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\DOI{10.5802/crchim.270}
\datereceived{2023-08-29}
\daterevised{2023-10-06}
\datererevised{2023-10-23}
\dateaccepted{2023-10-24}
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\dateposted{2024-01-11}
\begin{document}

\begin{noXML}

%\makeatletter
%\def\TITREspecial{\relax}
%\def\cdr@specialtitle@english{Chemical Ecology -- Chemical Mediation in the Environment}
%%\def\cdr@specialtitle@french{\'Ecologie chimique -- M\'ediation chimique dans l'environnement}
%\makeatother

\title{Impact of temporal and rearing environment variations on brood
ester pheromone production in the honey bee \protect\textit{Apis
mellifera}}

\alttitle{Impact des variations temporelles et de l'environnement
d'\'{e}levage sur la production de la ph\'{e}romone de couvain chez
l'abeille domestique \textit{Apis mellifera}}

\author{\firstname{Fanny} \lastname{Mondet}\CDRorcid{0000-0002-7737-0101}\IsCorresp}
\address{INRAE, UR 406 Abeilles et Environnement, 84914 Avignon,
France}
\email[F. Mondet]{fanny.mondet@inrae.fr}

\author{\firstname{Dominique} \lastname{Beslay}}
\addressSameAs{1}{INRAE, UR 406 Abeilles et Environnement, 84914
Avignon, France}

\author{\firstname{Marianne} \lastname{Cousin}}
\addressSameAs{1}{INRAE, UR 406 Abeilles et Environnement, 84914
Avignon, France}

\author{\firstname{Yves} \lastname{Le Conte}}
\addressSameAs{1}{INRAE, UR 406 Abeilles et Environnement, 84914
Avignon, France}

\keywords{\kwd{Chemical ecology}\kwd{\textit{Apis mellifera}}\kwd{Honey
bee}\kwd{Pheromone}\kwd{Brood}}

\altkeywords{\kwd{\'{E}cologie chimique}\kwd{\textit{Apis
mellifera}}\kwd{Abeille domestique}\kwd{Ph\'{e}romone}\kwd{Couvain}}

\shortrunauthors

\begin{abstract}
Chemical communication plays a key role in homeostasis within social
insect societies. The development of the superorganism relies on the
tightly regulated raising of new individuals, which necessitates an
efficient communication system between the brood and adult workers. In
the honey bee \textit{Apis mellifera}, a blend of ten fatty-acid esters
found on the cuticle of larvae ensures such successful brood
development. This brood ester pheromone (BEP) has both releaser and
primer effects on honey bee workers. In this study, we investigated the
temporal variations of BEP under natural conditions as well as the
developmental variations of the 10 compounds for in-hive versus in
vitro reared brood. We identified marked monthly changes in the
composition of BEP throughout the season, with an increase in total
amounts in the fall characterised by an enrichment in ethyl esters.
Drastic changes in the amount of BEP were confirmed during development,
with a peak during the last larval stage. Comparison between
in-hive and in vitro reared bees showed that individuals harbour very
similar BEP profiles during the larval stage, but distinct profiles
during the pre- and pupal stages. This study validates the use of in
vitro rearing methods for studying the chemical ecology of honey bee
larvae and increases our understanding of the ecophysiology of one of
the most important pheromones of honey bee colonies.
\end{abstract}

\begin{altabstract}
La communication chimique joue un r\^{o}le cl\'{e} dans
l'hom\'{e}ostasie des soci\'{e}t\'{e}s d'insectes sociaux. Le
d\'{e}veloppement du superorganisme repose sur un \'{e}levage
\'{e}troitement r\'{e}gul\'{e} des nouveaux individus, ce qui
n\'{e}cessite un syst\`{e}me de communication efficace entre le couvain
et les ouvri\`{e}res adultes. Chez l'abeille mellif\`{e}re,
\textit{Apis mellifera}, un m\'{e}lange de dix esters d'acides gras
pr\'{e}sents sur la cuticule des larves assure le succ\`{e}s du
d\'{e}veloppement du couvain. Il a \'{e}t\'{e} d\'{e}montr\'{e} que
cette ph\'{e}romone de couvain (BEP) avait \`{a} la fois des effets sur
la physiologie et et le comportement des ouvri\`{e}res d'abeilles
mellif\`{e}res. Dans cette \'{e}tude, nous avons \'{e}tudi\'{e} les
variations temporelles de la BEP dans des conditions naturelles ainsi
que les variations des 10 compos\'{e}s entre le couvain \'{e}lev\'{e}
dans la ruche et le couvain \'{e}lev\'{e} in vitro. Nous avons
identifi\'{e} des changements mensuels marqu\'{e}s dans la composition
de la BEP tout au long de la saison apicole, avec une augmentation des
quantit\'{e}s totales en automne caract\'{e}ris\'{e}e par un
enrichissement en esters \'{e}thyliques. Des changements importants
dans la quantit\'{e} de BEP ont \'{e}t\'{e} confirm\'{e}s au cours du
d\'{e}veloppement, avec un pic au cours du dernier stade larvaire. La
comparaison entre les abeilles \'{e}lev\'{e}es dans la ruche et celles
\'{e}lev\'{e}es in vitro a montr\'{e} que les individus pr\'{e}sentent
des profils de BEP tr\`{e}s similaires au cours du stade larvaire, mais
distincts au cours des stades pr\'{e}- et nymphal. Cette \'{e}tude
valide l'utilisation de m\'{e}thodes d'\'{e}levage in vitro pour
l'\'{e}tude de l'\'{e}cologie chimique des larves d'abeilles
mellif\`{e}res et am\'{e}liore notre compr\'{e}hension de
l'\'{e}cophysiologie de l'une des ph\'{e}romones les plus importantes
des colonies d'abeilles mellif\`{e}res.
\end{altabstract}

\maketitle
\vspace*{14pt}
\twocolumngrid

\end{noXML}

\section*{Introduction}

Similar to other social insects, the integrity of the superorganism
formed by a honey bee (\textit{Apis mellifera}) colony relies on
coordination of the tasks of the different individuals living in the
colony. Chemical communication through pheromones plays a major role in
the regulation of homeostasis, development, food sourcing, defences,
division of labour and \mbox{reproduction} of the social group~\cite{1,2}.
Pheromones are chemicals that are secreted externally and that produce
changes in behaviour and physiology, with the emitter and receiver
belonging to the same species~\cite{3}. Pheromones act as a chemical
language that is very diverse in terms of the chemical nature of the
semiochemicals involved and the complexity of the signal, but at the
same time very specific in relation to the state of the colony and the
environmental conditions. Two types of pheromones can be identified
depending on their effect on the receiver of the chemical message upon
reception: releaser pheromones initiate an immediate behavioural
response, whereas primer pheromones have long-term effects and modify
the physiology of the receiver~\cite{4}. One given pheromone can have
both primer and releaser effects~\cite{5}. 

The success of a honey bee colony not only relies on efficient
reproduction that is ensured by the queen but also on the development
of its population. Efficient population development requires new
individuals to be regularly raised because the life expectancy of
individual honey bee workers is limited to a few weeks~\cite{6}. Brood
care thus ensures the renewal of the colony population because immature
individuals at the larval stage are totally dependent on the care
provided by adult workers for their survival. The developing brood
produces a complex blend of pheromones that influence both the
behaviour and physiology of worker bees~\cite{5,7}. A mixture of ethyl
and methyl esters of fatty acids (10 in all) forms the main brood
pheromone and is known as brood ester pheromone (BEP)~\cite{8}.
Individual esters and/or subsets of BEP can be interpreted by worker
bees in various ways depending on the brood stage. In the last instar
worker larvae, four BEP compounds mediate brood cell capping
initiation. Differences in the proportion of different compounds of
BEP~and, more particularly, the ratio of ethyl to methyl esters provide
a signature that serves as a reliable indicator of larval
age~\cite{9,10}. However, the contribution and role of each of the 10
BEP compounds for other behaviours remains unknown.

BEP not only modifies the behaviour and physiology of workers but also
plays a critical role in the optimisation of nutrition provided to the
brood at the colony level. BEP can increase the amount of royal jelly
that is delivered to larvae~\cite{11}, as well as the amount of
proteins found in the hypopharyngeal glands~\cite{12,13}. BEP also
influences the transition of workers from nurses to foragers~\cite{14},
which provides the brood with additional control over the amount of
care that is delivered to them, thus ensuring that their development is
optimal. If the amount of brood within the colony increases, the levels
of BEP increase. As a result, the transition from nursing to foraging
is slowed down and the nurse/forager ratio is increased. This blend of
ten fatty-acid esters has also been identified as a kairomone that
attracts the parasitic mite \textit{Varroa destructor}~\cite{15,16,17}
and has been shown to serve as a defence pheromone in the hygienic
response of honey bee workers towards varroa-infested brood~\cite{18}.

These chemical cues that are found on the surface of the cuticles of
honey bee larvae and pupae are one of the few examples of primer
pheromones among more than 50 chemicals that are known to ensure the
efficient functioning of a honey bee society~\cite{5} and are at the
centre of the complex regulation of behaviour and physiology that
ensure the cohesion of the honey bee colony. 

Brood physiology is often studied using an in vitro rearing method that
allows the control of temperature and other environmental parameters,
such as pesticide exposure~\cite{19}. This OCDE-validated protocol has
been proven to provide broods with normal morphological development and
a high emergence rate into adults.

To improve our knowledge of the ecophysiology of the BEP pheromone, we
investigated the natural variations in the production of the 10
components of the BEP during the bee season using monthly measurements
from spring to fall. Using a comparative approach, we also investigated
the BEP chemical profile of in vitro- and in-hive-reared broods during
their development.

\section{Material and methods}\label{sec1}
\subsection{Honey bee colonies}\label{ssec11}

This study was conducted using \textit{Apis mellifera L.} hybrid honey
bee colonies located at the INRAE research centre of Avignon (France),
in 2017.

\subsection{In vitro brood rearing}\label{ssec12}

Larvae were reared in vitro according to the method developed by
Aupinel \etal~\cite{19}. To obtain larvae of a known age, three
days before the experiment, combs containing empty cells were
previously equipped with a queen excluder and placed in the hive for
egg laying. Four days later, 1st instar larvae were transferred into
plastic queen-starter-cells and placed in an incubator under controlled
conditions (34 ${\pm}$ 2~\textdegree C\ and 95 ${\pm}$ 5\% relative
humidity (RH)). On the 7{th} day, the RH was changed to 80\% for the
period. On the 8{th} day, each plate was individually sealed with a
thin layer of beeswax so that each cell was individually capped. In
each cell, orifices were made on the top to enable air exchange. Larvae
were provided with food at the daily intakes recommended for each
developmental stage~\cite{19}. The food was composed of 1 volume of
royal jelly and 1 volume of an aqueous solution containing 12\% (w/v)
glucose, 12\% fructose, and 2\% yeast extract (diet A); 15\% glucose,
15\% fructose, and 3\% yeast extract (diet B); or 18\% glucose, 18\%
fructose, and 4\% yeast extract. Daily feeding of larvae (volume per
diet and per day) was performed from the 1st day (grafting) to the 6th
day, except for the 2nd day, which was considered a period of
acclimatisation. The diet was: 20~${\upmu}$L of diet A on the 1st day,
20~${\upmu}$L of diet B on the 3rd day, and 30, 40, and 50~${\upmu}$L
of diet C on the 4th, 5th, and 6th days, respectively.

\subsection{Sampling of honey bee brood for chemical analyses}
\label{ssec13}

BEP production was studied across the beekeeping season, monthly from
April to October. Each month, frames containing open brood were brought
to the laboratory and ready to be capped last-instar larvae were
carefully sampled with soft tweezers. Six samples were collected (30
larvae) from each colony. The experiment was conducted on 3 colonies.

To study BEP production under different rearing environments and during
development, frames on which the queen had been caged (see above) were
retrieved each day from the colony, and larvae or pupae were carefully
sampled from the uncapped or capped cells. At the same time, brood
cells from the same colonies, but artificially reared in the lab (see
above), were sampled using the same procedure. Larval staging was
performed by weighing the larvae, whereas pupal staging was performed
by observing the colouration of the eyes, mandibles and body
parts~\cite{20}. The experiment was also conducted on 3 colonies in
April 2015, with a sample size between 6 and 15 larvae/prepupae per
stage and rearing condition and between 4 and 5 pupae per stage and
rearing condition. A total of 131 samples were analysed for this second
experiment.

\subsection{Brood ester pheromone (BEP) extraction}\label{ssec14}

The BEP compounds were extracted by crushing 10 individuals (larvae,
pre-pupae or pupae, only 5 larvae for L5 larvae as the level of
emission is very high) in 1900~${\upmu}$L of hexane (Sigma Aldrich) and
100~${\upmu}$L of C17 ester methyl heptadecanoate (10~ng/$\upmu$L;
internal standard, Sigma Aldrich) using an automatic grinder with a
metal bead (Fritsch Pulverisette 23). Crushed samples were immediately
placed on ice and stored at ${-}$30~\textdegree C. The tubes were then
centrifuged at 4000~rcf at 4~\textdegree C\ for 20 min. The supernatant
was transferred to new tubes and stored at ${-}$30~\textdegree C\ until
fractionation.

To facilitate analyses of the chemical profile of the samples, which
are composed of many compounds, fractionation of the samples was
performed. This allowed the separation of nonpolar compounds (e.g.
cuticular hydrocarbons) from compounds containing polar groups (e.g.
esters). Sample extracts were applied to a silica column (silica gel
60, particle size 40--63~mm, 230--400 mesh). The column was first
rinsed with a solvent mix (98.5\% isohexane, 1.5\% diethyl
ether---Sigma-Aldrich) until 3~mL of the mix was collected. Samples
were added to the column and eluted in a final volume of 3~mL of the
solvent mix containing 1.5\% diethyl ether. The second fraction was
eluted in a final volume of 3~mL of a second solvent mix (94\%
isohexane, 6\% diethyl ether). The 3~mL of the second fraction,
containing the BEP compounds, was concentrated to 30~$\upmu$L under a
nitrogen stream. 

\subsection{Gas chromatography analysis}\label{ssec15}

Quantitative analysis of the BEP compounds contained in the extracts of
the second fraction was performed on a fast gas chromatograph (Shimazu
GC-2014) equipped with a split/splitless inlet, a flame ionisation
detector, and a capillary Supelcowax column (15~m $\times$ 0.10~mm,
0.10~$\upmu$m film thickness). 1~$\upmu$L of each sample was injected
with a 30-ratio split mode and a column flow of 0.94~mL/min. The
carrier gas was hydrogen, and the temperatures of the injector and
detector were both set at 250~\textdegree C. The oven temperature was
programmed with the following conditions: 90~\textdegree C\ isothermal
held for 1~min, followed by temperature increases at a rate of
40~\textdegree C/min up to 195~\textdegree C, at a rate of
1~\textdegree C/min up to 201~\textdegree C, and at a rate of
40~\textdegree C/min up to 250~\textdegree C. The oven was finally held
at 250~\textdegree C\ for 3~min. The peaks corresponding to the internal
standard and each compound of the BEP were attributed to injection of
pure commercial compounds (Sigma Aldrich) diluted in isohexane at the
end of each batch of 10 samples. Profile comparison of each pure
synthetic compound injected individually and the sample profiles
allowed for identification of the 10 BEP compounds.

Quantification was obtained using a standard curve constructed for each
compound of the BEP, and normalised to the internal standard
concentration of each sample after correction of the slope differences
between the BEP standard curve and the C17 ester standard curve.
Standard curves were built using five concentrations (selected to cover
the concentration range of the samples) and five replicates of each
concentration. 18 samples were analysed for each date for the temporal
study, while 80 samples were taken from the hive and 51 from the in
vitro setting \mbox{($5 \leq n \leq 15$)}
to examine the effect of the rearing environment.

Further confirmation of the identification process of the BEP compounds
in the samples was performed on a gas chromatograph coupled with a mass
spectrometer (GC-MS, ThermoQuest Trace GC---Polaris) equipped with a
Phenomenex ZB-WAX column (30~m $\times$ 0.25~mm, 0.25~$\upmu$m film
thickness). 1~$\upmu$L of a selection of 10 representative samples was
injected with the inlet in splitless mode and a column flow of
0.7~mL/min. The carrier gas was helium, and the temperatures of the
injector and detector were set at 250~\textdegree C. The GC--MS
programme has been adapted to match the GC-FID programme. The oven
temperature was programmed with the following conditions:
60~\textdegree C\ isothermal held for 1~min, followed by temperature
increases at a rate of 30~\textdegree C/min up to 180~\textdegree C, at
a rate of 1.5~\textdegree C/min up to 210~\textdegree C, and at a rate
of 20~\textdegree C/min up to 250~\textdegree C. The oven was finally
held at 250~\textdegree C\ for 5~min. Compound identification was
confirmed by comparison of their mass spectra with those of synthetic
compounds, those available in libraries, and further comparisons of
their retention indices. 

\subsection{Statistical analyses}\label{ssec16}

All statistical analyses and figures were generated in the R
environment (Version 3.5.3). Redundancy analyses were performed to
identify overall differences in BEP profiles during different months in
the beekeeping season, stages, or according to the rearing environment
(package ``vegan''). Months was included as a fixed factor in the first
RDA, whereas Stage, Environment, and their interaction were included in
the second RDA. In both RDAs, ``Colony IDs'' were included as a random
factor, and pairwise comparisons were performed between group levels
with corrections for multiple testing using the
\textit{pairwise.factorfit} function (package ``RVAideMemoire''). 

Total BEP, ethyl/methyl ratio, and single compound amounts were
analysed using linear mixed models, with Months or Stage and
Environment as fixed factors and colonies as random factors. Wald tests
were used to assess the effect of independent variables, and pairwise
comparisons of estimated marginal means were performed using the
\textit{emmeans} function (package ``emmeans''). The $P$ values of
pairwise comparisons were adjusted using false discovery rate
correction~\cite{21}.

We also normalised the amount of each BEP compound according to the
maximum emitted during the 7 months of measures (temporal effect) or
along the 9 stages to represent the relative median values in a
heatmap.

\section{Results}\label{sec2}
\subsection{Temporal variations of brood ester pheromone (BEP)
production}\label{ssec21}

To highlight the main tendencies in the BEP pheromonal bouquet over
time, we performed multivariate redundancy analysis (RDA). The first
two components of the RDA explained 92.9\% of the variance. Emissions
varied throughout the beekeeping season (RDA; Season:
$F_{6,115}=22.51$, $p=0.001$). The first component of the RDA allowed
the separation of samples collected in July, August, and September from
those collected in April, May, June, and October, with the latter being
tightly clustered together (Figure~\ref{fig1}). The second component
separated samples collected in September from those collected in July
and August, which harbour highly similar profiles.

\begin{figure*}
{\vspace*{-2pt}}
\includegraphics{fig01}
{\vspace*{-4pt}}
\caption{\label{fig1}\fontsize{9.95}{12}\selectfont
Redundancy analysis (RDA) performed on brood ester
pheromone (BEP) compounds captured on last-instar larvae, monthly from
April to October. Compounds are represented by their IDs and grey
arrows. MP, methyl palmitate; MS, methyl stearate; MO, methyl oleate;
ML, methyl linoleate; MLN, methyl linolenate; EP, ethyl palmitate; ES,
ethyl stearate; EO, ethyl oleate; EL, ethyl linoleate; ELN, ethyl
linolenate.}
{\vspace*{-4pt}}
\end{figure*}

Samples collected in April, May, June, and October harboured similar
bouquets, whereas samples from July and August were grouped together;
September samples formed a third group of samples.

For each month, $n=18$. Compounds are represented by their IDs and grey
arrows. MP, methyl palmitate; MS, methyl stearate; MO, methyl oleate;
ML, methyl linoleate; MLN, methyl linolenate; EP, ethyl palmitate; ES,
ethyl stearate; EO, ethyl oleate; EL, ethyle linoleate; ELN, ethyl
linolenate.

Comparison of total BEP production over time showed a significant
difference in total BEP abundance (LMM; $X_{6,115}=447.6$, $p &lt; 2\times
10^{-16}$; Figure~\ref{fig2}A). In particular, BEP levels greatly
increased during summer (July, August and September). Following the
variable structuration visible on the RDA, we then examined the
ethyl/methyl ratio by estimating the proportion of the amount
represented by the ethyl compounds over the total amount. The ratio
significantly differed across time (LMM; $X_{6,115} = 126.8$,  $p &lt;
2\times 10^{-16}$), with a drastic increase in ethyl compound amounts
in September (Figure~\ref{fig2}B).

\begin{figure*}
\includegraphics{fig02}
\caption{\label{fig2}Temporal variations in brood ester pheromone (BEP)
compounds. (A) Total amount of BEP variations. (B) Ethyl/methyl ester
ratio variations. (C) Heat map showing the temporal variations of the 10
components of the BEP. $N=18$ per month. Different letters indicate
significant differences between groups. MP, methyl palmitate; MS,
methyl stearate; MO, methyl oleate; ML, methyl linoleate; MLN, methyl
linolenate; EP, ethyl palmitate; ES, ethyl stearate; EO, ethyl oleate;
EL, ethyl linoleate; ELN, ethyl linolenate.}
{\vspace*{2pt}}
\end{figure*}

When looking at the temporal evolution of each compound, the 10 BEP
compounds could be classified in two groups (Figure~\ref{fig2}C). The
first group, composed of all 5 methyl compounds (MP, MS, MO, ML, MLN)
reached their maximum in July or August. In particular, when compared
to the April level, MO, MP, MS, and MLN exhibited significantly higher
levels in July, August, September, and to a lesser extent in October
for MO and MLN (Figure~S1, Table~S1). ML displayed higher levels in
July and August, and significantly lower levels in May, June, and
October (Figure~S1, Table~S1). The second group, composed of all five
ethyl compounds, reached their maximum in September. EP and ELN indeed
displayed an isolated peak of production in September; EO and ES also
showed this peak, but with an intermediate increase in August
(Figure~S1, Table~S1). Ethyl linoleate (EL) displayed a peculiar
temporal dynamic, with marked production in spring and particularly in
May (Figure~\ref{fig2}C).

\subsection{Effect of the rearing environment on brood ester pheromone
production during development}\label{ssec22}

To highlight the main tendencies in the BEP pheromonal bouquet across
the development and the rearing environment (natural rearing conditions
in the hive {versus} in vitro rearing conditions in the laboratory),
we performed a multivariate redundancy analysis (RDA). The first two
components of the RDA explained 87.60\% of the variance. Production
varied along the development (stages) and according to the rearing
environment (RDA; Stage: $F_{8,113} = 17.69$, $p = 0.001$; Rearing:
$F_{1,113} = 23.26$, $p = 0.001$), and the interaction between factors
was significant ($F_{8, 113} = 4.74$,  $p = 0.001$). The first
component of the RDA allowed the separation of samples from the
last-instar larvae (L5) and sealed larvae (LS) groups from the rest of
the stages (Figure~\ref{fig3}). The second component separated samples
reared in vitro from those reared in hives. This separation seemed to
be linked with differential expression of MS, MO, and MP in the in
vitro reared group compared with the in-hive reared group, which was
more characterised by ML, EL, EN and to a least extent EP, MN, EO and~ES.

\begin{figure*}
\includegraphics{fig03}
\caption{\label{fig3}Redundancy analysis (RDA) performed on brood ester
pheromone (BEP) compounds captured at~9 different stages of honey bee
brood, reared under natural (hive) or in vitro (IV) conditions.
Compounds are represented by their IDs and grey arrows. MP, methyl
palmitate; MS, methyl stearate; MO, methyl oleate; ML, methyl
linoleate; MLN, methyl linolenate; EP, ethyl palmitate; ES, ethyl
stearate; EO, ethyl oleate; EL, ethyl linoleate; ELN, ethyl
linolenate.}
\end{figure*}

Comparison of total BEP productions according to stage and rearing
environment showed \mbox{significant} differences in total BEP
abundance ($F_{\mathrm{stage}} = 73.81$, $p &lt; 2\times 10^{-16}$; ddl
$=$\ 8, 113; $F_{\mathrm{environment}} = 73.82$, $p &lt; 10^{-14}$; ddl
$=$\ 1, 113; $F_{\mathrm{interaction}} = 7.25$, $p &lt; 10^{-8}$; ddl $=$\ 
8, 113; Figure~\ref{fig4}A). In particular, BEP amounts were similar
during the open brood stages (L3, L4 and L5). However, significant
differences were identified for the sealed larval stage (LS), prepupae,
and all pupal stages, with more production in vitro compared with the
hive-reared group \mbox{(Figure~\ref{fig4}A).} \mbox{Interestingly,} total BEP
amounts dropped dramatically in (pre)-pupae reared naturally, whereas
they remained \mbox{significant} in pupae reared in vitro. The ethyl/methyl
ratio varied according to the stage (\mbox{$F_{8,113}=7.21$}, 
\mbox{$p&lt;10^{-6}$}) and the rearing environment
(\mbox{$F_{1,113}=9.83$}, \mbox{$p=0.0023$}), but significant
differences were only detected at the PP and P3 stages, where more
ethyl was emitted by the hive group than by the in vitro reared group
(Figure~S2).

\begin{figure*}
\includegraphics{fig04}
{\vspace*{2pt}}
\caption{\label{fig4}Stage and rearing environment variations of brood
ester pheromone (BEP) compounds. (A)~Total amount of BEP variations. (B)
Heat map showing the variations in the components of the BEP according
to the stage in the hive-reared group. (C) Heat map showing the
variations of the components of the BEP according to the stage in the
in vitro reared group  $5 \leq n \leq 15$ per stage and rearing
condition. Different letters indicate significant differences between
groups. MP, methyl palmitate; MS, methyl stearate; MO, methyl oleate;
ML, methyl linoleate; MLN, methyl linolenate; EP, ethyl palmitate; ES,
ethyl stearate; EO, ethyl oleate; EL, ethyl linoleate; ELN, ethyl
linolenate.}
\end{figure*}

When looking at the evolution of each compound amount along
development, the 10 BEP compounds showed overall similar variation
patterns in both rearing environments, with low levels of production at
the L3 and L4 stages, followed by drastic increases at the L5 and LS
stages, before decreasing again to low levels during the pupal stage
(Figure~\ref{fig4}B, Figure~S2, Table~S2). However, in the hive group,
two groups of BEP compounds could be distinguished. The first group,
composed of MO, ML, and EP, reached their maximum at the L5 stage,
whereas the second group (MP, MS, MLN, ES, EO, EL and ELN) peaked at
the LS stage. All compounds had their most abundant amounts at the L5
and LS stages (Figure~S2). Even though the overall patterns were highly
similar in the in vitro reared group compared with the hive group, all
compounds reached their maximum at the LS stage in the in vitro group,
and ML and EL displayed overall low expression levels, which prevented
the study of their developmental evolution pattern. The most abundant
amounts were also quantified for the L5 and LS stages, similar to the
hive group for the ethyl compounds; however, the most abundant amounts
were observed for the LS and PP stages for the methyl compounds
(Figure~S2).\looseness=-1

\section{Discussion}\label{sec3}

This study investigated the ecophysiology of brood ester pheromone
(BEP) production in honey bee brood. We highlight marked temporal
differences in BEP produced by last instar larvae, with a peak at the
end of the beekeeping season towards fall. In \mbox{addition,} comparison of
BEP profiles during \mbox{development} and depending on the rearing conditions
highlight important quantitative variations, with a peak of production
in the last instar or sealed larvae. Larvae reared in vitro versus
under natural conditions in their colonies displayed similar BEP
profiles, whereas this was not the case at the pupal stage, where
production was more abundant when bees were reared in vitro.\looseness=-1

The developmental profile highlighted in this study corroborates
previous findings. Trouiller \mbox{\etal}~\cite{22} also identified a peak in
emission in \mbox{9-day-old} larvae reared in the field, which corresponds to
the last-instar larval stage (L5) described in the present study.
Differences in the ethyl/methyl ratios of young (L3) versus old (L5)
larvae have also been previously identified and are linked to the
ability of adult honey bees to distinguish young from old
larvae~\cite{9}. 

\break
When comparing rearing environments, our results confirm that in vitro
rearing methods seem suitable for studying the chemical ecology of
honey \mbox{larvae,} especially the relationship between adults and the brood,
as shown by highly similar BEP profiles in L3, L4, and L5 larvae reared
in their hives or in vitro (the only differences were found for ES and
MLN, and the L5 stage only). However, studying the chemical ecology of
brood at the pre- and pupal stage under controlled conditions might
require more caution. We observed a significant decrease in BEP
production towards zero in (pre-)pupae when they were reared in the
hive, which corresponds to earlier descriptions~\cite{22}.
Surprisingly, despite their apparent normal and healthy morphological
profiles, \mbox{(pre-)pupae} reared in vitro showed marked BEP production
until the P5 stage (80 to 200 ng/bee on average). This marked
production might be an indication of stress at this brood stage, as it
has been suggested that sick pupae may also display significant amounts
of BEP~\cite{18}.

The influence of the season on chemical communication has been
extensively investigated, both on the emission (pheromone production)
and reception (behavioural and/or physiological response) sides. Many
studies have identified seasonal differences in pheromone composition
and/or quantity in various species, from plants to insects and
birds~\cite{23,24,25,26,27,28,29,30}. Such variations have been
strongly linked with plant phenology and external environmental
parameters, particularly temperature. In honey bee colonies,
temperature inside the hive is a constant and tightly regulated
feature, which therefore cannot explain changes in chemical compound
production or emission. In social insects such as ants and termites,
temporal changes have been highlighted, particularly for cuticular
hydrocarbons~\cite{31,32,33,34,35}. However, these temporal changes
were frequently linked to changes in the reproductive status and/or the
social environment of the colonies. Seasonal variations (covering a
period from spring to fall) have been described in \textit{Solenopsis
invicta}~\cite{32} and \textit{Formica tremcorum}~\cite{31}, but
temporal and spatial factors were confounded in the latter case.
Temporal changes in pheromone emissions during the active season have
only been studied in bees for ethyl oleate (EO) in
worker~bees~\cite{36}. Interestingly, EO also increased at the end of
the beekeeping season in worker bees in July and August. This trend was
particularly strong in nectar foragers compared with nurses and pollen
foragers~\cite{36}. The trend observed in larvae was similar overall
for the 10 BEP compounds, but EO peaked in September in larvae and not
in August. This could be due to environmental condition variability
and/or caste differences between the brood and adults. Such an increase
in fall may be linked to the special physiology of bees that are raised
at this time of the year, when winter bees are produced. These bees
have a longer life expectancy, with a distinct physiology~\cite{37},
and such features might be initiated at the larval stage because
different BEP emissions may trigger differences in brood care provided
by adult bees~\cite{9}.

These changes in production during the summer months may also reflect a
different physiology at the colony level, as suggested by the influence
of BEP on the regulation of labour among adults and its impact on
colony growth rate~\cite{13}. Because BEP addition triggers an increase
in pollen foragers and pollen load weights~\cite{13,38,39}, such an
increase in larvae may reflect a shortage in proteins in the colonies,
as frequently reported by beekeepers at this time of the year. The bias
in the ethyl/methyl ratio observed at the end of the season, especially
in September (as reflected by the drastic increase in EO and the three
other quantifiable ethyl esters), may also influence social regulation
at the colony level. EO produced by adults delays the onset of foraging
in worker bees~\cite{40}. Therefore, increased levels of EO in larvae
may also produce the same effect and trigger a relative increase in
nurse bees within the hive. This may be the result of larvae requiring
higher care at this time of the year.

This descriptive study highlighted new features of the temporal
chemical profile of honey bee brood. Changes according to the season
(spring/summer/fall) and the rearing environment underline a knowledge
gap in the understanding of the physiology of larvae and pupae and the
link between stress and the chemical ecology profile of the brood. The
next steps should involve more functional studies to assess the
consequences of such changes in individual compounds or mixtures of
several molecules on the behaviour and physiology of individuals within
the colonies, using techniques such as electroantennography, similar to
those described in~\cite{41}. The consequences of such changes in the
brood ester pheromone profile at the colony level would be interesting
to investigate. It would also provide new knowledge about how bees
respond dynamically, both at the individual and colony levels, to
changing environments.

\section*{Author contributions}

FM, DB, and YLC designed the project. FM and MC performed in vitro
rearing. FM and DB performed the chemical analyses. FM analysed the
data and wrote the manuscript. YLC reviewed the manuscript. 

\section*{Declaration of interests}

The authors do not work for, advise, own shares in, or receive funds
from any organization that could benefit from this article, and have
declared no affiliations other than their research organizations.

\section*{Acknowledgments} 

We thank J. Senechal and D. Crauser for access to the honey bee
colonies, and A. Noel for R tricks.

\back{}

\section*{Supplementary data}

Supporting information for this article is available on the journal's
website under \printDOI\ or from the author.

\CDRsupplementaryTwotypes{supplementary-material}{\cdrattach{crchim-270-suppl.pdf}}

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