Revue suisse de Zoologie 108 (2): 303-316;juin 2001 A new tapeworm from the Amazon, Amazotaeniayvettae gen. n., sp. n., (Eucestoda: Proteocephalidea) from the siluriform fishes Brachyplatystomafilamentosum and B. vaillanti (Pimelodidae) CHAMBRIER Alain de Muséum d'histoire naturelle, P.O. Box 6434, CH-1211 Geneva6, Switzerland. E-mail: [email protected] A new tapeworm from the Amazon,Amazotaeniayvettae gen. n., sp. n., (Eucestoda: Proteocephalidea) from the siluriform fishes Brachy- platystomafilamentosum and B. vaillanti (Pimelodidae). - Amazotaenia yvettae gen. n. and sp. n. (Proteocephalidea, Monticelliidae, Peltidocoty- linae) is described from the intestine of the black-backed filhote, Brachy- platystoma filamentosum and from the piramutaba, B. vaillanti (Siluri- formes: Pimelodidae) from Itacoatiara, Amazonia State, Brazil. The new genus differs from all other members of Peltidocotylinae in the morpho- logy of the scolex, position of the vitelline follicles grouped in two elon- gated patches in the equatorial part of the proglottis, a smaller size of the strobila (less than 3 mm), smaller number of proglottides and smaller numberoftestes. The equatorial position of vitelline follicles, their shape in two elongated patches, the uterine development in Amazotaenia yvettae as well as the unusual behaviour of attachment in which individuals are grouped in compact clumps are peculiar within the Proteocephalidea. A key to the genera ofthe subfamily Peltidocotylinae is proposed. The sampling and processing methodology is also described in detail. Key-words: Eucestoda - Proteocephalidea - Amazotaenia yvettae gen. n., sp. n. - Brachyplatystoma spp. - Brazil - Techniques. INTRODUCTION Proteocephalidean tapeworms are among the most common platyhelminth parasites of freshwater fishes in neotropical area. Most proteocephalid species para- sitize siluriform fishes, particularly Pimelodidae (de Chambrier & Vaucher, 1999). As part of a study on the Proteocephalidea in South America, some minute tapeworms were collected from the pimelodid catfish Brachyplatystoma filamentosum. These new minute specimens display a series of peculiar and distinctive characters that Manuscriptaccepted 24.11.2000 304 A- DECHAMBRIER distinguish them from all known species and thatjustify the erection ofa new genus. The new species is described below. In this paper, I also give a detailed account of the preparation methods deve- loped by Vaucher and myselfsince 1971 (Vaucher, 1971; de Chambrier, 1987, 1990; & de Chambrier Vaucher, 1994, 1999) and progressively adopted by other authors working on the protecephalidean cestodes (Takemoto & Pavanelli, 1996; Scholz et al, 1997, Scholz & Hanzelova, 1998; Rego etal, 1999). MATERIALS AND METHODS Sample collection A total of29 Brachyplatystomafilamentosum and 4 B. vaillanti were collected from the Amazon River near Itacoatiara, State of Amazonas, Brazil. The hosts were dissected and examined for parasites immediately after death. The digestive tract was dissected along its entire length and searched for cestodes under a field stereo- microscope. A few cestodes were removed immediately from the intestine, gently washed into petri dishes with distilled water, then placed into 80% alcohol or in liquid nitrogen for subsequent izoenzyme and DNA analysis. Small pieces of each of the latter specimens were isolated as vouchers for identification. Both remaining whole cestodes and voucher specimens were placed separately into vials containing a small quantity of distilled water. After 1 minute, a hot (almost boiling) 4% v/v neutral formaldehyde solution was poured directly onto the worms. In order to collect also the remaining specimens (left in situ), the dissected digestive tract was placed into a vial (minimum 10 times the volume of tissues) with a small amount of water. After one minute, a hot (almost boiling) 4% v/v neutral formaldehyde solution was poured directly onto the digestive tract. The worms were subsequently stored (after a mini- mumof 1 week) in 75% (v/v) ethanol. Preparation ofspecimens Worms were either stained in Mayer's hydrochloric carmine solution or in Weigert's Haematoxyline solution. Mayer's hydrochloric carmine as follows: 5 g Carmine Certistain (Merck) in 10 ml of a 18% HCl solution (left in contact for 1 hour) to which 200 ml of a 95% ethanol solution and a small piece of iron were added; the solution was then left to simmer for two hours and filtered when the solution was cold (modified from Langeron, 1949). Staining with Carmine: worms were placed in Carmine for 15 minutes, rinsed with 75% ethanol, differentiated with acid 75% ethanol (HCl 0.5% in ethanol) until no carmine diffused from the worms (between 20 min and two hours), dehydrated in ethanol series (80, 95, twice 100% respectively - at least 30 minutes in each). Coiled specimens were placed carefully on a piece of Bristol board (about 25mm x 45mm) and soaked with 95% ethanol. On this surface, the worm was carefully spread, avoiding twisting, and then covered with a glass slide the same size. Bristol and glass slides will stick with one another. Specimens were placed in petri dishes with 95%, A NEWTAPEWORM FROM THEAMAZON 305 then 100% ethanol for 30 min each. In the latter solution, the glass slide was removed carefully and the specimen was left for further 10 min. Then worms were cleared in increasing concentrations of Eugenol (clove oil) diluted in absolute ethanol (50, 75, 90 and twice 100%, at least 15 min each) and mounted as permanent preparations in Canada balsam (Fluka) (Vaucher, 1971; de Chambrier, 1987). Staining with haematoxyline was done as follows: 5 ml 1% Haematoxyline (Fluka) in ethanol 95%, to which 5 ml of 1.2% FeC13 filtered solution in distilled water and 5 drops of 18% HCl were added (modified from Langeron, 1949). The worms were placed in distilled water for 5 minutes then in the staining solution for 15 minutes, destained with acid 75% ethanol, placed in tap water until a blue colour appeared (about 10 min) and then dehydrated in an ethanol series as described above forthe Carmine staining. For histological sections, pieces of strobila were embedded in paraffin wax, sectioned transversely or frontally at 13-19 urn, and after dissolution of paraffin in toluol and hydration with an ethanol series (respectively 100, 95, 75% and distilled water, 2 min each), stained in Weigert haematoxylin (10 min.), destained briefly with acid 75% ethanol, then placed in tap water until a blue colour appeared, counter- stained with 1% eosin B (Sigma) (2 min), immersed in increasing concentrations of ethanol for 2 min. each and finally in toluol before mounting in Canada balsam. Scoleces for scanning electron microscopy (SEM) were processed by following procedures: worms are deshydrated in an ethanol series (80, 95, twice 100% respec- tively) then transferred in amyl acetate, dried by critical point method, sputtered with gold andexamined in Zeiss 940A SEM (see also Scholz etal., 1998). Type material was deposited in the Helminthological Collection of the Insti- tuto Oswaldo Cruz (CHIOC), at the Natural History Museum (MHNG), Geneva, Switzerland and atthe Natural History Museum, London (BMNH). Measurements are in micrometers (mm) unless otherwise stated. The following abbreviations are used in the description: x = mean; n = numberofmeasurements; CV = coefficientofvariation (%). RESULTS Amazotaenia gen. n. Diagnosis: Proteocephalidea, Monticelliidae, Peltidocotylinae. Very small tapeworms (less than 3 mm), with acraspedote proglottides. Unarmed scolex with four uniloculate suckers. Testes cortical, in one continuous dorsal field. Ovary medullary, with projections into dorsal cortex. Uterus cortical, ventral, with lateral and dorsal outgrowths extending into dorsal cortex. Vitelline follicles ventral, in two elongated groups in equatorial part of proglottis, occupying less than half of proglottis length. Vagina always posterior to cirrus pouch, possessing vaginal sphincter. Genital pore near anterior proglottis margin. Parasites of neotropical siluriform fishes (Pimelo- didae). Type and only species: Amazotaeniayvettae sp. n. 306 A- DECHAMBRIER Remarks Amazotaenia gen. n. belongs to the subfamily Peltidocotylinae on the basis of the presence of a medullary ovary, cortical testes and vitellaria and cortical uterus with outgrowths penetrating the medulla and the dorsal cortex. Recent taxonomic works on this subfamily resulted into the erection of the genera Jauella Rego & Pavanelli, 1985 andMariauxiella de Chambrier & Rego, 1995 (see Rego & Pavanelli, 1985; de Chambrier & Rego, 1995), the redefinition of Peltidocotyle Diesing, 1850 and the supression of Othinoscolex Woodland, 1933 and Woodlandiella Freze, 1965 as its synonyms (de Chambrier & Vaucher, 1999; Zehnder & de Chambrier, 2000). Therefore, only three genera belong to this subfamily at present, i.e., Peltidocotyle Diesing, 1850, Jauella Rego & Pavanelli, 1985 and Mariauxiella de Chambrier & Rego, 1995. Amazotaenia differs from the three members of the Peltidocotylinae by the morphology of the scolex which is relatively small and without metascolex. In contrast, Peltidocotyle and Jauella are characterised by the presence of metascolex, and the species ofthe genus Mariauxiella have massive scoleces. The position ofthe ventral vitelline follicles of Amazotaenia is in two groups in the equatorial part of proglottis, compared to the lateral vitelline follicles in the other three genera. In addition, the new genus can be distinguished by the smaller size of the strobila (less than 3 mm), the smaller numberofproglottides (up to 7) and smallernumber oftestes (maximum 32) (see the diagnoses ofthe otherthree genera in the following key). On the basis of the present results and previous studies (Rego & Pavanelli, 1985; de Chambrier & Rego, 1995; de Chambrier & Vaucher, 1999; Zehnder & de Chambrier, 2000), the following key to the generaofthe subfamily Peltidocotylinae is proposed: la. Metascolex present 2 lb. Metascolex absent 3 2a. Suckers biloculate Peltidocotyle Diesing, 1850 Diagnosis: Proteocephalidea. Monticelliidae. Peltidocotylinae. Worm of me- dium size. Scolex with four biloculate suckers forming two separate cavities. Metascolex present. Ovary medullar. Uterus cortical, ventral, with diverticula occasionally protruding across longitudinal musculature into medulla. Testes in dorsal cortex. Vitelline follicles cortical, in two bands, dorsal and ventral. In South American siluroid fishes. Type species: Peltidocotyle rugosa Diesing, 1850 in Pseudoplatystoma corru- scans, P.fasciatimi. Other species: Peltidocotyle lenha (Woodland, 1933) in Paulicea luetkeni, Sorubimichthysplaniceps. 2b. Suckers uniloculate Jauella Rego & Pavanelli, 1985 Diagnosis: Monticelliidae, Peltidocotylinae. Worm of medium size, wide. Scolex small, retractile. Suckers uniloculate, small. Metascolex cone-shaped, with transverse circular folds. Ovary medullar. Uterus medullar, with nume- A NEWTAPEWORM FROM THEAMAZON 307 rous outgrowths from the diverticles to the ventral and dorsal cortex. Testes in cortex, in one dorsal field and in one or two layers. Vitelline follicles cortical, lateral, in two dorsal and ventral bands. Parasite ofNeotropical siluroid fishes. Type and only species: Jauella glandicephalus Rego & Pavanelli, 1985 in Paulicea luetkeni. 3a. Vitelline follicles lateral. Suckers uniloculate, with powerful circular musculature Mariauxiella de Chambrier & Rego, 985 1 & Diagnosis (modified from de Chambrier Rego, 1995): Monticelliidae, Pelti- docotylinae. Worm of medium size. Strobila acraspedote. Scolex without defined metascolex. Four uniloculate suckers with powerful circular muscu- lature in its distal parts. Vitelline field cortical, crescentic in cross sections, sometime extending posteriorly behind the ovary. Testes cortical in continous dorsal field and in one or two layers. Ovary medullar, with two lateral lobes, each lobulate with projections into the dorsal cortex. Uterus in ventral cortex, with numerous outgrowths from the diverticles to the dorsal cortex. Parasite of Neotropical siluroid fishes. Type species: Mariauxiella pimelodi de Chambrier & Rego, 1995 in Pime- lodus ornatus. & Other species: Mariauxiella piscatorum de Chambrier Vaucher, 1999 in Hemisorubimplatyrhynchos. 3b. Vitelline follicles equatorial. Suckers uniloculate, simple Amazotaenia gen. n. . . . Diagnosis: see above. Amazotaeniayvettae sp. n. Figs 1-18 Type host: Brachyplatystoma filamentosum (Lichtenstein, 1819), common name: filhote dacapaprêta [black backed filhote]. Other host: Brachyplatystoma vaillanti Cuvier & Valenciennes, 1840, com- mon name: piramutaba. Material studied: Brazil, State of Amazonas, Itacoatiara, Rio Amazonas, holotype CHIOC 34363, 6 paratypes MHNG, 29732-29737 INVE and 2 paratypes BMNH 2000.9.4.1., all collected on 13.10.1995 by the author. Othermaterial: MHNG, 29738-29741, 29743 INVE. 13.10.1995; 29742INVE(B. vaillanti), 02.10.1995, collectedbytheauthor. Prevalence: 1/29 = 3.4% forB.filamentosum and 1/4 = 25% forß. vaillanti. Intensity: 66 - more than 250 specimens. Site of infection: first twelfth of intestine, immediately posterior to the stomach. Always concentrated in small clumps of 14 to 66 individuals. Etymology: The generic name refers to the geographic region; the specific name in honourofYvette, the wife ofthe author. Description: Monticelliidae, Peltidocotylinae. Very small cestodes, 516-2900 long and 120-475 wide, flattened dorsoventrally, forming isolated compact groups of 14-66 (x = 32, n = 10) individuals, all situated in the first twelfth of intestine, immediately posterior to stomach (Fig. 18). Strobila acraspedote, apolytic, consisting of4-7 (n = 29) proglottides: 2-3 immature (up to appearance of spermatozoa in vas 308 A- DECHAMBRIER deferens), 1 mature (up to appearance of eggs in uterus), 1 pregravid (up to appea- rance ofhooks in oncospheres) and 1-2 gravid segments. Immature proglottides much wider than long (110-375 x 60-210; length/width ratio 1 : 0.40-1.00); mature proglottides slightly wider than long (200-430 x 140-400; ratio 1 : 0.48-1.60); pre- gravid proglottides wider than long to elongate (220-440 x 310-700; ratio 1 : 0.77- 2.19); gravid proglottides (attached and detached) longer than wide (260-490 x 450- 770; ratio 1 : 1.12-2.31) (Figs 1, 13). Tegument thick, covered with small, round microtriches on sucker margins (Fig. 14) and covered with dense filiform micro- triches on anterior margin ofmature proglottis (Fig. 16). Scolex aspinose, small, slightly flattened antero-posteriorly, 230-400 (x = 330, n = 29, CV = 14%) in diameter, much wider than neck (Figs 1, 15, 17). Scolex containing antero-lateral uniloculate suckers, 95-165 (x = 130, n = 116, CV = 12%) in diameter. Apical organ absent. Internal longitudinal musculature weakly developed, in particular in pregavid and gravid proglottides (Figs 11, 12), represented by fine bundles of muscular fibres (Figs 8-12). Fibres more numerous in immature and mature proglottides (Figs 8-10). Osmoregulatory canals situated at level of vitelline follicles, overlapping testes; ven- tral canals much wider than dorsals; at posterior extremity of each proglottis, one short, narrow secondary canal directed laterally (Fig. 2), separated from outside just by thin cytoplasmic layeroftegument. Testes cortical, numbering 19-32 (x = 25, n = 48, CV = 14%), spherical to oval, 45-70 (x = 55, n = 40, CV =11%) in diameterrepresenting 12-22% (x = 17%, n = 23) ofproglottis width, medially forming 1-2 layers, with some testes in third incomplete layer (Figs 2, 6, 7). Testes overlapping dorsal osmoregulatory canals and vitelline follicles, degenerate in gravid proglottides. Cirrus-sac pyriform to elongate (Figs 2, 5), 110-225 (x = 160, n = 47, CV = 10%), basal portion extending beyond mid-line of proglottis, representing 42-64% (x = 52, n = 47, CV = 9%) ofproglottis width. Internal vas deferens (sperm duct) forming few loops; ejaculatory duct thick-walled; unarmed cirrus occupying more than halfthe length ofcirrus sac. External vas deferens (sperm duct) strongly coiled, situated in anterior central part of proglottis, occupying up to 45% ofproglottis width and up to 48% ofproglottis length. (Figs 6, 7). Genital atrium present. Genital ducts passing between osmoregulatory canals. Genital pore irregularly alternating, situated anteriorly at 7 to 17% (x = 12%, n = 40; CV = 19%) ofproglottis length. Vagina always posterior (n = 76) to cirrus-sac, thin-walled, with higher concentration of chromophilic cells at proximal end (near genital pore). Circular vaginal sphincterpresent (Fig. 5). Ovary medullary, with dorsal outgrowth penetrating cortex, follicular, not clearly bilobed ventrally, occupying 42-71% (x = 57; N = 39; CV = 12%) of proglottis width (Figs 2, 4). Mehlis' glands 40-55 in diameter (Fig. 4). Vitelline follicles cortical, tightly grouped together, arranged in two elongated groups in equatorial part of proglottis, representing porally 25-48% (x = 39, n = 37; CV = 14%) and aporally 23-54 % (x = 42, n = 36; CV = 17%) of proglottis length, respectively, overlapping testes (Figs 2, 6-8). A NEWTAPEWORM FROM THE AMAZON 309 vt ut ov te Figs 1-5 Amazotaenia yvettae gen. n., sp. n. 1. Paratype 29733 INVE, entire worm, ventral view. 2. Holotype CHIOC 34363, premature segment, ventral view showing the disposition ofvitelline follicles. 3. Eggsdrawn indistilledwater. 4. HolotypeCHIOC 34363, detail ofposteriorpartof a pregravid proglottis, ventral view, uterus is not figured. 5. Paratype 29734 INVE, cirrus-sac and vagina, ventral view, uterus is not figured. Abbreviations in all the figures: ci, cirrus; cp. cirrus pouch; cs, capsule; cv, vaginal canal; ; em, embryophore; lm, longitudinal internal musculature; on, oncosphere; ov, ovary; sc, secondary osmoregulatory canals; te, testes; ut, uterus; vc, ventral osmoregulatory canal; vd, vas deferens; vs, vaginal sphincter; vt, vitelline follicles. Scalebars: 1, 2,4, 5 =250 urn; 3 =50urn. 310 A. DECHAMBRIER Figs6-7 Amazotaenia yvettae gen. n., sp. n., pregravid proglottides. 6. Paratype 29732 INVE, ventral view. 7. 29742 INVE, dorsal view. Scalebar: 250pm. Primordium ofuterine stem cortical, already present in immature proglottides. Formation of uterus: in immature proglottides, uterine stem straight, formed by tubular concentration of chromophilic cells (Fig. 2). Irregular lumen of uterine stem present in last immature and first mature proglottides, formed by lateral and dorsal outpocketings penetrating medulla. Diverticula formed before presence of first eggs in uterine stem. In pregravid proglottides, eggs completely filling uterine stem and thick-walled diverticula (Fig. 6). In gravid proglottides, diverticula occupying up to 90% of proglottis width (Fig. 12). Pregravid and gravid proglottides with dorsal outpocketings invading medulla, reaching dorsal cortex (Figs 11, 12). Uterus with 10- 18 (n = 32) lateral branches on each side. Terminal proglottides without uterine opening. Proglottidesreleased into the gutbefore opening ofuterus. Egg shell with hyaline membrane, irregular in shape, 45-55 in diameter, with spherical, bilayered embryophore, 19-21 in diameter, thick; diameter ofinternal layer containing granular material 17-18; oncosphere spherical to oval, 12-14 in diameter, with 3 pairs ofhooks 5-6 long (n = 10) (Fig. 3). DISCUSSION InAmazotaenia yvettae, the equatorial disposition ofthe vitelline follicles and their disposition in two elongated patches are rare within the Proteocephalidea. This disposition is observed only in Vennaiapseudotropii (Verma, 1928) (Gangesiinae) a A NEWTAPEWORM FROM THEAMAZON 311 Figs 8-12 Amazotaenia yvettae gen. n., sp. n., 29738 INVE 8, 9. Mature proglottides, cross sections at level of vitelline follicles and at level of ovary respectively. 10. Pregravid proglottis, cross sections at level of anterior part showing the penetration of uterus in the medulla. 11, 12. Gravid proglottis, cross sections showing uterine outgrowths crossing the medullaand reaching thedorsal cortex. Scalebars: 8-10= 250 urn; 11, 12= 100urn. 312 A. DECHAMBRIER parasite of Pseudeutropius garua (Siluriforme) in India, but in the latter, the dispo- sition is clearly lateral (Verma, 1928; Nybelin, 1942; Freze, 1965, Schmidt, 1986). In cross sections, the ventral position of vitelline follicles is similar to that found in another very peculiar proteocephalidean cestode, Vaucheriella bicheti de Chambrier, 1987 (Zygobothriinae) parasite of Tropidophis cf. taczanowskyi (Serpentes) in Ecua- dor, but the latter shows vitelline follicles in a posterior position (de Chambrier, 1987). The minute size ofAmazotaeniayvettae (500-2900 urn) is also remarkable and can be compared only with Proteocephalus microscopicus Woodland 1935, a parasite of Cichla spp. (Pisces: Cichlidae), which is 1540-2020 urn long according to Woodland (1935c) or 2050 to 2780 urn long according to Takemoto & Pavanelli (1996). The minute size is also linked with a very small number of proglottides in both species: 4-7 for Amazotaenia yvettae and 6-14 long according to Woodland (1935c) or6-12 according to Takemoto & Pavanelli (1996) forP. microscopicus. Amazotaenia yvettae is also characteristic by its unusual behaviour of attach- ment in which individuals are grouped in isolated compact clumps of 14 to 66 specimens. This behaviour is, to my knowledge, unique to the Proteocephalidea. They are all situated in most anterior part of the intestine (first twelfth) and are deeply embedded, perforating the epithelium into the lamina propria, generating a tissue reaction. Isolate attached specimens were never found. The specimens collected from B. vaillanti were identical to those from the type host and had the same site. Only one clump was found in B. vaillanti. The type-host is pararasitized by three other proteocephalidean species which occur in particular sites in the gut (see de Chambrier & Vaucher, 1997): Ampho- teromorphus piraeeba Woodland, 1934 occurs from the second twelfth to the five twelfth of the intestine; Endorchis piraeeba Woodland, 1934 occurs from the five twelfth to the eight twelfth ofthe intestine; Nomimoscolexpiraeeba Woodland, 1934 lives in the six twelfth to the ten twelfth of the intestine. As Amazotaenia yvettae occured in the first twelfth, it did not share its site with any other co-parasite. E. piraeeba shared a part of its habitat with A. piraeeba and N. piraeeba, but the two latter species occured in distinct sites. The uterine development is unusual among the Proteocephalidea and is similar to that ofMariauxiella (de Chambrier & Rego, 1995). The uterus possesses not only lateral branches, but also has outgrowths, fan-like in cross sections, crossing the me- dulla and reaching the dorsal cortex. Furthermore, another peculiarity is shared with Mariauxiella: the medullary ovary in Amazotaenia yvettae also possesses obvious outgrowths into the dorsal cortex. With regard to the classification of Woodland Figs 13-18 Amazotaenia yvettae gen. n.. sp. n. 13-17. Scanning electron micrographs. 13. Entire worm, (aiTow and white circle indicate regions oftegument illustrated in Figures 14 and 16. respec- tively). 14. Enlarged view ofmargin ofsucker. 15. Scolex. lateral view. 16. Enlarged view of tahnetergiuotrsmhaorwgiinngoafcmaotmupraectprcoglluomttpiso.f1t7h.eSnceowlexwo,rampsi.calSEviMew.pho18t.osDebtvailDro.fJt.hWeiainetsetri(oGrepnaervta)o.f Scale-bars: 13 = 100pm, 15. 17 =50pm. 14. 16= 1 pm, 18 = 1000pm.