Contraction-Expansion and the Effects on the Aquiferous System in the Demosponge Halichondria panicea

Contractile behavior is common among sponges despite their lack of nerves and muscles. As sessile filter-feeders, sponges rely on water with suspended food particles being pumped through their aquiferous system. During contractions, however, the water flow is being reduced and eventually shut down. Yet, purpose and underlying pathways of contractile behavior have remained largely unclear. Here, we document the external and internal morphology of contracted and expanded single-osculum explants of the demosponge Halichondria panicea. We show that contraction-expansion dynamics can occur spontaneously (in untreated explants) and can be induced by exposure to chemical messengers such as γ-aminobutyric acid (GABA, 1 mM) and L-glutamate (L-Glu, 1 mM), or to inedible ink particles (4 mg L–1). The neurotransmitter GABA triggered similar contraction-expansion dynamics in H. panicea as observed in untreated explants. The effects of GABA-induced contraction-expansion events on the aquiferous system were investigated using scanning electron microscopy (SEM) on cryofractured explants. Our findings suggest that contraction-expansion affects the entire aquiferous system of H. panicea, including osculum, ostia, in- and excurrent canals and apopyles.


INTRODUCTION
Sponges are sessile filter-feeders that pump water by means of choanocytes (Reiswig, 1975;Asadzadeh et al., 2019) which are specialized flagellated cells that efficiently retain particles down to ∼0.1 µm on their microvilli collars (Riisgård and Larsen, 2000). Ambient water is drawn through numerous inhalant openings (ostia) into an incurrent canal system, where it enters many choanocyte chambers (CCs) through prosopyles, passes microvillar collar-sieves, exits the CC through apopyles and leaves the sponge via an excurrent canal system that finally leads to an exhalant opening, the osculum (Larsen and Riisgård, 1994). Contractions can effectively restrict or stop the water flow through a sponge (Reiswig, 1971;Riisgård et al., 2016;Kumala et al., 2017;Ludeman et al., 2017;Goldstein et al., 2019) and have been suggested to protect the filter-pump, for instance during periods with high loads of re-suspended sediment (Elliott and Leys, 2010;Bannister et al., 2012;Leys, 2015;Grant et al., 2019).
Sponges lack conventional nerves and muscles but contract in response to environmental stimuli (Parker, 1910;McNair, 1923;Reiswig, 1971;Leys, 2007, 2010;Tompkins-MacDonald and Leys, 2008;Leys, 2015), or following endogenously controlled contraction-expansion cycles (Reiswig, 1971;Nickel, 2004). Signaling in demosponges is poorly understood but probably involves non-motile primary sensory cilia (Nickel, 2010;Ludeman et al., 2014;Leys, 2015), conduction pathways based on chemical messengers (Ellwanger and Nickel, 2006;Ellwanger et al., 2007) and an antagonistic contractile apparatus, most likely mediated by the pinacoderm as effector and the water pressure generated by the choanocytes (Nickel et al., 2011). Interaction of this "neural toolkit" (Leys, 2015) results in slow peristaltic-like waves of contraction traveling through the aquiferous system of a sponge (Elliott and Leys, 2007;Goldstein et al., 2019). An improved understanding of the functional morphology underlying contraction-expansion events in sponges may provide important insights into the control of water flow through the aquiferous system.
Halichondria panicea is a common marine demosponge widely distributed in temperate coastal regions of the northern hemisphere (Erpenbeck et al., 2004). Its contractile behavior regarding osculum closure (Riisgård et al., 2016;Kumala et al., 2017) and associated constriction of the excurrent canals  has been described, but the role of other aquiferous elements is unclear. Here, we determine the trigger potential of neurotransmitters and inedible particles compared to spontaneous (untreated conditions) contractionexpansion events in single-osculum explants of H. panicea. Subsequently we use these findings to induce similar contractionexpansion events as observed in untreated explants and study the resulting effects on the aquiferous system in explants fixed in different contraction-expansion phases using scanning electron microscopy (SEM).

Cultivation of Sponge Explants
Sponge explants were obtained from Halichondria panicea specimens collected in the inlet of Kerteminde Fjord, Denmark (55 • 26 59 N, 10 • 39 40 E). Small pieces (5-10 mm 3 ) were cut from the exhalant chimneys of collected sponges and individually placed on glass slides in flow-through aquaria with aerated bio-filtered seawater (15-25 psu, 15 • C). After attachment to glass slides and development of an osculum, the single-osculum explants (i.e. functional aquiferous modules; Kealy et al., 2019), were fed with Rhodomonas salina algae and bacteria growing in the algal culture (cf. Kumala et al., 2017).

Video-Microscope Observation of Contraction-Expansion Dynamics
The contractile responses of explants exposed to either γ-aminobutyric acid (GABA), L-glutamic acid (L-Glu), or to inedible particles (calligraphy ink) were compared to spontaneous contractions of untreated explants (i.e. seawater control). Contractile behavior of single-osculum explants was observed in 1 L aquaria with bio-filtered seawater (20 psu, 15 • C) under constant mixing with a magnetic stirrer. Only actively pumping explants, with fully expanded osculum at the start of experiments, were included. Contraction-expansion dynamics of explants under different treatments were documented in longterm (12-24 h) time-lapse image sequences of the osculum cross-sectional area (OSA, mm 2 ) and the side-view projected area (A, mm 2 ) of single-osculum explants using a video-microscope (Leica MZ8) with a USB 3.0 industrial camera (Imaging-source, DFK23UM021) and image acquisition software (IC Capture 2.3). We analyzed temporal changes in OSA and A with imaging software (ImageJ, 1.50f) to identify distinct contractionexpansion events. These events were defined by reduction in the initial (i.e. before contraction) maximum osculum cross-sectional area (OSA max ) and projected area (A max ), osculum closure (OSA = 0 mm 2 ) and corresponding minimum projected area (A min ), respectively, and subsequent expansion to a second maximum (i.e. after contraction). Spontaneous contraction-expansion events which occurred randomly within 24 h in untreated explants were selected from the time-lapse sequences. Trigger concentrations of GABA, L-Glu and ink which reliably induced contraction-expansion within <1 h were determined from dose-response experiments. We additionally tested the antagonistic effect of different concentrations of L-menthol (crystals dissolved in seawater) during GABA-induced contraction-expansion cycles (positive control). The intensity and duration of induced versus spontaneous contractionexpansion cycles was determined in replicate explants (n = 6 per treatment). For comparison across treatments, time (t, min) was standardized by setting t 0 = 0 as the mean time of osculum closure or as the mean time of minimum relative projected area defined by the confidence interval [(A min -0.025), (A min + 0.025)]. The relative osculum cross-sectional area and relative projected area were calculated as: where OSA max and A max correspond to OSA r = 1 and A r = 1, respectively. The duration of each contraction-expansion cycle was estimated as the total duration of the phase of contraction (I), the contracted phase (II), the phase of expansion (III), and the subsequent expanded phase until the onset of a new contraction (IV; Figure 1). Speeds of contraction (v I , OSA r min −1 and A r min −1 ) and expansion (v III , OSA r min −1 and A r min −1 ) were calculated for each explant from the slopes of linear regression lines for OSA r and A r during phase I and III, respectively. From relative changes in A during contraction ( A I ,%) and during expansion ( A III ,%) and the height (h, mm) of explants, the corresponding changes in explant volume during contraction ( V I ,%) and expansion ( V III ,%) were estimated using the volume (V, mm 3 ) equation for axisymmetric conical explants :

Scanning Electron Microscopy
Single-osculum explants (n = 4) with expanded oscula were transferred to small containers with 30 mL sterile filtered (0.2 µm) seawater from the cultivation tank. These explants were exposed to 1 mM GABA for various exposure times (t GABA , min). Exposure times to GABA were estimated based on the osculum-opening degree of explants to induce different phases of contraction-expansion, including I: contracting, t GABA = 41 min; II: contracted, t GABA = 48 min; III: expanding, t GABA = 54 min and IV: expanded phase, t GABA = 0 min (Figure 1). The external morphology of explants in these contractile phases was documented stereo-microscopically prior to and after fixation for scanning electron microscopy (SEM). Explants were fixed for SEM by addition of 2% OsO 4 in 0.1 M cacodylate buffer (6 mL) and left overnight in the fixative at 4 • C. Afterward they were transferred to containers with sterile filtered seawater from the cultivation tank and rinsed for 15 min; this step was repeated three times. After a final rinsing for 72 h in sterile filtered seawater, explants were dehydrated in a graded ethanol series, placed in a Parafilm sleeve with 100% ethanol and submerged in liquid nitrogen until the ethanol had solidified. The explants were then cryofractured with a razor blade, transferred to 100% ethanol at room temperature, critical point dried (EMS860) and sputter coated with a 45 nm thick gold layer (Edwards S150B). Cryofractured specimens were examined using a FEI NOVA NanoSEM 600 scanning electron microscope operated at 5 kV using an ETD detector. Diameter (D, µm) of osculum, ostia, canals and apopyles were measured using imaging software NIS-Elements D 4.10.04 (Nikon Instruments) and ImageJ, 1.50f. The degree of shrinkage due to fixation was estimated from measurements of the osculum diameter.

Statistical Analyses
Statistical data analyses were performed in R version 3.1.3 (R Core Team, 2015) using R package "lme4" (Bates et al., 2014). Generalized linear models (GLMs) were parameterized with Gamma error structure (meeting the assumptions of normal distribution, homoscedasticity and independence of residuals) to test for differences between the rates of contraction (v I ) and expansion (v III ) of the osculum cross-sectional area (OSA) or projected area (A) of untreated explants. Generalized linear mixed-effect models (GLMMs) with Gamma error structure were fitted to investigate variability in OSA or A across treatments (untreated, GABA, L-Glu and inedible particles; fixed effect) when correcting for individual variation (ID#; random effect).
Post hoc analysis was conducted by multiple comparisons of means using Tukey contrasts (significance level p < 0.05) from R package "multcomp" (Hothorn et al., 2013). While OSA was similar across the experimental conditions (GLMM, t = 1.6, p > 0.366), A varied significantly among the explants exposed to different treatments (GLMM, t = 10.3, p < 0.029; Tukey post hoc test, group 1: GABA, group 2: spontaneous and L-Glu, group 3: ink particles). Using GLMMs with Gamma error structure, we then compared the rates of contraction/expansion, duration of the contracted phase in OSA or A, and the volume change during contraction/expansion, respectively, after exposure to different treatments (GABA, L-Glu or inedible particles; fixed effect) to spontaneous (untreated) contraction-expansion dynamics by taking into account variable A (fixed effect) and individual variation of explants (ID#; random effect). Similar GLMMs were parameterized to investigate changes in the diameter (D) of ostia, canals and apopyles during different contractile phases of explants (fixed effect) by correcting for individual variation (ID#; random effect).

Contraction-Expansion of the Aquiferous System
External and internal morphology of single-osculum explants were investigated using SEM (Figure 4). Before fixation, 2 | Halichondria panicea. Spontaneous (i.e. untreated conditions) and induced (by exposure to the neurotransmitters GABA, L-Glu or ink particles) contraction-expansion events of sponge explants (n = 6, ID#1-24), expressed by changes in the relative osculum cross-sectional area (OSA r ). , v III : expansion rate (determined from the slope of linear regression for changes in OSA r during III). Means ± SD are shown and significant deviation from spontaneous contraction-expansion events is indicated by * (GLMM, p < 0.05).
Only the expanded and contracted explant could be used for measurements of internal structures due to poor quality of the other cryofractured specimens.

Functional Morphology of Contractile Behavior
The present study suggests that GABA-induced contractions of Halichondria panicea affect the entire aquiferous system. Similar contraction-expansion dynamics of untreated versus GABA-exposed explants indicate a general sequence of events underlying the contractile behavior of H. panicea, and possibly other sponge species. Contraction of H. panicea typically involves complete osculum closure for several minutes. Corresponding volume reductions by ∼15% after exposure to GABA, as derived from changes in the sponge surface (  Ostia D (µm) 7.7-14.5 ± 4.0-26.2 0.6-1.5 ± 0.5-2.7 2.6-7.9 ± 3.9-16.6 17.8-25.0 ± 3.8*-33.5 A (µm 2 ) 179 ± 107 2 ± 1 61 ± 57 502 ± 152 changes in internal morphology, as expressed by an estimated volume of (<0.6 × 15%) > 25% comprised by canals relative to the total sponge volume considering a reduction in canal diameters by up to 60% ( Table 5). Constriction of the canal system may cause considerable volume changes depending on the morphology of a sponge species. The demosponge Tethya wilhelma for instance, which has an extensive bypass-and recirculation-system that connects main excurrent canals with a lacunar system , reduces its entire volume by 42-73% during contractions, as expressed by much faster (∼10 3fold) contraction and expansion speeds of its projected area (Nickel, 2004) compared to our observations in H. panicea. Also, T. wilhelma contracts considerably faster than it expands (Nickel, 2004) which was not the case for H. panicea. Nevertheless, spontaneous contractions of T. wilhelma occur regularly at intervals of 1-10 h (Nickel, 2004) which is comparable to a mean expanded phase 4.2 ± 0.9 h across treatments for the present H. panicea explants.
Our study suggests a distinct temporal sequence of coordinated movement during contractions which may be comparable to that described for the freshwater sponge Ephydatia muelleri (Elliott and Leys, 2007). Contraction rates seem to be region-specific as a peristaltic-like wave of contraction travels through the different components of the aquiferous system in H. panicea ; in E. muelleri speeds range from 6 to 122 µm s −1 for the osculum, from 0.3 to 4 µm s −1 for peripheral/central canals and from 0.2 to 0.7 µm s −1 for the ostia (Elliott and Leys, 2007). Comparatively, the speed of propagation of the contraction in H. panicea explants appears ∼1000-fold slower after exposure to GABA for 48 min, as can be estimated from observed temporal changes in diameter between expanded and contracted state, i.  Table 5). The observed dimensions of ostia, canals and osculum of the expanded explant in this study are consistent with a previous study on H. panicea (Reiswig, 1975). Based on corresponding changes in the aquiferous system of a contracting explant, we hypothesize contractionexpansion events that initiate with contraction and closure of the osculum ( D = −100%) and ostia ( D = −94%), constriction of ex− and incurrent canals ( D = −60%), contraction of choanocyte chambers (CCs) by means of contractile apopyles ( D = −34%; Table 5) which may be followed by expansion of the osculum, relaxation of canals, CCs, and opening of ostia. Present observations on the aquiferous elements of cryofractured sponge explants represent a first overview of the morphological changes involved in contraction-expansion events. Follow-up studies may further unravel the temporal dynamics underlying coordinated behavior in sponges.
Observed changes in external and internal sponge volume during GABA-induced contractions may further provide support for pinacoderm-mediated contractions (Nickel et al., 2011) in H. panicea which seem to involve constriction of the exopinacoderm around the osculum and pronounced contractions of the endopinacoderm lining the canal system . Accompanying compression of the choanoderm in H. panicea is similar to that observed in T. wilhelma where especially larger canals and open cavities may disappear completely during contraction due to equally pronounced exo-and endopinacoderm contractions (Nickel et al., 2011). In comparison, the contractility of the exopinacoderm of H. panicea seems to be weaker, which is indicated by the presence of a spacious cavity characterized by many long fibers stretching between the exopinacoderm and the highly compressed choanosome in the contracted sponge ( Figure 7B). Radially arranged spicule bundles held together by collagen fibers protruding around the contracted osculum have previously been observed in the demosponge Haliclona sp. and could serve as an elastic force to reopen the osculum after contraction (Jones, 1962). Contractile behavior of H. panicea is therefore likely driven by an antagonist system which is based on the interplay between contractile pinacoderm cells, water pressure in the canal system and visco-elastic forces of a collagenous matrix with motile cells in the mesohyl (Elliott and Leys, 2007;Nickel et al., 2011;Hammel and Nickel, 2014). Recent work has further described a novel, yet abundant, choanocyte-related cell type in the mesohyl of a freshwater demosponge (myopeptidocyte) which is likely involved in contraction-expansion of sponges (Musser et al., 2019).

Ecological Role of Contractions in Sponges
Contractions may protect the sponge filter-pump from mechanical damage (Nickel, 2004;Elliott and Leys, 2007) caused by environmental disturbances such as overloading with resuspended sediment (Reiswig, 1971;Strehlow et al., 2016). In this study, exposure to high concentrations of inedible ink particles significantly prolonged the contracted phase of internal structures (expressed as changes in A and V; Table 3; Nickel, 2004) but not the duration of osculum closure. Also, we observed similar contraction and expansion speeds during ink-induced contractions compared to untreated conditions (Tables 2, 3), emphasizing the role of contractile behavior as possible protection mechanism. Compared to E. muelleri which has shown an osculum contraction rate of 18 ± 8 µm s −1 (entire cycle duration of "ink" fed sponges: 31 min; Elliott and Leys, 2007) after exposure to ∼6-fold higher ink concentrations as in the present study (4 × 10 3 -fold diluted ink; Elliott and Leys, 2007), the speed of propagation of the contraction across the pinacoderm of our H. panicea explants was 100-fold slower, i.e. v I = −155 nm s −1 [based on D (= 2 √ (0.24 mm 2 /π)-0 mm) = 0.55 mm within a contraction phase of 59 min; see Tables 1, 2]. Along with a prolonged contracted phase of internal structures (Tables 3, 4), our observations confirm previous observations that addition of inedible particles slows down contractile behavior (Elliott and Leys, 2007). The particle loads in this study are in the lower range of suspended sediment concentrations occurring in innershelf regions of the North Sea (Fettweis et al., 2010;Tjensvoll et al., 2013), emphasizing the ecological relevance of a contractile response mechanism to protect the sponge filter-pump from overloading with inedible particles. Coordinated peristaltic waves have additionally been shown to expel waste material from the aquiferous system of E. muelleri (Elliott and Leys, 2010). In H. panicea, however, cellular transport mechanisms seem to remove inedible particles from the aquiferous system .

Functional Pathways
The present study supports the presence of a glutamate and GABA receptor signaling system (Perovic et al., 1999) in H. panicea and emphasizes that chemical messengers specifically induce contraction (GABA, L-Glu, ink) or expansion (Lmenthol) of distinct aquiferous components of a sponge. While the effect of GABA resembled the spontaneous (untreated) contraction-expansion cycle, L-Glu significantly enhanced the rate of contraction and decelerated the rate of expansion of the external sponge surface (Table 3). Further, exposure to L-Glu prolonged the duration of osculum closure and subsequent contracted phase of the external surface (Tables 2, 3) and led to significantly increased reductions in sponge volume during contraction (Table 3). An inhibitory action of GABA and excitatory effect of glutamate has previously been observed in E. muelleri (Elliott and Leys, 2010). However, exposure of H. panicea explants to GABA did not inhibit contractions as observed in E. muelleri (Elliott and Leys, 2010) but triggered contractions at a final concentration of 1 mM, similarly as observed in T. wilhelma after exposure to 0.1 mM GABA (Ellwanger et al., 2007). As indicated by a ∼100-fold difference in the glutamate concentration to induce full contractions in E. muelleri (0.08 mM; Elliott and Leys, 2010) or T. wilhelma (10 mM; Ellwanger et al., 2007), respectively, physiological levels and effects of chemical messengers may vary across sponge species (Ellwanger and Nickel, 2006;Ellwanger et al., 2007;Elliott and Leys, 2010). Coordinated contractile responses of demosponges in part also seem to be regulated through nitric-oxide (NO) signaling (Ellwanger and Nickel, 2006;Elliott and Leys, 2010;Musser et al., 2019), which triggers relaxation of endothelial smooth muscle contraction in vertebrates (Palmer et al., 1987). An improved understanding of contractions in sponges on a cellular level may thus provide some fundamental insights into the evolution of nervous systems and muscle tissue in metazoans.

CONCLUSION
The excitatory effect of the neurotransmitter GABA made it possible to study the internal and external morphology of Halichondria panicea during induced contraction-expansion events. SEM of sponge explants in different contractile phases suggested gradual changes in the entire aquiferous system, including osculum, ostia, in-and excurrent canals and apopyles. The present findings emphasize the interplay between environmental and/or intrinsic cues, signal transduction via chemical messengers and functional cell morphology for coordinating contractile behavior in sponges.

DATA AVAILABILITY STATEMENT
All datasets generated for this study are included in the article/Supplementary Material.

ETHICS STATEMENT
The study was conducted on marine sponges (invertebrates) and therefore was exempted from this requirement.

Conflict of Interest:
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
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