Experimental Parasitology 128 (2011) 341–346

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Experimental Parasitology

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Development of the reproductive system of paraensei in 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 Reservoirs, Oswaldo Cruz Institute, Av. Brasil 4365, Manguinhos, 21040-360 Rio de Janeiro, Brazil b Laboratory of Pathology, Oswaldo Cruz Institute, Brazil c 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 article info abstract

Article history: This study was performed to gain insight into the maturation of the reproductive system of Echinostoma Received 14 January 2011 paraensei worms grown in an early of Mesocricetus auratus. Hamsters were infected with 100 Received in revised form 30 March 2011 metacercariae and necropsied on days 3, 5, 7, 10 and 14 post infection (dpi). Recovered flukes stained Accepted 26 April 2011 with hydrochloric carmine were preserved as whole mounts and analyzed by light and confocal scanning Available online 1 May 2011 laser microscopy. The average worm recovery was 43.7 per . Images of the male and female repro- ductive systems were taken. The ovary and anterior and posterior testis were evidenced on day 3, while Keywords: the ootype and cirrus sac were present on day 5. Confocal imaging showed primordium testis and ovary Echinostoma paraensei as a cluster of primordial cells from day 3 onward. The testes, ovary, cirrus sac and uterus organs were Reproductive system Morphology already present during the first week of life. The two testes were seen as individual structures on 7 dpi Mesocricetus auratus while the cirrus sac and vitelline glands were in development. The ovary was connected to the uterus Confocal laser scanning microscopy while the ootype was adjacent to it. Both testes were larger than the ovary, showing cells at different stages of development, but with few bundles of functional spermatozoa in 10 day-old worms. On day 14, eggs and spermatozoa were seen in the uterus and seminal vesicle, respectively, while oocytes appeared in the ootype as fertilized eggs. We conclude that the reproductive system of E. paraensei was functional on 14 dpi in the hamsters. Ó 2011 Elsevier Inc. Open access under the Elsevier OA license.

1. Introduction secluded Atlantic Forest regions of Rio de Janeiro state in Brazil (Maldonado et al., 2001a,b). Moreover, studies of E. paraensei Adult echinostomes are hermaphroditic digenetic flatworms populations from those different forest fragments have shown that generally parasitize the small intestine and bile ducts of all variations in biological parameters (Maldonado et al., 2005). Under classes of vertebrate hosts (Maldonado and Lanfredi, 2009). laboratory conditions, this parasite has been shown to be able to Echinostomes do not undergo tissue migration in the definitive complete its life cycle in the intermediate hosts B. glabrata, Physa host (Toledo, 2009). After infection of the definitive host, the marmorata and Lymnaea columella and the vertebrate hosts metacercariae excyst in the and the juvenile parasites Mesocricetus auratus and Mus musculus (Maldonado et al., 2001b). migrate to the small intestine, where they attach to the mucosa The most important and conspicuous organs of trematodes are by their ventral sucker (Fried and Huffman, 1996). those of the reproductive system (Nollen, 1997). This system is Echinostoma paraensei (Lie and Basch, 1967) (: extensively developed and although varying in detail, follows Platyhelminthes) was first described from natural infection of essentially the same pattern in most species (Smyth, 1994). The fe- Biomphalaria glabrata from Brazil shipped to the United States male reproductive system of E. paraensei comprises the ovary, (Maldonado et al., 2001a). The wild rodent Nectomys squamipes receptaculum seminal, ootype, oviduct, vitelline reservoir, vitelline (Rodentia: Sigmondontinae) is its natural vertebrate host in duct, and uterus (Madhavi and Rao, 1972). The male system com- prises two testes in tandem, the vas deferens leading to the cirrus sac containing seminal vesicle, along with the cirrus and prostate gland (Maldonado et al., 2001a). The cirrus is large and dominated ⇑ Corresponding author. by a highly muscular ejaculatory duct (Šebelová et al., 2004). E-mail address: [email protected] (J.R. Machado-Silva). 1 José Roberto Machado-Silva has a fellowship from the National Council for Laurer’s canal, vitellaria and Mehlis gland are structures associated Scientific and Technological Development (CNPq). in an intricate system.

0014-4894 Ó 2011 Elsevier Inc. Open access under the Elsevier OA license. doi:10.1016/j.exppara.2011.04.005 342 J.G.R. Souza et al. / Experimental Parasitology 128 (2011) 341–346

Echinostomes have long served as experimental model organ- day post infection. The ovary, uterus, ootype, vitelline glands, tes- isms, contributing in particular to knowledge of trematode biology, tes and cirrus sac were analyzed by light and confocal scanning la- parasite–host relationships, biochemical and parasite immune ser microscopy (LSM-510 META Zeiss), using a 543 nm He/Ne laser interactions and reproductive behavior (Fried, 1994; Fried and and a LP 570 filter under reflected mode. Huffman, 1996; Nollen, 1997; Trelis et al., 2011). In addition, morphological studies have examined features of eggs, miracidia, 2.4. Statistical analyses rediae, metacercariae and adult worms (Fried and Reddy, 1997; Fujino et al., 2000; Maldonado et al., 2003; Pinheiro et al., The one-way analysis of variance (ANOVA) and the post hoc test 2004a,b, 2005; Toledo et al., 2004). However, there is no informa- of Tukey were used for compare mean differences of body length tion concerning the morphology of the juvenile stage of this and organ measurements of E. paraensei. The accepted significant parasite, which corresponds to the pre-ovigerous stage of develop- level was P < 0.05. ment in vertebrate hosts. Previous studies have demonstrated that confocal microscopy provides detailed knowledge of the morphological structures, due 3. Results to its better resolution (Machado-Silva et al., 1998; Neves et al., 2005; Šebelová et al., 2004). In this study, both light and confocal In this experiment, necropsies performed between day 3 and scanning laser microscopy (CSLM) were used to describe details 14 dpi showed that all hamsters exposed to 100 E. paraensei meta- of the reproductive system development in E. paraensei from 3 un- cercariae became infected. The worms recovered data are summa- til 14 days post infection. rized in Table 1. Mean body length and width of 10 worms/day of observation are depicted in Table 1. Worm exhibited a significant growth in 2. Materials and methods body length after 7 days p.i. (P < 0.05) and in body width after 5 days p.i. onward (P < 0.001). 2.1. The host–parasite model Mean gonad length and width of worms are presented in Table 1. The ovary and testis were not able to be observed by light Encysted metacercariae of E. paraensei (Capitão Andrade – MG microscopy to measure at 3 days p.i. The length and width of ovary, isolate) were removed from the pericardial cavity-kidney region anterior and posterior testes were significant after 7 days p.i. of experimentally infected B. glabrata snails. Two-week old outbred (P < 0.05). M. auratus females were obtained from the Breeding Center The morphological study of the reproductive system, over time, of Oswaldo Cruz Foundation (FIOCRUZ, Rio de Janeiro, Brazil). The showed that on day 3, the ovary and testes were evidenced (Table hamsters were housed in polypropylene boxes (40 Â 33 cm) with 2). This early phase represented the youngest worms. By carmine stainless steel screen covers. Water and food (Nuvilab CR1, Colom- red-staining and CSLM it was possible to observe the development bo, Paraná, Brazil) were provided ad libitum. The experiments re- of the cirrus sac, testes and ovary anlages (Fig. 1A and B). Two sep- ported here complied with the ethical procedures for animal arate developing testes were close to each other within few germi- experiments (Ellery, 1985) and were approved by the local Ethical native cells as a cluster of cells surrounded by an elongated Committee on Animal Use (CEUA L-074/08). monolayer of cells, while the ovary anlage was individualized and not enveloped by a cell coat according to CSLM (Fig. 1B). How- 2.2. Experimental design ever, these structures were undifferentiated by light microscopy and seemed to be a single organ. On day 5, other structures, such To obtain the worms, 12 golden hamsters were each orally fed 100 metacercariae (Maldonado et al., 2001b). Two hamsters were Table 2 euthanized in a CO2 camera on each of days 3, 5, 7, 10 and 14 after Presence of reproductive structures of Echinostoma paraensei recovered from ham- infection. Worms were removed from the small intestine of the sters at various days after infection analyzed by light and confocal scanning laser hamsters and rinsed briefly in Locke’s solution to remove the deb- microscopy. ris and then were fixed in AFA solution (2% acetic acid, 3% formal- Age (days) Morphological characteristics dehyde, 95% and 70° ethanol) under slight cover slip pressure at room temperature. OV AT PT U OO SC VG SP EG 3+++–––––– 5++++++––– 2.3. Morphological study 7++++++––– 10 + + + + + + – – – The worms were stained with hydrochloric carmine, dehy- 14 + + + + + + + + + drated in a graded ethanol series, cleared with methyl salicylate +, present; –, absent. and mounted in Canada balsam (Neves et al., 1998). The body OV: ovary; AT: anterior testis; PT: posterior testis; U: uterus; OO: ootype; SC: cirrus and organ measurements of 10 worms (in mm) were taken on each sac; VG: vitelline glands; SP: spermatozoon; EG: eggs.

Table 1 Worm recovery and morphometric data (mm) of E. paraensei from experimental infection of Mesocricetus auratus. Different superscript letters (a–d) on each column indicate differences between values.

Days p.i. Worm recovery (%) Body Ovary Anterior testis Posterior testis Length ± S.E. Width ± S.E Length ± S.E. Width ± S.E. Length ± S.E. Width ± S.E. Length ± S.E. Width ± 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 0.03 ± 0.12a 0.01 ± 0.02a 0.02 ± 0.008a 0.01 ± 0.003a 0.02 ± 0.008a 7 37 1.56 ± 0.18b 0.37 ± 0.03b 0.05 ± 0.01a 0.05 ± 0.01a 0.08 ± 0.01a 0.06 ± 0.01a 0.04 ± 0.007a 0.06 ± 0.01a 10 48 4.78 ± 0.33c 0.91 ± 0.07c 0.12 ± 0.01b 0.15 ± 0.04b 0.19 ± 0.05b 0.23 ± 0.06b 0.21 ± 0.005b 0.22 ± 0.05b 14 36 7.53 ± 1.58d 1.19 ± 0.07d 0.26 ± 0.06c 0.43 ± 0.09c 0.36 ± 0.15c 0.62 ± 0.04c 0.56 ± 0.16c 0.59 ± 0.09c J.G.R. Souza et al. / Experimental Parasitology 128 (2011) 341–346 343

Fig. 1. Confocal scanning laser microscopy images of whole-mounts preparations of Echinostoma paraensei worms stained with hydrochloric carmine. (A) Anterior end showing 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] and anterior testis [at] – 5 days post infection; (F) anterior testis [at] and posterior testis [pt] – 5 days post infection. as the uterus, ootype and cirrus sac, were observed. At this point, On day 14, the worms had fully differentiated male and female the cirrus sac showed primordial cells and a muscular organ with- reproductive systems, in which vitelline glands, spermatozoa and out seminal vesicle, prostate gland and ejaculatory ducts (Fig. 1C). eggs were evidenced (Table 1). Confocal images showed a pore on The presence of cells surrounding a thin line of cells constituting the dorsal surface of the helminths (Fig. 3D), joined to the Laurer’s the uterus wall in the direction of the primordial ootype toward canal (Fig. 3E). The seminal receptacle arose from the oviduct the cirrus sac was observed (Fig. 1D and E). The oviduct was not (Fig. 3F). The ovary was mature, giving rise to a slender oviduct that present (Fig. 1E). The testes and ovary were clearly distinguished enlarged to become the ootype or egg chamber, which housed by light microscopy and CSLM (Fig. 1E and F). At this time, the tes- fecundated oocyte and vitelline cells forming eggs (Fig. 3F). The tes had larger bulk than at previous ages but differentiated cells in ovary had oogonial cells on the peripheral layer and primary oo- the gonads were still absent (Fig. 1F). cytes (Fig. 3F), which enlarged to form angular cells with large nu- The oviduct, seminal receptacle, vitelline ducts and reservoir clei filling the ovary. The cirrus sac was well developed and ovoid, were not observed on day 7 post infection. The cirrus sac contained with seminal vesicle, prostate gland and unspined cirrus being con- a muscular cirrus without seminal vesicle (Fig. 2A). Two testes spicuous. Sperm were stored in the seminal vesicle in worms 14 dpi were separated from each other (Fig. 2B). The uterus was joined (Fig. 4A). The vitelline glands were present, excreting vitelline cells to the ovary directly while the ootype was developing ahead of at 14 dpi (Fig. 4B) and irregular-shaped testes contained cells the ovary (Fig. 2C). CSLM images showed a rosette-shaped struc- undergoing division and a bundle of sperm (Fig. 4C). ture inside the ovary connected to the uterus (Fig. 2C). The ovoid cirrus sac was filled with secretory cells and was developing and clearly delimited on day 10 p.i. The seminal vesicle without sperm, 4. Discussion ejaculatory duct and gonopore were present in the cirrus sac (Fig. 2D). Remarkably, the testes were larger than the ovary Echinostomes have been considered good laboratory models for (Fig. 2E). This suggests an intensive proliferation of testis cells, host–parasite relationship studies due to their relative ease of indicating a maturation process was under way. A few bundles of maintenance in laboratory conditions (Fried and Huffman, 1996). mature spermatozoon were present on the testes (Fig. 2F). How- Based on worm establishment, survival and reproductive success, ever, oocytes were not observed in the ovary. Observation by CSLM hamsters have been indicated as compatible hosts for Echinostoma showed the ootype surrounded by Mehli’s gland (Figs. 2E and 3C). species (Toledo et al., 2006). Even though E. paraensei was de- The oviduct was present with a seminal receptacle and sphincter scribed some time ago (Lie and Basch, 1967), the E. paraensei–M. from the ovovitellin duct insertion (Fig. 3A). Vitelline ducts and auratus model has been little investigated. Knowledge of develop- reservoirs were present but there were no vitelline cells at this mental changes in reproductive system organization is important time (Figs. 2E and 3B). The ootype was formed and Mehli’s gland because these changes are the starting point of the life cycle occupied large space between the ovary and testes (Fig. 3C). of flatworms within vertebrate hosts. The arrangement of the 344 J.G.R. Souza et al. / Experimental Parasitology 128 (2011) 341–346

Fig. 2. Confocal scanning laser microscopy images of whole-mounts preparations of Echinostoma paraensei worms stained with hydrochloric carmine. (A) Anterior end showing 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.

reproductive system seems to have a conserved character within A clear-cut distinction of the ovary from ootype was not appar- hermaphroditic digeneans. For helminthological studies, bright field ent in E. caproni on days 2 and 4 p.i. (Manger and Fried, 1993). The and CSLM microscopy are complementary methods. The first gives uterus, ootype and cirrus sac were observed on day 5. In this exper- an overview of the anatomy, while the second allows precise iment, 3-day old E. paraensei specimens did not show ootype, observation of the structures at early development stages. uterus, vitelline gland, spermatozoa and eggs. Curiously, between Examination of whole-mount preparations has demonstrated that 7 and 10 days after infection, the worms had only slight genital confocal microscopy provides detailed knowledge of the reproduc- development. It seems that these worms only undergo a process tive system of Schistosoma mansoni (Machado-Silva et al., 1998; of cellular differentiation of organs. Whether this type of develop- Neves et al., 2005), Tanaisia bragai and Tanaisia inopina (D’ávila ment is the same for other hermaphroditic digeneans remains to et al., 2010) due to its better resolution. Confocal microscopy has also be elucidated. become an important tool to clarify the organization of the muscula- The development of the excysted metacercariae of E. caproni ture and innervations of Echinostoma caproni (Šebelová et al., 2004). until they reach the egg-laying adult stage is called the pre- By carmine red-staining and light microscopy, we observed that ovigerous period (Šebelová et al., 2004). Our findings showed that E. paraensei shows intensive morphogenesis of the reproductive the male reproductive system of E. parensei matures before the system, as does E. caproni (Šebelová et al., 2004). The present study female system, in contrast to other trematodes (Cable and Harris, and our previous ones (Biolchini et al., 2006; Silva-Leitão et al., 2002). In this study, the development of E. paraensei to the oviger- 2009) indicate that morphogenesis occurs over time. In the youn- ous stage in hamsters occurred later than for other species of the gest worms (recovered at 3 dpi) the beginning of the ovary and tes- genus. E. caproni, which parasitizes and , becomes tes could be observed CSLM. ovigerous between 7 and 10 days post infection (Chien and Fried, Maldonado et al. (2005) studied variations in the development 1992; Fried et al., 1988; Fried and Rosa-Brunet, 1991; Fried and of two E. paraensei isolate (SU and RB) from Rio de Janeiro State Huffman, 1996; Hosier and Fried, 1991; Manger and Fried, 1993; and observed differences in the dispersion of worms in the small Yao et al., 1991), while Echinostoma friedi, Echinostoma revolutum, intestine of mice. Worms from SU isolate reaching the preferential Echinostoma malayanum and Echinostoma hortense infecting niche after being dispersed along the small intestine at 2 weeks p.i. rodents became ovigerous earlier than E. paraensei (Franco et al., The statistical analyses of morphometric data of E. paraensei from 1986; Mohandas and Nadakal, 1978; Seo et al., 1985; Toledo M. auratus showed no significant difference in the growth variable et al., 2000). Egg production can begin on day 10 p.i. in pigeons in the initial analysis period. However, extremely significance infected with E. revolutum (Knav, 1994), even though both 12 development (P < 0001), mainly of gonads, was observed after and 14 dpi has been reported (Humphries et al., 1997). 1 week post infection. Our data suggest significant growth in the A previous study (Lie and Basch, 1967) showed E. paraensei worm occurred after it had arrived at this site. (Minas Gerais State) developed in hamsters, white mice and white J.G.R. Souza et al. / Experimental Parasitology 128 (2011) 341–346 345

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’s canal [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 sac showing 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 different stage of division – 14 days post infection.

. The eggs were found in the stools of hamsters and mice at 11– experimental infection of laboratory reared B. glabrata. Ten en- 13 days p.i. and in rats at 14–16. It suggests that helminthes be- cysted metacercariae fed albino mice (Swiss Webster). The first came mature with eggs in the uterus before these periods of time. uterine eggs appeared in the worms from the SU isolate at week Differently, E. parensei from the present study appeared eggs in the 2 p.i., similar to the present study, and from RB isolate at week 3 uterus at 14 days p.i. which can be an effect of 100 metacercariae p.i. However the present study had used 100 cysts of metacercariae cyst inoculum on the maturity of helminthes. Maldonado et al. (2005) used 10 cysts and both studies showed Maldonado et al. (2005) compared the biological features of two similar pre-patent period which suggest variability in the time of isolates of E. paraensei from Rio Bonito (RB) and Sumidouro (SU), development among distinct geographic isolates. Rio de Janeiro State, Brazil. Adult worms were colleted from At 14 dpi, worms showed elongated bodies and the lateral bor- naturally infected N. squamipes and the cysts were obtained after 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 eggs Madhavi, R., Rao, K.H., 1972. Anatomy of female reproductive system in digenetic in a long and coiled uterus, as also observed by Maldonado et al. trematodes: Part I. Echinostomatoidea. Rivista di Parassitologia 33, 173– 182. (2001a). Šebelová et al. (2004) reported that at this phase the sem- Maldonado Jr., A., Lanfredi, R.M., 2009. Echinostomes in the wild. In: Fried, B., inal receptacle arises from the oviduct and the mature ovary gives Toledo, R. (Eds.), The Biology of Echinostomes from the Molecule to the rise to a slender oviduct that enlarges to become the ootype or egg Community. Springer, New York, pp. 129–145. Maldonado Jr., A., Loker, E.S., Morgan, J.A.T., 2001a. Description of the adult worms chamber. In this study the ovary had oogonial cells on the periphal of a new Brazilian isolate of Echinostoma paraensei (Platyhelminthes: Digenea) layer and primary oocytes, which enlarged to form angular cells from its natural vertebrate host Nectomys squamipes by light and scanning with large nuclei filling the interior of the ovary, in agreement with electron microscopy and molecular analysis. Parasitology Research 87, 840– 848. a previous description (Nollen, 1983). The ootype opened into a Maldonado Jr., A., Vieira, G.O., Garcia, J.S., Rey, L., Lanfredi, R.M., 2001b. Biological coiled uterus, which proceeded anteriorly between the gut caeca aspects of a new isolate of Echinostoma paraensei (: to open ventrally via a common gonopore with the cirrus, as also Echinostomatidae): susceptibility of sympatric snails and the natural vertebrate host. Parasitology Research 87, 853–859. reported by Šebelová et al. (2004). While oocytes were not present Maldonado Jr., A., Vieira, G.O., Lanfredi, R.M., 2003. Echinostoma luisreyi n. sp. in the ovary at 10 dpi, the testes had partly developed cells. We (Platyhelminthes: Digenea) by light and scanning electron microscopy. Journal noted earlier male than female cell development, which is pro- of Parasitology 89, 800–808. posed as protandry in echinostomes, as also observed in S. mansoni Maldonado Jr., A., Zeitone, B.K., Amado, L.A., Rosa, I.F., Machado-Silva, J.R., Lanfredi, R.M., 2005. Biological variation between two Brazilian geographical isolates of (Silva-Leitão et al., 2009). 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