First report of Temnocephala haswelli ( Platyhelminthes : Temnocephalida ) in Pomacea canaliculata ( Mollusca : Ampullariidae ) from Brazil : description update based on specimens from the state of Rio Grande do Sul , Brazil

The impact of the discovery of Temnocephala haswelli Ponce de Léon, 1989, described as ectosymbionts of ampullariid apple snails outside of Uruguay, motivated us to collect a large number of specimens of Pomacea canaliculata (Lamarck, 1822) from several localities in the southern portion of the state of Rio Grande do Sul. This species was recorded three times after its description: in a study of chromosomes, in a study about the ultrastructure of the collar receptor cells, and in a study of the Haswell glands, all conducted in Uruguay. A total of 301 specimens of P. canaliculata were collected from 1999 to 2007. Temnocephalans found in the pallial cavity were identified as T. haswelli, which occurred in single infestations or concurrently with Temnocephala iheringi Haswell, 1893. Helminths usually showed a light-orange body pigmentation and conspicuous, intense red-eye pigment. Many taxonomic characters evidenced by several techniques were documented photographically for the first time. The typical curved cirrus, approximately 90°, typical of the species, showed some variation in the width of the shaft base, whereas the first longitudinal row of spines of the introvert appeared with shorter spines. The vagina was found to be thick-walled, but not very muscular, and to have a single, large and slightly asymmetrical sphincter, with the posterior portion of slightly larger diameter. Eggs were observed in the umbilicus and along the suture, but predominantly in the body whorl of the shell. Egg peduncles were found to be very short or, most of the time, the eggs were sessile, always with a long apical filament. The rounded shape of the dorsolateral ‘excretory’ syncytial epidermal plates had external margins reaching the ventrolateral region of the body and eccentric nephridiopores. This is the first record of the species outside Uruguay and in Brazil.

The present work records for the first time T. haswelli in Brazil and outside of Uruguay.A complete study (showing several specific characters) of this species has not yet been published.Thus, we present a complete characterization of the species following the methodology of AMATO et al. (2007) andSEIXAS et al. (2010).

MATERIAL AND METHODS
A total of 301 specimens of P. canaliculata were collected between 1999 and 2007, using dip nets and/or large sand sieves, and transported live to the Laboratório de Helmintologia, Universidade Federal do Rio Grande do Sul (UFRGS).Live typical of the species, showed some variation in the width of the shaft base, whereas the first longitudinal row of spines of the introvert appeared with shorter spines.The vagina was found to be thick-walled, but not very muscular, and to have a single, large and slightly asymmetrical sphincter, with the posterior portion of slightly larger diameter.Eggs were observed in the umbilicus and along the suture, but predominantly in the body whorl of the shell.Egg peduncles were found to be very short or, most of the time, the eggs were sessile, always with a long apical filament.et al. (2010) for internal morphometry.To preserve the eyespots' red pigmentation, body shape, and for the purpose of scanning electron microscopy studies (SEM), some specimens were flooded with hot, phosphate-buffered 10% formalin (HF) (AMATO et al. 2005(AMATO et al. , 2006)).The morphology of the dorsolateral 'excretory' syncytial epidermal plates (DLSPs) and the distribution of rhabditogen and disc glands followed AMATO et al. (2007).Photomacrographs of hosts showing egg distribution and live helminth pigmentation were taken with a Zeiss Stemi SV-6 stereomicroscope ( Figs 1-2, 4, and 5).Photomicrographs were taken with a Zeiss Axiolab microscope with phase contrast (or just the phase contrast condenser), a Leica DMR Hc microscope with Nomarski's differential interference contrast (DIC) prisms, and a Jeol (JSM-6060) SEM.Drawings were made with a drawing tube on a Nikon E-200 microscope.The photographic images and scanned line drawings were prepared using Adobe's Photoshop CS2 ® and CorelDRAW X4 ® .Measurements are in micrometers (µm) unless otherwise indicated; ranges are followed by the arithmetic mean, the number of specimens measured for a given character (when different than 30), and the standard deviation values (between parentheses).A number of internal organs were no longer measured and the terminology used followed SEIXAS et al. (2010).Voucher specimens fixed in AFA or silver nitrate (SN), as well as slides containing individual cirri in Faure's mounting medium (F) were deposited in the Coleção Helmintológica do Instituto Oswaldo Cruz (CHIOC), Rio de Janeiro, RJ, Brazil.Other voucher specimens stained in Delafield's hematoxylin, were deposited in the Colección de la Sección Helmintológica, División Zoología Invertebrados, Museo de La Plata (MLP), La Plata, Buenos Aires, Argentina.Some mollusk hosts were deposited in the Coleção Malacológica do Instituto Oswaldo Cruz (CMIOC).The remaining host specimens and temnocephalans are in the host and helminth collections of the UFRGS Coleção Helmintológica do Laboratório de Helmintologia, Porto Alegre, RS, Brazil.long, 190-450 (308, 80) wide; ratio between of maximum cirrus length/ adhesive disk diameter 3.15: 1. Body pigmentation absent (Fig. 6) or light-orange (Fig. 4) observed in live adults and juveniles.Eyespots' red pigmentation present (observations made on live specimens and fixed in HF) (Figs 5-7a, and 7b).Two dorsolateral, rounded epidermal 'excretory' syncytial plates, slightly wider than long (Figs 16 and 17); external margin reaching ventrolateral margin of body (Fig. 18), extending from basis of first and fifth tentacles, respectively; left plate 150-340 (263, n = 12, 49) long, 170-300 (254, n = 12, 38) wide; right plate 180-360 (254, n = 12, 58) long, 190-330 (255, n = 12, 48) wide; ratio between length of DLSPs/total body length, without tentacles, 5.35: 1. Nephridiopore (excretory pore) usually central, sometimes appearing off-centered (Figs 16 and 17).
Remarks.Like the majority of Neotropical species, T. haswelli has red-eye pigment, which in this species is conspicuous and intense (Figs 5-7b).The DLSP's are rounded, with external margins on the ventrolateral region of the body .The nephridiopores are eccentric and displaced to the inner region (Figs 16 and 17).As the female parts of the reproductive system of this species do not stain well with hematoxylin, measurements and photomicrographs were taken of specimens stained with aceto-carmine\fast green (Figs 26 and  27).Ootype and vagina (Figs 14 and 27) with thick walls, not very muscular, being thicker proximally.The vaginal sphincter is more difficult to observe, in some specimens it is symmetrical, but in the majority it is slightly asymmetrical (30 µm in average in the anterior portion, 39.5 µm in the posterior portion), with the anterior portion thinner, showing considerable variation within the sample (25-40 µm).Almost all eggs observed were sessile while others had minute peduncles.Temnocephala haswelli was found in single infestations, allowing the documentation of its eggs.The cirrus showed important intra-specific variation, on the curvature as well as on the width of the shaft's base (70-100 µm).The size of spines in the introvert of T. haswelli is not so uniform; first row has smaller spines than all the rest (Figs 22,arrow).

DISCUSSION
Temnocephala haswelli was described based on 23 specimens collected from Río Canelón Chico, in Uruguay (PONCE DE LEÓN 1989).Three papers published about T. haswelli also included information on T. iheringi (GONZÁLEZ et al. 1987 LEÓN (1989) described the body pigmentation as a variation from white to yellow, similar to that reported in the present work, in which this variation went from white to light-orange.There are differences in body size (without tentacles) in relation to the specimens of the present work when compared to the original description, but despite being of smaller size the specimens from Brazil (2.17 mm in Brazil and 3.82 mm in Uruguay) have the length as well as the shape of the cirri similar to those characteristic of the species (average length of cirri: 215 µm in Brazil and 200 µm in Uruguay).The red-eye pigment is conspicuous and intense in T. haswelli and 7b), whereas the vitellaria are very characteristic, which, according to PONCE DE LEÓN (1989), is arborescent with long branches which do not exceed 4-8 in number in the dorsal side of the body, agreeing with the observations made in the present work (Fig. 8), the vitellaria have, in average, four branches, with two more limited branches to the margins of the ventral region.Staining with aceto-carmine/fast green the cement glandular become evident around the genital pore, as PONCE DE LEÓN (1989), pointed out in the original description.
The eggs of T. haswelli were well studied herein (measurements, shape, filament position, and opercular plates) because we were able to collect mono-specific infestations of P. canaliculata with this species, although the opercular plates were sometimes difficult to see in all eggs.Measurements showed considerable differences when compared to those of the original description.PONCE DE LEÓN (1989) measured eggs of the interior of the "ootype" (= genital atrium) in his studies, but this method is likely to give inaccurate measurements.The measurement presented as being of the peduncle, is probably a measurement of the sub-apical or apical filament.This becomes clear when the average length of the peduncle (115 µm) is compared to the average of the length of the filament in Brazilian specimens (97.5 µm), which are quite similar.These values contrast with the average length of peduncles (53.3 µm), which, besides being minute, are only present in 30% of the eggs observed in the present study, as they are largely sessile.Although the original description indicates the limits of the rhabditogen glands and disk glands, two pairs of the large disk glands (paranephrocites?) are shown for the first time in the present work (Fig. 12).

Temnocephala haswelli (Platyhelminthes: Temnocephalida) in Pomacea canaliculata (Mollusca: Ampullariidae) from Brazil: description update based on specimens from the state of Rio Grande do Sul, Brazil Samantha A. Seixas 1 ; José F. R. Amato 1, 2 & Suzana B. Amato 1 1
Departamento de Zoologia, Instituto de Biociências, Universidade Federal do Rio Grande do Sul.Caixa Postal 15014, 91501-970 Porto Alegre, Rio Grande do Sul, Brazil.E-mail: samantha_bio@yahoo.com.br;sbamato@ufrgs.br 2 Corresponding author.E-mail: josefelipeamato@gmail.comABSTRACT.The impact of the discovery of Temnocephala haswelli Ponce de Léon, 1989, described as ectosymbionts of ampullariid apple snails outside of Uruguay, motivated us to collect a large number of specimens of Pomacea canaliculata (Lamarck, 1822) from several localities in the southern portion of the state of Rio Grande do Sul.This species was recorded three times after its description: in a study of chromosomes, in a study about the ultrastructure of the collar receptor cells, and in a study of the Haswell glands, all conducted in Uruguay.A total of 301 specimens of P. canaliculata were collected from 1999 to 2007.Temnocephalans found in the pallial cavity were identified as T. haswelli, which occurred in single infestations or concurrently with Temnocephala iheringi Haswell, 1893.Helminths usually showed a light-orange body pigmentation and conspicuous, intense red-eye pigment.Many taxonomic characters evidenced by several techniques were documented photographically for the first time.The typical curved cirrus, approximately 90°, Tramandaí Sub-water basin.All the above municipalities are in the state of Rio Grande do Sul, Brazil.These areas are included in the Atlantic Rain Forest (Brazilian Atlantic Forest Biome).Some helminths taken from live hosts were studied and photomicrographed alive or fixed and stained according to SEIXAS Ponce de León, 1989 , PONCE DE LEÓN & VOLONTERIO 2003, VOLONTERIO & PONCE DE LEÓN 2004).PONCE DE