Introduction
Echinostoma caproni Richard, 1964 (syn. E. liei) is a common model for the study of trematode life cycles and relationships with definitive and intermediate hosts (see Fried & Graczyk, Reference Fried and Graczyk2000; Fried & Toledo, Reference Fried and Toledo2009). Planorbids Biomphalaria glabrata and B. pfeifferi are usually used as its experimental molluscan hosts.
Abundant data on the development of parthenitae of E. caproni have been accumulated. The mechanism of infecting the mollusc, the migration of the mother sporocyst (MS), the dispersal of redioid generation and the reproductive dynamics of the parthenitae have been studied (Ataev et al., Reference Ataev, Dobrovolskij, Fournier and Jourdane1997, Reference Ataev, Isakova and Dobrovolskij2007; Fried & Graczyk, Reference Fried and Graczyk2000). The influence of the immune reactions of the molluscan host on these processes and the avoidance strategies employed by the parthenitae has also been in the focus of research (Ataev & Coustau, Reference Ataev and Coustau1999; Adema et al., Reference Adema, Sapp, Hertel, Loker, Fried and Graczyk2000; Bouchut et al., Reference Bouchut, Roger, Coustau, Gourbal and Mitta2006; Coustau et al., Reference Coustau, Gourbal, Mitta, Adema, Toledo and Fried2009; Adema et al., Reference Adema, Hanington, Lun, Rosenberg, Aragon, Stout, Richard, Gross and Loker2010; Hanington et al., Reference Hanington, Lun, Adema and Loker2010).
Echinostoma caproni is a convenient object for the study of reproduction of parthenitae of different generations. Particularly detailed information has been obtained on the reproductive mechanism of MS (Ataev et al., Reference Ataev, Dobrovolskij, Fournier and Jourdane1997; Ataev & Tokmakova, Reference Ataev and Tokmakova2018), with some of the studies carried out in vitro (Ataev et al., Reference Ataev, Fournier and Coustau1998).
Based on this information, the following scheme of the germinal material development in MS has been suggested. At the stage of the miracidium, the germinal material is localised caudally and represented by undifferentiated cells (UC) and germinal cells (GC). Together, these cells make up the primordium of the germinal mass. After the miracidium has penetrated into the mollusc and transformed into MS, the germinal elements are formed and develop in the germinal mass. In the sporocyst the germinal mass is also localised caudally but contains, in addition to UC and GC, embryos of various ages. If this scheme is correct, GC only divide by cleavage and their multiplication always results from the division of UC. This means that the germinal mass of E. caproni should be considered as the organ of reproduction.
It should be noted that we consider the life cycle of trematodes as heterogony and assume that rediae and sporocysts reproduce sexually by ameiotic parthenogenesis (apomixis). Our views are not in contradiction with the studies on cells of miracidia and sporocysts of Schistosoma mansoni using modern methods (Wang et al., Reference Wang, Collins and Newmark2013, Reference Wang, Lee, Li, Saberi, Yang, Liu, Zhao and Newmark2018; Attenborough et al., Reference Attenborough, Rawlinson, Diaz Soria, Ambridge, Sankaranarayanan, Graham, Cotton, Doyle, Rinaldi and Berriman2024). We also consider UC as stem cells whose potency is limited by the formation of GC and somatic elements of the germinal mass. At the same time, it is important to note that the germinal material of S. mansoni lacks mature GC (Tokmakova & Ataev, Reference Tokmakova and Ataev2022) and is thus fundamentally different from the germinal material of E. caproni (Ataev & Tokmakova, Reference Ataev and Tokmakova2018).
These conclusions are based on the histological and electron-microscopic studies of miracidia of E. caproni (Ataev & Tokmakova, Reference Ataev and Tokmakova2018) and the histological study of its sporocysts (Ataev et al., Reference Ataev, Dobrovolskij, Fournier and Jourdane1997, Reference Ataev, Fournier and Coustau1998). Only one transmission electron micrograph of GC in the sporocysts of E. caproni has previously been obtained.
The aim of this study was TEM analysis of all germinal elements of MS of E. caproni at the larval stage as well as the sporocystoid stage. Germinal material of the latter was analysed using 6-day-old sporocysts.
Materials and methods
We used the isolate of the trematode Echinostoma caproni maintained in the laboratory as previously described (Trouvé et al., Reference Trouvé, Renaud, Durand and Jourdane1996). A Brazilian laboratory isolate of snails Biomphalaria glabrata was used.
Infection of B. glabrata with Echinostoma caproni miracidia was carried out according to the method described previously (Jourdane, Kulo, Reference Jourdane and Kulo1981). Snails with a shell diameter of 9–11 mm were individually exposed to 9 in vitro-derived miracidia for 5 h at 26°С. Then the snails were transferred to 5 L tanks and maintained at ambient temperature (26°С) on a 12 L/12 D cycle.
Prior to fixation for SEM, newly hatched miracidia were subjected to sonication to remove adherent particles. Briefly, miracidia were sonicated for 5 min at 45 kHz using a Bransonic 321EH sonicator. Cultured miracidia were cleaned by careful successive resuspensions in filter-sterilized CBSS. Miracidia were fixed in glutaraldehyde-osmic acid (4°C, pH 7.4, 425 mOsm) and further prepared for SEM according to standard procedures (Fournier et al., Reference Fournier, Pagesr, Touassem and Mouahid1989).
For TEM studies, miracidia were fixed 6 h after hatching. For the study of MS, the hearts of the snails were fixed at 6 days post-infection (DPI). The material was thoroughly washed in Chernin’s solution and fixed in 4% glutaraldehyde (30 min at 4°С) and 1% OsO4 (1 hour at 4°С) diluted in the phosphate buffer (рН 7.4). The osmolarity of the solution was 425 mOsm. The material was embedded in Epon 812.
Thin sections were made with the use of Leica EM UC7 ultramicrotome, placed on copper grids, and contrasted with uranyl acetate and lead citrate. The material was examined with the use of JEM-1011 transmission electron microscope.
In total, four miracidia and two MS were analysed with the use of TEM. Series of longitudinal sections of two miracidia and series of transverse sections of two other miracidia were made. For an analysis of cell ultrastructure, 30–50 photographs of each cell were obtained and stitched with the use of Autopano Giga software. Morphometric analysis was performed with the help of ImageScope software.
Identification of UC, GC, and secretory cells was based on morphological characters described in earlier studies (Ataev et al., Reference Ataev, Dobrovolskij, Fournier and Jourdane1997; Ataev & Tokmakova, Reference Ataev and Tokmakova2018).
Results and discussion
Miracidia of E. caproni remain active for 4 hours after hatching. During this time, they have to find the molluscan host and, after shedding epithelial plates, penetrate it (Figure 1a). After penetration, the larva undergoes metamorphosis to become the sporocyst. This process takes up about 5 hr (resting period) (Ataev et al., Reference Ataev, Dobrovolskij, Fournier and Jourdane1997). Then MS migrate into the heart of the mollusc and attach to the walls of the ventricle or the aorta. After that, their localisation remains unchanged.
Miracidium of Echinostoma caproni. SEM micrograph (a), cross (b) and longitudinal (c) sections through the caudal part; sections through undifferentiated and germinal cells (d).
Abbreviations: am , accumulation of mitochondria; ep , epithelial plates; fc , flame cell; gc , germinal cell; n , nucleus; nep , nucleus of epithelial plate; nu , nucleolus; sc , secretory cells; uc , undifferentiated cell.
Our analysis of germinal material in miracidia of E. caproni confirmed the data obtained earlier with the use of different methods (Ataev et al., Reference Ataev, Dobrovolskij, Fournier and Jourdane1997; Ataev & Tokmakova, Reference Ataev and Tokmakova2018). In the posterior part of the larval body, 3–4 UC and 5–7 GC are localised (Figures 1b–d). The quantitative composition of germinal elements of the E. caproni miracidium has been studied in detail by various methods in previous works (Ataev et al., Reference Ataev, Dobrovolskij, Fournier and Jourdane1997, Reference Ataev, Fournier and Coustau1998, Reference Ataev, Dobrovolskij, Avanessian and Loker2001, Reference Ataev, Isakova and Dobrovolskij2007; Ataev & Tokmakova, Reference Ataev and Tokmakova2018). Together, these cells make up the primordium of the germinal mass. At this stage of the MS development, the germinal mass performs only the main function of the gonad, that is, multiplication of the number of GC. It should be noted that germinal elements in miracidia are arranged in a compact but irregular manner: in some individuals UC are located more posteriorly than GC, while in some others a reverse arrangement is observed.
GC exhibit a wide range of sizes and morphologies, often reflecting their level of specialization. Typically, they measure 8–10 μm in diameter, while their nuclei, which are irregularly shaped, reach 6–7 μm in diameter. These nuclei contain scattered patches of heterochromatin and feature a prominent nucleolus, signifying ongoing cellular growth and biosynthesis. A notable trait of GC is a vast accumulation of mitochondria at one of the cell poles.
UC are similar to GC but smaller, their diameter not exceeding 6–7 μm. Unlike GC, their nuclei have an irregular shape and typically measure less than 5 μm. A key distinction concerns their chromatin, which is densely packed, forming prominent clusters within the nucleus, particularly near the nuclear envelope. The cytoplasm contains several small mitochondria, abundant free ribosomes, and, sometimes, thin rough ER cisterns.
It is noteworthy that the germinal material in miracidia remains unchanged until 2 DPI. After that, all cells of MS are activated, including germinal elements (Ataev et al., Reference Ataev, Dobrovolskij, Fournier and Jourdane1997), and GC that were visible in the miracidium undergo cleavage. We have suggested the term ‘primary GC’ for these cells (Ataev & Tokmakova, Reference Ataev and Tokmakova2018). Division of UC results in multiplication of GC, and we refer to the cells formed in this way as secondary GC.
At 6 DPI, MS reaches 350 × 100 μm in size (Figure 2a). It is attached to the host tissues only with the posterior body end, which becomes overgrown with fibrous material (Ataev et al., Reference Ataev, Dobrovolskij, Fournier and Jourdane1997). By this time, somatic cells, which make up most of the sporocyst’s parenchyma, are degenerating actively. This process is accompanied by the emergence of numerous pyknotic bodies. As a result, a common brood cavity originating from the embryonic cavity (schizocoel) is formed (Figures 2d and 2e). It is lined with cytoplasmic outgrowths of stellate cells (in modern literature this lining is often referred to as the endocyst).
Mother sporocyst of Echinostoma caproni (6 days post-infection). SEM micrograph (a), cross section through the germinal mass (b), cross section through the cells of a redioid embryo (c), cross section through the wall of the mother sporocyst (d), cross section through an embryo of the mother redia (e), cross section through the primordium of the intestine of the mother redia (f).
Abbreviations: db , dividing blastomere; e , embryo of mother redia; ec , embryonic cavity; en , “endocyst”; gc , germinal cell; i , intestine; nem , nucleus of embryonic membrane; nu , nucleolus; sc , secretory cell; t , tegument of mother sporocyst; uc , undifferentiated cell. Arrows indicate the boundaries of the mother redia embryo.
About 11–14 embryos of mother rediae develop in the brood cavity. The diameter of the smallest is about 30 μm, while the largest ones reach the size of 130 × 70 μm. In addition, there are developing embryos in the germinal mass, which is located caudally in MS of this age (Figure 2b). The germinal mass is not distinctly segregated from the surrounding somatic cells, being embedded in the parenchymal matrix. At this stage of the MS development, germinal elements in the germinal mass are arranged in the order of reproductive maturity: UC are located in the posterior part, GC are located more anteriorly, and the embryos more anteriorly still, with the largest localising close to the anterior end of the germinal mass (Figure 2b). We found no significant differences between UC and GC developing in miracidia and those developing in sporocysts. They are similar in size and structure. Moreover, the same size and the same structural features are characteristic of UC and GC in redioid embryos (Ataev et al., Reference Ataev, Dobrovolskij, Fournier and Jourdane1997, Reference Ataev, Fournier and Coustau1998, Reference Ataev, Dobrovolskij, Avanessian and Loker2001, Reference Ataev, Isakova and Dobrovolskij2007; Ataev & Tokmakova, Reference Ataev and Tokmakova2018).
Altogether, 3–9 embryos develop in the germinal mass of MS at 6 DPI, the smallest of them consisting of 2–5 blastomeres (Figures 2b and 2c). This means that, in addition to its function as the gonad, the germinal mass of the sporocyst performs the function of the uterus. Redioid embryos, formed successively by cleavage of GC, develop in the germinal mass up to the stage of 20–25 blastomeres. Then they become enclosed in an embryonic membrane and pass into the embryonic cavity of MS, where their development continues (Figure 2e). This transition starts at 5 DPI (Ataev et al., Reference Ataev, Isakova and Dobrovolskij2007).
The total number of redioid embryos in MS at 6 DPI reaches 20 (16–24). The reproductive activity is manifested in numerous mitoses of the cells of both MS itself and developing redioid embryos (Figures 2c and 2e). The most advanced of the embryos undergo morphogenetic transformations, exhibiting the primordia of the pharynx and the gut (Figure 2f). Such embryos already contain 5–8 embryos of the daughter rediae.
The MS starts to release embryonic rediae at 8 DPI (Ataev et al., Reference Ataev, Dobrovolskij, Fournier and Jourdane1997) and proceeds actively for a week. One MS gives birth, on average, to six mother rediae formed from primary GC and approximately 20–25 rediae formed from secondary GC. However, the reproductive potential of MS is not implemented to the full. By the time of its death (at 21 DPI), it still contains 10–15 degenerating embryos.
To sum up, our ТЕМ study of the germinal material in miracidia and 6-day-old sporocysts of E. caproni confirmed our idea that the germinal mass is the reproductive organ combining the functions of the gonad and the uterus. The function of the gonad, expressed in the multiplication of GC resulting from the division of UC, starts at the time of embryogenesis of the miracidium. The function of the uterus is expressed in the cleavage of GC and subsequent development of redioid embryos up to the stage of the germinal ball. After that, the embryos leave the germinal mass and pass into the schizocoel cavity, which plays the role of the brood chamber.
The primordium of the germinal mass, already present in miracidia at the time of hatching, is represented by several UC and about 6 GC. The composition of the germinal elements remains unchanged until the metamorphosis of the larva into the sporocyst. Their activation is observed only at 2 DPI and results in multiplication of GC.
The classification of GC into primary and secondary is fairly contingent. We found no morphological differences between them. In fact, they differ only in the time of formation. Primary GC are formed during embryogenesis of miracidia, while secondary GC are formed at the sporocystoid stage.
In general, the germinal mass in MS of E. caproni is structurally similar to germinal masses of rediae of Echinostoma spp. described in an early study (Cort et al., Reference Cort, Ameel and Van der Woude1949). However, Cort and his co-authors did not study the reproduction of MS and considered it possible that GC could multiply by division. Our analysis of the germinal material in MS of E. caproni provides compelling evidence that this is not so. The number of GC increases only by division of UC. Germinal cells themselves cleave to form embryos.
Acknowledgements
The authors are grateful to Natalia Lentsman for her help with the preparation of the English version of the manuscript.
Financial support
The work was carried out in the Laboratory of Experimental Zoology of the Herzen State Pedagogical University with the support of a grant for the implementation of research work within the framework of the main scientific directions by scientific and pedagogical staff of the Herzen University No. 42-VG.
Competing interests
The authors declare no competing interests.
