We have used immunoelectron microscopy to investigate the subcellular location of Pfs16 in Plasmodium falciparum. It was detected in the outer membrane region of gametocytes and more specifically on the parasitophorous vacuole membrane (pvm), since, during gametogenesis when the pvm disintegrates, the majority of the antigen was detected on the remains of this membrane in multilaminated whorls and not on the gamete plasma membrane. The antigen was also present on other gametocyte cellular structures, including those which we believe to be Garnham bodies, present in the host cell cytoplasm of some gametocytes. The antigen was present too on the membrane surrounding cytostomes and the resulting food vacuoles in the parasite cytoplasm.

A striking feature of toxoplasmic seroconversion is the prominent and early IgM response to a low molecular weight antigen of 4–5 kDa. Two different monoclonal antibodies directed against the 4–5 kDa antigen have been generated and used to characterize this molecule. Using these monoclonal antibodies, we could demonstrate the surface localization of the low Mr antigen by immunofluorescence and immuno-electron microscopy assays. By immunoblotting, we observed that one of the monoclonal antibodies was unable to recognize the 4–5 kDa antigen in tachyzoites propagated in cell culture, indicating an epitope variability between Toxoplasma gondii tachyzoites grown in vivo and in vitro. We discuss the implications of this latter finding in the design of diagnostic reagents.

Small subunit ribosomal RNA (srRNA) genes of three Theileria species, one Cytauxzoon and four Babesia species were amplified using the polymerase chain reaction (PCR), cloned and sequenced. Our sequences were aligned with srRNA sequences previously published for eight species of Apicomplexa, one ciliate and one dinoflagellate, the last two being included as free-living outgroup species. Phylogenetic relationships between the organisms were inferred by four in-dependent methods of phylogenetic tree construction using the ciliate Oxytricha nova to root the trees. Our trees fail to show a consensus branching order. They do, however, clearly indicate that the theilerias form a monophyletic taxon derived from a paraphyletic group which includes the species B. equi, C. felis and B. rodhaini. The distance trees indicate that the babesias sensu stricto (B. canis, B. caballi, B. bigemina and B. bovis) form another monophyletic taxon which diverged before the theilerias separated from the above-mentioned paraphyletic group. The parsimony and maximum likelihood trees suggest that the babesias and theilerias are sister taxa, both of which were derived from the paraphyletic group.

Acetylcholinesterase (AChE) is present in all stages of the life-cycle of schistosomes and is located in muscle and on the surface of the parasite. Metrifonate is a drug that inhibits AChE. We compared the AChEs from three schistosome species (Schistosoma mansoni, Schistosoma haematobium and Schistosoma bovis) that have different susceptibilities to metrifonate in vivo. Sensitivities to AChE inhibitors were similar. The subunits of AChE were 110 kDa and 76 kDa and the dominant molecular form of AChE was a G2 form in all three species. This was the major form on the tegument while additional molecular forms were associated with the internal tissues. Differences in relative amounts of AChE activity between these species were found in the adults but not in the schistosomula. At the adult stage the major difference between species lay in the relative amounts of AChE activity in their teguments. S. haematobium teguments carried 20 times and S. bovis 6·9 times the activity present on S. mansoni teguments. These quantitative differences associate with the relative sensitivities of these species to metrifonate.

Autophosphorylation by protein kinases has been implicated as an important control mechanism in signal transduction and growth regulatory pathways in mammalian cells. We have set out to investigate whether any such autophosphorylating protein kinase activities can be found in Trypanosoma brucei. In order to do this, we have developed a system for characterizing such protein kinase activities using an in vitro assay. This assay was carried out by fractionation of trypanosome lysates using isoelectric focusing gel electrophoresis followed by incubation of the gel in γ32P-labelled nucleotide triphosphate and subsequent autoradiography. We have identified two classes of autophosphorylating protein kinase activities. In the first class all were dependent on ATP as the phosphate donor substrate and were all found to have a molecular size of 60 kDa. Differences in the activity of these protein kinases were observed between the bloodstream and procyclic life-cyle stages. Furthermore, the addition of mammalian epidermal growth factor to bloodstream stage lysates stimulated an additional activity. The second class of autophosphorylating protein kinases utilized GTP as the phosphate donor and were all found to be 90 kDa in size. Stage-specific differences were also observed in the activity of these protein kinases.

Gyrodactylus salaris has recently become a major pathogen of Atlantic Salmon (Salmo salar) in Norway. The survivorship, population age structure and pattern of insemination of G. salaris were studied to determine the extent to which this species reproduces sexually. The age-specific mortality schedule of G. salaris could be described by an exponential model but day to day variations were large, with an increase in mortality after each birth. Modelling population growth using the best fit mortality schedule indicated that, at stable age structure, 35% of the population would consist of newborn and pre-1st birth flukes. Using testis, penis and embryo development, pre-1st birth and immediately post-1st birth flukes could be unambiguously identified, and established infections were found to contain 35% pre-1st birth flukes, as predicted. The proportion of pre-1st birth flukes in newly established infections was significantly smaller, probably because of differences in the rate of transmission between newborn and older flukes. Gyrodactylus salaris is relatively long-lived, and more than 40% of the population may survive to give birth for the third time. As gyrodactylids are protogynous, and the first daughter is probably produced asexually, this long-lived strategy ensures that a large part of the G. salaris population possesses a functional male system, and that the asexually derived flukes are a smaller component of the total population in this species. Flukes with whorls of inseminated spermatozoa within the seminal receptacle were found in all age groups possessing a functional male system, and were interpreted as having been cross-inseminated. G. salaris on susceptible Norwegian salmon appears to regularly reproduce sexually, possibly accounting for its morphological variability and wide range of potential salmonid hosts.

A previous study has shown that, under natural conditions, energy reserves of the desert toad, Scaphiopus couchii, are negatively related to the density of infection by Pseudodiplorchis americanus. However, this was based predominantly on collections of active animals from breeding congregations and inevitably selected toads which were in good condition. The parasite, a blood-feeding monogenean, occurs in burdens of up to 30 worms/host (mean intensity 6 worms/host) and represents a significant drain on reserves because the host does not feed during a 10-month hibernation. Field studies cannot resolve the possibility that larger worm densities are not observed in nature due to parasite-induced host mortality. The present study was conducted during investigations of P. americanus development and survival under controlled laboratory conditions, utilizing experimentally infected hosts which created worm densities larger than those observed in natural populations. At all temperature regimes, infected animals had smaller fat bodies than those uninfected but differences were generally not statistically significant due to large individual variations, presumably resulting from variations in past feeding efficiency. At cool temperatures (15–20 °C) there was no density-dependent effect on host fat body weight, and at a diurnal temperature cycle of 20–34 °C (simulating that experienced by host and parasite during the summer months), the effects of high temperatures were greater than the effects of infection, due to increased toad metabolic rates. The most significant effects of P. americanus were observed in hosts that began hibernation in relatively poor condition and experienced moderate temperatures (25 °C) during hibernation. The toads generally maintained packed blood cell levels (PCV) levels even when fat body weights were low, but infected animals had a lower PCV irrespective of fat body levels. In animals unfed after field collection, PCV was reduced in uninfected toads and was even lower in infected animals. Although very heavily infected toads (burdens of 35–95 worms/host) were generally in poorer condition than uninfected toads they still survived long-term hibernation under extreme nutritional stress. This study therefore confirmed observations made in field studies that there is a density-dependent relationship between the hosts' survival prospects and P. americanus infection. However, given the large variability in feeding efficiency and stored resources between individual toads, there is no evidence that the most heavily infected toads would have been unrepresented in field samples due to parasite-induced mortality.

Populations of Pseudodiplorchis americanus infecting the desert toad, Scaphiopus couchii, have previously been shown to be remarkably stable from year to year, despite wide variation in conditions promoting invasion. The present study aimed to document the survival of both first-year and pre-existing adults under controlled laboratory temperatures. First-year worm survival in experimentally infected toads was shown to be very high for the first 5 months after migration to the definitive site, and there was no difference in survival at 25 °C or 15–20 °C. There was also no density-dependent survival during the same period: 20% of worms were recovered from the host urinary bladder, irrespective of initial intensities. After the first 5 months, there was a progressive loss of worms in toads maintained at 25 °C but not at 15–20 °C. Pre-existing adult populations were shown to be virtually identical to those under natural conditions for the first 4 months after toad capture. Following this, at 25 °C, populations declined and no pre-existing adults were recovered after 11–14 months. There was no such loss of pre-existing adults at 15–20 °C for up to 14 months. A diurnal temperature cycle of 20–34 °C (simulating temperatures during the desert summer) did not lead to a significant loss of worms. These observations suggest that during one annual cycle in the desert, when temperatures remain above 20 °C for less than 6 months, most adult worms established in the host urinary bladder will survive to the next transmission opportunity. However, the similar longevity of first-year and pre-existing adults in laboratory maintained toads shows that loss of worms cannot be due solely to parasite ageing. Temperature-dependent survival of P. americanus is suggestive of a host immune response. Low temperatures, which inhibit parasite growth and development, are essential for the survival of P. americanus.

Excretory/secretory (ES) products obtained by in vitro culture of infective-stage larvae of Trichinella spiralis and T. pseudospiralis were injected intramuscularly at various intervals into mice. Mini-osmotic pumps containing T. spiralis ES products were also implanted subcutaneously and intraperitoneally into rats. The introduction of ES materials into muscles elicited extensive lesions which included dissolution of myofibres, mobilization of mononuclear and polymorphonuclear leucocytes, angiogenesis, hypertrophy of myonuclei, myotube formation, mitosis, muscle bundles becoming rounded and separated from each other, disappearance of Z, I and A bands of sarcomeres, increase in endoplasmic reticulum and Golgi complexes, decrease in glycogen and relocation of mitochondria. These are considered as degenerative/regenerative changes of muscles to injury. Immunodominant epitopes of specific 45–53 kDa glycoproteins in ES antigens of T. spiralis could not be detected in hypertrophic nuclei of injected muscles by using polyclonal and monoclonal antibodies and immunocytochemical methods. ES products of T. spiralis failed to stimulate unsensitized lymphocytes in the lymphocyte transformation test. Infective-stage larvae of T. spiralis released from muscles were found capable of forming nurse cells after injection subcutaneously into rats. It is postulated that the invasion of muscles by trichinellids elicits two independent events, i.e. a general degenerative/regenerative response of muscles and a specific change in genomic expression of myonuclei. The two events are probably mediated by different effector molecules.

Foraging strategies of eight species of entomopathogenic nematodes were predicted from their response to host volatile cues and dispersal behaviour on 2-dimensional substrates. Positive directional response to chemical cues and similar distances travelled on smooth (agar) or nictation substrates (agar overlaid with sand grains) by Heterorhabditis bacterio-phora, Heterorhabditis megidis, Steinernema anomali, and Steinernema glaseri suggest their cruising approach to finding hosts. The absence of directional response and less distance travelled on nictation substrate, than on smooth agar by Steinernema carpocapsae and Steinernema scapterisci suggest their ambushing mode of foraging. Steinernema feltiae and Steinernema sp. responded directionally to host volatiles, but travelled less distance on the nictation substrate than on smooth agar; the two species also did not nictate. The cruiser species located hosts more effectively in the sand columns, whereas the ambushers were more effective at finding hosts on filter paper. Steinernema feltiae and Steinernema sp. performed equally on filter paper and in the sand column. We conclude that H. bacteriophora, H. megidis, S. anomali and S. glaseri cruise to find hosts, whereas S. carpocapsae and S. scapterisci ambush hosts. Steinernema feltiae and Steinernema sp. are intermediary in the search continuum sharing some characteristics of both ambush and cruise foragers.

Temporal changes in glucose transport capacity in excysted scoleces of Hymenolepis diminuta were 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 the Vmax increased 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 (log V versus 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.

The 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.

PF1 (SDPNFLRFamide) is a FMRFamide-like peptide extracted from the free-living nematode Panagrellus redivivus. Here we show that this peptide causes a hyperpolarization of somatic muscle cells of the parasitic nematode Ascaris suum and a relaxation of the somatic muscle strip preparation. We have assessed whether or not the relaxation of Ascaris dorsal muscle strip by PF1 is due to (i) inhibition of the release of the excitatory neuromuscular junction transmitter acetylcholine (ACh), (ii) potentiation of the release of the inhibitory neuromuscular junction transmitter γ-aminobutyric acid (GABA) or (iii) a direct inhibitory action of the peptide on the muscle cells. Under the experimental conditions described here, tonic ACh release does not seem to be involved in determining the resting membrane potential or resting tone of the Ascaris dorsal muscle strip and thus inhibition of tonic ACh release is unlikely to explain the relaxation elicited by the peptide. Furthermore, PF1 (100 nM–1 μM) inhibited the contraction of the muscle strip elicited by bath application of ACh, suggesting either a direct inhibitory action of the peptide on the muscle cells or a potentiation of GABA release. In electrophysiological experiments, the reversal potential for the PF1 hyperpolarization was not the same as that for GABA. Thus, PF1 hyperpolarizes Ascaris muscle by a mechanism that does not involve stimulation of GABA release from inhibitory pre-synaptic terminals.