New Species of Crossobothrium (Cestoda: Tetraphyllidea) from the Broadnose Sevengill Shark, Notorynchus cepedianus, in Argentina

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1 New Species of Crossobothrium (Cestoda: Tetraphyllidea) from the Broadnose Sevengill Shark, Notorynchus cepedianus, in Argentina Author(s): Verónica A. Ivanov Source: Journal of Parasitology, 95(6): Published By: American Society of Parasitologists DOI: URL: BioOne ( 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 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.

2 J. Parasitol., 95(6), 2009, pp F American Society of Parasitologists 2009 NEW SPECIES OF CROSSOBOTHRIUM (CESTODA: TETRAPHYLLIDEA) FROM THE BROADNOSE SEVENGILL SHARK, NOTORYNCHUS CEPEDIANUS, IN ARGENTINA Verónica A. Ivanov* Laboratorio de Helmintología, Departamento de Biodiversidad y Biología Experimental, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires. Ciudad Universitaria, Pabellón II, piso 4, C1428EHA, Buenos Aires, Argentina. ivanov@bg.fcen.uba.ar ABSTRACT: Two new species of Crossobothrium were found inhabiting Notorynchus cepedianus, the broadnose sevengill shark, collected from the coast of Buenos Aires Province, Argentina. Crossobothrium antonioi n. sp. is the smallest species in the genus and has at least 3 times as many testes per mature proglottid than any other congener. It can be further distinguished from other species of Crossobothrium by the possession of crenulated bothridial margins and a different microthrix pattern. Crossobothrium pequeae n. sp. is distinguished from all other species in the genus using the following combination of characters: worm size, number of testes per mature proglottid, bothridial margins, microthrix pattern, and the extension of ovary and testicular fields in cross section. Species in Crossobothrium, with the exception of C. laciniatum, seem to be restricted to hexanchid sharks, and a single species of shark can be parasitized by multiple species of Crossobothrium, as is the case with N. cepedianus, and probably Hexanchus griseus. Crossobothrium Linton, 1889 was synonymized with Phyllobothrium van Beneden, 1849 by Southwell in Some authors subsequently disregarded the synonymy (Euzet, 1959, 1994 [in part, as he accepted Phyllobothrium dohrni (Örley, 1885) as valid]; Williams, 1968), while others sustained it (Joyeux and Baer, 1936; Rees, 1946; Wardle and McLeod, 1952; Euzet, 1959 [in part]; Yamaguti, 1959; Schmidt, 1986). During this controversial history, at least 9 species were once included in Crossobothrium (see Caira and Healy, 2006), i.e., C. laciniatum Linton, 1889, C. angustum (Linton, 1889), C. dohrni (Örley, 1885), C. filiforme (Yamaguti, 1952), C. longicolle (Molin, 1858), C. triacis (Yamaguti, 1952), C. prionacis (Yamaguti, 1934), C. squali (Yamaguti, 1952), and C. campanulatum Klaptocz, 1906). Ruhnke (1996) made the most recent revision of Crossobothrium and presented an emended diagnosis of the genus. As a result, only 2 species, C. laciniatum and C. dohrni, were considered to belong to Crossobothrium; several species were transferred to Paraorygmatobothrium Ruhnke, 1994, i.e., P. prionacis (Yamaguti, 1934), P. filiforme (Yamaguti, 1952), P. triacis (Yamaguti, 1952), and probably C. angustum (see Ruhnke, 1994, 1996). Ruhnke (1996) proposed that the other species remain in the genera in which they were originally described, i.e., Phyllobothrium squali Yamaguti, 1952 and Tetrabothrius longicolle Molin, 1858, awaiting revision. No comments on the taxonomic status of C. campanulatum were included in Ruhnke s work (1996). This species has been considered a synonym of C. dohrni (as P. dohrni [Örley, 1885]) by some authors (Southwell, 1925; Euzet, 1959). The original description of C. campanulatum gives brief useful information on anatomy, but total length, scolex length, and number of testes per proglottid are not within the range given for C. dohrni (see Örley, 1885; Klaptocz, 1906). Therefore, C. campanulatum is considered a valid species in the present study. Since the original description of C. dohrni by Örley (1885) based on specimens from Heptranchias perlo (Bonnaterre, 1788), additional records and information on this cestode have been provided by several authors (Joyeux and Baer, 1936; Rees, 1946; Riser, 1955; Euzet, 1959; Robinson, 1959; Alexander, 1963; Ruhnke, 1996). However, with the exception of Joyeux and Baer (1936), all of these studies were based on specimens from different Received 16 March 2009; revised 12 June 2009; accepted 7 July * Researcher at: Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Argentina. DOI: /GE species of sharks. Most of these records belong to specimens from Hexanchus griseus (Bonnaterre, 1788) (see Rees, 1946; Riser, 1955; Euzet, 1959; Ruhnke, 1996), which happens to be the type host of C. campanulatum. Joyeux and Baer (1936) studied specimens from H. perlo, but also included 3 other species of unrelated sharks in the host list. Robinson (1959) and Alexander (1963) reported C. dohrni from Notorynchus cepedianus (Péron, 1807) in New Zealand. These records of the same species, C. dohrni, from different hosts and localities are the consequence of the taxonomic actions by Southwell (1925), who not only synonymized Crossobothrium to Phyllobothrium, but also C. laciniatum, C. campanulatum, and Orygmatobothrium velamentum Yoshida, 1917 to P. dohrni. As a result, all of these records of C. dohrni (as P. dohrni) from different sharks, other than the type host, should be considered doubtful. During a parasite survey of sharks from the coast of Argentina, 2 new species of Crossobothrium were collected from the broadnose sevengill shark, N. cepedianus. Detailed descriptions of both species based on whole mounts, cross serial sections, and study of the tegumental surface using scanning electron microscopy (SEM) are presented below. MATERIALS AND METHODS Cestodes were recovered from the spiral intestine of 18 specimens of N. cepedianus caught in February 2000, November 2002, and November 2005, in Puerto Quequén, Buenos Aires Province, Argentina (38u329S, 58u429W) and fixed in 10% formalin. Specimens prepared for light microscopy were hydrated in a graded ethanol series, stained with Harris hematoxylin, dehydrated in a graded ethanol series, cleared in methyl salicylate, and mounted in Canada balsam. Specimens prepared for SEM were hydrated in a graded ethanol series, post-fixed in 1% osmium tetroxide overnight at room temperature, dehydrated in a graded ethanol series, and dried using hexamethyldisilazane. After dehydration, the specimens were mounted on a stub with carbon tape, coated with 40 nm of gold/palladium in a Thermo VG Scientific Polaron SC 7630 (Quorum Technologies Ltd.; East Grinstead, West Sussex, England), and examined in a Philips XL 30 SEM (Fei Company; Eindhoven, Noord-Bravant, Netherlands). Scolices and mature proglottids were embedded in paraffin and transverse serial sections were cut at a thickness of 10 mm. All sections were stained with Harris hematoxylin and counterstained with eosin. Measurements include the range, followed in parentheses by the mean, standard deviation, number of worms examined (n), and the total number of observations when more than 1 measurement per worm was taken (n). All measurements are in micrometers, unless otherwise stated. Figures were drawn with the aid of a drawing tube on a Zeiss Axioskop microscope (Carl Zeiss Group; Jena, Thuringia, Germany). 1479

3 1480 THE JOURNAL OF PARASITOLOGY, VOL. 95, NO. 6, DECEMBER 2009 DESCRIPTIONS Crossobothrium antonioi n. sp. (Figs. 1 19) Diagnosis (based on 8 specimens: 6 whole mounts [all complete worms] and 2 worms observed with SEM and their posterior portion of the strobila sectioned): Worms euapolytic, (49.3 ± 2.0, n 5 6) mm long; greatest width at level of last proglottid 2,060 2,460 (2,252 ± 168, n 5 6); proglottids laciniate, with 4 posterior triangular laciniations (2 ventrolateral, 2 dorsolateral), (59 ± 2, n 5 6) per worm (Figs. 1 4, 14). Scolex composed of 4 stalked bothridia, lacking apical organ (Figs. 2, 5), 900 1,050 (983.3 ± 76.4, n 5 6) long, 1,000 1,500 (1,270 ± 251, n 5 6) wide. Bothridia (832 ± 79, n 5 6, n 5 6) long, (910 ± 35; n 5 6, n 5 6) wide, with anterior accessory sucker, bothridial margins crenulated (Figs. 2, 5, 6). Accessory sucker (91 ± 10, n 5 6, n 5 6) long, (96 ± 12, n 5 6, n 5 6) wide (Figs. 2, 5, 6). Bothridial stalks (221 ± 47, n 5 6, n 5 6) long. Neck and cephalic peduncle absent. Inner and outer surfaces of accessory sucker covered with short filitriches (0.23 ± 0.05) long, (0.14 ± 0.04) wide (Figs. 7, 8). Distal bothridial surface covered with conical spinitriches interspersed with filitriches (Figs. 9 12); conical spinitriches longer and denser at bothridial edges, (2.64 ± 0.47) long, (0.31 ± 0.05) wide at base (Fig. 10), becoming shorter and scarce at bothridial center, (2.19 ± 0.21) long, (0.37 ± 0.03) wide at base; short filitriches (0.28 ± 0.02) long, (0.10 ± 0.02) wide (Figs. 11, 12). Proximal bothridial surfaces covered with conical spinitriches (3.34 ± 0.37) long, (0.41 ± 0.05) wide at base, interspersed with long filitriches (0.97 ± 0.13) long, (0.08 ± 0.01) wide (Fig. 13). Papillose projections present on accessory suckers and distal bothridial surfaces, with central cilium, covered with short filitriches (Figs. 8, 9). Proglottid surfaces covered with short filitriches (0.42 ± 0.03) long, (0.15 ± 0.01) wide (Figs. 15, 16). Immature proglottids longer than wide, becoming as long as wide with maturity; (52 ± 4, n 5 6) in number (Fig. 1). Mature proglottids slightly wider than long, becoming longer than wide with maturity, 1,700 2,540 (2,114 ± 260, n 5 6, n 5 30) long, 1,880 2,420 (2,218 ± 184, n 5 6, n 5 30) wide; length to width ratio (0.96 ± 0.19):1; 5 8 (6 ± 2, n 5 6, n 5 30) mature proglottids per strobila (Figs. 1, 3). Gravid proglottids slightly longer than wide, 2,140 2,960 (2,500 ± 304, n 5 6, n 5 20) long, 2,060 2,460 (2,252 ± 167, n 5 6, n 5 20) wide; length to width ratio (1.11 ± 0.2):1; 1 2 (2, n 5 6) gravid proglottids per strobila. Velum (667 ± 83, n 5 6, n 5 50) long (not included in mature and gravid proglottid length). Deep bundles of longitudinal muscles dividing parenchyma in cortical and medullar, all genital organs and ducts medullary (Figs ). Testes oval, (83 ± 18, n 5 6, n 5 75) long, (78 ± 11, n 5 6, n 5 75) wide, 3 4 rows deep in cross-section (Figs. 3, 17); (736 ± 31, n 5 6, n 5 12) in number in mature proglottids, (69 ± 6, n 5 6, n 5 12) post-vaginal testes; testicular field extending laterally beyond osmoregulatory ducts in anterior third of proglottid (Fig. 3), testes degenerating in detached gravid proglottids. Cirrus sac elongated, curved anteriorly, (580 ± 64, n 5 6, n 5 24) long, (203 ± 18, n 5 6, n 5 24) wide in mature proglottids, occupying 26 33% (29 ± 2) of proglottid width (Figs. 3, 18); containing cirrus covered with short microtriches. Vas deferens extensive, highly coiled, extending anteriorly (Fig. 3). Ovary lobulated, bilobated in dorsoventral view (Fig. 3), multilobed in cross section at level of isthmus (Fig. 19); (417 ± 96, n 5 6, n 5 24) long, 830 1,050 (966 ± 71, n 5 6, n 5 24) wide at ovarian isthmus in mature proglottids. Vagina thick-walled, maximum diameter (144 ± 30, n 5 6, n 5 24), running parallel to cirrus sac, crossing vas deferens at posterior margin of cirrus sac, descending posteriorly, forming seminal receptacle at level of ootype region, vaginal sphincter present. Mehlis gland (121 ± 24, n 5 6, n 5 24) long, (145 ± 17, n 5 6, n 5 24) wide. Vagina and cirrus sac join into genital atrium; genital atrium (103 ± 14, n 5 6, n 5 30) deep; marginal genital pores alternate irregularly, 26 34% (29 ± 2, n 5 6, n 5 30) from posterior margin of mature proglottid. Vitelline follicles in 2 lateral fields, each field consisting of 4 8 columns of follicles (Figs ), follicles (39 ± 13, n 5 6, n 5 50) long, (46 ± 11, n 5 6, n 5 50) wide, vitelline field interrupted ventrally at level of cirrus sac and vagina, extending throughout entire proglottid length, becoming wider at level of ovary (Figs. 3, 17 19). Uterus saccate, extending anteriorly up to 29 47% (36 ± 7, n 5 6, n 5 20) from anterior margin of mature and gravid proglottids (Fig. 3). Unembryonated, intrauterine eggs spherical, (24.6 ± 0.8, n 5 3, n 5 20) in diameter. Taxonomicsummary Type host: Notorynchus cepedianus (Péron, 1807), broadnose sevengill shark (Hexanchiformes, Hexanchidae). Site of infection: Spiral intestine. Type locality: Puerto Quequén, Buenos Aires Province, Argentina (38u329S, 58u429W). Etymology: This species is named after my father, Antonio Ivanov. Specimens deposited: Holotype and 5 paratypes deposited in the Colección Parasitológica-Museo Argentino de Ciencias Naturales, Buenos Aires, Argentina, MACN-PA Nu 493/1-6. Additional specimens (whole mounts, histological sections, and specimens prepared for SEM) retained in my personal collection. Remarks Crossobothrium antonioi n. sp. is unique among species of Crossobothrium in having an incredibly large number of testes per mature proglottid and being the smallest species in the genus. Crossobothrium antonioi n. sp. possesses more than 700 testes per proglottid, whereas C. laciniatum, C. dohrni, and C. campanulatum were described as having up to 282, 200, and 150, testes per proglottid, respectively (Örley, 1885; Klaptocz, 1906; Ruhnke, 1996). Gravid specimens of C. antonioi can reach a length of 50 mm (with 62 proglottids), while all other species in the genus are at least double that size, i.e., C. laciniatum is up to 130 mm long (with 450 proglottids), C. dohrni is 100 mm long (up to 500 proglottids), and C. campanulatum is 500 mm long (with more than 330 proglottids). In addition, C. antonioi differs from C. laciniatum by having a smaller scolex (1,000 1,500 vs. 2,200 3,000 wide), bothridial margins that are crenulated instead of smooth, and different type of microtriches on proximal bothridial surface (conical spinitriches and filitriches vs. aristate spinitriches), distal bothridial surface (slender conical spinitriches vs. wide, large blade-like spinitriches), and on the strobila (filitriches vs. spinitriches). In addition, whereas the shape of proglottids is constant along the strobila (with a tendency of becoming longer than wide with maturity) in C. antonioi, the proglottids vary extensively in shape in different regions of the same strobila in C. dohrni and C. campanulatum. Crossobothrium pequeae n. sp. (Figs ) Diagnosis (based on 17 specimens: 15 whole mounts [all complete worms], and 2 worms observed with SEM and their posterior portion of the strobila sectioned): Worms euapolytic, (102.2 ± 26.6, n 5 15) mm long; greatest width at level of scolex or mature proglottids 1,150 2,570 (1,600 ± 563, n 5 15); proglottids laciniate, with 4 posterior triangular laciniations (2 ventrolateral, 2 dorsolateral), (460 ± 209, n 5 15) per worm (Figs. 20, 22, 32 36). Proglottid and laciniation shapes change conspicuously along the length of the strobila (Figs. 20, 32 36), from square to longer than wide with long laciniations (Fig. 33), to wider than long with short laciniations (Figs. 35, 36), becoming square to longer than wide with maturity (Fig. 22). Laciniations evident in all proglottids (Figs. 20, 22, 32 36). Scolex composed of 4 stalked bothridia, lacking apical organ, 810 1,100 (992 ± 121, n 5 15) long, 1,150 1,750 (1,434 ± 233, n 5 15) wide (Figs. 21, 23). Bothridia (822 ± 112, n 5 15, n 5 20) long, (835 ± 112; n 5 15, n 5 20) wide, with anterior accessory sucker, bothridial margins crenulated (Figs. 21, 23, 24). Accessory sucker (90 ± 13, n 5 15, n 5 30) long, (98 ± 6, n 5 15, n 5 30) wide (Figs. 21, 23, 24). Bothridial stalks (183 ± 15, n 5 15, n 5 30) long. Neck and cephalic peduncle absent. Inner and outer surfaces of accessory sucker covered with short filitriches (0.32 ± 0.06) long, (0.14 ± 0.03) wide (Figs. 25, 26). Distal bothridial surfaces covered with conical spinitriches interspersed with filitriches (Figs ); conical spinitriches longer and denser at bothridial edges, (2.83 ± 0.39) long, (0.34 ± 0.09) wide at base (Fig. 27), becoming shorter and scarce at bothridial center, (1.87 ± 0.21) long, (0.40 ± 0.03) wide at base; filitriches (0.27 ± 0.04) long, (0.14 ± 0.03) wide (Figs. 28, 29). Proximal bothridial surface covered with conical spinitriches (2.82 ± 0.23)

4 IVANOV NEW SPECIES OF CROSSOBOTHRIUM 1481 FIGURES 1 3. Crossobothrium antonioi n. sp. (1) Entire worm, scale bar mm. (2) Scolex, scale bar mm. (3) Mature proglottid, scale bar mm.

5 1482 THE JOURNAL OF PARASITOLOGY, VOL. 95, NO. 6, DECEMBER 2009 FIGURES Crossobothrium antonioi n. sp., scanning electron micrographs. (4) General view of scolex and immature proglottids, scale bar mm. (5) Scolex, scale bar mm. Small numbers indicate locations of details shown in Figures (6) Detail of accessory sucker, scale bar 5 50 mm. Small numbers indicate locations of details shown in Figures 7 and 8. (7) Inner surface of accessory sucker, scale bar 5 1 mm. (8) Outer surface of accessory sucker, scale bar 5 1 mm. (9) Distal bothridial surface showing gradual change on microthrix size and density, sale bar 5 10 mm. (10) Distal bothridial surface near the margin, scale bar 5 2 mm. (11) Distal bothridial surface at midway between margin and center, scale bar 5 2 mm. (12) Distal bothridial surface at the center of bothridium, scale bar 5 2 mm. (13) Proximal bothridial surface (a), scale bar 5 2 mm; detail of microtriches in close up (b), scale bar 5 1 mm. (14) Surface of immature proglottids, scale bar mm. Small numbers indicate locations of details shown in Figures 15 and 16. (15) Surface of laciniations in immature proglottid, scale bar 5 2 mm. (16) Surface of immature proglottid, scale bar 5 2 mm.

6 IVANOV NEW SPECIES OF CROSSOBOTHRIUM 1483 FIGURES Crossobothrium antonioi n. sp., cross sections of mature proglottid, scale bar mm. (17) At level of testes anterior to cirrus sac. (18) At level of genital pore. (19) At level of ovarian isthmus. Abbreviations: cs, cirrus sac; dlm, deep longitudinal musculature; dod, dorsal osmoregulatory duct; gp, genital pore; ov, ovary; sr, seminal receptacle; t, testis; u, uterus; vd, vas deferens; vf, vitelline follicle; vg, vagina; vod, ventral osmoregulatory duct. long, (0.38 ± 0.04) wide at base, interspersed with long filitriches (0.77 ± 0.01) long, (0.08 ± 0.01) wide (Fig. 31). Papillose projections present on accessory suckers and distal bothridial surfaces, with central cilium, covered with short filitriches. Proglottid surfaces covered with short filitriches (0.22 ± 0.02) long, (0.16 ± 0.02) wide (Figs. 37, 38). Immature proglottids longer than wide, becoming wider than long with maturity; (443 ± 214, n 5 15) in number (Figs. 20, 32 36). Mature proglottids wider than long, becoming longer than wide with maturity, 550 1,450 (920 ± 247, n 5 15, n 5 35) long, 910 2,570 (1,345 ± 575, n 5 15, n 5 35) wide; length to width ratio (0.80 ± 0.33):1; 6 32 (14 ± 11, n 5 15) mature proglottids per strobila (Figs. 20, 22). Gravid proglottids 600 1,600 (932 ± 359, n 5 15, n 5 20) long, 2,100 2,450 (2,330 ± 111, n 5 15, n 5 20) wide; length to width ratio (0.41 ± 0.17):1; 0 14 (3 ± 6, n 5 15) gravid proglottids per strobila (Fig. 20). Velum (318 ± 103, n 5 15, n 5 40) long (not included in mature and gravid proglottid length). Deep bundles of longitudinal muscles dividing parenchyma in cortex and medulla, testes and uterus medullary, ovary primarily medullary, protruding into cortex between muscle bundles, vitelline follicles medullary to paramuscular (Figs ). Testes oval, (76 ± 13, n 5 15, n 5 75) long, (73 ± 13, n 5 15, n 5 75) wide, 2 rows deep in cross-section (Fig. 39), (197 ± 30, n 5 15, n 5 30) in number in mature proglottids, (28 ± 5, n 5 15, n 5 30) post-vaginal testes; testicular field circumscribed laterally by osmoregulatory ducts (Figs. 22, 39 41), testes degenerating in detached gravid proglottids. Cirrus sac elongated, curved anteriorly, (467 ± 148, n 5 15, n 5 30) long, (188 ± 37, n 5 15, n 5 30) wide in mature proglottids, occupying 32 44% (37 ± 4) of proglottid width (Figs. 22, 40); containing cirrus covered with short microtriches. Vas deferens extensive highly coiled, extending anteriorly (Fig. 22). Ovary lobulated, bilobated in dorsoventral view (Fig. 22), X-shaped in cross section at level of isthmus (Fig. 41); (257 ± 81, n 5 15, n 5 30) long, 450 1,100 (650 ± 207, n 5 15, n 5 30) wide at ovarian isthmus in mature proglottids. Vagina thick-walled, maximum diameter (103 ± 17, n 5 15, n 5 50), running parallel to cirrus sac, crossing vas deferens at posterior margin of cirrus sac, descending posteriorly, forming seminal receptacle at level of ootype region, vaginal sphincter present. Mehlis gland (92 ± 26, n 5 15, n 5 30) long, (107 ± 28, n 5 15, n 5 30) wide. Vagina and cirrus sac join into genital atrium; genital atrium (68 ± 17, n 5 15, n 5 50) deep; marginal genital pores alternate

7 1484 THE JOURNAL OF PARASITOLOGY, VOL. 95, NO. 6, DECEMBER 2009 FIGURES bar mm. Crossobothrium pequeae n. sp. (20) Entire worm, scale bar mm. (21) Scolex, scale bar mm. (22) Mature proglottid, scale

8 IVANOV NEW SPECIES OF CROSSOBOTHRIUM 1485 FIGURES Crossobothrium pequeae n. sp., scanning electron micrographs. (23) Scolex, scale bar mm. Small numbers indicate locations of details shown in Figures (24) Detail of accessory sucker, scale bar 5 50 mm. Small numbers indicate locations of details shown in Figures 25 and 26. (25) Inner surface of accessory sucker, scale bar 5 2 mm. (26) Outer surface of accessory sucker, scale bar 5 2 mm. (27) Distal bothridial surface near the margin, scale bar 5 2 mm. (28) Distal bothridial surface at midway between margin and center, scale bar 5 2 mm. (29) Distal bothridial surface at the center of bothridium, scale bar 5 2 mm. (30) Distal bothridial surface showing gradual change on microthrix size and density, sale bar 5 10 mm. (31) Proximal bothridial surface, scale bar 5 2 mm.

9 1486 THE JOURNAL OF PARASITOLOGY, VOL. 95, NO. 6, DECEMBER 2009 irregularly, 28 45% (37 ± 4, n 5 15, n 5 50) from posterior margin of mature proglottid (Figs. 20, 22). Vitelline follicles in 2 lateral fields; each field consisting of 6 9 columns of follicles (Figs ), follicles (49 ± 9, n 5 15, n 5 50) long, (30 ± 9, n 5 15, n 5 50) wide, vitelline field interrupted ventrally at level of cirrus sac and vagina, extending throughout entire proglottid length, becoming wider at level of ovary (Figs. 22, 39 41). Uterus saccate extending anteriorly up to 22 29% (26 ± 2, n 5 15, n 5 30) from anterior margin of mature and gravid proglottids (Fig. 22). Unembryonated, intrauterine eggs spherical, (31 ± 1.1, n 5 6, n 5 35) in diameter. Taxonomicsummary Type host: Notorynchus cepedianus (Péron, 1807), broadnose sevengill shark (Hexanchiformes, Hexanchidae). Site of infection: Spiral intestine. Type locality: Puerto Quequén, Buenos Aires Province, Argentina (38u329S, 58u429W). Etymology: This species is named after my mother, María Angélica Peque Conti Ivanov. Specimens deposited: Holotype and 5 paratypes deposited in the Colección Parasitológica-Museo Argentino de Ciencias Naturales, Buenos Aires, Argentina, MACN-PA Nu 494/1-6. Additional specimens (whole mounts, histological sections, and specimens prepared for SEM) retained in my personal collection. Remarks Crossobothrium pequeae n. sp. is conspicuously smaller than C. campanulatum (up to 138 vs mm long) and it has a larger number of testes per mature proglottid ( vs ). Crossobothrium pequeae possesses a smaller scolex than C. laciniatum (1,150 1,750 vs. 2,175 3,050 wide), a different microthrix pattern on proximal bothridial surfaces (conical spinitriches and filitriches vs. aristate spinitriches), distal bothridial surfaces (slender conical spinitriches vs. wide large blade-like spinitriches), and strobila (filitriches vs. spinitriches), and the bothridial margins are crenulated instead of smooth. The brief information on the morphology of C. dohrni that is currently available (Örley, 1885) does not allow a clear distinction between C. pequeae and C. dohrni. Nevertheless, the bothridial margins seem to be smooth in C. dohrni (see Fig. 17 in Örley, 1885), whereas they are clearly crenulated in C. pequeae. Although C. pequeae and C. dohrni exhibit similar variation in proglottid shape along the length of their strobila, in the new species, the laciniations of the proglottids are always evident, even in the last proglottids on the strobila (Figs. 20, 22); they are not reduced as mentioned by Örley (1885) for C. dohrni. Finally, C. pequeae is larger than C. antonioi (up to 138 mm vs. up to 51 mm long) and it has a larger number of proglottids ( vs ). These 2 new species can also be distinguished from one another based on the number of testes per mature proglottid ( in C. pequeae vs in C. antonioi), the extension of the ovary in cross section (ovary medullar invading the cortex in C. pequeae vs. ovary completely medullar in C. antonioi), and in the lateral extension of the testicular field (circumscribed laterally by the osmoregulatory ducts in C. pequeae vs. testicular field extending laterally beyond osmoregulatory ducts in anterior third of proglottid in C. antonioi) (Figs. 3, 22). Detached proglottids FIGURES Crossobothrium pequeae n. sp., scanning electron micrographs. (32) Anterior region of strobila showing the variation in immature proglottid shape, from anterior to posterior (a c), scale bar 5 1 mm. (33 36) Detail of proglottid shape, from anterior (33) to posterior (36), scale bar mm. Small numbers in Figure 34 indicate locations of details shown in Figures 37 and 38. (37) Surface of laciniations in immature proglottid, scale bar 5 1 mm. (38) Surface of anterior half of immature proglottid, scale bar 5 1 mm. Detached gravid proglottids that clearly belong to one or both species of Crossobothrium described herein were found in the spiral intestine of N. cepedianus. Because these species differ in a combination of characters that are not visible in detached gravid proglottids, i.e., worm length, and number of testes and their distribution, which degenerate in gravid proglottids, it was not possible to determine the species to which these detached gravid proglottids belonged. Nonetheless, the following information is given, as it may be useful for future studies of the genus. Detached gravid proglottids 2,900 9,500 long, 1,540 5,100 wide; length to width ratio :1. Unembryonated eggs in diameter. DISCUSSION With the exception of C. laciniatum, which parasitizes a lamniform shark, Carcharias taurus Rafinesque, 1810 (see Linton,

10 IVANOV NEW SPECIES OF CROSSOBOTHRIUM 1487 FIGURES Crossobothrium pequeae n. sp., cross sections of mature proglottid, scale bar mm. (39) At level of testes anterior to cirrus sac. (40) At level of genital pore. (41) At level of ovarian isthmus. Abbreviations: cs, cirrus sac; dlm, deep longitudinal musculature; gp, genital pore; mg, Mehlis gland; nc, nerve cord; ov, ovary; t, testis; u, uterus; vf, vitelline follicle; vg, vagina; vod, ventral osmoregulatory duct. 1889; Ruhnke, 1996), species of Crossobothrium are parasites of hexanchid sharks (Klaptocz, 1906; Rees, 1946; Riser, 1955; Euzet, 1959; Williams, 1968; Ruhnke, 1996). A single species of shark can be parasitized by multiple species of Crossobothrium, as is the case of N. cepedianus, host for C. antonioi and C. pequeae. This might also be the case of H. griseus in the Mediterranean Sea. This shark is the type host of C. campanulatum (see Klaptocz, 1906) and it has also been reported as host for a species of Crossobothrium identified as C. dohrni by Euzet (1959). Even if the identification of the species described by Euzet (1959) is doubtful, there are some morphological differences with C. campanulatum that might indicate the presence of a second species of Crossobothrium parasitizing H. griseus. The Hexanchidae includes 4 species of sharks (Froese and Pauly, 2009), 3 of which already have been reported as hosts for at least 1 species of Crossobothrium (see Örley, 1885; Klaptocz, 1906; present study). If patterns of host specificity of tetraphyllidean species are assumed, and each host can harbor at least 2 species of Crossobothrium, there may be 4 additional species in this genus hosted in hexanchid sharks. Tazerouti et al. (2007) suggested that Calyptrobothrium chalarosomum Alexander, 1963 could be a species of Crossobothrium based on the possession of a post-vaginal field of testes, which is absent in all other species of Calyptrobothrium. However, Alexander (1963) described a very weak body musculature for C. chalarosomum, different from Crossobothrium. Actually, all species in Crossobothrium share the presence of a well-developed musculature composed of muscle bundles immediately below the tegument, in addition to conspicuous bundles of longitudinal musculature deeper inside, dividing the parenchyma into medullar

11 1488 THE JOURNAL OF PARASITOLOGY, VOL. 95, NO. 6, DECEMBER 2009 and cortical regions, a condition that was mentioned by Ruhnke (1996) as a putative synapomorphy for the genus. In addition, the scolex of C. chalarosomum is similar to species in Calyptrobothrium in having the accessory sucker partially covered by bothridial flaps that are absent in all species of Crossobothrium. Moreover, C. chalarosomum was reported from a scyliorhinid shark, an unusual host for a species of Crossobothrium. Even if the identifications of the worms studied by Rees (1946) and Ruhnke (1996) as C. dohrni are doubtful, the specimens clearly belong to Crossobothrium. Thus, a variety of bothridial margins has been reported in species of this genus. Rees (1946) referred to the bothridial margins of what she identified as C. dohrni as having temporary false loculi, not constant in number or arrangement that are formed as a consequence of the disposition and contraction of bothridial musculature (Rees, 1946). Ruhnke (1996) described the bothridial margins of C. dohrni as incompletely divided and irregularly divided, but no further explanation was given regarding this feature, leaving doubts as to what incompletely, irregular, or both actually mean. Each species described in this study, C. antonioi and C. pequeae, has crenulated bothridial margins, with short marginal notches in a regular disposition, which are constantly present regardless of the degree of contraction of the bothridia. The relevance of the degree of development of gravid proglottids when studying the eggs of species of Crossobothrium is worth mentioning. Undoubtedly, the eggs continue to grow once the gravid proglottid detaches from the strobila. In the present study, unembryonated intrauterine eggs of C. antonioi and C. pequeae ranged from to in diameter, respectively, whereas those from the detached proglottids recovered from the same host individual were even larger, i.e., in diameter. Therefore, it is critical to describe in detail the state of development of the proglottids from which the eggs were obtained for study. ACKNOWLEDGMENTS Special thanks are due to Leo Tamini, Gustavo Chiaramonte, and Jorge Pérez from Museo Argentino de Ciencias Naturales-CONICET for their invaluable help during the field trips; Leo Tamini for his wonderful meals during these trips and for providing lodging facilities at Necochea; Lic. Marcela Mastrocola, Prosecretaria General, Unidad de Enseñanza Universitaria Quequén, Universidad Nacional del Centro for providing lodging facilities at Puerto Quequén; Fabián Tricárico for his patience during SEM sessions; Janine Caira from the University of Connecticut for facilitating our work through the Global Cestode Database; and Margarita Ostrowski for her helpful translation of the original description of C. campanulatum (Klaptocz, 1906). This research has been supported by grant BID 1728 OC-AR PICT 2006 No. 825 from the Agencia Nacional de Promoción Científica y Tecnológica (Argentina), grant PIP No from CONICET (Argentina), grants X453 and X443 ( ) UBACyT from Universidad de Buenos Aires (Argentina), and NSF PBI Nos and (U.S.A.). LITERATURE CITED ALEXANDER, C. G Tetraphyllidean and diphyllidean cestodes of New Zealand selachians. Transactions of the Royal Society of New Zealand, Zoology 3: CAIRA, J. N., AND C. HEALY Order Tetraphyllidea Carus, In The Global Cestode Database [Internet]. J. N. Caira, K. Jensen, and C. J. Healy (eds.) [accessed 16 January 2006]. Available at www. cestodedatabase.org. EUZET, L Recherches sur les cestodes tétraphyllides des sélaciens de côte de France. Thèses présenteés à la Faculté des Sciences de Montpellier, France, 236 p Order Tetraphyllidea Carus, In Keys to the cestode parasites of vertebrates, L. F. Khalil, A. Jones, and R. A. Bray (eds.). CAB International, Wallingford, U. K., p FROESE, R., AND D. PAULY Fishbase. World Wide Web electronic publication. Available at version (09/2009). JOYEUX, C., AND J. G. BAER Cestodes. Fauna de France. Vol. 30. Lechevalier et Fils, Paris, France, 613 p. KLAPTOCZ, B Neue Phyllobothriden aus Notidanus (Hexanchus) griseus Gm. Arbeiten aus dem Zoologischen Institute der Universität Wien und der Zoologischen Station in Trieste 16: LINTON, E Notes on entozoa of marine fishes of New England. Annual Report of United States Commissioner of Fish and Fisheries 14: ÖRLEY, L A czápáknak és rájáknak belférgei. Természetrajzi Füzetek az Allat-, Növény-, Asvány- és Földtan Köréböl, Evnegyedes Folyóirat. Kiadja a Magyar Nemzeti Múzeum 9: REES, G The anatomy of Phyllobothrium dohrnii (Oerley) from Hexanchus griseus (Gmelin). Parasitology 37: RISER, N. W Studies on cestode parasites of sharks and skates. Journal of the Tennessee Academy of Science 30: ROBINSON, E. S Records of cestodes from marine fishes of New Zealand. 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