Single Miracidium Research Articles

Trematode genetic patterns at host individual and population scales provide insights about infection mechanisms.

Multiple parasites can infect a single host, creating a dynamic environment where each parasite must compete over host resources. Such interactions can cause greater harm to the host than single infections and can also have negative consequences for the parasites themselves. In their first intermediate hosts, trematodes multiply asexually and can eventually reach up to 20% of the host's biomass. In most species, it is unclear whether this biomass results from a single infection or co-infection by 2 or more infective stages (miracidia), the latter being more likely a priori in areas where prevalence of infection is high. Using as model system the trematode Bucephalus minimus and its first intermediate host cockles, we examined the genetic diversity of the cytochrome c oxidase subunit I region in B. minimus from 3 distinct geographical areas and performed a phylogeographic study of B. minimus populations along the Northeast Atlantic coast. Within localities, the high genetic variability found across trematodes infecting different individual cockles, compared to the absence of variability within the same host, suggests that infections could be generally originating from a single miracidium. On a large spatial scale, we uncovered significant population structure of B. minimus, specifically between the north and south of Bay of Biscay. Although other explanations are possible, we suggest this pattern may be driven by the population structure of the final host.

A simple and efficient miracidium hatching technique for preparing a single-genome DNA sample of Schistosoma japonicum.

In this study, a simple and efficient miracidium hatching technique (MHT) protocol for preparing a single-genome DNA of Schistosoma japonicum was proposed. The protocol was designed with 96-well plates to collect a miracidium for single-genome DNA preparation, and the effects of lighting conditions on hatching rates were evaluated. The highest hatching rate was recorded under sunlight (92.4%), followed by fluorescent light (88.0%), and the lowest rate was recorded under the dark condition (4.7%). The results suggested for the first time, to our knowledge, that sunlight was efficient for this simple MHT protocol. Successful amplification of microsatellite marker genes using DNA isolated from a single miracidium also confirmed the quality of the single-genome DNA for subsequent applications.

Interactions between Schistosoma haematobium group species and their Bulinus spp. intermediate hosts along the Niger River Valley

BackgroundUrogenital schistosomiasis, caused by infection with Schistosoma haematobium, is endemic in Niger but complicated by the presence of Schistosoma bovis, Schistosoma curassoni and S. haematobium group hybrids along with various Bulinus snail intermediate host species. Establishing the schistosomes and snails involved in transmission aids disease surveillance whilst providing insights into snail-schistosome interactions/compatibilities and biology.MethodsInfected Bulinus spp. were collected from 16 villages north and south of the Niamey region, Niger, between 2011 and 2015. From each Bulinus spp., 20–52 cercariae shed were analysed using microsatellite markers and a subset identified using the mitochondrial (mt) cox1 and nuclear ITS1 + 2 and 18S DNA regions. Infected Bulinus spp. were identified using both morphological and molecular analysis (partial mt cox1 region).ResultsA total of 87 infected Bulinus from 24 sites were found, 29 were molecularly confirmed as B. truncatus, three as B. forskalii and four as B. globosus. The remaining samples were morphologically identified as B. truncatus (n = 49) and B. forskalii (n = 2). The microsatellite analysis of 1124 cercariae revealed 186 cercarial multilocus genotypes (MLGs). Identical cercarial genotypes were frequently (60%) identified from the same snail (clonal populations from a single miracidia); however, several (40%) of the snails had cercariae of different genotypes (2–10 MLG’s) indicating multiple miracidial infections. Fifty-seven of the B. truncatus and all of the B. forskalii and B. globosus were shedding the Bovid schistosome S. bovis. The other B. truncatus were shedding the human schistosomes, S. haematobium (n = 6) and the S. haematobium group hybrids (n = 13). Two B. truncatus had co-infections with S. haematobium and S. haematobium group hybrids whilst no co-infections with S. bovis were observed.ConclusionsThis study has advanced our understanding of human and bovid schistosomiasis transmission in the Niger River Valley region. Human Schistosoma species/forms (S. haematobium and S. haematobium hybrids) were found transmitted only in five villages whereas those causing veterinary schistosomiasis (S. bovis), were found in most villages. Bulinus truncatus was most abundant, transmitting all Schistosoma species, while the less abundant B. forskalii and B. globosus, only transmitted S. bovis. Our data suggest that species-specific biological traits may exist in relation to co-infections, snail-schistosome compatibility and intramolluscan schistosome development.

Reduced infectivity inHimasthla elongata(Trematoda, Himasthlidae) cercariae with deviant photoreaction

Digenean larvae of hermaphroditic generation - cercariae - are known to be polymorphic at genetic and behavioural levels. Cercariae arise as a result of parthenogenetic reproduction of intramolluscan stages, and represent a clone if a snail was infected with a single miracidium. Here we investigated cercarial clones of Himasthla elongata - namely, the infectivity of cercariae with normal (negative) and deviant (positive) photoreaction. In our study, most H. elongata clones showed intraclonal variance in their response to light. The proportion of photopositive cercariae ranged between 0.2% and 60% in different H. elongata clones. Photopositive larvae demonstrated significantly reduced rates of encystment in Mytilus edulis haemolymph in vitro and in young mussels. We discuss the possible mechanisms behind intraclonal variations, such as non-specific genomic rearrangements.

Studies on resistance of Schistosoma to praziquantel XⅥ Biological characteristics of praziquantel-resistant isolates of Schistomoma japonicum in Oncomelania hupensis snails

To investigate the difference of biological characteristics between the praziquantel-resistant and -susceptible isolates of Schistosoma japonicum in intermediate host Oncomelania hupensis snails. Mice were infected with cercariae of praziquantel-resistant and -susceptible isolates of S. japonicum, and the parasite eggs were collected 37 days post-infection to hatch miracidium. Then, the snails were infected with the miracidium of each parasite isolate. The snail infection, survival rate of infected snails, prepatent period of cercariae, and the total number of cercariae shed from each infected snail were observed and compared between the praziquantel-resistant and -susceptible isolates of S. japonicum. If each snail was exposed to a single miracidium, there were significant differences between the praziquantel-resistant and -susceptible Jiangsu isolates in the snail infection (8.99% vs. 19.74%; χ2 = 3.948, P = 0.047) and the number of cercaria released from a single snail (1 460.2 vs. 1 039.3; t = 2.507, P = 0.02), and there were significant differences between the praziquantel-susceptible and -resistant Hunan isolates in the snail infection (10.00% vs. 21.52%; χ2 = 3.980, P = 0.046) and the number of cercaria released from a single snail (1 319.4 vs. 1 003.5; t = 2.566, P = 0.017). However, there were no significant differences between the praziquantel-resistant and -susceptible isolates of S. japonicum in the prepatent period of cercariae and the survival rate of infected snails (P > 0.05). The praziquantel-resistant isolate of S. japonicum has a higher susceptibility to O. hupensis but less cercaria released from each infected snail than the susceptible isolate.

Effect of Fascioloides magna (Digenea) on fecundity, shell height, and survival rate of Pseudosuccinea columella (Lymnaeidae).

Infection with Fascioloides magna (Digenea) causes serious damage to liver tissue in definitive hosts represented by ruminants, especially cervids. The distribution of F. magna includes the indigenous areas in North America, and the areas to which F. magna was introduced-Central Europe, Southeast Europe, and Italy. The North American intermediate host of F. magna, the freshwater snail Pseudosuccinea columella (Lymnaeidae), is an invasive species recorded in South America, the Caribbean, Africa, Australia, and west and Southeast Europe. In Europe, Galba truncatula is the snail serving for transmission, but P. columella has potential to become here a new intermediate host of F. magna. Little is known about interactions between F. magna and P. columella. In this study, the susceptibility of P. columella (Oregon, USA) to the infection by a single miracidium of the Czech strain of F. magna and the influence of F. magna on snail fecundity, shell height, and survival were evaluated. The data show that the Oregon strain of P. columella is a highly suitable host for the Czech strain of F. magna, with the infection rate of 74%. In addition, a negative effect on survival rate of infected snails was recorded only in the late phase of infection. The infection was accompanied by a major reduction in egg mass production and by a decrease in the number of eggs per egg mass. The shell height of infected snails did not significantly differ from that in unexposed controls.

Relative compatibility of Schistosoma mansoni with Biomphalaria sudanica and B. pfeifferi from Kenya as assessed by PCR amplification of the S. mansoni ND5 gene in conjunction with traditional methods.

BackgroundSchistosoma mansoni is hosted by several species of Biomphalaria spp. snails in Africa. We were interested in determining if there were differences in compatibility of S. mansoni with Biomphalaria sudanica from Lake Victoria, or with B. pfeifferi from streams and smaller water bodies in Kenya. Does this parasite develop with equal efficiency in both snail species, and does this have implications for transmission in different habitat types?MethodsPrimers for PCR amplification of the S. mansoni ND5 gene were designed and tested for sensitivity and specificity. We exposed laboratory-reared B. sudanica and field-derived B. pfeifferi to single miracidium infections and at 1, 2, 4, 8, 16 and 24 days post-exposure (dpe), snails were extracted for the PCR assay. Snails were also shed for cercariae and/or dissected prior to extraction. Additionally, B. sudanica and B. pfeifferi were collected from field locations and tested with the PCR assay.ResultsThe ND5 PCR assay was sensitive (>0.1 fg S. mansoni genomic DNA) and allowed S. mansoni to be differentiated from other relevant schistosome species or snails. The number of PCR positive snails at 1–4 dpe was higher for B. pfeifferi than for B. sudanica, but not significantly so (P = 0.052). From 8–24 dpe, more B. pfeifferi harbored successfully developing parasites (positive by both dissection and PCR) than did B. sudanica (P = 0.008). At 40 dpe, more B. pfeifferi than B. sudanica shed cercariae or harbored dissection positive/PCR positive infections (P < 0.001). Both immature and failed (dissection negative but PCR positive) S. mansoni infections could also be detected in naturally infected snails of both species.ConclusionsThe PCR assay detected S. mansoni infections in snails exposed to one miracidium for one day. Both B. sudanica and B. pfeifferi supported full development of S. mansoni, but B. pfeifferi was more compatible, with significantly more dissection positive/PCR positive or shedding infections, and significantly fewer failed infections (dissection negative/PCR positive). This highlights the relatively lower compatibility of B. sudanica with S. mansoni, and suggests the factors responsible for incompatibility and how they might affect transmission of S. mansoni in habitats like Lake Victoria deserve additional study.

Genetic diversity, multiplicity of infection and population structure of Schistosoma mansoni isolates from human hosts in Ethiopia.

BackgroundHuman intestinal schistosomiasis caused by Schistosoma mansoni and urinary schistosomiasis caused by Schistosoma haematobium are endemic in Ethiopia. Although schistosomes look morphologically uniform, there is variation in infectivity, egg productivity and virulence due to variation in their genetic make. Knowing the genetic diversity and population structure of S. mansoni isolates will enable to understand and consider the possible variability in terms of infectivity, egg productivity and virulence.MethodsBetween 2010 and 2011, genetic diversity and population structure of Schistosoma mansoni isolates from four endemic areas of Ethiopia was assessed using previously published 11 polymorphic microsatellite loci. Miracidia were hatched from eggs of S. mansoni collected from stools of human subjects residing in Kemissie, Wondo Genet, Ziway and Sille-Elgo villages. DNA was extracted from single miracidium and PCR was run following standard protocol. Allelic polymorphism and population genetic structure was analyzed using different software.ResultAt a population level (i.e. different villages), the mean number of alleles per locus, allelic richness, expected heterozygosity in Hardy–Weinberg equilibrium and pairwise FST values ranged from 8.5 to 11.5, 3.46–20.8, 0.66–0.73 and 3.57–13.63 %, respectively. All analyzes on population genetic structure reveals strong genetic structuration corresponding to the four sampled villages. At infrapopulation level (i.e. different hosts) the mean number of alleles per locus, allelic richness, expected heterozygosity in Hardy–Weinberg equilibrium and FIS values ranged from 3.09 to 7.55, 1–1.96, 0.59–0.73 and 0.1763–0.4989, respectively. Mean estimated genetically unique adult worm pairs within hosts ranged from 66 to 92 % revealing the occurrence of infection of a single host with genetically unique multiple S. mansoni strains. The data also indicated the occurrence of genetic variation within inter- and intra-hosts.ConclusionHigh level of genetic diversity and significant population differentiation characterized the S. mansoni isolates of Ethiopia. These results are quite different from previous studies demonstrating that it is difficult to generalize schistosome transmission patterns because epidemiological situation tends to vary. These are important factors to be considered in relation with morbidity, drug resistance or vaccine development.

A multiplex PCR for the detection of Fasciola hepatica in the intermediate snail host Galba cubensis

Fasciolosis is a snail-borne trematode infection that has re-emerged as a human disease, and is considered a significant problem for veterinary medicine worldwide. The evaluation of the transmission risk of fasciolosis as well as the efficacy of the strategies for its control could be carried out through epidemiological surveillance of the snails that act as intermediate hosts of the parasites. The present study aimed to develop the first multiplex PCR to detect Fasciola hepatica in Galba cubensis, an important intermediate host of the parasite in the Americas and especially in the Caribbean basin. The multiplex PCR was optimized for the amplification of a 340bp fragment of the second internal transcribed spacer (ITS-2) of F. hepatica rDNA, while another set of primers was designed and used to amplify a conserved segment of the nuclear 18S rDNA of the snail (451bp), as an internal control of the reaction. The assay was able to detect up to 100pg of the parasite even at high concentrations of snail DNA, an analytical sensitivity that allows the detection of less than a single miracidium, which is the minimal biological infestation unit. A controlled laboratory-reared G. cubensis – F. hepatica system was used for the evaluation of the developed multiplex PCR, and 100% sensitivity and specificity was achieved. This assay constitutes a novel, useful and suitable technique for the survey of fasciolosis transmission through one of the main intermediate hosts in the Western hemisphere.

Compatibility of Schistosoma japonicum from the hilly region and Oncomelania hupensis hupensis from the marshland region within Anhui, China.

Schistosome japonicum remains one main public concern in China. This is exemplified in the hilly region in Anhui Province, where rodents have served as reservoirs for the parasite and no effective intervention could target such wild animals. The closer relationship between the hilly region and the near marshland induces the worry of spread of the hill parasite to the marshland region. Therefore, the level of snail-parasite compatibility between the hill parasite and snail populations from the Yangtze River valley was investigated. The results of this study demonstrated that both the hill (Shitai, Anhui) and the marshland (Wuxi, Jiangsu) strains of parasite were more infective to the marshland strains of snail (Zongyang and Hexian, Anhui) than to the hill strain of snail (Shitai, Anhui). When snails were individually exposed to one single miracidium, the longest prepatent period for cercarial development was observed in the combination of Shitai schistosome/Shitai snail. A nocturnal cercarial emergence pattern was observed for the hill parasite, either harbored in the hill or the marshland strain of snails. The results suggested a high compatibility between the marshland strains of snail and both the hill and the marshland strains of parasite. This would have practical implications. Moreover, the fact of the lower compatible relationship between the hill parasite and its local intermediate hosts warranted more studies.

Approaches to genotyping individual miracidia of Schistosoma japonicum

Molecular genetic tools are needed to address questions as to the source and dynamics of transmission of the human blood fluke Schistosoma japonicum in regions where human infections have reemerged, and to characterize infrapopulations in individual hosts. The life stage that interests us as a target for collecting genotypic data is the miracidium, a very small larval stage that consequently yields very little DNA for analysis. Here, we report the successful development of a multiplex format permitting genotyping of 17 microsatellite loci in four sequential multiplex reactions using a single miracidium held on a Whatman Classic FTA indicating card. This approach was successful after short storage periods, but after long storage (>4years), considerable difficulty was encountered in multiplex genotyping, necessitating the use of whole genome amplification (WGA) methods. WGA applied to cards stored for long periods of time resulted in sufficient DNA for accurate and repeatable genotyping. Trials and tests of these methods, as well as application to some field-collected samples, are reported, along with the discussion of the potential insights to be gained from such techniques. These include recognition of sibships among miracidia from a single host, and inference of the minimum number of worm pairs that might be present in a host.

Detection of Early and Single Infections of Schistosoma japonicum in the Intermediate Host Snail, Oncomelania hupensis, by PCR and Loop-Mediated Isothermal Amplification (LAMP) Assay

Polymerase chain reaction (PCR) with the specific primer set amplifying 28S ribosomal DNA (rDNA) of Schistosoma japonicum was able to detect genomic DNA of S. japonicum, but not S. mansoni, at 100 fg. This procedure enabled us to detect the DNA from a single miracidium and a snail infected with one miracidium at just 1 day after infection. We compared these results with those from loop-mediated isothermal amplification (LAMP) targeting 28S rDNA and found similar results. The LAMP could amplify the specific DNA from a group of 100 normal snails mixed with one infected snail A PCR screening of infected snails from endemic regions in Anhui Province revealed schistosomal DNA even in snails found negative by microscopy. PCR and LAMP show promise for monitoring the early infection rate in snails, and they may be useful for predicting the risk of infection in the endemic places.

Susceptibility to praziquantel of male and female cercariae of praziquantel-resistant and susceptible isolates of Schistosoma mansoni

Praziquantel (PZQ) is now widely used for the treatment of human schistosomiasis. However, in recent years, there has been a growing concern about the resistance of Schistosoma to PZQ. The mechanisms of PZQ action against Schistosoma and resistance of Schistosoma to PZQ are poorly understood. Here, we report differential susceptibilities to PZQ between male and female cercariae in the PZQ-susceptible and PZQ-resistant isolates of Schistosoma mansoni, using tail loss as a measurement of PZQ action. The miracidia were collected by hatching eggs collected from faeces of infected mice. Single-sex cercaria lines were made by infecting a single Biomphalaria glabrata snail with a single miracidium. The sex of each single-sex cercaria line was identified by a direct W1-specific polymerase chain reaction (PCR) technique. Single-sex cercariae of two isolates were exposed to four different concentrations of PZQ, respectively. The tail shedding of cercariae was observed under a dissecting microscope for five time points up to 100 min after adding PZQ. The results showed that male cercariae have higher tail-shedding rates than that of female cercariae when PZQ-susceptible isolates of S. mansoni are exposed to the same concentration of PZQ. But this phenomenon was not observed in the PZQ-resistant isolates. This sexual differential resistance phenomenon of S. mansoni suggests that resistance to PZQ is induced by decreasing the PZQ susceptibility of male worms. The experiment described here may also be useful for developing tests to detect PZQ resistance in the field.

Efficient genotyping of Schistosoma mansoni miracidia following whole genome amplification

Small parasites and larval stages pose a problem for molecular analyses because limited amounts of DNA template are available. Isothermal methods for faithfully copying DNA have the potential to revolutionize studies of such organisms. We evaluated the fidelity of multiple displacement amplification (MDA) for amplifying DNA extracted from a single miracidium of Schistosoma mansoni. To do this we genotyped DNA extracted from 28 F1 miracidia following MDA using 56 microsatellite markers. Because these miracidia were obtained from a cross between a male and female worm of known genotypes, we were able to predict the alleles present in the progeny and quantify the genotyping error rate. We found just 8/1568 genotypes deviated from Mendelian expectations. Furthermore, because 1 of these resulted from a genuine mutation, the error rate due to MDA is 7/1568 (0.45%). We conclude that many hundreds of microsatellites or other genetic markers can be accurately genotyped from a single miracidium using this method, greatly expanding the scope of population genetic, epidemiological and evolutionary studies on this parasite.

Fasciola gigantica: Cercarial shedding pattern from Lymnaea natalensis after long-term exposure to cadmium at different temperatures

Little is known about the response of Fasciola spp., inside its snail host to pollutant toxicity. The effect of long-term exposure of Lymnaea natalensis to 0.1 μg/l, 10 μg/l, or 100 μg/l cadmium (Cd) on the infection rates with F. gigantica, percentage of cercariae-shedding snails and cercariae shedding pattern was investigated. The snails were exposed to Cd for 7 days either pre-infection with single Fasciola miracidium or during the late pre-patent period of the infection. The possible interaction between metal exposure and acclimatization temperature was also studied in three ranges; 16–18 °C, 23–25 °C and 30–32 °C. Results clearly showed that host exposure to 100 μg/l Cd significantly affected the pattern of Fasciola development inside its snail host, and that the acclimatization temperature was a key factor affecting the role played by Cd. Pre-infection exposure to Cd caused a significant reduction in the infection rates where the effect was temperature-dependent. Post-infection exposure to Cd significantly increased the percentage of cercariae-shedding snails; the effect was temperature-independent. Interestingly, post-infection exposure significantly altered the differential cercarial output pattern, while no clear impact on the total was found. The alteration in the differential cercarial output was represented as a significantly higher proportion of floating metacercaria as compared to fixed ones and also higher proportion of dead cercariae which may directly reduce the transmission of Fasciola to the final host. The mechanisms of cadmium impact are briefly discussed.

Multiple near-identical genotypes of Schistosoma japonicum can occur in snails and have implications for population-genetic analyses

We genotyped (using 16 or 17 microsatellite loci) numerous adult Schistosoma japonicum raised in rabbits exposed to pooled cercariae from small numbers of naturally infected snails from several localities in China. As expected, duplicate multi-locus genotypes (MLGs) were found among these worms. Additionally, many more MLGs, often near-identical, were found than snails used as sources of cercariae. Explanations for these results include (i) genotyping errors, (ii) development within each infected snail of multiple sibling miracidia and (iii) somatic mutation producing genetically varied cercariae from a single miracidium. To control for genotyping errors we re-analysed samples from many individual worms, including repeating the initial PCR. Explanations invoking the development of multiple sibling miracidia within a single snail are not likely to be correct because almost all duplicate MLGs fell within same-sex clusters in a principal coordinates analysis. We would expect both sexes to be represented in a multi-miracidium infection. In addition, we exposed several snails to infection by a single miracidium. One such snail, via an experimentally infected mouse, yielded 48 adult worms. The presence of at least nine near-identical MLGs among these worms was confirmed by re-genotyping. We regard somatic mutation as the most likely explanation for our results. The implications of multiple MLGs for population-genetic studies in S. japonicum are discussed.

Stage-specific differences in fecundity over the life-cycle of two characterized isolates of the liver fluke, Fasciola hepatica

The variability inherent in different isolates of Fasciola hepatica has been evident from reports in the literature but to date there has been no systematic examination of the relationship between these differences and the fecundity of the parasite. In this study we have attempted to remedy this situation by comparing the relative efficiencies with which 2 well-characterized isolates of the liver fluke (Oberon and Fairhurst) progress through both their definitive and intermediate hosts. We did not observe a reduction in fitness in the Oberon isolate which has been reported to be triclabendazole-resistant, compared to the triclabendazole-susceptible Fairhurst isolate, but considerable inter- and intra-isolate variability at different life-cycle stages was recorded. Thus the Oberon isolate gave 4-fold the number of cercariae when 100 snails were each challenged with a single miracidium and was more successful in establishing productive infections in rats. Fairhurst metacercariae excysted at a higher rate than those from the Oberon isolate and Fairhurst flukes produced 4-fold more eggs. The extent of the intra- and inter-isolate variability revealed in this work will provide a basis for the development of models of population dynamics aimed at predicting the response of the liver fluke to changing environmental conditions such as the use of anthelmintics or climatic change.

Compatibility of Schistosoma mansoni Cameroon and Biomphalaria pfeifferi Senegal.

The vectorial capacity of Biomphalaria pfeifferi from Ndiangue, Senegal, was investigated with an allopatric isolate of Schistosoma mansoni from Nkolbisson, Cameroon. The snail infection rate after exposure to a single miracidium per snail (MD1) was 56. 3 %, and 91.6%, for snails exposed to 5 miracidia per snail (MD5). The minimum pre-patent period was 21 days. The mean total cercarial production for the MDI group was 18,511 cercariae per snail, and 9757 cercariae for the MD5 group. The maximum production of cercariae for 1 day was 4892 observed in a snail from the MDI group at day 43 post-infection. The mean longevity of snails was higher in group MD1 (88 days p.i.) than in group MD5 (65 days p.i.). The chronobiological emergence pattern revealed a circadian rhythm with one shedding peak at mid-day. Comparisons are made with the vectorial capacity of the sympatric combination of B. pfeifferi Senegal/S. mansoni Senegal.

Snail size as a determinant of prepatent period duration in Helisoma anceps infected with Halipegus occidualis (Digenea: Hemiuridae).

Pulmonate snails (Helisoma anceps) of varying sizes were exposed to 2, 4, 8, or 16 eggs of Halipegus occidualis and observed over several months to determine the duration of the prepatent period. Infection probability was positively correlated with number of eggs ingested. The number of eggs ingested did not, however, have a significant effect on the duration of the prepatent period, presumably because low infectivity of the eggs (15%) dictated that most patent infections arose from a single miracidium. There was a significant, positive correlation between snail length (measured at time of exposure) and duration of prepatent period, suggesting that density-dependent effects are, in part, responsible for the initiation of cercariae production in H. occidualis.

Production and Chronobiology of Emergence of the Cercariae of Euparyphium albuferensls (Trematoda: Echinostomatidae)

The production and the chronobiology of emergence of the cercariae of Euparyphium albuferensis from Gyraulus chinensis experimentally infected with a single miracidium were established during 28 consecutive days from the first day of cercarial shedding. Moreover, the effect of a sudden change in light-dark cycling was investigated. Although the daily cercarial shedding rates show great variability, a progressive increase in cercarial production was observed in the first weeks of the cercarial shedding periods, probably in relation to the demography of intramolluscan larval stages. Under 12:12 light-dark cycling conditions, E. albuferensis cercariae emerged in the light, and the rhythm was circadian. The sudden change in the light-dark cycle resulted in corresponding alterations in emergence patterns, which shows that cercarial emergence was correlated to light-dark alternation. The ecological consequences of daily emergence by mobile cercariae whose target hosts are organisms that regularly occur in the same habitat with the molluscan emitting host, such as those of E. albuferensis, are discussed with reference to the hypotheses proposed to date, and an alternative hypothesis is proposed.