A New Flow-Regulating Cell Type in the Demosponge Tethya wilhelma – Functional Cellular Anatomy of a Leuconoid Canal System

Abstract

Demosponges possess a leucon-type canal system which is characterized by a highly complex network of canal segments and choanocyte chambers. As sponges are sessile filter feeders, their aquiferous system plays an essential role in various fundamental physiological processes. Due to the morphological and architectural complexity of the canal system and the strong interdependence between flow conditions and anatomy, our understanding of fluid dynamics throughout leuconoid systems is patchy. This paper provides comprehensive morphometric data on the general architecture of the canal system, flow measurements and detailed cellular anatomical information to help fill in the gaps. We focus on the functional cellular anatomy of the aquiferous system and discuss all relevant cell types in the context of hydrodynamic and evolutionary constraints. Our analysis is based on the canal system of the tropical demosponge Tethya wilhelma , which we studied using scanning electron microscopy. We found

Full text

A New Flow-Regulating Cell Type in the Demosponge
Tethya wilhelma
– Functional Cellular Anatomy of a
Leuconoid Canal System
Jo
¨rg U. Hammel*
¤a
, Michael Nickel
¤b
Institut fu
¨r Spezielle Zoologie und Evolutionsbiologie mit Phyletischem Museum, Friedrich-Schiller-Universita
¨t Jena, Erbertstr. 1, 07743, Jena, Germany
Abstract
Demosponges possess a leucon-type canal system which is characterized by a highly complex network of canal segments
and choanocyte chambers. As sponges are sessile filter feeders, their aquiferous system plays an essential role in various
fundamental physiological processes. Due to the morphological and architectural complexity of the canal system and the
strong interdependence between flow conditions and anatomy, our understanding of fluid dynamics throughout leuconoid
systems is patchy. This paper provides comprehensive morphometric data on the general architecture of the canal system,
flow measurements and detailed cellular anatomical information to help fill in the gaps. We focus on the functional cellular
anatomy of the aquiferous system and discuss all relevant cell types in the context of hydrodynamic and evolutionary
constraints. Our analysis is based on the canal system of the tropical demosponge Tethya wilhelma, which we studied using
scanning electron microscopy. We found a hitherto undescribed cell type, the reticuloapopylocyte, which is involved in flow
regulation in the choanocyte chambers. It has a highly fenestrated, grid-like morphology and covers the apopylar opening.
The minute opening of the reticuloapopylocyte occurs in an opened, intermediate and closed state. These states permit a
gradual regulation of the total apopylar opening area. In this paper the three states are included in a theoretical study into
flow conditions which aims to draw a link between functional cellular anatomy, the hydrodynamic situation and the regular
body contractions seen in T. wilhelma. This provides a basis for new hypotheses regarding the function of bypass elements
and the role of hydrostatic pressure in body contractions. Our study provides insights into the local and global flow
conditions in the sponge canal system and thus enhances current understanding of related physiological processes.
Citation: Hammel JU, Nickel M (2014) A New Flow-Regulating Cell Type in the Demosponge Tethya wilhelma – Functional Cellular Anatomy of a Leuconoid Canal
System. PLoS ONE 9(11): e113153. doi:10.1371/journal.pone.0113153
Editor: David J. Schulz, University of Missouri, United States of America
Received July 11, 2014; Accepted October 20, 2014; Published November 19, 2014
Copyright: ß2014 Hammel, Nickel. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper.
Funding: Funding for this research came from Deutsche Forschungs Gemeinschaft (www.dfg.de) research grant HA 6405/1-1 to JUH. The funder had no role in
study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* Email: joerg.hammel@uni-jena.de
¤a Current address: Center for Materials and Coastal Research, Helmholtz-Zentrum Geesthacht, Max-Planck-Straße 1, 21502, Geesthacht, Germany
¤b Current address: Bionic consulting, Bruckena
¨cker 4, 70565, Stuttgart, Germany
Introduction
Sponges are sessile filter-feeding animals. Accordingly, the canal
or aquiferous system is their most distinct anatomical feature.
Functionally speaking it can be considered the most important
organizational unit besides the skeletal elements which give the
sponge its structure. In accordance with their feeding habits, all
physiological processes in sponges rely on the ability to process
high volumes of water through the body. Only in this way are they
able to obtain the required nutrients and oxygen and get rid of
metabolic waste products.
Research into the biomechanics and fluid dynamics of filter-
feeding and into biological fluid transport systems in general has
revealed a close interdependence between hydrodynamic con-
straints, the micro- and macro-morphology of the cellular elements
involved and, indeed, the structure of the anatomy in its entirety
[1–6]. A number of hydrodynamic constraints and optimality
principles have been suggested to play a role in shaping the general
architecture of the canal system [3], but the key features appear to
be flow resistance and pressure drop [2]. Pressure drop can be
understood as the resistance which fluid encounters when it passes
through a filter. In the incurrent canal system in sponges, small
apertures in the form of ostia and prosopyles contribute
significantly to the pressure drop within the system (Figure 1).
Further on, the apopylar apertures and the microvilli collar of the
choanocyte chambers are also thought to play a significant role
(Figure 1). While the effect of pressure drop in sponges has been
considered to varying extents in general models of flow on an
organismal scale, almost nothing is known about the influence of
cell morphologies on local flow conditions or their implication for
hydrodynamics on an organismal scale. Local flow regimes are of
the utmost importance, however, especially when it comes to
functional considerations such as nutrient uptake and gas
exchange.
From a biological perspective resistance has a significant
influence on two central aspects of filter feeding. On the one
hand it determines the power required to move the fluid through
the system. On the other hand it determines, in the context of
morphological constraints and anatomy, the flow velocity of the
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fluid in the canal system. Particle capture rates are greatly
influenced by the prevailing Reynolds number and are therefore
related to flow velocity and anatomy [7,8]. We are consequently
faced with complex interdependencies between cellular morphol-
ogy and anatomy, energy expenditure and filter-feeding. In order
to understand these complex relationships in sponges we need
detailed information regarding the hierarchy and three-dimen-
sional architecture of the canal system, quantitative morphometric
data pertaining to individual canal segments, flow velocity
measurements and detailed morphological data regarding the
cellular entities involved in the canal system. The morphometric
and anatomical data pertaining to the architecture of the canal
system and the cell types involved then needs to be integrated into
basic fluid dynamic theory in order to gain a deeper and more
detailed understanding of the hydrodynamic situation as a whole
in sponge canal systems. Current understanding is based on
general information regarding leucon-type canal systems
[3,4,9,10] and recent specific morphometric and hierarchical data
pertaining to the aquiferous system [6]. Flow velocity within the
canal system is affected most prominently by the total available
cross-sectional area of every functional unit in it (Figure 1A–C)
[3,4,11]. Slower flow velocities are caused by an increase in total
available cross-sectional area on any given hierarchical level [4,9].
However, the cross-sectional area of single segments on a
hierarchical level is usually small. Overall increases in cross-
sectional area are related to increases in the number of small sized
segments on the respective level [6,12]. As the lower cross-
sectional area of small sized canals is a consequence of their
smaller diameter we can draw from the following two equations a
direct relationship between pressure drop and resistance:
R~
8:g:l
p:r4ð1Þ
DP~Q:Rð2Þ
Where R is resistance, gthe viscosity of the fluid, l the length of a
canal segment, r the canal diameter, DP is pressure drop and Q is
flow. According to equation (1), radius has the greatest influence
on resistance, which allows us to conclude that numerous small
sized canals will lead to high resistance and therefore necessitate a
high level of pumping power. Equation (2) describes the
relationship between pressure drop within the system and flow,
viz. resistance. Sites with high local resistance in the system
contribute significantly to pressure drop, especially when small
sized elements are involved (Figure 1D). All considerations so far
have remained on a local scale, however, focusing on single canal
system elements. In order to come up with a comprehensive
functional morphological interpretation, the complete architecture
of the canal system and the specific sub-elements defined in the
context of hydrodynamics as functional units need to be taken into
account on both the local and the organismal scale. In order to do
this, two fundamental principles of resistance theory have to be
considered. (1) Total resistance for serial segments is the sum of all
the segments included. (2) For segments arranged in parallel, total
resistance is given by the following equation.
Rtot~
1
Xn
k~1
1
Rk
ð3Þ
As a consequence, the high resistance of numerous small sized
canal segments - on any hierarchical level - turns out to make a
much smaller contribution to total resistance on the organismal
scale than indicated by the high individual values.
At present, the model for flow regimes in sponges [9] considers
some of the physical and hydrodynamic constraints mentioned
above [3,4,11], but with regard to morphological and architectural
information is restricted to statistical morphometric data [3].
Modern imaging and analysis techniques have made detailed and
even complete morphometric data available for biophysical
considerations of general canal system anatomy [6,12–14]. The
Figure 1. Scheme of hydrodynamic conditions in different
sections of the leuconoid canal system based on morphometric
and anatomical data on the sponge canal system as well as on
fundamental physical laws in hydrodynamics [3,4,6,9–11,46].
(A) Structural representation of the main canal system elements in the
direction of flow. (B) Schematic diagram of the change of available total
cross sectional area along the flow path. (C) Schematic diagram of flow
velocities in the canal system. (D) Schematic diagram of the change of
pressure drop along the flow path.
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studies in question have revealed that the architectural complexity
of the canal system anatomy in leucon-type sponges is much
higher than previously thought, featuring phenomena such as
bypass elements or highly asymmetric branching which need to be
included in an updated flow model in order to explain canal
system hydrodynamics on a local scale as well as an organismal
one. However, in order to obtain a sufficiently detailed picture of
the hydrodynamics of the canal system to put together a new
biophysical model of flow, data from a single species needs to be
available for all the prerequisites mentioned above. Flow inside the
canal system of sponges is influenced not only by the system’s gross
morphological architecture but subject too to constraints imposed
by cellular elements. Most studies into sponge aquiferous systems
have focused either on the architecture and morphology of the
canal system in general or on the way in which choanocytes work.
The present study aims to provide an overview, from a functional
morphological and hydrodynamic perspective, of all relevant
cellular structures within the leucon-type poriferan aquiferous
system of one exemplary species.
The tropical demosponge Tethya wilhelma SARA
`,SARA
`,NICKEL
&BRU
¨MMER 2001 was chosen as a model on which to assess the
way in which the morphology of cellular elements of the canal
system relates to functional morphological aspects derived from
hydrodynamic constraints. The general architecture of the canal
system had already been examined for this species on an
organismic scale [6,15]. Being one of the rare sponge species
continuously cultivable under laboratory conditions [16–19] and
even exhibiting regular asexual reproduction by budding [20], T.
wilhelma is an emerging model demosponge for various types of
functional investigation including physiological, genetic and
morphological studies.
Morphologically speaking, the following series of elements are
considered the functional modules of the aquiferous system [21]:
Ostia.(sub dermal lacunae).incurrent canals.prosopyls.cho-
anocyte chambers.apopyles.excurrent canals.oscule(s). Ostia
are the microscopic incurrent openings into the system, while the
oscule or oscules are the excurrent openings. The choanocyte
chambers act as displacement pumps and generate the pressure
differential which drives the water through the system [10]. Their
in- and excurrent openings are called the proso- and apopyle.
There are large discrepancies in our current morphological
understanding of the various elements of and cell types involved in
the aquiferous system. Although some cell types (e.g. endopina-
cocytes and choanocytes in particular) have been studied in detail,
thorough comparative cytological studies based on broad taxon
sampling are scarce. The most comprehensive review is to be
found in Simpson’s compendium of sponge biology [21], though
the information in it is unfortunately fairly general. A more recent
and detailed study into cell types in demosponges focuses on
systematic and evolutionary aspects of aquiferous system charac-
ters [22]. Detailed morphological studies of cell types which
contribute to functionally important elements of the aquiferous
system help us, when they consider the hydrodynamic environ-
ment in which such cells are found, to assess their functional role
[23,24]. This applies to apopylar cells (cone cells), central cells and
any other cell type located in hydrodynamically pivotal sites in
choanocyte chambers.
Theoretical and experimental investigations into choanocyte
chambers have shown on the basis of choanocyte arrangement
and orientation that the chambers can be understood as positive
displacement pumps or, in technical terms, as peristaltic pumps
[10,11]. Experimentally and theoretically consistent models for
filter feeding in sponges do exist, though definitive experimental
evidence is still lacking since science currently lacks the technical
observation methodologies for in vivo studies [1,25]. However, in
order to complement our understanding of functional morphol-
ogy, the present study is intended to provide a detailed analysis of
cell types within the canal system of T. wilhelma with respect to
their impact on local flow and consequences for hydrodynamics on
an organismic level.
Results
Canal system compartments and anatomical details
The canal system architecture in T. wilhelma is of the leucon
type with some striking manifestations of specific canal system
elements. The incurrent canal system features voluminous cortical
lacunar sub-dermal cavities. This cortical lacunar network is
connected to an underlying network of sub-lacunar cavities located
at the choanosome/cortex boundary. Both lacunar systems consist
of an extensive network of anastomosing oval-shaped/flat canals.
Branching off from the lacunar- and sub-lacunar cavities, high
numbers of ramifying canals lead into the choanosome. Due to the
roughly globular shape of the body, the canals of the incurrent and
excurrent canal systems are significantly intertwined in the
choanosome region. Within the excurrent canal system the atrium
region stands out by virtue of its volume and can be characterized
as a larger sized canal resembling a vestibule which opens directly
into the outflow opening (oscule) (Figure 2). Depending on the
state of morphological (re-)organization and environmental flow
conditions, varying numbers of oscules are present, from one in
the majority of cases to several in more rare cases.
Ostia
Specimens of T. wilhelma exhibit ostia of varying sizes, with no
direct correlation with body size discernible - at least not in the
specimens investigated here (Figure 3). The diameters of single
ostia in all the specimens studied (N = 10) ranged from fully closed
to a typical maximum of ,15 mm. Ostia greater than this in
diameter were present only in very low numbers. Depending on
environmental flow conditions, ostia appear as single openings, in
small groups or as ostia fields (Figure 3A). Smaller sized ostia are
formed by intracellular pores (Figure 3B, Figure S1), whereas
larger ones are made up like intercellular ostia by groups of several
cells (Figure 3C). In both cases the exopincocytes involved in the
formation of ostia are in direct contact with adjacent exopinco-
cytes and endopinacocytes. Where specimens of T. wilhelma had
been cultured under steady flow conditions over a long period of
time, ostia fields covering the topmost portion of the surface of the
sponge were observable. In this case, ostia were generally larger
(up to 43 mm). Tissue bridges between the ostia usually varied in
length between 5 mm and 20 mm.
Choanocyte chambers
Choanocyte chambers are almost globular in T. wilhelma and
possess one apopylar and one to several prosopylar openings
(Figure 4A). The number of choanocytes within a choanocyte
chamber is dependent on chamber size and body size (,50–90
choanocytes/chamber, 70613 choanocytes/chamber (N = 15
taken from 4 specimens)). The choanocytic prosopyle is formed
by an interstice between adjacent choanocytes which lack
filopodial extensions, which means that the prosopyle itself lacks
any kind of specialized choanocytic prosopylar structure (Fig-
ure 4A).
Prosopyles
Prosendopinacocytes form internal, single-cell pores known as
pinacocytic prosopyles (Figure 4F). The mean diameter of these
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pore-based openings into the choanocyte chambers is about
7.4 mm. The prosendopinacocytes which form the pinacocytic
prosopyle come into direct contact with the basal part of
choanocyte cell bodies (Figure 4G).
Apopyles
The choanocytic apopyle is formed by apopylar cells (Fig-
ure 4B–D), two to three of which (depending on the size of the
choanocyte chamber) form a ring-like structure (Figure 4B). Each
apopylar cell bears a single cilium 3.9 mm in length (Figure 4D). In
a cross-sectional view the ring formed by apopylar cells around the
apopylar opening displays a characteristic double cone shape [26]
(Figure 4C). On the choanocytic face the apopylar cells come into
contact with choanocytes by way of a thin velum which forms the
edge of the inner part of the ring/pore structure. This velum
comes into direct contact with the choanocyte microvilli collar.
The single cilium of the apopylar cells projects into the apopylar
opening (Figure 4B–D). Facing the apopyle the cells connect to an
apopylar pore-forming apendopinacocyte, which in turn touches a
hitherto undescribed cell type spanning the apopylar opening
(Figure 4B, Figure 5).
Figure 2. Schematic organization (A) and habitus (B) of
T. wilhelma
aquiferous system. (A) Potential flow directions in the canal system are
indicated with arrows (after [15]). A color gradient from light to dark blue in the canals indicates the allocation of the corresponding elements to the
incurrent and excurrent system. Due to the presence of bypasses in the canal system flow directions cannot be assigned with certainty to all sections.
This might even cause backflows from the excurrent to the incurrent system. Main features/structures of the canal system are labeled in the scanning
electron micrograph (B) as well as in the schematic drawing (A).
doi:10.1371/journal.pone.0113153.g002
Figure 3. Scanning electron micrograph of an ostia pore field (A), a single ostium (B) and details of ostia in an ostia pore field (C).
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A new mesh-forming cell type within the apopyle -
Reticuloapopylocyte
Reticuloapopylocytes – a previously unknown type of cell - have
a high number of small intracellular pores which give them a mesh
or grid-like morphology (Figure 5). These pores have openings of
about 0.53 mm60.07 mm (N = 82, taken from 1 specimen)
(Figure 5E–F) and are found in an opened and closed state
(Figure 5D). Reticuloapopylocytes, then, are able to adopt a
gradient of opening states from totally open and highly fenestrated
to partially or almost completely closed. When all reticuloapopy-
locyte pores are open, the functional cross-sectional area of the
Figure 4. Scanning electron micrograph of cellular structures in the choanocyte chamber. (A) Overview of a choanocyte chamber
connected to an incurrent- and excurrent canal with the relevant cellular prosopylar and apopylar elements and the location of the new cell type:
reticuloapopylocyte. (B) Circular arrangement of apopylar cells and the position adjacent to reticuloapopylocyte. Hydrodynamic sealing of apopylar
velum and microvilli collar. (C) Arrangement of cilium bearing apopylar cells, choanocytes and reticuloapopylocytes in the choanocytic apopyle. (D)
Detailed view of an apopylar cell with its cilium directing into the flow at the apopyle. (E) Detailed view of the apopylar velum and microvilli collar
contact side which results in a hydrodynamic sealing. (F) Overview of prosopylar openings in the incurrent canal system. (G) Pore cell forming a
prosopylar opening. In the background microvilli collars of choanocytes are visible.
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Figure 5. Scanning electron micrographs of reticuloapopylocytes. (A) View on reticuloapopylocytes from the excurrent canal with adjacent
endopinacocytes and most of the pores open. (B) View on reticuloapopylocytes from the excurrent canal with one cell having most of the pores
closed. (c) Overview of the position of reticuloapopylocytes in the apopyle (cross section through a choanocyte chamber). (D) Detailed view on pores
of reticuloapopylocytes in an open and closed state. (E) Color coded and labeled ferret pore diameter of reticuloapopylocyte. (F) Distribution of ferret
pore diameters in reticuloapopylocytes.
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apopyle equals approximately 50% of the total area which would
be present if the reticuloapopylocyte was absent. Typically, the
cross-sectional area available to flow is much lower. The cell itself
is very thin, usually below 0.5 mm, which is why the high level of
fenestration leads to a grid-like morphology. Where a single
reticuloapopylocyte spans the apopylar opening, it is almost
circular in shape. In the case of larger apopylar openings, two or
more reticuloapopylocytes form a mesh-like covering (Figure 5A–
C).
Using the pore measurements presented in figure 5E–F, we
calculated how reticuloapopylocytes contribute to the resistance of
flow. Taking as a basis the cross-sectional area of pores and entire
cells, we calculated the radius of pores and the radius of the
apopylar opening. For the sake of simplification, we assumed that
both were circular. By putting the measurements presented into
equations 1 and 3, we calculated reticuloapopylocyte resistance to
be 4.12N10
23
Pa s mm
23
. In order to compare this value, we then
calculated the resistance of the same apopyle opening without the
reticuloapopylocyte and found it to be 3.13N10
23
Pa s mm
23
.An
apopylar opening with the same available cross-sectional area as
the reticuloapopylocyte (12.87 mm
2
) would give rise to a single
apopyle with a radius of 2.03 mm and a resistance of
5.45N10
23
Pa s mm
23
. The resistance of an apopyle with a
reticuloapopylocyte is therefore 1316 times greater than that of
the same apopylar opening unaltered. A smaller apopyle with the
same available cross-sectional area as observed in the reticuloa-
popylocyte would lead to a 17-fold increase in resistance compared
to the reference apopyle.
Pinacocytes
The prosendopinacocytes lining the walls of the lacunar and
sublacunar cavities and the incurrent canal walls are less than
0.5 mm thick except for a small swelling incorporating the nucleus.
Their overall shape is irregular and adopted to the local canal
geometry (Figure 6A–D). The prosendopinacocytes in our study
never displayed the T-shaped or umbrella-like morphology
characteristic of exopincocytes (Figure 6F).
T. wilhelma possesses two types of apendopinacocytes which
line the walls of excurrent canals and the atrium region,
respectively. The type present in and around the atrium region
bears a single 5.5 mm60.79 mm (N = 16, taken from 4 specimens)
long cilium (Figure 6C,E). Monociliated apendopinacocytes ex-
hibit a fusiform cell morphology and appear to be arranged in a
highly ordered fashion within the atrium region (Figure 6A,C). As
in the case of prosendopinacocytes, the main cell body is very thin,
usually below 0.5 mm, with the exception of the part holding the
nucleus. Away from the atrium, monociliated apendopinacocytes
become less frequent and non-ciliated apendopinacocytes start to
dominate in lining the canal walls. Non-ciliated apendopinaco-
cytes are no different on the micro morphological level to non-
ciliated prosendopinacocytes.
Discussion
1. Morphology
Ostia. The diameters displayed by ostia in T. wilhelma were
highly variable, ranging from total closure to more than 40 mm
when open. The ability to open and close ostia within a relatively
short period of time for flow-regulating purposes has been
documented in a number of different sponge species (e.g.
[27,28]). For this reason ostia diameters and numbers within
specimens appear highly variable at any given time.
Pinacocytes. Biophysically, pinacocytes encounter a number
of mechanical forces including shear stress and drag which are
generated by flow along the canal system. Some of these forces
result from direct interactions between the fluid and the
pinacocyte surface which in turn contribute to general flow
resistance and the resulting velocity profile. The boundary layer of
the flow profile is particularly important in the context of particle
feeding as it is involved in the slowdown and sedimentation of
particles for phagocytosis along the canal walls [29].
The morphologies of apendopinacocytes, and most likely
endopinacocytes in general, might reflect local hydrodynamics
[30]. For the purposes of comparison, arterial endothelial cells
have been shown under pulsatile but unidirectional laminar flow
to align in the direction of flow [5]. In areas of flow separation
and/or flow reversal (e.g. branching), they adopt an unaligned
polygonal-shaped organization [5]. However, since our knowledge
of local flow regimes in canals is very limited, it cannot yet be
claimed with certainty that there is a direct correlation between
endopinacocyte morphology and flow. Nevertheless, the fact that
apendopinacocytes in T. wilhelma are aligned in an ordered way
in the atrium region in particular is of great interest, for it is
theoretically possible, taking fluid dynamics and morphometric
data into account [6], that flow there might develop a pronounced
unidirectional laminar profile.
T. wilhelma apendopinacocytes in and around the atrium
region are monociliated. A morphologically similar cell type is
characteristic of all Homoscleromorpha [22,31,32]. However, the
monociliated endopinacocytes of Homoscleromorpha bear a much
longer cilium and have been proposed to be actively involved in
flow generation, something which is highly unlikely in T. wilhelma
where the short cilium would make flow generation by
apendopinacocytes relatively inefficient compared to that by
choanocytes [10]. We propose as an alternative that the short
apendopinacocyte cilium in T. wilhelma functions as a stereocil-
ium and is involved in local flow sensing. The fact that the
monociliated apendopinacocytes of the freshwater sponge Ephy-
datia muelleri (LIEBERKU
¨HN, 1856), which are located in exactly the
same position as in T. wilhelma, have recently been demonstrated
to have a sensory function backs up this claim [30]. The nonmotile
primary cilium in Ephydatia muelleri consists of 9 circularly
arranged microtubule doublets (‘‘9+0’’ fashion), but lacks the
central ones (‘‘9+1’’ fashion) characteristic of motile cilia and
flagellae [30,33].
Choanocyte chambers. The choanocyte chambers in T.
wilhelma exhibit two specializations which are presumed to have a
substantial impact on local and global fluid dynamics: (1)
monociliated apopylar cells and (2) reticuloapopylocytes. In T.
wilhelma apopylar cells form a ring-shaped reduction of the
choanocytic apopylar opening which is double cone-shaped in
cross-section. A functional morphological interpretation of the
location of this cell type in a hydrodynamically pivotal site is
discussed below. Apart from their role in preventing back flow, the
function of apopylar cells is currently unclear, especially with
regard to the cilium. However, since the cilium projects freely into
the apopylar opening we propose that it is involved in flow sensing.
Verifying this experimentally, however, will be technically
challenging. As in the case of monociliated apendopinacocytes,
ultrastructural data pertaining to microtubule arrangement might
help to answer this question.
2. Functional Anatomy
Hydrodynamic situation in sections of the canal system
and implications for the function of cell types. The
development of ostia pore fields (see Figure 3), as observed in T.
wilhelma under steady state flow conditions, can be explained as a
result of fundamental fluid dynamic principles. As explained by
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equations 1 and 3 in the introduction, pore fields drastically reduce
the total resistance of the global influx and therefore reduce global
pumping energy costs. Even though the parallel arrangement of
small sized elements in the canal system reduces resistance on an
organismic scale, resistance in each single element remains high.
Therefore, the systemic resistance of individual canal segments
influences the amount of water passing through certain areas of
the sponge body. This can be quantified by the term perfusion, the
amount of water passing through a defined volume of the sponge
body over a given time interval. Consequently, resistance is a
factor which can be used directly to control the perfusion of certain
areas of the sponge body and to adjust local flow. No studies to our
knowledge have yet addressed this aspect of local flow regulation
from a detailed theoretical and experimental perspective. Howev-
er, it seems on the basis of all the available data and fluid dynamics
models that a local regulation of perfusion is possible within
Figure 6. Scanning electron micrographs of pinacocytes. (A) Highly ordered apendopinacocytes in the atrium region. (B) Monociliated
apendopinacocytes in the excurrent canal system. (C) Detailed view of a monociliated apendopinacocyte. (D) Prosendopinacocytes lining the walls of
the incurrent canal system. (E) Detail of the cilium of an apendopinacocyte. (F) Cross section of an exopinacocyte lining the outer surface of T.
wilhelma. Note the T-shaped umbrella like cross sectional morphology with the cell body of the pinacocyte sunk into the extra cellular matrix.
doi:10.1371/journal.pone.0113153.g006
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specific areas of the sponge, and that this is most efficient in
regions which (1) have a significant impact on flow resistance and
(2) can be mechanically modified by the sponge. Both require-
ments are fulfilled when it comes to ostia and the oscule, and in
principle in the case of small sized canal segments too. As T.
wilhelma normally only possesses one oscule, flow theory and the
continuity of flow would suggest that oscule contraction would
only cause very slight variation in local flow. This is supported by
studies into ostia contraction in T. wilhelma (unpublished data)
and other species, which have demonstrated that single ostia can
be contracted individually [34]. Unless new methodologies
become available, however, it will only be possible to demonstrate
this quantitatively and experimentally in a transparent sponge
species which permits in situ high resolution flow measurements to
be taken within the canal system. The question of whether and
how small sized canal segments influence perfusion is closely
related to the pronounced regular body contractions observed in
T. wilhelma. Predicting the effects of canal contractions on local
flow during a contraction and expansion cycle is difficult, as
information on the exact dynamics of canal contractions can only
be obtained indirectly from the overall kinetics inferable from
time-lapse sequences [13,35]. However, local body contractions
and contraction waves across the body have been reported both
for T. wilhelma and other sponges [36] and are presumed to be
related to local changes in canal diameter and to result in changes
in perfusion (see equation 1).
In terms of local hydrodynamics, the most complex functional
unit within the canal system is the choanocyte chamber. From
experimental and theoretical studies into sponges and choano-
flagellates, we know a good deal about particle filtering at the level
of choanocytes (e.g. [2,10,25,37]). However, we still lack detailed
knowledge of flow fields in choanocyte chambers. A schematic
drawing of simulated flow fields is given in figure 7. Hydrody-
namically pivotal sites within the choanocyte chamber are marked
with stars (Figure 7) and refer to structures with a significant
impact on flow resistance. These include the prosopylar openings
(Figure 1D), where resistance is determined by the diameter of the
opening. It is presumed that the small size of these openings causes
flow to accelerate compared to its velocity in adjacent canal
segments. Predicting the situation for choanocytes is difficult as we
lack information on how flow in the near field surrounding the
choanocytes is affected by neighboring cells. In choanoflagellates,
which are morphologically and functionally very similar to
choanocytes microvilli collar height, density, spacing, angle and
flagella length have been demonstrated to be interdependent [2].
The choanocytes in T. wilhelma have a smaller number of almost
erect microvilli which are oriented parallel to each other and can
be expected to reduce resistance to flow. This in turn can be
expected to reduce pressure drop at the level of choanocyte
chambers, if velocity is the fixed parameter or a slower flow an
pumping capacity compared to choanoflagellates if pressure drop
is the reference constant determining flow. Downstream in the
direction of flow apopylar openings form the next anatomical
structure crucial to pressure drop. In T. wilhelma, as in some other
sponges [23,26,28,31,38–43], apopylar cells directly adjacent to
the apopylar opening form a cone-shaped ring structure which
makes contact with the neighboring choanocytes. The exact
function of this structure is hard to pinpoint. Comparative
experimental studies into flow fields around sessile and free
swimming choanoflagellates might serve as a starting point. The
studies in question have demonstrated that the boundary layer
(e.g. the height above the substratum in a sessile choanoflagellate)
has a significant influence on far and near field flow in terms of the
development of eddies [16]. Applying these observations to
choanocyte chambers may suggest that if no additional structures
were present, eddies would develop between choanocytes and the
apopylar opening. The direction of flow of eddies in this location
would be opposite to the direction of outflow and would result in a
significant disturbance of flow at the apopylar opening. In order to
prevent the development of eddies in this location an additional
boundary structure is needed. In T. wilhelma, the cone-shaped
ring of apopylar cells around the opening fulfils this requirement
by forming a ceiling seal with the microvilli collar tips of adjacent
choanocytes, thus seeming to prevent backflow through eddies,
which would significantly reduce local and global pumping
efficiencies.
3. Functional aspects of the new cell type
From a hydrodynamic point of view, reticuloapopylocytes are
the second functional morphological extravagance to be found in
connection with T. wilhelma choanocyte chambers. Their location
in the canal system and their morphology give rise to a number of
hypotheses regarding their function. Reticuloapopylocytes might
(1) serve as filtering devices, (2) be related to passive flow, and (3)
serve as local flow-regulating devices.
A role in particle filtration, suggested by their sieve-like nature,
can very likely be ruled out. We have never observed particles
stuck on reticuloapopylocytes, nor witnessed any phagocytosic
events. Considering the size of the pore(s) (,0.5 mm) and the size
Figure 7. Schematic drawing of a choanocyte chamber with
indicated flow directions and hydrodynamically pivotal sites
(stars): 1. prosopyle, 2. microvilli collar, 3. contact side
between apopylar velum of monociliated apopylar cells and
microvilli collar of choanocytes at the apopylar opening, 4.
reticuloapopylocyte.
doi:10.1371/journal.pone.0113153.g007
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of a typical food particle (2 mm–5 mm), we would expect the pores
to be clogged by retained particles within a very short period of
time. From particle feeding experiments and our understanding of
hydrodynamic constraints, we know that the majority of particles
are restrained with great efficiency by the microvilli collar of
choanocytes at the latest [25,44]. In other words, in terms of
efficiency, an additional downstream filtering element in the form
of reticuloapopylocytes is simply not necessary, which renders this
potential function obsolete under parsimonious evolutionary
principles.
Experimental and theoretical studies into filter feeding animals,
including several sponge species, have demonstrated using a
Venturi tube principle how even actively pumping species benefit
from and make use of ambient flow-induced passive ventilation
[11,45,46]. A recent work on hexactinellids provides detailed
calculations of the dimensions of canal system elements (especially
canal segments, choanocyte chambers and their openings) in
relation to their role in fostering passive flows [46]. In this context
the presence of large bypass elements [15] and the highly
asymmetric nature of branching in T. wilhelma [6] could be
interpreted as factors which promote passive flows. However, this
hypothesis is speculative as the impact of bypass elements on flow
patterns inside sponges is not yet well understood on either the
local or the organismic scale. It is therefore currently impossible to
prove or reject this hypothesis for T. wilhelma. What is more, a
closer look at the morphology and dimensions of apopylar
openings in T. wilhelma in the context of resistance theory does
not support the hypothesis of passive ventilation by ambient flow.
This is underlined by the resistance values we calculated for
reticuloapopylocyte-bearing apopyles, which are about 1300 time
greater than in unchanged apopyles and 8000 times greater than
in the hexactinellid Aphrocallistes vastus [46], where ambient
current-induced passive flow has been demonstrated. We would
expect the much greater pressure drop/resistance generated at
fenestrated apopyles in comparison to non-specialized apopylar
openings to prevent the induction of passive flow through
choanocyte chambers in T. wilhelma.
The third hypothesis regarding local flow regulation is related to
the fact that individual reticuloapopylocyte intracellular pores
have been observed in both an open and a closed state, and to the
detection of a specific myosin-heavy chain expression pattern in
this new cell type [47] which indicates its ability to actively modify
its state of opening. In this it is strikingly reminiscent of
intracellular ostia, which possess the ability to open and close
relatively rapidly in order to regulate flow [48–50]. Altering the
available cross-sectional area of the apopyle by entirely or partially
closing individual pores changes the resistance of the apopyle.
Closing pores leads to (has the capacity to lead to?) a reduction in
the volume of flow and possibly even to a complete shutdown of
individual choanocyte chambers in distinct areas of the sponge
body. A reduction in the volume of flow at an apopyle will result in
a change in the perfusion of the portion of the sponge body in
question. The ability to alter flow rates on a local scale with
consequences on the regional and even organismal levels qualifies
the reticuloapopylocyte as a simple and highly precise fine-tuning
device. Theoretically, reticuloapopylocytes permit a gradual
adjustment of resistance at the apopyle by closing increasing
numbers of pores to create an almost continuous decrease in flow.
However, as these cells are to be found deep in the sponge body
and are thus not accessible to in vivo light microscopy, direct
experimental evidence to back up or refute this hypothesis will be
difficult to obtain.
4. Functional constraints in the evolution of apopylar
elements
Body contraction-expansion cycles have been demonstrated in
representatives of all four major lineages of sponges ([36] and
Nickel unpublished data). Of all the species studied so far, the
amplitude and frequency of body contractions have been highest
in T. wilhelma [13,36]. The primary effectors of body contraction
are endopinacocytes [35]. In the course of a body contraction
cycle the canal lumen disappears almost entirely. The change in
canal diameter leads to an increase in resistance in the canal
system. This change in the hydrodynamic situation in the canal
system during a body contraction cycle gives rise to three different
functional constraints with regard to the evolution of apopylar
elements: (1) Risk of damage to canal system elements caused by
increasing pressure in the contraction phase. (2) A need to modify
the perfusion of body parts, something which can be influenced by
contraction and expansion phases (3) A need to generate increased
Gauge pressure during the inflation of the canal system in the
second kinetic phase (see [13,35]) of the expansion cycle.
An increase in Gauge pressure within the canal system during
the relatively rapid contraction phase is the result of cumulative
resistance caused by the reduction in canal diameter and the
presence of just a single oscule through which all residual water
has to be expelled. The increased Gauge pressure leads to
constraint (1), which primarily affects all delicate structures in the
canal system (e.g. choanocytes). From a technical point of view the
solution would be a pressure regulator. In a very simple way in T.
wilhelma, the reticuloapopylocytes constitute just such pressure
regulators. A comparable role has been demonstrated for the
morphologically highly similar sieve plates in the phloem of plants
[51].
The exact role of body contractions in sponges is unclear. One
hypothesis proposes a physiological need to flush the canal system
by exchanging all the water in the aquiferous system in the course
of a body contraction cycle. Experimental studies into body
contraction cycles in different sponge species have demonstrated
the presence of contraction waves which travel over the sponge
body ([35] and own unpublished data) Over the course of a body
contraction cycle, canal diameters undergo alterations which result
in changes in resistance. These changes affect perfusion rates, as
formulated by constraint (2) on the principle described in section 2
above.
An analysis of body contraction kinetics in sponges has revealed
four different sub-phases [35]. The contraction and expansion
stages exhibit two distinct kinetic phases each. Endopinacocytes
have been identified as effectors of contraction [35]. The two
different kinetic phases of the T. wilhelma expansion cycle are
thought to have two effectors. In the early and more rapid
expansion phase elastic energy loaded into a distinct higher
ordered sub-volume of the extracellular matrix is released [52].
This results in a partial inflation of the aquiferous system which
enables the choanocyte chambers to start working again. In the
second, much slower kinetic phase, we propose that Gauge
pressure plays a role in fully inflating the canal system. Fulfilling
this functional constraint (3) basically requires the presence of two
specific components of the sponge aquiferous system - reticuloa-
popylocytes and bypass elements. Reticuloapopylocytes increase
Gauge pressure by increasing resistance, while bypass elements
form direct connections between the incurrent and excurrent
canal system [6,12,14,15]. Their function and impact on flow in
sponges is still under debate, but hydrodynamics and resistance
theory might shed light on their functional role in the context of
body contraction cycles in T. wilhelma. The increased back
pressure in the incurrent canal system generated by the presence of
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reticuloapopylocytes in pumping choanocyte chambers is coupled
to the excurrent canal system via bypass elements. This increases
Gauge pressure throughout the system, helping it to inflate.
A large number of the hypotheses and interpretations discussed
above are based on theoretical considerations and fundamental
physical rules, the morphology of specific cell types and the
morphometric information available on the canal system. Again,
experimental verification in vivo is not currently possible due to
the lack of optical live imaging techniques for structures deep
inside the sponge body. Non-destructive approaches, e.g. x-ray
videography and tomography or magnetic resonance imaging, do
not provide the required spatial and/or temporal resolution
needed to simultaneously analyze morphology, flow and the
kinetics of contraction. Furthermore, we are faced with highly
complex interdependencies between the phenomena in question -
e.g. pressure drop and gauge pressure being caused by bypasses
and reticuloapopylocytes. A solution to this dilemma might be
computational fluid dynamic modelling approaches based on
exact canal system geometries obtained from biological entities.
Depending on the effect to be studied, modeling approaches might
enable us to reject and formulate new hypotheses, or even test the
influence of specific structural elements by modifying the
geometries used (e.g. including/excluding bypass elements).
However, this would require detailed information on the
morphology of the canal system, volume flow and temporal
analysis data pertaining to the kinetics of body contractions.
Conclusions
Reticuloapopylocytes, described here in Tethya wilhelma,
represent a new and functionally distinct type of cell. On the
basis of related functional morphological and hydrodynamic
constraints, we evaluated a range of hypotheses pertaining to the
function of this new cell and its effect on local and organismic flow
conditions. Compared to our understanding of the functional
morphology and influence on fluid dynamics of the other cell types
discussed in the present study, our knowledge of the apopyle in
leuconoid canal systems is patchy, especially when it comes to
understanding its role in flow conditions on a local and organismic
scale and its relationship to particle filtration in general. All the
studies concerned with flow in sponges so far have focused mainly
on the relationship between flow conditions and the architecture of
the canal system in general, or concentrated on ecological aspects.
However, if we break groups of cells in the aquiferous system down
into functional units, the most interesting one is constituted by
choanocytes and apopyle-related cells. The fact that a putative
flow-regulating cell type is able to cut off every single choanocyte
chamber and connected canal system elements from a highly
parallelized canal system configuration raises the question of
whether the apopyle is in fact a general regulative element in all
sponges. Further research needs to focus on morphological
changes in apopyles which reflect functional plasticity, e.g. during
contraction events or pumping arrests. This will require a highly
differentiated fixation scheme for functional states which will have
to be characterized, analysed and understood in detail.
Materials and Methods
Sponge material
Individuals of T. wilhelma were sampled from the type location
in the aquarium of the zoological-botanical garden ‘Wilhelma’
(Stuttgart). As T. wilhelma is not considered an endangered or
protected species, no special sampling permits were required to
retrieve material for scientific experiments from the aquarium
section of the zoological-botanical garden. A continuous culture of
sponges was maintained in a 180 l aquarium at 26uC using
artificial seawater under a light/dark cycle of 12:12 h. The
sponges were fed regularly with commercial invertebrate food
(Artifical Plancton, Aquakultur Genzel) [13].
Scanning electron microscopy
Specimens of T. wilhelma were fixed overnight in a precooled
iso-osmolar solution of 1.25% glutaraldehyde, followed by a
contrasting step in iso-osmolar 1% OsO
4
solution for 1.5 h. They
were desilified in 5% hydrofluoric acid for 1 h and then embedded
in styrenemethacrylate [53]. After semi-thin sectioning, we
dissolved the plastic around the remaining sponge using xylene-
treatment and dehydrated the samples in increasing concentra-
tions of acetone. Specimens were critically point dried in an
Emitech K850 CPD system and sputter coated in an Emitech
K500 SC system. SEM images were taken on a Philips
XL30ESEM instrument.
Morphometric measurements
Morphometric measurements of reticuloapopylocytes and other
cells were performed using ImageJ [54]. For the analysis of
reticuloapopylocyte pore sizes pores were semi-automatically
segmented using the level sets algorithm in Fiji [55]. The ferret
diameters (min and max) and area of reticuloapopylocytes and all
segmented pores were measured using functions in ImageJ.
Supporting Information
Figure S1 SEM image showing cell borders of exopina-
cocytes around an ostia opening in the outer surface of
T. wilhelma.
(PDF)
Acknowledgments
We are grateful to Martin S. Fischer (Jena) for infrastructure and financial
support, Katja Felbel and Benjamin Weiss (Jena) for excellent technical
assistance, Isabel Koch and Alex Mendosa (Wilhelma Stuttgart) for
additional supply of sponges complementing our own cultures, Isabel Heim
(Neubulach) for aquaristic knowledge, Christopher Arnold, Florian Wolf,
Henry Jahn and Josefine Gaede for aquarium maintenance. David J.
Schulz (Missouri) and an anonymous reviewer provided valuable
comments to this manuscript.
Author Contributions
Conceived and designed the experiments: JUH MN. Performed the
experiments: JUH MN. Analyzed the data: JUH MN. Contributed
reagents/materials/analysis tools: JUH MN. Wrote the paper: JUH MN.
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