Trichodina modesta: an exotic ciliate in the Neotropical region parasitizing an unusual host

In this study, an important ornamental fish, Betta splendens (Osphronemidae), from three different Brazilian states was examined for parasitic infestations. Smears with parasites were impregnated with silver nitrate or stained using Giemsa for taxonomic evaluation. A disc-shaped trichodinid with a body diameter of 39.7 ± 3.3 μm, adhesive disc diameter of 32.9 ± 3.1 μm and denticulate ring diameter of 19.5 ± 2.0 μm was found. The morphological characteristics resembled those of Trichodina modesta Lom, 1970, a species that shows clear host specificity for Cypriniformes. Until now, its occurrence was restricted to the Eurasian region. In the present study, a new host for T. modesta is reported and therefore the first occurrence of this species in the Americas. The parasite was possibly introduced into the Neotropical region through the exotic fish trade, especially of Cypriniformes used by aquarists. The distribution of this ciliate is discussed and a checklist of localities and hosts for the species is provided.

Trichodinid parasitism may cause lesions on its hosts under farming conditions as a result of rapid trichodinid proliferation. Pathological alterations such as hyperplasia of the secondary lamellae (YEMMEN et al., 2010, cellular desquamation, lamellar fusion (ABDEL-BAKI et al., 2011), subepithelial edema with epithelial displacement of the secondary lamellae (VALLADÃO et al., 2014) and gill necrosis VALLADÃO et al., 2013) are commonly associated with trichodiniasis. These ciliates are frequently associated with lesions on the body surface that lead to opportunistic bacterial infection and outbreaks of mortality (KHAN, 2009;VALLADÃO et al., 2013VALLADÃO et al., , 2014. Since few studies have been done on fish lesions caused by trichodinids, epidemiological studies need to be encouraged in order to establish prophylactic measures for avoiding dispersion of these fish pathogens worldwide.
This study evaluated the trichodinid fauna of B. splendens and registers T. modesta in the Americas. The distribution of this ciliate is discussed and a checklist of hosts and localities for the parasite is presented.

Study area and fish
Adult males of Betta splendens from Muriaé, Minas Gerais state (21° 7' 49" S; 42° 22' 3" W) (n=42), from Ribeirão Preto, São Paulo state (21° 10' 40" S; 47° 48' 36" W) (n=28) and from Cascavel, Paraná state (24° 57' 20" S; 53° 27' 19" W) (n=23) were examined. The fish had standard lengths of approximately 3.0 cm and only adult males were used in this assay for sampling. Each fish was kept separately in a two liter aquarium, avoiding contamination with others, and fed twice a day with Tetra  Color Tropical Granules™. Partial water changes of about 50% was done each day. Water parameters were kept at a pH 6.2 and a temperature of 27 °C.

Parasitic diagnosis
The body surface and gills of fish were scraped for parasitological analysis and when the parasites were present the smears were impregnated with silver nitrate using Klein's method (KLEIN, 1958) or stained with Giemsa to observe the nuclear apparatus (LOM, 1958). The span was the measurement from the extremity of the blade to the extremity of the ray as described by Arthur & Lom (1984). All measurements are in micrometers and followed the recommendations of Lom (1958) and Van As & Basson (1989) and analyzed using a Nikon E200  photomicroscope equipped with the Moticam 2300  image capture system. The parasite measurements were made using ImagePro Plus  4.1 software. Minimum and maximum values are provided, followed in parentheses by arithmetic mean, standard deviation and number of specimens or structures measured. Schematic drawings of the denticles, as proposed by Van As & Basson (1989), were produced by means of vectorization using CorelDraw  X6 software.

Parasite diagnosis
The fish examined showed trichodinid prevalence of 55.9% and Piscinoodinium pillulare Lom, 1981 prevalence of 21.5%, only in the fish from the states of São Paulo and Minas Gerais. No parasites were found in the fish from the state of Paraná.
In silver nitrate impregnated material (Figure 1a), the center of the adhesive disc is of similar appearance to the adhesive disc. The blade is sickle-shaped with a tangent point that is slightly flat and not totally parallel to the y+1 axis. The blade has a rounded apex, almost touching the y+1 axis (Figure 1b-c). The blade apophysis is rarely observed but some specimens have a discrete apophysis situated between the blade and the central part (Figure 1c). The central part is triangular and rounded, filling half of the space between the Y and y-1 axes. An indentation below the X axis in the central part is present in some specimens. The connection between the central part and the ray is short. The ray is slender and straight with a rounded point, parallel to the Y axes. Ray apophysis is present. A few specimens showed discreetly anterior or posterior-directed rays, with tips surpassing the Y axes. The nuclear apparatus has a horseshoe-shaped macronucleus and oval micronucleus situated in the y+1 position.

Discussion
The present study reveals the presence of T. modesta, a parasite originally reported from the Eurasian region and now found in the Americas with a new and unusual host (B. splendens).
Since the first report of T. modesta in Hungary, Slovakia and the Czech Republic by Lom (1970), it has now also been found in several other Eurasian countries (Table 1). The morphological and morphometric characteristics of T. modesta described in the present study resemble the original description (LOM, 1970) and other records (WIERZBICKA, 1997;GAZE & WOOTTEN, 1998;. Despite the variation in the body diameter, T. modesta is classified as a small-sized (BASSON & VAN AS, 1994;GAZE & WOOTTEN, 1998;ZHAO & TANG, 2007) or medium-sized trichodinid (WIERZBICKA, 1997;ÖZTÜRK & ÖZER, 2007;present study). Morphometric variation may occur due to environmental and ecological factors in this ciliate group (KAZUBSKI, 1971). Previous studies found the parasite exclusively on the gills (LOM, 1970;ARTHUR & LOM, 1984;WIERZBICKA, 1997). In contrast, in the present study, we have reported the presence of this parasite both on the gills and on the body surface, in a similar way to what was found by Basson & Van As (1994), Özer (2007) and Öztürk & Özer (2007). Gaze & Wootten (1998) commented that T. modesta shows a high degree of host specificity, in comparison with other cosmopolitan trichodinids, for example T. heterodentata. From analyzing the reports in the literature, it seems that T. modesta shows specificity for the order Cypriniformes. Nevertheless, in the present study, the parasite was reported in an unusual fish, similar to that found by Basson & Van As (1994), who also reported T. modesta in Perciformes fish. Two Silurifomes hosts found by Kibria et al. (2010) and Liu & Zhao (2010) are further examples of its unusual occurrence.
Betta spp. is originally from Asia and it may have been responsible for the introduction of T. modesta into South America. On the other hand, because there are no reports of this trichodinid in the native area of this fish, this hypothesis could be challenged. Misgurnus anguillicaudatus has recently been found in natural Brazilian environments (GOMES et al., 2011), while Aphanius danfordii, Crossostoma lacustre and Rhinogobius brunneus are frequently found commercially in Brazilian pet shops. These are native fish in the endemic area for T. modesta and several studies have reported parasitism by this ciliate on these ornamental fish (BASSON & VAN AS, 1994;ÖZTÜRK & ÖZER, 2007;ZHAO & TANG, 2007;HAN & ZHAO, 2011;TANG et al., 2013).
From the above statements, it can be suggested that the introduction of T. modesta into the Neotropical region might be associated with large-scale commercial introductions of fish. It was also suggested by Van As & Basson (1989) that T. heterodentata has become dispersed worldwide as a result of cichlid introductions into new localities. In addition, cyprinid introductions have also been responsible for dissemination of Lernaea cyprinacea in Brazil (PORTZ et al., 2013). Dispersion of fish and their parasites into new localities may increase the host diversity for parasitic infestations, thus supporting the recent findings of T. modesta in non-Cypriniformes fish such as those described by Kibria et al. (2010), Liu & Zhao (2010) and in the present study. This study shows that most of the trichodinid parasites in South America need to be evaluated. It contributes towards improving the knowledge of the parasitic fauna of an important freshwater fish (B. splendens). Nevertheless, this is the first record of T. modesta in the Neotropical region and B. splendens is a new host for this ciliate. These data may infer the possibility of parasite introduction into the Americas via introductions of ornamental cyprinids.