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1. Extracts of the liver fluke, Fasciola hepatica from three different hosts (cow, sheep, rat) have been subjected to radioimmunoassay using antisera to 6 mammalian regulatory peptides. 2. Immunoreactivity was measured to pancreatic polypeptide, substance P, peptide histidine isoleucine and gastrin-releasing peptide. Levels of each peptide varied considerably in flukes from different hosts. 3. Reverse-phase HPLC of rat and sheep fluke extracts revealed three molecular forms of tachykinin immunoreactivity and single peaks of pancreatic polypeptide and peptide histidine isoleucine immunoreactivity. No GRP-immunoreactivity was detected by RIA of HPLC fractions.

Both the liver fluke Fasciola hepatica and the blood fluke Schistosoma mansoni have GTP-binding proteins which are part of the trans-membrane signalling system. These proteins require GTP in order to interact with the catalytic subunit of adenylate cyclase. The technique of immunoblotting was used in order to distinguish the GTP-binding proteins Gs, Gi, and Go. Immunoblotting was carried out using antisera prepared against peptides deduced from bovine cDNA clones specific for alpha or beta subunits of known G proteins. A 41-kDa Gs alpha has been identified in S. mansoni and a 42.5-kDa Gs alpha in F. hepatica. A 41-kDa Go alpha was found in both parasites. A 36-kDa G beta was identified in both parasites using antiserum made against bovine transducin.

Trematode parasites protect their eggs with a tough tanned eggshell. Eggshell precursor proteins are synthesized and stockpiled within the extensive vitellaria of the animal. A major eggshell precursor protein with an apparent molecular weight of 31,000 and pI of 7.4 was isolated from the vitellaria of Fasciola hepatica. This protein, which represents 6-7% of the total protein in mature Fasciola, is unique in containing rather high levels of the amino acid 3,4-dihydroxyphenylalanine (DOPA), i.e., 110 residues per 1000. Other prominent amino acids are glycine, aspartic acid, and lysine. A prominent DOPA-containing tryptic peptide derived from eggshell precursor protein has the sequence Gly-Gly-Gly-DOPA-Gly-Gly-DOPA-Gly-Lys. DOPA residues disappear during the maturation of the eggshell and by treatment in vitro with mushroom polyphenol oxidase. This disappearance may be related to the formation of cross-links in the eggshell protein.

Lewis blood group specificities are detected in the liver Fluke: Fasciola hepatica. Such specificities had never been reported in nature, other than in the human body. The investigation of this phenomenon might contribute to a better understanding of the mechanisms involved in parasite-host relationship.

The vitellaria are an extensive network of glandular cells and ducts distributed throughout the peripheral tissues of the liver fluke Fasciola hepatica. Eggshell precursor proteins are produced and stockpiled in the vitelline cells of mature flukes. Vitelline protein C has an extraordinary composition: the amino acid 3,4-dihydroxyphenyl-L-alanine (DOPA) and histidine each comprise about 20% of the residues, while glycine represents 41-42% in all variants of what appears to be a microheterogeneous protein family. Protein C has an apparent molecular weight of 16,000-17,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Although the protein appears homogeneous following polyacrylamide gel electrophoresis in Tris-glycine with SDS and a acetic acid-urea, electrophoresis in borate, however, suggests that the vitelline protein consists of four or more closely related proteins weighing from 16,000 to 18,500. Isoelectric focusing of the protein family in the presence of 8 M urea resolved only two species having pI values of 6.89 and 6.99. A single N-terminus having the sequence H-H-W-D-G-DOPA-G-DOPA-G was detected. The primary structure of vitelline protein C is characterized by a repeated motif consisting of (G-X)n, where X is Ser, DOPA, or His. Most of the His occurs as G-H repeats in a pepsin-resistant fragment of the protein. Previously, a 31-kDa protein, representing up to 6% of the total protein in the fluke, was reported [Waite, J. H., & Rice-Ficht, A (1987) Biochemistry 26, 7819-7825] to contain significant levels of DOPA.(ABSTRACT TRUNCATED AT 250 WORDS)

The localisation and distribution of 5-hydroxytryptamine (5-HT or serotonin) in the nervous system of Fasciola hepatica has been determined by an indirect immunofluorescence technique. Cell bodies and nerve fibres immunoreactive to 5-HT are present in the anterior ganglia, and the longitudinal nerve cords and their commissures in the central nervous system. In the peripheral nervous system, similar immunoreactivity occurs in the nerve plexuses supplying the sub-tegumental muscle layers and the muscular lining of various reproductive ducts, including the ootype, uterus and cirrus pouch. The significance of these results in the light of previous studies on the role of 5-HT in F. hepatica is discussed.

Maturation of newly excysted Fasciola hepatica juvenile flukes in mice is accompanied by distinct changes in the pattern of radiolabelling of surface proteins. The major proteins identified on the surface of newly excysted juvenile flukes have apparent molecular weights of 78,000, 45,500, 30,000, 26,000, 13,500, 13,000 and 10,500 as measured by sodium dodecylsulphate polyacrylamide gel electrophoresis. By day 7 after infection the 13,000 molecular weight protein is no longer expressed on the surface of the parasite. By day 14 after infection there is an additional loss of the 78,000, 30,000 and 26,000 molecular weight proteins. It is suggested that these changes may be associated with the temporal variation in distribution of the T0, T1 and T2 granules in the tegument syncytium.

A comparison of the 35S-methionine metabolically labelled immunoreactive glycoproteins of immature and mature F. hepatica was carried out by one-and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Sera of rabbits infected for 3 weeks reacted much more strongly with glycoproteins of immature flukes than with glycoproteins of mature flukes as compared to sera of rabbits infected for 9 weeks. Several of the immunoreactive glycoproteins were also released by immature F. hepatica into the culture medium. At least one was a component of the T1 type granules. Analysis of the in vitro translation products of mature F. hepatica indicated that the initial humoral immune response of rabbit hosts may be directed against carbohydrate moieties.

Dopamine was identified in extracts from Fasciola hepatica by the fluorometric method. It was found that the dopamine concentration in F. hepatica was 0.668 +/- 0.027 micrograms/g of wet weight. The head region (including the oral and ventral suckers) contained 1.658 +/- 0.275 micrograms/g of dopamine (0.016 +/- 0.002 micrograms per parasite), while the posterior region (located behind the ventral sucker) contained 0.278 +/- 0.070 micrograms/g of dopamine (0.014 +/- 0.004 micrograms per parasite). When the worms were treated with DL-DOPA, the concentration of dopamine increased. In helminths treated with reserpine a marked decline of dopamine level was observed. Adrenaline and noradrenaline were not detectable in F. hepatica.

1. The phospholipid composition of Fasciola hepatica, the common liver fluke, was compared to that of the liver of the host animals (rats and cattle). Considerable differences were found:monoacyl-sn-glycero-3-phosphorylcholine, hardly detectable in the liver, was found in significant amounts in the parasite. On the other hand, sphingomyelin, a normal constituent in the liver, appears to be absent in the liver fluke. Fasciola hepatica isolated from rat and cow liver had a strikingly similar phospholipid composition. 2. Qualitative and quantitative differences were also found between the fatty acyl constituents of the phospholipids of the parasite and the liver. The major difference was the presence of eicosaenoic and eicosadienoic acids in the parasite, whereas these acids were not detected in the liver. 3. In vitro incubations of Fasciola hepatica in the presence of (32P)phosphate and (2-3H)glycerol resulted in the labelling of all phospholipids of the parasite, except that the 3H label did not incorporate into ethanolamine plasmalogen. This is in agreement with the concept that in animals, glycerol is introduced into plasmalogens via dihydroxyacetonephosphate. 4. Homogenates of liver flukes were found to catalyze the synthesis of phosphatidylcholine from 1,2-diacyl-sn-glycerols and CDPcholine. 5. These results strongly suggest that Fasciola hepatica is capable of synthesizing at least part of its fatty acids and phospholipids.

The localization and distribution of neuropeptides in the nervous system of the liver fluke, Fasciola hepatica at different stages in the development of the adult fluke have been determined by an indirect immunofluorescence technique, using antisera to 19 vertebrate peptides and the invertebrate neuropeptide, FMRFamide. Positive immunoreactivity was obtained with antisera to pancreatic polypeptide (PP), peptide tyrosine tyrosine (PYY), substance P (SP) and FMRFamide. Cell bodies and nerve fibres immunoreactive to the 4 peptides are present in the anterior ganglia and the 3 pairs of longitudinal nerve cords and their commissures in the central nervous system. In the peripheral nervous system, immunoreactivity occurs in the nerve plexuses supplying the subtegumental musculature, the oral and ventral suckers, and the muscular lining of the male and female reproductive ducts, including the ootype, uterus, cirrus pouch and gonopore. Cells displaying immunoreactivity to PYY and FMRFamide lie amongst the Mehlis' gland cells that surround the ootype. Processes from these cells extend into the wall of the ootype. One group of PP-immunoreactive cells occurs at the junction of the vitelline and ovovitelline ducts, whilst another group is situated at the entrance to the uterus from the ootype. The results are discussed in relation to the possible roles of the peptides in the neurophysiology and egg production of the fluke.

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 liver fluke Fasciola hepatica has serotonin (5-hydroxytryptamine) receptors that function through a transmembrane signalling system requiring GTP which activates adenylate cyclase (ATP pyrophosphate-lyase (cyclising), EC 4.6.1.1). Non-hydrolysable GTP analogs and NaF activate adenylate cyclase in membrane particles of these organisms. The nature of GTP-binding proteins in these membranes was studied using bacterial toxins and photoaffinity labelling. Treatment of membrane particles from flukes with cholera toxin increased basal adenylate cyclase activity, but markedly decreased activation by serotonin, non-hydrolysable GTP analogs, and NaF. [32P]ADP-ribosylation by cholera toxin or photoaffinity labelling with [32P]-8-N3GTP identified a 53 kDa protein and a 45 kDa protein which appeared to be similar to the forms of the alpha-subunit of the GTP-binding protein associated with adenylate cyclase in mammals. Treatment of membrane particles by pertussis toxin did not significantly change basal adenylate cyclase activity and did not change the stimulation of cyclase by activators. A 43 kDa protein which was [32P]ADP-ribosylated by either cholera or pertussis toxin, depending on the conditions used, and photoaffinity labelled by [32P]-8-N3GTP may be part of the transmembrane signalling system in the liver flukes.