Asian Pacific Journal of Tropical Biomedicine (2011)200-204 200

Asian Pacific Journal of Tropical Biomedicine

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Document heading Larval stages of digenetic trematodes in praemorsa snails from freshwater bodies in Palestine Sami Bdir, Ghaleb Adwan * Department of Biology and Biotechnology, An-Najah N. University, P. O. Box (7)-Nablus, Palestine

ARTICLE INFO ABSTRACT

Article history: Objective: ( ) To detect the species of larval tremaMethods:todes cercariae in Melanopsis praemorsasnails Received 11 February 2011 from 5 different fresh water bodies in Palestine. A total of 1 880 Received in revised form 3 March 2011 2008 snails were collected from Melanopsisdifferent f rpraemorsaesh water bodies in Palestine from October, to Accepted 15 March 2011 November, 2010. Cercariae in snails were obtained by lighting and crushing Available online 30 March 2011 Results: methods. The behavior of cercariae was observed using a dMelanopsisissecting m praemorsaicroscope. Three different species of larval trematodes were identified from snails collected Keywords: only from Al-Bathan fresh water body, while snails from other water bodies were not infected. Melanopsis praemorsa These species were microcercoCercariaus cerca melanopsiria, xiphi dpalestiniaiocercari aI, aCercariand brev imelanopsifurcate lo palestiniaphocerco uIIs Digenetic trematodes cercaCercariaria. The smelanopsie cercaria epalestinia called III Cercariae and Melanopsis praemorsahave not been described before from this snail in Palestine. 5 7% Xiphidiocercaria The infection rate of collected from Al-Bathan fresh water body was . , 4 3% Brevifurcate lophocercous while the overall infection rate of snails collected from all fresh water bodies was . . Details are Microcercous presented Conclusions:on the morphology and behavior of the cercariae as well as their development within Palestine the snail. These results have been recorded for the first time and these cercariae may be of medical and veterinary importance.

1. Introduction Most freshwater snails can become intermediate hosts for trematode cercariae which may be transmitted to people and [3-5] Melanopsis praemorsa M. praemorsa . Trematodes have a complex life cycle involving (L. 1758, Buccinum) ( ) one or two (rarely three) intermediate hosts prior to infecting is a gastropod snail which belongs to the prosobranchia S the definitive host. nail species are the first intermediate to the superfamily and to the family S hosts for the majority group of digenetic trematode. nail . This specie is highly polymorphic; within the infection occurs through ingestion of the eggs or penetration same population, conically elongated, dark-brown to black by free-swimming miriacidia. Inside the first intermediate prosobranch snail which may exceed 2 cm in length. The host, the process of polyembryony occurs and several i.e. sculpture of the shell is highly varied, being either smooth different intramolluscan larval stages ( , sporocyst, redia, or ribbed to a greater or lesser extent. The growth and the ) A M. praemorsa and cercaria are formed by asexual reproduction. fter phenotypic enzyme profile varied as well. is emerging from the snail tissue, the larvae of trematodes widely prevalent in freshwater bodies of the Mediterranean (cercariae) may find the suitable secondary intermediate region[1,2]. In Palestine, it is also one of the most abundant host or definitive host by means of passive transmission snails present in fresh water bodies. Therefore, it is (metacercaria) or active penetration, respectively. expected that it may act as an intermediate host for many Mollusks are regularly found harbouring larval stages of digenetic trematodes of various vertebrates in the area. parasitic trematodes or digenea[6]. Various studies have been Melanopsis done on fauna of cercariae from snails [4,7-12].

*Corresponding author: Ghaleb Adwan, Department of Biology and Biotechnology, Following the discovery of human philophthalmiasis Philophthalmus An-Najah N. University, P.O. Box (7) - Nablus, Palestine. cases in Israel, it showed that cercariae Fax: +970-9-2383589 M. praemorsa E-mail: [email protected] were collected from [8,9,13]. In Palestine, the Sami Bdir et al./Asian Pacific Journal of Tropical Biomedicine (2011)200-204 201 3. Results knowledge on freshwater larval trematodes is poor. The purpose of the present study is to investigate the infection M. praemorsa rate of trematodes in and the type of cercariae Snails collected from water bodies of Al-Nassareya, Al- M. praemorsa released from collected from different fresh Oja, Al-Jeftlek and Wadi Qana were not infected. The water bodies in Palestine. overall infection rate of snails collected from all fresh M. water bodies was 4.3%. However, from the total of 1400 praemorsa collected from Al-Bathan fresh water body, 80 2. Material and methods (5.4%) were infected with various larval trematodes including Cercaria melanopsi palestinia I n Cercaria ( =56, 4%), M. praemorsa melanopsi palestinia II n Cercaria palestinia A total of 1 880 snails were collected from ( =14, 1%) and III n different freshwater bodies in Palestine from October, 2008 ( =10, 0.7%). Three different species of larval trematodes to November, 2010. These water bodies included Al-Bathan, were recorded, which were xiphidiocercaria, a brevifurcate Al-Nassareya, Al-Oja, Al-Jeftlek and Wadi Qana. The lophocercous cercaria and a microcercous cercaria. These Cercaria melanopsi palestinia I Cercaria number of snails collected from these water bodies were cercariae called , melanopsi palestinia II Cercaria melanopsi palestinia III 1400, 150, 150, 100 and 80, respectively. These water bodies and were used for bathing, drinking and washing by the people. have not been described before from this snail in Palestine. The collected snails were kept in a glass aquaria containing Details of measurements of the various structures and the water and thin layer of sediment from the same habitat of flame cell formula of each type of cercariae are presented in snails. Aquaria were continuously aerated using air pumps. Table 1. Examination of snails for larval trematodes was carried 3.1. Cercaria melanopsi palestinia I (Figure 1 A, B ; Table 1) out as described previously[4,14]. Snails were examined for larval trematodes within 2 days after collection. Cercariae in M. praemorsa snails were obtained by lighting and crushing It has a variable size and shape depending on the methods. In the lighting method snails were put in the dishes contraction and extension movements of the body, with containing distilled water and illuminated for 12 hours a simple tail not quite as long as the body. The body is in the room. In the crushing method, snails were broken covered with minute delicate spines, with a 2 毺m thick with tweezers and the soft tissues were placed between 2 tegument and has an oval and elongated shape. The anterior slides and squashed. Recovered larval trematodes were end of the body is armed with a stylet situated on the oral studied alive, unstained or vitally stained 0.5% neutral red sucker which was 14 毺m long and 3 毺m wide at the round or 0.5% brilliant cresyl blue. They were fixed in acetic acid- part in the posterior end. The stylet lacking conspicuous formalin-alcohol (AFA) solution and subsequently stained in lateral thickening. The oral sucker is larger than the ventral acetocarmine. Measurements were taken on a minimum of 10 one, while the later is located in the final third of the body. specimens of live and fixed larval trematodes. Figures were The genital premordium cell mass lies in the posterior of the drawn with freehand, from preparations examined under a ventral sucker. Alimentary canal is composed of a mouth light microscope. The behavior of cercariae was observed and a pharynx which is located just behind the oral sucker. using a dissecting microscope. The excretory system consists of a V-shaped excretory Table 1 Cercaria melanopsi palestinia I, II and III M. praemorsa Measurements (毺m) of the various structures of encountered in snails collected from Al-Bathan freshwater bodies, Palestine. Cercaria melanopsi Type of Structure I II III a 90-130 75-110 250-310 Body length 100-120 110-136 230-280 a 57-70 20-34 60-120 Body width 60-68 30-35 70-85 b a 40-120 220-300 50-80 Tail length 45-90 230-280 45-65 a 8-15 15-20 30-60 Tail width 9-16 17-19 32-45 Oral sucker (diam.) 30-35 _ 42-47 Ventral sucker (diam.) 21-23 _ 40-45 Pharynx 7暳7 _ 18暳14 Flame cell formula 2[(2)+(2)+(3)+(2)]=18 2[(1)+(1)+(1)+(1)]=8 2[(2)+(3)+(1)+(1)]=14 a M ( ) ( ) b easurements are given for live upper values and fixed lower values specimens. This measurement includes the length of tail rami which are 50-75 by 5-10 毺m (live) and 40-50 by 7-12 毺m (fixed). Sami Bdir et al./Asian Pacific Journal of Tropical Biomedicine (2011)200-204 202

Figure 1. Cercaria melanopsi palestinia I, II III M. praemorsa C. melanopsi palestinia I and eC.nc melanopsiountered i npalestinia II snails from Al-Bathan. C. melanopsi palestinia III and its sporocyst (A and B), and its sporocyst (C and D) and and its sporocyst (E and F). vesicle measuring 10 毺m to 18 毺m, opening into the canal was seen. It has a long tail which is contractile and posterior part of body through a canal extending to the tail. bifurcated into two rami. The length of the rami are less It also has two main lateral ducts, which run anteriorly to the than that of half of the tail stem. The cercaria is equipped oral sucker. Flame cells are arranged symmetrically in the with an anterior protrusible organ, delimited from the body body with a flame cell formula 2[(2) + (2) + (3) + (2)] = 18, but by a conspicuous constriction at about 18-20 毺m from the Cercaria melanopsi palestinia I they are missed in the tail. anterior edge. The typical median dorsal fin-fold extended has two pairs of well developed penetration glands arranged from the posterior end of the body nearly to the region of symmetrically on both sides of the ventral sucker. One pair the anterior protrusible organ, which is 40-60 毺m long and of these glands opens into two small pores located lateral to 13-17 毺m at its maximum width. The last third of each the stylet, while the other pair opens into two sacs located on ramus is surrounded by a thin fin-fold. The cercaria body both sides of the mouth. When the cercaria swims, the body is covered with minute spines, with a 2 毺m thick tegument. is strongly contracted and the tail lashes in all directions, The glandular system consists of two groups of glands. The rarely pelagic or with powers of floatation. Once the cercaria anterior glands, four on each side of the body, are located comes to the surface, it pauses and startes sinking slowly, behind the anterior protrusible organ. The posterior-lateral but often resumes swimming before hitting the bottom and glands, six on each side, lead into two glandular bundles. Cercaria melanopsi after a few seconds of resting period. Ducts of these glands open at the anterior edge of the palestinia I develops within ovoid sporocysts measuring protrusible organ. The excretory vesicle is small measuring 110-190 毺m long and 80-130 毺m wide. Sporocysts are filled 8-10 毺m by 4-6 毺m. It has two main lateral ducts which run with one or two fully developed cercariae and germ balls. anteriorly, while other two canals run posteriorly extending into the tail with each one entering into the excretory 3.2. Cercaria melanopsi palestinia II (Figure 1C, D; Table 2) pores at about 18 毺m from the branch tip. Flame cells are organized symmetrically in the body with a flame cell It is furcocercous cercaria and neither sucker nor alimentary formula 2[(1) + (1) + (1) + (1)] = 8, but they are missed in the Sami Bdir et al./Asian Pacific Journal of Tropical Biomedicine (2011)200-204 203 Cercaria melanopsi palestinia II tail stem. swims actively by glands and others. However, the author concluded that the action of its tail which shows great power of contractility. none of these characteristics have significant taxonomic This cercaria develops within a spherical sporocyst and value and the xiphidiocercariae group should be left measures 130-200 毺m in diameter. Birth pore is not without subdivisions. There are several families of digenetic discernible. Sporocysts contain 3-6 developed cercariae, trematodes have xiphidiocercariae as their larval forms, for Plagiorchiidae Telrchiidae Ochetosomatidae several developing cercariae and germ balls. example, , , , Lecithdendriidae and . The presence of well developed 3.3. Cercaria melanopsi palestinia III (Figure 1E, F; Table2) Cercaria melanopsi palestinia I stylet in is identified as Cercaria melanopsi palestinia I xiphidiocercaria. belongs to This cercaria has an elongate cylindrical body with microcotylae sub-group of the xiphididiocercariae due to stumpy knob-like tail which may developed as a sucker. the absence of the tail fin-fold, the small size of the body, T (2 毺 ) M he body is covered with a smooth tegument m thick . and the locat”ion and ”size of the ventral sucker. oreover, Both ventral and oral suckers are round and nearly equal in it belongs to Pusilla sub-type due to the possession of size with ventral one near the middle region of the body. The two pairs of penetration glands, undeveloped gut, and oral sucker has a stylet in the anterior margin measuring development within small ovoid sporocyst, except that vs Cercaria 10 毺m long and 4 毺m wide. The alimentary canal consists the number of flame cells is greater (18 12)[19]. melanopsi palestinia I Cercaria of a mouth, a pre-pharynx and a pharynx. The excretory is closely related to melanopsi I M. praemorsa vesicle is relatively large, located in the space between the described previously from snails posterior end of the body and ventral sucker. It measures collected from Yarmouk River, Jordan[15], except that the vs 60-75 毺m long and 34-50 毺m wide and is characterized by number of pairs of penetration glands (2 3) and the number vs having a thick wall. It has two main lateral ducts which run of flame cells (18 20) are different. Cercaria melanopsi palestinia II Furcocercous anteriorly. Other two canals run posteriorly to the end of the is an cercaria F I body. lame cells are arranged symmetrically in the body , aphar“yngeal, brevif“urcate monostome cercaria. t with a flame cell formula 2[(2) + (3) + (1) + (1)] = 14, but they belongs to the Lophocerca group which is characterized are missed in the tail. There are four pairs of penetration by the presence of a dorsal median fin-fold on the body, glands located above the ventral sucker. Gland ducts, on lacking ventral sucker, having excretory system with two each side, form two bundles. Each of the outer and the inner canals in the tail and absence of flame cells in the tail Cercaria cristata bundle consists of two ducts. All bundles run anteriorly to stem. It resembles which develops in Lymnaea stagnalis Cercaria mlanopsi open on both sides of the stylet. This specie of cercariae snails. However, II Cercaria cristata shows no swimming activity because it has a stumpy knob- is different from because it lacks gut, like tail. This cercaria develops from the elongate sporocyst has eight flame cells instead of six, 10 pairs of penetration varying greatly in size. The sporocyst is 500-2000 毺m long glands instead of one pair and develops within a small and 130-580 毺m wide and has a birth pore at its anterior spherical rather than in a thread-like sporocyst[19]. Also Cercaria melanopsi palestinia II Cercaria end. It is filled with many developed cercariae, developing is different from brevifurca Planorbis trivolvis cercariae and germ balls. which develops in as well as Cercaria cristata Cercaria brevifurca except that has four pairs of glands ventral to cecum and rami are not bearing 4. Discussion Cercaria melanopsi palestinia II fin-folds[23]. resembles Cercaria melanopsi IV that of described previously from Melanopsis Melanopsis praemorsa Freshwater gastropods of the snails are snails collected from Yarmouk River, apparently obligate intermediate hosts for many species Jordan [15], except that the number of pairs of the posterior- vs Sanguinicola of trematode parasites that may exist in several vertebrate lateral glands (6 7) are different. It resembles Potamopyrgus antipodarum hosts[2,4,7-9,13,15,16]. A detailed description of the various cercaria which develops in Sanguinicola types of cercariae has been reported previously[17-21]. snails and was recoverd in France[24]. cercaria Some of these cercariae are classified according to the is characterized by a body and furca fin-fold, suckerless, position and number of body suckers. Some are categorized the absence of a pharynx and development in spherical according to the shape and relative size of their tails, while sporocyst, but the flame cells are not discernible and the others are categorized morphologically by specialized number of penetration glands and other characteristics were Sanguinicola sp body structures like the xiphidiocercariae. In the present not mentioned. . is a digenean blood fluke of study, three types of cercariae have been described. brackish or fresh water fishes. Cercaria melanopsi palestinia III T hese are xiphidiocercaria, brevifurcate lophocercous and “ “belongs to a group microcercous. The characteristics for xiphidiocercariae of cercariae called Microcercous cercariae, whose differentiation have been reviewed previously[22]. These tail is short and stumpy. This cercaria resembles that of Cercaria melanopsi V Melanopsis characteristics include flame cells, shape of stylet, presence described previously from praemorsa or absence of fin-folds, number and location of penetration snails collected from Yarmouk River, Jordan[15], Sami Bdir et al./Asian Pacific Journal of Tropical Biomedicine (2011)200-204 204 vs Arch Razi Ins 59 except that the number of pairs of penetration glands (4 5) transmission diseases. 2005; : 113-119. vs and the number of flame cells (14 16) are different. It also [6] P oulin R, Mouritsen KN. Large-scale determinants of trematode Sphaerostoma bramae Mar Ecol Prog Ser 254 resembles that of which develops in infections in intertidal gastropods. 2003; : Bithynia tentaculata Cercaria a sporocyst in [19]. However, 187-198. melanopsi palestinia III Sphaerostoma differs from that of [7] M anafov AA. Two new species of virgulate cercariae from the bramae Melanopsis praemorsa (L.). Parazitologiia freshwater molluscs in that the former has two suckers of equal size and 42 its gut consists of only a pharynx and a short oesophagus. 2008; (6): 467-475. Microcercous cercariae The may develop into trematodes [8] R adev V, Kanev I, Nollen PM, Gold D. Life history and Allocreadiidae Philophthalmus lucipetus J Parasitol of several families, such as, the and identification of from Israel. Gorgoderidae 85 1999; (2): 291-294. , which are reported from a freshwater fish and Philophthalmus [19] [9] G old D, Lang Y, Lengy J. species, probably frog, respectively . P. palpebrarum In summary, due to the presence of various cercariae in , in Israel: description of the eye fluke from M. praemorsa Parasitol Res 79 experimental infection. 1993; (5): 372-377. snails and their potential for establishment of Melanopsis praemorsa [10] I smail NS. Two new furcocercariae from in zoonotic diseases in animals and may be in humans, these Helminthologia 261 H M O Jordan. 1989; : 15-20 findings may be concerned by ealth inistry fficials for Melanopsis [11] O lenev AV. Cercarial fauna in the freshwater mollusk, surveillance of fresh water snails in this fresh water body as praemorsa Ecol Exp Parasitol 1 well as others. in western Georgia Part 1. 1987; : 73-96. [12] G alaktionov KV. Two species of cyathocotylid cercariae from the Conflict of interest statement Melanopsis praemorsa Parasitology fresh water mollusk . 1980; 14 (4): 299-307. We declare that we have no conflict of interest. [13] D imitrov V, Kanev I, Panaiotova M, Radev V, Gold D. Argentophilic structures of miracidia and cercariae of Philophthalmus lucipetus (Philophthalmidae: Trematoda) from Acknowledgements J Parasitol 86 Israel. 2000; (2): 255-261. [14] A bdel-Hafez SK, Ismail NS. Larval stages of digenetic trematodes T M of Theodoxus jordani (Sowerby 1836) snails from Yarmouk River, he authors want to acknowledge sincere thanks to iss Z Parasitenkd 69 Nuha Daraghmi, Miss Duaa Zaid, Miss Ayat Abdel-Hafez, Jordan. 1983; : 789-796. M R M M M K M A [15] I smail NS, Abdel-Hafez SK. Larval sages of digenetic tematodes iss ooya elhim, r. outasim anaan and r. hmad Melanopsis praemorsa S T of (L. 1758, Bueeinum) () snails htiwi for their collaboration in sampling collection. he Z Parasitol 69 technical help of Miss Reem hamadneh, Mr. Monther Ismail from Yarmouk River, Jordan. 1983; : 613-626. M S A G [16] T areen IU. Cercaria orospinosa encountered in the snail and r. uhail bu hdeib is also appreciated. Melanopsis praemorsa Int L. from a mountain stream in Turkey. Rev Hydrol 61 1976; (5): 699-702. References Medical and economic malacology [17] M alek EA, Cheng TC. . London: Academic Press; 1974. General parasitology [1] M ouahid A, Idaghdour M, Ghamizi M, Mone H. Observation of [18] C heng TC. . New York: Academic Press; 1973. Melanopsis praemorsa J The trematodes spawn in (Prosobranchia: Melanopsidae). [19] D awes B. . London: Cambridge University Press; Moll Stud 62 1996; : 398-402. 1968. Melanopsis Ind J Med Res [2] E lkarmi AZ, Ismail NS. Allometry of the gastropod [20] S ewell RBS. Cercariae Indicae. 1922; Suppl 10: praemorsa (Thiaridae: Prosobranchia) from Azraq Oasis, Jordan. 1-370. Pak J Biol Sci 9 Die 2006; (7): 1359-1363. [21] L uhe M. Parasitische Plattwurmer I, Trematodes. Susswasserfauna Deutschlands 17 [3] U kong S, Krailas D, Dangprasert T, Channgarm P. Studies on the 1909; : 1-217. Zool Anz morphology of cercariae obtained from freshwater snails at Erawan [22] N asir P. Some aspects of xiphidiocercarial classification. Southeast Asian J Trop 189 Waterfall, Erawan National Park, Thailand. 1972; : 382-392. Med Public Health 38 J Parasitol 2007; (2): 302-312. [23] M cCoy OR. Note on cercariae from Maissouri. 1929; XV [4] F arahnak A, Vafaie-Darian R, Mobedi I. A faunistic survey of (3): 199-208. Melanopsis spp cercariae from fresh water snails: . and their role in [24] G erard C, Le Lannic J. Establishment of a new host- Iranian J Publ Health 35 disease transmission. 2006; (4): 70-74. parasite association between the introduced invasive species Potamopyrgus antipodarum Sanguinicola [5] F arahnak A, Setodeh A, Mobedi I. A faunistic survey of (Smith) () and Melanoides tuberculata J Zool Lond 261 cercariae isolated from and their role in sp. Plehn (Trematoda) in . 2003; : 213-216.