The Life Cycle of Pseudosellacotyla lutzi (Digenea: Cryptogonimidae), in Aylacostoma chloroticum (Prosobranchia: Thiaridae), and Hoplias malabaricus (Characiformes: Erythrinidae), in Argentina Author(s): Manuel G. Quintana and Margarita Ostrowski de Núñez Source: Journal of Parasitology, 100(6):805-811. Published By: American Society of Parasitologists DOI: http://dx.doi.org/10.1645/13-379.1 URL: http://www.bioone.org/doi/full/10.1645/13-379.1 BioOne (www.bioone.org) is a nonprofit, online aggregation of core research in the biological, ecological, and environmental sciences. BioOne provides a sustainable online platform for over 170 journals and books published by nonprofit societies, associations, museums, institutions, and presses. Your use of this PDF, the BioOne Web site, and all posted and associated content indicates your acceptance of BioOne’s Terms of Use, available at www.bioone.org/page/terms_of_use. Usage of BioOne content is strictly limited to personal, educational, and non-commercial use. Commercial inquiries or rights and permissions requests should be directed to the individual publisher as copyright holder. BioOne sees sustainable scholarly publishing as an inherently collaborative enterprise connecting authors, nonprofit publishers, academic institutions, research libraries, and research funders in the common goal of maximizing access to critical research. J. Parasitol., 100(6), 2014, pp. 805–811 Ó American Society of Parasitologists 2014 THE LIFE CYCLE OF PSEUDOSELLACOTYLA LUTZI (DIGENEA: CRYPTOGONIMIDAE), IN AYLACOSTOMA CHLOROTICUM (PROSOBRANCHIA: THIARIDAE), AND HOPLIAS MALABARICUS (CHARACIFORMES: ERYTHRINIDAE), IN ARGENTINA Manuel G. Quintana and Margarita Ostrowski de Núñez* Museo Argentino de Ciencias Naturales ‘‘Bernardino Rivadavia’’, Av. Angel Gallardo 470, C1405 DJR, Buenos Aires, Argentina. Correspondence should be sent to: ostrowskimargarita@gmail.com ABSTRACT: Pseudosellacotyla lutzi (Freitas, 1941), at present included in the Faustulidae, is redescribed, and its life cycle was resolved experimentally. The prosobranch snail Aylacostoma chloroticum Hylton Scott (Thiaridae), collected in the Yacyretá Dam, Province of Misiones, Argentina, was found naturally infected with cercariae that lacked pigmented eyespots, and possessed 7 pairs of penetration glands, 8 pairs of flame cells, and a V-shaped excretory vesicle. The cercariae developed in oval cysts, which were found on fin rays, vertebrae, and spines of poeciliid and tetragonopterid fish species. Adults were obtained experimentally from Hoplias malabaricus (Erythrinidae) infected with metacercariae from albino Gymnocorymbus ternetzi (Tetragonopteridae), which had been exposed to emerging cercariae. Adults were also found in naturally infected H. malabaricus collected in the Yacyretá Dam. The morphology of the cercariae, and the characteristics of the life cycle show that P. lutzi should be included in the Cryptogonimidae. MATERIALS AND METHODS Snails of the genus Aylacostoma Spix, 1827 (Prosobranchia, Thiaridae) are distributed in freshwater habitats of tropical and subtropical areas of Central and South America. At their southernmost distributional limit in Brazil, Paraguay, and Argentina, they occur exclusively in the Paraná River and a few major tributaries (von Ihering, 1902, 1909; Hylton Scott, 1953, 1954; Quintana and Mercado Laczkó, 1997; Simone, 2006). Aylacostoma chloroticum Hylton Scott, 1954 is the only species of the genus in Paraguay and Argentina surviving in the wild after the construction of the Yacyretá hydroelectric power plant. However, it is now seriously threatened by the alteration of its original habitat, the rapids of the Paraná River. During a parasite survey of the last 2 remaining relictual populations at the upstream section of the reservoir, we observed 6 different cercariae emerging from the snails, of which 4 were opisthorchioid-like, and 2 belong to the Echinostomatoidea. Recently, the life cycle of 1 of these, Stephanoprora aylacostoma Ostrowski de Núñez et Quintana, 2008, has been elucidated (Ostrowski de Núñez and Quintana, 2008). In the present article we describe the life cycle of Pseudosellacotyla lutzi (Freitas, 1941), which proved to be the adult of 1 of the ‘‘opisthorchioid’’ like cercariae. This species has been included in different families along its history. It was originally included in the Nanophyetidae, Dollfus, 1939 by Freitas (1941), considered to have affinities to the Heterophyidae, in the Heterophyidae by Yamaguti (1953), in the Microphallidae by Yamaguti (1958), in the Fellodistomidae (Baccigerinae) by Yamaguti (1971), and finally, in the Faustulidae by Bray (2008). Pseudosellacotyla lutzi was reported several times from Brazil and Colombia, always from the same fish host, Hoplias malabaricus (Bloch, 1794), by Kohn et al. (1985), Kohn and Fernandes (1987), Kohn et al. (2011), and Lenis Velez et al. (2010). We include P. lutzi in Cryptogonimidae, based on its morphological characteristics and the pattern of its life history. Specimens of A. chloroticum up to 37 mm in total shell length (TSL) were collected by hand by scuba diving in flooded areas of the Paraná River. Sampling dates and locations were as follows: September 2008 in the Heller Peninsula (27820 0 S, 55855 0 W) near Posadas City, and January 2010 in the Heller Peninsula and Candelaria, (27827 0 S, 55845 0 W), Province of Misiones. Snails were kept individually in vials with 50 ml of tap water and examined daily for the emergence of cercariae. Some snails were dissected to check for intramolluscan stages. Laboratory-raised Poecilia reticulata Peters, 1859, Cnesterodon decemmaculatus (Jenyns, 1842), and albino Gymnocorymbus ternetzi (Boulenger, 1895) were exposed to emerging cercariae. After identifying the infection site and morphology of the metacercariae, fishes belonging to the Tetragonopteridae, Moenckhausia dichroura (Kner, 1858), and Hyphessobrycon eques (Steindachner, 1882) were sampled at different sites along the dam to identify the natural second intermediate host. These fish were not used in experimental infections. Two albino G. ternetzi with cysts aged 20 days or older were offered alive or in pieces to individual presumptive definitive fish hosts every other day for 15 days; fishes used were 4 Pimelodus maculatus Lacepède, 1803, 2 Pimelodus albicans (Valenciennes, 1840), 4 Callichthys callichthys (Linnaeus, 1758), 4 Oxydoras eigenmanni Boulenger, 1895, 1 Luciopimelodus pati (Valenciennes, 1836), 6 Crenicichla lepidota Heckel, 1840, 10 Crenicichla vittata Heckel, 1840, 8 Oligosarcus jenynsii (Günther, 1864) and 17 juvenile (ca. 12 cm standard length, SL) H. malabaricus (9 exposed, 8 controls). They were obtained from local dealers, held in aerated aquaria and starved for several days prior to the experiments, to ensure that they would eat infected intermediate hosts. Following cold narcosis, fishes were killed and dissected at different times from day 2 postexposure (PE) onward; their feces were examined for parasite eggs from day 15 PE onward. In an attempt to determine the natural definitive hosts, piscivorous fishes common in the area were captured and examined; to this aim, the following fishes were collected from Candelaria in November 2012, January and March 2013: 4 H. malabaricus, 21 C. vittata, 2 Gymnogeophagus balzanii (Perugia, 1891), 42 Acestrorhynchus pantaneiro (Menezes, 1992), 3 Serrasalmus maculatus (Kner, 1858), 6 Galeocharax humeralis (Valenciennes, 1834), 1 Gymnotus inaequilabiatus (Valenciennes, 1839), 4 Roeboides sp., 18 Astyanax sp., and 22 M. dichroura. Rediae, cercariae, and metacercariae were studied alive or fixed in nearly boiling 4% formalin, cleared in lactophenol, and then mounted in glycerine jelly or formalin without applying pressure for measurements. Metacercariae were mechanically freed from cysts. Adults and some metacercariae freed from cysts were fixed in 4% formalin, stained with Semichon’s carmine or hematoxylin, and sometimes counterstained with light green or eosin, respectively. Then, the specimens were dehydrated in an ethanol series, cleared in creosote, and mounted in Canada balsam. For histological studies, pieces of pyloric ceca containing 1–4 parasites were embedded in paraffin wax, cut at 3 lm, and stained with Harris hematoxylin and eosin. Photographs were taken with an Olympus DP10 digital camera (Olympus, Tokyo, Japan), drawings were made with a Received 21 August 2013; revised 14 June 2014; accepted 16 June 2014. * Departamento de Biodiversidad y Biologı́a Experimental, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria, Pabellón II, 1428 Buenos Aires, Argentina. DOI: 10.1645/13-379.1 805 806 THE JOURNAL OF PARASITOLOGY, VOL. 100, NO. 6, DECEMBER 2014 drawing tube attached to an Olympus BH2-NIC microscope (Olympus), and details were added freehand. Some naturally infected snails and experimentally obtained adults were fixed in 96% ethanol for later possible molecular analysis. All measurements are given in micrometers, with the range followed by the mean and number (n) of specimens measured in parentheses. Specimens were deposited in the Parasitological Collection of the Museo Argentino de Ciencias Naturales ‘‘Bernardino Rivadavia,’’ Buenos Aires, Argentina (MACN-PA). REDESCRIPTION Pseudosellacotyla lutzi (Freitas, 1941) Yamaguti, 1953 (Figs. 1–17) Adult (Figs. 1–4, 15–17): Based on 90 mounted and several living specimens 13, 20, 48, and 54 days PE, and on 20 specimens obtained from natural infections. Measurements based on egg-bearing specimens 20 days PE in text and on other specimens in Table I. Body squat to oval, slightly longer than wide, 302–447 (395; n ¼ 21) 3 233–340 (307; n ¼ 20) at level of testes; width/length ratio 1:1.1–1.5 (1.3; n ¼ 20), with rounded posterior extremity. Forebody c. 41% of body length. Body spines of same length, 8–10 long, extend to posterior extremity, except around excretory pore. Oral sucker subterminal, 43–64 (54) 3 53–80 (66; n ¼ 21), without crown of spines. Ventral sucker in middle of body, smaller than oral sucker, 24–48 (38) 3 29–54 (42; n ¼ 17), embedded in tegument; ventral sucker to oral sucker width ratio 1:1.2–2.0 (1.6; n ¼ 17). Prepharynx short, generally indistinguishable, sometimes up to 14 (3; n ¼ 21) long; pharynx well developed, 30–56 (43) 3 32–48 (43; n ¼ 19); esophagus short, 0–24 (13; n ¼ 18), bifurcates short distance anterior to ventral sucker in 2 wide, short blind ceca, reaching level of ventral sucker. Testes 2, oval to spherical, nearly equal in size, symmetrical, in posterior half of body, left testis 109–152 (124) 3 77–112 (89; n ¼ 17); right testis 96–149 (125) 3 72–117 (91; n ¼ 17). Vasa efferentia fuse to form tubulosaccular seminal vesicle, occupying intertesticular space in immature specimens (Fig. 2); subsequently its proximal end develops into larger saccular vesicle, sometimes appearing bipartite, lying posterolaterally or posteromedially to ventral sucker. Pars prostatica and hermaphroditic duct very short, discernible only in histological sections and in living pressed specimens (Fig. 3). Genital pore medial, immediately anterior to ventral sucker. Gonotyl and ventrogenital sac absent. Ovary oval or irregular in shape, 43–91 (73) 3 40–88 (57; n ¼ 16), compact, between testes, near right testis (38 %), left testis (53 %), or middle line (9 %) (n ¼ 78) and near ventral sucker, sometimes overlapping. Canalicular seminal receptacle large, posterior to ovary. Laurer’s canal present, opening dorsally near posterior end of body (Fig. 4). Vitelline follicles oval to spherical, 14–48 (27) 3 16–51 (30; n ¼ 52) arranged in single group on each side of body at level of pharynx, extending to anterior level of testis. Uterus forms loops filling posterior half of body, with distal end opening into hermaphroditic duct. Eggs small, numerous, 32–40 (36) 3 14–21 (18; n ¼ 67), contain developed miracidium (Fig. 17). Excretory vesicle V-shaped, arms reach to posterior border of testes. Metacercariae (Figs. 5, 12, 13, 14): Cysts (n ¼ 28): 129–176 (157) 3 107–164 (137), in fin rays, vertebrae, and spines. Freed metacercariae similar in morphology to adult, except for development of vitelline follicles and absence of eggs. Body (n ¼ 3) 196 (144–256) 3 127 (109–160), tegumental spines conspicuous, oral sucker 35 (29–45) 3 40 (35–45), pharynx 32 3 32, ventral sucker 27 3 29 (26–32), short ceca, and testes symmetrical, at level of excretory vesicle. Cercariae (Figs. 6, 7, 10, 11): Body small, pigmented (n ¼ 20) 198 (183– 214) 3 61 (57–69), covered by tegumental spines on dorsal and ventral surface and fine sensory hairs; oral sucker 29 (26–32) 3 26 (22–29), 7 pairs of finely grained penetration glands, their ducts running forward to open into 4 sets of 3–4–4–3 pores at anterior extremity. Pigmented eyespots absent. Small pharynx 10 in diameter; esophagus and 2 short ceca observed in 1 pressed living specimen. Ventral sucker consisting of conspicuous cluster of cells. Presence of genital anlagen represented by small group of cells posterior to ventral sucker. Excretory vesicle Vshaped. Flame cells probably 4 groups of 2 flame cells each. Tail 322 (296– 340) 3 28 (25–38), with dorsoventral finfold (Fig. 11); inconspicuous short excretory canal sometimes observed in proximal 1/8; bifurcation or excretory pores not detected. Swimming behavior and resting position in water similar to those of other opisthorchioid species. Rediae (Figs. 8, 9): Occur as tangled masses, making it difficult to separate individual rediae. Rediae have elongate, slender yellow-brownish pigmented bodies of variable width (n ¼ 20) 426 (284–554) 3 52 (32–107), depending on development of germinal masses; pharynx small 24 (22–29) 3 22 (19–26); cecum short 70–122 (90), reaching 14–35% (24%) of body length. Rediae may contain 1–2 fully developed cercariae and numerous developing germ balls. Old rediae with bodies of variable width and several constrictions. Small living rediae (Fig. 9) with elongate body tapering at posterior end, without developing germ balls, 95–150 (128; n ¼ 10) 3 28–47 (36), pharynx 17–37 (27; n ¼ 10) 3 19–25 (22), with relatively longer cecum, reaching 50–75% of body length. Mother redia and sporocyst not detected. Taxonomic summary Type host and experimental definitive host: Hoplias malabaricus (Bloch, 1794) (Characiformes, Erythrinidae). Site of infection: Intestine and pyloric ceca. Prevalence: Two of 4 H. malabaricus (November 2012 and March 2013). Intensity: Fourteen and 12, respectively. First intermediate host: Aylacostoma chloroticum Hylton Scott, 1954 (Thiaridae). Prevalence: Forty-eight of 444 examined. Second intermediate host: Moenckhausia dichroura (Kner, 1858), H. eques (Steindachner, 1882) (Tetragonopteridae) (natural); P. reticulata Peters, 1859, C. decemmaculatus (Jenyns, 1842), (Poecilidae), albino G. ternetzi (Boulenger, 1895) (Tetragonopteridae) (experimental). Locality: Yacyretá Dam [Heller Peninsula (27820 0 S, 55855 0 W); Candelaria, (27827 0 S, 55845 0 W)], Paraná River, Province of Misiones, Argentina. Deposition of specimens: MACN-PA 565/1: metacercariae; 565/2–5: adults 13 days PE; MACN-PA 565/6–9: adults 20 days PE; MACN-PA 565/10–12: adults 48 days PE, MACN-PA 565/13: adults 54 days PE; MACN-PA 565/14–15: adults from natural infection. Remarks Forty-eight of 444 snails examined were found infected with cercariae of P. lutzi, and none of them showed dual or multiple infections with other digeneans. Sporocysts and mother rediae were absent, probably because of the age of infection. All laboratory-raised fish species exposed to cercariae became infected, with the longest survival of metacercariae being recorded for albino G. ternetzi individuals (up to 270 days PE, at the end of the experiments). None of the fish species used as presumptive definitive hosts became infected with P. lutzi after ingestion of metacercariae, except for the 9 exposed juvenile H. malabaricus; the remaining 8 used as controls were negative. As fish were obtained from local dealers, captured in localities far away from Yacyretá Dam, all of them were infected with at least 1 of the following parasites: cestodes and metacestodes, larvae of acanthocephalans, nematodes, digenean metacercariae, and digenean species different from P. lutzi. In the 9 exposed H. malabaricus, parasites were located in the proximal part of the intestine in 4 cases, of which 1 had 12 immature specimens at day 2 PE, and 3 had 15, 70, and 80 ovigerous specimens at days 11, 12, and 13 PE, respectively. In the other 4 cases, parasites were located exclusively in the pyloric ceca, which contained more than 100, 10, 70, and 20 specimens at days 20, 22, 48, and 54 PE, respectively. It is interesting to note that specimens were located in the intestine lumen of H. malabaricus juveniles before 13 days PE but in their pyloric ceca afterwards. Finally, 1 of the fish was kept alive in order to obtain parasite eggs, but only dead specimens without eggs could be recovered from its feces. When dissected on day 41 PE, the pyloric ceca and body cavity appeared to be heavily infected with larvae of cestodes and acanthocephalans, and a single specimen of P. lutzi filled with eggs was found in a pyloric cecum. This heavy infection with cestodes and acanthocephalans may account for the slight experimental infection by P. lutzi. Among the fishes collected from Candelaria, only 2 of the 4 H. malabaricus were parasitized with P. lutzi; these fish measured 200 and 234 mm SL and harbored 12 and 14 gravid specimens, respectively, in their pyloric ceca. QUINTANA AND OSTROWSKI DE NÚÑEZ—LIFE CYCLE OF PSEUDOSELLACOTYLA LUTZI 807 FIGURES 1–8. Pseudosellacotyla lutzi (Freitas, 1941): (1) Adult experimentally from Hoplias malabaricus 20 days postexposure (PE) (most eggs in central body area omitted). (2) Adult 13 days PE (without eggs), showing position of seminal vesicle. (3) Terminal genitalia. (4) Female genitalia (Figs. 3 and 4 reconstructed from living specimens). (5) Excysted metacercaria. (6) Cercaria, body with tail. (7) Body of cercaria, dark triangles: observed flame cells, open triangle: suspected position of missing flame cell. (8) Redia, fully developed, with 2 mature cercariae. Scale bars ¼ 100 lm. 808 THE JOURNAL OF PARASITOLOGY, VOL. 100, NO. 6, DECEMBER 2014 FIGURES 9–17. Pseudosellacotyla lutzi (Freitas, 1941): (9) Young redia. (10) Cercaria body. (11) Cercaria tail. (12) Metacercaria encysted on vertebrae (dark arrow). (13) Metacercaria encysted on spines. (14) Metacercaria freed from cyst. (15) Adult experimentally from H. malabaricus, 13 days postexposure, ventral view; vs: embedded ventral sucker. (16) Tegument of adult specimen. (17) Eggs. Scale bars ¼ 25 lm (Figs. 16, 17); 50 lm (Figs. 9, 10); 100 lm (Figs. 11, 14, 15); 200 lm (Fig. 13); 250 lm (Fig. 12). Figs. 9–14, 16–17 from living specimens, Fig. 15 from specimen mounted in glycerine jelly. QUINTANA AND OSTROWSKI DE NÚÑEZ—LIFE CYCLE OF PSEUDOSELLACOTYLA LUTZI 809 TABLE I. Pseudosellacotyla lutzi (Freitas, 1941). Values are shown in micrometer as min-max with the mean and number of specimens measured in parentheses. L: length, W: width, os: oral sucker, vs: ventral sucker. Measurements (lm) Body L Body W (testes level) Forebody % forebody/L Oral sucker L Oral sucker W Prepharynx Pharynx L Pharynx W Ventral sucker L Ventral sucker W Ovary L Ovary W Left testis L Left testis W Right testis L Right testis W Vitelline follicles L Vitelline follicles W Egg L Egg W vs:os width ratio Body L/body W 13 days PE 163–278 128–224 64–122 37–45 24–48 38–59 0–16 27–42 21–35 22–37 22–38 32–64 19–40 48–104 35–72 61–104 46–66 16–32 10–19 29–40 14–21 0.5–0.9 1.2–1.5 (244, 16) (191, 16) (102, 14) (41, 14) (38, 15) (49, 15) (4, 15) (34, 12) (29, 12) (30, 13) (32, 13) (43, 12) (33, 11) (73, 12) (57, 12) (76, 12) (58, 12) (20, 19) (16, 19) (33, 29) (17, 29) (0.7, 13) (1.3, 16) 48–54 days PE 265–410 183–315 96–160 33–46 42–64 50–72 0–21 32–51 29–48 32–48 30–43 48–72 29–64 72–112 56–88 80–112 48–88 13–32 10–35 32–37 14–21 0.5–0.8 1.1–1.5 (330, 33) (266, 33) (131, 28) (40, 27) (51, 28) (60, 28) (6, 21) (45, 25) (37, 25) (36, 26) (36, 25) (57, 13) (44, 13) (93, 12) (75, 12) (96, 12) (74, 11) (21, 51) (20, 51) (34, 52) (17, 52) (0.6, 25) (1.2, 32) Natural infection 372–466 271–384 128–192 33–46 48–64 64–80 0–16 32–48 38–51 27–48 27–48 56–96 37–72 96–144 83–128 112–136 83–120 16–48 19–42 32–38 16–21 0.4–0.7 1.1–1.4 (407, 15) (320, 15) (158, 12) (38, 12) (56, 14) (71, 14) (9, 14) (39, 14) (45, 14) (39, 9) (40, 9) (79, 12) (50, 12) (123, 14) (99, 14) (120, 14) (100, 14) (29, 42) (27, 42) (36, 46) (18, 46) (0.6, 9) (1.3, 15) Freitas (1941) Kohn et al. (1985) 340–590 240–444 510–650 370–480 50–59 59–63 75–89 75–99 46–50 34–42 38–42* 50–60 50–60 47–52 47–52 67–120 70–94 90–210† 70–140 42–105 42–80 67–105† 50–63 34–38 17 33–38 16–19 1:530–650 * Diameter. † Include both testes. Body shape, body spination, and the position of gonads were identical in experimental metacercariae, adults from experimental infections, and adults from naturally infected H. malabaricus. The fact that Hyphessobrycon eques (from Heller Peninsula) and M. dichroura (from Candelaria) were parasitized with mature metacercariae that had been identified with those of P. lutzi from experimental infections for comparison strongly indicates that both fish species serve as second intermediate hosts of this parasite in the Paraná River. Adults at 13 days PE were smaller, 163–278 (244) 3 128–224 (191, n ¼ 16) produced fewer eggs and had a more prominent seminal vesicle than those at 20 days PE. After 48 days PE, specimens were larger than those at 13 days PE, 265–410 (330) 3 183–315 (266, n ¼ 33) (Table I), but smaller than those at 20 days PE, 302–447 (395) 3 233–340 (307, n ¼ 20). The latter were filled with eggs obscuring the internal organs, except for the conspicuous seminal vesicle. The specimens from natural infections, 372– 466 (407) 3 271–384 (320, n ¼ 15), were larger than those from experimental infections (Table I). DISCUSSION This is the second report of Aylacostoma chloroticum infected with larval stages belonging to a digenean species whose life cycle was experimentally established. The experimentally and naturally obtained adults are morphologically indistinguishable from P. lutzi, and were considered identical to this species, included in the Faustulidae by Bray (2008). The family Faustulidae is a member of the superfamily Microphalloidea, which consists of 18 families, from which 8 are known to present xiphidiocercariae (Microphallidae, Prosthogonimidae, Lecithodendriidae, Leyogonimidae, Phaneropsolidae, Stomylotrematidae, Zoogonidae), 2 presumably present xiphidiocercariae as they are parasites of bats (Anenterotrematidae) or have odonates as second intermediate hosts (Eumegacetidae), from 6 the life cycle is not known (Diplangidae, Exotidendriidae, Gyrapsidae, Pachypsolidae, Renschetrematidae, Taiwantrematidae) (Bray, 2008), 1 presents at least some species with xiphidiocercariae (Renicolidae), and the remaining 1 gymnocephalous cercariae (Faustulidae). Bray (2008) recorded 14 genera in the Faustulidae, of which 2 (Pseudobacciger Nahhas and Cable, 1954 represented by 3 marine species and Pseudosellacotyla, represented by a single freshwater species) lack a cirrus sac, present in the others. In the life cycle of Pseudobacciger harengulae a marine clam acts as first, and crustaceans as second, intermediate host. The cercaria is of the trichocercous type, which emerged from sporocysts; definitive hosts are different marine fish (Kim and Chun, 1984; Dimitrov et al., 1999). On the other hand, the life cycle of Pseudosellacotyla lutzi presents cercariae of the ‘‘opisthorchioid’’ type, which originate in rediae, and emerge from a gastropod of the superfamily Cerithioidea, as do most of the opisthorchioid species (Cribb et al., 2001). Pseudosellacotyla lutzi has a V-shaped excretory vesicle with short arms, not reaching the testes, although in Faustulidae the arms reach into the forebody, but both share the overall position of organs, and the opening of the Laurer’s canal near the posterior extremity. These characteristics are also seen in heterophyids, and some cryptogonimids, as in Acanthostomum brauni Mañe Garzon and Gil, 1961, where the Laurer’s canal opens at the level of the anterior testis (Ostrowski de Núñez, 1987), and probably in other species of Acanthostomum Looss, 1899, where genitalia lie in the posterior extremity of the body. On the other hand, P. lutzi possesses an embedded ventral sucker, a characteristic of Cryptogonimidae, Heterophyidae, and some Opisthorchiidae, absent in Faustulidae. Yamaguti (1953) considered probable from the figures of Freitas (1941) ‘‘that the acetabulum is embedded in the body parenchyma, and the genital 810 THE JOURNAL OF PARASITOLOGY, VOL. 100, NO. 6, DECEMBER 2014 pore opens to the outside over the acetabulum,’’ and if this assumption be justified, ‘‘there would be no question to include Pseudosellacotyla in the Heterophyidae.’’ In the living pressed specimens, in whole mounts, and in histological sections no ventrogenital sac could be detected; therefore the inclusion in Heterophyidae as proposed by Yamaguti (1953) is excluded. The marked difference in the larval stages of P. lutzi (with opisthorchioid cercariae developing in rediae in gastropods) in comparison to the faustulid P. harengulae (with trichocercous cercariae developing in sporocyst in a marine clam), the embedded condition of the ventral sucker, and the similarity in the terminal genitalia, lead to the conclusion that P. lutzi is not a faustulid, and should be included in the Cryptogonimidae. Indeed, recent molecular studies of another species included in the Faustulidae show relationship with the Gymnophalloidea, and suggest the polyphyletic character of the Faustulidae (Sun et al., 2014). Miller and Cribb (2008) recognized 68 valid genera in the Cryptogonimidae, most of which show a Y-shaped excretory vesicle with a long stem, and arms reaching to the pharynx. The single exception is Acanthostomoides apophalliformis Szidat, 1956, with the arms of the Y-shaped excretory vesicle extending to the ovary. Although Miller and Cribb (2008) mentioned a V-shaped excretory vesicle in some squat species, its arms would be placed anterior to the midbody. Pseudosellacotyla mainly differs from all other genera in the family by its V-shaped excretory vesicle, with the arms reaching to posterior border of testes. However, the genera Gonacanthella Sogandares-Bernal, 1959; Caecincola Marshall and Gilbert, 1905; Tabascotrema Lamothe-Argumedo and Pineda-López; Campechetrema Lamothe-Argumedo, SalgadoMaldonado and Pineda-López, 1997; Paleocryptogonimus Szidat, 1954 Exorchis Kobayashi, 1921; and Metadena Linton, 1910 resemble Pseudosellacotyla in their body shape and in having symmetrical testes, vitelline follicles in two groups and in the absence of a gonotyl (Miller and Cribb, 2008), but they differ from it at least in 1 of the following characters: extension of ceca, position of seminal vesicle, intestinal bifurcation, vitelline follicles, elongate body, funnel-shaped oral sucker, shape and position of ovary. Gonacanthella and Metadena are parasites of marine fishes, whereas Caecincola, Tabascotrema, Campechetrema, and Paleocryptogonimus (all of which occur in America) and Exorchis parasitize freshwater fishes. Miller and Cribb (2008) mentioned that there are at least 25 known life cycles of Cryptogonimidae with typical ‘‘opisthorchioid’’ cercariae, which commonly lack body pigmentation and have 8 pairs of flame cells. Cribb (1986) suggested that cercariae with pigmented body and numerous flame cells may belong to Heterophyidae. Notwithstanding this, studies on the life cycles of A. brauni, Acanthostomum gnerii Szidat, 1954, and A. apophalliformis have revealed that species of Cryptogonimidae also possess cercariae with these features (Ostrowski de Núñez, 1987; Ostrowski de Núñez and Gil de Pertierra, 1991; Ostrowski de Núñez et al., 1999). On the other hand, colorless cercariae with 8 pairs of flame cells have been observed to occur in heterophyids (e.g., Euhaplorchis californiensis Martin, 1950, Ascocotyle gemina Font, Overstreed and Heard, 1984; Ascocotyle secunda, Ostrowski de Núñez, 2001; A. tertia Ostrowski de Núñez, 2001; see Martin, 1950; Font et al., 1984; Ostrowski de Núñez, 2001). The cercaria of P. lutzi fits that described for cryptogonimids and heterophyids, except for the lack of pigmented eyespots, unusual in opisthorchioid cercariae, but is shared by the heterophyid species Eurihelmis monorchis Ameel, 1938, which penetrates in amphibians (Yamaguti, 1975). The presence of intestinal ceca is rare, however, has been mentioned for the cryptogonimid Pseudexorchis major (Hasegawa, 1934), and for species of the heterophyid Apophallus Lühe, 1909 (Odening, 1970, 1973; Yamaguti, 1975). The rediae of P. lutzi are morphologically similar to those of both groups but may contain fully developed cercariae, as opposed to those of the other 3 Argentine cryptogonimid species (A. brauni, A. gneri, A. apophalliformis). With the exception of the man-introduced genera Melanoides Olivier, 1804 and Tarebia H. Adams and A. Adams, 1854, molluscs of the Thiaridae are distributed in tropical and subtropical areas of Africa, Asia, Australia, Central, and South America. It is striking that all thiarid intermediate hosts of opisthorchioid life cycles were found in Asia, especially the numerous human heterophyid parasites, which were studied beginning in the first half of 20th century (Yamaguti, 1975). In tropical and subtropical areas of South America life cycle studies were scarce, and thiarids were seldom included. Actually, the present life cycle is the first of a cryptogonimid that involves a thiarid intermediate host in South America. In contrast, the Hydrobiidae were mainly distributed in template and cold areas of the world, and were extensively studied for digenean life cycles, which included several cryptogonimid species (Yamaguti, 1975). The Cochliopidae, well distributed in South America and more recently studied, presented a rich fauna of cryptogonimid and heterophyid species (Ostrowski de Núñez and Gil de Pertierra, 2004). As research on life cycles in South America increases, more thiarids should be found to be involved. In actual fact 3 other cercariae of the Opisthorchioidea were observed emerging from A. chloroticum, one of them lacking body pigment, and eyespots (unpubl. data). ACKNOWLEDGMENTS We are indebted to Lic. D. C. Pérez (Entidad Binacional Yacyretá) for his kind help in snail collection by diving, and to the ichthyologists of the Universidad Nacional de Misiones, Lic. D. R. Aichino, Lic. M. F. Benitez, and A. S. Masin for their effort to capture fishes in the dam with the use of different fishing gear. Special thanks go to Dr. C. Ituarte and his staff for laboratory facilities for histological sections, to Lic. A. C. Mercado Laczkó for helping in the preparation of figures, to Dr. V. Ivanov for helping with some photographs, and to 2 anonymous referees, whose comments improved the manuscript, and 1 of which helped with the correct identification of the present species, and provided us with recent literature. Funds and facilities for this work were provided by EBY (Yacyretá Binational Entity). The authors are sincerely grateful for the continued support of the Aylacostoma Conservation Program in Yacyretá. LITERATURE CITED BRAY, R. A. 2008. Family Faustulidae Pche, 1926. In Keys to the Trematoda, Vol. 3, R. A. Bray, D. I. Gibson, and A. Jones (eds.). CABI Publishing, Wallingford, Oxfordshire, U.K., p. 509–525. CRIBB, T. H. 1986. The life cycle and morphology of Stemmatostoma pearsoni, gen. et sp. nov., with notes on the morphology of Telogaster opisthorchis Macfarlane (Digenea: Cryptogonimidae). Australian Journal of Zoology 34: 279–304 ———, R. A. BRAY, AND D. T. J. LITTLEWOOD. 2001. The nature and evolution of the association among digeneans, molluscs and fishes. International Journal for Parasitology 31: 997–1011. QUINTANA AND OSTROWSKI DE NÚÑEZ—LIFE CYCLE OF PSEUDOSELLACOTYLA LUTZI DIMITROV, G., R. A. BRAY, AND D. GIBSON. 1999. A redescription of Pseudobacciger harengulae (Yamaguti, 1938) (Digenea: Faustulidae) from Sprattus sprattus phalericus (Risso) and Engraulis encrasicholus ponticus Alexandrov off the Bulgarian Black Sea coast, with a review of the genus Pseudobacciger Nahhas and Cable, 1964. Systematic Parasitology 43: 133–146. FONT, W. F., R. M. OVERSTREET, AND R. W. HEARD. 1984. Life cycle of Ascocotyle gemina n. sp., a sibling species of A. sexidigita (Digenea, Heterophyidae). Transactions of the American Microscopical Society 103: 392–407. FREITAS, J. F. T. 1941. Sellacotyle lutzi n.sp., trematodeo parasito de Hoplias malabaricus Bloch. Anais da Academia Brasileira de Ciencias 13: 17–19. HYLTON SCOTT, M. I. 1953. El género Hemisinus (Melaniidae) en la costa fluvial argentina. Physis 20: 438–443. ———. 1954. Dos nuevos melánidos del Alto Paraná. Neotrópica 1: 45– 48. KIM, Y. G., AND S. K. CHUN. 1984. Studies on the life history of Bacciger harengulae. Bulletin of the Korean Fishery Society 17: 449–470. KOHN, A., AND B. M. M. FERNANDES. 1987. Estudo comparativo dos helmintos parasitos de peixes do Rio Mogi Guassu, coletados nas excursoes realizadas entre 1927 e 1985. Memorias do Instituto Oswaldo Cruz 82: 483–500. ———, ———, B. MACEDO, AND B. ABRAMSON. 1985. Helminths parasites of freshwater fishes from Pirassununga, SP, Brazil. Memorias do Instituto Oswaldo Cruz 80: 327–336. ———, F. MORAVEC, S. C. COHEN, C. CANZI, R. M. TAKEMOTO, AND B. M. M. FERNANDES. 2011. Helminths of freshwater fishes in the reservoir of the Hydroelectric Power Station of Itaipu, Paraná, Brazil. Check List 7: 681–690. LENIS VELEZ, C. A., I. VELEZ, M. BECHARA ESCUDER, AND A. PEREZ CAICEDO. 2010. Nuevo registro de Pseudosellacotyla lutzi (Digenea: Faustulidae) en Hoplias malabaricus (Pisces: Erytrinidae) Chocó, Colombia. Revista Institucional Universidad Tecnológica del Chocó 29: 110–112. MARTIN, W. E. 1950. Euhaplorchis californiensis n. g., n. sp. (Heterophyidae, Trematoda) with notes on its life cycle. Transactions of the American Microscopical Society 69: 194–209. MILLER, T. L., AND T. H. CRIBB, 2008. Family Cryptogonimidae Ward, 1917. In Keys to the Trematoda, Vol. 3., R. A. Bray, D. I. Gibson, and A. Jones (eds.). CABI Publishing, Wallingford, U.K., p. 51–112. ODENING, K. 1970. Der Entwicklungszyklus von Apophallus muehlingi (Trematoda: Opisthorchiida: Heterophyidae) in Berlin. Zeitschrift für Parasitenkunde 33: 194–210. ———. 1973. Der Lebenszyklus des Trematoden Apophallus donicus in Berlin im Vergleich zu A. muehlingi. Biologisches Zentralblatt 92: 455–494. 811 OSTROWSKI DE NÚÑEZ, M. 1987. Der Entwicklungszyklus von Acanthostomum brauni Mañe Garzon und Gil, 1961 (Trematoda, Acanthostomatidae). Zoologischer Anzeiger 218: 273–286. ———. 2001. Life cycles of two new sibling species of Ascocotyle (Ascocotyle) (Digenea, Heterophyidae) in the Neotropical Region. Acta Parasitologica 46: 119–129. ———, AND A. A. GIL DE PERTIERRA. 1991. The life history of Acanthostomum gnerii Szidat, 1954 (Trematoda: Acanthostomatidae), from the catfish Rhamdia sapo in Argentina. Zoologischer Anzeiger 277: 58–71. ———, AND ———. 2004. Ciclos Biológicos dulceacuı́colas de Digenea (Trematoda) y Proteocephalidea (Cestoda). In Sanidade de Organismos Aquáticos. M. J. T. Ranzani-Paiva, R. M. Takemoto, M. de los A. P. Lizama (eds.). Editora Varela, São Paulo, Brazil, p. 215–257. ———, AND M. G. QUINTANA. 2008. The life cycle of Stephanoprora aylacostoma n. sp. (Digenea: Echinostomatidae), parasite of the threatened snail Aylacostoma chloroticum (Prosobranchia, Thiaridae), in Argentina. Parasitology Research 102: 647–655. ———, L. SEMENAS, N. BRUGNI, G. VIOZZI, AND V. FLORES. 1999. Redescription of Acanthostomoides apophalliformis (Trematoda, Acanthostomidae) from Percichthys trucha (Pisces, Percichthyidae) with notes on its life cycle in Patagonia, Argentina. Acta Parasitologica 44: 222–228. QUINTANA, M. G., AND A. C. MERCADO LACZKÓ. 1997. Caracoles de los rápidos en Yacyretá. Ciencia Hoy, Buenos Aires 7: 22–31. SIMONE, L. R. L. 2006. Land and freshwater molluscs of Brazil. Editora Gráfica Bernard Ltda., Fundaçao de Amparo a Pesquisa do Estado de Sao Paulo. Sao Paulo, Brazil, 390 p. SUN, D., R. A. BRAY, R. Q. Y. YOUNG, S. C. CUTMORE, AND T. H. CRIBB. 2014. Pseudobacciger cheneyae n. sp. (Digenea: Gymnophallidea) from Weber’s chromis (Chromis weberi Fowler & Bean) (Perciformes: Pomacentridae) at Lizard Island, Great Barrier Reef, Australia. Systematic Parasitology 88: 141–152. VON IHERING, H. 1902. As melanias do Brazil. Revista do Museu Paulista 5: 653–682. ———. 1909. Les mélaniidés américains. Journal de Conchyliologie 57: 289–316. YAMAGUTI, S. 1953. Systema Helminthum. Part I. Digenetic trematodes of fishes. Satyû Yamaguti (published by author), Tokyo, Japan, 405 p. ———. 1958. Systema Helminthum. Vol. I. The digenetic trematodes of vertebrates. Interscience Publishers, New York, New York, 1575 p. ———. 1971. Synopsis of digenetic trematodes of vertebrates. Keigaku Publishing Co., Tokyo, Japan, Vol. I, 1074 pp.; Vol. II, 349 pl. ———. 1975. A synoptical review of life histories of digenetic trematodes of vertebrates. Keikagu Publishing Co., Tokyo, Japan, 591 p.