A group of monoclonal antibodies (MAb) was used to immunochemically characterize Babesia bigemina surface components. Two surface reactive MAb by an IFAT test were subsequently shown to bind to epitopes on the external surface of the parasite plasma membrane as evidenced by immunoelectron microscopy. Parasite components with relative sizes of 68, 62, 60, 58, 56, 54, 51, 49, 48, 47, 43, 36 kDa were identified with the group of MAb in at least 6 geographically different B. bigemina isolates. The antigenic components were demonstrated to be species-specific and apparently predominant. Both polyclonal antibodies (immune bovine serum) and highly diluted MAb specifically reacted with such bands producing a strong signal in Western blots.

Parasitism was recognized as a potent evolutionary force by Haldane as early as 1949 but was then largely ignored by evolutionary biologists until the mid-1970s. Interest has since been rekindled, and parasites have now been attributed major roles in areas of evolutionary biology as fundamental and diverse as social behaviour, sexual reproduction, ornamentation and speciation. These ideas have their roots in evolutionary theory, and parasitologists have as yet contributed little to their development and testing. Likewise, evolutionary biologists have been slow to assimilate the parasitological literature. This year's BSP Autumn Symposium was thus organized with the aim of promoting constructive interchange between the two disciplines.

Differentiation of epimastigotes and production of infective metacyclic forms of Trypanosoma congolense were examined in a culture system which enabled manipulation of the population density of insect forms. Scanning electron microscopy of cultures revealed the attachment sites of epimastigotes in detail, showing them to be attached as ‘clusters’ or ‘bundles’ and having associated fibrillar structures. Dividing epimastigotes were observed either within individual bundles or in association with two bundles. Metacyclic forms were detected by an immunofluorescence antibody test (IFAT) using metacyclic variable-antigen type (M-VAT) specific monoclonal antibodies, by day 7 after seeding cultures. Trypanosomes expressing M-VATs appeared singly in bundles, observed by both IFAT and an immunogold labelling method. Statistical analysis using Poisson calculations suggested that, in general, the distribution of metacyclics expressing individual M-VATs was random throughout cultures.

Light and transmission electron microscopy were used to study different stages of Perkinsus atlanticus (Apicomplexa) during induced zoosporulation, with fluid thioglycollate medium and seawater. Cytokinesis and nucleokinesis of different developmental stages were studied every 12 h during the incubation period of 72 h, at which time the zoospores became free. Uninucleated and flagellated zoospores present the apical complex formed by conoid, polar ring, micronemes, rhoptries and subpellicular microtubules observed at different sections. Ultrastructural details were compared with the other two species of the genus Perkinsus.

Using cloned lines of Plasmodium berghei producing mixed asexual and sexual (clone 234L) and purely asexual (clone 233L) parasitaemias, the courses of parasitaemia, gametocytogenesis, exflagellation, ookinete production in vitro and mosquito infectivity have been followed. For clone 234L mosquito infectivity is maximal at day 3 and has ceased by day 6 post-infection. Conversely, gametocytogenesis, exflagellation and ookinete production are at minimal levels at day 3 and rise to peaks between days 10 and 15 of infection (in TO mice infected with blood at mechanical blood passage 3). Sexual potential declines progressively with sustained mechanical passage (up to P14). Gametocyte conversion is highest early in infection and declines exponentially; however, upon each mechanical passage conversion is again raised but decreases more rapidly with succeeding passages. For practical mosquito transmission in the laboratory we consider P8 to be the useful limit for mechanical transmission for this parasite clone. Asexual parasite growth (virulence) is more rapid with increased mechanical passage inducing a rapid fall in haematocrit. By P14 the course of infection closely parallels that of the purely asexual clone 233L.

The pathogenic flagellate Blastocrithidia triatomae disrupts the digestion of Triatoma infestans; the midgut ultrastructure of bugs infected with the flagellate and of uninfected bugs is compared. Third or fourth instar larvae were dissected either unfed or 1 week after feeding.In all uninfected bugs extracellular membrane layers (e.m.l.) covered the apical microvillar border of the epithelial cells. Some midgut regions of bugs infected with B. triatomae appeared normal but often adjacent cells showed pathological effects. In affected cells the e.m.l. and the microvilli and finally the cells themselves were reduced or destroyed. Correlated with these observations of pathogenicity the method of attachment of parasites changed. When the e.m.l. were present only rarely were flagella found, but on extracellular membrane-free cells B. triatomae attached by flagellar enlargement to the microvillar border or, if this was reduced, to the apical host cell membrane. No hemidesmosome-like plaques were found at the attachment site. Although some flagella were inserted into the apical region of the cells no intracellular flagellates were observed.

(1) The evolutionary biology of molecular parasites by s. NEE and j. MAYNARD SMITH.

Hybrid proteins containing selected regions of the major surface antigens of the sporozoite and merozoite stages of Plasmodium falciparum were expressed in insect cells using baculovirus vectors. A recombinant protein containing the signal peptide from the precursor to the major merozoite surface antigens (PMMSA) fused to a fragment from the carboxy (C) terminus of the same gene was recognized by monoclonal antibodies specific for reduction-sensitive conformational epitopes within the C-terminal fragment, suggesting that correct disulphide cross-linking of cysteine residues within this region had occurred. Addition of 26 copies of the tetrapeptide repeat from the circumsporozoite protein (CSP) resulted in a protein recognized by anti-CSP antiserum as well as the conformation specific inonoclonal antibodies. Deletion of the C-terminal putative anchor sequence from both proteins resulted in secretion of protein in a fully soluble form antigenically indistinguishable from the anchor containing products. Correct conformation was not observed when the proteins were expressed as polyhedrin fusions without the signal peptide. These data indicate that the PMMSA signal peptide is recognized in insect cells and that correct assembly of disulphide cross-links is dependent upon targeting the protein to the endoplasmic reticulum.

The British Society for Parasitology gratefully acknowledges financial support for this meeting from the following organisations:

When bovine peripheral blood leucocytes were infected with Theileria parva sporozoites, immediate treatment with oxytetracycline (OTC) inhibited the development of sporozoites to mature schizonts. The extent of inhibition was dependent on drug concentration and duration of treatment. Concentrations of 5 μg/ml OTC, or higher, for 8 days completely inhibited the establishment of schizonts and their ability to transform host cells. A cytostatic effect on schizont-infected cell lines was found with three tetracyclines and was also demonstrated on uninfected lymphoblasts. The parasites were found to be sensitive t OTC during development to schizonts, but when mature and established within host cells, schizonts were not demonstrably affected. The infectivity of sporozoites and the binding of sporozoites to lymphocytes were not directly inhibited by OTC. The results may explain the action of tetracyclines when used prophylactically during immunization against East Coast fever, and also the reasons for the ineffectiveness of these drugs when used therapeutically during patent disease.

Malaria and filaria infection rates were determined for anopheline mosquitoes collected whilst biting and resting in village houses in Papua New Guinea. The number of anophelines infected with both parasites was greater than expected from the infection rates of each parasite and this difference was significant in resting collections. The excess of multiply infected mosquitoes is probably a result of a vector population composed of individuals with differing numbers of opportunities to become infected. Malaria-positive, Anopheles punctulatus from resting catches had a significantly greater number of Stage 3 Wuchereria bancroftilarvae than malaria-negative mosquitoes. However, multiply infected mosquitoes appear to suffer greater mortality than non-infected or singly infected mosquitoes when the filarial worm reaches the third stage. Any potential increase in transmission resulting from multiple infections is thereby offset by a greater mortality rate in these mosquitoes.

It was observed that uninfected red cells resuspended in supernatant from Plasmodium falciparum cultures, then examined between a glass slide and cover-slip, assumed varying morphologies. A series of experiments suggested that P. falciparum releases molecules which cause red cells to become stomatocytic (cupped). These molecules, some of which are heat- stable, have an apparent molecular weight < 12 kDa, are released at or about schizogony, and do not bind tightly to erythrocytes.

Blood-stage malaria parasites in the vertebrate host can develop either into the asexual, multiplying forms, called schizonts, or into gametocytes, the sexual stages of the parasite. In the present work we studied the differentiation into asexual parasites or gametocytes of the progeny of single, isolated schizonts of the clone 3D7A of Plasinodium falciparum, using monoclonal antibodies specific for the sexual or asexual stages of the parasite. We observed that schizonts obtained from a continuous culture undergoing serial cycles of growth and dilution with fresh red blood cells produced either only gametocytes or only asexual parasites, showing a high degree of commitment to one or the other developmental pathway.

The relative proportion of schizonts which produced gametocytes was very low at low parasite densities in culture, while at high parasite densities a much greater proportion of schizonts produced gametocytes. Nevertheless, at both low and high parasite densities individual schizonts were almost always fully committed to producing only gametocytes or only asexual parasites.

Subtherapeutic doses of chloroquine have been reported to enhance infectivity of drug-resistant Plasmodium species to their vectors. In this investigation, Plasmodium yoelii nigeriensis N67 strain showed enhanced infectivity to mosquitoes when stimulated by chloroquine. Both sensitive and resistant clones were derived from the N67 strain by dilution, showing that this strain is polymorphic for the resistant trait. A resistant subline was derived by selection under drug pressure from a chloroquine-sensitive clone, but neither the sensitive nor the resistant clones or sublines showed enhancement of infectivity in the presence of chloroquine. This suggests that the enhancement of infectivity shown by the N67 strain is a response to chloroquine stimulation shown only by certain of the genotypes within it, and that this response to chloroquine is not a trait causally connected with the genes coding for chloroquine resistance.

A parasite can be considered to be the device of a nucleic acid which allows it to exploit the gene products of other nucleic acid–the host organisms. In this view, all parasites are ‘molecular parasites’. But it is interesting to restrict our attention to nucleic acids which do not encode organisms, as these live in a purely molecular world which lacks emergent features such as fangs and ovipositors. Viruses and transposons are molecular parasites in this sense. Most viral nucleic acids do code for some proteins, such as replicases and the protein shell in which they travel between their cellular oases. Some, however, do not even have a shell and code for nothing at all–these are the ‘viroids’ (Reisner & Gross, 1985), the smallest parasites in the world.

Cyclosporin A administered to Schistosoma mansoni-infected mice at around day 20 of infection reduces the worm burden by > 60%, as assessed by portal perfusion on days 28 and 86. Those worms recovered at perfusion have been examined by light and electron microscopy for drug-induced changes in morphology. Gross parasite damage was characterized by massive bolus formation and subsequent herniation of the gut. This event was attributed to the abnormal accumulation of crystalline structures in the lumen; the crystals were closely associated with lipid droplets, and were shown by X-ray micro-analysis to contain iron. Such crystals were seen only rarely in the intestines of control worms, but they too gave small iron peaks when examined by X-ray micro-analysis. In some drug-treated worms the caecal epithelium had ruptured, thereby releasing luminal contents throughout the worm body. In addition, herniations of the gut were seen protruding through the tegument causing surface deformation and disruption of tegumental and parenchymal tissues. The structural integrity of the worm was ultimately compromised allowing access to cytotoxic effector cells of the host. The combined effects of drug action and cellular cytotoxicity presumably account for the very significant levels of worm killing achieved by CsA treatment of the host.

L-Amino acid esters, such as L-Leu-OMe, kill Leishmania amazonensis amastigotes by a mechanism which appears to involve ester hydrolysis by cysteine proteinases located in the parasite megasomes. We have examined the killing of isolated amastigotes by L-dipeptide esters and derived some structure-activity correlations. Toxicity of the compounds for the parasites was measured by a tetrazolium (MTT) reduction assay. The results show that active dipeptide esters contained at least I hydrophobic amino acid (Leu, Ile, Val, Phe or Trp). The activity of homodipeptide methyl esters depended on the nature of the amino acid, as indicated by the following series: Phe-Phe-OMe < Val-Val-OMe < Leu-Leu-OMe < Trp-Trp-OMe < Ile-Ile-OMe. The nature of the amino acids in Leu-X-OMe and X-Leu-OMe was relatively unimportant when X was Phe, Trp or Val. However, when X was Ala or Gly, Leu-X-OMe was several-fold more active than X-Leu-OMe.

The object of this paper is to suggest that there may be an unexpected connexion between parasites and the evolution of sex, using for illustration an unfamiliar type of parasite, the selfish chromosome. The major intellectual challenge of sexuality is to an environment which is continually getting worse. The elegant solution given by the Red Queen theory (Levin, 1975; Hamilton, 1980; Bell, 1982; Bell & Maynard Smith, 1988) is that the relevant aspect of the environment is provided by antagonists—pathogens, predators and competitors—which, because they can respond adaptively so as to negate any improvement that has been made, provide a constant stimulus for continued evolution. Sexuality and recombination are favoured because some of the new combinations of genes which they create are resistant to the current population of antagonists. In other respects, sex and recombination are probably highly disadvantageous: outcrossed sex is expensive because it halves the rate of transmission of genes, while recombination breaks up successful combinations of genes. It is only in certain circumstances that the necessity for continual counter-adaptation will overcome these disadvantages: in particular, the damage (reduction in fitness) caused by an antagonist must be substantial, and the amount of damage must depend on a genetic interaction between the antagonistic species. These requirements are often satisfied by host—parasite systems, where both the ecological and genetic interactions between the antagonists may be very severe and highly specific (see reviews by Day, 1974 and Burdon, 1987). It is possible, therefore, that sex and recombination are maintained in natural populations largely through the dynamics of the coevolution of hosts and their parasites. This is certainly compatible with the major ecological patterns shown by sexual systems, with outcrossed sex being more common in the sea than in freshwater, more common at low than at high latitudes, and generally more common in stable, complex, climax environments where interactions between species are expected to be more frequent and intense (Bell, 1982). However, there is as yet no evidence which conclusively supports a direct causal link between the incidence of parasitism and the rate of recombination. In particular, it has never been demonstrated that a particular parasite has the effect of eliciting, directly or indirectly, a greater rate of genetic recombination in its host. We suggest that such a parasite exists; both the parasite and its effects are well known, but have never been interpreted in the context of the evolution of recombination through host—parasite coevolution. It is in many respects a rather unusual parasite. We shall argue that B-chromosomes represent highly evolved parasitic DNA, transmitted through the germ line and often eliciting greater rates of recombination in the host genome.

An oligonucleotide mixture based on the 21 base pair repeat sequence of Plasmodium falciparum was covalently coupled to biotin and used as a probe to detect P. falciparum DNA. The limit of detection was 10 ng. This method was further developed as a fingerprint assay for parasite strain typing. After restrictian enzyme digestion, blotting and hybridization, distinct banding patterns were obtained for the strains tested and these were reproducible. In addition, discrete differences were found between PLF-3 S + /S-strains which may implicate genetic reorganization in the switching mechanism which occurs when parasites are passed from an intact to a splenectomized animal.

It has been shown elsewhere that the immunosuppressive drug cyclosporin A (CsA) exerts profound schistosomicidal activity when given to infected mice via a multiple administration regime. We show here that a single treatment regime also effects significant reductions in worm burden. Moreover, drug efficacy is maintained at CsA concentrations shown to be subimmunosuppressive in other systems. Subcutaneous treatment effected higher levels of schistosomicidal activity than intraperitoneal or oral treatment, irrespective of whether the drug was given prior to or during infection. Topical application of CsA to the skin site of cercarial penetration, prior to infection resulted in no reduction in worm burden. Systemic release of CsA from a slowly adsorbed, oil-based drug-vehicle combination effected greater levels of killing than when CsA was dissolved in an aqueous drug vehicle. All developmental stages of Schistosoma mansoni were susceptible to killing by a single dose of CsA but, in the case of liver-stage worms, an increased concentration of drug and a longer period between treatment and portal perfusion were needed for killing to be measurable as a reduction in worm numbers.