Department of Zoology,

Faculty of Science, Assiut  University, Assiut, Egypt.

LIGHT AND SCANNING ‎‎ELECTRON MICROSCOPY OF THREE PARASITIC HELMINTHS FROM     FRESHWATER  FISHES IN ASSIUT, EGYPT.

(With 5 Plates)

By

Nawal A.M. Mazen and Hasnaa M.Thabit

(Received at 29/9/2005)

استخدام الميکروسکوب الضوئى والمجهر الالکترونى الماسح فى وصف ثلاثة  من الديدان المتطفلة على أسماک المياه العذبة فى محافظة أسيوط

نوال عبد السميع محمد مازن ، حسناء محمد ثابت

تم اعادة وصف ثلاث أنواع من الديدان الطفيلية وهى: 1- بروکمالاناس ليفيکونکس (ويدل, 1862 وبيلاس, 1923) ، تم دراسة هذا النوع لأول مرة بأستخدام المجهر الالکترونى الماسح فى أسيوط. حيث أظهرت الدراسة وجود ستة شفاه أمامية وأربعة حلمات تحت وسطية فى الجزء الأمامى, وأسنان رفيعة تشبه أسنان المنشار فى قاع تجويف الفم, وثمانية حلمات مستديرة فى الجزء الخلفى. وهذه الصفات قد تکون ذات أهمية کبيرة فى التمييز بين الانواع المختلفة لجنس البروکمالينس. 2- تم اعادة وصف نوعين من ديدان  الرأس شوکيات ودراستها بالمجهر الالکترونى الماسح لتوضيح السطح الخارجى والاشواک الموجودة به لکلا الطفيلين وهما: أ- ريدينوريکس نيلوتيکس (محمدين 1989). ب- أکانثوجيرس (أکانثوسينتس) تيلابيا (بيلاس, 1947)0 وأظهرت الدراسة تفاصيل هامة وخاصة فى الأشواک التى تحيط بالبوز والجسم، والتى لها أهمية خاصة فى التمييز بين أجناس وأنواع الرأس شوکيات.

SUMMARY

During the present study, a nematode and two acanthocephalan parasites of freshwater fishes were collected from Assuit city to be examined by scanning electron microscope (SEM). The surface fine structure of the three parasites, was revealed in order to help their identification and distinction from other allied species. These worms are: 1-Procamallanus laeviconchus (Wedi, 1862), Baylis, 1923, in which SEM illustrated: six anterior elevations or lips, four submedian cephalic papillae, minute saw-like teeth at the bottom of the buccal capsule and eight blunt caudal papillae. These may be important features in differentiation between different Procamallnus spp. 2-A- Rhadinorhynchus niloticus Mohamadin, 1989. B- Acanthogyrus (Acanthosentis) tilapiae, Baylis, 1947. SEM of acanthocephala illustrated more detail structures in the proboscis and body spines which are of great importance in generic and species identifications.

Key words: SEM, Procamallanus, Rhadinochynchus, Acanthogyrus, fresh water fish, Assiut, Egypt.

INTRODUCTION

Scanning electron microscope (SEM) makes possible the study of the surface structure which is taxonomically highly significant in the different  species of parasites. During investigations of the parasite fauna of freshwater fishes carried out in Assuit Governorate, one nematode and two acanthocephalan parasites were collected from the small intestine and stomach of Synodontis schall, Lates niloticus, and Oreochromis niloticus. Thenematode Procamallanus (Spirocamallanus) neocaballeroi (Caballero-Deloya, 1977) is a specific intestinal parasite of freshwater characid, Astyanax fasciatus (Cuvier) in Mexico. It was originally described from Lake Catemaco in Veracruz (Caballero-Deloya, 1977) and recently  has been recorded from Cenotes (Sinkholes) in the coastal region of the State of Quintana Roo of Yucatan Peninsula (Moravec et al., 1995 a and b). Information on the development of these nematodes and their larval morphogenesis still remains insufficient (Anderson, 1992). The same concerns congeneric species of the subgenus Procamallanus, where the development of only three African and Asian species has so far been studied (Moravec, 1975; Wang and Ling, 1975; Den and Majumder, 1986 and Sinha, 1988). Moravec et al. (2000) described 3 new species of Procamallanus (spirocamallanus) from the intestine of freshwater fishes in Mexico. Moravec and Van As (2004) recorded five nematode species from stomach and rectum of Spotted Squeaker Synodontis nigromaculatus. Moravec (1974 & 1975) described Procamallanus laeviconchus from Clarias gariepinus. P. laeviconchus has been reported from Egypt, Ghana, Nigeria, Senegal, South Africa, Sudan, Uganda and Zaire (Vassiliades, 1975; Shotter, 1980 and Khalil & Polling, 1997). In south Africa, it has been recorded from Clarias gariepinus in dams of the Limpopo River drainage system (Mashego & Saayman, 1981; Boomker, 1982, 1994).

Acanthogyrus Thapr, 1927 (Acanthosentis Verma & Datta, 1929), belonged to family Quadrigyridae Van Cleave, 1920. Golvan (1959) synonymised Acanthosentis with Acanthogyrus and reduced Acanthosentis to subgenus, based on the number of hooks on the proboscis. The normal subgenus Acanthosentis has three circles of eight proboscis hooks. Amin, (2005) described two new species of Acanthocephala, in Japan, Acanthogyrus (Acanthosentis alternat n.sp & A.(A.) paraceptaclis n.sp from Lake Biwa drainage fishes.

           The genus Rhadinorhynchus Luhe, 1911, a synonym with genus Nipporhynchus Chandler, 1934 was related to family Rhadinorhynchidae Travassos, 1923. The characters of Rhadinorhynchus niloticus were encountered and decribed by Mohamadin (1989) for the first time in Egypt from the intestine of Latus niloticus & Bagrus bayad from the River Nile at Quena Province. The present worm was described  from the stomach (new habitat) and intestine of Synodontis schall & Oreochromis niloticus from the River Nile at Assiut province. The presence or absence of trunk spines and the number of cement glands  define the family Echinorhynchida (Amin, 1985 and Golvan, 1969).

MATERIALS  and  METHODS

The adults of the nematode Procamallanus laeviconchus  and the Acanthocephalans Rhadinorhynchus niloticus, Acanthogyrus (Acanthosentis) tilapiae were collected from the stomach and  intestine of Synodontis schall, Latus niloticus, Bagrus  bayad, Bagrus docmac and Oreochromis niloticus fishes that were purchased from Assiut fishmarket.The three species of worms were washed thoroughly in normal saline and fixed in AFA solution (2% acetic acid, 3% formaldehyde and 70% alcohol). Part of the samples was used for the worm identification by light microscopy.

For SEM studies, samples of the collected worms were fixed in 2.5% glutraldehyde solution in 0.1 phosphate buffer (PH 7.4) for 30 minutes. After washing in the same buffer solution, they were fixed in 1% osmium tetroxide for one hour. Eggs were collected from the uterus of female worms and prepared for SEM (Appleton and Belinda, 1989) and coated with gold palladium, using apolarone 500 sputter coating unit and viewed on Hitachi 5.570 Scanning Electron Microscope at SEM unit in Assiut University.

RESULTS

1-a) Light microscopic description of  Procamallanus laeviconchus:

The body is elongated, filiform and covered with smooth and thick cuticle with distinct transverse striations. The mouth aperture is provided with a chitinous buccal capsule. This is followed by a cylindrical oesophagus which is differentiated into a muscular anterior part of 0.29-0.43 mm length, (average 0.35mm) and a longer glandular posterior one which measures 0.43-0.70mm, (average 0.52mm) (Plate  I, Fig.1). The muscular oesophagus is surrounded by a nerve ring at distance 0.15mm from the anterior end in the male and at 0.227mm in the female. The glandular oesophagus opens into the intestine which leads to the anus.

Male:

            The males are usually smaller and more slender than the females and also fewer in number. The body measures 2.17-3.47mm (average 2.99mm) in length by 0.086-0.13mm (average 0.11mm) in width. The posterior end of the male is sharply curled ventrally and bears 8 pairs of pre-cloacal papillae and 4 pairs of post-cloacal papillae. There are two unequal spicules, the longer one measures 0.06-0.16mm (average 0.09mm) in length and the smaller one is hardly visible (Plate I, Fig 2).

Female:

           The female is usually longer than the male. Its body measures 3.47-5.86mm (average 4.7mm) in length by 0.13-0.21mm (average 0.18mm) in width The posterior extremity of the body is conical in shape and  provided with blunt processes or papillae. The anus is situated near the posterior end at distance 0.06-0.10mm (average 0.077mm). Vulva is situated at the middle of the body (Plate I, Fig 3). The egg is very small in size and measures 25.2-43.4 µ(32.4 µ) in length by 14.4-32.4 µ (average 24.17 µ) in width.

1-b)  SEM  description  of Procamallanus laeviconchus:

            The electron micrographs showed  that  the worm has rounded  mouth  aperature (Plate II; Fig.1) surrounded by six elevations or lips and four submedian cephalic papillae (Plate II; Fig. 2). There are minute, saw – like teeth at the bottom of the buccal capsule (Plate II; Fig. 3). Deirids are very small, simple and situated behind the anterior end  (Plate I I; Fig. 4).

The posterior extremity of the male body is conical in shape and  provided with eight blunt processes or papillae (Plate II; Fig. 5). The anus is situated near the posterior end (Plate II; Fig. 6). The posterior extremity of the male is sharply curled ventrally and bears caudal papillae (Plate II; Fig.7). SEM revealed that The body is covered with smooth and thick cuticle with distinct transverse striations (Plate II;    Fig. 8).

2-a) Light microscopic descrioption of Rhadinorhynchus  niloticus:

Male:

            The body is long, slender and measures 7.5-10.6mm (average  9mm) in length with a maximum width of 0.47-1mm (average 0.8mm). It is covered anteriorly by irregularly arranged spines which cover about 1.5-3.1mm (average  2.6mm) of the ventral surface but only 1.9-2.5mm (average  2.2mm) of the dorsal surface (Plate I; Fig. 4). Proboscis is 0.86-303mm (average 1.77mm) long by 0.26-0.56mm (average  0.39mm) wide, armed with 16-22 longitudinal rows of hooks, each of them cosists of 22-26 strong recurved hooks (Plate I; Fig. 6).

Female:

           Thefemale body (Plate I, Fig. 5) measures 9-12mm (average 10mm) in length and 0.56-1mm (average  0.79mm) in maximum width. Proboscis measures 0.47-4.6mm (1.46mm) in length by 0.2-0.3mm (average 0.29mm) in width. It bears 16-22 longitudinal rows of hooks of 22-26 hooks each. Each hook is strongly recurved and composed of two parts, blade and root. Eggs are elongated with polar prolongations. Each measures 0.12-0.16mm (average 0.14) in length by 0.014-0.018mm (average  0.017mm) in width (Plate I; Fig. 7).

2-b) SEM description of  Rhadinorhynchus  niloticus:

            SEM micrographs confirm the light microscopic desciption. The body consists of an anterior proboscis, a neck and a trunk. The proboscis is cylindrical, long and provided with numerous rows of strongly recurved hooks (Plate III; Figs 1,2,3). The anterior half of the trunk is armed with irregularly arranged short triangular  spines (Plate III; Fig 4). These spines are more extensive ventrally than dorsally. There are wrinkles or irregular striations at the trunk region which disappear near the last third of the body (Plate III; Fig. 5). The posterior end of the worm contains a genital pore which is terminal in position (Plate III;  Fig. 6).

3-a) Light microscopic description of Acanthogyrus (Acanthosentis) tilapiae:

The body is elongated, cylindrical, gradually enlarging from anterior end to its middle and narrowing from there to posterior extremity. The body is surrounded by spinose cuticle at the trunk region. Proboscis is globular, short, with three transverse rows of six hooks each. There is a pair of lemnisci projection posteriorly. They are slender, cylindrical, subequal and much longer than proboscis receptacle.

Male:

The body measures 2.21-3.56mm (average  3.03mm) in length by 0.78-1.17mm (average  0.97mm) in maximum width. Proboscis is 0.079-0.097mm long by 0.064-0.083mm wide, with an average (0.088mm X 0.074mm). Proboscis hooks of anterior row are 0.039-0.047mm long (average 0.043mm). The middle hooks of the anterior row are needle-shaped, while the peripheral ones are curved. Hooks of the second row are 0.022-0.034mm long (average 0.028mm). The hooks of basal row are 0.011-0.015mm long (average 0.013mm). There are two unequal lemnisci situated at the first body third; the shorter lemniscus is 0.59-0.80mm (0.72mm) long and longer one is 0.60-0.89mm(0.75mm) long. They are equal in width measuring 0.036mm. The testes are ovoid in shape, tandem, slightly touching each other, subequal and occupying about the middle third of the body (Plate IV;  Fig.1).

Female:

The body is elongated,with maximum width in the middle region (Plate IV, Fig. 2) . It measures 3.13-6mm (average  5.82mm) in length by 0.87-1.5mm (average 1.22mm) in width. The proboscis measures 0.079-0.126mm (average 0.098mm) in length by 0.072-0.079mm (average 0.076mm) in width. Proboscis hooks are similar to those of the male, with the same arrangement and number. The two lemnisci are easily observed. Eggs are small in size, elliptical or fusiform and measuring 12.6-25.2µ (average 18.72µ) in length by 5.4-10.8µ (average 7.56µ) in width (Plate IV; Fig. 3).

3-b) SEM description of Acanthogyrus (Acanthosentis) tilapiae: 

The worm body is elongated and cylindrical (Plate IV; Fig. 4). Proboscis is globular, with three transverse rows of six hooks each (Plate V; Fig. 2). There are wrinkles at the trunk region (Plate V; Fig. 3). The cuticle is provided with transverse rings of cuticular spines (Plate V; Fig. 4). Beneath the cuticle, there is a syncytial hypodermis which contains a number of nuclei, they appear as amoeboid structures on the body surface (Plate V; Fig. 5). The posterior end of the body contains the genital pore (Plate V; Fig. 6).

Explanation  of  figuers

Light microscope of Procamallanus laeviconchus and Rhadinorhychus niloticus.

Plate I:

Fig. 1: Anterior end of Procamallanus laeviconchus showing buccal capsule X100.

Fig. 2:Posterior end of male Procamallanus  laeviconchus showing spicule X100.

Fig. 3: Posterior end of female Procamallanus  laeviconchus  showing anus X100.

Fig. 4 : Photomicrograph of maleRhadinorhychus niloticus.

Fig. 5 : Photomicrograph of femaleRhadinorhychus niloticus.

Fig. 6: Photomicrograph of Proboscis  Rhadinorhychus niloticus X50.

Fig. 7: Photomicrograph of the egg of Rhadinorhychus niloticus X400.

SEM of the anterior end  of Procamallanus  laeviconchus.

Plate II:

Fig. 1: Lateral view of mouth opening.

Fig. 2: Top view showing cephalic papillae.

Fig. 3: Top view showing Saw-like teeth and the bottom of the buccal  capsule.

Fig. 4: Showing deirid (d).

Fig. 5: Top view showing caudal papillae in male.

Fig. 6 : Lateral view showing cloaca (c)and caudal papillae (P) in male.

Fig. 7: SEM micrograph of the lateral view of the posterior end of male showing caudal papillae.

Fig. 8:  SEM micrograph showing transverse striations of the cuticle.

SEM of Rhadinorhynchus  niloticus

Plate III:

Fig. 1: Proboscis and anterior part of the trunk.

Fig. 2&3: Magnification of Proboscis hooks.

Fig. 4:  Showing trunk spines  of  Rhadinorhynchus  niloticus .

Fig. 5:  Showing transverse striations on the trunk region.

Fig. 6: Showing  posterior end of the worm and genital pore (g.p.)

Light microscope of Acanthogyrus (Acanthosintis) tilapiae

Plate IV:

Fig. 1 : Photomicrograph of male.

Fig. 2 : Photomicrograph of female.

Fig. 3 : Photomicrograph of  eggs X400.

Fig. 4 : SEM of Acanthogyrus (Acanthosentis).

SEM of Acanthosentis (Acanthosentis) tilapiae

Plate V :

Fig. 1: Proboscis and trunk region.

Fig. 2: Magnification of proboscis and hooks.

Fig. 3: Showing the wrinkles of the trunk region.

Fig. 4: Magnification of trunk spines.

Fig. 5: Showing Amoeboid structures (cuticular pits)of the body surface.

Fig.6 : Posterior end of genital pore (gp).

DISCUSSION

           Procamallanus  comprises numerous species described from fresh water and marine fishes. Many authors consider forms with spiral ridges in the buccal capsule to represent a separate genus Spirocamallanus Olsen, 1952. From comparison of, the present material Procamallanus  laeviconchus with  specimens of Imam (1971), Al-Bassel(1990) and El-Ganiny (1995), it was found that both males and females of the present parasite are shorter than their specimens. The present males have two unequal spicules, the shorter spicule is hardly visible while  the longer one is longer than their specimens. Moreover minor differences in measurements of  buccal capsule, muscular and glandular oesophagus, distance to nerve ring and to vulva from anterior end were also observed.  

             As compared to the P. laeviconchus specimens from Clarias  gariepinus studied by Moravec (1974, 1975) in Egypt, specimens of the

present material from Synodontis  spp. show some differences, mainly in the number and arrangement of caudal papillae and the preequatorial situation of vulva in the gravid female .

             SEM showed detailed structures particularly in the anterior end and caudal end of the male in the form of 6 anterior lips, 4 submedian cephalic papillae, minute saw-like teeth at the bottom of the buccal capsule and 8 blunt caudal papillae. These might be of great importance in differentiation of different procamallanus spp.

              Acccording to Yamaguti (1958), the present first acanthocephalan parasite belongs to genus Rhadinorhychus  Luhe, 1911 which is related to family Rhadinorhynchidae Travassos, 1923 the characters of the present acanthocephalon worms coincide with those of Rhadinorhynchus  niloticus  encountered and described by Mohamadain (1989) for the first time in Egypt from the intestine of Lates niloticus and Bagrus bayad from River Nile at Qena province. Compared  with  previous species, we noticed that there are common characters between the present parasite and these specimens but, with minor differences in measurements. Moreover, the present females are little bigger than males while in all other species,  the females are twice or even more the length of males. Also, it is similar to the other species in the size, shape and arrangement of probocis hooks, testes and vagina shape.

              The egg shape of the present parasite is similar to that of Ebraheem (1992) specimen but is longer and thinner than that of other species. Also, the entire male genitalia of the present parasite is shorter than that of Ebraheem (1992) specimen while it has longer female genitalia. The present work added SEM study for this species which showed the shape of proboscis, hooks on the proboscis, striations of trunk region and shape of posterior end of worm. Moreover, the fishes Bagrus docmac, Synodontis schall & Oreochromis niloticus were for the first time recorded as hosts for the present material in Egypt. Also, the stomach of Lates niloticus is considered a new site of infection for this parasite. Golvan (1969) defined explicitly the Rhadinochynchids as possessing four cement glands and trunk spines. Amin (1985) proposed that several genera of  Rhadinorhynchidae with eight cement glands and no trunk spines which include Leptorhynchoide, Metacanthocephalus and Pseudoleptorhynchoides. Acanthogyrus (Acanthosentis) tilapiae  was described  from regions in Egypt by Sahlab (1982), El-Naffar  et al. (1983), Negum El-Din (1987), Abu-El Ezz (1988), Al-Bassel (1990),  Ebraheem (1992) and El-Gganiny (1995) since fish host in Egypt is mainly Tilapia niloticus, T. zilli and T. galiilaea except Abu-El Ezz  (1988) recorded this parasite from only a  female of Anguilla vulgaris.

       The present second acanthocephalan Acanthogyrus (Acanthosentis) tilapiae is nearly similar to those previously described from Egypt but differs in size of the body. proboscis hooks, lemnisci and testes. These differences in measurements might be due to age differences of the parasites examined. The cement gland nuclei are not seen in the present study, while, Ebraheem (1992) and  El Ganiny(1995) reported these nuclei. The egg size is smaller than that of other forms. From comparison of the present material with Acanthogyrus (Acanthosentis) malawiansis the proboscis hooks of the middle circle are longer than those in the anterior circle, and the larger number of sensory papillae found on the trunk are not reported in the present material, Acanthogyrus (Acanthosentis) tilapiae. The giant subcuticular nuclei of A. (A.) malawiansis including one ventral and one or two dorsal and complx vessel – shaped in profile or amoeboid  star shaped in face view (Amin and Sherman,1999) were not observed in the present material .

SEM of acanthocephala in the present work illustrated detailed structures in the proboscis and body spines which are of great importance in generic and species identification.

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