Lipid catabolism in the plerocercoids of Schistocephalus solidus (Cestoda: Pseudophyllidea)

All of the enzymes of the β-oxidation sequence have been demonstrated in the plerocercoids ofS. solidus. However, the plerocercoids could not oxidize exogenous [¹⁴C-U-]palmitate although labelled palmitate was readily taken up and encorporated into the neutral and phospholipid fractions. This suggests that despite the presence of all of the enzymes of β-oxidation, the pathway is not functional in S. solidus plerocercoids; possible roles of the β-oxidation enzymes in the metabolism of S. solidus are discussed.     

Carbohydrate catabolism in the plerocercoids of Schistocephalus solidus (Cestoda: Pseudophyllidea)

The plerocercoids of S. solidus possess a complete sequence of glycolytic and tricarboxylic acid cycle enzymes. The presence of phosphoenolpyruvate carboxykinase and fumarate reductase activity and the relatively low activities of aconitase and isocitrate dehydrogenase suggest that carbon dioxide fixation is an important pathway in this parasite. Carbon balances show that glycogen is the main energy source under both aerobic and anaerobic conditions and there is only a slight Pasteur effect. Aerobically 22·5% of the glycogen catabolized is excreted as acetate and propionate (4:1), anaerobically 70% of the glycogen utilized can be accounted for as acetate and propionate (1:3). The results indicate that anaerobically the plerocercoids fix carbon dioxide and have a partial reversed tricarboxylic acid cycle, whilst under aerobic conditions at least part of the carbohydrate may be oxidized via a functional tricarboxylic acid cycle.     

Phosphofructokinase in the plerocercoids of Schistocephalus solidus (Cestoda:Pseudophyllidea)

Beis I. and Theophilidis G. 1982. Phosphofructokinase in the plerocercoids of Schistocephatus solidus (Cestoda: Pseudophyllidea). International Journal for Parasitology12: 389–393. The Phosphofructokinase from the plerocercoids of Schistocephalus solidus was found to be inhibited by ATP. AMP relieves the ATP inhibition and activates the enzyme. In contrast to mammalian phosphofructokinase, the plerocercoid enzyme does not appear to be sensitive to inhibition by citrate at physiological ATP concentrations. Except for AMP and 3'–5' cyclic AMP no other monophosphate nucleotides were found to activate the enzyme. Succinate, α-ketoglutarate, malate, isocitrate, β-hydroxybutyrate, butyrate, acetoacetate and CoA all inhibit the plerocercoid enzyme at the concentrations tested. The significance of these results in the regulation of glycolysis and the tricarboxylic acid cycle in this parasite is discussed.     

The contents of adenine nucleotides and glycolytic and tricarboxylic acid cycle intermediates in activated and non-activated plerocercoids of Schistocephalus solidus (Cestoda: Pseudophyllidea)

The steady state content of adenine nucleotides, inorganic phosphate and glycolytic and tricarboxylic acid cycle intermediates were measured in freeze clamped plerocereoids of S. solidus taken from the body cavity of infected fish (non-activated plerocercoids) and the results compared with the metabolite levels in plerocercoids which had been cultured in vitro for 12 h (activated plerocercoids). Of the glycolytic intermediates, the levels of fructose-6-phosphate, fructose-1, 6-diphosphate, 3-phosphoglycerate, phosphoenolpyruvate and lactate all decreased on activation, the remainder of the glycolytic intermediates did not alter significantly. In contrast, the levels of the tricarboxylic acid cycle intermediates all increased 2–3 fold on activation, whilst the adenine nucleotides remained virtually unchanged. The differences in the steady state content in intermediary metabolites in activated and non-activated plerocereoids are discussed in relation to possible mechanisms of metabolic control.     

Schistocephalus solidus and Ligula intestinalis: Pinocytosis by the tegument

Using ruthenium red as a macromolecule, endocytosis was demonstrated in the plerocercoid of Ligula intestinalis and adult Schistocephalus solidus. Uptake, transport across the tegument, and exocytosis across the basal plasma membrane occurred within 6 min. The types of vesicles in the tegument of L. intestinalis are redescribed and their former classification is modified. The vertical and longitudinal distribution of pinosomes in the tegument of adult S. solidus and L. intestinalis plerocercoids were determined. The possible role of macromolecular uptake in the economy of pseudophyllidean tapeworms is discussed with particular reference to growth and defence of an unencysted larval stage in the tissues of the intermediate host.     

A novel phosphagen phosphotransferase in the plerocercoids of Schistocephalus solidus (Cestoda: Pseudophyllidea)

Neither phosphagens nor phosphagen phosphotransferase activity could be detected in Fasciola hepatica, Hymenolepis diminuta, Moniezia expansa or in the plerocercoids of Ligula intestinalis. The plerocercoids of Schistocephalus solidus, however, possess an active taurocyamine phosphotransferase, although it too contains no detectable phosphagens. The taurocyamine phosphotransferase of S. solidus has an absolute requirement for a divalent metal ion and ATP could not be replaced by ITP, GTP, CTP or UTP as the phosphate donor. The role of phosphagens in helminths is discussed.     

Apical end organ structure and histochemistry in plerocercoids of Proteocephalus ambloplitis

The end organ of Proteocephalus ambloplitis pleroceroids was studied by light microscopy and histochemistry. The end organ consists of a spherical sac filled with an amorphous, granular secretory product. The lining of the end organ is devoid of an epithelial cell layer. Protease was detected within the end organ by a gelatin-silver film technique. No aminopeptidase activity was detected. The end organ was PAS positive and diastase-fast. A positive test was obtained for neutral mucopolysaccharides and protein. The end organ is thought to assist migration of larvae through host tissue.     

Abundance and distribution of Diphyllobothrium ditremum Creplin (Cestoda: Pseudophyllidea) plerocercoids in benthic whitefish, in northern Finnish Lapland

The prevalence of Diphyllobothrium ditremum plerocercoids in whitefish Coregonus lavaretus ranged between 70 and 100% in Lake Kilpisjärvi and in three other lakes in Northern Lapland, Finland. The mean abundance in Lake Kilpisjärvi (age groups 1+-10+ years), ranged between 103·5± 71·3 in 1992–1993 to 110·9± 80·0 plerocercoids per fish in 1997. The asymptotic value of the infection levelled at 113 plerocercoids per host after age 3. No significant difference in abundance was detected between study years ( P >0·10). Abundances in other lakes ranged between 4·8±9·7 and 91·1±115·1. Two seasonal peaks of plerocercoid recruitment were observed in Lake Kilpisjärvi; between March and April ( P <0·002) and between September and October ( P =0·042). In autumn the numbers of larvae increased particularly in female fish. The invasion rate of the parasite was lower in other lakes studied, and the infection rate in whitefish was closely related to the copepod food eaten.     

The karyotype of Eubothrium rugosum (Cestoda: Pseudophyllidea)

The diploid chromosome set of Eubothrium rugosum contains 16 elements. The karyotype consists of three pairs of metacentric, three pairs of acrocentric and two pairs of submeta-metacentric chromosomes. Their mean absolute length ranges from 2.20 to 8.80 microns. The first two pairs of large metacentric chromosomes comprise over 45% of the total complement length.     

Expression of gonadotropin subunits in roach (Rutilus rutilus, Cyprinidae) infected with plerocercoids of the tapeworm Ligula intestinalis (Cestoda)

Plerocercoids of the tapeworm Ligula intestinalis (Cestoda: Bothriocephalidea) have been reported to inhibit gametogenesis of their intermediate fish hosts. However, mechanistic studies are rare and the proximate cues leading to impaired reproduction still remain unknown. In the present study we investigated the effects of infection by L. intestinalis on reproductive parameters of roach (Rutilus rutilus, Cyprinidae), a common fish host of this parasite. Field studies on roach demonstrated that in both genders infection prevented gonad development. As revealed by quantitative PCR, infection was accompanied by essentially lower pituitary expression of follicle-stimulating hormone β-subunit (FSHβ) and luteinizing hormone β-subunit (LHβ) mRNA compared with uninfected roach, providing clear evidence for gonadotropin-insufficiency as the cause of arrested gametogenesis. Under controlled laboratory conditions infected roach showed lower mRNA levels of FSHβ but not of LHβ, despite histology revealing similar gonad stages as in uninfected conspecifics. These findings indicate the involvement of FSH rather than LH in mediating effects of infection early during gonad development in roach. Moreover, the impact of L. intestinalis on reproductive parameters of roach appeared to be independent of the parasite burden. Together, these data provide valuable information on the role of FSH and LH as mediators of parasite-induced sterilization in a vertebrate and implicate the selective inhibition of host reproduction by L. intestinalis as a natural source of endocrine disruption in fish.     

Ligula intestinalis (Cestoda: Pseudophyllidae): Studies on the properties of proteolytic and protease inhibitor activities of plerocercoid larvae

The somatic extract of L. intestinalis plerocercoids reveals hydrolytic activity against N-Benzoyl-l-tyrosine ethyl ester (BTEE) and Azocoll, and inactivates the esterolysis by mammalian trypsin and chymotripsin. The proteolytic enzyme activity and the inhibitory effect were completely separated by Sephadex G-100 column chromatography. Gel chromatography of the somatic extract revealed two peaks of proteolytic activity : one is bound to macromolecular substances, the other appears to be in free form and has a molecular weight of approx 60,000–65,000. The proteolytic activity showed the following characteristics : Tris-HCl buffer provided the highest activity against BTEE, the pH optimum was 7·4–7·8; the enzyme was activated by 10^?5m-Ca²⁺, Mg²⁺ or Mn²⁺, it was inhibited by 10^?5m-Cu²⁺, but not by 10^?5m-Zn²⁺. 0.001% soybean trypsin inhibitor, 2 × 10^?3m-EDTA, 1 mm-tosyl-l-phenylalanyl chloromethane, 1000 KIU/ml Trasylol did not inhibit the proteolytic activity, but it was inhibited by 1 mm-phenylmethyl-sulphonyl fluoride. The enzyme activity completely ceased upon 5 % TCA treatment or incubation at 56°C for 30 min. The trypsin and chyrnotrypsin inhibitor activities were eluted from the Sephadex G-100 column in a single peak with an estimated molecular weight of 6700–7200. The inhibitory effect was not sensitive to pH changes, and treatment by 5% TCA or incubation at 80°C for 15 min was ineffective. The proteolytic activity of plerocercoid extract was not effected ‘in vitro’ by the inhibitors isolated from this parasite.     

Spermatological characters in Diphyllobothrium latum (Cestoda, Pseudophyllidea)

Spermiogenesis and the ultrastructural features of the spermatozoon of Diphyllobothrium latum (Cestoda, Pseudophyllidea) are described using transmission electron microscopy. Spermiogenesis is characterized by the development of two flagella of unequal length that grow asynchronously. When the first growing flagellum starts to rotate, the second one develops. Flagellar rotation is thus asymmetric and asynchronic. It is followed by proximodistal fusion with the median cytoplasmic process. Electron-dense material is present in the apical region of the zone of differentiation in the early stages of spermiogenesis. The intercentriolar body consists of seven plates: three are electron-dense. Four attachment zones occur in the median cytoplasmic process. An atypical arrangement of striated roots was occasionally observed. The mature spermatozoon possesses two axonemes of 9 + "1" trepaxonematan pattern, nucleus, cortical microtubules, electron-dense granules, and lacks mitochondria. The ultrastructure of the anterior extremity of the spermatozoon in D. latum clearly differs from that in the bothriocephalid pseudophyllideans, mainly in the absence of a crested body and a ring of electron-dense tubular structures. The spermatological data support the assumption that the order Pseudophyllidea is formed by two unrelated clades, "Bothriocephalidea" and "Diphyllobothriidea."     

Comparison of surface topography of three species of Diphyllobothrium (Cestoda,pseudophyllidea) by scanning electron microscopy

Plerocercoids of different sizes as well as adult worms of D. dendriticum, D. latum and D. ditremum were studied using scanning electron microscopy (S.E.M.). In the plerocercoids there were found distinct differences in appearance and length of microtriches between these three species, while the microtriches of adult worms were more similar. A regional difference in microtrix appearance was found in the larvae of D. ditremum and D. dendriticum. This was not apparent with S.E.M. in adult worms. The length of ‘body’ microtriches in D. dendriticum varied with the length of the larvae. The topography of the genital atrium of mature and gravid proglottids in adult worms of these three species is also described.     

Peroxide metabolism in the cestodes Hymenolepis diminuta and Moniezia expansa

The enzymes of hydrogen peroxide metabolism have been investigated in the cestodes H. diminuta and M. expansa. Neither catalase, lipoxygenase, glutathione peroxidase, NADH peroxidase nor NADPH peroxidase could be detected in homogenates of either species. However, both H. diminuta and M. expansa possessed a peroxidase which had a high affinity for reduced cytochrome c. The peroxidase was characterized by substrate and inhibitor studies and cell fractionation showed the enzyme to be located in the mitochondrial membrane fraction. The peroxidase could act as a substitute for catalase, by destroying metabolic hydrogen peroxide. Appreciable superoxide dismutase activity was found in M. expansa and H. diminuta and it is possible that this enzyme is the source of helminth hydrogen peroxide.     

Schistocephalus cotti n. sp. (Cestoda: Pseudophyllidea) plerocercoids from bullheads Cottus gobio L. in an Arctic river in Finland, with a key to the plerocercoids of the Palaearctic species of the genus

We compared plerocercoids of Schistocephalus Creplin, 1829 from Cottus gobio (n = 57) and Gasterosteus aculeatus f. semiarmatus (n = 45) from the River Utsjoki, Finland, taken only from single worm infections. Segment numbers in the two populations were distinct (G. aculeatus range 55–107, average 74 (SE 1.66), median 73; C. gobio range 122–189, average 146 (SE 1.78); median 144). The mean difference between populations, 71.47, t = 28.76 with 100 degrees of freedom, two-tailed p value <0.001, was considered extremely significant. Amplification of microsatellite loci that were originally designed for Schistocephalus from G. aculeatus was positive for all larvae from G.␣aculeatus (n = 20), whereas in no plerocercoids from C. gobio (n = 20) were any of the six microsatellites amplified, indicating that plerocercoids from G. aculeatus and C. gobio were two distinct genetic populations of Schistocephalus. The material from C. gobio is described as S. cotti n. sp. Plerocercoids of the Palaearctic species of Schistocephalus are identified as follows: S. nemachili Dubinina, 1959 with 228–235 or more segments, specific to Barbatula spp. (Balitoridae); S. pungitii Dubinina, 1959 with 62–92 (usually 70–80) segments, specific to Pungitius pungitius; S. solidus (Müller, 1776) in two forms, one in G. aculeatus f. leiurus and f.␣semiarmatus, with 48–100 (usually 65–75) segments, and the other in G. aculeatus f. trachurus, with 99–138 (usually 112–122) segments; and S. cotti n. sp. with 103–189 (usually 130–159) segments, probably specific to cottids. Nearctic Schistocephalus were not considered owing to the uncertain status of some North American records. Some other species of Schistocephalus of highly doubtful status were briefly noted. Cross-infection experiments and molecular studies are recommended to further elucidate the interrelationships between the various species of Schistocephalus.     

The reaction of nitro-blue tetrazolium with d,l-L-tetrahydrofolate: The effect of pH,oxygen, formaldehyde and palladium(II)

d,l-L-Tetrahydrofolate (d,l-L-FH₄) transfers two electrons to nitro-blue tetrazolium (NBT) in oxygen-free buffers to form the highly coloured nitro-blue formazan and oxidized folate. Both the rate and extent of this reaction are affected by the pH, the nature of the buffer and the oxygen concentration. Inhibition of both the rate and extent of this reaction in air-saturated solutions by superoxide dismutase (SOD) indicates that the superoxide anion is an intermediate in the reaction so that formazan can be produced by both superoxide independent and superoxide dependent routes in air-saturated solutions.In oxygen-free solutions several lines of evidence can be interpreted to mean that the reduced pteridine ring of tetrahydrofolate is the electron donor in the reaction with NBT. Ionization of the amide hydrogen of the pteridine ring and subsequent increase in electron density of that ring might explain the large increases observed in the rate and extent of the reaction of tetrahydrofolate with NBT as the pH increases. Formaldehyde reacts non-enzymatically with tetrahydrofolate to form a methylene bridge between nitrogens 5 and 10 of methylenetetrahydrofolate. This molecule is much less reactive with nitro-blue tetrazolium than tetrahydrofolate. Complexes formed between tetrahydrofolate and palladium(II) ions are also less reactive with NBT than the tetrahydrofolate alone. This result provides added evidence that palladium(II) ions interact with tetrahydrofolate at the nitrogen 5, nitrogen 10 site of the molecule.