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MALDI Imaging Analysis of Neuropeptides in Africanized Honeybee (Apis mellifera) Brain: Effect of Aggressiveness Marcel Pratavieira, Anally Ribeiro da Silva Menegasso, Franciele Grego Esteves, Kenny Umino Sato, Osmar Malaspina, and Mario Sérgio Palma J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.8b00098 • Publication Date (Web): 18 May 2018 Downloaded from http://pubs.acs.org on May 19, 2018

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Journal of Proteome Research

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MALDI Imaging Analysis of Neuropeptides in Africanized Honeybee (Apis mellifera) Brain: Effect of Aggressiveness Marcel Pratavieira‡, Anally Ribeiro da Silva Menegasso‡, Franciele Grego Esteves‡, Kenny Umino Sato‡, Osmar Malaspina ‡, Mario Sergio Palma ‡,* ‡

Institute of Biosciences, Department of Biology, Center of the Study of Social Insects,

University of São Paulo State (UNESP), Rio Claro, SP, Brazil

* Correspondence: Prof. Dr. Mario Sergio Palma. CEIS-IBRC- UNESP, Av. 24A nº 1515, Bela Vista - Rio Claro, SP, Brazil, CEP 13506-900, FAX: 55-(19)-35348523. Email: [email protected]

KEYWORDS:

MALDI-imaging;

honeybee;

brain;

aggressiveness.

ACS Paragon Plus Environment

neuropeptides;

ontogeny;


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ABSTRACT

The aggressiveness in honeybees seems to be regulated by multiple genes, under the influence of different factors, such as polyethism of workers, environmental factors, and response to alarm pheromones, creating a series of behavioral responses. It is suspected that neuropeptides seem to be involved with the regulation of the aggressive behavior. The role of allatostatin and tachykinin-related neuropeptides in honeybee brain during the aggressive behavior is unknown; thus, worker honeybees were stimulated to attack and to sting leather targets hanged in front of the colonies. The aggressive individuals were collected and immediately frozen in liquid nitrogen; the heads were removed, and sliced at sagittal plan. The brain slices were submitted to MALDI-Spectral-Imaging analysis, and the results of the present study reported the processing of the precursors proteins

into

mature

forms

of

the

neuropeptides

AmAST

A

(59-76)

(AYTYVSEYKRLPVYNFGL-NH2), AmAST A (69-76) (LPVYNFGL-NH2), AmTRP (88 – 96) (APMGFQGMR-NH2), and AmTRP (254 – 262) (ARMGFHGMR-NH2), which apparently acted in different neuropils of honeybee brain, during the aggressive behavior, possibly playing the neuromodulation of different aspects of this complex behavior. These results were biologically validated performing aggressiveness-related behavioral assays, using young honeybee workers that received 1 ng of

AmAST A

(69-76) or AmTRP (88 – 96) via hemocele. The young workers that were not expected to be aggressive individuals, presented a complete series of the aggressive behaviors, in presence of the neuropeptides, corroborating the hypothesis that correlates the presence of mature AmASTs A and AmTRPs in honeybee brain with the aggressiveness of this insect.

Keywords: honeybee brain; neuroproteomics; agressivity; neuropeptides; MALDI -Imaging; mass spectrometry.


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INTRODUCTION When the colonies of honeybees are disturbed or attacked, raids of guard workers respond aggressively pursuing the invaders/aggressors, in a tentative to sting, putting them far away from the limits of security of the colonies 1, 2. The guard bees are highly responsive individuals to visual cues and dark colors, which permit them to identify potential threats for the colonies 3. The colony defense begins when the presence of intruders is detected by the guard bees, which release alarm pheromones, triggering collective aggressive responses in the workers of each colony, attracting all them for a massive attack against the intruders 4. The honeybee nest contains stored food (honey, pollen, royal jelly), the brood, and the queen; therefore, the defensive behavior of honeybees aims to protect the biological patrimony and the survival of the colony 5. Aggression is a complex behavior, used as part of the defensive strategies of colonies of honeybees, which seems to be regulated by several genes, under the influence of the age of workers, environmental factors, and response to alarm pheromones 5, creating a repertoire of behavioral responses with a strong molecular signature of the brain 1, 6. There are studies reporting the correlation between the concentrations of Juvenile Hormones (JH) and aggressiveness; apparently the increase in the titers of JH with the age of workers is accompanied by the increasing in the production of alarm pheromones, which in turn potentiates the aggressiveness 1, 6. Metabolomic studies revealed that the honeybee brain of aggressive bee workers presented increased glycolysis and decreased oxidative phosphorylation in their neurons, possibly provoked by the action of alarm pheromones, causing a metabolic shift to this condition in honeybee brain

7, 8

. Apparently, neuropeptides and biogenic

amines seems to be involved with the regulation of the aggressive behavior. Physiological investigations demonstrated that the alarm pheromones could act as primers to trigger the releasing of neuropeptides and/or biogenic amines in honeybee brain, reinforcing the aggressive behavior 9. Tenths of potential neuropeptides were identified in honey bee genome 10, but up to now only a few of them have been reliably confirmed


11, 12, 13

.

Among these


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neuropeptides there are different forms of Apis mellifera Allatostatins A (AmASTs A), which are widely expressed in honey bee brain, mainly in the regions of antennal lobes and mushroom bodies, that are important for learning and memory acquisition and/or recall 14. It is well known that these neuropeptides are involved with the inhibition of biosynthesis of JH in corpora alata of insect brains in general

15

, as well in the

modulation of foraging activity16, and in the reduction of food intake

17

. It has been

suggested that AmASTs A play in insects similar role as that of somatostatin in mammals, i.e., in insects these neuropeptides can regulate developmental growth in youth forms

18

. Despite this importance, the role of AmASTs A in the brain of adult

honeybees remains largely unknown 12. Another family of important neuropeptides in insects are the tachykinin-related peptides, which in Apis mellifera is known as AmTRP; these neuropeptides seems to be common in the mushroom bodies of honeybee brain, where they act as neuromodulators for sensory processing

19

. As already mentioned for the AmASTs A, the detailed

functional role of AmTRPs is not completely known in honeybee brain. The level of these neuropeptides may change in honeybee brain according to the sex, age, and division of labor, apparently to modulate the social behaviors according to the polyethism 20. The function of

AmASTs A and AmTRPs in honeybee brain, and their

potential role in the aggressive behavior is not clearly known up today. We consider that honeybee brain represents a very attractive system for studying the involvement of these neuropeptides in the aggressive behavior. For this purpose, honeybees were submitted to a standard assay protocol for evaluating their stinging behavior. The brain of stinger individuals were removed and submitted to MALDI- Mass Spectral Imaging (MALDIMSI) analysis. The results of the present investigation revealed that some forms of AmASTs A and AmTRP neuropeptides were present exclusively in the brain of aggressive individuals; distributed in different neuropils, suggesting a potential involvement of them in the regulation of aggressive behavior of worker bees.

EXPERIMENTAL SECTION Chemicals Calibration standards ProteoMassTM ACTH Fragment 18-39 MALDI-MS Standard (2465.19 Da), ProteoMassTM Angiotensin II MALDI-MS Standard (1046.54 Da), P14R MALDI-MS Standard (1533.85 Da) and MALDI matrix α-cyano-4-


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hydroxycinnamic acid (CHCA) were purchased from Sigma-Aldrich, Germany. Acetonitrile (MeCN), Ethanol (EtOH), 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).

Honeybees Collection The honey bees (A. mellifera), were maintained in the apiary of the Bioscience Institute of University of São Paulo State, Campus of Rio Claro, SP, Southeast Brazil. 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 summer time (from december 2015 to march 2016). A colony of Africanized honey bees containing about 25,000 bees was used. The colony used in the experiments was well formed without any disease, well fed and with a laying queen. Initially, a honeycomb containing larvae (free of bees) were collected from the colony and maintained up 24 hours inside a Biochemical Oxygen Demand incubator (ELETROlab) previously set to 32°C and 70% of relative humidity. Newly emerged workers (~1000 individuals) were marked (day-0) on their thorax using a non-toxic paint (Posca PC-5M, 1.8-2.5 mm) and returned to the colony for later capture when they reached 20-days old. When the bees achieved 20-days old, the colony was submitted to the aggression assay.

Aggression (stinging) assay The aggression assay was performed using small modifications of the protocol originally described by Stort (1974) 21 for the assay the stinging behavior of worker honeybees 22. In summary, the assay was carried out using spherical black ball targets (50 cm2) made of soft leather; it was waved several times 1 m from the hive, to disturb the colony. Guard workers alarmed the colony and attacked the leather targets, stinging their surface; the barbed sting lancets penetrates the leather material, but cannot be removed, making the aggressor workers attached to the target surface. The time lasting since the targets presentation for the colony, and their stinging by the aggressor individuals was about 30 seconds. Amongst the stinger workers that remained attached to the target, those marked with non-toxic paint (20-days old individuals) were collected, immediately frozen in liquid nitrogen and transported to the laboratory. The non-aggressive individuals (previously marked with non-toxic ink), were collected from



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the nest surface, frozen in liquid nitrogen and transported to the laboratory, to be analyzed as control group. The experiment was repeated three times at different days, and groups (non-aggressive and aggressive) of five individuals were collected in each experiment.

The assay of aggression-related behaviors under effect of neuropeptides

To investigate the effect of the neuropeptides in the development of a series of aggression-related behaviors, three groups of twenty five 7-days old workers of Africanized honeybees (Apis mellifera) were caged into plastic boxes and incubated during five minutes at 10ºC into a refrigerator; this was enough to cause 10 minutes of cold-anesthesia in the insects. During this period of time, it was injected 2 uL of physiological solution containing 0.5 ng/uL of AmAST (69-76) or AmTRP (88 – 96) into the hemocele of each insect. The workers of the control group were manipulated the same way, except that they were inoculated only with physiological solution. After awakening, the insects were transferred to an arena of behavioral observations, constituted of transparent plastic boxes, of cylindrical format (25 cm diameter x 15 cm high), covered by a plastic cap. Inside the arenas it was offered candy (paste of carbohydrates) and water ad libidum. The arenas with the honeybee workers remained without any disturbance during 30 minutes at room temperature (25ºC) to permit the insects to acclimate and to recover from the stress of manipulation. After the period of acclimation and stress recovery, black leather targets (3 cm diameter) were exposed in the arenas, waving from a Nylon line (5 cm) from the center of the plastic cap of each arena. The aggressiveness in honeybees is characterized by a series of sequential behaviors, that precede the stinging action: alert, attraction, and aggression 21. These behaviors were recorded with a video-camera, and the videos were later analyzed in details; the behaviors described above were observed and registered during a period of 5 minutes. The assay described above was repeated with three different groups of honeybee workers. Statistical significance was determined using GraphPad Prism, version 3.03 (La Jolla, CA), with Student's test to compare the control and experimental groups.


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Tissue sectioning and brain sample preparation

Following the frozen process, the specimens had their heads removed from the body, and then sectioned on a freezing microtome (LEICA, CM1850). Frontal sagittal sections from 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 for 8 µm thick, and then mounted onto microscope slides. Sample preparation for MSI analysis were based on some modifications of the original protocol described by Seeley et al., (2008)

23

. Briefly, brain sections were laid down on MALDI plate, and

then first dehydrated during 10 sec in 70% (v/v) EtOH, followed by twice dehydrations steps in 95% (v/v) EtOH, with an interval of 30 seconds between the successive steps. This process allowed contaminants removal of from the sample, such as salts and the excess of lipids. Brain slice sections to be submitted to MSI analysis were vacuumed for 15 minutes at room temperature; subsequently, it was performed the automatic deposition of

α-cyano-4-hydroxycinnamic acid (CHCA) over each slice surface,

with a High Performance Chemical Printer ChIP 1000 (Shimadzu), used as a robotic reagents sampler onto tissue. The ChiP-1000 was programmed to apply the CHCA in a microarray constituted of 484 spots /mm2, where the points of sample application were spaced from each other by 200 µm from center to center. On each spot were applied 10 nL of a solution of 10mg/mL CHCA (in 40% (v/v) MeCN and 10% (v/v) isopropanol, containing 0.1% (v/v) TFA) 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 just on these coordinates.

Hematoxylin-eosin staining The hematoxylin-eosin (H&E) staining was used to compare the histochemical method 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 minutes, 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 during 20



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seconds. The slides were immediately rinsed with tap water and left under rest into distilled water for about 5 minutes at room temperature. The slices were then stained with a solution of 1 % (v/v) eosin for 20 seconds, and rinsed with an excess of distilled water under current, as described elsewhere 24. At the end of the process, the slides were subjected to a five steps of successive washings in 95% (v/v) EtOH for removal of the excess of 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 conditions described above were used to build a contour map for data interpretation.

Mass spectrometry conditions for MALDI spectral acquisition and peptide sequencing

After reagents application on to the brain slices the preparations were dried under vacuum during 10 min and submitted to the acquisition of MALDI spectra, in the positive mode using a MALDI-TOF-TOF instrument mod. AXIMA Performance (Shimadzu Corp., Kyoto, Japan), equipped with a laser SmartBeam system and controlled by Launchpad v2.8 software (Shimadzu) using the 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 700-4000, with the laser power set to 80% and accelerating voltage of 20 kV, adjusted to perform delayed extraction; the density of peaks was set at 50 for each 200 peaks presenting S/N ratio ≥ 10. The spectra were acquired with 50 shots per movement from the center of each spot, up to a distance of 50 µm straight, performing a total of 250 laser shots per spot. After data acquisition, molecular images were reconstructed from the raw data, using mass tolerance of ± 0.2 Da, with the aid of Launchpad v2.8 software (Shimadzu). The identity of each neuropeptide was confirmed by sequencing them using CID conditions in a MALDI-TOF-TOF instrument (Shimadzu Corp., Kyoto, Japan) mod. AXIMA Performance, as described elsewhere11.

Briefly, the setting

conditions were: positive mode with the reflectron device activated, CDL temperature adjusted to 200 °C, block heater temperature at 200 °C, TOF region pressure 1.5×10−4 Pa, ion accumulation time 50ms, helium was used as collision gas, collision energy set


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at 35% for MS/MS, and collision gas set to 30%.; under these conditions it was obtained error of 3.08 ppm and resolution 10,000 FWHM.

Peptide synthesis and purification The peptides were synthesized on solid-phase synthesis using N-9-fluorophenylmethoxycarbonyl (Fmoc) chemistry, with Novasyn TGS resin (NOVABIOCHEM, Germany) in an automated peptide synthesizer (PROTEIN TECHNOLOGY INC., mod. Prelude; USA). Sidechain protective groups included t-butyl and t-butoxycarbonyl for serine and lysine, respectively. Cleavages of the peptide resin complexes were performed with trifluoroacetic acid, 1,2-ethanedithiol, anisole, phenol, and water (82.5:2.5:5:5:5 by volume, respectively) for 2 h. The peptides were precipitated with cooled ethyl ether (4 ºC). The crude peptides were solubilized in water and purified in a RP-HPLC system mod. LC-8A (SHIMADZU, Kyoto, Japan), using a semi-preparative column (SHISEIDO C-18, 250 mm x 10 mm, 5 mm) under isocratic conditions with 55% (v/v) ACN (containing 0.1% (v/v) TFA) at a flow rate of 2 mL/min. The elution was monitored at 215 nm with a UV-DAD detector, mod. SPD-M10A (SHIMADZU, Kyoto, Japan), and each eluted fraction was manually collected; the purity was checked using mass spectrometry analysis. The synthetic peptides were used to perform all bioassays described in this manuscript.

Image Analysis

Molecular images of neuropeptides distribution in honey bee brain sections were constructed with the software Launchpad v2.8 (Shimadzu), using the corresponding m/z values of the quasi-molecular ions in the monoprotonated form [M+H]+ of each neuropeptide, as described in literature

25, 26

. The spectral images of allatostatin A and

tachikinin-related precursors were obtained using the overlapping of the quasimolecular ions of the tryptic fragments of each one of these proteins. The scale of color patterns in these images represent a semi-quantitative method of representation of molecules distribution in a snap-shot of sample collection. The images obtained were built based on the Extract of Individual Ion Chromatogram (XIC), to represent the distribution of each neuropeptide. For correlating neuropeptides with their location on brain neuropils was developed a contour map based on the Honeybee Standard Brain Atlas (HSB atlas) 27, in which were defined the boundaries of each region of a bee brain, aiming a better



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understanding of 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 brain anatomy in the sagittal section, and facilitating the alignment of the molecular images got by mass spectral analysis with the histochemical images. After contour map development, the structures were named according to HSB atlas (available in http://www.neurobiologie.fu-berlin.de/beebrain/).

RESULTS Honeybee heads were sliced at rostral position (Figure 1A) to obtain sagittal sections of brain, necessary to acquire MALDI Spectral Images (MSI). In order to map the distribution of neuropeptides through the different neuropils of honeybee brain, were constructed contour maps for the neuropils, based on the histology of this organ according to Rybak et al. (2010) 27. Thus, the Figure 1B shows the contour map of 20days old

A. mellifera brain, numbered to assign the twelve neuropils,

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, different neuropils were easily visible such as the antennal lobe (2), lobula (3), medulla (4), β-lobe (6), αlobe (7), pedunculus (8), lateral calyx (9), medial calyx (10), and even the lateral ocelli (12) occurring symmetrically in the right (R) and left (L) hemispheres of the brain. About 150,000 MALDI-TOF spectra were acquired to cover the entire brain section (~0.98 mm2). In order to demonstrate the sensitivity of the preparation for the detection of neuropeptides, were acquired the global MALDI-TOF spectra of honeybee brains, from non-aggressive (Figure 2A) and aggressive workers (Figure 2B). The global MALDI-TOF/MS

spectrum reveals the presence of some AmASTs and

AmTRPs in the brain of the aggressive individuals, which are absent from the brain of non-aggressive

workers.

The

nomenclature

of

insect

neuropeptides

is

not

straightforward, and to avoid making it more complex, in the present manuscript we used the consensus nomenclature given to these peptides

13, 14

. Apparently the results

shown in Figures 2A and 2B are indicating the potential presence of a series of neuropeptides in the honeybee brain. To demonstrate the sensitivity of this protocol, it was performed a comparison between the global MALDI-TOF spectra of the brain extracts from non-aggressive and aggressive workers (Figure 2A vs. Figure 2B), revealing that some neuropeptides were detected in the brain of the aggressive individuals as [M + H]+ ions, which were not present in the brain of non-aggressive ACS Paragon Plus Environment

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individuals: AmAST A (69-76) m/z 921.51, AmTRP (88-96), m/z 993.43, AmAST A (59-66) m/z 995.43, AmTRP (254-262) m/z 1061.52, AmTRP (69- 85) m/z 1974.98, and AmAST A (59-76) m/z 2182.13. In order to confirm the identity of these neuropeptides the CID spectra of the precursor-ions mentioned above were acquired and interpreted. The Figures S1 to S3 are showing the CID spectra of the precursor ions of m/z 995.43, m/z 2182.15 and m/z 921.51 as [M + H]+, corresponding to the sequences of the neuropeptides AmAST A (59-66) (AYTYVSEY-OH), AmAST A (59-76) (AYTYVSEYKRLPVYNFGL-NH2), and AmAST A (69-76) (LPVYNFGL-NH2), respectively. Meanwhile, the Figures S4 to S6 are showing the CID spectra of the precursor ions of m/z 1974.98, m/z 993.47 and m/z 1061.52 as

[M + H]+, corresponding to the sequences of the neuropeptides

AmTRP (69 – 85) (NSIINDVKNELFPEDIN-OH), AmTRP (88-96) (APMGFQGMRNH2),

and AmTRP (254 - 262) (ARMGFHGMR-NH2), respectively. Figure 3A is showing the sequence of the precursor neuropeptides AmAST

A and AmTRPs, which after maturation results in active neuropeptides (Figure 3B). Thus, in order to improve our understanding about the maturation process and diffusion/distribution of the precursors and mature neuropeptides, in the brain of worker honeybees during the aggressive behavior, were obtained the MSIs for the precursor AmAST A (Figures 4A and 4B) and AmTRP (Figures 4C and 4D), both in the brain of non-aggressive individuals and in the aggressive ones. The MSIs were built using the overlapping of all the tryptic peptides detected for the precursor AmAST A (m/z 1300.6644, 1170.4103, 1123.5306, 1116.5578, 1107.6197, 1077.4959, 1005.4636, 996.5149, 899.4621, 886.4305, and 821.4304), and the precursor AmTRP (m/z 1662.7129, 1655.8023, 1589.7369, 1495.7461, 1294.6426, 1271.7093, 1263.6514, 1218.6001, 1188.6358 and 1183.5524), making the mapping their distribution very reliable. Taking into account that apparently only the peptides amidated at their C-terminus correspond to the active forms amongst the brain neuropeptides

28

, were

selected two peptides amidated at their C-termini, from each family to be investigated by MSI: AYTYVSEYKRLVPYNFGL-NH2 and LPVYNFGL-NH2 amongst the AmASTs A, and APMGFQGMR-NH2 and ARMGFHGMR-NH2 amongst the AmTRPs. Thus, the figures 5A and 5B are showing the mapping of the neuropeptide AmAST A (59-76) AYTYVSEYKRLPVYNFGL-NH2 in the brain of non-aggressive and aggressive worker bees, respectively; meanwhile, the figures 5C and 5D are showing the mapping of



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AmAST A (69 -76) LPVYNFGL-NH2 in the brain of non-aggressive and aggressive worker bees, respectively.

Using the same approach, the figures 6A and 6B are

showing the pattern of distribution of AmTRP (88 - 96) APMGFQGMR-NH2 in the brain of non-aggressive and aggressive worker bees, respectively; meanwhile, the figures 6C and 6D are showing the mapping of AmTRP (254-262) ARMGFHGMR-NH2 through the section of the brain of non-aggressive and aggressive worker bees, respectively. In order to perform a proof of concept about the potential biological role of the AmASTs A and AmTRPs identified in the present proteomic analysis, we selected some of the neuropeptides derivatives of both protein precursors, apparently involved with the modulation of aggressiveness in the brain of Africanized honeybee workers, to perform the biological validations of the findings reported above. Thus, the peptides AmAST A (69-76) or AmTRP (88 – 96) were synthesized on solid-phase, purified and used in behavioral bioassays. For developing these assays 1 ng of each neuropeptide was injected into the hemocele of 7-days old honeybee workers. The results of these assays are shown in Figure 7, which reveals that AmTRP (88 – 96) modulated significantly all the characteristic aggressiveness-related behaviors (alert - flying around; attraction – landing over target surface, and target inspection with antennae; aggression - bites with the mandibulae, abdomen vibration, protrusion of sting, and target stinging), while the neuropeptide AmAST A (69 – 76) did not modulate significantly “target inspection with the antenna”, but also modulated all the other aggressive behaviors. AmTRP (88 – 96) seems to elicits all the aggressiveness-related behaviors with higher frequencies than AmAST

A

(69 – 76).

Meanwhile, the workers of the control group only

presented the behaviors of attraction to the target and its inspection with the antenna, in low frequencies.

DISCUSSION The aggressive behavior of honeybees is mainly characterized by the stinging inflicted against the intruders and/or predators of their colonies.

To simulate this

situation, black leather ball targets were waved in front of the nest entrance of Africanized A. mellifera. The guard workers alarmed the colony about the presence of some potential threats caused by the presence of intruders and/or predators within the area of security of the colony. As consequence, groups of aggressive workers (stingers) flew in direction of the target, landed over its surface, and stung it; these individuals


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remained attached to the target’s surface through their sting lancets (which contains many barbs, that contribute to this attachment). All these actions occurred within a time window of 30 seconds. The stinger workers (attached to the targets) were then collected, and immediately frozen in liquid nitrogen. The use of the sting for venom inoculation in the victims/predators, is the climax of the aggressive behavior, which in turn is part of the defensive response for protecting the colony

29

. The stinging behavior seems to present a very complex

regulation which includes colors, odors and movements of the intruders/predators, but also there are a series of nest factors, such as alarm pheromones and decoding of chemical signals by the guard worker bees

9, 30, 31

. The modulation of the stinging

behavior in honeybee brain seems to involve a series of biogenic amines acting at level of different neural receptors to shape the aggression of these insects

29, 30

. In insects’

brain in general, the levels of serotonin seems to be correlated to the fight-or-flight response

32

, while the concentrations of octopamine is correlated to a transient

increasing in aggressiveness33,

34

.

Specifically in honeybees the use of exogenous

antagonists of endogenous biogenic amines, seem to stimulate the sting extension reflex 35

. Apparently, during the aggressive behavior the brain of aggressive workers undergo

pronounced metabolic changes, such as the inhibition of the oxidative phosphorylation, but maintain active the aerobic glycolysis 1,8. Honeybees have been used as model of study of physiology of stress since these insects are susceptible to various stressing factors, such as climate changes, use of pesticides, occurrence of diseases, existence of parasites, and availability of suitable forage sources33. Apparently, the stressor factors may strongly influence the defensive behavior of honeybees, including the aggressiveness 36. The investigations about how the stress may be accessed in honeybees include the observation of behavioral responses such as the extension of the sting lancets, as well as physiological responses such as measurement of the levels of neurotransmitters, juvenile hormone, biogenic amines, and neuropeptides

33, 37, 38

. In this context the response “fight-or-flight” have been

investigated in the model of study. The role of neuropeptides is not very clear in these studies; apparently ASTs A and TRPs are involved in the modulation of the energetic metabolism, mainly the catabolism of glucose and threalose

38, 39, 40

. The action of these families of

neuropeptides in honeybee brain has not been studied yet.

A recent investigation

identified 158 different neuropeptides in honeybee brain, from which 80 of them were



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derived from 22 precursors41. A careful examination of the annotations of honeybee genome, reveals that the neuropeptides AmASTs A and AmTRPs are biosynthesized as large protein precursors, that potentially undergo many proteolytic cleavages and metabolic transformations, to produce a series of neuropeptides amidated at their Cterminus, which constitute the active forms of these compounds41. The Figures 4A and 4B show the distribution of the precursor AmAST A in the brains of non-aggressive and aggressive workers, respectively; meanwhile, Figures 4C and 4D show the distribution of the precursor AmTRP in the brains of non-aggressive and aggressive workers, respectively. These figures are revealing that the precursors AmAST A and AmTRP are concentrated in the central and left regions of the brains of non-aggressive workers (Figures 4A and 4C). In the central brain the neuropils in which the precursors are more concentrated are the central body, right antennal lobe, and the regions of α- and β-lobes. In the left brain the neuropils in which the precursors are concentrated correspond to the regions extending from the upper left lateral calyx until the upper of left medulla. The brains of aggressive individuals revealed very low concentrations of the precursors AmAST A and AmTRP (Figures 4B and 4D), after the aggression itself. The results above are suggesting that the precursor proteins were cleaved to produce the mature allatostatin A and tachykinin-related peptides; the Figures 5 and 6 are corroborating this hypothesis. The Figures 5A and 5C are showing the distribution of the mature neuropeptides AmAST A (59-76) (AYTYVSEYKRLVPYNFGL-NH2) and AmAST A (69-76) (LPVYNFGL-NH2) in the brains of non-aggressive workers, where it is possible to observe that the large most neuropils present no (or very reduced) concentration of these neuropeptides. However, the brain of aggressive workers presented several neuropils containing relatively high concentrations of AmAST A (59-76) (upper region of right antennal lobe, left/right sections of lobulla, peduncle, and α- /β-lobe, and part of the left medulla) (Figure 5B). The neuropeptide AmAST A (69 76) was observed in high concentrations in the most neuropils of the brains of aggressive workers, except in both medial calyxes (Figure 5D). The Figure 6 shows a similar situation for the matures neuropeptides AmTRP (88 - 96) (APMGFQGMR-NH2) and AmTRP (254-262) (ARMGFHGMR-NH2). Both neuropeptides were absent from the most regions of brains from non-aggressive workers; however, AmTRP (88 - 96) was present in the most neuropils of the brain of aggressive workers (Figure 6B). The neuropeptide AmTRP

(254 – 262) (ARMGFHGMR-NH2) was absent from the


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brain of non-aggressive workers, but occurred in low concentrations in the most neuropils of the brain of aggressive workers (Figure 6D). The results above are strongly supporting the hypothesis that the precursor AmAST A existing in the brain before the aggressive behavior, was cleaved, generating two mature allatostatins, AmAST A (59-76) and AmAST A (69-76). Meanwhile, and the precursor tachykinin-related peptide was cleaved resulting the AmTRP (88 - 96) and AmTRP (254 - 262), which acted directly in different neuropils, to modulate the aggressiveness of worker honeybees. The presence of the precursors AmAST A and AmTRP, as well their mature derivatives, have been previously reported in honeybee brain mostly by mass spectrometric analysis.10,

11, 26, 42

However, the present study

represents the first initiative considering the precursors and their mature neuropeptides together in a continuous physiological process, associated to the aggressive behavior in honeybees. Taking into account that MSI is a semi-quantitative technique, is possible to estimate the changes in the relative concentrations of the neuropeptides under study in each specific neuropils, during the lasting time between the initial targets presentation and the stinging action by the guard bees (about 30 seconds). Considering that both precursors and the mature neuropeptides are more concentrated in some neuropils, it is possible to compare the relative concentrations of the neuropeptides in specific brain regions (based on the color scale of each image), using the data obtained for nonaggressive and aggressive individuals. Thus, it is possible to observe that the relative concentrations of the precursors AmAST A and AmTRP decreased about 100-fold in the right antennal lobe, and 2-fold in the left medulla, in the 30 seconds that lasted each experiment. In the mean time, the concentrations of AmAST A (59 – 76) and AmAST A (69 – 76) increased from 4- to 55-fold in left brain region including the antennal lobe, lobulla,

medulla, and peduncle, of aggressive honeybees. The concentrations of

AmTRP (88 – 96) and AmTRP (254 – 262) increased from 4- to 25-fold, depending on the neuropil,

in the brain of aggressive honeybees.

The use of techniques of

immunohistochemistry already revealed the localization of neuropeptides in specific neuropils of honeybee brain, like those reported above 42. Thus, in previous studies Am ASTs A and AmTRPs were reported in parts of the mushroom bodies and in the antennal lobes (but no specific behavior was stimulated workers).43,

44

in the target honeybee

Frequently, these neuropeptides are localized in peptidergic neurons

(which also release classical neurotransmitters), in which the neuropeptides act as co-



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transmitters42,

modulating

in

the

role

of

specific

neurons/neuropils

in

neurophysiological processes associated to some behaviors. However, the actions of these neuropeptides in the whole honeybee brain, and/or in different neuropils still remains to be elucidated. The role of AmASTs and AmTRPs have been learned from different studies with a large number of animals from different taxa; the role of neuropeptides in these animals is very diverse, so that it is necessary to be careful in the extrapolation of roles fron one species to another 42. Another important aspect that must be emphasized is that the grouping of the neuropeptides into families is based on their sequence similarity and their common origin from a common precursor; as example, at least five AmASTs A and seven AmTRPs may be generated from the maturation of each respective precursors (Figure 3). However, this does not means that each one of the peptides belonging to the same family will present the same activity; they may be expressed in different neurons/neuropils, activating different target receptors, associated to different neurophysiological processes42. The Allatostatins refers to a group of neuropeptides presenting inhibitory action on brain-associated-glands, known as corpora allata and corpora cardiaca, which are responsible by the biosynthesis of juvenile hormone (JH) (sesquiterpenoids compounds that play central role in the metamorphosis and reproduction of insects in general) 45, 46 . The modulation in the production of juvenile hormone has been used as reference to define the activity of the allatostatins 47. Based on sequence similarity of neuropeptides from different insect species, the members of allatostatins family are classified into three subfamilies (A, B, and C): i) sub-type A for those peptides presenting the domain Y/FXFGL-NH2 at the C-terminal region; ii) sub-type B for the peptides presenting W(X)6W-NH2 at the C-terminal region; iii) sub-type C for the peptides presenting the domain -PISCF-

48 - 50

. In A. mellifera the presence of allatostatins A and C, as well

their specific receptors were previously reported 12. Despite the functional properties of allatostatins were initially associated to the inhibitory activity of the biosynthesis of JH; their functional role in adult insects still remains to be determined; in honeybees these peptides seem to be related to the modulation of learning12. The allatostatins identified in the present study correspond to sub-type A, and may include up to five peptides originated from the same protein precursor, as shown in Figure 3B.

Amongst the identified peptides, one of them presents the C-terminal

residue in the acidic form: AmAST A (59 – 66) AYTYVSEY-OH (as shown in figure


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3B). This neuropeptide was detected and identified in our study, but apparently it was not enough characterized in the literature to ensure its allatostatic activity; therefore, it was not investigated in the present study. The two most evidently detected allatostatins were AmAST A (59-76) (AYTYVSEYKRLPVYNFGL-NH2), and AmAST A (69-76) (LPVYNFGL-NH2); it is important to emphasize that the former peptide contain the sequence of the second one at its C-terminal regions. No peptide of sub-type C was detected in the present study. Tachykinins constitutes a well-conserved group of multifunctional peptides in the vertebrates, which acts in the brain and guts, presenting central roles in neurotransmission and/or neuromodulation of the nervous system

51

. In insects it is

reported the existence of a family of tachykinin-related peptides (TRP), isolated from the brain–corpora cardiaca–corpora allata–suboesophageal ganglion, which share some sequence similarity in relation to those

peptides from vertebrates

52

.

The insect

peptides induce motility of smooth muscles, as well myotropic activity on hindgut and foregut 53. Different TRPs have been identified in the nervous system of the honeybees 54

, and tachykinin-like receptors have been identified in these insects by sequence

homology comparison with Drosophila

55

.

In insects TRPs are known to be

51

myostimulatory of the midgut muscle ; in honeybees these peptides are designated as AmTRPs, and were previously reported in different brain neuropils according to the age of the insects, apparently correlated with brain maturation during aging

11

, and

playing the neuromodulation of a series of processes related to the division of labor

56,

57

. A careful examination of the precursor AmTRP sequence deposited in UNIPROT

(http://www.uniprot.org/) reveals seven potential peptides (Figure 3B); however, only three AmTRPs were reported in the present study (Figure 2B): AmTRP

(69-85)

(NSIINDVKNELFPEDIN-OH), AmTRP (88 – 96) (APMGFQGMR-NH2), and AmTRP (254 – 262) (ARMGFHGMR-NH2) (Figures S4 to S6). The neuropeptide AmTRP (6985) was not studied since it was detected in its acidic, inactive form, while the two active forms were investigated by MSI in the present study. Mature insects TRPs usually present the conserved motif FXGLM-NH2 at their C-terminal region 52; however, in honeybees the conserved motif seems to be -FXGMR-NH2.

Biological validation of the proteomic/peptidomic results Honeybees live in colonies organized into different castes regulated by a temporal polyethism, in which workers of different ages present different behavioral



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roles to maintain the colony under sustainable condition

58

.

The young workers are

responsible by the duties performed exclusively within the hive (mainly brood care and nest cleaning) – these workers are as “nurses”; the workers remain under this condition since they are new born until they become 15-days old, when they become in transition between inside

duties and foraging activities (performed externally to the hive).

Therefore, from the 15th to the 21st day of life the workers perform tasks of “guards and foragers” 59. Generally, workers older than 20-days old become “foragers” after behave a brief period doing colony surveillance (as “guards”); frequently foragers are engaged with colony defense

60

. Guards represent from 10 to 15% of the individuals of the

colony, and are responsible by alerting the colony about a threat in the neighborhood, such as the presence of intruders/predators of the colony 61. The number of guards at the entrance of hive seems to be related to the defensive intensity of the aggressive response to a specific disturbance. The aggressive response in honeybee workers (mostly guards) is characterized by a series of sequential behaviors, that precede the stinging action; they are known as alarm-behaviors, and are classified as: i) alert - when the workers are attracted to out-side nest; in this behavior type are included the actions of flying around the target/predator with rapid and random movements; ii) attraction – characterized by landing on the target/predator surface, and inspection of the threat with the antenna; and iii) aggression – characterized by bites of target/predator skin with the madibulae, positioning of the abdomen upward accompanied by pumping/vibration movements, extension and protraction of the sting lancets, and target/predator stinging 21. The synthesis of venom by young workers begins when they are 2-/3-days old, reaching the maximal production when bees are 14 - 21-days old (100 µg of dry weight); venom production apparently decreases after the 25th day of age. Honeybee worker venom glands produce venom in a single secretory cycle beginning at the end of pupation, and reaching the maximum around between the 16th – 20th day of the worker’s adult life. New born workers do not sting; and according to the temporal polyethism the workers will begin to use their sting by the age of 14 days, for defending their self against predators 62 . In order to investigate the correlation between the proteopeptidomic findings in honeybee brain with the aggressive behavior of worker bees, it was set up a bioassay based on the observation of a series of aggressiveness-related behaviors of the honeybee workers (flying around, attraction, target inspection with antenna, bites with the


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mandibulae, abdomen vibration, protrusion of sting, and target stinging) under effect of 1 ng of AmAST A (69-76) or AmTRP (88 – 96) (Figure 7). It was not expected that 7-days old worker honeybees would present any of the aggressive behaviors described above; however, when the young bees received each neuropeptide, via hemocele application, the neurohormones possibly reached the brain carried by the circulatory system, and within a period of five minutes the young honeybee workers presented a complete series of aggressive behaviors, including some stinging on the target. Meanwhile, the young workers that received physiological solution, only present two of the aggressive behaviors at low frequency. The neuropils which presented the highest concentration of AmASTs were: the upper region of right antennal lobe, left/right sections of lobulla, peduncle, and α- /βlobe, and part of the left medulla, which corresponded to the neuropeptidergic regions of honeybee brain. These neuropils correspond to the high-order processing centers, generally involved with the division of labor, including the colony defense

43

. Thus, it

is possible that the neuropeptides AmAST A (69-76) or AmTRP (88 – 96) may be related to the regulation of honeybee brain to promote the transition from the condition of “nurses” to “guards/foragers”. The role of the ASTs and TRPs have investigated in large number of different insect species, but not so intensively in honeybees; multiple physiological roles have been attributed to this neurohormones, such as controlling process of heart beating, gut motility, nutrient absorption, migratory preparedness, and even modulation of the circadian cycle 47. Tachykinins are involved in feeding regulation in insects, affect their olfactory and locomotor behaviors

63, 64

and also may influence the perception and

localization of a food source and its actual collection

25

. There are metabolomic and

transcriptomic evidences indicating that the aggressivity in honeybees seems to be strongly correlated with a decreasing of the oxidative phosphorylation, accompanied of an increase in the glycolysis at level of brain of the stinger workers 1,7, 8, 31. Taking into account the high concentrations of AmASTs A and AmTRPs in honeybee brain of the aggressive workers, it is possible that these neuropeptides are also involved in the metabolic regulation in these individuals. The mechanisms of action of the neuropeptides seem to be very complex in honeybee brain, and the downstream pathways involved in the mediation of the physiological processes related to the aggressiveness is still waiting to be clarified. Despite to this, in the



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present study it was clearly demonstrated a strong correlation between their presence and the aggressive behaviors of honeybee workers.

CONCLUSIONS The specific role of allatostatin A and tachykinin-related neuropeptides in honeybee brain during the aggressive behavior was unknown, however the results above are clearly demonstrating the processing of the precursors AmASTs A and AmTRPs into mature form of neuropeptides. The present investigation clearly reported the presence of some derivatives of the precursor neuropeptides, such as AmAST A (59-76) (AYTYVSEYKRLPVYNFGL-NH2), AmAST A (69-76) (LPVYNFGL-NH2), AmTRP (88 – 96) (APMGFQGMR-NH2), and AmTRP (254 – 262) (ARMGFHGMR-NH2) in several different neuropils of honeybee brain, during the aggressive behavior, possibly neuromodulating different aspects of this complex behavior. The biological validation of the findings reported above was done by using assays based on the observation of aggressiveness-related behaviors; 7-days old honeybee workers (biologically nonaggressive individuals) received 1 ng of AmAST A (69-76) or AmTRP (88 – 96), and during five minutes presented a complete series of the aggressive behaviors, corroborating the main hypothesis of the present study, that correlates the presence of mature AmASTs A and AmTRPs in honeybee brain with the aggressiveness of this insect.

ASSOCIATED CONTENT The supporting information is available free of charge on the ACS Publications website at DOI:…………

Supporting Information Figure S1. (A) CID spectrum obtained for the neuropeptide AmAST A (59 - 66) under CID conditions in the positive mode, already deconvoluted in the form of [M + H]+, with the respective sequence interpretation (B). It was selected the precursor ion of m/z 995.43 for fragmentation, and spectrum was characterized by the assignment of a series of daughter-ions of b-types. Figure S2. (A) CID spectrum obtained for the neuropeptide AmAST A (59 -76) under CID conditions in the positive mode, already deconvoluted


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in the form of [M + H]+, with the respective sequence interpretation (B). It was selected the precursor ion of m/z 2182.13 for fragmentation, and spectrum was characterized by the assignment of a series of daughter-ions of b-type. Figure S3. (A) CID spectrum obtained for the neuropeptide AmAST A (69-76) under CID conditions in the positive mode, already deconvoluted in the form of [M + H]+, with the respective sequence interpretation (B). It was selected the precursor ion of m/z 921.51 for fragmentation, and spectrum was characterized by the assignment of a series of daughter-ions of b-type. Figure S4. (A) CID spectrum obtained for the neuropeptide AmTRP (69-85) under CID conditions in the positive mode, already deconvoluted in the form of [M + H]+, with the respective sequence interpretation. It was selected the precursor ion of m/z 1974.98 for fragmentation, and spectrum was characterized by the assignment a series of daughter-ions of b-type (B). Figure S5. (A) CID spectrum obtained for the neuropeptide AmTRP (88 - 96) under CID conditions in the positive mode, already deconvoluted in the form of [M + H]+, with the respective sequence interpretation (B). It was selected the precursor ion of m/z 993.47 for fragmentation, and spectrum was characterized by the assignment a series of daughter-ions of b-type. Figure S6. (A) CID spectrum obtained for the neuropeptide AmTRP (254 - 262) under CID conditions in the positive mode, already deconvoluted in the form of [M + H]+, with the respective sequence interpretation. It was selected the precursor ion of m/z 1061.52 for fragmentation, and spectrum was characterized by the assignment a series of daughterions of b-type (B). AUTHOR INFORMATION Corresponding author: CEIS-IBRC- UNESP, Av. 24A nº 1515, Bela Vista - Rio Claro, SP, Brazil, CEP 13506-900, FAX: 55-(19)-35348523. E-mail: [email protected] ORCID Mario Sergio Palma: 0000-0002-7363-8211 Author Contributions M.S.P, M.P. and A.R.S.M designed research and analyzed the mass spectrometric analysis; O.M. supplied the honeybees and supervised the aggressiveness assays; F.G.E. performed the behavioral bioassays and treated the data; M.P. and A.R.S.M. acquired the experimental data of MALDI-MSI; K.U.S. synthesized and purified the neuropeptides; M.S.P. wrote the paper. All authors contributed to the edition of the paper. Notes The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

This work was supported by a grant from the São Paulo State Research Foundation (FAPESP,

BIOprospecTA

program

2016/1621205);


M.P.

was

fellow

from


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PIBITI/CNPq Program, A.R.S.M was PhD fellow from FAPESP (Proc. 2015/26025-5). M.S.P. is researching for CNPq (Proc. 300377/2003-5)

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(32) Edwards, D. H.; Kravitz, E. A. (1997). Serotonin, social status and aggression. Curr. Opin. Neurobiol. 1987, 7, 812-819. (33) Even, N.; Devaud, J.M.; Barron, A.B. General Stress Responses in the Honey Bee, Insects 2012, 3, 1271-1298; doi:10.3390/insects3041271. (34) Rillich, J.; Schildberger, K.; Stevenson, P. A. (2011). Octopamine and occupancy: an aminergic mechanism for intruder-resident aggression in crickets.Proc. R. Sco. B. Biol. Sci. 2011, 278, 1873-1880. (35) Tedjakumala, S. R., Aimable, M. and Giurfa, M. (2014). Pharmacological modulation of aversive responsiveness in honey bees. Front. Behav. Neurosci. 2014, 7, 221. (36) Khoury, D.S.; Myerscough, M.R.; Barron, A.B. A Quantitative Model of Honey Bee Colony Population Dynamics. PLoS One 2011, 6, e18491. (37) Corona, M.; Velarde, R.A.; Remolina, S.; Moran-Lauter, A.; Wang, Y.; Hughes, K.A.; Robinson, G.E.(2007). Vitellogenin, juvenile hormone, insulin signaling, and queen honey bee longevity. Proc. Natl. Acad. Sci. USA 104, 7128-7133.


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(38) Veenstra, J.A. Does corazonin signal nutritional stress in insects? Insect Biochem. Mol. Biol. 2009, 39, 755-762. (39) Crailsheim, K. Intestinal transport of sugars in the honeybee (Apis mellifera L.). J. Insect Physiol. 1988, 34, 839 - 845. (40) Balt, J.; Roces, F. Haemolymph sugar levels in foraging honeybees (Apis mellifera carnica): Dependence on metabolic rate and in vivo measurement of maximal rates of trehalose synthesis. J. Exp. Biol. 2001, 204, 2709 – 2716. (41) Honey Bee Genome Sequencing Consortium. Insights into social insects from the genome of the honey bee Apis mellifera. Nature 2006, 443, 931-949. (42) Han, B.; Fang, Y.; Feng, M.; Hu, H.; Qi, Y.; Huo, X.; Meng, L.; Wu,B.; Li, J. Quantitative Neuropeptidome Analysis Reveals Neuropeptides Are Correlated with Social Behavior Regulation of the Honeybee Workers. J. Prot. Res. 2015, 41, 4382 – 4393. (43) Galizia, C.G.; Kreissl, S. Neuropeptides in Honey Bees (Chapter 37) In: Honeybee Neurobiology and Behavior- A Tribute to Randolf Menzel , 1st ed.; Galizia, C.G; Eisenhardt, D.; Giurfa, M., Editors; Springer: London, UK, 2012; pp 211 – 226. (44) Takeuchi, H.; Yasuda, A.; Yasuda-Kamatani, Y.; Sawata, M.; Matsuo, Y.; Kato. A.; Tsujimoto, A.; Nakajima, T.; Kubo, T. Prepro-tachykinin gene expression in the brain of the honeybee Apis mellifera . Cell Tissue Res. 2004, 316, 281–293. (45) Lorenz, M.W.; Hoffmann, K.H. Allatotropic activity in the suboesophageal ganglia of crickets, Gryllus bimaculatus and Achaeta domesticus (Ensifera, Grullidae). J. Insect Physiol. 1995, 41, 191–196. (46) Li, G.; Granger, N.A.; Roe, R.M. The juvenile hormones: historical facts and speculations on future research directions. Insect Biochem. Mol. Biol. 2000, 30, 617 – 644. (47) Hernández-Martínez, S.; Li, Y.; Lanz-Mendoza, H.; Rodríguez, M.H.; Noriega, F.G. Immunostaining for allatotropin and allatostatin-A and -C in the mosquitoes Aedes aegypti and Anopheles albimanus. Cell Tissue Res. 2005, 321, 105–113. (48) Stay, B,.; Tobe, S.S.; Bendena, W.G. Allatostatins - identification, primary structures, functions and distribution. Adv Insect Physiol. 1994, 25, 267–337.

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(51) Nassel D.R. Tachykinin-related peptides in invertebrates: a review. Peptides 1999, 20, 141–158 (52) Van Loy, T.V.; Vandersmissen, J.V.; Poels, J.; Van Hiel, M.B.; Verlinden, H.; Broeck, J.V. Tachykinin-related peptides and their receptors in invertebrates: A current view. Peptides 210, 31, 520–524. (53) Schoofs, L.; Holman, G.M.; Hayes, T.K.; Kochansky, J.P.; Nachman, R.J.; Loof, A.D.L. Locustatachykinin III and IV: two additional insect neuropeptides with homology to peptides of the vertebrate tachykinin family. Regul Pept, 1990, 31, 199– 212. (54) Boerjan, B.; Cardoen, D.; Bogaerts, A.; Landuyt, B.; Schoofs, L.; Verleyen, P. Mass spectrometric profiling of (neuro)-peptides in the worker honeybee, Apis mellifera. Neuropharmacology 2010, 58, 248–258. (55) Hause, F.; Cazzamali, G.; Williamson, M.; Blenau, W.; Grimmelikhuijzen, C.J.P. A review of neurohormone GPCRs present in the fruitfly Drosophila melanogaster and the honey bee Apis mellifera. Prog. Neurobiol. 2006, 80, 1–19. (56) Nassel, D.R. Tachykinin-related peptides in invertebrates: a review. Peptides 1990, 20, 141–158. (57) Takeuchi, H.; Yasuda, A.; Yasuda-Kamatani, Y.; Kubo, T.; Nakajima, T. Identification of a tachykinin-related neuropeptide from the honeybee brain using direct MALDI-TOF MS and its gene expression in worker, queen and drone heads. Insect Mol. Biol. 2003, 12, 291 – 298. (58) Winston, M. L.. The Biology of the Honey Bee. Cambridge, MA: Harvard University Press 1987; 281 pp. (59) Nouvian, M.; Reinhard, J.; Giurfa, M. The defensive response of the honeybee Apis mellifera . J. Experim. Biol. 2016, 219, 3505-3517. (60) Paxton, R. J.; Sakamoto, C. H.; Rugiga, F. C. N. Modification of honeybee (Apis mellifera L.) stinging behavior by within-colony environment and age. J. Apic. Res. 1994, 33, 75-82. (61) Moore, A. J.; Breed, M. D.; Moor, M. J. The guard honey bee: ontogeny and behavioral variability of workers performing a specialized task. Anim. Behav. 1987, 35, 1159-1167. (62) Roat, T.C.; Nocelli, R.C.F.; Cruz-Landim, C. Ultrastructural modifications in the venom glands of workers of Apis mellifera L. (Hymenoptera: Apidae) promoted by topical application of juvenile hormone. Neotrop. Entomol. 2006, 35, 469-476.


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Legend of Figures

Figure 1. (A) schedule of the sagittal plan positioning in Africanized honeybee head, used as references for brain slicing; (B) frontal view of the brain slice at the sagittal plan of honey bee head, showing the contour maps of the different brain neuropils (Photograph was a courtesy of Mario Sergio Palma. Copyright 2018.). Figure 2. Global MALDI-TOF/MS spectra obtained in the positive mode, for the whole brain slice: (A) of non-aggressive workers, and (B) of aggressive workers.

Figure 3. (A) Sequences of the precursors of Allatostatin A and Tachykinin-RelatedPeptide; the underlined sequences are indicating the position of the mature neuropeptides along the precursor protein; (B) Sequences of the mature neuropeptides, with the indication of the respective form of the C-terminal residues (acidic or amidated), positioning numbers in the precursor sequence, and molecular weight values.

Figure 4. MALDI Spectral Imaging of the precursor of AmAST and AmTRP in the brain of non-aggressive (A) and aggressive (B) honeybee workers. The images of AmAST were produced by the overlapping of the m/z values of eleven tryptic peptides (m/z 1300.6644, 1170.4103, 1123.5306, 1116.5578, 1107.6197, 1077.4959, 1005.4636, 996.5149, 899.4621, 886.4305, and 821.4304). MALDI Spectral Imaging of the



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precursor of AmTRPs in the brain of non-aggressive (C) and aggressive (D) honeybee workers. The images of AmTRP were produced by the overlapping of the m/z values of ten tryptic peptides (m/z 1662.7129, 1655.8023, 1589.7369, 1495.7461, 1294.6426, 1271.7093, 1263.6514, 1218.6001, 1188.6358, and 1183.5524). The spatial distribution of the neuropeptides were semi-quantitatively represented in a relative scale, shown at the right side of the figure. Figure 5. MALDI Spectral Imaging of some mature AmAST neuropeptides in honeybee brain of non-aggressive workers (A, and C), and aggressive workers (B, and D). The spatial distribution of the neuropeptides were represented semi-quantitatively is a relative scale, shown at the right side of the figure. The neuropeptide AmAST (59-76) (m/z 2182.13) is represented in (A) and (B), for the brain of non-aggressive and aggressive workers, respectively. The neuropeptide AmAST (69 -76) is represented in (C) and (D), for the brain of non-aggressive and aggressive workers, respectively. The spatial distribution of the neuropeptides were semi-quantitatively represented in a relative scale, shown at the right side of the figure.

Figure 6. MALDI Spectral Imaging of some mature AmTRP neuropeptides in honeybee brain of non-aggressive workers (A, and C), and aggressive workers (B, and D). The spatial distribution of the neuropeptides were represented semi-quantitatively in a relative scale, shown at the right side of the figure. The neuropeptide AmTRP (88 96) is represented in (A) and (B), for the brain of non-aggressive and aggressive workers, respectively. The neuropeptide AmTRP (254-262) is represented in (C) and (D), for the brain of non-aggressive and aggressive workers, respectively. The spatial distribution of the neuropeptides were semi-quantitatively represented in a relative scale, shown at the right side of the figure. Figure 7. Results of the assays of aggressiveness-related behaviors of 7 days-old Africanized honeybee workers (A. mellifera), in presence 1 ng of the neuropeptides AmAST (69 - 76) and AmTRP (88 - 96) injected in the hemocele of the insects. The results are expressed as the mean ± SEM of three experiments. *p < 0.005, a significant difference compared with the mean values of the neuropeptides/control group.


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Table of contents (for TOC only)

Laser shot

MSI of honeybee brain Brain slice


Journal of Proteome Research 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 1. (A) schedule of the sagittal plan positioning in Africanized honeybee head, used as references for brain slicing; (B) frontal view of the brain slice at the sagittal plan of honey bee head, showing the contour maps of the different brain neuropils (Photograph was a courtesy of Mario Sergio Palma. Copyright 2018.).


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Figure 2. Global MALDI-TOF/MS spectra obtained in the positive for the brain slice obtained at the sagittal plan: (A) for non-aggressive workers, and (B) for aggressive workers.



Figure 3. (A) Sequences of the precursors of Allatostatin A and Tachykinin-RelatedPeptide; the underlined sequences are indicating the position of the mature neuropeptides along the precursor protein; (B) Sequences of the mature neuropeptides, with the indication of the respective form of the C-terminal residues (acidic or amidated), positioning numbers in the precursor sequence, and molecular weight values.


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Page 33 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 4. MALDI Spectral Imaging of the precursor of AmAST A and AmTRP in the brain of non-aggressive (A) and aggressive (B) honeybee workers. The images of AmAST A were produced by the overlapping of the m/z values of eleven tryptic peptides (m/z 1300.6644, 1170.4103, 1123.5306, 1116.5578, 1107.6197, 1077.4959, 1005.4636, 996.5149, 899.4621, 886.4305, and 821.4304). MALDI Spectral Imaging of the precursor of AmTRPs in the brain of non-aggressive (C) and aggressive (D) honeybee workers. The images of AmTRP were produced by the overlapping of the m/z values of ten tryptic peptides (m/z 1662.7129, 1655.8023, 1589.7369, 1495.7461, 1294.6426, 1271.7093, 1263.6514, 1218.6001, 1188.6358, and 1183.5524). The spatial distribution of the neuropeptides were semi-quantitatively represented in a relative scale, shown at the right side of the figure.



Figure 5. MALDI Spectral Imaging of some mature AmAST A neuropeptides in honeybee brain of non-aggressive workers (A, and C), and aggressive workers (B, and D). The spatial distribution of the neuropeptides were represented semi-quantitatively is a relative scale, shown at the right side of the figure. The neuropeptide AmAST A (59-76) (m/z 2182.13) is represented in (A) and (B), for the brain of non-aggressive and aggressive workers, respectively. The neuropeptide AmAST A (69 -76) is represented in (C) and (D), for the brain of non-aggressive and aggressive workers, respectively. The spatial distribution of the neuropeptides were semi-quantitatively represented in a relative scale, shown at the right side of the figure.


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Figure 6. MALDI Spectral Imaging of some mature AmTRP neuropeptides in honeybee brain of non-aggressive workers (A, and C), and aggressive workers (B, and D). The spatial distribution of the neuropeptides were represented semi-quantitatively in a relative scale, shown at the right side of the figure. The neuropeptide AmTRP (88 - 96) is represented in (A) and (B), for the brain of non-aggressive and aggressive workers, respectively. The neuropeptide AmTRP (254-262) is represented in (C) and (D), for the brain of non-aggressive and aggressive workers, respectively. The spatial distribution of the neuropeptides were semi-quantitatively represented in a relative scale, shown at the right side of the figure.



Figure 7. Results of the assays of aggressiveness-related behaviors of 7 days-old Africanized honeybee workers (A. mellifera), in presence 1 ng of the neuropeptides AmAST (69 - 76) and AmTRP (88 - 96) injected in the hemocele of the insects. The results are expressed as the mean ± SEM of three experiments. *p < 0.005, a significant difference compared with the mean values of the neuropeptides/control group.


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(Apis mellifera) Brain - ACS Publications

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