The first internal transcribed spacer (ITS1) of the nuclear ribosomal DNA repeat was sequenced for members of the Schistosoma japonicum species complex (S. malayensis, S. mekongi and 2 geographical isolates of S. japonicum). The ITS1 is composed of 3 distinct regions: the 5′ end (23 nucleotides); a tract of approximately 90–140 nucleotides, which occurs up to 7 times in tandem, the number varying even within an individual in all species investigated in this study; the 3′ region (378 nucleotides), which lacks repeats. There is size and sequence variation among copies of the ITS1 repeat within a single individual. The relative abundances of size variants of ITS1 in S. japonicum have been ascertained by hybridizing genomic digests with an ITS1 probe. Multiple repeats and intra-individual variation in numbers and abundance of these is a feature of the Asian schistosomes, but not generally of African schistosomes. Possible reasons for this difference in ITS1 between African and Asian schistosomes are discussed. The ITS1 repeat sequences described for African schistosomes are different to, and cannot be aligned with, those from the Asian species described here, whereas the remainder of the ITS1 can be aligned quite easily.

The initial infection process of nematode-trapping fungi is based on an interaction between the trapping structure of the fungus and the surface of the nematode cuticle. A bioassay was designed to investigate the predatory response of several isolates of nematode-trapping fungi against 3 mutants of Caenorhabditis elegans (AT6, AT10 and CL261), which have been reported to differ in the reaction of their cuticle to antibodies and lectins. The bioassay was also applied to infective larvae of animal (Haemonchus contortus, Teladorsagia (Ostertagia) circumcincta and Trichostrongylus axei) and plant (Meloidogyne spp.) parasitic nematodes. Differences in trapping ability were most marked in the first 24 h, and were density dependent. Although the isolate of Arthrobotrys responded very rapidly in the first 24 h, Duddingtonia flagrans was generally the most effective isolate and Monacrosporium responded relatively poorly throughout all experiments. All the fungi tested trapped the srf mutants of C. elegans more efficiently than the wild type, and there were differences between the different srf mutants of C. elegans. Differences in trapping ability were also observed between different isolates of D. flagrans; similarly, differences in trapping behaviour were observed not only amongst the different species of plant-parasitic nematodes, but also between the sheathed and exsheathed larvae of the animal-parasitic nematodes.

Two clones from separate isolates of Trypanosoma evansi in Indonesia were found by polymerase chain reaction (PCR) analyses to contain 3 different repeated nuclear DNA sequences of Trypanosoma brucei spp: the consensus sequence for a highly repetitive 177 base pairs and the gene repeats encoding procyclin and the spliced leader. In addition, the 994 bp minicircle sequence of one of the clones was determined, and PCR amplification primers specific for minicircles of T. evansi were identified that do not amplify minicircle sequences in the T. brucei spp. clones tested.

The population dynamics of insect–pathogen interactions are examined with the aid of simple mathematical models. Three concepts of central importance to the interpretation of population behaviour are discussed, namely the ability of the pathogen to persist within its host population, the ability to regulate and depress host population abundance, and the ability to induce non-seasonal cyclic changes in host density. The selection of pathogen species or strains to depress pest population growth is discussed and the optimal characteristics are shown to be intermediate pathogencity combined with an ability to reduce infected host reproduction, high transmission efficiency, including elements of vertical as well as horizontal transmission stages. When the pathogen plays a significant role in the regulation of host population growth, it is argued that many insect–pathogen interactions will exhibit non-seasonal oscilations in host and pathogen abundance. Mathematical models are used to explore the patterns of population behaviour that result from the continual introduction of a pathogen into a target pest population. It is shown that there exists a critical introdution rate, above which the eradication of the pest is theoretically possible. Significant reductions in pest population abundance will not occur until the introduction rate approaches this critical value, whereupon the oscillatory behaviour of the interaction between host and pathogen population will be suppressed.

A general dicussion is given of the problems arising from the combined use of chemical agents and pathogens for the control of pest species, and the evolutionary pressures acting on host and pathogen populations.

A method is described by which viable sporozoites of the sheep parasite, Eimeria arloingi, may be obtained in sterile solution and free of any extraneous vegetable matter. Faeces are washed through metal sieves and the oocysts separated by a gravity floatation technique. The oocysts are sterilized and floated free of any remaining faecal debris in a solution of sodium hypochlorite. Sporulation is induced by constant aeration in water. Excystation is effected by incubating firstly under an atmosphere of carbon dioxide and secondly in a solution of trypsin and sheep bile.

I wish to thank Professor H. R. Carne for encouragement and advice, and Mr R. F. Jones for skilful technical assistance.

The immune reaction of larvae of D. algonquin against P. bochei was characterized either by the melanization and encapsulation of the parasites or by the production of melanized particles. In successful host reactions the eggs of the parasite were killed before they were completely melanized and encapsulated by haemocytes. When host larvae were transferred during the early stages of infection to a diet that contained phenylthiourea (PTU), the incidence of melanization and encapsulation of the parasites was significantly reduced. However, if the hosts were transferred to the diet just before the eggs of the parasite were normally melanized, PTU had virtually no inhibitory effect, and the immune reaction rate remained very high. These results suggest that the biochemical reactions associated with melanization constitute an important part of the immune reaction of D. algonquin. The data provide additional evidence to support the idea advanced by other workers that the phenoloxidase system plays an important role in insect immunity against some internal metazoan parasites. It is suggested that, during infection, the phenoloxidase system of the haemocytes is activated by hormonal imbalance.

When immunoglobulin G (IgG) was incubated with Spirometra mansoni plerocercoid (sparganum), it was cleaved into Fab and Fc fragments. Fab/c fragments were also hydrolysed. The digestion was accelerated by dithiothreitol (DTT), indicating that cleavage of IgG heavy chain was due to a cysteine protease secreted into the medium. The responsible enzyme, of Mr 27 (± 0·8) kDa, was purified by a series of thiopropyl affinity, Sephacryl S-300 HR and DEAE-anion exchange chromatographies, either from worm extracts or from excretory–secretory products (ESP). The purified, thiol-dependent protease showed an optimal activity at pH 5·7 with 0·1 M sodium acetate but was active over the pH range 4·5–8·0. Its activity was inhibited completely by 10−5 M L-trans-epoxysuccinylleucylamido(4-guanidino) butane (E-64) and 1 mM iodoacetamide (IAA), but by only 53% using the specific cathepsin L inhibitor, Z-Phe-Phe-CHN2 (5 × 10−5 M). Partial NH2-terminal amino acid sequence was Leu-Pro-Asp-Ser-Val-Asn-Trp-Arg-Glu-Gly-Ala-Val-Thr-Ala-Val which showed 80% homology to human cathepsin S. Immunoblot analysis showed that sera from infected patients exhibited IgE antibody reaction. It is proposed that cleavage of immunoglobulin by an excreted–secreted, cathepsin S-like, allergenic protease is a mechanism of immune evasion used by the sparganum.

Restriction endonuclease digestion of genomic DNA generates DNA fragments of unique size, dependent upon the particular base sequence. Following fractionation by agarose gel electrophoresis, repetitive DNA can be visualized as distinct bands in stained gels and the restriction fragment length of such bands used as diagnostic characters. Restriction fragment length differences were detected between species within the genera Trichinella, Caenorhabditis, Romanomermis, Steinernema (syn. Neoaplectana) and Meloidogyne. This technique provides a new tool for the taxonomist, which is independent of phenotypic variation and it enables the rapid and reliable separation of closely related species.

With few exceptions, the specific activities of the glycolytic enzymes and the steady-state content of glycolytic and associated intermediates in protoscoleces of the horse (E.g.H) and sheep (E.g.S) strains of Echinococcus granulosus and the closely related E. multilocularis (E.m.) are very similar. Phosphorylase, hexokinase, phosphofructokinase and pyruvate kinase catalyse non-equilibrium reactions and the patterns of activity for pyruvate kinase, phosphoenolpyruvate carboxykinase and malic enzyme are similar in the three organisms. The levels of tricarboxylic acid cycle intermediates in E.g.H., E.g.S. and E.m. are of the same order as those reported in tissues with an active cycle. Each has a complete sequence of cycle enzymes but there are substantial differences between the three parasites with regard to the activity of individual enzymes, The activities of NAD and NADP-linked isocitrate dehydrogenases are significantly lower in E.g.H. than in E.g.S. and particularly in E.m. which suggests that the tricarboxylic acid cycle may play a more important role in carbohydrate metabolism and energy production in the latter parasites. Nevertheless, the three organisms utilize fermentative pathways for alternative energy production, fix carbon dioxide via phosphoenolpyruvate carboxykinase and have a partial reversed tricarboxylic acid cycle. It is speculated that in vivo more carbon will be channelled towards oxaloacetate than pyruvate at the phosphoenolpyruvate branch point. The steady state content of ATP and the ATP/AMP ratios are low in the three organisms, suggesting a low rate of ATP utilization in each.

This paper describes two approaches to evaluate the use of fish macroparasites as bioindicators of heavy metal pollution at selected river stretches in Austria. Firstly changes in the diversity and richness of endoparasites of the cyprinid barbel, Barbus barbus (L.), were tested in relation to heavy metal contents in the aquatic system. Secondly, the bioaccumulation potential of cadmium, lead and zinc was assessed in the acanthocephalan, Pomphorhynchus laevis (Müller, 1776), and compared with that in the muscle, liver and intestine of its barbel host. The present results indicated that in order to validate the role of parasite community patterns related to heavy metal pollution, more investigations on food web dynamics, interelationships between parasites and the presence/absence of intermediate hosts will be essential. Heavy metal concentrations differed significantly between the organs of barbel and P. laevis (P=0·001) with levels up to 2860 fold in the parasite. The high level of heavy metal accumulation in P. laevis compared with that in its barbel host, suggests that despite variability in the parasite infrapopulation, host mobility and feeding behaviour, P. laevis is a most sensitive indicator of heavy metals in aquatic ecosystems.

1. Membranes prepared from chicken skin provide a suitable medium through which Aëdes aegypti females may be induced to gorge.

2. Under suitable conditions the proportion of female A. aegypti which will gorge through membranes, though more variable than when a living chick is offered, is great enough for experimental purposes.

3. It is shown that the gorging reaction in A. aegypti is provoked by a heat gradient between the environment and the food-limiting membrane.

4. The feeding reactions of A. aegypti towards whole blood, fractions of blood, and other sub-stances have been studied. It was found that (a) whole blood, and red corpuscles in saline when ingested through membranes go directly into the stomach which becomes fully distended; (b) haemoglobin in plasma or distilled water is ingested to a lesser degree than whole blood or red corpuscles in saline, and plasma alone is rarely ingested, but all these pass to the stomach; (c) sweet solutions containing glucose or honey are seldom imbibed through membranes and pass to the stomach or diverticula, but only the diverticula are fully distended.

When offered as open drops (a) blood is seldom ingested, but if ingested passes to the stomach; (b) haemoglobin in plasma or water, or plasma alone, are very rarely ingested, but pass mainly to the stomach; (c) sweet solutions containing honey or glucose, or mixtures of blood and honey are readily ingested and pass mainly to the diverticula which become fully distended, though traces may be found in the stomach.

5. Aëdes aegypti may be infected with Plasmodium gallinaceum by allowing them to gorge on drawn infected chicken blood through a membrane. Infection rates comparable to those obtained when the mosquitoes are fed directly on living chickens may be obtained by this method.

6. If infected mosquitoes are allowed to gorge upon uninfected blood through membranes they eject viable sporozoites into the blood. When young chicks are injected intravenously with blood so infected, infections are produced which in period of incubation and intensity are comparable with those resulting from the bites of infected mosquitoes.

7. The ejection of sporozoites through membranes in this manner provides a ready means of obtaining sporozoites free from glandular tissue.

8. Sporozoites collected by this method will be suitable for in vitro experiments upon the action of drugs on sporozoites, and also as a source of material for studying in tissue cultures the developmental stages of the malaria parasite arising directly from the sporozoite.

We wish to thank Prof. D. Keilin, F.R.S., and Dr P. Tate for helpful criticism and advice during the course of the work.

Humoral encapsulation is an effective defence reaction against fungal pathogens. The development of injected spores in the haemocoele of Chironomus larvae may be completely (Aspergillus niger) or partially (Mucor hiemalis) prevented by this reaction. The encapsulation proceeds very rapidly; within 5 min of injection most of the spores are enclosed in a solid capsule. Disintegrating blood cells may participate in the formation of capsule substance, but this is not usually the case. Encapsulation also occurs within the cuticle against invading hyphae of Beauveria bassiana. Histochemical tests show that the capsule substance does not consist of polysaccharides; some tests for proteins and all tests for melanin were positive. The significance of melanin formation and the biochemistry of the phenoloxidase system in insects is discussed in detail. The data presented lead to the conclusion that humoral encapsulation is based upon an activation of phenoloxidases and that the capsule substance represents a polyphenol—protein complex. In contrast to current opinion, the formation of melanin in cellular as well as in humoral encapsulation is not considered to be a secondary process, independent of the actual encapsulation procedure. Instead, the authors interpret the presence of melanin as an indication of the activity of phenoloxidases which cause the formation of a capsule substance on the surface of the parasites.

Development and forward migration of Leishmania parasites in the sandfly gut is accompanied by morphological transformation to highly motile, non-dividing ‘metacyclic’ forms. Previous studies in vitro have demonstrated that this metacyclogenesis is associated with developmentally regulated changes in expression of two major surface glycoconjugates of Leishmania, the lipophosphoglycan (LPG) and the glycoprotein protease GP63. Studies presented here are the first to examine in situ the changes in expression of these two important surface molecules which occur during amastigote-initiated development of L. major in its natural vector Phlebotomus papatasi. Immunocytochemical analysis using a GP63-specific monoclonal (3.8). and others recognizing metacyclic-specific (3F12) and common (WIC79.3) epitopes of LPG on logarithmic and metacyclic promastigotes, demonstrates: (1) clear expression of LPG and GP63 from 2 and 7 days post-bloodfeeding, respectively; (2) developmental modification of the LPG molecule as parasites undergo forward migration and morphological changes associated with metacyclogenesis; and (3) striking deposition of large amounts of parasite-free excreted LPG on/in the epithelial cells of the gut wall.

The investigation of Price & Clancy (1983), which demonstrated a significant positive correlation between total helminth species number per host species and geographical range of freshwater fish host species in Britain, was re-examined using a different measure of parasite species richness. Re-calculation of the correlations between the two parameters after controlling for the effect of the composition of the list of fish by excluding, on biological and distributional grounds, 2 species of agnathans and 7 species of introduced teleosts, and for the effect of sampling effort by using helminth richness in the richest component community of each fish species rather than check-list data, reveals no significant relationship between helminth species richness and host range. Habitat and an omnivorous host diet now appear more significant determinants of helminth richness than the accumulation of parasites by predation. The findings provide little support for the interpretation of the relationship between helminth species richness and host range in terms of island biogeographic theory, but do support an alternative explanation in terms of the colonization time hypothesis, i.e. that helminth species richness is related to the time since the fish host arrived in Britain.

Biomphalaria glabrata snails are known to be castrated by infection with the trematode parasite Schistosoma mansoni 4–6 weeks post-infection. The pattern of oviposition in the first 35 days post-exposure (p.e.) was investigated, in snails aged 14 weeks and measuring 7–10 mm diameter which had not commenced egg-laying, by counting the numbers of eggs laid in 7-day intervals. A group of exposed snails was compared with a control non-exposed group. The exposed group included both parasitized and non-parasitized snails, and showed a significant increase in the median number of eggs laid during the periods 14–21 and 22–28 days p.e. Throughout the entire 35-day period exposed non-parasitized snails laid significantly more eggs than control snails, while parasitized snails laid significantly more eggs than controls during days 22–28 p.e. and significantly fewer during days 29–35 p.e. Parasitized snails also laid significantly more eggs/egg mass in the period 16–28 days p.e. than did control snails. Growth of the snails was measured. By day 28 p.e. the mean diameter of the exposed group was significantly greater than that of the control group. The increase in oviposition by snails soon after exposure is discussed in terms of a compensatory response for expected future suppression of egg-laying. The fact that parasitized and non-parasitized snails both show increased oviposition indicates that normal development of the parasite is not necessary to trigger the response.

Naturally infected Biomphalaria glabrata snails were collected at two sites near Belo Horizonte, Brazil, and Schistosoma mansoni cercariae isolated from single snails were used to infect individual mice. Genetic comparison of single worm DNA was accomplished by hybridization of Southern blots to a polymorphic repetitive DNA element. Genetic profiles of parasite individuals revealed a diverse array of parasite genotypes in naturally infected intermediate hosts. The observed distribution of schistosome genotypes among intermediate hosts indicates that over half of the infected snails harbour multiple miracidia. Snails were more likely to carry multiple infections than expected by chance. This degree of overdispersion combined with high levels of genetic variability facilitates multi-genotype transmission and helps maintain parasite genetic diversity.

Gene expression studies in adult females of Schistosoma mansoni cultured in vitro revealed that the transcription of female-specifically expressed genes is influenced by pairing. In contrast, the activity of genes that are expressed in both genders was not affected by contact with the male. The transcription of genes was monitored in paired, separated and remated females. The transcript level of female-specifically expressed genes decreases within a few days following separation from males. Remating of uncoupled females with males leads to the reinitiation of transcription. These results provide strong evidence for the influence of the male on gene transcription in the female and contribute a molecular basis for the classical histological observation that the maturation of females is male dependent. The data also show that the culture system is suitable to monitor gene expression and, furthermore, they indicate de novo RNA synthesis in vitro.

A Bayesian geostatistical model was developed to predict the intensity of infection with Schistosoma mansoni in East Africa. Epidemiological data from purpose-designed and standardized surveys were available for 31458 schoolchildren (90% aged between 6 and 16 years) from 459 locations across the region and used in combination with remote sensing environmental data to identify factors associated with spatial variation in infection patterns. The geostatistical model explicitly takes into account the highly aggregated distribution of parasite distributions by fitting a negative binomial distribution to the data and accounts for spatial correlation. Results identify the role of environmental risk factors in explaining geographical heterogeneity in infection intensity and show how these factors can be used to develop a predictive map. Such a map has important implications for schisosomiasis control programmes in the region.

Ligand-gated chloride channels, including the glutamate-(GluCl) and GABA-gated channels, are the targets of the macrocyclic lactone (ML) family of anthelmintics. Changes in the sequence and expression of these channels can cause resistance to the ML in laboratory models, such as Caenorhabditis elegans and Drosophila melanogaster. Mutations in multiple GluCl subunit genes are required for high-level ML resistance in C. elegans, and this can be influenced by additional mutations in gap junction and amphid genes. Parasitic nematodes have a different complement of channel subunit genes from C. elegans, but a few genes, including avr-14, are widely present. A polymorphism in an avr-14 orthologue, which makes the subunit less sensitive to ivermectin and glutamate, has been identified in Cooperia oncophora, and polymorphisms in several subunits have been reported from resistant isolates of Haemonchus contortus. This has led to suggestions that ML resistance may be polygenic. Possible reasons for this, and its consequences for the development of molecular tests for resistance, are explored.

This study analyses the anti-proliferative effect of lemongrass essential oil and its main constituent (citral) on all 3 evolutive forms of Trypanosoma cruzi. Steam distillation was used to obtain lemongrass essential oil, with chemical composition determined by gas chromatography (GC) and GC coupled to mass spectrometry (GC-MS). The IC50/24 h (concentration that reduced the parasite population by 50%) of the oil and of citral upon T. cruzi was determined by cell counting in a Neubauer chamber, while morphological alterations were visualized by scanning and transmission electron microscopy. Treatment with the essential oil resulted in epimastigote growth inhibition with IC50=126·5 μg/ml, while the IC50 for trypomastigote lysis was 15·5 μg/ml. The IC50/48 h for the Association Index (% macrophage infection×number of amastigotes per cell) was 5·1 μg/ml, with a strong inhibition of intracellular amastigote proliferation. Ultrastructural analysis demonstrated cytoplasmic and nuclear extraction, while the plasma membrane remained morphologically preserved. Our data show that lemongrass essential oil is effective against T. cruzi trypomastigotes and amastigotes, and that its main component, citral, is responsible for the trypanocidal activity. These results indicate that essential oils can be promising anti-parasitic agents, opening perspectives to the discovery of more effective drugs of vegetal origin for treatment of parasitic diseases. However, additional cytotoxicity experiments on different cell lines and tests in a T. cruzi-mouse model are needed to support these data.