Efficacy of seed extract of Bixa orellana against monogenean gill parasites and physiological aspects of Colossoma macropomum after bath treatment

Aquaculture 462 (2016) 40–46

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Efficacy of seed extract of Bixa orellana against monogenean gill parasites and physiological aspects of Colossoma macropomum after bath treatment Jaqueline Inês Alves de Andrade a, Gabriela Tomas Jerônimo a,b, Elenice Martins Brasil a, Cecilia Verônica Nunez c, Eduardo Luiz Tavares Gonçalves a, Maria Luiza Ruiz a, Maurício Laterça Martins a,⁎ a b c

AQUOS — Aquatic Organisms Health Laboratory, Aquaculture Department, Federal University of Santa Catarina (UFSC), Rod. Admar Gonzaga 1346, 88040-900 Florianopolis, SC, Brazil Post-Graduate in Aquaculture, Nilton Lins University, Av. Nilton Lins 3259, 69058-030 Manaus, AM, Brazil LABB — Bioprospection and Biotechnology Laboratory, National Research Institute of Amazônia (INPA), Av. André Araújo, 2.936, Petrópolis, 69067-375 Manaus, AM, Brazil

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Article history: Received 25 February 2016 Received in revised form 22 April 2016 Accepted 25 April 2016 Available online 30 April 2016 Keywords: Fish farming Physiology Phytotherapic Annatto Treatment Study model

a b s t r a c t This study evaluated the use of therapeutic baths with acetone extract of Bixa orellana seeds on the hematological, biochemical and hormonal parameters and plasma cortisol levels of tambaqui (Colossoma macropomum) parasitized by the monogenean Anacanthorus spathulatus. The extract showed in vitro anthelmintic activity against the parasites, and the fish tolerated the concentrations used in the toxicity test. Based on these results, an in vivo test was performed. A total of 180 juveniles of tambaqui were divided into six treatment groups in triplicate: group 1: basal (fish non-treated and non-parasitized), group 2: exposed to acetone 0.2% and parasitized fish, group 3: control (non-treated and parasitized fish), group 4: parasitized fish treated with 125 μg·mL−1 of annatto extract for 2 h bath for two consecutive days, group 5: parasitized fish treated with 250 μg·mL−1 of annatto extract for 2 h bath for two consecutive days, group 6: parasitized fish treated with 125 μg·mL−1 of extract for a single bath for 12 h. After the last bath, parasitological, hematological, biochemical and hormonal analysis were performed. Annatto extract showed 100% efficacy in all concentrations and times of bath evaluated. Hemoglobin concentration and hematocrit percentage were higher in treated fish with 250 μg·mL−1 2 h and 125 μg·mL−1 12 h than that observed in the non-treated fish groups. Glucose was significantly higher in annatto-treated fish and cortisol was significantly higher in acetone group fish compared to other groups. Significant decrease in thrombocyte number was observed in fish after bath with acetone 0.2% compared to basal group, 125 μg·mL−1 2 h and 125 μg·mL−1 12 h, as well as decreased number of circulating lymphocytes in fish after bath with acetone 0.2% and 125 μg·mL−1 12 h in relation to non-treated fish (control). On the other hand, significant increase in WBC count was found in fish treated with 125 μg·mL−1 12 h in relation to basal and acetone groups. This is the first report on the use of seeds of B. orellana against monogenean parasites of fish. In vitro study model used with gills in Petri dishes and their in situ observation was successful and could be a useful tool for testing substances to treat fish parasites. Annatto extract bath is an efficacious alternative for treating monogeneans. However, more studies must be carried out for better understanding of the mechanism of anthelmintic activity, isolation of bioactive substances and toxicological evaluation before testing in farming conditions. Statement of relevance: Chemotherapies have been mostly used to treat fish parasites. However, they present consequences to both to environment and human health. Alternatives have been studied to improve the fish health status and control fish parasites. Phytotherapy shows several advantages in controlling parasites and improving the fish health status. This study shows by the first time the use of B. orellana in controlling monogenean parasites and its effects on the hematological, biochemical and hormonal parameters. © 2016 Elsevier B.V. All rights reserved.

1. Introduction Colossoma macropomum (Osteichthyes: Characidae), commonly known as tambaqui or chachama is a native fish species of the Amazon region, and has been highlighted in fish farming due to its excellent ⁎ Corresponding author. E-mail address: [email protected] (M.L. Martins).

http://dx.doi.org/10.1016/j.aquaculture.2016.04.024 0044-8486/© 2016 Elsevier B.V. All rights reserved.

culture conditions, acceptance of dry ration, rapid growth and rusticity (Costa et al., 2004; Chagas et al., 2007). Actually is the second most cultivated species in Brazil. Nevertheless, the intensive production can result in stressful conditions and immunosuppression, disease outbreaks and economic losses (Carneiro et al., 2007; Gomes et al., 2012). Among the pathogens frequently associated with farmed tambaqui are the monogenean helminthes mainly the Dactylogyridae. They present direct life cycle and reproduce rapidly under adequate conditions

J.I.A. de Andrade et al. / Aquaculture 462 (2016) 40–46

causing high infestation levels in every stage of the fish life cycle (Eiras et al., 2010; Jerônimo et al., 2014), consequently controlling these parasites is essential. Several chemical substances like formalin, potassium permanganate, hydrogen peroxide, praziquantel and mebendazole have been used for their control (Pavanelli et al., 2008; Forwood et al., 2013). However, the continuous use of drugs can result in adverse effects such as resistance to drug, immunosuppression, environmental contamination and risks to human health (Tavechio et al., 2009; Matyar et al., 2010; Lôbo et al., 2010; Suhet et al., 2011). This fact has stimulated the search for new medicines and plant-based products showing a promising strategy to treat fish pathogens, including parasites (Reverter et al., 2014; Boijink et al., 2015; Bulfon et al., 2015; Hashimoto et al., 2016). Studies demonstrated that plants have parasiticide properties in fish (Claudiano et al., 2009; Wang et al., 2010; Dotta et al., 2015; Levy et al., 2015; Hashimoto et al., 2016; Soares et al., 2016). Among the vegetal species studied with therapeutic purpose is the annatto, Bixa orellana, which originated from Tropical forests in the Central and South Americas (Lorenzi and Matos, 2002). It is widely used in the food and pharmaceutical industries, especially in the food industry where it is used for the preservation of food quality (Braga et al., 2007). Annatto seeds contain flavonoids, and alkaloids (Fleischer et al., 2003) and are rich in carotenoids. Bixin, the most abundant carotenoid found in annatto, constitutes approximately 80% of the total carotenoids in the seeds (Preston and Rickard, 1980). The extract of annatto seeds presents several biological activities like antibacterial, antiparasitic and antioxidant. However, its use is mostly related with pathogens of clinical interest (Majolo et al., 2013; Fleischer et al., 2003) and so far no findings on its use to control fish parasites have been found. This study evaluated the use of in vitro and in vivo activity of acetone extract of B. orellana seeds against monogenean gill parasites and their influence on the blood parameters and plasma cortisol, glucose, total protein and total cholesterol levels of tambaqui.

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evaporator (Fisatom, model 801) and the extract kept in dark flasks at −20 °C until the time of use. 2.2.2. Chemical composition of the extract and bixin quantification The extract was analyzed by Nuclear Magnetic Resonance (NMR) of 300 MHz (Bruker — model Fourier 300). The sample was standardized by weighing 10 mg of extract and diluting it in 600 μL of dimethyl sulphoxide-d6 (DMSO-d6). The spectrum was analyzed and compared with the NMR of the substances isolated from the species in order to detect the presence of each substance in each sample. Briefly, for bixin quantification, the extract was diluted in chloroform and the solution had the volume corrected to 100 mL with the same solvent. An aliquot of 10 mL of solution was removed and diluted until 50 mL with CHCl3, to obtain a solution with 20 mg·L−1, and then analyzed by spectrophotometry using UV–Vis equipment. Bixin concentration was determined using the formula: A = acb, where A means absorbance of the chloroformic solution of the extract read by spectrophotometry, c is the bixin concentration in the solution (g/L), b is the optical way (1 cm) and a is the absorption coefficient of bixin in CHCl3 (2826) at λmax 470 nm as described by Tocchini and Mercadante (2001). The bixin concentration was determined by considering the extract mass used in the dilution. 2.2.3. Model to study the parasite immobilization The fish were randomly selected, anesthetized and euthanized for gill collection. For this test, the first three gill arches were separated in sterile Petri dishes for each concentration in triplicate: 500, 250, 125 and 62.5 μg·mL− 1 of annatto extract, two controls (one using acetone 0.2% and the other using only water). For each concentration, a total of 60 parasites were counted. The plates were observed each for 15 min and the parasites were considered dead when no movement was detected after stimulation by an entomological needle. 2.3. Bath with annatto extract

2. Material and methods 2.1. Fish and parasites Juveniles of tambaqui were obtained from a commercial fish farm located in Manaus, AM, Brazil. At the sampling site, 10 fish samples were anesthetized with clove oil (20 mg·L−1) (Inoue et al., 2011) and euthanized to confirm the presence of monogenean parasites using a dissection microscope (Zeiss Stemi 2000-C, Carl Zeiss, Oberkochen, Germany). The fish were kept for 15 days in 500 L tanks, in static systems before the assays, with constant aeration to improve the infestation level and during the night, water flow was turned on. The fish were fed to apparent satiation twice a day (9 a.m. and 4 p.m.) with commercial diet (NUTRIPISCIS 36% crude protein). During the assays, dissolved oxygen was measured with YSI-55 oximeter (Ohio, USA), pH, water temperature and electric conductivity were measured with a multiparameter YSI-63 (Ohio, USA), ammonia and nitrite measured by colorimetric method according to Verdouw et al. (1978) and Boyd and Tucker (1992), respectively, and alkalinity and hardness according to Boyd and Tucker (1992) method. Fish management was according to the ethics of animal use by the Brazilian Society of Laboratory Animal Science (COBEA). 2.2. In vitro assay 2.2.1. Extract preparation Annatto seeds were donated by the company Chr. Hansen Indústria e Comércio Ltda, Valinhos, SP, Brazil. The extract was prepared using 100% acetone, in the proportion of 25 g of seeds in 100 mL of solvent. For extraction, an ultrasound bath (Unique, model USC 3300) was used for 20 min. After that, the solvent was removed with a rotary

2.3.1. Tolerance test The tolerance test was performed to determine the concentration of extract used in the assay to ensure safety of fish. The following concentrations of annatto extract were utilized; 500, 250, 125 and 62.5 μg·mL−1, with five fish in each aquarium of 80 L capacity in triplicate. Two controls composed of acetone 0.2% and another with only water were used. The fish were exposed to the baths with the extract for 24 h to verify their behavior and mortality. The water quality was kept as follows: dissolved oxygen 6.93 ± 0.32 mg·L− 1, pH 6.29 ± 0.58, temperature 26.79 ± 0.35 °C, electric conductivity 25.65 ± 2.04, ammonia 0.09 ± 0.03 mg·L−1, nitrite 0.03 ± 0.02 mg·L−1, alkalinity 3.12 ± 0.65 mg CaCO3·L−1 and hardness 2.92 ± 0.77 mg CaCO3·L−1. When the fish turned upside down, they were immediately transferred to a clean water and the time registered. 2.3.2. In vivo assay A total of 180 juveniles of tambaqui (31.4 ± 3.12 g) were divided into six treatment groups in tanks of 80 L (10 fish per tank), in triplicate: Group 1: basal (fish non-treated and non-parasitized). Group 2: exposed to acetone 0.2% and parasitized fish. Group 3: control (non-treated and parasitized fish). Group 4: parasitized fish treated with 125 μg·mL−1 of annatto extract for 2 h bath for two consecutive days. Group 5: parasitized fish treated with 250 μg·mL−1 of annatto extract for 2 h bath for two consecutive days. Group 6: parasitized fish treated with 125 μg·mL−1 of extract for a single bath for 12 h.

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2.4. Hematological, biochemical and parasitological analysis

3. Results

The blood was withdrawn from the caudal vein with syringes containing a drop of EDTA 10% and used for blood smears stained with May Grunwald/Giemsa/Wright for white blood cell count using an indirect method by counting the total leukocytes number (WBC) in 2000 erythrocytes in the smears (Ishikawa et al., 2008) and total number of thrombocytes and leukocytes (WBC) were calculated by the indirect method (Ranzani-Paiva et al., 2013). Hematocrit percentage was measured by the microhematocrit method and red blood cell count (RBC) in a Neubauer chamber after dilution 1:200 in Natt and Herrick (1952) solution. Hemoglobin concentration was determined by spectrophotometry using a commercial kit (LabTest, Minas Gerais, Brazil). The hematimetric indices, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC) were calculated according to Ranzani-Paiva et al. (2013). The plasma obtained by centrifugation was used to determine the glucose levels, total protein and cholesterol by colorimetric method in a spectrophotometer using a commercial kit (In VitroHuman®, Minas Gerais, Brazil). Plasma cortisol level was determined by immunoassay kit (Diagnostics Biochem Canada Inc.). After blood collection and euthanization, the gills of 5 fish per triplicate were collected for immediate parasitological analysis and from the other 5 fish, the gill arches were removed and bathed in 60 °C to be fixed in alcohol, 70% for posterior counting. Parasites quantification followed the Jerônimo et al. (2011) method. Parasites were mounted in Hoyer's medium for identification according to Kritsky et al. (1979). The efficacy was calculated according to the formula: Efficacy = MNPC – MNPT × 100 / MNPC (MNPC: mean number of parasites in control group, MNPT: mean number of parasites in treated group) (Dotta et al., 2015). Prevalence, mean intensity and mean abundance were calculated according to Bush et al. (1997).

3.1. Chemical composition of the extract, bixin quantification and in vitro assay

2.5. Statistical analysis The data obtained were firstly evaluated with regard to the assumptions of homoscedasticity using the Levene's tests. These data were then analyzed using the Anova test (p b 0.05), and then Tukey's test.

NMR analysis and spectrophotometry by UV–Vis confirmed the presence of bixin in a concentration of 49% per gram of extract and geranylgeraniol (Fig. 1). The effect of annatto extract was evident after 1 h exposure which the parasites absorbed the extract showing swollen body and slow movements. The most efficient concentrations to cause 90% mortality were 500 and 250 μg·mL−1, after 2 and 3 h, respectively. At 125 and 62.5 μg·mL−1, no mortality was found in a period of 4 h, but the parasites showed reduced mobility. Parasites exposed to water and acetone 0.2% started to die only after 4 h (Table 1). 3.2. Tolerance test After annatto extract was added, the fish became agitated in all concentrations followed by gulping on the water surface and remaining quiet. At the concentrations 500 and 250 μg·mL−1, the fish lost movement after 1 and 3 h, respectively. In the other concentrations the fish remained under normal behavior for N24 h. 3.3. In vivo assay From the in vitro results and tolerance test, the therapeutic doses of annatto extract were defined as 125 and 250 μg·mL−1 in 2 h bath for two consecutive days and one bath of 125 μg·mL−1 for 12 h. Therapeutic baths showed 100% efficacy in all concentrations tested in relation to non-treated fish and acetone 0.2%-treated fish (Table 2). Parasites were identified as Anacanthorus spathulatus Kritsky et al., 1979. After extract or acetone was added, the water quality remained within the normal values for fish (Costa et al., 2004; Marcon et al., 2004; Aride et al., 2007). Dissolved oxygen 7.0 ± 0.35 mg·L− 1, pH 6.35 ± 0.54, temperature 26.86 ± 0.47 °C, electric conductivity 25.20 ± 2.46, ammonia 0.09 ± 0.02 mg·L−1, nitrite 0.04 ± 0.02 mg·L− 1, alkalinity 3.33 ± 0.94 mg CaCO3·L− 1 and hardness 3.04 ± 1.05 mg CaCO3·L−1.

Fig. 1. Nuclear Magnetic Resonance spectroscopy (1H-NMR) of acetonic extract of Bixa orellana seeds, in DMSO-d6, 300 MHz.

J.I.A. de Andrade et al. / Aquaculture 462 (2016) 40–46 Table 1 Mortality of Anacanthorus spathulatus exposed to different concentrations of Bixa orellana extract, acetone 0.2% and water during 4 h. Concentrations (μg·mL−1)

Death time (h)

Efficacy (%)

500 500 500 500 250 250 250 250 125 125 125 125 62.5 62.5 62.5 62.5 Acetone 0.2% Acetone 0.2% Acetone 0.2% Acetone 0.2% Water Water Water Water

1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4

50 90 90 90 0 50 90 90 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

3.4. Hematological, biochemical and hormonal analysis Reduced hemoglobin concentration and hematocrit percentage (p b 0.05) were observed in non-treated fish (control) when compared to the basal, acetone 0.2% and fish treated with 250 μg·mL− 1 2 h and 125 μg·mL − 1 12 h (Table 3). No significant difference was found in the hemoglobin concentration, hematocrit percentage and red blood cell counts between basal fish and those exposed to acetone. Significant decrease (p b 0.05) in thrombocyte number was observed in fish after bath with acetone 0.2% compared to basal group, 125 μg·mL−1 2 h and 125 μg·mL−1 12 h, as well as decreased number of circulating lymphocytes in fish after bath with acetone 0.2% and 125 μg·mL−1 12 h in relation to non-treated fish (control). Monocyte number was higher in fish treated with 125 μg·mL − 1 12 h than that found in fish after bath with acetone 0.2%. On the other hand, significant increase in WBC count was found in fish treated with 125 μg·mL− 1 12 h in relation to basal and acetone groups. Granular leukocyte PAS+ (LG PAS) showed a higher number (p b 0.05) in fish treated with 250 μg·mL−1 2 h than that observed in the other groups (Table 4). Glucose levels were significantly higher (p b 0.05) in fish treated with annatto extract. Total protein was lower (p b 0.05) in group basal than that control and annatto extract-treated fish. Cholesterol showed an increase in fish treated with 250 μg·mL−1 2 h, 125 μg·mL−1 12 h and acetone than that non-treated (control). On the other hand, the cortisol levels were significantly higher in fish exposed to acetone than that found in the other groups (Table 3).

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4. Discussion In this study, the efficacy of annatto seed extract against the monogenean parasites of tambaqui was demonstrated in the in vitro assay corroborating the findings of Levy et al. (2015). In evaluating the ethanolic extract of ginger Zingiber officinale against the monogenean Gyrodactylus turnbulli from Poecilia reticulata, Fu et al. (2014) have reported the antiparasitic activity of acetone extract and ethyl acetate of mulberry Morus alba against the ciliate protozoan Ichthyophthirius multifiliis. This study showed 100% efficacy in the in vivo assay as also reported by Boijink et al. (2011) using therapeutic bath with essential oil of basil (Ocimum gratissimum). Similar results were found in the gold fish (Carassius auratus) parasitized by Dactylogyrus intermedius after treatment with methanolic extract and ethyl acetate of hare's ear root Radix bupleuri chinensis (Wu et al., 2010), aqueous and methanolic extract of Chinese cinnamon Cinnamomum cassia, methanolic extract of evergreen Lindera agreggata and ethyl acetate extract of false larch Pseudolarix kaempferi (Ji et al., 2012). Reduced number of monogenean parasites was also reported in tambaqui after bath with eugenol (Boijink et al., 2015) and essential oil of Lippia alba (Soares et al., 2016). The studies of Tu et al. (2013) have demonstrated 100% efficacy of chloroformic extract of Indian sandal wood Santalum album (40 mg·L−1) against D. intermedius parasite of C. auratus. In contrast to that observed in this study, the use of herbal Sancti Mariae Chenopodium ambrosiodes extract at 3.9 mL·L−1 for 24 h against monogenean parasites of tambaqui presented 54.4% efficacy (Monteiro, 2012). In comparison with other studies, lower efficacies can be found with therapeutic bath containing 40 mg·L−1 of essential oil of peppermint Mentha piperita for 10 min during three days (41.63% efficacy) against monogenean parasites Cichlidogyrus tilapiae, Cichlidogyrus thurstonae, Cichlidogyrushalli and Scutogyrus longicornis of Nile tilapia Oreochromis niloticus (Hashimoto et al., 2016). Acetonic extract of burning bush Kochia scoparia showed 77.6% and 72.34% at 100 and 110 mg·L− 1 respectively against D. intermedius from C. auratus (Lu et al., 2012). Soares et al. (2016) observed 14% efficacy of the essential oil of L. alba against monogenean parasites of tambaqui. Annatto extracts present several biological activities including antibacterial, antiparasitic and antioxidant (Fleischer et al., 2003). Phytochemical analysis of this plant revealed the presence of flavonoids, alkaloids and terpenoids, geranylgeraniol and bixin (Fleischer et al., 2003; Braga et al., 2007). NMR analysis confirmed the presence of bixin and geranylgeraniol after acetonic extraction. Studies have related the terpenoids with parasiticide activities once lipophilic substances are capable of crossing the membrane surface of helminthes causing its rupture and killing the parasites (Wink, 2008). Braga et al. (2007) have demonstrated the in vitro antiparasitic activity of annatto seeds extract against Leishmania amazonenses and Leishmania chagasi. Recently, Ritter et al. (2012) reported in their ethno veterinary studies the use of annatto seeds to treat scabies in dog. Moreover, annatto has also presented antifungal activity against Cryptococcus neoformans (Braga et al., 2007) and some Gram-positive and Gram-negative bacteria (Fleischer et al., 2003). These results confirm the promising use and efficacy of annatto seeds extract against monogenean fish parasites. The toxicity of vegetal species is related to the substances present in its chemical composition, the concentration utilized, time of exposure

Table 2 Parasitological indices of Colossoma macropomum parasitized by Anacanthorus spathulatus after treatment with acetonic extract of Bixa orellana seeds and acetone 0.2%. Indices

Acetone

Non-treated

125 μg·mL−1 (2 h)

250 μg·mL−1 (2 h)

125 μg·mL−1 (12 h)

Prevalence (%) Mean intensity Mean abundance

100 169.5 ± 49.3 169.5 ± 49.3

100 165.2 ± 68.4 165.2 ± 68.4

0 0 0

0 0 0

0 0 0

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Table 3 Blood parameters of Colossoma macropomum after treatment with Bixa orellana seed extract and acetone. 125 μg·mL−1 and 250 μg·mL−1 2 h (2 h bathes for 2 consecutive days) and 125 μg·mL−1 12 h (1 bath for 12 h). Basal means before treatment, red blood cells count (RBC), mean corpuscular volume (VCM), mean corpuscular hemoglobin concentration (MCHC), mean corpuscular hemoglobin (MCH), glucose (GLU); total protein (TP), total cholesterol (CHOL). Parameters

Basal

Acetone 0.2%

Non-treated

125 μg·mL−1 2h

250 μg·mL−1 2h

125 μg·mL−1 12 h

Hemoglobin (g·dL−1) Hematocrit (%) RBC (×106·μL−1) MCV (fL) MCHC (g·dL−1) MCH (pg) GLU (mg·dL−1) TP (g.dL−1) CHOL (mg·dL−1) Cortisol (ng·mL−1)

9.7 ± 1.0c 21.9 ± 1.9b 1.6 ± 0.1b 137.0 ± 2.4a 46.1 ± 3.2b 60.9 ± 5.5a 38.8 ± 3.2a 1.7 ± 0.2a 58.2 ± 3.4ab 89.6 ± 21.0a

9.8 ± 0.7c 20.7 ± 1.3b 1.4 ± 0.1ab 145.6 ± 4.1ab 46.6 ± 5.3b 69.7 ± 9.0a 47.9 ± 4.8a 2.1 ± 0.1ab 67.3 ± 8.9b 157.5 ± 29.0b

6.8 ± 1.0a 17.7 ± 2.3a 1.2 ± 0.2a 148.4 ± 19.3ab 38.1 ± 1.3ab 55.5 ± 7.5a 44.3 ± 5.5a 2.8 ± 0.4bc 52.6 ± 8.8a 94.4 ± 38.7a

7.6 ± 0.8ab 22.4 ± 1.7b 1.3 ± 0.1a 172.5 ± 15.0b 34.9 ± 2.9a 58.3 ± 8.1a 80.5 ± 12.8b 3.1 ± 0.5c 57.8 ± 3.0ab 91.0 ± 30.1a

8.6 ± 0.9bc 22.1 ± 1.9b 1.4 ± 0.1ab 160.2 ± 17.2ab 39.0 ± 3.2ab 61.8 ± 9.0a 83.9 ± 23.9b 3.0 ± 0.2c 63.3 ± 10.6b 98.5 ± 24.2a

8.0 ± 0.5b 21.0 ± 1.9b 1.3 ± 0.2a 161.2 ± 19.9ab 38.8 ± 2.8ab 61.9 ± 6.7a 90.9 ± 9.5b 3.1 ± 0.4c 65.4 ± 8.2b 96.7 ± 13.2a

Values are means (±SD). Different letters indicate significant difference by Tukey test (p b 0.05) among treatments.

and animal sensitivity (Kumar et al., 2010). In the present study, the highest concentrations (250 and 500 μg·mL−1) showed elevated toxicity. Similar results were obtained in carp Cyprinus carpio after treatment with seed extract of drumstick tree, Moringa oleifera where the toxicity increased as the concentration increased (Kavitha et al., 2012). Fish exposed to stressful conditions like environmental variations, diseases or chemical compounds may present physiological changes and imbalance on their homeostasis. A decrease in the RBC count, hemoglobin concentration and hematocrit may suggest destruction of erythrocytes leading to anemia (Wintrobe, 1934). This explains the results found in parasitized and non-treated fish. To date, Jerônimo et al. (2014) observed decreased RBC count in Piaractus mesopotamicus highly parasitized by the monogenean Anacanthorus penilabiatus (Monogenea: Dactylogyridae), as well as Ghiraldelli et al. (2006) related decreased RBC count in O. niloticus parasitized by Cichlidogyrus sclerosus. Similarly, Prochilodus lineatus parasitized by Dactylogyrus showed a decrease in the hematocrit percentage (Ranzani-Paiva et al., 2000). In this study, annatto extract-treated fish showed a tendency in returning to the normal blood parameters, as observed in the non-treated and non-parasitized fish. In contrast, Hashimoto et al. (2016) did not observe alterations in the blood parameters of O. niloticus after treatment with M. piperita while in contrast, Soares et al. (2016) have reported a decrease in the hematocrit and RBC count in C. macropomum treated with essential oil of L. alba. In teleost fishes, the immune system can be activated depending on the degree of parasitism causing alteration in the percentage of circulating defense cells (Jerônimo et al., 2011). Thrombocytes are involved in the coagulation and in the organism defense (Martins et al., 2008). Contrarily to that found in this study, Hashimoto et al. (2016) observed decreased number of thrombocytes in Nile tilapia treated with L. sidoides. Increased number of eosinophils could be related to parasitic infections (Ranzani-Paiva et al., 2013) as observed in this study in parasitized and treated tambaqui. Cortisol and glucose are the most used in physiological responses to indicate stressful conditions in fish. In general, there is an increase of

these levels in order to supply the energy demand caused by stress (Barton and Iwama, 1991). Increased glucose levels herein observed in treated-fish can suggest stressful condition as a result of treatment. Similar findings were reported in rainbow trout Oncorhynchus mykiss exposed to propolis bath (Talas and Gulhan, 2009), in C. carpio exposed to M. oleifera bath (Kavitha et al., 2012), in O. niloticus after treatment with essential oil of pepper rosemary L. sidoides (Hashimoto et al., 2016) and in C. macropomum after L. alba therapeutic bath (Soares et al., 2016). The cortisol levels increased only in fish exposed to acetone bath confirming the glucose response. Glucose levels could be influenced by the carotenoids present in the extract as observed in other animals. Similarly to that found in this study, an increase in the glucose levels were found in male rats fed diet containing annatto with 27% of bixin (Bautista et al., 2004) and rats fed with annatto containing 50% of bixin and norbixin (Fernandes et al., 2002). According to Subczynski and Wisniewska (2000), carotenoids can interact with the cell membrane by affecting the glucose inlet. In fact, this could have caused a decrease in the glucose absorption by the cells and consequently hyperglycemia. However, studies must be done for better understanding of the interaction between carotenoids and cell membranes in fish to elucidate how homeostasis is affected. On the other hand, increased cortisol levels suggest stressful condition in acetone-treated fish and perhaps the bath with annatto extract could have promoted protection against the acetone stress. This is the first report on the use of annatto seeds to treat fish parasites and also on the fish physiology. 5. Conclusions This study reported by the first time the efficacy on the use of annatto seeds extract on both monogenean parasites and physiology of farmed fish tambaqui. The in vitro model by observing the parasites in situ on the gill arches of fish was successful and can be used safely to test medicines against fish parasites. A hundred percent efficacy was obtained in all concentrations tested. Nevertheless, we suggest more

Table 4 Total number of circulating thrombocytes, leukocytes (WBC), immature leukocytes (Imm Leuk) and differential counting of leukocytes in Colossoma macropomum after treatment with Bixa orellana seed extract and acetone. Basal means before treatment. LG-PAS: Granular leukocyte PAS+. Parameters

Basal

Acetone 0.2%

Non-treated

125 μg·mL−1 2h

250 μg·mL−1 2h

125 μg·mL−1 12 h

Thrombocytes (×103·μL−1) WBC (×103·μL−1) Imm Leuk (x103.μL−1) Eosinophils (×103·μL−1) Neutrophils (×103·μL−1) Lymphocytes (×103·μL−1) Monocytes (×103·μL−1) LG-PAS+ (×103·μL−1)

37.4 ± 19.7b 17.3 ± 16.1ab 1.1 ± 1.4a 0.6 ± 1.3a 12.5 ± 14.4a 2.7 ± 1.7ab 0.2 ± 0.4ab 0.1 ± 0.3ab

9.7 ± 9.9a 14.7 ± 7.1a 1.1 ± 0.8a 6.4 ± 6.7ab 3.8 ± 2.7a 1.5 ± 1.3a 0.0 ± 0.1a 0.2 ± 0.4ab

22.9 ± 16.1ab 26.3 ± 18.7abc 1.2 ± 1.6a 12.3 ± 12.7b 5.0 ± 2.7a 6.2 ± 6.6b 0.9 ± 1.0ab 0.4 ± 0.3ab

38.7 ± 14.0b 28.2 ± 8.6abc 3.6 ± 1.9a 12.7 ± 9.8b 8.0 ± 7.6a 3.1 ± 1.7ab 0.7 ± 1.3ab 0.2 ± 0.2ab

30.7 ± 27.4ab 32.7 ± 12.6bc 2.5 ± 1.6a 12.1 ± 9.1b 12.0 ± 8.0a 2.7 ± 1.7ab 0.5 ± 0.6ab 2.9 ± 1.9c

48.9 ± 31.5b 39.1 ± 17.4c 3.3 ± 2.6a 15.1 ± 17.0b 16.2 ± 12.4a 1.6 ± 1.2a 1.3 ± 1.2b 1.5 ± 1.5b

Different letters indicate significant difference by Tukey test (p b 0.05) among treatments.

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studies testing lower concentrations and/or time of exposure in order to cause no alterations in the blood parameters and fish physiology. Studies may elucidate the anthelmintic mechanism, isolation of bioactive substances and ecological risks before its use in fish ponds. Bath with annatto seed extract can be used for treating monogenean gill parasites as a method to reduce the parasitic load before fish transporting or before harvesting in ponds. Acknowledgements The authors thank CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior) for scholarship to J.I.A Andrade; CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico) for research grant to M.L Martins (CNPq 305869-2014-0) and C.V. Nunez; the project “Desenvolvimento da Aquicultura e de Recursos Pesqueiros na Amazônia” — DARPA, FINEP (01.09.0472.00) for financial support; and Dr. Felipe N. Vieira, Dr. Rosendo A. Yunes, Dr. Evoy Z. Filho (Federal University of Santa Catarina, SC, Brazil), Dr. Jonas C. 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