Scanning electron microscope evidence for diatom uptake by two Antarctic sponges

Scanning electron microscopy (SEM) investigation of two Antarctic sponges, Phorbas glaberrima and Tedania charcoti, showed that the exopinacoderm effects a direct uptake of benthic diatoms which settle on the sponge surface. In P. glaberrima, planktonic diatoms were also observed penetrating through the inhalant system, the primary way of feeding in sponges. Benthic diatoms which accumulate in the mesohyl underneath the exopinacoderm help to strengthen the sponge cortex and may be an alimentary source during oligotrophic periods in the Antarctic environment.     

Allograft rejection, autograft fusion and inflammatory responses to injury in Callyspongia diffusa (Porifera; Demospongia)

Sponges exhibit a variety of swift, cellular defence responses to protect self integrity. The sponge Callyspongia diffusa has been used to characterize the cytological changes that occur during allograft rejection, autograft fusion, and inflammation. Allogeneic contact results in fusion of the two exopinacoderms followed by an infiltration of mesohyl cells into the graft zone. As mesohyl cells accumulate, they form tissue bridges that span the graft interface. After a few days, the tissue bridges and the nearby cellular infiltrate become necrotic and slough off, which separates the allogeneic tissues. Autograft fusion begins similarly but cellular infiltration does not follow exopinacoderm fusion. Contacted exopinacocytes are redistributed, the endopinacoderms and choanosomes come into contact, and the grafted sponge tissues merge. Tissue damage exposes internal regions of the sponge to the external environment. In many areas of injury, exposed choanosome is sealed by infiltrating mesohyl cells. In other areas, exposed endopinacoderm appears to serve as new exopinacoderm. Cellular debris is removed by phagocytic archaeocytes and new exopinacoderm is regenerated over the damaged choanosome. No scars remain once the inflammatory infiltrate has dispersed. In general, mesohyl cells are involved in defence responses without an observed enrichment of any specific cell type. However, archaeocytes from rejecting sponges appear to line both sides of the allogeneic interface.     

Differences Between Bacterial Communities Associated with the Surface or Tissue of Mediterranean Sponge Species

Bacterial communities associated with the surfaces of several Mediterranean sponge species (Agelas oroides, Chondrosia reniformis, Petrosia ficiformis, Geodia sp., Tethya sp., Axinella polypoides, Dysidea avara, and Oscarella lobularis) were compared to those associated with the mesohyl of sponges and other animate or inanimate reference surfaces as well as with those from bulk seawater. Denaturing gradient gel electrophoresis (DGGE) analysis of PCR-amplified bacterial 16S ribosomal RNA genes obtained from the surfaces and tissues of these sponges demonstrated that the bacterial communities were generally different from each other. The bacterial communities from sponges were different from those on reference surfaces or from bulk seawater. Additionally, clear distinctions in 16S rDNA fingerprint patterns between the bacterial communities from mesohyl samples of "high-microbial abundance (HMA) sponges" and "low-microbial abundance sponges" were revealed by DGGE and cluster analysis. A dominant occurrence of particularly GC-rich 16S ribosomal DNA (rDNA) fragments was found only in the DGGE banding pattern obtained from the mesohyl of HMA sponges. Furthermore, sequencing analysis of 16S rDNA fragments obtained from mesohyl samples of HMA sponges revealed a dominant occurrence of sponge-associated bacteria. The bacterial communities within the mesohyl of HMA sponges showed a close relationship to each other and seem to be sponge-specific.     

Localization of Two Brominated Metabolites,Aerothionin and Homoaerothionin,in Spherulous Cells of the Marine Sponge Aplysina fistularis (=Verongia thiona)

Energy dispersive X-ray microanalysis was used to localize the two brominated natural products (aerothinonin and homoaerothionin) in the tissues of a marine demosponge, Aplysina fistularis. Virtually all of these compounds were localized within the spherules of the spherulous cells in the mesohyl. This is the first localization of any secondary metabolite at the cellular or sub-cellular level in any marine invertebrate. In Aplysina fistularis, as in other species of the same genus studied by Vacelet, the spherulous cells are concentrated just beneath the exopinacoderm and just beneath the endopinacoderm of the excurrent canals. Moreover, there is electron microscopic evidence for degeneration of some spherulous cells throughout the mesohyl. Presumably, this degeneration can release some aerothionin and homoaerothionin, which are known to have antibiotic properties. After release from the spherulous cells, these brominated natural products could function (1) within the mesohyl to exclude some types of bacteria or to aggregate ingested bacteria and/or (2) within the boundary layer of the surrounding seawater for defense or offense, as considered in the discussion section.     

Evidence for the existence of microtubule protein in the extracellular space of marine sponges

The medulla region of sponges (= mesohyl) is only rarely dispersed with cells (approximately 25% of the total tissue volume). In vivo studies with a series of anti-cytoskeletal drugs revealed that taxol caused a significant contraction of intact specimens. Therefore, we investigated the possibility that microtubules are one component of the dynamic extracellular network in sponges, especially in the mesohyl. Using the sponge Geodia cydonium we found that most cells are not intracellulary stained in indirect immunofluorescence microscopy. However, all cells were brightly stained by the monoclonal antibody (mAb) at their plasma membranes. After incubating cryostat sections with a buffer, containing ATP, large (length up to 75 mum) aster- to filamentous-like structures became visible in the extracellular space of the mesohyl region. By three cycles of assembly and disassembly microtubules could be isolated from the extracellular material which were composed of alpha- and beta-tubulin (M(r): 55,000), several polypeptides within the M(r) range 55,000-82,000 (presumably tau proteins) and one protein of an M(r) higher than 100,000. Electron microscopical analysis revealed morphologically intact microtubules with typical side projections (very probably composed of microtubule-associated protein). Some aspects of the possible involvement of microtubules in the extracellularly localized displacement and motion network are discussed.     

Do associated microbial abundances impact marine demosponge pumping rates and tissue densities?

The evolution of marine demosponges has led to two basic life strategies: one involving close associations with large and diverse communities of microorganisms, termed high microbial abundance (HMA) species, and one that is essentially devoid of associated microorganisms, termed low microbial abundance (LMA) species. This dichotomy has previously been suggested to correlate with morphological differences, with HMA species having a denser mesohyl and a more complex aquiferous systems composed of longer and narrower water canals that should necessitate slower seawater filtration rates. We measured mesohyl density for a variety of HMA and LMA sponges in the Florida Keys, and seawater pumping rates for a select group of these sponges using an in situ dye technique. HMA sponges were substantially denser than LMA species, and had per unit volume pumping rates 52–94% slower than the LMA sponges. These density and pumping rate differences suggest that evolutionary differences between HMA and LMA species may have resulted in profound morphological and physiological differences between the two groups. The LMA sponge body plan moves large quantities of water through their porous tissues allowing them to rapidly acquire the small particulate organic matter (POM) that supplies the majority of their nutritional needs. In contrast, the HMA sponge body plan is suited to host large and tightly packed communities of microorganisms and has an aquiferous system that increases contact time between seawater and the sponge/microbial consortium that feeds on POM, dissolved organic matter and the raw inorganic materials for chemolithotrophic sponge symbionts. The two evolutionary patterns represent different, but equally successful patterns and illustrate how associated microorganisms can potentially have substantial effects on host evolution. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

An unexpectedly sophisticated, V-shaped spermatozoon in Demospongiae (Porifera): reproductive and evolutionary implications

The demosponge Crambe crambe shows a peculiar spermatogenesis, hard to be reconciled with the basal position of sponges in the animal phylogeny. Early spermatogenesis stages showed most of the simple features expected in sponges. However, spermiogenesis departed from the anticipated process. Spermatids lengthened remarkably, forming a deep cytoplasmic pit around the cilium insertion, with the proximal axoneme bending to produce a V-shaped spermatozoon surprisingly similar to that known in the phylum Phoronida. The cytology was unexpectedly complex, with a needle-like nucleus of helically condensed chromatin, a conical acrosome with a subacrosomal rod, and a mitochondrion connected to the basal body by striated rootlets. These findings establish that the spermatozoon of broad-casting demosponges occurs in two structural categories ('primitive' and 'modified' type). This dualistic condition must necessarily have pre-dated the evolutionary apparition of higher metazoans, if we are to keep regarding sponges as the most primitive animals. We hypothesize that internal fertilization in C. crambe – and incidentally other demosponges – may depart from the general model assumed for spermcasting sponges. The V-shape of this spermatozoon suggests a design to favour autonomous penetration through the dense mesohyl to reach the oocytes, rather than engulfment and transportation by carrier cells towards the oocyte.  © 2009 The Linnean Society of London, Biological Journal of the Linnean Society , 2009, 97 , 413–426.     

Dynamic structure of the mesohyl in the sponge Chondrosia reniformis (Porifera, Demospongiae)

The common demosponge Chondrosia reniformis possesses the capacity to undergo an unusual creep process which results in the formation of long outgrowths from the parent body. These shape changes, which have been interpreted as adaptive strategies related to environmental factors, asexual reproduction or localised locomotor phenomena, are due mainly to the structural and mechanical adaptability of the collagenous mesohyl. This contribution describes the morphological correlates of mesohyl plasticisation in C. reniformis. The microscopic anatomy of the mesohyl was examined when it was in different physiological conditions: (1) standard ”resting” condition, (2) ”stiffened” condition and (3) dynamic ”creep” condition. In this last case four representative regions of the sponge body were analysed: the parent region, the elongation region, the transition region and the propagule region. The results show that the histological modification of the sponge mesohyl during plasticisation is limited and localised. The most significant structural changes involve mainly cytological features of specific cellular components characterised by granule inclusions (i.e. the spherulous cells) and the arrangement and density of the collagenous extracellular framework, though the integrity of the collagen fibrils themselves is not affected. Morphological and functional aspects of mesohyl plasticisation invite comparison with the mutable collagenous tissue of echinoderms. Possible functional analogies between these two tissues are hypothesised. Accepted: 29 June 2001     

Microscopic anatomy and pigment characterization of coral-encrusting black sponge with cyanobacterial symbiont, Terpios hoshinota

Terpios hoshinota is a black-colored sponge encrusting both live and dead corals. On examination, we determined that in the mesohyl, unicellular cyanobacteria were distributed extracellularly in dense populations; roughly half of the area in the histological sections was occupied by the cyanobacterial cells in the mesohyl of the inner zone. Pigment extraction tests of whole sponges and microspectrophotometrical analysis of cyanobacterial symbionts showed that the black coloration of the sponge is attributable to extremely densely packed cyanobacteria expressing R-phycoerythrin. Spermatic follicles were found in the mesohyl of specimens collected in summer, indicating seasonal reproduction, which is likely to play a crucial role in long-distance dispersal. We also found a possible oocyte that did not contain cyanobacteria in the ooplasm.     

The choanoderm of Sycettusa hastifera (Calcarea,Porifera) is able to generate new individuals

One of the main characteristics of sponges is their capacity for cell dedifferentiation. This capability can allow an impressive amount of asexual reproduction in these animals, because they are able to develop new individuals from just a few somatic cells. Studies of dedifferentiation, however, have focused mainly on sponges of the class Demospongiae. Therefore, we investigated here whether individuals of three different species of Calcarea are able to reconstitute new individuals following artificial fragmentation. We observed that fragmentation releases clumps of choanoderm able to initiate somatic embryogenesis. In Borojevia brasiliensis (asconoid aquiferous system, subclass Calcinea) and Paraleucilla magna (leuconoid aquiferous system, subclass Calcaronea), these clumps started to develop, but they did not pass through the first developmental phases. In Sycettusa hastifera (syconoid aquiferous system, subclass Calcaronea), the choanoderm was reorganized into primmorphs that fused to each other and formed an exopinacoderm. The first primmorphs’ spicules were triactines. Despite a large mortality rate, the primmorphs developed into olynthus stages. The somatic embryogenesis and the metamorphosis of the olynthus were similar to those observed during the sexual development of this and other calcareous sponge species. Our results show that in S. hastifera, and perhaps in other syconoid calcareous sponges, somatic embryogenesis occurs mainly from choanocytes, at least in vitro. However, primmorph development does not follow the same pattern observed in post‐metamorphic sexual development, as in that case diactines are always the first spicules to be synthesized in calcaronean species.     

Continuous cell movements rearrange anatomical structures in intact sponges.

Time-lapse cinemicrography was used to record the active movements of cells in living intact sponges. Each of the three main cell types (pinacocytes, mesohyl cells, and choanocytes) continuously moved and rearranged themselves so that the internal anatomy of the sponge was continuously remodeled. The shape and appearance of the sponges anatomical structures often changed substantially within a few hours. The most motile were the mesohyl cells, with many moving as fast as one cell-length per minute (15 microns/min). Mesohyl cell locomotion was often accompanied by displacements of spicules, canals, and choanocyte chambers; the patterns of these displacements suggested that the mesohyl cells were providing the motive forces for these rearrangements. The locomotion of the pinacocytes varied according to position: those along the outer sponge margins were most active, whereas those in other parts of the surface moved relatively little. Choanocytes were never observed to undergo independent locomotion but were always found grouped together in choanocyte chambers. These choanocyte chambers interacted with pinacocytes and mesohyl cells to form excurrent canals, which continuously moved, fused with, and branched from one another. These observations suggest that the experimental phenomenon of sponge cell-reaggregation and reconstitution, discovered by H. V. Wilson, represents an extreme version of morphogenetic processes that normally go on continuously within intact sponges. The results from the present study also suggest that these cellular rearrangements are controlled by active cell movements and behavioral responses that include but are not limited to selective cell adhesion.     

Variable tensility of the oral arm plate ligaments of the brittlestar Ophiura ophiura (Echinodermata: Ophiuroidea)

The oral arm plates of the brittlestar Ophiura ophiura L. are connected to lateral arm plates at distal and proximal ligamentous junctions. The distal junction is mobile and is disrupted during arm autotomy; the proximal junction is more rigid and does not participate in autotomy. Aspects of the morphology and mechanical properties of the distal and proximal oral arm plate ligaments have been investigated in order to determine if their tensility is under physiological control. By means of creep tests it was found that elevation of the external potassium (K⁺) ion concentration causes a decrease in the viscosity of the distal ligament which is either transient or continues until rupture intervenes. In forced vibration tests the distal ligament often shows a biphasic stiffening then softening response to excess K ^- ions. Anaesthetics block the softening phase but enhance the stiffening component of this response. This ligament is also softened by repetitive electrical stimuli but stiffened by excess calcium ions and by acetylcholine. The proximal ligament appears to have the capacity for only transient changes in mechanical properties. Both ligaments are penetrated by the processes of juxtaligamental cells whose perikarya are arranged in clusters innervated by hyponeural nerves. These cells are thought to modulate the interfibrillar cohesion of the ligaments. It is concluded that the distal and proximal ligaments are mutable collagenous structures which in their stiffened condition help to maintain arm posture without the need for continuous muscular activity, and that at autotomy the distal ligament undergoes a profound loss of tensile strength which facilitates arm detachment.     

Evolution of Metazoan Cell Junction Proteins: The Scaffold Protein MAGI and the Transmembrane Receptor Tetraspanin in the Demosponge Suberites domuncula

Until recently the positioning of the sponges (phylum Porifera) within the metazoan systematics was hampered by the lack of molecular evidence for the existence of junctional structures in the surface cell layers. In this study two genes related to the tight junctions are characterized from the demosponge Suberites domuncula: tetraspanin (SDTM4SF), a cell surface receptor, and MAGI (SDMAGI), a MAGUK (membrane-associated guanylate kinase homologue) protein. Especially the MAGI protein is known in other metazoan animal phyla to exist exclusively in tight junctions. The characteristic domains of MAGI proteins (six PDZ domains, two WW domains, and a truncated guanylate kinase motif) are conserved in the sponge protein. The functional analysis of SDMAGI done by in situ hybridization shows its expression in the surface epithelial layers (exopinacoderm and endopinacoderm). Northern blot studies reveal that expression of SDMAGI and SDTM4SF increases after formation of the pinacoderm layer in the animals as well as in primmorphs. These results support earlier notions that sponges contain junctional structures. We conclude that sponges contain epithelia whose cells are organized by cell junctions.The sequence from Suberites domuncula reported here, the protein membrane-associated guanylate kinase with an inverted arrangement (MAGI), is deposited in the EMBL/GenBank database under accession number AJ580406.     

Effects of Near-Threshold Stimulation of the Vestibular Apparatus on Postural Responses Evoked by Displacements of the Visualized Surrounding

In healthy subjects in the relaxed upward stance and perceiving a virtual visual environment (VVE), we recorded postural reactions to isolated visual and vestibular stimulations or their combinations. Lateral displacements of the visualized virtual scene were used as visual stimuli. The vestibular apparatus was stimulated by application of near-threshold galvanic current pulses to the proc. mastoidei of the temporal bones. Isolated VVE shifts evoked mild, nonetheless clear, body tilts readily distinguished in separate trials; at the same time, postural effects of isolated vestibular stimulation could be detected only after averaging of several trials synchronized with respect to the beginning of stimulation. Under conditions of simultaneous combined presentation of visual and vestibular stimuli, the direction of the resulting postural responses always corresponded to the direction of responses induced by VVE shifts. The contribution of an afferent volley from the vestibular organ depended on the coincidence/mismatch of the direction of motor response evoked by such a volley with the direction of response to visual stimulation. When both types of stimulations evoked unidirectional body tilts, postural responses were facilitated, and the resulting effect was greater than that of simple summation of the reactions to isolated actions of the above stimuli. In the case where isolated galvanic stimulation evoked a response opposite with respect to that induced by visual stimulation, the combined action of these stimuli of different modalities evoked postural responses identical in their magnitude, direction, and shape to those evoked by isolated visual stimulation. The above findings allow us to conclude that the effects of visual afferent input on the vertical posture under conditions of our experiments clearly dominate. In general, these results confirm the statement that neuronal structures involved in integrative processing of different afferent volleys preferably select certain type of afferentation carrying more significant or more detailed information on displacements (including oscillations) of the body in space.     

Pulmonary arterial strain- and remodeling-induced stiffening are differentiated in a chronic model of pulmonary hypertension

Pulmonary hypertension (PH) is a debilitating vascular disease that leads to pulmonary artery (PA) stiffening, which is a predictor of patient mortality. During PH development, PA stiffening adversely affects right ventricular function. PA stiffening has been investigated through the arterial nonlinear elastic response during mechanical testing using a canine PH model. However, only circumferential properties were reported and in the absence of chronic PH-induced PA remodeling. Remodeling can alter arterial nonlinear elastic properties via chronic changes in extracellular matrix (ECM) content and geometry. Here, we used an established constitutive model to demonstrate and differentiate between strain-stiffening, which is due to nonlinear elasticity, and remodeling-induced stiffening, which is due to ECM and geometric changes, in a canine model of chronic thromboembolic PH (CTEPH). To do this, circumferential and axial tissue strips of large extralobar PAs from control and CTEPH tissues were tested in uniaxial tension, and data were fit to a phenomenological constitutive model. Strain-induced stiffening was evident from mechanical testing as nonlinear elasticity in both directions and computationally by a high correlation coefficient between the mechanical data and model (R² = 0.89). Remodeling-induced stiffening was evident from a significant increase in the constitutive model stress parameter, which correlated with increased PA collagen content and decreased PA elastin content as measured histologically. The ability to differentiate between strain- and remodeling-induced stiffening in vivo may lead to tailored clinical treatments for PA stiffening in PH patients.     

Comparative study of the choanosome of Porifera: 1. The Homoscleromorpha

The choanoderm and pinacoderm of representatives of the two families of Homoscleromorpha sponges, the Oscarellidae and Plakinidae, have been examined by transmission and scanning electron microscopy. Different fixative procedures have shown the dramatic influence of fixation conditions on the morphology of choanocytes. These two families of sponges have the following morphological features in common: flagellated endopinacocytes with short apical microvilli and basal pseudopods; the presence of a very thin and dense sheet of matrix material which limits the mesohyl. There are, however, only minor differences in the flagellar morphology, granule content, and anchoring system of their choanocytes. Two findings are of particular interest: (1) the presence of glycocalyx bridges between the microvilli of the choanocyte collar; and (2) the discovery of a new cell type, the apopylar cell, which has a morphology intermediate between that of pinacocytes and choanocytes. The apopylar cells limit the apopylar opening of the choanocyte chamber and indicate the transition between choanoderm and pinacoderm.     

Dynamic mechanical properties of body-wall dermis in various mechanical states and their implications for the behavior of sea cucumbers

The dermis of the sea cucumber body wall is a typical catch connective tissue that rapidly changes its mechanical properties in response to various stimuli. Dynamic mechanical properties were measured in stiff, standard, and soft states of the sea cucumber Actinopyga mauritiana. Sinusoidal deformations were applied, either at a constant frequency of 0.1 Hz with varying maximum strain of 2%-20% or at a fixed maximum strain of 1.8% with varying frequency of 0.0005-50 Hz. The dermis showed viscoelasticity with both strain and strain-rate dependence. The dermis in the standard state showed a J-shaped stress-strain curve with a stiffness of 1 MPa and a dissipation ratio of 60%; the curve of the stiff dermis was linear with high stiffness (3 MPa) and a low dissipation ratio (30%). Soft dermis showed a J-shaped curve with low stiffness (0.3 MPa) and a high dissipation ratio (80%). The strain-induced softening was observed in the soft state. Stiff samples had a higher storage modulus and a lower tangent delta than soft ones, implying a larger contribution of the elastic component in the stiff state. A simple molecular model was proposed that accounted for the mechanical behavior of the dermis. The model suggested that stiffening stimulation increased inter-molecular bonds, whereas softening stimulation affected intra-molecular bonds. The adaptive significance of each mechanical state in the behavior of sea cucumbers is discussed.     

Characterization of a culturable alphaproteobacterial symbiont common to many marine sponges and evidence for vertical transmission via sponge larvae

A closely related group of alphaproteobacteria were found to be present in seven genera of marine sponges from several locations and were shown to be transferred between sponge generations through the larvae in one of these sponges. Isolates of the alphaproteobacterium were cultured from the sponges Axinella corrugata, Mycale laxissima, Monanchora unguifera, and Niphates digitalis from Key Largo, Florida; Didiscus oxeata and Monanchora unguifera from Discovery Bay, Jamaica; an Acanthostronglyophora sp. from Manado, Indonesia; and Microciona prolifera from the Cheasapeake Bay in Maryland. Isolates were very similar to each other on the basis of 16S rRNA gene sequence (>99% identity) and are closely related to Pseudovibrio denitrificans. The bacterium was never isolated from surrounding water samples and was cultured from larvae of M. laxissima, indicating that it is a vertically transmitted symbiont in this sponge. Denaturing gradient gel electrophoresis, 16S rRNA gene clone library analysis, and fluorescent in situ hybridization with probes specific to the alphaproteobacterium confirmed the presence of this bacterium in the M. laxissima larvae. The alphaproteobacterium was densely associated with the larvae rather than being evenly distributed throughout the mesohyl. This is the first report of the successful culture of a bacterial symbiont of a sponge that is transferred through the gametes.     

Comparative study of the choanosome of porifera: II. The keratose sponges

The aquiferous system of representatives of the orders Dictyocer-atida, Dendroceratida, and Verongida has been studied to note its relevance to the systematics of the groups. The volume of the choanocyte chamber, the size and shape of the choanocytes, the number of choanocytes per chamber, the relative development of the mesohyl, and the features of endopinacocytes are estimated from scanning and transmission electron microscopic observations of representatives of most families of the three orders. Although the Dysideidae have a reticulate skeleton and were classified in the order Dictyoceratida, they are actually closer to the Aplysillidae (Dendroceratida) than to dictyoceratids. The anatomy and cytology of the Halisarcidae differ profoundly from those of these three orders and are clearly more closely related to nonkeratose sponges. Some changes in classification lead to a pattern with highly homogeneous orders that clearly differ in their anatomic and cytologic features, which does not support the hypothesis of a common origin of the “keratose” sponges.