Previously it was shown that the content of the Notocotylidae egg has lost all features of miracidia and cannot even be called this way (Murrils et al., 1985a). Our results support these findings: indeed, the egg of Paramonostomum sp. contains a very small mother sporocyst, composed of just one tegument cell and two germ cells. This corresponds well with the reductive trends in all other miracidia following the passive strategy of infection (Galaktionov, Dobrovolskij, 2003; Świderski et al., 2010, 2013; Smirnov, Dobrovolskij, 2019, 2021; Smirnov, Gonchar, 2022), but Notocotylidae went the furthest. What prompted their larvae to achieve such an extreme level of simplification? We believe that it was the emergence of the external penetration apparatus.
The main tool for penetration in Notocotylidae larvae is an evertable opercular cord—a part of the eggshell enclosed in the vitelline membrane. In vitro experiments suggested that penetration coincides with the hatching (Murrils et al., 1985b), and our data do not contradict this hypothesis. Moreover, we found a layer of the opercular material of the eggshell expanding to the cord. These new data suggest that the opening of the operculum likely causes the extrusion of this material, forcing the cord to turn out in a glove-finger fashion. It seems coherent that one of the factors triggering the hatching is mechanical. The long opercular filament could serve as a strong lever that, after hitting some relatively firm barrier (e.g. the gut wall of a snail, radula), might create a force sufficient for the hatching and abrupt eversion of the cord. This movement should be forceful enough to pierce the gut wall of the snail. The hatching is likely to happen soon after the ingestion of the egg, since young mother sporocysts were found on the outer surface of buccal and oesophagal regions of the snail gut (Rohde, Onn, 1967). Obviously, the impact of the mechanical factor in these ‘muscular’ regions is huge. Once the cord is everted the inner surface of the tunnel described above should form the outer surface of the growing tube. The mechanism behind the growth of the tube (Murrils et al., 1985b) is unclear. Perhaps, there are some chemical/mechanical cues for its elongation.
Our description of the mother sporocyst is almost completely the same as that by Murrills (Murrils et al., 1985a), albeit there are some nuances. First, we could not find anything resembling the brood chamber: the space beneath the tegument is tightly filled with the germ cells. Second, the inner extensions of the tegument, considered to be the ‘epithelium’ (Murrils et al., 1985a), are in fact two groups of intertwining extensions of both the tegument and the germ cells. Apparently, this tight connection increases the surface of contact between the cells. Last, we found nonmembranous granules in the cytoplasm of the germ cells. We consider these granules to be nuage granules—ribonucleoprotein (RNP) complexes known by different names in different animals (e.g. chromatoid bodies, perinuclear P-granules).
Previously, nuage granules have been explored in the germ cells of sporocysts (Klag et al., 1997, Podvyaznaya, 2007) and rediae (Podvyaznaya, Galaktionov, 2014), establishing one of the ultrastructural hallmarks of this type of cells in the intramolluscan stages of digeneans. Apart from digeneans, RNP bodies are present in germ cells of numerous metazoans (reviewed in Eddy, 1976) and seem to be an instrumental component of any cell of this kind. Outstandingly, nuage-like structures are also found in neoblasts—the stem-cells of the free-living flatworms (Coward, 1974; Hori 1982). Conversely, the stem (undifferentiated) cells of digeneans lack nuage (Podvyaznaya, Galaktionov, 2014; Smirnov, Gonchar, 2022). This fact opens a topic for an interesting discussion on the nature of the digenean life cycle, but it is beyond the scope of the present article. Here, we only admit that although the content and functions of nuage are being explored in model species (e.g. Lim, Kai, 2007; Kotaja, Sassone-Corsi, 2007; Cassani, Seydoux, 2022), in Digenea neither has been defined yet.
Our interpretation of the germ cells in the Paramonostomum sp. miracidium matches the described fate of the mother sporocyst after infection of a snail host (Murrils et al., 1988, Kanev et al., 1994, Skala et al., 2014). Each of the germ cells seems to develop into a single mother redia, the next intramolluscan generation of worms. The number of mother rediae was reported as stable for several notocotylid species: one in Notocotylus attenuatus (Skala et al., 2014) and N. stagnicolae (Herber, 1942); two in Pseudocatatropis joyeuxi (Joyeux, 1922); four in Quinqueserialis quinqueserialis (Herber, 1942). These rediae develop synchronously, and apparently the number of germ cells in the eggs of these species equals the observed number of offspring. However, there is also a record of varying number of rediae in the first generation: four to eight in N. urbanensis (Herber, 1955). The reason for these variations is unknown, but it seems that in some species the number of offspring is more strictly determined than in others.
The notocotylid mother sporocyst does not develop its somatic elements and is essentially a thin tegumental sac for the whole of its short life (Murrils et al., 1988, Kanev et al., 1994, Skala et al., 2014). This is not surprising since it has no stem (undifferentiated) cells. The intriguing feature of the sporocyst is the connection between the germ cells and the tegument, which also remains after the penetration (Murrils et al., 1988). It is likely that this connection serves for the nutrition of the embryos, but another function may be assumed. Once the sporocyst enters the haemocoel of a snail, it should get some cue and launch the germ cells cleavage. Given that the sporocyst lacks neurons, it is likely that some kind of chemical signal is transmitted through the tegument extensions directly to the germ cells, initiating the development of rediae within a sporocyst. Increased contact area between the two groups of extensions is likely to amplify this signal.