Journal of Insect Physiology xxx (2009) xxx–xxx

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Identification of major royal jelly proteins in the brain of the honeybeeApis mellifera

Leonardo Gomes Peixoto a, Luciana Karen Calabria a, Liudy Garcia b,c, Fausto Emılio Capparelli a,Luiz Ricardo Goulart a, Marcelo Valle de Sousa c, Foued Salmen Espindola a,*a Instituto de Genetica e Bioquımica, Universidade Federal de Uberlandia, 38400-902 Uberlandia/MG, Brazilb Centro de Aplicaciones Tecnologicas y Desarrollo Nuclear, Ciudad Habana, Cubac Instituto de Ciencias Biologicas, Universidade de Brasılia, 70910-900 Brasılia/DF, Brazil

A R T I C L E I N F O

Article history:

Received 17 September 2007

Received in revised form 5 May 2009

Accepted 7 May 2009

Keywords:

Major royal jelly protein

Honeybee

Brain

Hypopharyngeal gland

Royal jelly

A B S T R A C T

The consumption of royal jelly (RJ) determines the differences between castes and behavioral

development in the honeybee Apis mellifera. However, it is not known whether the proteins of RJ are

related to these differences, or which proteins are responsible for the changes. To understand the

functions of RJ proteins that are present in other tissues of the bee, in addition to hypopharyngeal gland,

we used a polyclonal antibody anti-MRJP1 to investigate the presence of this protein in nervous system

of honeybee. This study showed the presence of three polypeptides (p57, p70 and p128) in specific

tissues of bee brain. Mushroom body, optic lobe and antennal lobe neuropils all contained proteins

recognized by anti-MRJP1. Proteomic analysis showed that the three polypeptides are correlated with

proteins of the MRJP family. p57 is correlated with MRJP1, p70 with MRJP3, while p128 may be an

oligomeric form or a new polypeptide. Immunostaining of the brain and hypopharyngeal gland revealed

differential expression of MRJPs in various brain regions and in different honeybee castes and subcastes.

The identification and localization of these MRJPs contribute to the elucidation of the biological roles of

this protein family.

� 2009 Elsevier Ltd. All rights reserved.

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1. Introduction

The fate of an adult honeybee is determined at the larval stage,as its caste, development, phenotypical plasticity and behavior arerelated to an early consumption of royal jelly (O’Shea and Schaffer,1985; Tublitz et al., 1991; Nassel, 1993; Schmitzova et al., 1998).Royal jelly (RJ) is secreted by hypopharyngeal glands of nurse beesand constitutes a nutritious complex, in which the major royal jellyproteins (MRJPs) are the most abundant proteins (Lensky andRakover, 1983; Knecht and Kaatz, 1990). The MRJP genes expressevolutionarily unique proteins, wich present homology with genespreserved in dipterans (Drosophila melanogaster and Anopheles

gambiae), known as the Yellow–MRJP protein family. Severalstudies have been published using proteomic analyses of royal jellyas well as of the MRJPs (Li et al., 2007; Furusawa et al., 2008; Quet al., 2008; Li et al., 2008). Besides the expression these proteins inhypopharyngeal glands, MRJP1 (mRNA) (Kucharski et al., 1998)and MRJP2 (cDNA) (Kucharski and Maleszka, 2002) have also beenfound in the brain of worker bees (Garcia et al., 2009).

* Corresponding author. Tel.: +55 34 3218 2477; fax: +55 34 3218 2203.

E-mail address: foued@ufu.br (F.S. Espindola).

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0022-1910/$ – see front matter � 2009 Elsevier Ltd. All rights reserved.

doi:10.1016/j.jinsphys.2009.05.005

The molecular and functional features of MRJPs in honeybeetissues outside the hypopharyngeal gland are almost unknown.However, studies on the transcriptome of Apis mellifera L.(Whitfield et al., 2002; Nunes et al., 2004) revealed contigs ofMRJPs, principally MRJP1, in libraries of the brain of this honeybee.The complete sequencing of the honeybee (A. mellifera) genomehas revealed additional genes in the MRJP family. Nine of thesegenes have apparently evowed from one ancestral yellow gene, andnow are functionally related to queen and brood nursingsuggesting achievement of new functions during the evolutionof sociality (The Honeybee Genome Sequencing Consortium,2006). Moreover, microarray studies that compared young adultfunctionally sterile (wild-type), bees with a stain in wich workerswere reproductivelly active, found that two genes (MRJP2 and 7)were expressed at significantly higher levels in the heads of wild-type bees (Thompson et al., 2006). Given the assumed role RJ playsin modulating caste hierarchy, the possibility that RJ proteinsmight be endogenous participants in brain activities seems quiteinteresting (Garcia et al., 2009).

The aim of this study was to apply immunopurified antibodiesagainst MRJP1 to investigate the distribution of MRJP1 in the headand brain of the A. mellifera castes and subcastes, comparing withthree others species of hymenopterans. This approach allowed theidentification of 57 and 70 kDa polypeptides, which are related to

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MRJP1 and MRJP3, respectively, as well as an additional 128 kDapolypeptide which may correspond to an oligomeric form, or a notyet described MRJP. We also present immunohistochemistry datashowing MRJP1 localization in different areas of the brain of thenurse and worker honeybees.

2. Materials and methods

2.1. Biological specimens

Bees (A. mellifera, Mellipona scutellaris, Scaptotrigona postica)and ants (Camponotus atriceps) used in the biochemical assay werecollected at the University’s experimental garden. To distinguishbetween honeybee nurse and forager workers anatomical features,i.e., coat condition, damage to the wings, and development of thehypopharyngeal gland were considered. Rabbits and mice used inthe production of the polyclonal antibodies for this study werekept at the University’s animal facilities under supervision of theAnimal Experiments Review Board at our University.

2.2. Preparation of the samples

Head homogenates were prepared with aid of a hand blender incold homogenization buffer (40 mM Hepes at pH 7.7 containing10 mM EDTA, 2 mM EGTA, 5 mM ATP, 2 mM DTT, 1 mMbenzamidine, 0.1 mM aprotinin and 0.5 mM PMSF). The samebuffer was also used to prepare brain and hypopharyngeal glandhomogenates (n = 30). The supernatant fraction was obtained bycentrifugation at 40,000 � g for 40 min at 4 8C. Total proteinconcentration (Bradford, 1976) of these samples was determinedprior to SDS-PAGE and Western blotting analysis as describedbelow. Alternatively, A. mellifera brain regions were dissected(n = 30) to obtain the optic lobe, antennal lobe, peduncle, andmushroom body neuropils. Each sample of these neuropils wasalso prepared using same buffer and centrifugation as describedabove.

2.3. Production and purification of polyclonal antibodies

Rabbits and mice were inoculated with water soluble proteins(WSP), isolated from RJ homogenates. The RJ was provided byClaudio Franco Lemos (Apiarios Girassol Ltda of Uberlandia/MG,Brazil). To prepare WSP, the royal jelly sample was homogenized inPBS pH 8.0 containing 20 mM EDTA, centrifuged at 10,000 � g,10 min at 4 8C, supernatant fraction was dialyzed in Tris–HClbuffer pH 8.0 containing 0.5 mM EDTA, after dialysis the finalsupernatant was obtained by another centrifugation step. Tochallenge animals with WSP antigen samples of 500 mg/mL(rabbit) or 100 mg/mL (mouse) of a mixture of WSP and Freund’scomplete adjuvant (1:1), and subsequent 15-day reinforcement of250 mg/mL (rabbit) or 25 mg/mL (mouse) were employed forgeneration of polyclonal antibodies. Animals were bled three daysafter the last inoculation. Serum was obtained by centrifugation at3000 rpm for 3 min and stored at �80 8C. For purification ofpolyclonal antibodies for MRJP1 (anti-MRJP1), 2 mg/mL WSPfraction was separated in SDS-PAGE 5–22% (300 V, 2 h) andstained (Coomassie brilliant blue—R250, at 1 h). The bands ofinterest were excised and electrotransferred onto a nitrocellulosemembrane (300 V, overnight). Membranes were incubated in TBS-T (Tris–HCl pH 7.4, 0.5% Tween 20) containing 5% of dried milkovernight at room temperature, prior to incubation with dilutedserum at TBS (1:1) at 2 h. Subsequently membranes were washedthree times for 5 min with TBS-T and bound antibodies (anti-MRJP1) were eluted with 1.4% triethylamine (1 min) andtransferred to vials containing 1 M Tris–HCl pH 8.5 at roomtemperature. After elution antibodies were dialyzed in TBS

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containing 0.1% sodium azide, quantified (Bradford’s method)and stored at �20 8C. The specificity of the antibodies wasdetermined by ELISA.

2.4. Enzyme-linked immunosorbent assay (ELISA)

Micro-titration plates were coated overnight at 4 8C with 50mL/well of WSP at a 10 mg/mL dilution in phosphate-bufferedsaline (PBS) and then blocked with 1% bovine serum albumin (BSA)in PBS. Subsequently, 50 mL of anti-MRJP1, serially diluted (1/100to 1/512,000) with 0.1% BSA in PBS were incubated in the coatedwells for 2 h at room temperature. The binding of anti-MRJP1 withantigen was visualized by using 50 mL of an anti-rabbit IgGconjugated to peroxidase at 0.4 mg/mL. The color reaction wasinitiated by adding 50 mL/well of orthophenylene diaminechromogen at 0.4 mg/mL in substrate buffer (citric acid 0.1 M,sodium acetate 0.1 M, pH 5.4, in H2O2 at 0.33% final concentration).The reaction was stopped after 15 min by adding 25 mL of 4 NH2SO4 to the wells. Optical density was measured at 492 nm withan ELISA plate reader. Two rinses with PBS were performedbetween each step described above.

2.5. Immunoprecipitation of MRJPs from nervous system

Protein extracts were incubated overnight at 4 8C with20 mg/mL of anti-MRJP1 or non-immune rabbit IgG and150 mM NaCl 0.2% Triton X-100. Protein A Sepharose beads(2 mg/mL) were added and the suspensions were gently mixedon a rocking platform for 30 min at 4 8C. The immunoprecipita-tion fractions were then collected by centrifugation at 14,000 �g for 10 min. Supernatants were kept for further analysis, andpellets were washed once with TBS supplemented with 0.5 MNaCl and then six times in TBS without salt addition. Finally, thepellets and the initial supernatants were diluted in SDS samplebuffer, boiled at 100 8C for 5 min and analyzed for MRJP1content by immunoblot using rabbit anti-MRJP1 and mouseanti-MRJP1 (negative control).

2.6. Immunohistochemical analysis of MRJPs in the bee brain

Brains were dissected and fixed in 4% paraformaldehyde, asdescribed by McLean and Nakane (1974). Fixed tissues weredehydrated in ethanol, cleared in xylene and embedded inparaffin. Five micrometer sections were pretreated with 4 mMcitrate buffer (pH 6.0), containing 0.025% Tween 20, in amicrowave for 5 min. Thereafter, sections were incubated withMRJP1 antibody for 16 h followed by incubation with theNovoLinkTM Max Polymer Detection System-Post Primary Block(Novocastra Laboratories Ltd, Newcastle Upon Tyne, UK). Afterthree washes with Tris buffer with Tween, the sections wereincubated with the NovoLink polymer for 30 min at 37 8C.Chromogen development was performed with 3,30-diaminoben-zidine, and the material was counterstained with Harrishematoxylin, dehydrated, and mounted with Permount andanalyzed using a light microscope (Zeiss Axiolab, MC80911258, Germany). Negative controls consisted of the omissionof the primary antibody in the reaction.

2.7. MALDI-TOF MS and database search

Protein fragments corresponding to the previously immuno-precipitated MRJP1 were excised and digested for furtheridentification by peptide mass fingerprinting (PMF). The methodemployed was based on the reduction of proteins with DTT,alkylation with iodoacetamide and digestion with trypsin. Theyielded fragments were submitted to microchromatography using

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C18 Zip Tips (Millipore, Billerica, USA), followed by matrix assistedlaser desorption ionization-time of flight (MALDI-TOF) in a BrukerReflex IV mass spectrometer. Known trypsin autolysis peaks andkeratin contaminants were removed. Mascot software, assumingp < 0.05 (Perkins et al., 1999) was used to search the PMF-identified proteins in the NCBI nr database. No restrictions weremade with respect to the molecular mass or the taxonomy of theproteins. The fragment mass tolerance was <0.2 Da for MH+

monoisotopic data. The protein sequence identified by MASCOTwas aligned with other MRJPs sequences using CLUSTALW (http://www.ebi.ac.uk/Tools/clustalw2/index.html).

2.8. Analysis of MRJP1 secondary structure

A 96-well microtitter plate was coated with 100 mg/mL of anti-MRJP1 in bicarbonate–carbonate buffer at pH 8.6 (BCB) overnightat 4 8C. After coating solution was poured off each well was filledwith BCB and 6.5 mg/mL bovine serum albumin (BSA) for 1 h at4 8C. Subsequently, 100 mL of TBS-T with 4 � 1010 phage wereadded (Ph, D – 12 mer – New England Biolabs) and incubated atroom temperature for 1 h. Bound phages were eluted at roomtemperature for 10 min with 100 mL of 200 mM glycine–HCl pH2.2, 1 mg/mL BSA, and immediately neutralized with 1 M Tris–HCl,pH 9.1. E. coli cells (ER2738 strain) were then infected with 100 mLof eluted during the rounds of phage amplification (inputinoculum).

Colonies deriving from the fourth cycle of biopanning (non-amplified eluted) were used for the amplification process in deep-well plates. After amplification, clones were tested by ELISA fortheir ability to bind to serum-purified antibodies. Positive cloneswere sequenced according to a cut-off value of 0.127 (averagereadings of the negative control).

The nucleic acid sequences of positive clones were translatedusing DNA2PRO12 software (http://relic.bio.anl.gov/dna2-pro12.aspx). Amino acid frequency was calculated with AAFREQSsoftware (http://relic.bio.anl.gov/aafreqs.aspx). To further inves-tigate the importance of the identified residues or motifs, asecondary structure prediction of the MRJP1 protein was madeusing the PredictProtein server (http://www.predictprotein.org).For selected peptides, biochemical properties, such as antigenicity,surface exposure, and hydrophilicity, were assessed throughDnaStar-Lasergene software (subprogram Protean). Every peptidehomology with MRJP1 was tested with MATCH (http://relic.

Fig. 1. Immunoprecipitation of p57 polypeptide from worker bee brain homogenate usi

PAGE (a) and Western blotting (b) using anti-MRJP1 produced at mouse as positive cont

fold more concentrated than IgG immune (anti-MRJP1). Numbers at left indicate the s

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bio.anl.gov/match.aspx) and CLUSTALW (18.1) (http://services.-bioasp.nl/blast/cgi-bin/clustal.cg) softwares.

3. Results

3.1. Identification of brain MRJP1

Immunoprecipitation of A. mellifera brain homogenate withrabbit anti-MRJP1 allowed partial purification of a p57 polypeptide(Fig. 1a). Controls were carried out in presence of non-immune IgG.Unspecific bands failed to cross-react with mouse anti-MRJP1 andwere discarded from our analysis. These results confirm thespecificity of p57 immunoprecipitation (Fig. 1b). For furtheridentification the p57 band was excised and processed for MALDI-TOF mass spectrometry measurements. Analysis revealed fourteenpeptides with mass m/z ranging from 582.3367 to 2854.6471, inaccordance to the predicted mass of in silico digested MRJP1(Fig. 2a). Searches using these peptides mass in the MASCOTprotein database covered 39% of the A. mellifera MRJP1 sequence(Fig. 2b). The MRJP1 identified in this search has accession number58585098, estimated molecular mass (mw) of 49.311 kDa andisoelectric point (pI) 5.1 (Fig. 2c). Protein multi alignment studiesof the MRJP1 with the MRJP2 and MRJP3 expressed in the A.

mellifera nervous system presented 44% of identity. Furthermore,MRJP1 has more than 44% identity with all other major RJ proteins(Fig. 3a).

Phage display and bioinformatic analyses showed that thelinear sequence of the brain MRJP1 exhibit seven hydrophilicregions with high identity to other MRJPs. Although hydrophilicregions in the linear sequences of MRJP proteins (as showed bythe phage display data) were recognized by anti-MRJP1 (Fig. 3band c), only the MRJP1 native form was immunoprecipitated bythis antibody. MRJP multiple alignments showed similarity withYellow proteins of the D. melanogaster. In order to predict thefunctions of the honeybee brain MRJP1 we compared itssequence with D. melanogaster paralogues using FatiGO analysisof the MRJP1 sequence based on cellular content, molecularfunction and biological process (Al-Shahrour et al., 2004, http://fatigo.bioinfo.cnio.es/). This ontology analysis suggests thatMRJP1 is probably located in intracellular organelles, potentiallybinds nucleic acids, and is potentially involved in morphogenesis,embryonic development, metabolism, cell differentiation and sexdetermination.

ng anti-MRJP1 antibody. Immunoprecipitated proteins (IP) were analyzed by SDS-

rol, in supernatant (S) and pellet (P) fractions. The IgG non-immune was loaded 10-

tandard molecular mass.

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Fig. 2. MALDI-TOF MS spectrum of the MRJP1 immunoprecipitated. (a) Spectrum of the peptide masses generated from the digested immunoprecipitated band (p57) showed

the m/z – mass to charge ratio – (x-axis) and the intensity of the molecular ions (y-axis). From the results of MS/MS analysis, peaks with asterisk were identified as MRJP1. The

peaks with k/t indicate keratin/trypsin. (b) MRJP1 sequence with access number 58585098 indicates the fourteen peptides of the spectrum (underlane/bold). (c) Table of the

main characteristics of the MRJP1 identified in database.

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3.2. Analysis of the expression of MRJPs in honeybee tissues

SDS-PAGE revealed differential protein content in headhomogenates of A. mellifera, M. scutellaris, S. postica and C.

atriceps (Fig. 4a). Remarkably, Western blots showed that only A.

mellifera homogenates contain bands reactive to MRJP1, asshown in Fig. 4. Honeybee workers presented three mainpolypeptides bands, termed p57, p70 and p128 while inhoneybee drones the p70 band was absent. We observed apredominance of p70 in the honeybee subcaste of nurse worker(Fig. 4b). The expression levels of each one of the immunor-eactive bands also seemed to be caste-specific, especially innurses, whose p57 levels were higher than in foragers (Fig. 4c).The anti-MRJP1 antibody also recognized all three polypeptidesin hypopharyngeal gland homogenates, regardless of caste.

Fig. 3. Phage display and bioinformatics analysis of the MRJPs immunodetected in the ho

six exons (rectangle), respectively. (b) Indices of antigenicity predicted for the recognized

program (Lasergene softwares). (c) Alignment and identity of the linear sequences of the

recognized by anti-MRJP1.

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However, p70 was clearly more expressed in brain samplesand p128 in hypopharyngeal glands samples (Fig. 4d). As theremaining insects failed to display any immunoreactions,regardless of antibody titration or span of incubation, furtherlocalization experiments were carried out only for A. mellifera.

Supernatant fractions of the homogenates from differentregions of A. mellifera brain such as optic lobe, antennal lobe,peduncles and mushroom body showed variable cross-reaction tothe three polypeptides described above with a predominance ofp57 in all regions (Fig. 4e). Immunolocalization of MRJP1 indicatedthat the protein is localized similarly in specific regions of thenurse and forager worker brain (Fig. 5). Kenyon cells were stronglyimmunoreactive to anti-MRJP1, represented by a brown coloring,as well as the calyx. On the other hand, anti-MRJP1 strongly stainedthe non-compacted Kenyon cells (Fig. 5b and c). In the optic lobe,

neybee brain. (a) The MRJP1, MRJP2 and MRJP3 genes showed five introns (lane) and

regions of the MRJP1 with the antibody anti-MRJP1 in accordance with the Protean

se MRJPs. Boxes indicate the regions in the linear sequence of these proteins that are

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Fig. 4. Immunodetection of major polypeptides related with MRJPs in head, brain and hypopharyngeal gland of A. mellifera. Supernatant fractions of head, brain and

hypopharyngeal gland were analyzed by SDS-PAGE (left panel) and Western blotting (right panel) with immune detection of p57, p70 and p128 by anti-MRJP1. (a)

Supernatant fractions of head: C. atriceps (Ca), S. postica (Sp), M. scutellaris (Ms), A. mellifera (Am). (b and c) Supernatant fractions of A. mellifera head: drone (D), forager (F),

nurse (N). (d) Supernatant fractions from worker A. mellifera brain (br) and hypopharyngeal gland (hg). (e) Supernatant from different regions of A. mellifera brain: optic lobe

(ol), antennal lobe (al), peduncles (ped), mushroom body (mb). Numbers at left indicate the standard molecular mass.

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anti-MRJP1 diffusely stained cells in the retina, monopolar neuronsin the fenestrated layer and fibers in the lamina (Fig. 5f and g). Thiscould be distinguished from the pigmented cells of the retina thatwe seen in control section (Fig. 5h). In the antennal lobe, anti-

Fig. 5. Immunolocalization of MRJP1 in honeybee brain section. (b, c, f, g, j and k) labeled w

worker; (c, g and k) forager worker. (a–d) mushroom body; (e–h) optic lobe; (i–l) antenn

compacted Kenyon cells (nkc) and in the mushroom body calyx (cx). (f and g) labeling o

photomicrograph shows the pigmented layer (pc). (j–k) densely stained of anti-MRJP1 in

Kenyon cells, fenestrated layer and interneurons (inset box) show the MRJP1 localizati

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MRJP1 showed considerable staining among interneurons and inthe glomerulus. In detail was possible to observe anti-MRJP1staining in fibers and pericellular regions of the interneurons(Fig. 5j and k).

ith anti-MRJP1, bar: 55 mm; (a, d, e, h, i and l) control, bar: 55 mm. (b, f and j) nurse

al lobe. (b and c) anti-MRJP1 immunodetection in internal compacted (ikc) and non-

f anti-MRJP1 in retina (re), fenestrated layer (fl) and lamina fibers (la). In detail, the

antennal lobe interneurons (in) and glomerulus (gl). Detail of the non-compacted

on in fibers and pericellular regions, bar: 22 mm.

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4. Discussion

The major royal jelly proteins (MRJP1-5) of honeybee (A.

mellifera) are highly expressed and secreted by the hypophar-yngeal gland of nurse bees to feed the queen and the growinglarvae. We described in this study the immunodetection andimmunolocalization of MRJPs with affinity-purified polyclonalantibodies. These antibodies specifically recognized protein bandsin A. mellifera that were absent in S. postica, M. scutellaris and C.

atriceps. A caste-specific pattern of staining was observed for theMRJP1 antibody. Proteomic analysis revealed that the mainpolypeptides recognized by anti-MRJP1 in the A. mellifera headhomogenates are well correlated with the MRJPs.

The hypopharyngeal gland is localized in the bee head andproduces most of the royal jelly proteins (Patel et al., 1960;Klaudiny et al., 1994; Kubo et al., 1996). The protein profile of thisgland from Melipona and Apis bees reported by Silva de Moraeset al. (1996) showed four polypeptides with similar molecularmass. Also morphological analysis failed to clarify whether there ishypopharyngeal gland secretion in the larval food by S. postica bee(Costa and Cruz-Landim, 2002a,b). Considering the cross-reactivityof the anti-MRJP1 and the phylogenetic profile of this family, ourresults suggest that the proteins expressed in the head of the A.

mellifera differ considerably from those expressed in the heads ofM. scutellaris and S. postica.

We detected in the honeybee brain homogenate threepolypeptides, p51, p70 and p128, wich cross-reacted to anti-MRJP1 with some particularities regarding the caste and thesubcastes. The p57 polypeptide was immunoprecipitated from beebrain and identified as MRJP1. MRJP1 and MRJP3 were detectedand localized in different honeybee brain neuropils. In addition, ap128 detected polypeptide is suggested were to be a potentialMRJP1 dimer, or a new not yet reported MRJP-like protein.Remarkably of the p70 polypeptide was differentially expressed,the amount of this protein being considerably greater in nursebrain homogenates than in queen, drone or forager brainhomogenates. Nurse bee brains also presented higher expressionof p57 polypeptide than the others. This protein was immuno-precipitated suggesting that the anti-MRJP1 antibody recognizedthe native form of this brain protein since immunoprecipitationassay works with soluble protein instead of immobilized poly-peptides in the nitrocellulose filter. Mass spectrometry analysisidentified the immunoprecipitated protein as A. mellifera MRJP1,with a molecular mass of 49 kDa. Other studies have also reportedthat MRJP1 may present variant forms of 49–60 kDa (Santos et al.,2005).

Immunoreactive p70 in the nervous system and hypophar-yngeal gland of nurse and forager worker bees co-migrated in thesame range as MRJP3 in SDS-PAGE. The polypeptide p70 has alsobeen differentially detected in brain supernatants of queen,drone, nurse and forager worker bees. This finding indicates thatp70 is a good marker to distinguish forager from nurse workerbees. Furthermore, Albert et al. (1999) have previously shownthat MRJP3 exhibits length polymorphism. In fact, our observa-tions of p70 identified in distinct samples of honeybee brainvalidate this finding. Strikingly, our results suggest the existenceof a queen/nurse-specific isoform of MRJP3 suggesting a lengthpolymorphism of p70 in caste and subcaste.

The p128 band was immunodetected in the honeybee’s head,brain and gland tissues. Compared to the two polypeptidesdescribed earlier, p128 was less immunoreactive, except in head ofdrones and head of nurse worker. There were variations in itspresence in the brain for different castes and brain regions. Thispolypeptide seems to be more expressed in the hypopharyngealgland than in brain tissue; although we did not compare the glandtissues from different castes and subcastes, the data from the head

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homogenates suggest that nurse workers express more p128 in thehypopharyngeal gland. These MRJPs are secreted solely by thehypopharyngeal gland of A. mellifera and are evolutionarilyconserved, presenting a high identity among each other and,together with the Yellow proteins of D. melanogaster, theyconstitute a new protein family as described by Albert andKlaudiny (2004). The polypeptide p128 could be an oligomericform or an unknown MRJP, since this polypeptide was ignored in aprevious proteomic study of the hypopharyngeal gland ofAfricanized A. mellifera (Santos et al., 2005). In their study, Santoset al. (2005) identified 27 isoforms of MRJPs in hypopharyngealgland homogenates, which presented relative molecular massesfrom 49 to 87 kDa. Because p128 has a molecular mass unlike theones reported above and is immunoreactive with anti-MRJP1, evenunder the denaturated conditions of the immunoblot analysis, wesuggest that p128 is a new member of the A. mellifera MRJP familyand in future studies should be immunoprecipitated andsequenced.

Analogous antigenic regions among MRJPs found in phagedisplay and bioinformatic analyses show that MRJP family proteinscontain seven antigenic conserved regions which probablycorrespond to the epitopes for anti-MRJP1. The identities amongMRJP sequences have been suggested by hybridization assay andsequence alignment (Klaudiny et al., 1994; Schmitzova et al., 1998;Albert et al., 1999; Drapeau et al., 2006). This antibody recognizesmembers of the MRJP family in contiguous sequences in thedenatured protein. In addition, sequence analysis agree withavailable data showing that all of the MRJP family members havean N-terminal hydrophobic sequence that would function as acleavable signal peptide as well as a putative N-linked glycosyla-tion site, suggesting that these proteins are also secreted by the cell(Klaudiny et al., 1994; Drapeau et al., 2006).

Immunolocalization data showed intense staining of MRJP1 indifferent regions of the worker’s brain. In the mushroom bodies,MRJP1 was localized in both internal compacted and non-compacted Kenyon cells, and in the calyx. Western blots revealedthe presence of polypeptides corresponding to MRJP1 and MRJP3,as well as p128 in this region. In situ hybridization data onmushroom bodies indicate the presence of this MRJP in specificneurons, the intrinsic Kenyon cells, whose function is unknown(Kucharski et al., 1998). It is known that the mushroom bodies arecentral for visual and mechanosensory (primary), and olfactory(secondary) integration. They are constituted of densely packedparallel neurons called internal and external compact Kenyon cells.These neuropils constitute the brain centers for processing ofinformation and memory in the bee’s brain (Hammer and Menzel,1995; Capaldi et al., 1999).

Immunohistochemical analysis of the optic lobe showedMRJP1 to be present in the retina, fenestrated layer and fibersof the lamina. These neuropils are formed by axonal, pigmentcells, photoreceptors, and monopolar and laminar neurons(Nassel et al., 1985). The localization of MRJP1 in the optic lobesuggests multiple functions of these proteins in the bee nervoussystem. In the antennal lobe both antibodies localized MRJPs inthe interneurons and glomeruli. These primary neuropils receiveimpulses from chemosensory axons, and transmit this informa-tion to terminal olfactory receptors, which finally carry it to themushroom bodies and the alpha and beta lobes (Nassel et al.,1986; Kloppenburg, 1995; Galizia and Menzel, 2000; Menzel andGiurfa, 2001). Other studies reporting MRJP transcripts in thebrain consider that their function remains unknown (Kucharskiet al., 1998; Albert and Klaudiny, 2004). Notably, anti-MRJP1stained the fibers and pericellular regions of interneurons moreintensely than other regions in the honeybee brain, and theimmunolocalization was coincident between the workers, nurseand forager.

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Other studies have shown the presence of MRJP1 mRNA andMRJP2 cDNA in the honeybee brain (Kucharski et al., 1998;Kucharski and Maleszka, 2002; Albert and Klaudiny, 2004). Brainhistological sections stained with the anti-MRJP1 revealed specificdistributions of MRJP protein, confirming the results of immuno-blotting experiments and eliminating the possibility of cross-contamination by hypopharyngeal gland tissue in the brainsamples. These data indicate that the honeybee brain expressesMRJP family members, although their functions remain open tospeculation. FatiGO analysis also suggests their involvement indevelopmental processes in the A. mellifera nervous system.

In conclusion, the affinity-purified antibodies for the MRJPs inthe brain and hypopharyngeal gland of A. mellifera revealeddifferential expression of MRJPs regarding brain regions anddifferent castes and subcastes. The identification and localizationof these MRJPs in various honeybee brain regions may contributeto elucidate the biological role of this protein family.

Acknowledgements

This work was supported by grants from CNPq and FAPEMIG toFSE, LRG and MVS, CAPES fellowship to LGP and LKC, CNPqfellowship to FEC, and TWAS to LG. Also, we thank Pablo MarcoVeras Peixoto (New York University) for critically reading themanuscript.

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