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The liver fluke, Fasciola hepatica , is an obligate blood-feeder, and the gastrodermal cells of the parasite form the interface with the host's blood. Despite their importance in the host-parasite interaction, in-depth proteomic analysis of the gastrodermal cells is lacking. Here, we used laser microdissection of F. hepatica tissue sections to generate unique and biologically exclusive tissue fractions of the gastrodermal cells and tegument for analysis by mass spectrometry. A total of 226 gastrodermal cell proteins were identified, with proteases that degrade haemoglobin being the most abundant. Other detected proteins included those such as proton pumps and anticoagulants which maintain a microenvironment that facilitates digestion. By comparing the gastrodermal cell proteome and the 102 proteins identified in the laser microdissected tegument with previously published tegument proteomic datasets, we showed that one-quarter of proteins (removed by freeze-thaw extraction) or one-third of proteins (removed by detergent extraction) previously identified as tegumental were instead derived from the gastrodermal cells. Comparative analysis of the laser microdissected gastrodermal cells, tegument, and F. hepatica secretome revealed that the gastrodermal cells are the principal source of secreted proteins, as well as showed that both the gastrodermal cells and the tegument are likely to release subpopulations of extracellular vesicles (EVs). Microscopical examination of the gut caeca from flukes fixed immediately after their removal from the host bile ducts showed that selected gastrodermal cells underwent a progressive thinning of the apical plasma membrane which ruptured to release secretory vesicles en masse into the gut lumen. Our findings suggest that gut-derived EVs are released via a novel atypical secretory route and highlight the importance of the gastrodermal cells in nutrient acquisition and possible immunomodulation by the parasite.

Cytochemical staining techniques were carried out en bloc with in vitro excysted and gut-penetrated Fasciola gigantica larvae in order to visualise the glycocalyx of the tegument, a structure which comprises the parasite component of the host-parasite interface, yet is incompletely preserved by conventional fixation and preparation techniques for electron microscopy. Positive reactivity with ruthenium red and periodic acid-thiocarbohydrazine-osmium (PATCO) techniques revealed that the glycocalyx is polyanionic and carbohydrate-rich throughout its depth. It comprises a trilaminate arrangement, with a thin dense zone and fibrillar layer closely apposed to the outer aspect of the apical plasma membrane, invested by an irregular thick mucopolysaccharide capsule. The latter, not recorded in adult flukes, may represent a specific adaptation to facilitate invasion in the face of host immunity, and may also protect the parasite surface from the action of host- and parasite-derived proteases. Early in the invasion of a naïve host, the glycocalyx may be partly responsible for triggering the responses of innate immunity, while later in infection, or when an anamnestic response is initiated in an immunocompetent host, the antibodies and activated lymphocytes of specific acquired immunity are invoked to interact with the parasite surface. The cytochemical properties of the glycocalyx, together with its potential for dynamic turnover due to exocytosis of the T0 tegumental secretory bodies, are likely to aid neutralisation of potentially damaging immune effectors and ensure their removal from the vicinity of the parasite by sloughing in complex with glycocalyx components., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The effects of glucose, yeast extract, fetal bovine serum albumin, and ruthenium red on endocytosis of smooth micropinocytotic vesicles (pinosomes) in the tegument of the cysticercus of Taenia crassiceps have been investigated stereologically. Glucose has been shown to stimulate pinocytosis, whether it was used alone or in combination with yeast extract or bovine serum albumin. Yeast extract was a stimulant of endocytosis. Bovine serum albumin was the most potent stimulant of all the substances investigated in this study. Although the time of incubation in ruthenium red was the same for all incubation experiments, varied numbers of ruthenium red-containing pinosomes were observed in different experiments. The role of ruthenium red as a stimulant and/or initiator of endocytosis and the possible explanations for differences in ruthenium red uptake are discussed.

The fine structure of the nervous system in the scolex and neck region of Hymenolepis nana has been investigated by transmission electron microscopy. A description of the gross neuroanatomy in these regions of the worm is presented. The ganglia, commissures and nerve cords consist of an incomplete cortex of nerve cell bodies, and a core of nerve fibres. A delimiting sheath or capsule is absent. The nerve cell bodies contain a single nucleus with a single nucleolus, mitochondria, many ribosomes, Golgi complexes and vesicles formed within the Golgi cisternae. Numerous sub-surface cisternae are present beneath the outer plasma membrane of the nerve cell body, and the inner surfaces of these cisternae are studded with ribosomes. Some of the cisternae run tangentially into the cytoplasm of the perikaryon, particularly in the vicinity of the Golgi complexes; both sides of these cisternae are studded with ribosomes. From each neuronal perikaryon arise one or more neurites that contain neurotubules, mitochondria, ribosomes and electron-lucent or dense-cored vesicles. Five types of vesicle have been distinguished on the basis of their size and content. The neurites are unmyelinated and form synapses in the neuropile; the synapses possess features typical of those where mechanical strength is of importance. Three types of sensory receptors have been observed in H. nana, two ciliated and one non-ciliated; the latter typically form double or triple nerve endings which terminate within the tegument.

Regional variations in the thickness of the tegument, in the morphology of microtriches and mitochondria, in the distribution of dense bodies, smooth micropinocytotic vesicles (SMVs), and coated micropinocytotic vesicles (CMVs) have been shown for the cysticercus of Taenia crassiceps. The number of SMVs and CMVs present in the syncytial layer are in inverse proportion to each other, the former being more numerous in the bladder wall and upper part of the invagination canal and the latter in the lower part of the canal and the rostellar region. Tegumental cells contain numerous granular endoplasmic reticulum-Golgi complexes involved in the synthesis of both primary lysosomes and dense bodies. Vesicles characteristic of various stages of heterolysosomes are present and show regional variations in numbers and size. Acid phosphatase activity (EC 3.1.3.2) is present on the tegumental surface, and within the Golgi complex, primary lysosomes, and heterophagosomes of the tegumental cells. CMVs are reported for the first time in the tegument of any helminth and have characteristics similar to CMVs in other tissues. T. crassiceps, therefore, because of the presence of both SMVs and CMVs, is a unique model system for the study of basic mechanisms of endocytosis.

The fine structure of the oncosphere of Hymenolepis nana has been investigated by transmission and scanning electron microscopy, together with light microscope observations of JB-4 embedded material. The outer surface of the oncosphere is covered by an epithelial layer, termed the embryonic epithelium. Cell types present within the oncosphere include the penetration gland cell, oncoblast, or hook-forming cells, nerve cells, muscle cells (both somatic and hook), and undifferentiated 'stem' cells. The penetration gland is a large, U-shaped structure, situated in the anterior region of the oncosphere, and filled with secretory granules of 2 distinct morphological types. Histochemically, the secretory material yields reactions characteristic of an acid mucopolysaccharide. A proteinaceous-substance and small amounts of glycogen are also present. Up to 4 pairs of ducts from the penetration gland have been observed. They pass through the basal lamina and the epithelial layer to open against the polar filament layer at the anterior end of the oncosphere. Nerve cells are described in a cestode oncosphere for the first time. The cells are paraldehyde-fuchsin-positive and show a high level of secretory activity, as evidenced by the large numbers of dense-cored vesicles produced by the Golgi apparatus in the perikarya; consequently, they are tentatively regarded as possible neurosecretory cells. The vesicles are transported down the axon to be stored in specialized swollen axon terminals, which form definite junctions with the muscle cells.

Adult flukes, Fasciola hepatica, incubated in Hedon - Fleig saline containing concanavalin A (Con A) for 10 and 45 min, respectively, exhibited severe alterations to tegumental morphology involving increased secretory activity, blebbing of the apical plasma membrane, increased total surface area, and swelling of the basal infolds . The effects of Con A were prevented by the addition of alpha-methyl-D-mannoside to the incubating medium. Similar, but less pronounced, effects were caused by wheat germ agglutinin (WGA) binding. Con A and WGA binding indicate the presence of mannose, glucosamine, or glucose moieties and of N-acetylglucosamine. The effects of lectin binding were similar to the early effects of antibody attachment, and it was considered that accelerated membrane turnover was occurring in both cases. Swelling of the basal infolds was thought to be a result of increased apical surface membrane and/or increased permeability due to lectin binding.

The effects of a wide range of fasciolicides on the in vitro motility of Fasciola hepatica have been determined by means of an isometric transducer system. Carbon tetrachloride and diamphenethide do not affect movement at concentrations up to 500 and 100 micrograms/ml, respectively; at 1000 micrograms/ml, however, carbon tetrachloride induces a rapid tonic paralysis. Brotianide and the deacetylated metabolite of diamphenethide cause a rapid flaccid paralysis of the fluke at concentrations of 1.0 micrograms/ml and above. In contrast, the effect of MK-401 is a long-term one, a flaccid paralysis occurring after 20 hr only at 200 micrograms/ml. Praziquantel also produces a flaccid paralysis of the fluke, but this follows an initial increase, then decrease in muscle tone. The effect is rapid at 500 micrograms/ml, but long-term at 100 and 200 micrograms/ml; at these lower concentrations there is also a stimulation of activity. Oxyclozanide , rafoxanide, niclofolan , bithionol, and hexacholorophene induce a rapid spastic paralysis of the fluke at concentrations of 1.0 micrograms/ml and above. Both phasic and tonic components are evident in the response at concentrations of 1.0 micrograms/ml and below; the phasic component disappears at higher concentrations. Nitroxynil produces a similar effect, evident at higher concentrations. Among the benzimidazoles, mebendazole, oxfendazole, and albendazole sulphoxide cause a suppression of motility, whilst thiabendazole and albendazole produce a stimulation of movement. The effects are not rapid, however, for only mebendazole at 500 micrograms/ml causes total inactivity of the fluke within a 12-hr period. Possible explanations for these effects on fluke motility are discussed.

The fine structure of the envelopes surrounding hatched and unhatched oncospheres of Hymenolepis nana has been investigated by transmission and scanning electron microscopy (SEM), together with light microscope histochemical observations of JB-4 embedded material. The oncosphere is surrounded by 3 layers--the capsule, the outer envelope and the inner envelope, the latter giving rise to the embryophore and the 'oncospheral membrane'. An additional layer--the polar filament layer--lies between the 'oncospheral membrane' and the oncosphere. Shell material is deposited on the capsule as a thin layer. It is secreted by the outer envelope, which degenerates once shell formation is complete. The uterus may also contribute to shell formation. The embryophore forms a thin incomplete and peripheral layer within the inner envelope. In the basal region of this envelope, partial development of an 'oncospheral membrane' takes place, but it does not become detached as a separate layer. The polar filaments, which are characteristic of the oncosphere of H. nana, are derived from the epithelial covering of the oncosphere itself, which delaminates to form a separate polar filament layer. The filaments arise from knob-like projections at opposite poles of this layer. The design of the embryonic envelopes in H. nana show a number of modifications from the basic cyclophyllidean pattern, and these can be related to the demands of its 'direct' life-cycle.

Opaque or darkened areas (DA) of variable size and position occur on Hymenolepis diminuta in mice and rats. In mice DA normally first appear in the neck region of the worm but subsequently they appear elsewhere and increase in number until destrobilation or worm expulsion. The posterior of destrobilated worms is often darkened. In the more immunogenic infections with six cysticercoids there are more DA per worm than in infections with one cysticercoid. DA are areas of the tegument with a homogeneous increase in electron density; abnormal mitochondria; reduced granular endoplasmic reticulum, Golgi complexes and discoidal secretory bodies; and accumulation of lipid droplets. DA disappear from worms maintained for up to 4 h in Hanks' balanced salt solution and can be induced by mechanical damage to the worms. As the numbers of DA increase with the duration and intensity of infection and have similarities with types of cell injury, they are probably sites of worm pathology induced by host immunity.

The effect of various temperatures, poly-L-lysine, and poly-L-glutamic acid on endocytosis of smooth micropinocytotic vesicles (pinosomes) in the tegument of the cysticercus of Taenia crassiceps has been investigated stereologically. The temperature regimes used were 0, 5, 10, 20, 30, and 40 C. Maximum volume, surface density, and number per unit volume were found at 40 C, and minimum surface-to-volume ratio and numbers at 10 C. At 10 C, mean pinosome volume and mean surface area per pinosome were maximal, but volume and surface density did not differ significantly from 40 C. It is proposed that this anomalous finding for 10 C incubations was due to this being a critical temperature at which a slower rate of pinosome formation was compensated for by the formation of larger individual pinosomes. Poly-L-lysine was shown to be a stimulant of pinosome formation, leading to a significant increase in numbers per unit volume. However, volume and surface density, surface-to-volume ratio, mean volume, and mean surface area per pinosome were not significantly different in poly-L-lysine-incubated samples, when compared to controls (fresh from the mouse) or incubations in medium only or samples returned to medium after poly-L-lysine incubation, the only exception being surface to volume ratio and mean volume of pinosomes in the 75-min incubation. These anomalous results were explained by a marked reduction in the form ellipse values, which indicated the production of more elliptical-shaped pinosomes under poly-L-lysine stimulation. Incubation in poly-L-glutamine acid did not have any significant effect at any incubation time.

The ultrastructure of the seminal vesicle, ejaculatory duct, cirrus sac and cirrus is described. The epithelium of the seminal vesicle consists of a single layer of squamous to cuboidal cells. The apical ends of the cells have thin polymorphic lamellae and long narrow pits, both of which enclose normal spermatozoa. The cells have a moderate amount of GER and Golgi complexes which produce a lucid secretory body. The ejaculatory duct epithelium is composed of cuboidal to columnar cells between or through which project the terminal parts of the ducts of the unicellular prostate glands. The apical surfaces of the epithelia are extended into triangular or filiform projections having thin sinuous lamellae. The cytoplasm contains GER cisternae and Golgi complexes which synthesize a dense ovoid secretion. The cirrus sac and cirrus are covered by a thin modified tegument. The cirrus has many spines and the normal ratio of T1 and T2 type of secretory bodies, whereas the cirrus sac has few spines and the T2 type of secretory body predominates over the T1 type. The significance and possible functions of the structures observed in the three tissues are discussed.

The tegument of Fasciola hepatica was treated with 0.5% pepsin (EC 3.4.4.1), 0.5% alpha-amylase (EC 3.2.1.1), and 0.5% sheep bile solutions both without and following preincubation in poly-L-lysine. Without poly-L-lysine preincubation, pepsin appeared to be breaking down limited areas of the tegumental surface but had no other marked effects on tegumental structure. alpha-Amylase and bile had no major effects on the tegument except for a reduction in matrix density by the latter. Incubation in poly-L-lysine alone resulted in some changes in surface morphology of the tegument and a limited amount of swelling of the basal infolds. When poly-L-lysine was followed by pepsin treatment, blebbing, microvillus-formation, and swelling of the basal infolds was greatly enhanced and led to surface destruction in some areas. alpha-Amylase following poly-L-lysine resulted in complete destruction and loss of the tegument, and left the basal lamina as the external surface, Incubation in bile after poly-L-lysine preincubation resulted in little change in tegumental morphology.

The effect of the deacetylated (amine) metabolite of diamphenethide (10 mugm/ml) on the tegument of Fasciola hepatica over a 24 hr period in vitro has been determined by means of transmission electron microscopy. In the tegumental syncytium, there is an initial accumulation of T2 secretory bodies at the apical surface (after 6 hr), together with increased exocytosis of secretory bodies and blebbing of the surface membrane. After 9 hr, the two surfaces of the fluke show different tegumental responses to drug treatment with a marked swelling of the basal infolds in the dorsal tegument, while the ventral tegument remains normal. By 18 hr, the swelling in the dorsal tegument is very severe, the entire basal region becoming edematous. In some areas, the tegument becomes detached to expose the basal lamina. The ventral tegument retains a fairly normal morphology, although there is a slight swelling of the basal infolds. The edema spreads internally to the cell bodies, beginning after 9 hr on the dorsal side of the fluke and 18 hr on the ventral side. By 18 hr, the flooding on the dorsal side is very severe and the cells attenuated, retaining few contacts with the surrounding parenchyma. From 9 hr onwards, there are progressive changes in cell structure, including a decrease in amount of granular endoplasmic reticulum and extent of its ribosomal covering, a decrease in numbers of secretory bodies, a swelling of the trans-most Golgi cisternae and disruption of the release of secretory bodies, and a swelling and disorganization of the mitochondria. The results are discussed in relation to the postulated activity of the deacetylated (amine) metabolite of diamphenethide as a Na+ ionophore.

An apparatus utilizing a force and displacement transducer is described for the direct and long-term recording of the motility in vitro of Fasciola hepatica. Normal movement is typically rhythmical, with bursts of more powerful contractions alternating with periods of lesser activity. Such rhythms and the overall level of activity are maintained for more than 30 hr. The fluke remains active for much longer periods of time: recordings of fluke movements have been made for up to 4 days. Potential damage to the fluke caused by the attachment system within the recording apparatus has been determined by the Evans' Blue Technique and scanning electron microscopy. It is restricted to the attachment sites, and does not spread to other parts of the body over the 30-hr normal activity period. Transmission electron microscope studies have shown that the tegument retains its structural and functional integrity over this period of time. There are advantages of the recording apparatus over previous kymographic methods for studying fluke motility.