; New Temnocephalans from the Branchial Chamber of Australian Euastacus and Cherax Crayfish Hosts Kim B. Sewell1 2 and Lester R.G. Cannon1 1Queensland Museum, PO Box 3300, South Brisbane, Qld, Australia, 4101 [address forcorrespondence] 2Also: (a) The DepartmentofAnatomical Sciences and (b) The Departmentof Parasitology, The University ofQueensland, Brisbane, Qld, Australia, 4072 Sewell, K.B. and Cannon, L.R.G. (1998). New Temnocephalans from the branchial cham- ber ofAustralian Euastacus and Cherax crayfish hosts. Proceedings ofthe Linnean SocietyofNewSouthWales119,21-36. Australian freshwater crayfish are hosts to ectosymbiotic, turbellarian worms ofthe temno- cephalan subfamily Craspedellinae Baer, 1931 which are found in the the branchial chamber and are characterised by possession of one or more transverse papillate ridgesacrossthedorsalbodyandcrenulate(papillate)tentacles.TheCraspedellinaeis enlarged to accommodate four species viz. Gelasinella powellorum gen. et sp. nov. fromEuastacusspiniferandthree new species ofCraspedellafrom Cheraxspp. The definition ofthe Craspedellinae is emended to include a description ofthe organisa- tionoftheepidermal syncytial mosaic. Thepatternoftheepidermal syncytial mosaic ofCraspedellinaeisdiagnostic forthe subfamily, butnotusefultodiscriminateeither generaorspecies within thetaxon. The biogeography ofthe Craspedellinae suggests an origin in Australia/New Guinea after the separation of South America and Australia from Antarctica (c. 45 mya) and coevolution and radiation with Cherax crayfish,followedbyhostswitchingtoEuastacus. Manuscriptreceived23June 1997,acceptedforpublication 19November 1997. KEYWORDS: Australia, Cherax, Craspedella, Craspedellinae, crayfish, ectosymbionts, Euastacusspinifer,Gelasinella,Parastacidae,taxonomy,Temnocephalida. INTRODUCTION Australian crayfish are noted for their association with ectosymbiotic turbellarian worms, the Temnocephalida, of which one group, the Craspedellinae, are found in the branchial chamber. For more than 100 years Craspedella spenceri described by Haswell (1893) from the branchial chamber of the indeterminate Australian crayfish species, Astacopsis bicarinatus (= Cherax sp.), remained the only temnocephalan species recog- nised with papillate posterior dorsal ridges and the only described species in the genus. Recently Cannon and Sewell (1995) described a new genus Heptacraspedella from the crayfish Euastacus bispinosus from the Grampians, five new species of Craspedella from eastern Australian Cherax spp. crayfish and three new species in a new genus Zygopella from Western Australian Cherax spp. These temnocephalans which inhabit the branchial chamber oftheir respective hosts share a common facies ofcrenulate tentacles and dorsal posterior ridges. Cannon and Sewell (1995) recognised the subfamily Craspedellinae to include these three temnocephalan genera and predicted that further examination ofAustralian crayfish hosts would reveal a greater diversity forthe subfam- ily. Moreover, they suggested that examination of large specimens of Euastacus may yield new species of gill-dwelling temnocephalans. Examination of the branchial cham- ber of Euastacus spinifer and Cherax spp. hosts as part of a Ph. D. study by KBS has revealed newtaxa. Proc.Linn.Soc.n.s.w., 119. 1998 22 NEWTENNOCEPHALANSFROMCRAYFISH MATERIALS AND METHODS Euastacus and Cherax crayfish were collected from freshwater habitats using bait- ed collapsible minnow traps or occasionally by dip netting. To obtain temnocephalan worms, the carapace of crayfish was detached using strong forceps inserted anteriorly through the articular membrane and under the dorsal carapace, and the carapace and car- cass were then placed into a shallow vessel containing filtered fresh waterfrom the habi- tat. The inner surface of each branchiostegite (i.e. the branchiostegal membrane), the gills and the body wall were searched with the aid of a dissecting microscope. Worms were allowedto detach themselves spontaneously. Worms for wholemounts were routinely fixed by flooding with hot (c. 90°C) 10% formalin buffered to pH 7.0 with phosphate (HF) for c. 30 s and then transferred to 10% phosphate buffered formalin (Form.) at ambient room temperature. Worms were then rinsed in distilled water, stained with either Mayer's or Harris's Haematoxylin (Hx), dehydrated in ethanol, cleared in xylene and mounted in Canada balsam. Worms for seri- al sections were fixed in either Bouin's fluid (Bouin) or Form, at ambient room tempera- ture, dehydrated in ethanol, embedded in 'Paraplast' at 56°C, sectioned at 4-7 m and stained with Mayer's Haematoxylin and eosin (H&E), cleared and mounted in Depex. To show the epidermal mosaic, live temnocephalans were fixed by flooding with a solution of2% silver nitrate heated to c. 60°C, washed in distilled water then exposed to either bright sunlight or incident light from a Volpi 'cold light' source for c. 15-30 min, dehydrated in ethanol and mounted in Euparol. For scanning electron microscopy (SEM) specimens were fixed by flooding with HF, washed for 30 s to remove surface contami- nation in a 20% solution ofDecon 90 detergent and rinsed c. six times in filtered distilled water. Worms were then dehydrated in ethanol, critical point dried, mounted on stubs, coated with gold, and examined with a Hitachi S-530 SEM operating at 25 kilovolts. Photographs for figures were scanned from 35 mm negative film onto Kodak PhotoCD™ and edited, then assembled into plates using Adobe Photoshop™. Lineart illustrations for figures were prepared using Adobe Illustrator™ from templates of scanned sketches prepared by pen and ink with the aid ofa drawing tube. Terminology and Measurements Descriptive terminology essentially follows the conventions established by Cannon and Sewell (1995). The term vesicula resorbiens used in error by Cannon and Sewell (1995) is corrected to vesicularesorbens (see Cannon 1993). Names forthe syncytia which comprise the epidermal mosaic follow those established by Joffe et al. (1995a, b). Taxonomic material is deposited in the collections ofthe Queensland Museum (QM) and specimen slide preparations are designated as either: wholemount (WM); de Faure's mounting medium (deF) (see Evans, Sheals and MacFarlane (1961) for recipe details) cir- rus preparation (CP); longitudinal serial section (LS); oblique serial section (OS): the num- QM berofslides in the registered series is given in square brackets. Specimen datafor reg- istered material examined is listed in the order: registration number; specimen/slide prepa- ration details (in parentheses); location on host; host scientific name; locality details; date collected; collector(s); histological fixation/staining procedures. Where the crayfish ho—st was collected by those other than the coll—ector of the worms the labelling convention host collector name/worm collector name is observed. Full registration details are pro- vided for each holotype specimen. For all subsequent specimens listed in the material QM examined, the registration numberand specimen/slide preparation details are provided, followed by only those data which are differentfrom that ofthepreceding registration. Forclarity, discrete blocks ofregistration dataare separated by semi-colons. Terminology applied to the male copulatory organ is derived from Cannon and Sewell (1995j. In addition, the arrangement and orientation of the spined ridges of the cirrus introvert is characterised as approximately either parallel or diagonal in relation to Proc. Linn. Soc. n.s.w.. 119. 1998 K.B.SEWELLANDL.R.G.CANNON 23 the longitudinal axis of the inverted introvert. Descriptions of the cirrus refer to the inverted state ofthe organ and exclude fine details ofthe introvert spines. The measure- ments provided for soft structures and the cirrus are taken only from the worms which comprise respectively the taxonomic type series [WM, LS and OS] and CP series unless otherwise stated, for example, the seminal receptacle was rarely discernable in WM. All taxonomic measurements were made with the aid ofa drawing tube and are presented in ^m as a range followed usually by the mean in parentheses. Where no range is provided the measurements were the same for all worms, and where measurements are only approximate they are preceded by the qualifierc. For measurement of the cirrus, live worms were placed on a microscope slide in a drop offiltered fresh waterthen passedoveraflame forafew seconds to heatthe waterand kill the specimen in an extended position. After fixation in hot water (HW) by this method, excess waterwasremovedby pipette, adropofdeFadded, andthe specimencovered with a coverslip. Cirrus dimensions were measured as described previously by Cannon and Sewell (1995) ondrawingtubedrawings usingametal rulerforstraightdimensions andamapmea- sure (Western-Germany) for curved dimensions. The registration number of any voucher QM specimens ofhostcrayfish lodgedinthe Crustaceancollectionisprovided. SYSTEMATICS Gelasinella gen. nov. Diagnosis Craspedellinae with two posterior, dorsal, low, transverse papillate ridges and pos- teriorally four short ridges consisting of raised points radiating towards the posterior body margin. The most posteriortransverse ridge has a slightcentral indentation. Type species Gelasinellapowellorum sp. nov. Etymology Latin, gelasinus, dimple (masculine), a reference to the central indentation on the mostposteriordorsal transverse ridge. Remarks The number and form of the dorsal ridges differ from those of other members of the Craspedellinae described by Cannon and Sewell (1995). The exact configuration of the transverse and posterior ridges was only possible to determine using SEM, although the central indentation was clearly observed on live worms, wholemounts and silver nitrate stained preparations (Fig. 1). The SEM also revealed a semi-regular, transverse line of large multiciliated papillae distal to the most anterior transverse dorsal ridge which were notraised on adorsal ridge (Fig. 1). Gelasinellapowellorum sp. nov. (Figs 1;2A, B;3A) Material examined Holotype: QMGL18659 (WM), ex branchial chamber Euastacus spinifer from Mammy Johnsons River (tributary of Karuah River), NSW (32°19'S; 151°57'E) 31/Aug/1995 Powell J. and Powell R./Sewell K.B. HF/Hx. Proc.Linn.Soc.n.s.w., 119. 1998 24 NEWTENNOCEPHALANSFROMCRAYFISH Figure 1. ScanningelectronmicrographofGelasinellapowellorumgen. etsp. nov.fromthetype localityfixed in HFand shown in dorsal view. The most anteriorofthe two dorsal ridges (arrowhead) is continuous across the body. The posterior dorsal ridge (arrow) has a central indentation or 'dimple' from which papillae are absent. Scale=200^m. Paratypes: QMGL18660-18661 (WM); QMGL18666 (LS[1]) Bouin/H&E. Other material: QMGL18662-18663 (WM) HF/Hx; QMGL18667 (LS[1]) Bouin/H&E; QMGL18669 cirrus inverted (CP[6]) HW/deF; QMGL18664-18665 (WM), from Karuah River at Washpool Bridge, NSW (32°21'S; 151°55'E) 28/Aug/1995 Powell J. and Powell R./Sewell K.B. HF/Hx; QMGL18668 cirrus part everted (CP[6]) HW/deF; QMGL18675 (LS[1]) Bouin/H&E. Description External Body from posterior margin to tip of tentacles 607-667 (647), to eyes 409-442 (424) long and 266-270 (268) wide. Posterior disc 145-172 (156) in diameter; peduncle 87-100 (93) in diameter. Epidermis c. 2-3 high dorsally and ventrally. Proc Linn. Soc. n.s.w., 119. 1998 K.B.SEWELLANDL.R.G.CANNON 25 Excretory ampulla Pharynx Rhabdite Testes Sucker "glands Postero-lateral glands Peduncle Sucker Vesicula resorbens Vitelline ducts Seminal receptacle Copulato Prostate secretio .Common genital Cirrus Gonopor atrium Figure2. Gelasinellapowellorumgen.etsp.nov.(A)=HolotypeQMGL18659indorsalview.Scale= 100/jm, (B)=Reproductivestructuresfromlivespecimenindorsalview.Scale=50yum. Proc.Linn. Soc.n.s.w., 119. 1998 26 NEWTENNOCEPHALANSFROMCRAYFISH A Ill IS1, * D Figure 3. Nomarski interference photomicrographs ofcirri of adult worms from the type host and locality. Scale= 100/ym, 'A)= Gelasinellapowellorumgen.etsp. nov., inverted. Note: the introvertswellingis unusu- ally obvious for this particular specimen ofthe species; and the distal region ofthe introvert is typically con- stricted and reflexed, (B) = Craspedella bribiensis sp. nov., (i) inverted, (ii)everted, (C) = Craspedellacoora- nensissp.nov.,inverted,(D)=Craspedellajoffeisp. nov., inverted. Proc. Linn. Soc. n.s.w.. 119. 1998 K.B.SEWELLANDL.R.G.CANNON 27 General Anatomy Pharynx 37^-5 (41) long, 24-28 (27) wide. Gastrodermis c. 25-40 high. Excretory ampullae 39—45 (43) long and 22-31 (27) wide. Eyes c. 11 across. Posterior glands pre- sent and discharging from two postero-lateral regions. Reproductive System. Female Ovary 33-41 (37) long, 30-34 (31) wide. Vesicularesorbens 47-58 (54) long, 42-55 (47) wide, wallc. 3-10thick. Seminal receptaclec. 60long, 15 wide (from live worms). Reproductive System. Male Anterior testes 62-81 (67) long, 39-62 (48) wide. Posteriortestes 67-81 (78) long, 45-67 (59) wide. Seminal vesicle 56-66 (62) long, 27-31 (30) wide. Copulatory bulb 36-41 (38) long, 39-44 (42) wide, with semi-discrete ejaculatory sac. Prostate duct QMGL reservoirs parallel. Cirrus (based on 6 part-everted adult specimens ex 18668) 153-167 (160) long in total. Shaft cone-shaped, curved; proximal opening 43-57 (54) wide, with narrow or thickened rim. Introvert with constricted and reflexed distal region, 11-12 (11) wide at base, longer side 72-80 (76) long, shorter side 39-41 (40) long (i.e. introvert c. 4 times longer than width of introvert base), with asymmetrical swelling i.e. longer side much wider and extending proximally well past the base ofthe introvert, dis- tal opening c. 15 wide. Rows of inverted spines, except for those in the reflexed distal region, oriented parallel to long axis ofthe introvert. Hosts Euastacus spinifer: Parastacidae. Locality Karuah Riversystem, NSW. Etymology For both Robyn Powell and her husband Jules who provided the host from which the first specimen was recognised. Remarks The cirrus ofthis species has a distinctive form when alive, as the distal region of the inverted introvert is reflexed and the region immediately proximal to the everted region is constricted (Figs 2B, 3A). This makes theexact form anddimensions ofthe dis- tal region ofthe inverted introvert difficult to determine. The introvert is revealed clearly however when everted to have the form of the typical cirri of the Craspedellinae. Measurements of introvert length were all derived from partially everted cirri. The swelling on the longer side of the introvert extends proximally further past the introvert base than in any other species in the Craspedellinae. The ejaculatory sac ofthis species is more discrete than in any other species ofCraspedellinae (Fig. 2B). Host voucher speci- men QMW20765. Craspedella Haswell, 1893 Diagnosis Craspedellinae with three dorsal papillate ridges in the posterior half of the body and, behind the last ridge, four short posteriorpapillate ridges radiating towards the pos- teriorbody margin. Proc.Linn.Soc.n.s.w., 119. 1998 28 NEWTENNOCEPHALANSFROMCRAYFISH :?/ ^^IV^I ; • • s Ji ^ / « ''• • ;wKS :?;.«'<)*:-- 'Ilk-.'--.™ ' W :-:|g;.:MS *:?:»"s'1:?;;:.-*H.f:! Figure4. (A) =Nomarski interference micrograph ofthe distal tipofthe cirrusandthevaginaofCraspedella bhbiensis sp. nov. cleared in deF to reveal the shape of the vaginal cavity (arrow). Scale = 50 yum, (B) = Nomarski interference micrograph of the distal tip ofthe cirrus and the vagina of C. cooranensis sp. nov. cleared in deFto reveal the shape ofthe vaginal cavity (arrow). Note the introvertswelling is unusually obvi- ousforthisparticularspecimen ofthe species. Scale = 50pm, (C) =Nomarski interferencemicrographofthe distal tip ofthe cirrus and the vagina of Craspedellajoffei sp. nov. cleared in deF to reveal the shape ofthe vaginalcavity(arrow)andthe uniquedistal 'pockets' (arrowheads). Scale=50pm, (D)=Lightmicrographof the posteriorendofCraspedellajoffei sp. nov. QMGL18654in dorsal view stainedinH&Eshowing the large sizeofthecopulatorybulb(arrow)relativetothesizeofthecirrus(arrowhead). Notethe lackofanejaculatory sac.Scale=50^m. Type species Craspedella spenceri Haswell, 1893 Other species Craspedella bribiensis sp. nov.; C. cooranensis sp.nov.; C. gracilis Cannon and Sewell, 1995; C. joffei sp. nov.; C. pedum Cannon and Sewell, 1995; C. shorti Cannon and Sewell, 1995; C. simulatorCannon and Sewell, 1995; C. yabba Cannon and Sewell, 1995 Craspedella bribiensis sp. nov. (Fig. 3Bi-ii;4A) Material examined Holotype: QMGL18627 (WM) ex branchial chamber Cherax robustus from Bribie Island, pool beside McMahon Road, Qld, Australia (27°02.5'S; 153°10.3'E) 31/Jan/1995 Sewell K.B., Cannon L.R.G., Khalil Z. and ShortJ. HF/Hx. Yy<>< . Linn. Soc. n.s.w., 119. 1998 K.B.SEWELLANDL.R.G.CANNON 29 Paratypes: QMGL18628-18629 (WM); QMGL18631 (LS[1]) Bouin/H&E. Other material: QMGL18630 (WM) HF/Hx; QMGL18632 (WM); QMGL18633 (0S/LS[1]) Bouin/H&E; QMGL18634 (LS[1]); QMGL18635 cirrus inverted (CP[6], 6 adult specimens) HW/deF; QMGL18636 cirrus everted (CP[6], 6 adult specimens). Description External Body from posterior margin to tip of tentacles 638-667 (654), to eyes 428-437 (431) long and 230-274 (256) wide. Posterior disc 120-145 (132) diameter; peduncle 77-80 (79) diameter. Transverse body ridges do not form lamellae. Epidermis c. 2 high dorsally and ventrally. General Anatomy Pharynx 30-34 (32) long, 25-30 (28) wide. Gastrodermis c. 25 high. Excretory ampullae45-53 (50) long, 28-34 (31) wide. Eyes c. 11 across. Reproductive System. Female Ovary 31-50 (41) long, 23-44 (35) wide. Vesicular resorbens 56-69 (61) long, 44-59 (49) wide, wall c. 3-11 thick. Seminal receptacle c. 30 long, 15 wide (from live worms). Reproductive System. Male Anterior testes c. 50-81 (62) long, 39-75 (52) wide. Posterior testes c. 66-94 (80) long and 41-86 (62) wide. Seminal vesicle 53-69 (60) long, 27-30 (28) wide. Copulatory bulb 34-36 (35) long, 39-44 (41) wide, with ejaculatory sac. Prostate duct reservoirs parallel. Cirrus (based on 6 fully inverted adult specimens ex QMGL18635) 138-145 (141) long in total. Shaft narrow, funnel-shaped, curved, thick-walled, with distal region less than length ofintrovert; proximal opening 35-48 (41) wide, with narrow rim. Introvert slightly curved, 8-10 (9) wide at base, longer side 46-53 (49) long, shorter side 41-44 (42) long (i.e. introvert c. 5.5 times longer than width of introvert base), with narrow swelling slightly wider on longer side, dis- tal opening 12-15 (14) wide. Rows of inverted spines oriented obliquely to long axis ofthe introvert. Hosts Cherax robustus: Parastacidae. Locality Bribie Island, south-east Qld. Etymology From Bribie, referring to the type locality, the areaofBribie Island. Remarks The prominent and oblique rows ofspines on the inverted introvert distinguish this species clearly from C. simulator(see Cannon and Sewell 1995: 407, Fig. 3Ei-iii), which itotherwise resembles closely (Fig. 3Bi-ii). Proc.Linn.Soc.n.s.w., 119. 1998 30 NEWTENNOCEPHALANSFROMCRAYFISH Craspedella cooranensis sp. nov. (Figs 3C; 4B) Material examined Holotype: QMGL18637 (WM) ex branchial chamber Cherax depressus from Mt. Mothar foothills, creek crossing Shadbolt Road, Qld, Australia (26°13.6'S; 152°46.3'E) 8/Nov/1995 Sewell K.B. and ShortJ. HF/Hx. Paratypes: QMGL18638-18639 (WM); QMGL18640-18641 (LS[1,1]) Bouin/H&E. Other material: QMGL18642 (OS[l]) Bouin/H&E; QMGL18643 cirrus inverted (CP[6], 6adultspecimens) HW/deF; QMGL18644cirruseverted(CP[1], 1 adultspecimen). Description External Body from posterior margin to tip of tentacles 563-594(575), to eyes 374^115 (393) long and 164-296 (242) wide. Posterior disc 119-164 (149) diameter; peduncle 94-129 (109) diameter. Transverse body ridges do not form lamellae. Epidermis c. 2 high dorsally and ventrally. General Anatomy Pharynx 30-37 (33) long, 22-31 (28) wide. Gastrodermis c. 25 high. Excretory ampullae 42-56 (51) long, 22-31 (28) wide. Eyes c. 14 across. Reproductive System. Female Ovary 37-47 (42) long, 28-34 (32) wide. Vesicular resorbens 62-114 (80) long, 44-75 (55) wide, wall c. 3—10thick. Seminal receptacle c. 19 long, 9 wide. Reproductive System. Male Anterior testes 39-67 (53) long, 34^7 (40) wide. Posterior testes c. 58-87 (73) long and 34-67 (47) wide. Seminal vesicle 75-97 (84) long, 30-34 (32) wide. Copulatory bulb 39-4-2 (40) long, 36-41 (38) wide, with ejaculatory sac. Prostate duct QMGL reservoirs parallel. Cirrus (based on 6 fully inverted adult specimens ex 18643) 148-169 (161) long in total. Shaft narrow, goblet-shaped, curved, thick-walled, with dis- tal region greater than length of introvert; proximal opening 43-59 (51) wide, with nar- row rim. Introvert not curved, 10-12 (11) wide at base, longer side 47-48 (48) long, shorter side 41—45 (43) long (i.e. introvert c. 4.5 times longer than width of introvert base), with wide symmetrical swelling, distal opening 19-29 (25) wide. Rows ofinverted spines oriented parallel to long axis ofthe introvert. Hosts Cheraxdepressus complex sensu Riek, 1951: Parastacidae. Locality Cooran Tableland, south-east Qld. Etymology From Cooran which refers to the type locality; the areaofthe Cooran Tableland. Remarks The extremely wide distal opening and the almost symmetrical shape of the cirrus introvert serve to distinguish clearly the species from Craspedella bribiensis (Figs 3Bi-ii; Proc.Linn.Soc.n.s.w., 119. 1998