10_Zitova_05_21.indd UDC 594:591.437.616.995.1 SUBMICROSCOPIC CHANGES IN THE HEPATOPANCREAS OF FRESHWATER MOLLUSKS INFECTED WITH PARTHENITES OF TREMATODES ECHINOPARYPHIUM ACONIATUM (ECHINOSTOMIDA) AND PLAGIORCHIS ELEGANS (PLAGIORCHIIDA) O. Zhytova1*, T. Kot2, S. Huralska3, O. Andreieva4, V. Moroz5 Polissia National University, Stary Blvd., 7, Zhytomyr, 10008 Ukraine O. Zhytova (https://orcid.org/0000-0003-2572-4163) T. Kot (https://orcid.org/0000-0003-0448-2097) S. Huralska (https://orcid.org/0000-0001-7383-1989) O. Andreieva (https://orcid.org/0000-0003-0851-800X) V. Moroz (https://orcid.org/0000-0002-1457-4641) *Corresponding author E-mail: elmi1969@meta.ua Submicroscopic Changes in the Hepatopancreas of Freshwater Mollusks Infected with Parthenites of Trematodes Echinoparyphium aconiatum (Echinostomida) and Plagiorchis elegans (Plagiorchiida). Zhytova, O., Kot, T., Huralska, S., Andreieva, O., Moroz V.— Th e study contains the results of the electron microscopic research of the hepatopancreas of Lymnaea stagnalis (Linné, 1758) molluscs infected with Plagiorchis elegans (Rudolfi , 1802) Braun, 1902 and Echinoparyphium aconiatum Dietz, 1909 trema- todes. With a high degree of invasion, fi brous connective tissue growth between lime and liver cells was observed. Th e number of vacuoles in the cell cytoplasm increased, and the structural organization of the plasma membrane was disrupted. Heterochromatin content decreases in the nucleus, karyorrhexis could occur. Th e cytoplasm contained single organelles and a large number of electron-dense granules, some cells were destroyed. At a high degree of invasion of L. stagnalis by partenites and cercariae of P. elegans, the nature of the destructive changes in hepatic and lime cells of the hepatopancreas had same orientation as in mollusks with parasitic trematode E. aconiatum. However, the severity of the destructive changes in the hepatopancreas acini of mollusks infected with trematode P. elegans was much smaller, as evidenced by the absence of complete destruction of hepatic and lime cells. K e y w o r d s : freshwater mollusks, trematodes, liver cells, lime cells, acinus. Zoodiversity, 55(5):431–438, 2021 DOI 10.15407/zoo2021.05.431 432 O. Zhytova, T. Kot, S. Huralska, O. Andreieva, V. Moroz Introduction In the life cycle of the vast majority of trematodes, freshwater gastropods are intermediate hosts, due to the formation of their initial life cycle, which coincides with the period of formation of modern freshwater mollusk fauna (Shakarbaev et al., 2013; Zhytova, 2015; Akimova, 2016). Th e links that exist between the components of the trematode-freshwater gastropods system are practically not disturbed; physiological balance is established in the system. However, no matter how smooth these relations are, they are still built on the principle of dynamic equilibrium (Kornyushin, 2011; Zhytova, 2015). Histopathological studies have been used for investigation the parasite-host relationship in the trem- atode-mollusk system long enough. Suffi cient data was collected on morphofunctional, histopathological changes in the hepatopancreas of freshwater and marine mollusks infected with trematodes (Stadnichenko, 2005; Usheva, Frolova, 2006; Stadnichenko, Leichenko, 2010; Zhytova, Khomych 2011; Choubisa et al., 2012; Nacheva, Sumbaev, 2013; Akyildiz et al., 2019). Parasite invasion is the cause of signifi cant destructive changes in this mollusk organ. Various trematode species, due to the peculiarities of their reproduction and the degree of adaptation to the organism of a certain host species, cause diff erent degrees of damage (Rizk et al., 2014). It was revealed (Zhytova, 2015) that the severity of microscopic changes in the hepatopancreas of freshwater gastropods infested with parthenites and cercariae of trematodes directly depends on the intensity of invasion and the type of the trematode life cycle, depending on the generation (redia or sporocyst) that follows the mather sporocyst. A suffi cient number of papers is devoted to the electron microscopic study of the hepatopancreas of mollusks infacted with various species of trematodes (Adam et al., 1995; Luchtel et al., 1997; Silva, 2003; Rizk et al., 2014; Paviotti-Fischer et al., 2019). However, information on the eff ect of these parasites on the host organism at the subcellular level is still insuffi cient; in particular, this concerns the comparative aspect of the infl uence of specifi c species of trematodes with diff erent life cycles on the hepatopancreas of the host mollusk. Th is prompted us to study profound changes in the hepatopancreas of mollusk Lymnaea stagnalis (Linné, 1758), infested with one of the most widespread in the Ukrainian Polissia trematodes Echinoparyphium aconia- tum Dietz, 1909 and Plagiorchis elegans (Rudolphi, 1802) with a high degree of invasion. Th ese studies made it possible to analyze the degree of changes in the hepatopancreas of freshwater mollusks in the case of redioid or sporocystoid types in trematode life cycle. Material and methods For electron microscopic studies, the hepatopancreas from L. stagnalis, both free from parasites (control) and infested with parthenites and cercariae of redioid (E. aconiatum) and sporocystoid (P. elegans) trematodes was selected. Th e eff ect of parthenitis and cercariae of trematodes on L. stagnalis hepatopancreas was studied at a high degree of invasion. Th e infection of mollusks with L. stagnalis was determined by the emission of cercariae (Zhytova, Kho- mych, 2011; Zhytova, 2015). Aft er the end of the fi rst stage of the experiment, the hepatopancreas was removed from the body of the mollusk and examined, starting from its color and consistency. Assessment of the he- patopancreas degree of lesion was carried out visually according to certain criteria: weak invasion — damage by parasites up to 1/10; moderate — from 1/10 to 1/2; high invasion — more than 1/2 of the organ volume (Kirichuk, Stadnichenko, 2010). Hepatopancreas from 15 specimens of L. stagnalis (shell height 40–56 mm), 5 specimens of each category (uninfected and highly infested) was selected for the research. Electron microscopic studies were carried out on the basis of the laboratory of electron microscopy of the National Medical University named aft er A. A. Bogomolets (Kyiv). When making sections for electron micros- copy, hepatopancreas was crushed in a drop of fi xative, 2.5 % glutaraldehyde solution, additional fi xation of the material was carried out using Caulfi eld’s reagent (based on a 1 % Os3O4 solution) during 2 h at 40 °C; then it was thoroughly washed with distilled water and dehydrated in ethanol of increasing concentrations (700, 800, 900, 960, 1000), and later in acetone for 15 min. Next, the material was impregnated with acetone and epoxy resins (a mixture of eponym and araldite), gradually transferring it to glasses with diff erent resin ratios (from 3 : 1 to 1 : 1 and 1 : 3, respectively), keeping for 1 h in each mixture. In the fi nal stages, the material was em- bedded in resin (Epon-Araldite processing method). Polymerization took place at a temperature of 56 °C for 7–10 days (Karupu, 1984). Aft er targeted orientation the tissue was sectioned with a Reichert Jung Ultracut E Ultramicrotome into semi-thin sections, which were then stained with toluidine blue and contrasted with a saturated solution of uranyl acetate in 70 °C ethyl alcohol and lead citrate for 15 min each. Sections were exam- ined and photographed using a TEM-125K electron microscope. Research results and discussion Th e purpose of our electron microscopic studies was to identify pathological disorders in the hepatopancreas of L. stagnalis caused by a high degree of invasion by parthenitis and cercariae of the trematode E. aconiatum. For comparison, the structure 433Submicroscopic Changes in the Hepatopancreas of Freshwater Mollusks Infected with Parthenites… of the hepatopancreas of uninfected mollusks L. stagnalis was studied. Th e hepatopan- creas of such mollusks had a brownish-brown color; its consistency was moderately dense. It consisted of the acini surrounded by loose fi brous connective tissue in which collagen fi bers prevailed over reticular ones. Th e acini of the L. stagnalis hepatopan- creas were predominantly oval in cross section, less oft en rounded. Th e wall of acini was formed by the basement membrane with two types of epithelial cells: hepatic and lime (fi g. 1, A). Hepatic cells were elongated-oval or round, or irregularly polygonal. Th e contours of these cells were uneven; their membrane had a characteristic structure. Th e nuclei were rounded, elongated-oval, located in the basal part of the cell. Th e contours of the nucleus were equal. Th e nuclear envelope was formed by two elementary biologi- cal membranes, with perinuclear space of uneven thickness between them (fi g. 1, B). Nucleoplasm was of low electron density. Heterochromatin in the form of lumps, grains of diff erent electron density and size was determined in the nucleoplasm. A small por- tion of the heterochromatin was attached to the inner surface of the inner membrane of the nuclear envelope. Th e nucleolus was predominantly one, but there could be two of them; they were located on the periphery of the nucleus, sometimes in its center. Th e electron density of the cytoplasm was average. It contained many mitochondria, mainly oval, ribosomes, vacuoles of low electron density, a cell center located near the nucleus, the Golgi complex, granular and agranular endoplasmic reticulum. Liver cells were larger than lime ones. Th e apical parts of the liver cells reached the lumen of the acini. Unlike liver cells, lime cells were low, multifaceted, pyramidal, less oft en elongated- oval, they did not reach the lumen of the acinus. Structurally, lime cells were similar to liver cells. Th e nuclei of lime cells could be elongated-oval, round and pyramidal in shape, their contours were uneven, and the electron density was much higher than that of the hepatic cell nucleus. Th e perinuclear space was well expressed. Th e nucleoplasm contained signifi - cantly more heterochromatin than the liver cells. Th e nucleolus was predominantly one, lo- cated in the center of the nucleus. A signifi cant amount of heterochromatin was fi xed to the inner surface of the inner membrane of the nuclear envelope. In the cytoplasm, there were many oval and small mitochondria, the Golgi complex, ribosomes. We did not observe lime granules. Th e endoplasmic reticulum was well expressed, elements of its granular and agranular form were revealed. Fig. 1. Acinus of L. stagnalis hepatopancreas: A: Cells of L. stagnalis hepatopancreas acinus: 1 — hepatic cell; 2 — lime cell. (Electronogram ×4800); B: Hepatic and lime cells of a L. stagnalis hepatopancreas fragment undamaged by trematode parthenitis: 1 — hepatic cell; 2 — nucleus; 3 — heterochromatin; 4 — pore in the nuclear envelope; 5 — perinuclear space; 6 — the lumen of the acinus; 7 — lime cell; 8 — the nucleus of the lime cell. (Electronogram ×13000). 434 O. Zhytova, T. Kot, S. Huralska, O. Andreieva, V. Moroz Our electron microscopic studies of hepatopancreas tissues of L. stagnalis mollusks with a high degree of invasion by parthenites and larvae of E. aconiatum revealed the proliferation of fi brous connective tissue not only between the hepatopancreas acini, but also between lime and hepatic cells (fi g. 2, A). Destructive changes in the hepatopancreas of L. stagnalis with a high degree of invasion by parthenitis and cercariae of E. aconiatum were enhanced due to long-term reproduction of parthenitis of trematodes. Fragments of destroyed liver cells and cellular debris were visible between the cells of the acini. Intact hepatic cells had an indistinct structure of the plasma membrane and a large number of vacuoles. Th e nucleus in some of these cells appeared to be spotty due to the formation of clumps of heterochromatin. Th ere were also liver cells in which the nucleus was electroni- cally transparent, its membrane was fragmented or absent (fi g. 2, B). In some cells, the nucleus broke down into fragments (karyorrhexis). Th e nucleolus was displaced to one of the poles of the nucleus or was not noticeable. Th e content of heterochromatin decreases; it was almost not found near the nuclear envelope. Th e cytoplasm contained solitary organelles and a large number of electron-dense granules of a round and elongated shape. Liver cells in the last stages of destruction were identifi ed; the presence of destructed nuclei and vacuolization of the cytoplasm testifi ed to this. In most lime cells, the amount of cytoplasm was signifi cantly reduced; it surrounded the nucleus with a thin strip, which became irregular, elongated due to invaginations. In most lime cells, Fig. 2. Changes in the cells of the L. stagnalis hepatopancreas acinus with a high degree of invasion with parthenitis: A: Walls of a hepatopancreas acinus of a mollusk infected with E. aconiatum: 1 — collagen fi bers; 2 — hepatic cell; 3 — lime cells; 4 — karyorrhexis. (Electronogram ×1000); B: hepatic cells of the hepatopancre- as of a mollusk infected with E. aconiatum: 1 — fragments of a destroyed hepatic cell. (Electronogram ×10000); C: Lime cells of the hepatopancreas of the mollusk infected with P. elegans: 1 — interlobular fi brous connective tissue; 2 — hepatic cell; 3 — lime cell. (Electronogram × 6500); D: Cells of the hepatopancreas acinus of the mollusk infected with P. elegans: 1 — hepatic cell; 2 — lime cell. (Electronogram ×15000). 435Submicroscopic Changes in the Hepatopancreas of Freshwater Mollusks Infected with Parthenites… the amount of cytoplasm was signifi cantly reduced; its thin strip surrounded the nucleus, which acquired an irregular, elongated shape due to invaginations. In separate lime cells, the nuclei were reduced in size, placed marginally (near plasmalemma). As a rule, such nuclei form protrusions, which suggest the presence of karyorrhexis. The accumulation of heterochromatin was observed throughout the nucleoplasm. An extreme manifestation of destructive changes in lime cells was the processes of vacuolization and karyolysis of their nuclei. In the latter, the amount of heterochromatin decreased, the nucleolus disappeared, and the nucleoplasm became electronically transparent. Heterochromatin was found at the surface of the inner membrane of the nuclear envelope. The cytoplasm was electron-dense. Its microclasmatous outgrowths separating from the cells were noted, what led to a decrease in their size. At the same time, lime cells with an increased volume of cytoplasm were observed, what changed relation between the nucleus and the cytoplasm. The cytoplasm of such cells was of moderate electron density. Mitochondria had lysed matrix and cristae. The cytoplasm contained small vacuoles with and without granules. Lime cells differed in the number of granules, which could be a sign of their different functional state. At the same time, the presence of a significant number of lime cells with an insignificant volume of cytoplasm, and the absence of granules in it, suggested that with a high degree of invasion, the processes of synthesis and secretion in them were reduced in comparison with the norm (control). So, our electron microscopic studies of L. stagnalis hepatopancreas, infested with parthenites and larvae of E. aconiatum, showed microscopic changes in most of the organ. Th e detection of signifi cant changes in the microstructure of L. stagnalis hepatopancreas recorded at a high degree of invasion (E. aconiatum) made it necessary to obtain comparative data on changes in the microstructure of L. stagnalis hepatopancreas infected with parthenites and larvae of the trematode P. elegans with the same degree of infection. With a high level of L. stagnalis infection with parthenites and cercariae of P. elegans, the nature of destructive changes in the hepatic and lime cells of the hepatopancreas had the same direction as in the case of mollusks invasion with E. aconiatum trematode. However, the severity of destructive changes in the acini of the hepatopancreas of mollusks infected with P. elegans trematode was much less, as evidenced by the absence of complete destruction of hepatic and lime cells (fi g. 2, C; fi g. 2, D). Heterochromatin, which in the form of clusters was located both at the cell’s periphery and throughout the nucleoplasm, predominated in the liver cells. Th e cytoplasm varied in volume and contained few organelles. Th e nuclei of lime cells had slightly higher electron density than the nuclei of hepatic cells. Lime cells diff ered somewhat in the size of the cytoplasm. Th e number of inclusions in these cells, even with a large volume of cytoplasm, was insignifi cant. Also, with a high level of infection with L. stagnalis partenits and P. elegans larvae, we did not fi nd a continuous proliferation of collagen fi bers between the cells of the acinus. Th e manifestation of the pathogenic eff ect of parasites on the host organism depends on the relationships formed between them in the process of ontogeny and phylogenesis. Th e intensity of this eff ect depends on the direct eff ect of the trematodes on the intermediate host, the mollusk, and on the nature of its reaction to the presence of helminths. Th e presence of parasites in the host body leads to changes in its organs and organ systems (Nevyadomska et al., 2007; Choubisa et al., 2012). Having settled mainly in the hepatopancreas of mollusks, trematodes use its tissues for nutrition and poison the host body with the products of their metabolism, what undoubtedly has a negative eff ect of the parasite on the intermediate host. Redia cause mechanical damage to the tissues of the host mollusks hepatopancreas (Stadnichenko, 1977). In the intestines of redia, we observed in large numbers fragments 436 O. Zhytova, T. Kot, S. Huralska, O. Andreieva, V. Moroz of hepatopancreas tissues, separate whole cells, and cellular detritus, which is consistent with the data of A. P. Stadnichenko (Stadnichenko, 1972) and S. L. Choubisa (Choubisa, 2008). The degree of destruction of the hepatopancreas of mollusks largely depends on the species of Trematoda. According to T. A. Ginetsinskaya (Ginetsinskaya, 1968) sporocysts of Cercaria roscovita Stunk. caused the destruction of 80 % of hepatopancreas tissues in two months, while those of C. lebouri Stunk. only 15 % of the entire mass of this organ was destroyed in eight months. According to N. E. Serbina (Serbina, 2008), parasitizing redioid species of trematodes requires more energy from the host than sporocystoid. According to our electron microscopic studies of L. stagnalis hepatopancreas, E. aconiatum trematode, in comparison with P. elegans, caused greater destruction of the hepatopancreas structure in mollusks. Our information on changes in tissues and cells of the hepatopancreas infected with L. stagnalis is confi rmed by other studies (Stadnichenko, 1972; Souza et al., 1995). In mollusk Viviparus viviparus (Linné, 1758) infected with Neoacanthoparyphium echinatoides (de Filippi, 1854), under the mechanical impact of parasite larvae on the glandular epithelium, the liver cells were destroyed more than lime ones. In hepatic cells of the aff ected hepatopancreas, changes in the topography of their nuclei, which moved to the medial part, were noted. Not only cells adjacent to parasites, but those that were distant from them were subjected to destruction. Th e damage of liver cells was also noted in mollusks Bithynia siamensis goniomphalus Morelet, 1866, infected with trematode Opisthorchis viverrini (Adam et al., 1995) According to C. P. Souza (Souza et al., 1995), no proliferation of fi brous connective tissue was observed in the digestive gland of mollusks Biomphalaria tenagophila and B. straminea infected with the trematode Schistosoma mansoni. Some researchers (Silva, 2003) noted a signifi cant de- struction of acini and presence of cells with a large number of vacuoles in the hepatopan- creas of L. columella (Say, 1817) infected with larvae of trematode Echinostoma paraensi Lie and Basch, 1967. In mollusks infected with parthenites and larvae of redioid trematodes (E. aconiatum), we identifi ed virus-like particles, which in shape and size (12.5 μm) were attributed to Baculoviruses group (baculovirus, or rod-like viruses) of the polyhedrosis subgroup, in particular, cytoplasmic polyhedrosis (Zhytova et al., 2013). Th e detection of virus-like particles in L. stagnalis infected with E. aconiatum is probably due to the fact that the parasitization of virus-like particles (cytoplasmic polyhedrosis) in mollusks infected with redioid trematodes is a manifestation of a signifi cant weakening of the animal body de- fenses and the transition of the virus from a latent state to an active one. Th is case, in our opinion, is a manifestation of an opportunistic infection. Сonclusions Thus, the results of our electron microscopic studies showed that infection with parthenitis of redioid trematode E. aconiatum leads to significant destructive changes in the hepatopancreas of the host L. stagnalis. At the same time, changes in the hepatopancreas acini of L. stagnalis infested with parthenites of sporocystoid P. elegans, are significantly less, as evidenced by the absence of complete destruction of hepatic and lime cells. 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