Morphological and molecular characterization of the metacercaria of Paragonimus caliensis, as a separate species from P. mexicanus in Costa Rica.

The trematode Paragonimus mexicanus is the etiological agent of paragonimiasis, a food-borne zoonotic disease in Latin America. This species, as well as Paragonimus caliensis, have been reported from Costa Rica, but it is not known if the two are synonymous. Two types of Paragonimus metacercariae from freshwater pseudothelphusid crabs from several localities in Costa Rica were recognized by light microscopy. Morphologically, these corresponded to descriptions of P. mexicanus and P. caliensis. Metacercariae of the former species lacked a membrane or cyst and their bodies were yellow in color. Those of P. caliensis were contained in a transparent thin cyst and were pink in color. Morphotypes of metacercariae were determined using scanning electron microscopy (SEM). Based on the number and distribution of papillae in the ventral sucker, three morphotypes were found for P. mexicanus and two for P. caliensis. Analysis of DNA sequences (nuclear ribosomal 28S and ITS2 genes, and partial mitochondrial cox1 gene) confirmed the presence of P. mexicanus and provided the first molecular data for P. caliensis. The two species are phylogenetically distinct from each other and distant from the Asian species. The confirmation of P. caliensis as a separate species from P. mexicanus raises several questions about the ecology, biological diversity, and epidemiology of the genus Paragonimus in Costa Rica.

Ultrastructure of spermatogenesis in the testis of Paragonimus heterotremus.

Lung fluke, Paragonimus heterotremus, is a flatworm causing pulmonary paragonimiasis in cats, dogs, and humans in Southeast Asia. We examined the ultrastructure of the testis of adult P. heterotremus with special attention to spermatogenesis and spermiogenesis using scanning and transmission electron microscopy. The full sequence of spermatogenesis and spermiogenesis, from the capsular basal lamina to the luminal surface, was demonstrated. The sequence comprises spermatogonia, spermatocytes with obvious nuclear synaptonemal complexes, spermatids, and eventual spermatozoa. Moreover, full steps of spermatid differentiation were shown which consisted of 1) early stage, 2) differentiation stage representing the flagella, intercentriolar body, basal body, striated rootlets, and electron dense nucleus of thread-like lamellar configuration, and 3) growing spermatid flagella. Detailed ultrastructure of 2 different types of spermatozoa was also shown in this study.

Indochinamon ou (Crustacea: Potamidae) as a new second intermediate host for Paragonimus harinasutai in Luang Prabang Province, Lao PDR.

Paragonimus harinasutai metacercariae were found in a species of freshwater crab, Indochinamon ou, collected in a small stream of Namback District, Luang Prabang Province, Lao PDR. Adult flukes were recovered after experimental infection of the metacercariae to dogs. Metacercariae were round or slightly elliptical, 0.666 x 0.626 mm in average size, and had a thin cyst wall of about 20 microm in thickness, a black excretory bladder, convoluted ceca, and some pinkish materials in the body. Adults were somewhat elongated, 95.2 x 36.5 mm in average size, covered with single-tipped tegumental spines, had a smaller oral sucker than the ventral sucker, a moderately branched ovary, and 5-6 lobulated testes. Eggs were ovoid and bilaterally symmetrical in shape, 79 x 45 microm in average size, and had a uniformly thickened shell. By the present study, it has been confirmed that I. ou is a new second intermediate host for P. harinasutai.

Surface fine topography and PCR-based determination of metacercaria of Paragonimus sp. from edible crabs in Arunachal Pradesh, Northeast India.

In several mountainous regions of Northeastern India, foci of Paragonimus infection reportedly involving species that are known to prevail in China have been identified. The present study was undertaken to demonstrate the surface fine topography and sequence analysis of the ribosomal deoxyribonucleic acid (rDNA; second internal transcribed spacer, ITS2) of the metacercarial stages of the lung fluke collected from a mountain stream of the area (Miao, Changlang District in Arunachal Pradesh). The encysted metacercariae were oval in shape and had a smooth surface. The newly excysted metacercaria had a ventral sucker larger than the oral; the body surface was covered with numerous single-pointed and thorn-like tegumentary spines, of which those on the anterior part of the body were bigger in size and showed a gradual reduction in length and number towards the posterior end; dome-shaped papillae in variable numbers were seen around the rim of the oral sucker and were sparsely distributed all over the body surface. The polymerase chain reaction-amplified rDNA ITS2 sequences of the metacercariae were aligned with known sequences for the various species of Paragonimus, and the expectation value was found to be most significant with P. westermani, revealing an absolute match. The surface topography including the number and distribution of papillae and spination patterns and the ITS2 sequences of the metacercariae strongly suggest that the Paragonimus species, prevalent in the region of India, is in fact P. westermani.

[Scanning electron microscopy of Paragonimus proliferus].

OBJECTIVE: To identify the species of Paragonimus proliferus with scanning electron microscopy (SEM) based on the surface structure of excysted metacercariae, adult worms and eggs. METHODS: Crabs were collected from the endemic area of P. proliferus and excysted metacercariae were separated. Adult worms at different ages and eggs were obtained from the experimentally infected rats. After being fixed by 2.5% glutardialdehyde and 1% osmic acid, alcohol dehydration, gilded by ion spatter, the specimens were observed under SEM by STEREOSCAN-100. RESULTS: The cuticular spines of excysted metacercariae distributed in single pattern, bayonet-shaped or scale-shaped. There were 6 dome-shape papillae around the rim of the ventral sucker symmetrically arranged. The cuticular spines of different age adult worms distributed in group pattern, relatively denser and more regularly arranged in the anterior part than the posterior part of the worm body. The shape and arrangement of the cuticular spines on adult worms at different ages were basically uniform. The surface of eggshell including the operculum was generally smooth. The shell rim joining the operculum was thick and prominent. A knot-like prominence was observed at the aboperculum end. CONCLUSION: The cuticular spines of both excysted metacercariae and adult worms of P. proliferus show its own characteristics, but the size and shape of the cuticular spines among individuals or different parts of the same specimen show certain differences.

[Effect of triclabendazole on the ultrastructure of body wall and vitelline cells of Paragonimus westermani].

AIM: To observe the ultrastructural changes in the body wall and the vitelline cells of Paragonimus westermani in vitro and in vivo before and after triclabendazole treatment. METHODS: The worms were obtained from in vitro and in vivo tests. All of the samples were processed by conventional techniques, and observed by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). RESULTS: The external plasma membrane and matrix were cracked or disappeared after the treatment. The necrosis of the muscular layer differed. The cell membranes of cortex and vitelline cells were damaged. Nuclear membrane was damaged partially, heterochromatin solidified and condensed to brim and dissolved. The Golgi complex disappeared, endoplasmic reticulum expanded, mitochodria denatured and dissolved. The damage was more serious in vivo than in vitro. CONCLUSION: Triclabendazole is remarkablely effective against Paragonimus westermani by damaging the body wall and vitelline cells, mainly affecting the nuclei, membrane structures and microtubular system.

Ultrastructure of the foregut and associated glands in the lung fluke, Paragonimus miyazakii (Digenea: Troglotrematidae), with particular reference to their functional roles.

The foregut and associated glands of a digenetic trematode, Paragonimus miyazakii, were examined in the forebody by transmission and scanning electron microscopy as well as by light microscopy, and their functional roles were discussed. The foregut is lined with a general tegument without spines and sensory receptors throughout its length, although it consists of the mouth, pharynx, and esophagus. This foregut tegument is regionally and intraregionally modified in appearance, suggesting the performance of auxiliary functions in digestion. This appearance is characterized by long, frequent cytoplasmic extensions of the apical tegument around the middle portion of the mouth and the anterior esophagus. Electron-dense granules and multimembranous and multilamellar bodies are developed in the tegument to various degrees, and elaborately in the apical layer of the prepharynx. A single type of unicellular gland is embedded in the antero-middle part of the worm in small groups. The gland cells synthesize clear secretory granules as a chief product, each granule with a pleomorphic, dense, core-like inclusion. Mature granules are elliptical in shape, approximately 500 nm in diameter, and are subsequently discharged into the prepharyngeal foregut lumen after passing through the elongated cytoplasm of the gland cell. In the prepharynx and pharynx, host blood cells are apparently processed for digestion. In the wide lumen of the esophagus, foodstuff could undergo sufficient digestion prior to absorption by the cecal epithelium.

Two types of parenchymal cells in the lung fluke Paragonimus ohirai (Digenea: Troglotrematidae) characterized by the cytochemistry of their mitochondria.

Morphology and respiratory function were studied in situ and in the isolated mitochondria of Paragonimus ohirai. Two types of parenchymal cells (i.e., Pc1 and Pc2 cells), whose mitochondria differ in terms of morphology and staining for cytochrome c oxidase activity, were found in fluke tissues. Enzymatic and spectrophotometric analyses of the isolated mitochondria showed that fluke mitochondria possess both aerobic and anaerobic respiratory chains. These results suggest that there are two mitochondrial populations in fluke parenchymal cells, one possessing an aerobic respiratory chain and the other an anaerobic respiratory chain.

Localization of peroxidase activity in tegumental, muscle, and parenchymal cells of the lung fluke Paragonimus miyazakii (Digenea: Troglotrematidae).

The distribution and localization of peroxidase activity were examined in adults of Paragonimus miyazakii in sections stained with 3,3'-diaminobenzidine in the presence of hydrogen peroxide. Peroxidase activity was detected in tegumental cell mitochondria. With cell development, the reaction intensity became greater. Activity extended from the inner membrane (undifferentiated stage) to the intermembrane space (differentiating stage) and then to the matrix (differentiated stage). Mitochondria with the most intense reaction were densely distributed in the outer syncytial region. Strong activity was also present in mitochondria of muscle cells and in those of Type II parenchymal cells. Intermembrane spaces were the predominant site of this activity. Characteristically, both 10 mM azide and 1 mM cyanide were mildly effective in the inhibition of mitochondrial peroxidase activity in each cell type.

Ultrastructure of the ootype of the lung fluke Paragonimus ohirai (Digenea, Troglotrematidae) in mated and single-infection worms.

The ootype of Paragonimus ohirai was studied by both scanning and transmission electron microscopy. The ootype wall in 20-week-old worms from single infections was similar in its epithelial cell architecture and components to that of mated worms at 10 weeks postinfection. The lining epithelium consisted of a single layer of nucleated cells. The cytoplasm displayed a variety of organelles such as occasional Golgi complexes, well-developed annulate lamellae, frequent lysosomes, abundant mitochondria, and numerous ribosomes, suggesting high activities of intracellular synthesis and digestion. The former three organelles were generally located in the apical cytoplasm protruding into the lumen and may be significant in participating in regulation of egg formation. The present comparative studies suggest that the ootype epithelium can mature even by single infections and that the organized intracellular activities remain developed in single worms even after prolonged infection.

[Scanning electron microscopic observation on the encysted and excysted metacercariae of Paragonimus heterotremus].

The surface ultrastructure of metacercariae of Paragonimus heterotremus was studied by scanning electron microscope. Encysted metacercaria was egg-shaped with a button-like structure on its end. The oral sucker of the newly excysted metacercaria was larger than the ventral sucker. Single-pointed spines were covered densely on all of the tegumental surface. There were two and three rings sensory papillae on the oral and ventral suckers, respectively. Six papillae of the most inner ring were distributed on the ventral sucker inner-pore symmetrically. A few sensory papillae were dispersed on each side of the anterior part of the worms. These papillae of some excysted metacercariae were arranged irregularly in two rows with 5 to 6 papillae in each row (Figs. 1-8).

[Observation on metacercariae of Paragonimus westermani by transmission electron microscopy].

The ultrastructure of metacercariae of Paragonimus westermani was observed by transmission electron microscopy. The worm is composed of integument, parenchyma, gut and excretory sac. The integument consists of tegument, muscular layer and tegumental cell. The tegument, a syncytium, includes tegumental membrane, tegumental cytoplasm, and basal plasma membrane. On the surface of the tegument, many finger-like processes with branches and spines can be readily identified. The spines projecting beyond the surface of the tegument are completely enclosed within the outer tegument membrane and the basal plasma membrane; in the transverse section, the spine shows a crystalline lattice structure. The tegumental cytoplasm consists of an electron dense granular matrix, in which two types of inclusion bodies, spherical and rod-like bodies may be recognised, both of them comprise a unit membrane and dense granular contents, the rod-like bodies are arranged along the edge of the spines. The tegument nuclei are located not within the surface syncytium but within sunken tegumental cells situated beneath the muscle layer, the tegumental cells contain a few cell organelles, and the two types of characteristic tegument inclusion bodies. The cells are joined to the tegument by one or more long, tortuous cytoplasmic tubules, responsible for the synthesis of the tegumental inclusion bodies, and that these inclusion bodies are translocated into the tegument by the cytoplasmic tubules. It suggests that the tegument of metacercaria is the main absorptive site of nutrients.

A comparative ultrastructural and histochemical study of the metacercarial cyst walls of four species of Paragonimus (Troglotrematidae: Trematoda).

The metacercarial cyst walls of Paragonimus westermani, P. miyazakii, P. ohirai, and P. iloktsuenensis were examined using ultrastructural and histochemical techniques. The cyst walls of P. westermani, P. miyazakii, and P. ohirai were found to have five distinct layers, whereas the wall of P. iloktsuenensis had only two. The so-called outer cyst wall recognized on light microscopy by Miyazaki (1961) was composed of layers I-III and the so-called inner cyst wall comprised layers IV and V. The outermost layer (I) consisted of collagen fibers and probably originated from the host. Layer III detached easily from layer IV on removal of the cysts from the host tissues. Layer IV was composed of proteoglycans, and layer V consisted of protein alone. A regular hexagonal pattern in layer V appeared to be characteristic of P. westermani. Although the basic morphological patterns observed in the layers constituting the cyst walls were similar in all species examined, the thickness of each layer was apparently different. This characteristic seems to be a suitable criterion for the identification of metacercarial cysts of Paragonimus species.

Morphological features of spermatozoa of Paragonimus ohirai (Trematoda: Platyhelminthes) examined by a silver nitrate staining technique.

The mature thread-like spermatozoon of Paragonimus ohirai was shown to be composed of 7 distinct segments which have morphologically different characteristics, following staining with silver nitrate. The first segment (1S) is a needle-shaped structure with a perforatorium. The 2S is composed of two filaments loosely paired; 3S consists of a somewhat coarse thread (vestige of the cytoplasmic projection); 4S is composed of two flagella tightly paired; in 5S, two silver-positive filaments are independently seen as in 2S; 6S has a 'spiral' structure composed of two flagella; in 7S, two filaments are seen dissociated from each other. The sperm nucleus exists within the 'spiral' structure of 6S and this structure may serve to hold and/or protect the nucleus. The technique presented here is useful for gross morphological observations of the long sperm of flatworms.

[Antibody-dependent rat macrophage-mediated damage to the excysted metacercariae of Paragonimus westermani in vitro].

An in vitro immune effector mechanism against the target excysted metacercariae of Paragonimus westermani was demonstrated in the rat system. Peritoneal exudate cells, mainly macrophages from normal rats, showed adherence to and killing of excysted metacercariae of P. westermani in the presence of complement-independent serum from rats infected with Paragonimus metacercariae. These reactions were specific for the excysted metacercariae, as tissue-migrating juvenile worms were not affected. Damage of excysted metacercariae of P. westermani due to antibody and macrophages was assessed by morphological observation, by cell adherence reaction and by the use of vital dyes. Trypan blue dye exclusion proved to be a reliable indicator of judging metacercarial viability. Electron microscopic studies demonstrated that macrophages reacted with fuzzy material on the tegumental surface and fine structures in the syncytium of the parasites. The tubular tunnels formed between the basement membrane and muscle layers of the damaged parasites were also noticeable. The relevance of these findings to cellular immunity in the early paragonimiasis was discussed.

Scanning electron microscopy of cuticular surface of the adults Paragonimus heterotremus in Phitsanulok, Thailand.

Scanning electron microscope observation of surface structure was made on adult Paragonimus heterotremus (Phitsanulok, Thailand) and the surface topography of the anterior end, suckers, body cuticle and papillae was described. The numerous thorn-like tegumentary spines were distributed all over the surface. The spines were well-developed and branched. The papillae were divided into two types: dome-shaped and ciliated. Two pairs of papillae were seen around the lip of the oral sucker, while those around the ventral sucker were not evident.

Ultrastructure of Mehlis' gland in the lung fluke, Paragonimus ohirai (Trematoda: Troglotrematidae).

Mehlis' gland of a digenetic trematode, Paragonimus ohirai, is composed of two types of secretory cells, DB and CB. The less abundant type (DB) produces dense bodies, with the cytoplasm characterized by greatly distended cisternae of rough endoplasmic reticulum. The other type (CB) synthesizes clear, vesicular bodies. Its cytoplasm contains numerous mitochondria, rough endoplasmic reticulum with narrow cisternae, and abundant Golgi complexes. Processes of the two cell types converge on the ootype-proximal uterine wall, pass through the epithelium, and finally open into the lumen. These proximal processes contain longitudinally arranged microtubules whose luminal ends are anchored to the epithelium by ring-form septate desmosomes. According to the distribution of the two types of processes, three different zones (DB, mixed, and CB) can be recognized within the epithelia. As the CB processes enter the lumen predominantly beyond the uterine valve region, this cell may produce secretions required for egg shell maturation or hardening. The role of DB cells (which enter the lumen more commonly in the ootype near the oviduct) remains unknown.

[Ultrastructural study on the vitelline cells of Pagumogonimus skrjabini].

This paper reported on the ultrastructure of the developing vitelline cells of Pagumogonimus skrjabini by using transmission electron microscope. The vitelline glands were isolated under dissecting microscope from the adult worm of P. skrjabini before 2.5% glutaraldehyde fixation. Four developmental stages of the vitelline cell were described. In stage 1. the nucleus contained many heterochoromatins. The mitochondria, ribosomal complex, rough endoplasmic reticulum and glycogen granules were observed in the cytoplasm. In stage 2. the typical characteristic of the developing cell was the grouping together of the vitelline globules. The junctional complexes were seen between the vitelline cells. In stage 3, that represented the initial stage of vitelline droplet formation, the vitelline cells were filled with rough endoplasmic reticulum. The immature vitelline cells included stage 1-3. In stage 4. the mature cells were divided into two phases. In the early phase of stage 4, the cytoplasm contained abundant glycogen granules, lipid droplets and a lot of ribosomal complexes and rough endoplasmic reticulum, while in the later phase part of the perinuclear cisterna showed vacuolization. The cytoplasm contained a large number of glycogen granules and the vitelline droplets were distributed on the inner border of the cell. In the developing egg, the vitelline granules adhered to the egg-shell.