Redescription of the Redia and Cercaria of Haplorchis pumilio Using Light and Scanning Electron Microscopy

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Author: A. A. Taima 1, H. El-Sheikh 2, M. Q. Wanas (peace be upon him) 3, S. A. Noor El-Din 4 and M. A. Tawf

Redescription of the Redia and Cercaria of Haplorchis pumilio Using Light and Scanning Electron Microscopy

A. A. Taima 1, H. El-Sheikh 2, M. Q. Wanas (peace be upon him) 3, S. A. Noor El-Din 4 and M. A. Tawfik (peace be upon him) 5.

Abstract:

This study aims to know the ultrastructure of the tegument of redia and cercaria, which represent the intramolluscan stages of Haplorchis pumilio parasite, in an attempt to know how the cercaria penetrates the second intermediate host (fish); and also to know more details on the redia tegument. So 1866 snail of Melania tuberculata, which represent the main first intermediate host for H. pumilio parasite in Egypt, were collected from drains and canals of old planted lands and canals of newly reclaimed lands from Beheira governorate, Egypt. The snails were then examined for redia and cercaria of the parasite. The results showed that the percentage of infection in snails of old planted lands was 4.75% in drains and 1.3% in canals, whereas the percentage of infection in canals of newly reclaimed lands was 5.5%. In the light of detailed morphology of redia and cercaria as observed by light microscope (LM) and scanning electron microscope (SEM) structural adaptations were recognized on the surface of cercaria that help in infecting fish. These adaptations are represented by ventrally concaved body, spines located on the dorsal anterior portion of the cercarial body and the large preoral spines. These structures, in coordination with the penetration glands, help in attaching and penetrating the second intermediate host. SEM micrographs exhibited details on the cercarial body that LM did not, such as sensory papillae and openings of cystogenous glands. No more details were observed on the redia tegument when examined by SEM as compared to those recorded by LM.

1 Corresponding Author: Dr. A. A. Taima, Faculty of Science, Sebha University, Libya. Email Address: dr_ahmedtaima@yahoo.com 

2 Prof. of Parasitology, Faculty of Science, Tanta University, Egypt.

3 Ass. Prof. of Parasitology, Faculty of Science, Al-Azhar University, Egypt.

4 Ass. Prof. of Parasitology, Faculty of Science, Tanta University, Egypt.

5 Prof. of Parasitology, National Research Center, Egypt. Introduction The larval stages of Haplorchis pumilio, which is a fish eating birds and mammals parasite, were described by many authors using light microscope, among these: Khalil (1932), Price (1940), El-Gindy and Hanna (1963), Khalifa et al. (1977), Hassan (1987), Al-Shareef (1988) and Saad and Abed (1995). In this work the tegument of the redia and cercaria of this parasite was studied by LM & SEM to illustrate more details, in order to know how the cercaria penetrates the skin of the second intermediate host (fish) and to know more details on the redia tegument.

Material and Methods

Collection of Snails Melania tuberculata snails, the intermediate host of H. pumillio parasite, were collected from canals and drains of old planted lands and canals of newly reclaimed lands from Beheira governorate, Egypt during the period from December 1997 to November 1999. The net used for snail collection consists of a suitable wire net fixed to a strong square iron frame with the dimensions 33.33 cm. The depth of the net is 10 cm and provided with wooden handle about three meters in length. The scooping started at the edge of the water towards midline, where the net was shaken in the water, after it has been filled with plants, the snails then losen their hold on the plants and dropped to the bottom of the net. For collecting M. tuberculata snails found on the mud, the net was pulled on the mud surface, then washed and the contained snails were collected. The collected snails were placed in a container and transferred to the laboratory, where they were identified on the basis of the morphological characters of the shell and the body according to keys proposed by Abdel-Malek (1974), WHO Snails Identification Center (1973) and (1980) and Nada (1983).

Examination of Snails for Larval Stages of Haplorchis pumilio

The snails were maintained in plastic aquaria containing dechlorinated tap water and small amount of vegetations from the field for a period not more than a week. During this period they were examined for larval trematodes. Two methods were used for examination of snails for infection with larval trematodes: (a ) By exposure of snails to natural or artificial light for one hour at 25o C for cercarial shedding. (b) Crushing of snails for detection of the intra-molluscan stages using dissecting microscope.

Staining of Fixed Specimens

Different stains such as acetocarmine, boraxcarmine and Gowers were tested for permanent whole mount preparations. Acetocarmine gave the best results with rediae and cercariae. The procedure was carried out as follows: After fixation in 70 % ethanol, few drops of the stain were added to the fixed specimens for about 2-5 minutes. Dilute solution of acid alchohol (½ ml conc. HCl per 100 ml 70 % ethanol) was used for differentiation, specimens were dehydrated in ascending grades of alcohol (80 %, 90 %, 96 % and 100 % ethanol). Clearing was carried out by terpineol or clove oil and mounted in Canada Balsam.

Drawing, Measurements and Photomicroscopy

Camera Lucida was used for drawing mounted specimens. The measurements (10 specimens) in micrometer of the cercariae were taken from the longest and widest parts of body, tail and other organs. The minimum and maximum values were recorded for each organ with the average in parenthesis. Photomicroscopy was done using automatic camera of Carl Zeiss photomicroscope III with different magnifications.

Preparation of Specimens for SEM Examination

The specimens were fixed in 2.5% glutaraldehyde, dehydrated in ascending grades of ethanol and critical point dried by carbon dioxide. The specimens were then mounted on stubs, coated with gold and examined with Jeol, JSM, 5300 SEM at accelerating voltage at 30 K. V. (King and Van As, 1997).

Identification of the Cercaria

Identification of the cercariae was based on keys proposed by Luhe (1909), Dawes (1946) and Frandsen and Christensen (1984).

Results

In this work 1866 Melania tuberculata snail were collected from drains (358) and canals (154) of old planted lands and canals of newly reclaimed lands (1354 snails). The percentage of infection of snails with larval stages of Haplorchis pumilio in old planted lands was 4.75 % in drains and 1.3 % in canals, whereas the percentage of infection in canals of newly reclaimed lands was 5.5 %. The larval stages of this parasite was commonly found in aged (20-24 mm in height) M. tuberculata snails during all seasons of the year.

Redia (Plates 1 and 2)

The mature redia is cylindrical in shape without lappets, measuring 787-1138 (944) m in length and 160-208 (190) m in maximum breadth. It has a well developed oral sucker measures 53 - 62 (57) m and short sacular intestine. The rediae contain cercariae in different developmental stages and germ balls which are located at the posterior end of the rediae.

Cercaria (Plates 1 - 4)

Biocellate and parapleurolophocercous cercaria, with oval to elongate body, measuring 217-253 (240) m in length and 98-128 (110.83) m in width. The tegument is covered with minute spines which are pronounced on the anterior third of the body (Figs 10 and 13). Oral sucker is oval in shape and subterminal in position. It measures 35- 51 (43.33) m in length and 33-40 (36) m in breadth, provided with a muscular protrusible organ (sacrificing or penetration organ). The ventral sucker can not be detected. There are two quadrangular dark eye spots located anteriorly at either sides of the body. The distance between any of which and the anterior border of the body is 66-80 (72.33) m . The pharynx which is circular in shape, is present in between or directly behind the eye spots, measuring 11-20 (15.5) m in diameter. The remainder of the alimentary canal is not detected in all specimens. The body contains seven pairs of big penetration glands arranged vertically in two rows extending from just below the level of pharynx to posterior extremity of the body. Penetration glands occupied 133-160 (148) m throughout the length of the body, leading to two bundles of ducts that open on the terminal edge of oral sucker. The excretory vesicle fills about the posterior fifth of the body, enclosed with the posterior three pairs of penetration glands. Its shape varies according to the state of contraction from oval to globular. The wall of the vesicle is composed of cells with granular cytoplasm and relatively large nuclei. The exact pattern of the collecting ducts and flame cells formula could not be traced due to the thick tegument. The primordium of the genital system occurs just anterior to the excretory vesicle and is partially surrounded by the cell bodies of penetration glands. It is oval or pyramidal heavily-stained mass of cells. The tail fits into a socket in the posterior end of the body. It is slender and provided with transparent lateral and dorsoventral fins. The lateral fins extend nearly to the anterior third of the tail, while the dorsal fin begins at a short distance from the end of the lateral fin, passing around the tip of the tail to terminate after the two fifth of the tail on the ventral side. Bristle-like structure can be better detected on the tail fins during the life span of cercaria. The tail measures 511-577 (535) m in length and 26-44 (32.83) m in maximum breadth (excluding the fins). The general appearance of cercaria, as revealed by SEM, is characterized by concave ventral surface with projected subterminal oral sucker. The tegument bears conspicuous transverse folds which are connected with each other to form anastomosing reticulum (Fig. 15). The openings of cystogenous glands are numerous and are located between tegumental folds on the lateral walls of the body, below the level of eye spots (Fig. 16). There are two rows of spike-like spines on the inner margin of oral sucker (Fig. 17). A number of sensory papillae are dispersed on the ventral surface of the cercaria (Figs. 15 and 16). There are also sensory papillae on the anterior region of the oral sucker (Fig. 17). The tail is provided with conspicuous finfolds armed with spine-like bristles (Figs. 14 and 15). Biological Characters The cercariae are moderately active swimmers. They swim randomly in all directions in the water of the container; they remain active for about 2-3 days at room temperature. The cercariae showed positive phototaxis where they are concentrated at the more illuminated part of the container. The cercariae relax to rest for a short period from time to time.

Discussion

Khalifa et al. (1977) mentioned that their cercaria was similar to the cercaria of Kasr Al Aini, described by Khalil (1932) and which synonymized by Price (1940) as the cercaria of H. pumilio. Also Khalifa et al. (1977) stated that the parapleurolophocercous cercaria (A) descibed by El-Gindy and Hanna (1963) was identical with cercaria of H. pumilio. Fahmy et al. (1976) in Assiut governorate recorded two parapleurolop-hocercous cercaria from fresh water snail Melania tuberculata. Their finding disagreed with the present finding especially in the measurements and structures. Hassan (1987) in Qena observed two types of cercariae in M. tuberculata. He claimed that the first type was identical with the cercaria of Haplorchis pumilio, while the second type was similar to the cercaria of Haplorchis yokogawai. Al-Shareef (1988) in Qualiobyia and Giza recorded parapleurolophocercous cercaria from M. tuberculata and identified it as cercaria of H. pumilio. Saad and Abed (1995) in Aswan described all the stages of the life cycle of H. pumilio. By comparing the present cercaria with the cercariae described by the above mentioned authors, it is obvious that the handled cercaria is the cercaria of H. pumilio (Table 1). On the other hand, the general architecture of the present cercaria, as revealed by SEM, shows structural adaptations for locating and infecting fish, that serves as the second intermediate host. Owing to the weak developed ventral sucker, attachment to the host skin is probably facilitated by the spined and ventrally concaved body. The attachment and penetration of the host skin may be due to the coordinated action of the preoral spines. The sensory papillae on the ventral surface of the body are probably involved in the navigation and recognition of the host and site of penetration. Similar observations were noticed by Abdul-Salam and Sreelatha (1993 a and b).

Explanation of plates

Abbreviations:

B: body. dff: dorsal fin fold. Dpg: ducts of penetration glands. E: eye. EV: excretory vesicle. G: gut. gb: germ balls. gp: genital primordia. Lff: lateral fin fold. M: mouth. Ocg: openings of cystogenous glands. OS: oral sucker. P: papillae. Pg: penetration glands. Ph: Pharynx. R: redia. So: sacrificing organ. Sp: spines.

Plate 1

Camera Lucida drawing of redia and cercaria of H. Pumilio recovered from Melania tuberculata snail. Fig.(1): Redia showing oral sucker, gut and germ balls in different developmental stages. Figs. (2, 3): Cercariae showing oral sucker, eye spots, pharynx, penetration glands, genital primordia, excretory vesicle and the tail with lateral and dorsoventral fin folds.

Plate 2

Redia and cercaria of H. pumilio recovered from M. tuberculata snail and stained with acetocarmine. Fig.(4): Entire cercaria, X=125. Figs. (5, 7): Rediae showing oral sucker and germ balls in different developmental stages, the redia in Fig. (5) cleared in clove oil, while the redia in Fig. (7) cleared in terpineol, X=125. Fig. (6): Body of the cercaria showing oral sucker, penetration glands and genital primordia, X=500. Fig. (8) Body of the cercaria showing eye spots, penetration glands, genital primordia and excretory vesicle, X=500. Fig. (9): Body of the cercaria showing sacrificing organ, X=500.

Plate 3

Cercaria of H. pumilio stained with acetocarmine. Specimens in Figs.(10, 11) cleared in terpineol, while specimens in Figs. (12, 13) cleared in clove oil. Fig. (10): Enlarged anterior part of cercarial body showing sacrificing organ and the spines which cover this region, X=1250. Fig. (11): Enlarged posterior part of cercarial body showing the posterior four pairs of penetration glands, genital primordia and excretory vesicle, X=1250. Fig (12): Anterior part of the body, X=500. Fig. (13): Enlarged anterior part of the body showing spination of this region, oral sucker and mouth opening, X=1250.

Plate 4

Micrographs of cercaria of H. pumilio. Fig. (14): Entire cercaria (ventral view) showing oral sucker, lateral and dorsoventral fin folds, X=200. Fig. (15): Cercarial body and anterior third of the tail showing oral sucker, sensory papillae and lateral fin folds provided with bristle like structure, X=500. Fig. (16): Ventral, view of cercarial body showing mouth, oral sucker, papillae and openings of cystogenous glands on both sides of the body, X=750. Fig. (17): Oral sucker showing spines and papillae, X=3500.

Acknowledgement

The corresponding author would like to express his thanks to Dr. Yousef Shahrour from Geology Department, Sebha University, Faculty of Science, for his help in editing and revising this paper.

Table (1):

Comparison between rediae and cercariae of Haplorchis pumilio described from Melania tuberculata by different authors and the present cercaria from the same snail.

Khalifa et al (1977) killed by heat fixed in 10 % formaline Cercaria Body length 180 - 350 120 - 220 (160) 240 - 258 180 - 204 243.2-326.4 (262.41) 200 - 210 (204) 200 - 215 217 - 253 (240) Body breadth 100 50 - 95 (72) 78 - 90 102 - 114 70.4 - 134.4 (92.48) 65 - 85 (75.1) 65 - 75 98 - 128 (110.83) Oral sucker 39 × 29 20 - 35 29.9-34.5 × 27.5-33.8 43.7-48.3 × 32.2-34.5 33.6-49 × 30.8-43.4 (40.39X35.91) 30 - 38.5 (35.2) 35 - 40 × 30-35 35-51 × 33-40 (43.33 × 36) Pharynx near oral sucker - after eye spots after the eye spots after the eye spots 10 11-20 (15.5) Eye spots from anterior end - - - - - 59 - 66-80 (72.33) Eye spots - - 9.2-11.5 × 4.6-5.2 - - - 11-15 × 8-11 (14.33 × 10) Tail length 486 390 - 560 (448) 480 - 552 430 - 510 448 - 576 (523.84) 362.5 - 455 (431.5) 420 - 430 511-577 (535) Tail width 39 20 - 35 (30) 30 - 36 24 - 30 25.6 - 57.6 (34.24) 24 - 30 (27) 30 26 - 44 (32.83) Patternsofof penetration glands 7 pairs in 2 rows similar to preceding similar to preceding similar to preceding similar to preceding similar to preceding Similar to preceding Redia Redia length - - - - - 700 - 765 (732.5) 600 - 1000 787 - 1138 (944) Redia width - - - - - 110 - 175 (142.5) 180 - 200 160 - 208 (190) Oral sucker - - - - - 40 - 50 (45) 30 - 35 53 - 62 (57)

Plate 2 References

Abdel-Malek, E.(1974): Medical and economic malacology. A subsidiary Harout Brace Jovanovich. Academic press New York and London.

Abdul-Salam, J. and Sreelatha, B. S. (1993 a): Studies on cercariae from Kuwait Bay. 1- Description and surface topography of cercaria kuwaitae I n. sp. (Digenea: Cyathocotylidae). Res. Rev. Parasit. 53 (3-4): 117 - 123.

Abdul-Salam, J. and Sreelatha, B. S. (1993 b): Studies on cercariae from Kuwait Bay. V- Description and surface topography of cercaria kuwaitae V n. sp. (Digenea: Heterophyidae). Jap. J. Med. Sci. Biol. 46: 155 - 164.

Al-Shareef, A. M. F. (1988): Biological studies on some larval helminths associated with some fresh and brackish water snails in Egypt. Ph. D. Thesis, Faculty of Science, Al - Azhar University, Cairo, Egypt.

Dawes, Ben (1946): The trematoda with special reference to British and other European forms. Cambridge University Press.

EI-Gindy, M. S. and Hanna, F. Y. (1963): Larval trematodes from snails Pirenella conica and Melania tuberculata with special reference to heterophyiasis. Bull. End. Dis. Bag. 5 (2): 33-58.

Frandsen, F. and Christensen, N. O. (1984): An introductory guide to identification of cercariae from African fresh water snails with special reference to cercariae of trematode species of medical and veterinary importance. Act. Trop. 41: 181-202.

Fahmy, M. A. M.; Mandour, A. M.; Arafa, M. S. and Omran, L. A. M. (1976): Larval trematodes recovered from Melanina tuberculata in Assiut province. Assiut Vet. Med. J. 3: 241 - 247.

Hassan, I. M. (1987): Studies on the role played by some snails in transmitting parasites to animals and man in Qena province. Ph. D. Thesis, Faculty of Science, Assiut University, Egypt.

Khalifa, R.; El-Naffar, M. K. and Arafa, M. S. (1977): Studies on heterophyid cercariae from Assiut province, Egypt. 1 - Notes on the life cycle of Haplorchis pumilio Looss, with a discussion on previously described species. Act. Parasit. Pol. 25: 25 - 38.

Khalil, M. (1932): The life history of a heterophyid parasite in Egypt. C. R. Congr. Int. Med. Trop. Hyg. 4: 137-147.

King, P. H. and Van-As, J. G. (1997): Morphology and scanning electron microscopy of cercariae shed by Bulinus tropicus (Krauss, 1848) in the free state, South Africa. J. Afr. Zoo. 111 (4) : 301 - 312.

Luhe, M. (1909): Parasitche plattwurmer Die susswasser fauna Dentschland. Heft 17, Trematodes: 1-217.

Nada, M. S. M. (1983): Biological and ecological studies on some snails in Sharkia governorate. Ph. D. Thesis, Fac. Vet. Med. Zagazig University.

Price, E. W. (1940): A review of the heterophyid trematodes with special reference to those parasitic in man. Int. Congr. Microbial. Rep. Proc. : 440 - 447.

Saad, A. I. A. and Abed, G. H. (1995): Studies on the life cycle of Haplorchis pumilio (Looss, 1896) Looss, 1899 with morphological redescription of larval and adult stages. J. Egypt. Soc. Parasit. 25 (3): 795 - 806.

W. H. O. (1973): Snail Identification Centre. A field guide to African freshwater snails 3- North East African species. Danish Bilharziasis Laboratory.

W. H. O. (1980): A practical guide to the identification of African freshwater snails Danish Bilharziasis Laboratory.

إعادة وصف ريديا وسركاريا طفيل هابلوركس باميليو باستخدام المجهر الضوئي والإلكتروني الماسح د. أحمد عبد الله طعيمة 1، أ. د. حسين الشناوي الشيخ 2، المرحوم أ. م. محمد قاسم ونس 3، أ. م. سعيد نور الدين 4، المرحوم أ. د. محمد عادل عباس توفيق 5

ملخلص البحث: تهدف هذه الدراسة إلى معرفة التركيب الدقيق لجليد رديا redia وسركاريا cercaria طفيل هابلوركس باميليوHaplorchis pumilio اللذان يمثلان مراحل الطفيل داخل القوقع، وذلك في محاوله لمعرفة كيف تستطيع السركاريا اختراق جلد العائل الوسيط الثاني (السمك)، ومعرفة تفاصيل أكثر عن جليد الرديا. وقد تم تجميع 1866 قوقع ميلانيا يتوبركيولاتا Melania tuberculata، وهي تمثل العائل الوسيط الأول الأساسي لطفيل هـ. باميليو في مصر، من مصارف وقنوات الأراضي القديمة وقنوات الأراضي المستصلحة حديثاً من محافظة البحيرة، مصر، بهدف الحصول على رديا ودسركاريا الطفيل. وقد أظهرت النتائج أن نسبة إصابة القواقع المجمعة من الأراضي القديمة كانت 4.75 % في المصارف و1.3 % في القنوات، في حين أن نسبة الإصابة في قواقع الأراضي المستصلحة حديثاً كانت 5.5 %. وبعد الوصف الدقيق للشكل الخارجي للرديا والسركاريا بالمجهر الضوئي والإلكتروني الماسح، وجدت تركيبات تكيفية تساعد السركاريا على عدوى السمك متمثلة في جسمها المقعر من الناحية البطنية، والأشواك الموجودة على الجزء الأمامي الظهري لجسم السركاريا وكذلك الأشواك الكبيرة التي توجد أمام الفم. وهذه التركيبات تساعد على الإلتصاق بجسم العائل الوسيط الثاني واختراقه بالإشتراك مع غدد الإختراق. وقد أظهرت صور المجهر الإلكتروني تفاصيل لم تظهر بالمجهر الضوئي على جسم السركاريا، مثل الحلمات الحسية وفتحات غدد التكيٌّس. ولم يُظهر المجهر الإلكتروني الماسح تفاصيل على جليد الرديا أكثر من تلك التي ظهرت باستخدام المجهر الضوئي. محاضر الطفيليات بكلية العلوم، جامعة سبها، ليبيا. ب. أ.: dr_ahmedtaima@yahoo.com 2 أستاذ الطفيليات بكلية العلوم، جامعة طنطا، مصر. 3 أستاذ الطفيليات المساعد بكلية العلوم، جامعة الأزهر (سابقاً)، مصر. 4 أستاذ الطفيليات المساعد بكلية العلوم، جامعة طنطا، مصر. 5 أستاذ الطفيليات بالمركز القومي للبحوث (سابقاً)، مصر.