Modification of the brain proteome of Africanized honeybees (Apismellifera) exposed to a sub-lethal doses of the insecticide fipronil

T. C. Roat • J. R. A. dos Santos-Pinto •

L. D. dos Santos • K. S. Santos • O. Malaspina •

M. S. Palma

Accepted: 6 August 2014

� Springer Science+Business Media New York 2014

Abstract Fipronil is a phenylpyrazole insecticide that is

widely used in Brazilian agriculture for pest control.

Although honeybees are not targets of fipronil, studies

indicate that this pesticide can be harmful to honeybees. To

assess the effects of fipronil in the brain of Africanized

Apis mellifera workers, this study focused on the toxico-

proteome profiling of the brain of newly emerged and aged

honeybee workers that were exposed to a sub-lethal dose

(10 pg fipronil per day. i.e. 1/100 of LD50/bee/day during

5 days) of the insecticide. Proteomic analysis identified 25

proteins that were differentially up-regulated or down-

regulated when the fipronil-exposed and non-exposed

groups were compared. These proteins are potentially

related to pathogen susceptibility, neuronal chemical stress,

neuronal protein misfolding, and occurrence of apoptosis,

ischemia, visual impairment, damaged synapse formation,

brain degeneration, memory and learning impairment. The

exposure of honeybees to a very low dose of fipronil, even

for a short period of time (5 days), was sufficient to cause a

series of important neuroproteomic changes in the brains of

honeybees.

Keywords Brain proteome � Phenylpyrazole �Insecticide � Proteomic analysis � 2-DE � Mass

spectrometry

Introduction

Honeybees Apis mellifera L., 1758 (Hymenoptera: Apidae)

are considered excellent pollinating insects in agroecosys-

tems (Kremen et al. 2007) giving to this specie a great

agronomic, environmental and economic importance.

However the reduction in the number of pollinators,

especially bee species, has become a major problem for

beekeepers, and it may threat food security and ecosystem

integrity due to reduced pollination. The causes of this high

mortality are not well understood. Many stress factors,

acting alone or together, can weaken the colony including

the decreased availability of resources due to the degra-

dation and fragmentation of the honeybee habitat, patho-

gens and parasites, beekeeping management, and the

extensive use of chemical pesticides on crops (vanEn-

gelsdorp and Meixner 2009; Bromensshenk et al. 2010;

Wratten et al. 2012).

Honeybees can be exposed to insecticides during their

foraging activities and while carrying out activities within

the colony through contact with contaminated pollen and

nectar. Consequently, different larval stages and adults of

different ages can be exposed to these chemical products

(Pham-Delegue et al. 2002; Malaspina and Silva-Zacarin

2006; Desneux et al. 2007). There are different viewpoints

in the discussion about the exposure of honeybees to dif-

ferent levels of insecticide residues and whether this

T. C. Roat (&) � J. R. A. dos Santos-Pinto � O. Malaspina �M. S. Palma

Institute of Biosciences of Rio Claro, Department of Biology,

Center of the Study of Social Insects, University of Sao Paulo

State (UNESP), Avenida 24-A, N.1515-Bela Vista, Rio Claro,

SP, Brazil

e-mail: thaisaroat@yahoo.com.br

L. D. dos Santos

Center for the Study of Venoms and Venomous Animals

(CEVAP), University of Sao Paulo State (UNESP), Botucatu,

SP, Brazil

K. S. Santos

Discipline of Allergy and Immunology (HC/Incor/FMUSP),

Sao Paulo, SP, Brazil

123

Ecotoxicology

DOI 10.1007/s10646-014-1305-8

exposure directly causes honeybee death (Greig-Smith

et al. 1994; Rortais et al. 2005; Rondeau et al. 2014).

The fipronil is the active component of different agricul-

tural products (Tingle et al. 2003). Fipronil belongs to the

phenylpyrazole group and acts on the nervous system of

insects by blocking GABA receptors and inhibiting ionotro-

pic glutamate-gated chloride channels, inhibiting the central

nervous system (El Hassani et al. 2005). GABA receptors are

located in several neuropils of the honeybee brain (Schafer

and Bicker 1986), and they are involved in modulating

learning, memory and sensory perception (El Hassani et al.

2005; Aliouane et al. 2009; Bernadou et al. 2009).

Studies assessing the toxicity of fipronil to honeybees

reported that it is harmful to these insects (Decourtye et al.

2005; Pochi et al. 2012). It can modulate insects’ sensi-

tivity to sucrose (Aliouane et al. 2009), affect learning

(Decourtye et al. 2005), orientation and foraging behavior

(Decourtye et al. 2009), and cause hyperactivity followed

by paralysis and death (Barbara et al. 2005; Gunasekara

et al. 2007). The results from studies of the oral toxicity of

fipronil against the stingless bee Melipona scutellaris

showed that these insects exhibited signs of intoxication,

such as initial tremors followed by paralysis, before their

death (Lourenco et al. 2012). It was reported that low doses

of fipronil impaired the olfactory learning (0.5 ng/bee) and

memory abilities (0.1 ng/bee) in A. mellifera through the

action of multiple targets, including glutamatergic and

GABAergic inhibitory signaling (El Hassani et al. 2005)

Roat et al. (2013) reported the lethal dose (LD50) and

lethal concentration (LC50) values of fipronil for newly

emerged Africanized honeybees showing that this insecti-

cide may be harmful to these bees independently of exposure

route: LD50 = 1.06 ng fipronil/bee and LC50 = 1.27 ng

fipronil/lL of food, respectively. Besides, this study assessed

the side effect of a sublethal dose of fipronil on neuron

metabolic activity from Africanized honeybees and showed

that the insecticide causes metabolic changes by increasing

the respiratory activity of mitochondria.

A series of discussions about the use and restrictions of

different classes of pesticides has been reported in the lit-

erature; however, despite the fact that many studies have

been published, there is still a lack of knowledge on the

relationship between the exposure to insecticides and the

possible effects on the honeybee physiology (Maxim and

van der Sluijs 2007). Studies performed using neonicoti-

noids already showed effects of sublethal doses on hon-

eybee behaviors, e.g. antifeedant effect (Han et al. 2010a),

and reduced visual learning capacities and decreased

olfactory learning performances (Decourtye et al. 2009;

Han et al. 2010b).

The most studies that were performed with phenylpy-

razoles were conducted by evaluating acute toxicity, i.e.,

determining the LD50 values for these pesticides (OEPP/

EPPO 2003). The assessment of risk to honeybee popula-

tions using this approach has been contested because the

pesticides are generally used at sub-lethal doses (Halm

et al. 2006), which would require the evaluation of chronic

toxicity caused by continued use of low doses of pesticide

(Rortais et al. 2005); there is a need of more accurate

methods for risk assessment of pesticides on bees (De-

courtye et al. 2013).

Because fipronil is widely used in Brazilian agriculture,

we used it as a pesticide model in a proteomic approach

aiming to analyze the biochemical effects of sublethal

doses in the brains of Africanized A. mellifera. We

developed a toxicoproteomic approach using an experi-

mental protocol; it combined 2-DE gel electrophoresis with

in-gel protein digestion, followed by mass spectrometry

analysis to assess the effects of fipronil by profiling the

protein expression pattern in the brains of newly emerged

and aged workers.

Materials and methods

Honeybees

Honeybees were obtained from queen-right colonies of

Africanized A. mellifera at the experimental apiary of the

Institute of Biosciences of Rio Claro, Department of

Biology, University of Sao Paulo State (UNESP), Rio

Claro, SP, Brazil. To obtain newly emerged workers, three

sealed brood combs from three different colonies were

placed in a climatic room at a temperature of 34 ± 2 �C

and a relative humidity (RH) of 70 ± 10 %, and the adults

were collected after emergence. Of the 740 newly emerged

honeybees that were collected, 240 were used in bioassays

and 500 were marked with ink on their thorax and were

returned to the colony for capture when they reached

20 days of age (considered here as aged honeybees).

Honeybee toxicity assays

The honeybee toxicity assay was performed using a sub-

lethal dose of fipronil that was based on the LD50 value for

newly emerged honeybees, which was determined by Roat

et al. (2013) to be 1.06 ng fipronil per bee. Fipronil

(95.0 %, analytical grade) was supplied by Dalian Raiser

Pesticides (China). To intoxicate the honeybees, a stock

solution of fipronil (1,000 ng/lL) was prepared in acetone,

and from this solution, several dilutions were prepared and

applied to a syrup preparation (50 % m/v sucrose) to obtain

a solution containing 1 pg fipronil per lL diet and

0.0001 % of acetone. Groups of 60 newly emerged and 60

aged honeybees (from three different colonies-20 of each

colony) were equally divided into three disposable cages

T. C. Roat et al.

123

(20 workers per cage). The honeybees were collectively fed

with the sucrose syrup containing fipronil, and the total

volume was adjusted to ensure that each honeybee could

consume approximately 10 lL sucrose solution daily.

Therefore, each bee ingested 10 pg fipronil per day (1/100 of

LD50), during 5 days. After ingestion of the contaminated

syrup, the bees were fed with syrup (the same that was

furnnihed for the control group). The experimental control

used in these experiments consists of honeybees that were

fed with syrup. The food was provided in a small plastic

cup that was placed on the bottom of the experimental

cages and covered with a plastic mesh. Water was supplied

ad libitum through impregnated cotton. The cages were

kept in a climatic room at 32 ± 2 �C and 60 ± 10 % RH.

Samples of newly emerged and aged honeybees were

collected to assess the alterations that were induced by

fipronil 5 days after the beginning of exposure to fipronil.

Thus, newly emerged bees were 5 days old at the time of

collection and aged bees were 25 days old.

Protein assay

For the analysis of the protein fraction, the brains of 30

bees derived from each treatment were homogenized in

1 mM PMSF (phenylmethylsulphonyl fluoride) in dis-

solved bi-distilled water (containing 1 % v/v isopropanol).

Protein quantification was determined by the Bradford

method (Bradford 1976) using bovine serum albumin

(BSA) as a standard.

Two-dimensional gel electrophoresis

Samples (250 lg proteins) were applied by rehydration to

7 cm IPG strips, pH 3–10. Isoelectric focusing (IEF) was

carried out on an IPGphor system (GE Healthcare) at

3,500 V for 7,100 Vh. Immobilized pH Gradient (IPG)

strips were incubated in equilibration buffer [50 mM Tris–

HCl, pH 8.8, 6 M urea, 30 % (v/v) glycerol, 2 % (w/v) SDS]

containing 0.5 % (w/v) dithiothreitol (DTT) for 15 min,

followed by equilibration buffer containing 4 % (w/v)

iodoacetamide for 15 min. The second dimension was run on

self-cast SDS-PAGE gels [12.5 % (w/v) polyacrylamide and

0.8 % (w/v) bis-(N,N’-methylenebisacrylamide)] at 15 mA/

gel for 15 min and 30 mA/gel for 1 h at 10 �C in a Mini-VE

system (GE Healthcare). The gels were stained overnight

with Coomassie Brilliant Blue R-250 (CBB) and stored at

21 �C in preserving solution [7 % (v/v) acetic acid].

Image acquisition, protein quantitation and statistical

analysis

For each treatment or control group were carried out three

gels, representing a triplicate. 2-DE gels stained with

Coomassie Brilliant Blue (CBB) were scanned and digi-

tized (BioImage, GE Healthcare) in the transparency mode

with 24-bit red-green-blue colors and 400 dpi resolution for

documentation. The gel staining was performed as indi-

cated by the manufacturer of the staining system (GE

Healthcare) as follows: gels were fixed for 30 min in a

solution of 40 % (v/v) methanol containing 7 % (v/v)

acetic acid; the gel was then stained overnight in Coo-

massie (0.02 % PhastGel Blue R, 60 % methanol, 10 % (v/

v) acetic acid). The destaining was conducted by incubat-

ing the gel for 30 min in 40 % methanol containing 7 % (v/

v) acetic acid, and a solution containing 7 % (v/v) acetic

acid and 5 % (v/v) methanol, until the background was

clear. All images were analyzed by ImageMaster 2-DE

Platinum 7.0 software from GE Healthcare (Uppsala,

Sweden) using a white light tray; the f/stop was set 22

(minimal exposure). The integrated optical densities were

determined for the full width of each protein spot and the

parameters for image capture of the CBB stained gel were:

time = 0.28 s, gain = 1, b.lev = 7, and gamma = 0.55;

the duration of exporting the image to a computer was

0.45 s, gain = 0.1, b.lev = 6 and gamma = 1.0. The gel

with the most spots was chosen as a reference. The refer-

ence gel was then used to match the corresponding protein

spots between gels. After an average mode of background

correction, manual subtraction of the ‘‘joined valleys’’ was

performed. The rate values of individual spot volume

intensity and the total volume intensity (sum of the inten-

sity volumes obtained from all spots in the same 2-DE gel)

were calculated. Then, a comparison was made between

the control (honeybees not exposed to fipronil) and the

fipronil-exposed group; thus, the rate values of individual

spot volume intensity were transformed to inter-experi-

mental normalized spot volume intensity (INSVI), and the

values of this rate were used to determine the differential

protein expression between the control and fipronil-

exposed groups, which were considered as up- or down-

regulated when the INSVI values were greater than 1.50

and less than 0.50, respectively. One-way ANOVA was

used for the analysis of the INSVI value differences of the

spots between both groups; a probability of p \ 0.05 was

considered to be statistically significant.

In-gel digestion

Pieces (*1 mm3) were excised from the protein spots of

the 2-DE gel, destained twice for 30 min at 25 �C with

50 mM ammonium bicarbonate/50 % acetonitrile (MeCN),

dehydrated in the presence of 100 lL pure MeCN at 25 �C,

dried in a speed-vac system for 30 min at 38 �C, and

treated with 25 lL trypsin solution (20 lg/mL, Promega,

Madison, USA) in 50 mM ammonium bicarbonate, pH 7.9

at 37 �C for 18 h. Digests were extracted from each gel

Brain proteome of Africanized honeybees

123

piece with 30 lL 50 % (v/v) acetonitrile containing 5 %

(v/v) formic acid, desalted and cleaned with PerfectPure

C18 pipet tips (Eppendorf, Hamburg, Germany) according

to the manufacturer’s instructions, and the desalted digests

were vacuum dried. The concentrated digests were mixed

with 0.5 lL matrix [10 mg/mL a-cyano-4-hydroxycin-

namic acid in methanol/acetonitrile (1:1, v/v) mixed with

an equal volume of 0.2 % (v/v) aqueous trifluoroacetic acid

(TFA)] and spotted onto a MALDI plate target.

Mass spectrometry analysis

Mass spectrometric analysis was performed with a MALDI

ToF/ToF–MS (matrix-assisted laser desorption ionization

time of flight/time of flight-mass spectrometry) instrument

(Shimadzu, Axima Performance). The MS and MS/MS

spectra were acquired in the positive ion reflectron mode

using a N2 laser. Typically, 250–500 laser shots were

acquired for the MS and MS/MS mode, and spectra were

obtained at a laser power that was maximized for the

highest possible resolution and peak intensity in each

analytical condition. CID spectra were acquired using a

dual-timed ion gate at a laser power that was approximately

20 % higher than for MS acquisition. MS data were

acquired in the m/z range of 700–3,500, with an acceler-

ating voltage of 20 kV, delayed extraction, a peak density

of a maximum 50 peaks per 200 Da, a minimal S/N ratio of

10 and a maximum peak at 60. MS/MS data were acquired

in the mass range from 60 Da to each precursor mass with

a minimum S/N ratio of 10; the maximum number of peaks

was set to 65 and a peak density of a maximum of 50 peaks

per 200 Da.

Protein identification

Launchpad 2.8 (Shimadzu) was used to submit the com-

bined MS and MS/MS data to the MASCOT protein search

engine v. 2.2 (http://www.matrixscience.com) for search-

ing against a publicly available A. mellifera protein

sequence database deposited in the National Center for

Biotechnology Information non-redundant protein database

(NCBInr) (http://blast.ncbi.nlm.nih.gov/Blast.cgi). The

following search parameters were used: no restrictions on

protein molecular weight, one tryptic missed cleavage was

allowed, and peptide mass tolerances for the searches were

0.5 Da for MS spectra and 0.3 Da for MS/MS spectra.

Iodoacetamide derivatized cysteine and oxidation of

methionine were specified in MASCOT as fixed and vari-

able modifications, respectively. Scaffold (v. 2.04.00,

Proteome Software Inc., Portland, OR) was used to validate

MS/MS-based peptide and protein identifications. The

False Discovery Rate (FDR) assessment was estimated

using the original decoy FDR approach from Mascot; a

separate decoy database was generated from the protein

sequence database with the decoy.pl Perl script provided by

Matrix Science. This script randomizes each entry, while

retaining the average amino acid composition and length of

the entries. The maximum protein and peptide FDR rates

were set to 0.01 and the maximum peptide FDR to 0.1;

FPR was calculated based on the Mascot score. Proteins

were considered identified when at least two peptides were

assigned to the respective sequence. All peptides and

proteins were also confirmed by manual examination of the

spectra.

Results

To investigate the effects of a sub-lethal doses of fipronil

on the protein expression profile of the honeybee brain,

proteomic profiles of this organ from newly emerged and

aged workers were compared and analyzed without (con-

trol) and with exposure to a dose of 10 pg fipronil/day for

5 days; under these conditions no death of bees was

observed. The electrophoretic patterns of samples from

three 2-DE gels were analyzed for each experimental

condition. The patterns were highly similar, as reflected by

the high scatter plot correlation coefficient ([98 %)

between the three gels. Image analysis revealed 96 protein

spots in newly emerged brains from the control group and

the fipronil-treated group (Fig. 1a, b respectively), while

116 protein spots were observed in the aged worker brains

of both groups in the 14.74–67.90 kDa MW range and the

3.79–9.93 pI range (Fig. 1c, d respectively).

Considering that no apparent qualitative differences

were observed in the triplicates of 2-DE gels, and in order

to accurately compare the quantities of each protein in each

spot of the triplicate from each group of newly emerged

bees, or in the group of aged bees, the volume of each

protein in the spot was normalized and automatically cal-

culated by the software as a single spot volume. Differ-

ential expression analysis was performed using the

algorithm ImageMaster 2-DE Platinum 7.0 for the gels that

were representative of each experimental condition. This

analysis revealed a total of 25 proteins spots that were

differentially expressed in all the experiments. From these,

20 proteins spots characterized the proteomic differences

between the brains of the control and fipronil-treated

groups of newly emerged honeybees (Fig. 1a vs. b), and 15

differentially expressed proteins characterized the differ-

ences in the brains of aged honeybees in the presence and

absence of fipronil (Fig. 1c vs. d). Thus, these 25 protein

spots were assigned as numbers from 1 to 25 (Fig. 1), and

the corresponding proteins were identified with the protein

engine search MASCOT, with scores from 61 to 449, with

sequence coverages from 5 to 32 %.

T. C. Roat et al.

123

From the 25 selected proteins spots (Table 1), ten were

common to the newly emerged and aged honeybee work-

ers: calreticulin (spot 1), enolase-like protein (spot 4),

transcription factor IIIA (spot 5), aldolase-like protein (spot

8), glyceraldehyde-3-phosphate dehydrogenase (spot 11),

14-3-3 protein epsilon (spot 13), putative dual specificity

tyrosine-phosphorylation-regulated kinase 3 (spot 15),

protein lethal essential for life (spot 16), pyruvate dehy-

drogenase E (spot 18), and fatty acid binding protein (spot

20). Among these proteins, six were up-regulated

(1.63–2.92 fold) in the group that received a sub-lethal

dose of fipronil (calreticulin, enolase-like protein, aldolase-

like protein, glyceraldehyde-3-phosphate dehydrogenase,

protein lethal essential for life, and pyruvate dehydroge-

nase E), while four proteins were down-regulated (0.41 to

0.46-fold) by fipronil (transcription factor IIIA, 14-3-3

protein epsilon, tyrosine kinase 3, and fatty acid binding

protein).

Ten out of the 25 selected proteins were characteristic of

the brains from newly emerged honeybee workers: actin 87

E (spot 2), arginine kinase (spot 3), enolase-like protein

(spot 6), glutamine synthetase (GS) (spot 7), aldolase-like

protein (spot 9), glyceraldehyde-3-phosphate dehydroge-

nase (spot 10), annexin IX (spot 12), 14-3-3 Leonardo

protein (spot 14), thioredoxin peroxidase (spot 17), and

vascular endothelial growth factor receptor 3-like (VEGF-

3) (spot 19). Among these proteins, six were up-regulated

(1.60 to 2.16-fold) in the presence of fipronil (enolase-like

protein, GS, aldolase-like, glyceraldehyde-3-phosphate

dehydrogenase, thioredoxin peroxidase, and VEGF-3-like),

whereas four were down-regulated (0.35 to 0.48-fold) by

effect of fipronil (actin-87E, arginine kinase, annexin IX,

and 14-3-3 Leonardo protein). Five of the selected proteins

were characteristic aged honeybee worker brain proteins

that were up-regulated (1.53 to 1.99-fold) in the presence

of fipronil: defensin precursor (spot 21), neuroligin 2 (spot

22), protein lethal essential for life (spot 23), protein kinase

(spot 24), and amyloid precursor protein (spot 25).

Discussion

The present investigation was carried out to evaluate the

effects of honeybee exposure to a sub-lethal dose (1/100 of

LD50/day/bee) of the insecticide fipronil for 5 days and to

determine the proteomic profile of the honeybee brain. Up

to now, no previous studies have been performed to

investigate some potential biochemical damage to the

Fig. 1 Representative 2-DE

gels from the newly emerged

(a–b) and aged (c–d) honeybee

brain. a Control sample, 5 days

and b fipronil sample, 5 days.

c Control sample, 25 days and

d fipronil sample, 25 days

Brain proteome of Africanized honeybees

123

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Brain proteome of Africanized honeybees

123

honeybee’s nervous system that can be caused by fipronil.

Two groups of workers were used, i.e., 5 day old (newly

emerged workers) and 25 day old (aged workers), and their

exposure to the insecticide was biochemically character-

ized by the differential expression of 25 proteins in their

brains. These proteins can be organized into three groups:

i) ten proteins that are common to the newly emerged and

aged honeybee workers; ii) ten proteins that are charac-

teristic of the newly emerged honeybee workers; and iii)

five proteins that are typical of the aged honeybee workers.

The structural and functional features of these groups

are described as follows

i) The group of ten proteins that are common to the newly

emerged and aged honeybee workers was composed of six

proteins that are up-regulated by the effect of fipronil on

the brain (calreticulin, enolase-like protein, aldolase-like

protein, glyceraldehyde-3-phosphate dehydrogenase, pro-

tein lethal essential for life, and pyruvate dehydrogenase

E), and four proteins that are down-regulated in the hon-

eybee brain due to the action of fipronil (transcription

factor IIIA, 14-3-3 protein epsilon, tyrosine kinase 3, and

fatty acid binding protein).

Among the up-regulated proteins, the enzymes enolase,

aldolase, and glyceraldehyde-3-phosphate dehydrogenase,

are directly involved in glycolysis, while pyruvate dehy-

drogenase links glycolysis to the acid citric cycle (within

the mitochondria) releasing energy via NADH; thus, the

over-expression of these proteins is likely related to the

requirement for metabolic energy in the neurons of hon-

eybees that are effected by fipronil. According to Vidau

et al. (2009), in addition to being neurotoxic, fipronil also

exerts cytotoxic action on other tissues, acting on common

cellular targets that are components of mitochondrial

energy metabolism. The ability of fipronil to induce cyto-

toxicity is related to its ability to prevent the production of

ATP by mitochondrial disruption (Vidau et al. 2011).

The two other up-regulated proteins, calreticulin and

lethal protein (Efl21) are most likely related to neuronal

protection against the action of fipronil. Calreticulin binds

to misfolded proteins, preventing them from being expor-

ted from the endoplasmic reticulum to the Golgi apparatus

(Michalak et al. 2012), whereas the lethal protein Efl21 is

related to the heat shock protein family, playing the role of

a chaperonin (Kurzik-Dumke and Lohmann 1995).

According to Ki et al. (2012), oxidative stress plays a

central role in fipronil-induced cytotoxicity. Thus, both

proteins are likely related to neuronal protection against

protein misfolding that is caused by fipronil-induced

chemical stress in the neuronal soma.

This group also contains four proteins that are down-

regulated in the honeybee brain due to the action of

fipronil: transcription factor IIIA, 14-3-3 protein epsilon,

tyrosine kinase 3, and fatty acid binding protein. The

transcriptional repressor factor IIIA plays a role in regu-

lating the G2/M transition of the cell cycle (access code:

XP_001121169) 0.14-3-3 protein epsilon is involved in

neural signaling, neuronal development and neuroprotec-

tion and are known to promote cell survival by inhibiting

apoptotic processes via multiple mechanisms (Foote and

Zho 2012). Tyrosine kinase-3 plays a significant role in a

signaling pathway that regulates cell proliferation and is

involved with brain development (Galceran et al. 2004).

Fatty acid-binding protein (FABP) belongs to a family of

small proteins that act as cytoplasmic fatty acid trans-

porters, transferring fatty acids to membranes, and medi-

ating the effects of fatty acids on gene expression in

different tissues, including the brain. Thus, the group of

four down-regulated proteins is likely to be related to

neuronal development and neuroprotection; the fipronil-

induced decreased level of expression of these proteins in

the honeybee brain could be associated with neuronal

damage in these bees.

ii) The group of ten proteins that are characteristic of the

newly emerged honeybee workers was composed of six

proteins that are up-regulated by the action of fipronil

(enolase, GS, aldolase, glyceraldehyde-3-phosphate dehy-

drogenase, thioredoxin peroxidase, and VEGF-3-like pro-

tein) and four down-regulated proteins (actin-87E, arginine

kinase, annexin IX, and 14-3-3 Leonardo protein).

The overexpression of enolase, aldolase, and glycer-

aldehyde-3-phosphate dehydrogenase clearly indicates an

activation of glycolysis that is caused by the action of

fipronil in the honeybee brain, which was also observed in

the group of proteins that are common to the newly

emerged and aged honeybee workers, as discussed in the

above paragraphs. Because the proteomic identification of

these enzymes was based on the same accession numbers,

although the proteins had different MW and pI values

(Table 1), it is likely that they are different forms/isoforms

of the same proteins.

Glutamine synthetase (GS) acts in the brain by regu-

lating the levels of glutamate and detoxifying the neurons

by removing excess ammonia (Suarez et al. 2012); the up-

regulation of GS indicates that the honeybee’s brain may

be over-producing glutamate and/or ammonia as a result of

the action of fipronil. The enzyme thioredoxin peroxidase

is also related to protection against oxidative stress (Santos

et al. 2010); thus, the up-regulation of this enzyme is also

likely to be related to the protection of the honeybee’s

brain against the metabolic toxicity caused by the action of

fipronil. The VEGF-3-like protein stimulates vasculogen-

esis and angiogenesis to restore oxygen supply to the tis-

sues with inadequate blood circulation (Shin et al. 2010);

thus, the over-expression of this protein suggests that the

T. C. Roat et al.

123

fipronil-affected honeybee brain could have suffered from

some damage that restricts hemolymph circulation,

requiring the over-production of VEGF-3 to protect the

brain against possible ischemia that is caused by fipronil.

Additionally, four other proteins were down-regulated in

the newly emerged honeybee brains that were affected by

fipronil: actin-87E, 14-3-3 Leonardo protein, arginine

kinase, and annexin IX. It was recently reported in hon-

eybees that the depolymerization of actin in the brain is

related to the enhancement of the associative olfactory

memory and the increase of long-term memory retention in

insects (Ganeshina et al. 2012). The 14-3-3 Leonardo

protein is involved in forming olfactory learning and

memory structures that are necessary for scent recognition

and learning abilities (Skoulakis and Davis 1996). The

down-regulation of these two proteins in the brains of

newly emerged bees that are exposed to fipronil suggests

that the low level of expression of these proteins could be

related to the impairment of learning and/or decreased

memory. The arginine kinases (AK) from the honeybee

brain belongs to the family of ATP:guanidino phospho-

transferases. They are important components of the energy

shuttle that delivers ATP generated by the mitochondria to

biochemical processes of the visual system that require

high energy, such as pigment regeneration in the retina

(Kucharski and Maleszka 1998). The down-regulation of

this protein could contribute to the occurrence of visual

problems in worker honeybees that are affected by fipronil.

The annexins are a large family of calcium- and

phospholipid-binding proteins that regulate cellular growth

and a series of different signal transduction pathways

(Gerke and Moss 2002). However, currently, there are no

known processes that involve annexin IX in animal brains.

iii) The group of five proteins that are up-regulated in

the brains of aged honeybee workers is composed of a

defensin precursor, neuroligin 2, protein lethal essential for

the life (Efl21), a protein kinase, and amyloid precursor

protein. These proteins could potentially be related to the

age differences that characterize the changes of the work-

ers’ function in the nest, according to the insect’s age.

Defensins are non-specific proteins that mediate the innate

immune defense of insects and protect tissue surfaces from

invading pathogens (Bulet and Stocklin 2005). Thus, the

up-regulation of a defensin could indicate that fipronil

affects the honeybee immune system and thus the honey-

bee became more susceptible to microorganism infection in

the presence of fipronil. Neuroligins are a family of pro-

teins that mediate the synapse formation between neurons,

which affects the signaling of neural networks; in mam-

mals, structural alterations of these proteins are involved in

the occurrence of cognitive disorders (Sudhof 2008). The

overexpression of neuroligin 2 in the brains of aged worker

bees that are exposed to fipronil may indicate that the

insecticide could have affected synapse formation in the

bee’s neurons. The lethal protein essential for life (Efl21) is

related to a family of small heat shock proteins that play

the role of chaperonins (Kurzik-Dumke and Lohmann

1995). The up-regulation of this protein suggests that some

important neuronal proteins in these insects are misfolded,

which requires the action of the chaperonin to protect the

neural proteins against denaturation that can result in loss

of biological activity. The cAMP-dependent protein kinase

catalytic subunit mediates cAMP-dependent signaling that

is triggered by receptor binding to GPCRs. Its activation

regulates a series of cellular processes such as cell prolif-

eration, cell cycle, differentiation and regulation of

microtubule dynamics, and it is important for brain plas-

ticity (Qi et al. 1996). The overexpression of this protein

could be related to neuronal proliferation and/or differen-

tiation that are induced by fipronil in the brains of aged

honeybee workers. The amyloid protein is an important

component of senile plaques in animal brain; the overex-

pression of this protein is strongly associated with neuronal

degeneration (Murakami et al. 1998). Therefore, the up-

regulation of the proteins discussed above indicates that

fipronil rendered the aged bees more susceptible to path-

ogenic infection, and likely damaged the formation of

synapses, protein misfolding and neuronal degeneration.

The proteins identified in the present investigation were

already reported in previous studies of honeybee brain

proteomics; as exemple Li et al. (2010) and Hernandez et al.

(2012) reported the presence of calreticulin, actin-87E,

arginine kinase, enolase, GS, aldolase, glyceraldehyde-3-

phosphate, annexin, 13-3-3 protein epsilon, Leonardo pro-

tein, protein lethal, thioredoxin peroxidase, pyruvate

dehydrogenase, fatty acid binding protein, cAMP-depen-

dent protein kinase and amyloid protein precursor-like in

the brains of honeybees submitted to differential protein

expression in the caste differentiation studies and compar-

ative proteomic analysis between nurse and forager subc-

astes, respectively. The cAMP-dependent protein kinase,

for example, contributes to the induction of a long-term

memory formation in honeybees (Fiala et al. 1999). Garcia

et al. (2009) also reported the proteins such as calreticulin,

GS and protein lethal in the honeybee brains upon onto-

genetic and behavioral development. Neuroligins have been

previously reported in studies of the honeybee brain syn-

aptic development, since this family of proteins is involved

in synapse formation in A. mellifera, and thus it is used as a

model for comparison with synaptic development in ver-

tebrates (Biswas et al. 2008). However, in none of these

studies honeybee brain proteomics was used as strategy to

investigate the toxicological effects of an insecticide in the

nervous system, as done in the present study.

Figure 2 summarizes the interpretation of the effects of

honeybee exposure to a sub-lethal dose of fipronil in the

Brain proteome of Africanized honeybees

123

brains of newly emerged and aged honeybees. The effects

were detected as differential protein expression changes by

comparing the control versus fipronil-treated groups of 5

and 25 day old honeybees. Considering the results that

were obtained for both ages, fipronil initially causes

structural damage that requires the activation of the

anaerobic phase of glycolysis to supply energy to repair the

damage. The presence of fipronil causes chemical stress in

the brain, which leads to the increased expression of pro-

teins that are related to neuronal protection; fipronil also

renders the aged bees more susceptible to pathogenic

infection. The chemical stress in neurons causes protein

misfolding, which results in apoptosis, brain ischemia, and

possibly visual problems that result from impaired pigment

regeneration after oxidation/reduction. These processes are

followed by damage to synapse formation and neurode-

generation, which could result in memory and learning

impairment in aged honeybee workers.

Conclusions

Honeybees have fewer xenobiotic detoxifying enzymes

than other non-social insects, which render honeybees

highly sensitive to a series of insecticides, when compared

to Drosophila melanogaster and Anopheles gambiae

Fig. 2 Flow chart indicating

the interpretation of the possible

effects of a sub-lethal dose of

fipronil on the brains of newly

emerged and aged honeybees.

The actions resulting from the

up-regulation ( ) or down-

regulation ( ) of proteins are

assigned outside of the dashed

square blocks, while the

identified proteins that are

differentially expressed are

indicated within the

corresponding action block

T. C. Roat et al.

123

(Claudianos et al. 2006). This aspect makes the honeybee

an interesting living model for testing novel pesticides and/

or xenobiotics. Recently, a review that was conducted by

the European Food Safety Authority stated that phenylpy-

razoles pesticides present a high risk to the survival of

honeybees (European Food Safety Authority 2013). How-

ever, the degree of risk that is presented by this class of

compounds requires further investigation. To obtain more

details regarding the effect of fipronil on the honeybee

brain, the present investigation focused on a toxicoprote-

omic analysis of newly emerged and aged honeybee

workers that were subjected to a sub-lethal dose of fipronil

for 5 days.

This study demonstrated the differential brain protein

expression profile of the Africanized A. mellifera, an insect

that is not targeted by fipronil. Our results suggest that low

doses of fipronil (sub-lethal doses) may cause changes in

the expression pattern of certain proteins that are involved

in pathogen susceptibility, neuronal chemical stress, neu-

ronal protein misfolding, a higher occurrence of apoptosis,

ischemia, visual problems, damage to synapse formation,

and brain degeneration, that could result in memory and

learning impairment. Taken together, these actions can

induce several types of injuries to honeybee physiology.

Besides, this study demonstrates that honeybees chroni-

cally exposed to sublethal doses of fipronil could have a

reduction of their life span, showing the importance of

establishing more accurate methods for risk assessment of

pesticides on bees as discussed by Decourtye et al. (2013).

Acknowledgments This work was supported by Grants from FA-

PESP (Proc. 2008/05018-7 and 2012/13370-8), BIOprospecTA/FA-

PESP program (Proc. 11/51684-1), CNPq and CAPES. M.S.P. and

O.M. are researchers from the National Research Council of Brazil-

CNPq. T.C.R., L.D.S. and K.S.S. were Postdoctoral fellows from

FAPESP, and J.R.A.S.P is Ph.D. student fellow from FAPESP.

Conflict of interest The authors declare that they have no conflict

of interest.

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