Cryptobia neghmei sp . n . ( Protozoa : Kinetoplastida ) in two species of flounder , Paralichthys spp . ( Pisces : Paralichthydae ) off Chile Cryptobia neghmei sp . n ( Protozoa : Kinetoplastida ) en dos especies de lenguados Paralichthys spp . ( Pisces : Paralichthydae ) de la costa de Chile

Cryptobia neghmei sp.n. is described from the blood oftwo species of flounder, Paralichthys microps and P. adspersus, inhabiting the Chilean coast in the southern Pacific Ocean. Flagellates were elongate, slender, with two flagella and a conspicuous undulating membrane. It was distinguished from previously described species on the basis of its unusual shape and dimensions. All of 97 flounder were infected upon examination. Developmental stages of kinetoplastid protozoans, perhaps C. neghmei sp. n., were observed in some leeches Glyptonotobdella sp. that were found attached to flounder, which probably represent a mode for transmission among piscine hosts.


INTRODUCTION
Among kinetoplastid protozoans of fish, two morphologically similar genera ( Cryptobia Leidy 1846 and Trypanoplasma Laveran and Mesnil 1901) are recognized mainly on the basis of their location on (or in) the host body.Their life cycles can involve direct transmission or by blood sucking leech species.Species of the genus Cryptobia are kinetoplastid flagellates which possess two free-moving flagella, one located anteriorly, the other one posteriorly attached, and invariably an undulating membrane extending the full length of the body.They have been reported from some invertebrates but more often in the blood of fish (Becker 1977, Khan 1991).
This study was conducted on the blood parasites of two species of flounder, Paralichthys microps (Gunther 1881) and P. adspersus (Steindachner 1867) living off the coast of Chile.An ectoparasitic leech was also taken from some of the fish and examination of its intestinal contents revealed a number of developmental stages of kinetoplastid protozoans.Since morphometric criteria of this organism were not comparable to those of any species described previously, it is considered a new species.A description of the parasite in flounder and other developmental stages of kinetoplastid protozoans found in the leech are reported herein.

MATERIAL AND METHODS
Flounder were captured by gillnet and by otter trawl off the coast of Chile at three locations: Concepcion bay, San Vicente bay, and Gulf of Arauco, at depths 8-15 m during November and December, 1998 (Fig. 1).The fish were bled from the caudal artery after capture using a 3.0 ml syringe that was heparinised and 23 gauge needle.The fish varied in length from 20 to 55 em and weighed 65 to 2,100 g.Smear made from living specimens were exposed to iodine vapour for about one minute to prevent contraction.Fresh smears in phosphate buffer (pH 7 .6)from the gills and skin of these fish were also examined for parasites.Thick (one drop of blood) and thin smears were prepared from all other fish, air-dried, incubated at 60 °C for 30 min before fixation with methanol and staining with Giemsa (1:10 dilution with phosphate buffer, pH 7.6) for about one hour.Some smears were also stained with "diff quik" (Baxter Scientific Products, Illinois, U.S.A.).Three specimens of a leech, collected from the bodies of three flounder, were dissected and smears prepared on slides were stained with Giemsa.Ilustrations of the parasite were made from photomicrographs using a Zeiss "ultraphot" with a micrometer and measurement (mm) were made from stained smears.Means and standard errors were determined.

RESULTS
Description of the parasite is based on five living (which were also recorded on a video tape) and 25 stained (both Giemsa and "diff quik") specimens which were obtained from five hosts (5 per fish).None was seen in smears made from the surface (both dorsal and ventral) of the body or from the gills.Diagnosis (blood stream forms, mean ± SE measurements in mm; based on living and Giemsastained specimens).Body elongate 41.9 ± 0.4 x 3.8 ± 0.6, narrow, tapering toward the posterior extremity of body flexing continually in live specimens; anterior flagellum prominent, moving rapidly, 10.3 ± 1.1 mm long; distinct undulating membrane extending the full length of the body (Fig. 2), beating rapidly in a wave-like manner, posteriad; posterior flagellum short, 5.2 ± 0.4 long, flicking but moving less rapidly than the anterior flagellum; nucleus ovoid to elongate, 3.8 ± 0.2 x 3.2 ± 0.1, located 6.8 ± 0.1 from anterior extremity, appearing translucent in living specimens and light gray when stained; in one stained specimen, an extension of the nucleus was observed, kinetoplast elongate, 9.7 ± 0.8 x 3.6 ± 0.2, magenta-stained at the anterior extremity, lying near the flagellum in stained specimens but ovoid and translucent in live specimens.

Kinetoplastid stages in the leech Glyptonotobdella sp. Sawyer and White, 1969, an ectoparasite of flounder
Several developmental stages ofkinetoplatid protozoans were observed in stained smears prepared from the digestive tract of three specimens of a leech Glyptonotobdella sp., that were taken from the body surface of flounder.Some were ovoid, ( 11.6 ± 0.9 x 7.1 ± 0.4) with a nucleus (Fig. 3A), and kinetoplast and appeared to be diving with pale-stining flagella that were faintly visible (Fig. 3B).Others were ovoid to pyriform, nucleated with an anteriorly located kinetoplast and a flagellum 12.5 ± 1.1 x 8.6 ± 1.0 (Fig. 3C, 3D).
Site of infection in host: blood.Geographical location: coast of Chile (near to 36° 44'S, 73° 09'W) in the South Pacific Ocean.
Etymology: the species is named after Professor Amador Neghme in recognition of his contributions to parasitology in Chile.Etymology: the species is named after Professor Amador Neghme in recognition of his contributions to parasitology in Chile.

DISCUSSION
Two types of kinetoplastid flagellates are recognized in Paralichthys (see Sypek & Burreson 1983, Khan 1991): Cryptobia and Trypanoplasma are morphologically similar.Based on their location, an ectocomensal group is referred to as species of Cryptobia and another living in the blood stream as Trypanoplasma.Transmission of the former is direct (host to host) without other developmental changes whereas the latter are transmitted by hematophagous leeches in which a number of development stages occur (Burreson 1979b(Burreson , 1982)).However, a review by Woo (1984), with which we concur, considered the genus Trypanoplasma a synonym of Cryptobia, based not only on the morphological similarity of the genera but also on observations that at least one hematozoic species could be transmitted both directly and by a blood-sucking leech.Recent molecular evidence revealed that T. borreli was more closely related to C. salmositica than to C. bullocki or C. catostomi, raising doubts about the validity of the genus Trypanoplasma (Wright et al. 1999).However, Lorn & Dykova (1992) continue to use Trypanoplasma for biflagellated haematozoans transmitted by leeches.
Cryptobia neghmei sp.n. can be distinguished from other described species on account of its unusual morphology and dimensions.While it appears similar to Cryptobia (= Trypanoplasma) beckeri, it is about half of the total length of the latter [mean: 109 (43-165) ] species (Burreson 1979a).A species of Cryptobia was observed in the blood of a flounder, Limanda aspera, in the North Pacific Ocean adjacent to British Columbia, but no description of the parasite was made (Sloan et al. 1984).Since 90 % of the piscicolid leech, Notostomum cyclostomum were infected, it was suggested this was probably the vector.A similar parasite was described from an Antarctic eelpout, Rhigophila dearborni, but only one flagellum was observed (Becker & Halloway 1968).Subsequent examination of the parasite by Burreson (1979a) revealed a recurrent flagellum, confirming its biflagellated status.We concur with Burreson (1979a) that in air-dried blood films, the recurrent flagellum might not be detected unless living specimens are examined.
The life cycle of C. neghmei sp.n. might be similar to that of C. ( = T.) beckeri if developmental stages observed in the piscicolid leech, Glyptonotobdella sp.prove to be conspecific to C. neghmei.Burreson (1979a) demonstrated that flagellates ingested by the leech, M almiana diminuta, became rounded and underwent binary fission in the crop.Eventually, small infective stages entered the proboscis sheath and were transmitted following the next blood meal of the leech.Burreson (1979aBurreson ( , 1979b) ) suggested that growth of the infective forms in the piscine host was rapid as only large individuals were observed 8 to 9 days after transmission in laboratory experiments.The absence of any small forms in the Paralichthys spp. in the present study, provides support for this view.Woo (1987) noted that at least two hematozoic species also had ectoparasitic stages and one was transmitted directly via water (see also Brower & Margolis 1983, Woo & Wehnert 1983).One of these included C. bullocki which infects several species of flatfish inhabiting the eastern seaboard of North America (Burreson & Zwerner 1982).Although we did not observe C. neghmei on the gill or skin of Paralichthys spp., it is likely that the infections were too low to detect.Further studies should focus on direct transmission, especially in juvenile flounder which tend to exhibit higher parasitemias than adults (Burreson & Zwerner 1984).Moreover, a knowledge of the life cycle and pathogenicity of C. neghmei is relevant since there is evidence that C. bullocki, causes morbidity and mortality in juvenile summer flounder, Paralichthys dentatus, both in the laboratory and in the field (Burres on & Zwerner 1982, 1984 ).The fish were anemic had abdominal distension with ascites and splenomegaly specially when the water temperature was low (Sypek & Burreson 1983 ).Although anemia was observed in the infected flounder, this condition might have been the result of synergism between C. neghmei sp.n. and exposure to a variety of contaminants (George-N ascimento et al. 2000).Further studies on juvenile flounder inhabiting uncontaminated areas along coastal Chile coupled with experimental infections in the laboratory might reveal the impact of this parasite on fish health.Additional studies should focus on its mode of transmission since at least one species, C. salmositica can be transmitted by a leech and directly via seawater.