Accumulation and excretion of neutral lipids in the metacercaria of Leucochloridiomorpha constantiae (Trematoda) maintained in vitro.

Histochemical and thin-layer chromatographic analyses were made on neutral lipids in Leucochloridiomorpha constantiae (Trematoda) metacercariae incubated in non-nutrient Locke's solution at 37.5 or 42 C for 24 hr. As determined by Oil Red O staining, lipid droplets accumulate mainly in the intestine of incubated worms. Attempts to distinguish specific neutral lipid fraction of metacercariae obtained directly from snails is free sterol. During incubation, metacercariae accumulate triglycerides and to a lesser extent cholesterol esters, and excrete free sterol into the medium.     

Histochemical and thin layer chromatographic analyses of neutral lipids in Echinostoma revolutum metacercariae cultured in vitro

Excysted metacercariae of Echinostoma revolutum cultured in vitro in the defined medium, NCTC 135 supplemented with 20% hens' egg yolk, doubled their mean relative body area and showed significant sucker growth within 14 days. Histochemical Oil Red O staining showed neutral fat mainly in the excretory system of excysted metacercariae and in adults grown in the domestic chick. In vitro cultured worms showed neutral fat in the intestine, parenchyma, and excretory system. As detected by TLC the major neutral lipid fractions were free fatty acids for excysted metacercariae; free sterols for adults grown in chicks; and triglycerides, free fatty acids, and free sterols for cultured worms. Excysted metacercariae excreted free fatty acids into a nonnutrient incubation medium, whereas cultured worms excreted diglycerides, triglycerides, and free fatty acids into the medium.     

Neutral lipids in cercariae, encysted metacercariae, and rediae of Zygocotyle lunata

High-performance thin-layer chromatography was used to analyze the neutral lipids in the rediae, cercariae, and encysted metacercariae of the paramphistomid trematode Zygocotyle lunata. Visual observations of the chromatograms showed that the most abundant lipid fractions were free sterols and free fatty acids in all larval stages and triacylglycerols in the metacercariae and rediae. The weight of free sterols (x +/- SE) was 120+/-20 ng/cercaria, 56+/-3.8 ng/redia, and 5.9+/-1.5 ng/encysted metacercaria; the weight of triacylglycerols was 13+/-0.88 ng/encysted metacercaria, 6.3+/-0.063 ng/redia, and was not detectable in the cercaria; the weight of free fatty acids was 160+/-17 ng/ cercaria, 76+/-9.1 ng/redia, and 4.2+/-0.46 ng/encysted metacercaria. Oil red O staining of whole larvae showed the presence of neutral lipids in the rediae but not in the cercariae or encysted metacercariae. A dramatic reduction was seen in the quantity of free sterols and free fatty acids in the encysted metacercariae as compared with the cercariae, suggesting that these neutral lipids are used in some way during the transformation from cercaria to metacercaria.     

Presumptive neurosecretion in Leucochloridiomorpha constantiae (Trematoda) and its possible role in governing maturation

A review of literature dealing with neurosecretion in the Platyhelminthes is presented and presumptive neurosecretory cells associated with the cerebral ganglia and longitudinal nerve cords ofLeucochloridiomorpha constantiae metacercariae and adults are described. Both the mean number of aldehyde-fuchsin stained presumptive neurosecretory cells and the intensity of staining are less in adults than in metacercariae. This suggests that maturation in this trematode may be directly or indirectly associated with a reduction in neurosecretion as in nereid annelids. Results of cytochemical studies indicate the neurosecretory product contains a glycoprotein.     

Studies on the viability of metacercariae of Fasciola gigantica.

The viability of metacercariae of Fasciola gigantica was tested by in vitro and in vivo methods. In vitro testing was based upon the motility of juvenile flukes within the inner cyst as examined under the light microscope. In vivo testing was undertaken through experimental infections of rabbits (two groups) and natural definitive hosts, lambs (one group). In the first group, out of six rabbits each given 25 metacercariae, worm establishment only took place in one rabbit with a single fluke recovery on 60 days post infection. In the second group of six rabbits each given 200 metacercariae, five were infected, with two or three flukes per host. All the lambs given 250 metacercariae became infected showing prevalences of 7.2-40% in comparison with rabbits in which low prevalences (0-4%) were recorded. The results indicated that even viable metacercariae which were already tested in vitro could not readily establish in rabbits. Such variability in worm establishment suggests that immunological and chemotherapeutic studies in rabbits infected with F. gigantica are likely to be unreliable.     

Parasites of commercial shrimps and fishes in Argentine sea: on the adult and metacercaria of Opecoeloides feliciae n. sp. (Digenea: Opecoelidae).

Opecoeloides feliciae n. sp., first record of opecoelid metacercariae in commercial shrimps of South Atlantic Ocean, parasitizing Cynoscion striatus (adults) and Artemesia longinaris (metacercariae), is described, illustrated and compared with related species of the genus. Adults, immature worms and metacercariae are compared, and rates of prevalence and intensity of infection are also given.     

Effect of cobalt-60 irradiation on the infectivity of Paragonimus westermani metacercariae.

A study was made to observe the effect of cobalt-60 irradiation on the viability of Paragonimus westermani metacercariae in Sinopotamon chekiangense crabs. The crabs were collected in mountain regions of the Zhejiang Province of China in which paragonimiasis is endemic. Adult cats and albino mice were infected with metacercariae irradiated at different doses. Dissection of the host animals was conducted 90 or 30 days, respectively, after infection for recovery of lung flukes. Anti-metacercariae antibody in infected mice was measured by enzyme-linked immunosorbent assay (ELISA). Results showed that metacercariae were unable to grow into adult worms in cats after exposure to gamma irradiation at a dose of 0.10 kGray. However, a small number of metacercariae exposed to a dose of 2.0 kGray excysted and survived in 1 mouse for 30 days. No worm was recovered from mice when the metacercariae were irradiated at a dose of 2.5 kGray. Seropositive results by ELISA were obtained when the mice were infected with metacercariae irradiated at doses ranging from 2.0 to 3.5 kGray.     

Neutral lipids in cercariae, encysted metacercariae, and rediae of Echinostoma caproni

High performance thin-layer chromatography (HPTLC) was used to analyse the neutral lipids in the rediae, cercariae, and encysted metacercariae of Echinostoma caproni from Biomphalaria glabrata snails. Visual observations of the chromatograms showed that the most abundant lipid fraction in all stages was free sterol. Quantification of the free sterol revealed mean weights of 2.7 +/- 0.64 ng per redia, 0.53 +/- 0.023 ng per cercaria, and 0.081 +/- 0.0098 ng per encysted metacercaria. Oil Red O staining of the larval stages confirmed the presence of lipids within the rediae and cercariae but did not show lipids in the encysted metacercariae. The dimunition in neutral lipids from the cercarial to the encysted metacercarial stage does not support a previous observation that fat increases in successive phases of the digenean life cycle.     

Activities of scavenging enzymes of oxygen radicals in early maturation stages of Paragonimus westermani.

In early maturation stages of Paragonimus westermani (metacercariae, 4-, 8-, 12-week old worms), activities of antioxidant enzymes, such as superoxide dismutase, catalase, peroxidase and glutathione peroxidase, were examined. Specific activity of catalase was the highest in metacercariae and decreasing with age. That of superoxide dismutase was higher in metacercariae and 4-week worms. Specific activity of peroxidase was at its peak in 4-week worms while that of glutathione peroxidase was in 8-week worms. Specific activities of all these antioxidant enzymes were decreased to their lowest in 12-week old adults.     

Taxonomic status, redescription, and surface ultrastructure of Ascocotyle (Phagicola) pindoramensis n. comb. (Digenea: Heterophyidae)

Pygidiopsis pindoramensis Travassos, 1928, is redescribed from the holotype and specimens obtained from experimental infections of hamsters with metacercariae of the naturally infected poeciliids Poecilia vivipara and Phalloptychus januarius from Rio de Janeiro, Brazil. Ascocotyle (Phagicola) pindoramensis (Travassos, 1928) n. comb. is proposed, based on the presence of a solid, tapering muscular posterior prolongation of the oral sucker and Ascocotyle (Phagicola) mollienisicola (Sogandares-Bernal and Bridgman, 1960) is synonymized with A. (P.) pindoramensis because of the identical morphology and measurements of adults and metacercariae, similar spectrum of fish intermediate hosts (poeciliids), and the same site of infection of the metacercariae. Trematodes designated as Pygidiopsis pindoramensis, previously reported from Argentina and Mexico, represent another species of Pygidiopsis Looss, 1907, because they possess circumoral spines and their oral sucker is devoid of the posterior muscular prolongation. Data on the surface morphology of metacercariae and adults of A. (P.) pindoramensis are inferred from scanning electron microscopy observations.     

Cultivation of excysted metacercariae of Echinostoma caproni to ovigerous adults in the allantois of the chick embryo.

Chemically excysted metacercariae of Echinostoma caproni inoculated into the allantois of domestic chick embryos became ovigerous in that site within 9 days postinoculation. The egg preparation technique of Saville and Irwin was markedly better than that of a modified Zwilling procedure for obtaining large numbers of postinoculation embryos with worm infections. Adults of E. caproni from the allantois were larger and became ovigerous sooner than worms grown on the chorioallantois. Only worms from the allantois produced eggs with fully developed miracidia. Miracidia were released from these eggs, but an insufficient number was available to attempt infections in Biomphalaria glabrata snails.     

Standardization of the Papanicolaou stain. I. A comparison of five nuclear stains.

The staining characteristics of five nuclear stains used in a Papanicolaou staining procedure were investigated. Alcohol-fixed cervical smears were stained with a modified Papanicolaou procedure using hematoxylin, alcoholic thionin bromide, alcoholic Victoria blue B, gallocyanin or the thionin Feulgen reagent (thionin-SO2) as the nuclear stain. The same anionic counterstain was used for all slides, and the optical densities of cell nuclei and cytoplasm were measured with the IBAS 2000 image analyzer. Alcoholic thionin gave the most intense nuclear stain, with a very high reproducibility of the staining pattern. Hematoxylin showed the highest coefficient of variation of the staining intensity. Both hematoxylin and gallocyanin gave some nonspecific cytoplasmic staining. Thionin-SO2 allowed a quantitative assessment of DNA, but gave a low staining intensity. Staining with the metal complex dyes interfered with subsequent staining with the pararosaniline Feulgen reagent. Alcoholic thioinin is thus recommended as a nuclear stain for cervical cytology in the Papanicolaou procedure, both for image analysis and for visual microscopy.     

First record and description of metacercariae of Curtuteria arguinae n. sp. (Digenea: Echinostomatidae), parasite of cockles Cerastoderma edule (Mollusca: Bivalvia) in Arcachon Bay, France

A new Himasthlinae species, Curtuteria arguinae, is described as metacercariae from the cockle Cerastoderma edule (L.), collected at Banc d'Arguin (southwestern France). These metacercariae encysted preferentially in the mantle and also in the foot of cockles. Encysted and chemically excysted metacercariae were studied by light microscopy and scanning electron microscopy, respectively. Excysted metacercariae were elongated and curved ventrally. They bore a 33-spine circumoral collar. Sensory papillae were arranged around the oral sucker and also symmetrically along the ventral surface body, from the collar to the acetabulum. The dorsal and ventral tegument surfaces were densely packed with similar pointed spines. The posterior end of the body was without any spines. Among the Curtuteria species described previously, only Curtuteria haematopodis Smogorjewskaja and Iskova, 1966 had the same number of circumoral collar spines. A 6-yr field survey showed that the cockle population at Banc d'Arguin was subjected to a summer infection of C. arguinae. Curtuteria arguinae phenology of infection is characterized by interannual variability and seasonality (beginning in July-August and maximum in autumn). The first intermediate and final hosts remain unknown.     

Prevalence of two site-specific populations of Diplostomum spp. in eye infections of rainbow trout, Salmo gairdneri Richardson, from lakes in Washington State, U.S.A.

Two distinct site-specific populations of Diploslomum spp., occurring concurrently in the lens and retina of naturally infected rainbow trout, were different morphologically. They represent separate populations epidemiologically. The lens and retina metacercariae did not differ immunologically when tested by an enzyme-linked immunosorbent assay against immune rabbit and trout antisera. Antibody titres became positive by September 1982 in a natural population of yearling rainbow trout infected with lens and retina metacercariae and remained positive for at least 8 months. The antibodies formed may offer protection against reinfections with Diplostomum spp. cercariae.     

Studies on host specificity in Paragonimus westermani: II. Histochemical and cytochemical characterization of metacercariae and worms from rats and dogs

Histochemical tests were done on newly excysted metacercariae and worms recovered from an abnormal host (rat) and the definitive host (dog) for some oxidoreductases, phosphatases and glycosidases. The results demonstrate that rat worms have enzymatic distribution and intensities more similar to those of metacercariae than to adult worms from dogs. Ultracytochemical examination of acid and alkaline phosphatase and Mg-ATPase activity was also carried out. Acid phosphatase activity occurred exceptionally in the excretory bladder and caeca of dog worms. No activity was observed in rat worms except for lysosomal granules in the tegument. Alkaline phosphatase activity was exhibited in the excretory bladder in both dog and rat worms. Mg-ATPase activity occurred in the tegument and parenchymal cells in dog worms and in the excretory bladder in rat worms. In metacercariae, little or no reaction for these enzymes was present except for Mg-ATPase activity on the excretory ducts. These observations, together with the histochemical results, indicate that metabolic activity in rat worms is higher than in metacercariae although it is strongly reduced compared with dog worms.     

Encystment and metacercariae development of Echinostoma cinetorchis cercariae in an in vitro culture system

For some species of 37-collar-spined Echinostoma, their cercariae successfully encyst and develop to metacercariae in vitro. In our study, we cultured Echinostoma cinetorchis cercariae in 12 different media to study the formation of metacercariae. Locke's solution, medium 199, and RPMI 1640 were used as media for culture. RPMI 1640 produced the highest encystment and normal metacercariae development. The osmolality of the media was related to their ability to encyst and develop. The 0.5 X media induced higher encystment and normal metacercaria formation than the 1x media. The addition of fetal bovine serum to RPMI 1640 increased the level of encystment and normal metacercariae development. In the mixture of 0.5 x RPMI 1640 and 10% fetal bovine serum, encystment was highest, at 96.0%, and the development ratio of normal metacercariae was also the highest, at 91.5%, after 48 hr of cultivation. In a viability test, 7 day- and 14 day-cultured metacercariae were successfully matured to adult worms in experimentally infected rats. These results showed that E. cinetorchis cercariae could be cultured to viable metacercariae in an in vitro culture system and that 0.5 x RPMI 1640 plus 10% fetal bovine serum was the most useful medium for cultivation. This culture system can be adapted for additional studies on the E. cinetorchis life cycle, especially to supply large numbers of metacercariae for other studies on this echinostome.     

Studies on the excystment of trematode metacercariae in vitro.

Excystment of metacercariae of Parorchis acanthus, Posthodiplostomoides leonensis, Posthodiplostomum sp. and Clinostomum tilapiae was found to be an active process initiated by the metacercariae when the correct physico-chemical conditions exist in the environment; but in the excystment of Posthodiplostomum nanum the metacercaria seems not to take part in the process. Temperature, osmotic pressure, pH and eH are factors which influence all the five species studied. Fresh ox bile was found not to be an absolute necessity for the excystment of the five species studied but its presence in the medium increased the rate of excystment of three species.     

The epidemiology of the metacercariae of Diplostomum baeri and D. spathaceum in perch (Perca fluviatilis) from the warm water effluent of a nuclear power station

Thermal effects on the dynamics of infection with metacercariae of Diplostomum baeri and D. spathaceum were monitored between May and September, in a year-class of perch Perca fluviatilis, in an artificial lake receiving warm water discharges from a nuclear power station and in an unheated reference site, for a two year period. In the heated area the prevalence of infection of the retinal form, D. baeri, was always 100%, whereas in the unheated site there was an increase from 93% in May to 100% in June 1986. The relative density of D. baeri was found to increase gradually during the first summer in both areas, although the accumulation rate of metacercariae was significantly increased in the heated area. In August of the first year the relative density of D. baeri peaked in the heated area, whereas it continued to increase in the unheated control. However, in September of the following year, the relative densities were at the same level in both thermal regimes. A concomitant decline in the degree of overdispersion of metacercariae within the host population was observed in the heated area as the population density of metacercariae decreased, whereas the index of dispersion remained at the same level throughout the study in the unheated area. However, it was not possible to sample perch in the unheated area between October 1986 and May 1987 and changes in the parasite population could have occurred during this period. The prevalence and relative density of D. spathaceum, the lens form, was on the contrary low, especially in the unheated site where it was recorded only occasionally. The infection of D. baeri exhibited a convex pattern in both thermal regimens, although the peak infection was noticed earlier in the heated area. Regulation of the parasite infrapopulation may have been achieved by the combined effects of a decreased transmission rate of cercariae with increasing age of the host, the mortality of metacercariae as a natural termination of the life span and to selective predation of heavily infected hosts. However, regardless of an increased accumulation rate of metacercariae in the heated area, the relative densities of D. baeri became equal in both thermal regimes at the end of the study. The mortality rate of metacercariae in the heated area was therefore presumably increased as compared with the unheated reference site.     

Two species of goby, Boleophthalmus pectinirostris and Scartelaos sp., as the new second intermediate hosts of heterophyid fluke in Korea

A survey was performed in order to determine the infection status of the metacercariae of heterophyid fluke in two goby species, Boleophthalmus pectinirostris and Scartelaos sp., collected from Gangjin-gun, and Shinangun, Sooncheon-shi, Jeollanam-do, Republic of Korea. A total of three metacercariae of Heterophyopsis continua was found in only one B. pectinirostris (10.0%) from Gangjin-gun. Heterophyes nocens metacercariae were detected in 24 B. pectinirostris (96.0%) and 14 Scartelaos sp. (63.6%) from Shinan-gun. Heterophyopsis continua metacercariae were found in 11 B. pectinirostris (44.0%) and 21 Scartelaos sp. (95.5%) from Shinan-gun. Stictodora fuscata metacercariae were detected in 18 B. pectinirostris (72.0%) from Shinan-gun. No metacercariae were detected in 20 B. pectinirostris from Sooncheon-shi. From the above results, this study is the first to prove that B. pectinirostris and Scartelaos sp. serve as the second intermediate hosts of some heterophyid flukes in Korea.     

Rapid hypotonic method for flow cytofluorometry of monolayer cell cultures. Some pitfalls in staining and data analysis.

The rapid hypotonic staining procedure developed by Krishan for DNA determinations by flow cytofluorometry has been proven accurate for in vivo cell samples and for cell lines growing in suspension culture. We show that the unmodified procedure may produce distorted DNA histograms when used for staining cells growing in monolayer cultures, however. To eliminate these distortions, it was necessary to avoid the use of trypsin by staining the attached cells directly, using a hypotonic fluorochrome solution to which nonionic detergent was added. Two sublines of HeLa S3 cells are shown to exhibit major differences in their staining characteristics. By using our revised staining procedure, the two sublines appear to produce very satisfactory DNA histograms. However, in only one subline does the S phase fraction calculated from the histograms agree with the autoradiographical labeling index. Mitotic cells remain intact under these staining conditions, and the principal observed effect of nonionic detergents in this case is to decrease the coefficient of variation of fluorescence intensity.