Trichodina colisae (Ciliophora: Trichodinidae): new parasite records for two freshwater fish species farmed in Brazil

Family Trichodinidae comprises ciliate protozoa distributed worldwide; they are considered some of the main parasitological agents infecting cultivated fish. However, the trichodinidae parasitizing important fish species cultured in Brazil are unknown, and more taxonomic studies on this group of parasites are required. This research morphologically characterizes Trichodina colisae Asmat & Sultana, (2005) of pacu ( Piaractus mesopotamicus ) and patinga hybrid ( P . mesopotamicus × P . brachypomus ) cultivated in the central and southeast regions of the country. Fresh assemblies were made from mucus scraped from the skin, fins and gills, fixed with methanol and, subsequently, impregnated with silver nitrate and stained with Giemsa for assessment under light microscopy. This research reports not only the second occurrence of T . colisae in the world, but also its first occurrence in South America


Introduction
Trichodinids are ciliate protozoa widely known as ectocommensals and/or parasites of shes. Members of the family Trichodinidae, they present complex structures on the aboral face, composing the adhesive disk (BASSON; VAN AS, 2006), which presents fast circular movements that can cause abrasive lesion on the host when at high levels of parasitism, even being able to cause mortality (KHAN, 2004). Situations like these usually occur in sh breeding, since changes in the environmental quality directly in uence the occurrence of these protozoa (HOSSAIN et al., 2008).
Knowledge on the fauna of trichodinidae infecting sh in Brazil is little known. So far, only the occurrence of Tripartiella pseudoplatystomae in pintado (Pseudoplatystoma corruscans) (PINTO et al., 2009) and Trichodina heterodentata in pacu (Piaractus mesopotamicus) (PÁDUA et al., 2012) were reported in Brazilian native shes. Nevertheless, Trichodina compacta and Trichodina magna have been reported in Nile tilapia (GHIRALDELLI et al., 2006;GHIRALDELLI, 2008) and T. heterodentata in channel cat sh (MARTINS et al., 2010); they are both exotic species farmed in Brazil. e industrial production of native freshwater sh in Brazil is concentrated in the central and north regions, being less in the northeast, southeast and south regions of the country. Round sh are among the species farmed in Brazil, including pacu (Piaractus mesopotamicus), tambaqui (Colossoma macropomum), pirapitinga (Piaractus brachypomus), and the hybrids tambacu (C. macropomum × P. mesopotamicus), tambatinga (C. macropomum × P. brachypomus) and patinga (P. mesopotamicus × P. brachypomus). Little information is available regarding parasite infecting these shes in commercial production systems.
is study contributes to the description of Trichodina colisae Asmat and Sultana (2005) as a new parasite for pacu and patinga hybrid farmed in Brazil.

Materials and Methods
Pacu were captured in sh ponds in the southeast region of the country (20° 20' 47.1" S and 48° 11' 27.1" O) and were denominated Population A; hybrid patinga (P. mesopotamicus × P. brachypomus) farmed in sh ponds were captured in the southeast region (22° 14' 22.7" S and 48° 17' 33.4" O) and denominated Population B; whereas pacu were captured in sh ponds in the central regions (22° 19' 42.7" S and 54° 43' 55.2" O) and were denominated Population C. Fresh assemblies from mucus scraped from the skin, ns and gills were assessed using light microscopy for the searching of trichodinidae. When positive, the slides were dried; 30% were xed with methanol and stained with Giemsa (1 drop: 1 mL of distilled water, from 120 to 180 minutes) to highlight the nuclear apparatus. Others (70%) were subjected to impregnation with silver nitrate (2%) for observation of the adhesive disk (KLEIN, 1958). e span of the denticle was measured from the blade tip to the ray tip, as described by Arthur and Lom (1984). All measures are in micrometers and follow the recommendations by Lom (1958) and Van As and Basson (1989), using photomicrographies taken with Nikon E200 ® light microscope, equipped with Moticam 2300 ® image capture system. e measurement of the parasite was performed with the assistance of Image-Pro Plus ® software. In addition, the schematic diagram of the denticles, as proposed by Van As and Basson (1989), was built through vectorization, using CorelDRAW ® X5 software.

Results
In this study, medium-sized, disk format trichodinids were found on the skin, ns and gills in 100% of pacu (Population A) and hybrid patinga (Population B) in southeastern Brazil, as well as on the skin and ns of 52% the analyzed pacu (Population C) from central region (Figure 1a measurements of Trichodinidae populations A, B and C were set based on the assessment of 30, 45 and 28 specimens, respectively. is trichodinid presents wide blade, forming a moderately closed angle, tting itself in the quadrant delimited by the axes y and y + 1. e anterior margin of the blade is convex and its apex, in most cases, surpasses the axis y + 1. However, in some specimens, the blade apex touches the axis y + 1 without surpassing it. e distal surface of the blade is at, sometimes round, parallel to the membrane of the border. e point of tangency is oblong in most individuals and may present acute angle in others, it is situated slightly above or at the same height of the distal point of distal surface in populations A and B, but below or at the same height from the distal point of distal surface in population C. Blade apophysis is not present. e blade tting with the central part is thin on populations A and B, and robust in population C (Figure 2). No posterior projection was observed in the sample. e central part is thin, triangular, with no conical part, with an oblong tip and free from indentation below the X axis. In some specimens, the presence of discrete apophysis near the tting with the ray was noticed. e ray is moderately thick, in ated, straight, and oblong-tipped, usually accompanying the Y axis in most individuals; some may present di erent lengths in the same specimen, but this di erence is discreet. In population A, few individuals showed discreetly posterior-directed rays, with its proximal portion surpassing the Y axis. On the other hand, in population B, in some cases, the radius is projected anteriorly, with its proximal portion surpassing the axis y+1. e nuclear apparatus, evidenced by the Giemsa staining, revealed the presence of horseshoe-shaped macronucleus, with oval micronucleus located in the +y portion. However, the micronucleus was only detected in some specimens (Figure 1d). 20.9 ± 1.6 (17-25) 20.5 (18-23) Denticle span 9.6 ± 1.0 (7.6-11.7) 10.4 ± 1.2 (7.8-13.9) 9.6 (5.5-11.8) Denticle L 4.7 ± 0,6 (3.7-5.7) 6.1 ± 0.7 (4.
Trichodina pagoda (Basson and Van As, 1994) and T. erbilensis (Shwani et al., 2010) are similar to the material of this study regarding the number of denticles, pins per denticle, length of denticle, and ray length. T. ngoma (Van As and Basson, 1992) is similar concerning body measurement, border membrane, number of denticles, radial pins by denticle, denticle length, central part and ray length. On the other hand, T. minuta Basson et al. (1983) di ers from the species found in this research in denticle length and ray length, which were smaller in this species. Similarly, T. nkasa (Van As and Basson, 1992) shows ray of smaller size, and this species also has a prominent apophysis in the tting of the central part with the ray. In this research, it was possible to observe only a discrete apophysis in some specimens. e border membrane is broad in T. kazubski (Van As and Basson, 1989), which can be con rmed in the description by Tang et al. (2007) for specimens collected from cyprinids in China. Additionally, this species di ers from the others by presenting apophysis in the anterior blade margin and prominent apophysis of the ray (VAN AS; BASSON, 1989). In the central part, T. kazubski (Van As and Basson, 1989) shows discreet indentation below the X axis, opposite to the ray apophysis of the posterior denticle, which was not perceived in this research.
Two species of trichodinidae showed 100% of morphometric similarity with the specimens analyzed in this study, namely T. matsu (Basson and Van As, 1994) and T. colisae (Asmat and Sultana, 2005). Asmat and Sultana (2005) recognized the similarity between these species and have set some criteria to distinguish the two of them. Among these criteria, the following were observed in the sample specimens, namely: 1-absence of indentation in the inferior part of the X axis of the central part of T. colisae; 2-thick, in ated, round-tipped ray in T. colisae, while in T. matsu it is thin and sharp tipped; 3-the posterior blade margin forms a triangular curve in T. matsu, while in T. colisae, it is growing and shallow. ese three main characteristics of the species based the di erentiation of trichodinid species found in the Brazilian sh of this research, coinciding with T. colisae Asmat and Sultana (2005).
Trichodina colisae was initially reported on gills of colisa Polyacanthus fasciatus (synonym Trichogaster fasciatus), an ornamental sh from Bangladesh. is is the second record of this species of trichodinid in the world and the rst one in Brazil. Pacu (P. mesopotamicus) and hybrid patinga (P. mesopotamicus × P. brachypomus) are new records of hosts for this parasite.