2.3.2.1 Pinacoderm

The pinacoderm is represented by the exo‐, baso‐, and endopinacoderm. Exopinacoderm forms the external cover of the sponge. Basopinacoderm develops at the sponge base, attaching it to the substrate. Endopinacoderm forms the walls of the subdermal cavities and the aquiferous system canals, excluding regions of choanocyte chambers (which are lined by choanoderm; see below). There are, correspondingly, several types of pinacocytes.

The exopinacoderm consists of the exopinacocytes – the covering cells of the sponge, that may be T‐shaped or spindle shaped in cross‐section (Figures 2.1a, 2.2a,b, 2.3c,d,f). The spindle‐shaped exopinacocytes are described in many Demospongiae from the orders Spongillidae and Poecilosclerida (Bagby 1970; Weissenfels 1989), in all the Homoscleromorpha (Muricy et al. 1996, 1999; Ereskovsky et al. 2014) and in some Calcarea (Borojevic 1969; Eerkes‐Medrano and Leys 2006). The T‐shaped exopinacocytes are described in part of Demospongiae and Calcarea (see section 2.4.1) (Boury‐Esnault 1973; Willenz and Hartman 1989; Ereskovsky et al. 2011; Lavrov et al. 2018).

In most sponges, exopinacocytes lack specialized cell junctions, but are united with a well‐developed adhesive system (Blumbach et al. 1998; Schütze et al. 2001); for example, in Hippospongia communis, Ephydatia fluviatilis, Sycon coactum, S. ciliatum, and Leucosolenia variabilis, the sites of exopinacocyte contacts have electron‐dense thickenings of the membranes resembling zonula adhaerens (Pavans de Ceccatty et al. 1970; Pottu‐Boumendil 1975; Eerkes‐Medrano and Leys 2006; Lavrov et al. 2018). In Homoscleromorpha exopinacocytes have specialized cell junctions, and the exopinacoderm in general has all the structural features of eumetazoan epithelium (Ereskovsky and Tokina 2007). Unique characteristics of the homoscleromorph exopinacocytes are the flagellum (Muricy et al. 1996, 1999; Ereskovsky et al. 2014), and the ability to synthesize spicules (Maldonado and Riesgo 2007). Exopinacoderm contains ostia – numerous microscopic structures, 4–100 μm in diameter, through which water is drawn into the aquiferous system of the sponge.

Exopinacoderm exhibits many functions characteristic of the typical eumetazoan epithelia, such as absorption, secretion, transport, excretion and protection (Harrison and de Vos 1991; Meyer et al. 2006; Leys and Hill 2012). Moreover, exopinacocytes are capable of contractile responses (Pavans de Ceccatty 1986; Wachtmann and Stockem 1992a, b; Adams et al. 2010), amoeboid movement (Ereskovsky et al. 2015), incorporation of exogenous silica particles (Bavestrello et al. 1998), and phagocytizing of food particles (Willenz and van de Vyver 1982).

The basopinacoderm consists of the basopinacocytes – flattened cells, which are located at the basal surface of the sponge and function in attachment of the sponge to the substrate. Synthesizing basal spongin and fibronectin, basopinacocytes function as spongocytes (Garrone and Rozenfeld 1981; Labat‐Robert et al. 1981). During sponge growth, marginal basopinacocytes actively secrete proteins that make up spongin (Garrone 1978).

In the demosponges with a massive calcareous skeleton, such as Acanthochaetetes wellsi (order Clionaida), Ceratoporella nicholsoni and Stromatospongia norae (order Agelasida), basopinacocytes participate in the formation of this skeleton (Willenz and Hartman 1989; Reitner and Gautret 1996). In calcareous sponge Petrobiona massiliana basopinacocytes participate in formation of the basal massive skeleton by producing an extracellular organic framework that might guide the assemblage of submicronic amorphous Ca‐ and Mg‐bearing grains into higher structural units (Gilis et al. 2012).

Basopinacocytes of the freshwater demosponges have a well‐organized cytoskeleton (Wachtmann et al. 1990; Wachtmann and Stockem 1992a, 1992b; Kirfel and Stockem 1997; Adams et al. 2005). Actin is located in the cortical layer and in the fibrils in the cytoplasmic matrix. Microtubules radiate from the perinuclear zone, finishing at the cell periphery. At the same time, intermediate filaments have not been described. In Ephydatia muelleri, basopinacocytes were shown to have desmosome‐like junctions (Pavans de Ceccatty 1986).

The endopinacoderm consists of the endopinacocytes – flattened, polygonal cells, spindle shaped on cross‐section (Figures 2.1a, 2.2b, 2.3c; see also Figure 2.7a,b,f). The endopinacocytes are divided into prosopinacocytes, lining the inhalant canals, and apopinacocytes, lining the exhalant canals of the aquiferous system. The external surface of the endopinacocytes is covered with a glycocalyx layer (Boury‐Esnault et al. 1984; Vacelet et al. 1989; Harrison and de Vos 1991). The basal surface often forms numerous projections (pseudopodia) for anchoring in the extracellular matrix. In all Homoscleromorpha and in some Demospongiae, endopinacocytes bear flagella (Boury‐Esnault et al. 1984; Vacelet et al. 1989; Ereskovsky et al. 2014). In particular, this is the case in most studied representatives of the orders Dictyoceratida and Dendroceratida (Thiney 1972; Donadey 1982; Vacelet et al. 1989; Boury‐Esnault et al. 1990). The presence of flagella appears to be associated with the involvement of endopinacocytes in the generation of water currents through the aquiferous system. However, in some demosponges, the endopinacocytes lining the oscular tube (large exhalant opening) have short nonmotile cilia, presumably with sensory function, which may be involved in the coordination of simple sponge behavior (Ludeman et al. 2014).

Generally, endopinacocytes contact each other by simple overlap. However, in the oscular tubes of freshwater sponges they are united by desmosome‐like junctions (Masuda et al. 1998). Endopinacocytes of Homoscleromorpha are joined by zonula adhaerens junctions and underlined with basement membrane (Ereskovsky and Tokina 2007; Ereskovsky et al. 2009).

Endopinacocytes and choanocytes of some demosponges, homoscleromorphs, and calcareans are thought to be functionally and ontogenetically interrelated. For example, during reparative regeneration the choanocytes of different species from these taxa can differentiate into endopinacocytes by reduction of the flagellum and the microvilli and the subsequent flattening of the cell (Diaz 1974; Borisenko et al. 2015; Ereskovsky et al. 2015; Lavrov et al. 2018).

The apopylar cell is a particular cell type forming the boundary between apopinacoderm (epithelium lining the exhalant canal) and choanoderm (see section 2.3.2.2) and linking choanocytes and apopinacocytes (de Vos et al. 1990). Apopylar cells display morphologic characteristics intermediate between those of choanocytes and pinacocytes. In Homoscleromorpha, these cells possess a flagellum and an unfolded collar of microvilli (Boury‐Esnault et al. 1984). In demosponges, the apopylar cells were observed in all investigated species of Dictyoceratida and Dendroceratida, in some Chondrosida (Halisarca dujardinii, Thymosia guernei) and Haplosclerida, in the freshwater sponge E. fluviatilis and in Tethya wilhelma (Tethyida) (Langenbruch et al. 1985; de Vos et al. 1990; Hammel and Nickel 2014). Earlier, these cells were referenced as “cone cells” and “cell‐ring” (de Vos et al. 1990). In T. wilhelma, the apopyle (the exit from the choanocyte chamber) has also a reticuloapopylocyte, a modified apopylar cell, which has numerous small intracellular pores, which give them a mesh or grid‐like morphology (Hammel and Nickel 2014).

2.3.2.2 Choanoderm

The choanoderm consists only of choanocytes, which form the choanocyte chambers (or tubes in asconoid and solenoid sponges) (see section 2.4.2) (Figure 2.2b; see also Figure 2.6a–e). Contrary to the pinacoderm, the choanoderm has a cubic or palisade epithelium. Choanocytes can be cylindrical, cubic, trapezoid, or slightly flattened. These cells bear a flagellum surrounded with a collar of cytoplasmic microvilli interconnected by glycocalyx bridges. In some demosponges choanocytes have a periflagellar sleeve, such as in Suberitidae, Polymastiidae, Acanthochaetetidae, and Halisarcidae (Connes et al. 1971; Boury‐Esnault et al. 1990, 1994; Ereskovsky et al. 2011).

Another cell type associated with the choanocyte chambers of some demosponges is the central cell (Reiswig and Brown 1977; Diaz 1979; Langenbruch and Scalera‐Liaci 1986; Langenbruch and Jones 1989; Sciscioli et al. 1997; Ereskovsky et al. 2017a). Central cells have an irregular, branched shape with numerous projections and holes. The cell is perforated with a vast canal, into which flagella of the choanocytes enter. The central cells participate in the regulation of beating of the choanocyte flagella within a chamber and thus in the regulation of water currents through the aquiferous system.

2.3.3.1 Supportive‐Connective Tissue

This tissue comprises a variety of cells participating in the formation of organic and mineral skeleton and ground substance of the mesohyl.

Collencytes (lophocytes) are mobile cells participating in the secretion of collagen and formation of its fibrils (Figure 2.4a). These cells are frequently characterized by a nucleus without nucleolus, a well‐developed rough endoplasmic reticulum (RER), few nonspecific inclusions, and specific vacuoles containing collagen, which can be dense and homogenous or contain clear fibrillar material. Collencytes are devoid of phagosomes. These cells can be found all over the mesohyl. The secretory activity of these cells is evident from the deposition of oriented collagen fibrils near the cells, sometimes attached to the cell membrane (Borojevic 1966; Bonasoro et al. 2001).

Two names are used for this type of cell (Lévi 1970; Simpson 1984; Boury‐Esnault and Rützler 1997): collencyte usually refers to a stellar‐like or spindle‐like cell, while lophocyte refers to an obviously motile cell with anterior–posterior polarity and often forming a collagen bundle, which is associated with its posterior pole (Garrone 1978; Bonasoro et al. 2001).

Spongocytes are amoeboid cells responsible for the secretion of spongin in different forms (see section 2.4.3.2). Spongocytes always form groups of several cells during spongin secretion. They are characterized by a nucleus with nucleolus, well‐developed RER, perinuclear cisterns of Golgi complex and vesicular cytoplasm, containing numerous homogenous dense inclusions with spongin precursor (Garrone 1978).

Special types of spongocytes participate in the development of gemmules (a resistant asexual reproductive body, composed of internal mass of archaeocytes [thesocytes] charged with reserves and enclosed in a noncellular protective envelope) in freshwater sponges from the family Spongillidae. These spongocytes form a palisading epithelium around the developing gemmule and secrete collagenous shell and chitin for its coat (de Vos 1971, 1977; Langenbruch 1981, 1982; Ehrlich et al. 2013).

Sclerocytes are mobile cells, secreting elements of the mineral skeleton – spicules. Depending on the size and hence type of spicule produced (megasclere or microsclere) (see section 2.4.3.1), the sclerocytes are divided into megasclerocytes and microsclerocytes. Megasclerocytes have a nucleus with nucleolus, prominent Golgi complex, free ribosomes, mitochondria, few phagosomes and cisterns of RER (Figure 2.4b). Microsclerocytes are characterized by smaller size and a nucleus without nucleolus (Wilkinson and Garrone 1980; Garrone et al. 1981; Custodio et al. 2002).

Microscleres and microsclerocytes are characteristic only for Demospongiae and Hexactinellida. The mineral skeleton of Homoscleromorpha and Calcarea consists of megascleres of different size, so representatives of these classes have only megasclerocytes.

Silica spicules of Demospongiae, Hexactinellida, and Homoscleromorpha are synthesized intracellularly in vacuoles around an organic axial filament (Uriz et al. 2003; Uriz 2006; Leys et al. 2007; Maldonado and Riesgo 2007). During synthesis of large spicules, which are bigger than sclerocytes, several cells join (Uriz et al. 2003). In Demospongiae and Hexactinellida the membrane forming the spicule vacuole has a specific structure and is called a silicalemma (Uriz et al. 2003; Uriz 2006; Leys et al. 2007). It pumps silica inside the vacuole, producing a higher concentration inside for effective deposition around the organic axial filament (Uriz 2006). No information exists about the structure of analogous membranes in Homoscleromorpha.

In contrast, in calcareous sponges spicules are always synthesized extracellularly by the group (2–6 cells) of sclerocytes, which are joined by septate junctions and form an extracellular vacuole, where increased concentration of calcium ions is produced (Jones 1970; Ledger and Jones 1977; Uriz 2006).

Transport cells are peculiar amoeboid cells of the mesohyl described from the freshwater sponge E. fluviatilis (Nakayama et al. 2015). Transport cells are attached to the newly synthesized megascleres and transport a spicule from its place of synthesis to the final position in skeletal framework. No specific structural features of the transport cells are yet known. This cell type is defined only by its location on the newly synthesized megascleres, motile behavior, and specific expression of the gene EflSoxB1 (Nakayama et al. 2015).

2.3.3.2 Protective‐Secretory Tissue

Various amoeboid cells and cells with specific inclusions compose this tissue. The functions of protective‐secretory tissue include transfer and distribution of nutrition and oxygen, excretion, immune protection, and secretion of specific substances.

Amoebocytes sensu lato are common motile cells of the mesohyl. There is no clear definition of these cells and at various times they were called thesocytes (Sollas 1888), spherulous cells (Topsent 1892), polyblast or hyaline cells (Tuzet and Pavans de Ceccatty 1958), amoebocytes (Müller 1911), or nucleolated amoebocytes (Wilson and Penney 1930; Faure‐Fremiet 1931; Efremova 1972). They occur in all regions of the mesohyl and often are the main cell type in it. The amoebocytes have a large nucleus with nucleolus associated with Golgi complex, numerous RER cisterns and unspecific inclusions, especially phagosomes, and symbiotic zoochlorellae in the cytoplasm of freshwater sponges (Figure 2.4c) (Gilbert and Allen 1973; Williamson 1979). Amoebocytes are traditionally considered to execute several functions including digestion and distribution of nutrition, immune response in the form of phagocytes, elimination of refractory leftovers, and functioning as stem cells. Considering the broad variety of executed functions and absence of obvious structural features, amoebocytes could represent a highly heterogenous cell group and should be further researched. Currently, one subpopulation of amoebocytes can be distinguished – archaeocytes.

Archaeocytes are amoebocytes with a high nuclear/cytoplasmic ratio, with cytoplasm reach in RER and ribosomes and devoid of special cytoplasm inclusions (Figure 2.4d) (Smith and Hildemann 1990; Harrison and de Vos 1991). They occur in Demospongiae and Hexactinellida (in which they are the only cellular elements independent from the main syncytial tissues) and represent one of the stem lines in sponges of these classes (Lévi 1970; Korotkova, 1981, 1997; Simpson 1984; Harrison and de Vos 1991; Funayama, 2008, 2018).

In addition, participation of some amoebocytes in various immune reactions is well known (Smith and Hildemann 1986, 1990), and some attempts to distinguish this subpopulation have been made, using molecular markers (Funayama et al. 2005).

Bacteriocytes represent mobile cells with specific vacuoles, containing various symbiotic prokaryotes. This cell type is known only in demosponges. Bacteriocytes can contain single large or several small vacuoles with symbionts (Figure 2.4e) (Vacelet 1970; Vacelet and Donadey 1977; Bigliardi et al. 1993; Vacelet and Boury‐Esnault 1996; Maldonado 2007). Bacteriocytes participate in food digestion in carnivorous sponges (Vacelet and Duport 2004) and are responsible for vertical transmission of symbionts in some demosponges (Ereskovsky et al. 2005; Maldonado 2007), as they penetrate the embryos during their development and remain intact until the larval settlement and metamorphosis (Lévi and Lévi 1976).

Cells with specific inclusions are an important element in sponge mesohyl. This heterogenous cell group includes various cells types, which are united by the presence of specific inclusions in the cytoplasm but differ by structure of these inclusions. Currently, the function of most cells with specific inclusions is unknown. Some evidence indicates that these cells can realize the content of their vacuoles in the mesohyl, participate in metabolism of glycogen, excrete metabolic by‐products, and produce metabolites with antibiotic functions, which may be involved in the regulation of symbiotic bacteria or defense against foreign bacteria. Cells with inclusions can be found in most demosponges and homoscleromorphs but are rare in calcareous sponges and hexactinellids. According to Simpson (1984), cells with specific inclusions are subdivided into two major categories: cells with larger inclusions and cells with smaller inclusions.

Cells with larger inclusions. Vacuolar cells (cystencytes) are characterized by the presence of one or several large transparent vacuoles, occupying almost all cytoplasm of these cells (Figure 2.4g; see also Figure 2.7b,f). The free cytoplasm is reduced to a thin film around inclusions and nucleus. The nucleus may be displaced to the cell periphery by the inclusions. In addition, cytoplasm may contain Golgi complex, some RER and smooth endoplasmic reticulum, few mitochondria, and small phagosomes. Cystencytes are vacuolar cells of freshwater sponges, having one large inclusion with amorphous material of a polysaccharide nature (Tessenow 1969; Pottu‐Boumendil 1975; Ereskovsky et al. 2016). Vacuolar cells can be used as a diagnostic characteristic in closely related species of sponges without a skeleton, for example Oscarella and Halisarca (Muricy et al. 1996; Ereskovsky 2006, 2007).

Spherulous cells have several large membrane‐bounded inclusions (0.8–8 μm diameter). Free cytoplasm is reduced to narrow strands between the inclusions and on the cell periphery. The cytoplasm contains few mitochondria and rare cisterns of RER (Figure 2.4h; see also Figure 2.8f). The nucleus is usually small, anucleolated, and deformed by the inclusions. Inclusion content is usually homogenous but can be paracrystalline, fibrillar, or lamellar (Diaz 1979; Thompson et al. 1983; Bonasoro et al. 2001; Ereskovsky et al. 2017b). In some species, spherulous cells are localized near the sponge surface or aquiferous system canals, although they can lie diffusely in the mesohyl (Uriz et al. 1996; Bonasoro et al. 2001; Ereskovsky 2007; Maldonado 2016). The presumable function of the spherulous cells varies in different species, indicating possible heterogeneity of this cell type. The spherulous cells were reported to participate in storage of various metabolites (including toxic ones) (Thompson et al. 1983; Uriz et al. 1996; Becerro et al. 1997), immune response against nonsymbiotic bacteria, defense against fouling, predation by release of metabolites (Thompson et al. 1983; Ternon et al. 2016), excretion (Vacelet 1967; Donadey 1978; Maldonado 2016; Ereskovsky et al. 2020), and mesohyl extracellular matrix synthesis and maintenance (Donadey and Vacelet 1977; Donadey 1982; Bretting et al. 1983; Smith and Hildemann 1990).

Granular cells have numerous membrane‐bound inclusions (0.5–2 μm diameter) in their cytoplasm. The inclusions of the granular cells are smaller, and their number is higher in comparison with spherulous cells (Figure 2.4i). The shape of inclusions varies from round to irregularly ovoid. Using electron microscopy, the inclusions are usually homogenous, but they also can be fine‐grained or have a fine‐grained periphery with a homogenous central region. The content of the inclusions is often separated from the surrounding membrane by the transparent space. The nucleus is round with or without a nucleolus. A few phagosomes, unspecific inclusions and vacuoles can appear in the cell cytoplasm (Pomponi 1976; Ereskovsky et al. 2011, 2017a, 2017b; Willenz et al. 2016). The exact functions of granular cells are unknown, but they may be involved in the immune response, as the inclusion contents show antimicrobial activity (Krylova et al. 2003). In addition, in some demosponges maternal granular cells penetrate into the forming larvae (Ereskovsky and Gonobobleva 2000; Rützler et al. 2003) and can be retained there until the beginning of metamorphosis (Gonobobleva and Ereskovsky 2004).

Granular and spherulous cells can be used as a diagnostic characteristic in closely related species (Pomponi 1976; Boury‐Esnault et al. 1994; Bergquist 1996; Muricy et al. 1996; Reveillaud et al. 2012; Gazave et al. 2013; Willenz et al. 2016).

Microgranular cells are characterized by cytoplasm filled with minute dense granules (~0.09–0.3 μm diameter). The nucleus is often anucleolated and cytoplasm contains few mitochondria and RER cisterns (Figure 2.4j) (Sciscioli et al. 2000; Pinheiro et al. 2004). These cells could contribute to the synthesis of glycoprotein components of the extracellular matrix. Others functions of the microgranular cells remain unknown.

Cells with smaller inclusions. Gray cells (glycocytes) contain numerous small ovoid membrane‐bounded inclusions (~0.2–0.8 μm diameter). The inclusions are acidophilic and osmiophilic. Another characteristic feature of these cells is glycogen rosettes in the cytoplasm (Figure 2.4k). Besides the glycogen rosettes, the cytoplasm of gray cells contains well‐developed RER and Golgi complex (Boury‐Esnault 1977). The nucleus usually contains a small nucleolus. Presumably these cells participate in glycogen metabolism (Boury‐Esnault 1977) and also have been considered as immunocytes, responsible for the allogeneic response (Humphreys 1994; Yin and Humphreys 1996; Sabella et al. 2007).

Rare type of cells with inclusions. The types of cells with inclusions described above are widespread and found in many sponge species. In addition to these, several rarer types of cells with inclusions occur in some sponges: rhabdiferous cells (Simpson 1968; Smith 1968; Smith and Lauritis 1969; Ereskovsky et al. 2011), sacculiferous cells (Smith 1968; Smith and Lauritis 1969), spumeuse cells (Donadey and Vacelet 1977; Donadey 1982), globoferous cells (Borojevic and Lévi 1964; Simpson 1968) and styllocytes (Harrison et al. 1974). These rare cell types have been found in one or several sponge species, consequently additional studies of their structure and functions are required. Moreover, these rare cells with inclusions could possibly be species‐specific modifications of common types of cells with inclusions.

Contractile cells of the mesohyl, myocytes, are found in the mesohyl of many demosponges and calcareous sponges. In some sponges, the myocytes can occur in large concentric multilayered structures (sphincters) around large exhalant canals and oscula, while in other species they lie sparsely in the mesohyl near the aquiferous system canals and/or dermal membrane (Bagby 1966). The myocytes are fusiform cells with an ovoid nucleus, lying in the central part of the cell (Figure 2.4f). Other organelles (Golgi complex, mitochondria, unspecific inclusions) are usually located near the poles of the nucleus. The bundles of myofilaments are concentrated at the cell periphery. In some sponges, the myocytes have two types of filaments, which are spatially organized, forming regular patterns (e.g., Tedania ignis) (Bagby 1966; Thiney 1972). Considering their position and ultrastructural features, the myocytes are thought to be contractile cells, which regulate the diameter of large canals of the aquiferous system, thus participating in the regulation of water flow.