IBN AL- HAITHAM J. FOR PURE & APPL. SCI VOL.22 (2) 2009 The Life Cycle and Larval Development of Neoechinorhynchus iraqensis (Acanthocephala: Neoechinorhynchidae) in the Intermediate Host R. S. Al-Sady Department of Biology¸ College of Education Ibn Al-Haitham, University of Baghdad Abstract For the first time, the life cycle of Neoechinorhynchus iraqensis, a parasite of Liza abu and other freshwater fishes in Iraq and its larval development in the intermediate host, the copepod Cyclops hyalinus was given. Hatching took place in the intestine of the copepod host within 10 minutes of exposure to the infective eggs. Motile acanthor penetrated the wall of the intestine within 2-4 hr. after exposure and was found free in the hemocoel where it metamorphoses through the acanthella stage. Acanthella stage revealed changes in the entoblast where the anterior section gave rise to brain, proboscis and proboscis receptacle. The posterior section differentiation gave rise to the genital system. By 12 days, development in the copepod apparently was completed and by 13-14 days, the juvenile was infective for the definitive host. Introduction The family Neoechinorhynchidae, order Eoacanthocephala includes eight species of the genus Neoechinorhynchus from freshwater fishes of Iraq (1). N. iraqensis, an intestinal parasite of the mugilid fish Liza abu, was described for the first time by Amin et al. (2). All acanthocephalans have the same fundamental life cycle and development stages. All require an arthropod as intermediate host for the larval development and all utilize a vertebrate definitive host (3). Species of the family Neoechinorhynchidae, whose life cycles had been elucidated, include N. cylindratus by Ward (4), N. emydis by Hopp (5), N. rutili by Merritt and Pratt (6), Octospinifer macilentis by Harms (7), Paulisentis fractus by Cable and Dill (8), N. saginatus by Uglem and Larson (9) and N. cristatus by Uglem (10). All these species use ostracods as intermediate hosts to complete their life cycles except P. fractus which uses a copepod. Al-Sady (11) used 13 species of crustaceans: one copepod (Cyclops vernalis), one amphipod (Parhyla sp.), one peracarid (Mysis sp.), two cladocerans (Daphnia magna and Simocephalus vetulus) and eight ostracods (Stenocypris malcolmsoni, Potamocypris variegata, Cyprinotus putei, Cyprinotus salinus, Cyclocypris cruciata, Candona sigmoides, Cypricercus reticulates and Eucypris cisternina) for experimental life cycle of N. iraqensis. None of them was an appropriate intermediate host in the life cycle. IBN AL- HAITHAM J. FOR PURE & APPL. SCI VOL.22 (2) 2009 Materials and Methods Mugilid fishes (Liza abu) were brought to laboratory from a fish market at Baghdad city. Adult worms were obtained from the intestine of these fishes. Female worms were used as a source of eggs by rupturing the body in the posterior region. The eggs were removed from gravid females, washed with tap water and then were stored in the refrigerator at 4 o C in tap water. Copepods were collected from Al-Zawraa lake, Baghdad city, with the aid of zooplankton net (mesh size 335µ). Copepods were taken to the laboratory and kept in well- aerated aquaria at 25 o C. They were exposed to the infective eggs by allowing them to feed on a suspension of eggs for 1-2 hr. after being isolated from their source of food for one day. Several hundreds of copepods were removed with a pipette and examined for infection by putting them on a slide with a drop of 0.9 saline. Drawings were made with the aid of a camera lucida. All measurements were taken in micrometer unless otherwise indicated. Results Eggs and Acanthors: Fresh infective eggs from adult females ranged from 38-42 × 17-20 wide (Fig. 1). Four envelops enclosed the acanthor. These are the inner membrane, the fertilization membrane, the inner shell membrane and the outer shell. The hatching of eggs occurred within 10 minutes after exposure. The hatching embryos (acanthors) rapidly increased in length to about 43-50 × 17-20 and showed contractile movement. After ingestion, the inner membrane ruptured near the anterior pole releasing the acanthor. In the anterior end, the tube-like structure observed, extended from the apex to the entoblast Fig. (2). The peripheral syncytium was granular and contained several large nuclei. After 2-4 hr. postfeeding, the acanthor was slightly large, 48-53 × 18-20. At this stage, the acanthor was able to penetrate the copepod gut wall. Following penetration, acanthors were unattached and immobiled in the hemocoel Fig. (3). Acanthellas: One day after exposure, the parasite increased little in length to reach 50-60 × 40-42. Little changes occurred in the entoblast, but the giant nuclei become more distinct. This stage is called early acanthella Fig. 4). By 3-5 days, the acanthella began to be of a round shape, 75- 100 × 69-75 Fig. (5). The giant nuclei were increased in size. These were four dorsals and one ventral. By 5-8 days, the larvae now 125-180 × 88-92. Five hypodermal nuclei were distinguished. The epidermis was apparent as a lighter area surrounding the internal organs which were as a number of small cells Fig. (6). At 8-10 days, the larvae reached 280 × 93. They were elongated and the proboscis, proboscis receptacle and brain had become well differentiated Fig.( 7). The central nuclear mass was differentiated into the anterior primordial of the proboscis apparatus and the posterior primordium of the reproductive system. In this stage, the sex of a larva cannot be determined. The brain was then an evident as a group of small cells in the posterior end of the proboscis sheath. In 12 days, larvae are 0.5-1 mm (Fig. 8). Primordials of the lemnisci appeared, proboscis was not inverted and its hooks were sometime formed. Genital organs were differentiated. The two testes appeared in lateral IBN AL- HAITHAM J. FOR PURE & APPL. SCI VOL.22 (2) 2009 position of the body. In females of the same age, ligament sacs appeared with mass of cell to be the ovarian balls. Juveniles Larvae by 13 days of morphological differentiation which happened became anatomically adult-like. Females were slightly longer than males. The larval phase was completed and became infected to the definitive host. Discussion Neoechinorhynchus iraqensis has a wide spread among fishes in Iraq as 15 species of freshwater fishes were records as definitive hosts for this species in Iraq (12). In general, the development of this parasite in its intermediate host (Cyclops hyalinus) is similar to that of other neoechinorhynchids with minor variations. The acanthor of N. saginatus is similar to that of N. iraqensis in its increment in size and in showing contractile movement. Such increase in size seems to be characteristic for this group of worms, although Cable and Dill (8) noted the reduction in size in case of P. fractus. In the anterior end of the acanthor of N. iraqensis, the rostellar hooks appeared, such as in P. fractus (8) and N. saginatus (9). On the other side, the absence of these hooks has been noted in N. cylindratus (4) and N. rutili (6). The infective stage of N. iraqensis was reached in about 13-14 days, in comparison with 21 days in case of N. emydis, a parasite of map turtle (5), 48-57 days in case of N. rutili (6), 30 days in case of O. macilentis (7), 13 days in case of P. fractus (8), 16 days in case of N. saginatus (9) and 20 days in case of N. cristatus (10). All these species used ostracods to complete their life cycles, except P. fractus which used a copepod. Al-Sady (11) used 13 species of crustaceans belonging to five orders to complete the life cycle of N. iraqensis. No evidence of penetration of gut wall was apparent. In the previous studies about the life cycles of the genus Neoechinorhynchus, the necessity of second intermediate host can be explained as an adaptation to the feeding habits of the definitive host. In her work with N. cylindratus, Ward (4) detected light infection in ostracods (Cypria globula), so she used small fishes (Lepomis pallidus) as a second intermediate host. N. emydis used ostracods (Cypria maculata) as the first host and snails (Campeloma rufum) as second intermediate host (5). Since Liza abu is relatively a small fish and feed on small crustaceans (13), this fish can get the infection with N. iraqensis directly from consuming crustaceans. Acknowledgements Special thanks are due to Dr. F. T. Mhaisen, College of Education (Ibn Al-Haitham), University of Baghdad for his suggestions while reading this article and to Dr. O. M. Amin, Institute of Parasitic Disease, Tempe, Arizona, USA for providing valuable literature. References 1.Al-Sady, R. S.; Al-Saadi, A. A. J. J. and Ali,W.R. (2009). Neoechinorhynchus elongatus (Acanthocephala: Neoechinorhynchidae), a new record from freshwarer fishes in Iraq. (Unpublished). IBN AL- HAITHAM J. FOR PURE & APPL. SCI VOL.22 (2) 2009 1. 2. Amin, O. M.; Al-Sady, R. S. S.; Mhaisen, F. T. and Bassat, S. F. (2001). Comp. Parasitol., 68(1): 108-111. 3. Kennedy, C. R. (2006). Ecology of the Acanthocephala. Cambridge Univ. Press: 249 pp. 4. Ward, H. L. (1940). Trans. Amer. Microsc. Soc., 59: 327-347. 5. Hopp, W. B. (1954). J. Parasitol., 40: 284-299. 6. Merritt, S. V. and Pratt, I. (1964). J. Parasitol., 50(3): 394-400. 7. Harms, C. E. (1965). J. Parasitol., 51(2): 286-293. 8. Cable, R. M. and Dill, W. T. (1967). J. Parasitol., 53(4): 810-817. 9. Uglem, G. L. and Larson, O. R. (1969). J. Parasitol., 55(6): 1212-1217. 10. Uglem, G. L. (1972). J. Parasitol., 58(6): 1071-1074. 11.Al-Sady, R. S. (2000). Description of a new species of acanthocephala (Neoechinorhynchus iraqensis) and some ecological aspects of its infection to the mugilid fish Liza abu from Al-Faluja region, Al-Anbar province with observations on the experimental infection. M. Sc. Thesis, Univ. Baghdad: 84 pp., (In Arabic). 12.Mhaisen, F. T. (2002). Al-Mustansiriya J. Sci., 13(1): 13-25. 13.Yousif, U. H. (1983). Eco-biological study of Carasobarbus luteus (Heckel) and Liza abu (Heckel) from Mehaijeran canal, south of Basrah. M. Sc. Thesis, Univ. Basrah: 192 pp., (In Arabic). IBN AL- HAITHAM J. FOR PURE & APPL. SCI VOL.22 (2) 2009 Fig. (1-8): Life cycle and larval development of N. iraqensis. 1. Egg. 2. Ten minutes after exposure. 3. Acanthor, 2-4 hours after exposure. 4. Early acanthella (1 day). 5. Acanthella (3-5 days). 6. Acanthella (5-8 days). 7. Acanthalla (8-10 days). 8. Female acanthella (12 days). Abbreviations: BP: Brain primordium, DN: Dorsal giant nucleus, EN: Entoblast, FM: Fertilization membrane, GN: Giant nucleus, GP: Genital primordium, IM: Inner membrane, ISM: Inner shell membrane, LS: Ligament sac, OS: Outer shell, OV: Ovarian sphere, PH: Proboscis hooks, PP: Proboscis primordium, PR: Proboscis receptacle, SP: Spine, VN: Ventral giant nucleus. 0 .0 3 m m 4 O S FM IS M EN M IM M 0 .0 2 5 m m 1 2 0 .0 2 5 m m SP GN EN 3 0 .0 2 5 m m 6 PP GN GP 0 .0 5 m m 0 .0 5 m m 5 7 DN BP VN LS 0 .0 6 m m PH 8 PR O V 0 .1 m m 2002( 2) 22مجلة ابن الهيثم للعلوم الصرفة والتطبيقية المجلد Neoechinorhynchus دورة الحياة والتطور اليرقي للدودة شوكية الرأس iraqensis في المضيف الوسطي رنا صاحب السعدي جامعة بغداد ،ابن الهيثم -قسم علوم الحياة ، كلية التربية الخالصة Neoechinorhynchus شملت الدراسة الحالية ألول مرة وصف دورة الحياة والتطور اليرقي للدودة شوكية الرأس iraqensis ي، وهو طفيلي يصيب أسماك الخشني في المضيف الوسطLiza abu وأنواع أخرى من أسماك المياه العذبة فقست .Cyclops hyalinusهو أحد أنواع القشريات وهو مجذافي األقدام ي العراق. المضيف الوسطي المستعملف المخصبة.الجنين الفاقس )يسمى دقائق من تناولها عند تعريضها لبيوض الطفيلي 10البيوض داخل أمعاء مجذافي األقدام بعد حراً في الجوف ساعات من الفقس وبعدها يتوافر 4-2 يخترق جدران أمعاء القشري بعد مرور Acanthorأكانثور( . تحصل Acanthella الجسمي وهنا يعاني من تغيرات مظهرية وصوالً إلى المرحلة الثانية وهي المشوكة الصغيرة أما الجزء الخلفي فيتمايز إلى .يتمايز الجزء األمامي منها ليكّون الدماغ والخطم وغمد الخطمذ ،اتغيرات في الكتلة النووية يوماً من إصابة مجذافي األقدام تكتمل دورة حياة الطفيلي في المضيف الوسطي 22أجزاء الجهاز التناسلي. بعد مرور وهذه المرحلة هي Juvenileة المتكيسة أو اليافعة من اإلصابة يصل الطفيلي إلى مرحلة المشوك 24و21وخالل اليومين الطور المعدي للمضيف النهائي.