Aquaculture 462 (2016) 4046

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Aquaculture
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Efcacy of seed extract of Bixa orellana against monogenean gill parasites

and physiological aspects of Colossoma macropomum after
bath treatment
Jaqueline Ins Alves de Andrade a, Gabriela Tomas Jernimo a,b, Elenice Martins Brasil a,
Cecilia Vernica Nunez c, Eduardo Luiz Tavares Gonalves a, Maria Luiza Ruiz a, Maurcio Latera 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 Amaznia (INPA), Av. Andr Arajo, 2.936, Petrpolis, 69067-375 Manaus, AM, Brazil

a r t i c l e

i n f o

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 sh 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 (sh non-treated and non-parasitized), group 2: exposed to acetone 0.2% and parasitized sh, group 3: control (non-treated and parasitized sh), group 4: parasitized sh treated with 125 gmL1 of annatto extract for
2 h bath for two consecutive days, group 5: parasitized sh treated with 250 gmL1 of annatto extract for 2 h
bath for two consecutive days, group 6: parasitized sh treated with 125 gmL1 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% efcacy in all concentrations and times of bath evaluated. Hemoglobin concentration
and hematocrit percentage were higher in treated sh with 250 gmL1 2 h and 125 gmL1 12 h than that
observed in the non-treated sh groups. Glucose was signicantly higher in annatto-treated sh and cortisol
was signicantly higher in acetone group sh compared to other groups. Signicant decrease in thrombocyte
number was observed in sh after bath with acetone 0.2% compared to basal group, 125 gmL1 2 h and
125 gmL1 12 h, as well as decreased number of circulating lymphocytes in sh after bath with acetone
0.2% and 125 gmL1 12 h in relation to non-treated sh (control). On the other hand, signicant increase in
WBC count was found in sh treated with 125 gmL1 12 h in relation to basal and acetone groups. This is
the rst report on the use of seeds of B. orellana against monogenean parasites of sh. 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 sh parasites. Annatto extract bath is an efcacious 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 sh parasites. However, they present consequences to both to environment and human health. Alternatives have been studied to improve the sh health
status and control sh parasites. Phytotherapy shows several advantages in controlling parasites and improving
the sh health status. This study shows by the rst 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 sh species of the Amazon
region, and has been highlighted in sh farming due to its excellent
Corresponding author.
E-mail address: mauricio.martins@ufsc.br (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) 4046

causing high infestation levels in every stage of the sh life cycle (Eiras
et al., 2010; Jernimo 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; Lbo 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 sh 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 sh
(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 avonoids, 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 ndings on its use to control sh 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
inuence on the blood parameters and plasma cortisol, glucose, total
protein and total cholesterol levels of tambaqui.

41

evaporator (Fisatom, model 801) and the extract kept in dark asks at
20 C until the time of use.
2.2.2. Chemical composition of the extract and bixin quantication
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 L 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. Briey, for bixin
quantication, 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 mgL1, and then analyzed by spectrophotometry using UVVis 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 coefcient of bixin in CHCl3 (2826) at
max 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 sh were randomly selected, anesthetized and euthanized
for gill collection. For this test, the rst three gill arches were
separated in sterile Petri dishes for each concentration in triplicate: 500, 250, 125 and 62.5 gmL 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 sh farm
located in Manaus, AM, Brazil. At the sampling site, 10 sh samples
were anesthetized with clove oil (20 mgL1) (Inoue et al., 2011) and
euthanized to conrm the presence of monogenean parasites using a
dissection microscope (Zeiss Stemi 2000-C, Carl Zeiss, Oberkochen,
Germany). The sh 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 ow was turned on. The sh
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 Indstria
e Comrcio 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 sh. The following
concentrations of annatto extract were utilized; 500, 250, 125 and
62.5 gmL1, with ve sh in each aquarium of 80 L capacity in triplicate. Two controls composed of acetone 0.2% and another with only
water were used. The sh 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 mgL 1, pH 6.29
0.58, temperature 26.79 0.35 C, electric conductivity 25.65 2.04,
ammonia 0.09 0.03 mgL1, nitrite 0.03 0.02 mgL1, alkalinity
3.12 0.65 mg CaCO3L1 and hardness 2.92 0.77 mg CaCO3L1.
When the sh 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 sh per tank), in
triplicate:
Group 1: basal (sh non-treated and non-parasitized).
Group 2: exposed to acetone 0.2% and parasitized sh.
Group 3: control (non-treated and parasitized sh).
Group 4: parasitized sh treated with 125 gmL1 of annatto
extract for 2 h bath for two consecutive days.
Group 5: parasitized sh treated with 250 gmL1 of annatto
extract for 2 h bath for two consecutive days.
Group 6: parasitized sh treated with 125 gmL1 of extract for a
single bath for 12 h.

42

J.I.A. de Andrade et al. / Aquaculture 462 (2016) 4046

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 sh per
triplicate were collected for immediate parasitological analysis and
from the other 5 sh, the gill arches were removed and bathed in
60 C to be xed in alcohol, 70% for posterior counting. Parasites
quantication followed the Jernimo et al. (2011) method. Parasites
were mounted in Hoyer's medium for identication according to
Kritsky et al. (1979). The efcacy was calculated according to the formula: Efcacy = 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 quantication and

in vitro assay

2.5. Statistical analysis

The data obtained were rstly 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 UVVis conrmed 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 efcient concentrations to cause 90% mortality
were 500 and 250 gmL1, after 2 and 3 h, respectively. At 125 and
62.5 gmL1, 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 sh became agitated in all concentrations followed by gulping on the water surface and remaining
quiet. At the concentrations 500 and 250 gmL1, the sh lost movement after 1 and 3 h, respectively. In the other concentrations the sh
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 dened as 125 and 250 gmL1 in 2 h bath for
two consecutive days and one bath of 125 gmL1 for 12 h.
Therapeutic baths showed 100% efcacy in all concentrations tested
in relation to non-treated sh and acetone 0.2%-treated sh (Table 2).
Parasites were identied as Anacanthorus spathulatus Kritsky et al.,
1979. After extract or acetone was added, the water quality remained
within the normal values for sh (Costa et al., 2004; Marcon et al.,
2004; Aride et al., 2007). Dissolved oxygen 7.0 0.35 mgL 1, pH
6.35 0.54, temperature 26.86 0.47 C, electric conductivity
25.20 2.46, ammonia 0.09 0.02 mgL1, nitrite 0.04
0.02 mgL 1, alkalinity 3.33 0.94 mg CaCO3L 1 and hardness
3.04 1.05 mg CaCO3L1.

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) 4046

Table 1
Mortality of Anacanthorus spathulatus exposed to different concentrations of Bixa orellana
extract, acetone 0.2% and water during 4 h.
Concentrations (gmL1)

Death time (h)

Efcacy (%)

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 sh (control) when compared to the basal, acetone 0.2% and sh treated with 250 gmL 1
2 h and 125 gmL 1 12 h (Table 3). No signicant difference
was found in the hemoglobin concentration, hematocrit percentage
and red blood cell counts between basal sh and those exposed to
acetone.
Signicant decrease (p b 0.05) in thrombocyte number was observed in sh after bath with acetone 0.2% compared to basal group,
125 gmL1 2 h and 125 gmL1 12 h, as well as decreased number
of circulating lymphocytes in sh after bath with acetone 0.2% and
125 gmL1 12 h in relation to non-treated sh (control). Monocyte
number was higher in sh treated with 125 gmL 1 12 h than
that found in sh after bath with acetone 0.2%. On the other hand,
signicant increase in WBC count was found in sh treated with
125 gmL 1 12 h in relation to basal and acetone groups. Granular
leukocyte PAS+ (LG PAS) showed a higher number (p b 0.05) in sh
treated with 250 gmL1 2 h than that observed in the other groups
(Table 4).
Glucose levels were signicantly higher (p b 0.05) in sh treated
with annatto extract. Total protein was lower (p b 0.05) in group basal
than that control and annatto extract-treated sh. Cholesterol showed
an increase in sh treated with 250 gmL1 2 h, 125 gmL1 12 h
and acetone than that non-treated (control). On the other hand, the
cortisol levels were signicantly higher in sh exposed to acetone
than that found in the other groups (Table 3).

43

4. Discussion
In this study, the efcacy of annatto seed extract against the monogenean parasites of tambaqui was demonstrated in the in vitro assay
corroborating the ndings of Levy et al. (2015). In evaluating the
ethanolic extract of ginger Zingiber ofcinale 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
multiliis.
This study showed 100% efcacy 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 sh
(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% efcacy
of chloroformic extract of Indian sandal wood Santalum album
(40 mgL1) 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 mLL1 for 24 h against monogenean parasites of tambaqui presented 54.4% efcacy (Monteiro,
2012). In comparison with other studies, lower efcacies can be found
with therapeutic bath containing 40 mgL1 of essential oil of peppermint Mentha piperita for 10 min during three days (41.63% efcacy)
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 mgL 1 respectively against D. intermedius from C. auratus (Lu
et al., 2012). Soares et al. (2016) observed 14% efcacy 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 avonoids,
alkaloids and terpenoids, geranylgeraniol and bixin (Fleischer et al.,
2003; Braga et al., 2007). NMR analysis conrmed 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 conrm the promising use and efcacy of
annatto seeds extract against monogenean sh 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 gmL1
(2 h)

250 gmL1
(2 h)

125 gmL1
(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

44

J.I.A. de Andrade et al. / Aquaculture 462 (2016) 4046

Table 3
Blood parameters of Colossoma macropomum after treatment with Bixa orellana seed extract and acetone. 125 gmL1 and 250 gmL1 2 h (2 h bathes for 2 consecutive days) and 125
gmL1 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 gmL1
2h

250 gmL1
2h

125 gmL1 12 h

Hemoglobin (gdL1)
Hematocrit (%)
RBC (106L1)
MCV (fL)
MCHC (gdL1)
MCH (pg)
GLU (mgdL1)
TP (g.dL1)
CHOL (mgdL1)
Cortisol (ngmL1)

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 signicant 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 gmL1) 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 sh. To date, Jernimo
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 sh showed a tendency in returning
to the normal blood parameters, as observed in the non-treated and
non-parasitized sh. 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 shes, the immune system can be activated depending on
the degree of parasitism causing alteration in the percentage of circulating defense cells (Jernimo 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 sh. 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-sh can suggest stressful condition as a result of treatment. Similar ndings 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 sh exposed
to acetone bath conrming the glucose response.
Glucose levels could be inuenced 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 sh to elucidate how homeostasis is affected.
On the other hand, increased cortisol levels suggest stressful condition
in acetone-treated sh and perhaps the bath with annatto extract
could have promoted protection against the acetone stress. This is the
rst report on the use of annatto seeds to treat sh parasites and also
on the sh physiology.
5. Conclusions
This study reported by the rst time the efcacy on the use of annatto seeds extract on both monogenean parasites and physiology of
farmed sh tambaqui. The in vitro model by observing the parasites in
situ on the gill arches of sh was successful and can be used safely to
test medicines against sh parasites. A hundred percent efcacy 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 gmL1
2h

250 gmL1
2h

125 gmL1
12 h

Thrombocytes (103L1)
WBC (103L1)
Imm Leuk (x103.L1)
Eosinophils (103L1)
Neutrophils (103L1)
Lymphocytes (103L1)
Monocytes (103L1)
LG-PAS+ (103L1)

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 signicant difference by Tukey test (p b 0.05) among treatments.

J.I.A. de Andrade et al. / Aquaculture 462 (2016) 4046

studies testing lower concentrations and/or time of exposure in order to

cause no alterations in the blood parameters and sh physiology.
Studies may elucidate the anthelmintic mechanism, isolation of bioactive substances and ecological risks before its use in sh ponds. Bath
with annatto seed extract can be used for treating monogenean gill parasites as a method to reduce the parasitic load before sh transporting
or before harvesting in ponds.
Acknowledgements
The authors thank CAPES (Coordenao de Aperfeioamento
de Pessoal de Nvel Superior) for scholarship to J.I.A Andrade; CNPq
(Conselho Nacional de Desenvolvimento Cientco e Tecnolgico) 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 Amaznia DARPA, FINEP (01.09.0472.00) for nancial support;
and Dr. Felipe N. Vieira, Dr. Rosendo A. Yunes, Dr. Evoy Z. Filho
(Federal University of Santa Catarina, SC, Brazil), Dr. Jonas C. Espndola,
Dr. Robert Lenoch (IFC, Araquari, SC, Brazil) for critical review of the
manuscript prior to submission.
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