Barson_cestodes.indd INTRODUCTION Fish helminthology in southern Africa is not as wide- ly studied as other aspects of aquatic parasitology and fish biology. This is probably because helminths mainly infect the internal organs, predominantly the gastrointestinal tract which, for humans, does not com prise the edible portion of the fish. Although fishermen and anglers regularly encounter encysted “grubs” (metacercariae) in the skin and muscles of fish (B. Marshall, personal communication 2002), they regard them as just a nuisance, notwithstand- ing the biological and economic impact they may have on the fish species. Only a few studies on cestode and trematode para- sites of fish in South Africa have been documented (Whitfield & Heeg 1977; Boomker, Huchzermayer & Naude 1980; Mashego 1977, 1981, 1982, 2001; Ma- she go & Saayman 1989; Brandt, Van As, Schoon- bee & Hamilton-Attwell 1981; Van As, Schoonbee & Brandt 1981; Britz, Van As & Saayman 1985; Saay- man, Mashego & Mokgalong 1991, Luus-Powell & Mashego 2003). In 1984, Van As and Basson com- piled a checklist of South African freshwater fish parasites, but many new species have been discov- ered since then (Khalil & Polling 1997). Paperna (1996) published a concise update of the parasitic diseases of fish in Africa, in which the occurrence and geographical distribution, life cycles, pathology, epizootiology and control of the parasites is de- scribed. The tapeworms (Cestoda) and flukes (Digenea) in- fect internal organs of fish (Roberts & Janovy 2000) and their life cycles involve more than one interme- diate host, including planktonic copepods, molluscs and fish. Piscivorous birds, in which some helminths 101 Onderstepoort Journal of Veterinary Research, 73:101–110 (2006) On cestode and digenean parasites of Clarias gariepinus (Burchell, 1822) from the Rietvlei Dam, South Africa M. BARSON1, 2 and A. AVENANT-OLDEWAGE1 ABSTRACT BARSON, M. & AVENANT-OLDEWAGE, A. 2006. On cestode and digenean parasites of Clarias gariepinus (Burchell, 1822) from the Rietvlei Dam, South Africa. Onderstepoort Journal of Veterinary Research, 73:101–110 Sharptooth catfish, Clarias gariepinus, from the Rietvlei Dam near Pretoria, South Africa were exam- ined for internal platyhelminth parasites. Two adult cestodes, Polyonchobothrium clarias (stomach) (prev alence 71 %, mean intensity = 5, n = 7) and Proteocephalus glanduliger (anterior intestine) (prev- alence 14 %, mean intensity = 2, n = 7), were found in the gut while metacercariae of one larval dige- nean, Ornithodiplostomum sp. (prevalence 14 %, mean intensity = 140, n = 7), were found encysted in the muscles. The morphology of these species, based on light and scanning electron microscopy as well as histological analysis, and how they differ from previously described specimens, are dis- cussed. Ornithodiplostomum is a new record in southern Africa. Infection levels of the host fish were mild compared to records from previous surveys. Keywords: Cestode, Clarias gariepinus, digenean, platyhelminth, Rietvlei Dam, South Africa 1 Department of Zoology, University of Johannesburg, Kingsway Campus, P.O. Box 524, Auckland Park, Johannesburg 2006, South Africa 2 Author to whom correspondence is to be directed. E-mail: barson@science.uz.ac.za. Present address: Department of Biological Sciences, University of Zimbabwe, P.O. Box MP 167, Mt Pleasant, Harare, Zimbabwe Accepted for publication 23 January2006—Editor 102 Cestode and digenean parasites of Clarias gariepinus (Burchell, 1822) from Rietvlei Dam, South Africa develop into adult stages, are important in that they can disseminate parasite eggs over long distances, making it difficult to control the spread of infections between water bodies in different catchments (Saay- man et al. 1991). In wetland systems such as the Riet- vlei Dam locality, there is a high diversity of aquatic birds, both resident species (e.g. ducks) and migra- tory species (e.g. cormorants) (M. Barson, personal observation 2003). While there is much need to un- derstand the interactions between fish and birds in the transmission of helminth infections, only a few parasitological studies on piscivorous birds in Africa are documented (Beverly-Burton 1963; Ukoli 1968; Saayman et al. 1991; Mokgalong 1996; Barson 2004; Barson & Marshall 2004). The sharptooth catfish, Clarias gariepinus, (Burchell, 1822) investigated in this study, is a widely distrib- uted food fish in Africa (Safriel & Bruton 1984; Skel- ton 2001) and is one of the best species being tar- geted for aquaculture and biological research. This study was carried out as a survey of the inter- nal parasites that are found in C. gariepinus from the Rietvlei Dam, which can be used in the fish health assessment index that has been developed for South African fish by Avenant-Oldewage (2001). The ob- jectives of this paper were to specifically identify and classify the internal platyhelminth parasites col- lected from the host fish (Clarias gariepinus) based on their morphological features, and to note their prevalence and mean intensity in the Rietvlei Dam. FIG. 1 The location of the Rietvlei Dam inside the Rietvlei Nature Reserve. X indicates point of sam- pling. Bar = 2 km Pretoria Rietvlei Nature Reserve Johannesburg Rietvlei Dam Marais Dam Sesmylspruit South Africa 0 0 ,1 0 ,5 1 km 2 km 103 M. BARSON & A. AVENANT-OLDEWAGE MATERIALS AND METHODS Study area The Rietvlei Nature Reserve (25°41’22” S, 26°37’48” E) lies between Pretoria and Johannesburg in the Gauteng Province, the economic hub of South Afri- ca (Fig. 1). It is solely responsible for conservation of the Sesmylspruit catchment area, and the Rietvlei Dam (25°32’30” S, 28°16’46” E) currently supplies 27 % of Pretoria’s water requirements (Wessels 1998). The dam is supported by the smaller Marais Dam, which lies approximately 4 km upstream, and the two are separated by a wetland (Fig. 1). Further upstream, just before the Sesmylspruit enters the reserve, effluent from a number of industries and a wastewater treatment plant is discharged into the stream, directly affecting the two dams. Collection of fish and parasites Fish were collected from the Rietvlei Dam using large mesh gill nets in May 2003. The gastrointestinal tract was dissected from the rectum to the oesophagus and parasites encountered were carefully detached from the stomach or intestinal mucosa. Portions of the skin between the lateral line and dorsal fin of each fish were peeled off with forceps and fillets of muscle tissue were cut and examined for encysted parasitic forms. The liver, spleen, gall bladder and kid neys of each fish were also examined for para- sites or cysts. Trematode cysts from the muscle were teased man- ually to release metacercariae, which were fixed in hot alcohol-formal-acetate (AFA) and preserved in 70 % ethyl alcohol. Cestodes from the intestinal tract were swirled in 0.1 % sodium chloride (saline) to re- lax them, fixed in hot AFA and preserved in 70 % ethyl alcohol. Cestodes were stained with aqueous acetocarmine solution as described by Khalil (1991). Digenean trematode metacercariae were stained in Delafield’s haematoxylin and counterstained in eosin. Standard microtechnique procedures were used to prepare transverse serial sections of cestodes, which were embedded in synthetic resin (Transmit LM) with a curing point of 70 °C. The specimens were sectioned with a rotary resin microtome (Anglio Scientific) at 5 μm thickness, and the sections were stained with AZAN, a trichrome stain. The parasites were identified based on their mor- phology, and using drawings and light micrographs taken with a Zeiss Axioplan microscope. Scanning electron micrographs were taken with a JEOL 6100 scanning electron microscope (SEM). Drawings and measurements were done with a Zeiss Standard 25 microscope equipped with a drawing tube. The fol- lowing keys were consulted for identification: Ya ma- guti (1958) and Gibson, Jones & Bray (2002) for the trematodes, and Yamaguti (1959), Freze (1965), Schmidt (1986), Khalil, Jones & Bray (1994) and Rego (1994) for the cestodes. Parasite prevalence and mean intensities were calculated as defined by Margolis, Esch, Holmes, Kuris & Schad 1982. Voucher specimens were deposited in the zoologi- cal collection of the University of Johannesburg (for- merly Rand Afrikaans University), South Africa. RESULTS AND DISCUSSION The platyhelminth endoparasites found in C. gariepi- nus include two adult cestode species, Poly oncho- bothrium clarias and Proteocephalus glan duliger, and digenean metacercariae of the genus Ornitho- dip lostomum (Table 1). Cestoda Polyonchobothrium clarias (Woodland, 1925), (Fig. 2 and 5) Scolex rectangular with a flat to slightly raised rostel- lum armed with a crown of 26–30 hooks (mean 28; n = 6). Rostellum divided into two semicircles each bearing 13–15 hooks. Hooks at the end of each semi- circle smaller than the others. Two longitudinally elongated bothria in line with the gaps between the crowns of hooks. Immature proglottids of strobila not completely segmented. Some mature segments ap- parently fused as shown by SEM (Fig. 5E). Testes medullary; uterus anterior to ovary, highly folded and occupying the greater portion of gravid proglottids. Vitellaria cortical. Eggs unoperculate and embryo- nated. Measurements of structures are given in Table 2. Polyonchobothrium clarias is widely distributed in siluroid fishes from African freshwater fishes, hav- ing been recorded from Nigeria in the North African catfish Clarias lazera (= C. gariepinus) Cuvier & Valenciennes, 1840 (Aderounmu & Adeniyi 1972). It was also reported in the Bagrid catfish Chrysichthys thonneri Steindachner, 1912 from Gabon, the mud- fish Clarias anguillaris (Linnaeus, 1758) and Hetero- branchus bidorsalis Geoffroy Saint-Hilaire, 1809 from Senegal (Khalil 1973), and in C. anguillaris from Egypt (Amin 1978). In southern Africa, P. clarias was first observed and recorded by Mashego (1977) from C. gariepinus in seven dams in the Le bowa region, Limpopo Province, South Africa. The only 104 Cestode and digenean parasites of Clarias gariepinus (Burchell, 1822) from Rietvlei Dam, South Africa other record of the parasite was from the Middle Letaba Dam (Saayman et al. 1991), also in the Lim- popo Province. Its high prevalence in the Rietvlei system, as well as in the Vaal Dam (M. Barson & A. Avenant-Oldewage, unpublished data 2003), seems to suggest that the cestode is widely distributed in C. gariepinus in the country. In Zimbabwe, Chishawa (1992) and Douëllou (1992) recorded an intestinal cestode from the brown squeak- er, Synodontis zambezenis Peters, 1852, and C. ga- riepinus from Lake Kariba. Although they mistook it for larval P. clarias, it was apparently an adult with 38 hooks on its apical crown. Larvae only occur in copepod intermediate hosts. The low intensity of P. clarias in hosts from the Riet- vlei Dam (up to 11 worms per fish) would be expect- ed to inflict minimal damage on the host tissue (Pa- perna 1996), whereas high parasitic loads in the gall bladder have been shown to cause granulomatous nodules and fibrosis (Wabuke-Bunoti 1980). Mashego (1977) recorded intensities of up to 200 in C. gariepi- nus from Lebowa. In Nigeria, Aderounmu & Adeniyi (1972) reported a heavy infection of 123 worms per host in C. gariepinus, causing nodules at the point of attachment. Barson & Avenant-Olde wage (unpub- lished data 2003) recorded more than 100 individu- als infecting one specimen of C. gariepinus in the Vaal Dam. The fact that the tapeworms physically FIG. 2 Polyonchobothrium clarias (A) mature proglottid, (B) scolex, (C), (D) gravid proglottids (scale bar = 200 μm), (E) eggs (scale bar = 50 μm), b = bothrium, ec = excretory canal, gp = genital pore, hk = hook, ov = ovary, r = rostellum, t = testis, ut = uterus, vt = vitel laria gp ut ec t ov vt ut eggs A B C D E r hk b ov 105 M. BARSON & A. AVENANT-OLDEWAGE resisted detachment from the gut mucosa suggests that the suction created by the bothria and the clasp of the apical hooks could cause severe pathological effects in heavy infections. Proteocephalus glanduliger (Janicki, 1928) Fuhrmann, 1933 (Fig. 3 and 5F) Scolex unarmed, with four cup-shaped suckers ar- ranged symmetrically around a protrusible rostel- lum; neck region not differentiated into well-formed proglottids. Glandular organ present in one speci- men but not apparent in the other, approximately similar in size to suckers. All proglottids broader than long. Genital pores lateral and alternating. Testes medullary as shown in resin sections (Fig. 3E and 5F). Specimens much larger than those de- scribed by Freze (1965) and Mashego (2001) (Table 3). Despite the abundance and diversity of proteo- cephalid cestodes in African freshwater fish (Khalil & Polling 1997), only one species, Proteocephalus glanduliger has been recorded in South Africa from C. gariepinus (Mashego 1977, 2001; Van As & Bas- son 1984; Saayman et al. 1991). Only two speci- mens of this cestode were recovered from one of the seven catfish from the Rietvlei Dam. Mashego TABLE 1 Prevalence and intensity of endohelminths of C. gariepinus from Rietvlei Dam Parasite species Location in host n* Prevalence (%) Intensity Mean intensity Polyonchobothrium clarias Stomach mucosa, anterior intestine 7 71.4 1–11 5.0 Proteocephalus glanduliger Anterior and mid- intestine 7 14.0 1–2 2.0 Ornithodiplostomum sp. metacercariae Mid-dorsal muscles between dorsal fin and lateral line 7 14.0 0–140 140.0 *n = sample size TABLE 2 Polyonchobothrium clarias measurements (in μm, unless otherwise stated) n = 6 Range Mean Length (mm) Maximum width Number of proglottids Scolex size (L*W)1 Number of hooks in apical rostellum Mean size of hooks Mean size of immature proglottids (L*W) Mean size of mature proglottids (L*W) Mean size of gravid proglottids (L*W) Mean egg size 23–43 280–701 133–230 420–680*160–230 26–30 35–57 80–180*146–170 122–179*129–222 240–378*280–710 28–42.5*21–38 29.8 506 183.0 527*198 28 44 150*151 151*186 330*506 32.8*27.0 1 L*W : L = length, W = width TABLE 3 Proteocephalus glanduliger measurements (in mm, unless otherwise stated) Measurement 1 2 Mean Strobila length Strobila maximum width No. of proglottids Scolex (L*W) 1 Size of apical organ (L*W) Size of suckers (mean L*W) Size of mature proglottid (L*W) Size of gravid proglottid (L*W) Size of cirrus sac (L*W) Size of testes 7.04 0.52 58 1.08*0.82 0.36*0.28 0.36*0.32 0.13*0.45 0.14*0.03 – 13.0 1.08 45 1.28*0.92 – 0.48*0.40 – – – 10.02 0.80 51.5 1.20*0.88 0.36*0.28 0.44*0.36 – – – 1 L*W : L = length, W = widths 106 Cestode and digenean parasites of Clarias gariepinus (Burchell, 1822) from Rietvlei Dam, South Africa FIG. 3 Proteocephalus glanduliger (A, D) scolex (scale bar = 400 μm), (B) gravid proglottid, (C) mature proglottid, (E) transverse section (scale bars = 200 μm), ec = excretory canal, go = glandular organ, cs = cirrus sac, lm = longitudinal muscles, s = sucker, sg = shell gland, t = testis, tg = tegument, ut = uterus, vt = vitellaria A B C D E go ut ec t vt vt ut ov S sg cs ec cs ec t vt lm tg 107 M. BARSON & A. AVENANT-OLDEWAGE (1977) recorded it in 11 of 337 hosts and Mashego (2001) in 11 of 115 hosts, with a mean intensity of seven parasites per fish. While the present speci- mens were much larger (Table 3) than those de- scribed by Janicki (Freze 1965) and Mashego (2001) from C. anguillaris and C. gariepinus, respectively, the glandular organ at the apex of the scolex was much smaller and almost equal in size to the suckers (Fig 3A). Serial sectioning of the present material confirmed the medullary positioning of the testis and vitellaria (Fig. 3E and 5F), a characteristic of the ge- nus Proteocephalus (Freze 1965; Schmidt 1986; Rego 1994). The histology of the worm as observed from the sec- tions resembles the description of P. glanduliger by Mashego (2001) in C. gariepinus from four South African dams. Proteocephalid cestodes have been found in C. gariepinus from the neighbouring Zim ba- bwe (Barson 2004; Chishawa 1991; Douëllou 1992) but as they were not specifically identified, compari- FIG. 4 Ornithodiplostomum sp. Metacercariae (A) encysted (scale bar = 400 μm), (B) drawing showing main features (scale bar = 350 μm), (C) whole worm (scale bar = 250 μm), (D) anterior end showing oral sucker and pharynx (left arrow) and ventral sucker (right arrow), (E) posterior end showing rudi- mentary genital/excretory opening (scale bars = 150 μm), eo = excretory opening, ic = intestinal caecum, os = oral sucker, ph = pharynx, t = testes, to = tribocytic organ, vs = ventral sucker A B C ED os ph ic vs to t eo to t 108 Cestode and digenean parasites of Clarias gariepinus (Burchell, 1822) from Rietvlei Dam, South Africa FIG. 5 Polyonchobothrium clarias (photographs) (A) scolex (SEM) showing the arrangement of hooks around the rostellum (scale bar = 200 μm); (B) mature proglottid (LM); (C) gravid proglottid (LM) (scale bars = 400 μm); (D) embryonated egg (LM) (scale bar = 50 μm); (E) strobila (SEM) showing genital openings (go) and fused segment (fs) on the ventral surface; (F) light micrographs of AZAN stained transverse section through early mature proglottid (scale bar = 60 μm), t = testes, vt = vitellaria A B C ED F son with their South African counterparts cannot be made. In African fish species other than the Clariidae, many proteocephalid species have been described from the Sudan (Khalil 1963, 1973; Jones 1980), the Democratic Republic of Congo, Egypt, Liberia and Senegal (Khalil 1973; Khalil & Polling 1997). Trematoda Ornithodiplostomum sp. metacercariae (Fig. 4) Metacercariae coiled and encysted in tough white cysts formed by the host in the dorso-lateral mus- cles and a thin transparent wall, secreted by the par a- site (Fig. 4A), thus difficult to identify. The few suc- cessfully excysted metacercariae were identified as belonging to the family Diplostomidae and closely resemble Ornithodiplostomum sp. Body indistinctly bipartite with no pseudosuckers (as in other diplo- stomes), but with a large circular holdfast organ (tribo- cytic organ). Developing, immature testes apparent; Subterminal eversible genital apparatus also clear from photomicrographs (Fig. 4C–E). The present specimens match the description of adult Ornithodiplostomum sp. that occurs in the Pod- icipedidae (grebes) and Anatidae (ducks) from the TABLE 4 Ornithodiplostomum sp. measurements (μm) n = 3 Range Mean Length Maximum width Size of oral sucker (L*W)1 Size of pharynx (L*W) Size of ventral sucker (L*W) Size of tribocytic organ (L*W) Size of anterior testes Size of posterior testes 544–656 320–356 97–115*60–75 48–55*45–52 75–82*64–70 138–150*115–124 44–60*31.5–42.5 62–73.3*50–58 602 336 108*66.5 50*49.5 80–68 142.3*119.5 53.3*40 67*52.5 1 L*W : L = length, W = width 109 M. BARSON & A. AVENANT-OLDEWAGE Holarctic region and Africa (Niewiadomska 2002). This is the first record of this genus in South Africa, neither appearing in the latest checklist (Khalil & Pol- ling 1997) nor in Canaris & Gardner’s (1967) check- list of helminths of African vertebrates. How ever, re- lated genera such as Diplostomum, Neodiplo stomum and Postodiplostomum are quite common in south- ern Africa (Mashego 1977; Prudhoe & Hus sey 1977; Khalil & Polling 1997). The prevalence of this para- site was low (14 %) but the intensity of infestation was very high (Table 1). Only three metacercariae were successfully recov- ered and measured (Table 4) from the cysts. Some were indistinct and difficult to identify (e.g. Fig 4A), thus it cannot be concluded that all the metacercar- iae found were Ornithodiplostomum sp., or even ex- clusively diplostomid. Experimental infection of suit- able bird hosts with these metacercariae is the only way to obtain adult parasites which can then identi- fied to species level. ACKNOWLEDGEMENTS We acknowledge the assistance of the University of Johannesburg (UJ) and the Water Research Fund for Southern Africa (WARFSA) in funding this study; Dr R. Greenfield, G. O’Brien, Prof. V. Wepener, Drr M.A. Tsotetsi, M. Mathonsi & T. 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