Schistosoma mansoni is the main causative agent of intestinal schistosomiasis which affects millions of people worldwide. At the larval stage, miracidia are released into bodies of water where they utilize their motility to successfully infect their intermediate host, snails. Here, we revisit the motility and survival of S. mansoni miracidia throughout its life span. Briefly, miracidia motility was monitored at 30-min and 60-min intervals under the presence/absence of natural/artificial light. Based on a subjective evaluation of activity, body shape and transparency, 6 categories of miracidia activity were established from its fully active stage to its immobile larva stage. The estimated life span of miracidia was 5.8 and 3.5 h in the experiments with 60-min and 30-min observation intervals, respectively. Death was defined by an absence of cilia and body movement. When mobility was used as a proxy for infectivity, infective miracidia were detected at 2.5 and 4.5 h, respectively. The present miracidia motility and survival re-evaluation supports parameters optimization for computational modelling of schistosomiasis transmission dynamics. Target control interventions, especially at late stages next to transmission interruption, may greatly benefit from improved modelling studies.

Adult schistosomes live in the blood vessels and cannot easily be sampled from humans, so archived miracidia larvae hatched from eggs expelled in feces or urine are commonly used for population genetic studies. Large collections of archived miracidia on FTA cards are now available through the Schistosomiasis Collection at the Natural History Museum (SCAN). Here we describe protocols for whole genome amplification of Schistosoma mansoni and Schistosome haematobium miracidia from these cards, as well as real time PCR quantification of amplified schistosome DNA. We used microgram quantities of DNA obtained for exome capture and sequencing of single miracidia, generating dense polymorphism data across the exome. These methods will facilitate the transition from population genetics, using limited numbers of markers to population genomics using genome-wide marker information, maximising the value of collections such as SCAN.

Temperature is an important factor influencing the biology of organisms and is intrinsically linked to climate change. The establishment of trematodes in target hosts is potentially susceptible to temperature changes effecting parasite infectivity or host susceptibility, and therefore in order to develop predictive frameworks of host–parasite dynamics under climate change large-scale analyses are required. The present study analyses the thermodynamics of the infectivity of larval trematodes including miracidia, cercariae and metacercariae from experimental data contained in the scientific literature using the Arrhenius critical incremental energy of activation (E*), an accurate measure of temperature-driven reaction rates. For miracidia and cercariae, infectivity increases as the temperature rises reaching a plateau over optimal thermal ranges before declining at higher temperatures. In contrast, metacercarial infectivity is at its greatest at low temperatures, declining with increasing temperature.

Miracidia are short-lived, non-feeding (lecithotrophic) free-living stages of trematodes, whose survival is potentially influenced by temperature. Climate change may result in elevated temperatures affecting trematode transmission. Therefore understanding their thermobiology forms an important step in determining the future dynamics of parasite populations. An empirical relationship exists between the mean expected life span of lecithotrophic larvae and the half life of their population (t0·5) and therefore t0·5 is a good indicator of glycogen utilization. In this study experimental data on the effects of temperature on miracidial survival were compiled from the scientific literature and evaluated in terms of metabolism using Q10 and Arrhenius activation energy (E* or μ). Temperature poorly influenced survival/metabolism with all miracidia having distinct zone(s) of thermostability. Overall there were few differences in Q10 and E* values between most species temperature ranges whilst there were only limited strain-specific variations in thermal responses of laboratory-maintained Schistosoma mansoni. Miracidia demonstrated a trend of greater thermal resistance than cercariae. In particular, comparative studies on 4 strains of the same species of miracidia and cercariae showed little correlation in thermal biology between the 2 life-history stages. The importance of these results for trematode transmission under global climate change is discussed.

Experiments were performed to study the effect of age and lifespan on the infection success of Diplostomum spathaceum miracidia. Specimens of Lymnaea peregra and L. stagnalis were individually exposed to single miracidia of D. spathaceum of different ages under conditions of constant illumination at 20°C. The life-span of the free-swimming miracidium of D. spathaceum is 24 h at 20°C. It was observed that as the miracidium ages, its ability to successfully infect snails declines rapidly.

The effects of water temperature on the ability of Diplostomum spathaceum miracida to infect and establish patent infections in Lymnaea peregra and L. stagnalis were investigated. Snails were infected over a range of temperatures (6–20°C) and kept thereafter at 20°C or were infected at 20°C and kept at either 14, 20, or 25°C. Infection success was determined after 8 weeks by either observing cercarial shedding or examining snail viscera for sporocysts. The establishment of miracidia declined at lower water temperatures despite maintenance for 8 weeks at 20°C while exposure of snails to miracidia at 20°C and maintenance at different temperatures had little apparent effect. Infection success under these conditions was related more to the numbers of miracidia to which the snails were exposed. However, under this latter experimental regime, the time taken for the infection to become patent clearly depended upon maintenance temperature.

Schistosoma haematobium miracidia were collected from a locality with a high prevalence of human infection with the animal parasite, S. mattheei, which hybridizes with S. haematobium, and from 2 localities with negligible infection rates. The terebratoria of the miracidia from these localities were compared with each other, with laboratory maintained S. haematobium and with four populations of S. mattheei by means of scanning electron microscopy. It was found that the terebratorial membrane of certain of the S. haematobium miracidia from the locality with a high S. mattheei prevalence in humans, resembled the more intricate membrane of S. mattheei. This suggests introgressive hybridization between S. haematobium and S. haematobium x S. mattheei.

The effect of experimental non-host snail (NHS) communities on the infection of Lymnaea truncatula with Paramphistomum daubneyi and Fasciola hepatica miracidia was studied. The results obtained indicated that NHS had variable effects on basic population parameters of host infection. Specifically, NHS were able to produce aggregation of sporocyst number/snail in the experimental host populations. Proposed hypotheses for decoy effect mechanisms failed to explain completely the experimental results. A new mechanism is suggested, based on a distance response of miracidia to snail chemo-attractants, to explain effects of NHS on miracidial host finding behaviour. It is assumed that miracidia have evolved the ability to discriminate among individual snail chemical attractants in a community already at a distance. This attribute of miracidial behaviour allows NHS to induce variations in the effectiveness of individual host localization by miracidia. The resulting aggregation of successful infections/host is assumed to be the specific component of snail decoy effect at the community level. It was also established that NHS affected trematode reproduction in the host inducing a compensatory increase in the intramolluscan population. Intraspecific competition among developmental stages is suggested to be the mechanism of this compensatory reaction.

Infection of the snail, Biomphalaria glabrata, by the free-swimming miracidial stage of the human blood fluke, Schistosoma mansoni, and its subsequent development to the parasitic sporocyst stage is critical to establishment of viable infections and continued human transmission. We performed a genome-wide expression analysis of the S. mansoni miracidia and developing sporocyst using Long Serial Analysis of Gene Expression (LongSAGE). Five cDNA libraries were constructed from miracidia and in vitro cultured 6- and 20-day-old sporocysts maintained in sporocyst medium (SM) or in SM conditioned by previous cultivation with cells of the B. glabrata embryonic (Bge) cell line. We generated 21 440 SAGE tags and mapped 13 381 to the S. mansoni gene predictions (v4.0e) either by estimating theoretical 3′ UTR lengths or using existing 3′ EST sequence data. Overall, 432 transcripts were found to be differentially expressed amongst all 5 libraries. In total, 172 tags were differentially expressed between miracidia and 6-day conditioned sporocysts and 152 were differentially expressed between miracidia and 6-day unconditioned sporocysts. In addition, 53 and 45 tags, respectively, were differentially expressed in 6-day and 20-day cultured sporocysts, due to the effects of exposure to Bge cell-conditioned medium.

The presence of antigenic carbohydrate epitopes shared by Biomphalaria glabrata as well as by the sporocysts and miracidia representing snail-pathogenic larval stages of Schistosoma mansoni was assayed by immunohistochemical staining of paraformaldehyde-fixed tissues. To this end, both polyclonal rabbit antiserum raised against soluble egg antigens (SEA) of S. mansoni and monoclonal antibodies recognizing the carbohydrate epitopes LDN [GalNAc(β1-4)GlcNAc(β1-)], F-LDN [Fuc(α1-3)GalNAc(β1-4)GlcNAc(β1-)], LDN-F [GalNAc(β1-4)[Fuc(α1-3)]GlcNAc(β1-)], LDN-DF [GalNAc(β1-4)[Fuc(α1-2)Fuc(α1-3)]GlcNAc(β1-)] and Lewis X [Gal(β1-4)[Fuc(α1-3)]GlcNAc(β1-)] were used. Intriguingly, anti-SEA serum as well as anti-F-LDN antibodies displayed significant binding in the foot region, anterior tissue and the hepatopancreas of uninfected snails, whereas the Lewis X epitope was only weakly detectable in the latter tissue. In contrast, increased binding of antibodies recognizing LDN, LDN-F and LDN-DF was observed in infected snail tissue, in particular in regions involved in sporocystogenesis, in addition to an enhanced binding of anti-SEA serum and antibodies reacting with F-LDN. A pronounced expression of most of these carbohydrate antigens was also observed at the surface of miracidia. Hence, the detection of shared carbohydrate determinants in uninfected snail tissue, sporocysts and miracidia may support the hypothesis of carbohydrate-based molecular mimicry as a survival strategy of S. mansoni.

Molecular interplay during snail-schistosome interactions is poorly understood and there is much to discover concerning the effect of snail host molecules on molecular processes in schistosomes. Using the Biomphalaria glabrata – Schistosoma mansoni host-parasite system, the effects of exposure to haemolymph, derived from schistosome-resistant and susceptible snail strains, on protein tyrosine phosphorylation in miracidia have been investigated. Western blotting revealed several tyrosine phosphorylated proteins in this larval stage. Exposure of miracidia to haemolymph from susceptible snails for 60 min resulted in a striking, 5-fold, increase in the tyrosine phosphorylation of a 56 kDa (p56) S. mansoni protein. In contrast, haemolymph from resistant snails had little effect on protein tyrosine phosphorylation levels in miracidia. Confocal microscopy revealed that tyrosine phosphorylation was predominantly associated with proteins present in the tegument. Finally, treatment of miracidia with the tyrosine kinase inhibitor genistein significantly impaired their development into primary sporocysts. The results open avenues for research that focus on the potential importance of phospho-p56 to the outcome of schistosome infection in snails, and the significance of protein tyrosine kinase-mediated signalling events to the transformation of S. mansoni larvae.

The ability of the larval forms of Schistosoma mansoni to invade and parasitize their molluscan host, Biomphalaria glabrata, is determined by a multitude of factors. In this study we sought to elucidate the possible mechanisms by which the invading larvae are able to counteract the potentially harmful oxidative environment presented by the host upon initial miracidial infection. This was attempted by examining the gene expression profile of parasite antioxidant enzymes of the linked glutathione-(GSH) thioredoxin (Trx) redox pathway during early intramolluscan larval development. Three such enzymes, the peroxiredoxins (Prx1, Prx2 and Prx3) were examined as to their activity and sites of expression within S. mansoni miracidia and in vitro-cultured mother sporocysts. Results of these studies demonstrated that the H2O2-reducing enzymes Prx1 and 2 are upregulated during early mother sporocyst development compared to miracidia. Immunolocalization studies further indicated that Prx1 and Prx2 proteins are expressed within the apical papillae of miracidia and tegumental syncytium of sporocysts, and are released with parasite excretory-secretory proteins (ESP) during in vitro larval transformation. Removal of Prx1 and Prx2 from larval ESP by immunoabsorption significantly reduced the ability of ESP to breakdown exogenous H2O2, thereby directly linking ESP Prx proteins with H2O2-scavenging activity. Moreover, exposure of live sporocysts to exogenous H2O2 stimulated an upregulation of Prx1 and 2 gene expression suggesting the involvement of H2O2–responsive elements in regulating larval Prx gene expression. These data provide evidence that Prx1 and Prx2 may function in the protection of S. mansoni sporocysts during the early stages of infection.

Non-availability of adult worms from living hosts remains a key problem in population genetic studies of schistosomes. Indirect sampling involving passage through laboratory animals presents significant ethical and practical drawbacks, and may result in sampling biases such as bottlenecking processes and/or host-induced selection pressures. The novel techniques reported here for sampling, storage and multi-locus microsatellite analysis of larval Schistosoma mansoni, allowing genotyping of up to 7 microsatellite loci from a single larva, circumvent these problems. The utility of these assays and the potential problems of laboratory passage, were evaluated using 7 S. mansoni population isolates collected from school-children in the Hoima district of Uganda, by comparing the associated field-collected miracidia with adult worms and miracidia obtained from a single generation in laboratory mice. Analyses of laboratory-passaged material erroneously indicated the presence of geographical structuring in the population, emphasizing the dangers of indirect sampling for population genetic studies. Bottlenecking and/or other sampling effects were demonstrated by reduced variability of adult worms compared to their parent field-collected larval samples. Patterns of heterozygote deficiency were apparent in the field-collected samples, which were not evident in laboratory-derived samples, potentially indicative of heterozygote advantage in establishment within laboratory hosts. Genetic distance between life-cycle stages in the majority of isolates revealed that adult worms and laboratory-passaged miracidia clustered together whilst segregating from field miracidia, thereby further highlighting the utility of this assay.

Allelic variation in 6 microsatellite markers was compared between frozen Schistosoma mansoni eggs and laboratory-passaged worms originating from the same 5 fecal samples obtained from Brazilian residents. Based on allelic richness values, the number of alleles detected per locus did not differ between egg and worm DNA templates. However, our ability to score loci differed between these DNA templates, with worms providing more scored loci per individual than eggs. Differences also existed between the worms and eggs in the identity of the specific alleles that were detected. Additionally, we observed a reduction in homozygous genotypes among laboratory-passaged worms relative to the eggs. Allelic diversity curves were calculated by genotyping all worms from a representative host sample to determine the relationship between the number of alleles detected at a locus and the number of worms genotyped. Curves for the 5 residents' worm infrapopulations for each of the loci were very similar. The equation y=19·55×ln(x)+9·992 explained the association between sampling effort (x) and number of alleles detected (y) with an R2 of 0·775. In conclusion, egg DNA templates and allelic diversity curves can benefit efforts to discern the sociological, ecological and evolutionary forces impacting the genetic diversity and disease epidemiology of human schistosomes.

The multi-host lifestyle of parasitic trematodes necessitates their ability to communicate with their external environment in order to invade and navigate within their hosts' internal environment. Through recent EST and genome sequencing efforts, it has become clear that members of the Trematoda possess many of the elaborate signal transduction systems that have been delineated in other invertebrate model systems like Drosophila melanogaster and Caenorhabditis elegans. Gene homologues representing several well-described signal receptor families including receptor tyrosine kinases, receptor serine tyrosine kinases, G protein-coupled receptors and elements of their downstream signalling systems have been identified in larval trematodes. A majority of this work has focused on the blood flukes, Schistosoma spp. and therefore represents a narrow sampling of the diverse digenean helminth taxon. Despite this fact and given the substantial evidence supporting the existence of such signalling systems, the question then becomes, how are these systems employed by larval trematodes to aid them in interpreting signals received from their immediate environment to initiate appropriate responses in cells and tissues comprising the developing parasite stages? High-throughput, genome-wide analysis tools now allow us to begin to functionally characterize genes differentially expressed throughout the development of trematode larvae. Investigation of the systems used by these parasites to receive and transduce external signals may facilitate the creation of technologies for achieving control of intramolluscan schistosome infections and also continue to yield valuable insights into the basic mechanisms regulating motility and behaviour in this important group of helminths.

Two strains of Schistosoma mansoni were used to investigate the hereditary basis of species-specific host recognition by analysing behavioural responses of miracidia to snail-conditioned water. An Egyptian strain of S. mansoni, capable of distinguishing its host snail Biomphalaria alexandrina from other snails was cycled repeatedly through Biomphalaria glabrata, the intermediate host of a Brazilian strain known to respond even to non-susceptible snails with high intensity. After 5 cycles in the non-natural host, miracidia of the Egyptian strain still retained their preference for the original host snail. In a second experiment, host-finding behaviour of hybrids between these two parasite strains was studied. In the F1 generation, hybrids of both parental combinations showed the same low degree of specificity as the pure-bred Brazilian strain. Approximately one quarter of F2 hybrids proved to be as discriminatory as the Egyptian strain, confirming dominant Mendelian inheritance of non-specificity in schistosome miracidial host-finding behaviour. Moreover, hybrids seem to have lost the ability to develop in B. alexandrina, possibly suggesting a link between host recognition and host compatibility. The heredity of this behavioural trait is of evolutionary and epidemiological significance, since a shift to low host-finding specificity might have been a prerequisite for S. mansoni to acquire new host snails after being introduced to South America by the slave trade.

The increased occurrence of pollutants in ecosystems is a continuing area of concern. It is known that numerous diseases of wild aquatic animals can occur with decreased or increased prevalences in areas associated with high or chronic levels of pollution. This may have serious implications for environmental health. There has consequently been an increasing number of laboratory and field studies on disease transmission under polluted conditions, especially focusing on digeneans of medical or economic importance. The effect of pollutants to the transmission of larval digeneans (miracidia, cercariae, metacercariae) and snail-digenean interactions is therefore considered. An overview and interpretation of the published literature on laboratory and field studies is provided. It is apparent from these studies that the influence of pollutants on digenean transmission is highly complex with much of the observed effects in the laboratory often masked by a complexity of other factors in the field. Future studies would benefit from a standardisation of experimental procedures, increasing the number of combined laboratory and field studies, and increasing the complexity of the experiments undertaken.

The specific objectives of this study were to elucidate metal toxicity to hatching, survival and avoidance behaviour of Schistosoma mansoni miracidia. The toxicity of cadmium, zinc, and cadmium/zinc mixtures at concentrations ranging from 10000 to 10μg/l was investigated. Metal mixture toxicity investigation was undertaken with equal concentrations of the metals. The hatching of miracidia from eggs was inhibited by concentrations of 1000–10000μg/l of single metals. Metal mixtures had no effect on egg hatching. Survival of miracidia was reduced by increasing metal concentration except at concentrations of 10μg/l for single metal toxicity where survival was increased above the control. Miracidia demonstrated a rapid avoidance behaviour when briefly exposed to heavy metals. The mechanisms of metal toxicity to miracidia are briefly discussed.

The snail host signals releasing host-finding responses in miracidia and cercariae of Echinostoma caproni were analysed by fractionation of snail-conditioned water (SCW). Cercariae responded non-specifically to organic and hydrophilic, low molecular weight components of SCW showing their typical turning response. Hydrolysis of peptides in SCW had no effect on cercarial responses. An artificial mixture of amino acids in concentrations determined from SCW as well as glycine alone in a concentration corresponding to the total concentration of amino acids in SCW showed nearly the same efficacy as SCW itself. Miracidia responded to a high molecular weight glycoprotein fraction, which could be isolated from SCW by ion-exchange and size-exclusion chromatography. In contrast to an Egyptian Schistosoma mansoni strain, the echinostome miracidia were not able to differentiate between different snail species. The results show for the first time that miracidia and cercariae of the same species may use different signals to identify the same snail host species. This indicates an independent evolution of host-finding mechanisms in the two parasite stages.

A novel method for the isolation of schistosome eggs and miracidia from livers of mice infected with Schistosoma japonicum or S. mansoni is described. The method employed collagenase B to degrade the interstitial matrix of mouse liver tissue, after which the schistosome eggs were separated from the liver cells by 2 single-step density centrifugations through Percoll. Using this procedure sufficient quantities of miracidia were obtained to generate a cDNA library. Southern blot analysis demonstrated that miracidia isolated by this method were free from contaminating host DNA.