A NEW GENUS AND SPECIES OF PROTEOCEPHALIDEAN (CESTODA) FROM CLARIAS CATFISHES (SILURIFORMES: CLARIIDAE) IN AFRICA

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1 J. Parasitol., 95(1), 2009, pp American Society of Parasitologists 2009 A NEW GENUS AND SPECIES OF PROTEOCEPHALIDEAN (CESTODA) FROM CLARIAS CATFISHES (SILURIFORMES: CLARIIDAE) IN AFRICA Alain de Chambrier, Tomáš Scholz*, Moges Beletew, and Jean Mariaux Département des Invertébrés, Muséum d Histoire Naturelle, P.O. Box 6434, CH-1211 Geneva 6, Switzerland. tscholz@paru.cas.cz ABSTRACT: A new proteocephalidean cestode is described from 2 catfishes, Clarias gariepinus (type host) and C. cf. anguillaris (Siluriformes: Clariidae), from Ethiopia (type locality), Sudan, Tanzania, and Zimbabwe, and a new genus, Barsonella, is proposed to accommodate it. The genus belongs to the Proteocephalinae because its genital organs (testes, ovary, vitellarium, and uterus) are situated in the medulla. Barsonella lafoni, the type and only species of the new genus, is characterized mainly by the possession of an additional opening of each sucker; circular musculature on the anterior margin of suckers, serving as a sphincter; a small thin-walled glandular apical organ; absence of well-developed osmoregulatory canals in mature, pregravid, and gravid proglottids; and a large strobila, up to 173 mm long and 3.2 mm wide. Species of Marsypocephalus Wedl, 1861 (Marsypocephalinae), other large-sized proteocephalidean tapeworms occurring sympatrically in African catfishes (Clarias and Heterobranchus) and also possessing a sphincter-like, circular musculature on the anterior part of suckers, differ from B. lafoni in the absence of an additional sucker opening and glandular apical organ, the cortical position of the testes, well-developed osmoregulatory canals throughout the strobila, and a large cirrus sac. Proteocephalus glanduligerus (Janicki, 1928), another cestode parasitic in Clarias spp. in Africa, is much smaller than B. lafoni (maximum length 15 mm), has suckers without additional opening and circular musculature on the suckers, a large-sized glandular organ, much larger than suckers, and well-developed osmoregulatory canals. Comparison of partial sequences of the 28S rrna gene for 7 samples of B. lafoni from 2 different hosts and 4 localities in Ethiopia, Sudan, and Tanzania has shown a very low genetic variability. In a limited phylogenetic analysis, B. lafoni formed a clade with Corallobothrium solidum Fritsch, 1886 (Proteocephalidae: Corallobothriinae), an African electric catfish parasite. This clade was the sister group of almost all Neotropical taxa from pimelodid and other catfishes. In total, 18 species of proteocephalidean tapeworms of the following genera have been described from freshwater catfishes, bichirs, and other freshwater fishes in Africa: Corallobothrium Fritsch, 1886 (1 species), Electrotaenia Fritsch, 1886 (1), Marsypocephalus Wedl, 1861 (5), Proteocephalus Weinland, 1858 (10), and Sandonella Khalil, 1960 (1) (Khalil, 1971; Schmidt, 1986; Khalil and Polling, 1997). Most taxa were described in the 19th century or in the first half of the 20th century; the most recent description of new species of African proteocephalidean tapeworms was published by Troncy (1978). During a parasitological survey of freshwater fish in the Sudan and Ethiopia, numerous large-sized proteocephalidean tapeworms (Cestoda) were collected from catfishes of the genus Clarias Scopoli. The study of these cestodes, including light microscopy, scanning electron microscopy (SEM), and histological observations, has shown that they differ from all other proteocephalidean cestodes hitherto described. This evaluation has also revealed that the tapeworms from Clarias possess unique morphological characteristics that warrant erection of a new genus, the description of which is provided herein. MATERIALS AND METHODS The present study was based on the examination of numerous tapeworms found in Clarias gariepinus (Burchell, 1822) and Clarias cf. anguillaris (Linnaeus, 1758) collected in several localities in Ethiopia and Sudan from March 2006 to February 2007 (see Beletew, 2007; de Chambrier et al., 2007, 2008). The catfishes were dissected as soon as possible after their capture and parasites were isolated from the host intestine, fixed immediately in hot 4% neutral formaldehyde solution (with pieces of several worms placed in 99% pure ethanol for DNA analysis) and subsequently stored in 70% ethanol. The specimens were then stained with Mayer s hydrochloric carmine solution, dehydrated in Received 7 February 2008; revised 26 May 2008; accepted 5 June * To whom correspondence should be addressed. Institute of Parasitology, Biology Centre of the Academy of Sciences of the Czech Republic, Branišovská 31, České Budějovice, Czech Republic. Department of Biology, Faculty of Science, Addis Ababa University, P.O. Box 1176, Addis Ababa, Ethiopia. a graded ethanol series, cleared with eugenol (clove oil), and mounted in Canada balsam. In addition, several tapeworms collected from C. gariepinus from Chivero Dam Lake (Chivero Reservoir) in Zimbabwe in October 2001 by Maxwell Barson were studied. Because these specimens were fixed with cold 4% formaldehyde solution and thus were unnaturally contracted, their measurements were not used in the morphological description. One tapeworm from the same host (C. gariepinus) from Mwanta Bay, Victoria Lake, Tanzania, collected by C. Mwita in 2001, now deposited in the Natural History Museum in Geneva, Switzerland (MHNG INVE 32821), was also examined. Pieces of tapeworms were embedded in paraffin wax, sectioned at m (cross sections of the strobila and longitudinal sections of scolices), stained with Weigert s hematoxylin and counterstained with 1% eosin B (de Chambrier, 2001). Eggs were studied in distilled water. Four scolices of tapeworms from Ethiopia and Sudan were used for SEM observations following the procedure outlined by de Chambrier et al. (2008). Intraspecific genetic variability of the new species was assessed on the basis of comparison of partial sequences (about 1 kb of the 5 portion) of the 28S rrna gene of the following 7 samples fixed in 99% ethanol: (1) 2 specimens from C. gariepinus from Tana Lake, Ethiopia (host field numbers ET 38 [type series] and ET 27); (2) 2 specimens from C. gariepinus from Langano Lake, Ethiopia (ET 91 and ET 174); (3) 2 specimens from C. cf. anguillaris, Nubia Lake, Sudan (water reservoir on the Nile River in southern Egypt and northern Sudan; Sud 178 and Sud 180); and (4) 1 specimen from C. gariepinus from Lake Victoria, Tanzania (AF 001). The methodology described previously by Zehnder and Mariaux (1999) and de Chambrier et al. (2008) was used. Sequences were edited and aligned with Sequencher v4.6 (Gene Codes Corporation, London, United Kingdom) with the use of default parameters (dirty data), and minor corrections were done by hand. The sequences are publicly available through EMBL, accession numbers FM FM To assess phylogenetic position of the new genus among other proteocephalideans, a preliminary comparative analysis of its partial 28S rrna gene sequences was performed. Briefly, the sequences of the new species were aligned with a subset of our Proteocephalidea 28S database (see de Chambrier et al., 2004, 2008) composed of 15 representative taxa, 1 tetraphyllidean outgroup (Acanthobothrium sp. Onchobothriidae) and 3 sequences of the new taxon. A parsimony analysis was performed in PAUP* (Swofford, 2002) with the following parameters: heuristic search with uninformative characters removed from the analysis and 100 replicates, no maxtree, acctran, gaps considered as missing. One thousand bootstrap replicates, with 3 repeats each, were performed to measure branch support. 160

2 DE CHAMBRIER ET AL. B. LAFONI N. GEN. AND N. SP. 161 FIGURES 1 6. Scanning electron micrographs of scolex of Barsonella lafoni n. gen., n. sp. (1,2) Dorsoventral and sublateral views, respectively; specimen from Clarias gariepinus, Tana Lake, Ethiopia (INVE 60351). (3,5) Dorsoventral and apical views, respectively; specimen from Clarias cf. anguillaris, Nubia Lake Reservoir, Sudan (INVE 60352). Black arrows show the additional sucker opening (Figs. 1 3). (4) Detail of additional sucker opening; specimen from C. cf. anguillaris, Nubia Lake Reservoir, Sudan (INVE 60350). (6) Microtriches on scolex apex (INVE 60351). Scale bars: 1 3, m; 4 20 m; 6 2 m. DESCRIPTION Barsonella n. gen. Diagnosis: Proteocephalidea, Proteocephalidae, Proteocephalinae. Testes, ovary, vitelline follicles, and uterus medullary. Large tapeworms, with massive strobila and well-developed inner longitudinal musculature. Main osmoregulatory canals thin-walled, disappearing in last immature proglottids. Scolex large, subspherical, without metascolex, with small, elongate, thin-walled glandular apical organ. Suckers large, deeply embedded, with small additional, slit-like or transversely oval orifice, situated at short distance posterior to main opening of sucker cavity; anterior part of suckers with circular musculature serving as sphincter. Testes in 1 2 layers. Cirrus sac pyriform, thin-walled, small in relation to proglottid width. Genital pores irregularly alternating, preequatorial. Genital atrium small. Ovary bilobed, lobulated. Vagina posterior or anterior to cirrus sac, with small, weakly developed vaginal sphincter near genital atrium. Vitelline follicles arranged in 2 lateral bands, slightly wider at ovarian level. Uterine development of type 1 according to de Chambrier et al. (2004). Taxonomic summary Etymology: The genus is named after Maxwell Barson, fish parasitologist from Zimbabwe, who first collected the tapeworms and kindly provided them to the present authors; generic name should be treated as feminine. Remarks The new genus belongs to the Proteocephalinae because genital organs (testes, ovary, vitelline follicles, and uterus) are situated in the medulla (Rego, 1994). The genus is characterized by (1) the presence of an additional slit-like or transversely oval orifice on suckers, a short distance posterior to the main opening of the sucker cavity (Figs. 1 5, 7 10); (2) circular (instead of radial) musculature on the anterior margin of suckers serving as a sphincter closing a deep sucker cavity (Figs. 7 10); (3) the absence of well-developed osmoregulatory canals in mature, pregravid, and gravid proglottids (only very narrow, sinuous canals are present on the ventral side of the medulla at the level of vitelline follicles); (4) the presence of a small, elongate, thin-walled glandular apical organ (Figs. 7, 8, 10); and (5) a massive strobila, up to 173 mm long and 3.2 mm wide. The only other proteocephalidean cestode possessing 2 openings on the suckers, Zygobothrium megacephalum Diesing, 1850, is a parasite of the Neotropical catfish Phractocephalus hemioliopterus (Bloch and Schneider, 1801). As a member of the Zygobothriinae, Z. megacephalum has vitelline follicles situated in the cortex, whereas the other organs (testes, ovary, and uterus) are situated in the medulla (Rego, 1994). It also differs from the type species of the new genus by the shape of the scolex, absence of an apical organ, and morphology of proglottids (Woodland, 1933; Freze, 1965). Marsypocephalus Wedl, 1861 (Marsypocephalinae), species of which are of similar size as the new species described here, also possess circular, sphincter-like musculature on the anterior margin of suckers and may occur sympatrically in African catfishes (Clarias and Heterobranchus Khalil and Polling, 1997). Species of Marsypocephalus can be easily differentiated from Barsonella by the lack of an additional sucker opening; the cortical position of the testes; the possession of well-developed osmoregulatory canals in mature, pregravid, and gravid proglottids; and a markedly larger cirrus sac (Freze, 1965). Proteocephalus glanduligerus (Janicki, 1928) Fuhrmann, 1933, which also parasitizes Clarias spp. in Africa and has genital organs positioned in the medulla, is much smaller than the new species described here (maximum length 15 mm; maximum width 0.59 mm Mashego, 2001), has suckers without an additional opening and circular

3 162 THE JOURNAL OF PARASITOLOGY, VOL. 95, NO. 1, FEBRUARY 2009 FIGURES Barsonella lafoni n. gen., n. sp. (7) Dorsoventral view of scolex (IPCAS C-485), (8) Sublateral view of scolex (INVE 60358), specimens from Clarias cf. anguillaris, Nile, Nubia Lake Reservoir. (9,10) Longitudinal section of scolices; specimens from Clarias gariepinus, Tana Lake, Ethiopia (INVE and 60354, respectively). Abbreviations: ad, additional opening; ao, apical organ; cm, circular musculature. Scale bars: m; m. sphincterlike musculature, possesses a large-sized glandular organ much larger than suckers, and has osmoregulatory canals that are well developed throughout the strobila (Freze, 1965; Mashego, 2001; Scholz et al., 2009). Barsonella lafoni n. sp. (Figs. 1 18) Diagnosis (based on 11 specimens from Clarias gariepinus from Ethiopia and Clarias cf. anguillaris from Sudan; measurements are given in Table I): Proteocephalidae, Proteocephalinae. Testes, ovary, vitelline follicles, and uterus medullary (Figs. 16, 17). Strobila acraspedote, covered with filiform microtriches, consisting of proglottids: immature, 7 11 mature, and pregravid and gravid. Internal longitudinal musculature well developed, formed by large bundles of muscle fibers (Figs. 14, 16, 17). Osmoregulatory canals well developed in scolex and anterior part of strobila, thin-walled, disappearing in last immature proglottids. In mature, pregravid, and gravid proglottids, dorsal canals absent; ventral canals sinuous, very narrow, thick-walled, ventral to vitelline follicles, difficult to observe even in cross sections. Scolex large, subspherical, without metascolex, wider than proliferative zone (neck), with elongate, thin-walled glandular apical organ (Figs. 7, 8, 10). Suckers large, uniloculate, deeply embedded, with small additional, slit-like or transversely oval orifice, situated short distance

4 DE CHAMBRIER ET AL. B. LAFONI N. GEN. AND N. SP. 163 FIGURES Barsonella lafoni n. gen., n. sp. from Clarias gariepinus, Tana Lake, Ethiopia. (11) Holotype, mature proglottid, ventral view (INVE 60346). (12) Holotype, gravid proglottid, dorsal view (INVE 60346). (13) Paratype, sketch of gravid proglottid with 10 uterine pores (up), ventral view (INVE 49395). Scale bars: m; 12,13 1 mm. posterior to main opening of sucker cavity (Figs. 1 5, 7 10). Anterior part of suckers with circular (instead of radial) musculature serving as sphincter closing deep sucker cavity (Figs. 7 10). Scolex, including its apex and regions between suckers, and proliferative zone covered uniformly with filiform microtriches (Fig. 6). Testes medullary, spherical to oval, densely packed in 1 complete layer, with a few testes in second incomplete layer (Figs. 11, 17); testes forming 1 field reaching laterally up to vitelline follicles (Fig. 11), present also in gravid proglottids (Fig. 12). Vas deferens (external sperm duct) strongly coiled, occupying large field between cirrus sac and median line of proglottid. Cirrus sac small, pyriform, thin walled (Figs , 15). Internal sperm duct wide, coiled; cirrus occupying about

5 164 THE JOURNAL OF PARASITOLOGY, VOL. 95, NO. 1, FEBRUARY 2009 FIGURES Barsonella lafoni n. gen., n. sp. (14) Cross section through uterine pore; specimen from Clarias gariepinus, Langano Lake, Ethiopia (INVE 49398). (15) Terminal genitalia, dorsal view; specimen from C. gariepinus, Langano Lake, Ethiopia (INVE 60359). (16,17) Cross sections at level of ovary and uterine pore, respectively; specimens from Clarias cf. anguillaris, Nile, Reservoir to Nubia Lake (INVE and 60352). (18) Eggs in distilled water, with collapsed outer hyaline envelope (INVE 60361). Abbreviations: cc, chromophilic cells; cs, cirrus sac; em bilayered embryophore; lm, longitudinal internal musculature; ln, longitudinal nerve cord; mic, microtriches; oe, outer envelope; on, oncospheres; ov, ovary; te, testes; up, uterine pore; ut, uterus; va, vagina; vi, vitelline follicles; vs, vaginal sphincter. Scale bars: 14, m; 16,17 1 mm; m. ⅓ ½ of length of cirrus sac (Fig. 15). Genital pores irregularly alternating, pre-equatorial (Figs ). Genital atrium small. Ovary medullary (Fig. 16), bilobed, with lobulated lateral wings (Figs. 11, 12). Vagina anterior or posterior to cirrus sac (anterior in 50% proglottids of specimens from Ethiopia, n 517 [in 61% of holotype; n 93], 36% proglottids from Sudan, n 329; 26% from Tanzania, n 48; 54% from Zimbabwe, n 33); distal part of vagina with a small vaginal sphincter near genital atrium, formed by diffuse muscle fibers (Fig. 15). Vitelline follicles medullary, small, arranged in 2 narrow lateral bands, slightly wider at ovarian level, with follicles missing near anterior margin of proglottid (Figs ); poral band interrupted ventrally and sometimes dorsally at level of cirrus sac (Figs. 11, 12). Uterus medullary, type 1 development (de Chambrier et al., 2004): uterine stem with numerous intensely staining cells concentrated along its wall in immature proglottids. Uterine lumen appears in last immature proglottids similarly as dorsally situated lateral outgrowths (diverticula), where then first eggs appear in pregravid proglottids; lateral branches (diverticula) asymmetrical, occupying up to 60% of width of gravid proglottids (Fig.

6 DE CHAMBRIER ET AL. B. LAFONI N. GEN. AND N. SP. 165 TABLE I. Comparative measurements (in micrometers unless otherwise stated) of Barsonella lafoni n. gen., n. sp. Host N* Clarias gariepinus Clarias gariepinus Clarias cf. anguillaris Country Ethiopia Ethiopia Sudan Host field numbers ET 38 (holotype) (Tana Lake) ET 27, 38 (Tana Lake) 144, 186, 187 (Langano Lake) Sud 112 (Kostí), Sud 180 (Nubia Lake) Total length (mm) Maximum width 3 1,785 3,235 2,580 Scolex width 9 1,505 1,090 1,585 1,100 1,425 Diameter of suckers Apical organ Length Width Width of neck 9 1, , ,270 Immature proglottids Length Width 14 1,645 1,865 1,725 2,165 1,390 2,205 Mature proglottids Length ,380 Width 13 1,785 1,885 2,560 2,960 1,430 2,340 Pregravid proglottids Length , ,820 Width ,235 1,250 3,235 1,130 2,580 Gravid proglottids Length ,190 1,630 2,185 Width 17 1,350 3,235 1,190 1,630 Testes Diameter Number (n 4) (n 2) (n 4) Cirrus sac Length (l) Width (w) l:w ratio Relative size % 14 18% 14 21% Position of genital pore % 18 35% 24 35% Ovary Length Width Relative size % 60 64% 56 71% Mehlis s glands Relative size % 8 10% 8 12% Vaginal sphincter Vitelline follicles# Poral % 90 95% 89 96% Aporal % 89 95% 87 98% Uterine diverticula Eggs** Outer envelope 9 Ripe eggs not observed Not measured Embryophore Oncosphere Embryonic hooks * Number of measurements. Length of cirrus sac as percent of width of mature proglottid. Position of genital pore (cirrus pore) as percent of length of mature proglottid from anterior end. Width of ovary as percent of width of mature proglottid. Diameter of Mehlis gland as percent of width of mature proglottid. # Length of lateral band of vitelline follicles as percent of length of mature proglottid. Number of lateral branches (diverticula) on each side. ** Diameter. Length.

7 166 THE JOURNAL OF PARASITOLOGY, VOL. 95, NO. 1, FEBRUARY ). In last gravid proglottids, internal side of uterine wall with numerous chromophilic cells (Fig. 14). Several uterine pores present along median line on ventral side (Fig. 14), 7 13 in number (8 10 in holotype; Fig. 13). Eggs spherical, with hyaline outer envelope (collapsed in distilled water Fig. 18) and thick, round embryophore, consisting of 2 layers, outer layer and nuclei-containing inner layer. Oncospheres spherical to oval, with 3 pairs of embryonic hooks (Fig. 18). Taxonomic summary Type host: Clarias gariepinus (Burchell, 1822) (Siluriformes: Clariidae). Other host: Clarias cf. anguillaris (Linnaeus, 1758). Site of infection: Anterior part of the intestine. Type locality: Tana Lake at Bahir Dar, Ethiopia (11 36 N, E); collection date of the type series 4 April 2006 (field sample ET 38); other tapeworms sampled on 3 6 April 2006, May 2006 and 3 November Other localities: Ethiopia: Langano Lake (11 12 April 2006 ET 082 to ET 104, and April 2006 ET 136 to ET 195), Zeway Lake (26 August 2006 ET 242 to ET 252); Sudan: Khartoum (22 March 2006), Kostí (26 March 2006 Sud 069 to Sud 164), Nubia Dam Lake (28 March 2006 Sud 166 to Sud 193); Tanzania (collected by C. Mwita): Mwanta Bay, Victoria Lake (2001) (AF 001); Zimbabwe (collected by M. Barson): Chivero Dam Lake (October 2001 H 04/4). Deposition of specimens: Holotype (1 slide with mounted specimen and 6 slides of its cross sections from the host with the field number ET 38) Natural History Museum in Geneva (MHNG INVE 60346, 1 slide with cross sections in the Department of Zoology, University of Addis Ababa, Ethiopia); 1 paratype (1 slide with mounted specimen, 10 slides with cross sections and 3 slides with longitudinal sections of scolex; ET 27) in The Natural History Museum, London, United Kingdom (Coll. No. BMNH ) and 2 slides in Geneva (MHNG INVE 60349); 2 paratypes (2 slides with 2 mounted specimens, 10 slides with cross sections and 1 scolex on stub for SEM MHNG INVE 60351; ET 27 and ET 38), Natural History Museum in Geneva, Switzerland (MHNG INVE and 60348, 2 slides with cross sections in the Institute of Parasitology, BC AS CR, České Budějovice, Czech Republic, Coll. No. IPCAS C-485). Voucher specimens were deposited in the Natural History Museum in Geneva (MHNG INVE 32821, , , 50024, , 60538, 60347, , 60986; SEM samples MHNG INVE and 60371), Institute of Parasitology, BC AS CR, České Budějovice, Czech Republic (IPCAS C-485 ET 187, ET 187, H 04/4, and Sud 178), The Natural History Museum, London, United Kingdom (Collection Nos. BMNH Sud 166, BMNH ET 192), and the U.S. National Parasite Collection, Beltsville, Maryland (Collection No. USNPC Sud 180). Vouchers of sequenced specimens were deposited in the Natural History Museum Geneva (host field numbers ET 38 MHNG INVE 49395, ET 27 MHNG INVE 49397, ET 91 MHNG INVE 49398, ET 174 MHNG INVE 49399, Sud 178 MHNG INVE 49394, Sud 180 MHNG INVE 49393, AF 001 MHNG INVE 32821). Etymology: The specific name lafoni is dedicated to Dominique Lafon, world-famous winemaker from Meursault, Burgundy, France, who produces 1 of the most inspiring white wines in the world. Prevalence: Because it was not possible to count all the specimens found precisely, the values of intensity of infection are not provided. Most catfishes were infected with a few specimens (usually 1 3 tapeworms), but a few hosts harboured several 10s of specimens, mostly juvenile and immature. Overall prevalence in individual localities was as follows: Ethiopia: 55% in Tana Lake (number of fish examined 65), 28% in Langano Lake (n 83), and 12% in Zeway Lake (n 25), catfish from Chamo Lake (Shelle River; n 8) and Awasa Lake (n 25) were negative; Sudan: 7% in Khartoum (n 14), 6% in Kostí (n 16), and 20% in Nubia Dam Lake (n 15); Tanzania: 0.07% in Mwanta Bay, Victoria Lake (n 1,400). Data on the prevalence in catfish from Chivero Dam Lake in Zimbabwe were not available. Remarks Barsonella lafoni is the type and only species of Barsonella and differs from other proteocephalidean cestodes by the characteristics listed in the generic diagnosis of the genus (see above). In juvenile or immature specimens, the apical organ is widely elongated and flask shaped, and its diameter represents up to 12% of the scolex width, whereas that of mature tapeworms is more elongated and its width is almost identical to that in juveniles, and thus represents only about 3.5 5% of the scolex width (data not shown). Genetic variability and phylogenetic position Sequences of the 28S rrna gene (1,028 bp long) of all 7 specimens of B. lafoni were identical, except for the 2 following differences: (1) both samples from Nubia Lake differed from the 5 remaining samples in an insertion of 10 nucleotides (all other proteocephalideans lack this sequence, and its absence is thus plesiomorphic in the order), and (2) both samples from Tana Lake differed from the 5 other samples by a single point mutation (transversion). To determine the position of B. lafoni within the order Proteocephalidea, we aligned its 28S rrna sequence (all genotypes) with a representative selection of 15 published proteocephalidean taxa in a 19- taxa matrix, 1,072 bp long. The Acanthobothrium sp. (Tetraphyllidea: Onchobothriidae) sequence was used to root the tree. The final matrix was composed of 201 informative characters and a parsimony analysis resulted in 6 shortest trees 565 steps long, with a consistency index (CI) of Their strict consensus tree (not shown) is fully compatible with the general Proteocephalidea topology recovered by de Chambrier et al. (2004). In the strict consensus tree, B. lafoni formed an African clade with Corallobothrium solidum Fritsch, 1886 (Proteocephalidae: Corallobothriinae), a parasite of the electric catfish, Malapterurus electricus (Gmelin), from the Nile River in Egypt and Sudan (Freze, 1965; Schmidt, 1986). This clade was the sister group of almost all Neotropical taxa from pimelodid and other catfishes (see de Chambrier et al., 2004). DISCUSSION Catfishes of the genus Clarias Scopoli, 1777 are common in Africa, with over 30 species recognized as valid (Froese and Pauly, 2008). Some of them, especially C. gariepinus (syn. C. lazera Valenciennes in Cuvier and Valenciennes, 1840), are also economically important for local fisheries and aquaculture. Numerous studies on helminth parasites of these catfishes have been carried out, especially in Egypt, Ethiopia, and South Africa (e.g., Khalil, 1963, 1971; Amin, 1978; Wabuke-Bunoti, 1980; Faisal et al., 1989; Mashego and Saayman, 1989; Imam and El Askalany, 1990; Wondim, 1990; Khalil and Polling, 1997; Mashego, 2001; Dayhoum and Al-Bassel, 2003; Yimer and Enyew Mulualem, 2003; Barson, 2004; Oniye et al., 2004; Barson and Avenant-Oldewage, 2006; Beletew, 2007). It is, therefore, surprising that B. lafoni, a very large and relatively frequent parasite, has not been recorded previously. A possible explanation could be that B. lafoni was misidentified as Marsypocephalus rectangulus Wedl, 1861, a largesized proteocephalidean cestode, which also parasitizes Clarias spp. catfishes in Africa (Freze, 1965; Khalil and Polling, 1997). This is plausible because, based on gross morphology, B. lafoni is actually difficult to distinguish from M. rectangulus and other species of Marsypocephalus that occur sympatrically in some localities (Tana Lake in Ethiopia and the Nile River in the Sudan; data not shown). However, none of the many Clarias catfishes examined during this study (206 from Ethiopia and 45 from Sudan) harbored tapeworms of both genera simultaneously, whereas other species of considerably smaller size, i.e., Tetracampos ciliotheca Wedl, 1861 (Bothriocephalidea, previously part of the Pseudophyllidea; see Kuchta, Scholz, Brabec, and Bray, 2008; Kuchta, Scholz, and Bray, 2008) or P. glanduligerus, were found together with B. lafoni. In Lake Tana, 25 of 65 Clarias specimens examined harbored

8 DE CHAMBRIER ET AL. B. LAFONI N. GEN. AND N. SP. 167 only B. lafoni, whereas another 6 fish harbored only T. ciliotheca (twice with P. glanduligerus), and 13 Clarias specimens were simultaneously infected with both B. lafoni and T. ciliotheca. Two catfish hosted Marsypocephalus tapeworms only. In Langano Lake (n 83), monospecific infections were more frequent (19 Clarias spp. specimens with B. lafoni and 11 with T. ciliotheca), whereas concurrent infections were recorded only in 4 hosts. Barsonella lafoni occurs in a large area of Africa and has been found in Clarias spp. catfishes from northern Sudan (Nubia Lake on the Nile River near the border with Egypt) to southern Africa (Zimbabwe). In the localities studied by the present authors, B. lafoni was more prevalent and abundant than species of Marsypocephalus or P. glanduligerus. Specimens of B. lafoni from C. gariepinus from Ethiopia and those from C. cf. anguillaris from the Sudan were morphologically almost identical in all but 1 characteristic (see Figs. 1 18; Table I) and are considered to be conspecific. The only marked difference was in the number of testes, which were more numerous in Sudanese specimens ( ) than in those from Ethiopia ( ). This difference, as well as slight differences in the shape of proglottids, and length:width ratio of the cirrus sac (Table I), may be accounted for by intraspecific variability of populations from different definitive hosts. However, variation in the number of testes may also reflect small sample size, because testes in only 10 segments were counted. Because testes are very numerous and form more than 1 layer, their precise number could be only counted from line drawings of each segment measured. The genetic variability of B. lafoni samples from 2 different fish host species and 4 distant localities is extremely low ( 0.1%, not considering the insertion); only a single point mutation was recorded within our samples, suggesting that populations of B. lafoni from different geographical localities have not significantly diverged genetically. This is another argument in favor of conspecificity, because interspecific distances known for other proteocephalid genera are much larger even in relatively conserved genes like 28S rdna, e.g., from 0.4% in Peltidocotyle to 2 3.4% in Kapsulotaenia, Nomimoscolex, or the Proteocephalus aggregate. It remains to be seen whether the presence of the 10-bp insertion/deletion reflects a geographic separation or is possibly the signature of a strain linked to a specific host, as all parasites of C. gariepinus share this insertion, whereas parasites of C. cf. anguillaris have the plesiomorphic condition without insert. Sequences from additional samples would allow us to confirm whether such parasite populations, easily distinguishable by the presence or absence of this insertion/deletion, are specific to a particular host species. We have recently shown that another cestode parasite of freshwater fish in Africa, Sandonella sandoni (Lynsdale, 1960) (Proteocephalidea: Sandonellinae), which parasitizes the osteoglossiform fish Heterotis niloticus Cuvier (Arapaimidae), is even more genetically homogeneous, with identical sequences of the partial 28S rrna gene in 4 specimens from 2 very distant parts of Africa (Senegal and Sudan) (de Chambrier et al., 2008). Thus, the situation in Africa, with a limited number of parasite species showing wide geographic distributions, appears to be quite different from the one prevailing in South America, where parasite taxa are much more numerous, but generally seem to be restricted to limited distributions (de Chambrier and Vaucher, 1999; Rego et al., 1999). As for many other proteocephalidean cestodes, the life cycle of B. lafoni is not known. Based on the shape of eggs and analogy with the Holarctic fish proteocephalideans (see Freze, 1965 and Scholz, 1999 for review), it can be assumed that cyclopid copepods serve as the first intermediate hosts. The presence of a glandular organ, although it is fairly small in B. lafoni, indicates that second intermediate or paratenic hosts, probably small prey fish, may be involved in the life cycle of this tapeworm (Freze, 1965; Fischer and Freeman, 1973; Scholz, 1999). ACKNOWLEDGMENTS The stay of A. de C. and T.S. in Ethiopia would not have been possible without the support and advice of Dr. Abebe Getahun Gubale and Dr. Seyoum Mengistou, Department of Biology, Faculty of Science, Addis Ababa University, and Dr. Eshete Dejen Dresilign, Director of Fisheries & Livestock Branch, Amhara Regional Agricultural Research Institute, Bahir Dar. A. de C. and T.S. thank Dr. Dia-Eldin Elnaiem (Davis, California) for help in organizing their stay in Sudan; André Piuz for providing SEM photomicrographs; Janik Pralong, Florence Marteau, and Gilles Roth (all Geneva) for their help with drawings and laboratory assistance; and Alicia Gil de Pertierra (Buenos Aires) and Kirsten Jensen (Kansas) for valuable suggestions on the text. The research stay in Sudan would not have been possible without the invaluable help of Dr. Zuheir N. Mahmoud, Ali Adam and Sayed Yousif Osman Elsheikh (University of Khartoum), Khalid Bashir Abaker and Ammar Osmar (White Nile Fisheries Research Station in Kostí). The support of the Embassy of Switzerland in Khartoum (Andrea Reichlin) is also acknowledged. A. de C. is also deeply indebted to the Donation Georges et Antoine Claraz for financial support, as is T.S. to the Grant Agency of the Czech Republic (Projects 524/04/0342 and 524/08/0885) and the Institute of Parasitology (Projects Z and LC 522). 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