Histological and electron microscopic analysis of germinal material in miracidia of Schistosoma mansoni.

We performed histological and electron microscopic analysis of miracidia of Schistosoma mansoni in order to examine their germinal elements. In total, about 20 germinal cells at different stages of maturation were found. We described their ultrastructure and proposed a scheme of reproduction of mother sporocysts of S. mansoni based on our data and literature information. According to this scheme, the only germinal elements present in the miracidia are germinal cells (undifferentiated cells were not found). Regardless of their size and localisation, none of the germinal cells in the miracidia has undergone full differentiation. This process is completed after the metamorphosis of the larva into the sporocyst.

Reproduction of Echinostoma caproni mother sporocysts (Trematoda).

The localisation and the composition of germinal material in miracidia and mother sporocysts of Echinostoma caproni were studied with the use of histological and electron microscopic methods. Germinal material in miracidia was localised in the posterior body half and was represented by 3-4 undifferentiated cells and 5-7 germinal cells. Taken together, these cells are referred to as the primordium of the germinal mass. In the mother sporocyst, germinal elements also form and develop in the germinal mass, which is located caudally. It comprises undifferentiated cells and germinal cells as well as embryos of various ages (up to the stage of 30-50 blastomeres). Germinal cells divide only by cleavage. New germinal cells are formed only from undifferentiated cells, which can proliferate in the germinal mass and nowhere else. This indicates that the germinal mass is the reproductive organ of E. caproni mother sporocyst.

[REPRODUCTION OF SCHISTOSOMA MANSONI MOTHER SPOROCYST].

The development of generative elements of Schistosoma mansoni mother sporocysts (MS) was examined by histological methods. About 20 large cells, on average, determined as germinal cells (GC) were found in the miracidium. These cells formed a C-shape cellular aggregation (a band) beginning in the caudal part of the larva, and reaching the nerve ganglion in the anterior part. At the level of the 3d tier of epithelial plates of the miracidium, this band shifted to the external body wall, bypassing the zone of excretory channels. Apparently, this shift resulted in the subdivision of a single pool of GC into two structurally associated groups. A group of several undifferentiated cells (UC) was also revealed in the caudal part of the body. After the metamorphosis of the miracidium into sporocysts, GC had increased in size and on the 3d day started to divide, forming first embryos of daughter sporocysts. During the same time, germinal masses were being formed in the subtegumental area of the MS body. Since this time point, proliferation of UC occured only in germinal masses. A part of UC also differentiated there into GC. These cells formed sporocystoid embryos, developing as far as the germinal ball, and then came out into the sporocyst schizocoel (approximately in 10 days p. i.). Thus, in S. mansoni, the formation of generative elements into MS occurs in two stages. Primary GC are formed during the development of the miracidium into the egg, whereas secondary GC develop in germinal masses of the sporocyst.

[SEASONAL CHANGES IN THE BIOLOGY OF LEUCOCHLORIDIUM PARADOXUM (TREMATODA, LEUCOCHLORIDIOMORPHIDAE)].

Infection of molluscs Succinea putris by trematodes Leucochloridium paradoxum was studied in the region of Vyritsa (Leningrad Province) during the period of 2008-2014. On the basis of the obtained data, seasonal dynamics of infection of molluscs can be presented as follows. Infection of S. putris occurs during the whole warm period from May to August. Young sporocysts of L. paradoxum overwinter and the metacercariae that develop in their extensions mature during spring becoming infective for birds. In the second half of summer, sporocysts start degenerating and die in late August-September. Each sporocyst can form 2-3 mature broodsacs (maximum 5) simultaneously. In cases of multiple infections, their number can reach 19. Several cases of independent release of sporocysts from molluscs were observed. They survive in environment for about an hour, retaining the ability to infect definitive hosts. Additionally, birds can be infected by pecking of horns of infected snails.

[REACTION OF HAEMOCYTES OF THE MOLLUSK PLANORBARIUS CORNEUS TO A XENOTRANSPLANT].

Tissue reaction of the mollusk Planorbarius corneus to the introduction of a transplant (cat vibrissa) was examined. The transplant was introduced into mollusk tissues with the use of an injection needle. After a day, flattened haemocytes were found on the surface of the transplant. The wound channel formed by the needle was arrested by a capsule formed of 5-15 layers of flattened cells. The cavity of the wound channel and the core of the vibrissa were also filled with haemocytes. During incubation of the vibrissa in vitro, adhesion and sedimentation of haemocytes on its surface was observed.

Identification of species Leucochloridium paradoxum and L. perturbatum (Trematoda) based on rDNA sequences.

The full nucleotide sequences of DNA ribosome cluster of Leucochloridium paradoxum Carus, 1835 and L. perturbatum Pojmanska, 1967 were obtained. rDNA was extracted from 40 isolates of Leucochloridium sp. and analyzed using specific primers. The intraspecific genetically identity of morphologically detected L. paradoxum and L. perturbatum sporocysts was proven. A noticeable interspecific divergence between L. paradoxum and L. perturbatum was indicated. Using rDNA genotyping a case of double infection of snail Succinea sp. with L. paradoxum and L. perturbatum sporocysts was detected.

The study of the sporocyst broodsacs coloring in Leucochloridium paradoxum (Trematoda: Brachylaemidae).

The secretory cells were found in the subtegument of the sporocysts Leucochloridium paradoxum by histological assay. Pigment granules are formed by these cells. The movement of granules from secretory cells to the tegument external layer was observed. These pigment granules provide the yellow color of sporocysts broodsacs and the brown color of protuberant spots in the terminal part of broodsacs. It was shown that the pigment granules did not contain proteins, nucleotides, lipids and carbohydrates. The positive result was received while staining on bile pigments. The question on the nature of the green pigment remains open. The paletot on the surface of sporocyst formed by spreading hemocytes was observed. This structure was not described before in brachylaemid parthenites.

[Reproduction of trematode Leucochloridium paradoxum sporocysts (Trematoda: Leucochloridiidae)].

The histological study of the trematoda sporocysts Leucochloridium paradoxum confirmed the presence of three morphological zones in it: 1) central part (reproductive), where embryos are forming, 2) narrow tubes through which the embryos penetrate in colored broodsacs (3), where the development of metecercaria completes. It was found that germinal mass only is the reproduction organ of the sporocysts, located in reproductive zone. There are young (without embryos), mature (with embryos) and degenerated germinal masses. So, in the process of sporocysts development the centre of multiplication of germinal elements was changed. The old parts of central part are degenerated, but the new ones with young germinal masses appear. The multiplication of generative elements does not take place in the broodsacs which are breeding cameras functionally.

The transcriptomic analysis of Planorbarius corneus hemocytes (Gastropoda) naturally infected with Bilharziella polonica (Schistosomatidae).

The study of molluscan innate immunity is essential for understanding the evolution of the immune system. An advance in the knowledge of their immune system can be achieved by increasing the number of model species. Our study focuses on the immunity of Planorbarius corneus, a pulmonate snail widely distributed in Eurasia. These snails are intermediate hosts of many trematodes, including Bilharziella polonica (Schistosomatidae). In this paper we obtained and analyzed transcriptomes of hemocytes of uninfected snails Planorbarius corneus and snails naturally infected with Bilharziella polonica. The transcriptomes were found to contain transcripts encoding all major groups of immune factors previously described for other gastropods. Pathogen-recognition molecules were the most diverse group of immune factors. Comparison of the transcriptomes of the infected and the uninfected molluscs showed that the expression of some genes changed during infection. Our results extend the knowledge of immune responses of pulmonate snails to trematode invasion and promote P. corneus as a new model for the study of molluscan defence reactions.

[Molecular genetic analysis of trematodes of the genus Leucochloridium dwelling in the territory of Leningrad Province].

The color of the broodsac sporocyst traditionally serves as the main taxonomic criterion for distinguishing of trematodes of the genus Leucochloridium. Broodsacs of L. paradoxum (Cams, 1835) are green, while broodsacs of L. perturbation (Pojmanska, 1969) are brown. We used molecular genetic analysis of sporocyst rDNA for verifying the accuracy of the mentioned morphological criteria. Trematode infected snails Succinea sp. were collected in Vyritsa and Lyuban (Leningrad Province, Russia). Nucleotide sequences of L. paradoxum (n = 18) and L. perturbatum (n = 10) rDNA including transcribed spacers (ITS1 and ITS2) and 5.8 S rRNA gene were received, rDNA fragments of Leucochloridium sp. sporocysts of the same color were identical. The difference in the ITS1 (2.6%) and ITS2 (6.7%) between sequences of L. paradoxum and L. perturbatum was revealed. Specific nucleotide sequences are deposited at the GeneBank.