Germinal elements and their development in Echinostoma caproni and Echinostoma paraensei (Trematoda) miracidia.

Among the large cells located in the posterior of Echinostoma caproni and E. paraensei miracidia are secretory cells, germinal cells (GC), and undifferentiated cells. Secretory cells do not give rise to progeny, whereas GC do. Undifferentiated cells develop into GC that can also divide to produce embryos. Cleavage of GC of E. caproni occurs only after the parasite has entered the snail host and develops into a sporocyst. With E. paraensei, GC are larger than noted for E. caproni, and in 3 of 23 miracidia examined, germinal cell cleavage had occurred in the miracidium such that an embryo containing 20-25 blastomeres was present. Observations on the germinal elements of miracidia help to explain previous results showing that (1) E. paraensei sporocysts release mother rediae a few days earlier than do sporocysts of E. caproni, and that (2) a single mother redia is produced ahead of all others by sporocysts of E. paraensei, but not by sporocysts of E. caproni. The present study adds support to the concept that E. caproni and E. paraensei are distinct species.     

Development of the partenitae infrapopulation of Echinostoma caproni (Digenea: Echinostomatidae)

The first generation of Echinostoma caproni partenitae is represented maternal sporocysts developing in the cardium of the Biomphalaria mollusk. During all their life, they produce rediae of maternal generation. Rediae of Echinostoma caproni of all generations are similar. The first generation consists of maternal rediae forming only redoid embryos. Due to this process, the number of partenitae increases very fast. The next generations are represented by daughter rediae. In the beginning of their life they produce redoid embryos but later begin forming cercariae. The number of rediae produced before this shift of production depends on the population density. Further, the partenitae retain their potential ability to form rediae but realize it in exceptional cases. Generative organs of all generations are germinal masses. Proliferation of generative elements and beginning stages of redia and cercaria development take place within these masses. The infrapopulation of E. caproni belongs to the "prolonged type", because it is a complete microhemipopulations; its existence is limited by a lifespan of the mollusk host.     

Reproduction of the trematode Echinostoma caproni parthenites (Digenea: Echinostomatidae)

Dynamics of the reproduction in the trematode Echinostoma caproni parthenites (Echinostomatidae) was observed. Early laying and maturation of the generative cells are for the first time shown to be characteristic for all parthenogenetic generations. Really the process of reproduction had been finishing to the beginning of the generating of new age by parthenites. Mother sporocysts, as well as redia of different generations, in fact stop producing new generative cells with the beginning of the generating of new age, and assume the function of a brood pouch. This feature was considered previously as peculiar mainly to mother sporocysts. Data on the autotomy of the anterior body end in mother sporocysts are verified. In our opinion, these data are an evidence of an early manifestation of the evolutionary trend to the morpho-functional regress and disintegration of the parasitic stage of mother sporocyst.     

Germinal mass of the rediae of Trematoda

Dynamic of the reproduction of the trematodes Notocotylus imbricatus (Notocotylidae), Echinostoma caproni (Echinostomatidae), Sphaeridiotrema globulus and Psilotrema tuberculata (Psilostomatidae) parthenites was observed. The formation of generative cells takes place only as a result of undifferentiated cells proliferation and following differentiation. These processes and early stages of embryogenesis are taking place only in the special reproductive organ--germinal mass. The germinal mass is always formed at the posterior part of the body. The process of reproduction had been finishing to the beginning of the generating of cercariaea of new age by parthenites. Rediae of different generations in fact stop producing new generative cells with the beginning of the appearance of a new generation.     

The organizational characteristics of the generative material and the the proliferative dynamics of trematode mother sporocysts

Analysis of miracidia germinal material organization and proliferation dynamics of mother sporocysts enabled us to divide them into three well-defined groups. The first one includes species whose miracidia possess only differentiated (mature) generative cells and embryos of earlier stages of cleavage. In this case during parasite phase of development of maternal sporocysts the generative function is not performed. To the first group therefore trematode species with pedogenetic larvae could be attributed also. The next group embraces species whose miracidia as well as mature generated cells have some undifferentiated cells; thus the parasitic phase of mother sporocyst development acquires restricted proliferative capacity. The third group consists of species with higher trematodes dominating. They perform generative function exclusively at parasitic phase of mother sporocyst development. Representatives of more archaic and ancient species are the bases of the first two groups on the contrary. Such type of distribution can not occur occasionally and apparently reflects first steps of emergenes of parthenogenetic generations of trematodes.     

The influence of Echinostoma caproni metacercariae (Trematoda) on the survival of biomphalaria molluscs (Pulmonata)

The infrapopulation of the Echinostoma caproni partenites has a development of prolong character (Ataev et al, 2005). However, in laboratory conditions, Biomphalaria molluscs infested with this parasite die within 1--3 weeks after the beginning of cercariae emission. It has been suggested that autoinvasion of the mollusc host with the cercariae, which use it as second intermediate host, is the cause of this phenomenon. Studying the dynamics of metacercariae accumulation in the host (both infected and non-infected with the Echinostoma caproni rediae) and experiments where quantity of cercariae around molluscs reduced by different ways, confirmed this hypothesis. Evidently, pathogenicity of metacercariae for molluscs is lesser in nature, because the concentration of cercariae reduces to the values, which do not result in lethal effect: some part of cercariae dies, but another part uses other animals as a host (Haas, 2000).     

Excystation of the encysted metacercariae of Echinostoma trivolvis and Echinostoma caproni in a trypsin-bile salts-cysteine medium and morphometric analysis of the excysted larvae

A trypsin-bile salts-cysteine (TBC) medium was used to excyst the encysted metacercariae of Echinostoma caproni and Echinostoma trivolvis, 2 allopatric species of Echinostoma. This medium was used to replace a previously used trypsin-bile salts (TB) medium that was no longer effective because of the unavailability of the original stocks of trypsin. The TBC medium maintained at 41 C allowed for 68.6% excystation of E. caproni at 1 hr and 57.5% excystation of E. trivolvis at 2 hr. The cysteine reductant in the TBC medium was necessary; if it was omitted, excystation was nil. Morphometric analysis was done on the excysted metacercariae following fixation of the larvae in hot, 5% neutral buffered formalin and mounting them on slides in glycerin jelly. Body and organ measurements were made on these larvae. The diameter of the acetabulum of E. caproni was significantly greater than that of E. trivolvis. Likewise, the number and diameter of the excretory concretions found in E. caproni were significantly larger than those of E. trivolvis. Morphometric analysis can be used to distinguish structural differences in closely related allopatric species of Echinostoma.     

Age of adult worms of Echinostoma caproni does not affect development of miracidia

The effect of the age of adult Echinostoma caproni on egg development was studied. The percentage of fully developed miracidia was determined in eggs derived from adult worms obtained from laboratory mice at 2, 4, 6, and 8 wk postinfection (PI). Regardless of the age of worms from which the eggs were obtained, the percentage of fully developed miracidia was always >90%, and 60-80% of the eggs hatched. Several previous studies have shown that eggs derived from 2- to 4-wk-old E. caproni yielded miracidia that infected Biomphalaria glabrata snails. Results of the present study on E. caproni were in marked contrast to previous results with Echinostoma friedi, for which viable eggs were not obtained at 2 and 3 wk PI and maximal infectivity of miracidia in snails was obtained from eggs derived from worms collected at 8 and 9 wk PI. Further studies are needed to determine if the egg viability of other species in the "revolutum" group follow that of E. caproni or E. friedi.     

Electrophoretic analysis of proteases in Echinostoma Caproni and Echinostoma trivolvis

Gelatin substrate sodium dodecyl sulfate polyacrylamide gel electrophoresis was used to analyze proteases in 14 day-old adults of Echinostoma caproni and Echinostoma trivolvis. At pH 8.0, E. caproni adults showed 2 protease bands at 36 kDa and 58 kDa, whereas E. trivolvis adults showed 6 bands at 39, 64, 77, 96, 120, and 168 kDa. Each species also showed distinct protease banding patterns in their excretory/secretory (E/S) products. The E. caproni E/S proteases were at 36 and 58 kDa, whereas those of E. trivolvis were at 120 and 168 kDa. Further characterization of E. caproni adult proteases revealed 2 bands (58 and 66 kDa) with optimal activity at pH 3.0-4.5 and 3 bands (38, 61, and 96 kDa) that were most active at pH 7.0-8.0. Four low molecular weight bands (19, 21, 25, and 30 kDa) appeared when E. caproni worm extracts were incubated in the presence of CaCl2 at pH 8.0 but were inhibited with ethylenediaminetetraacetic acid and 1,10-phenanthroline. Echinostoma caproni protease bands at 58 and 38 kDa in the whole worm samples and the E/S products and the 36-kDa band in the whole worm samples were inhibited with phenylmethylsulfonyl fluoride. By showing protease differences in addition to recent work on nucleotide differences, this study helps distinguish these 2 related allopatric species of 37-collar-spined Echinostoma.     

A comparison of Echinostoma caproni and Echinostoma trivolvis (Trematoda: Echinostomatidae) adults using isoelectrofocusing.

An isoenzymatic analysis using thin-layer agarose gel isoelectrofocusing on laboratory strains of Echinostoma trivolvis and Echinostoma caproni adults showed characteristic monomorphic phenotypes for phosphoglucomutase and glucose phosphate isomerase. The fixed allelic variation observed between these 2 taxa is consistent with their current classification as distinct species.     

Echinostoma caproni: mating behavior and the timing of development and movement of reproductive cells

The development and movement of reproductive cells were determined in Echinsostoma caproni on autoradiograms by labeling nuclei of stem cells during exposure to [3H]thymidine and then transplanting the worms to mice for various times. The development and movement of sperm, primary oocytes, and vitelline cells were much more rapid in E. caproni than other digenetic trematodes investigated previously. Mating behavior was determined by labeling the sperm of 1 adult by in vitro exposure to [3H]tyrosine and transplanting alone or with unlabeled worms to mice for 4 and 6 days. Echinostoma caproni adults self-inseminated when isolated and self- and cross-inseminated when in groups. This behavior is similar to that found for the frog rectal fluke Megalodiscus temperatus but unlike that determined for eyeflukes in the genus Philophthalmus. Worm size was not a barrier to insemination in E. caproni. Cross-insemination could not be detected in 6-day transplants probably because of dilution or elimination of radioactive sperm due to rapid turnover or frequent sperm transfer. An increased number of structural anomalies was noted in the transplanted worms. The most common anomaly was an accumulation of vitelline cells in the vitelline reservoir and ducts.     

Echinostoma caproni: identification of enolase in excretory/secretory products, molecular cloning, and functional expression

In order to investigate molecules that could be involved in host-trematode relationships, we have analysed the excretory/secretory products (ESP) of Echinostoma caproni following a proteomic approach. Actin, Gluthathione S-transferase (GST) and enolase have been identified in the ESP. Enolase, observed to be one of the most abundant proteins, was further characterized. The molecular cloning and in vitro expression in Escherichia coli of E. caproni enolase allowed us to determine that the protein contains 431 amino acids and a theoretical MW of 46272 Da. E. caproni enolase shows high homology to other trematode enolases. The recombinant protein binds specifically to human plasminogen in vitro, as observed for the native protein, confirming its properties as a host-interacting molecule.     

Kinetics of Echinostoma caproni (Trematoda: Echinostomatidae) antigens in feces and serum of experimentally infected hamsters and rats

This study reports on the kinetics of antibody production to Echinostoma caproni and the dynamics of antigens in feces and sera in 2 experimental hosts (hamsters and rats) that display different degrees of susceptibility with this echinostome. Echinostoma caproni produced chronic infections in hamsters, whereas rats lost the infection at 49-56 days postinfection (DPI). Hamsters developed higher antibody responses than rats, probably in relation to different intestinal absorptions of worm antigens in each host species. The levels of coproantigens were indicative of the course of infection in each host. Positive coproantigen levels were detected at 1-2 DPI in both hosts, and the values remained positive until the end of the experiment in hamsters; in rats, the coproantigen levels reverted to negative values, coinciding with the loss of infection. High levels of circulating antigens were detected in hamsters from 21 DPI to the end of the study. In contrast, low levels of E. caproni seroantigens were detected in rats only. These observations may reflect the differences in local inflammatory responses induced by E. caproni in each host species.     

Concurrent infections of Echinostoma caproni and Echinostoma trivolvis in ICR mice.

ICR female mice, 6- to 8-weeks old, were exposed concurrently to 25 metacercarial cysts of Echinostoma caproni and 25 metacercarial cysts of Echinostoma trivolvis and necropsied 10 and 14 days post-infection. Controls consisted of mice exposed singly to either 25 or 50 E. caproni or E. trivolvis cysts. All 23 mice exposed to E. caproni cysts were infected with a total of 331 worms (37.8%), whereas only 11 (37.9%) of 29 mice exposed to E. trivolvis cysts were infected with a total of 77 (6.4%) worms. In the concurrent infections, 13 (59.1%) of 22 mice were infected with both species and the percentage of worm recovery was 72.6% for E. caproni and 14.2% for E. trivolvis. There was no difference in worm distribution of either species in single vs concurrent infections. In concurrent infections at 14 days PI, there was a significant decrease in the body area of worms of both species, when compared to single worm species.     

Antibodies in the serum of golden hamsters experimentally infected with the intestinal trematode Echinostoma caproni.

The serum antibody response in golden hamsters (Mesocricetus auratus) infected with the intestinal trematode Echinostoma caproni was examined with ELISA, SDS-PAGE and Western blot, and IFAT techniques. All methods showed that the hamsters responded slowly but developed a clear positive humoral response to the infection. In most hamsters, an antibody response to infection could not be detected earlier than 11-13 weeks after infection with 6 or 25 metacercariae, and responses were weak when compared to previous results from mice infected with the same parasite. IFAT with positive hamster sera on live juvenile E. caproni showed only fluorescence at the posterior tip, which is a different pattern from that seen using from infected mice, indicating a different response to antigens on the juvenile parasites by these two hosts. The results are discussed in relation to the limited selfcure and development of resistance which is observed in golden hamsters infected with E. caproni.     

Histochemical demonstration of acetylcholinesterase in sporocysts of Schistosoma mansoni (Trematoda).

The distribution of acetylcholinesterase in mother and daughter sporocysts of Schistosoma mansoni was studied histochemically. In young mother sporocysts derived from miracidia cultured in vitro the miracidial neural mass and flame cells were shown to persist. The nerve trunks and commissures, as well as papillae, are apparently lost in the transformation process. In young daughter sporocysts freshly dissected from mother sporocysts there was little enzyme activity except for a sparse distribution in the tegument. After cultivation, intense enzyme activity was associated with developing cercarial embryos. A similar distribution of activity was observed in older daughter sporocysts obtained from the digestive gland of snails. No evidence of flame cells, neural mass, or commissures was detected in daughter sporocysts by the methods employed.     

Exposure of Dugesia tigrina (Turbellaria) to cercariae of Echinostoma trivolvis and Echinostoma caproni (Trematoda)

Laboratory-reared planarians, Dugesia tigrina, were exposed to cercariae of Echinostoma trivolvis or Echinostoma caproni. Of 100 D. tigrina exposed to 2,750 cercariae of E. trivolvis, 29 were infected with a total of 85 encysted metacercariae 24 hr postinfection (PI). None of 40 D. tigrina exposed to 1,100 cercariae of E. caproni was infected with metacercariae 24 hr PI. Cyst structure of E. trivolvis from the parenchyma of D. tigrina varied from normal to abnormal. Metacercariae removed from planarians could be excysted in an alkaline trypsin-bile salts medium. Cercariae of E. trivolvis were not attracted to dialysate material from D. tigrina. Cercariae of E. trivolvis that penetrated, but did not encyst in planarians, voided the contents of their paraesophageal glands. Cercariae of E. caproni lack paraesophageal glands. Secretions of those glands probably are involved in tissue penetration of D. tigrina by E. trivolvis cercariae.     

Schistosoma mansoni: emergence of progeny-daughter sporocysts in monoxenic culture

Daughter sporocysts of Schistosoma mansoni released from mother sporocysts from Biomphalaria glabrata were cultured monoxenically for 21 days at 27.4 C in a medium consisting of a balanced salt-sugar solution supplemented with lactalbumin hydrolysate, inactivated fetal calf serum, serum Fraction A, and egg ultrafiltrate. The sporocysts were retained in transparent porous membranes so that their germinal development could be observed without interference from the underlying cells of Aedcs albopictus tissue cultures; the membrane permitted interchange of medium between sporocysts and cell cultures. The greatest increase in length, fivefold, occurred by 18 days. Progeny-daughters began to emerge at 9 days.The effects of pH, osmolality, and antibiotics were evaluated in axenizing solutions. Optimal conditions were a pH range of 6.5–7.6 and 113–153 mOsm. Antibiotics were not toxic.     

Echinostoma caproni in mice: shedding of antigens from the surface of an intestinal trematode

The surface antigens, which induce a serum antibody response during infection of mice with the intestinal trematode Echinostoma caproni, were examined. It was demonstrated that antigens are shed from the surface of juvenile and 4-week old adult E. caproni during in vitro culture. SDS-PAGE and Western blot analysis of in vitro shed and detergent solubilized surface antigens indicated that the four major antigens released from the surface of adult parasites had molecular masses of approximately 26,000, 66,000, 75,000 and 88,000. A modified ELISA technique showed the in vitro turn-over rate of the surface antigens to be very high, with a half-life of 8-15 min in both juvenile and adult E. caproni trematodes. Transmission electron microscopy of the surface of adult parasites revealed a highly active secreting tegument which was densely packed with membrane-bound vesicles, reflecting the high rate of shedding of the surface antigens. An attempt to immunize mice with detergent solubilized adult surface antigens failed to induce resistance to infection with metacercariae of E. caproni.     

Infectivity, growth, and distribution of Echinostoma caproni (Trematoda) in the ICR mouse

Host-parasite interactions of the intestinal trematode Echinostoma caproni were studied in ICR laboratory mice. All of 40 mice, each fed 25 metacercarial cysts of Echinostoma caproni, were infected 1-20 wk postinfection (PI) with a mean of 17.2 worms/host. At 24 and 29 wk PI only 2 of 6 mice (33%) were infected, with a mean of 4.2 worms/host. Mean body area of worms increased rapidly to about 5 mm2 by week 2, increased less rapidly to 8.8 mm2 by week 12, plateaued until week 24, and then declined. Mean dry weight of worms increased rapidly to about 0.5 mg by week 2, less rapidly to 1.4 mg by week 12, and then plateaued until week 24 PI. From 1 to 8 wk PI most worms localized in the jejunum and ileum; later most worms were in the jejunum and duodenum. Considerable differences were seen in the growth and distribution of E. caproni in the ICR mouse compared with previous studies on this echinostome species in the NMRI mouse.