Future Microbiology

ISSN: (Print) (Online) Journal homepage: www.tandfonline.com/journals/ifmb20

Geraniol and Linalool Anticandidal Activity,

Genotoxic Potential and Embryotoxic Effect on
Zebrafish

Junya L Singulani, Reginaldo S Pedroso, Arthur B Ribeiro, Heloiza D Nicolella,

Karoline S Freitas, Jacqueline L Damasceno, Tatiana M Vieira, Antônio EM
Crotti, Denise C Tavares, Carlos HG Martins, Maria JS Mendes-Giannini &
Regina H Pires

To cite this article: Junya L Singulani, Reginaldo S Pedroso, Arthur B Ribeiro, Heloiza D
Nicolella, Karoline S Freitas, Jacqueline L Damasceno, Tatiana M Vieira, Antônio EM Crotti,
Denise C Tavares, Carlos HG Martins, Maria JS Mendes-Giannini & Regina H Pires (2018)
Geraniol and Linalool Anticandidal Activity, Genotoxic Potential and Embryotoxic Effect on
Zebrafish, Future Microbiology, 13:15, 1637-1646, DOI: 10.2217/fmb-2018-0200

To link to this article: https://doi.org/10.2217/fmb-2018-0200

Published online: 27 Nov 2018.

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Short Communication

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Geraniol and linalool anticandidal activity,

genotoxic potential and embryotoxic effect
on zebrafish
Junya L Singulani1 , Reginaldo S Pedroso2,3 , Arthur B Ribeiro2 , Heloiza D Nicolella2 ,
Karoline S Freitas2 , Jacqueline L Damasceno2 , Tatiana M Vieira4 , Antônio EM Crotti4 ,
Denise C Tavares2 , Carlos HG Martins2 , Maria JS Mendes-Giannini1 & Regina H Pires*,2
1
Universidade Estadual Paulista Julio de Mesquita Filho, 14800-903, Araraquara, SP, Brazil
2
Universidade de Franca, 14404-600, Franca, SP, Brazil
3
Universidade Federal de Uberlândia,38400-902, Uberlândia, MG, Brazil
4
Universidade de São Paulo, 14040-901, Ribeirão Preto, SP, Brazil
*Author for correspondence: regina.pires@unifran.edu.br

Aim: Geraniol and linalool are major constituents of the essential oils of medicinal plants. Materials &
methods: Antifungal activity of geraniol and linalool were evaluated against five Candida species. The
genotoxicity of these compounds was evaluated by the cytokinesis-block micronucleus test, and the em-
bryotoxic assays use zebrafish model. Results: Geraniol and linalool inhibited Candida growth, but geran-
iol was more effective. The geraniol at concentration of 800 μg/ml and the linalool at concentration of
125 μg/ml significantly increased chromosome damage. Geraniol was more toxic to zebrafish embryo
than linalool: LC50 values were 31.3 and 193.3 μg/ml, respectively. Conclusion: Geraniol and linalool have
anticandidal activity, but they also exert genotoxic and embryotoxic effects at the highest tested concen-
trations.

Graphical abstract:

MIC
determination

OH

OH Genotoxicity

Linalool Geraniol

Embryotoxicity

10.2217/fmb-2018-0200 
C 2018 Future Medicine Ltd Future Microbiol. (2018) 13(15), 1637–1646 ISSN 1746-0913 1637
Short Communication Singulani, Pedroso, Ribeiro et al.

First draft submitted: 8 July 2018; Accepted for publication: 31 October 2018; Published online:
27 November 2018

Keywords: antifungal • cytotoxicity • embryotoxicity • geraniol • linalool

Candida species have been listed among invasive infection agents since the late 1970s. Until 2000, about 90% of
the invasive infections caused by Candida were attributed to five species: C. albicans, C. glabrata, C. parapsilosis
(current C. parapsilosis sensu stricto), C. tropicalis and C. krusei (current Issachenkia orientalis) [1,3]. Other Candida
species such as C. guilliermondii (Meyerozyma guilliermondii), C. inconspicua, C. norvegensis (Pichia norvegensis), C.
dubliniensis, C. pelliculosa (Wickerhamomyces anomalus), C. rugosa and C. lipolytica have been also recovered and
are considered as emerging species in some countries [4]. In addition, these infections are often associated with high
morbidity and mortality rates [5].
Extended use of immunosuppressive drugs, broad-spectrum antibiotics, antifungals for prophylaxis and increasing
number of immunocompromised patients have resulted in appearance of drug-resistant clinical Candida species
isolates, a phenomenon designated multidrug resistance (MDR). In recent years, clinical isolates of Candida auris,
an emergent species with simultaneous reduced susceptibility to different classes of antifungal, such as azoles,
echinocandins and polyenes, have been described in different countries [6,7]. This is cause for great concern because
a limited number of antifungal drugs are available [8]. Therefore, the search for new antifungals in natural sources
such as plants is urgent.
Linalool and geraniol have been reported as major constituents of the essential oils of various medicinal plants
such as Peperomia pellucida [9], Acorus calamus [10], Pelargonium graveolens [11] and Lippia alba [12,13]. These
monoterpenes inhibit 3-hydroxy-3-methylglutaryl coenzyme A reductase, cell growth and signaling modulators,
apoptosis initiators and cell cycle arrest inducers [14,16]. Taken together, these inhibition effects contribute to cell
death and point to the antimicrobial activity of these monoterpenes against pathogens that cause several pathologies,
including respiratory, intestinal and urinary infections and septicemia, as well as to the action of these monoterpenes
against biofilm formation in medical devices or prostheses. Linalool and geraniol exhibit antimicrobial properties
against fungal species like Candida species, Cryptococcus species, Saccharomyces cerevisiae [12], Candida tropicalis in
both the planktonic and biofilm forms [12], and Candida albicans and nonC. albicans strains [15], not to mention their
activity against bacteria [9,13,17]. In addition, they have been widely used in cleaning products, foods and antitumoral
and/or cosmetic formulations [18–22], so knowing the in vitro and in vivo toxicity of these two monoterpenes is
paramount.
In vitro assay systems are often based on cell cultures, enzyme systems or cloned receptors, whereas in vitro
genotoxicity assays primarily include assessment of DNA damage, mutations and chromosomal aberrations by the
Ames test [23], the micronucleus test and the Comet test [24]. However, in vitro tests cannot completely replace
animal tests [25]: despite their high sensitivity (and therefore low false-negative rate), in vitro tests have relatively
low specificity, and the rate of false-positive (‘misleading’) results is high, which requires in vivo confirmation of the
data [26,27]. Unfortunately, validation in mammalian models is usually laborious and expensive. The zebrafish (Danio
rerio) is a vertebrate model organism that has been extensively employed for this purpose [28]. Its characteristics such
as transparency, small size (which enables drug screening in 96-well microplates), development in 5 days in small
quantities of fish water, an attached yolk that provides nutrients (no feeding is needed during the first week), and
simple drug administration (small molecules can be dissolved in fish water and diffuse into the embryo) enables
the use of zebrafish embryos as model to assess drug-induced changes. Entire body-plan development within 24-h
postfertilization (hpf ) and full development of internal organs (heart, liver, intestine and kidney) within 96 hpf are
other attractive characteristics that make the zebrafish a valuable model organism to predict drug toxicity [29].
This paper describes the anticandidal effect, cytotoxic and genotoxic potential of linalool and geraniol, and shows
the changes these compounds induce in vivo with the aid of the zebrafish model.

Materials & methods

Chemicals
(±)-Linalool ((±)3,7-dimethyl-octa-1,6-dien-3-ol), geraniol (trans-3,7-dimethyl-octa-2,6-dien-1-ol), Dimethyl
sulfoxide (DMSO), morpholinepropanesulfonic acid, methyl methanesulfonate (MMS), amphotericin B, strep-
tomycin, penicillin, cytochalasin-B and sodium bicarbonate were purchased from Sigma-Aldrich (MO, USA).

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 Geraniol & linalool anticandidal activities Short Communication

To check the degree of purity, linalool and geraniol were previously analyzed on a Shimadzu 2010 Plus gas
chromatograph equipped with flame ionization detection. Both compounds had purity higher than 98%.

Microorganisms
Candida albicans ATCC 5314, C. glabrata ATCC 2001, C. parapsilosis ATCC 22019, C. krusei ATCC 6258 and
C. tropicalis ATCC 13803 strains were used in the experiments. These strains were stored in 30% glycerol at -80◦ C
and were subcultured on Sabouraud Dextrose agar (Difco, MI, USA) plates at 37◦ C to confirm their viability.

Antifungal tests
Anti-Candida activity was evaluated in terms of the minimal inhibitory concentration (MIC); the broth microdilu-
tion method in 96-well plates was used [30]. Stock solutions of the compounds (linalool or geraniol) at 4000 μg/ml
were prepared in DMSO. Twofold and serial dilutions of the compounds were prepared in Roswell Park Memorial
Institute (RPMI 1640) medium with L-glutamine without sodium bicarbonate and buffered with morpholine-
propanesulfonic acid 0.165 M. The inoculum was diluted in RPMI to obtain a final concentration of 5 × 103
cells/ml. Compound-free and yeast-free controls were included. The microtiter plates were incubated at 35◦ C
for 48 h and were visually inspected. Amphotericin B (Sigma, concentrations ranging from 0.06 to 8 μg/ml), C.
krusei ATCC 6258 and C. parapsilosis ATCC 22019 were used to validate the MIC of the compounds. MIC was
determined in the well containing the lowest concentration of the tested compound that inhibited microorganism
growth as compared with the compound-free growth control. Experiments were performed independently, in
triplicate.

Cells & culture conditions

Chinese hamster lung fibroblast cells (V79 cells), kindly provided by the Laboratory of Mutagenesis, Department of
Biological Sciences, State University of São Paulo, Araraquara, São Paulo, Brazil, were used during the experiments.
V79 cells were maintained in culture flasks (25 cm2 ) containing Ham’s F10 media (HAM-F10, Sigma-Aldrich)
and Dulbecco’s modification of Eagle medium (DMEM), (Sigma-Aldrich, 1:1) supplemented with 10% fetal
bovine serum (Nutricell), sodium bicarbonate 1.2 g/ml (Sigma-Aldrich), streptomycin 0.1 g/ml (Sigma-Aldrich)
and penicillin 0.06 g/ml (Sigma-Aldrich) at 37◦ C in a Bio-Oxygen Demand (BOD) incubator. Under these
conditions, the average cell cycle time was 12 h.

Genotoxicity assay
The cytokinesis-block micronucleus assay was employed to assess genotoxicity. The geraniol and linalool concen-
trations were chosen on the basis of their cytotoxicity, according to Souza et al. [11]. The following concentrations
were evaluated: geraniol at 200, 400 and 800 μg/ml and linalool at 62.5, 125 and 250 μg/ml. Negative (no
treatment), solvent (DMSO, 5.5 μg/ml; Sigma-Aldrich) and positive (MMS, 44 μg/ml; Sigma-Aldrich) controls
were also included. The protocol was carried out in triplicate on three different days to ensure reproducibility. The
treatment designs and cell collection and fixation procedures described by Furtado et al. [31].
The criterion established by Fenech [32] was used to analyze the micronuclei. To this end, 3000 binucleated
cells were scored per treatment, for 1000 cells/treatment/repetition. To evaluate treatment cytotoxicity, the nuclear
division index (NDI) was determined for 1500 cells analyzed per treatment (500 cells/repetition). Cells with well-
preserved cytoplasm and containing between one and four nuclei were scored. The NDI was calculated according
to Eastmond and Tucker [33]; the following formula was applied:

[ M 1  2( M 2)  3( M 3)  4( M 4]
NDI  ,
N

where M1–M4 is the number of cells with one, two, three and four nuclei, respectively, and N is the total number
of viable cells.
Additionally, the cytotoxicity index (CI) was calculated as previously described [34]:

 NDIT  1 
CI  100  100  ,
 NDIC  1 

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Short Communication Singulani, Pedroso, Ribeiro et al.

Table 1. Geraniol and linalool minimal inhibitory concentration results against Candida species.
Microorganisms Geraniol (μ g/ml) Linalool (μ g/ml)
Candida albicans ATCC 5314 1000 ⬎1000
Candida tropicalis ATCC 13803 250 500
Candida krusei ATCC 6258 250 250
Candida parapsilosis ATCC 22019 37.5 125
Candida glabrata ATCC 2001 125 250

Table 2. Frequency of micronuclei and nuclear division index obtained for V79 cell cultures treated with geraniol or
linalool and the respective controls.
Treatment (μ g/ml) MN frequency† NDI‡
Negative control 5.00 ± 2.00 1.80 ± 0.03
MMS 21.30 ± 5 8.00 ± 1.73 1.81 ± 0.05
MMS 21.30 ± 5.69§ 1.68 ± 0.08
GE 200 μ g/ml 13.00 ± 4.00 1.77 ± 0.05
GE 400 μ g/ml 11.30 ± 3.21 1.77 ± 0.02
GE 800 μ g/ml 16.70 ± 2.52§ 1.70 ± 0.02
LI 62.5 μ g/ml 9.50 ± 0.70 1.82 ± 0.00
LI 125 μ g/ml 16.70 ± 1.15§ 1.74 ± 0.11
LI 250 μ g/ml 9.70 ± 2.08 1.57 ± 0.11§
Values are the mean ± standard deviation.
† A total of 3000 binucleated cells were analyzed per treatment group.
‡ A total of 1500 cells were analyzed per treatment group.
§ Significantly different from the negative control group (p ⬍ 0.05).

DMSO: Dimethylsulfoxide (5.5 μ g/ml); GE: Geraniol; LI: Linalool; MMS: Methyl methanesulfonate (44 μ g/ml); MN: Micronuclei; NDI: Nuclear division index.

where NDIT is the NDI found for the different treatments, and NDIC is the NDI of the negative control.

Embryotoxicity assay
Adult zebrafish (Danio rerio) were maintained at 28 ± 1◦ C in a 14/10 h (light/dark) photoperiod regime. Male and
female fish (at 1:1, 1:2 or 2:1 ratio) were placed in a spawning tank overnight, and embryos were collected and washed
with embryo medium (NaCl, KCl, CaCl2 .2H2 O and MgCl2 .6H2 O) plus 0.00003% methylene blue. The assay
was accomplished according to the OECD guideline for the Fish Embryo Toxicity test [35]. Fertilized embryos were
exposed to different linalool or geraniol concentrations (31.25, 62.50, 125, 250, 500 and 1000 μg/ml), prepared
by dilution with embryo medium. The assay was performed in 96-well plates at 28◦ C; two embryos/well and
ten embryos/concentration were employed. Embryos and larvae were analyzed with a stereomicroscope connected
to a camera at 5, 24, 48 and 120 hpf. Embryo coagulation and malformations were assessed. Three independent
experiments were conducted.

Statistical analysis
Statistical analyses were performed with the GraphPad Prism 5 software (GraphPad Software Inc., CA, USA).
Cytokinesis-block micronucleus assays were analyzed by analysis of variance. Treatment means were compared by
the Tukey test. Zebra fish survival curves were plotted by using the Kaplan–Meier method and analyzed by log-rank
(Mantel–Cox). In every case, differences were considered statistically significant at a p-value lower than 0.05.

Results
We examined how geraniol and linalool affected the in vitro growth of Candida ATCC strains by following the
Clinical & Laboratory Standards Institute (CLSI) standard. The results were shown in Table 1; overall, geraniol
was more effective than linalool. C. albicans and C. parapsilosis were the most resistant and most susceptible to the
compounds, respectively.
Table 2 lists the frequency of micronuclei, the NDI and the CI obtained for V79 cells treated with geraniol or
linalool. The highest geraniol concentration (800 μg/ml) and the intermediate linalool concentration (125 μg/ml)
assayed herein significantly increased chromosome damage as compared with the negative control, which demon-

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 Geraniol & linalool anticandidal activities Short Communication

Geraniol Linalool
100 100

80 80

% survival
% survival

60 60

40 40

20 20

0 0
5 24 48 120 5 24 48 120
Hours post fertilization Hours post fertilization

Control Control
31.25 μg/ml 62.5 μg/ml 125 μg/ml 31.25 μg/ml 62.5 μg/ml 125 μg/ml
250 μg/ml 500 μg/ml 1000 μg/ml 250 μg/ml 500 μg/ml 1000 μg/ml

100 100
Effects (%) at 120 hpf

Effects (%) at 120 hpf

80 80

60 60

40 40

20 20

0 0
Control 31.25 62.5 125 250 500 1000 Control 31.25 62.5 125 250 500 1000
Normal embryos Malformed embryos Dead embryos

Figure 1. Zebrafish survival after exposure to geraniol and linalool. Survival curves of zebrafish embryos exposed to concentrations
ranging to 31.25–100 μg/ml of geraniol (A) or linalool (B) for 120 hpf. Rate of normal, malformed and dead embryos after exposure for
120 hpf to geraniol (C) or linalool (D). N = 30 embryos/group.
hcf: Hour postfertilization.

strated a genotoxic effect. Furthermore, the culture treated with the highest linalool concentration of (250 μg/ml)
exhibited significantly lower NDI as compared with the negative control; CI was equal to 29.0%.
The survival rate and malformations in zebrafish were measured up to 120 hpf. Geraniol and linalool exerted
dose-dependent effects on embryo survival for 120 hpf (Figure 1). The survival curves constructed for all the
tested geraniol concentrations were significantly different from the survival curve constructed for the control group
(p < 0.05). In contrast, the survival curves constructed for linalool at 31.25 and 62.50 μg/ml did not differ
significantly from the survival curve constructed for the control group, but the higher linalool concentrations tested
herein decreased embryo survival (p < 0.05 vs control). The letal concentration 50% (LC50 ) was also calculated from
the survival curves (Figure 1A & B). Geraniol was more toxic than linalool: LC50 values were 31.3 and 193.3 μg/ml,
respectively. The embryotoxicity of the compounds included mortality and malformations (Figure 1C & D). The
control group presented normal embryonic development of the body and heart rate as well as pigmentation on
the body and eyes (Figure 2A, D, G & J). On the other hand, toxic geraniol and linalool concentrations caused
embryo coagulation (Figure 2B, C & K). In addition, some concentrations of the compounds were teratogenic,
and embryos presented malformations such as yolk sac edema (Figure 2E & H), slow pigmentation (Figure 2I) and
tail deformity (Figure 2L).

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Short Communication Singulani, Pedroso, Ribeiro et al.

Control Geraniol Linalool

5 hpf

24 hpf

48 hpf

120 hpf

Figure 2. Embriotoxicity after exposure to geraniol and linalool. Normal embryonic development of the control
group (A, D, G & J). Embryo coagulation (B & K) and yolk sac edema (E & H) caused by geraniol. Embryo coagulation
(C), slow pigmentation (I) and tail deformity (L) caused by linalool. Teratogenic effects were not observed at 24 hpf
for linalool (F). Scale bar: 2 mm.
hcf: Hour postfertilization.

Discussion
The search for new drugs with antifungal activity has intensified in recent years in view of the more frequent diagnoses
of invasive fungal infections, especially in immunocompromised and hospitalized individuals [4,5]. Associated with
this, the growing number of reports on isolates of the genus Candida with increased resistance to currently available
antifungal agents has increased the need for research in this field. In fact, the resistance of these fungi to some
drugs has emerged as a major challenge for modern medicine and public health. News reports on C. auris species
in several countries worldwide provide an example of such challenge [6,7].
Here, we evaluated two oxygenated monoterpenes, geraniol and linalool. We analyzed the activity of these
compounds against Candida species by determining their MIC, in vitro genotoxic and cytotoxic potential, and in
vivo toxicity to the zebrafish. Given the Candida species included in this study, geraniol afforded lower MIC values
than linalool; the largest and the lowest MIC values were determined for C. albicans and C parapsilosis, respectively.
Previous studies have pointed out the therapeutic potential of geraniol. This compound needs to be explored
more deeply [36], so that the mechanisms of antifungal activity and the underlying pathways can be elucidated
with a view to practical applications. [37] showed that geraniol inhibits C albicans growth in vitro. Other studies
have also demonstrated geraniol antifungal activity against other Candida species, such as C. glabrata, C. tropicalis,
C. guilliermondii and C. krusei, among others, not to mention its action against Trichophyton rubrum [38–40] and
antibacterial activity against Streptococcus mutans and Shigella sonnei [36,41]
The antimicrobial action of linalool-containing essential oils has been demonstrated against Gram-positive and
Gram-negative bacteria and yeasts [42,43]. Geraniol gave lower MIC values than linalool. Additionally, except for C.
albicans, geraniol showed antimicrobial activity at concentrations that did not have a genotoxic or cytotoxic effect
on mammalian cells. Sasaki et al. [44] and Doppalapudi et al. [45] also verified the absence of geraniol genotoxicity

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 Geraniol & linalool anticandidal activities Short Communication

to Chinese hamster fibroblasts when they conducted sister-chromatid exchanges assay with 5.13, 15.40 and
51.36 μg/ml and chromosomal aberrations assays with 39.1–156.3 μg/ml, respectively. The micronucleus test
revealed that geraniol at 375, 750 or 1500 mg/kg was not genotoxic to mouse bone marrow erythrocytes [45].
Although literature studies have demonstrated the absence of geraniol genotoxic activity in mammalian cells,
in the present study, the genotoxicity assessment of geraniol demonstrated that the highest concentration tested
(800 μg/ml) was able to induce damage to the genetic material in V79 cells. However, it is worth mentioning that
the methodologies and concentrations used in the literature [44,45] differ from those employed in the present study.
Despite the genotoxic results obtained for geraniol in in vitro assays, the concentrations used herein were much
higher than the concentrations expected at human plasma levels: metabolism, exposure and pharmacokinetic and
pharmacodynamic factors alter the distribution and concentration of compounds in more complex organisms than
in cellular units. In addition, the results demonstrating the absence of genotoxicity in vivo at extremely high doses
suggested that geraniol has minimal genotoxic risk for use in mammals.
The zebrafish embryo in the control group presented normal development. However, embryo development
or mortality was altered in a concentration-dependent manner in the groups exposed to geraniol or linalool.
Embryo alterations were coagulation, yolk sac edema, slow pigmentation and tail deformity. All the zebrafish
embryos died within 5 hpf at higher concentrations of the compounds (500 and 1000 μg/ml). Geraniol exhibited
higher toxicity than linalool: LC50 values were 31.3 and 193.3 μg/ml, respectively. According to the literature,
various compounds, like antibiotic, disinfectants, antifouling agents, herbicides and candidates for pharmaceutical
products, among others, have had their toxic effects tested by means of the zebrafish model. Results have shown
that different concentrations of the assayed compounds have distinct effects on embryo development [46–50]. Drug
combinations have also been analyzed; for instance, ethanol and trichostatin is a lethal combination for zebrafish
embryos, whereas trichostatin alone is not lethal and ethanol alone has only a minor effect [51].

Conclusion
Geraniol and linalool have anticandidal activity, but they also exert a genotoxic effect. At high concentrations,
both compounds induce organism deformities and embryonic death. Despite causing some developmental defects,
these compounds deserve further studies so that their effects on animal health can be understood. Future studies
will involve the search for synergy between these compounds and different antifungal agents by analysis of the
antifungal activity in vitro as well as their action on some virulence factors, which is important for mucocutaneous
or invasive infections caused by Candida species.

Summary points
• Geraniol and linalool are commonly found in plant-derived essential oils and display a wide range of
antimicrobial activity.
• The results point that geraniol and linalool displayed activity against selected Candid a species, but geraniol was
mostly more effective than linalool.
• Antimicrobial activity of geraniol against Candida parapsilosis (minimal inhibitory concentration = 37.5 μg/ml) is
very promising.
• Geraniol and linalool at concentrations of 800 and 125 μg/ml, respectively, significantly increased chromosome
damage.
• Geraniol was also more toxic than linalool to zebrafish embryo (LC50 = 31.3 and 193.3 μg/ml, respectively).
• Both geraniol and linalool displayed anti-Candida activity, but they also exert genotoxic activity and embryotoxic
effects at the highest tested concentration.

Author contributions
TM Vieira and AEM Crotti contributed with reagents, material and GC analyses. JL Singulani, JL Damasceno, RS Pedroso, AB
Ribeiro, HD Nicolella and KS Freitas performed the experiments. DC Tavares, CHG Martins and MJS Mendes-Gianini supervised the
experiments and laboratory work. RH Pires designed the experiments and drafted the manuscript. All the authors approved the
final version of the manuscript.

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Short Communication Singulani, Pedroso, Ribeiro et al.

Financial & competing interests disclosure

This work was funded by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, Proc. 2007/54241-8). The research fel-
lowship provided by Conselho Nacional de Desenvolvimento Cientı́fico e Tecnológico (CNPq) to AEM Crotti is duly acknowledged.
The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in
or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.
No writing assistance was utilized in the production of this manuscript.

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