ARTICLE IN PRESS European Journal of
PROTISTOLOGY European Journal of Protistology 40 (2004) 213–218 www.elsevier.de/ejop
Ultrastructural description of Agarella gracilis Dunkerly, 1915 (Myxozoa, Chloromyxidae) parasite of the dipnoan Lepidosiren paradoxa from the River Amazon Pedro Vitaa,b, Laura Corrala,b, Edilson Matosc, Carlos Azevedoa,b,* a
Department of Cell Biology, Institute of Biomedical Sciences, University of Oporto, Lg. A. Salazar, no. 2, Porto 4099-003, Portugal b CIIMAR-Center for Marine and Environmental Research, University of Oporto, Porto, Portugal c Research Laboratory Carlos Azevedo, University Federal Rural of the Amazonia, Bel!em (PA), Brazil Received 27 January 2004; accepted 20 March 2004
Abstract The myxosporean, Agarella gracilis, a parasite in Lepidosiren paradoxa collected in the estuarine region of the River Amazon, is described using light and transmission electron microscopy. The most evident life cycle stages were the spores, found in both the testis and ovary, which appeared scantily dispersed in the lumina. The prevalence of infection was 10/30 (33.3%). In mean dimensions the mature spore was 59.7 mm in total length, composed of an elongated body 17.7 mm long, 6.6 mm wide and 5.9 mm thick, with two long slightly divergent tail-like projections of the valves. There were four pyriform polar capsules, two longer (7.0 2.4 mm) and two shorter (3.8 2.4 mm), located anteriorly in the spore body; the polar filaments of all capsules had 6–7 turns. There was a posterior binucleate sporoplasm. The morphology agrees with the original description by light microscopy, and the taxonomy of the genus is discussed. The presence of the same genus of myxosporean parasites in both dipnoan and anuran species perhaps reflects their close phylogenetic relationship. r 2004 Elsevier GmbH. All rights reserved. Keywords: Ultrastruture; Myxozoa; Agarella gracilis; Parasite; Dipnoi; Lepidosiren
Introduction In the course of his work using the spermatogenesis of Lepidosiren paradoxa Fitzinger, 1837 (Sarcopterygii: Dipnoi), the South American lung-fish, as a model for mitosis and meiosis, because it has beautifully clear chromosomes with very pronounced size differences, Agar (1911) discovered certain bodies in the testis which he took to be parasitic Protozoa. Studying the same collected material, Dunkerly (1915) described a new
*Corresponding author. Fax: +351-22-2062232/33. E-mail address:
[email protected] (C. Azevedo). 0932-4739/$ - see front matter r 2004 Elsevier GmbH. All rights reserved. doi:10.1016/j.ejop.2004.03.001
genus and species of a myxosporean parasite, Agarella gracilis (Chloromyxidae). The phylum Myxozoa includes numerous species, some of which are well-documented pathogens, mainly parasitizing different organs and tissues of fish (Lom and Dykova! 1992). While considerable information has been collected on myxozoan species from different geographical locations (Lom and Dykova! 1992), little is known about South American species, particularly from the River Amazon, where a diverse assemblage of several hundred species of fish live (Azevedo and Matos 2002). The material used by Dunkerly (1915) was obtained from the swamps of the Chaco in Paraguay, and his diagrammatic illustrations made by light microscopy
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provide the only description of the genus Agarella. Mutschmann and Mutschmann (2002) report a case of infection with a myxosporean morphologically identified as an Agarella sp. in two poison arrow (or poison dart) frogs Dendrobates tictorius (Anura: Dendrobatidae) from Surinam. No other original references were found in the literature. In the present paper, we present data obtained by light and transmission electron microscopy on the spores of A. gracilis, found infecting the testis and ovary of L. paradoxa from the River Amazon, Brazil, a location intermediary between Paraguay and Surinam.
Material and methods Thirty specimens of L. paradoxa, known by the Brazilian common name ‘‘Moreia’’, were collected from the estuarine region of the Amazon River (01 110 3000 S and 47 180 5400 W) near the city of Bele! m (Para! ), Brazil. The lungfishes were dissected and small fragments of infected testes and ovaries were examined under the light microscope with Nomarski differential interference contrast (DIC) optics for spore description and measurement. For ultrastructural studies, small fragments of infected tissues were excised and fixed in 3% glutaraldehyde in 0.2 M sodium cacodylate buffer (pH 7.2) for 5 h at 4 C, washed overnight in the same buffer at 4 C, and postfixed in 2% OsO4 buffered with the same solution for 4 h at the same temperature. After dehydration in a graded ethanol-series followed by propylene oxide (8–10 h in each change), samples were embedded in Epon. Semithin sections were stained with methylene blue-Azur II. Ultrathin sections were cut with a diamond knife, double-stained with uranyl acetate and lead citrate, and observed in a JEOL 100CXII TEM operated at 60 kV.
Results The mature spores appeared scantily dispersed in the lumina of both testes and ovaries (Fig. 1) when observed under Nomarski optics. Small groups of spores arranged
in pseudo-cyst structures, i.e. without a defined wall, were observed within the tissues surrounding the lumina (Fig. 4), suggesting the possibility of polysporic trophozoites. The spores were composed of an elongated ovoid pyriform body (Figs. 2 and 3) and two slightly divergent caudal projections of the valves (tails) (Fig. 1). There were four pyriform polar capsules, two a little anterior to the other two (Figs. 2 and 3), located in the anterior portion of the spore. These morphological characteristics suggest that this myxosporean parasite belongs to the previously described genus Agarella Dunkerly, 1915.
Spore characteristics The spore body was composed of four polar capsules and a binucleated sporoplasm enclosed by two valves, each with a long ‘tail’ (Figs. 1, 5-8, 10, 11). There were two polar capsules, one with a longer ‘neck’, the other with a shorter ‘neck’, for each valve (Figs. 6-8 and 12). The ‘necks’ of all four polar capsules terminated at the apex of the spore (Figs. 6, 8, 9 and 12), and in each capsule the polar filament made 6–7 turns. Typical dimensions of spores and polar capsules are given in the diagnosis below.
Diagnosis Host: L. paradoxa Fitzinger, 1837 (Sarcopterygii: Dipnoi) Locality: Estuarine region of the River Amazon (01 110 3000 S and 47 180 5400 W) near the city of Bele! m, Brazil. Site of infection: Spores were located in the tissues surrounding the lumina of testes and ovaries. Prevalence and intensity: 10/30 (33.3%) Measurements from fresh spores (n ¼ 20): Total length =59.772.6 mm. Spore body: length =17.771.0 mm, width =6.67 0.5 mm, thickness =5.970.3 mm. Longer polar capsules: length=7.070.8 mm, width= 2.470.3 mm. Shorter polar capsules: length=3.870.6 mm, width= 2.470.3 mm. Number of polar filament turns of all capsules: 6–7
Figs. 1–7. Figs. 1–3 show A. gracilis observed with Nomarski optics. 1, Isolated mature spore showing the spore body and the bifurcated tail. 2, Lateral view of spore body where three of four polar capsules are visible. 3, Longitudinal aspect of the spore body showing the anterior four polar capsules and the posterior sporoplasm. Fig. 4, Semithin section of part of the testicular lumen of L. paradoxa containing some life cycle stages, including several mature spores (Sp) stained with methylene blue-Azur II. Figs. 5–7. Transmission electron micrographs of A. gracilis. 5, Longitudinal ultrathin section of a mature spore showing the general ultrastructural organisation of the spore body, its sporoplasm (S) and three of the four polar capsules (PC). 6, Longitudinal section of the apical region of the spore showing the coiled polar filament (arrowheads) inside three of the four polar capsules (PC) located at two levels. 7, Transverse oblique section of the apical region of the spore body showing the 4 polar capsules side-by-side (PC). Some aspects of the suture between the 2 valves (V) and a transverse section of the tails (T) are also visible.
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Table 1. Agarella spp. comparative data Species
TL
BW
PCL
PCW
FC
Host
Locality
Site of infection
Source
A. gracilis
28–35
4–5.5
5–7.7
n
n
L. paradoxa
Paraguay
Testis
Agarella sp.
14–18
6.5–8
2.5–3.2
1.3–1.6
6–8
D. tictorius
Surinam
A. gracilis
57.1–62.3
6.1–7.1
6.2–7.8/ 3.2–4.4
2.1–2.7
6–7
L. paradoxa
Brazil
gall bladder, liver, kidney and urinary bladder Testis and ovary
Dunkerly (1915); Lom and Dykov!a (1992) Mutschmann and Mutschmann (2002) Present study
TL: total length; BW: body width; PCL: polar capsule length; PCW: polar capsule width; FC: number of filament coils; n: no data.
Discussion These results provide the first ultrastructural data on the genus Agarella. Although it belongs to the family Chloromyxidae, the spores of Agarella closely resemble those of Henneguya of the family Myxobolidae (Nemeczek 1926; Azevedo and Matos 2003; Casal et al. 2003; Vita et al. 2003), except for the presence of four polar capsules, instead of two. No reports of A. gracilis have been published since Dunkerly’s (1915) drawings from light microscopy, and this is still the only species of the genus. Mutschmann and Mutschmann (2002) identified a myxosporean from the poison arrow frog as belonging to the genus Agarella using light microscopy, and published an abstract with three micrographs. This species of Agarella is smaller; it has a very different lemon-like body shape, and is almost round, in contrast with the slender shape in both descriptions of A. gracilis (Dunkerly 1915, present study). Table 1 provides a comparison of the available descriptions of Agarella spp. Spore morphology is still the principal taxonomic tool for discriminating between Myxozoa (Lom and Dykova! 1992; Vita et al. 2003). There are some pronounced differences between the previous description of A. gracilis (Dunkerly 1915) and the present results, but, taking account of the fact that specimens were collected nearly 2000 km and almost 90 years apart, we do not consider the species described in this paper as new. Our reasons for this conclusion are as follows: (1) previous measurements are based only on illustrations of material fixed some years before examination; (2) the host species is the same; and (3) the site of infection is the same. Nevertheless, a species diagnosis is provided, according to Lom and Noble (1984), as if the species was being newly described (Figs. 13 and 14).
Fig. 14. A. gracilis. Schematic drawing of a spore.
Figs. 8–13. Electron micrographs of spores of A. gracilis. 8, Longitudinal section of the spore body, with its valves (V), polar capsules (PC) and sporoplasm (S). Note the longer and the shorter polar capsules (PC) with polar filament (PF) inside. 9, Detail of the apex of spore showing the polar capsule (PC) insertion. Note the suture between valves (V). 10, Another detail of the suture between valves (V). Note the transverse section of tails (T) and the section of the filament inside the polar capsule (PC). 11, Longitudinal section of the posterior spore body showing the sporoplasm (S) and the beginning of the bifurcation of the tails (T). 12, Detail of a transverse section at the tip of the spore showing the insertions of the four polar capsules (arrowheads) and the suture line between the two valves (arrow). 13, The middle region of the tails in longitudinal oblique section.
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Lung-fishes are important due to their phylogenic position between aquatic and terrestrial animals. The presence of members of the same genus of myxosporean parasite in both dipnoan (Dunkerly 1915, present study) and anuran species (Mutschmann and Mutschmann 2002) may reflect their close phylogenetic relationship, which is probably closer than that between dipnoans and teleosts. However, due to the unexplored parasitological biodiversity of South American fishes, especially in the Amazon basin (Azevedo and Matos 2003), we cannot exclude the possibility of finding an infection by the genus Agarella in a teleost host.
Acknowledgements The authors would like to thank Jo*ao Carvalheiro for his excellent photographic work. This study was ! partially supported by Eng. Antonio de Almeida Foundation, Porto, Portugal.
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Azevedo, C., Matos, E., 2003. Fine structure of Henneguya pilosa sp. n. (Myxozoa: Myxosporea), parasite of Serrasalmus altuvei (Characidae), in Brazil. Folia Parasitol. 50, 37–42. Casal, G., Matos, E., Azevedo, C., 2003. Light and electron microscopic study of the myxosporean, Henneguya friderici n. sp. from the Amazonian teleostean fish, Leporinus friderici. Parasitology 126, 313–319. Dunkerly, J.S., 1915. Agarella gracilis, a new genus and species of myxosporidian, parasitic in Lepidosiren paradoxa. Proc. R. Soc. Edinburgh Sect. A 19, 213–219. Lom, J., Dykova! , I., 1992. Myxosporidia (phylum Myxozoa). In: Lom, J., Dykova, I. (Eds.), Protozoan Parasites of Fishes. Developments in Aquaculture and Fisheries Science, Vol. 26. Elsevier, Amsterdam, pp. 159–235. Lom, J., Noble, E.R., 1984. Revised classification of the class Myxosporea Butschli, 1881. Folia Parasitol. 31, 193–205. Mutschmann, F., Mutschmann, C., 2002. A case of an infection with an Agarella sp. (Myxosporidia: Chloromyxidae) in the poison arrow frog Dendrobates tinctorius (Anura: Dendrobatidae) from Surinam, In: McKinell, R.G., Calson, D.L. (Eds.), Proceedings of the Sixth International Symposium on Pathol. Rept. Amphib., Saint Paul, MN, pp. 149–152. Nemeczek, A., 1926. Beitr.age zur Kenntnis der Myxosporideinfauna Brasiliens. Arch. Protistenkd. 54, 137–150. Vita, P., Corral, L., Matos, E., Azevedo, C., 2003. Ultrastructural aspects of the myxosporean Henneguya astyanax n. sp. (Myxozoa: Myxobolidae), a parasite of the Amazonian telost Astyanax keithi (characidae). Dis. Aquat. Org. 53, 55–60.
