Revue suisse deZoologie 111 (2): 371-380;juin 2004 Redescription ofOphiotaenia hylae Johnston, 1912 (Eucestoda: Proteocephalidea), parasite ofLitoria aurea (Amphibia: Hylidae) from Australia Alain DE CHAMBRIER Muséum d'histoire naturelle, Département des Invertébrés, PO Box 6434, CH-1211 Geneva 6, Switzerland. E-mail: [email protected] Redescription ofOphiotaenia hylae Johnston, 1912 (Eucestoda: Proteo- cephalidea), parasite of Litoria aurea (Amphibia: Hylidae) from Australia. - Type material of the proteocephalidean cestode Ophiotaenia hylae Johnston, 1912 is redescribed. It is characterised by a globular scolex with uniloculate suckers, a prominent apical organ covered by spiniform microthriches and containing round to oblong cells offinely granular cyto- plasm, andby the internal longitudinal musculature composedby 4-5 dorsal and 4-5 ventral bundles of fibres. A similar taxon, Ophiotaenia sp. from a closely related host species, Litoria moorei, is also studied and compared. Key-words: Eucestoda - Proteocephalidea - Ophiotaenia hylae - Litoria aurea - Litoria moorei - Hylidae - Australia. INTRODUCTION Since its original description in 1912, nobody made aredescription ofO. hylae. Prudhoe & Bray (1982, p. 33, figs 8a, b, c) only published drawings of Ophiotaenia hylae (BMNH 1968.4.19.1-15) from Litoria (Hyla) moorei, Cannington, Australia. This material has been examined and is here considered as belonging to Ophiotaenia sp. (see below) and not to O. hylae Johnston. New material ofthe true O. hylae was unavailable due to the extreme scarcity ofits host, Litoria (Hyla) aurea. This species is actually considered threatened with extinction inAustralia (Pyke etal., 2002; Pyke, 2002). I had the opportunity to study Johnston's type material deposited in different Australian museums (Queensland Museum, Brisbane; South Australian Museum, Adelaide). This allowed me to redescribe this material, to add new information to the original description and clarify its taxonomic status. MATERIALAND METHODS The worms, conserved in museum collection in alcohol, were stained with Weigert'shaematoxylin solution,dehydratedinanethanol series, clearedwithEugenol (clove oil), and mounted in Canada balsam. Pieces of strobila were embedded in paraffin wax, cross sectionned (thickness 12-15 pirn), stained with Weigert's haema- Manuscript accepted 15.10.2003 372 A. DECHAMBRIER toxylin and counterstained with 1% eosin B according to the method recently published by de Chambrier (2001). All measurements are given in micrometres unless otherwise stated. Abbreviationsusedindescriptions: x=mean,n=numberofmeasurements,CV = coefficient of variability (%), OV = ovary width to proglottis width ratio, PG = position of the genital pore in relation to proglottis length (%), PC = cirrus pouch length to proglottis width ratio (%), JNT = Johnston original description; BMNH = NaturalHistoryMuseum, London; MHNG=NaturalHistory Museum, Geneva; INVE = Geneva Museum, Invertebrates Collection; SAM = South Australian Museum, QM Adelaide; = Queensland Museum, Brisbane. RESULTS Ophiotaenia hylae Johnston Figs 1-6 Ophiotaenia hylaeJohnston, 1912: 63. Batrachotaenia hylae\ Rudin, 1917: 366. Batrachotaenia hylae; Freze, 1965: 385. Type host: Litoriaaurea (Lesson, 1829) (Amphibia: Hylidae). Materialstudied: SyntypematerialofOphiotaeniahylae: 1 slideV4141 (SAM44141); S 689 (SAM 20689), four slides: a) 2 immature pieces, one with scolex; b) 1 immature piece, 9 mm; c) 1 gravid proglottis, 1 mm, bad conservation state; d) 11 gravid proglottides, 15 mm. Oneimmaturespecimenwithascolex,G 16/423,QM,fromHylaaurea,Sydney,NSW,37°41'S, 144°40'E. Othermaterial: Inone separateboxcontainingalotofJohnston'soriginalmaterial, 1 slide with 12 immature pieces, withone scolex, SAM 28407. Siteofinfestation: Intestine. Type-locality: NeighbourhoodofSydney, NSW,Australia. Redescription (based on syntypes and Johnston's original material) Proteocephalidea, Proteocephalidae. Testes, ovary, uterus diverticles in me- dulla, uterine stem cortical. In the whole mounted syntype material, one fragment 18 mm long. Strobila acraspedote, anapolytic, consisting of 33 immature and mature proglottides (JNT = 60 mm long). Immature proglottides 540-635 long and 405-500 wide, mature proglottides 695-750 long and 710-865 wide, gravid proglottides 1250- 1540 long and 865-920 wide (Figs 3, 4). Tegument thick and wrinkled in mature proglottides. Presence ofnumerous small dorso-ventral muscles. Scolex 340-390 (JNT = 320) in diameter (Figs 1-2), covered by small dense microtriches about 1 long, suckers 130-135 (JNT=110) in diameter. Apical organ, 65- 80 in diameter, covered by small dense spiniformmicrotriches 2-3 long (Fig. 2) above anetworkofsmallandpoorlydefinedcanals filled withagranularcontent, andending beneathtegument surface. Presence ofsmallretractormusculature atthe margin ofthe apical organ (Fig. 2). Beneath the apical organ, a concentration of cells with a finely granular cytoplasm is present in two zones, one just beyond the apical organ and another made oftwice bigger cells situated at the level ofthe suckers (Fig. 2). Longi- tudinal internal musculature dense, formed by 4-5 thick bundles of fibres on both dorsal and ventral sides (Fig. 5). Ventral and dorsal osmoregulatory canals between vitelline follicles and testes, crossing cirrus pouch at level of its two/third part (Fig. 3). Ventral canal, twice the REDESCRIPTION OFOPHIOTAENIA HYLAE 373 Figs 1-2 Ophiotaenia hylae Johnston, 1912. Syntype, 20689 SAM. 1. Scolex, general view. 2. Detailed viewofthe apicalorganregion.Abbreviations: cl, smallcanals- filledwithgranularcontent; gc, glandcells;lm,internallongitudinalmusculature;rm,retractormuscles;ro,rostellum-likeapical organ; sm, spiniformmicrotriches (hooklets). Scale-bars: 1 = 200/mi; 2 =100firn. 374 A. DECHAMBRIER Fig. 3 Ophiotaenia hylae Johnston, 1912. Syntype, 44141 SAM, dorsal view ofa mature proglottis. Scale-bar: 500pim. diameter of the dorsal canal, with narrow secondary canals directed externally ven- trally. Testes 74-106 in number (x = 86, n = 12, CV =13%, JNT = numerous) in two dorsal field, with tendency to converge anteriorly and posteriorly, in one ortwo layers dorsally, not reaching laterally to vitelline follicles (Fig. 3), 35-60 in diameter. Testes 15-22 preporal, 16-25 postporal and 38-53 aporal in number. Testes degenerated in gravid proglottides (Fig. 4). Genital pores irregularlyalternating, openingbetween44and55 % (n= 12, CV = 7%) ofproglottis length. Small genital atrium present. Cirrus pouch pyriform, US- MS long (JNT 140). PC = 17-19% (x = 18%, n = 11, CV = 5%). Cirrus occupying up to 70% ofcirrus pouch length. Evaginated cirrus covered by numerous minute spini- form microtriches, 2-3 long. Vagina anterior (38%) orposterior (62%) to cirrus pouch (JNT = antero-ventrally), with a small subterminal vaginal sphincter (Fig. 3). When anterior, passing ventrally to the cirrus pouch. Mehlis glands 60-85 in diameter. Vas deferens coiled, between base of cirrus pouch and median part of proglottis, rarely extending beyond body midline in mature and premature proglottides, extending anteriorly. REDESCRIPTION OFOPHIOTAENIAHYLAE 375 Figs4-7 4-6. OphiotaeniahylaeJohnston, 1912. Syntypematerial,20689SAM.4. Schemeofdorsalview ofagravidproglottis showingtheuterinediverticledevelopment. 5. Schemeoftheinternal lon- gitudinal musculature at level ofimmature proglottides. 6. Scheme ofa cross section showing thedispositionofgenitalorgansrelatedtotheinternal longitudinalmusculature. 7. Ophiotaenia sp. from Litoria moorei. BMNH 1968.4.19.1-15, eggs drawn in distilled water. Abbreviations: em,embryophore;lm,internallongitudinalmusculature;oe,outerenvelope;on,oncosphere;ov, ovary;te,testes; ut, uterus; vi, vitellaria; Scale-bars: 4= 500ptm; 5 = 250/<m; 6=noscale; 7 = 20pirn. Ovary bilobate, medullary, folliculate, with numerous dorsal outgrowths (Figs 3,7). OV=68-71% (x=70%,n= 11,CV=2%). Vitellinefollicles,intwolateral bands, occupying porally 91-97% of proglottis length, and aporally 94-97% of pro- glottis length (Fig. 3). 376 A. DE CHAMBRIER Primordium ofuterine stem cortical, already present in immature proglottides, with diverticles in medulla. Formation of uterus of type 2 (see de Chambrier et al., 2004): in immature proglottides, chromophil cells concentrated laterally on both sides of uterine stem; in the first mature proglottides, lateral ramified digitations without a lumen, occupying at this stage already about 35% of proglottis width; in gravid proglottides, lateral diverticles occupying upto 91% ofgravidproglottis width. Uterus with 10-17 (JNT = numerous) lateral medullar ramified diverticles on each side (Fig. 4) and one or sometimes several ventral apertures as described for Crepidobothrium spp (de Chambrier, 1989a, b). Eggs, measuredinwhole preparations, withoncosphere 11-12 in diameter (JNT = 7.5-11), hooklets 5-6 long; embryophore 13-14 in diameter (JNT = 15-19); outerenvelope 60-75 in diameter. Ophiotaenia sp. Figs 7-10 Proteocephalushylae; Prudhoe & Bray, 1982: 33, Figs 8a, b, c. [Not Ophiotaenia hylaeJohnston]. Host: Litoria moorei (Copland, 1957) (Amphibia Hylidae). : Locality: Neighbourhood ofPerth (Cannington andDarlington),W.A.,Australia. Materialstudied: 9 whole mountpreparations andmaterial inalcohol(fromwhereSEM microphotographs come from) ex Litoria moorei, Cannington, Western Australia, 17.04.1966: BMNH 1968.4.19.1-15; 1 wholemountpreparation SAM21402,Darlington,W.A., 12.11.1980. Site ofinfestation: Intestine. Description Proteocephalidea, Proteocephalidae. Testes, ovary, uterus diverticles in medulla, uterine stem cortical. Strobila acraspedote. anapolytic. Tegument thick and wrinkled in mature proglottides. Presence ofnumerous small dorso-ventral muscles. Scolex 260-340 (n = 4, x = 290) in diameter, suckers 105-130 in diameter (Fig. 9). Apical organ, 50-80 in diameter, covered by small dense spiniform micro- triches (Fig. 10). Longitudinal internalmusculaturedense,formedby4-5 thickbundles offibres on both dorsal and ventral sides (Fig. 8). Ventral and dorsal osmoregulatory canals crossing cirrus pouch at its middle part, situated between vitelline follicles and testes (Fig. 8). Ventral canal, twice the diameter of the dorsal canal, with numerous narrow secondary canals directed externally. Testes 46-76 in number (x = 59, n = 30, CV = 14%) in two dorsal field, with tendency to converge anteriorly and posteriorly, in one or two layers dorsally, not reaching laterally to vitelline follicles (Fig. 8), 50-80 in diameter. Testes 14-26 pre- poral, 6-18 postporal and 23-44 aporal in number. Testes degenerated in gravid pro- glottides. Genital pores irregularly alternating, openingbetween46 and57 % (n= 17, CV = 6%) ofproglottis length. Small genital atrium present. Cirrus pouch pyriform, 175- 215 long, PC = 27-33% (x = 29%, n = 17, CV = 4%). Cirrus occupying up to 85% of cirrus pouch length. Vagina anterior (54%) or posterior (46%) to cirrus pouch, with a sub-terminal vaginal sphincter (Fig. 8). When anterior, passing ventrally to the cirrus pouch. Mehlis glands 45-65 in diameter. Vas deferens coiled, between base ofcirrus pouch and median part ofproglottis, often extending beyond body midline in mature and premature proglottides, extending anteriorly. REDESCRIPTIONOFOPHIOTAENIA HYLAE 311 Fig. 8 Ophiotaenia sp. from Litoria moorei. 21402 SAM. Mature proglottis, dorsal view. Note the secondarycanals endingbeneath the tegument. Scale-bar: 500}im. Ovary bilobate, medullary, folliculate, with dorsal outgrowths. OV = 55-63% (x=60%, n= 17,CV=3%) (Fig. 8). Vitellinefollicles, intwolateralbands, occupying porally 87-91% ofproglottis length and aporally 86-88% ofproglottis length. Primordium ofuterine stem cortical, already present in immature proglottides, with diverticles in medulla. Formation of uterus of type 2 (see de Chambrier et al., 2004). 378 A. DECHAMBRIER .: ^ ._:_.„ Figs 9-10 BMNH Ophiotaenia sp., 1968.4.19.1-15, from Litoria moorei. Scanning electron micrographs ofthe scolex. 9. Dorsoventral view. 10. Apical view, detail ofthe apical organ. Scale-bars: 9 = 50firn; 10= 10pirn. Eggs, measured in distilled water, with oncosphere 12-18 in diameter, hooklets 5-9 long; embryophore 18-23 in diameter; outerenvelopeupto 55 indiameter(Fig. 7). Remarks This taxon is similarto Ophiotaenia hylae onthe basis ofthe following charac- ters: similar apical organ, position ofthe genital pore, presence of4-5 dorsal and 4-5 ventral longitudinal internalbundles ofmusculature. Itdiffersfromitbythenumberof testes (46-76 versus 74-106), by the cirrus pouch length/width ofproglottis ratio (27- 33% versus 17-19%), and by the ovary width / proglottis width ratio (55-63% versus 68-71%). Although these observations suggest that it could belong to a new distinct species, the material studied is fragmented and not in suitable conditions for an accurate description. The scolex particularly is badly fixed. In order to confirm that it represents a new species, it would be necessary to collect new material from Litoria moorei. DISCUSSION Johnston (1912) situated the ovary and the vitelline follicles of Ophiotaenia hylae in the cortex. My observations show the ovary to be clearly medullary (see scheme, Fig. 6).Asforthe vitellinefollicles, theirpositionis difficulttoassess asthere are no clear lateral muscle bundles (Fig. 6). The uterus stem is cortical with further development of diverticles into the medulla (Fig. 6). Contrary to the opinion of Johnston (1912, p. 64), the uterus does not arise as "athin duct..." but is clearly ofthe type 2 ofuterine development as described by de Chambrier etal. (2004). According to Johnston (1912), the vagina is situated anterior and ventral to the cirrus pouch. I observed aposition mainly posterior (62%) ofthe vagina. I also observed a small sub- REDESCRIPTION OF OPHIOTAENIAHYLAE 379 terminal vaginal sphincter, secondary canals emerging from the ventral osmo- regulatory canal ending under the tegument and the position of vas deferens which extends anteriorly (see Fig. 3). The structure ofthe internal longitudinal musculature is also uncommon within the Proteocephalidea. It is composed by 4-5 dorsal and 4-5 ventral powerful isolate bundles ofmusculature easy to observe in immature and mature proglottides but less so in gravidproglottides. Given the stability ofthis character, the numberofbundle in matureproglottides couldbediscriminantatthe specific level asI alreadyproposedfor Crepidobothrium species (de Chambrier, 1989b, p. 369). Theapicalorganispeculiarbecauseofthepresenceofsmall spiniformhooklets covering its surface, retractor-like muscles andnetworkofsmall canals surrounding it. This morphology shows some similarities with that of the Gangesiinae and looks intermediatebetweenthe apical organsfoundintheNomimoscolexpiraeeba aggregate (Zehnderetal., 2000) andthose inthe Gangesiinae (deChambrieretal., 2003). Tomy knowledge, no other Proteocephalidea have this kind of apical organ. It would be interesting to analyse the twoAustralian Ophiotaenia species described in the present paper using DNA sequences, and compare them with the taxa cited above in order to see iftheirrespective apical organs could represent a possible evolutionary trend orif this structure is homoplastic. ACKNOWLEDGEMENTS IamindebtedtoLesterCannon(Brisbane), IanWhittington andLeslieChilsom (Adelaide) for loan ofthe type material, David I. Gibson and Eileen Harris (London) for loan ofthe comparative material. I am deeply indebted to the "Donation Georges et Antoine Claraz" for supporting this study. 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