A NEW NEMATODE SPECIES Goezia leporini n . sp . ( ANISAKIDAE ) FROM CULTURED FRESHWATER FISH Leporinus macrocephalus ( ANOSTOMIDAE ) IN BRAZIL

This paper describes nematode infection in the cultured freshwater fish Leporinus macrocephalus (Osteichthyes: Anostomidae) collected at Batatais, São Paulo State, Brazil. Of a total of 32 examined fish, 21 (65%) were infected with Goezia leporini n. sp. (Nematoda: Anisakidae) with mean infection intensity of 4.1 parasites. The nematodes presented total length greater than G intermedia, G. holmesi, G. pelagia, G. minuta, G. kliksi, G. sinamora, G. nonipapillata, G. alii, G. moraveci, G. brasiliensis, and G. brevicaeca. The main difference was a great number of preanal papillae in males when compared to G. brasiliensis and G. brevicaeca. The present description also differs from that of G. brasiliensis with respect to spicule length and distance of vulva from the anterior extremity.

A new species of Goezia was identified in Brazil in the farmed freshwater fish Leporinus macrocephalus.

MATERIAL AND METHODS
Thirty-two specimens of L. macrocephalus Garavello & Britski, 1988 (Osteichthyes: Anostomidae), 121.9 (20.5 to 537.0) g in weight and 18.4 (11.0 to 33.0) cm in total length, were cultured in ponds with a 6.7 fish/m 2 density at a fish farm in Batatais, São Paulo State, Brazil.Observed fish were captured in July and August 1999.At the same time, water quality was assessed using Corning portable equipment for measuring electric conductivity, and pH and YSI equipment for measuring dissolved oxygen and water temperature.
Before dissection the fish were sacrificed by immersion in a 0.1% benzocaine solution.Body mucus and pieces of gills, kidney, liver, spleen, and heart were then compressed between a glass slide and a coverslip with a drop of 0.65% saline solution for microscopic observation.Stomach and intestines were opened and observed in a Petri dish containing saline solution.Nematodes were carefully collected from the stomach, fixed in AFA at 65°C, and preserved in alcohol 70% with 5% glycerine.Helminths were dehydrated and cleared in Faia creosote or glycerine, and en face preparations were performed.For uterus and ovijector observation and egg measurement, five females were carefully dissected.Twenty males, 20 females, and 40 eggs from dissected females were studied in a camera lucida for measurement.For scanning electron microscopy, the nematodes were fixed at room temperature in a 3% glutaraldehyde solution with a 0.1 M phosphate buffer (pH 7.4).Afterwards, they were dehydrated with serial concentrations of alcohol, dried with CO 2 , assembled, and coated with 20 nm gold paladium, following which they were examined with a JEOL JSM-5410 microscope.Parasite identification was made according to Anderson et al. (1989).Prevalence and mean intensity were calculated according to Bush et al. (1997).All measurements are in millimeters (average and range in parentheses) unless otherwise stated.

RESULTS
Throughout the sampling period, water parameters showed normal values: electric conductivity 5.0-20.0µS/cm, pH 6.3-7.3,dissolved oxygen 7.4-9.4mg/I, and water temperature 14.0-19.0°C.Observed fish showed a 65% infection prevalence and a mean intensity of 4.1 parasites in the stomach.After dissection of infected fish, some nematodes could be seen passing through the stomach wall, in which helminths were sometimes found embedded.

Goezia leporini n.sp. (Nematoda: Anisakidae)
Description.Cylindrical and stout whiteyellowish nematode.Females larger than males.Body cuticle with distinct transverse striations and cuticular spines.Spine rows more separated and spines longer when observed at ventriculus level.Anterior end flattened with three demarcated lips of approximately equal size.A smooth cuticle covering each lip was present.Dorsal lip with two double papillae situated laterally; each ventrolateral lip with one double papilla, one single papilla, and one lateral amphid.Internal edges of each lip provided with two protruding projections.Mouth separated from body by a slight constriction.One pair of cervical papillae (deirids) between the nerve ring and ventriculus.Esophagus clavate, ventriculus small (bulb) narrower than the widest level of esophagus.Excretory pore at the level of the nerve ring.Nerve ring surrounding the esophagus at the first third of its length.Ventricular appendix narrow, two to three times longer than the esophagus.Intestinal caecum reaching anteriorly certain level of esophagus.Male with two similar spicules.Female with didelphic and opisthodelphic uteri.Vulva without prominent labia slightly anterior to midbody and vagina directed posteriorly.Tail conical with digitiform process.

Taxonomic summary
Type Etymology: the specific name of this species is proposed from the genus of the host

DISCUSSION
The fish studied belong to the most cultivated freshwater fish species in Brazil.Endoparasites of cultivated fish have previously enjoyed little research interest in Brazil and this is the first recording of mortality in farmed fish in Brazil probably caused probably by nematodes in the digestive tract, although fish was not specifically diagnosed for bacterial and viral disease.This may be affirmed because of macroscopical observation and the fact that nematodes were found perforating the stomach wall.There are several studies of the Goezia, but most of these focus on parasite description.Only low prevalences were reported in P. corruscans infected with G. spinulosa (Hamann, 1984) (8.33%), Mastacembelus armatus infected with G. moraveci De & Dey, 1992 (0.09%), and Goezia larvae from Rhamdia guatemalensis (Moravec et al., 1995) (0.12%).In this study, the massive presence of G. leporini n. sp.(prevalence 65%) was similar to that found by El-Darsh & Whitfield in 1999 (66.7%).
Here, the nematodes were firmly attached to the stomach wall and evidence of their presence was associated with feeding and occurrences of secondary lesions in the host, as discussed by Deardorff & Overstreet (1980).
Extensive mortality in a freshwater population of Morone saxatilis was also related to the presence of Goezia sp.(Gaines & Rogers, 1972), as also observed in this work.Features important in species differentiation have been based on size, shape, cuticular spines, caudal papillae of male, ratio between caecum/ ventricular appendix and caecum/esophagus, excretory pore and nerve ring positions, distance of vulva from anterior end, and egg morphology.
There are three species described in Brazil: G. spinulosa in A. gigas collected from Amazonas and Pará (Travassos et al., 1928;Santos et al., 1979) and in A. ocellatus from Ceará (Freitas & Lent, 1946), and the G. brasiliensis in B. hilarii and P. corruscans and G. brevicaeca in B. hillarii from the Paraná River (Moravec et al., 1994).
The position and number of caudal papillae in males were different when compared with several other species (Table 1) and similar to one of G. brevicaeca (Moravec et al., 1994), although these authors did not report the presence of adanal papillae.The present description showed higher measurements of esophagus length, ventricular appendix length, caecum appendix, and caecum/oesophagus ratio in male specimens than those observed in G. brasiliensis and G. brevicaeca by Moravec et al. (1994).On the other hand, G. leporini n. sp.showed also higher measurements of esophagus length, ventricular appendix length, caecum appendix, and caecum/oesophagus ratio in females than those observed in G. kliksi Deardorf &Overstreet, 1980, andG. brasiliensis andG. brevicaeca (Moravec et al., 1994).It can also be inferred that G. leporini n. sp. has such a number of genital papillae in males, the highest distance of the vulva from the anterior extremity, and egg size in females when compared to G. brasiliensis described by Moravec et al. (1994).
The single prominent median papilla situated immediately anterior to the cloaca of G. leporini n. sp. was present only in G. spinulosa studied by Baylis (1927), and by scanning electron microscopy as shown by Costa et al. (1995).In addition, the present description showed lower spicule length than that observed in G. kliksi (Deardorff & Overstret, 1980) and in G. brasiliensis (Moravec et al., 1994).Nevertheless, a thin membrane surrounding the proximal spicule extremity was in according with that reported in G. spinulosa studied by Baylis (1927) and Santos et al. (1979), andG. nonipapillata Osorio-Sarabia, 1982.On the other hand, G. holmesi Sprent, 1978, andG. moraveci (De &Dey, 1992) have presented spicules with proximal and distal alae.
In accordance with these characteristics, the authors have defined a new species of Goezia, helpful in defining the effects and the seasonality of the nematode in the host.