International Journal of Biological Macromolecules 225 (2023) 848–860

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International Journal of Biological Macromolecules

journal homepage: www.elsevier.com/locate/ijbiomac

Major royal jelly proteins influence the neurobiological regulation of the

division of labor among honey bee workers
Yu Fang a, Mao Feng a, Chuan Ma a, Olav Rueppell b, Jianke Li a, *
a
Institute of Apicultural Research/Key Laboratory of Pollinating Insect Biology, Ministry of Agriculture, Chinese Academy of Agricultural Sciences, Beijing 100093, China
b
Department of Biological Sciences, University of Alberta, Edmonton, Alberta T6G2L3, Canada

A R T I C L E I N F O A B S T R A C T

Keywords: Age-based division of labor among workers is a fundamental life-history trait of many social insects, including
Task choice the Western honey bee, Apis mellifera L. Extensive studies of the causation of the most pronounced transition from
Royal jelly protein performing tasks in the nest to outside foraging indicate hormonal regulation of complex physiological changes.
Honeybee
However, the proximate neurobiological mechanisms that cause the behavioral repertoire to change are still not
Brain
Neurological regulation
understood and require novel approaches to be fully characterized. Thus, we established the first comprehensive
monoclonal antibody microarray in honey bees with 16,320 antibodies to directly identify proteins in the brain
that regulate the transition to foraging. Major royal jelly protein (MRJP) 1 and MRJP3 were identified as po­
tential protein effectors and further investigated. A series of experimental manipulations of the workers'
behavioral transition led to changes in MRJP1 and MRJP3 quantities in accordance with their presumed func­
tional role. Injection of MRJPs into the brain resulted in increased task-reversal from foraging to nursing and
decreased task-progression from nursing to foraging, while the latter was increased by injection with MRJP
antibodies. Finally, down-regulation of MRJP1 and MRJP3 expression via RNAi injection into the brain increased
the transition from in-hive nursing to outside foraging, confirming a causal role of these two proteins in the
proximate regulation of behavior and life-history of honey bee workers. Interaction partners of MRJP1 and
MRJP3 in the honey bee brain included other regulators of honey bee behavior and life history. Thus, our
transformative methodological advancement of proteome analysis in honey bees reveals novel regulators of
honey bee behavior, extends our understanding of the functional pleiotropy of MRJPs, and supports a general
nutrition-based model of the regulation of the age-based division of labor in honey bees.

1. Introduction
Social groups gain much of their competitive advantage over solitary
competitors because their members can efficiently divide labor by
specializing in particular tasks [1,2]. In addition to the most funda­
mental division between reproductives and non-reproductive workers,
further specialization among workers characterizes most social insect
societies, including the colonies of the Western honeybee (Apis mellifera
L.). Honeybee workers go through an age-based division of labor with a
pronounced transition from performing various in-hive tasks to outside
foraging [3]. When young, workers perform various behaviors inside the
hive but one major task is nursing, providing food and other care for the
brood, which develops inside wax cells and is completely dependent on
the nursing. Foraging is equally essential and the worker transition from
nursing to foraging has profound consequences for individual and
colony life-history [4–6]. The causation of this complex transition has
been extensively studied across different levels of biological organiza­
tion [7–11].
Intrinsic and extrinsic influences determine the age at which the
transition from in-hive tasks to foraging occurs: Specifically, the tran­
sition to foraging is influenced by brood and brood pheromone [12],
queen mandibular pheromone [13], and foragers and their pheromone
[9,14]. Various forms of stress at the individual and colony levels can
accelerate the transition to foraging [10,15,16]. Major endocrine regu­
lators, including insulin-like signaling, juvenile hormone, and vitello­
genin, play a central role in translating the external influences into the
concerted physiological changes of transitioning from nursing to
foraging [6,8,17], and foragers revert only under exceptional circum­
stances back to nursing [5]. Pronounced genotypic differences exist in
the age of this transition among honey bee populations [18,19] and

* Corresponding author.
E-mail address: apislijk@126.com (J. Li).

https://doi.org/10.1016/j.ijbiomac.2022.11.150
Received 8 October 2022; Received in revised form 13 November 2022; Accepted 14 November 2022
Available online 20 November 2022
0141-8130/© 2022 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-
nc-nd/4.0/).
Y. Fang et al.
genetic studies indicate multiple influential loci [20], as well as large-
scale shifts in the brain transcriptome [10] that accompany significant
neuronal reorganization during the transition [21]. At the proteomic
level, several differences between nurses and foragers have also been
identified that correlate with the alternative behavioral states [22–24].
Among other tasks, the in-hive nurses feed the growing brood with a
complex, proteinaceous secretion that contains several major royal jelly
proteins (MRJPs) [25]. In addition to their primary function as brood
food, MRJPs may also have other functions in honey bee life-history,
including influences on behavior [26–28]. Brood feeding entails a
physiological cost for nurses by depleting internal nutrients, including
the egg-yolk protein vitellogenin [29]. This storage protein also has
pleiotropic signaling effects and its depletion leads to an increase in
juvenile hormone and presumably an interaction with insulin-like
signaling to cause the onset of foraging [7,8]. Downstream mecha­
nisms that underlie the transition from nursing to foraging in the brain
include protein kinase G and biogenic amine changes [30,31] and
neuronal growth [32]. However, the causal links between these findings
remain to be elucidated and it is unclear whether nutritional influences
can also act directly on the brain to influence the onset of foraging in
honey bee workers. Thus, we developed the first monoclonal antibody
(mAb) array for the honeybee and used it to initiate an in-depth study of
the brain proteome changes that influence the complex behavioral
transition from in-hive nurse bees to outside foragers. Our novel
approach suggests a regulatory role of MRJPs in the division of labor in
honey bees, which we causally confirmed in extensive follow-up ex­
periments. These findings confirm the utility of the mAb array, shed
novel light on the regulation of age polyethism in honey bees, and
extend the functional pleiotropy of MRJPs.
2. Materials and methods
All experiments were conducted with honeybees (Apis mellifera lig­
ustica) raised at the Institute of Apicultural Research, Chinese Academy
of Agricultural Sciences, Beijing, China. After the construction of the
first comprehensive monoclonal antibody (mAb) array for honeybees
(Experiment 1), we used this new tool to compare the proteome of nurse
and forager brains in a series of observational and manipulative exper­
iments (Experiments 1–3). Based on the result that major royal jelly
proteins (MRJPs) show the biggest differences among the successfully
identified and validated proteins, we proceeded to characterize their
spatial expression in the brain (Experiment 2) and their protein inter­
action partners (Experiment 4). To investigate the causal involvement of
MRJP1 and MRJP3, we manipulated the levels of these proteins via
brain injections of RNAi, antibodies, or the MRJPs themselves and
observed the resulting changes in the transition from nursing to foraging
or its reversal (Experiments 5, 6).
2.1. Experiment 1: nurse – forager comparison using monoclonal
antibodies (mAbs) array
Newly emerged workers were obtained from brood frames of several
colonies that were transferred prior to emergence into an incubator
(34 ◦ C and 80 % relative humidity). Within 24 h, emerged workers were
labeled with a paint dot on their thorax and returned to respective hives.
From each cohort (= replicate), 80 nurses were collected from brood
frames while performing brood care, defined as poking their heads into
cells containing young larvae for at least 10 s. Correspondingly, 80
foragers were sampled at the hive entrances when returning with pollen
loads. All sampled bees were immediately anesthetized at 4 ◦ C, followed
by brain dissection according to previously described methods [33]. It
was noteworthy that the gland tissues coupled with the brain should not
be completely stripped out. The isolated brains were immediately
soaked in protease inhibitor (Roche, Basel, Switzerland) solution and
stored at − 80 ◦ C until further use.
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International Journal of Biological Macromolecules 225 (2023) 848–860
2.1.1. Establishment of mAb library and microarray for honey bees
To generate an in-depth antibody library, mAbs were produced
through the immunization of mice according to standard protocols [34].
Representative samples of multiple developmental stages (embryos on
day 3, larvae on day 6, pupae on day 12, and a blend of adult worker
brains) were selected as antigen sources to increase the coverage of the
library. The frozen honeybee samples were pooled together and then
ground into a fine powder with liquid nitrogen in the presence of 2 %
polyvinylpolypyrrolidone. All chemicals were purchased from Sigma-
Aldrich (St. Louis., MO., USA) unless otherwise specified. Five vol­
umes of ice-cold extraction buffer [pH 8.0, ingredients: 200 mM Tris-
HCl, 200 mM NaCl, 50 mM sodium ascorbate, 5 mM Na4EDTA, 10 %
glycerol, 1 % NP-40, 1 % Triton X-100, 1 % Tween-20, 0.25 % sodium
metabisulfite, protease inhibitor cocktail (Roche, Basel, Switzerland), 2
mM PMSF] were added to lyse the samples. Samples were gently
agitated at 4 ◦ C for 2 h. The resulting solutions were centrifuged at
15000g for 30 min to recover the supernatant.
The extracted proteins in the supernatant were mixed with complete
Freund's adjuvant to serve as antigens. BALB/c mice were immunized 4
times at 14-day intervals with the antigen mixture. Mice spleen cells
were isolated and fused with the P3X63Ag8.653 cell line to generate
hybridoma cells. Using polyethylene glycol as an adjuvant, hybridoma
cells were screened by ELISA to ensure that all selected clones were able
to detect an antigen at a concentration of <1 × 10− 8 M. Selected clones
were cultured for expansion, and antibodies were commercially har­
vested using standard protocols (Abmart Ltd., Shanghai, China). Each
antibody was printed with 10 nl of a 1 μg/μl solution per spot on the
surface of microscope slides coated with nitrocellulose (FAST chip,
Capitalbio, Beijing, China; Arrayjet Ltd., Roslin, UK) at 4 ◦ C and stored at
− 80 ◦ C before use. Before incubation, the antibody microarray slides
were washed and then blocked with 10 % BSA overnight at 4 ◦ C.
2.1.2. Brain proteome comparison
To compare the brain proteome between nurses and foragers, pro­
teins were extracted from pooled, frozen brains of 50 nurses and 50
foragers per biological replicate. Three biological replicates were
analyzed per trial, and the experiment included three independent trials.
After initial technical replicates indicated high reproducibility, no
further technical replicates were performed.
The diluted protein solution (1 μg/μl) was labeled with NHS-biotin
(Molecular Probes, Carlsbad, CA, USA). The labeling efficiency was
verified by Western Blotting (WB; see below). The labeled protein
samples were further diluted to a final concentration of 0.1 μg/μl with
TBS in the presence of 0.1 % Tween-20 (TBS + 0.1 % T20) and 10 %
BSA. This dilution was used to incubate the microarrays at room tem­
perature for 60 min. After three subsequent washes, the antigen-
conjugated microarray chips were incubated with Cy3-labeled Strepta­
vidin (1:5000) at room temperature for 60 min. After four additional
washes in TBS + 0.5 % T20 and two final rinses in ddH2O, the slides
were centrifuged to remove the remaining buffer and dried. Slides were
scanned by Genepix (Axon Instruments, Union City, CA, USA) and the
Cy3 fluorescence signals were detected at 570 nm.
To assess the differential abundance of proteins, Cy3 fluorescence
intensity was quantified by GenePix 6.0 software (Axon Instruments,
Union City, CA, USA) and further analyzed with the limma package
(http://bioconductor.org/). Raw data were normalized through back­
ground correction and quantile normalization using the “Background
Correct” and the “Normalize Between Arrays” modules, respectively. A
fold-change cutoff of 1.5 and a p-value cutoff of 0.05 were applied to
identify differences.
2.1.3. Protein isolation via immunoprecipitation
To isolate and subsequently identify the candidates with the most
significant differences between nurses and foragers, the mAbs corre­
sponding to the top 500 array spots were used for immunoprecipitation
(IP) experiments: For each mAb, 1 mg of extracted total protein from
Y. Fang et al.
pooled honey bee worker heads was prepared with protein G resin for 4
h at 4 ◦ C (Invitrogen, Carlsbad, CA, USA). After centrifugation, the su­
pernatant was incubated with cyanogen bromide-conjugated antibody
at 4 ◦ C overnight. The IP complex was collected after centrifugation,
redissolved in 0.2 M glycine solution (pH 2.5), and analyzed by LC-MS/
MS for identification (see below). Additional samples were separated by
SDS-PAGE followed by WB analysis (see below) to confirm the affinity of
the precipitated protein to the corresponding mAb.
2.1.4. Protein identification with LC-MS/MS
The eluted protein complexes were subjected to trypsin digestion.
The trypsin-digested proteins were analyzed by an EASY-nLC 1000
system (ThermoFisher Scientific, Waltham, MA, USA) coupled with a Q-
Exactive HF mass spectrometer (ThermoFisher Scientific, Bremen, Ger­
many). Acetonitrile with linear gradients from 2 % to 40 % was used for
the chromatograph separation over 60 min. Tandem mass spectra were
processed by PEAKS Studio (version 8.5, Bioinformatics Solutions Inc.,
Waterloo, Canada). PEAKS DB was used to search the NCBI Apis mellifera
database (downloaded April 2020, containing 23,478 entries), coupled
with the common Repository of Adventitious Proteins database (cRAP,
downloaded from The Global Proteome Machine Organization, April
2020). The search parameters were: parent ion tolerance, 15 ppm;
fragment tolerance, 0.05 Da; enzyme, trypsin; maximum missed cleav­
ages, 2; maximum variable PTM per peptide, 3; fixed modification,
carbamidomethyl (C, +57.02 Da); and variable modification, oxidation
(M, +15.99 Da). A protein was identified only if it contained at least one
unique peptide with at least two spectra (false discovery rate (FDR) ≤
1.0 %) in a fusion-decoy database search, a more conservative FDR
estimation than that of the target decoy approach [35]. The PEAKS Q
module was used for quantitative analyses of peptide and protein
abundance. Feature detection and alignment were performed with al­
gorithms of expectation-maximization and high-performance retention
time, respectively. Normalization to compute sample ratios was per­
formed by dividing each value by the total ion current (TIC) and
multiplying by the TIC of the reference sample. Based on the m/z shift
distribution, RT shift distribution, and ratio quality, the mass error
tolerance, retention time range, and feature quality were set, respec­
tively. Protein abundances in all samples were determined from the
three most abundant precursor ion peak intensities of the trypsin-
digested peptides. Proteins among different samples were regarded as
significantly different in abundance levels only when they exhibited a
fold-change of >1.5 and a p-value of <0.05.
2.1.5. Western blotting for confirming protein identity and quantitative
protein differences
To confirm the identity of the proteins identified by IP and LC-MS/
MS, Western blotting (WB) was performed according to standard pro­
tocols [36]. Additionally, WB also served the purpose to confirm dif­
ferences in target protein quantities between nurse and forager brains,
as well as quantify protein levels in other experimental groups (see
below). Briefly, equal amounts of proteins from select samples were
separated on SDS poly-acrylamide 12.5 % gels and transferred electro­
phoretically onto polyvinylidene fluoride (PVDF) membranes at 42 V at
room temperature (RT) for 2 h. The efficiency of transferring was
checking for the presence of prestained low molecular weight marker
bands on the membranes. Then the membranes were blocked with 5 %
skim milk at room temperature for 1 h, followed by incubation with
primary antibodies and HRP-anti-mouse IgG antibodies. The immune
reactive bands were visualized by an enhanced chemiluminescence
system (Biouniquer, China) and exposed to X-ray film. Signal intensities
were quantified by ImageJ and normalized to the corresponding tubulin
signal.
2.1.6. 2D SDS-PAGE for differentiation of MRJPs
Due to the similar molecular masses of major royal jelly proteins
(MRJPs) and their isoforms, 2-dimensional gel electrophoresis was
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International Journal of Biological Macromolecules 225 (2023) 848–860
performed to distinguish the specific proteins following established
protocols [37]. In brief, 100 μg of proteins extracted from the nurse
brain were loaded on an IPG strip (NL, pH 3–10, GE Healthcare) and the
conditions for the first dimension of electrophoresis were applied: 30 V
for 12 h, 500 V for 1 h, 1000 V for 1 h, 8000 V for 8 h, and 500 V for 4 h.
The 2nd dimension of the electrophoresis was performed as a standard
10 % SDS-PAGE. The gels were transferred onto PVDF membranes for
WB analysis (analogous to the analysis of one-dimensional gels
described above). Preparative gels were run in parallel with 1 mg of
proteins loaded on the IPG strip. After silver-staining the targeted pro­
tein spots were cut from the preparative gel for further MS identification
(as described above).
2.2. Experiment 2: candidate protein comparisons among experimental
nurse and forager cohorts
To obtain nurses and foragers of different physiological states, two
normal colonies that had been maintained next to each other for at least
seven days under standard conditions were subjected to the following
manipulation: During the day, while foraging activity was observed, the
queen was removed from one of the colonies with one frame of honey,
one frame of pollen, and two empty frames to establish a new colony.
This old colony (OC) was left in a hive in the original location to collect
returning foragers, while the remainder of the two colonies, including
the brood, queen, and all non-foraging bees, were combined into a new
colony (NC) and transferred to a new location.
After new brood emerged in the OC, some of the foragers were forced
to revert to nursing [38], while the absence of foragers in the NC forced
young bees to prematurely start foraging [14]. As soon as these behav­
iors were observed after the manipulation, reverted nurses and control
foragers were collected from the OC and precocious foragers and control
nurses were collected from the NC. Foragers were identified as returning
to the hive entrance with visible pollen and nurses were identified by
displaying nursing (head in brood cell for at least 10 s) on brood comb.
Samples from these four groups were either used to quantify the
levels of 42 protein candidates that were determined in the first exper­
iment via SDS-PAGE (see Results) or to further characterize the spatial
distribution of MRJP1 and MRJP3 in worker's brains via immune-
histochemical in-situ staining, following a previously established pro­
tocol [39]. Honeybee brains were dissected and immediately transferred
into a cold acetone/methanol solution before they were fixed in 6.7 mM
PBS containing 4 % paraformaldehyde. The tissue was dehydrated in a
50 %–100 % ethanol series before fixation in xylene, then embedding in
paraplast (Leica, Wetzlar, Germany). Mounted tissues were cut into 5 μm
sections, and the slides were rehydrated in ethanol in a decreasing series
from 100 % to 50 %. Subsequently, the slides were washed 3 times with
0.01 M PBS and transferred to 3 % hydrogen peroxide solution for 10
min incubation in the dark. Afterward, the slides were transferred to a 5
% BSA solution for 20 min to block nonspecific binding. Primary anti­
bodies (mouse anti-bee MRJP1/MRJP3, Abmart Co., Shanghai) were
used in concentrations of 1:500 in BSA to incubate the slices overnight at
4 ◦ C. Thereafter, the slices were washed 3 times for 5 min in PBS, then
transferred to anti-rabbit IgG (diluted 1:50 in BSA) with Cy5 and incu­
bated for 1 h at RT. The washed slices were incubated with DAPI (4′ , 6-
diamidino-2-phenylindole) for 5 min at RT in the dark. Finally, the slices
were washed 3 times for 5 min in PBS and covered with a drop of
Fluoromount-GTM for fluorescence quenching. The localization of
MRJP1 and MRJP3 were examined and photographed under inverted
microscopy (Olympus Corp., Tokyo, Japan) using an excitatory wave­
length of 405 nm. The target intensities were processed using ImageJ
software (National Institutes of Health, Bethesda, Maryland, USA.)
2.3. Experiment 3: MRJP responses to manipulation of worker ontogeny
To further investigate the correlation of MRJP1 and MRJP3 with the
transition from in-hive nursing to foraging, we manipulated the rate of
Y. Fang et al.
worker ontogeny by either treating workers with the pheromone ethyl
oleate (EO) to retard the transition to foraging [9] or with 8-Br-cGMP to
accelerate this transition [40]. In short, a total of 6000 newly emerged
bees with age-specific color labels were added into 6 parallel colonies (3
EO-treated and 3 control): each colony contained two open brood
frames, three capped brood frames, two frames with small amounts of
food, and a corresponding number of workers with one queen. After two
weeks (15–17 days after eclosion of the focal cohorts), treatment col­
onies were fed with 50 % syrup containing 2.1 mg of EO per gram of
sugar, while control hives were fed with 50 % syrup. Normal-aged for­
agers were removed from treatment colonies while foraging 18–20 days
after emergence. Labeled in-hive workers were collected from EO-
treated hives when the control hive no longer contained any labeled
individuals. Altogether about 300 bees with delayed caste switches were
harvested.
In a similar set-up, six colonies were treated with 500 μM of 8-Br-
cGMP in 50 % sugar syrup 7–10 days after the emergence of focal co­
horts to activate the foraging gene [30,41] and thus promote the tran­
sition to foraging, the control hives were fed with 50 % untreated sugar
syrup. One to five days later, workers with accelerated ontogeny from
treated colonies and control workers from untreated colonies were
identified by conducting proboscis extension reflex (PER) tests. The PER
testing followed previously described protocols [42], using 50 % sucrose
solution. Positive PER responses in workers from treated colonies were
interpreted as individuals with a likely accelerated behavioral ontogeny
and negative responses from untreated colonies were considered non-
accelerated controls. These individuals were collected for brain dissec­
tions and subsequent determination of MRJP quantities via WB as
described above.
2.4. Experiment 4: co-immunoprecipitation to identify MRJP partners
A total of 9 μg/μl of total proteins extracted from honeybee brains as
described above were used for co-immunoprecipitation (Co-IP) experi­
ments, performed with the Pierce Co-IP Kit (Thermo Scientific) ac­
cording to the manufacturer's recommendations. Briefly, antibodies of
MRJP1 and MRJP3 were each independently incubated with the resin
matrix to bond covalently. Then 1 mg total brain protein (homogenate in
500 μl PBS) was purified by centrifugation at 20,000 g for 30 min and
incubated overnight with the antibody-coupled resin at 4 ◦ C. Subse­
quently, the resin beads were magnetically removed from the solution
and the proteins bound to the MRJP1 or MRJP3 antibody were eluted in
an elution buffer. The eluted proteins were neutralized and then iden­
tified by MS as described above.
2.5. Experiment 5: in-vivo injection of MRJPs and corresponding mAbs
To study the causal role of MRJPs, the behavioral consequences of
experimentally increasing or decreasing the abundance of MRJP1 and
MRJP3 were studied. Worker bees were injected either with MRJP1,
MRJP3, or the respective antibodies, following an established brain in­
jection protocol [43]. Firstly, the proteins of MRJP1 (monomer) and
MRJP3 were isolated and purified. In brief, 100 mg RJ was dissolved in
phosphate buffer (pH 8.0). The suspension was centrifuged for 20 min at
4 ◦ C, and the supernatant was filtered with a 0.45 μm membrane filter.
The filtrate was added to 1 mL 20 mM Tris-HCl (pH 8.0), then concen­
trated and desalted by cellulose membrane (Ultracel-50, Amicon Ultra-
0.5, Millipore, USA.). The concentrated solution was purified by Q
Sepharose column (GE Healthcare) chromatography and eluted by a
linear gradient of NaCl from 0 to 1.0 M. The elution was separated by
SDS-PAGE, and the bands containing MRJPs were collected and purified
further on a Superdex 75 10/300 GL column (GE Healthcare) that was
equilibrated with a buffer containing 20 mM Tris-HCl (pH 8.0) and
150 mM NaCl. The eluted peak was collected and concentrated for
subsequent injection experiments. Once the proteins for injection were
ready, worker bees were anesthetized on ice for 10 mins. Then, they
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International Journal of Biological Macromolecules 225 (2023) 848–860
were injected with a custom-made glass injection needle (50 μm inner
diameter) into the median ocellus (puncture depth 1 mm) with one of
the following treatments (1 μl per bee): 500 ng MRJP1 antibody (AP04)
or 500 ng MRJP3 antibody (AP01) at 10–12 days after emergence (group
1), 500 ng MRJP1 or MRJP3 15–17 days after emergence (group 2), or
500 ng MRJP1 and MRJP3 in foragers that were 18–20 days old (group
3). In group 3, also a 100 ng treatment was included to study the dose-
dependence of reversal from foraging to nursing tasks. Furthermore,
500 ng BSA or PBS injections, and a “no injection” treatment were tested
as controls in these groups (only BSA/PBS control data are shown). Each
group overall contained 1500 individually paint-marking workers
among all treatments, which were evenly distributed into 3 colonies. For
five days following the treatments, we recorded the foraging activity of
tagged bees by monitoring the hive entrances, recording and collecting
all bees that were observed returning to the hive with visible pollen load
on their hind legs. Nurses were identified when they were observed
poking their heads into cells containing young larvae for at least 10 s.
2.6. Experiment 6: RNAi-mediated knockdown of MRJPs
To silence mrjp1 and mrjp3 gene expression, three siRNAs were
synthesized (BGI, China) for each gene (for sequences see Table S1) and
mixed in equal amounts. Honeybee workers between ten and twelve
days of age were collected upon exhibiting nursing behavior. In three
independent replicates, 260, 330, and 100 honeybees were injected with
siRNAs against mrjp1, and 210, 100, and 80 were injected with siRNAs
against mrjp3. In each case, tagged workers were injected at 10–12 days
of age with 1 μl of PBS, containing 500 ng/μl of total siRNA or no siRNA
as control, into the brain through the median ocellus. For each group, 50
honeybees injected with 1 μl of PBS and 50 non-injected honeybees from
the same cohort were used as controls. The focal individuals were
returned to their native hive, and their foraging behavior was monitored
for five days after injection as described above. The number of honey­
bees that switched from nurses to foragers was recorded daily.
At the end of the experiment, MRJP1 and MRJP3 levels were quan­
tified in untreated nurse (n = 30) and forager bees (n = 30), as well as
RNAi-treated bees that remained nurses (n = 30) and that transitioned to
foraging (n = 30) by WB after collection and brain dissection as
described above.
2.6.1. Quantitative real-time PCR
To examine the effect of RNA interference on mRNA levels of MRJP1,
the total RNA of 30 RNAi-injected nurses that converted to foraging and
30 that remained nurses after RNAi injection, as well as of 20 unma­
nipulated nurses and 20 unmanipulated foragers was extracted (TRLzol
reagent, Invitrogen, CA) and quantified by NanoDrop ND1000 spectro­
photometer (ThermoFisher Scientific, Wilmington, DE, USA). Corre­
spondingly for MRJP3, we processed 30 converted and 30 unconverted
nurses after RNAi treatment, as well as 20 control nurses and 20 control
foragers. PrimeScript RT reagent kit, RR037 (Takara Bio, Kyoto, Japan)
was used to generate cDNA as per the manufacturer's recommendations
(500 ng total RNA was used per cDNA reaction). The primer sequences
used to amplify the MRJP1 and MRJP3 targets are listed in Supple­
mental Table S2. The PCR amplification and data retrieval was con­
ducted on iQ5 Multicolor Real-Time PCR Detection System (Bio-Rad,
Hercules, CA). Each PCR was performed in a 20 μl reaction: 95 ◦ C for 30
s, followed by 40 cycles of 95 ◦ C for 5 s and 58 ◦ C for 30 s, with the melt
curve tracking from 55 to 95 ◦ C. Glyceraldehyde-3-phosphate dehy­
drogenase (GAPDH) was selected as a reference gene, and the differ­
ences in gene expression were calculated by the 2− △△Ct method [44].
An error probability p < 0.05 was considered a statistically significant
difference in gene expression by one-way ANOVA (SPSS version 18.0,
SPSS Inc. Chicago, IL).
Y. Fang et al.
3. Results
3.1. Brain protein differences between nurse and forager bees
To facilitate proteomic studies in the honeybee (Apis mellifera), a
comprehensive monoclonal antibody (mAb) library was generated from
a wide spectrum of samples involving different organs and tissues from
different developmental stages. The resulting 16,320 mAbs were printed
on microarrays that could then be used to directly assess quantitative
differences between the brain proteome of nurse and forager bees by
hybridization. Approximately 10 % of the mAbs spots (1622) displayed
significantly different signal intensities (Fig. 1; Table S3). The proteins
bound to the 500 array spots with the greatest differences were identi­
fied by IP and MS, resulting in 83 unique, successfully identified proteins
(Table S4).
Among the identified proteins, 40 were validated by WB results
(Fig. 2; Table S5). Among the most different proteins (fold change >5),
several were highly abundant, which were identified as MRJPs. Four
antibodies recognizing MRJPs were identified by IP and MS analysis and
to differentiate among the similar MRJPs, two-dimensional gel electro­
phoresis and WB analyses were performed (Fig. S1A). In total, 32 mAb-
recognized spots were labeled and sliced from the gel and 18 spots were
successfully identified by MS (Table S6). The majority of spots revealed
that the antibody “AP04” recognized different MRJP1 and MRJP2 iso­
forms (Fig. S1B) while “AP03” recognized MRJP1 isoforms and MRJP4
(Fig. S1C), and “AP01” was specific for MRJP3 isoforms (Fig. S1D).
Despite “AP03” binding MRJP1 and MRJP4 isoforms, we interpreted
this antibody as an indicator of MRJP1 because MRJP4 was over ten
times less abundant than MRJP1, which is also true for other tissues
[45,46]. The specificity of “AP01” and “AP04” was further confirmed by
WB analysis with purified MRJP3 and MRJP1, respectively (Fig. S1E).
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International Journal of Biological Macromolecules 225 (2023) 848–860
3.2. Validation of MRJP differences between nurse and forager states
To further examine the relationship between the abundance of
MRJP1 and MRJP3 and the behavioral state of workers, demographic
manipulations in experiment 2 were used to generate precocious for­
agers and nurses that reverted from foraging activities to be compared to
regular nurses and foragers for brain MRJP levels. Consistently, MRJP1
and MRJP3 were more abundant in nurses than in foragers (Fig. 3). In
the additional experiment 3, colony-level feeding of ethyl oleate, known
to delay the transition from nursing to foraging [9], increased the
abundance of MRJP1 and MRJP3 in worker brains (Fig. 3). Corre­
spondingly, colony-level feeding of 8-Br-cGMP, which induces foraging
[30], was associated with a decrease in MRJP1 and MRJP3 (Fig. 3).
To exclude the possibility that measured protein differences were
due to contamination with glandular tissues, the distinctive expression
patterns of MRJP1 and MRJP3 were further confirmed by immunohis­
tochemical staining of mushroom body and antennal lobe sections of
brains from control nurses and foragers, as well as precocious foragers
and reverted nurses (Fig. 4). MRJPs were consistently and significantly
(p < 0.05) more abundant in both brain regions of the workers in the
nursing state compared to the foraging state.
In-vivo manipulations of MRJPs indicate their causal role in the
behavioral transition from nursing to foraging.
The causal involvement of MRJPs in regulating the nurse-to-forager
transition was tested by investigating the behavioral effect of in-vivo
brain injections of MRJP1 (monomer), MRJP3, or their respective an­
tibodies. The workers injected with purified MRJP1 (monomer), and
MRJP3 transitioned less to foraging (Fig. 5A), while injection of the anti-
MRJP1 and anti-MRJP3 caused a significantly higher percentage of bees
to transition to foraging (Fig. 5B). Correspondingly, brain injection of
MRJP1 (monomer) and MRJP3 caused a higher proportion of foragers to
revert to nursing behavior (Fig. 5C).
In-vivo manipulations of mrjp1 and mrjp3 gene expression in the
Fig. 1. Quantitative protein differences between
nurse and forager bee brains discovered by mono­
clonal antibody array. The overall comparison results
between brains of nurses and foragers revealed 1622
of 16,320 mAbs signals as different (blue dots up-
regulated in nurses, red dots up-regulated in for­
agers). This does not correspond to the number of
unique proteins because different antibodies can
recognize the same protein. (For interpretation of the
references to color in this figure legend, the reader is
referred to the web version of this article.)
Y. Fang et al. International Journal of Biological Macromolecules 225 (2023) 848–860

Fig. 2. Validation of quantitative brain protein differences between behavioral states of honey bee workers. Western blot analyses compared the abundance of 40
candidate proteins identified by our mAbs array experiments among normal nurses and foragers and precocious foragers and reversed nurses (induced by de­
mographic manipulations). Behavioral states were identified by direct observation. Actin and tubulin were used for normalization. Major royal jelly proteins
exhibited the most consistent and strong abundance differences among the nursing and foraging states.

honeybee brain confirms the role of MRJPs in regulating the behavioral
state of workers.
To further validate the causal role of MRJPs in the behavioral tran­
sition of honeybee workers from nursing to foraging, the mrjp1 and
mrjp3 genes were down-regulated with an RNAi-based approach by
injecting a mixture of small inhibitory RNAs into nurse bees and
observing their subsequent transition to foraging. As predicted, a
significantly higher proportion of mrjp-injected nurses transitioned to
foraging than nurses of a negative or a PBS-injected control (Fig. 6A).
Down-regulation of mrjp1 and mrjp3 was confirmed by qPCR. The
injected bees displayed much lower expression levels than unmanipu­
lated nurses, with an additional significant difference between injected
nurses that transitioned to foragers and those that did not transition
(Fig. 6B). However, the experimental down-regulation of either gene
was weaker than the natural differences observed between nurses and
foragers. The resulting protein level of MRJP1 and MRJP3 in these four
groups were quantified by western blot analysis, exhibiting parallel re­
sults with significantly higher levels in nurses that did not transition to
foragers than nurses that did transition (Fig. 6C, D). The abundance of
MRJP1 and MRJP3 in behavior-switched workers exhibited a similar
level to regular foragers, while the unswitched bees expressed almost an
identical pattern to control nurse bees (Fig. 6D).

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Y. Fang et al.
Fig. 3. Further association of specific MRJPs in the brain with behavioral state of honey bee workers. (A) Representative western blot of MRJP1 (AP04 and AP03)
and MRJP3 (AP01) in brains of regular nurses, nurses from a single-cohort colony, precocious foragers from a single cohort colony, regular foragers and old foragers,
and nurses that reverted from foraging behavior, as well as ethyl oleate (EO) treated and 8-Br-cGMP treated workers with corresponding control groups. (B)
Quantitative comparison of the western blot signals among the same groups. Signals were quantified with ImageJ and normalized by the signal of actin. Values
represent means ± SD, n = 3.
3.3. Identification of proteins that interact with MRJPs
To identify how MRJPs relate to other known mechanisms to exert
their influence on the behavioral transition from nursing to foraging, the
proteins that interact with MRJP1 and MRJP3 were selected by co-IP
and identified and quantified by LC-MS/MS. Over 100 proteins were
identified overall, and 19 proteins interacted with both MRJP1 and
MRJP3 (Table 1). Among them, hexamerin 110 (Hex) and vitellogenin
were the major interactive proteins with MRJPs in the brains of nurses.
Moreover, other MRJPs, such as MRJP2, MRJP4, and MRJP7, were also
identified as interactors with MRJP1 and MRJP3 (Table 1).
4. Discussion
The complex social regulation of the division of labor among honey
bee workers, particularly the pronounced transition between the in-hive
and forager life-history stages, has been extensively studied at the mo­
lecular, organismal, and societal levels, revealing hormonal, phero­
monal, and nutritional regulation [8,9,47,48]. Numerous correlates of
this transition have also been characterized at the transcriptomic, epi­
genomic, and proteomic levels [5,10,23]. Our studies identify the
pleiotropic effects of MRJPs as a novel, proximate link between nutri­
tional and hormonal signaling in the causation of the onset of worker
foraging, based on the interrogation of the first monoclonal antibody
(mAb) array for honey bees. Subsequently, we strengthen the argument
for a functional role of MRJPs in the life-history regulation of honey bee
workers via a series of experiments that manipulate the transition be­
tween in-hive bees and foragers and demonstrate a causal role by
downregulating MRJPs via RNAi, which causes the predicted increase in
foraging. These results show the utility of the mAb array as a novel tool
for advanced proteomic study and are a major advance in our under­
standing of one of the most complex and best-studied life-history
transitions.
Due to the considerable development efforts for comprehensive mAb
arrays, this technique has been used mostly in human disease prote­
omics [49]. However, mAb arrays have several advantages over mass-
spectrometry-based proteomics, including better sensitivity, resolu­
tion, accuracy, dynamic range, and reproducibility [50]. We have
collaborated with Abmart Ltd. to generate the first mAb array for honey
bees, the most important managed pollinator and an important scientific
model with a long tradition of “-omics” approaches [51]. Overall,
16,320 mAbs were developed and printed onto arrays to be able to
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International Journal of Biological Macromolecules 225 (2023) 848–860
simultaneously interrogate a large part of the honey bee proteome.
However, we do not know how many unique proteins our array in­
terrogates. Our study of 500 specific array spots that exhibited the
greatest differences between nurse and forager brains resulted only in 83
unique protein identification, and only half of those could be verified by
Western blots. These results are due to a combination of factors: Some
antibodies recognized the same protein, and other proteins could not be
unambiguously identified by mass spectrometry. Nevertheless, this
novel tool represents a major advance in extending the functional pro­
teomics tools in honey bees, and antibody solutions can be made
available upon request.
MRJPs are the main components of brood food that nurse bees feed
to developing larvae [52], 51]. MRJPs are quantitatively important for
the nutrition of developing larvae, and their production is crucial to the
nursing task of workers, which produce MRJPs in their hypopharyngeal
glands in a task-dependent manner [28]. Our results indicate that two of
the nine functional MRJPs [54] (MRJP1 and MRJP3) exert an additional
regulatory function, influencing the behavioral specialization of workers
into nurses or foragers. Thus, the same proteins that are important in the
performance of nursing are also regulating the nursing stage. Such
recruitment of effector proteins to signaling functions has evolved in a
variety of contexts [55], including honey bees [6]. MRJP1 and MRJP3
are major components of brood food, representing 31 % and 26 % of the
RJ proteins, respectively [54,55]. Other functions have been described
for both proteins, including antibacterial activity [58] of MRJP1 and
immune elicitor-binding [59] of MRJP3, and they are expressed in tis­
sues outside the hypopharyngeal glands [25]. Specifically, both proteins
are also known to be expressed in the brain [60], which we confirm in
this study.
Regarding to the molecular structure of MRJP1, the monomer and
oligomer are usually existed together [61], and the latter one is pre­
dominant form due to its heat resistance and long storage stability in
physiological conditions [62–65]. Furthermore, the MRJP oligomer
contains 24-methylenecholesterol molecules, which act as a hormone
regulator for promoting the growth of honeybee larvae [66]. Moreover,
monomer MRJP1 may play a crucial role in prolonging longevity of
queens. Importantly, only monomer MRJP1 is crucial in determination
of the caste fate [67] due to the aggregated MRJP1 in RJ that reduces the
digestion and utilization of nutrition in honeybee larvae [68]. This is in
line with the present's findings that monomer MRJP1 play key roles in
regulation of honeybee labor transition.
Our studies show that MRJP1 and MRJP3 levels in the brain
Y. Fang et al. International Journal of Biological Macromolecules 225 (2023) 848–860

Fig. 4. MRJP localization in the honeybee brain. Immunohistochemistry staining of MRJP1 with mAb AP04 (A) and MRJP3 with AP01 (B) in the mushroom body
and antennal lobe of nurse, forager, reversed nurse, and old forager shows that these MRJPs are present in two functionally important brain regions.

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Y. Fang et al. International Journal of Biological Macromolecules 225 (2023) 848–860

Fig. 5. Direct manipulation of MRJPs in the brain affects the behavioral state of workers. (A) The proportion of honeybees that delayed switching from nursing to
foraging after injection with MRJP1 (monomer) and MJRP3 proteins is significantly increased relative to control-injected workers. The shown proportions are low
because they were calculated based on the total number of manipulated individuals, many of which died as a consequence of experimental treatment (B) The
proportion of honeybees that switched from nurse to forager state after injection with monoclonal antibodies against MRJP1 and MRJP3 was increased relative to the
PBS-injected control group. (C) The proportion of workers that reverted from forager to nurse state after injection with either MRJP1 (monomer) or MRJP3 was
increased relative to bees from a BSA-injected control group. The behavioral state of all groups was determined during five days after injection. The different letters
above each bar indicate statistically significant differences (p < 0.01).

correlated with the behavioral state of honey bee workers, confirming
previous discoveries [60,61]. The results of a series of experimental
manipulations to accelerate the transition from nursing to foraging, to
delay this transition, or to revert this transition strengthens the argu­
ment that the levels of both proteins are closely linked to behavioral
state: Regardless of whether behavioral manipulations were performed
demographically or pharmacologically, nursing was always associated
with increased levels of MRJP1 and MRJP3 while foraging was always
related to low levels. These quantitative protein differences in the brain
were confirmed by immunohistochemical staining, excluding the pos­
sibility that the brain samples with closely associated glandular tissue
could explain the observed differences. Specifically, MRJP1 and MRJP3
levels in the functionally important antennal lobes and mushroom
bodies reflected the overall quantitative brain differences between
nurses and foragers. Both of these brain regions have been implicated in
the behavioral specialization of nurses and foragers [70]. The mushroom
bodies are involved in many higher-order functions and it experiences a
significant expansion in arborization when honey bee workers transition
to foraging [32]. The Kenyon cells are intrinsic neurons of the mush­
room bodies involved in associative learning and memory, specifically
expressing MRJP1 [71], although its neurobiological function has not
been clarified. MRJP3 and MRJP1 may serve as a reserve for amino acids
in the brain during the ontogenetic and behavioral development from
nurse to forager [69] and also has the ability to aggregate RNA and could
mediate high-order ribonucleoprotein (RNP) assemblies [59], which
have in turn be associated with synaptic plasticity in Drosophila [72].
Thus, declining MRJPs may affect the neuronal changes to adapt to the
different challenges of foraging and nursing in several ways.
The causal effects of MRJP1 and MRJP3 on the behavioral state of
honey bee workers are demonstrated by the direct manipulation of these
proteins in the brain that influenced worker behavior as predicted: In
separate experiments, injection of MRJPs increased the proportion of
nurses that did not transition to foraging and increased the proportion of
foragers that reverted to foraging. In contrast, the injection of MRJP

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Y. Fang et al. International Journal of Biological Macromolecules 225 (2023) 848–860

Fig. 6. RNAi-mediated knock-down of major royal jelly genes accelerates the transition from nurse to forager. (A) The proportion of nurses that switched to foraging
after injection with a mixture of siRNAs for either mrjp1 (left) or mrjp3 (right) was significantly higher than in unmanipulated or PBS-injected control groups. (B) RT-
qPCR analysis of mrjp1 and mrjp3 expression in siRNA-injected nurses that either remained in the nursing stage (“unconverted”) or transitioned to foraging
(“converted”) showed significantly lower mrjp expression in converted individuals. Gene expression in unmanipulated nurse and forager bees showed more pro­
nounced differences in the same direction. (C) Protein levels in the four groups (unmanipulated nurses and foragers, and converted and unconverted siRNA-injected
nurses were measured by western blot analysis and (D) showed corresponding differences, albeit not as pronounced. The experiment was repeated with three
biological replicates. Values represent means ± SD, n = 3.

Table 1
MRJPs interactants identified by co-immunoprecipitation and mass spectrometry.
Accession No. Description Gene name Anti-MRJP1 Anti-MRJP3 Cellular localizationa

Nurse Forage Ratio Nurse Forage Ratio

O18330 Major royal jelly protein 1 MRJP1 90 4 22.5 87 6 14.5 EC

O77061 Major royal jelly protein 2 MRJP2 41 NA 55 4 13.8 EC
Q17061 Major royal jelly protein 4 MRJP4 548 14 39.1 29 1 29.0 EC
A0A088AU27 Major royal jelly protein 3 MRJP3 35 NA 29 NA EC
D3Y5T0 Major royal jelly protein 7 MRJP7 50 2 25 1360 122 11.1 EC
A0A088A1A1 Hexamerin 110 Hex110 396 1 396 396 3 132 EC
Q868N5 Vitellogenin Vg 17 NA 11 NA EC
A0A088ADL8 Vitellogenin Vg 299 33 9.1 171 29 5.9 EC
A0A088AS56 Apolipophorins apolpp 344 118 2.9 240 108 2.2 EC
Q5BLY4 Icarapin 12 4 3 18 2 9 EC
A0A088ASG4 Molybdenum cofactor synthesis protein cinnamon 92 39 2.4 15 7 2.1 Cyt
A0A088ALS8 Glycogen debranching enzyme 86 21 4.1 85 9 9.4 Cyt
H9K5X4 Dehydrogenase/ reductase SDR family member 19 8 2.4 19 8 2.4 Cyt
A0A088ACJ2 Dynactin subunit 2 dcnt2 26 12 2.2 27 13 2.1 CS
A0A088A9R3 Non-specific lipid-transfer protein 24 9 2.7 17 8 2.1 Cyt; Mt.; PO
A0A088AC74 40S ribosomal protein S18 28 8 3.5 23 10 2.3 Cyt; Rib
A0A088ADQ6 40S ribosomal protein S14 13 4 3.3 11 5 2.2 Cyt, Rib
A0A088ARI3 Nicotinate phosphoribosyl transferase 14 6 2.3 14 4 3.5 Cyt
A0A088A4N3 Eukaryotic translation initiation factor 3 subunit H eIF3h 11 5 2.2 14 4 3.5 Cyt
a
EC = Extracellular, CS = Centrosome, Cyt = Cytoplasm, Mt. = Mitochondria, PO = Peroxisome, Rib = Ribosome.

857
Y. Fang et al.
antibodies to decrease the quantity of active MRJPs increase the pro­
portion of nurses that transitioned to foraging. Likewise, the down­
regulation of MRJP expression by injecting dsRNA into the brain led to
lower MRJP1 and MRJP3 protein levels and more workers transitioning
from nursing to foraging than PBS-injected or unmanipulated control
groups. While the opposite genetic manipulation to increase MRJP
expression was hampered by the practical difficulties of gene engi­
neering of honey bees, these experiments together strongly support a
causal role of MRJP1 and MRJP3 in regulating honey bee division of
labor.
The mechanisms of MRJP function during the transition from
nursing to foraging are likely different from the proposed link of MRJP1
to epidermal growth factor receptor (Egfr) signaling in the fat body
during larval development [73]. During larval development, royal jelly
consumption causes the simultaneous upregulation of juvenile hormone
(JH) and vitellogenin (Vg) [73], in contrast to the mutually repressive
relation between JH and Vg that is well-established in the transition
from nursing to foraging [7]. At the end of the nursing stage, Vg titers
decline, presumably as a direct result of brood feeding when Vg is used
as a precursor for MRJPs [48,74]. The decline is accompanied by
increased insulin-like signaling (ILS) and JH production from the
corpora allata [8,17]. The directional causality between declining nu­
trients, MRJPs, Vg, JH, and ILS is unclear and could involve several
additional mediators of gene expression [75]. However, our study of
proteins interacting with MRJPs indicates direct interactions at the
protein level between MRJP1/3 and vitellogenin, which could be an
immediate connection between these two components of nutrient
sensing. The possibility of direct indicators of nutrient status by MRJPs is
supported by the interaction of MRJP1/3 with other nutrition-related
proteins (hexamerin 110 and apolipophorins). Hexamerins, known pri­
marily as larval storage proteins, are expressed in the worker fat body
and inducible by JH [76] but in turn, may also be involved in JH binding
in conjunction with apolipophorins [77]. Whether MRJPs are interact­
ing with JH transport or sequestration remains to be elucidated.
Overall, our results for MRJP1 and MRJP3 were remarkably similar
throughout several experiments involving the regulation of nursing
versus foraging. Furthermore, we found them co-precipitating and thus
interacting with MRJP2, MRJP4, and MRJP7. These MRJPs evolved
from a single progenitor gene via gene duplication [25], and partial
redundancy can therefore be expected. Even though evolutionary
divergence among these genes has led to considerable sequence differ­
ences and varying functions [25,52,78], their biochemical properties
and immuno-labeling can be quite similar, and thus we have taken great
care to distinguish the individual MRJPs throughout this study. Whether
the similarity of MRJP1 and MRJP3 in the context of behavioral regu­
lation translates into functional redundancy remains to be clarified.
In conclusion, we established the first honeybee antibody library
with comprehensive coverage of 16,320 mAbs, which provides a
powerful platform for honeybee functional proteomics that enables
large-scale antibody generation and target profiling. MRJP1 and MRJP3
were identified as potential neural protein effectors in the brain of
honeybee workers of the reversible subcaste-switch between nurses and
foragers. We experimentally confirmed that downregulation of MRJP1
and MRJP3 in the honeybee brain shifts worker life history toward
foraging, while upregulation has the opposite effect. This novel pleio­
tropic function of MRJP1 and MRJP3 may act in concert with JH, Vg,
and other nutrient storage and sensing pathways that have previously
been implicated in regulating the division of labor in honey bees. The
increased repertoire of MRJPs exemplifies the promiscuity of nutritional
proteins and the evolutionary potential of gene duplication and func­
tional diversification.
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.ijbiomac.2022.11.150.
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International Journal of Biological Macromolecules 225 (2023) 848–860
CRediT authorship contribution statement
YF and J-K L conceived and designed the experiments; YF and MF
performed the experiments; YF and CM analyzed the data; YF, J-K L, and
OR drafted and revised the manuscript.
Declaration of competing interest
The authors declare no competing interests.
Data availability
Data will be made available on request.
Acknowledgments
We thank Boris Baer (Department of Entomology, University of
California, Riverside) for his valuable comments on this study. This work
was supported by the Agricultural Science and Technology Innovation
Program (CAAS-ASTIP-2015-IAR), Modern Agro-Industry Technology
Research System (CARS-44) in China, the National Project for Upgrad­
ing Overall Bee-Product Quality of the Beekeeping Industry of China, the
Alberta Beekeepers Commission, and the University of Alberta.
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