Cellular aspects of early development of Ornithodiplostomum ptychocheilus metacercariae in the brain of fathead minnows, Pimephales promelas

Trematode metacercariae typically are regarded as nonfeeding and metabolically inactive. However, the metacercariae of many trematode species undergo complex and prolonged periods of development within their intermediate hosts. In the present study, we used electron microscopy to document chronological changes in development of the tegument of Ornithodiplostomum ptychocheilus metacercariae recovered from the brains of experimentally infected fathead minnows (Pimephales promelas). Commencing at 4 days postinfection (PI), the smooth, thin, syncytial tegument transforms into a complex microlamellar and microvillar system that encircles the entire body surface. The microvilli are oriented in parallel in an extended pattern, reaching directly away from the parasite and toward the receding host tissue. The microvilli disappear at approximately 28 days PI, followed by deposition of the cyst wall and further transformation of the tegument into the spinose, a glandular structure typical of an immature adult. To our knowledge, the progressive disaggregation of host cells at the leading edge of elongating parasite microvilli has not been demonstrated previously for any trematode. These results provide morphological evidence that the metacercariae of some trematode species undergo complex developmental changes associated with feeding in their intermediate host.     

The pathogenicity, localization, and cyst structure of echinostomatid metacercariae (Trematoda) infecting the kidneys of the frogs Rana clamitans and Rana pipiens

Light and scanning electron microscopy were used to examine the localization and pathogenicity of echinostomatid metacercariae infecting the kidneys of leopard frogs, Rana pipiens, and green frogs, Rana clamitans. Cysts occurred predominantly in the ventrolateral renal cortex, and at least some were confined to the lumen of the Bowman's capsules. Each vermiform metacercarial body was enclosed by a spherical cyst wall that had a uniform thickness. The wall was composed of a homogeneous material containing basic and keratinlike proteins, with sulfated acid mucopolysaccharides on the outer surface. Most cysts were enclosed by a fibrous capsule of host origin, or were surrounded by an inflammatory focus. Fibrosis was always focal, but its degree varied between individual hosts and between different cysts within the same host. Some heavily encapsulated cysts were darkened and contained disintegrating worms. In heavily infected kidneys, confluence of fibrotic or inflammatory foci resulted in the displacement of functional renal tissue. These data suggest that infection by echinostomatids may impair renal function and that the host's response affects parasite viability.     

In vitro excystment of the metacercaria of Maritrema arenaria (Digenea: Microphallidae)

Metacercarial cysts of Mantrema arenaria were subjected to a solution containing trypsin and bile salts at 41°C. This treatment induced intense metacercarial activity and after 15 min metacercariae burst through their cyst walls and emerged. Electron microscopy demonstrated that during the process of excystment the inner layer of the cyst wall changed from a compact to a loose fibrous state. Experiments showed that only cysts containing viable metacercariae underwent this change whereas cysts which had been forcibly vacated before treatment did not. This indicated that the structural change of the inner layer of the cyst wall could not be attributed to the excystment medium. Also there was much less acid phosphatase activity in and on the surface of newly excysted metacercariae compared with encapsulated specimens. It was concluded that the excystment medium induced physical activity in, and the release of enzymic material by, the metacercariae. Together these activities rendered the cyst wall soft and susceptible to rupture by physical pressure.     

Prevalence,geographic distribution,and fitness effects of Microphallus turgidus (trematoda: Microphallidae) in grass shrimp (Palaemonetes spp.) from coastal Georgia

Grass shrimp, Palaemonetes pugio Holthuis and P. vilgaris (Say), were collected at 11 localities along the coast of Georgia and surveyed for digenetic trematode metacercariae. The effect of trematode infection on grass shrimp fitness was also examined. Microphallus turgidas (Leigh) was the only trematode observed. The prevalence of metacercarial cysts of this parasite in P. pugio (75%) was higher than in P. vulgaris (24%), as were the mean intensity, abundance, and population density (no. cysts/cm host body length) of the parasite. Infected shrimp were found at every collection locality and parasite prevalence and density were greatest in P. pugio from higher salinity localities (> or = 20 parts per thousand [ppt]). There was no relation between host body size and parasite density in P. vulgaris, and parasite density increased with host body size in P. pugio, suggesting that the parasite does not affect host survival. There was no relation between parasite density and shrimp egg mass, but nonovigerous female P. pugio were more heavily infected than ovigerous ones. In addition, 1 metacercaria of M. turgidus in each of 2 specimens of P. vulgaris was parasitized by the haplosporidian Urosporidium crescens De Turk. This represents a new shrimp host record for this hyperparasite.     

Histochemistry and ultrastructure of the metacercarial cyst of Bolbogonotylus corkumi (Trematoda: Cryptogonimidae).

Histochemical and ultrastructural studies were conducted on the metacercarial cyst of the cryptogonimid trematode Bolbogonotylus corkumi from the muscle tissue of fantail darters Etheostoma flabellare. The metacercarial cyst consisted of an outer host capsule and an inner parasite cyst. The host capsule was composed of an outer region of fibroblasts, collagen, macrophages, and unidentified cells, and an inner region containing degenerating cells. The parasite cyst was thin, homogenous, and noncellular in nature. The host capsule stained strongly for connective tissue and proteins and moderately for lipids, nucleic acids, nonspecific esterase activity, and acid and alkaline phosphatase activities. The parasite cyst stained intensely for acid mucopolysaccharides and moderately for acid phosphatase activity. A thick glycocalyx occurred between the parasite cyst and metacercarial tegument.     

The responses of Ligia oceanica (L.) (Crustacea: Isopoda) to infection by Maritrema linguilla Jäg. 1908 and Microphallus similis (Jäg. 1900) (Digenea: Microphallidae) and to abiotic implants

The initial and principal encapsulation response of Ligia oceanica to Araldite implants and to encysted metacercariae of Maritrema linguilla is hemolymph coagulation followed by limited hemocyte agglutination. Granules secreted by isolated granulocytes and semigranulocytes may catalyze coagulation. Isolated hyaline cells explode and make an insignificant contribution to the initial cyst wall. Later, hemocytes agglutinate and some granulocytes retain their granules which become melanized. Eventually, a wide multilayered hos capsule is formed. Unencysted metacercariae of M. linguilla transplanted from the pleopods into the dorsal hemocoel of another specimen of L. oceanica encyst and become encapsulated but are not damaged by encapsulation. Transplanted encysted metacercariae are also encapsulated and unharmed. Cercariae implanted directly into the dorsal hemocoel, however, fail to encyst, become encapsulated, die, and lyse within the capsule. Implanted cercariae and encysted metacercariae of Microphallus similis are also encapsulated and destroyed in the hemocoel of L. oceanica. The absence of host response to the naturally infecting unencysted parasite in the pleopod sinuses may be attributed to rhythmic movement, mucopolysaccharide secretions and to the retention of excreta within the excretory bladder. Once the excreta is released during cyst formation in the dorsal hemocoel, encapsulation occurs but this does not appear to harm the parasite. On the contrary, considerable growth occurs within the cyst which suggests that the parasite may absorb nutrients released from necrotic hemocytes.     

Expression of the proteinase specialized in bone resorption, cathepsin K, in granulomatous inflammation

BACKGROUND: The cysteine proteinase cathepsin K has aroused intense interest as the main effector in the digestion of extracellular matrix during bone resorption by osteoclasts. The enzyme is not a housekeeping lysosomal hydrolase, but is instead expressed with striking specificity in osteoclasts. In this work, we present evidence for the association of cathepsin K with the granulomatous reaction. Granulomas are inflammatory tissue reactions against persistent pathogens or foreign bodies. We came across cathepsin K while working on Echinococcus granulosus, a persistent tissue-dwelling, cyst-forming parasite that elicits a granulomatous response. MATERIALS AND METHODS: The walls of hydatid cysts from infected cattle were solubilized. Strong proteolytic activity was detected in the extracts. The proteinase responsible was purified by anion exchange and gel filtration. The purified protein was subjected to N-terminal sequencing, and its identity further confirmed by Western blotting, with a cathepsin K-specific antibody. The same antibody was used to localize the proteinase in paraffin-embedded sections of the parasite and the local host response. RESULTS: A proteinase was purified to near homogeneity from hydatid cyst extracts. The enzyme was unequivocally identified as host cathepsin K. Both the proenzyme and the mature enzyme forms were found. Cathepsin K was then immunolocalized both to the parasite cyst wall and to the epithelioid and giant multinucleated cells of the host granulomatous response. CONCLUSIONS: In the granulomatous response to the hydatid cyst, cathepsin K is expressed by epithelioid and giant multinucleated cells. We propose that, by analogy with bone resorption, cathepsin K is secreted by the host in an attempt to digest the persistent foreign body. Both processes, bone resorption and granulomatous reactions, therefore tackle persistent extracellular material (the bone matrix or the foreign body), and utilize specialized cells of the monocytic lineage (osteoclasts or epithelioid/giant cells) secreting cathepsin K as an effector.     

Explanation for naturally occurring supernumerary limbs in amphibians

The occasional occurrence of high frequencies of limb abnormalities, including extra limbs, in natural populations of amphibians has long been a puzzle. In this paper we report the discovery of a population in which such limb abnormalities appear to be caused by a parasitic flatworm (trematode) that uses amphibians as intermediate hosts. The cercarial larval stage of the trematode attacks amphibians, penetrating the skin to form cysts (metacercariae). The cysts are preferentially localized in the cloacal region, including the developing hind limb regions in larvae of both frogs (Hyla regilla) and salamanders (Ambystoma macrodactylum). A wide range of limb abnormalities are seen, including duplicated limb structures ranging from extra digits to several extra whole limbs. We hypothesize that these limb abnormalities result from localized regulatory responses of developing and regenerating limb tissues to mechanical disruption caused by the trematode cysts. We have tested this idea by implanting inert resin beads into developing limb buds of frogs and salamanders. Since this treatment can cause supernumerary limb structures, our hypothesis is sufficient to explain the naturally occurring extra limbs.     

Chemical, histochemical, and histopathological studies on corals. Porites spp., parasitized by trematode metacercariae

The occurrence of the metacercaria of Plagioporus sp. encysted in two species of corals, Porites compressa and P. lobata, from Kaneohe Bay, Oahu, Hawaii, is reported. This parasite is covered with a bilayered metacercarial cyst the chemical compositions of which have been ascertained by histochemistry. Both of these cyst walls are believed to be secreted by the parasite. In addition, the cytochemical properties of the metacercaria and the adjacent host cells are reported.Histo- and cytopathological alterations in the epidermis and gastrodermis of the coral hosts were studied and such changes have been interpreted to have resulted from mechanical pressure exerted by the parasite.Because gross observations had revealed the presence of elevated nodules at the sites where the metacercariae occurred, chemical analysis was conducted to ascertain whether this larval trematode had induced hypercalcification in the hosts. However, calcium determinations of Decal in which pieces of parasitized coral tissue had been decalcified revealed that hypercalcification had not occurred.Examination of the cellular reactions in both species of Porites revealed that if the gastrodermis had been ruptured, partial encapsulation of the parasite occurs. The reaction cells include free cells of the mesoglea that become fused, accidentally trapped cnidoblasts and nutritive-muscular cells, and gastrodermal interstitial cells. Despite the partial encapsulation, the encysted metacercariae did not appear to be injured.     

The influence of habitat on the distribution and abundance of metacercariae of Macravestibulum obtusicaudum (Pronocephalidae) in a small Indiana stream

Snails (Elimia livescens) from a central Indiana stream were examined for encysted metacercariae of Macravestibulum obtusicaudum (Pronocephalide) to determine the distribution and abundance of this parasite on its second intermediate host. Five samples of snails were collected, with 2 samples being restricted to high-flow (riffle) or low-flow (pool) areas of the stream. Snails (n = 386) were measured for shell length; the shell and the inner and outer surfaces of the operculum (in most samples) were examined for metacercariae. Seventy-five percent of snails (overall) had encysted metacercariae (range, 52-97%), primarily on the opercula. A significantly lower proportion of snails from riffles were infected, and these snails had significantly fewer cysts as well. Snails collected from pools showed up to 10 times as many metacercariae than those from riffles, although there was no significant difference in the proportion of snails with intramolluscan infections of M. obtusicaudum. The inner opercular surface appeared to be the preferred site of encystment in both flow regimes. Differences in microhabitats, in terms of both snails in the stream and metacercariae on the snails, clearly must be considered when evaluating the infection patterns of this trematode.     

Tributyltin and copper effects on encystment and in vitro excystment of Parorchis acanthus larvae

Effects of tributyltin (TBT) and copper (Cu) on cercariae and metacercariae of the trematode Parorchis acanthus (Digenea: Philophthalmidae) were investigated. Cercariae released by the dogwhelk, Nucella lapillus were maintained in natural seawater (SW) or solutions of TBT or Cu ranging from 0.001-100 microg l(-1) and 1-6 mg l(-1) respectively before they encysted. Over 79% of the cercariae encysted in control and test solutions. Low concentrations of TBT reduced encystment success more than low concentrations of Cu. The percentage of cercariae that formed cysts in the highest concentrations of both pollutants was higher than in the controls, perhaps representing an 'emergency response' to the pollutants. Before being induced to excyst in vitro, metacercariae were left in the heavy metal solutions for 3 weeks. Metacercariae exposed as cercariae to TBT and Cu achieved lower percentage excystment success than those that had encysted in SW. Cyst walls provided greater protection against Cu than TBT. It was concluded that TBT and Cu had a detrimental effect on the larval stages of P. acanthus at the higher concentrations used but the cyst wall afforded an element of protection if formed in unpolluted seawater before the larval stages were subjected to the pollutants.     

Histopathological changes in fish gills infected with myxosporidian parasites of the genus Henneguya

Tissue reaction to invasion by two Henneguya species was studied in gills of three species of fish. Henneguya psorospermica and Henneguya creplini develop in secondary lamellar arterioles in perch ( perca fluviatilis ) and ruff ( Acerina cernua). In pike ( Esox lucius), H. psorospermica develops in the artery. During the growth of the vegetative stage, the gill tissue may undergo local atrophy or hyperplasia of the epithelium and displacement and disfiguration of the lamellae. After the myxosporidian cyst, surrounded by a host tissue capsule has reached maturity and contains fully-formed spores, an inflammatory reaction may set in which reduces the cyst to granulomatous tissue. This inflammatory process is essentially of the same type as that observed in mammals. There is evidence that inflammatory responses are of regular occurrence in the final stages of most myxosporidian infections. Transfer from low to room temperature provokes a rapid elimination of the cysts by granulomatous inflammation.     

High-resolution immunogold localization of Giardia cyst wall antigens using field emission SEM with secondary and backscatter electron imaging

We describe here the ultrastructural localization of Giardia cyst antigens in the filaments associated with the outer portion of intact cysts and on developing cyst wall filaments in encysting trophozoites. Post-embedding immunogold labeling of thin sections of intact Giardia cysts with polyclonal and monoclonal antibodies specific for cyst wall antigens (major protein bands of approximately 29, 75, 88, and 102 KD on Western blots) showed strong labeling of the filamentous cyst wall, whereas no labeling was seen on the membranous portion. High-resolution field emission scanning electron microscopy (FESEM) of Giardia cysts revealed that the cyst wall-specific polyclonal rabbit antisera and monoclonal mouse antibody produced gold labeling of 20-nm filaments in the cyst wall as detected with secondary electron imaging (SEI) and backscatter electron imaging (BEI) at 10 kV, despite coating of the cells with platinum by ion sputtering. FESEM studies of encysting Giardia trophozoites demonstrated that immunostaining with antibodies to cyst wall antigens produced colloidal gold labeling of developing cyst wall filaments on the cell surface; however, the intervening membrane domains were unlabeled. Substitution of normal serum for cyst wall-specific antibodies, or preabsorption of specific antibodies with Giardia cysts, eliminated immunolabeling of the filaments.     

Features of pathogenesis of Diplostomum infection in riverine Abbottina rivularis (Cypriniformes: Cyprinidae) from Primor'e

Autopsies of Abbottina rivularis from southern Primorye (drainage-basin of Artyomovka River, Razdolnaya River and Khanka Lake) and southern Sakhalin Island (Maloye Chibisanskoye Lake) revealed high indices of diplostomum-infected lenses of these fishes. The metacercariae have been identified as Diplostomum parviventosum Dubois, 1932, D. huromense (La Rue, 1927), D. helveticum (Dubois, 1929), D. mergi Dubois, 1932, Diplostomum sp. The most lenses of parasitized eyes possessed dorsally situated sperical protrusions of the lens capsule ("cyst"). Earlier, this phenomenon was found by Larson (1965) in naturally infected bullheads (Ictalurus). The metacercarial infection level and its dynamics, age-composition of metacercariae in both lenses and "cysts", and "cysts" production are discussed.     

The organization of the wall filaments and characterization of the matrix structures of Toxoplasma gondii cyst form

The encystation process is a key step in Toxoplasma gondii life cycle, allowing the parasite to escape from the host immune system and the transmission among the hosts. A detailed characterization of the formation and structure of the cyst stage is essential for a better knowledge of toxoplasmosis. Here we isolated cysts from mice brains and analysed the cyst wall structure and cyst matrix organization using different electron microscopy techniques. Images obtained showed that the cyst wall presented a filamentous aspect, with circular openings on its surface. The filaments were organized in two layers: a compact one, facing the exterior of the whole cyst and a more loosen one, facing the matrix. Within the cyst wall, we observed tubules and a large number of vesicles. The cyst matrix presented vesicles of different sizes and tubules, which were organized in a network connecting the bradyzoites to each other and to the cyst wall. Large vesicles, with a granular material in their lumen of glycidic nature were observed. Similar vesicles were also found associated with the posterior pole of the bradyzoites and in proximity to the cyst wall.     

Spatial variation in parasite-induced mortality in an amphipod: shore height versus exposure history

Characterizing the causes of spatial and temporal variation in parasite-induced mortality under natural conditions is crucial to better understanding the factors driving host population dynamics. Our goal was to quantify this variation in the amphipod Paracalliope novizealandiae, a second intermediate host of the trematode, Maritrema novaezealandensis. If infection and development of trematode metacercariae are benign, we expected mature metacercariae to accumulate within amphipods inhabiting high infestation areas. In field samples, intensity levels of mature metacercariae decreased linearly when amphipods harbored >5 immature metacercariae. This finding is consistent with the hypothesis that the parasite can be detrimental at high intensities of infection. Short-term field experiments showed that host survival also declines with the intensity of new infections and drops below 80% when early stage metacercariae reach 10 amphipod^?1. However, parasite effects varied over space and time. High-shore amphipods suffered an increased risk of infection in the summer and a lower likelihood of survival: there was a 10–30% decrease in survivorship for any given infection intensity at high- versus low-shore locations. We also tested for differences in the susceptibility of naive and exposed populations using transplant experiments, and found that naive amphipods acquired greater parasite loads (on average, 4.7 vs. 2.8 metacercariae amphipod^?1). Because survival decreases rapidly with infection intensity of both early- and late-stage metacercariae, naive populations would suffer considerably if the parasite were to increase its range. Our results indicate that trematode infections cause high mortality in amphipods during summer months under natural conditions, and emphasize that the effects of parasitism vary at local spatial scales and with exposure history.     

Phenology of recruitment and infection patterns of Ascocotyle pachycystis, a digenean parasite in the sheepshead minnow, Cyprinodon variegatus

In northwest Florida, Cyprinodon variegatus are parasitized by Ascocotyle pachycystis, a digenean parasite that forms metacercarial cysts in the lumen of the bulbus arteriosus. Field experiments revealed that fish accumulated parasites at an uneven rate within the highly seasonal trematode recruitment period. Older (= larger) fish had higher rates of parasite recruitment and higher parasite prevalences and densities (numbers of metacercariae per individual fish) than did younger (= smaller) fish. Nearly all adults were parasitized (prevalence range 70-100%), and parasite densities ranged from zero to 6800 per fish. Parasite distributions were clumped (= aggregated) in fish of all age classes but were less heterogeneous in early juveniles and adults than they were in late juveniles. Parasites affected the population dynamics of sheepshead minnows by causing reduced winter survivorship, as evidenced by an increase in the average size of fish and a decrease in the average infection intensity over the winter. This revised version was published online in July 2006 with corrections to the Cover Date.     

Histopathological and ultrastructural observations of metacercarial infections of Diplostomum phoxini (Digenea) in the brain of minnows Phoxinus phoxinus

The spatial distribution and histopathological changes induced by metacercariae of the digenean trematode Diplostomum phoxini (Faust, 1918) in the brains of European minnows Phoxinus phoxinus (L.) from the River Endrick, Scotland, were studied by light and electron microscopy. Post-mortem examination of a sample of 34 minnows revealed that 50% (n = 17) of the population was infected with 13.7 +/- 2.6 (mean +/- SE; range 1 to 38) metacercariae per infected host. Serial histological sections of the infected minnow brains revealed that the metacercariae were unevenly distributed throughout the brain, with aggregations occurring in the cerebellum, the medulla oblongata and the optic lobes. In fish with highest intensities of infection, over 40% of the cerebellar area and about 30% of the medulla oblongata area were occupied by larvae. Metacercariae disrupt the integrity of brain tissue, with individuals being found in small pockets surrounded by cellular debris. Metacercariae were rarely encountered on the surface of the brain. Electron microscopic examination of infection sites revealed that the granular layer surrounding metacercariae was necrotic, exhibited nuclear degradation and was marked by vacuolation of the cytoplasm. Rodlet cells, the only inflammatory cell types recorded in this study, were found only in parasitized brains and in close proximity to the teguments of metacercariae. It is hypothesised that secretions released from the teguments of metacercariae are a counter response to protect the metacercariae from the fish brain's cellular defence mechanisms.     

A trematode metacercaria encysted in the nerves of the dungeness crab, Cancer magister

Of 188 Dungeness crabs, Cancer magister, 8 contained unidentified trematode metacercariae encysted in various components of the nervous system, including the thoracic ganglion, brain, lamina ganglionaris of the eyestalk, and major nerves arising from the thoracic ganglion. A single cyst was present in the available tissue sections of 7 of the crabs and no behavioral abnormalities were exhibited. One crab with multiple cysts in major nerves near the thoracic ganglion was markedly ataxic. The cysts and included worms distorted, compacted, and destroyed nervous tissue, and occupied most of the nerves where present. Host response was minimal, but some cysts invoked massive hemocytic accumulations near the infection site. Infections probably seriously affect nerve impulse transmission and accounts for the lethargic behavior of the crab with multiple cysts in major nerves. The present report is the first record of digenetic trematode infections in the Dungeness crab and the apparent restriction of the worm to nervous tissue is unusual if not unique in the Digenea. Because of the absence of grossly recognizable lesions and the small samples excised at necropsy, both the incidence and intensity of infection in Dungeness crab populations are almost certainly higher than indicted by our data.     

Helminth parasites of the tropical gar, Atractosteus tropicus Gill, from Tabasco, Mexico

A total of 8 helminth species were recorded in an examination of 43 tropical gar, Atractosteus tropicus Gill, collected at the Pantanos de Centla Biosphere Reserve, Tabasco, Mexico. The parasite species included 1 adult trematode, 3 metacercariae, 1 cestode, 1 adult nematode, and 2 nematode larvae. Six of these 8 species were rare, with low prevalence (< 17%) and abundance (< 1.0 helminths per examined fish). The larvae of Contracaecum sp. were the most abundant in the sample, constituting 60% of the total helminths (64% prevalence, 3.8 +/- 5.2 abundance), followed by the cestode Proteocephalus singularis, constituting 18% of the worms (30.5% prevalence, 1.1 +/- 3.0 abundance). Species richness, individual parasite abundance, and diversity were low in the infracommunities. The recording of 3 specialist species in the tropical gar confirms that the helminth fauna of gar has an appreciable degree of specificity. This study indicates the importance of ecological determinants of richness in helminth communities of the tropical gar.