Histochemistry and ultrastructure of the metacercarial cysts of blackspot trematodes Uvulifer ambloplitis and Neascus pyriformis.

Cysts of Uvulifer ambloplitis from green sunfish, Lepomis cyanellus, and Neascus pyriformis from red shiners, Notropis lutrensis, were studied with light-level histochemistry and scanning and transmission electron microscopy. Cysts of both species are bilayered, consisting of an outer host capsule and an inner parasite cyst; the space between these layers is filled with a viscous material. The outer portion of the host capsule of both species is composed of fibrocytes, melanin granules, and collagen fibrils, and the inner portion of layers of flattened fibrocytes. The parasite cyst of U. ambloplitis is formed of 2 layers, an outer dense layer and an inner light layer, whereas the parasite cyst of N. pyriformis is made of 3 layers. A thin outer light-staining layer is present in addition to the 2 layers observed in U. ambloplitis. Results of histochemical staining were the same for both species. The host capsule stained positively for proteins and neutral and acid mucopolysaccharides. The viscous material was positive for neutral and acid mucopolysaccharides but not for proteins. The parasite cyst gave a strong positive reaction for neutral mucopolysaccharides but was negative for acid mucopolysaccharides and proteins.

A comparative study of the protein content of some helminths and the suitability of assay methods.

The protein content of fresh homogenates and their corresponding TCA precipitated fractions of 10 different species of helminths was estimated by the methods of Lowry et al. and Spector using the Folin phenol reagent and Coomassie brilliant blue G-250 respectively. The former method gives exaggerated values as compared to the latter method. The parasite phenols, phenolic proteins and catecholamines could be responsible for interference in the Lowry's procedure. The TCA noln-precipitable moieties also give colour only with the Folin phenol assay. The pronounced intra-specific differences in the total protein content of helminths reflect their metabolic variations and adaptations. Habitat does not appear to influence the protein content of parasites, however, the effect of host variation was evident in the pouched amphistome G. crumenifer. It is concluded that the dye binding method gives more consistent results and it can be conveniently applied to crude tissue homogenates of helminths.

The serotoninergic, cholinergic and peptidergic components of the nervous system in the monogenean parasite, Diclidophora merlangi: a cytochemical study.

Confocal scanning laser microscopy has been employed with immunocytochemical techniques to map the distribution of serotoninergic and peptidergic components in the nervous system of the monogenean gill-parasite, Diclidophora merlangi; results are compared with the distribution of cholinergic components, following histochemical staining for cholinesterase activity. While all three neurochemical elements are present in the central and peripheral nervous systems, the cholinergic and peptidergic systems dominate the CNS, whereas the PNS has a majority of serotoninergic nerve fibres. The cholinergic and peptidergic neuronal pathways overlap extensively in staining patterns, suggesting possible co-localization of acetylcholine and neuropeptides. Within the peptidergic nervous system, immunoreactivity to the pancreatic polypeptide family of peptides and FMRFamide were the most prevalent. Gastrin/cholecystokinin (CCK)-, neuropeptide Y-, substance P-, neurokinin A- and eledoisin-like immunoreactivities have been demonstrated for the first time in a monogenean parasite. The gastrin/CCK- and tachykinin-like immunoreactivities had an apparently restricted distribution in the worm.

Mammalian regulatory peptide immunoreactivity in the trematode parasite Haplometra cylindracea and the lung of its frog host, Rana temporaria: comparative chromatographic characterisation using reverse-phase high-performance liquid chromatography.

1. Extracts of Haplometra cylindracea and lung tissues of its host, Rana temporaria, were subjected to radioimmunoassay using antisera to nine mammalian regulatory peptides. 2. In these extracts, immunoreactivity was measured to pancreatic polypeptide, substance P, neurokinin A, gastrin-releasing peptide and glucagon. The levels of each peptide varied considerably with some marked differences between those demonstrable in parasite and host (notably pancreatic polypeptide). 3. Reverse-phase HPLC fractionation of extracts revealed general chemical differences between parasite and host peptides, with some peptides present in more than one molecular form.

Occurrence and distribution of putative neurotransmitters in the frog-lung parasite Haplometra cylindracea (Trematoda: Digenea).

The localisation and distribution of the cholinergic, serotoninergic and peptidergic components of the nervous system of the frog-lung fluke Haplometra cylindracea have been determined by the application of standard enzyme cytochemical and immunocytochemical techniques to cryostat sections and whole-mount preparations. Cholinesterase activity (ChE), as indicative of acetylcholine, has been demonstrated cytochemically in the CNS and PNS; however, the anterior ganglia were notably unreactive. The occurrence of serotonin was examined by an indirect immunofluorescence technique, and immunoreactivity (IR) was demonstrable in small, paired anterior ganglia and in fine nerve fibres associated with the somatic muscle, cirrus and gonopore. The peptidergic portion of the nervous system was investigated using antisera to 17 mammalian regulatory peptides and the invertebrate peptide FMRFamide, and was visualised by both indirect immunofluorescence and confocal scanning laser microscopy. Positive immunostaining occurred with antisera raised against pancreatic polypeptide (PP), peptide tyrosine tyrosine (PYY), substance P (SP), peptide histidine isoleucine (PHI) and FMRFamide. Immunoreactivity to PP, PYY and FMRFamide was widespread throughout the nervous system and was evident in large, paired anterior ganglia, the dorsal commissure, main nerve tracts and the extensive array of small fibres that constitute the PNS. In contrast, the distribution of nerves immunoreactive to SP and PHI was less apparent, with PHI-IR occurring exclusively within the fibrous neuropile of the ganglia and in fibres of the ventral nerve cord. Results are discussed with respect to the distribution of the various neurochemical elements and their roles as putative neurotransmitters and/or regulatory molecules.

Distribution of zinc in parasitic helminths.

The distribution of zinc in representative groups of parasitic helminths was determined by the use of the atomic absorption spectrophotometer. The results of these analyses have shown that growing flukes (smaller forms) with active oogenesis and spermatogenesis contained more zinc than old (large) or very old adults with an empty uterus and large lobulated testes. In cestodes, the neck region and immature proglottids showed more zinc concentration than mature and gravid proglottids and fully grown cyst walls. Similarly, the youngest endogenous daughter cysts of Echinococcus granulosus contained more zinc in their walls than those of larger/older forms. Zinc was concentrated more in nematode eggs than in adult females.

Immunocytochemical demonstration of neuropeptides in the fish-gill parasite, Diclidophora merlangi (Monogenoidea).

Using the indirect immunofluorescence technique, immunoreactivity (IR) to three mammalian and one invertebrate regulatory peptide has been demonstrated in the nervous system of the monogenean gill parasite Diclidophora merlangi. IR to pancreatic polypeptide (PP), peptide tyrosine tyrosine (PYY) and FMRFamide was evident throughout central and peripheral nervous tissues, whereas vasoactive intestinal polypeptide (VIP)-IR was confined to a portion of the longitudinal ventral nerve cords. Staining patterns revealed the orthogonal arrangement of the nervous system consisting of paired cerebral ganglia, connecting post-pharyngeal commissure, three pairs of longitudinal nerve cords and associated neurones. PP-IR, PYY-IR and FMRFamide-IR were intense throughout the central nervous system of the worm. A small plexus of nerve fibres and somata in each peduncle was immunoreactive for FMRFamide and provided innervation to each of the eight posterior clamps. In the peripheral nervous system, PP-IR, PYY-IR and FMRFamide-IR occurred in an extensive nerve-net with fine, possibly sensory nerve endings in the tegument. PP-IR was also present in nerve fibres in the walls of the ootype, seminal vesicle and uterus. PYY- and FMRFamide-IRs, while evident in nerve fibres of the ootype wall, were also present in a distinct population of cells that encircles the ootype, and which are linked to it by fine cytoplasmic connectives. The majority of these somata were bipolar or multipolar. PYY-IR and FMRFamide-IR were also associated with nerve fibres and bipolar cells in the wall of the vitelline reservoir. Regulatory peptides would appear to play an integral role in neuronal functioning and egg development in D. merlangi.

[Serotonin and dopamine in the tissue extracts of Polystoma integerrimum (Monogenea)]. / Serotonin i dofamin v ékstraktakh iz tkanei Polystoma integerrimum (Monogenea).

Spectrofluorometric analysis has revealed substances with spectral characteristics identical to those of serotonin and dopamine in tissue extracts of Polystoma integerrimum. A possible function of the substances is discussed.

Immunocytochemical localization of regulatory peptides in six species of trematode parasites.

1. Frozen and paraffin sections of six species of trematodes: Schistosoma mansoni, S. mattheei, S. japonicum, Schistosomatium douthitti, Echinostoma paraensei and Fasciola hepatica have been incubated with antisera against leu-enkephalin, FMRF-amide, gastrin-17, luteinizing hormone releasing hormone, neurotensin, oxytocin, prolactin, substance P, thyroid stimulating hormone and cholecystokinin, using indirect immunofluorescence and biotin-avidin horseradish peroxidase detection systems. 2. Of the ten antisera tested, six (leu-enkephalin, FMRF-amide, gastrin-17, luteinizing hormone releasing hormone, substance P and cholecystokinin) showed significant immunoreactivity, primarily in the central and peripheral nervous system, and also perhaps in the osmoregulatory system of the three species of Schistosoma. 3. Immunopositive nerve fibers extended from ganglia to gut wall, uterus and vitelline follicles, and especially from subtegumental nerve plexi to sensory receptors on the surface or in dorsal nippled tubercles.

Demonstration of calcium salts in excretory corpuscles of larval stages of trematodes.

Calcium salts were demonstrated by Kóssa's method in excretory corpuscles of five species of cercariae and six species of metacercariae. The interference of calcium salts in excretory corpuscles in Gomori's method for the detection of alkaline phosphatase is discussed.

Characterization of sterols of three digenetic trematodes of buffalo.

Sterols of three digenetic trematodes were isolated and characterized by infrared and 1H nuclear magnetic resonance spectroscopies, and gas-liquid chromatography and gas chromatography-mass spectrometry. Sterols identified were cholesterol, cholestanol, 24-methylcholesterol, 24-methylcholestanol, 24-ethyl-22-dehydrocholesterol, 24-ethyl-22-dehydrocholestanol, 24-ethylcholesterol and 24-ethylcholestanol.

Preliminary studies of lipids of the trematodes Eurytrema pancreaticum, Calicophoron erschowi and the turbellarian Penecurva sibirica.

The amount of total lipids, phospholipids, neutral lipids and fatty acids was determined in cattle parasites, namely the trematodes Eurytrema pancreaticum, Calicophoron erschowi, and in the free-living turbellarian Penecurva sibirica. Neutral lipids of these flatworms contained sterols, sterol esters, triacylglycerols and free fatty acids. P. sibirica also contained diacylglycerols. Parasitic and free-living flatworms differed in phospholipid composition: the turbellarian did not contain phosphatidylserine, and the trematodes had practically no sphingomyelin or lysophosphatidyl choline. E. pancreaticum, C. erschowi and P. sibirica contained high levels of phosphatidylcholine and phosphatidylethanolamine as well as lysophosphatidylethanolamine and cardiolipin. The fatty acid composition of total lipid extracts of flatworms and of the pancreas and rumen of the host animals were determined. The fatty acid composition of the flatworm lipids reflected the fatty acid composition of the host tissue but was not identical to it.

Cytochemical characteristics of the neurosecretory cells of Ceylonocotyle scoliocoelium (Trematoda: Digenea).

Two types of neurosecretory cells (NSC), large alpha cells and small beta cells were detected on the basis of their size and characteristics as shown by aldehyde fuchsin and chrome haematoxylin-phloxin staining. A histochemical survey of the neurosecretory material indicates that it is a glycoprotein. Phosphatases and esterases present in NSC probably help in the transfer of metabolites and their hydrolysis and in the control of secretory material produced in a NSC, while SDH provides the necessary energy for the synthetic activity of the latter. The role of neurosecretion in governing the reproduction of this parasite is discussed.

Cytochemical studies on the prostate glands of the trematodes, Paramphistomum epiclitum and Paradistomoides orientalis.

Pear-shaped prostatic gland cells which surround the ejaculatory duct of Paramphistomum and Paradistomoides, secrete two types of products: (i) granules which are cytochemically complex containing lipids, proteins and polysaccharides (1:2, glycol groups) and (ii) globules which contain phospholipids only. The cytoplasm of these cells, in addition to high acid phosphatase activity, also contains RNA, proteins and polysaccharides.

Study of phosphagens in two trematodes.

Extracts from the adults of two trematodes Fasciola gigantica and Paramphistomum explanatum have been shown to contain three phosphagens, phosphoarginine, phosphocreatine, and phosphoglycocyamine. The three phosphokinases involved in the transphosphorylation of these phosphagens in both worms were inhibited by EDTA and mercuric ions while iodoacetate had no effect.

Comparison of carbon and nitrogen content of infected and uninfected snails, Succinea ovalis, and the trematode Leucochloridium variae.

In June, 6.7% of adult Succinea ovalis collected near Urbana, Ohio, were infected with the trematode, Leucochloridium variae. The effects of parasitism were assessed as total organic carbon (equivalent to calorific values) and as total nitrogen. The parasite represents 23.8% of total (parasite + snail tissue) dry tissue weight, 21.4% of total carbon and 19.8% of total nitrogen of infected snails. The higher C : N ratio for parasite tissue indicates a higher proportion of nonproteinaceous compounds (e.g., fats and/or carbohydrates) as compared to host tissue. There is less snail tissue in parasited S. ovalis. The C : N ratios for parasitized and nonparasitized snail tissue suggest identical percentage compositions of proteins, fats, and carbohydrates.

Identification of helminth species by means of disc electrophoresis.

Soluble proteins of 25 helminth species of the classes Trematoda, Cestoidea and Nematoda, were separated by disc electrophoresis using polyacrylamide gel columns. Differences between the species were investigated on the basis of Rm values of the bands. Protein spectra were complemented by the detection of lipoproteins and glycoproteins and by identification of LDH, SHD, peroxidase, esterase and alkaline phosphatase. On the basis of comparison of protein spectra of parasitic worms belonging to three taxonomic classes it was found by means of numerical taxonomy that individual classes are characterized by a certain number of proteins of the same migration properties.