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.

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.

[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.

Pathological and parasitological aspects of the first autochthonous case of human paragonimiasis in Venezuela.

A worm found in histopathologic sections of a lung piece of a young Venezuelan male was identified as a Paragonimus sp. Definitive identification of the species was not possible since only a deteriorated segment of the worm was recovered, nevertheless comparison with other known species is discussed. This is the first report of an indigenous case of human paragonimiasis in Venezuela.

Ultrastructure of spermatozoa of the lung fluke, Paragonimus ohirai (Trematoda: Troglotrematidae), in the seminal receptacle.

The seminal receptacle of Paragonimus ohirai contains not only mature spermatozoa, but also atypical and degenerate ones, suggesting that abnormal spermatozoa are retained in this organ. The spermatozoon is of a parallel biflagellar type with cortical microtubules, consisting of the anterior region, first mitochondrial region, intermediate (amitochondrial) region, second mitochondrial region, posterior nuclear region (PNR) and tail region (TR). The first third of the spermatozoon exhibits typical undulatory movement, while the middle part shows vibratory movement. At the area between head and midsections (H-M area) the peripheral doublets of axonemes are interrupted, and the external ornamentation is distributed widely around this portion. Throughout the immotile PNR and TR, the axonemes lack the dynein arms of their peripheral doublets. H-M, PNR, and TR ultrastructural characteristics are specific in P. ohirai spermatozoon and seem to be closely related to its pattern of movement.

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.

Ultrastructure of the oviduct of the lung fluke, Paragonimus ohirai (Trematoda: Troglotrematidae).

The oviduct of Paragonimus ohirai is lined by an epithelium composed of cells that rest on a prominent basal lamina and exhibit extensive basal infoldings. Apical surfaces of the epithelial cells are lamellated, and height and abundance of lamellae vary in different regions. Cilia are confined to two separate areas and are variable with respect to their axonemal pattern. There are five principal regions of the oviduct: infundibular (IR), distal ciliary (DCR), preseminal receptacle (PSR), proximal ciliary (PCR), and junctional (JR). The pattern of organization of cytoplasmic organelles such as mitochondria, Golgi complexes, and lysosomes is distinctive within each region. The remainder of the wall consists of circularly arranged myofiber bundles, together with a plexus of nerve fibers. The structure of the oviduct in relation to passage of oocytes, movement of spermatozoa, and fertilization is discussed.

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.

Development of the excretory bladder of the lung fluke Paragonimus ohirai (Trematoda: Troglotrematidae).

The excretory bladder of Paragonimus ohirai was studied during development in rats. Parasites were obtained at each development stage, and single worms were prepared for both transmission and scanning electron microscopy (SEM). During early transformation (1, 2 days), excretory materials such as calcareous concretions and lipid droplets filled the bladder, and Golgi complexes and various types of cytoplasmic bodies were abundant. The bladder epithelia of late transformation (3, 5 days) were characterized by dense granules and glycogen concentration around lipid droplets. In differentiating bladders (7, 10 days), rough endoplasmic reticulum, ribosomes, and alpha-glycogen were extensive in the epithelia, whereas concretions had disappeared. The apical surfaces of the epithelia were highly folded and lamellated, and lamellae were seen associated with fingerlike cytoplasmic projections (10 days) and spherical cytoplasmic bulges (15, 20 days). During maturation, SEM revealed nuclear profiles (20 days) and a muscular framework (30 days) in relief.

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.

Viruslike inclusions in the cecal epithelial cells of Pa agonimus kellicotti (Digenea, Troglotrematidae).

Rodlike or tubular inclusions are described from the cytoplasm and nuclear matrix of the cecal epithelial cells of Paragonimus kellicotti. These inclusions are 3.4 mu or more long, 350 A in diameter, and comprised of a wall of helically arranged subunits and a dense, possibly filar, central core. They occur in organized bundles of up to 17 members. Cells containing these inclusions manifest a series of nucleolar modifications typical of inclusions is discussed, noting especially their resemblance to known rodlike viruses from other systems.

Ultrastructural studies on spermatogenesis in a parthenogenetic type of Paragonimus westermani (Kerbert 1878) proposed as P. pulmonalis (Baelz 1880).

Spermatogenesis in a parthenogenetic type of P. westermani (Kerbert 1878) called P. pulmonalis (Baelz 1880) throughout this study, was observed by light microscopy (LM), scanning (SEM), and transmission (TEM) electron microscopy. During spermatogenesis, most of the cells became degenerated or malformed as a result of aberrations during spermatogenesis. Vacuolated cells were often found in the testicular lumen. In some nuclei of spermatocytes, synaptonemal complexes were formed and this indicated that some pairing of homologous chromosomes did occur, but only rarely. Cytophores in some rosettes were broken down into small fragments and the cells separated from each other. Norman spermatozoa were very rarely found in the testis and never in the seminal receptacle, where egg and vitelline cells were present instead. Throughout spermatogenesis, Golgi complexes, mitochondria, ribosomes, and endoplasmic reticulum were not abundant, and this suggested that cell activities and protein synthesis were greatly reduced.

Development of the ovary and the female reproductive cells of the lung fluke, Paragonimus ohirai (Trematoda: Troglotrematidae).

The ultrastructure of the ovary of Paragonimus ohirai was investigated in different developmental stages of experimental infection in rats, from the metacercarial stage to the adult stage. The female reproductive cells were observed in order to understand the development of the ovary. During its development in the definitive host, the ovarian primordium and the ovary increased in size and cell number and underwent morphological changes. The blind end of the female genitalia was an undifferentiated primordium at the metacercarial stage, but became the bud of an ovary on day 3. Germ cells and supporting cells were observed on day 5. Oogonia were identified in the 15-day-old ovary, followed by the appearance of young oocytes at 17 days. Large oocytes were found on day 19, but the 21-day-old ovary contained degenerated oocytes. Mature ovaries were observed in the 26-day-old worms and egg formation was seen to arise on day 28. The development of the ovary and female reproductive cells was discussed in relation to the physiology of P. ohirai.

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.

Surface ultrastructure of newly excysted metacercariae of Paragonimus heterotremus.

The newly excysted metacercariae of P. heterotremus were examined by scanning electron microscopy (SEM). Large dome-shaped papillae, small ones with a pit, and small ones with a smooth surface were observed around the suckers. The number of the small dome-shaped papillae with a pit was about 30 around the oral sucker and that of the small ones with a smooth surface varied from 9 to 13 around the ventral sucker. The present report is the first record of excysted metacercariae of P. heterotremus examined by SEM.

[Ultrastructural observation on the body wall of Pagumogonimus skrjabini].

This paper deals with the ultrastructure of the body wall of adult Pagumogonimus skrjabini by transmission electron microscopy. Infected crabs were collected from Siyen, Hubei Province, and adult worms were obtained from the lungs of experimentally infected dogs 90 days post-infection. The normal structure of body wall of the P. skrjabini is composed of tegument, tegument cell, muscle muscle cell and protoplasmic tubules, all of which form together syncytium. The tegument contains external plasma membrane, tegument matrix and basal plasma membrane. The cell coat in fine granules is distributed over the whole external plasma membrane surface. The tegument matrix contains various secretory bodies, vesicles and mitochondria. The tegument cell is irregular in shape. Golgi complex, ribosome, autolysosome are seen in the cytoplasm. There are two layers of muscle, the external circular muscle and the inner longitudinal muscle layers. The nucleus of immature muscle cell has many heterochromatins, while the nucleus of mature muscle cell is large and round in shape. Mitochondria and glycogen granules are transmitted from muscle cell proper to the muscle by protoplasmic tubules (Figs. 1 approximately 7).

[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).