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Since isolates of Hymenolepis nana infecting humans and rodents are morphologically indistinguishable, the only way they can be reliably identified is by comparing the parasite in each host using molecular tools. In the current study, isolates of H. nana from rodent and human hosts from a broad geographical range were sequenced at the ribosomal first internal transcribed spacer (ITS1), the mitochondrial cytochrome c oxidase subunit 1 (C01) gene and the nuclear paramyosin gene loci. Twenty-three isolates of H. nana were sequenced at the ITS1 locus and this confirmed the existence of spacers which, although similar in length (approximately 646 bp), differed in their primary sequences which led to the separation of the isolates into 2 clusters when analysed phylogenetically. This sequence variation was not, however, related to the host of origin of the isolate, thus was not a marker of genetic distinction between H. nana from rodents and humans. Sequencing of a 444 bp fragment of the mitochondrial cytochrome c oxidase 1 gene (C01) in 9 isolates of H. nana from rodents and 6 from humans identified a phylogenetically supported genetic divergence of approximately 5% between some mouse and human isolates. This suggests that H. nana is a species complex, or 'cryptic' species (=morphologically identical yet genetically distinct). A small segment of the nuclear gene, paramyosin, (625 bp or 840 bp) was sequenced in 4 mouse and 3 human isolates of H. nana. However, this gene did not provide the level of heterogeneity required to distinguish between isolates from rodent and human hosts. From the results obtained from faster evolving genes, and the epidemiological evidence, we believe that the life-cycle of H. nana that exists in the north-west of Western Australia is likely to involve mainly 'human to human' transmission.

The free radical nitric oxide (NO) is a neuronal messenger which is synthesized from L-arginine and O2 by nitric oxide synthase (NOS). In the synthesis NO and L-citrulline are produced in a stoichiometric 1[ratio ]1 relation. The activity of NOS was analysed in homogenates of the rat tapeworm Hymenolepis diminuta by measuring the formation of L-[3H]citrulline after incubation with L-[3H]arginine. The nature of NOS in H. diminuta was determined by studying the effect of 3 types of NOS inhibitors: (1) L-NAME, (2) EGTA, (3) 7-nitro-indazole. All inhibitors caused a significant but not complete reduction in the formation of L-[3H]citrulline. The results are discussed against the background of nerve cells and fibres positive for NADPH-diaphorase staining in H. diminuta.

Three hymenolepidid tapeworms, Hymenolepis diminuta, H. nana and H. microstoma, are commonly maintained in laboratory rodents and used in many experimental model systems of tapeworm infections. We examined partial sequences from the mitochondrial cytochrome c oxidase subunit 1 (CO1) gene and nuclear ribosomal internal transcribed spacer 2 (ITS2) sequences to infer phylogenetic relationships of the 3 hymenolepidid species. Parts of the CO1 gene and ITS2 were amplified by PCR and sequenced directly. The CO1 gene sequence obtained was the same in length (391 bp) among all specimens. In the case of ITS2, however, several insertions and deletions were detected (671–741 bp) not only among species but also between an American isolate and a Japanese isolate of H. diminuta. Percentage nucleotide differences between H. diminuta and H. microstoma, or H. diminuta and H. nana were 16·6–18·2% for the CO1 gene and 21·3–22·9% for ITS2. The differences in both sequences between H. microstoma and H. nana were about 14%. Phylogenetic trees inferred from both of the nucleotide sequences showed similar topology, and suggest that H. diminuta may have diverged from the common ancestral line the earliest, and that H. nana is closer to H. microstoma than to H. diminuta.

Interactions between adult Hymenolepis diminuta and rat C-reactive protein (CRP) were investigated in vivo and in vitro. Using an ELISA technique, serum levels of CRP were monitored in rats infected with 100 cysticercoids. Although infection increased the level of this protein in the early stages of parasitization, the increase was not significant until 35 days post-infection (p.i.). Secondary infections did not enhance the response. When H. diminuta was cultured in the presence of CRP, reduced worm motility and opaque areas were observed and electron microscopical studies revealed shedding of microtriches and lysis of the tegument. Initially, damage was restricted to the strobila which correlated with the regional distribution of phosphorylcholine as visualized using immunofluorescence.

SUMMARYVitellogenin synthesis by the fat body has been monitored using in vitro culture and immunoprecipitation. This system was found to be efficient for measuring vitellogenin production in both non-infected Tenebrio molitor and those infected with Hymenolepis diminuta. In fat bodies from infected beetles, vitellogenin production was decreased by up to 75% (day 24 post-infection) and, at all times investigated, vitellogenin synthesis was significantly below control levels (days 3–30 post-infection). Incubating fat bodies from control insects with isolated metacestodes indicated that this may be a direct effect by the parasite which is developmental stage-specific. Stage II, but not stage III–IV, nor heat-killed parasites could bring about this decrease in vitellogenin. In addition, these effects may be density dependent within the range of 2–20 parasites per fat body; only 2 metacestodes were necessary to cause a significant decrease. Since metacestodes do not take up vitellogenin, nor limit the amount of [14C] leucine available to the fat body for vitellogenin production, it is conceivable that the parasite produces a potent inhibitor of vitellogenin synthesis, or a molecule which induces cells within the fat body to do so.

SUMMARYThe metamorphosis of oncospheres of the rat tapeworm, Hymenolepis diminuta, to mature metacestodes induces several pathophysiological effects in the intermediate host, Tenebrio molitor (Coleoptera). Previous investigations have failed to elucidate the mechanism responsible for changes in the host reproductive physiology and behaviour. This work forms part of an assessment of the degree to which nutrient resource management may be involved in these interactions. We report that developing metacestodes exert a pronounced effect upon host carbohydrate reserves and also alter circulating carbohydrate titres at some stages post-infection. Biochemical studies of fat body glycogen demonstrated a significant depletion of reserves as early as 3 days post-infection in males and 5 days post-infection in females. Gas chromatography revealed trehalose to be the predominant haemolymph carbohydrate, titres being significantly elevated in male beetles 5 and 9 days post-infection and in females 12 days post-infection. Metacestodes had no effect on glucose concentrations in male or female beetles.

SUMMARYDensity-dependent constraints on parasite growth, survival or reproduction are thought to be important in preventing the unchecked increase in parasite numbers within individual hosts or host populations. While it is important to know where, and with what severity, density dependence is acting within the parasite life-cycle, interpretation of data from natural infections is difficult. In this paper, we present a Monte Carlo simulation technique for examining such data for evidence of density dependence. We also describe how this technique may be used to distinguish among mechanisms hypothesized to generate density-dependent phenomena.

SUMMARYWhen incubatedin vitrofor 24 h, oncospheres ofHymenolepis diminutaabsorb and metabolize radioactive glucose. Between 0 and 12 h post-activation, oncospheres absorb glucose, but glucose is neither metabolized into other carbohydrates nor incorporated into the ethanol-precipitable fraction (which would contain glycogen). Between 12 and 24 h post-activation glucose is incorporated into a number of higher molecular weight carbohydrates that are demonstrable in ethanol extracts of the larvae, as well as the incubation media. Furthermore, measurable amounts of radioactivity are incorporated into the ethanol-precipitable carbohydrate fraction of oncospheres. To determine if these temporal changes in carbohydrate metabolism occurred spontaneously following activation, oncospheres were pre-incubated for 12 h (0–12 h post-activation) in the absence or presence of glucose, and then transferred to media containing radioactive glucose for an additional 12 h (12–24 h post-activation). In these latter experiments, glucose absorption and metabolism between 12 and 24 h post-activation were virtually identical to glucose metabolism in oncospheres that were incubated in radioactive glucose for 0–12 h immediately following activation. Thus, these data do not support the hypothesis that the temporal shift in carbohydrate metabolism occurs spontaneously.

SUMMARYWhen incubatedin vitrofor 24 h, intact eggs, chemically shelled eggs (obtained by treating intact eggs with NaOCl), activated larvae (eggs in which the outer shell and inner envelope were removed), and oncospheres (activated larvae treated with papain to remove the embryophore) absorb and metabolize radioactive glucose. Intact eggs, which are covered by the impermeable shell, absorb only small amounts of exogenous radioactive glucose, while chemically shelled eggs, activated larvae, and oncospheres absorb much larger amounts. Only very small amounts of the exogenous glucose are incorporated into the ethanol-precipitable carbohydrate fraction (which would include glycogen) by any of the preparations of eggs/larvae. However, the glucose is incorporated into higher molecular weight end-products that are liberated into the incubation medium. There is a temporal shift in the ability of activated larvae and oncospheres to metabolize exogenous glucose. Activated larvae and oncospheres absorb but do not metabolize glucose during the first 8 h post-activation. Between 8 and 16 h post-activation, however, virtually all of the absorbed glucose is metabolized into higher molecular weight end-products that are liberated into the incubation media. This temporal shift suggests that activation of oncospheres and cysticercoid morphogenesis are accompanied by distinct changes in carbohydrate metabolism.

Cyclosporin A (CsA) induced significant changes in parasite morphology when administered to mice infected with Hymenolepis microstoma. Gross morphological damage consisted of proglottis swelling and the formation of protuberances from the worm surface, visible with a low-power dissecting microscope, occurring most frequently in the posterior third of the strobila. Gross morphology and ultrastructure were examined further using scanning and transmission electron microscopy. Swollen proglottides exhibited areas covered in small pits and fissures (diameter approximately 1–2 μm) but it was not possible to establish the significance of this damage. The brush-border and distal cytoplasm appeared largely intact although some evidence of swelling of the basal membrane invaginations and possible fluid accumulation was seen in drug-treated TEM sections. The apparent oedematous condition of many of the proglottides from drug-treated mice may indicate that CsA treatment mediates permeability changes in the worm surface membrane but the mechanisms by which this may occur remain to be elucidated. The effects of CsA on the morphology of H. microstoma correlate with the previously described anthelmintic activity of the drug against this parasite where CsA treatment dramatically reduces worm growth, retards migration into the bile duct and limits parasite survival.

SUMMARYHymenolepis diminutamigrates up the small intestine in response to feeding the host 1 g of glucose. Locomotion during migration may result from fixed patterns of retrograde peristaltic-like waves in the strobila of the tapeworm which propel the organism against the normal expulsive forces in the small intestine. The peristaltic-like locomotory waves occur in a gradient along the strobila with a frequency of 24·9±0·9 cycles/min in the anterior segments of the worm, decreasing linearly to 6·6±1·4 cycles/min in the posterior segments of the worm. Chemical signals, isolated from the small intestine of fed hosts, which stimulate migration behaviourin vivodo not alter the behaviour of the scolex or strobilain vitro. Removal of the scolex containing the cerebral ganglia does not alter the frequency or pattern of locomotory activity in the strobila. After the worm is cut into pieces, each region generates the pattern of locomotory activity that is appropriate for that region. These data suggest that the peripheral nervous system, and not the central nervous system, is responsible for the coordination of the fixed patterns of locomotory activity in these tapeworms.

Standard indirect immunocytochemical techniques have been interfaced with confocal scanning laser microscopy (for whole-mount preparations) and epifluorescence microscopy (for cryosections) to investigate the occurrence and distribution of serotoninergic and peptidergic nerve elements in adultH. diminuta. Serotonin (5-HT)-immunoreactivity (IR) was widespread throughout the worm, occurring in the paired cerebral ganglia, transverse commissure, the 10 longitudinal nerve cords and in a plethora of small nerve fibres of the peripheral nervous system. An abundance of serotoninergic nerve cell bodies was found in association with the lateral nerve cords. The genital atrium and accessory reproductive ducts were richly innervated with serotoninergic nerve fibres. Thirty-five antisera to 20 vertebrate regulatory peptides and 1 invertebrate peptide (FMRFamide) were used to screen the worm for neuropeptide IR. Immunostaining was obtained with antisera raised to pancreatic polypeptide (PP), peptide YY (PYY), neuropeptide Y (NPY), substance P (SP), peptide histidine isoleucine (PHI), xenopsin (XP) and FMRFamide. The most extensive pattern of IR occurred with antisera to PP and PYY, IR being evident in the cerebral ganglia, transverse commissure, longitudinal nerve cords and in small nerve fibres that ramified throughout the parenchyma. A series of bipolar nerve cell bodies between the median nerve cords displayed PP/PYY-IR. The distribution of FMRFamide-IR was reminiscent of the PP/PYY pattern but was less extensive. Comparison of the serotoninergic and peptidergic nervous systems has revealed general similarities and some distinct differences, especially with regard to the distribution of immunoreactive nerve cell bodies. Quantitative data are presented on the levels of PP-, SP-, PH1-, and gastrin-releasing peptide (GRP)-immunoreactivities demonstrable in acid-alcohol extracts of whole worms. The highest level of peptide IR determined was recorded for PP.

SUMMARYAntineoplastic cyclophosphamide (CY) significantly reduced the survival and the weight ofHymenolepis microstomain mice. Corticosteroid dexamethasone (DEX), on the other hand, did not influence the worm survival and the worms in DEX-treated mice became significantly heavier. Both immunosuppressants abolishedH. microstoma-induced bile duct hypertrophy and submucous enteritis and lowered the number of mast cells (MC) in these regions. CY appeared to be more effective in depleting the bile duct MC whereas DEX seemed to affect more the duodenal MC population.

Cyclosporin A (CsA) is a widely investigated experimental anti-parasitic drug whose mode of action remains unresolved. The immunosuppressive action of CsA depends on the drug binding to the intracellular receptor cyclophilin (CyP). This study investigates whether complexing of CsA with parasite CyP is equally essential for its anthelmintic action. The correlation between initial cyclosporin-induced damage in vitro and drug binding to parasite CyP has been examined. CsA and the analogues B-5-49, CsH and CsA-acetate all induced similar damage to the tapeworm Hymenolepis microstoma in vitro in incubations between 2 h and 4 days. The initial foci of drug damage were the parasite surface and mitochondria in the syncytium. In a competitive binding assay only B-5-49 displaced [II 3 ]-CsA from either crude parasite cytosolic CyP P or affinity-purified CyP while CsH and CsA-acetate had no effect. 'I'hese data suggest strongly that cyclosporins act on the surfaces of helminth parasites but that drug action does not involve complex formation with CyP An alternative drug-binding site must therefore be identified which may lead to the rational design of novel anthelmintic drugs.

Previous laboratory studies have indicated that tenebrionid beetles infected with Hymenolepis diminuta display behavioural alterations. These are assumed to increase the likelihood of this intermediate host being predated by the Rattus sp. definitive host, and hence provide support for the Manipulation Hypothesis. We tested this hypothesis by both assessing beetle behaviour and predation rates in semi-naturalistic environments. Behavioural assays showed that infected beetles were more often exposed (not concealed under boxes) than uninfected beetles. However, there were no differences in predation rates between infected and uninfected beetles. We discuss this in terms of the historical and evolutionary contexts under which such altered behaviours could evolve and their implications for the Manipulation Hypothesis.

Regional differences of Hymenolepis diminuta to immune lysis have been investigated by monitoring surface membrane fluidity utilizing fluorescence recovery after photobleaching (FRAP). Although the surface membrane of the newly excysted stage was completely immobile the molecular fluidity of the strobila membrane of 4-day-old parasites was greater than that associated with the scolex/neck region. Significant differences ( P >0·001) occurred in the mobility of the strobila membrane of 4-day-old H. diminuta from 100-worm infections compared with 7- and 21-day-old parasites and 4-day-old individuals from 10-worm infections. Exposure to 50% normal rat serum and 1 mg/ml rat C reactive protein decreased or eliminated membrane fluidity. The significance of membrane mobility is discussed with reference to resistance to complement-mediated lysis and destrobilation.

The defensive glands of beetles, Tenebrio molitor, infected with metacestodes (cysticercoids) of Hymenolepis diminuta are everted less frequently upon stimulation, and contain less toluquinone (methylbenzoquinone) and m-cresol, than glands of uninfected controls. These differences, as shown in predation trials with wild rats, increase the likelihood that both cysticercoids and beetles will be ingested by the tapeworm's definitive host. This is the first documented case of a parasite inhibiting the chemical defence of an intermediate host, and one of only a few reports of parasite-induced manipulation of host biology supported by empirical evidence implicating facilitated parasite transmission between host species.

The drug cyclosporin A (CsA) exerts its immunosuppressive action by binding to the cytosolic protein, cyclophilin (CyP) and, as a complex, binding to and inhibiting the calcium/calmodulin-dependent serine threonine phosphatase, calcineurin. It is unknown whether a similar mode of action occurs during the drug's antiparasite activity. Calmodulin-binding proteins from the helminth parasites Hymenolepis microstoma and H. diminuta were purified by affinity chromatography, yielding single polypeptide bands of 60000 Mr, according to SDS–PAGE. These proteins were tested for calcineurin activity by the dephosphorylation of the RII peptide (part of the catalytic subunit of cAMP-dependent protein kinase). Both proteins were calcium- and calmodulin-dependent and were inhibited by mammalian cyclophilin complexed with cyclosporin A (IC50 values of 0·75 μg CyP for H. microstoma and 0·90 μg CyP for H. diminuta). However, neither of the parasite calcineurins was inhibited by H. microstoma cyclophilin/CsA. These data suggest the anthelmintic mode of action of CsA in these helminth models does not involve the inhibition of a signal transduction pathway requiring interaction with calcineurin.

SUMMARYThe free radical nitric oxide (NO), which is synthesized by nitric oxide synthase (NOS), has recently been discovered to function as a neuronal messenger. The presence of NOS was detected in the nervous system of adult Hymenolepis diminuta with NADPH-diaphorase (NADPH-d) histochemistry. The NADPH-d histochemical reaction is regarded as a selective marker for NOS in neuronal tissue. NADPH-d staining was observed in nerve fibres in the main and minor nerve cords and the transverse ring commissures, and in cell bodies in the brain commissure, along the main nerve cords, in the suckers and the rostellar sac. NADPH-d staining was also observed in the wall of the internal seminal vesicle and the genital atrium. The pattern of NADPH-d staining was compared with that of the 5-HT immunoreactive nervous elements. The NADPH-d staining reaction and the 5-HT immunoreactivity occur in separate sets of neurons. This is the first time the NADPH-d reaction has been demonstrated in the nervous system of a flatworm, indicating that NOS is present and that NO can be produced at this level of evolution.

SUMMARYCyclosporin A (CsA) acts as a powerful immunosuppressant through its binding to the cytosolic isomerase, cyclophilin (CyP), forming a complex which inhibits the phosphatase activity of calcineurin. The drug is also selectively anti-parasitic but its mode of action remains unknown. The mouse tapeworm,Hymenolepis microstomais sensitive to CsA, but the rat tapeworm,H. diminutais not susceptible either in rats, mice orin vitro. Using these two tapeworm models, the uptake and binding of CsA were examined in relation to parasite cyclophilins. Uptake and compartmentalization of the drug were markedly different in the two species:H. microstomatakes up more drug than doesH. diminutaand sequesters more drug into intracellular compartments. Characterization of cyclophilins using both CsA binding and isomerase activity assays reveals thatH. microstomapossesses two cyclophilin isoforms (Mr17700 and 21400) with isomerase activity that is inhibited by CsA. Using identical assays, we have been unable to demonstrate CsA-binding proteins or CsA-sensitive isomerase activity inH. diminuta. These data suggest that the anthelmintic action of CsA relates in some way to the presence and function of parasite cyclophilins.

SUMMARYThe carbohydrate constituents of whole eggs and fractions derived from the eggs of Hymenolepis diminuta were partly characterized. Whole eggs contained 9·6 ng of carbohydrate (phenol-sulphuric acid-positive material) per egg, and 3·6 ng of glucose. Analysis of hydrolysed eggs by HPLC demonstrated the presence of glucose, galactose, glycerol, N-acetylgalactosamine, and 2-deoxyribose. Isolated egg shells and the KOH-stable, ethanol-precipitable fraction (putative glycogen) of eggs contained 26 and 76%, respectively, of the total carbohydrate associated with eggs. Glucose and galactose were present in both of these samples, but only glucose was present in the KOH-stable, ethanol-precipitable fraction isolated from chemically deshelled eggs; thus, the source of the galactose in the KOH-stable, ethanol-precipitable fraction isolated from whole eggs was the egg-shells. These data confirm earlier reports that the shells contain carbohydrate; the data demonstrate further that only two monosaccharides (glucose and galactose) are present in the shells, and that they are present as high molecular weight polymers. The ethanol-soluble fraction of eggs contained 2·5% of the total carbohydrate in eggs, and glucose accounted for < 1% of the ethanol-soluble carbohydrate. Analysis of the ethanol-soluble fraction by HPLC demonstrated that the predominant monosaccharides were glycerol and mannose, with smaller quantities of 2-deoxyribose and 2-deoxyglucose. The absence of a ‘free pool’ of glucose in eggs, and the presence of large amounts of glycerol and mannose, suggest that the pathways of intermediary carbohydrate metabolism in eggs might be very different from those in adult tapeworms.

SUMMARYFour-day-old worms of the tapewormsHymenolepis microstoma,H. diminutaandH. nanaand newly excystedH. microstomawere exposed in vitro at 37 °C to immune serum from mice infected for 4–1 2 weeks withH. microstoma. Worms were fixed for electron microscopy after intervals of 5 min to 96 h. Within 10–15 min an homogeneous precipitate occurred between the microtriches of 4-day-oldH. microstomaandH. nana, while on some areas ofH. microstomathe precipitate extended distal to the microthrix border and contained small vesicles (30 nm in diameter) and shed microtriches. InH. diminutaprecipitates were not found until 2 h post-incubation. The thickness of the precipitate and the number of small vesicles and shed microtriches increased with time after incubation. Since a similar precipitate occurred on worms kept in complement-depleted immune serum, antibodies alone may induce immune damage. The precipitate on newly excystedH. microstomalacked microthrix fragments. After 48 h an extensive precipitate was found protruding from the rostellar glands on someH. microstoma, and within the culture vessel. Antibodies may therefore be complexing with tapeworm secretory products.

SUMMARYThe transport of [14C]glucose by Hymenolepis microstoma in vitro following in vivo treatment with cyclosporin A (CsA) was determined over a range of concentrations. For untreated (control) worms glucose uptake showed saturation kinetics with a small diffusion component. Estimates of the maximum velocity of glucose uptake (Vmax) and the affinity of substrate for the glucose transporter (Kt) revealed that untreated 8-day-old worms had a Vmax twice that of 15-day-old worms and that younger worms had a lower Kt. An inverse relationship was demonstrated between log10 worm weight and the rate of uptake of [14C]glucose, reflecting the relatively greater number of glucose transporters due to the larger surface area: volume ratio of smaller worms. Treatment of H. microstoma with CsA in vivo significantly increased the diffusion component of glucose uptake in vitro. Parasites from drug-treated mice had a significantly lower Vmax for glucose uptake than size-matched controls. The affinity of glucose for its transporter in CsA-treated worms (Kt) was not significantly different from size-matched controls. Both juvenile and adult worms underwent transient depletion in total glycogen content after CsA treatment in vivo. The data confirm that CsA treatment in vivo disrupts the functional integrity of the worm tegument, one facet of which is impaired acquisition of glucose.

SUMMARYTemporal changes in glucose transport capacity in excysted scoleces ofHymenolepis diminutawere examined. Assays involved incubation for 1 min in [3H]glucose after pre-incubation for 1 min to 8 h in saline. There were two abrupt increases in uptake velocity, a relatively small one between 15 and 75 min, and a large one between 5 and 6 h, during which theVmaxincreased from 0·36 to 2·49 nmol/25 larvae/h. The second increase was unaffected when the pre-incubation saline contained 5 mM glucose, but it was completely blocked when the excysted larvae were pre-incubated in Ca2+-free saline. Abrupt glucose transport changes did not occur in intact cysticercoids or in scoleces when the substrate was [3H]leucine or [3H]uracil. Arrhenius plots (logVversus 1/temperature, 10–42 °C) were linear for intact cysticercoids, but were biphasic for both scoleces and adults with discontinuities at 20 ± 1 °C. Thus, ‘activation’ of the excysted scolex seemed to involve a specific, Ca2+-dependent increase in number of glucose transporters functioning in the worm surface. The Arrhenius plots indicated that development in the final host does not involve a major change in lipid composition of the parasite's membranes.

SUMMARYThein vivoeffects of the anthelmintics taenifugin, VÚFB 14170 and VÚFB 15269 on the tegument ofHymenolepis fraternahave been examined by SEM, TEM and cytochemistry. The drugs were given toH. fraterna-infected mice on the 14th day post-infection in a single oral dose of 150, 200 and 200 mg/kg, respectively. By 72 h post-treatment, the drug-induced pathomorphological changes in the tegument indicated that all three drugs had a significant effect. Changes were most pronounced on the brush border and in the intercellular material. On the apical surface, there was blebbing as well as accumulation of membrane fragments over the microthrix tips and erosion of the brush border. The intercellular material was changed in structure, showing increased electron density in some areas and oedema of the intercellular spaces in other areas. There were also fractures of the tegument of variable depth, sometimes reaching to the parenchyma. These results suggest altered tegumental integrity and, occasionally, complete disruption of the selective permeability barrier created by the normal tegument. This suggestion is further supported by the penetration of ruthenium red into some tegumental areas and its distribution into the intercellular spaces, down to the parenchyma. The intrategumental lysosomes also appeared to be significantly activated. There was evidence of autophagy in both distal cytoplasm and tegumental cells. Mature and gravid proglottides were more susceptible to drug damage than those in the anterior strobila and neck.

SUMMARYIn the absence of exogenous divalent cations, the isolated brush-border plasma membrane of Hymenolepis diminuta possesses alkaline phosphatase activity (APA). APA is stimulated in the presence of exogenous Mg2+ and inhibited by low concentrations of Zn2+ or high concentrations of Ca2+, and inhibition of APA by Zn2+ is reversed by both Mg2+ and Ca2+. APA is inhibited by ethylenediamine tetraacetic acid (EDTA), ethyleneglycol-bis-(β-aminoethyl ether) N, N′-tetraacetic acid, and 1, 10-phenanthroline in time- and concentration-dependent fashions, with EDTA being the most effective inhibitor. Following treatment with EDTA, APA is restored by Mg2+ and, to a lesser extent, by Ca2+, but not by Zn2+. Thus, APA represents a Mg2+-dependent enzyme that can be partly activated by Ca2+ but only in the absence of Mg2+.

SUMMARYCyclosporin A (CsA), administered in 5 daily subcutaneous doses of 50 mg/kg to MF1 mice immediately following infection withHymenolepis diminutaenhanced parasite growth relative to controls. Drug administered at 24 h intervals for 10 days, and thereafter every 48 h to MF1 and CBA/Ca mice infected withH. diminuta, increased worm survival and growth, delayed host-mediated expulsion of the parasite and enabled some worms to develop to patency. Worm survival and weight both increased in a dose-dependent manner following daily CsA treatment of infected CBA/Ca and BALB/c mice (0–150 mg/kg CsA/day). Delay in parasite elimination was accompanied by increased frequency of worm-attachment in the anterior small intestine (MF1 mice given 5 daily doses of CsA [0–150 mg/kg] following infection); posteriad migration of worms was restricted in a dose-dependent manner. The data presented contrast markedly with the action of the same drug onH. microstomain mice. Thus CsA treatment acts in opposing ways on two closely related parasites in the same host; this possibly reflects the mechanistic antagonism between immunosuppression and anthelmintic activity. This paper reports the first use of a specific T cell-suppressive drug onH. diminutain the mouse, implicating the role of T cells in protective immunity to this parasite.

SUMMARYMetacestodes of Hymenolepis diminuta cause a perturbance of vitellogenesis in the intermediate host Tenebrio molitor. The reduction in host reproductive output associated with infection may be due to this pathophysiology. Many of these events are regulated by host juvenile hormone (JH). A comparison of the titre of JH and its rate of degradation in female control and parasitized 15-day-old insects has been made. Haemolymph from female beetles contained 1·27 pMol JH equivalents/100 µl. No significant difference was associated with infection. However, topical application of a JH analogue, methoprene, at the time of infecion or 8 days post-infection reduced the significant accumulation of vitellogenin usually found in the haemolymph of females 12 days or more post-infection. These findings indicate that parasite-induced alteration of host vitellogenesis is not mediated via alteration in JH titres, although observations made after hormone supplementation suggest some form of interaction between the parasite and the host endocrine system.

SummaryFour acidic glutathione (GSH) transferase forms were isolated from the cytosol of the adult cestodeHymenolepis diminutaby hydroxylapatite chromatography, glutathione-affinity chromatography and chromatofocusing, pH 7–5. The enzymes were dimers of subunit size approximately 24 kDa and accounted for at least 3% of the total soluble protein. The major GSH transferase had limited catalytic activity but may interact with a range of ligands and function as a binding/passive detoxification protein. An endogenous factor interfered with the binding of the crude cytosolic GSH transferase activity to glutathione-dependent affinity matrices but, following partial purification, the GSH transferase activity successfully interacted with the glutathione affinity matrix.

Similar survival characteristics were demonstrated for hexacanths of Hymenolepis diminuta incubated in Tyrode's solution with or without a glucose supplement (0–50 mg/ml).The survival rate of hexacanths in all media tested was shown to be age-dependent and led to a maximum life-span of approximately 11 h.The amount of energy reserves, as measured by microdensitometric determinations of PAS + material, declined rapidly in time to a plateau at approximately 8 h. Residual PAS + matter present beyond that period was interpreted as structural and thus non-utilizable material.The rate of activity as measured by hook movements declined more rapidly, and continuous hook cycles were rarely observed after 2 h and ceased after 4 h. A close correlation was demonstrated between the decline in PAS + material and the total number of hook cycles completed per unit of time.The quantitative results on survival, energy reserves and activity are discussed in relation to the penetration of hexacanths into the haemocoele of the intermediate host.

SUMMARYHymenolepis citellicaused a marked mortality inTribolium confusumduring the first 14 days of infection. The mortality was higher when the beetles were starved for 6 days immediately prior to infection (73–93%) than when they were starved for 1 day (51%). Regardless of the starvation time, the majority of beetle deaths occurred between days 8 and 11 post-infection (p.i.) and the precise survival time for individuals tended to vary inversely with the number of parasites they contained. With few exceptions beetles that survived until day 15 p.i., when the experiments were terminated, contained 14 or fewer cysticercoids, whereas those that died on or before day 14 p.i. contained 20 or more parasites. Mortality was equally high in both sexes but the mean survival time was significantly shorter for female beetles (8·8 days) than for males (9·6 days). Also, parasite development rate varied with the sex of the host. When parasite populations recovered from beetles that died on the same day were compared, the degree of development attained by those from males was significantly higher than in the populations grown in female hosts. The line ofH. citelliused in these experiments has been maintained in the laboratory for the past 5 years. The results obtained with the current generation of this line were compared with those obtained with it 2 years earlier (approximately 16 generations ago). Mortality was lower with the current generation (67%) than with the earlier generation (93%). However, with the latter generation, the mean parasite population size in beetles that survived to day 15 p.i. was significantly higher (14·1 cysticercoids/beetle) than in those infected with the recent generation (7·3–7·9) cysticercoids/beetle). These findings suggest that relatively high selection pressures have been applied byT. confusumtoH. citelli, thereby reducing the number of parasite lines with high infectivity and consequently improving host survivorship. The host also responded to artificial, directed selection for reduced cysticercoid number (heritability estimates of 0·30 ± 0·12 and 0·26 ± 0·12) and increased survival (heritability estimates of 0·28 ± 0·12 and 0·15 ± 0·083). The realized heritability was 0·37 for cysticercoid number and 0·17 for increased survival.

SUMMARYThe 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.

In one (1c) and six (6c) cysticercoid primary infections of Hymenolepis diminuta in NIH (inbred) and CFLP (outbred) male mice 6±1 weeks old > 85% of the worms established but were rejected (destrobilated or expelled) subsequently. Rejection occurs more quickly in 6c infections than in 1c infections. Considerable worm growth occurs in 1c and 6c primary infections but worms from 6c infections weighed less than worms from 1c infections on all days studied.Expulsion of H. diminuta does not occur more rapidly in secondary infections than in primary infections; loss of 6c secondary worms occurs at the same rate as 6c primary worms but 1c secondary worms survive longer than 1c primary worms. Although worms are not lost more quickly in secondary than in primary infections, they are affected at an early age by the immune response which stunts their growth. Increasing the intensity of primary and secondary infections increases the severity of stunting of secondary worms.The results are discussed and it is suggested that immune responses to Hymenolepis spp. in rodents are common but that thresholds of worm numbers exist below which appreciable worm loss does not occur. Stunting due to crowding, which generally is attributed to inter-worm competition, may be in part immunologically mediated. For future immunological studies attempting to induce secondary responses to H. diminuta in mice, worm growth, not survival, is the criterion to evaluate.

SUMMARYThe effects of the direction of gut flow, of injections of glucose and saline into different regions of the small intestine and of surgical re-routing or ligature of gastric, biliary and pancreatic secretions into the small intestine have been correlated with changes in the migratory response of the rat tapeworm Hymenolepis diminuta. Reversing the normal anterior to distal flow of luminal contents in the small intestine did not affect worm migration following feeding. Injections of a glucose-saline solution into the duodenum did not initiate a migratory response; similar injections into the mid- and posterior regions of the small intestine resulted in migrational responses similar to those following intragastric glucose feeding. Re-routing gastric secretions to the distal duodenum inhibited anterior migration of the worms beyond the new point of entry of gastric juices. Results following re-routing and ligation of the biliary and pancreatic secretions suggest that there is a potent cue to anteriad migration in the pancreatic secretions. Biliary secretions also appear to contain an additional migratory cue to worm migration. In order of importance the factors stimulating/inhibiting worm migration are pancreatic > gastric > biliary > glucose. The results support the hypothesis that the factors affecting worm distribution in the small intestine are interactive and synergistic, involve other luminal factors, such as 5- hydroxytryptamine and the physico-chemical gradients, and are of a regional nature such that the migratory response of a particular worm is directly related to its position in the small intestine when the cues to relocation are received.

In CFLP male tapeworm-free mice, from 2 to 7 weeks old at infection, at least 80% of singleHymenolepis diminutaestablish and grow but then are rejected; day of rejection defined as the first day when ≦ 50% of the worms were recovered. Young mice, 2 to 4 weeks old, usually reject their worms during days 16–20 while older mice, 5 to 7 weeks old, reject them during days 12–14. Biomass (total dry weight of all worms recovered on a given day from a group of mice) varied markedly with host age and was consistently greatest in 4-week-old mice. The position of the worm in the intestine did not vary with host age. The quality of mice (categorized by the Medical Research Council Laboratory Animals Centre) did not appear to affect the course of a primary, singleH. diminutainfection although, undoubtedly, the mice had varied immunological histories. Variations in time of rejection and biomass of worms recovered are accounted for by both immunological and physiological mechanisms.

SUMMARYThe anthelmintic effect of an alcoholic extract from a shrub, Diospyros mollis, popularly known as Ma-Klua in Thailand, on the adults and larvae of the dwarf tapeworm, Hymenolepis nana, in mice was studied in comparison with that of flubendazole. The experimentally infected mice were given a single oral dose of 10–1000 mg of Ma-Klua extract or flu-bendazole/kg body wt 1, 2, 3, 4, or 12 days post-infection and autopsied 14 days post-infection. Ma-Klua extract was effective in the elimination of adults (ED50 = 79 mg/kg) but not larvae. Drastic effects of Ma-Klua extract on the motility and structure of adults were observed and the number of the adults in mice decreased with time after administration of the drug 12 days post-infection. The small numbers of adults remaining in the host intestine 2 days after the drug administration showed severe damage in the gravid segments. These facts were thought to be responsible for the significant reduction in egg output observed 1 and 2 days after medication. Fresh eggs exposed to Ma-Klua extract in vitro and in vivo, or in vivo alone showed reduced infectivity. The effect of flubendazole on adults and larvae was minimal.

SummarySeven and 56-day-oldHymenolepis diminutawere exposed to complement by incubation in 50% normal rat serum (NRS) in modified Hanks' saline. Ultrastructural studies revealed that the scolex/neck region remained relatively intact whilst in the strobila region microthrix denudation and loss of distal cytoplasm were observed. When complement-mediated damaged worms were incubated in vitro in 50% heat-inactivated normal rat serum (hiNRS) plus M199 or implanted into the duodenum of NMRI mice repair occurred, although destrobilated parasites were only foundin vivo. The regions undergoing repair contained tegumental protrusions, vesicles, large electron-lucent areas and large quantities of lipid. Microtriches were formed parallel to the parasite surface and were raised into a perpendicular position. It is suggested that the regenerative process exhibited after complement-mediated damage does not mimic totally the embryological development of the surface layer.

Rats harbouring 35-day-old primary infections ofMoniliformis moniliformisandHymenolepis diminutawere inoculated with equal doses ofNippostrongylus brasiliensisand were autopsied 10 days later. Significant reductions were found in the dry weight ofMoniliformisandHymenolepisand in the numbers ofNippostrongylusrecovered compared with single infections. Similarly, in infections with two parasites, the numbers ofNippostrongyluswere reduced when concurrent withMoniliformisand the dry weights ofMoniliformisandHymenolepisdecreased in the presence of each other. A comparison of the concurrent infections themselves revealed thatMoniliformisweighed significantly less in the presence ofHymenolepisthan in the three parasite infections. Parasite interactions and their possible mechanism are discussed and comparisons are made with the relevant single infection for each parasite.

This study was based on the experimental exposure of beetles to homologous and heterologous infections of Hymenolepis diminuta and Raillietina cesticillus. The results demonstrated that, for both species over the range of parasite densities employed, the presence of a primary infection had no effect on the establishment success of an homologous challenge infection. The establishment success of R. cesticillus cysticercoids was not affected by the presence of an H. diminuta infection. The existence of an R. cesticillus infection, however, severely reduced the establishment success of an H. diminuta infection. The reduction in H. diminuta establishment was greatest in R. cesticillus infections less than 10 days old. The plausibility of some of the mechanisms that are potentially responsible for the reduction in H. diminuta establishment success is discussed, as is the possible ecological significance of these results.

SUMMARYSurvival of Hymenolepis diminuta eggs (as measured by their infectivity to the intermediate host) was found to be age-dependent with an expected value of 11 days when the eggs were retained within the faecal pellet at 10 °C. The expected life-span of eggs under experimental conditions (i.e. extracted from faecal material and placed on filter paper at 30 °C) was estimated as 33 min. The mean parasite burden of populations of Tribolium exposed to known densities of H. diminuta eggs was found to rise to a plateau with increasing exposure time. The experimental results provided a method of estimating the instantaneous rate of parasite transmission: a value of 0·0004/egg/min/host/13 cm2 was obtained. The mean parasite burden of populations of T. confusum exposed to known densities of H. diminuta eggs was found to decrease exponentially with increasing host density. A second estimate of the instantaneous rate of parasite transmission of 0·004/egg/min/host/13 cm2 was obtained from the experimental results. No differences in susceptibility to infection between 2-week-old male and female beetles were found. A marked decrease, however, was apparent with increasing beetle age up to 14 weeks post-eclosion.

SUMMARYGrowth of Hymenolepis nana in mice is analysed by identifying designated stages of proglottid maturation in the strobila, and by determining the numbers of proglottids between them and the posterior terminus of the worm, as a function of time after cysticercoid infection. The proglottid maturation rate was fairly constant between stage A (onset of the somatic primordium formation) and stage B (first appearance of sperms in the seminal vesicle), but gradually fell after stage B, during pre-patent development of the worm. The actual rate of proglottization during this period coincided well with that estimated from the number of proglottids between stages A and B on an assumption of a certain maturation rate. This procedure is extended to estimate the rates of proglottid formation and shedding during later development. It is deduced that the absolute rate of proglottization increases until day 12, gradually decreasing afterwards, and that from 2300 to 2800 proglottids are shed by day 20, until which from 4000 to 4500 proglottids have passed stage A. It is shown that the proportionate increase of the gravid region in older worms depends not only on a decrease in the rate of proglottization, but also on the prolonged period during which proglottids stay in the strobila.

SUMMARYPreparations of isolated brush border plasma membrane of Hymenolepis diminuta and H. microstoma possess the following enzymatic activities: alkaline phosphohydrolase (E.C. 3.1.3.1); Type I phosphodiesterase (E.C. 3.1.4.1); ribonuclease (E.C. 3.1.4.22); adenosine triphosphatase (E.C. 3.6.1.3); and 5′-nucleotidase (E.C. 3.1.3.5). The following enzymatic activities could not be demonstrated in either membrane preparation: Type II phosphodiesterase (E.C. 3.1.4.18); cyclic adenosine-3′, 5′-monophosphate phosphodiesterase (E.C. 3.1.4.17); leucine aminopeptidase (E.C. 3.4.11.1); maltase (α-glucosidase; E.C. 3.2.1.20); and lactase (β-galactosidase; E.C. 3.2.1.23). These data generally agree with those of previous studies in which similar membrane-bound enzymes were demonstrated in intact (living) worms.

The hydrolytic, reductive and oxidative enzyme systems involved in the phase I biotrans formation of xenobiotic compounds have been investigated inHymenolepis diminuta. AdultH. diminutaare able to carry out a range of hydrolytic and reductive reactions, but in common with other helminths oxidative detoxification reactions were absent (oxidative demethylation, aniline hydroxylation, nitrobenzene hydroxylation, biphenyl hydroxylation). These oxidative reactions were readily demonstrated in rat liver. Extracts ofH. diminutahydrolysed nitrophenylphosphates and inorganic pyrophosphate, but not arylsulphates, nor could epoxide hydratase activity be detected.N-Deacetylaseactivity was present. However,O-deacetylase activity could not be demonstrated, although butyrate and palmitate, but not benzoate, esters were hydrolysed.H. diminutawas capable of hydrolysing a range of a-and β-glycosides, but not β-glucuronides. Extracts ofH. diminutareduced azo-compounds, aldehydes and disulphides, but ketones and aromatic nitro-compounds were not reduced. The phase I detoxification systems ofH. diminutadiffer considerably from those of its rat host; the results also suggest that, within the cestodes, there may be considerable species variation in detoxification reactions.

Oncospheral agglutination given by sera immunized with Hymenolepisnana eggs is described as a new way of assessing H. nana infection. All sera of mice which possessed acquired protective immunity against reinfection by H. nana eggs had the potency to induce oncospheral agglutination in vitro. Only oncospheres, which had been hatched, agglutinated, no agglutination occurred in sera from uninfected mice. Oncospheral agglutination was carried out by mixing 0·1 ml of serial two-fold dilutions of serum and 0·1 ml of Hanks' balanced salt solution containing about 600 hatched oncospheres. Titre of agglutinins was indicated as a reciprocal of the final dilution capable of giving agglutination clusters made of three or more oncospheres. Agglutinins developed within 14 days after a primary infection with 500 shell-free eggs. There was no rapid increase of agglutinins within 4 days following a secondary infection. The titre increase coincided with the increase in dosages of eggs. Agglutinins were thought to be immunoglobulins, because the potency of the serum to agglutinate oncospheres was extinguished after absorption of globulins with rabbit anti-mouse globulin serum.Agglutinins were produced in rabbits by intravenous injections of shell-free eggs. The titres of the rabbit sera were much higher than those of mouse sera.

SUMMARYInfection withHymenolepis microstomasignificantly affected the lipid metabolism of young male Balb/C mice. Infection increased the rates of hepatic fatty acid and cholesterol synthesis and cholesterol synthesis by the gut. Decreases were recorded in testicular fatty acid synthesis and in the weights of testes and white epididymal adipose tissue. Plasma glucose decreased rapidly during infection. The observed changes in lipogenesis could not be attributed to changes in food intake or body temperature. The changes are discussed in relation to nutritional interactions between host and parasite and the possible effects on host hormone levels. The presence of newly synthesized fatty acid inH. microstomais also reported.

SUMMARYThe effects of the phenothiazine, Stelazine, on Hymenolepis diminuta were investigated. The cestode was incubated for 10 min at 37 °C with 1 mM trifluoperazine, in the presence and absence of Ca2+. Assay of brush border enzymes showed that drug treatment lowered the activities of alkaline phosphatase, Ca2+-ATP'ase, 5′-nucleotidase and type 1 phosphodiesterase. This occurred in parallel with a significant reduction in tegumental protein. Under these conditions gross changes in ultrastructural appearance and cellular organization were observed. There was a lack of ordered microtriches and the distal cytoplasm was absent. Glycogen granules were scattered throughout the cytoplasm within the subtegumental layer. The connective tissue also appeared to be in some disarray. The effects of Stelazine appeared to be dependent on time and were significantly increased when Ca2+ was included in the incubation medium. Incubation with the less hydrophobic phenothiazine trifluoperazine sulphoxide had minimal effect on the integrity of the cestode. The results reported here support the premise that certain phenothiazines may be considered as potential cestocidal agents.

SUMMARYThe effect of the intestinal changes brought about by the expulsion ofTrichinella spiralisin rats was studied in relation to the growth and survival of a concurrent infection withHymenolepis diminuta, a cestode not normally rejected by the rat in low-level infections. Growth ofH.diminutawas stunted in rats givenT.spiralisjust before, or after, infection withH.diminuta, the stunting being more pronounced when the cestode was given closer to the period of inflammation. There was no loss of the cestode from dual-infected rats and no evidence for destrobilation was found. LowerT.spiralisburdens had a correspondingly weaker effect on growth ofH.diminuta, and stunting was abolished by administration of the anti-inflammatory drug cortisone acetate. It is concluded that the stunting ofH.diminutais probably due to the non-specific inflammatory component of the rat's response toT.spiralisinfection.

SUMMARYA series of development stages (I–XI) have been devised to describe the development of the cyst wall of the metacestode ofHymenolepis diminuta. The cyst wall possesses tegumentary, muscular, fibrous and inner cyst tissues, the developmental rates and differentiation patterns of which are not identical. The tegumentary tissue differentiates posteriorly. Its microvillus-bearing distal cytoplasm remains simple until scolex retraction, after which rapid increase in depth followed by vacuolation occurs and basal membrane infoldings surround Phase 3 fibrogenesis fibrils. Senescence, which also affects the tegumentary cytons, then ensues. The muscle system development is posteriad and maturation, completed before scolex retraction, is followed by myocyton senescence. Posteriorly differentiated fibroblasts commence Phase 1 fibrogenesis after scolex retraction and the primary fibrous zone is fully established within approximately 6 days. Phase 2 and 3 fibrogenesis develop centrifugally, the fibrils of Phase 2 surrounding the tegumentary cytons and myocytons prior to their senescence, and those of Phase 3 lying more peripherally. The inner cyst tissue, established posteriorly, differentiates anteriorly, centripetally and early, the penultimate stage commencing just before scolex retraction, about 6 days after which the final maturation junctional complexes start development. Neitherin vitroexcystment nor infectivity of the definitive host can be satisfactorily achieved before the initial development of the primary fibrous zone. This may play a skeletal role during excystment, and is shown to be unaffected by the digestive enzymes which cause loss of cytoplasmic integrity in the outer regions of the cyst.

SUMMARYA membrane-bound monoamine oxidase (EC 1.4.3.4) was demonstrated in homogenates of Hymenolepis diminuta. The enzyme oxidized a variety of biologically active amines (in decreasing order: dopamine, adrenaline, noradrenaline, tryptamine, tyramine, octopamine), there was, however, no activity with 5-hydroxytryptamine or benzylamine. No diamine oxidase (EC 1.4.3.6) could be detected in H. diminuta (using histamine, cadaverine or putrescine as substrates). The monoamine oxidase from H. diminuta was not inhibited by azide, hydroxylamine or semicarbazide, but was inhibited by cupferron, α-α dipyridyl and iodoacetamide, and by the specific monoamine oxidase inhibitors pargyline, nialamide and iproniazid. Several anthelmintics were also found to be inhibitors of monoamine oxidase. The possible roles of monoamine oxidase in H. diminuta are discussed.