J. EirhTo-vot. Microbiol.. 49(3), 2002 pp. 197-200 8 2002 by the Society of Protozoologists

Fine Structure of the Myxosporean, Henneguya curimata n. sp., Parasite of the Amazonian Fish, Curimata inormata (Teleostei, Curimatidae)

CARLOS AZEVEDO'J and EDILSON MATOSh nDepartment of Cell Biology, Institute of Biomedical Sciences, and CIIMAR, University of Oporto, 4099-003 Porto, Portugal, and

bhboratoni of Animal Biology, Faculty of Agricidtiwd Sciences, 66.000 Belhn, Brazil

ABSTRACT. Henneguya curinzata n. sp. (Myxozoa, Myxobolidae) is described from the kidney of the teleost Curimata inormata collected in an estuarine region of the Amazon River, near BelCrn. Brazil. This myxosporean produces large cysts (0.6-1.2 mm in diam.) that represent plasmodia containing all life cycle stages. including spores. The spore body is ellipsoidal (- 16.6 ym in length and - 6.2 pm in width), and each valve presents a tapering tail (- 19.1 ym in length). These valves surround the binucleate sporoplasm cell and two ellipsoidal polar capsules located side-by-side at the same level, measuring 6.5 X 1.2 Frn each and containing 10-1 1 coils of the polar filament. On the basis of its host specificity and on data collected by light and electron microscopy, the organism, H. curimafn n. sp. is distinguished as a new species. The taxonomic affinities and morphological comparisons with other similar species of the same genus are discussed.

Key Words. Life cycle, South America, spore, taxonomy, ultrastructure.

ESIDES the considerable information on the species of B Myxozoa collected in different geographical areas (Lom and DykovA 1992), little is known about those from South America, particularly about the Amazon River, where a diverse assemblage of several hundred species of fish live. Recently, a Brazilian myxosporidian check-list was published (Gi6ia and Cordeiro 1996; Molntir et al. 1998) in which the great majority of the results were based on light microscopy (Cordeiro et al. 1984; Gidia and Cordeiro 1996; Pinto 1928; Walliker 1969). Nevertheless, light and ultrastructural data are available for some Brazilian myxosporeans (Azevedo and Matos 1989, 1995, 1996a, b, Azevedo, Corral, and Matos 1997; Casal, Matos, and Azevedo 1996, 1997; Moln5r et al. 1998; Rocha, Matos, and Azevedo 1992; Torres, Matos, and Azevedo 1994).

Within the Phylum Myxozoa, the family Myxobolidae is one of the largest families (Lom and Dykov6 1992; Sprague 1982) and the genus Henneguya ThClohan, 1892, includes about 120 species (Lom and Dykovfi 1992). Some Brazilian species of Hennegriya have been described recently (Azevedo and Matos 1989, 1995, 1996a, Azevedo, Corral, and Matos 1997; Casal, Matos, and Azevedo 1997; Cordeiro et al. 1984; Kent and Hoff- man 1984; Rocha, Matos, and Azevedo 1992). In this paper we provide light and electron microscopical data of some life cycle stages and spores of a new species of myxosporidian parasite of an Amazonian fish.

MATERIALS AND METHODS Small fragments of kidney containing cysts removed from

the economically important freshwater fish, Curimata inomata Vari, 1987 (Teleostei, Curimatidae) (Brazilian common name "Coaca"), were collected in the Amazon River (00" 35' 38"S, 47" 35' OO'W), near BClem (Brazil). The parasitized fishes had a total length of 11.2 cm (9.1-12.3). The released spores were observed using Nomarski differential interference contrast mi- croscopy. For transmission electron microscopy (TEM), the cysts were fixed in 3% (w/v) glutaraldehyde in 0.2 M sodium cacodylate buffer (pH 7.2) for 6 h at 4 "C, washed overnight in the same buffer at 4 "C, and post-fixed in 2% OsO, buffered with the same buffer for 4 h at 4 "C. After dehydration in a graded ethanol series and propylene oxide, the fragments con- taining cysts were embedded in Epon. Semi-thin sections were stained with toluidine blue. Ultrathin sections, cut with a dia- mond knife, were double-stained with aqueous uranyl acetate and lead citrate and observed in a JEOL lOOCXTI TEM at 60 Kv .

I Corresponding Author: C. Azevedo-Telephone number: +35 1.22.2062200; FAX number: +351.22.2062232/33; E-mail: azeve- doc@icbas.up.pt

RESULTS

Kidneys of 28.5% of specimens (n = 42) of C. inormata were parasitized by a myxosporidian that belongs to the genus Henneguya ThCloham, 1892, because the spores were ellipsoi- dal in shape with two equal valves, each one containing a cau- dal projection forming a tail. Based on the morphological as- pects and host specificity, we propose the establishment of a new species. Following Lorn and Noble (1984), it is classified as follows: Phylum Myxozoa

Class Myxosporea Order Bivalvulida

Family Myxobolidae Henneguya curimata n. sp. (Figs. 1-7)

Life history stages. Ellipsoidal plasmodia, containing some hundred spores and all other developmental stages were ob- served among kidney host cells. Development was asynchro- nous, with younger stages occurring at the periphery and spores in the central zone of the plasmodium. The xenoma wall sur- rounding the plasmodia ranged from 2-6 pm in thickness and contained numerous collagen-like fibrils. More externally, some fibroblasts were present (Fig. 1).

At the periphery of the plasmodia, beneath the plasmalemma, numerous pinocytotic channels extended from the inner mem- brane into the ectoplasmic zone of the plasmodium. Beneath this zone, lay numerous mitochondria and some small vacuoles (Fig. 3).

Description. Life cycle stages, including spores, with mor- phological characters of the genus Henneguya Thtlohan, 1892: ellipsoidal spores with tails enclosing two pyriform polar cap- sules. Sporonts, sporoblasts, and spores were present in the same plasmodium (Fig. 4, 5) that varied 50-110 pm in the longest dimension. Mature fresh spores were ellipsoidal with a total length - 35.4 p,m (34.2-36.1) (n = 50), body length 16.6 pm (16.0-17.4), body width - 6.2 pm (5.8-6.6), and tail length - 19.1 km (18.3-19.9) (Fig. 2), with polar capsules 6.5 ? 0.3 pm long and 1.2 2 0.2 pm (n = 25) wide (Fig. 7, and in- set).The number of polar filament coils was 10-1 1 (Fig. 7, and inset).The spore wall, thin and smooth, comprised 2 equal valves, each one with a caudal projection forming the tail (Fig. 2, 5-7).

Type Host. Plasmodia observed only in the kidney of the fish Curimata inomata Vari, 1987 (Teleostei, Curimatidae).

Type Locality. Estuarine region of the Amazon River, near BelCm (Parti), Brazil (00" 35' 38"S, 47" 35' OOW).

Prevalence. Twelve of 42 C. inomata. Type Specimens. The slides with holotypes were deposited

197

198 J. EUKARYOT. MICROBIOL., VOL. 49, NO. 3, MAY-JUNE 2002

Fig. 1-4. Life cycle of the myxosporean Henneguya curiniara n. sp. 1. Semi-thin section of part of a plasmodium with cyst wall and all different developmental stages of the parasite. The mature spores are located more internally (*). 2. Differential interference contrast image of the spore, showing the spore body and the two tails with complete tapering processes. 3. Ultra-thin section of the periphery of the plasmodium showing some of he youngest life cycle stages (*). The xenoma wall (W), formed by numerous collagen-like fibrils, is surrounded more externally by fibroblasts (F). 4. Disporoblastic pansporoblast showing a binucleate sporoplasrn cell (SC) and polar capsules (PC) with their polar filaments. All bars in p,m.

in the International Protozoan Type Slide Collection at the Smithsonian Institution, Washington, D.C. 20560, USA.

Etymology. The specific epithet derives from the name of the genus of the host.

DISCUSSION

The myxosporidian described in this paper has the distin- guishing features of the genus Henneguya ThClohan, 1892 (i.e.

AZEVEDO & MATOS-ULTRASTRUCTURE OF HENNEGUYA CURIMATA N. SP. 199

Fig. 5-7. Life cycle of the myxosporean Henneguya curimata n. sp. 5. and 6. Details of two sequential development stages of the tails, showing longitudinal microtubules (arrows). 7. Ultra-thin section of an internal zone of the plasmodium showing some life cycle stages (*) and mature spores (S). Inset: Detail of a longitudinal section of a mature polar capsule and its polar filament (PF). All bars in pm.

ellipsoidal spores with tails enclosing two pyriform polar cap- sules), and the ultrastructural details of the various life cycle stages did not differ significantly froin the majority of other species previously described (Ali 1999; Kent et al. 2001; Loin and Dykovfi 1992; Lorn and Noble 1984).

Our study revealed that the sporogenesis was similar to that discovered in other species of the genus Heizneguya (Lom and Dykovi 1992). We compared our results with those reported for Henneguya spp. from different geographical areas, taking

into account the stage and dimensions of the spore body, tails, and polar filament coils, including number of coils. None of these characters was simultaneously equal. In Comparing the Brazilian Henrzeguya spp. with sheath structure surrounding the spores (Table I ) , only H. adherens (Azevedo and Matos 1995) has approximately the same total length. However, comparing the other measurements and other characters for other species, all of them are different from our isolate (Table 1). Moreover, comparing our results with those previously reported for Hen-

Table 1. Comparative measurements (in pm) and other characters of the spores of different species of the genus Heniieguyu from South America.

Species TL BL BW TaL PCL PCW PF VT H S Authors * + * * * -

* = H. theca 48.0 3.5 23.2 11.1 1.4

H. piscifoime 20.4 6.1 10.6 4.3 I .7 (40.6-52.6) (3.0-4.1) (20.3-24.2) (9.8-12.5) (1.0-1.6)

( 1 7.3-23.2) (4.5-6.7) (8.4-12.8) (3.1-6.1) (1.2-2.4) H. amazonica 59.3 13.9 5.7 45.4 3.3 1.3 6 = -

(56.0-65.9) (1 1.5-14.9) (5.2-6.3) (41.7-52.1) (2.7-3.6) (1.1-1.9) H. adherens 32.3 12.4 5.8 20.5 3.1 1.2 3-4 0 + H. malabarica 28.3 12.6 4.8 17.1 3.7 1.8 6-7 = +

(30.7-35.1) (10.5-13.8) (5.1-6.5) (18.0-21.7) (2.8-3.5) (1 .O-1.6)

(26.6-29.8) ( I 1.8-13.1) (16.2-18.9) (3.0-4.3) (1.6-2.2) H. striolata 42.2 15.8 5.3 25.9 6.8 1.2 13-14 = +

(39.3-45.6) (4.4-17.0) (4.9-5.9) (23.6-29.8) (5.1-7.0) (1.1-1.3) H. testiculures 27.5 14.0 6.5 13.5 9.0 2.0 12-13 0 +

(27.0-28.5) (14.0-14.5) (6.0-6.5) (13.0-14.5) (8.5-9.5) (2.0-2.5) H. curimata n. sp. 35.4 16.6 6.2 19.1 6.5 1.2 10-11 = -

(34.2-36.1) (16.0-17.4) (5.8-6.6) (18.3-19.9) (6.2-6.8) (1.0-1.4)

Kent and Hoffman 1984

Cordeiro et al. 1984

Rocha, Matos and Azevedo 1992

Azevedo and Matos 1995

Azevedo and Matos 1996a

Casal, Matos and Azevedo 1997

Azevedo, Corral and Matos 1997

Present study

Abbreviations: TL-total length; BL-body length; BW-body width; Tal-tail length; PCL-polar capsule length; PCW-polar capsule width: PF-polar filaments (coils); VT-valve type (= equal; 0 unequal); HS---external homogeneous sheath (+ present; - absent); * without references.

200 J. EUKARYOT. MICROBIOL., VOL. 49, NO. 3, MAY-JUNE 2002

neguyu spp. without sheath structure surrounding the spores shows this new isolate was very different in all measurements and other characters of the spores (Table 1). Furthermore, the newly described species of parasite differs from the previously reported Henneguya spp. (Table 1) in terms of its host speci- ficity and its site of infection (Lom and DykovA 1992) and is the only described Hennegziyu sp. parasitizing the kidney of the fish. Thus, on the basis of morphological measurements and ultrastructural differences (i.e. in spore size and shape, arrange- ment of the polar filament coils, and host specificity), we have established a new species. Because of the very large species diversity in the several hundred species of fish fauna in the Amazon River, we expect that a large number of myxosporeans remain to be discovered.

ACKNOWLEDGMENTS This works was partially supported by the A. Almeida Foun-

dation (Porto, Portugal). We would like to thank Ms. Laura Corral and Mr. J. Carvalheiro for their excellent technical as- sistance. I thank the anonymous reviewers for their most helpful comments and suggestions.

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Received 10/31/01, 02/20/02; accepted 02/23/02

48:61-69.