The organization of germinal material and dynamics of mother sporocyst reproduction in the genus Echinostoma (Trematoda: Echinostomatidae)

The reproduction in the first parthenogenetic generation--mother sporocyst (MS) in two species of echinostomes (E. caproni, E. paraensei) is investigated. A group of densely packed cells, which noticeably differ from others, occupies the posterior part of the miracidium. They are characterized by large sizes and a large bubble-shaped nucleus with heterogeneous nucleolus and strong dispersed chromatin. The use of histological and electron microscopic methods has shown that with observed similarity these cells are classified in two tyoes and have a completely different origin. First of all, large secretory cells stand out. In E. caproni miracidia their number averages 6.8 +/- 0.2 and linear sizes is 10-12 microns. Secretory cells possess a large bubble-shaped nucleus. The caryoplasm looks optically empty because of strong dispersion of chromatin. A large nucleolus occupies a bit eccentric position. Eosinophilic cytoplasm contains poorly noticeable at light-optical level accumulation of small granules. The second group of cells is represented by typical germinal cells (GC). The number of GC does not exceed six. Their polymorphy is well above that of secretory cells. The sizes of GC vary from 5.4 to 9 microns. The largest cells (8.1-9 microns) occupy the front position and usually are located between secretory cells. Intensively basophilic cytoplasm surrounds bubble-shaped nucleus with a large nucleolus like border with uniforming thickness. The heterochromatin is evenly distributed over the caryoplasm. Its content of nuclei is more than that in nuclei of secretory cells. That is why they do not look optically empty. So, it is "mature" germinal cells. Four or five cells are located directly behind "mature" cells. Their sizes are gradually decrease towards the posterior of the miracidium (the diameter of the smallest cells reaches 5.4 microns). Nuclei with a centrally located nucleolus are characterized by larger amount and more condensation of the heterochromatin than those in "mature" cells. Meanwhile, they concern to nuclei of bubble-shaped type. In general, all cells of second type represent the primary germinal cells distinguished by the stage of their differentiation. Also, 2-3 undifferentiated cells occupy the most posterior part of the miracidium. Their sizes average 5.55 +/- 0.18 microns. The nucleus contains a lot of densely packed heterochromatin. On parasitic phase of MS development undifferentiated cells give rise to secondary GC. Electron microscopic data in details confirm the situation described above. The essentially similar results were received during the investigation of E. Paraensei miracidia. The differences are observed in parameterical characteristics of germinal material and in small variability of the extent of germinal material development. With E. paraensei, germinal material may be represented by not only GC and undifferentiated cells, but one germ as well. So, our investigation has shown that germinal material of echinostomes represents typical germinal mass. The germinal material condition does not change on parasitic phase of E. caproni MS development during the first day of post infection (PI). The activization of germinal material coincides in the time with the beginning of schizocoel formation in 2 Days PI. On the 3rd day of PI, the proliferation of undifferentiated cells begins and the first germs are free to float in enormous schizocoel. After 8 days of PI, MS release the first rediae. During the following 2-3 days the other rediae formed by primary GC left MS. The release of rediae derived from secondary GC was observed later. So, E. caproni MS give rise to 12-16 rediae which is much less than the number of GC formed in MS. The earlier release of the first E. paraensei rediae by MS is predetermined by the difference in the structure of germinal mass in E. paraensei miracidia. Therefore, Echinostomatidae is intermediate between two groups of trematodes. The first group has MS that completely realize reproductive function in the time of miracidial formation; but the second group includes higher trematodes characterized by the transfer of reproductive time on a parasitic phase of MS development. The question concerning to so-called "pedogenetic larvae of trematodes" is discussed.     

Development of mother sporocysts of Echinostoma caproni (Trematoda: Echinostomatidae)

New data on the migration and development of Echinostoma caproni mother sporocysts in two mollusk species of the genus Biomphalaria are obtained. It is confirmed, that the formation of primary and second generative cells takes place only as a result of undifferentiated cells' proliferation and following differentiation of some of them. These processes in miracidium, as well as in the parasitic stage of mother sporocyst, take place in a special organ, germinal mass, which occupies caudal position in both cases. The supposition of the role of germinal mass as the universal centre of multiplication and development of generative elements in all generations of Echinostoma caproni parthenites is confirmed. It is established, that mother sporocysts do not relize their reproductive potential completely, and the degree of its realization depends on the conditions arising in the host organism.     

Analysis of messages expressed by Echinostoma paraensei miracidia and sporocysts, obtained by random EST sequencing

A lambdaZAP Express cDNA library was constructed with mRNA obtained from immature miracidia within eggs, hatched miracidia, and sporocysts of Echinostoma paraensei. This cDNA library was amplified and 213 expressed sequence tag (EST) sequences (averaging 466 nucleotides in length) were obtained. The mean percentage of unresolved bases within the EST sequences was 0.4%, ranging from 0 to 4.6%. The 213 ESTs represent 151 unique messages. BLAST (version 2.0.8) analysis disclosed that 64 unique E. paraensei messages (42.4%) had significant similarities (BLAST score < or =e-5), at deduced amino acid or nucleotide levels, with known sequences in the nonredundant GenBank databases or the dbEST database (NCBI). The remainder, 57.6% of the unique EST-encoded messages, scored nonsignificant hits. Most of the E. paraensei messages that could be assigned a cellular role based on sequence similarities were involved in gene/protein expression. Several ESTs scored highest similarities with sequences obtained from trematode species. A total of 22,560 nucleotides present in open reading frames from ESTs that aligned with known sequences was used to determine codon usage for E. paraensei. Analysis of a subset of eight ESTs that contained full-length open reading frames did not reveal a bias in codon usage. Also, EST sequences were found to contain 3' untranslated regions with an average length of 69.9 +/- 88.4 nucleotides (n = 46). The EST sequences were submitted to GenBank/dbEST, adding to the 51 available Echinostoma-derived sequences, to provide reference information for both phylogenetic analysis and study of general trematode biology.     

Differential adherence of M line Biomphalaria glabrata hemocytes to Schistosoma mansoni and Echinostoma paraensei larvae, and experimental manipulation of hemocyte binding

The ability of M line strain Biomphalaria glabrata hemocytes to adhere to mother sporocysts (MS) of PR1 Schistosoma mansoni or to MS or daughter rediae (DR) of Echinostoma paraensei was studied using an in vitro hemocyte adherence assay. Hemocytes were significantly more likely to bind to S. mansoni MS than to E. paraensei MS or DR. Hemocyte adherence to E. paraensei MS or DR was significantly increased if glutaraldehyde-fixed larvae were used as targets. Also, E. paraensei MS pretreated with the lectin concanavalin A (Con A) were more likely to be bound by hemocytes than MS pretreated with Con A in the presence of the competing sugar, alpha-methyl mannoside. Pretreatment of hemocytes with Con A increased their ability to bind E. paraensei sporocysts, but the effect was small compared to that achieved by pretreatment of MS with Con A. The lectin probably did not function as a bridging molecule between hemocytes and MS but, rather, altered the MS surface in a way that facilitated adherence. Similarly, adherence to E. paraensei MS was significantly increased if the MS were pretreated with cell-free M line plasma prior to use in adherence assays. Our results indicate that the two parasites provoke fundamentally different responses from M line hemocytes in vitro and that the living tegument can be modified by host humoral factors and by lectins such that hemocyte binding is significantly increased.     

Lectin-binding properties of the surfaces of in vitro-transformed Schistosoma mansoni and Echinostoma paraensei sporocysts

As carbohydrates on the surfaces of sporocysts of digenetic trematodes may be targets of attack by the molluscan internal defense system, the lectin-binding patterns of living, in vitro-transformed sporocysts of Schistosoma mansoni and Echinostoma paraensei were characterized. Schistosoma mansoni sporocysts specifically bound 8 and E. paraensei 6 of 11 lectins examined. Sporocysts of the 2 species responded differently to 7 of the 11 lectins. Lectins inhibitable by mannose, galactose, and N-acetylgalactosamine were bound by both species. Lectins inhibited by fucose and N-acetylglucosamine bound uniquely to S. mansoni, and an N-acetylneuraminic acid (NeuNAc)-inhibitable lectin bound only to E. paraensei. Preincubation of sporocysts of either species in the plasma of the host snail Biomphalaria glabrata for as long as 24 hr only marginally altered the subsequent binding of lectins. Pretreatment of S. mansoni sporocysts with pronase E and trypsin substantially altered subsequent lectin binding, but similar treatment of E. paraensei sporocysts had little effect. A neuraminidase enzyme derived from Clostridium perfringens diminished binding of the NeuNAc-inhibitable lectin to E. paraensei sporocysts. This study indicates that lectin-binding monosaccharides are expressed abundantly on sporocyst surfaces, they vary considerably between 2 species parasitizing the same host, and they are not obscured readily or altered by exposure to host plasma.     

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.     

The effect of size of M line Biomphalaria glabrata on the course of development of Echinostoma paraensei

M line Biomphalaria glabrata snails of 4-, 6-, 8-, 10-, 12-, or 20-mm shell diameter were individually exposed to 10 miracidia each of Echinostoma paraensei. Snails 10 mm in size or larger were found to be significantly less likely to harbor intraventricular sporocysts than snails in smaller size categories. The percentage of snails with intraventricular sporocysts that also developed hemocyte encapsulation responses generally increased with snail size, whereas the number of snails that ultimately became heavily parasitized with large numbers of daughter rediae decreased significantly with snail size. However, at least some snails in each size category developed such disseminated infections. Comparative histological study of 6- and 12-mm snails revealed that parasites readily penetrated both groups of snails, but were more likely to be encapsulated and destroyed in larger snails. Encapsulation reactions were noted from 1 to 15 days postexposure (dpe) in 12-mm snails, indicating that unlike other commonly studied models of trematode-gastropod interactions, snail resistance is not always manifested during the first few days following exposure. Upon infection with E. paraensei, both 6- and 12-mm snails showed significant increases in the number of circulating hemocytes/mm3 of hemolymph. In 6-mm snails, such increases occurred concurrently with successful parasite development. Hemocyte counts in 6-mm snails were significantly elevated from 4 to 15 dpe whereas in 12-mm snails they were significantly elevated from 2 to 30 dpe. A significant degree of resistance to E. paraensei develops as B. glabrata grows and attains sexual maturity. A mechanistic understanding of this phenomenon awaits further investigation.     

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.     

Responses of Echinostoma caproni miracidia to gravity, light, and chemicals.

In a four-tube vertical system, Echinostoma caproni miracidia exhibited a strong negative geotaxis which was dominated by a positive phototaxis. In horizontal chambers a positive phototactic response was also demonstrated. These miracidia showed a positive chemoresponse, as determined in phi-chambers, to glutamic and aspartic acids but not leucine. Positive responses were also elicited to snail-conditioned water and sulfuric and acetic acids. Ammonia, Mg2+, and HCl produced no significant reactions. Responses of E. caproni and Schistosoma mansoni miracidia, both of which develop in Biomphalaria glabrata snails, were similar providing further evidence that miracidia mimic the behavioral patterns of compatible snail species.     

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.     

Effects of excretory-secretory products of Echinostoma paraensei larvae on the hematopoietic organ of M-line Biomphalaria glabrata snails.

Responses of the hematopoietic organ (HO) in Biomphalaria glabrata snails to extracts and excretory-secretory (E-S) products of Echinostoma paraensei larvae were studied to understand the HO-activating mechanism. M-line B. glabrata snails were injected with materials from E. paraensei larvae, and the size of the HO was ascertained in histological sections. The size of HO in snails injected with extracts and E-S products from sporocysts and rediae was significantly larger than that in snails injected with culture medium. E-S products of sporocysts were fractionated using ultrafiltration membranes, polyacrylamide gel electrophoresis, and electrophoretic elution. Examination of fractionated E-S products of sporocysts revealed that specific components of E-S products were responsible for HO-stimulating activity.     

Schistosomatium douthitti: Exposure of Lymnaea catascopium to irradiated miracidia

Irradiation of Schistosomatium douthitti miracidia (4000, 5000, or 6000 rad) did not substantially alter their behavior or ability to penetrate their snail host. Treatment with 4000 rad was not sufficient to prevent all miracidia from establishing patent infections in Lymnaea catascopium, although significantly fewer snails exposed to these miracidia shed cercariae than did controls exposed to normal miracidia. Irradiation of miracidia with either 5000 or 6000 rad totally prevented cercarial production. Although destruction of irradiated mother sporocysts by encapsulating amebocytes was occasionally observed, most expanded without concomitant multiplication of germinal cells and embryo production and then collapsed. They generally persisted in this state throughout the period of observation (32 days). Snails sensitized by exposure to irradiated miracidia and challenged 2 or 10 days later with normal miracidia were as likely to develop patent infections as were snails exposed only to normal miracidia. Double sensitization of snails with irradiated miracidia also failed to confer protection upon challenge with normal miracidia. Most challenge sporocysts developed normally, often in close proximity to collapsed irradiated sporocysts.     

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.     

Cultivation of excysted metacercariae of Echinostoma caproni to ovigerous adults in the allantois of the chick embryo.

Chemically excysted metacercariae of Echinostoma caproni inoculated into the allantois of domestic chick embryos became ovigerous in that site within 9 days postinoculation. The egg preparation technique of Saville and Irwin was markedly better than that of a modified Zwilling procedure for obtaining large numbers of postinoculation embryos with worm infections. Adults of E. caproni from the allantois were larger and became ovigerous sooner than worms grown on the chorioallantois. Only worms from the allantois produced eggs with fully developed miracidia. Miracidia were released from these eggs, but an insufficient number was available to attempt infections in Biomphalaria glabrata snails.     

Echinostoma paraensei: hemocytes of Biomphalaria glabrata as targets of echinostome mediated interference with host snail resistance to Schistosoma mansoni

Earlier in vivo work by Lie et al. (1977) indicated that the innate resistance of the 10R2 strain of Biomphalaria glabrata to PR1 Schistosoma mansoni could be interfered with if the snails were infected previously with another trematode, Echinostoma paraensei. We have studied this interference phenomenon using in vitro methods in an attempt to understand its mechanistic basis. Hemolymph, derived from 10R2 snails infected with E. paraensei for 14-28 days, killed 25% of S. mansoni sporocysts in vitro, significantly less (P less than 0.001) than the 90% killing rate observed with hemolymph from uninfected, control 10R2 snails. Hemolymph from the infected 10R2 snails and from schistosome susceptible M line snails did not differ significantly (P greater than 0.1) in their relative inability to kill S. mansoni sporocysts in vitro. The defect in sporocyst killing exhibited by echinostome infected 10R2 snails was traced to the cellular, rather than the humoral, component of the hemolymph. Preparations containing uninfected 10R2 snail hemolymph and echinostome daughter rediae exhibited significantly less (P less than 0.001) killing of S. mansoni sporocysts than did controls containing only 10R2 hemolymph and S. mansoni sporocysts. Our results suggest that echinostome larvae release factors that interfere with the ability of B. glabrata hemocytes to kill S. mansoni sporocysts.     

Observations on the host-parasite relations between Echinostoma revolutum and lymnaeid snails.

Echinostoma revolutum from Taiwan was studied in lymneid snails at 29 +/- 0.5 degrees C. Given 3-5 miracidia, 95% of Lymnaea ollula and 40% of Lymnaea swinhoei became positive; the prepatent periods were 18 and 25 days, respectively. The following are based on the observations in Lymnaea ollula: The time required for miracidial penetration was about one hour. The sporocysts developed only in the ventricle of the snail but mother rediae developed in the heart and other organs. Mature daughter rediae were not found in the heart cavity. The sporocysts reached the ventricle within 3 days. Mother rediae were released after 6 days and daughter rediae after 8 days. Given 5 miracidia, 1-3 sporocysts reached the heart and 2-20 mother rediae were found per snail. The number of mature daughter rediae was usually 100-200 although more than a thousand may develop in a snail. The sporocysts and mother rediae attained maximal size 9 days postinfection and started degeneration 13 days postinfection. Daughter rediae were largest at the beginning of cercarial emergence and decreased in size thereafter. Simultaneous production of daughter rediae and cercariae by the mother redia was seen only once in this snail mature cercariae were obtained in 10 days postinfection. The cercariae emerged from a small area of mantle collar near the posterior corner of shell aperture. They were negatively phototactic and positively geotactic. An estimation showed that each snail shed about 350 cercariae a day. The cercariae reached the pericardial cavity of snail in one hour via the renal orifice and metacercariae were seen 4-5 hours after exposure. The infectivity of cercariae at various times after shedding, as expressed by cyst recovery rates, were: 51.6%, O-hr old; 76.1%, 2-hr; 68% 4-hr; 32%, 6-hr; 3%, 8 and 10-hr. Cyst recovery rates were not different within the dosage of 50-500 cercariae per snail. Most metacercariae recovered 1-2 days after cercarial exposure were viable; only 5 among 6,533 cysts were dead.     

Development and pathology of Echinostoma caproni in experimentally infected mice

In the present article, several parasitological features of mice, each experimentally infected with 75 metacercariae of Echinostoma caproni (Trematoda: Echinostomatidae), were studied during the first 12 wk postinfection. Moreover, the early pathological responses also were analyzed and compared with data previously published on other host species of E. caproni to gain further insight into the factors determining worm rejection or establishment of chronic infections. The results obtained show that the pattern of E. caproni infection in mice is consistent with a highly compatible host-parasite system. This combination is characterized by a high worm establishment, high egg output, and long survival of the worms. However, some differences with respect to other highly compatible hosts have been observed, particularly in relation to the survival of the adult worms. Histological studies suggest that the kinetics of goblet cells, mucosal neutrophils, and mononuclear inflammatory cells in the mesentery seem to be essential in determining the course of E. caproni infection in mice.     

Protein kinase C signalling during miracidium to mother sporocyst development in the helminth parasite, Schistosoma mansoni

For schistosomes, development of the miracidium to mother sporocyst within a compatible molluscan host requires considerable physiological and morphological changes by the parasite. The molecular mechanisms controlling such development have not been explored extensively. To begin to elucidate the importance of kinase-mediated signal transduction to this process, the phosphorylation (activation) of protein kinase C (PKC) in larval stages of Schistosoma mansoni undergoing in vitro transformation was explored. Mining of the S. mansoni genomic database revealed two S. mansoni PKC proteins with high homology to human PKCβ and containing the conserved autophosphorylation (activation) site represented by serine 660 of human PKCβ_II. Western blotting with anti-phosphospecific antibodies directed to this site demonstrated that miracidia freshly-hatched from eggs possessed PKC (78 kDa) which was phosphorylated (activated) when miracidia were exposed to phorbol ester, and dephosphorylated (inhibited) following exposure to the PKC inhibitor GF109203X. Miracidia treated with the phospholipase C (PLC) inhibitor U73122 also displayed decreased PKC phosphorylation. S. mansoni PKC was phosphorylated during the initial 24 h development of miracidia into mother sporocysts; after 31 h and 48 h development, phosphorylation was reduced by 72% and 86%, respectively. Confocal microscopy of miracidia revealed phosphorylated PKC associated with the neural mass, excretory vesicle, tegument, ciliated plates, terebratorium and germinal cells; in larvae undergoing transformation for 31 h, phosphorylated PKC was only occasionally detected, being present in regions likely corresponding to the ridge cyton. Inhibition of PKC in miracidia by GF109230X resulted in accelerated transformation, particularly to the postmiracidium stage; ciliated plates were also shed from developing larvae more rapidly. These results highlight the dynamic nature of PKC signalling during S. mansoni postembryonic development and support a role for active PKC in restricting transformation of S. mansoni miracidia into mother sporocysts.     

Single and multiple worm infections of Echinostoma caproni (Trematoda) in the golden hamster

Six of 10 hamsters fed a single metacercarial cyst of Echinostoma caproni (single-worm infections) and 13 of 19 hamsters fed either 2 or 5 cysts (multiple-worm infections) were infected with adult echinostomes at necropsy 22 days post-infection. Considerable histopathological changes to the small intestine occurred in hamsters carrying single-worm infections. There were no differences in either mean length, width or wet weight of echinostomes in single- versus multiple-worm infections. The mean number of eggs/worm from single-worm infections (525) was significantly greater than that from multiple-worm infections (288). The average percentage of fully developed miracidia/worm from single worms (94%) was similar to that from worms in multiple infections (92-95%). Single worms of E. caproni were capable of self-fertilization and production of viable eggs. Miracidia derived from single worms were as capable of infecting laboratory-reared Biomphalaria glabrata and producing patent rediae as were those from multiple infections.     

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.