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Apr 17, 2014 - and this increase was accompanied by an increase in the number of in-hive activities performed by the nurse bees, followed by a decreas...
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MALDI Imaging Analysis of Neuropeptides in the Africanized Honeybee (Apis mellifera) Brain: Effect of Ontogeny Marcel Pratavieira, Anally Ribeiro da Silva Menegasso, Ana Maria Caviquioli Garcia, Diego Simões dos Santos, Paulo Cesar Gomes, Osmar Malaspina, and Mario Sergio Palma* Institute of Biosciences, Department of Biology, Center of the Study of Social Insects, University of São Paulo State (UNESP), Avenue 24A 1515, Bela Vista, Rio Claro, 13506-900 SP, Brazil ABSTRACT: The occurrence and spatial distribution of the neuropeptides AmTRP-5 and AST-1 in the honeybee brain were monitored via MALDI spectral imaging according to the ontogeny of Africanized Apis mellifera. The levels of these peptides increased in the brains of 0−15 day old honeybees, and this increase was accompanied by an increase in the number of in-hive activities performed by the nurse bees, followed by a decrease in the period from 15 to 25 days of age, in which the workers began to perform activities outside the nest (guarding and foraging). The results obtained in the present investigation suggest that AmTRP-5 acts in the upper region of both pedunculi of young workers, possibly regulating the cell cleaning and brood capping activities. Meanwhile, the localized occurrence of AmTRP-5 and AST-1 in the antennal lobes, subesophageal ganglion, upper region of the medulla, both lobula, and α- and β-lobes of both brain hemispheres in 20 to 25 day old workers suggest that the action of both neuropeptides in these regions may be related to their localized actions in these regions, regulating foraging and guarding activities. Thus, these neuropeptides appear to have some functions in the honeybee brain that are specifically related to the age-related division of labor. KEYWORDS: MALDI imaging, mass spectrometry, honeybee brain, neuropeptides, ontogeny, proteomics

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INTRODUCTION Division of labor is considered an important factor for the evolution of eusociality among Hymenoptera insects; it increases the efficiency of social organization, permitting different tasks to be performed simultaneously by different individuals.1 The most common form of the division of labor in honeybees is related to reproduction: the queen reproduces exclusively, while the workers specialize in the routine operations and maintenance of the colony.1,2 In many insect societies, there is a further division of labor among the nonreproducing members, as observed in termites and ants, in which the morphologically distinct individuals have specialized activities, for example, soldiers that defend the colonies. Meanwhile, other individuals known as workers may execute a series of age-related specialized tasks, such as nest cleaning, nursing, and foraging.3 In honeybees, the division of labor is an age-related process: workers become specialized in different tasks according to their age in a process known as “age polyethism”.4 During the first week of their lives, the honeybee workers generally perform brood rearing activities. Between the second and third weeks of age, these individuals become engaged in hive maintenance tasks, such as guarding, nectar processing, and wax secretion. In the transition from 2 to 3 weeks of age, the workers change their duties to foraging and colony defense.5−7 The interaction of the workers with the environment around the nest may interfere internally in the colony, accelerating, delaying, or even © 2014 American Chemical Society

reverting their behavioral development, adapting the execution of tasks to the needs of the entire colony.8,9 In the search for the biochemical stimuli that drive the behavioral changes characteristic of this polyethism, it was demonstrated that juvenile hormone (JH) titers are higher in older bees compared with young ones, suggesting that these compounds may play some role in this process.10 Additionally, it was reported that octopamine and dopamine could also play roles in the division of labor among worker honeybees.11 Apparently, the corpora allata gland in the honeybee (HB) brain produces a series of neuropeptide hormones, which may play different metabolic and developmental roles in honeybees, including the inhibition of the release of JH.12 Furthermore, some of these neuropeptides are likely involved with the regulation of honeybees’ behavioral experience.12 Whether neuropeptides control the age-related behavioral changes remains unknown, but this hypothesis is very attractive. The publication of the honeybee genome predicted the existence of ∼100 different neuropeptides, while mass spectrometry analysis of the honeybee brain confirmed the presence of ∼67 of these compounds.13−15 Neuropeptides likely play important roles in the coordination of the physiological process related to insect communication as well in learning and memory acquisition.15 The current knowledge Received: March 6, 2014 Published: April 17, 2014 3054

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Temporal Polyethism

about the most studied neuropeptides in other insect species, associated with the recent reports of several of such peptides in the honeybee brain, raises the possibility that these compounds may be involved with complex functions in the brain and possibly with social behavior. Among the neuropeptide compounds previously reported in insect brains, including honeybees, the members of the allatostatin and tachykinin families are likely the most common.15−17 Some of the allatostatins appear to be involved with the release of neuroendocrine signals for regulating food intake and age-related behavioral changes.12 Furthermore, some tachykinin peptides function as neuromodulators or hormones associated with age-related behavior of labor division in honeybees.18 Thus, considering the importance of the recent advances in mass spectrometry for screening brain tissue to identify predicted and discover novel neuropeptides, in this study, MALDI spectral imaging (MSI) protocols were used for the first time to obtain honeybee brain images, showing the distribution and the relative concentrations of two different neuropeptides according to honeybee ontogeny. For this purpose, one mature allatostatin and one tachykinin peptide were selected. The results obtained by MSI analysis indicate that the selected neuropeptides reach their maximum relative concentrations in the brains of 10−15 day old honeybee workers when they were observed to be widely distributed throughout most of the analyzed brain sections. Then, their relative concentrations decreased significantly, and by the 25th day the distribution of these neuropeptides becomes restricted to small regions near the subesophageal ganglion, antennal lobe, and lobula of both sides of the brain. Apparently, this profile can be correlated with some age-related behaviors of labor division in honeybees.

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The experiments were conducted at the apiary of the Bioscience Institute of University of São Paulo State, Campus of Rio Claro, SP, Southeast Brazil, during the summer (from December 2011 to March 2012). A colony of Africanized honeybees containing ∼15 000 bees was used. The colony was housed in a framed observation hive, connected via tubes extending through the wall to the outside of the observation room, where the bees were able to forage naturally. A transparent Plexiglas plate (divided into 250 grid squares) was set over one face of each colony during the observation time; this plate was used to record the location of each worker bee in the comb under observation. Combs were manipulated to permit cohorts of newly emerged worker bees to emerge overnight in an incubator set at 28 °C to be later introduced into the observation hives 2 h after marking. Approximately 600 newly emerged workers were marked every day, with different colors for 5 consecutive days. These newly emerged workers were introduced daily into the observation hives (200 workers/ hive) until 1000 workers/hive were acquired. Marked workers were observed 4 h/day every day for 45 days for the identification and quantification of behavioral activities performed in-hive and outside the nest to characterize the temporal polyethism. The observations were conducted following “ad libitum” (sensu Altmann, 1974).19 The repertoire of behaviors observed were as follows: cell cleaning, brood capping, brood tending, queen care, nectar receiving, pollen packing, ventilating, guarding, and foraging. Hematoxylin-Eosin Staining

Hematoxylin-eosin (H&E) staining was used to compare the histochemical method results with the data obtained by MSI as well to generate histological data to support the interpretation of spectral images. The slides were submerged into 95% (v/v) EtOH for 20 min and rinsed with tap water without allowing the material to come off the slides. Brain slices were then stained with a solution of 1% (v/v) hematoxylin for 20 s. The slides were immediately rinsed with tap water and allowed to remain in distilled water for ∼5 min at room temperature. The slices were then stained with a solution of 1% (v/v) eosin for 20 s and rinsed with an excess of distilled water, as described elsewhere.20 At the end of the process, the slides were subjected to five steps of successive washings in 95% (v/v) EtOH to remove the excess dye. In the final mounting of the histological preparations, the stained slices were consecutively washed in 100% xylol and protected with a coverslip. Digital microscopic images of stained tissue sections were generated with a BX51TF Olympus microscope connected to a U-LH100HG Olympus camera. The images of H&E stained sections acquired under the previously described conditions were used to build a contour map for data interpretation.

EXPERIMENTAL SECTION

Chemicals

As calibration standards, ProteoMass ACTH Fragment 18−39 MALDI-MS Standard (2465.19 Da), ProteoMass Angiotensin II MALDI-MS Standard (1046.54 Da), P14R MALDI-MS Standard (1533.85 Da), and MALDI matrix α-cyano-4hydroxycinnamic acid (CHCA) were purchased from SigmaAldrich (Germany). Acetonitrile (MeCN), ethanol (EtOH), and isopropanol were purchased from TEDIA (Brazil). Trifluoroacetic acid (TFA), xylol, hematoxylin, and eosin were obtained from Vetec (Brazil). Liquid nitrogen (N2) was purchased from White Martins (Brazil). Honeybee Collection

Honeybees (A. mellifera) were maintained in the apiary of the Bioscience Institute of the University of São Paulo State, Campus of Rio Claro, SP, Southeast Brazil. The colony used in the experiments was well formed, free of disease, well fed, and in possession of a laying queen. Initially, a honeycomb containing larvae (free of bees) was collected from the colony and maintained for 24 h inside a Biochemical Oxygen Demand incubator (ELETROlab) previously set to 32 °C and 70% relative humidity. Newly emerged workers were marked (day 0) on their thorax using a nontoxic paint and returned to the colony for later capture when they reached 5, 10, 15, 20, and 25 days of age. After collection, bees were immediately frozen in liquid nitrogen, where they remained stored until head slicing for sample preparation.

Tissue Sectioning and Brain Sample Preparation

Following the freezing process, the specimen heads were removed from the bodies and then sectioned on a freezing microtome (LEICA, CM1850). Frontal sagittal sections from the frozen whole brains of workers were obtained at −25 °C to a thickness of 14 μm and mounted onto the MALDI plate. For histochemical analyses, the tissue was sliced 8 μm thick and then mounted onto microscope slides. Sample preparation for MSI analysis was based on modifications of the original protocol described by Seeley et al. (2008).21 In brief, brain sections were laid on a MALDI plate and then dehydrated for 10 s in 70% (v/v) EtOH, followed by two dehydration steps in 3055

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Figure 1. (A) Representation of the sagittal plan in honey bee (Africanized A. mellifera) head, indicating the projection plan of the brain. (B) Frontal view of the brain slice at the sagittal plan of honey bee head, showing the different brain regions, indicated by numbers from 1 to 12, with the respective legend.

95% (v/v) EtOH with an interval of 30 s between the successive steps. This process allowed the removal of sample contaminants, such as salts and excess lipids. Brain slice sections to be submitted to MSI analysis were vacuumed for 15 min at room temperature. Subsequently, the automatic deposition of α-cyano-4-hydroxycinnamic acid (CHCA) onto the tissue was performed with the use of a high-performance Chemical Printer ChIP 1000 (Shimadzu) used as a robotic reagent sampler. The ChiP-1000 was programmed to apply the CHCA in a microarray consisting of 484 spots/mm2, where the sample application points were separated from each other by 200 μm from center to center. On each spot, 10 nL of a 10 mg/mL CHCA solution [in 40% (v/v) MeCN and 10% (v/v) isopropanol, containing 0.1% (v/v) TFA] was applied in five applications of 2 nL each. The planar coordinates (x and y) created by the ChiP-1000 were saved and exported to the mass spectrometer workstation. The laser shots were performed only under these coordinates.

equipped with a laser SmartBeam system and controlled by Launchpad v2.8 software (Shimadzu) using a reflectron device. The instrument was calibrated using a standard calibration mixture of ACTH (fragment 18−39), Angiotensin II, and P14R. MS spectra were acquired in the m/z range of 700− 4000, with the laser power set to 80% and an accelerating voltage of 20 kV adjusted to perform delayed extraction. The peak density was set to 50 for all 200 peaks, with an S/N ratio ≥10. The spectra were acquired with 50 shots per movement from the center of each spot to a linear distance of 50 μm, performing a total of 250 laser shots per spot. After data acquisition, molecular images were reconstructed from the raw data using a mass tolerance of ±0.2 Da with the aid of Launchpad v2.8 software (Shimadzu). The identity of each neuropeptide was confirmed by sequencing using CID conditions in an AXIMA Performance MALDI-TOF-TOF instrument (Shimadzu, Kyoto, Japan), as described elsewhere.22 In brief, the setting conditions were as follows: positive mode with the reflectron device activated, CDL temperature adjusted to 200 °C, block heater temperature set at 200 °C, TOF region pressure 1.5 × 10−4 Pa, ion accumulation time 50 ms, helium as the collision gas, collision energy set at 35% for MS/MS, and collision gas set at 30%. Under these conditions, a mass error of 3.08 ppm and a resolution of 10 000 fwhm were obtained.

Mass Spectrometry Conditions for MALDI Spectral Acquisition

After reagent application, the preparation was dried under vacuum for 10 min and submitted to the acquisition of MALDI spectra in the positive mode using a MALDI-TOF-TOF AXIMA performance instrument (Shimadzu, Kyoto, Japan) 3056

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Figure 2. Frontal view of a brain slice at the sagittal plan of a 15 day old worker honey bee head, showing the different brain regions, and the MALDI-TOF/MS spectra obtained at the right pedunculus (R-Pe), left medial calyx (L-MC), right medulla (R-ME), and left antennal lobe (L-AL).

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RESULTS Honeybee brain samples were obtained from sagittal sections of the rostral position, as represented in Figure 1A. The contour map of the honeybee brain is shown in Figure 1B, where it is also possible to observe the brain’s positioning in the bee’s head in relation to the insect body, as indicated by the arrows. It is important to emphasize that the sections were not obtained after brain dissection but rather by cutting the entire head with its exoskeleton to avoid any structural deformation during sample manipulation. The organization of the honeybee brain into specialized regions, according to the histology of this organ, as suggested by Rybak et al., 2010,24 in the HSB atlas for the sagittal section, resulted in a contour map presenting 12 different regions. Thus, it is possible to observe that the honeybee brain presents some symmetry, considering a virtual axis running from the subesophageal ganglion (1), passing by the central body (5), and reaching the central ocelli (12). In this manner, structures such as the antennal lobe (2), lobula (3), medulla (4), β-lobe (6), α-lobe (7), pedunculus (8), lateral calyx (9), medial calyx (10) and the ocelli (12) occur symmetrically in the center, as well in the right (R) and left (L) hemispheres of the brain (Figure 1B); even the compounded eyes (11) have a symmetrical distribution in relation to the brain. To cover the entire section of the brain (∼1.1 mm2) under study, we acquired 133 000 MALDI-TOF spectra. The sample preparation previously described was sensitive enough for the reliable detection of a series of neuropeptides in the honeybee brain. To demonstrate the sensitivity of this protocol, Figure 2 shows a MALDI spectral image with an overlapping contour map of the honeybee brain. Some snapshots of MALDI-TOF spectra from four different brain regions are also shown,

Neuropeptide sequences were identified by manual interpretation of the spectra obtained under CID conditions. Image Analysis

Molecular images of the neuropeptide distribution in honeybee brain sections were constructed with Launchpad v2.8 (Shimadzu) using the corresponding m/z values of the quasimolecular ions in the monoprotonated form [M + H]+ of each neuropeptide, as previously described in the literature.17,23 The scale of the color patterns in these images represents a semiquantitative method of representing the molecule distribution in a snapshot of sample collection. Two types of images were built: one based on the total ion spectrum (TIS), used to represent the overall accumulation of peptides in brain section, and another using the extracted individual ion spectrum (XIIS), used to represent the distribution of each neuropeptide. To correlate neuropeptides with their locations on brain neuropils, we developed a contour map based on the Honeybee Standard Brain Atlas (HSB atlas)24 in which the boundaries of each region of the bee brain were defined to aid the interpretation of the MSI data. The contour map was then plotted over the image obtained with the histochemical protocol (stained with H&E) of each section, allowing the visualization of the brain anatomy in the sagittal section and facilitating the alignment of the molecular images obtained by mass spectroscopic analysis with the histochemical images. After contour map development, the structures were named according to the HSB atlas (available in http://www. neurobiologie.fu-berlin.de/beebrain/). 3057

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Figure 3. (A) MS2 spectrum obtained for the neuropeptide AST-1 under CID conditions in the positive mode, already deconvoluted in the form of [M + H]+, with the respective sequence interpretation. The precursor ion of m/z 995.451 for fragmentation was selected, and the spectrum was characterized by the assignment of a series of daughter ions of y type and a few of them of b type. (B) MS2 spectrum obtained for the neuropeptide AmTRP-5 under CID conditions in the positive mode, already deconvoluted in the form of [M + H]+, with the respective sequence interpretation. The precursor ion of m/z 1061.622 was selected for fragmentation, and the spectrum was characterized by the assignment a series of daughter ions of b type and a few of them of y type.

more complex, in the present manuscript, we use the original nomenclature given to these peptides. Considering the high sensitivity of the MSI protocol previously described, which permits the detection of a series of neuropeptides from different families, as well the potential importance of these neuropeptides for the regulation of agerelated behavior of labor division in honeybees, we decided to monitor the presence and the spatial distribution of two important families of neuropeptides for the biology and social behavior of A. mellifera, that is, allatostatin (AST) and tachykinin (AmTRP) in the honeybee brain. For this purpose, the peptides AST-1 and AmTRP-5 were monitored using the MSI technique. The identities of these peptides were confirmed by mass spectrometry analysis under CID conditions, as shown in Figure 3. The peptides AST-1 and AmTRP-5 were detected

revealing the presence of different neuropeptides in each of these regions: (i) in the right pedunculus (Pe-R), three allatostatin peptides (AST-1, -3, and -5), two tachykinins (AmTRP-5 and -9), one neuropeptide-like precursor (NPLP1.8), and one MVPV peptide can be observed; (ii) in the right medulla (ME-R), two allatostatin peptides (AST-1 and -5), two tachykinin (AmTRP-5 and -9), one neuropeptide-like precursor (NPLP-1.8), and apidaecin-2 were observed; (iii) in left medial calyx (MC-L), three allatostatins (AST-1, -3, and −5), two tachykinins (AmTRP-6 and -9), and orkokinin-1 were observed; (iv) in the left antennal lobe (AL-L), two allatostatins (AST-1 and -5), two tachykinins (TAmTRP-6 and -9), perivisc peptide, one neuropeptide-like precursor (NPLP-1.8), and apidaecin-2 were detected. The nomenclature of insect neuropeptides is not straightforward, and to avoid making it 3058

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Figure 4. Molecular images of honeybee brain obtained from the treatment of MALDI spectral data got for worker bees presenting ages from 0 to 25 days old. The upper line of Figures were built based on the TIS; it was used to represent the overall accumulation of peptides in brain section. The two other rows of images were based on the XIIS used to represent the distribution of the neuropeptides AST-1 and AmTRP-5. The scale of colors represented in the right side of the images represents the relative concentrations of the peptides based on the use of an internal standard peak, that is, the peak of highest intensity was taken as 100% reference. The dashed lines represent the mask used to identify each brain region as contour maps built based on the section stained with H&E.

Figure 5. Frequency of age-related activities of worker honey bees from the colonies used for neuropeptides assay.

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as quasi-molecular ions with m/z values of 995.451 and 1061.622 as [M + H]+, respectively (Figures 2 and 3). The mass spectral characterization of AST-1 is shown in Figure 3A, where the precursor selected for fragmentation corresponded to the ion with m/z 995.451. This spectrum is dominated by a series of y-type fragment-ions from y2 to y7 and complemented by a set of b ions (b1, b2, b6, and b7). The interpretation of this CID spectrum resulted in the sequence AYTYVSEY−OH, which confirmed the identity of the allatostatin peptide AST-1. The tandem mass spectrum of the neuropeptide AmTRP-5 is shown in Figure 3B, where the precursor-ion of m/z 1061.622 was selected for fragmentation, generating a long series of b-type of fragment-ions, from b1 to b8, and complemented by a small number of y-type ions (y2, y4, y7, and y9). The interpretation of this spectrum confirmed the sequence ARMGFHGMR-NH2 and the identity of the tachykinin peptide (AmTRP-5). The MALDI spectral images were constructed and presented as global images formed by the TIS and also the molecular images of the peptides AST-1 and AmTRP-5 in the honeybee brain at different insect ages (Figure 4). The images generated by the TISs correspond to the overlapping of 133 000 spectra acquired for each brain section at ages from 0 to 25 days old; thus, the most intense colors represent the accumulation of proteins and peptides in each brain region. When the honeybees were newly emerged (0 days old), the neuropeptide AmTRP-5 was concentrated in the left and right pedunculi, which later (5 days old) expanded to the lateral calyx and covered the entire medial calyx. The relative concentration of the neuropeptide increased, and its distribution extended until it covered almost the entire brain when the insects reached 10 days of age. Then, the relative concentration of AmTRP-5 decreased significantly at 15 days of age, but it remained widely spread throughout the brain. When the bee workers reached 20 days of age, an increase in the relative concentration of AmTRP-5 was observed, which became restricted to some small regions of the brain: the antennal lobes, subesophageal ganglion, upper region of both medulla, lateral calyces of both hemispheres, and β-lobes. When the workers reached 25 days of age, the neuropeptide AmTRP-5 had nearly disappeared from the brain, remaining only in reduced concentrations in the regions of the antennal lobes and both lobula. A careful observation of the distribution profile of the neuropeptide AST-1 reveals that it was not present in the brain of newborn (0 days old) honeybee workers. At the age of 5 days, the neuropeptide was concentrated in the regions of the lateral and medial calices. When the honeybee workers reached 15 days of age, AST-1 was distributed throughout the brain, with a very similar pattern of distribution and concentration to that observed for AmTRP-5 at the same age. The spatial pattern of distribution of AST-1 changed at the age of 20 days, becoming concentrated mainly in the left medulla, upper region of the left lobula, and left antennal lobe. The peptide AST-1 almost disappeared from the upper brain regions, becoming fairly concentrated in the inferior brain, including the left and right antennal lobes, α-lobes, and lobula. Considering that the duration and the transition from each labor division behavior characteristic of the temporal polyethism in worker honeybees may be very plastic, depending on a series of biological events inside the colony, and characteristic of the interaction of the colony with some environmental factors, we decided to evaluate the duration of a series of age-related activities performed by individuals from the

same colony for which the pattern of spatial distribution of the neuropeptides AmTRP-5 and AST-1 in worker brains was investigated. The results are shown in Figure 5, which indicates that 1. Cell cleaning was mainly performed by workers from 1 to 10 days old. 2. Brood capping was mainly performed by workers from 3 and 10 days old. 3. Brood tending was mainly performed by workers between 6 and 14 days old. 4. Queen care was mainly performed by workers from 6 to 15 days old. 5. Nectar receiving was mainly performed by workers from 10 to 17 days old. 6. Pollen packing was mainly performed by workers from 11 to 20 days old. 7. Colony ventilating was mainly performed by workers from 16 to 21 days old. 8. Colony guarding was mainly performed by workers from 16 to 24 days old. 9. Foraging was mainly performed by workers from 17 to 29 days old.

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DISCUSSION Honeybees perform several different age-related tasks inside in the nest during their first weeks as adult workers, including brood care, hive cleaning, food storage, and queen care, among other tasks, which shift to other activities outside the nest, such as foraging and guarding.24,25 However, the transition between these behaviors may be very plastic in response to the social context and the needs of the colony, which increase or decrease the rate of development between each behavioral stage.25 To determine the age period in which each behavioral activity was performed in the colony under investigation, the profile of agedependent behaviors (Figure 5) was analyzed, and it can be summarized as follows: (i) between 0 and 5 days of age, the major behaviors performed were cell cleaning and brood capping, and (ii) between 5 and 10 days of age, in addition to the previous activities, novel behaviors began to be performed, such as brood tending and queen care, while between 10 and 15 days age, the worker honeybees also performed nectar receiving and pollen packing. All of these activities were typically performed inside the nest. Meanwhile, from 15 to 20 days of age, the worker honeybees began to perform behavioral activities outside the nest, such as ventilating the colony, guarding, and foraging. At the age period from 20 to 25 days of age, the major behaviors performed were guarding and foraging. During the transition from in-hive tasks to those performed outside the nest, the worker honeybees underwent changes from an environment presenting controlled conditions to an environment that was far more heterogeneous. Inside the nest, there is reduced luminosity, the temperature is controlled by the workers between 33 and 35 °C, and the humidity is ∼70%.25,26 Meanwhile, outside the nest, the temperature may fluctuate from 10 to 50 °C, the worker bees may be exposed to severe weather conditions such as wind and rain, and they also become susceptible to predation.26 This transition is characterized by pronounced physiological changes, such as the regression of hypopharingeal glands, reduction of body weight, increase in water content, and increase in the metabolic capacity.25,27−30 Foraging requires the development of a wide repertoire of navigational skills due to the spatial complexity 3060

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Tachykinins were the first neuropeptides identified in insects that cause the activation of gut movement. These peptides cause excitatory actions in some systems and inhibitory actions in others. There are also reports regarding the effects of tachykinins on the regulation of food intake in honeybees.49 Their actions in different regions of the honeybee brain are unknown.12 In honeybees, mature tachykinins constitute a family of neuropeptides that originate from specific cleavages of a precursor protein referred to as Prepro-Apis mellifera tachykinin-related peptides (Prepro-Am TRP), which result in the formation of seven different peptides:18 APMGFQGMRNH2 (AmTRP3), APTGHQEMQ-NH2 (AmTRP-1), ALMGFQGVR-NH2 (AmTRP-2), APMGFYGTR-NH2 (AmTRP-4), ARMGFHGMR-NH2 (AmTRP-5), SPFRYLGAR-NH2 (AmTRP-6), and NPRWEFRGKFVGVR-NH2 (AmTRP-7). The prepro-AmTRP form was detected in much higher concentrations in the brains of forager bee workers than in the nurses, suggesting that the function of AmTRPs may be related to honeybee social behavior.50 The literature has referred to them generically as AmTRP peptides, making it difficult to ascertain the individual role of each mature peptide in the honeybee brain. The peptide under study in the present manuscript corresponds to AmTRP-5. The levels of some neuropeptides, including AmTRP-4, are much higher in the brains of nectar foragers than in pollen foragers, suggesting that neuropeptide signaling may be important in regulating differences of behavior, even for a short period of time, depending on the type of behavioral experience of the insect.49 In a manner similar to that described above for the tachykinins, the allatostatins correspond to a family of ten neuropeptides originating from the controlled proteolysis of a precursor prepro-protein (UNIPROT -http://www.uniprot. org/uniprot/P85797#PRO_0000341453): AYTYVSEY-OH (AST-1), LPVYNFGI-NH2 (AST-2a), LPVYNF-OH (AST2b), GRDYSFGL-NH2 (AST-3), RQYSFGL-NH2 (AST-4), NDNADYPLRLNLDYLPVDNPAFHSQENTDDFLEE-OH (AST-5), GRQPYSFGL-NH2 (AST-6), AVHYSGGQPLGSKRPNDMLSQRYHFGL-NH2 (AST-7a), AVHYSGGQPLGSOH (AST-7b), and PNDMLSQRYHFGL-NH2 (AST-7c). Again, the literature has referred to these molecules generically as AST peptides, making it difficult to determine the individual role of each neuropeptide in the honeybee brain. The peptide studied in the present manuscript corresponds to AST-1. Allatostatins were reported to inhibit the release of juvenile hormone by the corpora alata, consequently influencing the modulation of the rate of development of age-related behaviors in insects.51 These neuropeptides also inhibit visceral and skeletal muscles in Drosophila52 and decrease synaptic transmission at neuromuscular junctions in crustaceans.53 The physiological actions of ASTs in bees have not yet been studied. Establishing a parallel between the age-related behaviors and the profile of expression of the neuropeptides in specific brain regions, it becomes clear that the activities of cell cleaning and brood capping seem to be performed in the presence of AmTRP-5 but in the absence of AST-1 in young worker bees. Another aspect that must be emphasized is that most behaviors typically performed inside the nest occur in the age period from 10 to 15 days of age, which is the period in which the neuropeptides AmTRP-5 and AST-1 achieve their maximum relative concentrations and are spatially distributed throughout the workers’ brain, suggesting the possible involvement of these neuropeptides in the regulation of the

and the acquisition of novel sensory information from the environment outside the nest.31 The proteome profile of bee brain also follows the physiological changes of this period; for example, the expression of the royal jelly proteins such as MRJP-1 was identified in the mushroom body, while the MRJP2 and -7 were detected in the central brain region during the transition from nursing to foraging tasks;32 apparently, these proteins may have functions related to caste determination as well as amino acid reserves for the brain metabolism.33,34 The overexpression of guanosine 3′,5′-monophosphate (cGMP)dependent protein kinase, 35 alpha-glucosidase, transferrin,32,36,37 and a putative orphan receptor protein (HR38) involved with ecdysteroid-signaling in the mushroom body38 in workers’ brain was also an important proteomic characteristic of this transition. This behavioral development could also be influenced by the genetic background of each colony, which makes the colony more or less sensitive to the weather, season, and nutritional status.39−41 Despite its small size and reduced number of neurons, the honeybee brain can be considered morphologically and anatomically complex, playing an important role in the development of the temporal polyethism of these insects.24 The main regions of the brain neuropils are the optic lobes and antennal lobes, the mushroom bodies, and the central complex. The optic lobe appears to be responsible for processing vision, while the antennal lobes are the main neuropils involved with the processing of olfaction.35 Mushroom bodies (MBs) are prominent structures that consist of the lateral and medial calices in each hemisphere,42,43 which are involved with sensory integration, learning, memory acquisition, and spatial orientation.12,42,44 The honeybee brain contains other structural parts such as the subesophageal ganglion, tritocerebrum, and ventral cord, about which very little information is known in terms of their functional roles. The mushroom bodies are larger in the bees that perform activities outside the nest than the ones that perform in-hive activities, suggesting that these neuropils could be involved in the learning and acquisition of sensorial and navigational memory.45 The modulation of age-related behaviors in honeybees appears to involve the actions of juvenile hormone, octopamine, and possibly neuropeptides in specific brain neuropils. Juvenile hormone apparently modulates the rate of behavioral development; however, this compound does not appear to be required for foraging.46 Corroborating this observation, the use of proteomic analysis associated with in situ hybridization techniques identified the up-regulation of the enzyme juvenile hormone diol kinase (involved with the degradation of juvenile hormone) in the mushroom bodies during the behavioral transition from nurse to forager bees.47 The majority of the current knowledge about the roles of neuropeptides was obtained through studies carried out in insect species other than A. mellifera because little research has been carried out regarding the specific effects of neuropeptides in bees. Considering their large chemical diversity and wide variety of mechanisms of action in different insect species, it is necessary to be cautious in extrapolating their potential roles from one species to another one.12 The functions of a few peptides have been studied in honeybees. For example, insulin-like peptides (ILPs) influence the release of juvenile hormone, increasing the longevity of queens.48 Among the worker bees, these peptides also appear to regulate the development of nurses to foragers through the inhibition of the ILP-related rapamycin pathway.49 3061

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aforementioned behaviors. In fact, the profile of AST-1 fits the profile of behavioral activity in this period of age much better than the profile of AmTRP-5. The age period of the transition from performing activities intranest to outside the nest activities in the colony under study (from 15 to 20 days old) is characterized by an increase in the relative concentration of AmTRP-5 and a decrease in the relative concentrations of AST1. At 20 days of age, the spatial distributions of both neuropeptides are similar to each other; that is, both neuropeptides become concentrated in small regions of the brain: the antennal lobes, subesophageal ganglion, upper regions of both medulla, both lobula, and α- and β-lobes of both brain hemispheres. Thus, it could be speculated that these brain regions would be important in modulating behaviors such as guarding and foraging at this age, which appear to be the major behavioral activities of 20 day old worker bees. At the age of 25 days, the major activities of the workers are still guarding and foraging, while both neuropeptides have almost disappeared from the brain, with only a few clear spots remaining in the left and right antennal lobes, α- and β-lobes, and both lobula. The distribution of allatostatin in the brains of forager honeybees was examined via immunohistochemistry using specific antibodies for different AST peptides in a singlelabeling experiment and using a specific antibody for GABA in a double-labeling experiment.47 A series of small and localized areas of immune reactive neurons were detected in different brain regions brain such as in the antennal lobes, mushroom bodies (medial and lateral calices), and optic lobes, indicating localized actions of different neuropeptides, which may play distinct roles in different subsets of neurons in each brain region. Thus, the authors suggested potential roles of AST peptides in regulating the vision, odor perception, and sensorial memory of forager bees. Doubly reactive neurons were observed in the mushroom bodies, which were labeled for AST-5 and GABA, suggesting that AST-5 acts on GABAergic neurons at the level of lateral and medial calices. The immune reactive neurons for AST-1 were localized in the α- and β-lobes of both brain hemispheres. In the present investigation, AST-1 was detected in the mushroom bodies of 5 day old workers, throughout the brain in the age period from 10 to 15 days old, and spatially located in the antennal lobes, α- and β-lobes, and both lobula in the period of age from 20 to 25 days old (corresponding to the period in which the worker bees are foragers). Thus, the result of the present investigation partially corroborate the reports of Kreissl et al., 2010.54 The expression of prepro-AmTRP in the honeybee brain was previously reported to occur predominantly in the mushroom bodies, suggesting that AmTRPs could be mainly involved with the development of learning and memory. AmTRPs were also observed in a few neurons of the optic and antennal lobes, which may suggest some involvement of these peptides with vision and odor perception.50 It has been suggested that distinct populations of neurons appear to express different neuropeptides that act in particular regions of the brain, mediating different physiological processes.50 This observation fits the majority of the results obtained in the present investigation well, in that AmTRP-5 and AST-1 were clearly detected to be concentrated in some specific brain regions, except at the age of 10 days, when both peptides were spread throughout the most brain regions. Apparently, the profile of age-related activities (Figure 5) fits the profile of the relative concentrations of both neuropeptides

well; that is, the successive increase in the number of tasks performed in the hive by the worker bees is followed by a gradual increase in the relative concentrations of both neuropeptides. The transition from these activities to those performed outside the nest was characterized by a decrease in the relative concentrations of these neuropeptides as well as by a pattern in the spatial distribution of AmTRP-5 and AST-1 in the brain of foragers bees that differed from that observed in the nurse bees. This observation suggests that the same peptides may present different functions in each brain region, which seem to be differentially activated or required at different ages, according to an age-related process. This multifunctionality seems to be common among the neuropeptides and may be used, at least in part, to explain their involvement with the complex regulation of the age-related polyethism in honeybees.55 It is also important to recognize the participation of other neuropeptides that are not the subject of the present investigation in this mechanism. The honeybee brain is also the target of peptides produced by other organs and glands, which were not under consideration in the present study. The increase in the abundances of neuropeptides could be due to the novel synthesis and maturation of these peptides in brain neurons or even the inflow of peptides into the bee brain (through the circulatory system) from neurons located in other parts of the nervous system. Meanwhile, the decrease in neuropeptide abundance could be due to the release of these components from the brain to other parts of the nervous system or their proteolytic degradation in the brain.49

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CONCLUDING REMARKS

The neuropeptides AmTRP-5 and AST-1 appear to have important functions in the honeybee brain that are specifically related to age-related division of labor. Their occurrence was monitored through MALDI spectral imaging according to the ontogeny of Africanized A. mellifera. The levels of these peptides increased in the honeybee brain in the age range from 0 to 15 days old, accompanying the increase in the number of in-hive activities performed by the nurse bees, followed by a decrease during the age range of 15 to 25 days old in which the workers began to perform outside nest activities (guarding and foraging). The period of transition from nursing to guarding and foraging activities (10 to 15 days old) corresponds to that in which AmTRP-5 and AST-1 were distributed throughout the brain. Considering the highly localized distribution of AmTRP5 in the upper region of both pedunculus of 0-days-old workers and the absence of AST-1, it might be suggested that the action of AmTRP-5 in this brain region could be related to the regulation of cell cleaning and brood capping. Additionally, the highly localized occurrence of AmTRP-5 and AST-1 in the antennal lobes, subesophageal ganglion, upper regions of both medullae, both lobula, and α- and β-lobes of both brain hemispheres in 20- to 25-day-old workers suggests that the action of both neuropeptides in these regions could be related to their localized action in the regulation of foraging and guarding activities. Therefore, AmTRP-5 and AST-1 could be multifunctional neuropeptides that act at different sites of the honeybee brain at different ages to regulate a series of different age-related behaviors characteristic of the process of polyethism in honeybees. 3062

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AUTHOR INFORMATION

Corresponding Author

*Phone: 55-(19)-35264163. Fax: 55-(19)-35348523. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by grants from the BIOprospecTA/ FAPESP program (Proc. 2011/51684-1). M.S.P. and O.M. are researchers from the National Research Council of BrazilCNPq.

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ABBREVIATIONS MeCN, acetonitrile; CHCA, α-cyano-4-hydroxycinnamic acid; EtOH, ethanol; HE, hematoxylin-eosin; MALDI, matrixassisted laser desorption/ionization; TIS, total ion spectrum

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REFERENCES

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