Cercariae developing in Lymnaea natalensis Krauss , 1848 collected in the vicinity of Pretoria , Gauteng Province , South Africa

MOEMA, E.B.E, KING, P.H. & BAKER, C. 2008. Cercariae developing in Lymnaea natalensis Krauss, 1848 collected in the vicinity of Pretoria, Gauteng Province, South Africa. Onderstepoort Journal of Veterinary Research, 75:215–223 Freshwater snails are known to serve as first intermediate hosts for various parasitic diseases such as schistosomosis and fasciolosis. Snails were collected on several occasions in the proximity of Pretoria, South Africa and their cercarial sheddings were studied. This article describes three different types of cercariae shed by the freshwater snail, Lymnaea natalensis, viz. a fork-tailed cercaria of a Trichobilharzia sp., an avian parasite belonging to the family Schistosomatidae, an echinostomatid cercaria of the family Echinostomatidae, also avian parasites and a xiphidiocercaria of the family Plagiorchiidae which parasitise avians and amphibians. The morphology of these cercariae was studied by light and scanning electron microscopy.


INTRODUCTION
Molluscs, usually snails, serve as first intermediate hosts for a large number of trematode parasites, e.g.Schistosoma spp. in humans and animals (Appleton 1996).Brown (1994) mentioned that approximately 400 species of freshwater snails (including limpets) occur in Africa, some of which form an important part of freshwater ecosystems for inland fisheries where they provide a valuable source of food.
Lymnaeid snails are the first intermediate hosts for several Fasciola spp.The main snail host for the liver fluke, Fasciola gigantica, is Lymnaea natalensis which is widely distributed in Africa (Brown 1994).Another study by Van Eeden, Brown & Oberholzer (1965) indicated that this species also has a wide distribution range in South Africa that extends into the Western Cape (as far as the Knysna district), Kwa-Zulu Natal and the eastern Mpumalanga provinces.Surveys executed over the past 2 years in farm dams and other waterbodies around Pretoria showed L. natalensis to be the most abundant freshwater snail in this area.
The aim of this study was to examine different cercariae shed by L. natalensis in dams surrounding Pretoria, Gauteng Province, as well as to gather information on other life stages within the life cycles of these parasites.

MATERIALS AND METHODS
Lymnaea natalensis specimens were collected over a period of 2 years using metal scoops as described by Van Eeden (1960).Snails found under water lilly leaves were also collected manually.In the laboratory they were kept in plastic containers and fed fish flakes.They were exposed individually to daylight (but not direct sunlight) to stimulate the natural shedding of cercariae.The cercariae were stained with nile blue sulphate or neutral red vital stains and studied by light microscopy while alive.Drawings were made with a drawing tube and measurements were derived from these drawings.In all cases 20 specimens were measured and all measurements given in the descriptions are in micrometres (μm).Minimum and maximum values are indicated with the mean value and standard deviation in brackets.No other intra-molluscan stages were collected.
For scanning electron microscopy (SEM), specimens were fixed overnight in 2.5 % glutaraldehyde, washed in Millonig's phosphate buffer (pH = 7.2) and dehydrated through a graded ethanol series for 30 s to 1 min in each concentration.Thereafter, they were critical point dried (Polaron, UK) overnight, mounted on stubs, sputter coated with gold (Emscope, UK) and examined using a Leica Stereoscan 420 scanning electron microscope (Leica Electron Optics, UK) at the Electron Microscope Unit of the University of Limpopo (Medunsa Campus).
In a preliminary life cycle study, two Mallard ducks and two Wisstar rats were exposed to the fork-tailed cercariae.A small number of cercariae were placed with the experimental animals and left for 1 h.Dissection of the animals took place 3 weeks later.

RESULTS
Three types of cercariae were identified, namely the fork-tailed type, and two single-tailed types, one of which is an echinostome and the other a xiphidiocercaria.
The excretory bladder is Y-shaped measuring 6-13 (9 + 3) x 7-16 (10 + 3) (Fig. 1A).From the bladder an excretory duct extends posteriorly into the tail that terminates in an opening on the tip of each caudal ramus (Fig. 2H).The main excretory duct on either side of the bladder extends anteriorly and divides opposite to the acetabulum to form anterior and posterior collecting ducts.Six pairs of flame cells were observed in the body, three occurring posteriorly and three anteriorly to the acetabulum.The flame cell formula is 2 (3 + 3) = 12.

Remarks
The cercaria is classified as a brevifurcate-apharyngeate distome cercaria according to the taxonomic key of Frandsen & Christensen (1984).It is placed in the family Schistosomatidae and is commonly known as an avian cercaria as the adult parasites are mostly found in ducks and geese (Appleton 1986).This cercaria with its characteristic schistosome morphology has one unique feature, i.e. a pair of dark pigmented eyespots, which differentiates it from human schistosome cercariae.Three weeks after infection only two spindle-shaped eggs were recovered from the faeces of one of the Mallard ducks.The other duck and the rats remained uninfected.The eggs were identified as those belonging to the genus Trichobilharzia according to the descriptions of avian schistosome eggs (type 3) by Appleton (1982Appleton ( , 1986)).This avian cercaria was thus identified as a Trichobilharzia sp.It has been recorded that this type of cercaria is able to penetrate human skin, and in so doing causes cercarial dermatitis, as a result of an allergic response (Picard & Jousson 2001).Since humans are not suitable hosts the cercariae do not enter the blood stream to mature but perish after penetrating the skin (Cheng 1986).
An oval-shaped and very protrusible oral sucker measures 36-60 (47 + 10) x 26-67 (45 + 12) at the anterior end of the body.This sucker is surrounded by many long-ciliated receptors (Fig. 3C, E and G) as well as multi-ciliated (cluster) receptors (Fig. 3E and G) consisting of groups with 6 to 12 short cilia.These receptors are situated mostly dorsal and dorso-laterally to the oral sucker (Fig. 3G).
The excretory bladder is bipartite with the most posterior part larger and oval, measuring 18-45 (32 + 9) x 15-33 (26 + 6), and preceded by a smaller oval part measuring 7-10 (9 + 1) x 19-22 (21 + 1) (Fig. 1C).The main excretory ducts on either side of the bladder extend anteriorly towards the pharyngeal region where they fold back posteriorly thus forming anterior and posterior collecting ducts in the midbody.These collecting ducts branch to form capillaries ending in flame cells.Eight flame cells were observed anteriorly and six posterior to the acetabulum.The flame cell formula is as follows: 2 [(2 + 1+ 2 + 2) + (1 + 1 +2 + 1 + 2)] = 28.A caudal excretory duct was also observed to extend into the anterior part of the tail stem where it branches at a quarter of the tail length into two ducts that open to the outside through two excretory openings (Fig. 1C).
Locomotion is provided by a single muscular tail (Fig. 1D) that is slightly longer than the head, measuring 297-306 (300 + 4) x 30-59 (37 + 9).It displays various long ciliated papillae arranged bilaterally on the tail stem (Fig. 3H).Finfolds are absent.The tegument of both the body and tail is aspinous and unevenly ridged (Fig. 3F and H).

Remarks
The cercaria is classified as an echinostome cercaria according to the keys provided by Frandsen & Christensen (1984).Porter (1938) described an echinostomatid cercaria with a collar of 26 spines, Cercaria middelburgensis that was shed by L. natalensis collected from a lake in Middleburg, Mpu malanga Province.This cercaria differs from the description of the present cercaria in respect of size, most body structures and the number and arrangement of the collar spines.Fain (1953) described Cercaria bruynoghei and Cercaria decora with 27 spines in the collar.These cercariae differ from the present specimen in the species of the snail host, general sizes of all structures, position of the acetabulum and flame cell formula.
Similarly, Jansen van Rensburg (2001) described a 27-spined echinostomatid cercaria from L. natalensis from the Okavango Delta, Botswana.Most body structures are similar to those of the present material but the prepharyngeal sac is absent.In contrast to Porter (1938), King & Van As (1997) described a 27-spined echinostomatid cercaria that was shed by Bulinus tropicus from the Free State Province.
Most of the cercarial features also appear similar to those of this study, with the exception of the content of the prepharyngeal sac and the species of the first intermediate host.
Locomotion is provided by a short single tail (Fig. 1H and 4H) measuring 148-164 (152 + 6) x 22-24 (23 + 1), that is aspinous and lacks dorso-ventral fin-folds.The body tegument is evenly granulated and covered by numerous minute backwardly pointing spines (Fig. 4G).Sensory receptors are few, but receptors with long cilia were observed on the sides of the body (Fig. 4F).Short ciliated receptors were also observed on the ventral surface of the body (Fig. 4E).Most receptors with short cilia were observed surrounding the oral sucker (Fig. 4C), whereas nonciliated receptors were observed alongside the acetabulum (Fig. 4D).

Remarks
This cercaria is classified as an Armatae xiphidio cercaria according to the classification keys proposed by Frandsen & Christensen (1984) and Haseeb (1984).Frandsen & Christensen (1984) are of the opinion that xiphidio cercariae belonging to the Armatae group develop into intestinal parasites in all groups of vertebrates.
In an earlier work, Porter (1938)

DISCUSSION
The apharyngeal-brevifurcate distome fork-tailed cer caria was found to be an avian schistosome cercaria.
The life cycle studies in the Mallard ducks and Wisstar rats unfortunately produced only two Trichobilharzia eggs in the faeces of one of the Mallard ducks.According to Appleton (2003) 16 adults and 12 cercariae of avian schistosomes have been described from the Afrotropical Region.Nine Trichobilharzia species have been described from birds in central Africa and Madagascar, and Appleton (1982Appleton ( , 1986) ) recorded five different types of avian schistosome eggs from faecal samples collected at various localities in South Africa.Three Trichobilharzia cercariae were described from L. natalensis in Africa, but since the cercaria in the present study is the smallest and the resulting eggs clearly fall within Trichobilharzia type 3 as was classified by Appleton (1982Appleton ( , 1986Appleton ( and 2003)), this cercaria may well be different from those described earlier.The life cycle therefore needs to be repeated using a larger number of ducks of different species in order to find the adult form and to verify the characteristic eggs.
The echinostomatid cercaria with its collar with 27 spines shed by L. natalensis was recovered from most localities.Although the life cycle of this parasite is unknown, L. natalensis was found to host echinostomatid metacercarial cysts in the mantle.This snail therefore also serves as second intermediate host for this parasite.
The xiphidiocercaria described in this study was found to be the most abundant single-tailed cercaria in the habitats examined in the proximity of Pretoria.

FIG. 4
FIG. 4 Xiphidio cercaria.Light micrographs of (A) whole mount; (B) anterior stylet (S); (C-H) scanning electron micrographs of cercaria: (C) oral sucker (Os) and stylet opening (So); (D) acetabulum (Ac); (E) short ciliated receptors (Scr); (F) long ciliated receptors (Lcr); (G) tegumental features of the body; (H) tail (T) features The life cycle of this parasite is also unknown, but other xiphidio stages were also found during this study.Xiphidio metacercariae were found encysted in various second intermediate hosts, i.e. in the gill chambers of freshwater shrimps, in the mantle of L. natalensis and occasionally also in the intestine of Barbus paludinosus.The cysts found in freshwater shrimps were bigger in size and the cyst wall was thinner compared to the cysts in L. natalensis and B. paludinosus.Although it appears as if freshwater shrimps are the most suitable second intermediate host, surveys have shown that they were not always present, especially during winter months.Studies have also shown that the highest infection with cysts occurs in L. natalensis.