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Sponges (Porifera), basal nonbilaterian metazoans, are well known for their high regenerative capacities ranging from reparation of a lost body wall to whole-body regeneration from a small piece of tissues or even from dissociated cells. Sponges from different clades utilize different cell sources and various morphological processes to complete the regeneration. This variety makes these animals promising models for studying the evolution of regeneration in Metazoa. However, there are few publications concerning the regenerative mechanisms in sponges. This could be partially explained by the delicacy of sponge tissues, which requires modifying and fine adjusting of common research protocols. The current chapter describes various methods for studying regeneration processes in the marine calcareous sponge, Leucosolenia. Provided protocols span all significant research steps: from sponge collection and surgical operations to various types of microscopy and immunohistochemical studies.

Sponges (phylum Porifera) are early-branching animals, whose outwardly simple body plan is underlain by a complex genetic repertoire. The transition from a mobile larva to an attached filter-feeding organism occurs by metamorphosis, a process accompanied by a radical change of the body plan and cell transdifferentiation. The continuity between larval cells and adult tissues is still obscure. In a previous study, we have produced polyclonal antibodies against the major protein of the flagellated cells covering the larva of the sponge Halisarca dujardini , used them to trace the fate of these cells and shown that the larval flagellated cells transdifferentiate into the choanocytes. In the present work, we identified the sequence of this novel protein, which we named ilborin. A search in the open databases showed that multiple orthologues of the newly identified protein are present in sponges, cnidarians, flatworms, ctenophores and echinoderms, but none of them has been described yet. Ilborin has two conserved domains: triosephosphate isomerase-barrel, which has enzymatic activity against macroergic compounds, and canonical EF-hand, which binds calcium. mRNA of ilborin is expressed in the larval flagellated cells. We suggest that the new protein is involved in the calcium-mediated regulation of energy metabolism, whose activation precedes metamorphosis.

BackgroundTissues of multicellular animals are maintained due to a tight balance between cell proliferation and programmed cell death. Phylum Porifera is an early branching group of metazoans essential to understanding the key mechanisms of tissue homeostasis. This paper is dedicated to the comparative analysis of proliferation and apoptosis in intact tissues of two sponges belonging to distinct Porifera lineages, Halisarca dujardinii (class Demospongiae) and Leucosolenia variabilis (class Calcarea).ResultsLabeled nucleotides EdU and anti-phosphorylated histone 3 antibodies reveal a considerable number of cycling cells in intact tissues of both species. The main type of cycling cells are choanocytes - flagellated cells of the aquiferous system. The rate of proliferation remains constant in areas containing choanoderm. Cell cycle distribution assessed by the quantitative DNA stain reveals the classic cell cycle distribution curve. During EdU pulse-chase experiments conducted in H. dujardinii, the contribution of the choanocytes to the total amount of EdU-positive cells decreases, while contribution of the mesohyl cells increases. These findings could indicate that the proliferation of the choanocytes is not solely limited to the renewal of the choanoderm, and that choanocytes may participate in the general cell turnover through migration. The number of apoptotic cells in intact tissues of both species is insignificant. In vivo studies in both species with TMRE and CellEvent Caspase-3/7 indicate that apoptosis might be independent of mitochondrial outer membrane permeabilization.ConclusionsA combination of confocal laser scanning microscopy and flow cytometry provides a quantitative description of cell turnover in intact sponge tissues. Intact tissues of H. dujardinii (Demospongiae) and L. variabilis (Calcarea) are highly proliferative, indicating either high rates of growth or cell turnover. Although the number of apoptotic cells is low, apoptosis could still be involved in the regular cell turnover.

The phenomenon of whole-body regeneration means rebuilding of the whole body of an animal from a small fragment or even a group of cells. In this process, the old axial relationships are often lost, and new ones are established. An amazing model for studying this process is sponges, some of which are able to regenerate into a definitive organism after dissociation into cells. We hypothesized that during the development of cell aggregates, primmorphs, new axes are established due to the activation of the Wnt and TGF-beta signaling pathways. Using in silico analysis, RNA-seq, and whole-mount in situ hybridization, we identified the participants in these signaling pathways and determined the spatiotemporal changes in their expression in demosponge Halisarca dujardinii. It was shown that Wnt and TGF-beta ligands are differentially expressed during primmorph development, and transcripts of several genes are localized at the poles of primmorphs, in the form of a gradient. We suppose that the Wnt and TGF-beta signaling cascades are involved in the initial axial patterning of the sponge body, which develops from cells after dissociation.

International audience; While virtually all animals show certain abilities for regeneration after an injury, these abilities vary greatly among metazoans. Porifera (Sponges) is basal metazoans characterized by a wide variety of different regenerative processes, including whole-body regeneration (WBR). Considering phylogenetic position and unique body organization, sponges are highly promising models, as they can shed light on the origin and early evolution of regeneration in general and WBR in particular. The present review summarizes available data on the morphogenetic and cellular mechanisms accompanying different types of WBR in sponges. Sponges show a high diversity of WBR, which principally could be divided into (1) WBR from a body fragment and (2) WBR by aggregation of dissociated cells. Sponges belonging to different phylogenetic clades and even to different species and/or differing in the anatomical structure undergo different morphogeneses after similar operations. A common characteristic feature of WBR in sponges is the instability of the main body axis: a change of the organism polarity is described during all types of WBR. The cellular mechanisms of WBR are different across sponge classes, while cell dedifferentiations and transdifferentiations are involved in regeneration processes in all sponges. Data considering molecular regulation of WBR in sponges are extremely scarce. However, the possibility to achieve various types of WBR ensured by common morphogenetic and cellular basis in a single species makes sponges highly accessible for future comprehensive physiological, biochemical, and molecular studies of regeneration processes.

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Origin and early evolution of regeneration mechanisms remain among the most pressing questions in animal regeneration biology. Porifera have exceptional regenerative capacities and, as early Metazoan lineage, are a promising model for studying evolutionary aspects of regeneration. Here, we focus on reparative regeneration of the body wall in the Mediterranean demosponge Aplysina cavernicola. The epithelialization of the wound surface is completed within 2 days, and the wound is completely healed within 2 weeks. The regeneration is accompanied with the formation of a mass of undifferentiated cells (blastema), which consists of archaeocytes, dedifferentiated choanocytes, anucleated amoebocytes, and differentiated spherulous cells. The main mechanisms of A. cavernicola regeneration are cell dedifferentiation with active migration and subsequent redifferentiation or transdifferentiation of polypotent cells through the mesenchymal-to-epithelial transformation. The main cell sources of the regeneration are archaeocytes and choanocytes. At early stages of the regeneration, the blastema almost devoid of cell proliferation, but after 24 hr postoperation (hpo) and up to 72 hpo numerous DNA-synthesizing cells appear there. In contrast to intact tissues, where vast majority of DNA-synthesizing cells are choanocytes, all 5-ethynyl-2'-deoxyuridine-labeled cells in the blastema are mesohyl cells. Intact tissues, distant from the wound, retains intact level of cell proliferation during whole regeneration process. For the first time, the apoptosis was studied during the regeneration of sponges. Two waves of apoptosis were detected during A. cavernicola regeneration: The first wave at 6-12 hpo and the second wave at 48-72 hpo.

Sponges (phylum Porifera) traditionally are represented as inactive, sessile filter-feeding animals devoid of any behavior except filtering activity. However, different time-lapse techniques demonstrate that sponges are able to show a wide range of coordinated but slow whole-organism behavior. The present study concerns a peculiar type of such behavior in the psychrophilic demosponge Amphilectus lobatus: stolonial movement. During stolonial movement, sponges produce outgrowths (stolons) that crawl along a substrate with a speed of 4.4 ± 2.2 μm min

It was found that cells of different color morphs of the cold-water marine sponges Halichondria panicea (Pallas, 1766) of the class Demospongiae differ in the content of epibionts of bacteria of the genus Pseudoalteromonas. The sponge cells with elevated levels of epibionts of bacteria of the genus Pseudoalteromonas showed an increased expression of Hsp70 proteins but had a reduced level of the proteasomal catalytic beta 5 subunit, which was accompanied by a change in their activity. Probably, epibionts of bacteria of the genus Pseudoalteromonas may affect the ubiquitin-proteasome system in the cells of cold-water marine sponges and, thereby, ensure their adaptive plasticity.

The ability of sponge cells to reaggregate and reconstruct intact functional organism is known for more than 100 years. This process was studied in numerous species of sponges, and its interspecific variability is well described. However, some data also indicate the existence of a certain intraspecific variability of the cell reaggregation. The present study deals with the cell reaggregation in two demosponges, Halichondria panicea and Halisarca dujardinii, during different periods of their sexual reproduction. In both species, cell reaggregation shows a common pattern at all studied periods of reproduction. However, the course of the reaggregation process significantly depends on the reproduction period of an individual used in the experiment, thus demonstrating pronounced intraspecific variability, which concerns the rate of the cell reaggregation and the final stage of the process. This variability occurs due to tissue rearrangements that accompany reproduction and changes cell composition and amount of available somatic stem cells in sponge tissues, and consequently alters morphogenetic potencies of a cell suspension and multicellular aggregates. In both Halichondria panicea and Halisarca dujardinii, the growth period is the most favorable for the reaggregation process, while the cell reaggregation is depressed during periods of embryogenesis and restoration of somatic tissues after the reproduction. At the same time, the structure of a particular stage of reaggregation and morphogenetic processes underlying the development of multicellular aggregates are always identical, independently from the period of the reproductive cycle.

Sponges (phylum Porifera) are one of the most ancient extant multicellular animals and can provide valuable insights into origin and early evolution of Metazoa. High plasticity of cell differentiations and anatomical structure is characteristic feature of sponges. Present study deals with sponge cell reaggregation after dissociation as the most outstanding case of sponge plasticity. Dynamic of cell reaggregation and structure of multicellular aggregates of three demosponge species (Halichondria panicea (Pallas, 1766), Haliclona aquaeductus (Sсhmidt, 1862), and Halisarca dujardinii Johnston, 1842) were studied. Sponge tissue dissociation was performed mechanically. Resulting cell suspensions were cultured at 8-10°C for at least 5 days. Structure of multicellular aggregates was studied by light, transmission and scanning electron microscopy. Studied species share common stages of cell reaggregation-primary multicellular aggregates, early-stage primmorphs and primmorphs, but the rate of reaggregation varies considerably among species. Only cells of H. dujardinii are able to reconstruct functional and viable sponge after primmorphs formation. Sponge reconstruction in this species occurs due to active cell locomotion. Development of H. aquaeductus and H. panicea cells ceases at the stages of early primmorphs and primmorphs, respectively. Development of aggregates of these species is most likely arrested due to immobility of the majority of cells inside them. However, the inability of certain sponge species to reconstruct functional and viable individuals during cell reaggregation may be not a permanent species-specific characteristic, but depends on various factors, including the stage of the life cycle and experimental conditions.

Sponges (Porifera) demonstrate prominent regeneration abilities and possess a wide variety of mechanisms, used during this process. In the current study, we combined in vivo observations with histological, immunohistochemical, and ultrastructural technics to elucidate the fine cellular mechanisms of the regeneration in the calcareous sponge Leucosolenia cf. variabilis. The regeneration of Leucosolenia cf. variabilis ends within 4-6 days. The crucial step of the process is the formation of the transient regenerative membrane, formed by the epithelial morphogenesis-spreading of the intact exopinacoderm and choanoderm. The spreading of the choanoderm is accompanied by the transdifferentiation of the choanocytes. The regenerative membrane develops without any contribution of the mesohyl cells. Subsequently, the membrane gradually transforms into the body wall. The cell proliferation is neither affected nor contributes to the regeneration at any stage. Thus, Leucosolenia cf. variabilis regeneration relies on the remodeling of the intact tissues through the epithelial morphogenesis, accompanied by the transdifferentiation of some differentiated cell types, which makes it similar to the regeneration in homoscleromorphs and eumetazoans.

International audience; Sponges (phylum Porifera) possess the remarkable regenerative abilities and great diversity of the regeneration mechanisms. The current study dealt with the regener-ation of calcareous sponge Leucosolenia complicata. Two types of experiments on L. complicata regeneration were performed: 1) the regeneration of the body wall, and 2) the regeneration of the amputated oscular tube. We have combined in vivo light microscopy and histological studies to reveal morphogenetic mechanisms and determine the cell types involved in the reparative regeneration in this sponge. The wound healing followed by complete restoration of lost body parts have been observed in both types of the experiments. The regeneration in Leucosolenia has a mode in which lost body parts are replaced by the remodeling of the remaining tissue. Epithelial morphogenesis, mainly spreading (flattening) and fusion of exo-and endopinacoderm sheets accompanied by the transdifferentiation of the choanocytes to the endopinacocytes was found to be the key morphogenetic process during regeneration in this species. The epithelial nature of the regeneration in Leucosolenia makes it similar to the regeneration in homoscleromorphs sponges and Eumetazoans.

Sponges (Porifera) are lower metazoans whose organization is characterized by a high plasticity of anatomical and cellular structures. One of the manifestations of this plasticity is the ability of sponge cells to reaggregate after dissociation of tissues. This review brings together the available data on the reaggregation of sponge cells that have been obtained to date since the beginning of the 20th century. It considers the behavior of dissociated cells in suspension, the mechanisms and factors involved in reaggregation, and the rate and stages of this process in different representatives of this phylum. In addition, this review provides information about the histological structure of multicellular aggregates formed during reaggregation of cells and the regenerative morphogenetic processes leading to the formation of normal sponges from these multicellular aggregates.

Baculoviruses are large DNA viruses that infect insect species such as Lepidoptera and are used in biotechnology for protein production and in agriculture as insecticides against crop pests. Baculoviruses require activity of host proteasomes for efficient reproduction, but how they control the cellular proteome and interact with the ubiquitin proteasome system (UPS) of infected cells remains unknown. In this report, we analyzed possible changes in the subunit composition of 26S proteasomes of the fall armyworm, Spodoptera frugiperda (Sf9), cells in the course of infection with the Autographa californica multiple nucleopolyhedrovirus (AcMNPV). 26S proteasomes were purified from Sf9 cells by an immune affinity method and subjected to 2D gel electrophoresis followed by MALDI-TOF mass spectrometry and Mascot search in bioinformatics databases. A total of 34 homologues of 26S proteasome subunits of eukaryotic species were identified including 14 subunits of the 20S core particle (7 α and 7 β subunits) and 20 subunits of the 19S regulatory particle (RP). The RP contained homologues of 11 of RPN-type and 6 of RPT-type subunits, 2 deubiquitinating enzymes (UCH-14/UBP6 and UCH-L5/UCH37), and thioredoxin. Similar 2D-gel maps of 26S proteasomes purified from uninfected and AcMNPV-infected cells at 48hpi confirmed the structural integrity of the 26S proteasome in insect cells during baculovirus infection. However, subtle changes in minor forms of some proteasome subunits were detected. A portion of the α5(zeta) cellular pool that presumably was not associated with the proteasome underwent partial proteolysis at a late stage in infection.