occupied by canals)
was calculated and expressed as a proxy for the contraction of canals.
2.2.3 Fixation for fluorescence and confocal microscopy
Juvenile sponges on glass coverslips (Fisher #1) were placed directly into a mixture of
3.7% paraformaldehyde and 0.3% gluteraldehyde in phosphate buffered saline (PBS; 100
mM) for 24 hr at 4°C. After fixation, preparations were washed in cold buffer and
incubated in 1% sodium borohydride for 5 min to remove autofluorescent free aldehyde
groups. Sponge tissues were permeabilized with 0.2 % Triton-XlOO in PBS for 2
minutes and washed in cold PBS. To label the actin cytoskeleton, coverslips were
inverted onto a drop of solution containing Bodipy 591 Phalloidin, Alexa 594 Phalloidin
41
or Bodipy 505 FL Phallacidin (Molecular Probes-Invitrogen, USA) in PBS with 10%
BSA (Bovine Serum Albumin). A 300 uL depression was made in a Parafilm-covered
Petri dish to prevent damage to the soft tissue by the gemmule. After 3 hrs at room
temperature, preparations were rinsed 3 times in cold PBS. For mounting, sponges were
incubated in a 50:50 v/v glycerine:PBS solution, and mounted in 100% glycerin or in
Mowiol with Dabco (antifade reagent), and allowed to harden overnight. For best results
slides were stored at 4°C. Preparations were viewed with a Zeiss Axioskop
epifluorescent microscope or a Leica 2 photon confocal microscope.
2.2.4 Fixation for scanning electron microscopy
Juvenile sponges on plastic or glass coverslips were fixed in a cocktail consisting of 1 %
Os0 4 , 2% gluteraldehyde in 0.45 M Sodium Acetate buffer (pH6.4) with 10% sucrose for
24 hr at 4°C (Harris and Shaw, 1984). The following day, preparations were washed with
cold distilled water and dehydrated in cold 70% ethanol for 24 hr at 4°C. Sponges on
glass coverslips were desilicified in 4% HF in 70% ethanol for 2 hr at 4°C. Once the
sponge had lifted off the coverslip, it was placed into a new Petri dish with fresh 4% HF
in 70%) ethanol at 4°C until spicules were dissolved. After desilicification, the loose
sponges were dehydrated to 100% ethanol and, while still in the vial of ethanol, fractured
in liquid nitrogen. Sponges on plastic coverslips and fractured pieces of loose sponge
were critical point dried, mounted on aluminum stubs with silver paste or nail polish,
gold coated, and viewed in a field emission scanning electron microscope (SEM).
42
Figure 2-1. Scanning electron micrograph fracture (A) and schematic diagram (B)
illustrating the principal features of Ephydatia muelleri: the apical pinacoderm (apd), subdermal cavity (sdc), choanocytes (ch), and basal pinacoderm (bpd). The apical
pinacoderm consists of an inner layer of endopinacocytes (enp) and outer layer of
exopinacocytes (exp); porocytes (p), which form the ostia (os), are sandwiched between
the two layers. The choanosome contains incurrent (in) and excurrent (ex) aquiferous
canals, choanocyte chambers (cc) and spicule tracts (sp) that support the apical
pinacoderm. A thin collagenous middle region (mesohyl, me) houses mobile cells.
Prosopyles (pp), the entrance to chambers are formed by perforate 'sieve'-like cells.
Apopyles (ap) vent water from chambers. Scale bar, 20 urn.
43
f ; PP. %Mr
,?f;:
Fjgwire 2-1.
44
2.3 Results
2.3.1 Description of the juvenile sponge
Gemmules of the freshwater sponge Ephydatia muelleri hatched in sterile culture dishes
at room temperature (18-23°C) in the laboratory within 2-4 days of plating. Within 4
days of hatching, the apical pinacoderm (surface epithelium), choanocyte chambers,
canal system, and incipient osculum had begun to develop, and by 7-10 days, a filtering
juvenile sponge was formed. Week to ten day old sponges typically had a single osculum
arising from two large excurrent canals that bifurcated around the gemmule and branched
successively into finer canals lined by choanocyte chambers. In other specimens, the
osculum was positioned directly over the gemmule and arose from a highly branched
network of smaller excurrent canals (Figure 2-lA,B).
2.3.2 Description of the inflation-contraction behaviour
The response triggered by stimulation of the sponge, either by adding ink to the water or
by agitation of the dish, consisted of three phases (Figure 2-2A-E; Movie SI;
http://jeb.biologists.org/cgi/content/full/210/21/3736/DCl): an inflation phase in which
the major excurrent canals dilated; a plateau phase involving dilation of smaller diameter
canals - this phase was most pronounced in larger specimens; and a contraction phase in
which the excurrent canals constricted and there was a rapid contraction of the osculum.
The sequence of events following either stimulus was similar, but changes in the
morphology were more readily measured in the absence of ink.
Every response consisted of eight identifiable events: 1) initial contraction of the
45
Figure 2-2. The response of Ephydatia muelleri to mechanical agitation. (A-D) Light
micrographs illustrating the changes to the excurrent aquiferous system (black arrows)
during one inflation-contraction cycle. Choanosome (cho), excurrent canals (ex),
gemmule (g), incurrent canals (in), and osculum (osc). Scale bar, 1mm. (A) Initial
contraction of the osculum: immediately after stimulation the base of the osculum
contracts but the tip remains slightly open. (B) Inflation phase: excurrent canals dilate
(black arrows); the base of the osculum begins to dilate, but the tip remains constricted
(white arrows). (C) Contraction phase: excurrent canals contract (black arrows) and the
base of the osculum dilates (white arrow). (D) Contraction of the osculum (arrow) and
return of canals to their original diameter. A-D correspond to phases a-d, respectively,
in (E-G) below. (E-G) Changes in diameter of the largest excurrent canal and osculum
(E) during the inflation-contraction cycle, and of all canals on the right (F) and left (G)
sides of the sponge. R1-R4 and L1-L4 in D indicate locations of measurements plotted
in F and G. (See Movie SI in supplementary material).
46
1800
2400
3000
4200
Time (s)
Figure 2-2.
47
4800
osculum and apical pinacoderm (Figure 2-2A); 2) Inflation phase: expansion of the subdermal cavity raising the apical pinacoderm and dilation of the excurrent canals travelling
from the base of the osculum back along the aquiferous canal system; 3) dilation of the
smallest peripheral canals; 4) contraction of porocytes (closure of ostia) in the apical
pinacoderm; 5) Contraction phase: lowering of the apical pinacoderm forcing the water
into the aquiferous canals; 6) a peristaltic-like wave of contraction that travelled along the
excurrent canals from the distal edge of the sponge to the base of the osculum and caused
the contraction of the choanocyte chambers (Figure 2-2B); 7) a rapid contraction
propagating from the base to the tip of the osculum (Figure 2-2C); and 8) relaxation of
the canals to their original diameter and extension of the osculum back to its original
length. This sequence followed a predictable time course, and up to 3 such sequences
occurred after a single stimulus, each separated by a recovery period.
Although the velocity of the propagated contraction varied as it progressed from
the periphery of the sponge towards the osculum, both the left and right hand sides of the
sponge inflated and contracted equally (Figure 2-2F,G).
2.3.3 Response to the addition of inedible ink particles
The inflation-contraction cycle was first recorded in response to the addition of inedible
ink to the culture dish. Ink taken into the sponge became lodged in the choanocyte
chambers making them black. Addition of the ink into the culture medium resulted in
several events. The first was identical to the orchestrated series of responses triggered by
agitation of the sponge as described above, but resulted in ejection of clumps of ink
(Figure 2-3A-E; Supplementary Movie S2). Additional responses followed minutes to
48
Figure 2-3. Uptake and clearing of inedible ink by a 7-day-old sponge. (A) Prior to
addition of ink the choanosome (cho) is white, the yellow gemmule (g) husk is in the
centre of the sponge, and the osculum (osc) is to the right. Scale bar, 1 mm. (B) 5
minutes after the addition of ink. (C) One hour after ink was added the sponge has
undergone a one inflation-contraction cycle. (D) 24 hours after ink was added, 4-6
inflation-contraction cycles have removed some of the ink. (E) 48 hours after ink was
added the choanosome is cleared of all ink particles.
49
H Ihip
Sm
'i
it
r'
JSTTES
: •
4 8 Hair
Ftgiire:
50
hours later; up to 3 additional peristaltic-like waves traversed the entire sponge over a 48
hour period.
The ink treatment also generated brief contractions that occurred simultaneously
in different parts of the choanosome (like twitches), as well as short waves of contraction
that propagated across portions of the choanosome in a linear direction - ripples. There
were also local non-propagating inflations and contractions - local events. Addition of
too little ink to the dish did not trigger the full 'inflation-contraction behaviour', but
twitches and ripples still occurred. Similarly, too little agitation of the dish failed to
trigger a full inflation-contraction cycle, but twitches, ripples and local events were
common after any amount of agitation. Attempts to trigger the full inflation-contraction
cycles by focal tactile stimuli (pin pricks) and electrical stimuli were so far unsuccessful.
2.3.4 The kinetics of the inflation-contraction cycle.
Comparison of the duration of the entire cycle from start of inflation to end of contraction
for ink 'fed' (30 min 45 s ± 2:01; n=8) and shaken sponges (19 min 9 s ± 2:45; n=12)
shows that addition of ink slows down the process but it was not significantly different
(Table 2-1). Estimates of rates at which the events occur in different regions of the
sponge (across the choanosome, along a canal, and up the osculum) depended upon the
type of preparation (whole mount or sandwich) and type of stimulus applied.
Contractions of the osculum were fastest, but contractions propagated across the
choanosome occur more slowly during a full 'inflation-contraction' cycle (2-5 um-s"')
than during ripples that occurred between cycles (4-11 um-s"1) (Table 2-2). During an
'inflation-contraction' cycle contractions usually propagated across all tissues from the
51
Table 2-1. Duration of the phases in the inflation contraction cycle in response to
different stimuli. AtT Duration of inflation cycle; A t c h c , duration of choanosome
contraction; A t 0 s C , duration of oscular contraction, At I C , duration of inflationcontraction cycle. (Values are mean ± SEM).
Stimulus
Af'(min:sec)
AtChC(min:sec)
A/0sC(min:sec)
Ink'fed'
11:30±4:43, n=8 15:55 ± 3:19, n=8 1:43 ± 0:24, n=5
Shaken
8:31 ± 1:38, n=12 10:43 ± 1:34, n=12 0:41 ±0:19, n=3
p
0,47
014
0J3
p - p-value for ink fed versus shaken.
At'c (min:sec)
30:45 ±2:01, n=8
19:09 ±2:45, n=l2
0.21
52
Table 2-2. Rates of contraction in different regions of E. muelleri juveniles
Region (type of response; stimulus and preparation)
Choanosome
Canals (Full cycle; agitation; whole mount)
Incurrent canal (inflation phase)
Excurrent canal (contraction phase)
(mean ± SE).
Contraction rate (urn s~')
Range (urn s~')
2.91 ± 0.46 (n=3)
3.48 ± 0.79 (n=3)
2.28-2.63
2.31-4.99
Canals (Full cycle; ink; sandwich preparation)
Incurrent canal (inflation phase)
Excurrent canal (contraction phase)
1.68 ± 0.78 (n=6)
0.49 ± 0.13 (n=7)
0.25-5.0
0.32-1.0
Canals (Ripple; agitation; whole mount)
7.09 ±0.95 (n=7)
4.5-11.72
Osculum (full cycle; agitation; whole mount)
(full cycle; ink; whole mount)
71.85 ±32.4(n=3)
17.68 ±8.26 (n=5)
11.59-122.80
6.31-50.34
Values are means ± s.e.m.; TV values are given in
parentheses.
53
periphery of the sponge to the base of the osculum; in some cases the waves travelled
along the long axis of canals, but in others an entire canal expanded in unison as the wave
propagated across it. All told, rates appeared to be very dependent on the resulting effect
of the contraction.
Canals: The full inflation-contraction behaviour had stereotypical 'inflation', 'plateau'
and 'contraction' phases, but the duration of the entire event varied depending on the
initial (resting) diameter of the aquiferous canals: sponges with larger resting canal
diameter (e.g., 64.9, 76.9, 103.52 , 154.13 and 213.35 urn) had a longer overall inflationcontraction phases (500, 899, 1399, 2052, 2988 s; Figure 2-4 and Figure 2-5), extending
the duration of the entire cycle from 15 to 40 minutes. Otherwise, the events occurred
almost identically in sponges with quite different patterns of canals. The rates of dilation
and contraction of the large excurrent canals were similar (2.80 ± 0.26 um-s"1, n=5 and
3.30 ± 0.45 um-s"1, n=5, p=0.23 respectively, Table 2-2). Interestingly, the rate of the
peristaltic-like contraction, measured from preparations in which specific points on the
canals could be accurately tracked at high resolution, depended upon which region of the
aquiferous system it moved through. In the peripheral canals it traveled at 0.03 - 1
um-s"1, in the central canals at 1-4 um-s"', and up osculum at 6-122 um-s"' (Table 2-2).
Sandwich preparations allowed clear observations of the waves of peristalticlike contraction, and use of the ink stimulus provided a clear marker for incurrent and
excurrent aquiferous canals. This preparation revealed that during the inflation phase,
dilation of the excurrent canals was caused by a wave of contraction travelling along the
incurrent canals. Ink entered incurrent canals rapidly filling choanocyte chambers (Figure
2-6A,B). Contraction of the incurrent canals condensed the ink in the chambers and
54
incurrent canals and even forced some ink-filled water through the choanocyte chambers
into the excurrent canals during the plateau phase (Figure 2-6C,D). During the
contraction phase, a wave of contraction propagated along the excurrent canals so as to
cause the dilation of the incurrent canals (Figure 2-6E,F). As the excurrent canals
contracted, the flow of water (as seen by movement of ink) briefly reversed direction, and
then remained stationary for up to 6 minutes (Figure 2-6G; Supplementary Movie S3).
After one inflation-contraction cycle the sponge returned to a relaxed state (Figure 2-6H).
This type of preparation also illustrated that the wave of contraction propagated along
two vectors, both along and across the incurrent and excurrent canals (Figure 2-7A-D,
Supplementary Movie S3). Contractions traveled across canals that were 310 urn apart at
a delay of 300 s (approximately 1 um-s"1). Furthermore, cells crawling through the
mesohyl arrested forward motion for about 10 minutes (approximately 600 s) as the wave
of contraction passed by (Figure 2-7B). The two cells tracked here were 1053 um apart,
and they arrested with a delay of 600s (Figure 2-7B).
In sandwich preparations stimulated with ink, the contractions propagated along
the incurrent canals slightly faster than along the excurrent canals (Table 2-2; Figure 27C,D). Time-lapse images of these events in sandwich preparations suggest that cells in
the mesohyl between the two canals shorten causing the choanocyte chambers to
compress. These images also show that cells crawling through the mesohyl stop moving
as the waves of contraction pass over them (Supplementary Movie S3).
Osculum: Immediately after agitation or addition of ink, the osculum contracted
downwards. Then, as the aquiferous canals contracted, the base of the osculum dilated to
55
Figure 2-4. The duration of the inflation-contraction cycle depends on the resting
diameter of the largest excurrent canals. Responses to agitation were measured in 5
sponges with the same sized (diameter) choanosome, but with varying sizes (1-5) of
excurrent canals: 64.9, 76.9, 103.52 , 154.13 and 213.35 um respectively. Sponges with
larger excurrent canal diameters have a longer inflation and contraction period 500, 899,
1399, 2052, 2988 s respectively, but the rate of propagation of contractions along
incurrent and excurrent canals was not significantly different (In: 2.80 ± 0.26 um-s"1, n=5;
Ex: 3.30 ± 0.45 urns"1, n=5, p= 0.23). Bars indicate standard error of three
measurements from one sponge.
56
450
600
1200
1800
2400
3000
3600
4200
Time (s)
Figure 2-4.
57
Figure 2-5. Stereomicrographs showing the sponges, and enlargements of the regions of
the principal canals, measured in Figure 2-4.
58
Sponge. 1
Sponge 2
Sponge 3
igure 2-5.
59
Figure 2-6. Contraction of incurrent and excurrent canals in a sandwich preparation.
Incurrent (in, white arrows) and excurrent (ex, black arrows) canals can be identified by
the movement of ink along the incurrent canals in (B). Scale bar, 100 um. (A-H)
Inflation phase: Uptake of ink into the canals and chambers triggers contraction of the
incurrent canals dilating excurrent canals. The wave of contraction moves from right to
left along each canal and across canals from bottom to top of the image. At maximum
contraction of the incurrent canals, some ink is forced through the choanocyte chambers
(double black arrows, E). (F-H) Contraction phase: Excurrent canals begin to contract,
dilating the incurrent canals. Water is stagnant for a period of 6 minutes (F,G), and as the
incurrent canals dilate, some ink flushes backwards along the incurrent canals (double
black arrows, G). (H) Excurrent canals continue to contract. (Movie S3 in supplementary
material).
60
Figure 2-6.
61
become almost balloon-like. Only when the entire choanosome had completely
contracted did a wave of contraction run from the base to the tip of the osculum (Figure
2-8A-D). The final oscular contraction took 71.85 ± 32.4 um-s"1, n=3, in agitated
sponges and 17.68 ± 8.7 um-s"1, n=5, in ink-fed sponges (range 6 - 122 um-s"1) and was
always followed by a slow extension (Supplementary Movie S4). Because precise
changes in diameter of the osculum were difficult to track in ink-fed animals,
measurements for those animals present a conservative estimate of the duration of the
contraction event.
Apical pinacoderm: Upon agitation the apical pinacoderm contracted down towards the
choanosome, lowering 50-200 um within 60 s. This contraction occurred after the initial
response of the osculum, but before the inflation of the canals. The apical pinacoderm
moved as a single unit, like a diaphragm, reducing the volume of the sub-dermal space.
During the inflation phase, the apical pinacoderm moved back to its relaxed position
(Figure 2-8A,B), and just before the excurrent canals contracted, it lowered again. For
sponges with a diameter of 3-5 mm, these waves of contraction traveled at 50-80 um-s"1
propagating from the periphery of the sponge to the base of the osculum. In some
instances a series of twitches occurred across the entire surface of the sponge just before
the main wave of contraction that lowered the entire apical pinacoderm (Supplementary
Movie S4).
Porocvtes: In relaxed sponges, fields of porocytes - flat cells that formed the ostia,
incurrent openings for water - littered the apical pinacoderm (Figure 2-9). The margin of
each cell was anchored in a collagenous extracellular matrix between the inner and outer
62
Figure 2-7. Analysis of the kinetics during the inflation-contraction cycle showing
waves of contraction that propagates along and across incurrent and excurrent canals.
Analysis of the kinetics of the contraction of incurrent (A,B) and excurrent (C,D) canals
shows that waves of contraction propagate along and across canals. Sponges grown as
sandwich preparations were stimulated by addition of inedible ink. Measurements of
incurrent (A) and excurrent (C) canal diameters over time are plotted in (B) and (D)
respectively. In (B) the wave of contraction propagated between sites 1 and 2 (100 urn
apart) in 300 s, a rate of 0.33 urns" . The wave of contraction reached site 3 with a delay
of 150 s. Cells crawling through the mesohyl (indicated by white and black stars on A
and B) arrest movement for approximately 10 minutes (B, white arrows) while the wave
of contraction passes. In (D) the wave of contraction propagated between sites 3 and 4
(300 um apart) in 940 s, rate of 0.32 um-s"1. In, incurrent; Ex, excurrent, arrows indicate
direction of water flow in the canal. Scale bars, A, 100 um; C, 300 um. (B, D). (Movie
S3 in supplementary material.)
63
w
Distance Travelled by Cell (pm)
Figure 2-8. Stereomicrograph images (A-D) and changes in diameter (E) of the tip and
base of the osculum (at positions indicated by arrows in A) during contraction of the
osculum. (A,a) immediately after stimulation by agitation, (B,b) during the contraction
phase when its base is fully inflated, (C,c) fully contracted; and (D,d) when relaxed at the
end of the cycle; insets show enlargements of the position of the apical pinacoderm.
Scale bar, 1 mm. A-D corresponds to phases a-d, respectively, in (E). Between (A) and
(D) the canals inflate and the apical pinacoderm raises. The wave of contraction
propagates from base to tip, a distance of 1473 urn, in 12 s, a rate of 122.8 um-s"1. Bars
denote the standard error of an average of three measurements from one sponge. (Movie,
S4 in supplementary material).
65
590
Figure 2-8.
620
650
680
710 740
Time (s)
770
800
830
Figure 2-9. Closure of a field of porocytes in the apical pinacoderm correlates with
contraction of the choanosome after stimulus by agitation. (A-C) Stereo microscope
images show that individual ostia (arrows) close within 50 s, and a field of 20 porocytes
closes over a period of 83 s. Scale bar, 100 |j,m. (D) Constriction (closing) of 8 ostia in
the field shown in A is correlated with the contraction of the choanosome (dashed line).
Shortly after the sponge was stimulated the ostia closed and the choanosome contracted.
A few ostia opened briefly at 1200 s during an expansion of the choanosome, but these
closed again as the choanosome contracted. The field opened again with the expansion
of the choanosome at t=1500 s (here approximately 22 minutes later), and closed just
prior to contraction of the choanosome at 2400 s.
67
1200
1800
Time (s)
2400
Figure 2-9.
68
epithelia of the apical pinacoderm. Each porocyte was surrounded by 3-4 plate-like
exopinacocytes. In a relaxed sponge there were 10-12 porocytes per 1 mm2 of apical
pinacoderm (Figure 2-9A). After stimulus by agitation, fields of up to 35 ostia closed
synchronously (Figure 2-9A-C; Supplementary Movie S5). Individual ostia took
approximately 40 s to close, and a field of ostia closed just before the contraction of
excurrent canals in the choanosome. Ostia re-opened as canals relaxed (Figure 2-9D). In
ink-fed sponges the ostia also closed just before canals contracted, and remained closed
until the contractions had finished. Use of the ink as a stimulus revealed that in all cases
a few ostia near the base of the osculum remained open, allowing a small amount of
water to flush back out through the sub-dermal cavity (observed as puffs of ink in
Supplementary Movie S6).
2.3.5 Kinetics of twitches, ripples and local contractile events
In between sequential inflation-contraction cycles, brief propagating and non-propagating
contractions took place. In many experiments, waves of contraction rippled across
portions of the sponge choanosome at a rate of 7.09 ±0.95 um-s"1 n=7; these contractions
did not travel towards the osculum and occurred without periodicity. Local contractile
events also occurred in the interval between major inflation-contraction cycles. Here, a
small region of the choanosome, usually no more than several hundred micrometers in
diameter, inflated and contracted independently of any other activity of the sponge. In
some experiments, the entire sponge choanosome contracted rapidly and apparently
simultaneously like a twitch. These quick, global contractions of less than 20 s duration
occurred nearly simultaneously (<5 s difference) in very different regions of the
69
Figure 2-10. Rapid contractions ('twitches') occur simultaneously in distinct regions of
the choanosome after uptake of inedible ink. (A) Projected area (density of the tissue)
was measured as a proxy for the extent of contraction of the tissue; a contraction was
observed as a decrease in the area occupied by tissue (a decrease in blackness). (B) The
change in projected area of each region shows that two contraction events occur within
seconds of each other in Areas I and II of A, 700 um apart. (Movie S2)
70
^
n
i
era
e
Projected Area (%)
choanosome (Figure 2-10A,B; Supplementary Movie S2).
Typically, an unstimulated sponge exhibited occasional ripples, twitches, and
local inflation-contraction events; however only one full inflation-contraction cycle
occurred during every 8 hours of 48 hours of observation.
2.3.6 The contractile apparatus of the sponge.
Phalloidin-labelled sponges revealed dense tracts of actin in endopinacocytes of the
apical pinacoderm, canals, and the osculum. In the apical pinacoderm, 2-3 bundles of
filamentous actin traversed individual pinacocytes (Figure 2-11A,B). Contacts between
neighbouring cells labelled brightly, like adhesion plaques, and actin bundles in adjacent
cells continued in the same direction so as to form tracts that were continuous for up to 3
mm (Figure 2-1 IB). These tracts of actin stretched across the apical pinacoderm, around
the perimeter of the sponge, and from the perimeter of the sponge to the top of the
gemmule converging at the pinnacle of shafts of spicules that supported the overlying
apical pinacoderm. Endopinacocytes lining the canals labelled much less intensely with
phalloidin, and fine tracts of actin were visible only in sandwich preparations in which
sponges grew in a 50 um thick space between two coverslips (Figure 2-11C). In
unstimulated sponges, excurrent canals were lined by thin (1-3 urn) endopinacocytes, and
choanocyte chambers were spherical (30 um in diameter) (Figure 2-12A,B). In sponges
fixed in a contracted state, endopinacocytes lining the excurrent canals were thicker (5-7
um and choanocyte chambers were compressed to the extent that their flagella projected
out through the apopyle (Figure 2-12C,D).
72
Figure 2-11. Actin distribution and morphology of pinacocytes. (A) Scanning electron
microscopy shows that endopinacocytes (enp) are elongated cells that form the underside
of the apical pinacoderm. Dotted lines indicate cell boundaries. Scale bar, 10 um. (B)
Epifluorescence microscopy of Bodipy 505 FL Phallacidin - labelled tissue shows
extensive tracts of actin in endopinacocytes of the apical pinacoderm, a region equivalent
to that shown in (A). Actin is brightly labeled in focal adhesion plaques between cells
(arrows). Dotted lines indicate cell boundaries demonstrating that the actin tracts
continue in adjacent cells. Scale bar, 50 um. (C) In cells lining excurrent canals of a
sandwich preparation, actin tracts (black arrows) are much less brightly labelled. The
preparation was fixed as a wave of contraction passed through the field of view. Dense
packing of choanocyte chambers (cc) indicates that the lower canal was contracted. Scale
bar, 100 um.
73
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4-
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Figure 2-12. Morphology of the aquiferous canals and choanocyte chambers studied by
scanning electron microscopy of sponges preserved when relaxed (A,B), and after
stimulus when contracted (C,D). When relaxed the canals are broad, endopinacocytes
(end) form a thin layer lining the canals and choanocyte chambers (chc) are round, with
well-spaced choanocytes (ch, arrows) and broad apopyles (ap). In contrast, canals in
contracted specimens are narrower, endopinacocytes are slightly thicker (double arrows)
and choanocyte chambers are compressed such that the flagella (fl) project out of the
apopyle into the excurrent canal (C,D). Scale bars: A, 10 um, B-D, 5 um.
75
N
s
76
2.4 Discussion
In response to a mechanical stimulus Ephydatia muelleri initiates a series of slow
contractions (summarized in Figure 2-13) that effectively expel water and wastes from
the aquiferous system. The periodicity of contractions seen in unstimulated sponges, and
reported in other species and genera, suggests this behaviour may also function to assist
the sponge feeding and/or respiratory activity (Weissenfels, 1990; Nickel, 2004). There
is growing evidence that sponges, like other metazoans, possess a broad repertoire of
signaling molecules (e.g., Perovic et al., 1999; Nichols et al., 2006; Adell et al., 2007;
Sakarya et al., 2007) and experiments have demonstrated that many of these substances
trigger contractile responses in a variety of sponges (Emson, 1966; Prosser, 1967;
Ellwanger and Nickel, 2006; Ellwanger et al., 2007). The exact nature of the contractile
response has nevertheless been rather unclear, largely due to the difficulty of watching
the animal at the cellular level. The small size and transparent tissues of E. muelleri
however, allow high magnification observations of specific regions of the sponge which
illustrate that it is not the speed of contraction but rather the temporal and spatial
coordination of all the events that allows the sponge canal system to form an effective
peristaltic-like pump. Given the basal position of sponges within Metazoa, and the
absence of nerves and true muscle in this group (Pavans de Ceccatty, 1989; and
references above), it can be inferred that what we see in sponges today represents a
coordination system that predated the evolution of neuromuscular systems observed in
higher Metazoa.
77
Figure 2-13. Summary diagram illustrating the temporal coordination of contractions by
the aquiferous canals, apical pinacoderm, ostia, osculum, during a single inflationcontraction event in Ephydatia muelleri. During the inflation phase, the apical
pinacoderm, canals and osculum gradually dilate. The ostia contract for the duration of
the inflation phase. The contraction of the apical pinacoderm and canals lead to the full
inflation of the osculum and its rapid contraction. Ostia open only after all other
components have relaxed.
78
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Figure 2-13.
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1200
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1500
r
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1800
Canals
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Apical Pinacoderm
2.4.1 Rates of contraction
Rates of waves of contraction reported in cellular sponges are several orders of
magnitude slower than electrically controlled contractile systems (for review see Mackie
1979; Mackie et al., 1983). Contractions tend to be slightly slower in freshwater than
marine sponges (presumably due to the lower calcium available), but rates of endogenous
contractions reported in the literature largely depend on what region of a sponge was
observed. For example, waves of endogenous contractions cross the surface (including
the choanosome) of marine sponges {Tethya wilhelma, Tethya lyncurium, Euspongia
officinalis) at 12 - 30.5 um-s"1 (Pavans de Ceccatty, 1969; ,1971; Nickel, 2004), and
freshwater sponges (Ephydatia fluviatilis, Eunapius fragilis, Spongilla lacustris) at 8 - 11
um-s"1 (de Vos and Van de Vyver, 1981; Weissenfels, 1990). Electrical stimuli applied
to the base or tip of the osculum of Ephydatia fluviatilis triggered faster waves of
contractions up and down (170 and 350 um s"1 respectively) the osculum (McNair, 1923);
Prosser (1962) reports a similarly quick contraction of the oscula (1-5 s for 1mm diameter
oscula) in several marine species. Furthermore, precise measurements of rates are
difficult to calculate from video recordings or a series of still images. For example, in T.
wilhelma periodic contractions have been documented by measuring the decrease in area
of a projection of the sponge (Nickel, 2004). Subcontractions (= equivalent to ripples)
propagate at 12.5 um-s"1 over the surface of the sponge, yet full contractions take 20-50
minutes to encompass the entire sponge; relaxation (inflation) takes somewhat longer.
Although it is proposed that the contraction travels through the pinacoderm, because
Tethya is an opaque sphere, the route that the contractile wave travels cannot be easily
determined.
80
I encountered similar difficulty in determining precisely when contractions
initiate at two points 100 um apart along a canal. Cells in the mesohyl around the canal
begin to change shape long before changes to the diameter of the canal are evident. Also,
in some instances entire canals seemed to widen uniformly along their entire length, such
that no 'rate' of propagation could be measured. In general however, contractions
propagated very slowly through the canals at the periphery of the sponge (0.3-1 urns"1),
slightly faster through the large exhalent canals (1-4 u r n s ' ) , and even faster up the
osculum (6-122 um-s"1); these were part of the overall 'inflation-contraction' behaviour,
while ripples and spasms occurred separately. Thus my study indicates that the actual
speed of propagation of a contraction depends on the function of the contractile tissue
(the effector). From this I infer that because each region comprises part of a hydrostatic
skeleton whose function is to expel water from the aquiferous system, the rates observed
indicate control of the body of water rather than the absolute ability to propagate a signal.
The individual rates observed result from coordination of these regions.
2.4.2 Coordination of effectors
Coordination of the series of effectors is seen most acutely in the synchronous closure of
fields of ostia independently of, and usually just before the contraction of the apical
pinacoderm. It has long been known that individual porocytes contract (Emson, 1966;
Kilian and Wintermann-Kilian, 1979), but this is the first data showing that whole fields
of porocytes contract and relax in unison. Synchronous closure of ostia is a remarkable
event. The contraction of each porocyte sphincter takes some 60 s, but the fact that up to
50 ostia close over the same time frame, and just before the choanosome contracts points
81
either to some fairly rapid coordinating signal traversing the apical pinacoderm, or
suggests that inflation of the entire sponge stretches the apical pinacoderm triggering
simultaneous closure of ostia (presumably by entry of calcium into each porocyte). It is
interesting to note that in all experiments a few ostia remained open around the base of
the osculum allowing ink to be flushed back and out of the sub-dermal cavity. Reversal
of flow by sponges has only been described by Storr (1964) and likely refers to a similar
back-flushing event during a periodic (cyclical) contraction event.
Contraction of inhalant and exhalant canals also demonstrates coordination of
effectors. Initially it seemed that dilation of the exhalent canals occurred by passive
inflation when the osculum closed in response to the initial stimulus. However, careful
observation of videos shows that the osculum is never entirely closed - the tip constricts,
but a fast stream of water continues to flow from it at all times (e.g., ink flows from the
constricted osculum prior to expulsion of ink from the choanosome in Supplemental
Movie S2). Sponges treated with cytochalasin B did not inflate the choanosome (dilate
the incurrent or excurrent canals) even though the osculum did a small initial contraction
when the dish was vigorously shaken; thus passive inflation of the choanosome is
unlikely (data not shown). Because videos of sandwich cultures show that cells in the
mesohyl bridging adjacent exhalent canals contract during the inflation period, we
suggest that dilation of the exhalent canals seems at least partly due to the active
contraction of inhalant canals. These observations explain why the rates of inflation and
contraction are very similar regardless of the diameter of the canal (Table 2-1). What can
also be seen is that water is absolutely stagnant for the duration of the plateau phase (the
ink front in the excurrent canal remains completely stationary for up to 6 minutes in one
82
instance; Supplementary Movie S3) - that is the sponge uses contractions to control the
movement of water in its canals. This observation is the first precise visual
demonstration that cellular sponges can stop their feeding current.
2.4.3 Evidence for effector tissue and signal propagation
Most studies suggest that endopinacocytes - the cells that line the inside of the sponge are responsible for propagated contractions (de Vos and Van de Vyver, 1981; Pavans de
Ceccatty, 1986; Nickel, 2004), but in sponges with a denser mesohyl, it is implied that
either myocytes - cells in the mesohyl - or pinacocytes form sphincters to constrict flow
through canals (Parker, 1910; Pavans de Ceccatty, 1960; Jones, 1962; Prosser et al.,
1962; Bagby, 1965; Pavans de Ceccatty et al., 1970). The contractile apparatus has been
difficult to pin down. The actin cytoskeleton is only known from stationary
basoendopinacocytes of freshwater sponges (Pavans de Ceccatty, 1986; Wachtmann and
Stockem, 1992), and from myocytes in one marine sponge (Microciona proliferd). In
basoendopinacocytes, the cytoskeleton is much like that of a fibroblast in which
microfilaments form stress fibers across and around the cell. Actin filaments are slightly
denser between neighboring cells, and between cells adhesion plaques reminiscent of
early stage desmosomes in fish embryos (Lentz and Trinkaus, 1971), which can be seen
in freeze fracture electron micrographs (Pavans de Ceccatty, 1986). In contrast,
myocytes in sphincters in the canals are well-endowed with both thick and thin filaments
(Bagby, 1965).
These images show that a substantial actin network exists in the cells that form
the lower portion of the apical pinacoderm, the endopinacocytes. Bundles of actin
83
filaments form tracts traversing endopinacocytes, and each tract connects to another in
neighboring cells through a dense plaque of actin; together these form the longest semicontinuous tracts known in sponges (1-3 mm). Continuity of the cytoskeleton in the
apical pinacoderm is presumably necessary for the entire tent-like structure to lower in a
single diaphragm-like movement in less than 60 s. Actin microfilaments appear as
'rings' around the circumference of the aquiferous canals; in both cases tracts connect to
others in neighboring cells as in the apical pinacoderm.
Earlier researchers favored mechanical tugging of one cell on another as the
explanation of contractile waves (Parker, 1910; Pavans de Ceccatty et al., 1960; Emson,
1966; Pavans de Ceccatty, 1969). This hypothesis is difficult to test because damage to
any portion of the sponge disrupts flow and interrupts contractions throughout the
sponge. Furthermore, although mechanical 'tugging' might explain how waves of
contraction propagate along canals, it does not readily explain how the waves propagate
across canals or between completely distinct regions of the sponge as during twitches. It
is possible that a change in pressure could result in signals being transmitted to a distant
site, but how ink building up in the chambers could generate a pressure wave causing the
osculum to constrict (the first event to occur), is unclear. Moreover, how pressure waves
could orchestrate the spatio-temporal coordination of contractions in different regions is
difficult to imagine.
Recent evidence that diffusible chemical messengers including amino acids
(glutamate and GABA), biogenic amines, and short-lived gases (e.g., nitric oxide) trigger
or modulate contractions in Tethya wilhelma strongly suggest that signals travel through
the mesohyl in a paracrine-like manner or through the aquiferous system (Ellwanger and
84
Nickel, 2006; Leys and Meech, 2006; Ellwanger et al., 2007). Perhaps the most
definitive evidence that a diffusible chemical messenger is involved in contractions in
Ephydatia is that cells crawling through mesohyl stop moving as contractions pass by
(Figure 2-5) as also noted by other authors (de Vos and Van de Vyver, 1981). Since
these cells are wandering through the mesohyl, not in contact with pinacocytes, it can be
inferred that a signal passes through the mesohyl at least at 1.75 fims"1 (a distance of
1053 um in 600 s). It is quite possible that chemical and mechanical signalling function
together to coordinate the propagation of contractions. Nevertheless, the rapid lowering
of the apical pinacoderm and rapid contraction of the osculum are faster events than can
be explained by calcium signalling, which is generally up to 20 um-s"1 (Nedergaard,
1994).
2.4.4 Comparison with other contractile systems
I have described contractions in E. muelleri as 'peristaltic-like', but this is the first time
the term peristalsis would be applied to an animal that lacks muscle. Peristalsis is usually
considered to involve neurogenic modulation of myogenic contraction to propel a fluid
through a tube (Randall et al., 2002). In the sponge the canals behave as a single motor
complex in which a period of dilation is followed by a propagated contraction that
squeezes the water forward towards the osculum. Except that neuronal modulation is
absent, the system does not appear much different from those composed of multi-unit
smooth muscles (Randall et al., 2002).
Peristalsis seems to be a central feature of body plans in all animals. It is
involved in moving fluid for nutrient transfer in the gastrovascular cavity (GVC) of the
85
sea pansy Renilla koellikeri (Anctil, 1994), for burrowing by anemones, nemerteans,
polychaetes and bivalves (Ansell and Trueman, 1968). Peristalsis is also involved in the
contraction of the heart in tunicates and amphioxus (Holland et al., 2003). In each of
these instances control is thought to be myogenic, although the role of nerves is not well
understood. It is interesting to note that while contractions of the GVC of Renilla
propagate at 1-1.3 mms" 1 (Anctil, 1994; Anctil et al., 2005), contractions of the body
wall of the sessile anemone Metridium senile propagate at -500 um-s "' (Batham and
Pantin, 1950) and even slower in the tiny burrowing starlet anemone Nematostella
vectensis (at 3-20 um-s" ) (S.P. Leys, unpublished). Cnidarians have the advantage of
both muscle (epitheliomyocytes) and neurons, yet contractions are still slow. As
suggested by Batham and Pantin (1950) this is presumably due to the load the muscle
acts against rather than intrinsic limitations, because when stimulated electrically, the
same region of the body wall can contract much faster. My observations suggest this is
also true for sponges. In order to expel water, the tissues contract in a controlled and
coordinated manner; but when water is not being pushed out of the aquiferous system
faster contractions are possible, as when ripples run across portions of the sponge or the
osculum contracts down in response to mechanical agitation. Evidently sponges have,
without nerves or true muscle, evolved a way of coordinating contractions of cells to
generate an effective mechanism of controlling water flow.
The next step is to determine what signal or mechanism controls each type of
contraction. Because of its small size and transparency, the freshwater sponge promises
to be an excellent model system for further study of the role of signalling molecules in
inducing, controlling, and modulating behaviour in these 'simple animals'.
86
2.5 Supplementary movies.
(http://jeb.biologists.org/cgi/content/full/210/21/3736/DCl)
Movie SI (Figure 2-2): Response of Ephydatia muelleri to 1 minute of agitation (2-3 Hz)
of the Petri dish. The 'inflation-contraction' behavior of the sponge consists of three
phases - inflation (0-1140 s), plateau (1150-1880 s) and contraction (1890-3240 s).
Upon stimulation, the sponge constricts the osculum (lower centre), contracts the
incurrent canals thereby dilating the excurrent canals, and then contracts the excurrent
canals venting all the water out of the osculum. At the final oscular contraction (26002740 s) the water is expelled out of the osculum. The choanosome is white, and the
empty gemmule from which the sponge hatched is yellow. Duration: 61.9 min; time
interval: 10 s; and frame rate: 10 fps.
Movie S2 (Figure 2-10): Response of Ephydatia muelleri to the addition of inedible ink
particles. The 'inflation-contraction' cycle (840-2240 s) lasts approximately 30 min,
successfully expelling unwanted ink. During this cycle back-flushing occurs through a
few open ostia in the apical pinacoderm (1260-2180 s). Note that during the 'inflationcontraction' cycle the osculum remains slightly open expelling inedible ink. At 6400 s, a
twitch occurs across the entire choanosome. Duration: 164.7 min; time interval: 20 s; and
frame rate: 10 fps.
Movie S3 (Figure 2-7 & 2-6): Sandwich preparation of Ephydatia muelleri showing the
response of the sponge to the addition of inedible ink. The 'inflation-contraction'
behaviour consists of three phases - inflation (590-1490 s), plateau (1500-1790 s) and
contraction (1800-3290 s). Cells crawling in the mesohyl arrest as the incurrent canals
87
contract during the inflation and plateau phases (600-1700 s). During the inflation phase,
back-flushing occurs through the choanocyte chambers (1200-1850 s) and water in the
incurrent canals stops flowing for 6 minutes (2390-3340 s) as indicated by the static ink
front. During the 'inflation phase', the canals contract from right to left along the canals
and from the bottom to top across canals; contractions run in the opposite directions
during the 'contraction phase'. Duration: 74.5 min; time interval: 10 s; and frame rate: 6
fps.
Movie S4 (Figure 2-8): Lateral view of the final contraction of the osculum in Ephydatia
muelleri in response to agitation (2-3 Hz) of the Petri dish (530-610 s). Trembling of
apical pinacoderm occurs in advance of a second 'inflation-contraction' cycle (16252995 s). The apical pinacoderm contracts initially (prior to the inflation phase); then it
begins to raise as the canals inflate. Duration: 49.9 min; time interval: 5 s; and frame
rate: 10 fps.
Movie S5 (Figure 2-9): Contraction of ostia in the apical pinacoderm of Ephydatia
muelleri in response to agitation (2-3 Hz) of the Petri dish. Arrows indicate position of
ostia. Ostia close in unison just before the choanosome (darker tissue on the right)
contracts. As the choanosome expands again the ostia re-open. Duration: 49.9 min; time
interval: 5 s; and frame rate: 6 fps.
Movie S6: Contraction of ostia in the apical pinacoderm after stimulation by Sumi Ink in
Ephydatia muelleri. Ink is taken into the choanosome stimulating a series of quick
contractions and a final massive contraction that expels ink in clumps over the sponge.
The ostia contract in unison twice, always just before the contraction of the choanosome
88
(the dark region located to the top left). Arrows indicate ostia that close in unison; a
small amount of ink squeezes back through 2 ostia that remain open near the choanosome
(between 5810-6190 s). Duration: 166.4 min; time interval: 20 s; and frame rate: 6 fps.
89
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Chapter 3 : Morphology of the juvenile sponge, Ephydatia muelleri: A
phylotypic body plan
3.1 Introduction
As one of the most ancient groups of multicellular animals, Porifera (sponges) hold
important clues to how multicellularity arose, yet very few studies on this group exist,
and our understanding of their body plan is rudimentary at best. In most animal phyla,
embryonic development of practical model species is compared to seek evidence for
ancestral traits. One of the main difficulties in interpreting sponge and cnidarian
ancestry, however, is that patterns of embryogenesis are highly varied (Gilbert and
Raunio, 1997; Byrum and Martindale, 2004) making it difficult to generalize about
relationships between groups. For many years Hydra was the principal cnidarian model
species studied, but most work focused on regeneration and formation of the adult body
plan since sexual development via embryogenesis is not common (Bode and Bode, 1984;
Martin et al., 1997; Steele, 2002). Recent use of the anthozoan Nematostella vectensis
has allowed study of cnidarian embryogenesis, and the resulting larva and juvenile have
been found to share many of the same characters as the adult Hydra, two layers, a gut and
oral-aboral polarity (Hand and Uhlinger, 1992; Fritzenwanker et al., 2007).
The same difficulty of finding practical model species has limited study of the
origin of the sponge body plan. Studies on larvae of the demosponge Amphimedon
queenslandica have quickly changed this idea (Leys and Degnan, 2001; 2002). The
genome project (www.jgi.doe.gov/sequencing/why/3161.html) shows that sponges share
a substantial molecular heritage with other animals (Larroux et al., 2007) and gene
A version of this chapter has been submitted to the Elliott GRD, and Leys SP. Journal of
Morphology.
98
expression patterns show signalling and polarity genes are present in the larva (Adamska
et al., 2007; Richards et al., 2007; Sakarya et al., 2007). Sponge larvae may therefore
share more characteristics with other metazoans than do adult sponges, and that the
ancestral metazoan likely resembled a sponge larva (Nielsen, 2008). Larvae have a
ciliated epithelium, a clear anterior-posterior polarity, tissue layers, cilia with crossstriated rootlets, form cell layers with polarized epithelia, a basement membrane, and
septate and desmosome-like junctions (Leys and Degnan, 2001; Leys et al., 2002; BouryEsnault et al., 2003; Maldonado, 2004; Degnan et al., 2005; Maldonado and Riesgo,
2008). However, the larvae are non-feeding, and metamorphosis into the young adult
involves a radical reorganization of cells to form a feeding epithelium (Leys and Degnan,
2002; Leys, 2004; Leys and Eerkes-Medrano, 2005; Eerkes-Medrano and Leys, 2006;
Leys and Ereskovsky, 2006). The juvenile and adult stages of sponges comprise most of
the life cycle of the sponge. In addition to possessing a polarized body, differentiated
epithelia and sensory cells, they show a far greater behavioural repertoire than do sponge
larvae (Elliott et al., 2004), especially of activities that are seen in eumetazoans including
slow but highly coordinated peristaltic-like contractions and rapid convulsions (Nickel,
2004; Leys and Meech, 2006; Elliott and Leys, 2007). Adult sponges have physiological
signalling systems that imply a substantial level of organization (Ellwanger and Nickel,
2006; Ellwanger et al., 2007).
Relationships among sponge groups are still controversial. Most molecular
analyses suggest sponges are paraphyletic (Kruse et al., 1998; Zrzavy et al., 1998;
Borchiellini et al., 2001; Medina et al., 2001; Peterson and Butterfield, 2005).
Hexactinellids (glass sponges) and demosponges form a clade of siliceous sponges
99
(Silicea) while the calcareous sponges and homoscleromorphs are more closely related to
other metazoans. Nevertheless, all of these hypotheses imply that a sponge-like animal
(with canal-like gut) must have given rise to all other metazoans. A better understanding
of the basic morphology of the adult sponge is direly needed.
The textbook 'classification' of sponges into asconoid, syconoid, and leuconoid
body types may have much value, but has unfortunately also generated much confusion.
This system describes the placement of the filtering units (choanocytes) - from lining a
simple tube, to lining outpockets of a tube, to forming alveolar-like buds from multiple
branches of a tube - so as to increase the cross-sectional area or filtering capacity (Vogel,
1977). Since only the Calcarea has all three forms, and this is likely one of the most
derived sponge groups (Sollas, 1888; Hyman, 1940; Eerkes-Medrano and Leys, 2006;
Ruppert et al., 2004), grades in filtering complexity were either lost in other sponge
groups, or came about much more recently during sponge evolution. At any rate, the
most 'complicated' branched morphology (leuconoid) is what needs to be understood as
it describes 95% of all sponge species. Admittedly, the external morphology of adult
sponges though often beautiful, can be extremely variable making it difficult to
generalize across species. The growth and form of the external framework of sponges is
directed by environmental forces such as water flow, food source or oxygen availability;
but even though they can be spherical, encrusting or branching the morphology of the
filtration system is essentially the same internally.
I examined the morphology of Ephydatia muelleri, a species belonging to the
genus that has been important in the study of sponge feeding, reproduction, cell biology,
and sponge behaviour. My goal is to provide a clearer understanding of the phylotypic
100
sponge body plan, tissue organization, and body polarity, building on the work of
numerous researchers (Kilian, 1952; Weissenfels, 1975; 1976; De Vos, 1977;
Weissenfels, 1978; De Vos, 1979; Kilian and Wintermann-Kilian, 1979; Weissenfels,
1980; 1981; 1982; 1983; Simpson, 1984; Weissenfels, 1984; Langenbruch et al., 1985;
Weissenfels, 1992). My interpretation is that juveniles are organized into three distinct
and readily identifiable regions: ectosome (apical pinacoderm), choanosome (choanocyte
chambers, mesohyl, aquiferous canals and basopinacoderm) and an osculum, which
interact with each other to coordinate contractile behaviour, a trait observed in cellular
sponges (Elliott and Leys, 2007). The results illustrate that sponges have epithelia
organized into a single functional sheet with an apical-basal polarity of pinacocytes and a
proximal-distal polarity centered on the position of an osculum. These traits allow the
use of Ephydatia muelleri as an effective model to understand physiological and
developmental signalling during embryogenesis, metamorphosis and re-aggregation as
the sponge body plan is formed.
3.2 Materials and methods
3.2.1 Sponge collecting and culturing
Gemmules of the freshwater sponge Ephydatia muelleri (Lieberkuhn, 1955) were scraped
from sunken trees or submerged rocks from Frederick Lake, B.C. (48° 47' 51.7559"; 125°
2' 58.5600") in winter months at a depth of 0-3 m and stored plastic bags with unfiltered
lake water at 4°C in the dark until use; bags were aerated monthly. Gemmules were
mechanically separated from their spicule skeletons by gently rubbing the 1-2 cm2
fragments between two sheets of wet corduroy without crushing the gemmules. Loose
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gemmules were separated from debris on ice, sterilized with 1% hydrogen peroxide
(H2O2) for 5 minutes, and rinsed with cold distilled water to remove excess H2O2.
Gemmules were transferred using sterile pipettes to Petri dishes containing
either Strekal's medium (0.9 mM MgS0 4 -7H 2 0, 0.5 mM CaC0 3 , 0.1 mM Na 2 Si0 3 -9H 2 0,
0.1 mM KC1) (Strekal and McDiffett, 1974) or M-medium (0.5 mM MgS0 4 -7H 2 0, ImM
CaCl2-2H20, 0.5 mM NaHC0 3 , 0.05 mM KCl, 0.25 mM Na 2 Si0 3 -9H 2 0) (Funayama et
al., 2005). Single gemmules were placed on an ethanol-washed, flamed glass 22 mm2
coverslip in Petri dishes and allowed to hatch undisturbed at room temperature (18-25°C)
in the dark. Gemmules hatched after 2-3 days of incubation at room temperature and the
culture media was changed every 48 hours until the juvenile stage was reached 7-10 days
post hatching (dph). Live juvenile sponges were viewed as whole-mounts on an
Olympus SZX 12 stereomicroscope and images were captured with a QiCam camera
using Northern Eclipse software (Empix Imaging Inc., Mississauga, CAN).
3.2.2 Fixation for scanning electron and light microscopy
Juvenile sponges (7-10 dph) were fixed by direct immersion of the coverslip with the
sponge into a cocktail consisting of 1% OSO4, 2% gluteraldehyde in a 0.45 M Sodium
Acetate buffer (pH 6.4) with 10% sucrose for 24 hr at 4°C (Harris and Shaw, 1984).
Several other fixatives and techniques were tested, including gradual addition of fixative
to the sponge in culture medium, changing fixative ratios, and use of pre-post fixation
protocols, but none provided better results. For scanning electron microscopy, whole
specimens were desilicified, dehydrated to 100% ethanol and fractured in liquid nitrogen.
Fractured pieces were critical point dried, mounted on aluminum stubs with silver paste
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or nail polish, gold coated and viewed in a JOEL field emission scanning electron
microscope (SEM).
For thick (1 (xm) sections, sponges were fixed as above, stained with 0.5%
uranyl acetate en bloc, and embedded in Epon (Taab 812). Thick sections were cut with
a Reichert Ultramicrotome and stained with Richardson's stain. Thick sections were
viewed with a Zeiss Axioskop microscope; images were captured with a QiCam camera
with Northern Eclipse software, and were manipulated with Adobe Photoshop CS2. All
measurements of structures were preformed by ImageJ (NIH, USA vl.37c) and Northern
Eclipse.
3.2.3 Fixation for fluorescence and confocal microscopy
Juvenile sponges on glass coverslips were placed directly into a mixture of 3.7%
paraformaldehyde and 0.3% gluteraldehyde in phosphate buffered saline (PBS; 100 mM)
for 24 hr at 4°C. Coverslips with sponges were rinsed and processed as described
previously (Elliott and Leys, 2007). The actin cytoskeleton was labeled with either
Bodipy 591 Phalloidin, Alexa 594 Phalloidin, or Bodipy 505 FL Phallacidin (Molecular
Probes-Invitrogen, USA) with 10% bovine serum albumin (BSA). The tubulin
cytoskeleton was labeled by incubation in a mouse anti-tubulin antibody at 1:100 dilution
(clone E7; from Developmental Studies Hybridoma Bank) in PBS with 0.1% Triton X100 (PBTX-100) and 10% goat serum for 24 hr or overnight at room temperature. The
next day preparations were washed 3 times in PBS and incubated for 5 hours at room
temperature in a secondary Alexa 488 goat anti-mouse antibody at a dilution of 1:100 in
PBS and 10% goat serum. Preparations were viewed with a Zeiss Axioskop
103
epifluorescent microscope or a Leica 2 photon confocal microscope and a maximum
projection image was compiled by 1 um image stacks.
3.2.4 Pharmacological
manipulations
To determine the role of actin in epithelial integrity, sponges were treated with
cytochalasin B to disrupt the actin filaments by inhibition of actin polymerization (CB,
Sigma) at a final concentration of 20 uM. Sponges were not disturbed for 1 hour, then
transferred to dishes with 20 uM CB for 30 min and fixed as above. Some sponges were
returned to normal growth medium following CB treatment and allowed to recover
overnight before fixation. Control sponges were fixed after treatment with 0.2% DMSO
and after a 24 hour recovery period. All sponges were processed for actin and viewed as
described above.
3.3 Results
Live adult sponges of Ephydatia muelleri were green, greenish-yellow or grey (due to the
presence of symbiotic algae), with a flat or encrusting form, and an irregular surface
devoid of tubes or projections. The sponges were found predominately on or inside
submerged woody debris and on the underside of rocks that were partially or fully
shaded. The sponge formed disks up to 10 cm in diameter or narrow long patches, never
more than 0.5 cm thick (Figure 3-1A). The apical pinacoderm was usually aspiculous,
but could have a hispid surface from spicules emerging from choanosome (Figure 3-1 A).
The skeleton of the adult sponges was organized in a reticulate, anisotropic pattern;
104
Figure 3-1. General morphology of the adult and gemmulated sponges by light (LM)
and scanning electron (SEM) microscopy. A) LM image of an adult sponge on a
submerged log. Scale bar, 5 cm. B) LM image of the spicule skeleton with embedded
gemmules before mechanical dissociation. White arrows indicate gemmule and black
arrows indicate skeleton. Scale bar, 1 mm. C) SEM image of the smooth (sm) and spiny
(sp) amphioxea of the adult skeleton that can be bent in the middle 164°-180° (inset).
Scale bar, 50 urn; inset 20 um. D) LM image of gemmules that have been mechanically
dissociated from the dead adult skeleton (micropyles, m). Scale bar, 1 mm.
105
Figure 3-1.
106
paucispicular primary tracts were connected to monospicular secondary tracts by spongin
(Figure 3-IB) (Ricciardi and Reiswig, 1993; Boury-Esnault and Rutzler, 1997; Hooper
and Van Soest, 2002). Spicule tracts were formed of amphioxea megascleres.
Megascleres (length 195.1 - (244.3) - 291.4 um, width 7.4- (14.2) - 18.8 urn; n=10) were
either stout or slender and were usually covered with short conical spines everywhere
except the tips (Figure 3-1C). Megascleres could also be smooth, with a slight bend in
the middle of the shaft of 164-180° (Figure 3-1C inset); both spiny and smooth
megascleres can also exist in the same specimen (Figure 3-1C). No microscleres were
present in the adult skeleton.
Like other freshwater sponges, Ephydatia muelleri was ephemeral. Animals
hatched in the spring from over-wintering cysts called gemmules, grew in the summer to
maximum of 10 cm diameter, reproduced sexually, and then in the autumn the tissue
regressed into cysts that lasted over the winter until they hatched next spring. (Figure 31D & 3-2A). Gemmules were abundant from November to December as yellow or
yellow-green, oblong cysts (length 327.9-(560.9)-833.0 um; width 299.4-(503.2)-707.1
um; n=20), scattered throughout the scaffold of the adult skeleton (Figure 3-1B).
3.3.1 Morphology of the gemmule
The gemmule coat or husk, called a theca, was a tri-layered structure with a small
opening called the micropyle or foramen at one side (Figures 3-1D & 3-2A,B,D). The
foramen (38.2±2.3 urn diameter; 80.0±0.6 um outer foramen diameter) was simple, flat
without a ridged collar (Figure 3-2A-D). The gemmular theca consisted of three layers of
spongin: outer, pneumatic, and inner layers (Figure 3-2E,F). The outer layer was well
107
Figure 3-2. The morphology of the gemmule viewed by scanning electron microscopy.
A) Whole-mount of a gemmule husk showing the arrangement of spicules and location
of micropyle (m). Scale bar, 200 urn B) Open micropyle (m) of a hatched gemmule
showing the smooth collar (c). Scale bar, 25 um. C) High magnification of the
orientation of the gemmuloscleres projecting from the pneumatic layer of the gemmule.
Scale bar, 25 um. D) A closed micropyle (m) and collar (c) on a gemmule prior to
hatching. Scale bar, 25 um. E) Cross-section through the gemmule theca showing the
smooth inner layer made of compact spongin. Scale bar, 200 um. F) High
magnification of the gemmule theca showing the inner, pneumatic and outer layers.
White arrow indicates bacteria on the outer layer of the gemmule. Black arrows indicate
the location of the gemmuloscleres. Scale bar, 3 um. G and H) Two forms of the
gemmuloscleres either spiny (G) or birotulated (H and inset). Scale bar, 5 um. I)
Pockets of bacteria (white arrow) on the outer layer of the gemmule that were covered by
exopinacocytes (exp). Black arrows indicate the location of the gemmuloscleres. Scale
bar, 5 um. J) The gemmule covered by thesocytes, the first cells to emerge out of the
micropyle at the time of hatching. Scale bar, 200 um.
108
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developed and covered all but the distal portions of gemmuloscleres (Figure 3-2C). The
middle (pneumatic) layer ranged in thickness and had irregular chambers (Figure 3-2F).
The spicules embedded in the theca, called gemmuloscleres, serve as the diagnostic
character for species identification for freshwater sponges (Figure 3-2G,H). As described
by Riccardi and Reiswig (1993), gemmuloscleres were flat birotulates, umbonate, deeply
and irregularly incised, with 12 or fewer rays (Figure 3-2G,H). The shaft was typically
smooth and was shorter than the rotule diameter (gemmulosclere length 15-(16.3)-18.4
um, shaft width 4.1-(4.3)-4.4 urn, rotule diameter 23.6-(18.7)-14.8 u.m; n=4).
Gemmuloscleres were radially embedded in the middle layer with the proximal rotule in
contact with the inner layer and distal portion exposed (Figure 3-2C). The inner layer
was composed of a layer compact spongin (Figure 3-2E,F). Bacteria coated the outside,
and were later covered by the exopinacoderm of the developing sponge (Figure 3-21). At
hatching, thesocytes (amoebocytes rich in vitellogenin - yolk platelets) had crawled out
of the micropyle (Figure 3-2 J) and the juvenile developed within 4-7 days post hatching
(dph).
3.3.2 General anatomy of the juvenile sponge
The juvenile sponge body was organized into three functional regions - the ectosome
consisting of the apical pinacoderm and subdermal cavity; the choanosome, consisting of
the aquiferous system including choanocyte chambers, mesohyl and basal pinacoderm;
and the osculum - all of which were centered around an empty gemmule husk (Figure 33A-E). The tent-like apical pinacoderm draped over a choanosome and spicule skeleton
with a protruded osculum to the distal end of the animal (Figure 3-3 A-E). Within the
110
Figure 3-3. General anatomy of the freshwater juvenile sponge by light (LM) and
scanning electron (SEM) microscopy. A) Side view LM image of a sponge showing the
osculum (osc), apical pinacoderm (apd), gemmule (g) and choanosome (cho). Scale bar,
1 mm. B) Top view LM image of a sponge showing the location of the incurrent canals
(in), horizontal branching excurrent canals (ex), osculum (osc) and gemmule (g). The
excurrent canals consisted of peripheral canals (p), two main canals (m) which converge
on a central canal (c) at the base of the osculum. Scale bar, 1 mm. C) SEM image of a
sponge showing the apical pinacoderm (apd) supported by spicule tracts (sp), osculum
(inset; osc), locations of the ostial fields (os), and the basopinacoderm (bpd). Scale bar, 1
mm; inset 0.5 mm. D) Merged SEM image of a fracture through the sponge showing
locations of the incurrent and excurrent canals. Two paired central excurrent canals (exc)
were flanked by the main excurrent canals (exm) and peripheral excurrent canals (exp).
The apical pinacoderm (apd) was supported by vertical tracts of spicules (sp) rising from
the choanosome (cho) and the basal pinacoderm anchors the sponge to the substrate.
Water enters the sponge through ostia (os) into the subdermal cavity (sdc), and from there
through prosopyles (pp) to choanocyte chambers (cc). Water exits via the apopyles (ap)
into excurrent canals. Scale bar, 50 um. E) General diagram depicting the major
structures and direction of water flow within a juvenile sponge.
Ill
Figure 3-3.
112
choanosome, the most striking feature was the branching nature of the aquiferous canal
system that looked reminiscent of the branching patterning of bronchia, trachea in lungs
of mammals, and the choanocyte chambers appeared as alveoli budding off the excurrent
canals (Figure 3-3B,D). Water entered the sponge through the ostia in the apical
pinacoderm (Figure 3-3C) and entered the choanosome via prosopyles that led to
incurrent canals and choanocyte chambers (Figure 3-3D,E). Filtered water flowed from
the choanocyte chambers via apopyles into the excurrent canals and vented out of the
osculum (Figure 3-3D,E).
3.3.3 Subdermal cavity and choanosome
The choanosome of the juvenile sponge contains the aquiferous system with incurrent
canals, choanocyte chambers and excurrent canals. The most obvious feature of the
aquiferous system was a pair of large horizontal excurrent canals (Figure 3-3B,D). The
form of the excurrent canals dictates the overall body plan of the sponge. Two distinct
forms developed: in most (-70% of sponges), the osculum arose beside the gemmule
husk and the two main canals branched around it. In the remaining sponges, the osculum
arose from the top of the gemmule and many smaller branches converged vertically on it
from all sides. The largest central canals at the base of the osculum had a mean diameter
of 217.7±14.9 um, (n=10); canals in the middle of the sponge had a mean diameter of
107.3±3.7 (im, (n=10); and those at the periphery arising from choanocyte chambers had
a mean diameter of 40.1±1.3 urn, (n=10) (Figure 3-3B,D). A cross section of the sponge
shows the relationship of the ectosome to the choanosome and classification of the canals
(Figure 3-3D).
113
Figure 3-4. Description of the choanosome - subdermal cavity, aquiferous canal system,
and mesohyl - viewed by thick section (TS) and scanning electron microscopy (SEM).
A) TS image through the apical pinacoderm (apd), choanosome and basopinacoderm
(bsp). The apd had 3 layers with crawling cells in the mesohyl. The choanosome was
covered by a continuous layer of endopinacocytes (enp) and canals (incurrent - in and
excurrent - ex) were lined with a continuous layer of endopinacocytes. Scale bar, 5 um.
B) SEM image of a fracture through the choanosome showing the placement of the
choanocyte chambers (cc) in relation to the excurrent canal (ex) and incurrent canals (in)
and showing the position of the prosopyles (pp) and apopyles (ap). Scale bar, 30 urn. C)
SEM image of the endopinacoderm (enp) layer of the choanosome (incurrent epithelium)
showing primary and secondary sieve cells (sc and sdc) and entrances to the incurrent
canals (in). Scale bar, 50 um. D) SEM image of a primary sieve cell (sc) that surrounds
the back of the choanocyte chamber (cc) full of choanocytes (ch). Scale bar, 70 um. E)
SEM image of a secondary sieve cell (sdc) that leads into the incurrent canals surrounded
by endopinacocytes (enp). Scale bar, 10 um. F) SEM stereo pair image of secondary
sieve cells (sdc in previous part E) to show the reticular nature of this cell type.
114
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115
The choanosome was lined by two pinacoderm epithelia: an outer layer of cells
exposed to the subdermal cavity and an inner layer lining incurrent and excurrent canals
(Figure 3-4A,B). The surface of the choanosome was formed by a single layer of flat
cells ('endopinacocytes') 3-5 of which surrounded openings to choanocyte chambers or
the entrances to incurrent canals (Figure 3-4B-C). Water entered the canals and
choanocyte chambers through prosopyles: these were unusual flat cells with many
perforations (like swiss cheese) termed secondary sieve cells (Figure 3-4C,E,F). A
similar reticulate cell type formed the immediate opening to choanocyte chambers,
surrounding and supporting choanocytes, termed a primary sieve cell (Figure 3-4C,D).
There was substantial depth of these reticulate cells that extend down from the surface
into the incurrent canals seen in the stereo pair image (Figure 3-4F).
Choanocyte chambers opened directly into the excurrent canal, a form known as
eurypylous (Figure 3-5A). Chambers were 22.1±1.3 um (n=13) in diameter with roughly
70-100 choanocytes each. Choanocytes were squat cells 5.1±0.1 um wide (n= 12) and
only 2.5±0.2 um (n=7) tall (Figure 3-5A-E). Many choanocytes had thin filopodial
connections that connected neighboring cells and in cases stretched across the prosopyle,
thereby creating a choanocytic prosopyle (Figure 3-5C). A collar of microvilli (4.6±0.2
um long, n= 15), which labelled brightly with phalloidin, surrounded a long flagellum
(9.3±0.2 um), which labeled brightly with anti-tubulin antibodies (Figure 3-5B,D,E).
The collar microvilli varied in length, number and even shape. Microvilli were 87.8±2.3
nm in width and were spaced 100±30 nm apart at the base of collar; adjacent microvilli
were connected by fine threads of a glycocalyx mesh (Figure 3-5B).
Choanocyte chambers vented into peripheral excurrent canals via the apopyle, a
116
Figure 3-5. The choanocyte chamber and apopyle viewed by scanning electron (SEM)
and epifluorescent (EP) microscopy. A) SEM image of the choanocyte chamber (cc)
with choanocytes (ch) showing the location of the incurrent canals (in), prosopyle (pp),
apopyle (ap), excurrent canal (ex), amoeboid cells (amb) and support cells (sup) of the
mesohyl. Scale bar, 10 um. B) High magnification SEM image of the collar microvilli
(mv); arrow shows the fine glycocalyx mesh between adjacent microvilli. Scale bar, 1
um. C) SEM image of the choanocytic prosopyles (cpp) created by extensions of
choanocyte cell bodies (arrows). Scale bar, 1 um. D) EP image of the tubulin
cytoskeleton of the choanocytes showing the flagellum (fl) and tubulin cytoskeleton in
the cell bodies (cb). Scale bar, 50 um. E) EP image of a phalloidin label of the actin
cytoskeleton of choanocytes showing the microvilli of collars (mv) and actin
cytoskeleton in the cell bodies (cb). Scale bar, 50 um. F) SEM image of apopyle (ap)
fields of the excurrent canals. Scale bar, 30 um G) SEM image of an apopyle torn from
the endopinacoderm epithelium shows it was composed of two tightly adherent cells
whose smooth edge connected to endopinacocytes and ruffled edge projected into the
middle of the chamber to form a gasket-like structure. Scale bar, 5 um. H) High
magnification SEM image of the apopyle cells (ape) showing a central cilium (ci), free
edge (fr) and the connection between the two apopylar cells (arrows). Scale bar, 5 um.
Inset: close-up SEM image of the cilium of the apopylar cell. Scale bar, 1 um.
117
Figure 3-5.
118
Figure 3-6. Ciliated cells in the excurrent canal system viewed by scanning electron
microscopy (SEM). A) SEM image of a fracture through a main canal showing an
endopinacocyte with a pair of cilia (c; inset). Scale bar, 40 um; inset 1, 2 um; and inset
2, 1 um B) SEM image of a fracture through a peripheral canal showing two
endopinacocytes each with a pair of cilia (c; inset). Scale bar, 10 um; and inset 1 um.
119
15 urn diameter opening formed by 2-4 cells that were tightly joined to create a conical
gasket-like structure (Figure 3-5F,G). Each apopyle cell had a single cilium 4.3±0.1 um
long (n=4) (Figure 3-5G,H inset). Observations on live specimens indicated that these
cilia beat continuously with a slow whip and recovery stroke, unlike the flagella of the
choanocyte chambers which beat in sinusoidal waves (data not shown).
The excurrent canals were lined by a layer of tightly juxtaposed pentagonal cells
(endopinacocytes) 27.2±1.9um (n=16) in diameter (Figure 3-6A). In the peripheral
canals a single cell reached around the entire circumference of the canal; whereas, the
large central canals were formed by up to 15 cells. Interestingly, some pinacocytes in the
peripheral excurrent canal system had a single pair of 4 um-long cilia (Figure 3-6A,B).
3.3.4 Apical pinacoderm
The outer surfaces of the sponge had a continuous epithelium formed by the apical and
basal pinacoderms; the former is presumed to function as a defensive barrier and the
latter for attachment to a substrate. The apical pinacoderm, albeit slim, was made of
three layers - two pinacocyte layers and a middle layer of a collagenous extracellular
matrix with amoebocytes (Figure 3-7A-D). Exopinacocytes (barrier cells) were
pentagonal cells 43.7±1.4 \am in diameter (n=20), and only 1.5 um thick, except where
the nucleus was located (Figure 3-7A,D). Endopinacocytes, cells forming the underside
of the apical pinacoderm were long, thin cells (42.4±2.6 |im long, 12.1±0.5 urn wide,
n=18) (Figure 3-7B,D). A sheet of collagen layer over the endopinacocytes separating
them from amoeboid cells crawling in the mesohyl, and formed an anchoring substrate
for both endopinacocytes and porocytes (Figure 3-7C,D).
121
Figure 3-7. The apical pinacoderm viewed by light (LM), scanning electron (SEM) and
epifluorescent (EP) microscopy. A) SEM image of the exopinacoderm - outer layer - of
the apical pinacoderm showing the pentagonal exopinacocytes (exp) with arrows
indicating the corresponding layer in part (D). Scale bar, 30 urn. B) SEM image of the
endopinacoderm - inner layer- of the apical pinacoderm composed of holly-leaf shaped
endopinacocytes (enp) and corresponding layer in part (D). Scale bar, 30 um. C) SEM
image of a sheet of collagenous extracellular matrix in the apical pinacoderm and
corresponding layer in part (D). Scale bar, 1 um. D) LM image of a thick section
through the apical pinacoderm showing the exopinacoderm (exp), endopinacoderm (enp)
and amoeboid cells (amb) of the thin mesohyl. Scale bar, 10 um. E) EP image of a
whole sponge showing the distribution of actin filaments over the entire apical
pinacoderm and location of spicule tracts (sp). Scale bar, 1 mm. Inset: High
magnification EP image of the actin filaments converging on the top of the spicule tract
"tent pole". Scale bar, 5 um F) SEM image showing the top of a spicule tract that
supports the apical pinacoderm. Scale bar, 10 um. G) EP image of the multiple actin
tracts and the adhesion plaques (arrows) in the endopinacocytes of the apical pinacoderm.
Scale bar, 10 um.
122
Figure 3-7.
123
Figure 3-8. Actin cytoskeleton labeled with 548 Bodipy phallacidin after disassembly by
Cytochalasin B (CB) and subsequent recovery viewed by epifluorescence microscopy.
A) Whole-mount of sponge treated with CB for one hour showing the absence of long
actin tracts across the apical pinacoderm. Scale bar, 1 mm. B) High magnification of
endo- and exopinacocytes shows actin only at the periphery of cells. Scale bar, 30 urn.
C) After recovery, actin tracts appear again. Scale bar, 30 um. D) Control preparation
treated with DMSO shows long tracts of actin across the apical pinacoderm. Scale bar,
50 um.
124
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Phalloidin labelling of the actin cytoskeleton revealed dense tracts of actin in
endopinacocytes of the apical pinacoderm (Figure 3-7E). In the apical pinacoderm, 2-3
bundles of actin fibers traversed individual endopinacocytes (Figure 3-7G). Contacts
between neighboring cells labelled brightly, like adhesion plaques (Figure 3-7G) and
tracts in adjacent cells lay in the same direction, carrying on from cell to cell so as to
form semi-continuous tracts 1 -3 mm across the apical pinacoderm running both parallel
to the perimeter of the sponge and from the edge of the sponge to the top of the gemmule
(Figure 3-7E, inset). These actin tracts converged at the top projections caused by spicule
tracts (Figure 3-7E inset,F). Cytochalasin B (CB) treated sponges lacked the dense actin
tracts in the apical pinacoderm (Figure 3-8A). Actin labeled only at the cell membrane
(Figure 3-8B). After CB was washed out, sponges recovered actin tracts in the apical
pinacoderm (Figure 3-8C). Actin was more disorganized than in control sponges because
the epithelium had remodeled itself during the recovery period (Figure 3-8C,D).
3.3.5 Porocytes
Porocytes were single cells with a central hole, the ostium, which allowed water to enter
the sponge via the subdermal cavity. Fields of ostia covered the apical pinacoderm
(Figure 3-9 A) and ostia varied in diameter depending on the state of contraction of the
whole sponge. Porocytes were 32.6±1.4 um across, and the ostium when fully open was
30.6±1.1 \im wide (n=33) (Figure 3-9B-D). Porocytes were anchored to the extracellular
matrix by minute filopodia (Figure 3-9D; inset). In some cases a single porocyte formed
multiple ostia (Figure 3-9E). It was unclear whether the pores fused when completely
closed or disappeared under the pinacocyte; no actin ring could be found in contracted
126
Figure 3-9. Porocytes in the apical pinacoderm seen by scanning electron (SEM),
epifluorescent (EP) and light microscopy (LM). A) SEM image of the apical pinacoderm
(apd) showing the porocytes with open ostia (os) around a spicule tract (sp). Scale bar,
50 um. B) High magnification SEM image of a porocyte (p) surrounded by five
exopinacocytes (exp). Scale bar, 5 um. C) LM image of a porocyte (p) showing the
ostium (os) and amoebocytes (amb) crawling in the mesohyl of the apical pinacoderm.
Scale bar, 20 um. D) High magnification SEM image of a porocyte from which
exopinacocytes have torn away revealing the role of filopodia (inset) of the porocyte in
hooking on to the collagen sheet (co). Scale bar, 10 um. E) SEM image of a single
porocyte (p) with three ostia (os). Scale bar, 20 um. F) EP image of the actin
cytoskeleton of porocytes showing the ring of actin that surrounds the ostium (os). Scale
bar, 50 um.
127
128
Figure 3-10. The basal pinacoderm viewed by scanning electron (SEM) and
epifluorescent (EP) microscopy. A) SEM image of the substrate attachment surface of
the basal pinacoderm. Individual basopinacocytes (bsp) were outlined by a dashed line.
Arrows indicate crystals associated with the bsp after critical point drying. Scale bar, 100
um. B) SEM image of a fracture across the basal pinacoderm showing amoebocytes
(amb) just above the basal pinacocytes (bsp). Scale bar, 50 um. C) EP image of the actin
cytoskeleton of the basopinacocytes showing the typical distribution of actin around the
periphery of cells. Scale bar, 50 um. D) EP image of the tubulin cytoskeleton of the
basopinacocytes (outlined by a dashed line). Scale bar, 50 um.
129
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porocytes (Figure 3-9F).
The basal epithelium was formed by a single layer of pentagonal cells
(basopinacocytes) 57.6±1.7 um (n=59) in diameter (Figure 3-10A). In processing the
tissue for SEM, crystals of potassium and aluminum were always associated with the
basal pinacoderm and may be involved in the chemical cohesion to the substrate (Figure.
3-10A). Directly above the basopinacocytes there was a layer of amoebocytes with
elongate shape and a uniform orientation suggesting they were actively crawling prior to
fixation (Figure 3-10B). The actin cytoskeleton outlined the perimeter of
basopinacocytes; tubulin formed a network radiating from the nucleus of each cell to its
outer edge (Figure 3-10C,D).
3.3.6 Osculum
The osculum in relaxed state was 1-1.5 mm long and 1-1.2 mm wide (Figure 3-11A), but
much smaller when fully contracted (Figure 3-1 IB). The inner and outer epithelia were
formed by a continuation of exopinacocytes of the canals and apical pinacoderm. Cells
lining the inside of the osculum were rectangular, and each cell possessed a pair of cilia
that were 4 um long, 1 urn apart (Figure 3-11C, inset). The actin cytoskeleton labelled
the perimeter of the cells lining the outside of the osculum. (Figure 3-1 ID). No
sphincters were visible in central canals that gave rise to the osculum (Figure 3-1 IE).
131
Figure 3-11. The osculum viewed by scanning electron (SEM) and confocal (CF)
microscopy. A) SEM image of an osculum in relaxed and (B) contracted states. Scale bar
A, 200 urn; and B, 100 um. C) SEM image of a fracture through the osculum showing
the endopinacoderm (enp), exopinacoderm (exp) and the collagenous extracellular matrix
(co). On the inside of the osculum endopinacocytes (enp) each had a pair of cilia. Scale
bar, 10 um Inset: high magnification SEM image of the pair of cilia. Scale bar, 1 um.
D) CF maximum projection image of the actin cytoskeleton of the osculum showing actin
at the periphery of cells but no tracts across cells. Scale bar, 50 um. E) SEM image of a
view of a specimen in which the osculum has been removed shows there are no special
sphincters at the base of the osculum (area indicated by the dashed line). Scale bar, 250
um.
132
Figure 3-11.
133
3.4 Discussion
Here I present a detailed description of the anatomy of a freshwater sponge. Although
others have tried to pull together a succinct description of sponge anatomy, few previous
studies were able to find and describe similarities between sponge morphology and that
of other metazoans - because sponges appear to have a structure and function that is
unique among all other animals. I found that this not to be the case. I took the approach
of Pavans de Ceccatty (1974) and Weissenfels (1984), in trying to interpret the structure
of a sponge in terms of its behavioural function. I found that Ephydatia muelleri, like
many sponges, can be understood to have three principal anatomical regions - the outer
surface or skin, consisting of the apical and basal pinacoderms, the inner feeding tissue or
choanosome consisting of aquiferous canals, mesohyl, and choanocyte chambers, and the
vent or osculum. Tissue layers of the sponge are formed by monolayers of cells and
show differentiation depending on function. Such regional differentiation allows
coordination of contractile events by the sponge - much as contractions run down the
length of a hydroid stolon to move fluid through the gastrovascular cavity - and implies a
substantial level of complexity in what is otherwise thought to be a 'simple' animal.
3.4.1 The sponge body plan
One difficulty in describing sponge anatomy is the problem of terminology; because of
the apparent uniqueness of these animals, cells and regions have names exclusive to the
phylum even though similar cells and tissues are found in other animals. The outer
epithelial cells are called pinacocytes because of their 'tablet' or pavement appearance.
134
Fish gill epithelia are also lined by 'pavement' cells - and the function of ion control by
these cells is likely reflected in the similar morphology. While appropriate terminology
is important to be able to adequately describe features of an animal, it is also important to
seek similarities and provide comparisons for appropriate translation wherever possible.
A good example of terminological confusion is the classification of sponges into
asconoid, syconoid, and leuconoid organization of their aquiferous canal system. This
classification refers to the increasing cross-sectional area of the sponge filtering units
(chambers), but is only found in calcareous sponges that have all three forms, which
comprise less than 5% of the phylum. These grades are thought to reflect evolutionary
transitions in elaboration within the Calcarea (Borojevic et al., 2002). This kind of
elaboration of filtering capacity may be thought of as equivalent to the transition of the
sac-like frog lung, to the highly branched mammalian lung, as the need for efficient
exchange increases. It would probably be more useful however, to compare the highly
branched and presumably more efficient leuconoid sponge structure with the flowthrough lung of a bird. Another set of potentially confusing terminology surrounds the
structure of choanocyte chambers. Chambers are usually classified as eurypylous,
aphodal or diplodal depending on the proximity of the entrances (prosopyles) and exits
(apopyles) to the canals (Sollas, 1888; Hyman, 1940). It has been suggested that
chamber types vary depending on the development of the mesohyl and food capturing
ability of the chamber, however the functionality of these different chambers has not been
tested.
Another difficulty in understanding sponge morphology arises in the apparent
lack of comparable anatomical regions, e.g., polarity (anterior-posterior, dorsal ventral),
135
gut, skin, skeleton and even epithelia, with other animals. Polarity is a functional
property of all tissues, and allows the animal to maintain outer defensive and inner
metabolic surfaces. Clearly sponges cannot function if their canals and chambers are not
polarized with respect to the direction of water flow. I find that the best way to
understand the sponge body plan is to characterize it by functional body regions - the
ectosome, comprised of the apical pinacoderm and the subdermal cavity, which forms the
outer barrier to the elements, and permits water in through specially designed openings;
the choanosome, an area where choanocyte chambers are located and food extraction and
waste excretion largely occurs; and the osculum which ultimately controls water flow out
of the sponge.
3.4.2 Epithelia
Epithelia are functional tissues sealing the internal environment, and though function has
not been tested in any sponge, by measurements of transepithelial resistance, many
authors claim sponges lack epithelia. Here again, a suite of ultrastructural characteristics
known from animals with a tough integument is used as the benchmark (Tyler, 2003). As
I present here, the apical pinacoderm of Ephydatia muelleri is a three layered tissue that
extends over a subdermal cavity that behaves as a single unit. The outer layer is
composed of exopinacocytes whose margins are adherent to one another, and the inner
layer is composed of contractile endopinacocytes whose cytoskeleton forms continuing
tracts from one cell to the next, suggesting contractile function across the entire sheet.
Between the two epithelia is a collagenous extracellular matrix that resembles the lamina
reticularis zone of the vertebrate epithelial basal lamina (Harrison et al., 1985).
136
Amoebocytes crawl along the upper surface of the collagenous matrix in both Ephydatia
muelleri (Elliott, personal observation) and Eunapius fragilis (Harrison et al., 1985). The
three layered morphology of the apical pinacoderm is also found in many freshwater
(Fjerdingstad, 1961; De Vos, 1977; 1979; Weissenfels, 1980; Harrison et al., 1985) and
marine (Langenbruch and Scalera-Liaci, 1986) sponges indicating that this is a common
body part of a sponge. The position of porocytes in the apical pinacoderm is also similar
in other sponges. Porocytes straddle the extracellular matrix and make contact with both
exo- and endopinacocytes. Details as to how the ostia close however are still vague and
require closer study.
3.4.3 Choanosome -flow control and feeding
The bulk of any sponge is the choanosome, or feeding chambers. The overall structure of
the choanosome depends on the function and life-history of a particular sponge. Thick
(massive) sponges tend to have a thick mesohyl of collagen, skeleton, and cells, in which
case the sponge is less compressible and contractile units are concentrated into sphincters
that line canals. Slim sponges (encrusting or branching) tend to have a thinner mesohyl,
and the contractile units line the canals as pinacocytes (Pavans de Ceccatty, 1974). In
most cases, contractile cells tend to be epithelial (e.g., Nickel, 2004). Fewer instances of
contractile mesohyl cells are known, but it is also more difficult to observe and record
contraction of cells in the sponge mesohyl. Further investigation on the ultrastructure and
protein complement of the contractile pinacocytes is needed to determine if they are in
fact similar to primitive smooth muscle cells with 2 types of contractile fibrils, motor
proteins and adhesion plaques composed of a-actinin and basal bodies.
137
Sponges may also be able to control flow by cells forming the entrances and
exits of chambers. In Ephydatia muelleri two types of cells permit water to enter
chambers. Those that lead to incurrent canals are highly reticulate, in essence a flat cell
with holes throughout that has been drawn out vertically to fill a cylindrical entrance to
an incurrent canal. These large incurrent canals can be seen at the light microscope level,
and close simultaneously in response to different stimuli (Elliott, personal observation).
Presumably this cell closes the entrance to the canal. The other 'entrance' cell
(prosopyle), covers the back of choanocyte chambers, and is reticulate but flat. The
distinctions between the two types of prosopyles have not yet reported in any sponge.
In Ephydatia muelleri the exit of chambers (apopyle) is a cone-shaped structure
composed of 2-4 cells each with a single cilium. A similar structure is known from other
haplosclerids (Weissenfels, 1980; Langenbruch, 1983; Langenbruch and Scalera-Liaci,
1986) and a variety of homoscleromorph species (Boury-Esnault et al., 1984; De Vos et
al., 1985). Weissenfels (1980) suggested that the cone cells were modified choanocytes,
but my observations suggest that the appendage beats like a cilium, not a flagellum. The
function of the cone cell ring is uncertain, but Langenbruch et al. (1985) suggested it
could have a mechanical role in stabilizing the chamber, or that it is involved in
regulation of water flow out of the choanocyte chamber. Given the whip-like beat of the
cilia on each apopyle cell away from the chamber, I have proposed that this ring of
apopyle cells functions as a gasket between the chamber and excurrent canal preventing
uptake of expelled material from contracted chambers during an inflation-contraction
cycle (Elliott and Leys, 2007).
138
3.4.4 Osenium
The osculum of Ephydatia muelleh is the most responsive part of the sponge with the
ability to rapidly contract, and extend by relaxation. Although the osculum is seamlessly
attached to the apical pinacoderm and excurrent canal system, it can behave in isolation
of the contracting apical pinacoderm and excurrent canals. This implies a level of
regionalization for the precise control of contractions without specialized cells - nerves.
In particular the finding that every cell lining the osculum has a pair of short cilia
suggests that - presuming these are sensory cilia -information regarding the precise flow
moving through the osculum can fed back to epithelia elsewhere in the sponge. My
continued work aims to determine how the osculum is controlled physiologically.
3.5 Conclusion
Juveniles of Ephydatia muelleri exhibit a stereotypical body plan that can be considered
as phylotypic of demosponges. The sponge is composed of definable body regions ectosome, choanosome, and osculum - that behave in coordination with each other.
Sponges have a continuous tissue layer or epithelium covering the entire aquiferous canal
system that provides a separation of the internal mesohyl from the environment and a
possible conduit for a communication system to coordinate contractions. The finding that
sponges have functional epithelia, differentiated tissues including regions with sensory
cells, and complex cellular structures that control flow through the animal for feeding and
respiration, allows the formulation of new hypotheses regarding the evolution of similar
traits in other phyla. The demonstrated practicality of studying these features in the
freshwater sponge suggests that this animal is an excellent model system for future study
139
of the physiology and development of sponges in light of new genomic information.
140
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Chapter 4 : Evidence for Glutamate, GABA and NO in coordinating
behaviour in the sponge, Ephydatia muelleri (Demospongiae,
Spongillidae)
4.1 Introduction
Sponges are benthic filter feeders that are often described as lacking a tissue level of
organization, sensory cells or coordinated behavior - features that are characteristic of
eumetazoans. The absence of these features is usually attributed to the early branching of
sponges from other metazoans. Intriguingly, recent molecular and physiological studies
generated by the new genome project on the encrusting demosponge Amphimedon
queenslandica suggest that Porifera possess many of the molecules that are involved in
cell signalling systems in higher metazoans (for review see Adamska et al., 2007;
Richards et al., 2007; Sakarya et al., 2007). The genomic findings force us to ask
whether these components reflect a primitive pre-nervous system or whether sponges
have a signalling and coordination system similar to that of other animals, but without the
use of conventional nerves and muscle.
While the absence of nerves and of rapid behaviour in sponges has intrigued
researchers for over a century, at the same time it has posed difficulties for testing
hypotheses using conventional techniques that require observation of responses to
stimuli. The general activities of cellular sponges include slow contractions across part
and occasionally all of the body, closure of the osculum (the most distinct morphological
region for observation) and slowing or cessation of pumping (filtering). Three
mechanisms of signalling have been proposed to explain these behaviours: electrical,
A version of this chapter is planned to be submitted to Elliott, GRD and Leys SP. Evidence
for Glutamate, GABA and NO in coordinating behaviour in the sponge, Ephydatia muelleri
(Demospongiae, Spongillidae). Journal of Comparative Physiology Part A.
i AQ
mechanical, or chemical (Jones, 1962). Electrical signalling is only known from glass
sponges, presumably because they are syncytial, which means that action potentials can
move throughout the animal unimpeded by cell boundaries (Leys and Mackie, 1997;
Leys and Meech, 2006). Mechanical signalling, through tugging on each cell by
neighbor cells, (Emson, 1966) and chemical signalling, through diffusible molecules
either in the aquiferous canal system or through paracrine signaling in the mesohyl, have
been investigated extensively in cellular sponges with few concrete results except to
show that sponges respond by slow contraction to touch, electric shock, temperature
change, sediment and chemical agents (reviewed in Leys and Meech, 2006). The
possibility that contractions propagate by mechanical tugging of cell on cell is difficult to
test because severing any portion of the aquiferous system deflates the canals and leads to
fairly rapid remodeling of the tissues and reorganization of the canals. Depolymerization
of the actin by cytochalasin B causes cells to lose close contact, but contractions are
eliminated entirely, so their use of cell contacts to propagate contractions cannot be
determined (Chapter 3). Two observations strongly suggest that paracrine chemical
signaling is the most likely mechanism of signal transduction. First, cells in the mesohyl
stop crawling as contractions pass over a region (de Vos and Van de Vyver, 1981; Elliott
and Leys, 2003; Elliott, 2004; Elliott and Leys, 2007), and the choreography of the entire
inflation and contraction response involves coordination of regions hundreds of
micrometers apart, at the same moment. Paracrine chemical signalling mechanisms in
sponges rely on a molecule binding to a ligand-receptor system that controls the
movement of ions directly (ionotropic) or indirectly (metabotropic) via g-protein coupled
pathways, possibly by an amino acid (Glutamate, GABA), biogenic amine (epinephrine),
150
peptide (RF-amides, melatonin) or short-lived gas (nitric oxide, carbon monoxide).
Glutamate (Glu), y- aminobutyric acid (GABA), and nitric oxide (NO) are
important chemical messengers that are found in plants, protists, and metazoans where
they function in feeding, sensory systems, development and also act as neuro-active
compounds (Chiu et al., 1999; Moroz, 2001; Bouche et al., 2003). This suggests that
they were already present in the common ancestor to metazoans. The first
characterization of any of these receptors from a sponge was a putative dual Glu/GAB A
receptor from Geodia cyndonium (Perovic et al., 1999). However, the recent work from
the genome of the demosponge Amphimedon queenslandica (http://www.jgi.doe.gov/
sequencing/why/3161.html) has shown there to be no ionotropic, but some metabotropic
Glutamate, GABA, NO receptors. Interestingly the post-synaptic scaffolding proteins
required for signaling have been found in sponges, yet morphological and behavioural
demonstration of'proto-synaptic' complexes is still lacking (Sakarya et al., 2007;
Richards et al., 2008). The effect of stimulation by Glutamate, GABA, and NO have
been tested in Tethya wilhelma (Ellwanger and Nickel, 2006), and found to play a role in
stimulating and modulating contractions. Furthermore, GABA-immunoreactive proteins
have been localized in cells associated within the aquiferous canal system in Chondrilla
nucula (Ramoino et al., 2007), and NO has been implicated in temperature stress
activation in Axinellapolypoides and Petrosia ficiformis (Giovine et al., 2001).
Molecular evidence suggests that receptors for Glu, GABA and NO were present before
plants and animals diverged (Chiu et al., 1999; Moroz, 2001). In the absence of more
direct mechanisms of signaling between cells, the slowness of the coordinated behaviour
shown by sponges makes a Glu-GABA signaling systems and modulation by a NO
151
system a most likely hypothesis.
Ephydatia muelleri has coordinated contractions of the aquiferous canal system
that function to expel wastes and to flush the canal system on a regular basis (Elliott and
Leys, 2003; Elliott, 2004; Elliott and Leys, 2007). I hypothesize that these sponges are
able to coordinate or modulate contractions by the use of small diffusible molecules such
as amino acids (Glutamate- L-Glu, y-aminobutyric acid - GABA) or short lived
diffusible gas (Nitric oxide - NO).
Using a combination of microscopy techniques, immunocytochemistry and
pharmacological manipulations I provide a description of possible signalling systems of
the freshwater sponge Ephydatia muelleri. I have found that glutamate triggers the
inflation-contraction cycle of the sponge in a dose-dependent manner that varies with
amplitude and duration. Nitric oxide synthase (NOS) is found in choanocytes, in
dendritic cells of the apical pinacoderm, osculum, and in cells that line the excurrent
canal system. Upon stimulation by a NO donor cGMP was localized to cells in the
osculum, which also contracted. GABA on the other hand generated spasms of the
openings to incurrent canals, and may be involved in inhibiting the inflation-contraction
cycle. These results confirm a role for a chemical messenger signalling system in
sponges, and offer insight into the mechanisms by which coordination of contractile
behaviour occurs in the absence of a conventional nervous system.
4.2 Materials and methods
4.2.1 Collection and culturing of sponges.
Gemmules of the freshwater sponge Ephydatia muelleri (Lieberkuhn 1855) and Spongilla
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lacustris were collected from sunken trees in Frederick Lake, BC, Canada
(48°47'51.7559"N, 125o2'58.5600"W) at a depth of 0-3 m and Rousseau Lake, BC,
Canada (48°50'20.05"N, 125° 1'26.05"W) at a depth of 1-4 m. They were stored in
unfiltered lake water at 4°C in the dark until ready to use. The gemmules in sealed bags
were aerated once a month and were viable for up to one year. The gemmules were
mechanically dissociated from the dead sponge skeleton and sterilized before placing
upon glass coverslips as described by Elliott and Leys (2007). Gemmules were grown in
M-medium (0.5 mmol T1 MgS0 4 -7H 2 0, 1 mmol T1 CaCl 2 H 2 0, 0.5 mmol l"1 NaHC0 3 ,
0.5 mmol l"1 KC1, 0.25 mmol l"1 Na 2 Si0 3 -9H 2 0) (Funayama et al., 2005), and media was
replaced every 48 hours. Whole-mount preparations consisted of a single gemmule on an
ethanol flamed sterilized 22 mm2 coverslip in Petri dishes, which allowed for easy
transfer of the sponge into test substances and rinses. Only sponges 7-10 days post
hatching (dph) with a fully functional aquiferous canal system were used for
experimentation and care was taken not to use sponges in the process of regression.
4.2.2 High performance liquid chromatography (HPLC)
For HPLC, 150-200 gemmules were hatched and sponges were grown in mass cultures
on 5 cm diameter Petri dishes. The growth medium was change every 24 hours. At 7
dph, the medium was aspirated off and tissue was scraped from the bottom of the Petri
dishes with a sterile, plastic tissue scraper, and transferred with a sterile glass pipette to
1.6 ml plastic microfuge tubes. The tissue was centrifuged for 1 min at 1000 g to remove
excess M-media. The final weight of each preparation was -200 mg. The sponge tissue
was immediately frozen in liquid nitrogen, weighed, and stored at -80°C until analyzed.
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HPLC coupled with fluorimetric detection and the derivitization procedure of
Grant et al. (2006) was used to separate and detect amino acids at the Neurochemical
Research Unit, University of Alberta. Sponge tissue samples of both E. muelleri and S.
lacustris were homogenized in 5 volumes of distilled water. The homogenate was diluted
1:3 in methanol to precipitate proteins, immersed in ice for 10 min, and centrifuged
(13000 g) at 4°C. Stock solutions of amino acid standards (L-glutamate, L-glutamine, Laspartate, L-asparagine, L-alanine, D-serine, L-serine, L-serine-O-phosphate, Ltryptophan, glycine, y-amino-butyric acid (GABA), L-threonine, L-taurine, L-valine;
Sigma-Aldrich, Oakville, ON, CAN) were prepared in 20% (v/v) methanol at a
concentration of 1.0 mg mL"1 and were used to identify peaks in the samples based on
retention time, and to quantify amino acids in the samples based on a 9-point calibration
curve. The actual standard concentrations of GLU, ASP, SER, GLN, ALA, GLY, TAUR
and TRYP curves were 6, 4, 2, 1, 0.5, 0.2, 0.1 and 0.04 ug mL"1; ASN and GABA curves
were 3, 2, 1, 0.5, 0.25, 0.1, 0.5, and 0.2 ug mL"1; THREO and VAL were 1.5, 1, 0.5, 0.25,
0.125, 0.05, 0.025, and 0.01 ug mL"1. Derivitization reagent solutions were prepared by
dissolving 1 mg o-phthaldialdehyde (OPA; Sigma-Aldrich, Oakville, ON, CAN) and 2
mg of N-isobutyryl-L-cysteine (IBC; Sigma-Aldrich, Oakville, ON, CAN) in 0.1 mL of
methanol followed by the addition of 0.9 mL 0.2 M sodium borate buffer (pH 10).
Automated pre-column derivitization was carried out on 5 uL of sample, standard, or
blank and 5 uL of derivitizing agent that was injected into a HPLC system for analysis.
The HPLC system comprised of a Waters Alliance 2690XE instrument equipped with a
Waters 474 programmable fluorescence detector (Waters Corporation, Milford, MA,
USA) that pumped a mobile phase containing 15% methanol in 0.04 M Sodium
154
phosphate buffer (pH 6.2) through a Waters Symmetry CI8 column (3.9 x 20 mm, 5 fim).
Samples were detected with an excitation wavelength of 344 nm and an emission
wavelength of 443 nm.
4.2.3 Digital time-lapse and data acquisition
For all pharmacology experiments, digital images of whole-mount sponges were viewed
on a stereomicroscope (Olympus SZX-12) captured with a Ql-Cam monochrome CCD
camera with a color filter. Images were captured using Northern Eclipse version 7
(Empix Imaging Inc., Mississauga, ON, Canada) from live video feed every 5 seconds.
For image analysis, changes in the diameter of canals was taken for every first, fifth,
tenth or twentieth image of the aquiferous canal system (perpendicular to the long axis of
the canal) were measured in triplicate using either Northern Eclipse or Image J 1.31
(NIH, Washington, USA) and data were logged into MS Excel 2003. Center, middle and
peripheral canals were classed based on location and resting diameter as characterized in
Elliott and Leys (2007). Graphs were created using Sigma Plot version 10 (Systat
Software, Inc., San Jose, USA).
4.2.4 Test substance application
The contractile mechanism, response and coordination of the aquiferous canal system in
Ephydatia muelleri was characterized and tested by the use of Ca Free EGTA/EDTA
buffered medium (CF: calcium free and CMF: calcium magnesium free media) (M-media
without MgS0 4 & CaCl, but with 1 mM NMDG-Cl, 0.5 mM NMDG-S0 4 , 0.5 mM
155
EGTA, and/or 0.005 mM EDTA), L-glutamate (Sigma-Aldrich, Oakville, ON, CAN),
and GABA (Sigma-Aldrich, Oakville, ON, CAN) with specific blockers to the
metabotropic glutamate receptors (AP3: 2-Amino-3-phosphonopropionic acid and KYN:
Kynurenic Acid; Sigma-Aldrich, Oakville, ON, CAN). Stock solutions (50 mM L-Glu,
50 mM GABA, 50 mM AP3 and 50 mM Kynurenic acid; Sigma-Aldrich, Oakville, ON,
CAN) were prepared in distilled water or DMSO and were added into the medium to
reach the final concentrations listed in each experiment. Great care was taken to always
add solutions at the side of the Petri dish opposite to the sponge and to mix by gently
pipetting 3-4 times for each test substance. The sponges did not respond to equivalent
additions of M-medium and mixing. For blockers, sponges were placed into the final
diluted medium in Petri dish and allowed to sit undisturbed for 10-60 minutes depending
on experiment. For each experiment, t=0 was defined as the time filming began or after 1
min of shaking (Elliott and Leys, 2007), not the time of injection of the test substance.
4.2.5 Fluorescence
microscopy
Juvenile sponges were fixed for fluorescence microscopy by direct immersion into a
mixture of 3.7% paraformaldehyde and 0.3% gluteraldehyde in phosphate-buffered saline
(PBS; 100 mmol l"1) for 24 hours at 4°C (Elliott and Leys, 2007). After fixation, samples
were washed in cold PBS and permeabilized with 0.2% Triton-XlOO in PBS. For
cytochalasin experiments, the actin cytoskeleton was labeled with Alexa 594 Phalloidin
or Bodipy 505 FL Phallacidin (Molecular Probes-Invitrogen, Carlsbad, CA, USA) in PBS
with 10% bovine serum albumin (BSA). After 3 hours at room temperature sponges
were rinsed in cold PBS. For NOS immunolabelling, three nitric oxide synthase (NOS)
156
antibodies were used: neuronal (monoclonal-nNOS), epithelial (monoclonal-eNOS) and
universal (polyclonal-uNOS) (1:1000) (Millipore-Chemicon, Billerica, Massachusetts,
USA) with 3% goat serum (Sigma-Aldrich, Oakville, ON, CAN), and 0.1% Triton-XlOO
in cold PBS and placed on a shaker at 4°C overnight. Preparations were rinsed in PBS
and labelled with a solution containing Alexa 488 goat anti-rabbit (1:100) (Molecular
Probes-Invitrogen, Carlsbad, CA, USA) with 10% goat serum in cold PBS on a shaker
for 3 hours. Preparations were either mounted in Mowiol with Dabco (Sigma-Aldrich,
Oakville, ON, CAN) or 100% glycerin, sealed with nail polish, and allowed to harden
overnight at 4°C. Preparations were viewed and imaged with a Zeiss Axioskop
epifluorescence microscope with a Qicam CCD camera operated by Northern Eclipse.
4.2.6 NADPH-diaphorase histochemical detection of nitric oxide synthase
Juvenile sponges were fixed with paraformaldehyde:gluteraldehyde mixtures as above,
rinsed three times in cold PBS, rinsed twice in 0.1 M Tris-HCl buffer (pH 8.0) for 5 min
each, and permeabilized for 10 min in 0.1 M Tris-HCl buffer (pH 8.0) with 0.25% TritonXI00. For histochemical detection of nitric oxide synthase, sponges were incubated with
1 mM P-NADPH (Sigma-Aldrich, Oakville, ON, CAN), 0.5 raM Nitro Blue tetrazolium
(Sigma-Aldrich, Oakville, ON, CAN), 0.1 mM dicumarol (Sigma-Aldrich, Oakville, ON,
CAN), 0.25% Triton-XlOO in 0.1 M Tris-HCl buffer (pH 8.0) in the dark until color
development was reached. The reaction was stopped by rinsing 4 times in cold PBS.
Sponges were post-fixed in 4% paraformaldehyde in methanol for 1 hour at 4°C and
rinsed in 100% dry ethanol. The ethanol was allowed to evaporate and coverslips with
sponges were inverted, mounted slightly raised on Vaseline legs in 100% glycerol.
157
Edges were sealed with nail polish. Images were captured as indicated above.
4.2.7 cGMP assay for nitric oxide reaction
Juvenile sponges were incubated in 1 mM 3-Isobutyl-l-methylxanthine (IBMX:
phosphodiesterase inhibitor; Sigma-Aldrich, Oakville, ON, CAN) in Strekal's medium, an
equivalent culture medium to M-medium (Strekal and McDiffett, 1974) diluted from a
0.5 M stock solution in DMSO for 30 minutes. Sponges were exposed to 0.1 mM Snitroso-N-acetyl-DL-penicillamine (SNAP: Nitric oxide donor; Sigma-Aldrich, Oakville,
ON, CAN) for 2 minutes and fixed in 4% paraformaldehyde in PBS for 2 hours at 4°C.
Coverslips with sponges were rinsed (30 min) and inverted on a solution containing
primary rabbit anti-cGMP (1:3000) (Millipore-Chemicon, Billerica, Massachusetts, USA)
with 3% goat serum (Sigma-Aldrich, Oakville, ON, CAN), and 0.1% Triton-XlOO in cold
PBS and placed on a shaker at 4°C overnight. Preparations were rinsed in PBS and
labelled with a solution containing Alexa 488 goat anti-rabbit (1:100) (Molecular ProbesInvitrogen, Carlsbad, CA, USA) with 10% goat serum in cold PBS on a shaker for 3
hours. Sponges were rinsed in PBS, mounted in 100% glycerol, and imaged as indicated
above.
4.3 Results
4.3.1 Description of the freshwater sponge
The young sponge of Ephydatia muelleri was organized into three functional regions the ectosome consisting of the apical pinacoderm and subdermal cavity; the choanosome,
158
consisting of the aquiferous system including choanocyte chambers, mesohyl and basal
pinacoderm; and the osculum - all of which were centered around an empty gemmule
husk. The tent-like apical pinacoderm draped over a choanosome and spicule skeleton
with an osculum protruding to the distal end of the animal. Within the choanosome, the
most striking feature was the branching nature of the aquiferous canal system that looked
reminiscent of the branching patterning of bronchia, trachea in lungs of mammals, and
the choanocyte chambers appeared as alveoli budding off the excurrent canals. Water
entered the sponge through the ostia in the apical pinacoderm and entered the
choanosome via prosopyles that led to incurrent canals and choanocyte chambers.
Filtered water flowed from the choanocyte chambers via apopyles into the excurrent
canals and vented out of the osculum.
4.3.2 Evidence of neurotransmitter molecules in sponge tissues: HPLC-MS Analysis
Samples of sponge tissue analyzed by HPLC revealed chromatographs that co-eluted
with standards for the putative amino acid neurotransmitters aspartic acid, glutamic acid,
asparagines, serine, glutamine, glycine, threonine, taurine, alanine, GABA, tryptophan,
and valine (Figure 4-1; A- E. muelleri, B- S. lacustris, and C-standard trace). All amino
acids were found in each sample with a detection limit ranging from 0.01-0.04 |j,g ml"1 of
supernatant. As shown in Table 4-1, the amino acid levels were low except for glutamic
acid, glutamine, aspartic acid, glycine, and valine. Similar high concentrations were
found for glutamate in Spongilla lacustris, however all other amino acids in that sponge
were low compared to Ephydatia muelleri (Table 4-1; Figure 4-1). When single sponges
were exposed to 30 mM KC1 in an attempt to activate release of possible transmitters, no
159
detectable amino acids were released into the supernatant.
4.3.3 Evidence for a metabotropic glutamate signalling system
Glutamate (L-Glu) caused contractions of the aquiferous canal system and could induce a
full inflation-contraction cycle (as defined by Elliott and Leys, 2007) at a threshold
agonist concentration of 75 - 80 uM L-Glu (Figures 4-2 & 4-3). All sponges were
acclimated to the experimental chamber for 1 hour to ensure that they were at rest before
the application of L-Glu. Sponges were filmed for 10 minutes prior to application of LGlu in order to make sure an inflation-contraction cycle was not spontaneously occurring.
If a sponge started to inflate it was allowed to complete the contraction and was left to
relax for 1 hour. Only responses to agonist concentrations between 3 0 - 1 0 0 uM L-Glu
were measured because concentrations above 100 uM were fatal. A sub-threshold
agonist concentration (30-60 uM) application of glutamate triggered a gradient of
behavior. The first response was an initial lowering of the apical pinacoderm, pushing
down on the subdermal cavity and choanosome, and its return to normal diameter.
Following this, incurrent canals contracted, expanding excurrent canals, but it was not
until the application of 60 uM L-Glu that a consistent inflation of the excurrent canals
was generated by the contraction of the incurrent canals. At the threshold level of 70 uM
L-Glu a full inflation contraction cycle occurred (Figure 4-2), which was similar to the
response of sponges stimulated by ink and agitation (see Elliott and Leys, 2007), except
that the plateau phase was reduced or absent. At an agonist concentration 80 - 100 uM
L-Glu the apical pinacoderm had severe contractions that destroyed its morphology
(Figure 4-3). Interestingly, the osculum and canals could contract without the intact
160
Figure 4-1. HPLC-MS chromatographs of amino acids in tissue of Ephydatia muelleri
and Spongilla lacustris. Peaks represent putative active neuronal amino acids. A)
Ephydatia muelleri; B) Spongilla lacustris; C) Amino acid standards. GLU, ASP, SER,
GLN, ALA, GLY, TAUR and TRYP were used at 2 ng ml"1, ASN and GAB A were used
at 1 u£ ml"1, and THREO and VAL at 0.5 ug ml"1. Aspartate (ASP), glutamate (GLU),
asparagine (ASN), serine (SER), glutamine (GLN), glycine (GLY), threonine (THREO),
taurine (TAUR), alanine (ALA), tryptophan (TRYP), and valine (VAL).
161
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162
Table 4-1. Amino acid levels for tissue from Ephydatia muelleri and Spongilla lacustris
analyzed by HPLC. Values are expressed in fxg/ml of sample as means ± SE (n=5
colonies of 150-200 sponges).
Amino Acid
Aspartic acid
Glutamic acid
Asparagine
Serine
Glutamine
Glycine
Threonine
Taurine
Alanine
y-Aminobutyric acid (GABA)
Tryptophan
Valine
E. muelleri
24.98±3.23
48.21±6.03
1.58±0.14
4.67±1.09
23.16±2.59
12.38±0.98
9.88±0.90
2.97±0.57
28.16±3.84
6.09±1.28
7.13±1.94
18.58±3.06
S. lacustris
4.67±0.33
116.94±3.94
2.76±0.20
16.40±2.39
10.40±1.38
14.77±1.24
3.28±0.13
1.97±0.26
19.56±1.43
2.67±0.16
2.59±0.11
4.86±0.17
163
Figure 4-2. Response of Ephydatia muelleri to 80 uM L-glutamate. (A-D) Time series
of images showing the contractile events at the start of experiment (A) with gemmule (g),
osculum (osc), excurrent canals (ex), and incurrent canals (in); (B) contraction of the
osculum; (C) maximum expansion and (D) maximum contraction of the excurrent canals.
Scale bar, 1 mm. (E) Percent of the maximum expansion of the excurrent canals at times
shown in A-D.
164
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165
Figure 4-3. Concentration dependence of contractions of Ephydatia muelleri treated
with L-glutamate. Response is calculated as the percent expansion of the excurrent
canals over time. For each concentration (30-100 uM), three independent experiments
are shown in black, dark grey, and grey.
166
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Time (sec)
Figure 4-3.
167
Table 4-2. Kinetics of the excurrent and incurrent canal contractions.
Concentration
Amplitude 1
Duration
of glutamate
(HM)
(s)
30
0.00
0.00
40
0.22
264.67
50
0.12
372.00
60
0.28
542.50
70
0.41
1231.00
75
0.49
1618.33
80
0.43
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1. Percent maximum diameter of canal
Rate of contraction of canals (fim-s-l)
Incurrent
Excurrent
0.00
0.47
0.16
0.30
0.19
0.15
0.26
0.24
0.00
0.00
0.19
0.13
0.14
0.13
0.18
0.31
0.20
0.00
168
structure of the over-lying apical pinacoderm. At an agonist concentration of 80 uM LGlu, two types of events occurred: either the sponge went into a defibrillation-like
behaviour leading to full contraction with no inflation-contraction cycle or it went
through an inflation-contraction cycle leading into full contraction of the excurrent
canals. At an agonist concentration of 100 uM L-Glu both the incurrent and excurrent
canals contracted at the same time which prevented the excurrent canals from full
contraction (Figure 4-3). As the agonist concentration increased from 30 to 100 uM the
amplitude and duration of the inflation-contraction cycle increased, however the rate of
contraction of the canals was similar at all concentrations (Table 4-2 - data analyzed
from Figure 4-3). The results are presented in nine concentration-dependent contraction
kinetic graphs ranging from 30 to 100 uM for L-Glu, based on 27 measurements (Figure
4-3).
4.3.4 Evidence for function ofglutamate receptors
A response at 80 uM L-Glu was considered to be the maximum concentration that
elicited a full response and so concentration of 75 and 80 \\M were used in the following
experiments to investigate the type of receptor involved, its competitive ability, and role
of calcium stores (internal and external). The receptor type was evaluated by using a
metabotropic glutamate receptor blocker AP3 (2-amino-3-phosphoproprionic acid).
When sponges were treated with 75 (part A) and 80 uM (part B) L-Glu, they exhibited
normal L-Glu induced inflation-contraction cycles (Figure 4-4i). When sponges were
incubation in a concentration of 100 uM AP3 for 30 minutes, it was enough to abolish the
inflation-contraction cycle induced by 75 uM and 80 uM L-Glu (Figure 4-4ii). When the
169
concentration of AP3 was reduced by half to 50 uM, both 75 uM (part A) and 80 uM LGlu (part B) induced an inflation-contraction cycle in part A, but delayed in part B.
(Figure 4-4ii). After the AP3 was washed out for one hour, all sponges carried out a
normal inflation-contraction cycles in response to L-Glu (Figure 4-4iii). Interestingly,
Sponges that were incubated in 100 or 50 uM AP3 and then stimulated by agitation,
exhibited convulsions or twitches, but no inflation-contraction cycles (Figure 4-4iv).
To further test the functionality of the metabotropic glutamate receptor system
an allosteric competitive inhibitor Kynurenic acid (KYN) was applied to the sponges
(Figure 4-5). Sponges were incubated in 25, 50, 100, 150, 200 uM KYN and L-Glu was
added after 10, 20, or 30 minutes to test for competition of the two molecules.
Concentrations of KYN above 200 uM were cytotoxic and concentrations below 25 uM
had little to no effect. All sponges were washed with fresh M-media and left for 24 hours
to test viability after experiments took place. Sponges incubated in all concentrations of
KYN showed either no response at all or only slight contractions when stimulated by
shaking (Figure 4-5). Application of 80 uM L-Glu generated different responses
depending on the concentration of inhibitor and length of incubation. At a concentration
of 200 uM KYN no inflation-contraction cycles were induced by either application of 80
uM L-Glu or agitation (Figure 4-5). Sponges incubated in 150 uM KYN and stimulated
by shaking initially contracted the excurrent canals and then carried out rapid
uncoordinated contractions; there was no full inflation-contraction cycle. In contrast,
application of 80 uM L-Glu after incubation in 150 uM KYN did trigger an inflationcontraction cycle but it was delayed by 10 minutes and was abolished with longer
incubation times in the inhibitor (Figure 4-5). Sponges incubated in 100 uM KYN did
170
Figure 4-4. Effect of the allosteric inhibitor of metabotropic glutamate receptors L-AP3
has on glutamate triggered contractions. Sponges (E. muelleri) were treated with (i) Lglutamate alone at t=600 s with 75 uM (column A; circles) or 80 uM (column B; squares)
indicated by black arrow showing normal induced response (black lines), (ii) Sponges
were then pre-incubated with 50 uM (open symbol) or 100 uM AP3 (closed symbol) for
30 minutes, at t=600s 75 uM (circles) or 80 uM (squares) L-glutamate was applied
reducing and eliminated the inflation-contraction cycle respectively (blue line), (iii)
Sponges in which AP3 was washed out for 1 hour and then treated with L-glutamate
showed normal inflation-contraction cycles (green line), (iv) Sponges were preincubated in 50 uM (open symbol) or 100 uM (closed symbol) AP3 for 30 min and then
agitated for 1 minute had no inflation-contraction cycles (red line), only spasms.
171
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AP3 + Agitation
600 1200 1800 2400 3000 3600 4200 4800 5400
Time (sec)
AP3 + Agitation
0
600 1200 1800 2400 3000 3600 4200 4800 5400
Time (sec)
Figure 4-4.
172
Figure 4-5. Effect of the competitive inhibitor of glutamate receptors, Kynurenic acid
(KYN) on glutamate triggered contractions. Sponges were incubated in 25-200 uM KYN
(i-v), shaken (black line) or exposed to 80 uM L-Glu 10 min (red line), 20 min (green
line), and 30 min (blue line) thereafter (columns A-C). Arrows indicate application of
glutamate to the preparation. A) When L-Glu was applied 10 minutes after incubation in
the competitive inhibitor contractions were delayed by 2, 5, and 10 minutes with
increasing concentration of KYN. B) Application of L-Glu 20 minutes after incubation
in the competitive inhibitor delayed contractions by 5 and 10 minutes in 25 and 50 mM
KYN, but no contractions occurred in concentrations of 100 mM and above. C) L-Glu
applied 30 minutes after incubation in the competitive inhibitor failed to elicit any
contractions.
173
A
B
600 1200 1800 2400 3000
600 1200 1800 2400 3000
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600 1200 1800 2400 3000
not contract when stimulated by shaking (Figure 4-5). At the same concentration of
KYN application of 80 uM L-Glu induced an inflation-contraction cycle but with 10
minute delay (Figure 4-5); again incubations of 20 minutes in the inhibitor abolished all
responses to L-Glu (Figure 4-5). At a concentration of 50 uM KYN stimulation by
shaking did not trigger inflation-contraction cycles (Figure 4-5), but 80 uM L-Glu
triggered an inflation-contraction cycle even after 30 minutes of incubation in the
inhibitor (Figure 4-5). At a concentration of 25 uM KYN, no inflation-contraction cycles
occurred by shake stimulation (Figure 4-5), but 80 uM L-Glu triggered an inflationcontraction cycle at 10 and 20 minutes of incubation; however, no inflation-contraction
cycle was induced at 30 minutes (Figure 4-5).
4.3.5 Role of calcium in contractions
When sponges were incubated in CF media, the application of 80 uM L-Glu stimulated
an inflation-contraction cycle, but at a lower amplitude and duration as compared to
sponges stimulated in normal media (Figure 4-6A). When sponges were incubated in
CMF media, the application of 80 uM L-Glu stimulated no inflation-contraction cycles
(Figure 4-6A). When sponges were incubated in CF media and stimulated by agitation,
no inflation-contraction cycle occurred; only irregular contractile events (Figure 4-6B).
When sponges were incubated in CMF media and stimulated by agitation, no inflationcontraction cycles were elicited, in contrast to those triggered in sponges stimulated in
normal calcium-containing media (Figure 4-6B).
175
Figure 4-6. A) Response of Ephydatia muelleri to 80 uM L-Glu in Ca -free medium
(green solid line) and Ca2+Mg2+-free medium (red solid line). Control shows normal 80
uM L-Glu response (black line). B) Response of Ephydatia muelleri to mechanical
agitation in Ca2+-free (green line) and Ca2+Mg2+-free (red line) medium. Control shows
normal response to shaking (black line). 80 uM L-Glu was applied at t=600 s indicated
by black arrow and 1 min agitation occurs at t=0 s.
Ca Free + 80 uM L-Glu
CaMg Free + 80 uM L-Glu
80 uM L-Glu normal media
600
1200
1800
2400
3000
3600
Time (sec)
100
Ca Free + 1 min agitation
CaMg Free + 1 min agitation
1 min agitation normal media
600
1200
1800
2400
3000
3600
Time (sec)
Figure 4-6
177
4.3.6 Evidence for a GABA signalling system
GABA induced rapid twitches (as defined in Elliott and Leys, 2007) and contraction of
the incurrent canals depending on the agonist concentration 25-1000 uM. All sponges
were acclimated to the experimental chamber for 1 hour to ensure that the sponges were
at rest before the application of GABA. Sponges were filmed for 10 minutes prior to
application of GABA in order to make sure an inflation-contraction cycle was not
spontaneously occurring. At doses of 250 uM, GABA triggered rapid 'twitches' of the
incurrent canals in 5 experiments, while most treatments show minor twitches of the
choanosome (example Figure 4-7). Only one sponge out of 18 responded to 1000 uM
GABA with an inflation-contraction event (data not shown), but the contraction occurred
well after the agonist was applied.
4.3.7 Evidence for nitric oxide signalling system
Nitric oxide synthase (NOS) activity was visualized by the use of 3 different protocols:
NAPDH-diaphorase staining (Figure 4-8), nitric oxide synthase antibodies (neuronal and
universal-epithelial, neuronal and immune) (Figure 4-9), and cGMP accumulation assay
(Figure 4-10). Using the NADPH-diaphorase staining protocol, cells that stained blue
indicated possible sites of active nitric oxide synthase in the cells of the choanocyte
chambers, osculum, excurrent canals and apical pinacoderm (Figure 4-8). Cells that lined
the osculum stained the darkest and showed a gradient of lighter staining at the base as
compared to the tip of the osculum (Figure 4-8A). The pinacocytes of the excurrent
canals stained for NOS activity as well. Within the apical pinacoderm only a few cells
178
Figure 4-7. Contraction of the choanosome and excurrent canals ofEphydatia muelleri
in response to 250 uM GABA. In 5 out of 18 of sponges treated, the choanosome
contracted rapidly at least once after application (arrow) of GABA. The red arrow
represents the width measurement of the choanosome.
179
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Excurrent Canal
Choanosome
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1200
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2400
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Figure 4-7.
180
Figure 4-8. Localization of nitric oxide synthase (NOS) in Ephydatia muelleri by
NADPH-diaphorase staining. Cells showing NOS activity (blue stained cells, white
arrows) were located in the osculum (A), cells that lined the excurrent canals (B), and
cells in the mesohyl of the apical tissue (C). Scale bar, 500 um. The black arrow in (A)
indicates the top of the osculum.
181
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182
that formed a loose network in the mesohyl and the outer epithelium stained darkly for
NOS activity (Figure 4-8C).
Immunofluorescence of cells labeled with commercially available NOS
antibodies were observed in cells of the osculum, apical pinacoderm, and in the
choanosome (Figure 4-9A). Cells that labeled with the nNOS antibody were found in the
osculum (Figure 4-9C) and throughout the apical pinacoderm (Figure 4-9B), but no clear
label was observed in the choanosome due to the density of cells. Mesohyl cells of the
apical tissue and porocytes had what appeared to be both a membrane and cytoplasmic
associated label of nNOS (Figure 4-9AB). When cells of the choanosome were labelled
with uNOS fluorescence increased but no discernable pattern was found (Figure 4-9D);
however, the choanocyte cell bases showed slightly increased labelling (Figure 4-9E).
Both pinacocytes and porocytes showed a cytoplasmic distribution of the label (Figure 49F-I).
Using a cGMP live assay, cells immunoreactive for cGMP were found within
the osculum and in the apical tissue indicating these are active targets for nitric oxide.
Young sponges were treated with 1 raM IBMX to prevent the breakdown of cGMP
produced in cells responsive to nitric oxide, and then challenged by the nitric oxide donor
SNAP (Figure 4-10). When exposed to SNAP cells in the osculum accumulated cGMP
and the osculum contracted (Figure 4-10AB). No detectable staining was observed
within the choanosome (Figure 4-1 OB). However, when the pinacoderm was exposed to
SNAP numerous a cytoplasmic staining was found even after the incubation period
(Figure 4-IOC).
183
Figure 4-9. Localization of nitric oxide synthase in Ephydatia muelleri by neuronalNOS (A-C) and universal-NOS antibodies (D-F) compared to control images (G-I). A)
Whole-mount of a sponge showing the choanosome (cho) and cells in the outer apical
tissue (apd) labeled with anti-nNOS. Scale bar, 100 um. B) A higher magnification
view of the apical pinacoderm and mesohyl shows the porocytes (p) and mesohyl cells
(mc) (arrows) are immunoreactive for nNOS. Scale bar, 50 um. C) Elongate cells
(arrow) in the osculum (osc) show nNOS immunoreactivity; the shape of these cells
suggests they are in the mesohyl of the osculum. Gemmule (g). Scale bar, 500 um. D)
Low magnification image shows many cells in the choanosome are immunoreactive for
uNOS. Scale bar, 500 um. E) High magnification of the apical pinacoderm and
mesohyl shows cytoplasmic immunofluorescence of uNOS in amoeboid looking cells
that are likely in the mesohyl. Scale bar, 10 um. F) Choanocytes of the choanocyte
chambers (cc), indicated by arrows, are highly immunoreactive for uNOS. Scale bar, 25
um. G, H) Image of a control sponge showing autofluorescence in the choanosome
(cho), but no label in the apical pinacoderm or mesohyl. Scale bar, 200 urn. H) Image
of the apical pinacoderm in a control sponge showing little autofluorescence. Scale bar,
25 um. I) Choanocyte chambers (cc) in a control sponge showing some
autofluorescence. Scale bar, 10 um.
184
0- ^0«;
Figure 4-9,
185
Figure 4-10. NOS activity in Ephydatia muelleri. Activation by the nitric oxide donor
SNAP in the presence of a phosphodiesterase inhibitor IMBX is detected by a rabbit anticGMP antibody. A) Cells in the osculum show strong cGMP immunoreactivity in the
presence of the nitric oxide donor SNAP. Scale bar, 100 um. B) Pinacocytes (arrows)
are immunoreactive for cGMP in the presence of the nitric oxide donor SNAP. Scale bar,
100 um. C) In the absence of the nitric oxide donor SNAP there is fluorescence in the
choanosome but no cGMP accumulation in the pinacoderm, indicated by arrows. Scale
bar, 100 urn.
186
Fie
4=10.
187
4.4 Discussion
Neuroactive molecules are defined by their action in the nervous system, and specifically
by their role in the chemical synapse (Kandel et al., 1991). But what about activity in an
organism without a conventional nervous system, like sponges? Pavans de Ceccatty
(1974b) suggested that sponges have the ability to form different adjoining contacts:
close appositions giving visible exchange areas, press-button-like articulations, and
punctate tight junctions, which could carry out directional spreading of signals from cell
to cell. Recent molecular analysis has shown that the demosponge Amphimedon
queenslandica possesses the proteins involved in forming a postsynaptic scaffold in other
animals (Sakarya et al., 2007). Although there is still no morphological or behavioural
evidence for true chemical synapses in sponges, it is important to determine what
molecules could be active at a potential 'proto'-synapse, and whether these have
molecules play a role in controlling sponge behaviour. I report evidence for the presence
of signaling molecules in the tissues of the freshwater sponge Ephydatia muelleri and for
their role in triggering and mediating contractions of the sponge body.
4.4.1 Neuroactive amino acids in sponge tissue
My first approach was to screen Ephydatia muelleri tissue lysate for all available
molecules using HPLC-MS. This analysis revealed 12 free amino acids (AA) that are
considered neuroactive compounds in the vertebrate nervous system. However, analysis
of whole sponge lysate does not differentiate between possible metabolically active
molecules (e.g. osmolytes) and neuroactive substances (neurotransmitters,
188
neuromodulators, or their precursor molecules), and since osmolytes can be dominant
AA's that are mobilized from intracellular pools in response to a hypo- or hyperosmotic
stress or for maintenance of cellular osmolarity (example Abe et al., 2005), it is possible
that these sponges use AA for osmotic protection in freshwater systems. However, some
of the free AA may also be used for signalling.
Eight potential neuroactive AA and metabolites were found with the HPLC
screen: Glutamate, GABA, glycine, taurine, serine (D-serine), tryptophan (biogenic
amines), aspartate (precursor for NMD A: N-methyl-D-aspartic acid) and glutamine
(precursor for GABA and Glutamate) (Figure 4-1; Table 4-1). Glutamate and GABA are
neuroactive amino acids that are involved in both the CNS and peripheral nervous system
of vertebrates, and in invertebrates are typically associated with control of muscle
contraction in either an excitatory action (L-Glu) or an inhibitory action (GABA).
Glutamine is the precursor molecule or reserve store for glutamate and the GABA
metabolic pathway. The high activity of GABA and glutamate as signalling molecules
requires storage of an inactive glutamine for proper function within a cell. Glycine is a
major inhibitory AA neurotransmitter in the vertebrate central nervous system that works
by inducing a hyperpolarizing chloride current when bound to a post synaptic receptor,
but can also be a modulator in excitatory ionotropic glutamate receptors. In Hydra,
glycine has been identified and localized to the nerve net where it functions in pacemaker
activity of peristaltic contractions (elongations and contractions) of the muscle in the
body column; it is also involved in the chemosensory response during feeding by
inhibiting the closure of the mouth upon stimulation by glycine (Pierobon et al., 2001;
Ruggieri et al., 2004). Similar effects of the above responses in Hydra are observed by
189
the application of alanine and taurine (Pierobon et al., 2001). In the demosponge Tethya
wilhelma, glycine has been shown to stimulate a contraction, increase contraction rhythm
and decrease contraction amplitude (Ellwanger and Nickel, 2006). It was proposed that
this action occurs via a metabotropic glycine receptor, but unfortunately no antagonists
(e.g., strychnine) were used to block the response to confirm receptor-ligand binding.
Taurine (Tau) is increasingly acknowledged to be a biologically active substance
in both invertebrate and vertebrate nervous systems (Strang et al., 1990; Pirvola and
Panula, 1992). Carlberg et al. (1995) showed that Tau was abundant in many cell types
that function both neurally and metabolically in cnidarians. Anctil and Minh (1997) have
shown that Tau is the dominant AA found in endodermal contractile cells in the sea
pansy Renilla, but within the spicule cells they suggest it functions as an osmolyte. In
marine flatworms, Tau had the highest free AA concentration indicative of an osmolyte
(Barrett, 1981). Tau was detected in E. muelleri, but no functional data is available to
suggest if it has a signaling or osmolyte function in the Porifera.
Other interesting metabolites found in the sponge tissue are tryptophan and
aspartate both of which are used as precursor molecules for biogenic amine metabolism
(Kandel et al., 1991).. Trytophan is the precursor molecule for the production of
serotonin, which has been found in sponges (Weyrer et al, 1999) and has some reactivity
(Ellwanger and Nickel, 2006). Aspartate is a modulator of NMD A glutamate receptors
and its involvement as a chemical transmitter is tentative (Kandel et al., 1991), but since
it is also a major product of the Krebs cycle its role in chemical transmission in sponges
is uncertain without further experimentation. We did not examine the amount of free
arginine, but its presence would indicate a pool that could be used for nitric oxide
190
production. Future work is required to develop protocols to measure biogenic amines
present in specific sponge tissues, as has been shown for cnidarians and some mollusks
by microhistochemistry. This precise characterization of spatial and temporal
localization of AA would help confirm the role of each of these molecules in the
sponge's physiology.
Although presence/absence data cannot confirm the function of amino acids as
chemical transmitters in sponges, the role of amino acids as transmitter molecules has
likely arisen from the simple gustatory behavior of protists to control feeding. Cellular
responses to specific AA are speculated to have evolved into triggers for feeding in
cnidarians - stimulating the gut and entrances to the feeding system - in sponges, this is
equivalent to the aquiferous canal system - ostia, canals, and choanocytes. I focused my
experimental work on two principal AA found in Ephydatia and Spongilla tissue lysate,
Glutamate and GAB A, because of their ubiquity in signaling in plant and animal systems.
4.4.2 Evidence for metabotropic glutamate signalling system
Glutamate (Glut) is an important chemical messenger that acts both through metabotropic
and ionotropic receptors in sensory systems as a neurotransmitter (Fagg and Foster,
1983). Molecular evidence indicates that glutamate receptors evolved well before plants
and animals diverged (Chiu et al., 1999), and its ubiquity in signaling in plant, protists,
invertebrate and vertebrate systems has prompted examination of its role in sponges (Van
Houten, 1998; Nedergaard et al., 2002; Forde and Lea, 2007). In Tethya wilhelma,
glutamate was found to regulate body contractions in a dose dependent manner
(Ellwanger and Nickel, 2006), but desensitization and spasm-like behavior were also
191
observed, and it was impossible to determine the precise effects since Tethya is opaque to
light microscopy. In E. muelleri I found that glutamate triggered contractions of the
incurrent and excurrent canal systems (the 'inflation-contraction' response) in a dosedependent manner. Contractions increased in duration and amplitude with increasing
concentration of L-Glu, and could be inhibited by blockers and competitive agonists of
glutamate receptors, 2-Amino-3-phosphonopropionic acid (AP3) and Kynurenic acid
(KYN).
AP3 and KYN blocked the inflation-contraction event at an effective
concentration of 100 uM and 200 uM respectively, but did not prevent the contraction of
the osculum and apical pinacoderm. This suggests that control of the 'inflationcontraction' response is separated regionally into a primary (1°: apical pinacoderm and
osculum) and a secondary (2°: in- and excurrent canals) system. Addition of glutamate,
after the sponge was incubated in AP3, caused the primary system to contract, but no
inflation or contraction occurred in the secondary system, suggesting that receptors in the
canal system were blocked.
Incubation of sponges in the competitive agonist KYN also prevented the
'inflation-contraction' response of the secondary system in both shake stimulated and
glutamate stimulated sponges. Longer incubation times and higher concentrations of
KYN were more effective at blocking the contractions of canals; with lower
concentrations of KYN and shorter incubation periods, glutamate triggered a delayed
response of the 2° system suggesting that it eventually is able to out compete and bind to
the receptor. Therefore, the coordination of the inflation-contraction cycle in the sponge
must depend on the release of glutamate between cells much like a chemical synapse,
192
generating a pool that eventually is able to bind to receptors on neighboring cells.
This is the first work to identify physiologically distinct regions in the sponge
body, and suggests that sponges may have different receptor populations corresponding
to the two contraction systems. The new genome project on Amphimedon queenslandica
has identified 8 separate sequences of metabotropic glutamate receptors (Sakarya et al.,
2007), and thus it would be interesting to determine if they have regionalized expression
boundaries in the young sponge. It is unclear however, how universally these data may
apply to other sponges. Molecular characterization of a receptor from the demosponge
Geodia cyndonium showed putative affinity to both metabotropic glutamate and
metabotropic GABA receptor families (Perovic et al., 1999), and the authors inferred
from this that in sponges the glutamate/GABA system might not have yet diverged.
However, application of glutamate to dissociated cells from Geodia caused an increase in
calcium in only 20% of cells (Perovic et al., 1999), indicating that a limited set of effector
cell types are equipped with specific receptor systems.
I also tested for the ability of glutamate-triggered contractions to propagate in
the absence of calcium and of all divalent cations. In calcium-free media, the effective
dose of glutamate (75 uM) caused the osculum to contract and the incurrent canals to
inflate, but no further events. In CaMg-free media the incurrent canals did not even
inflate. These results confirm that Mg2+ can substitute for Ca2+ in contractions as shown
by Prosser et al. (1962), but that an external calcium store is required for propagation of
contractions. A link between the glutamate receptor and an internal calcium store is
suggested by the fact that an initial contraction of the osculum and apical pinacoderm can
occur in CaMg-free media. However, no blockers have been identified that will block the
193
internal calcium stores in the Porifera (Lorenz et al., 1996). Experiments with BAPTA
(10-25 uM) are promising because BAPTA is more effective at chelating free Ca2+ and
will not scavenge the Ca2+ involved in cell adhesion. When sponges were incubated in
25 uM BAPTA and stimulated with 80 uM L-glutamate, however the effect was
inconsistent. Future work is required to isolate which secondary messenger system is
involved in the inflation-contraction cycle in the freshwater sponge.
4.4.3 Evidence for a metabotropic GABA signalling system
GABA (y-amino butyric acid) is a chemical messenger that functions primarily as an
inhibitory signal in feeding, growth, metamorphosis and as a neurotransmitter (Fagg and
Foster, 1983; Chebib and Johnston, 1999; Bouche et al., 2003). GABA controls ciliary
activity and swimming behavior in Paramecium (Ramoino et al., 2003), and modulates
the feeding behavior in Hydra, Asterias rubens and Aplysia californica, and is involved
in rhythmic contractions in holothuroids (Newman and Thorndyke, 1994; Concas et al.,
1998; Diaz-Rios et al., 1999). In nematodes, GABA is involved in modulating foraging,
defecation, and muscle relaxation of the body wall during movement (White et al., 1986).
The precise role of GABA in sponge physiology is somewhat unclear. Using
antibodies to mammalian GABA, Ramoino et al. (2007) showed localization to most cells
of the sponge. Although cross-reactivity of the antibodies was confirmed by western
blotting, the implication of the rather vague immunostaining obtained is difficult to
interpret. It was shown Chondrilla possesses the enzymes to synthesize and transport
GABA, and HPLC-MS analysis showed that GABA was released from cells stimulated
by KC1 (Ramoino et al., 2007); although no experimental work was carried out it was
194
speculated that GAB A played a role in the control of feeding, water circulation and body
contractions. Experiments in Tethya wilhelma have shown that GABA is 100 fold more
potent than L-Glu in triggering contractions (Ellwanger et al., 2007), which lends support
to the idea of a possible combined glutamate/GABA receptor system in that sponge.
In contrast to both of these studies, I found no evidence for GABA release after
depolarization of E. muelleri cells with 30 mM KC1. I also found that GABA did not
trigger inflation-contraction responses in E. muelleri; its effect was almost the opposite:
there was no movement of the apical pinacoderm, and it is possible that propagated
contractions were prevented. GABA treatment generated only twitches and slow
inflation of the incurrent canals suggesting that its function is different from the action of
glutamate; I did not test for its activity in conjunction with L-Glu treatment, but based on
our analysis, this should now be done. Emson (1966) also found that GABA treatment
had no effect on the behaviour of the demosponge Cliona celata, and suggested that its
role was inhibitory. The varied effects of GABA suggest a different localization of
GABA receptors in different groups of sponges. Other effects of GABA, such as those
observed on the incurrent canal system in Ephydatia cannot be observed in opaque
sponges like Tethya and Geodia, so its diverse roles in sponges are not known. However,
it is possible that in Tethya both glutamate and GABA receptors are combined as they are
in Geodia. Although I did not attempt to stimulate sponges with both GABA and
glutamate, the lack of any inflation/contraction by the aquiferous system is highly
indicative of an inhibitory effect on those tissues. In contrast, doses of 250 uM GABA
did trigger rapid 'twitches' of the incurrent canals in 5 experiments. Further work is
required to observe an inhibitory signal in the presence of glutamate in order to
195
separation its function in specific behaviors.
4.4.4 A potential role for nitric oxide signalling
Nitric Oxide (NO) is an ancient messenger molecule that is a short-lived, diffusible gas
essential in the regulation of numerous physiological processes including nervous
(particularly sensory and motor circuits), vascular and immune systems (Michel and
Feron, 1997; Moroz, 2001; Cristino et al., 2008). NO is released by soluble nitric oxide
synthase (NOS) during the conversion of L-arginine to citrulline, a process that is
constitutively regulated by intracellular calcium, and diffuses directly from cell to cell in
either an autocrine or paracrine manner with a range limited by its half life (Michel and
Feron, 1997; Cristino et al., 2008). NO acts upon soluble guanylate cyclase to increase
the concentration of cyclic guanosine monophosphate (cGMP) that then initiates cellular
processes (Michel and Feron, 1997). Its short life makes localization of NO in living
things impossible, and thus indirect experimental procedures (e.g. of nitrates or nitrites
and radiometrically labeled metabolites, and immuno or pharmacological localization)
are used to detect NOS activity.
Using nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase
staining, NOS has been located in fusiform, so-called dendritic cells of the sponges
Axinella polypoides and Petrosiaficiformis, and NO has been shown to be produced in
response to a heat stress (Giovine et al., 2001). Here I show by NADPH diaphorase
staining that in Ephydatia muelleri, NO activity is located in the cells of the osculum,
canals, and choanocytes and cells similar to the dendritic shaped cells in A. polypoides
located the apical pinacoderm. However it is difficult to dissociate a stress (immune
196
type) response from a contraction inducing or modulating factor because no live
experiments were preformed. Only the osculum in E. muelleri contracted with
application of the NO donor SNAP. In Tethya wilhelma NO (produced by NOC-12)
induced contractions and at the same time modulated endogenous contraction rhythm and
amplitude (Ellwanger and Nickel, 2006). These results are similar to the involvement of
NO in the feeding response in Hydra (De Petrocellis et al., 1999) and in Molluscs
(Korneev et al., 1998), control of swimming in Aglantha (Moroz et al., 2004), and
peristaltic contractions in Renilla (Anctil et al., 2005).
Another method to detect if a NO system is active is to use an assay to induce
production of cGMP by activation of guanylate cyclase by a NO donor (SNAP).
Typically tissue that stains positively for NADPH diaphorase does not accumulate cGMP
with this assay, so it allows for the identification of target cells or tissues that react to
NO. In E. muelleri NO activity was found the osculum and some mesohyl cells of the
apical pinacoderm. Thus in E. muelleri NO may function in modulating contraction of
mesohyl cells of the apical pinacoderm and osculum. In Amphimedon queenslandica a
gene for NOS has been found; however it does not share similarities with either neuronal
or immune NO signaling (Sakarya et al., 2007). Future work is required to develop a
protocol for a behavioral assay that will allow the dissection of the NO signaling system
in sponges to determine whether it is directly involved, responsible for, or modulates, the
propagation of contractions across the sponge.
4.4.5 Evolution ofligand based coordination pathways
The evolution ofligand based receptor systems is of interest because sponges predate the
197
evolution of a nervous system (Jones, 1962; Pavans de Ceccatty, 1962; Mackie, 1970;
Pavans de Ceccatty, 1974b; 1974a; Mackie, 1979; Pavans de Ceccatty, 1979; Mackie,
1990; Nickel, 2004). As most sponges are cellular (only glass sponges are syncytial), it
would likely have been easier for a mechanical or chemical messenger system to evolve
by adapting existing chemical molecules and membrane receptors instead of an electrical
system that would require completely novel proteins such as connexins or innexins. In
his discussion of sponge coordination systems, Jones (1962) favored a mechanical
mechanism of signal propagation such as a pressure front over chemical messenger
mechanisms. However, many researchers working on large sponges describe a lack of
transmission of injury or mechanical stimuli through the body of the sponge (Parker,
1910; 1919; McNair, 1923; Emson, 1966). One problem with mechanical signalling
system is the restriction on body size as it relies on a direct neighbour-neighbour contact
in a local area to pass on information; a chemical messenger system on the other hand,
would allow a faster mode of information transfer, and one that could reach distant points
in the sponge body that were not in direct contact. My results show that the sponge uses
at least in part a diffusible chemical signalling system that coordinates (glutamate,
GABA) or locally modulates (NO) contractions.
4.5 Conclusions
My study demonstrates that Ephydatia muelleri is a useful model system for the
investigation of coordination systems in sponges (Meech, 2008; Kosik, 2009).
Experimental evidence for chemical messenger systems has until recently involved the
response of sponges to the application of agonists or antagonists (for review see Leys and
198
Meech, 2006), and for this a small diffusion system (<400 uL) is required for further
analysis of the signalling pathways and second messenger systems; the freshwater sponge
represents an excellent model system for this work. The availability of genomic
information has provided direct identification of specific signaling molecules and
receptors present in sponges. Future expression studies to demonstrate the localization of
receptors in sponge tissues combined with the kind of pharmacological and behavioural
studies shown here promise offers powerful new tools for understanding how sponges
coordinate behaviour.
199
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Chapter 5 : General Discussion
5.1 Overview
All multicellular organisms have mechanisms to coordinate cells to facilitate growth,
reproduction, homeostasis, and behaviour. In higher animals, the whole organism uses a
mixture of hormones, growth factors, transmitters, or electrical signaling to control and
coordinate these processes. At the cellular level, amino acids, peptides and a collection
of diffusible molecules are employed. In plants, electrical signaling controls leaf closure
(Sibaoka, 1962; Wildon et al., 1992) and release of hormones or transmitters control
germination, root growth and regulate stomatal pore closure (Kang et al., 2004). In
unicellular organisms, cell cycle arrests and subsequent morphological alterations for
mating in yeast Saccharomyces cerevisiae occur by the release of mating factors. The
aggregation of slime mold Dictyostelium occurs via the release of cAMP gradient (Segall,
1993; Xu et al., 2005). These examples illustrate that intercellular signaling is
evolutionarily ancient and phylogenetically diverse. There is growing evidence that the
molecules used in communication between unicellular and colonial organisms like
choanoflagellates gave rise to systems in higher animals (King et al., 2003; King, 2004).
Propagated cell-cell signalling systems affect contractile and flagellar systems in
sponges, and may explain how contractions are propagated in the Porifera, the only
metazoans to lack nerve and muscle.
Freshwater sponge juveniles have an easily studied behavior that is very much
like a slow sneeze: in response to specific stimuli they slowly expand and contract the
aquiferous canal system. The contractions are propagated and are comparable in many
208
ways to peristaltic contractions of the vertebrate gut and to contractions involved in
movement, gamete release and feeding in tubular invertebrates (e.g., the hydrozoan
gastrovascular cavity). Because sponges are the only metazoans in which conventional
muscle and nerves are not known, the topic is one that has intrigued scientists for over
two centuries. However technical limitations have hampered earlier research such that
mechanisms of coordination in cellular sponges are still largely obscure to this day. In
this thesis I have taken advantage of a previously established system - the sponge
juvenile - to gain insights into the origin of the communication and coordination
mechanisms of higher metazoans. Specifically I have: a) described how contractions are
propagated throughout the sponge body and detailed the roles of different regions of the
sponge in orchestrating the behavior of the whole animal; b) re-examined the morphology
of the sponge to understand what cellular components are available for carrying out
coordination and contraction; and c) tested the function of several possible signalling
molecules in mediating the propagation of contractions of the aquiferous canal system.
5.2.1 Behaviour in cellular sponges
A great variety of movements of the larva, juvenile and the adults fall under the broad
description of sponge behaviour. Sponge larvae respond to stimuli, gravity, light, or a
chemical plume, by changing the position of cilia on one pole (Leys and Degnan, 2001;
Elliott et al., 2004); this behaviour is considered to be for habitat selection, and will not
be discussed any further in this thesis. For the adults, activity or behaviour may involve
either contractions of the whole body or crawling by individual cells, and is involved in
maintenance in good flow regimes for feeding, growth and reproduction.
209
Adult sponge behaviour includes orientation to flow or to a chemical gradient,
cell rearrangements, locomotion by crawling or by tissue extensions, creeping, and
mechanical adaptability of mesohyl stiffness. Spongia sp. has been observed to orientate
to uni-directional flow rotating as much as 90°, which results in significantly higher
growth rates (McDonald et al., 2003). Numerous sponges have been observed to migrate
towards higher flow in order to maximize food capture (Bond, 1992; McDonald et al.,
2003) or towards a chemical gradient such as cAMP (Gaino and Magnino, 1996), which
requires movement of the sponge as it grows. Bond and Harris (1988) showed that
marine and freshwater sponges actively locomote across the substrate at 4 mmday"1 and
cells can crawl up to 160 um-hr"1. Bond (1992) found that cells in the sponge
continuously moved and rearranged themselves, which caused the internal morphology to
change substantially within hours. The most active cells were mesohyl cells archeocytes or lophocytes, and the outer margin pinacocytes, but choanocytes and
pinacocytes, which line the aquiferous canals, were always grouped together and moved
as a unit. In Tethya wilhelma, the movement of the sponge is by specialized contractile
body extensions that allow the sponge to crawl around the aquarium, without
reorganizing the whole sponge, at up to 186 um-h"1 (Nickel, 2006). Chondrosia
reniformis, forms long outgrowths from the adult body, which allow the sponge to adapt
to environmental changes by localized creeping locomotion and asexual budding
(Bonasoro et al., 2001). The mechanism that controls this behavior is the ability of the
sponge to stiffen its mesohyl when touched, but without the use of contractile cells. This
biomechanical change is under the physiological control of cells releasing a stiffening
factor that is calcium dependent (Wilkie, 2002; Wilkie et al., 2004; Wilkie et al, 2006).
210
However, in adult sponges these behaviors are slow processes that require great
remodeling of the sponge body, as in migration of the whole animal, or contraction of
processes, neither of which allow for examination of fast responses to external stimuli.
The principal sponge 'behaviour' this thesis describes involves a whole organism
event. Other activities are also documented, and it is not known if those are reflexes or
convulsions, or represent an endogenous and responsive behaviour that is not yet fully
understood. The principal behaviour exhibited by Ephydatia muelleri consists of a highly
orchestrated series of events starting with constriction of the tip of the osculum, which
leads to dilation of excurrent canals and is followed by closing (in unison) of fields of
ostia in the apical tissue just prior to contraction of the choanosome, apical tissue and
osculum. Relaxation of the animal returns the osculum, canals and the apical tissue to
their normal state, and three such coordinated 'inflation-contraction' responses typically
follow a single stimulus.
Whole organism contractions had only previously been described in sponges by
Gaino et al. (1991) in calcareous sponges and Weissenfels (1990) in demosponges as
"condensation" rhythms. Most reports concluded that the phenomenon was a local event
and was not propagated or coordinated across the whole animal (Mackie, 1990). Another
sponge has been shown to have very similar contractile behavior to that of Ephydatia.
Tethya wilhelma a spherical marine sponge carries out rhythmic contractions of the entire
body (Nickel, 2004), contractions occur in a day-night periodicity and propagate over the
pinacoderm at maximum rate of 12.5 u m s 1 . Bond (2003) has also shown that explants
of both calcareous sponges and demosponges exhibit contractions, but it is unknown if
there is coordination in these events.
211
Given that sponges seem to use the myocyte lined canals as a type of hydrostatic
skeleton, the evidence provided by all of the above observations suggests that control
over a hydrostatic skeleton evolved prior to the origin of nerves and true muscle. The
uniformity of sponge behaviour (global contractions) may not have previously been
appreciated in part due to the great diversity of methods used to test and document
endogenous and stimulated responses in sponges.
5.2.2 A note on methodology for behavioural assays.
Although researchers have tried using electrical, chemical and mechanical methods to
stimulate sponges for over two centuries, it is still not known exactly how the sponge
responds to these stimuli other than by contracting the osculum. In this thesis I have
shown that shaking of the sponge and addition of ink into the medium stimulates
contractions; however, these methods are not possible to use in a perfusion chamber for
testing small volumes of molecules or for stimulating specific areas of the sponge.
Different sponge species may have different thresholds for particles, which makes the use
of stimulation by Sumi Ink not easily transferable to other species. My field observations
(Appendix 1) suggest that newly logged drainage basins typically have only one species
of sponge (Spongilla lacustris), whereas older logged drainage basins have up to 4 or 5
species in a lake suggesting that sediment load dictates species abundance.
In later experiments I was able to use 75-80 uM glutamate as a trigger of the
inflation-contraction behaviour. However it would be useful to develop electrical
stimulation by either spot or field application because this would be an objective method
to stimulate the behavior and would also be amenable to use in a perfusion rig. The
212
application of arachadonic acid would also be interesting to experiment with as it is
hypothesized to mimic the effects of mechanically disturbing the sponge by chemically
deflecting the membrane (Zocchi et al., 2001). Another measurement technique that
requires some development is the use of thermistor flow probes that are small enough to
detect the flow out of an osculum or near the ostia. The ability to measure flow would
allow an objective measure that could be quickly compared to the time consuming frame
by frame measurement of diameters of the excurrent canals.
5.3 Sponge morphology.
To truly understand how coordinated contractions occur one must have a sound grasp of
the construction of the sponge body. Because of the complicated nomenclature of sponge
anatomy, their problematic taxonomy and only partial descriptions of sponge structure
the present understanding of the basic morphology of the adult sponge is poor. Using a
combination of light, scanning electron, epifluorescent and confocal microscopy I have
re-examined the morphology of the juvenile freshwater sponge Ephydatia muelleri
(Demospongiae, Haplosclerida, Spongillidae). The sponge is composed of three distinct
functional regions - ectosome, choanosome, and osculum - whose behavioural
interactions are coordinated to carry out contractions of the whole animal. Functional
regionalization of the sponge has not been previously emphasized. The functional
integrity of sponge epithelia has not previously been recognized. In the apical
pinacoderm a vast network of condensed actin tracts traverse the entire length of the
sponge. The nature of contractile cells in sponges is very poorly understood; this new
perspective offers a clear view of how the surface tissue of the sponge can be so active.
213
The osculum vents water from the choanosome and interestingly all endopinacocytes in
the osculum, and a few within the excurrent canals possess a pair of short presumed
sensory cilia that may be used to detect water flow. These cell types in the choanosome
and osculum somewhat remarkably have not previously been reported, despite many
years of investigations by other researchers. These various cell types illustrate the sponge
has a far more differentiated cellular and tissue complement than previously appreciated.
5.5.7 Cytology of contractile cells.
All contractile cells have structural (tubulin, actin) and motor (dynein, myosin) proteins
that allow cell crawling, cytoplasmic streaming, anterograde transport and cell
contractility to occur (Lodish et al., 2003). The concept of multi-unit smooth muscle
represents a useful analogy for understanding sponge myocytes or contractile pinacocytes
(epitheliomyocytes). Note that some authors also call contractile cells actinocytes due to
the presence of actin filaments; but, considering all cells have actin this term is
inappropriate and the original term, myocyte, which is better suited for this cell type.
Similarities exist in the structure of sponge contractile cells, and in their arrangement into
a single tissue or epithelium, to that of the smooth muscle lining of the intestines of
vertebrates (Randall et al., 2002). Smooth muscle that is not coupled by gap junctions is
used to regulate the diameter of vessels, and is involuntarily controlled by hormones,
diffusible gases or the autonomic nervous system. Interestingly, smooth and striated
muscle have been identified in Cnidaria and Ctenophora, whereas Placozoa possess only
a syncytial contractile cell type, the fibre cell (Hernandez-Nicaise et al., 1980; Behrendt
and Ruthmann, 1986; Cario et al., 1995; Seipel and Schmid, 2005). Sponge myocytes
214
may be considered similar to primitive smooth muscle because of the presence of two
sizes of filaments (thick and thin) as in smooth muscle fibers (Bagby, 1965; Nickel,
2004), and by the adhesion plaques connecting neighboring endopinacocytes that were
identified in Chapter 3. In thick-walled sponges, myocytes are organized into sphincters
in canals that control flow of water into and within the aquiferous canal system (Pavans
de Ceccatty, 1962; Reiswig, 1971; Pavans de Ceccatty, 1974a; Figure 5-1). Thin-walled
sponges - like Ephydatia muelleri - lack sphincters in the canals, but the epithelial cells
that line the canals, the pinacocytes, and cells in the apical pinacoderm, porocytes, are
thought to serve the same function of regulating water flow (Pavans de Ceccatty, 1974b;
de Vos and Van de Vyver, 1981; Elliott and Leys, 2003; Elliott, 2004; Elliott and Leys,
2007; Figure 5-1). Nevertheless, while both actin and myosin have been identified in
different sponges (Kanzawa et al., 1995), so far the correlation of myosin with actin
filaments has not been shown, largely due to the minute diameter of pinacocytes (1-200
nm) and thus the difficulty of visualizing actin and myosin. Actin may be labeled with
phalloidin or labeled with heavy meromyosin (e.g., Bagby, 1965), but immunolabelling
of myosin is not so easily done due to lack of cross reactivity. The best evidence for an
actin-myosin-based contractile system comes from the distribution of actin filaments
along presumed lines of tension in the apical tissue, and the result that depolymerization
of actin with cytochalasin B prevents contraction of the apical tissue and canal system as
shown in Chapter 2.
The cytology of the pinacocytes varies depending on their location in the sponge
body. Exopinacocytes form the outer layer of the sponge lining the apical and
basopinacoderms. Endopinacocytes line the inside of the sponge, the inner layer of the
215
Figure 5-1. Theoretical diagram showing the body organization of the contractile and
coordination systems in a thin versus thick walled sponge based on Figure 2 from Pavans
de Cecatty (1974b). Sponges are lined with exopinacocytes on the exterior and
endopinacocytes line the incurrent (IC) and excurrent canals. Choanocytes (ch) are the
main filtering cells and are responsible for water movement in the sponge (arrows).
Three sectors represent, from left to right and symmetrically with respect to a horizontal
axis, the increasing volume of the mesenchyme which is: 1) slightly developed in thinwalled species, 2) thicker in other species, 3) well developed in thick-walled species. The
mesenchyme contains amoeboid, fixed and contractile cells (black). The extracellular
matrix within each sector is indicated by density of stippling.
216
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217
tent and aquiferous canal system. The exopinacocytes have a pentagonal morphology
with an actin cytoskeleton only robust at the cell periphery. The endopinacocytes of the
apical tissue have an elongated, holly-leaf like morphology with actin bundles that
connect to adjacent cells through adhesion plaques. Endopinacocytes of the aquiferous
canal have a pentagonal morphology similar to the exopinacocytes, but differ in function
as they are contractile. However, it is still unclear how the actin cytoskeleton is
organized in these cells. It may be a more diffuse network rather than a few condensed
tracts. The porocytes of the apical tissue are remarkable cells that have a hole (ostium) in
the middle of the cell body with a ring of actin that closes of the ostium.
In future work it would be important to identify myosin (myosin II heavy chain)
thick filaments and characterize the adhesion plaques. It would be very beneficial to
know if these plaques contain focal adhesion components such as a-actinin and vinculin
in the freshwater sponge, which would provide support for the hypothesis that sponges
have primitive smooth muscle that is similar to the smooth muscle in observed in higher
metazoans.
5.3.2 Ciliated and sieve cells.
The main cell types that can be observed using SEM, TEM, thick section and video light
microscopy are archeocytes, pinacocytes (exo-, endo- and basopinacocytes), sclerocytes,
choanocytes, myocytes and porocytes. Unfortunately it is difficult to ascertain the
morphology of the collagen producing cells because they look like archeocytes except
they have a collagen fibril in a vacuole. In sandwich preparations, cell behavior can be
well documented as described in Chapter 2. Although most cells types in sponges have
218
been well described, these descriptions come from a range of sponges (e.g., Simpson,
1984), and do not necessarily apply to what is seen in the freshwater sponge with its thin
walls and slight body. This is important for experiments that deal with release of
transmitter molecules, and for developmental studies looking at sponge body
construction.
In studying the tissues of Ephydatia muelleri I found two new cell types, ciliated
pinacocytes and sieve cells. The ciliated pinacocytes are found sporadically in the
excurrent canal system, but completely cover the inside of the osculum. These cells
resemble primary cilia from invertebrates such as protuberance bivalves (Schaefer, 2000)
and the vertebrate kidney and brain sensory cells (Arkett and Mackie, 1988; Pazour and
Witman, 2003). The number of cilia varies from 1 to 2 in vertebrate cells (Pazour and
Witman, 2003); in various sponge oscula, there are up to 4 cilia per cell (unpublished
data, Elliott and Leys). Sensory cells described along the manubrium of jellyfish, tunic
of ascidians, and dorsal papillae of holothuroids all have a collar of microvilli around a
cilium, and are all thought to detect water movement (Arkett and Mackie, 1988;
VandenSpiegel et al., 1995; Pazour and Witman, 2003; Caicci et al., 2007; Burighel et
al., 2008). The ciliated sponge cells have no collar around the cilium and in this way
much more resemble vertebrate sensory cells.
The other new cell type I have described is a cell with multiple holes I term a
sieve cell. Sieve cells are located in the incurrent canals and at the entrances to
choanocyte chambers, where they form a mesh-like structure across the canal. These
cells may be involved in contraction of the incurrent canals or possibly of the choanocyte
chambers; however, it is unknown how they modify flow into the chambers. Future work
219
is required to confirm that the ciliated cells are sensory and to determine the function of
the sieve cells.
5.3.3 Do sponges have an epithelium?
The absence of extensive septate junctions and basement membranes are the principal
reasons why sponges are not considered to possess conventional epithelia. Sponges are
not thought to have tissues because those arise during gastrulation and sponges aren't
thought to undergo gastrulation. Generally, sponge epithelia are delicate, and difficult to
fix for electron microscopy. However, although many electron micrographs show that
cell contacts are tenuous (e.g., Uriz et al., 2001), good fixation techniques have illustrated
dense cell-cell interactions and a number of cell junction complexes that may contain
components of septate junctions as in other invertebrates (Pavans de Ceccatty et al.,
1970; Garrone et al., 1980; Lethias et al., 1983; Boury-Esnault et al., 2003). However,
many non-spongologists do not consider these as good enough evidence as they lack
robust cytoskeletal structure associated with the junction and most cells are still thought
to be loosely attached (Tyler, 2003).
Future work to determine whether sponges have septate junctions that function to
seal a tissue for compartmentalization should combine the current morphological
evidence with new physiological and molecular biological data. The physiology of
sealing must be pursued in order to understand how these junctions create a sealed
epithelium to allow for communication signals not to be lost to the outside environment.
Fluorescent tracers have now shown that for the most part the tissues are static (Adams
and Leys, unpublished data).
220
Figure 5-2. Diagrammatic drawings of possible synaptic morphologies found in the
freshwater sponge primitive synapse based on the summary of data from the Cnidaria
from Westfall (1996). A) Interneuronal synapses found in the Cnidaria. B)
Neuromuscular synapses found in the Cnidaria. C) Unusual synapses found in the
cnidaria.
A
Interneuronal Synapses
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2-way
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B Neuromuscular Synapses
dense-cored
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postsynaptic
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222
5.3.4 Morphology of a 'primitive synapse'.
A true chemical synapse has been identified in all four cnidarians classes (Westfall,
1996), but the simplest synapse formed is a neuromuscular type with a one-way
transmission (Figure 5-2B). The chemical synapse has vesicles associated with one
(unidirectional) or both sides (bi-, reciprocal- or multidirectional) of an electron dense
plasma membrane with a separation of 13-25 nm creating an intracleft region (Westfall,
1996; Figure 5-2A-C). It is difficult to tell if sponges possess either a neuromuscular or
neuronal type of chemical synapse because the presence of postsynaptic specializations is
only hypothesized from molecular data (Sakarya et al., 2007). It seems unlikely that a
novel structure like a chemical synapse appeared de novo without deriving from a
primitive synapse; the primitive synapse may be present in both the Porifera and
Placozoa. The description of genes that form the post synaptic scaffold in Amphimedon
queenslandica indicates that sponges contain half of the genes involved in the chemical
synapse of cnidarians and other animals. The freshwater sponge presents a unique model
for testing whether this 'half complement' of genes can function as a chemical synapse.
Chemical synapses usually have vesicles associated with one or both sides of a
pair of electron dense membranes (Westfall, 1996), but it is unknown what a synapse
would look like in a sponge (Figure 5-2). Lentz (1966) described vesicles next to an
electron dense plaque on a membrane in a calcareous sponge, but could not identify the
transmitter molecules, if any, in the vesicles. Development of new fixation techniques
such as microwave or cryofixation may allow better preservation of these structures.
Given that my experiments in Chapter 4 suggest there is an active glutamate signaling
system, which should require vesicles for proper release, future work should be done to
223
determine localization of glutamate by immunogold and transmission electron
microscopy.
5.4 Sponge Physiology
5.4.1 Physiology of peristalsis in sponges
Sponge contractions can be considered to have the features of a peristaltic system
because the animal behaves as a single motor complex that has a period of relaxation and
a propagated contraction that moves water forward to be expelled out of the osculum.
Peristalsis is a distinctive pattern of contractility whereby smooth muscle contractions
propel food distally through the intestines (Olsson and Holmgren, 2001 ; Randall et al.,
2002). Control of peristaltic gut motility is best explained by the hypothesis of moving
motor complexes controlled by the enteric nervous system. In vertebrates, this comprises
an excitatory signal such as acetylcholine, tachykinin, serotonin, or substance P that
stimulates contraction of the smooth muscle behind the bolus and an inhibitory signal
such as nitric oxide, vasoactive peptide or ATP that stimulates the relaxation of smooth
muscle cells in front of the bolus via motor neurons (Hansen, 2003).
Peristalsis in invertebrates is involved in gut motility: (Leech - (Wrong et al.,
2003; Stent et al., 1979; Anctil et al, 1984), release of gametes {Renilla and Lymnaea De Lange et al., 1998; Renilla and Lymnaea - Tremblay et al., 2004), locomotion
(Calliactis - McFarlane, 1969), and movement of circulation fluids (Renilla - Tremblay
et al., 2004). The closest relative to the sponges in which peristalsis has been studied is
the sea pansy, Renilla where peristalsis occurs in the gastrovascular cavity, a
hydrovascular system that traverses the entire animal, and which is important for feeding
and gamete release (Tremblay et al., 2004). Rates of peristalsis in both Renilla and the
anemone Calliactis vary from 3-100 cms' 1 , depending on the location in the body, and is
under neuronal control (McFarlane, 1969; Anderson and Case, 1975; McFarlane, 1982).
Peristalsis in Renilla is modulated by glutamate, melatonin, RFamides, and GNRH
(Anctil, 1989; 1991; Mechawar and Anctil, 1997; Tremblay et al., 2004; Anctil et al.,
2005). Nitric Oxide (NO) modulates the amplitude and frequency of contractions, while
serotonin and cAMP potentiate contractions (Anctil, 1989; 1991; Mechawar and Anctil,
1997; Tremblay et al., 2004; Anctil et al., 2005).
In Chapter 1,1 showed that cells in the mesohyl arrest crawling as a wave of
contraction passes, suggesting an extracellular signal may pass between cells (Elliott and
Leys, 2003; Elliott, 2004; Elliott and Leys, 2007). This observation was also made by
Ellwanger and Nickel (2006) and suggests that a released molecule is involved in
signalling. This hypothesis is supported by the slow rate of apparent signal propagation
through the tissues (0.3-1 u-m-s"1 m the peripheral canals, 1-4 um-s"1 in central canals, and
6-122 um-s"1 in the osculum), and the known requirement for divalent cations (Chapter 4
and other references therein). In Chapter 4,1 provided evidence for mechanism of
glutamate, GABA and nitric oxide signaling. There are other possible signaling systems
and I address these possibilities here.
5.4.2 Mechanism of signal propagation and contraction.
A signal could be propagated within the freshwater sponge in three ways: mechanical
'tugging' of cell upon cell, via aqueous cell junctions, or by way of a diffusible molecule
(Figure 5-3). However, it is uncertain if the signal propagation of the inflation-
225
Figure 5-3. Proposed pathways of signal propagation in freshwater sponge contractions.
1) Mechanical pathway of signal transmission that involves cells tugging on each other
and activating a calcium (Ca ) stretch receptor. 2) Transmission through aqueous cell
junctions by passage of inositol triphosphate (IP3) or calcium through a gap junction
complex between to cell membranes. 3) Transmission by a diffusible molecule either
adenosine triphosphate (ATP), glutamate, y-Aminobutyric acid (GABA), nitric oxide
(NO), or adenosine 3',5'-cyclic monophosphate (cAMP).
226
Ca2
Ca2
Cell 1
(1) Mechanical
Cell 2
Pulls on
Next Cell,
Stretch
Stretch
Receptor
Receptor
f [Ca
| [Ca2+]
Halothane/Octanol
Disrupts Channels
(2) Aqueous Cell Junctions
/
2
• f[Ca 2+ ]
J
IP3 or Ca
ATP
(3) Diffusible Molecules
ATP
P2Y G-Protein Counted Pathway
M G-Protein—• | [IP3]
•|[Ca2+]
ATP
• •
[Ca2+]
Ca
/
ATP
P?Y
P2X Ca]
Calcium
Ion Channel
NO
|[Ca 2 + ]
NO S y n t h a s e — • j r N O l -
Guanylyl Cyclase
NO
Glutamate
/GABA
"•|[Ca2+]
L-glutamate •
GABA
L-glutamate/GABA
Receptor
cAMP
CAMP
Figure 5-3.
>
?-
•>
9
[Ca2
or [C1]
t
contraction cycle is by one mechanism or involves a combination of all three.
Mechanical signal propagation can be defined as a mechanical pull on the adjacent cell
that pulls on the next. This is probably not the primary mechanism, but its involvement is
unknown during the inflation-contraction cycle because it is difficult to dissect or damage
portions of the sponge body due to the requirements of a hydrostatic skeleton for signal
transmission. An intracellular signal passed through an aqueous junction could propagate
a signal, not in the same way as an action potential, but through the passage of IP3 or Ca
+
through a hemi-channel or an innexin-like channel. This is not likely since no ultrastructural evidence for gap junctions exist in the demosponges. Therefore, the most
likely candidates for possible propagation of a signal between cells is by way of a
diffusible molecule such as glutamate, GABA, NO, ATP, or cAMP.
Indirect evidence, from experiments that have simply added molecules and
observed a reaction, suggests that sponges possess neurotransmitter and neuromodulator
molecules (Emson, 1966; Pavans de Ceccatty, 1971; Ellwanger and Nickel, 2006; 2007).
Histochemical assays of sponge tissue have demonstrated the presence of
acetylcholinesterase, monoamine oxidase, epinephrine, norepinephrine, 5hydroxytryptamine, and serotonin (Lentz, 1966; Weyrer et al., 1999). Pavans de
Ceccatty (1971) found that adrenaline and acetylcholine increase the number of localized
contractions in Euspongia officinalis. However, these drugs did not initiate contraction
waves nor cause the closure of the osculum and it was interpreted that they work as
synergistic exciters of spontaneous rhythmic contractions, actions similar to those in
smooth muscle of both invertebrates and vertebrates (Pavans de Ceccatty, 1971). A
functional experiment performed on cells isolated from Geodia cyndonium showed that
228
in response to glutamate intracellular calcium increased; further molecular and
pharmacological work identified a metabotropic glutamate/GABA receptor (Perovic et
al., 1999). In vitro experiments on both Tethya wilhelma and Ephydatia muelleri suggest
that a separate receptor exists for GABA and glutamate signaling systems. This is
corroborated by the genetic analysis of Amphimedon queenslandica in which 8 separate
glutamate receptor and 3 GABA receptor sequences have been identified, but as of yet
not isolated (Sakarya et al., 2007; Richards et al., 2008).
5.4.2.1 Metabotropic glutamate signalling pathway
In my research I was able to show that glutamate works as a signaling molecule in the
sponge, both by triggering and propagating the contractions (Chapter 4). When blocked
by L-AP3, calcium free media or Kynurenic acid, no propagation of inflation-contraction
cycle occurred. When the sponge was incubated in AP3 no propagation of the inflation
contraction cycle occurred, but interestingly the osculum and apical pinacoderm could
still contract. The difference in the reaction to the addition of glutamate suggests that the
osculum and apical pinacoderm may possess different glutamate receptor populations.
Thus glutamate is a good candidate for propagation of the signal for the inflationcontraction cycle; however, more experiments are required to further elucidate its
involvement in this behavior. Although I know that glutamate induces the inflationcontraction cycle in a dose dependent manner and is blocked by both an antagonist and
competitive agonist, the family these receptors belong to is still unknown. Unfortunately,
due to the high cost to use class-specific drugs in a volume needed to test on sponges it
may be useful to both dissociate them into single cells and hope that they keep their
229
phenotype or to use a whole sponge in a perfusion rig by reducing the volume of the
testing chamber to 400 uL. Another tantalizing thought is that if glutamate is used as a
signaling molecule it must be in vesicles to be an effective transmitter. Therefore it is
possible that use of a gluteraldehyde-glutamate conjugated antibody will show pools of
glutamate ready to be released. Along with the presence of vesicles, glutamate
transporters must take up the glutamate for recycling so an exchanger or transporter must
be present on cells that contract or on cells adjacent to the pinacocytes. Finally, further
behavioral experiments are required to find blockers that prevent the contraction of the
osculum and pinacoderm and to investigate independence between the osculum and
apical pinacoderm.
5.4.2.2 Metabotropic GABA signalling pathway
In Chapter 4,1 carried out experiments that showed a very different role for GABA than
glutamate. When sponges were exposed to GABA no inflation-contraction cycle
occurred, rather only a fast twitch covered the sponge body or the incurrent canals
contracted (Figure 5-4). There are different possible interpretations to this. First clearly
GABA does not trigger contractions in the sponge (only a gradual 'cringe' was seen in a
couple of instances), in contrast to that observed by Ellwanger et al. (2007). Second,
GABA receptors could line incurrent canals or be on the unusual sieve cells in the
incurrent canals, and cause those to contract. Third, and more intriguingly, GABA could
in fact inhibit contraction, and as such may oppose the glutamate signal in the freshwater
sponge. The pharmacological experiments are not simple to do because of the constant
need to determine the osmotic effects of the drug and counter that with an appropriate
230
Figure 5-4. Possible pathways for signal propagation to occur in the freshwater sponge.
The glutamate system consists of the metabotropic glutamate receptor (mGluR) that is
stimulated by the glutamic acid (L-Glu) which is connected to a G-Protein pathway to
release calcium (Ca2+) by a secondary messenger system. This system is blocked by the
application of 2-amino-3-phosphonopropionic acid (AP3) and kynurenic (KYN). The yaminobutyric acid (GABA) system consists of a metabotropic GABA receptor
(mGABA) that prevents the contractions from occurring through a secondary messenger
system. Contractions stimulated by the addition of glutamate and shaking are reduced in
Ca2+ free media and abolished in CaMg Free media.
231
I GAB A
Ca2* Channel
Figure 5-4.
232
control, and thus I did not manage to test a number of antagonists, such as Bicuculline or
Picrotoxin that could have been used. It would also be useful to observe production by
nuclear chemistry (radio-labelled) of glutamate and localize it to specific cells by
immunolabelling. Future work would involve blocking GABA receptors with
antagonists and with the removal of chloride from the medium. If the GABA system
does indeed function in the inflation-contraction cycle by inhibiting body regions in the
sponge from contracting, the result of disrupting the receptors would be spastic
contractions throughout the sponge body. As with glutamate receptors, western blots to
test antibodies and to isolate and clone proteins involved in GABA synthesis this would
be worthwhile. Proteins that would be worthwhile seeking are glutamate decarboxylase,
vesicular GABA transporter, and the metabotropic GABA receptor. It is noteworthy that
in a related sponge Amphimedon queenslandica one GABA receptor was cloned
suggesting a limited role of GABA in the behavioral response in sponges.
5.4.2.3 Nitric oxide signalling pathway
Nitric oxide (NO) is a good candidate for a diffusible molecule involved in the
propagation of a signal for contraction because it is a ubiquitous signaling molecule in
contraction responses (Figure 5-4). In vertebrates, nitric oxide is involved in neuronal
signaling, aneuronal signaling in vascular tone control, and is a potent molecule in
immune response signaling cascade. It is known that nitric oxide is produced in response
to a heat stress in sponges (Giovine et al., 2001) and is involved in modulating rhythmic
contractions (Ellwanger and Nickel, 2006). I was able to show that when sponges were
exposed to a nitric oxide donor the osculum contracted, but no other activity was
233
observed in the sponge. A cGMP assay showed that addition of a nitric oxide donor
caused an increase in cGMP in the mesohyl cells of the osculum, apical pinacoderm, and
excurrent canals. It is likely that NO is a candidate for modulating contraction of
mesohyl cells and osculum that needs to be confirmed by a behavioral assay, which
would allow us to confirm if it is involved in the contractions or is a possible modulator
(Figure 5-4). If these transmitters are active in the sponge I hypothesize that they work
through a paracrine mechanism in a primitive synapse as suggested by the molecular
characterization of post synaptic scaffolding proteins in a related sponge (Sakarya et al.,
2007).
To concretely show that nitric oxide is active in the freshwater sponge, behavioral
experiments are needed. The addition of a NO donor such as SNAP or L-NMMA nitric
oxide synthase inhibitor to bath and perfusion experiments should be tested to observe
the effect NO has on the inflation-contraction cycle. From the initial results of the cGMP
and NADPH diaphorase staining further investigations should examine the exact cells
that label and seek whether co-localization occurs. Further development of an antibody
from the one sequence of nitric oxide synthase described from Amphimedon is required to
understand the evolution of the nitric oxide system and determine whether the nitric
oxide system shows affinity to the neuronal, epithelial or immune family of vertebrate
class genes.
5.4.2.4 Other signaling molecules
Other signaling molecules that may be involved in sponge contractions should be
explored. Prime candidates are ATP, glycine, taurine, melatonin, GNRH, RFamides,
234
biogenic amides, and cAMP. All of these compounds have activity in cnidarians (KassSimon and Pierobon, 2007) and further investigation is required in the Porifera and
Placozoa. Future work relies on a combination of strategies such as HPLC-MS and
pharmacological manipulations to understand if and how these molecules function in the
freshwater sponge.
ATP could be involved in signal propagation in a manner similar to the way it
diffuses between astrocytes (Nedergaard, 1994). ATP signaling could exist in the sponge
by a mechanism of either mechanical or chemically induced release of ATP that diffuses
to other cells that are not in direct contact. This diffusion in astrocyte signalling is on the
same scale as the rate of propagation occurs during the inflation-contraction cycle. I
suggest that ATP could work through a G-protein coupled purinergic receptor (P2Y)
propagating the contraction through the sponge body. Future work requires the
application of ATP to the medium to observe a behavioral response, followed by the
removal of naturally released ATP by the use of apyrase. Lastly, the effects of
antagonists that interfere with ATP signal propagation such as PPADS (pyridoxalphosphate-6azophenyl-2,4-disulfonic acid tetrasodium), which blocks P2X calcium ion
channels, and Suramin, which blocks P2Y G-protein coupled release of IP3 increasing the
cytosolic calcium concentration, should be explored.
5.4.2.5 Calcium dynamics
Evidence for the role of calcium in regulating cell movement and contraction in sponge
cells is based on the absence of cell crawling or contraction if calcium is omitted from the
external medium (Prosser, 1967; Lorenz et al., 1996). It is suggested that a calcium wave
235
is transmitted from cell to cell either mechanically by tugging on adjacent cells or
chemically by a diffusible molecule (Jones, 1962; Pavans de Ceccatty, 1989; Leys and
Meech, 2006). These effects are similar to those observed during contractions in vascular
smooth muscle cells and in astrocytes in the absence of gap junctions (Nedergaard, 1994;
Anderson, 2003). The rate of propagation of the contractile wave (1-4 urns-')
muelleri is slightly slower than that of astrocytes especially if they are gap junction
coupled. The development of a protocol using the calcium indicator Fura and a diffusion
chamber mounted on a microscope is required to evaluate this hypothesis in either the
whole animal or cell preparations. My initial work on this has shown that Fura-2 AM
ester is taken up by cells; however, the calcium signals from the contractile pinacocytes
are lost due to the high cellular activity of the choanocytes. This may require sponge
cells to be dissociated and imaged as was done with the first astrocyte cell culture
preparations to allow visualization of calcium dynamics in an artificial preparation.
5.5 Concluding remarks
The novelty and significance of the results presented in this thesis along with the future
work described above will make the juvenile sponge, Ephydatia muelleri, an important
model system for testing hypotheses of sponge physiology and development, which is at
the forefront of molecular revolution of sponge research. This research shows that even a
simple organism like a sponge has multiple signaling systems that provide insight into the
type of signalling systems that evolved in the descendents of the earliest metazoans on
earth and which predated the existence of nerves. Due to its many attributes, the juvenile
sponge is a promising aneural preparation for the study of signaling systems, which is
236
paramount to the understanding the evolution of cellular communication used in higher
metazoans today.
237
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Appendix 1 : Field observations of freshwater sponges in lakes
surrounding Bamfield Marine Sciences Center
The gemmules of the freshwater sponge Ephydatia muelleri were collected in Frederick
Lake 30 minutes from Bamfield just before the turn-off for the Bamfield Main logging
road. The lake is accessed from the south boat launch area just off the main road. The
sponges are typically located along the south facing part of the lake flanking both sides of
the land that juts out on the right-hand side of boat launch. The water level can vary 2-5
feet from the summer level depending on the amount of rain that has fallen in the wet
period (Oct-Dec). The highest densities of sponges occur in the high fetch regions where
deadfalls have accumulated into snags on the south facing shore (Figure 1).
Sponges that gemmulate regularly are found in the first ten feet between the
shoreline and the soft sediment weed bed on fallen logs or rocks. Sponge gemmule
patches of E. muelleri tend to grow half exposed or completely covered from the sun
under trees or under rocks. The patch form can be circular, encrusting or long encrusting
tracks along the tree limbs. The patches are identified by loosed packing of the yellow or
yellow-green gemmules in a single monolayer with about 1-2 mm spacing. Gemmule
patches over 3 cm in diameter are deemed harvestable and only a lA to % of the patch is
removed. Sponge gemmule patches are collected into Ziploc bags with a flat sharpened
dive knife below the water to prevent the entrapment of air in the sponge skeleton. A
typical snorkel lasts one hour where 0-10 collected sponges are found and a collection
bag will tend to have 1000 gemmules per 4 cm2 patch. However, usable sponge
gemmules compose 40-70% of the patch collected after sterilization.
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The gemmules were identified to species in the field by a crude spicule
preparation and size of gemmules from sponge skeleton. The spicules of E. muelleri are
spiky not smooth when viewed under the microscope. The gemmules will typically range
in size from 100 um to 700 urn.
Sponge sampling of lakes and rivers around Bamfield Marine Science Center
occurred in an area within 2hr from the main logging road. Eighteen lakes and 2 rivers
were sampled for Spongilla lacustris, Ephydatia muelleri, and Eunapius fragilis (Table
1). Only Frederick Lake contained all three species and the other lakes had only
Spongilla lacustris in low to high densities depending on water quality.
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Table 1. Sponge Survey of lakes and streams around Bamfield Marine Sciences Center.
Site
Number
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
Water Body
Frederick Lake
Pachena Lake
Sugsaw Lake
Rosseau Lake
Black Lake
Newstead Lake
Blue Lake
Dorothy Lake
Flora Lake
Arthurs Lake
Crown Lake
Sarita Lake
Darlington Lake
Francis Lake
Blue Creek
Hawthorn Lake
Lizard Lake
Lizard Pond
Sarita River
Pachena River
Ephydatia
muelleri
Abundant
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
Spongilla
lacustris
Abundant
Abundant
Abundant
Abundant
Few
Abundant
Abundant
Few
Abundant
Abundant
Abundant
Few
Abundant
Abundant
Abundant
Abundant
Abundant
Abundant
Abundant
None
Eunapius
fragilis
Few
None
None
None
Few
None
None
None
None
None
None
None
None
None
None
None
None
None
Few
None
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Figure 1. Bathymetric map of Frederick Lake indicating the boat launch and the green
line indicates where the sponges are to be found.
252