Among closely related taxa of trematodes such as Plagiorchioidea, Microphalloidea and Lecithodendroidea organisation of the daughter sporocysts has been described in detail only for some representatives of the plagiorchioidea and microphalloidea (see Cort et al. 1954; Schell 1962, 1965; Dobrovolskij 1975; Dobrovolskij et al. 1983; Galaktionov and Dobrovolskij 2003 and others). First of all it concerns the spatial organization of the brood cavity which could be compartmentalized by laminated outgrowths of the brood cavity lining. Such compartmentalization was shown for mother sporocyst of Haplometra cylindracea (Plagiorchiidae) by Dobrovolskij (1975) and daughter sporocysts of Microphallus ‘pygmaeus’ group (Microphallidae) by Galaktionov and Dobrovolskij (2003). Secondly it refers to morphofunctional organization of the germinal mass (i.e. organ of the parthenogenetic reproduction of the sporocysts and rediae, see Dobrovolskij and Ataev 2003) which has been studied in detail for some daughter sporocysts of the Plagiorchiidae (Mukhamedov 1979; Dobrovolskij et al. 1983; Dobrovolskij and Ataev 2003; Galaktionov and Dobrovolskij 2003) and Microphallidae (Microphallus ‘pygmaeus’ group, Dobrovolskij et al. 1983). It was demonstrated that in daughter sporocysts of Xiphidiocercaria sp. VII Odening, 1962 (Plagiorchiidae) proliferation of the undifferentiated cells in the germinal mass occurs during almost whole life of the individuals (Mukhamedov 1979; Dobrovolskij et al. 1983). Whereas all mitoses in germinal mass of the daughter sporocysts of Microphallus ‘pygmaeus’ group (Microphallidae) terminate at the early stages of the ontogeny of these parthenitae (Dobrovolskij et al. 1983).
It should be noted that spatial organization of the brood cavity of the daughter sporocysts of Lecithodendriidae, Pleurogenidae and Prosthogonimidae families have never been a topic of a particular study. All our current knowledge concerning parthenitae of these taxa is based mainly on the studies of their life-cycles (Knight and Pratt 1955; Hall 1959; Etges 1960; Madhavi et al. 1987; Goodman 1989; Brinesh and Janardanan 2013). The only detailed morphological research is the TEM description of the body wall organization of Cercaria helvetica XII (Lecithodendriidae) daughter sporocysts by Reader (1975). But even in this article no information regarding the spatial organization of the brood cavity of the studied parthenitae is given. Moreover, germinal mass of the Lecithodendriidae, Pleurogenidae and Prosthogonimidae daughter sporocysts hasn’t been described in literature yet.
Taking into account close phylogenetic relationship of the Microphalloidea and Lecithodendroidea (see for example Sokolov and Shchenkov 2017) it can be expected that sporocysts of these taxa share similar principle of the morphofunctional organization. However without thorough study of parthenitae from widely spread groups of Lecithodendriidae, Pleurogenidae and Prosthogonimidae this assumption can not be confirmed.
According to conventional histological technique we describe the anatomy of Cercaria etgesii Shchenkov, 2017 (Pleurogenidae) daughter sporocysts with emphasis on the organization of the brood cavity lining and germinal mass morphology.
Materials and Methods
457 specimens of Bithynia tentaculata (Mollusca: ‘Prosobranchia’) were collected on the territory of Kruglen’koe lake (53°10′45,1″N, 49°25′49,9″E) near the Mordovo village in September 2012. Only 49 gastropods were emitting larvae described by Shchenkov (2017) as C. etgesii (Pleurogenidae). Four infected molluscs were fixed in Bouin’s solution. The material was then sequentially dehydrated in 70% and 96% ethanol, isobutanol and chloroform. After dehydration samples were embedded in Histomix™. Histological sections (five micrometres thick) were made using Leica RM2235 microtome. Prepared sections were deparaffinised and stained with Erlich’s acid haematoxylin/azure-eosin. Stained sections were embedded in the synthetic medium Biomount™ and studied using a Leica DM4000B microscope. All microphotographs were taken with a Leica DFC365 FX camera. Schemes (Fig. 5 and 6) were implemented in CorelDRAW™ graphic editor, on the basis of illustrations made with drawing apparatus ‘RA–7’.
For a 3D reconstruction, a series of microphotographs of longitudinal sections through a sporocyst were taken (Fig. 4A). Obtained images were aligned with Amira® software. Then the set of aligned microphotographs was united in a stack using Fiji software (Schindelin et al. 2015). The stack was uploaded in Bitplanelmaris® software where 3D reconstruction was performed (according to the following steps). All regions of interest (the sporocyst body wall, embryos, etc.) were outlined manually on each microphotograph using ‘Surface’ mode. After that the program automatically generated volumetric images of the allotted structures. The prepared 3D model (and some of it inner parts) were photographed using the ‘Snapshot’ function of the Bitplanelmaris® software (Fig. 1A and 2A, C, E). On the basis of this snapshot schemes were implemented in CorelDRAW™ graphic editor (Fig. 1B and 2B, D, F). Reconstruction was made using the computer performance of the Research park of St. Petersburg State University ‘Center for Molecular and Cell Technologies’.
The sporocysts were mainly detected inside haemal system lacunae located in the mollusc’s hepatopancreas (Fig. 4A). Connective tissue that forms lacunae closely adjoins the outer tegumental layer of the sporocysts (Fig. 4A, 5). Single parthenitae are occasionally found in the gill vessels of the molluscs’ ctenidium. According to observation on the living material the sporocysts are immobile.
The body form of daughter sporocysts ranges from oval to egg-shaped (Fig. 1, 4A, 5). The anterior end is dilated while the posterior is rather narrowed (Fig. 1, 4A, 5). Body length varies within 179–331 microns, body width within 182–259 microns. The birth pore is located at the anterior end of the sporocysts (Fig. 1, 3A).
The outer tegumental layer is unevenly covered by the mollusc’s amebocytes (Fig. 3C, 5). These cells are flattened in the middle region of the sporocysts’ body (Fig. 5) and possess a cylindrical form when located on its ends (Fig. 3C).
Beneath the outer tegumental layer the contractible elements of circular and longitudinal musculature are located (Fig. 3B). Below the contractible elements the layer of cytons is situated (Fig. 3B, C, 4C). The cytons possess nuclei with little amount of heterochromatin (it distributed as small inclusions across the nucleoplasm; Fig. 3A-C, 4C).
The lining of the brood cavity is formed by flattened structures which in total constitute the continuous layer (Fig. 1, 3–6). As it seen at the light microscopy level these laminated structures are derived from the cells located beneath the layer of cytons (Fig. 3A, 4B, C, 5 and 6). We designate these cells neither ‘parenchyma’ nor ‘lining cells’ but as the ‘endocyst cells’ (see ‘Discussion’ part for details). Heterochromatin in the nuclei of the endocyst cells is distributed as peripheral aggregations and small inclusions in nucleoplasm (Fig. 4C, 5 and 6).
Elements of nervous and excretory systems of sporocysts were not identified on histological sections.
The brood cavity of sporocysts contains from two to five cercariae at the stages of late morphogenesis and from two to five germinal balls (Fig. 1, 4A, 5). Larvae at the stages of late morphogenesis are floating freely in anterior part of the sporocysts’ brood cavity. The germinal balls are developing within separate chambers formed by the laminated structures of the lining (Fig. 1, 2A–D, 4B, C, 5). Along with the developing cercariae the disintegrating embryos are frequently found inside the sporocysts. Such disintegrating embryos do not float freely in a unified space of the brood cavity. Disintegration products are enclosed in separate chambers also formed by the laminated structures of the lining (Fig. 1, 2E, F, 4B, C).
Germinal mass is situated subterminally at the posterior end of the sporocysts’ body (Fig. 1, 3D, 4A, 6). It is constituted by structural cells and germinal elements (Fig. 3D, 6). Outgrowths of the structural cells form the ‘soma’ of the germinal mass and divide it into separate compartments with developing embryos or germinal cells inside (Fig. 3D, 6). Also the outgrowths of these cells closely adjoin laminated structures of the lining located in the posterior part of the parthenitae body and provide the binding of the germinal mass with brood cavity lining (Fig. 3D, 4A, 6). Thus the germinal mass of C. etgesii daughter sporocysts refers to the ‘attached type’ according to terminology of Galaktionov and Dobrovolskij (2003).
Germinal mass is heteropolar: germinal elements are unevenly located within it (Fig. 3D, 4A, 6). Anterior part of the germinal mass is occupied by several embryos (from two to three) at the early stages of embryogenesis (Fig. 3D, 6). The embryos’ development is asynchronous (Fig. 6). Along with the embryos, from two to five physiologically mature germinal cells are located in the central part of germinal mass (Fig. 3D, 6): these cells possess highly basophilic cytoplasm, a ‘lucid’ nucleus almost devoid of heterochromatin, and a conspicuous nucleolus (Fig. 6). Heterochromatin in the nucleus of physiologically mature germinal cells is distributed as peripheral aggregation and threads crossing the nucleoplasm (Fig. 6). In the posterior part of the germinal mass there are from two to four cells with less basophilic cytoplasm (compared with mature germinal cells), a nucleus containing a considerable amount of heterochromatin and noticeable nucleolus (Fig. 3D, 6): these are physiologically maturing germinal cells. Mitotic divisions in the germinal mass of all studied sporocysts were not detected.
Body wall elements
Our results concerning body wall organization of C. etgesii daughter sporocysts in general correspond to the TEM data obtained by Reader (1975) for morphologically close species of C. Helvetica XII daughter parthenitae. In both cases the sporocysts body wall is formed by the outer tegumental layer, contractible elements of body wall musculature, the layer of tegumental cytons and myocytons and laminated structures lining the brood cavity. Reader (1975) also described the terminal cells of the excretory system of C. helvetica XII daughter sporocysts that lay between the outgrowths of the tegumental cytons. As noted above we were unable to found any parts of the excretory system of C. etgesii daughter sporocysts on the studied histological sections.
Neither in article of Reader (1975) nor in other studies on the Lecithodendroidea parthenitae (Knight and Pratt 1955; Hall 1959; Etges 1960; Madhavi et al. 1987; Goodman 1989; Brinesh and Janardanan 2013) elements of the nervous system have not been described. On the histological slides we also have not trace any fragments of the C. etgesii daughter sporocysts nervous system. But according to confocal microscopy data of Shchenkov et al. (2016) near the birth pore of C. etgesii daughter sporocysts one serotoninergic neuron is located. Evidently, further detailed studies of the both nervous and excretory systems of Lecithodendroidea parthenitae is needed.
Although presence of the laminated structures lining the brood cavity of pathenitae have been shown in numerous TEM studies (Rees 1966; Bibby and Rees 1971; Køie 1971a, b; Reader 1975; Zdárská 1979; Nesterenko et al. 1980; Dobrovolskij et al. 1983; Klag et al. 1997; Galaktionov and Dobrovolskij 2003; Pinheiro et al. 2004; Franco-Acuña et al. 2011; Podvyaznaya and Galaktionov 2011 and others) all authors designate this part of sporocysts and rediae body differently.
Some researchers don’t apply any special term to denote the cells with numerous outgrowths that lay beneath a layer of cytons (Køie 1971a, b; Pinheiro et al. 2004). Whereas other authors designate this laminated structures and cells from which they originate as ‘parenchyma’ (Bibby and Rees 1971), ‘overlapping cells’ (Rees 1966), ‘macrophage cells’ (Klag et al. 1997) or ‘lining cells’ (Reader 1975; Podvyaznaya and Galaktionov 2011). Moreover there is one more term – the endocyst – which is traditionally used to refer to the peculiar multilayered lining of the brood cavity of Dicrocoelidae daughter sporocysts (see for example Tang 1950; Zdárská 1979; Nesterenko et al. 1980; Franco-Acuña et al. 2011).
Though no comparative researches on the organization of these cells and their outgrowths among parthenitae have been performed all TEM data obtained to date suggests that laminated structures lining brood cavity of sporocysts and rediae are ultrastructurally similar (Rees 1966; Bibby and Rees 1971; Køie 1971a, b; Reader 1975; Zdárská 1979; Nesterenko et al. 1980; Dobrovolskij et al. 1983; Klag et al. 1997; Galaktionov and Dobrovolskij 2003; Pinheiro et al. 2004; Franco-Acuña et al. 2011; Podvyaznaya and Galaktionov 2011). Considering this similarity we designate the totality of the laminated structures lining the brood cavity of C. etgesii daughter sporocysts (along with all rest parthenitae) as the endocyst and the cells from which this laminated structures originate as the endocyst cells.
The only evident for now distinction is the thickness of the endocyst. In C. etgesii daughter sporocysts as well as in daughter sporocysts of C. helvetica XII (Reader 1975), daughter sporocysts of Cercaria buccini (Køie 1971b), daughter sporocysts of Diplostomum pseudospathaceum (Klag et al. 1997), sporocyst of Prosorhynchoides borealis (Podvyaznaya and Galaktionov 2011) and rediae of Neophasis anarrhichae (=lageniformis) (Køie 1971a) the laminated outgrowths of the endocyst constitute one continuous layer. While in rediae of Parorchis acanthus (Rees 1966) and especially in daughter sporocysts of the Dicrocoelidae (Zdárská 1979; Nesterenko et al. 1980; Franco-Acuña et al. 2011) the endocyst is multilayered.
Spatial organization of the brood cavity
In C. etgesii daughter sporocysts the laminated structures of the endocyst serve several purposes. Firstly they form the lining of brood cavity. Secondly the laminated structures of the endocyst provide compartmentalisation of the brood cavity by forming separate chambers around developing and disintegrating embryos (Fig. 1, 4B, C, 5). Thus there is no uniform brood cavity in C. etgesii daughter sporocysts. Instead the brood cavity is represented by several compartments isolated from each other. The widest one contains cercariae at the stages of late morphogenesis (Fig. 1, 5). Inside the rest of compartments (denoted by us as the endocyst chambers) germinal balls or disintegrating embryos are located (Fig. 1, 4B, C, 5).
Embryos’ development under protection of the laminated outgrowths of the brood cavity lining has been shown for both mother and daughter parthenitae of different taxa of trematodes (James and Bowers 1967; Sannia et al. 1978; Galaktionov and Dobrovolskij 2003; Skála et al. 2014; Krupenko et al. 2016) including the daughter sporocysts of the Microphallus ‘pygmaeus’ group (see for example Galaktionov and Dobrovolskij 2003). Although cited authors do not use the term ‘endocyst chamber’ (except Krupenko et al. 2016) it can be supposed that in all cases embryos development under protection of the outgrowths of the brood cavity lining are similar to those described by us for daughter sporocysts of C. etgesii, i.e. proceeds inside the chambers, formed by laminated structures of the endocyst.
In the available literature concerning other parthenitae there is no mention of chambers with disintegrating embryos inside.
Presence of the mitotic divisions is proposed to be an important criterion for defining the existence of the oogonia in the ovary of adult trematodes (Koulish 1965; Kremnev et al. 2017). Consequently we suggest that the absence of mitoses gives evidence for the expenditure of all undifferentiated cells in the germinal mass of the studied C. etgesii daughter sporocysts. Therefore it can be proposed that generative function of the germinal mass of C. etgesii daughter sporocysts is exhausted quite early, just after the termination of the undifferentiated cells’ proliferation. All mitotic divisions in the germinal mass presumably take place at the previous stages of the daughter sporocysts’ ontogeny. Thus the germinal mass of the studied C. etgesii daughter sporocysts serves as peculiar ‘brood chamber’ where only physiological maturation of the germinal cells and their subsequent cleavage is realised.
The rapid depletion of the generative function (resulting from the completion of the undifferentiated cells’ divisions) is known for the germinal mass of some rediae of Echinostomatidae (Ataev et al. 2007), Psilostomatidae (Isakova 2011a) and for the germinal mass of daughter sporocysts from the Microphallus ‘pygmaeus’ group (Dobrovolskij et al. 1983). But in contrast to C. etgesii there is strict synchronisation of the germinal cells’ maturation and their following cleavage in the germinal masses of Microphallus ‘pygmaeus’ daughter parthenitae (Dobrovolskij et al. 1983). Therefore all embryos inside these sporocysts develop almost simultaneously. Mentioned steps are more prolonged in the case of C. etgesii daughter sporocysts.
Finally it should be emphasized that the analogous plan of daughter sporocysts organisation is characteristic for the studied species of Pleurogenidae and Microphallidae. Representatives of these highly specialized taxa possess a number of considerable differences in morphology, ecology, and strategies of the life-cycles realization. Nevertheless, the morphofunctional principle of the organisation of the daughter sporocysts is surprisingly uniform for certain species of Pleurogenidae (C. etgesii) and Microphallidae (Microphallus ‘pygmaeus’ group). It manifests in the compartmentalization of the brood cavity by the formation of chambers around developing embryos and rapid completion of the generative function of the germinal mass.
The work was performed at the Research park of St. Petersburg State University ‘Center for Molecular and Cell Technologies’. Authors are grateful to Dr. A. A. Dobrovolskij (St. Petersburg State University, department of Invertebrate zoology) for his advising during the writing of manuscript.
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About the article
Published Online: 2018-04-13
Published in Print: 2018-06-26
Conflict of interest: The authors declare that they have no competing interests.