Experimental Parasitology 128 (2011) 341–346

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Experimental Parasitology

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Development of the reproductive system of Echinostoma paraenseiin Mesocricetus auratus analyzed by light and confocal scanning laser microscopy

Joyce G.R. Souza a,c, Juberlan S. Garcia a, Pedro Paulo de A. Manso b, Renata H. Neves c,Arnaldo Maldonado Jr. a, José Roberto Machado-Silva c,⇑,1

a Laboratory of Biology and Parasitology of Wild Mammal Reservoirs, Oswaldo Cruz Institute, Av. Brasil 4365, Manguinhos, 21040-360 Rio de Janeiro, Brazilb Laboratory of Pathology, Oswaldo Cruz Institute, Brazilc Romero Lascasas Porto Laboratory of Helminthology, Department of Microbiology, Immunology and Parasitology, School of Medical Science, Biomedical Center,Av. Prof. Manoel de Abreu 444/5� andar, Vila Isabel, 20511-070 Rio de Janeiro, Brazil

a r t i c l e i n f o a b s t r a c t

Article history:Received 14 January 2011Received in revised form 30 March 2011Accepted 26 April 2011Available online 1 May 2011

Keywords:Echinostoma paraenseiReproductive systemMorphologyMesocricetus auratusConfocal laser scanning microscopy

0014-4894 � 2011 Elsevier Inc.doi:10.1016/j.exppara.2011.04.005

⇑ Corresponding author.E-mail address: machado@uerj.br (J.R. Machado-Si

1 José Roberto Machado-Silva has a fellowship frScientific and Technological Development (CNPq).

Open access under the Els

This study was performed to gain insight into the maturation of the reproductive system of Echinostomaparaensei worms grown in an early infection of Mesocricetus auratus. Hamsters were infected with 100metacercariae and necropsied on days 3, 5, 7, 10 and 14 post infection (dpi). Recovered flukes stainedwith hydrochloric carmine were preserved as whole mounts and analyzed by light and confocal scanninglaser microscopy. The average worm recovery was 43.7 per host. Images of the male and female repro-ductive systems were taken. The ovary and anterior and posterior testis were evidenced on day 3, whilethe ootype and cirrus sac were present on day 5. Confocal imaging showed primordium testis and ovaryas a cluster of primordial cells from day 3 onward. The testes, ovary, cirrus sac and uterus organs werealready present during the first week of life. The two testes were seen as individual structures on 7 dpiwhile the cirrus sac and vitelline glands were in development. The ovary was connected to the uteruswhile the ootype was adjacent to it. Both testes were larger than the ovary, showing cells at differentstages of development, but with few bundles of functional spermatozoa in 10 day-old worms. On day14, eggs and spermatozoa were seen in the uterus and seminal vesicle, respectively, while oocytesappeared in the ootype as fertilized eggs. We conclude that the reproductive system of E. paraenseiwas functional on 14 dpi in the hamsters.

� 2011 Elsevier Inc. Open access under the Elsevier OA license.

1. Introduction

Adult echinostomes are hermaphroditic digenetic flatwormsthat generally parasitize the small intestine and bile ducts of allclasses of vertebrate hosts (Maldonado and Lanfredi, 2009).Echinostomes do not undergo tissue migration in the definitivehost (Toledo, 2009). After infection of the definitive host, themetacercariae excyst in the duodenum and the juvenile parasitesmigrate to the small intestine, where they attach to the mucosaby their ventral sucker (Fried and Huffman, 1996).

Echinostoma paraensei (Lie and Basch, 1967) (Echinostomatidae:Platyhelminthes) was first described from natural infection ofBiomphalaria glabrata from Brazil shipped to the United States(Maldonado et al., 2001a). The wild rodent Nectomys squamipes(Rodentia: Sigmondontinae) is its natural vertebrate host in

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secluded Atlantic Forest regions of Rio de Janeiro state in Brazil(Maldonado et al., 2001a,b). Moreover, studies of E. paraenseipopulations from those different forest fragments have shownvariations in biological parameters (Maldonado et al., 2005). Underlaboratory conditions, this parasite has been shown to be able tocomplete its life cycle in the intermediate hosts B. glabrata, Physamarmorata and Lymnaea columella and the vertebrate hostsMesocricetus auratus and Mus musculus (Maldonado et al., 2001b).The most important and conspicuous organs of trematodes arethose of the reproductive system (Nollen, 1997). This system isextensively developed and although varying in detail, followsessentially the same pattern in most species (Smyth, 1994). The fe-male reproductive system of E. paraensei comprises the ovary,receptaculum seminal, ootype, oviduct, vitelline reservoir, vitellineduct, and uterus (Madhavi and Rao, 1972). The male system com-prises two testes in tandem, the vas deferens leading to the cirrussac containing seminal vesicle, along with the cirrus and prostategland (Maldonado et al., 2001a). The cirrus is large and dominatedby a highly muscular ejaculatory duct (Šebelová et al., 2004).Laurer’s canal, vitellaria and Mehlis gland are structures associatedin an intricate system.

342 J.G.R. Souza et al. / Experimental Parasitology 128 (2011) 341–346

Echinostomes have long served as experimental model organ-isms, contributing in particular to knowledge of trematode biology,parasite–host relationships, biochemical and parasite immuneinteractions and reproductive behavior (Fried, 1994; Fried andHuffman, 1996; Nollen, 1997; Trelis et al., 2011). In addition,morphological studies have examined features of eggs, miracidia,rediae, metacercariae and adult worms (Fried and Reddy, 1997;Fujino et al., 2000; Maldonado et al., 2003; Pinheiro et al.,2004a,b, 2005; Toledo et al., 2004). However, there is no informa-tion concerning the morphology of the juvenile stage of thisparasite, which corresponds to the pre-ovigerous stage of develop-ment in vertebrate hosts.

Previous studies have demonstrated that confocal microscopyprovides detailed knowledge of the morphological structures, dueto its better resolution (Machado-Silva et al., 1998; Neves et al.,2005; Šebelová et al., 2004). In this study, both light and confocalscanning laser microscopy (CSLM) were used to describe detailsof the reproductive system development in E. paraensei from 3 un-til 14 days post infection.

Table 2Presence of reproductive structures of Echinostoma paraensei recovered from ham-sters at various days after infection analyzed by light and confocal scanning lasermicroscopy.

Age (days) Morphological characteristics

OV AT PT U OO SC VG SP EG

3 + + + – – – – – –5 + + + + + + – – –7 + + + + + + – – –

10 + + + + + + – – –14 + + + + + + + + +

+, present; –, absent.OV: ovary; AT: anterior testis; PT: posterior testis; U: uterus; OO: ootype; SC: cirrussac; VG: vitelline glands; SP: spermatozoon; EG: eggs.

2. Materials and methods

2.1. The host–parasite model

Encysted metacercariae of E. paraensei (Capitão Andrade – MGisolate) were removed from the pericardial cavity-kidney regionof experimentally infected B. glabrata snails. Two-week old outbredM. auratus females were obtained from the Animal Breeding Centerof Oswaldo Cruz Foundation (FIOCRUZ, Rio de Janeiro, Brazil). Thehamsters were housed in polypropylene boxes (40 � 33 cm) withstainless steel screen covers. Water and food (Nuvilab CR1, Colom-bo, Paraná, Brazil) were provided ad libitum. The experiments re-ported here complied with the ethical procedures for animalexperiments (Ellery, 1985) and were approved by the local EthicalCommittee on Animal Use (CEUA L-074/08).

2.2. Experimental design

To obtain the worms, 12 golden hamsters were each orally fed100 metacercariae (Maldonado et al., 2001b). Two hamsters wereeuthanized in a CO2 camera on each of days 3, 5, 7, 10 and 14 afterinfection. Worms were removed from the small intestine of thehamsters and rinsed briefly in Locke’s solution to remove the deb-ris and then were fixed in AFA solution (2% acetic acid, 3% formal-dehyde, 95% and 70� ethanol) under slight cover slip pressure atroom temperature.

2.3. Morphological study

The worms were stained with hydrochloric carmine, dehy-drated in a graded ethanol series, cleared with methyl salicylateand mounted in Canada balsam (Neves et al., 1998). The bodyand organ measurements of 10 worms (in mm) were taken on each

Table 1Worm recovery and morphometric data (mm) of E. paraensei from experimental infectiondifferences between values.

Days p.i. Worm recovery (%) Body Ovary

Length ± S.E. Width ± S.E Length ± S.E.

3 15 0.40 ± 0.06a 0.11 ± 0.03a –5 36 0.58 ± 0.08a,b 0.15 ± 0.01a 0.01 ± 0.003a

7 37 1.56 ± 0.18b 0.37 ± 0.03b 0.05 ± 0.01a

10 48 4.78 ± 0.33c 0.91 ± 0.07c 0.12 ± 0.01b

14 36 7.53 ± 1.58d 1.19 ± 0.07d 0.26 ± 0.06c

day post infection. The ovary, uterus, ootype, vitelline glands, tes-tes and cirrus sac were analyzed by light and confocal scanning la-ser microscopy (LSM-510 META Zeiss), using a 543 nm He/Ne laserand a LP 570 filter under reflected mode.

2.4. Statistical analyses

The one-way analysis of variance (ANOVA) and the post hoc testof Tukey were used for compare mean differences of body lengthand organ measurements of E. paraensei. The accepted significantlevel was P < 0.05.

3. Results

In this experiment, necropsies performed between day 3 and14 dpi showed that all hamsters exposed to 100 E. paraensei meta-cercariae became infected. The worms recovered data are summa-rized in Table 1.

Mean body length and width of 10 worms/day of observationare depicted in Table 1. Worm exhibited a significant growth inbody length after 7 days p.i. (P < 0.05) and in body width after5 days p.i. onward (P < 0.001).

Mean gonad length and width of worms are presented in Table1. The ovary and testis were not able to be observed by lightmicroscopy to measure at 3 days p.i. The length and width of ovary,anterior and posterior testes were significant after 7 days p.i.(P < 0.05).

The morphological study of the reproductive system, over time,showed that on day 3, the ovary and testes were evidenced (Table2). This early phase represented the youngest worms. By carminered-staining and CSLM it was possible to observe the developmentof the cirrus sac, testes and ovary anlages (Fig. 1A and B). Two sep-arate developing testes were close to each other within few germi-native cells as a cluster of cells surrounded by an elongatedmonolayer of cells, while the ovary anlage was individualizedand not enveloped by a cell coat according to CSLM (Fig. 1B). How-ever, these structures were undifferentiated by light microscopyand seemed to be a single organ. On day 5, other structures, such

of Mesocricetus auratus. Different superscript letters (a–d) on each column indicate

Anterior testis Posterior testis

Width ± S.E. Length ± S.E. Width ± S.E. Length ± S.E. Width ± S.E.

– – – – –0.03 ± 0.12a 0.01 ± 0.02a 0.02 ± 0.008a 0.01 ± 0.003a 0.02 ± 0.008a

0.05 ± 0.01a 0.08 ± 0.01a 0.06 ± 0.01a 0.04 ± 0.007a 0.06 ± 0.01a

0.15 ± 0.04b 0.19 ± 0.05b 0.23 ± 0.06b 0.21 ± 0.005b 0.22 ± 0.05b

0.43 ± 0.09c 0.36 ± 0.15c 0.62 ± 0.04c 0.56 ± 0.16c 0.59 ± 0.09c

Fig. 1. Confocal scanning laser microscopy images of whole-mounts preparations of Echinostoma paraensei worms stained with hydrochloric carmine. (A) Anterior endshowing cirrus sac [cs] – 3 days post infection; (B) posterior testis [pt], anterior testis [at] and ovary [ov] – 3 days post infection; (C) anterior end showing cirrus sac [cs] –5 days post infection; (D) anterior end showing cirrus sac [cs] and uterus [u] in the medium region of body – 5 days post infection; (E) uterus [u], ootype [oo], ovary [ov] andanterior testis [at] – 5 days post infection; (F) anterior testis [at] and posterior testis [pt] – 5 days post infection.

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as the uterus, ootype and cirrus sac, were observed. At this point,the cirrus sac showed primordial cells and a muscular organ with-out seminal vesicle, prostate gland and ejaculatory ducts (Fig. 1C).The presence of cells surrounding a thin line of cells constitutingthe uterus wall in the direction of the primordial ootype towardthe cirrus sac was observed (Fig. 1D and E). The oviduct was notpresent (Fig. 1E). The testes and ovary were clearly distinguishedby light microscopy and CSLM (Fig. 1E and F). At this time, the tes-tes had larger bulk than at previous ages but differentiated cells inthe gonads were still absent (Fig. 1F).

The oviduct, seminal receptacle, vitelline ducts and reservoirwere not observed on day 7 post infection. The cirrus sac containeda muscular cirrus without seminal vesicle (Fig. 2A). Two testeswere separated from each other (Fig. 2B). The uterus was joinedto the ovary directly while the ootype was developing ahead ofthe ovary (Fig. 2C). CSLM images showed a rosette-shaped struc-ture inside the ovary connected to the uterus (Fig. 2C). The ovoidcirrus sac was filled with secretory cells and was developing andclearly delimited on day 10 p.i. The seminal vesicle without sperm,ejaculatory duct and gonopore were present in the cirrus sac(Fig. 2D). Remarkably, the testes were larger than the ovary(Fig. 2E). This suggests an intensive proliferation of testis cells,indicating a maturation process was under way. A few bundles ofmature spermatozoon were present on the testes (Fig. 2F). How-ever, oocytes were not observed in the ovary. Observation by CSLMshowed the ootype surrounded by Mehli’s gland (Figs. 2E and 3C).The oviduct was present with a seminal receptacle and sphincterfrom the ovovitellin duct insertion (Fig. 3A). Vitelline ducts andreservoirs were present but there were no vitelline cells at thistime (Figs. 2E and 3B). The ootype was formed and Mehli’s glandoccupied large space between the ovary and testes (Fig. 3C).

On day 14, the worms had fully differentiated male and femalereproductive systems, in which vitelline glands, spermatozoa andeggs were evidenced (Table 1). Confocal images showed a pore onthe dorsal surface of the helminths (Fig. 3D), joined to the Laurer’scanal (Fig. 3E). The seminal receptacle arose from the oviduct(Fig. 3F). The ovary was mature, giving rise to a slender oviduct thatenlarged to become the ootype or egg chamber, which housedfecundated oocyte and vitelline cells forming eggs (Fig. 3F). Theovary had oogonial cells on the peripheral layer and primary oo-cytes (Fig. 3F), which enlarged to form angular cells with large nu-clei filling the ovary. The cirrus sac was well developed and ovoid,with seminal vesicle, prostate gland and unspined cirrus being con-spicuous. Sperm were stored in the seminal vesicle in worms 14 dpi(Fig. 4A). The vitelline glands were present, excreting vitelline cellsat 14 dpi (Fig. 4B) and irregular-shaped testes contained cellsundergoing division and a bundle of sperm (Fig. 4C).

4. Discussion

Echinostomes have been considered good laboratory models forhost–parasite relationship studies due to their relative ease ofmaintenance in laboratory conditions (Fried and Huffman, 1996).Based on worm establishment, survival and reproductive success,hamsters have been indicated as compatible hosts for Echinostomaspecies (Toledo et al., 2006). Even though E. paraensei was de-scribed some time ago (Lie and Basch, 1967), the E. paraensei–M.auratus model has been little investigated. Knowledge of develop-mental changes in reproductive system organization is importantbecause these changes are the starting point of the life cycleof flatworms within vertebrate hosts. The arrangement of the

Fig. 2. Confocal scanning laser microscopy images of whole-mounts preparations of Echinostoma paraensei worms stained with hydrochloric carmine. (A) Anterior endshowing cirrus sac [sc] and cirrus [ci] – 7 days post infection; (B) posterior testis [pt], anterior testis [at] – 7 days post infection; (C) ovary [ov] connected by uterus [u] (detail)and ootype adjacent [oo] – 7 days post infection; (D) anterior end showing cirrus sac [cs], seminal vesicle [sv] and gonopore [gp] – 10 days post infection; (E) uterus [u],ootype [oo], ovary [ov], vitelline ducts [vd] and anterior testis [at] – 10 days post infection; (F) inner of testis showing few cells in process of division – 10 days post infection.

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reproductive system seems to have a conserved character withinhermaphroditic digeneans. For helminthological studies, bright fieldand CSLM microscopy are complementary methods. The first givesan overview of the anatomy, while the second allows preciseobservation of the structures at early development stages.Examination of whole-mount preparations has demonstrated thatconfocal microscopy provides detailed knowledge of the reproduc-tive system of Schistosoma mansoni (Machado-Silva et al., 1998;Neves et al., 2005), Tanaisia bragai and Tanaisia inopina (D’ávilaet al., 2010) due to its better resolution. Confocal microscopy has alsobecome an important tool to clarify the organization of the muscula-ture and innervations of Echinostoma caproni (Šebelová et al., 2004).

By carmine red-staining and light microscopy, we observed thatE. paraensei shows intensive morphogenesis of the reproductivesystem, as does E. caproni (Šebelová et al., 2004). The present studyand our previous ones (Biolchini et al., 2006; Silva-Leitão et al.,2009) indicate that morphogenesis occurs over time. In the youn-gest worms (recovered at 3 dpi) the beginning of the ovary and tes-tes could be observed CSLM.

Maldonado et al. (2005) studied variations in the developmentof two E. paraensei isolate (SU and RB) from Rio de Janeiro Stateand observed differences in the dispersion of worms in the smallintestine of mice. Worms from SU isolate reaching the preferentialniche after being dispersed along the small intestine at 2 weeks p.i.The statistical analyses of morphometric data of E. paraensei fromM. auratus showed no significant difference in the growth variablein the initial analysis period. However, extremely significancedevelopment (P < 0001), mainly of gonads, was observed after1 week post infection. Our data suggest significant growth in theworm occurred after it had arrived at this site.

A clear-cut distinction of the ovary from ootype was not appar-ent in E. caproni on days 2 and 4 p.i. (Manger and Fried, 1993). Theuterus, ootype and cirrus sac were observed on day 5. In this exper-iment, 3-day old E. paraensei specimens did not show ootype,uterus, vitelline gland, spermatozoa and eggs. Curiously, between7 and 10 days after infection, the worms had only slight genitaldevelopment. It seems that these worms only undergo a processof cellular differentiation of organs. Whether this type of develop-ment is the same for other hermaphroditic digeneans remains tobe elucidated.

The development of the excysted metacercariae of E. caproniuntil they reach the egg-laying adult stage is called the pre-ovigerous period (Šebelová et al., 2004). Our findings showed thatthe male reproductive system of E. parensei matures before thefemale system, in contrast to other trematodes (Cable and Harris,2002). In this study, the development of E. paraensei to the oviger-ous stage in hamsters occurred later than for other species of thegenus. E. caproni, which parasitizes birds and mammals, becomesovigerous between 7 and 10 days post infection (Chien and Fried,1992; Fried et al., 1988; Fried and Rosa-Brunet, 1991; Fried andHuffman, 1996; Hosier and Fried, 1991; Manger and Fried, 1993;Yao et al., 1991), while Echinostoma friedi, Echinostoma revolutum,Echinostoma malayanum and Echinostoma hortense infecting

rodents became ovigerous earlier than E. paraensei (Franco et al.,1986; Mohandas and Nadakal, 1978; Seo et al., 1985; Toledoet al., 2000). Egg production can begin on day 10 p.i. in pigeonsinfected with E. revolutum (Knav, 1994), even though both 12and 14 dpi has been reported (Humphries et al., 1997).

A previous study (Lie and Basch, 1967) showed E. paraensei(Minas Gerais State) developed in hamsters, white mice and white

Fig. 3. Confocal scanning laser microscopy images of whole-mounts preparations of Echinostoma paraensei worms stained with hydrochloric carmine. (A) Oviduct [ovd]forming seminal receptacle [rs] and insertion of ovovitellin duct by a sphincter [sf] – 10 days post infection; (B) junction of vitelline duct [vd] to form reservoir vitelline [rv] –10 days post infection; (C) ovary [ov] connected by uterus [u] though ootype [oo] – 10 days post infection; (D) pore at dorsal surface [p] – 14 days post infection; (E) Laurer’scanal [Lc] connected to oviduct – 14 days post infection; (F) seminal receptacle [sr], oviduct [ovd] and ootype [oo] containing an early stage of development of the egg –14 days post infection.

Fig. 4. Confocal scanning laser microscopy images of whole-mounts preparations of Echinostoma paraensei worms stained with hydrochloric carmine. (A) Detail of cirrus sacshowing seminal vesicle [vs] containing sperm [sp] – 14 days post infection; (B) vitelline glands [vg] – 14 days post infection; (C) inner of the testis showing cells in differentstage of division – 14 days post infection.

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rats. The eggs were found in the stools of hamsters and mice at 11–13 days p.i. and in rats at 14–16. It suggests that helminthes be-came mature with eggs in the uterus before these periods of time.Differently, E. parensei from the present study appeared eggs in theuterus at 14 days p.i. which can be an effect of 100 metacercariaecyst inoculum on the maturity of helminthes.

Maldonado et al. (2005) compared the biological features of twoisolates of E. paraensei from Rio Bonito (RB) and Sumidouro (SU),Rio de Janeiro State, Brazil. Adult worms were colleted fromnaturally infected N. squamipes and the cysts were obtained after

experimental infection of laboratory reared B. glabrata. Ten en-cysted metacercariae fed albino mice (Swiss Webster). The firstuterine eggs appeared in the worms from the SU isolate at week2 p.i., similar to the present study, and from RB isolate at week 3p.i. However the present study had used 100 cysts of metacercariaeMaldonado et al. (2005) used 10 cysts and both studies showedsimilar pre-patent period which suggest variability in the time ofdevelopment among distinct geographic isolates.

At 14 dpi, worms showed elongated bodies and the lateral bor-ders curved ventrally, the reproductive system occupied great part

346 J.G.R. Souza et al. / Experimental Parasitology 128 (2011) 341–346

of the body and almost all worms (89%) were ovigerous, with eggsin a long and coiled uterus, as also observed by Maldonado et al.(2001a). Šebelová et al. (2004) reported that at this phase the sem-inal receptacle arises from the oviduct and the mature ovary givesrise to a slender oviduct that enlarges to become the ootype or eggchamber. In this study the ovary had oogonial cells on the periphallayer and primary oocytes, which enlarged to form angular cellswith large nuclei filling the interior of the ovary, in agreement witha previous description (Nollen, 1983). The ootype opened into acoiled uterus, which proceeded anteriorly between the gut caecato open ventrally via a common gonopore with the cirrus, as alsoreported by Šebelová et al. (2004). While oocytes were not presentin the ovary at 10 dpi, the testes had partly developed cells. Wenoted earlier male than female cell development, which is pro-posed as protandry in echinostomes, as also observed in S. mansoni(Silva-Leitão et al., 2009). We conclude that the reproductive sys-tem of E. paraensei is functional on 14 dpi in hamsters.

Acknowledgments

We thank Dr. Marcelo Pelajo of the Pathology Laboratory ofOswaldo Cruz Institute (FIOCRUZ) for providing access to the con-focal microscopy equipment. Souza received financial support fromCAPES (Brazil).

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