35-41 35 H. F. Hassan & I. S. Saeed Bull. Iraq nat. Hist. Mus. (2001) 9 (3): 35-41 LIGHT AND ELECTRON MICROSCOPE STUDIES OF THE ADULT OF PLEUROGENOIDES MEDIANS (OLSSON, 1876) (TREMATODA: LECITHODENDRIIDAE) FROM IRAQI MARSH FROGS RANA RIDIBUNDA Husain F. Hassan and Isam S. Saeed Department of Biology, College of Education, University of Salahaddin, Arbil, Iraq. ABSTRACT The morphology of the Pleurogenoides medians from Rana ridibunda in Iraq was studied by light and scanning electron microscopy. The light microscopical studies confirmed the original observations with the ventral sucker being smaller than the oral sucker and the genital pore being laterally situated. Electron micrographs of adult P. medians revealed that the body surface had a rough appearance by hand-like spines, which are more sparsely distributed towards the posterior end with a presumed function in nutrient absorption. INTRODUCTION Pleurogenoides medians (Olsson, 1876), a digenian lecithodendriid trematode , parasitizes numerous aquatic vertebrate species including frogs, fresh water fishes, urodeles and anurans (Smyth and Smyth, 1980). Frogs infection with P. medians has been reported worldwide and this trematode is the most common encountered frog intestinal parasite in Europe and Asia (Dawes, 1968; Cox, 1971; Hristovski and Less, 1973; Brooks, 1976; Gupta and Chopra, 1985). The life cycle of P. medians had been studied in detail by Buttner (1951), while that of other related species in the same genus namely P. japonicus, P. tener and P. orientalis were studied by Shibue (1953), Macy (1964) and Madhavi et al. (1987) respectively. Certain differences concerning the intermediate host and the structure of larval stages have been visualized. In fact to date, the helminth parasites occuring in Iraqi frogs Rana ridibunda are poorly known. Except of some early works on trematodes (Saoud and Roshdy, 1970; Dauood, 1974; Hamad, 1985), no comprehensive list of Pleurogenoides species has been published. The present study undertaken to open the way for more detailed investigations of trematodes of frogs from Iraq to help in the elucidation of the morphology of adult worms and to establish the special adaptations of individuall species within their host. MATERIALS AND METHODS Adults of P. medians were obtained from the intestine of frogs Rana ridibunda Sulaimanya, Iraq. The flukes were fixed in AFA solution under coverglass pressure and stained with alum carmine according to standard protocols outlined by Humason (1972). Diagrams were drawn with the aid of a camera lucida and all measurements were in millimeters and were based on stained specimens. Scanning electron microscopy (SEM): Adult worms were fixed for 2-3 hr in 2.5% glutaraldehyde buffered with cacodylate to pH 7.4, washed in buffer and postfixed for 1-2 hr 36 Light and electron microscope studies in cacodylate-buffered osmium tetraoxide, dehydrated in ethanol and freeze-dried. The mounted specimens were coated with gold and examined in a Jeol 840 scanning electron microscope. RESULTS The morphology of the adult form of P. medians is best illustrated in fig. 1. The body is small, oval and 1.52-2.16 mm by 1.10-1.43 mm in size. The tegument is spinulate with spines densely distributed over the anterior surface. The oral sucker is subterminal and 0.27 mm; the ventral sucker measures 0.14 mm is smaller than the oral sucker and situated slightly above the middle of the body. The pharynx is globular and measures 0.06 mm in diameter; the oesophagus is short (0.07 mm) and bifurcates into two short ceca (0.51 mm) which are terminated at level of the ventral sucker. Testes symmetrical, situated near the cecal termination, right testis measures 0.3X0.28 mm whereas the left testis measures 0.29X0.27 mm. The genital pore is lateral and situated near the right body margin at the level of pharynx. The cirrus sac is elliptical, running obliquely from the front margin of the ventral sucker to the genital pore and encloses the seminal vesicle, the well developed prostatic complex and the cirrus. The ovary measures 0.22X0.20 mm, is submedian to left testis and situated to the left of the ventral sucker. The vitellaria follicular are extracaecal and extends from the level of pharynx to a level slightly beyond the ovary. Uterus is much coiled and the coils occupy the posterior half of the body. Eggs are small, oval, operculate, amber coloured and measures 0.018X0.010 mm. As illustrated in figs. 2-10 throughout the worm body. The tegument is carpeted with regularly arranged flattened hand-like spines and each spine is composed of 5-8 finger-like appendages (figs. 9-10). No structures corresponding to the microvillus-like projections or knobs were seen. It appears that the spines are more densely arranged towards the anterior end and more sparsely distributed towards the posterior end, although the spines all over the body surface are of similar size. It is to be note that the spines occur in association with suckers as well as genital pore but not with excretory pore at the extreme posterior end. DISCUSSION The digenean trematode belonging to Pleurogenoides found in the present study resemble and corresponds morphologically to P. medians described by Mathias (1924), Neuhaus (1941), Vojtkova (1974) and Smyth and Smyth (1980). The present form, however, differs from those described by these authors as well as from the rest known species of Pleurogenoides in having: (i) an ovary which is situated to the left of the ventral sucker, (ii) a genital pore which is present near the right body margin at the level of pharynx. The differences noted above appear to be sufficient to treat the form described here as a new variety of P. medians for which the name Pleurogenoides medians var. sulaimanialis is being proposed. This is the first time that P. medians has been recovered in Rana ridibunda in Iraq. There have not, however, been any reports on the tegumental surface of P. medians nor indeed of its function, although there is an indication that the morphologically similar tegument of the echinostome trematodes and nematodes have pinocytic activity and a digesting effect on the host cells (Smales and Blankespoor, 1984; Koie, 1986, 1987; Imai et al., 1989). Nevertheless, this is the first attempt to study the tegumental surface in P. medians with scanning electron microscope and various conclusions can be made. The highly increased tegumental surface due to the densely arranged spines suggests that the tegument is likely to be active in absorption of nutrients and probably also in digestion of host cells. It is possible that the digestive system of the adult worm probably does not function 37 H. F. Hassan & I. S. Saeed or only to a very limited degree, in the absorption of nutrients as indicated by the presence of short intestinal caeca and this may result in the increment of the tegumental surface area that could probably have a possible role in Pleurogenoides nutrition. Future studies should give more critical attention to this possibility . ACKNOWLEDGEMENT We are grateful to Mrs. E. A. Harris, Parasitic worm section, British Museum (Natural History) for identification of the worm and to Dr. D. Tetley (Glasgow University, Scotland) for suggestions in the preparation of the material for scanning electronscopy and to the Dept. Biology, Coll. Education, Univ. Salahaddin, Iraq for financial support. LITERATURE CITED Brooks, D. R. 1976 Parasites of amphibians of the Great Plains. Part 2. Platyhelminthes of amphibians in Nebraska,USA. Bull. Univ. Nebr. State Mus., 10: 1-92. Buttner, A. 1951 La progenese chez le trematodes digenetiqes. Research personelles sur deux especes progenetiques deja connues. Ratazia joyeuxi (Brumpt, 1922) et Pleurogenes medians (Olss., 1876). Ann. Parasitol. Hum. Comp., 22: 319-323. Cox, F. E. 1971 Parasites of British amphibians. J. Biol. Educ., 5: 35-51. Dauood, K. S. 1974 Studies on the protozoan and trematode parasites of some amphibians. M. Sc. Thesis, Mosul University. Dawes, B. 1963 The Trematoda. Cambridge University Press, London. Gupta, N. K. and Chopra, R. 1985 On digenetic trematodes of amphibians from India. Part II. Res. Bull. Punjab Univ.(Science), 35: 11-18. Hamad, N. R. 1985 Taxonomic study of digenetic trematodes of some vertebrates, Northern Iraq. M. Sc. Thesis, Salahaddin University. Hristovski, N. D. and lees, E. 1973 The helminth fauna of Rana temporaria in relation to that of Europe generally. Acta Parasit. Jugosl., 4: 93-98. Humason, G. L. 1972 Animal tissue techniques. WH Freeman and Co., San Francisco . H . F . Hassan &I . S . Saeed Imai, J., Akahane, H., Horiuchi, S., Maruyama, H. and Nawa, Y. 1989 Gnathostoma doloresi: development of the larvae obtained from snakes, Agkistrodon hyals, to adult worms in a pig. Jpn. J. Parasitol., 38: 221-225. Imai, J. ; Akahane , H . ; Horiuchi , S . ; Maruyama ; H . and Hawa , Y . 1989. Gnathostoma doloresi : Development of the larvae obtained from snake , Agkistrodon halys , to adult worms in a pig . Jpn. J . Parasitol. , 38 : 221 – 225 . Koie, M. 1986 The life history of Mesorchis denticulatus (Trematoda, Echinostomatidae). Z. Parasitenkd, 72: 335-343. 38 Light and electron microscope studies Koie, M. 1987 Scanning electron microscopy of redia, cercariae, metacercariae and adults of Mesorchis denticulatus (Trematoda, Echinostomatidae). Parasitol. Res., 73: 50- 56. Macy, R. W. 1964 Life cycle of the digenetic trematodes Pleurogenoides tener (Looss, 1898)(Lecithodendriidae). J. Parasitol., 50: 564-568. Mahadiv, R., Dhanumakumari, C. and Ratnakumari, T. B. 1987 The life history of Pleurogenoides orientalis (Srivastava, 1934)(Trematoda, Lecithodendriidae). Parasitol. Res., 73: 41-45. Mathias, P. 1924 Contribution a letude du cycle evolutif d un trematode de la famille de Pleurogenetinae Loss. Bull. Soc. Zool. Fr., 49: 375-377. Neuhaus, W. 1941 Entwickung and Bioloie von Pleurogenes medians Olss. Zool. Jahrb. Syst., 74: 207-242. Saoud, M. F. A. and Roshdy, M. A. 1970 On Halipegus alhaussaini n. sp. (Trematoda: Halipegidae) from Rana esculenta in Iraq with notes on Halipegus and related genera. J. Helminthol., 44: 349-356. Shibue, H. 1953 The first intermediate host of a frog trematode Pleurogenes japonicus Yamaguti. Jpn. J. Med. Sci. Biol., 6: 213-220. Smalea, L. R. and Blankespoor, H. D. 1984 Echinostoms revolutum and Isthiophora melis (Echinostomatinae:Digenea).: Scanning electron microscopy of the tegumental surfaces. J. Helminthol., 58: 187-195. Smyth, J. D. and Smyth, M. M. 1980 Frogs as host-parasite systems. The macmillan Press Ltd., London. Vojtkova, L. 1974 The trematodes of amphibia in Czechoslovakia. Folia Fac. Sci. Nat. UJEP Brun Biol., 45: 1-131. 39 H. F. Hassan & I. S. Saeed Bull. Iraq nat. Hist. Mus. (2001) 9 (3): 35-41 بالمجهر الضوئي واإللكتروني Pleurogenoides mediansدراسة المثقبة البالغة Rana ridibundaالمتوسم من الضفادع العراقية حسين فاضل حسن و عصام سعد اهللا سعيد العراق –أربيل –جامعة صالح الدين –كلية التربية –قسم علوم الحياة الخالصة وطن أمعـــاء الـــيت تســـت Pleurogenoides mediansمت دراســـة بنيـــة الديـــدان البالغـــة باســــتخدام ا هــــر الضــــوئي وا هــــر Pleurogenoides medians الضــــفادع العراقيــــة اإللكرتوين املتوسم وقد تبني باستخدام ا هر الضوئي بان للدودة البالغة حمجماً بطنيـاً وأمـــــا باســـــتخدام ا هـــــر . أصـــــغر مـــــن احملجـــــم الفمـــــي وفتحـــــة تناســـــلية جانبيـــــة املوقـــــع بــان اجلســم مغطــى بأشــواك شــبيهة بأصــابع اليــد والــيت تقــل يف اإللكــرتوين فقــد لــوحظ كثافتهــــا باجتــــاه النهايــــة اخللفيــــة ويعتقــــد بأ ــــا تــــؤدي دوراً وظيفيــــاً يف امتصــــاص املــــواد .الغذائية 40 Light and electron microscope studies Fig. 1: Adult worm Pleurogenoides medians var. sulaimanialis drawn from testis; TL, left testis Fig. 2: Scanning electron micrograph of Pleurogenoides medians var. sulaimanialis showing spinulate surface (bar=0.5um). gp, genital pore; os, oral sucker; vs, ventral sucker. 41 H. F. Hassan & I. S. Saeed Fig. 3-6: Scanning electron micrograph of leurogenoides medians var. sulaimanialis. Fig. 3 (bar=1um) showing the ventral side; Fig. 4