Parasitic fauna and histopathology of farmed freshwater ornamental fish in Brazil

Aquaculture 470 (2017) 103–109

Contents lists available at ScienceDirect

Aquaculture journal homepage: www.elsevier.com/locate/aquaculture

Parasitic fauna and histopathology of farmed freshwater ornamental fish in Brazil Monyele Acchile Santos a, Gabriela Tomas Jerônimo a,b, Lucas Cardoso a, Karen Roberta Tancredo a, Paula Brando Medeiros a, José Victor Ferrarezi a, Eduardo Luiz Tavares Gonçalves a,c, Guilherme da Costa Assis d, Maurício Laterça Martins a,⁎ a

AQUOS - Aquatic Organisms Health Laboratory, Aquaculture Department, Federal University of Santa Catarina (UFSC), Rod. Admar Gonzaga 1346, 88040-900 Florianópolis, SC, Brazil Post Graduate in Aquaculture, Nilton Lins University, Av. Nilton Lins 3259, 69058-030 Manaus, AM, Brazil c Federal Institute of Santa Catarina, Campus São Carlos, R. Aloisio Stoffel, 1271, 89885-000 São Carlos, SC, Brazil d Vale dos Bettas Fishfarm, Sorocaba of Fora s/n, 88160-000 Biguaçu, SC, Brazil b

a r t i c l e

i n f o

Article history: Received 7 November 2016 Received in revised form 21 December 2016 Accepted 23 December 2016 Available online 26 December 2016 Keywords: Ornamental fish Epidemiology Parasites Cestodes Histopathology Necrosis

a b s t r a c t Ornamental fish farming represents a consolidated market over the world. However, confinement is a factor that favors the occurrence of diseases. This study aimed to report the parasitic fauna of ornamental fish from three facilities, as well as to observe the histological pathogenesis caused by the parasites. Between May 2015 and February 2016, a total of 781 ornamental fishes were used for parasitological and histopathological analysis. Water quality was measured in fishponds from each facility. Ciliate protozoans Ichthyophthirius multifiliis; Trichodina sp.; the monogeneans Dactylogyrus extensus, D. minutus and Diaphorocleidus kabatai; metacercariae of the digeneans; the cestode Bothriocephalus acheilognathi; the nematode Rhabdochona sp.; and the branchiuran Argulus japonicus were found in the examined fish. The greatest prevalence and mean intensity was observed in the gills of Gymnocorymbus ternetzi parasitized by D. kabatai, followed by the protozoan parasite I. multifiliis on the body surface of Xiphophorus maculatus. Histopathological analysis showed epithelial interlamellar hyperplasia of the secondary lamellae, partial fusion of the secondary lamellae, telangiectasia, justalamellar edema and eosinophilic inflammatory infiltrate. The intestine of cestode parasitized fish showed necrosis in the submucosa, intestinal obstruction and lymphoeosinophilic inflammatory infiltrate. It is important to know the parasitic fauna of farmed fish and the pathogenesis caused by the parasites in order to ensure fish production and the health of the hosts. Statement of relevance: Ornamental fish production as a consolidate activity around the world faces problems of parasite infection leading to fish mortality and economic losses. To ensure farming production, it is important to monitor the status of fish health. Parasitic fauna and histopathological analysis are used as important tools for the diagnosis of tissue lesions. © 2016 Elsevier B.V. All rights reserved.

1. Introduction Fish farming is rapidly growing and ornamental fish farming is an important economic activity (Santos et al., 2014). According to Lima et al. (2001), Brazil is recognized as the main supplier of ornamental fish species, however most of the production is as a result of capture. These captured ornamental fish are exported, while the internal market is supplied mainly by allocthonous species produced in captivity (Nottingham and Ramos, 2006). In 2007, the amount of ornamental fish imports in Brazil was relatively low, yielding about US$5 million (Monticini, 2010). This low amount can be explained by the enthusiastic entrance into the market of new domestic producers of ornamental fish in recent years. The market entry of fish farmers is stimulated by the ⁎ Corresponding author. E-mail address: [email protected] (M.L. Martins).

http://dx.doi.org/10.1016/j.aquaculture.2016.12.032 0044-8486/© 2016 Elsevier B.V. All rights reserved.

rapid growth of fish, well adapted to culture conditions and by the desire to diminish the extractive capture of the native fish species, since many of them are threatened by extinction (Tlusty, 2002; Zuanon et al., 2011). Intensive fish farming has favored the occurrence and dissemination of parasitic diseases as a result of imbalances in the host/parasite/ environment relationship (Jerônimo et al., 2012), which predispose the fishes to disease outbreaks (Portz et al., 2013). The equilibrium in this triad is easily disturbed by increased numbers of parasites, high levels of nitrogen compounds from excessive feeding, high stocking density, poor water quality, inadequate handling and lack of the best management practices (Garcia et al., 2003; Eiras, 2004; Giorgiadis et al., 2001). The pathogenic action of parasites, especially those that cause lesions on the hosts, has been studied mainly in fish of economic importance (Lom and Dyková, 1992; Martins et al., 2015). Depending on the

104

M.A. Santos et al. / Aquaculture 470 (2017) 103–109

mode of parasite attachment, focal, multifocal and diffuse lesions can be found (Khan, 2012). Previous studies have shown that Ichthyophthirius multifiliis Fouquet, 1876 (Mohammadi et al., 2012); trichodinids (Yemmen et al., 2011); monogeneans (Fujimoto et al., 2014); digeneans (Omrani et al., 2010); nematodes (Menezes et al., 2006); cestodes (Dezfuli et al., 2011); and branchiurans (Saha and Bandyopadhyay, 2015) are responsible for tissue damage in ornamental fishes. Monitoring of parasitism and histological analysis of the organs in farmed fish can ensure the early diagnosis of pathogens prior to their dissemination. Histological examination of fish organs is an important tool for rapid diagnosis (Takashima and Hibiya, 1995; Genten et al., 2009). For example, gill alterations such as hypertrophy, edema, necrosis, epithelial desquamation, hyperplasia, fusion of the secondary lamellae and telangiectasia are reported in parasitized fish (Roberts, 2001; Campos et al., 2011). Intestinal helminths can provoke an inflammatory reaction at their attachment site. Depending on the intensity of the parasitic infestation, they can also provoke intestinal hemorrhages, inflammation and loss of gastrointestinal function (Molnár, 2005; Alvarez-Leon, 2007; Dezfuli et al., 2007, 2011; Alvarez-Pellitero et al., 2008). The aim of this study was to assess the parasitic fauna in farmed ornamental fishes from three facilities in Southern Brazil, as well as to evaluate the pathogenesis caused by the parasites through histological analysis.

Fish farm B: blood swordtail (Xiphophorus helleri) (1.8 ± 1.2 g, 5.2 ± 1.5 cm, n = 57); black swordtail (Xiphophorus helleri) (2.3 ± 0.8 g, 6.0 ± 0.8 cm, n = 15); wagtail platy (Xiphophorus maculatus) (0.6 ± 0.1 g, 3.3 ± 0.2 cm, n = 15); hawaii platy (Xiphophorus maculatus) (1.3 ± 0.4 g, 4.4 ± 0.5 cm, n = 30); blue platy (Xiphophorus maculatus) (1.4 ± 0.8 g, 4.3 ± 0.7 cm, n = 60); mickey mouse platy (Xiphophorus maculatus) (1.3 ± 0.4 g, 4.3 ± 0.4 cm, n = 45); black tetra (Gymnocorymbus ternetzi Boulenger, 1895) (4.0 ± 1.7 g, 6.0 ± 0.5 cm, n = 15); pink tetra (Gymnocorymbus ternetzi) (4.3 ± 1.0 g, 6.0 ± 0.7 cm, n = 60); zebrafish (Danio rerio Hamilton, 1822) (0.5 ± 0.2 g, 3.4 ± 0.6 cm, n = 60); jewel tetra (Hyphessobrycon eques Steindachner, 1882) (1.2 ± 0.4 g, 4.3 ± 0.4 cm, n = 60); goldfinned barb (Puntius sachsii Ahl, 1923) (2.6 ± 2 g. 6.0 ± 0.9 cm, n = 60). Fish farm C: Koi carp (Cyprinus carpio Koi Linnaeus, 1758) (4.4 ± 1.2 g, 6.8 ± 1.9 cm, n = 229). The ornamental fishes were collected with net and kept alive in plastic bags to be transported to the laboratory for parasitological and histopathological analysis. In each fish collection, the water quality parameters were measured: transparency with Secchi disc, ammonia and pH measured with commercial kit ammonia freshwater Hanna (HI 38049, São Paulo, Brazil), dissolved oxygen, water temperature and salinity measured with a multiparameter Hanna (HI 9828, São Paulo, Brazil). Concomitantly to the collections was asked to the producers which management practices usually were adopted for better understanding the data.

2. Material and methods

2.2. Parasitological analysis

2.1. Fish collection

The fish were anesthetized in eugenol (75 mg·L−1) and euthanized by cerebral concussion. These procedures were previously approved by the Ethics Committee on Animal Use from the Federal University of Santa Catarina (CEUA/UFSC PP00928). Macroscopic observation of the body surface and organs was performed to verify any lesions and/or alterations caused by pathogens. Parasitological analysis was performed according to Jerônimo et al. (2013). Scrapings from the body surface and fresh samples of the internal organs were mounted on glass slides with a drop of saline solution 0.65% for microscopic observation. The eyes were placed in Petri dishes, dissected and observed under a stereomicroscope, while the gill arches were placed in flasks containing hot water at 55 °C, agitated and fixed in 70% ethanol for later parasite quantification. Parasites were counted according to Jerônimo et al. (2016) and parasitological indices (prevalence and mean intensity) were calculated as recommended by Bush et al. (1997). The trichodinid protozoans were impregnated with silver nitrate using the method of Klein (1958) and identified according to Pádua et al. (2012), Valladão et al. (2013) and Dove and O'Donoghue (2005). Monogeneans were mounted in Hoyer's medium between a slide and coverslip for observation of sclerotized structures and the copulatory complex (Eiras et al., 2006), for the purpose of identification according to Dzika et al. (2009) and Sujan and Shameem (2015). Cestodes were stained with carmine according to Eiras et al. (2006) and identified according to Brandt et al. (1981) and Scholz (1997). Nematodes were clarified with Amann's lactophenol, mounted in Canada balsam and identified according to Moravec (1998, 2001). Branchiurid crustaceans were clarified with lactic acid and identified according to Cressey (1978), Mousavi et al. (2011), Rushton-Mellor (1994) and Soes et al. (2010).

Between May 2015 and February 2016, a total of 781 ornamental fishes were collected each three months from three facilities located in the State of Santa Catarina, Brazil: fish farm A (FA) (26° 22′ 12″ S 48° 43′ 20″ W), fish farm B (FB) (27° 29′ 39″ S 48° 39′ 20″ W) and fish farm C (FC) (26° 49′ 24″ S 49° 16′ 18″ W), characterized in Table 1. The number of sampled fish (n) and biometry are as follows: Fish farm A: blood swordtail (Xiphophorus helleri Heckel 1848) (4.1 ± 0.8 g, 6.9 ± 0.5 cm, n = 30); wagtail platy (Xiphophorus maculatus Gunther 1866) (1.6 ± 1.1 g, 4.5 ± 0.7 cm, n = 30); common platy (Xiphophorus maculatus) (0.8 ± 0.6 g, 3.1 ± 2.0 cm, n = 15). Table 1 Characteristics of the fish farms used in this study. Characteristics

FA

FB

FC

Fish farm size Culture system Pond size Water source Water exchange rate Fish source Stocking density Feeding frequency Larval diet (powder) Breeding diet (pellet) Aeration Control of water quality Fertilization Water renewal Mortalities TR (cm) AM (mg·L−1) pH DO (mg·L−1) TE (°C) SAL (‰)

0.0018 ha Semi intensive 0.0004 ha Rain water 5–15% Own production No control 2 times a day 36% CP 2–3% (biomass) No Yes Yesa Yes No 25 ± 8.5 0.4 ± 0.3 6.5 ± 1.2 6.0 ± 3.4 21 ± 1.5 0.06 ± 0.0

22 ha Semi intensive 0.03 ha Velho River 5% Own production No control Once a day 55% CP 3% (biomass) No No Yesa Yes 20% 18 ± 10 0.1 ± 0.2 6.0 ± 3.2 5.3 ± 1.6 22.4 ± 2.6 0.02 ± 0.0

0.28 ha Intensive 0.02 ha Fortuna River No exchange Own production 1 fish/m3 2 times a day 46% CP 3% (biomass) Yes No Yesa Yes No 23.2 ± 26.3 0.1 ± 0.1 7.2 ± 0.9 6.8 ± 2.0 23.6 ± 3.7 0.02 ± 0.0

FA: Araquari. FB: Biguaçu. FC: Timbó. CP: crude protein. Mean values ± standard deviation of water quality in the fish farms studied from Southern Brazil. TR: transparency, AM: ammonia, DO: dissolved oxygen, TE: temperature, SAL: salinity. a Fertilization only when needed.

2.3. Histopathological analysis Fragments of the gills and intestine of 260 fishes with the highest mean intensities of parasitism were fixed in 10% buffered formalin solution to observe the tissue alterations caused by the parasites. The organs were dehydrated in serial solutions of alcohol, cleared in xylol, embedded in paraffin at 60 °C for posterior cross sections of 5 μm thickness and

M.A. Santos et al. / Aquaculture 470 (2017) 103–109

105

justalamellar edema and eosinophilic inflammatory infiltrate were observed in the gill tissue (Fig. 1). In fish parasitized by the cestode B. acheilognathi, necrosis in the intestinal submucosa and lymphoeosinophilic inflammatory infiltrate were observed (Fig. 2), but there was no significant difference among the samples. No significant difference was observed on the histological alteration in both the gills and intestine of fish from fish farms A and B. Light intensity alterations were found in the gills of Koi carp from fish farm C. The results showed significant differences in the following lesions: fusion (p = 0.004712) and epithelial hyperplasia of the secondary gill lamellae (p = 0.02020).

stained with hematoxylin and eosin (HE). Slides were mounted in Entellan® and analyzed in differential interference contrast microscope (DIC) (ZEISS, Axio Imager A.2, Gottingen, Germany). Histological alterations in the gills and intestines were semiquantitatively evaluated according to the severity degree of lesions: 0 (absence of lesion), 1 (light lesion), 2 (moderate lesion) and 3 (severe lesion) according to the method of Schwaiger et al. (1997) with slight modifications. 2.4. Statistical analysis Comparison of the presence and absence of histological lesions was performed using Fisher's exact test using the software Openepi (Dean et al., 2013), for bicaudal comparison (p b 0.05). It was not possible to apply parametric analysis because the data did not show normality and homoscedasticity.

4. Discussion The prevalence values of I. multifiliis in X. maculatus (36 to 40%) were higher than those found in X. maculatus (4%) from Sri Lanka (Thilakaratne et al., 2003), in Hyphessobrycon copelandi (Durbin, 1908) (21%) from the Negro River, Amazonas (Tavares-Dias et al., 2010) and in X. maculatus (6%) commercialized in Southern Brazil (Piazza et al., 2006). The proliferation of this parasite in fish ponds can be favored by poor water quality, high stocking density, inadequate water temperature and nutritional deficiencies in fish (Piazza et al., 2006). Prevalence rates found in this study may have been influenced by the lack of control of the fish population and water quality, although low parasite intensities were observed, signaling that control measures should be taken to prevent their proliferation, especially in fish farm B. Few studies have been reported on histological alterations in ornamental fish. Hyperplasia, fusion of the secondary lamellae, edema and telangiectasia are mechanisms of host defense related to the presence of I. multifiliis in the gill epithelium. Much of the histological changes in gills are associated with the non-specific defense mechanism (Buchmann et al., 2001; Paruruckumani et al., 2015), triggering responses that make up the organism's first defense mechanisms against infections (inflammation, phagocytosis, accessory phagocytosis cell and non-specific cytotoxicity). However, there is little evidence that the lesions observed in this study were caused by parasitism by I. multifiliis. The prevalence rates of T. heterodentata (23%) in wagtail platy X. maculatus and Trichodina sp. (10%) in X. helleri reported in this study were lower than those observed by Trichodina spp. (51.57%) in Carassius auratus (Linnaeus, 1758), X. maculatus and in Poecilia reticulata (Peters, 1859) (Martins et al., 2012). In contrast, T. heterodentata was registered at high levels when compared with Trichodina sp. (16.6%) from C. auratus (Iqbal and Hussain, 2013). As argued by Jerônimo et al. (2012), trichodinids are considered parasites of high dispersion all over the world. Its proliferation is strongly associated with poor water quality, high stocking density, high organic matter content and increased temperature (Martins et al., 2010, 2015). These damages might be strongly associated with the circular movement of the parasite on the gill surface. Damage is not only a result of mechanical injuries — bacteria can colonize the trichodinids, with mechanical injuries

3. Results 3.1. Parasitological analysis This study revealed great parasite diversity in ornamental fishes cultured in the South of Brazil comprising the causative agents I. multifiliis (Fouquet, 1876) and tricodinids, monogeneans, digeneans, nematodes, cestodes and branchiurids. All parasite species were observed at a low degree of prevalence and mean intensity (Tables 2, 3 and 4). The highest prevalence rate found in the examined fish was monogenean Diaphorocleidus kabatai (Molnar, Hanek and Fernando, 1974) Jogunoori, Kritsky and Venkatanarasaiah, 2004 in the gills of G. ternetzi with 45% prevalence and mean intensity of 3.7 ± 2.0. Dactylogyrus extensus Mueller and Van Cleave, 1932 and D. minutus Kulwiec, 1927 parasitized 40% of Koi carp examined, with mean intensity of 4.3 ± 4.2 parasites per host. For protozoa, the ectoparasite I. multifiliis was observed on the body surface of X. maculatus, with prevalence of 40% and mean intensity of 1.0 ± 0,0. Trichodinids were found in six species of ornamental fish and Trichodina heterodentata Duncan, 1977 showed the highest prevalence and mean intensity on the body surface of X. maculatus. Metacercariae of the digeneans were only observed in the muscle of H. eques with 1.7% prevalence and mean intensity 5.0 ± 0.0. Nematodes of the genus Rhabdochona parasitized the intestine of 6.6% of G. ternetzi with a mean intensity 1.2 ± 2.1. In the intestine of Koi carp Bothriocephalus acheilognathi Yamaguti, 1934 was found at a prevalence of 13% and mean intensity of 23.9 ± 22.8. On the other hand, the branchiuran Argulus japonicus Thiele, 1900 was found to be parasitizing the body surface at 5.7% prevalence and mean intensity of 1.0 ± 0.05. 3.2. Histopathological analysis Regarding histological alterations, epithelial hyperplasia of the secondary lamellae, partial fusion of the secondary lamellae, telangiectasia,

Table 2 Parasitological indices of trichodinids, monogeneans and cestodes in farmed ornamental fishes from fish farm A, Brazil. Fish species

Black swordtail (Xiphophorus helleri)

Wagtail platy (Xiphophorus maculatus)

Southern platy (Xiphophorus maculatus)

Trichodinids

Monogeneans

SI

P (%)

MI

G M I G M I G M I

10.0 13.3 – 10.0 23 – 6.6 0.0 –

0.7 0.7 – 1.3 1.9 – 1.0 0.0 –

± 0.9 ± 0.8 ± 0.3 ± 1.7 ± 0.0 ± 0.0

Cestodes

P (%)

MI

0.0 0.0 – 6.6 0.0 – 0.0 0.0 –

0.0 0.0 – 0.5 0.0 – 0.0 0.0 –

± 0.0 ± 0.0 ± 0.7 ± 0.0 ± 0.0 ± 0.0

Site of infestation/infection (SI); prevalence (P %); mean intensity (±standard deviation); gills (G); mucus from the body surface (M) and intestine (I).

P (%)

MI

– – 6.7 0.0 0.0 3.3 – – 0.0

– – 1.2 0.0 0.0 2.5 – – 0.0

± ± ± ±

1.7 0.0 0.0 3.5

± 0.0

106

M.A. Santos et al. / Aquaculture 470 (2017) 103–109

Table 3 Parasitological indices of Ichthyophthirius multifiliis, trichodinids, monogeneans, digeneans and nematodes in farmed ornamental fishes from fish farm B, Brazil. Fish species

Blood swordtail (Xiphophorus helleri) Black swordtail (Xiphophorus helleri) Wagtail platy (Xiphophorus maculatus) Hawaii platy (Xiphophorus maculatus) Blue platy (Xiphophorus maculatus) Mickey mouse platy (Xiphophorus maculatus) Zebrafish (Danio rerio) Goldfinned barb (Puntius sachsii)

Ichthyophthirius multifiliis

Trichodinids

SI

P (%)

MI

P (%)

MI

P (%)

MI

G M G M G M G M G M G M G M G M

0.0 3.4 26 13 6.6 40 3.3 36 0.0 0.0 0.0 0.0 0.0 0.0 0.0 3.0

0.0 0.5 3.3 1.0 1.0 1.0 4.0 7.8 0.0 0.0 0.0 0.0 0.0 0.0 0.0 1.7

0.0 5 0.0 0.0 0.0 0.0 6.6 20 0.0 0.0 0.0 20 0.0 3.3 0.0 0.0

0.0 ± 0.0 1.4 ± 0.6 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 7.2 ± 6.2 10 ± 4.3 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 4.4 ± 3.5 0.0 ± 0.0 0.5 ± 0.9 0.0 ± 0.0 0.0 ± 0.0

0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 1.7 0.0 2.2 0.0 0.0 0.0 0.0 0.0

0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.2 0.0 0.4 0.0 0.0 0.0 0.0 0.0

Fish species

Pink tetra (Gymnocorymbus ternetzi) Black tetra (Gymnocorymbus ternetzi) Jewel tetra (Hyphessobrycon eques)

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.0 0.8 0.0 0.0 0.0 0.0 5.6 6.9 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.2

Monogeneans

Ichthyophthirius multifiliis

Trichodinids

SI

P (%)

MI

P (%)

MI

G M G M G M

1.7 0.0 0.0 0.0 1.7 15

0.0 0.0 0.0 0.0 0.0 2.8

1.7 0.0 0.0 0.0 1.7 10

0.0 0.0 0.0 0.0 0.0 3.9

Fish species

± ± ± ± ± ±

0.0 0.0 0.0 0.0 0.0 0.2

Jewel tetra (Hyphessobrycon eques)

0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0

± ± ± ± ± ±

2.0 0.0 0.0 0.0 0.0 0.0

Monogeneans

± ± ± ± ± ±

Digeneans

Pink tetra (Gymnocorymbus ternetzi)

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.0 0.0 0.0 0.0 0.0 0.2

P (%)

MI

45 0.0 0.0 0.0 0.0 0.0

3.7 0.0 0.0 0.0 0.0 0.0

Nematodes

SI

P (%)

MI

MU I G M MU

0.0 0.0 0.0 – 1.7

0.0 0.0 0.0 – 5.0

± 0.0 ± 0.0 ± 0.0 ± 1.5

P (%)

MI

0.0 6.6 – – 0.0

0.0 ± 0.0 1.2 ± 2.1 – – 0.0 ± 0.0

Site of infestation/infection (SI); Prevalence (P %); Mean intensity (±standard deviation); gills (G); mucus from the body surface (M), muscle (MU) and intestine (I).

providing a portal of entry for infections that result in mortality (Valladão et al., 2013, 2014). Similar to the present report, the monogenean Diaphorocleidus sp. can be found parasitizing hosts of six genera, including Gymnocorymbus sp. (Almeida and Cohen, 2011). Gymnocorymbus ternetzi examined in the present study showed a higher prevalence than previously reported in ornamental fish (22.2%) (Piazza et al., 2006). Similar prevalences were found in the gills of Koi carp examined by Kritsky and Heckmann (2002) with a prevalence of 39% for five species of Dactylogyrus sp., and lower prevalences (1.2%) of D. minutus were reported by Kritsky and Heckmann (2002). The prevalence values of the monogenean D. kabatai (45%) from the gills of G. ternetzi and Dactylogyrus spp. (40%) from the gills of Koi carp C. carpio were slightly lower than for Urocleidoides sp. (100%) from Xiphophorus sp. (Garcia et

al., 2003). Monogeneans are considered one of the most important metazoan parasites in farmed fish (Martins et al., 2002; Garcia et al., 2009; Eiras et al., 2011; Tavares Dias et al., 2010; Portz et al., 2013). Histological alterations as edema and telangiectasia related to the presence of these parasites in the gills were similar to those observed in ornamental fish from the Guamá River, Pará, Northern Brazil (Fujimoto et al., 2014). To date, fusion and hyperplasia of the secondary lamellae has also been reported in C. carpio parasitized by D. vastator (Paperna, 1964; Vinobaba, 1994) and Dactylogyrus sp. (Shinn et al., 2004; Hossain et al., 2007). Such lesions were associated with the attachment mode of the parasite in terms of the haptor and the feeding preference on the body and gill epithelium, causing desquamation and portals of entry for secondary infection (Xu et al., 2007). As a result of parasitism, excessive mucus production can inhibit respiratory function and cause

Table 4 Parasitological indices of Ichthyophthirius multifiliis, trichodinids, monogeneans, nematodes, cestodes and branchiurans in farmed ornamental fishes from fish farm C, Brazil. Fish species

Koi carp (Cyprinus carpio)

Ichthyophthirius multifiliis

Trichodinids

SI

P (%)

MI

P (%)

MI

P (%)

MI

G M

1.7 18

2.8 ± 2.3 3.6 ± 2.1

2.6 22

3.0 ± 1.8 3.6 ± 4.0

40 0.07

4.3 ± 4.2 2.2 ± 2.0

Nematodes

Koi carp (Cyprinus carpio)

Monogeneans

Cestodes

Branchiurans

SI

P (%)

MI

P (%)

MI

P (%)

MI

M I

– 2.6

– 0.75 ± 0.3

– 13

– 23.9 ± 22.8

5.7 –

1.0 ± 0.05 –

Site of infestation/infection site (SI); prevalence (P %); mean intensity (±standard deviation); gills (G); mucus from the body surface (M) and intestine (I).

M.A. Santos et al. / Aquaculture 470 (2017) 103–109

107

Fig. 1. Histological sections of the gills of koi carp Cyprinus carpio (fish farm C). A – Normal gill filament; B – hyperplasia of the secondary lamellae (*); C – interlamellar hyperplasia (arrow); D – justalamellar edema (arrow); E – telangiectasia (T); F – fusion of the secondary lamellae (F); G – eosinophilic inflammatory infiltrate (arrow). Stained with hematoxylin/eosin.

death in farmed fish. The eosinophilic inflammatory infiltrate observed in the gill epithelium is probably related to parasitism (Kantham and Richards, 1995). Metacercariae of the digeneans are more pathogenic than the adult forms. A low prevalence of metacercariae was observed in H. eques, similar to that reported in Hyphessobrycon sp. (Alvarez-Leon, 2007), in X. maculatus (Piazza et al., 2006) and by Centrocestus formosanus (Nishigori, 1924) in G. ternetzi (Acampo, 2012). Apart from the appearance of cysts that prejudice commercialization, gill lesions such as hyperplasia, fusion of the secondary lamellae (Shoaibi et al., 2010), edema, and telangiectasia (Fujimoto et al., 2014) have been reported. No lesions in the gill epithelium were associated with the presence of metacercariae in this study and this might be related to low degree of parasitism. Bothriocephalus acheilognathi has also been reported in Brazil and its introduction could be related to C. carpio introduced from Asia (Rego, 2000). Studies have shown that the cestode B. acheilognathi parasitizes mainly cyprinid fishes (Scholz et al., 1997). Similar findings were reported in C. carpio from Lake Kovada, Turkey (Kir and Ozan, 2007). Necrosis was also related in the intestine of C. carpio parasitized by B.

acheilognathi (Scholz et al., 2012). In carp, a strong lymphocytic inflammatory reaction was found by Britton et al. (2011). Intestinal obstruction was also observed in fish infected by B. acheilognathi (Han et al., 2010). According to Scott and Grizzle (1979) the presence of bothriocephalid cestodes reduces growth and in severe infections can lead to mortality. No lesions associated with nematode parasitism were found in the intestines of the examined fish as a result of the low degree of infection and the mode of attachment of this nematode. At high parasitic levels, nematodes can attach to the intestinal mucosa of the hosts provoking an inflammatory reaction or bleeding (Heckmann et al., 1987). They can also cause anemia, malnutrition, intestinal obstruction, and decreased growth culminating in host death (Thatcher, 2006). There are some genera that cause significant damage in ornamental fish such as camallanids (Menezes et al., 2006; Iyaji and Eyo, 2008) and the capilariid Pseudocapillaria tomentosa (Dujardin, 1843) (Martins et al., 2016). The branchiuran parasite Argulus is frequently found in farmed fish and presents low host specificity (Gama et al., 2015). This parasite is normally found on the body surface, and histological analysis of the

108

M.A. Santos et al. / Aquaculture 470 (2017) 103–109

Fig. 2. Histological sections of the intestine of koi carp Cyprinus carpio (fish farm C). A – Normal intestine; B – necrosis of the intestinal submucosa (*); C – lymphocytic inflammatory infiltrate (arrow); D – intestinal obstruction by Bothriocephalus acheilognathi (B); E – eosinophilic inflammatory infiltrate (E). Stained with hematoxylin/eosin.

gills of Koi carp C. carpio did not show any alterations. In contrast, Noga (2010) has observed petechiae and necrosis caused by the presence of argulids feeding on the fish epithelium. As the fish farms in this study are located in the South of Brazil, where temperatures are relatively low compared to other regions, a small influence of temperature on parasite reproduction could be expected. High temperatures allied to a lack of the best management practices and poor water quality normally favor parasite reproduction (Martins and Romero, 1996; Martins et al., 2015). In addition, it must be emphasized that the tissue alterations could be associated with the farming conditions, as the gills are directly exposed to changes in the water quality and pollutants (Cantanhêde et al., 2014). Further studies should be carried out to evaluate the influence of abiotic factors on gill tissue structures in ornamental fish. It must be emphasized that the parasite diversity can be related to the lack of routine water quality monitoring and uncontrolled fish population, especially in fish farms A and B. Acknowledgements The authors thank National Council for Scientific and Technological Development (CNPq) for financial support (CNPq 446072/2014-1), for grant to M.L. Martins (CNPq 305869/2014-0), for Post-Doctoral scholarship to G.T. Jerônimo (CNPq 506263-2013-4); Coordination for the Improvement of Higher Education Personnel (CAPES/EMBRAPA no. 15/ 2014) for Master scholarship to M.A. Santos; Dr. Adolfo Jatobá (IFC, Instituto Federal Catarinense, SC, Brazil), fishfarm Valle dos Bettas, SC, Brazil and Dr. Silvio Negrão (Fundação Integrada de Pisciculturas do Vale do Itajaí/FUNPIVI, SC, Brazil) for fish donation, MSc. Patrícia Garcia for help in the histological analysis and PhD Aline Brum Figueredo (AQUOS, Aquaculture Department, UFSC, SC, Brazil) for critical review of the manuscript prior to submission. References Almeida, K.S.S., Cohen, S.C., 2011. Diversidade de monogenea (Platyhelminthes) parasitos de Astyanax altiparanae do reservatório da usina hidrelétrica de Itaipu. Saúde & Amb. Rev. 6 pp. 31–41.

Alvarez-Leon, R., 2007. Asociaciones y patologías en los peces dulceacuícolas, estuarinos y marinos de Colombia: aguas libres y controladas. Bol. Cient. 11 pp. 81–129. Alvarez-Pellitero, P., Palenzuela, O., Sitja-Bobadilla, A., 2008. Histopathology and cellular response in Enteromyxum leei (Myxozoa) infections of Diplodus puntazzo (Teleostei). Parasitol. Int. 57, 110–120. Brandt, F.W., Van As, J.G., Schoonbee, H.J., Hamilton, A.V.L., 1981. The occurrence and treatment of bothriocephalosis in the common carp, Cyprinus carpio in fish ponds with notes on its presence in the largemouth yellow fish Barbus kimberleyensis from the Vaal Dam, Transvaal. Water SA 7, 35–41. Buchmann, K., Sigh, J., Nielsen, C.V., Dalgaard, M., 2001. Host responses against the fish parasitizing ciliate Ichthyophthirius multifiliis. Vet. Parasitol. 100, 105–116. Bush, A.O., Lafferty, K.D., Lotz, J.M., Shostak, A.W., 1997. Parasitology meets ecology on its own terms: Margolis et al. revisited. J. Parasitol. 83, 575–583. Campos, C.M., Moraes, J.R.E., Moraes, F.R., 2011. Histopathology of gills of Piaractus mesopotamicus (Holmberg, 1887) and Prochilodus lineatus (Valenciennes, 1836) infested by monogenean and myxosporea, caught in Aquidauana River, State of Mato Grosso do Sul, Brazil. Braz. J. Vet. Parasitol. 20, 67–70. Cantanhêde, S.M., Medeiros, A.M., Ferreira, F.S., Ferreira, J.R.C., Alves, L.M.C., Cutrim, M.V.J., Santos, D.M.S., 2014. Uso de biomarcador histopatológico em brânquias de Centropomus undecimalis (Bloch, 1972) na avaliação da qualidade da água do Parque Ecológico Laguna da Jansen, São Luís - MA. Arq. Bras. Med. Vet. Zootec. 62, 593–601. Cressey, R.F., 1978. Marine flora and fauna of the Northeastern United States. Crustacea: Branchiura. NOAA Technical Report NMFS Circular. 413, pp. 1–10. Dean, A.G., Sullivan, K.M., Soe, M.M., 2013. OpenEpi: Open Source Epidemiologic Statistics for Public Health (http://www.openepi.com/Menu/OE_Menu.htm, accessed in 2016/ 05/16). Dezfuli, B.S., Giari, L., Simoni, E., Menegatti, R., Shinn, A.P., Manera, M., 2007. Histopathology and ultrastructure of Platichthys flesus naturally infected with Anisakis simplex s.l. larvae (Nematoda: Anisakidae). J. Parasitol. 93, 1416–1423. Dezfuli, B.S., Giari, L., Squerzanti, S., Lui, A., Lorenzoni, M., Sakalli, S., Shinn, A.P., 2011. Histological damage and inflammatory response elicited by Monobothrium wageneri (Cestoda) in the intestine of Tinca tinca (Cyprinidae). Parasit. Vectors 4, 1–11. Dove, A.D.M., O'Donoghue, P.J., 2005. Trichodinids (Cilophora: Trichodinidae) from native and exotic Australian freshwater fishes. Acta Protozool. 44, 51–60. Dzika, E., Dzikowiec, M., Hoffmann, R.H., 2009. Description of the development of the attachment and copulatory apparatus of Dactylogyrus extensus from Cyprinus carpio var. Koi. Helminthology 46, 39–44. Eiras, J.C., 2004. Aspectos Gerais da Patologia das Parasitoses de Peixes Marinhos. In: Ranzani-Paiva, M.J.T., Takemoto, R.M., Lizama, M.A.P. (Eds.), Sanidade de Organismos Aquáticos. Varela, São Paulo, pp. 143–156. Eiras, J.C., Takemoto, R.M., Pavanelli, G.C., 2006. Métodos de estudo e técnicas laboratoriais em parasitologia de peixes. 2ª ed. EDUEM, Maringá. Eiras, J.C., Takemoto, R.M., Pavanelli, G.C., Adriano, E.A., 2011. About the biodiversity of parasites of freshwater fish from Brazil. Bull. Eur. Assoc. Fish Pathol. 31, 161–168. Fujimoto, R.Y., Neves, M.d.S., Santos, R.F.B., Cruz, C., Diniz, D.G., Eiras, J.C., 2014. Histopathological evaluation of seven Amazon species of freshwater ornamental armored catfish. Acta Sci. 36, 349.

M.A. Santos et al. / Aquaculture 470 (2017) 103–109 Gama, C.S., Vieira, I.M., Júnior, A.C.S.S., Tavares-Dias, M., Sobrinho, A.F., 2015. Argulus juparanensis (Branchiura) em raias Potamotrygon spp. (Potamotrygonidae) na foz do Rio Amazonas (Brasil). Biota Amazon. 5, pp. 134–136. Garcia, F., Fujimoto, R.Y., Martins, M.L., Moraes, F.R., 2003. Urocleidoides sp. parasitism in Xiphophorus spp. and its relation to the aquatic parameters. Bol. Inst. Pesca. 29, 123–131. Garcia, F., Fujimoto, R.Y., Martins, M.L., Moraes, F.R., 2009. Protozoan parasites of Xiphophorus spp. (Poeciliidae) and their relation with water characteristics. Arq. Bras. Med. Vet. Zootec. 61, 156–162. Genten, F., Terwinghe, E., Danguy, A., 2009. Atlas of fish histology. Enfield. Science Publisher-SP, USA, pp. 4–5. Giorgiadis, M.P., Gardner, I.A., Hedrick, R.P., 2001. The role of epidemiology in the prevention, diagnosis and control of infectious diseases of fish. Prev. Vet. Med. 1, 287–302. Han, J.E., Shin, S.P., Kim, J.H., Choresca Jr., C.H., Jun, J.W., Gomez, D.K., Park, S.C., 2010. Mortality of cultured Koi Cyprinus carpio in Korea caused by Bothriocephalus acheilognathi. Afr. J. Microbiol. Res. 4, 543–546. Heckmann, R.A., Kimball, A.K., Short, J.A., 1987. Parasites of Mottled Sculpin, Cottus bairdi Girard, From Five Locations in Utah and Wasatch Counties, Utah, Great Basin Nat. 43 pp. 13–21. Hossain, M.K., Hossain, M.D., Rahman, M.H., 2007. Histopathology of some diseased fishes. J. Life Earth Sci. 2, 47–50. Iyaji, F.O., Eyo, J.E., 2008. Parasites and their freshwater fish host. Bio-Res. 6, 328–338. Jerônimo, G.T., Marchiori, N.C., Pádua, S.B., Neto, J.D., Pilarski, F., Ishikawa, M.M., Martins, M.L., 2012. Trichodina colisae (Ciliophora: Trichodinidae): new parasite records for two freshwater fish species farmed in Brazil. Braz. J. Vet. Parasitol. 21, 366–371. Jerônimo, G.T., Ventura, A.S., Pádua, S.B., Satake, F., Ishikawa, M.M., Martins, M.L., 2013. Parasitofauna de cachara cultivado em tanque-rede no rio Paraguai. Pesq. Agrop. Brasileira 48, 1164–1166. Jerônimo, G.T., Pádua, S.B., Ventura, A.S., Gonçalves, E.L.T., Ishikawa, M.M., Martins, M.L., 2016. Parasitological assessment in the hybrid surubim (Pseudoplatystoma reticulatum × P. corruscans), with uncommon occurrence of Monogenea parasites. Braz. J. Vet. Parasitol. 25, 179–186. Kantham, K.P.L., Richards, R.H., 1995. Effect of buffers on the gill structure of common carp, Cyprinus carpio L., and rainbow trout, Oncorhynchus mykiss. J. Fish Dis. 18, 411–423. Khan, R.A., 2012. Host-parasite interactions in some fish species. J. Parasitol. Res. 2012, 1–7. Kir, I., Ozan, T.S., 2007. Helminth infections in common carp, Cyprinus carpio L., 1758 (Cyprinidae) from Kovada Lake (Turkey). Türk. Parazitol. Derg. 31, 232–236. Klein, B.M., 1958. The dry silver method and its proper use. J. Protozool. 5, 99–103. Kritsky, D.C., Heckmann, R., 2002. Species of Dactylogyrus (Monogenoidea: Dactylogyridae) and Trichodina mutabilis (Ciliata) infesting Koi Carp, Cyprinus carpio, during mass mortality at a commercial rearing facility in Utah, U.S.A. Comp. Parasitol. 269, 217–218. Kulwiec, Z., 1927. Ubtersuchungen an Arten der Genus Dactylogyrus Diesing. Bull. Internat. Acad. Polon. Sci.et Lettres, Cracovie, Cl. Sci. Math. Et Nat., ser. B: Sci. Nat. pp. 113–144. Lima, A.O., Bernardino, G., Proença, C.E.M., 2001. Agronegócio de peixes ornamentais no Brasil e no Mundo. Panor. Aquic. 11 pp. 14–24. Lom, J., Dyková, I., 1992. Developments in Aquaculture and Fisheries Science. Protozoan Parasites of Fish. 26. Elsevier, Amsterdam, pp. 159–235. Martins, M.L., Romero, N.G., 1996. Efectos del parasitismo sobre el tecido branquial em peces cultivados: Estudo parasitológico e histopatológico. Rev. Bras. Zool. 13, 492. Martins, M.L., Onaka, E.M., Moraes, F.R., Bozzo, F.R., Mello, A., Paiva, F.C., Gonçalves, A., 2002. Recent studies on parasitic infections of freshwater cultivated fish in the state of São Paulo, Brazil. Acta Sci. 24, 981–985. Martins, M.L., Azevedo, T.M.O., Ghiraldelli, L., Bernardi, N., 2010. Can the parasitic fauna on Nile tilapias be affected by different production systems? Acad. Bras. Cienc. 82, 493–500. Martins, M.L., Marchiori, N., Roumbedakis, K., Lami, F., 2012. Trichodina nobilis Chen, 1963 and Trichodina reticulata Hirschmann et Partsch, 1955 from ornamental freshwater fishes in Brazil. Braz. J. Biol. 72, 281–286. Martins, M.L., Cardoso, L., Marchiori, N., Pádua, S.B., 2015. Protozoan infections in farmed fish from Brazil: diagnosis and pathogenesis. Braz. J. Vet. Parasitol. 24, 1–20. Martins, M.L., Watral, V., Rodrigues-Soares, J.P., Kent, M.L., 2016. A method for collecting eggs of Pseudocapillaria tomentosa (Nematoda: Capillariidae) from zebrafish Danio rerio and efficacy of heat and chlorine for killing the nematode's eggs. J. Fish Dis. http://dx.doi.org/10.1111/jfd.12501. Menezes, R.C., Tortelly, R., Tortelly-Neto, R., Noronha, D., Pinto, R.M., 2006. Camallanus cotti Fujita, 1927 (Nematoda, Camallanoidea) in ornamental aquarium fishes: pathology and morphology. Mem. Inst. Oswaldo Cruz 101, 683–687. Mohammadi, F., Mousavi, S.M., Rezaie, A., 2012. Histopathological study of parasitic infestation of skin and gill on Oscar (Astronotus ocellatus) and discus (Symphysodon discus). AACL Bioflux. 5, pp. 88–89. Molnár, K., 2005. Histopathological changes caused by the metacestodes of Neogryporhynchus cheilancristrotus (Wedl, 1855) in the gut of the gibel carp, Carassiusgibelio. Acta Vet. Hung. 5, 45–52. Monticini, P., 2010. The ornamental fish trade. Production and commerce of ornamental fish: technical-managerial and legislative aspects. Globefish Res. Programme. 102, pp. 17–30. Moravec, F., 1998. Nematodes of Freshwater Fishes of the Neotropical Region. Academia Praha. Moravec, F., 2001. Thichinelloid Nematodes Parasitic in Cold-Blooded Vertebrates. Academia Praha.

109

Mousavi, H.E., Behtash, F., Rostami-Bashman, M., Mirzargar, S.S., Shayan, P., Rahmatiholasoo, H., 2011. Study of Argulus spp. infestation rate in goldfish, Carassius auratus (Linnaeus, 1758) in Iran. HVM Bioflux. 3, pp. 198–204. Noga, E.J., 2010. Fish Disease: Diagnosis and Treatment. Wiley-Blackwell, Iowa, pp. 107–129. Nottingham, M.C., Ramos, H.A.C., 2006. Exploração de peixes ornamentais no Brasil com ênfase sobre a introdução de espécies exóticas. IBAMA (9 p). Omrani, S., Mousavi, H.A., Sharifpour, I., 2010. Occurrence and histopathology of Ascocotyle tenuicollis metacercaria in gill of platyfish (Xiphophorus maculatus) imported to Iran. Iran. J. Fish. Sci. 9, 472–477. Pádua, S.B., Martins, M.L., Carraschi, S.P., Cruz, C., Ishikawa, M.M., 2012. Trichodina heterodentata (Ciliophora: Trichodinidae): a new parasite for Piaractus mesopotamicus (Pisces: Characidae). Zootaxa 34, 62–66. Paperna, I., 1964. Competitive exclusion of Dactylogyrus extensus by Dactylogyrus vastator (Trematoda, Monogenea) on the gills of reared carp. J. Parasitol. 50, 94–98. Paruruckumani, P.S., Maharajan, A., Ganapiriya, V., Narayanaswamy, Y., Raja Jeyasekar, R., 2015. Surface ultrastructural changes in the gill and liver tissue of Asian sea bass Lates calcarifer (Bloch) exposed to copper. Biol. Trace Elem. Res. 168, 500–507. Piazza, R.S., Martins, M.L., Guiraldelli, L., Yamashita, M.M., 2006. Parasitic diseases of freshwater ornamental fishes. Bol. Inst. Pesca 32, 51–57. Portz, L., Antonucci, A.M., Ueda, B.H., Dotta, G., Guidelii, G., Roumbedakis, K., Martins, M.L., Carniel, M.K., Tavechio, W.L.G., 2013. Parasitos de peixes de cultivo e ornamentais. In: Pavanelli, G.C., Takemoto, R.M., Eiras, J.C. (Eds.), Parasitologia de peixes de água doce do Brasil. Maringá: Eduem, pp. 85–114 (ORG). Rego, A.A., 2000. In: Salgado-Maldonado, G., García Aldrete, A.N., Vidal-Martínez, V.M. (Eds.), Cestode Parasites of Neotropical Teleost Freshwater Fishes, in Metazoan Parasites in the Neotropics: A Systematic and Ecological Perspective. Instituto de Biología, Universidad Nacional Autónoma de México, Mexico, pp. 135–154. Roberts, R.J., 2001. Fish Pathology. W.B. Saunders, London. Rushton-Mellor, S.K., 1994. The genus Argulus (Crustacea: Branchiura) in Africa: identification keys. Syst. Parasitol. 28, 51–63. Saha, M., Bandyopadhyay, P.K., 2015. First report of three species of Argulus (Crustacea: Branchiura) infesting on redcan Oranda gold fish (Carassius auratus auratus) in India. Biolife 3, 813–819. Santos, E.L., Lira, R.C., Souza, C.A., Santos, I.V.V.S., Silva, S.C.B., Albuquerque, I.C.M., 2014. Performance of Betta splendens associates the different alimentary frequencies. Rev. Cient. Prod. Anim. 16, 10–16. Scholz, T., 1997. A revision on the Bothriocephalus Rudolphi, 1808 (Cestoda: Pseudophyllidea) parasitic in American freshwater fishes. Syst. Parasitol. 36, 85–107. Scholz, T., Kuchta, R., Williams, C., 2012. In: Woo, P.T.K., Buchmann, K. (Eds.), Bothriocephalus acheilognathi in fish parasites: pathobiology and protection. CAB International, Wallingford, UK, pp. 282–297. Schwaiger, J., Wande, R., Adm, S., Pawert, M., Honnen, W., Triebskorn, R., 1997. The use of histopathologic indicators to evaluate contaminant related stress in fish. J. Aquat. Ecosyst. Stress. Recover. 6, 75–86. Scott, A.L., Grizzle, J.M., 1979. Pathology of cyprinid fishes caused by Bothriocephalus gowkongensis Yea, 1955 (Cestoda: Pseudophyllidea). J. Fish Dis. 2, 69–73. Shinn, A.P., Hansen, H., O., K., Bachmann, L., Bakke, T.A., 2004. The use of morphometric characters to discriminate specimens of laboratory-reared and wild populations of Gyrodactylus salaris and G. thymalli (Monogenea). Folia Parasitol. 51, 239–252. Shoaibi, O.B., Ebrahimzadeh, M.H.A., Sharifpour, I., 2010. Occurrence and histopathology of Ascocotyle tenuicollis metacercaria in gill of platyfish (Xiphophorus maculatus) imported to Iran. Iran. J. Fish. Sci. 9, 472–477. Soes, D.M., Walker, P.D., Kruijt, D.B., 2010. The Japanese fish louse Argulus japonicas new for the Netherlands. Lauterbornia. 70, pp. 11–17. Sujan, M., Shameem, U., 2015. Monogenoidean parasites of some cyprinid fishes from noth coastal andhra pradesh. Int. J. Rec. Sci. Res. 6, 3147–3155. Takashima, F., Hibiya, T., 1995. An Atlas of Fish Histology: Normal and Pathological Features. 2. ed. Kodansha, Tokyo, pp. 1–5. Tavares-Dias, M., Lemos, J.R.G., Martins, M.L., 2010. Fauna parasitária de oito espécies de peixes ornamentais de água doce do médio Rio Negro na Amazônia brasileira. Braz. J. Vet. Parasitol. 19, 103–107. Thatcher, V.E., 2006. Nematoda. Amazon Fish Parasites, second ed. Sofia, Moscow, pp. 235–236 (Editora). Thilakaratne, I.D.S.I.P., Rajapaksha, G., Hewakopara, A., Rajapakse, R.P.V.J., Faizal, A.C.M., 2003. Parasitic infections in freshwater ornamental fish in Sri Lanka. Dis. Aquat. Org. 54, 157–162. Tlusty, M., 2002. The benefits and risks of aquaculture production for the aquarium trade. Aquaculture 205, 203–219. Valladão, G.M.R., Pádua, S.B., Gallani, S.U., Menezes-Filho, R.N., Dias-Neto, J., Martins, M.L., Ishikawa, M.M., Pilarski, F., 2013. Paratrichodina africana (Ciliophora): a pathogenic gill parasite in farmed Nile tilapia. Vet. Parasitol. 197, 705–710. Valladão, G.M.R., Gallani, S.U., Pádua, S.B., Martins, M.L., Pilarski, F., 2014. Trichodina heterodentata (Ciliophora) infestation on Prochilodus lineatus larvae: a host-parasite relationship study. Parasitology 141, 662–669. Vinobaba, P., 1994. Some Aspects of the Biology of Dactylogyrus vastator Nybelin, 1924 (Monogenea) a Gill Parasite of Cyprinus carpio L. (PhD Thesis). Institute of Aquaculture, University of Stirling, Stirling, U.K., pp. 1–46. Xu, D.H., Shoemaker, C.A., Klesius, P.H., 2007. Evaluation of the link between gyrodactylosis and streptococcosis of Nile tilapia, Oreochromis niloticus (L.). J. Fish Dis. 30, 233–238. Yemmen, C., Quilichini, Y., Ktari, M., 2011. Morphological, ecological and histopathological studies of Trichodina gobii Raabe, 1959 (Ciliophora: Peritrichida) infecting the gills of Solea aegyptiaca. Protistology 4, 258–263. Zuanon, J.A.S., Salaro, A.L., Furuya, W.M., 2011. Production and nutrition of ornamental fish. Rev. Bras. Zootec. 40, 165–174.