Redescription of Temnocephala iheringi ( Platyhelminthes : Temnocephalida ) based on specimens from Pomacea canaliculata ( Mollusca : Ampullariidae ) of the state of Rio Grande do Sul , Brazil : the possible type host and type locality

The original description of Temnocephala iheringi Haswell, 1893 was based on specimens collected by Hermann von Ihering from undetermined ampullariid apple snails, which at that time were identified as Ampullaria sp., and sent to William H. Haswell, with the type locality simply indicated as Brazil. The type specimens studied by Haswell were not found in the scientific collections of Brazil, Europe or Australia, and should be considered lost. In 1941, Pereira & Cuocolo collected specimens from apple snails, identified as Pomacea lineata (Spix in Wagner, 1827), at two localities (Guaicurús and Salobra) in the state of Mato Grosso do Sul, central Brazil. These specimens could not be located either and should, thus, be considered lost as well. Pomacea canaliculata (Lamarck, 1822), the main host of T. iheringi in southern Brazil, is known to have a geographical distribution that reaches Uruguay and 400 km beyond the province of Buenos Aires, Argentina. Three hundred and one mollusks were collected from 1999 to 2007. Temnocephalans found in the pallial cavity presented a greenish body pigmentation (adults) and lacked eye pigment of any color, including the red-eye pigment, typical of Neotropical species of Temnocephala Blanchard, 1849; straight cirrus, with a thick band at base of the introvert’ swelling; and a single, circular, asymmetric vaginal sphincter, wider in diameter in the posterior portion. As the species occurs concurrently with two other species, at least in P. canaliculata from Rio Grande do Sul, the eggs of T. iheringi could not be reliably distinguished. A redescription of the species is provided. A comparison of data from the present work with those of earlier papers published on T. iheringi from Argentina showed that the Argentinean specimens had the smallest measurements.

ZOOLOGIA 27 (2): 245-257, April, 2010 bution and abundance of temnocephalans was performed using infrapopulations of T. iheringi from P. canaliculata.This study extended the known distribution of the species to more than 400 km south of province of Buenos Aires, the southernmost natural limit of the host species (MARTÍN et al. 2005).
Four species of Temnocephala Blanchard, 1849 are presently known from ampullariid snails: T. iheringi (HASWELL 1893), Temnocephala rochensis Ponce de León, 1979(PONCE DE LEÓN 1979), T. haswelli (PONCE DE LEÓN 1989), and Temnocephala lamothei Damborenea & Brusa, 2008(DAMBORENEA & BRUSA 2008).Only T. iheringi has been recorded in Brazil.HASWELL (1893) did not specify a type locality, indicating Brazil as the origin of the specimens.PEREIRA & CUOCOLO (1941) redescribed the species for the first time, but based on ectosymbiont specimens from Pomacea lineata (Spix in Wagner, 1827) collected in Salobra and Guaicurús, both localities in the state of Mato Grosso, now Mato Grosso do Sul.HYMAN (1955) made an unsatisfactory redescription of T. iheringi based on specimens from Argentina, collected from ampullariid snails identified as A. platae, from Arroyo Malabrigo, province of Santa Fe.This was the first record of T. iheringi in another genus of Ampullariidae and it was also the first record of the species outside of Brazil, although the correct identification of the mollusk cannot be confirmed.DAMBORENEA et al. (1997), in a paper about the benthonic fauna of Isla Martín García, province of Buenos Aires, Argentina, recorded the species in a new host, Asolene megastoma Sowerby, 1825 (= currently Pomella megastoma).Temnocephala iheringi was also recorded by DIONI (1967) but this record is incorrect since he described the cirrus with strong curvature ("fuerte curvature" on page 483) and illustrated (figs 7a, b and c, page 480) a curved cirrus.
To date, there are no records of any species of Temnocephala from correctly identified ampullariid snails in precisely identified geographical localities in the state of Rio Grande do Sul, southern Brazil.Even with several published records of this species, a complete study of T. iheringi (showing several specific characters) through an array of special techniques, as we have been doing since 2003, has not yet been undertaken.
Some helminths taken from live hosts were studied and photomicrographed alive or fixed in cold AFA under slight cover slip pressure (the traditional procedure).Some specimens were stained in Delafield's hematoxylin (AMATO & AMATO 2005) or aceto-carmine/fast green (GALIGHER & KOZLOFF 1971, HUMASON 1972), cleared in cedar oil, and mounted in Canada balsam for internal morphometry.To preserve body shape and for the purpose of scanning electron microscopy (SEM), some specimens were flooded with hot, phosphate buffered 10% formalin (HF) (AMATO et al. 2006).The morphology of the dorsolateral 'excretory' syncytial epidermal plates (DLSPs) was studied in specimens fixed with silver nitrate (SN) (ROMEIS 1948, JOFFE et al. 1995) or observed with SEM (AMATO et al. 2005, AMATO et al. 2007).Cirrus morphology (Fig. 2a) has been studied after micro dissection and mounting in Faure's medium (CANNON 1993, CANNON & SEWELL 1995, SEWELL et al. 2006).The distribution of rhabditogenic and disc glands was studied by clearing juvenile specimens in lactophenol (AMATO & AMATO 2005).Photomacrographs of hosts showing the egg distribution and the helminth pigmentation were taken with a Zeiss Stemi SV-6 stereomicroscope 7,and 8).Photomicrographs were taken with a Zeiss Axiolab microscope with phase contrast (or just the phase contrast condenser), and a Leica DMR Hc microscope with Nomarski's differential interference contrast (DIC) prisms, or 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 ® .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).Some internal organs were not measured following the suggestion by SEWELL et al. (2006).
Voucher specimens and slides containing individual cirri of T. iheringi 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 invertebrate collection of the "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 "Coleção Helmintológica do Laboratório de Helmintologia, Departamento de Zoologia", UFRGS, Porto Alegre, RS, Brazil.
Glands.Rhabditogenic glands small, numerous, with a granular appearance, forming bunches (average of 22 cells), in lateral fields of body (Figs 13 and 15),11) in diameter, extending on the sides of the intestinal sac, with conspicuous ducts (Figs 13 and 15), best observed in juvenile specimens cleared in lactophenol, before complete development of vitellarium.Two groups of four Haswell glands (Figs 12 and 17) showing little affinity with hematoxylin, in front of the brain transverse band (Fig. 17), diameter of the largest cell 50-97 (66, n = 24, 15).Disc glands between adhesive disc and genital complex forming two lateral bunches extending from posterior testis to margin of adhesive disc (Fig. 16), including two pairs of large, round, more central cells (paranephrocytes?), 37-72 (53, 9) in diameter (Figs 14 and 16).
Site of infestation.Adults and juveniles in mantle cavity, eggs in umbilicus, suture, and spire, sometimes in larger numbers in the body whorl of the shell; never present on the operculum.
Host specimens were deposited in the CMIOC under the Nº 5820.
Remarks.Examination of a large number of specimens of T. iheringi collected from the probable type locality consistently showed that they did not present red-eye pigmentation when observed alive (Figs 8 and 9a).The adhesive secretions produced by the disc glands oozing from pores in the ventral surface of the disk can be observed for the first time in figure 22.The vagina has a muscular wall on the distal portion (Figs 18 and 33), difficult to observe, as it has the same thickness as the middle and proximal portions.The vaginal sphincter has the anterior portion with a smaller average diameter than the posterior portion (Fig. 18).The apparent large variation of the shaft's base width of the cirrus may represent only intra-specific variation.These differences are not related to collection localities, thus excluding the possibility that these variations would be related to a given component population of each collection area.Further, another peculiar aspect never before recorded for the cirrus of this species is the circular 'thickening' at the base of the introvert' swelling, only visualized in samples prepared in Faure's medium and observed with SEM (Figs 25 and 30).The eggs of T. iheringi were not measured because this species, in Rio Grande do Sul, has, to date, always been found in concurrent infestations with either T. haswelli or T. rochensis making it impossible, at present, to separate the eggs of T. iheringi which are always found in the same areas of the shell.

DISCUSSION
Based on historical documentation (ANONYMOUS 1930, ODEBRECHT 2009), Herman von Ihering lived in Rio Grande do Sul, Brazil until 1892, when he moved to São Paulo, SP, Brazil.Sometime before 1893 (publication year of the original description) he collected mollusk specimens that were received by Haswell through Charles Chilton of New Zealand.A plausible possibility of a collection locality is the area of the delta of Rio Camaquã, where von Ihering lived for seven years (1885-1892), on an island known then as "Ilha do Doutor" (Doctor's Island is one of the three islands: Quebra Mastro, Santo Antônio, and Três Bocas, in the delta of Rio Camaquã, presently in the municipality of Camaquã).
Remarks by HASWELL (1893) suggest that the type locality of T. iheringi could only be the state of Rio Grande do Sul.As far as we know, only in Rio Grande do Sul is the host P. canaliculata found with concurrent infestations by two species of temnocephalan.In his remarks he reports receiving a sample which included specimens of distinct species.Although he did not realize that he had more than one species in the sample, he reported that: "T.Jheringi was found by Dr v. Jhering in Brazil in the branchial cavity of a species of Ampullaria.It differs from T. Semperi Weber: (1) in the absence of eyes; (2) in the form of the cirrus, which is very much wider at its proximal end, curved instead of straight, and with a terminal introvert of a different form; (3) in the first pair of testes being placed much further back; and (4) in the shape of the ovary."HASWELL (1893), however, described and illustrated (Plate XIII, Fig. 19 and Plate XV, Fig. 2) the cirrus straight.The curved cirrus, with a much wider shaft base belonged to a species that would only be described in 1989, by Ponce de León in Uruguay.Several characters described by HASWELL (1893) such as testes position and vitellarium distribution, agree with the observations in the present study.The lack of the red pigmentation of the eyes, if it existed, could not have been observed by him, since he received specimens already fixed (probably in some grade of ethanol), which, as we now know, dilutes the red pigment.
PEREIRA & CUOCOLO (1941) justified their redescription of the species based on the fact that in the original description of HASWELL (1893) the precise type locality was not defined.These authors attributed the report of absence of the red pigmentation of the eyes to a technical deficiency, as well as, the "absence" of seminal vesicle.In fact, HASWELL (1893) did not recognize the seminal vesicle and mistook it for the prostatic bulb.The absence of red pigmentation of the eyes in T. iheringi , indicated by Haswell, was confirmed in the present work, although he received dead specimens, possibly in ethanol (which, as mentioned above, dilutes the red pigment).The Brazilian populations of T. iheringi are the only ones (up to now) in the Neotropical Region without such pigmentation.This aspect calls for careful examination of live specimens.HYMAN (1955) also confirmed the lack of the red-eye pigmentation, although she also did not work with live specimens.This author described the bulb of the cirrus as "a prostatic vesicle with a muscular wall containing a mass of elongated cells around a central lumen".This type of prostatic bulb has been observed in the present study in which the central lumen in the proximal portion of the bulb dilates forming a prostatic vesicle (Fig. 35) that also contained spermatozoids.
DAMBORENEA (1992) stated that the prostatic glands are external to the bulb and that they surround the cirrus, free in parenchyma, disagreeing with HYMAN (1955), who studied longitudinal and frontal histological sections and affirmed that there was no evidence to postulate external prostatic glands.The statement by Damborenea also agrees with the drawings made by HASWELL (1893).In this respect, Damborenea may be right.
DAMBORENEA & CANNON (2001) reviewed the Neotropical species of Temnocephala.It was the first work to show the cirrus and DLSPs of several species in detail.The diagrammatic representation of the shape of the DLSPs of T. iheringi is similar to that found in the present study, although the greater richness of observations (SN and SEM) has shown details not revealed by DAMBORENEA & CANNON (2001), including the pores with secretions oozing out of the adhesive disk's syncytial plate (Fig. 22).These authors considered that all Neotropical species had red-eye pigmentation (examined alive by them) defining this character as an autapomorphy of Temnocephala.The absence of this pigment is so uncommon that there is only one species (Temnohaswellia pearsoni Sewell, Cannon & Bray, 2006) without eye pigmentation.This putative autapomorphy should be re-evaluated since, as in the present study, other species or populations in other ampullariid may be found to lack red-eye pigmentation.
A vesicle was observed inside the prostatic bulb, which due to its localization was called 'prostatic vesicle' (Fig. 35).This same vesicle was observed and illustrated in the description of Temnocephala mertoni Volonterio, 2007, ectocommensal of Aegla platensis Schmitt, 1942, by VOLONTERIO (2007), who called it 'contractile vesicle'.
Comparing measurements of the specimens of T. iheringi published to date (Tab.I), there are appreciable differences between the Brazilian and the Argentinean specimens, mainly in the total body length.This character was measured in almost all previous studies (even in the oldest ones), but has no taxonomic value.The Argentinean specimens showed the smallest measurements when compared to the Brazilian specimens.This could be indicative of intra-specific variation between the Brazilian and Argentinean populations, or may be the representation of a latitudinal cline, making it necessary that, in the future, a complete study should also be conducted with specimens collected in different latitudes in Argentina.