Nematodes of the genus Heterorhabditis are insect parasites that are widely used as biological control agents. When conditions are unfavourable for reproduction in H. bacteriophora, a long-lived, non-feeding, survival and dispersal stage, the dauer juvenile (DJ), is formed. This DJ stage is also adapted for host finding and infection. When it infects a suitable host, the DJ recovers and resumes growth and development. We describe a series of methods for improved detection of recovery in H. bacteriophora. We also describe some of the physiological changes that occur immediately after the onset of recovery in these nematodes as revealed using fluorescent nucleic acid binding SYTO dyes. Although recovery could be monitored using morphological changes, we found that observation of the uptake of fluorescent latex microspheres by recovering nematodes was a far more sensitive and efficient means of detecting recovery. SYTO dyes were also found to be useful indicators of recovery, binding to the pharyngeal glands and genital primordia as little as 3 h after the onset of recovery. The use of SYTO dyes also indicated that the pharyngeal glands produce large quantities of RNA following the onset of recovery, implying that these structures may produce proteins important in the infection and/or feeding process of H. bacteriophora.

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.Nucleotide sequence data published here have been submitted to GenBank™ and are available under accession numbers AF461124 and AF461125 (18S–28S); AY121842 and AY121843 (cytochrome c oxidase 1); AY1844 and AY121845 (paramyosin). 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 effect of cadmium and zinc at concentrations ranging from 0.1 to 10000 μg/l on tail loss in cercariae of Diplostomum spathaceum was investigated at 3 temperatures (12, 20 and 25 °C) and 3 levels of water hardness (distilled water, soft water and hard water). Increasing tail loss over time was found to be linked with a parallel decrease in cercarial survival in controls. Exposure to the heavy metals induced, especially at high concentrations, a change in the relationship between cercarial tail loss and survival, causing either stimulation or inhibition of tail loss dependent on the individual toxic exposure. Under most environmental conditions the rate of tail loss over time was reduced by increasing metal concentrations. Inhibition of tail loss occurred in a limited number of both control and metal-exposed cercariae, with a number of low metal concentrations inducing greater inhibition than in controls. Stimulation of tail loss causing an increased tail loss rate above controls also occurred at certain high metal concentrations. Increasing water hardness and decreasing water temperature caused a reduced tail loss rate over time in both control and metal-exposed cercariae. However, with decreasing temperature a reduced rate of tail loss over time in metal-exposed cercariae compared to controls occurred at some low metal concentrations. When tail loss was compared against cercarial death of the experimental population toxic exposure induced changes in the parallel relationship of these parameters, dependent on individual metal concentration, water temperature and hardness. Differences in the relative effects of cadmium and zinc on cercariae were dependent on the environmental conditions of exposure. Both metals showed limited effects during the period of maximum cercarial infectivity (0–5 h). The mechanisms and importance of metal toxicity to cercarial tail loss are discussed.

A mermithid nematode emerging from a male ant, Prenolepis henschei Mayr, in Baltic amber is the first fossil record demonstrating nematode parasitism of ants. This parasite, described as Heydenius formicinus sp. n., is compared to extant mermithid parasites of ants, especially Allomermis myrmecophila Baylis 1921. The present fossil, together with earlier reports of braconid and mite parasitism of ants in Baltic amber, indicates that several diverse groups had already evolved parasitic associations with ants by the Eocene.

Parasites are able to influence intermediate hosts in a way that optimizes their growth and transmission to the next host. Macrocyclops albidus (Copepoda) suffer from a reduced escaping ability and an increased level of general activity, when infected with Schistocephalus solidus (Cestoda). This facilitates predation by the subsequent host, the three-spined stickleback. However, instead of adaptive host manipulation by the tapeworm, the altered copepod behaviour might be explained more simply as a constraint of the infection. Energy depletion could lead to decreased muscle performance and increased food searching activity. Furthermore, resource allocation among host tissues might change after infection. We therefore analysed the amount of storage lipids and muscle tissue before and after experimental infection. To determine the amount of muscles, we developed a new polarization-microscopic technique. Irrespective of infection, lipids and muscles were predictors of copepod survival. However, we found no effect of the parasite infection on muscles or lipids, and no indication of a change in resource allocation between these tissues. Our study suggests that behavioural changes in infected copepods are mediated by a mechanism different from energy depletion or a re-allocation of resources between muscles and lipids. We rather propose that the tapeworms directly manipulate copepod behaviour.

The identification of protective helminth antigens remains the most important challenge in the development of parasitic vaccines. To identify protective antigens of Ostertagia ostertagi, an important abomasal parasite of cattle, parasite-specific local antibodies from the abomasal mucus and from the draining lymph nodes were collected from calves immunized with multiple infections and from ‘primary infected’ animals. With these probes, Western blots of extracts and excretion/secretion (E/S) material from L3, L4 and adult life-stages as well as cDNA expression libraries were screened to identify antigens that were exclusively recognized by antibodies from ‘immunized’ calves. In the adult stage, a protein of 32 kDa was specifically detected on Western blot by mucus antibodies from ‘immunized’ animals. In the L3 and L4 larval stages, proteins situated in the regions of 28–29 kDa were recognized by mucus antibodies and a 59 kDa antigen was specifically recognized by lymph node antibodies from ‘immunized’ animals. Screening E/S material revealed no specific difference in recognition pattern between ‘immunized’ and ‘primary infected’ animals. Screening of the cDNA libraries revealed 26 relevant clones, coding for 15 proteins, among these several with potential protective capacity, immunodominant properties or functional and physiological importance e.g. metalloproteases, an aspartyl protease inhibitor and collagen.

The entomopathogenic nematode Heterorhabditis megidis may undergo several rounds of reproduction within a single host. Infective juveniles (IJs) are formed within each generation during a process referred to as endotokia matricida, which involves the progressive consumption of the parent hermaphrodite or female by the developing IJs prior to emergence from the host cadaver. The present study examines the extent to which within-host population dynamics exhibit density-dependent variation. Particular attention is paid to the effect of infection density on the relative production of IJs and ‘normal’, non-infective offspring within each generation and on the emergence of the IJs from the host. Fecundity was found to be negatively density dependent across generations. However, at high infection density the first generation hermaphrodites invested relatively more in IJs at the expense of producing non-infective offspring. It is suggested that this pattern resulted from an adaptive, phenotypically plastic allocation of reproductive investment between offspring types in response to increased competition. The F1 and F2 IJs were also shown to emerge from the host in relatively discrete pulses.

If parasitic infection is a driving force in female mate choice, then females should preferentially select parasite-free males. The role of infection on female mate choice in mammals was assessed using a 3-chambered apparatus. A female CD-1 mouse was allowed to choose between 2 tethered male mice, 1 uninfected and 1 infected with 200 larvae (L3) of the intestinal nematode Heligmosomoides polygyrus. Both uninfected and infected males were equally receptive to the oestrous females, and females did not differ in the number of visits and time spent exploring the 2 males. Female time preference was not a useful predictor of ultimate mate choice, whereas first mount preference of the female was a reliable indicator. Results indicate that female mice preferentially mated with uninfected males as evidenced by first ejaculation choice, but that male infection status did not significantly affect female reproductive success. Interestingly, litters sired by infected males contained a significantly higher percentage of females suggesting that parasite-induced hormonal changes may alter the sex ratio of the offspring. This study provides the first direct evidence of the impact of parasitic infection on mammalian mate choice.

This is the first demonstration of the in vitro development of the 3rd-stage larvae (L3) of Hysterothylacium aduncum to the adult. This was achieved in a semi-defined medium that is easy to prepare and to reproduce. The L3, collected from the peritoneal cavity of horse mackerel (Trachurus trachurus), were individually inoculated into RPMI-1640 medium +20% heat-inactivated fetal bovine serum (IFBS). It has been demonstrated that the optimum temperature for development is around 13 °C and is stimulated by the presence of 5% CO2 in the growth atmosphere, increasing the percentage moulting to the 4th larval stage (L4) by 1.9-fold (from 44 to 82%) and the average survival of the nematodes by 1.6 times (from 60 to 96 days). When the larvae were grown at different pHs, optimum development occurred at pH 4.0. Under these conditions, all the larvae moulted to the L4 and more than two-thirds transformed to the adult stage – in which 25–30% of the females laid eggs – and reached an average survival of over 4 months. When this medium was supplemented with 1% (w/v) of commercial pepsin, all the larvae reached the adult stage, at least 45% of the females oviposited, laying around 12-fold more eggs per female than in the medium without pepsin. The mean size of the eggs (non-fertilized) obtained was 56.8×47.6 μm. The mean length of the adult males obtained was between 3.2 and 5.2 cm and the females were between 3.0 and 6.5 cm. The adult specimens were morphologically identified as Hysterothylacium aduncum aduncum. This culture medium (RPMI-1640+20% (v/v) IFBS+1% commercial pepsin, at pH 4.0, 13 °C and 5% CO2 in air) could facilitate the identification of at least some of the larvae of the genus Hysterothylacium – and perhaps other anisakids – for which the specific identification and the biological study of these parasites is often difficult.

The effects of macrocyclic lactone anthelmintics (MLs) on feeding by Trichostrongylus colubriformis nematodes in vitro were examined using inulin uptake as a measure of ingestion and electropharyngeograms as a record of the electrical events associated with pharyngeal pumping. Inulin uptake was inhibited by the 4 MLs tested (EC50s 0.045–4.57 nM), with an order of potency of eprinomectin (most potent), ivermectin, ivermectin monosaccharide, and ivermectin aglycone. The MLs caused both the frequency and amplitude of pharyngeal electrical events to decrease. In individual worms the inhibition of pump frequency preceded the inhibition of pump amplitude. The order of potency of the MLs as inhibitors of frequency was ivermectin aglycone, ivermectin, ivermectin monosaccharide and eprinomectin. The difference compared with the inulin assay results are probably due to the dynamics of drug uptake in the two systems. It was possible that the nematodes in the electrophysiology experiments were effectively orally ligated by enclosure of the worm's head in the recording pipette which contained no drug. Despite this difference in relative potencies, both the ingestion assays and the electrical events indicate that MLs are potent inhibitors of the pharynx in T. colubriformis in vitro. The sequence of effects on pharyngeal electrical activity suggests that ML action involves an initial inhibitory effect on the rate of pharyngeal contractions, followed by a decrease in the amplitude of the potentials associated with pharyngeal pumping events.

Cyclophilins are a family of proteins found ubiquitously in eukaryotes, many of which bind to the immunosuppressive drug cyclosporin A (CsA). CsA has been found to have anti-parasitic effects against a variety of helminth and protozoan parasites and this activity could be mediated via cyclophilin. In this study we characterize a full length cyclophilin gene from Echinococcus granulosus, the associated natural gene and expression pattern, and investigate the functional properties of the recombinant E. granulosus cyclophilin protein. In addition, the effects of CsA were investigated on E. granulosus protoscoleces in in vitro culture. The full length E. granulosus cyclophilin cDNA encodes a protein of 20 kDa and is encoded by a single gene (EGCyP-1) comprising 2 exons separated by a 31 bp intron. The gene is expressed constitutively in all E. granulosus life-cycle stages examined. Recombinant E. granulosus cyclophilin (egCyP-1) exhibited functional enzyme activity as an isomerase. Treatment of in vitro cultures of E. granulosus protoscoleces with CsA was found to be lethal to the parasites. No protoscoleces survived treatment with 10 μg/ml of CsA over 7 culture days, as determined by observing motility and the uptake of toluidine blue dye. Untreated protoscoleces remained viable for the duration of experiments. The survival of protoscoleces was CsA dose dependent. A concentration of 10 μg/ml CsA was 100% lethal while doses of 8 μg/ml and 5 μg/ml resulted in 82% and 32% killing, respectively, after 7 days in culture. The anti-parasitic activity of CsA may have the potential to be developed as a new therapeutic agent for treatment of cystic hydatidosis in humans.