J. Parasitol., 94(3), 2008, pp.

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A NEW SPECIES OF POLYSTOMOIDES WARD, 1917 (MONOGENEA: POLYSTOMATIDAE) FROM FRESHWATER CHELONIANS (TESTUDINES: CHELIDAE) IN BRAZIL
Fabiano M. Vieira, Iara A. Novelli, Bernadete M. Sousa, and Sueli de SouzaLima
Laboratorio de Taxonomia e Ecologia de Helmintos, Departamento de Zoologia, Instituto de Ciencias Biologicas, Universidade Federal de Juiz de Fora, Campus Universitario, Bairro Martelos, Juiz de Fora, MG, Brasil 36036-330. e-mail: fmatosvieira@gmail.com
ABSTRACT: This report describes the rst occurrence of Polystomoides brasiliensis n. sp. (Monogenea: Polystomatidae), a new monogenean species in the buccal and pharyngeal cavities of the freshwater turtles in Brazil. Live monogeneans were collected from Hydromedusa maximiliani and Phrynops geoffroanus at the Mariano Procopio Museums lake, in Juiz de Fora, Minas Gerais, Brazil. Polystomoides brasiliensis differs from all other species of this genus in having 89 genital spines, except for Polystomoides uruguayensis, which has 810 genital spines. However, the new species differs morphometrically from P. uruguayensis in the greater size of the outer and inner hamuli, as well as having a testis that is proportionally greater than the pharynx and oral sucker. The current study is the rst report of monogeneans in chelonians of Brazil, and the rst record of helminths in H. maximiliani.

Polystomatidae (Monogenea) includes 3 parasitic genera exclusive to chelonians, i.e., Polystomoides Ward, 1917, Neopolystoma Price, 1939, and Polystomoidella Price, 1939. Species in this genus differ mainly in the number of hamuli they possess. Thus, Polystomoides spp. have 2 pair of hamuli, Polystomoidella spp. have 1 pair, and Neopolystoma spp. have none (Price, 1939; Sproston, 1946; Yamaguti, 1963; Rohde, 1965; Pichelin, 1995; Platt, 2000). Species of Polystomoides occur in the buccal and pharyngeal cavities, or the urinary and accessory bladder, of terrestrial tortoises, as well as freshwater and sea turtles (Sproston, 1946; Yamaguti, 1963; Knoepfer and Combes, 1977). In South America, species in this genus have been recorded only in the buccal cavity of freshwater chelonians of the Chelidae in Uruguay (Mane-Garzon, 1958; Mane-Garzon and Gil, 1961, 1962; Yamaguti, 1963; Mane-Garzon and Holcman-Spector, 1968; Knoepfer and Combes, 1977; Kohn and Cohen, 1998). In Brazil and other South American countries, there are no records of monogeneans in chelonians (Kohn and Cohen, 1998). The freshwater turtle, H. maximiliani (Mikan, 1820) (Testudines, Chelidae), or Maximilians snake-necked turtle, has been reported only in Brazil, where it is endemic to the eastern and southeastern regions; in these areas, the distribution is always associated with mountains (Souza, 2004; Novelli and Sousa, 2007). Phrynops geoffroanus (Schweigger, 1812) (Testudines, Chelidae), also known as Geoffroys side-necked turtle, occurs in several countries of South America. It can be found from Colombian Amazonia to the Brazilian state of Rio Grande do Sul, and from Uruguay to the northern parts of Argentina (Lema and Ferreira, 1990; McCord et al., 2001). It inhabits the shallow areas of both lakes and rivers, under the trunks of trees and rocks (Medem, 1960; Pritchard and Trebbau, 1984; Ernst and Barbour, 1989). In Brazil, this species is also common in rivers of urban areas (Souza and Abe, 2001a, 2001b; Brites, 2002; Lisboa et al., 2004). The present report describes a new species of monogenean Polystomoides from both H. maximiliani and P. geoffroanus, and is the rst occurrence of this monogenean genus in Brazil and from this host species.

MATERIALS AND METHODS

Live monogeneans were collected from the buccal and pharyngeal cavities of a single specimen of H. maximiliani and 4 specimens of P. geoffroanus from the Mariano Procopio Museums lake, in Juiz de Fora, Minas Gerais, Brazil, in 2007. On removal from their hosts, specimens were killed in distilled water kept in a refrigerator (approximately 7 C) for 24 hr and xed in AFA (70% ethanol, 93 parts; 37% formalin, 5 parts; glacial acetic acid, 2 parts), under slight cover glass pressure for 48 hr, and preserved in 70% ethanol (Amato et al., 1991). Monogeneans were stained in Delaelds hematoxylin or Mayers carmalum following the procedure of Amato et al. (1991) and mounted in Canada balsam for examination as whole mounts. Parasites (31 collected) were observed with an Olympus BX 41 light microscope (Olympus America Inc., Melville, New York). Drawings were made with the aid of photomicrographs derived from an Olympus micrographic system; measurements were made using an ocular micrometer. Identication and classication of the monogeneans to the generic level were in accordance with Price (1939), Sproston (1946), and Yamaguti (1963). Measurements are given as ranges in micrometers ( m), with mean standard deviation in parentheses.

DESCRIPTION
Polystomoides brasiliensis n. sp. (Figs. 15) Measurements of 8 adult specimens (1 holotype and 7 paratypes): Body elongate, oval, 2,4506,062 (3,600 1,479) in length, including opisthaptor; 5451,875 (1,100 592) in width at level of vaginal apertures. Opisthaptor digitate, 9982,200 (1,325 503) in length, 1,100 2,325 (1,503 484) in width, bearing 6 haptor suckers of type 2 (Pichelin, 1995), 300813 (455 179) in diameter (n 46). One marginal hook in interior of each haptor sucker. Hamuli, 2 pairs; inner pair 55.0 71.3 (65.1 4.9) in length (n 8), outer pair 62.580.0 (72.1 5.5) in length (n 8). Marginal hooks, 16; ventral, of similar shape and size, 25.036.3 (31.9 3.4) in length (n 63). Mouth subterminal, ventral. Oral sucker 245490 (312 102) in length by 290520 (364 89) in width. Pharynx oval, 175415 (249 96) in length by 205 460 (290 99) in width; esophagus short. Intestinal caeca without anterior diverticula, extending to near anterior margin of opisthaptor; ends not contiguous. Common genital aperture median, posterior to intestinal bifurcation; genital coronet with 89 spines with similar sizes and curved, 11.317.5 (12.8 1.6) in length. Testis circular or oval, postovarian, median, prequatorial; with regular margin, 3701,125 (633 308) in length by 3301,050 (583 298) in width, with lateral regions in contact with intestinal caeca. Ovary pretesticular, submedian, 95250 (161.5 63) in length, to right of median line. Uterus absent. Ootype short, not opposite ovary, conned between ovary and testis. Two vaginal apertures ventral, near margins of body, extra caeca, at level of anterior margin of testis. Vitellaria follicular, extend from midpharyngeal level and anterior margin of opisthaptor, surround intestinal caeca. Egg ovoid, 195245 (220 35) in length by 175195 (185 14) in width.
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Received 9 August 2007; revised 2 October 2007; accepted 3 October 2007.

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FIGURES 15. Polystomoides brasiliensis n. sp. (1) Whole mount, ventral view; (2) genital coronet; (3) egg; (4) marginal hook; (5) inner hamuli (left) and outer hamuli (right).

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TABLE I. Polystomoides spp. described from South AmericaNumber and size of genital spines. Genital spines Species P. P. P. P. uruguayensis fuquesi rohdei brasiliensis Number 810 2 2932 89 Size ( m) 10 56 3452 1117 Author Mane-Garzon and Gil (1961) Mane-Garzon and Gil (1962) Mane-Garzon and Holcman-Spector (1968) Current study

Testis length/oral sucker length ratio 2.02:1.0; testis width/oral sucker width ratio 1.64:1.0; testis length/pharynx length ratio 2.54: 1.0; testis width/pharynx width ratio 2.01:1.0; outer hamuli length/ inner hamuli length ratio 1.1:1.0; outer hamuli length/marginal hook length ratio 2.26:1.0; inner hamuli length/marginal hook length ratio 2.04:1.0; oral sucker length/pharynx length ratio 1.25:1.0; oral sucker width/pharynx width ratio 1.25:1.0. Taxonomic summary Type host: H. maximiliani (Mikan, 1820) (Testudines, Chelidae). Other host: P. geoffroanus (Schweigger, 1812) (Testudines, Chelidae). Site of infection: Buccal and pharyngeal cavities. Type of locality: Juiz de Fora, Minas Gerais, Brazil, 21 41 20 S, 43 20 40 W. Prevalence: H. maximiliani: 100%; P. geoffroanus: 100%. Intensity of infection: H. maximiliani: 6 parasites; P. geoffroanus: 7.75 12.2. Type specimens: Holotype CHIOC no. 36902 (whole mounts); 4 paratypes CHIOC no. 36903 ad (whole mounts), deposited in the Helminthological Collection of the Instituto Oswaldo Cruz (CHIOC), Rio de Janeiro, Brazil. Etymology: The specic name was chosen because this species was the rst Polystomoides described and reported from Brazil. Remarks Polystomoides brasiliensis n. sp. differs from all other species in the genus by possessing 89 genital spines. The only exception is P. uruguayensis Mane-Garzon and Gil, 1961, which has 810 genital spines (Table I). However, P. brasiliensis differs morphometrically from the latter species because the greater size of the outer and inner hamuli (Table II) and because its testis is approximately twice the size of its oral sucker, while in P. uruguayensis, the 2 structures are approximately the same size (Table II). Another morphometric difference between these 2 species is the size of the testis when compared with the size of

the pharynx. Testis size of P. brasiliensis is greater than twice that of the pharynx, while in P. uruguayensis, the testis size is less than double the size of the pharynx (Table III). We also observed a morphometric difference between the genital spines of the 2 species (Table I).

DISCUSSION In the present study, the characters used for differentiation of P. brasiliensis sp. included morphometric data for the outer and inner hamuli, testis, pharynx, and oral suckers (Table II), as well as quantitative and morphometric data for the genital spines (Table I). The morphology of the outer and inner hamuli, the relation of sizes of these structures with size of the haptoral sucker, the morphology and morphometric data for marginal hooks (Table II), the testis size/pharynx size and oral sucker size/pharynx size (Table III) ratios are characters used by Rohde (1965) in the distinction of the several species. The number and size of genital spines are the characters more frequently used in the identication of Polystomoides spp. (Rohde, 1965). Thus, the validity of these characteristics in the differentiation of the species of this genus is evident. In the current study, it was veried that absolute sizes of outer and inner hamuli, marginal hooks, and of haptor suckers of P. brasiliensis are greater than those of P. uruguayensis (Table II). However, when ratios of the structures of the opisthaptor were compared, it was determined that only the relative size of the inner hamuli of P. brasiliensis is signicantly different from the inner hamuli of P. uruguayensis (Table III). This became evident in the analysis of the ratios of outer hamuli length/inner hamuli length, haptor sucker diameter/inner hamuli length, and
TABLE III. Comparative ratios of Polystomoides uruguayensis and Polystomoides brasiliensis. Species

TABLE II. Comparative measurements* of Polystomoides uruguayensis and Polystomoides brasiliensis. Species P. uruguayensis (Mane-Garzon and Gil, 1961) 345 260 350 270 210 210 21 38 52 315 P. brasiliensis (Current study) 633 583 312 364 249 290 31.9 65.1 72.1 455

Measures Testis length Testis width Oral sucker length Oral sucker width Pharynx length Pharynx width Marginal hook length Inner hamuli length Outer hamuli length Opisthaptor sucker diameter
* Mean size, shown as m.

Ratios

P. P. uruguayensis brasiliensis (Mane-Garzon (Current and Gil, 1961) study) 0.98:1 0.96:1 1.64:1 1.23:1 1.67:1 1.28:1 1.36:1 6.05:1 8.28:1 2.47:1 1.8:1 2.02:1 1.64:1 2.54:1 2.01:1 1.25:1 1.25:1 1.10:1 6.30:1 6.98:1 2.26:1 2.04:1

Testis length/oral sucker length Testis width/oral sucker width Testis length/pharynx length Testis width/pharynx width Oral sucker length/pharynx length Oral sucker width/pharynx width Outer hamuli length/inner hamuli length Haptor sucker diameter/outer hamuli length Haptor sucker diameter/inner hamuli length Outer hamuli length/marginal hook length Inner hamuli length/marginal hook length

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TABLE IV. South American chelonian hosts of monogeneans. Family Chelidae Species Phrynops hilarii (Dumeril and Bibron, 1835) Phrynops geoffroanus (Schweigger, 1812) Hydromedusa maximiliani (Mikan, 1820) Trachemys dorbignyi (Dumeril and Bibron, 1835) Parasite P. uruguayensis P. fuquesi P. brasiliensis P. brasiliensis P. coronatus P. rohdei Site of infection buccal cavity pharyngeal cavity buccal and pharyngeal cavities buccal and pharyngeal cavities buccal cavity buccal cavity Country Uruguay Uruguay Brazil Brazil Uruguay Uruguay Author Mane-Garzon and Gil (1961) Mane-Garzon and Gil (1962) Current study Current study Mane-Garzon (1958) Mane-Garzon and Holcman-Spector, 1968

Emydidae

inner hamuli length/marginal hook length. The other ratios between the structures of opisthaptors of both species are similar in their relative sizes (Table III). Finally, the morphology of inner and outer hamuli in P. brasiliensis (Fig. 5) differs from that of the hamuli of P. uruguayensis represented in the study of Mane-Garzon and Gil (1961). Prior to the present study, Polystomoides included 30 species. Of these, 15 had been described as parasites collected in the buccal cavity and pharynx of freshwater turtles in Africa, Asia, Europe, South America, and North America (Ozaki, 1935; Paul, 1938; Mane-Garzon and Gil, 1961, 1962; Rohde, 1965; Mane Garzon and Holcman-Spector, 1968; Gonzalez and Mishra, 1977; Knoepfer and Combes, 1977; Combes and Rohde, 1978; Timmers and Lewis, Jr., 1979). In South America, P. uruguayensis, P. fuquesi Mane-Garzon and Gil, 1962, and P. rohdei Mane-Garzon and Holcman-Spector, 1968, had been previously described, all in Uruguay (Knoepfer and Combes, 1977; Kohn and Cohen, 1998) (Table IV). Since the study by Mane-Garzon and Holcman-Spector (1968), there have been no descriptions of new monogeneans from chelonians in South America. Polystomoides coronatus (Leidy, 1888) was also identied in a freshwater chelonian from South America (Table IV) (Mane Garzon, 1958), but this species was originally described from North America. The current study is the rst to describe a monogenean in chelonians from Brazil, and the rst to identify helminths in H. maximiliani. ACKNOWLEDGMENTS
We are grateful to Dr. Simone C. Cohen, Dr. Klaus Rohde, Dr. Claude Combes and Dr. Olivier Verneau for sending copies of papers that were used as references in this work.

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