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.

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.

Multiple infection of amber Succinea putris snails with sporocysts of Leucochloridium spp. (Trematoda).

Amber Succinea putris snails were collected in the Leningrad Region (Russia). Some of them were infected with trematodes Leucochloridium paradoxum, Leucochloridium perturbatum and Leucochloridium vogtianum. One snail had triple infection with all these species. Genotyping of sporocysts by ITS1-5.8S-ITS2 nucleotide sequences of ribosomal DNA (rDNA) and phylogenetic analysis were performed. The results confirmed the species identification of sporocysts of Leucochloridium based on the shape and colour of mature broodsacs. Sporocyst broodsacs could leave the host snail on their own, remaining viable in the environment for up to an hour. This ability of sporocysts may prevent the excessive infection of the molluscan host.

[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 daughter rediae Echinostoma caproni (Trematoda) in vitro cultivation].

Methods of in vitro cultivation were used to examine the feeding and reproductive behaviors of daughter rediae of Echinostoma caproni. It was noted that under conditions of in vitro cultivation, rediae fed on tissues of the mollusc Biomphalaria glabrata, on rediae and cercariae of E. daikenaensis and E. congoensis, and on sporocysts and cercariae of Schistosoma mansoni. No cases of cannibalism of daughter rediae E. caproni by their offspring rediae were observed, although they could feed on their own cercariae. When kept in mediae containing (B. glabrata embryonic) Bge cells, rediae E. caproni gradually turned to feeding on these cells and stayed away other objects. Under conditions of in vitro cultivation, daughter rediae E. caproni were capable of forming redial and cercarial embryos. However, no cases of return from producing of cercariae to producing of rediae were observed. These in vitro data confirm the results of previous studies of this species's parthenithae performed in vivo (ATaeB and dp., 2007).

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.

[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.

[Expression of genes encoding defense factors in the snail Planorbarius corneus (Gastropoda, Pulmonata) infested with trematodes].

Because many species of gastropods are intermediate hosts for trematodes, these molluscs are often used as model-organisms in the studies of invertebrate immune system. Revealing of the ways in which the defense factors functioning became possible due to the use of the methods of molecular biology. Contemporary molecular methods allow analyzing the defense factors allocations and levels of their expression. We investigated the expression of genes encoding defense factors in gastropods by the example of the snail Planorbarius corneus from water bodies of the Leningrad Oblast under infestation with trematods. The snails naturally infested with the parthenites of trematode species belonging to the families Strigeidae, Notocotylidae, Plagiorchiidae, and Schistosomatida were used as the experimental sample. Uninfested snails were used as a control sample. Several genes encoding the factors, which have been recently found involved in the anti-trematode defense reactions in pulmonates, were chosen, namely fibrinogen-related protein, C-lectin, calcium-binding protein, and cystatin-like protein. The genes' expression was analyzed on total mRNA samples by the reverse transcription with the polymerase chain reaction. It was shown than expression levels of the genes under consideration are different in uninfested snails and in the snails infested with different trematode species. Thus, in the mollusks infested with the parthenites of Cotylurus sp. and Bilharziella polonica, the expression levels of the genes of all factors under study were increased, while in the infested Notocotylus sp. n Plagiorchis sp., only expression levels of C-lectin and cystatin-like protein were increased. Results of the expression analysis confirm the role of hemocytes and cells of hepatopancreas in the production of humoral defense factors. In the snails infested with trematodes, the expression levels of C-lectin and calcium-binding protein genes are increased in haemocytes, while the genes of fibrinogen-related and cystatin-like proteins are activated in the hepatopancreas. Our data also confirm the role of the factors examined in the anti-trematode defense reactions in pulmonates.

[The influence of Echinostoma caproni metacercariae (Trematoda) on the survival of biomphalaria molluscs (Pulmonata)].

The infrapopulation of the Echinostoma caproni partenites has a development of prolong character (Ataev et al, 2005). However, in laboratory conditions, Biomphalaria molluscs infested with this parasite die within 1--3 weeks after the beginning of cercariae emission. It has been suggested that autoinvasion of the mollusc host with the cercariae, which use it as second intermediate host, is the cause of this phenomenon. Studying the dynamics of metacercariae accumulation in the host (both infected and non-infected with the Echinostoma caproni rediae) and experiments where quantity of cercariae around molluscs reduced by different ways, confirmed this hypothesis. Evidently, pathogenicity of metacercariae for molluscs is lesser in nature, because the concentration of cercariae reduces to the values, which do not result in lethal effect: some part of cercariae dies, but another part uses other animals as a host (Haas, 2000).

[Reproduction of the trematode Echinostoma caproni parthenites (Digenea: Echinostomatidae)].

Dynamics of the reproduction in the trematode Echinostoma caproni parthenites (Echinostomatidae) was observed. Early laying and maturation of the generative cells are for the first time shown to be characteristic for all parthenogenetic generations. Really the process of reproduction had been finishing to the beginning of the generating of new age by parthenites. Mother sporocysts, as well as redia of different generations, in fact stop producing new generative cells with the beginning of the generating of new age, and assume the function of a brood pouch. This feature was considered previously as peculiar mainly to mother sporocysts. Data on the autotomy of the anterior body end in mother sporocysts are verified. In our opinion, these data are an evidence of an early manifestation of the evolutionary trend to the morpho-functional regress and disintegration of the parasitic stage of mother sporocyst.

[Development of mother sporocysts of Echinostoma caproni (Trematoda: Echinostomatidae)].

New data on the migration and development of Echinostoma caproni mother sporocysts in two mollusk species of the genus Biomphalaria are obtained. It is confirmed, that the formation of primary and second generative cells takes place only as a result of undifferentiated cells' proliferation and following differentiation of some of them. These processes in miracidium, as well as in the parasitic stage of mother sporocyst, take place in a special organ, germinal mass, which occupies caudal position in both cases. The supposition of the role of germinal mass as the universal centre of multiplication and development of generative elements in all generations of Echinostoma caproni parthenites is confirmed. It is established, that mother sporocysts do not relize their reproductive potential completely, and the degree of its realization depends on the conditions arising in the host organism.

[Development of the partenitae infrapopulation of Echinostoma caproni (Digenea: Echinostomatidae)].

The first generation of Echinostoma caproni partenitae is represented maternal sporocysts developing in the cardium of the Biomphalaria mollusk. During all their life, they produce rediae of maternal generation. Rediae of Echinostoma caproni of all generations are similar. The first generation consists of maternal rediae forming only redoid embryos. Due to this process, the number of partenitae increases very fast. The next generations are represented by daughter rediae. In the beginning of their life they produce redoid embryos but later begin forming cercariae. The number of rediae produced before this shift of production depends on the population density. Further, the partenitae retain their potential ability to form rediae but realize it in exceptional cases. Generative organs of all generations are germinal masses. Proliferation of generative elements and beginning stages of redia and cercaria development take place within these masses. The infrapopulation of E. caproni belongs to the "prolonged type", because it is a complete microhemipopulations; its existence is limited by a lifespan of the mollusk host.

[Defensive reactions of gastropod molluscs. Humoral reactions].

Humoral protective reactions and their mechanisms in gastropod mollusks are considered based on the results of various investigations. It is important to note that lectines in molluscs, as well as in other invertebrates, are functional analogues of immunoglobulins in vertebrates.

[Protective reactions of gastropod molluscs. 1. Cell reactions]. / Zashchitnye reaktsii briukhonogikh molliuskov. 1. Kletochnye reaktsii.

Protective reactions of molluscs are traditionally considered in cell and humoral aspects. The paper analyses original data and recent reference data oncell (phagocytic) reactions of gastropod molluscs. The main attention is paid to peculiarities of hemopoiesis, cell elements of hemolymph, and dynamics and mechanics of encapsulating the parasites.

[Dynamics of infection of Bithynia tentaculata (Gastropoda: Prosobranchia) with trematodes]. / Dinamika zarazhennosti Bithynia tentaculata (Gastropoda: Prosobranchia) trematodami.

The dynamics of infection of Bithynia tentaculata with 7 trematode species was examined during 5 years. Stability of parasite fauna with significant changes of infection rate has been recorded. During the period of observations the infection rate of Sphaeridiotrema globulus, Notocotylus imbricatus, Holostephanus volgensis, Pleurogenoides medians and Metorchis intermedius has decreased, while that of Psilotrema tuberculata has increased. The infection rate of Plagiorchis sp. slightly fluctuated. It was found out that the infection rate of S. globulus, P. tuberculata, Plagiorchis sp., H. volgensis and M. intermedius increases by the age of hosts. Maximal infection rate of N. imbricatus was observed in mollusks of 2-3 years old. Based on peculiarities of infection dynamics during the year, 3 groups of parasites have been recognized. 1. S. globulus, P. tuberculata and N. imbricatus show an increase of infection rate from April to August with subsequent decrease. 2. Infection rate of H. volgensis increases during the Summer and reaches maximum in Autumn. Age group of host 2+ and older ones showed some decrease of infection in the beginning of Summer. 3. First cases of infection with M. intermedius occur in May, then the infection rate increases and reaches maximum in the end of July. The infection rate gradually decreases and in the end of October the mollusks infected with M. intermedius are usually absent. An emission of cercariae is usually observed in June-August. The difference in infection rate of Bithynia tentaculata males and females was not found. Based on a complex analysis of infection dynamics and population dynamics of mollusks, different aspects of the life cycle of parasites (periods of emission, maturity and longevity of local microhemipopulations) are discussed.