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\DOI{10.5802/crchim.228}
\datereceived{2023-01-12}
\daterevised{2023-03-06}
\dateaccepted{2023-03-06}
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\dateposted{2023-04-26}
\begin{document}

\begin{noXML}

%\TopicEF{Chemical Ecology -- Chemical Mediation in the
%Environment}{\'Ecologie chimique -- M\'ediation chimique dans
%l'environnement}

\title{Chemical profiles of venom glands in queens, foundresses,
pre-wintering gynes, and workers in the hornet \protect\textit{Vespa
velutina nigrithorax}}

\alttitle{Profils chimiques des glandes \`{a} venin chez les reines,
les fondatrices, les gynes pr\'{e}-hivernantes et les ouvri\`{e}res du
frelon \textit{Vespa velutina nigrithorax}}

\author{\firstname{Laurence} \lastname{Berville}\CDRorcid{0000-0001-9540-9869}}
\address{IRBI, UMR CNRS 7261, University of Tours, 37200 Tours, France}
\email[L. Berville]{laurence-berville@hotmail.fr}

\author{\firstname{Christophe} \lastname{Lucas}\CDRorcid{0000-0003-2839-7583}}
\addressSameAs{1}{IRBI, UMR CNRS 7261, University of Tours, 37200
Tours, France}
\email[C. Lucas]{christophe.lucas@univ-tours.fr}

\author{\firstname{M\'elissa} \lastname{Haouzi}\CDRorcid{0000-0002-8851-9223}}
\addressSameAs{1}{IRBI, UMR CNRS 7261, University of Tours, 37200
Tours, France}
\email[M. Haouzi]{melissa.haouzi@univ-tours.fr}

\author{\firstname{Ali} \lastname{Khalil}}
\addressSameAs{1}{IRBI, UMR CNRS 7261, University of Tours, 37200
Tours, France}
\address{Oniris, Ecole Nationale V\'{e}t\'{e}rinaire, Agroalimentaire
et de l'Alimentation, 44322 Nantes, France}
\email[A. Khalil]{khalile\_a@hotmail.com}

\author{\firstname{J\'er\'emy} \lastname{G\'{e}var}\CDRorcid{0000-0001-9084-6445}}
\address{iEES Paris, INRAE, 78026 Versailles, France}
\email[J. G\'{e}var]{Jeremy.Gevar@inrae.fr}

\author{\firstname{Anne-Genevi\`eve} \lastname{Bagn\`{e}res}\CDRorcid{0000-0002-1729-379X}}
\address{CEFE, CNRS, University of Montpellier, EPHE, IRD, Montpellier,
France}
\email[A.-G. Bagn\`{e}res]{ag.bagneres@cefe.cnrs.fr}

\author{\firstname{Eric} \lastname{Darrouzet}\CDRorcid{0000-0002-7224-6937}\IsCorresp}
\addressSameAs{1}{IRBI, UMR CNRS 7261, University of Tours, 37200
Tours, France}
\email[E. Darrouzet]{eric.darrouzet@univ-tours.fr}

\keywords{\kwd{Yellow-legged hornet}\kwd{Venom gland}\kwd{Invasive
species}\kwd{Chemical communication}\kwd{Vespidae}}

\altkeywords{\kwd{Frelon \`{a} pattes jaunes}\kwd{Glande \`{a}
venin}\kwd{Esp\`{e}ces invasives}\kwd{Communication chimique}\kwd{Vespidae}}

\shortrunauthors

\begin{abstract}
Invasive species are permanently modifying the distribution and
diversity of native species worldwide. For nearly two decades, a
hornet, \textit{Vespa velutina nigrithorax} (Hymenoptera: Vespidae),
has been spreading in Europe. Due to its marked invasiveness, this
yellow-legged hornet is of great economic and ecological concern,
mainly because of the damage it causes to insects in general and bees
in particular. Current management methods are sparse and ineffective.
Naturally produced by insects, semio-chemicals have been proposed as
integrated management tools in this context, either for disruption or
mass trapping, as an alternative tool to conventional non-selective
traps. Here, we focused on the venom gland, which produces the alarm
pheromone. In previous studies, individuals showed marked diversity in
their chemical profiles. However, to successfully conduct targeted
pheromone-based trapping, the hornet's chemical ecology must be
thoroughly characterized. Therefore, it was necessary to better
understand the chemical composition of the alarm pheromone of not only
workers but also other \textit{V. v. nigrithorax} females.
%{\pagebreak}
 First, we
evaluated the differences in venom gland profiles between the four
types of females: queens, foundresses, pre-winter gynes, and workers.
Next, we experimentally explored the venom gland profiles of \textit{V.
velutina nigrithorax} workers and pre-wintering gynes by \textit{in
vivo} and \textit{in vitro} approaches. We found 13 new compounds in
the venom gland, of which 9 were identified (chain lengths: C\tsub{8}
to C\tsub{12}). Two compounds were found exclusively in reproductive
females. Profiles differed among pre-wintering gynes, foundresses, and
queens but not between pre-wintering gynes and workers.
 This result
indicates that the chemical signature of the female venom gland changes
over the course of life history: from pre-wintering gynes to
foundresses to queens. 
\end{abstract}

\begin{altabstract}
Les esp\`{e}ces invasives modifient en permanence la r\'{e}partition et
la diversit\'{e} des esp\`{e}ces indig\`{e}nes dans le monde entier.
Depuis pr\`{e}s de vingt ans, un frelon originaire d'Asie du Sud-Est,
\textit{Vespa velutina nigrithorax} (Hymenoptera : Vespidae), se
r\'{e}pand en Europe. En raison de son caract\`{e}re invasif
marqu\'{e}, ce frelon \`{a} pattes jaunes suscite de vives
inqui\'{e}tudes sur le plan \'{e}conomique et \'{e}cologique,
principalement en raison des d\'{e}g\^{a}ts qu'il cause aux insectes en
g\'{e}n\'{e}ral et aux abeilles en particulier. Les m\'{e}thodes de
gestion actuelles sont rares et inefficaces. Naturellement produits par
les insectes, les produits s\'{e}mio-chimiques ont \'{e}t\'{e}
propos\'{e}s comme outils de gestion int\'{e}gr\'{e}e. Dans ce
contexte, soit pour la perturbation, soit pour le pi\'{e}geage de
masse, en tant qu'outil alternatif aux pi\`{e}ges non s\'{e}lectifs
conventionnels. Ici, nous nous sommes concentr\'{e}s sur la glande
\`{a} venin, qui produit la ph\'{e}romone d'alarme. Des \'{e}tudes
ant\'{e}rieures ont montr\'{e} que les femelles pr\'{e}sentaient une
variabilit\'{e} dans les profils chimiques. Cependant, pour mener \`{a}
bien un pi\'{e}geage cibl\'{e} \`{a} base de ph\'{e}romones,
l'\'{e}cologie chimique du frelon doit \^{e}tre caract\'{e}ris\'{e}e de
mani\`{e}re approfondie. Il \'{e}tait donc n\'{e}cessaire de mieux
comprendre la composition chimique de la ph\'{e}romone d'alarme non
seulement des ouvri\`{e}res, mais \'{e}galement des autres femelles de
\textit{V. v. nigrithorax}. Tout d'abord, nous avons examin\'{e} les
diff\'{e}rences dans les profils des glandes \`{a} venin entre les
quatre types de femelles : les reines, les fondatrices, les gynes
pr\'{e}-hivernantes et les ouvri\`{e}res. Ensuite, nous avons
explor\'{e} exp\'{e}rimentalement les profils des glandes \`{a} venin
des ouvri\`{e}res de \textit{V. velutina nigrithorax} et des gynes
pr\'{e}-hivernantes par des approches in vivo et in vitro. Nous avons
constat\'{e} la pr\'{e}sence de 13 nouveaux compos\'{e}s dans la glande
\`{a} venin, dont 9 ont \'{e}t\'{e} identifi\'{e}s (longueur des
cha\^{i}nes : C8 \`{a} C12). Deux compos\'{e}s ont \'{e}t\'{e}
trouv\'{e}s exclusivement chez les femelles reproductrices. Les profils
diff\`{e}rent entre les gynes pr\'{e}-hivernantes, les fondatrices et
les reines, mais pas entre les gynes pr\'{e}-hivernantes et les
ouvri\`{e}res. Ce r\'{e}sultat indique que la signature chimique de la
glande \`{a} venin femelle \'{e}volue au cours de l'histoire de vie :
des gynes pr\'{e}-hivernantes aux fondatrices, en passant par les
reines. 
%{\vspace*{-1pt}}
\end{altabstract}

\maketitle

%{\vspace*{-6pt}}

\break

\twocolumngrid

\end{noXML}

\section{Introduction}

{\vspace*{-2pt}}

Invasive species are increasingly affecting the Earth's
ecosystems~\cite{1}. They are permanently modifying the distribution
and diversity of native species worldwide~\cite{2} at a variety of
biological scales and with differing degrees of severity~\cite{3}. In
many different habitats, social insects are highly successful invaders
because of their excellent ability to disperse, compete for resources,
mount effective defenses, reproduce at high rates, and exploit a broad
range of habitats and diets~\cite{4}. Native to China, the invasive
yellow-legged hornet, \textit{Vespa velutina nigrithorax}~\cite{5}, 
was accidentally introduced into southwestern France
around 2004~\cite{6}. It is a hardy, adaptable species whose flexible
dietary requirements have allowed it to invade and establish itself
within a variety of habitats~\cite{7}.  \textit{Vespa v. nigrithorax}
has successfully expanded its range and is now found throughout most of
France~\cite{8} as well as in several other European countries
\cite{9,10,11,12,13}. Ecosystems invaded by this hornet are impacted in
various ways since \textit{V. v. nigrithorax} preys upon several insect
and arthropod taxa~\cite{14}, including the domestic honey bee,
\textit{Apis mellifera}~\cite{15}. Finally, \textit{V. v. nigrithorax}
presents human health risks as hornets can aggressively spray liquid in
people's eyes~\cite{16}; also, their stings are painful and sometimes
even fatal~\cite{17,18}. Consequently, medical practitioners and
researchers are increasingly seeking information on \textit{V. v.
nigrithorax} venom for diagnostic and treatment purposes~\cite{17,19}.
The hornet's introduction into Europe led to calls for control efforts,
which have largely focused on passively trapping adults using homemade
or commercial poison baits~\cite{20}. Unfortunately, this approach has
done little to limit \textit{V.~v.~nigrithorax} population
sizes~\cite{20,21}. Furthermore, because they lack specificity, these
traps can have significant ecological impacts on a wide range of
non-target species~\cite{22,23}. As a result, work is currently
underway~\cite{20} to develop new species-specific systems that are
based on pheromones~\cite{24,25,26,27}. 

Chemical signals are an essential component of animal communication
systems~\cite{28,29} and mediate a variety of behaviors, including
social organization. In insects, long-distance signals play an
important role in intra- and interspecific communication~\cite{29} and
elicit species-specific responses. Such communication provides fitness
benefits by enhancing colony cohesion, defense, and alarm systems.
There is a long history of research on chemical signals (e.g.,
pheromones) in social insects (e.g., ants, bees, hornets, and
termites); these signals often trigger different behaviors
(recruitment, dispersal, aggressiveness, or aggregation) that can
affect colony survival~\cite{30,31}. Thus, a promising pest control
approach is to develop species-specific attractants (e.g., trap bait)
or repellents that target specific life stages. Alarm pheromones are of
key importance because they are deployed when colonies are threatened:
they prompt nestmate recruitment and defense behaviors~\cite{32,33}.
They can also be used by workers to signal danger in areas farther away
from the nest~\cite{34}. The most effectively disseminated signals are
made up of highly volatile compounds that act quickly and then
dissipate, limiting further responses. Most alarm pheromones in social
insects are multicompound blends~\cite{24,35,36} that are generally
involved in both offensive and defensive reactions by workers. The
venom gland tends to be the primary source of alarm
pheromones~\cite{33,37}, although interspecific variation exists. Thus,
during aggressive interactions, alarm signals are released at the same
time as venom~\cite{38}. Such is the case in several wasp and hornet
species, including \textit{Polistes dominulus}~\cite{39},
\textit{Vespula squamosa}~\cite{40}, \textit{Vespa crabro}~\cite{41},
\textit{Vespa mandarinia}~\cite{37}, and \textit{Vespa simillima
xanthoptera}~\cite{37}. \looseness=-1

The first study conducted on alarm pheromones in a subspecies of the
yellow-legged hornet characterized the volatile compounds found in
worker venom glands in a native \textit{V.~velutina auraria} population
in China~\cite{42}. A second study~\cite{43} did the same in an
invasive \textit{V. v. nigrithorax} population in France. In both
cases, researchers identified some venom gland compounds that elicited
hornet attacks when colonies were threatened. When the alarm pheromones
were released, they attracted other workers and encouraged stinging
behavior. However, compound abundance differed between the two
subspecies. Next, researchers explored the chemical signals associated
with caste and species' reproductive status using four colonies in an
invasive population in Italy; the study looked at compounds in volatile
venom gland mixtures in gynes and workers~\cite{27}. Twelve major
compounds were observed, which contrasted with the 16 and 17 compounds
observed previously (Refs.~\cite{42,43} respectively). Five of these 12
compounds had been seen in the first two studies. However, others had
never been reported before, including 4 unknown acetates of aliphatic
alcohols and citronellyl acetate, which had the second highest
abundance~\cite{27}. Finally, work on an invasive population in Spain
observed 8 compounds~\cite{26}, collected \textit{in vivo} by SPME from
seven hornets, including 3 previously seen ketones~\cite{27,42,43}.
However, in this most recent study, individuals displayed marked
diversity in their chemical profiles since 2 of the 8 compounds were
found in only one hornet and 3 others in fewer than 3
hornets~\cite{26}; only one compound occurred across all the profiles.
There is clearly a need for further research given this range of
results. First, a better understanding of the chemical composition of
\textit{V. v. nigrithorax}'s alarm pheromone is needed. Second, since
gynes, foundresses, and queens alike use alarm pheromones, we also must
clarify associations between hornet caste and alarm pheromone
composition. However, in the latter three cases, the volatile compounds
involved remain poorly characterized. Here, we hypothesized that
females from different castes and/or with different tasks might differ
in their venom gland profiles. 

This study thus had two goals. First, we assessed differences in venom
gland profiles among the four types of females: queens, foundresses,
pre-wintering gynes, and workers. Second, we experimentally explored
venom gland profiles of \textit{V.~velutina nigrithorax} workers and
pre-wintering gynes via an \textit{in vivo} approach (collection of
volatiles, solid-phase microextraction (SPME)) and an \textit{in vitro}
approach (liquid extraction of compounds from the venom gland).

\begin{figure*}
{\vspace*{2pt}}
\includegraphics{fig01}
{\vspace*{-3pt}}
\caption{\label{fig1}\textit{Vespa velutina nigrithorax} collection
sites.}
%{\vspace*{-3pt}}
\end{figure*}

\section{Materials and methods} 
\subsection{Study sites and hornet sampling} 

Hornet colonies were collected at 44 different sites in the
administrative department of Indre-et-Loire, which is part of the
Centre-Val-de-Loire region of France (Figure~\ref{fig1}). The
yellow-legged hornet is \mbox{classified} as an invasive species, and all
research was carried out in compliance with relevant national
guidelines. To determine hornet caste (worker versus
reproductive), mass and wing spacing were measured~\cite{44}. The
reproductives were either pre-wintering gynes (females without nest),
foundresses (mated females with small nest and some eggs), or queens
(mated females with workers). To characterize the venom gland's
compounds, hornets of each caste were stored at ${-}$80~\textdegree C
until they could be dissected. For the second experiment, individuals
from a given colony were placed in a mesh breeder box and fed an
\textit{ad libitum} mixture of honey and water, which was replenished
daily. The hornets underwent SPME within three days of collection. 

\subsection{Analysis of volatile compounds} 

To characterize the venom gland's compounds, hornets ($n=75$ workers,
40 pre-wintering gynes, 30 foundresses, and 30 queens) were dissected
under a binocular microscope. Within each group, a set of 5 undamaged
venom sacs ($n=15$, 8, 6, and 6 replicates for the workers,
pre-wintering gynes, foundresses, and queens, respectively) was placed
in a vial containing heptane (1~mL). The vials were then stored at
${-}$20~\textdegree C. Glands were perforated right before performing the
gas chromatography-mass spectrometry (GC-MS) analysis; the vials were
vortexed at 300~rpm for 1~min. Gland extract (2~$\upmu$L) from each
vial were analyzed using a gas chromatograph (Agilent Technologies
7890B) coupled to a mass spectrometer (Agilent Technologies 7000C GC/MS
Triple Quad, Les Ulis, France) equipped with an HP-5 capillary column
(Agilent Technology, USA; 30~m $\times$ 250~$\upmu$m $\times$ 0.25
$\upmu$m) and using a Gerstel MPS autosampler (electron impact at
70~eV). Helium was the carrier gas (flow rate $=$\ 2.3~mL/min).
Injectors were used in splitless mode (splitless time $=$\ 2~min) at a
constant temperature of 250~\textdegree C. The oven temperature was set
to ramp up from 50~\textdegree C to 200~\textdegree C at a rate of
8~\textdegree C/min and then from 200~\textdegree C to 315~\textdegree
C at a rate of 5~\textdegree C/min (held constant for 5~min). Masses
were scanned between 15 and 550 amu at 0.1 scan/s. Immediately before
the analysis, 10~$\upmu$L of internal standard ($n$-eicosane at
$10^{-3}$~g/mL diluted in heptane) was added to each sample.

To identify and quantify the compounds released by workers or
pre-wintering gynes in response to stress, two experimental stimuli
were assessed. The experiments were conducted with sets of four live
co-workers or four live pre-wintering gynes. The hornets were cold
anesthetized (${-}$18~\textdegree C for 10~min) and were subsequently
placed in a clean 350~mL glass vial at room temperature
(19--21~\textdegree C for 10~min). The hornets were still able to move
but not to fly. Each~hornet was exposed once to either a stressed
hornet group (SH, replicates: pre-wintering gynes $n=4$; workers $n=8$)
or a wounded hornet group (WH, replicates: pre-wintering gynes $n=4$;
workers $n=8$). The SH treatment consisted of gently shaking the hornet
group inside the glass vial. The WH treatment consisted of applying
pressure to a hornet's thorax using metal forceps; wounded hornets were
then resealed before closing the glass vial. We assumed that the WH
treatment induced a higher stress level than the SH treatment. To
extract the resulting volatile compounds, a 40-min procedure was
carried out using red solid-phase microextraction (SPME) fibers coated
with polydimethylsiloxane (PDMS, 100~$\upmu$m; Supelco). Immediately
after the treatments, the fibers were placed within a metal wire to
prevent any physical contact with the hornets. First, for each caste,
two fibers were desorbed, and the resulting compounds were identified
using the temperature program and GC-MS procedure described above.
Second, individual fibers representing different castes and treatment
groups were desorbed ($\mathrm{WH}_{\mathrm{workers}}\,n=8$;
$\mathrm{SH}_{\mathrm{workers}}\,n=8$; $\mathrm{WH}_{\mathrm{gynes}}\,n=4$;
$\mathrm{SH}_{\mathrm{gynes}}\,n=4$) using a GC system (CPG
Agilent Technologies 7820A) equipped with a flame-ionization detector
(FID) and a capillary column (HP-5 Agilent Technology, Santa Clara,
USA; 30~m ${\times}$ 0.32~mm ${\times}$ 0.25~${\upmu}$m); helium served
as the carrier gas (1.7 ml/min). The temperature program was the same
as above. The relative area of each compound was calculated based on
the proportions represented by each peak within the chromatogram using
ChemStation (v. 04.02, Agilent). 

%tab1
\begin{table*}
\caption{\label{tab1}Compounds found in venom glands of \textit{V.
velutina} subspecies}
\begin{tabular}{c}
\tbody
\inlinefig{fx01}
\end{tabular}
\fontsize{9}{12}\selectfont
\tabnote{\protect\inlinefig{fx02} Peaks present in both
\textit{V.~v.~auraria}~\cite{42} and \textit{V. v.
nigrithorax}~\cite{43}. Peaks present in \textit{V. v. nigrithorax}
populations in \protect\inlinefig{fx03} Italy~\cite{27} and
\protect\inlinefig{fx04} Spain~\cite{26}. All the compounds observed in
this study (\textbf{a} to \textbf{z}) underwent MS analysis
(\textbullet) and were identified by comparing their mass spectra with
those in the NIST 2017 database ($\varepsilon$) and/or in the
literature (\protect\inlinefig{fx05}); final validation was performed
using available standards ($\Delta$). The major $m/z$ fragments are
sorted based on their occurrence frequency (highest to lowest). The
Kovats retention index values were calculated and obtained from PubChem
and the literature. Chemical Abstracts Service (CAS) of the American
Chemical Society. (!) compound (Figure~\ref{fig3}B) identified by
Rodr\'{i}guez-Flores \etal~\cite{26}
on the basis of the HS-SPME chromatogram
and mentioned as eluting after undecan-2-one. However, its MS spectra,
retention time, and KRI value differed from those of the
4,8-dimethyl-1,7-nonadiene standard. $^{*}$ Mass spectra of unknown
compound \textbf{s} (Figure~\ref{fig3}A) isolated from the venom gland
(Figure~\ref{fig3}). $n$: \textit{Vespa velutina nigrithorax. a: Vespa
velutina auraria.}}
\end{table*}

\subsection{Compound identification}

The data were analyzed using MassHunter (v. B.07.00; Agilent) and AMDIS
(v. 2.0g; NIST, 2011). Compounds were identified based on their mass
spectra, which were interpreted via fragmentation analyses~\cite{45}.
The results were then compared to published \mbox{spectra} and/or confirmed
using commercial standards (Table~\ref{tab1}) purchased from different
suppliers (Sigma-Aldrich, Saint-Louis, Missouri, USA; GreenPharma,
Orl\'{e}ans, France; and ChemSpace, Monmouth Junction, New Jersey,
USA). The molecules 4,8-dimethyl-7-nonen-2-one and
4,8-dimethyl-1,7-nonadiene were synthesized by Synthenova
(H\'{e}rouville-Saint-Clair, France). Kovats retention index (KRI)
values were calculated based on the retention times of C8--C20 n-alkane
standards (Fluka, 94234) that were analyzed under the same set of GC
and GC-MS conditions.

\subsection{Statistical analyses} 

Venom gland profile composition was analyzed using multivariate
principal component analysis (PCA) performed in Statistica (v. 10;
Statsoft). The analysis used the percentage representation of
identified compounds that occurred above a minimum threshold of 0.5\%.
The worker and gyne profiles obtained in the stress experiment were
explored using another PCA, which employed the percentage
representation of all the compounds collected via PDMS-SPME. To assess
profile-based grouping patterns, we conducted a K-means cluster
analysis~\cite{46}. Because the number of groups is unknown ahead of
time, the choice of initial group number is somewhat arbitrary. To
optimize this number, we used the Cali\~{n}ski-Harabasz
index~\cite{47}.

\section{Results}
\subsection{Identification of venom gland compounds} 

The venom gland profiles contained 38 compounds with chain lengths
ranging from 8 to 12 carbons (Figure~\ref{fig2}, Tables~\ref{tab1} and 
\ref{tab2}). Of these, 26 occurred at levels higher than the 0.5\%
threshold (Table~\ref{tab2}). This group included 11 ketones, 8 esters,
4 alcohols, and 3 unknown molecules. The MS spectrum were investigated,
and the identities of the compounds were confirmed using chemical
standards (Table~\ref{tab1}). The unknown compounds were subject to MS
interpretation. The third unknown compound (\textbf{s},
Table~\ref{tab2}, Figure~\ref{fig3}A) was thought to be
4,8-dimethyl-1,7-nonadiene (Figure~\ref{fig3}B, Table \ref{tab1}).
Indeed, this compound was identified~\cite{26} based on the HS-SPME
chromatogram. However, its MS spectra, retention time, and KRI value
differed from those of the 4,8-dimethyl-1,7-nonadiene standard
(Figure~\ref{fig3}). The MS spectra for compound \textbf{s}
(Figure~\ref{fig3}A) and \mbox{citronellyl} acetate (compound \textbf{r})
shared some similarities. However, they differed in the presence of a
fragment at $m/z$ 152 fragment and a trace of $m/z$ 170. \mbox{Furthermore,}
compared to the citronellyl acetate, compound \textbf{s} had higher
peaks at $m/z$ 137 (instead of at $m/z$ 138), $m/z$ 96, and $m/z$ 109.
Unfortunately, spectra for the citronellyl family remain quite rare,
limiting more detailed comparisons. Additionally, we found no other
commercial compounds with more similar spectra, thus we were not able
to confirm our hypothesis.\looseness=-1

\begin{figure*}
\includegraphics{fig02}
\caption{\label{fig2}Chemical profiles of hornet workers, gynes,
foundresses, and queens (unit of analysis $=$\ pools of five venom
glands). The letters refer to the different compounds observed
(Table~\ref{tab2}).}
%{\vspace*{-8pt}}
\end{figure*}

\begin{figure*}
{\vspace*{-1pt}}
\includegraphics{fig03}
{\vspace*{-1pt}}
\caption{\label{fig3}(A) Mass spectra for unknown compound \textbf{s}
(Kovats retention index $=$\ 1310), isolated from the venom gland of
\textit{V. v. nigrithorax}. (B) Mass spectra for the
4,8-dimethyl-1,7-nonadiene standard (Kovats retention index $=$\ 1149).
(C) Combined GC-MS profiles for C10-C18 and 4,8-dimethyl-1,7-nonadiene.
The blue arrow represents the molecule \textbf{s} location (RT), in the
gland profile.}
{\vspace*{-2pt}}
\end{figure*}

%tab2
\begin{table*}
\caption{\label{tab2}Chemical composition of the venom glands of
\textit{V. v. nigrithorax} queens, foundresses, workers, and
pre-wintering gynes (percentages ${\pm}$ standard error), which were
obtained either via solvent- or fiber-based extraction{\vspace*{-2pt}}} 
\fontsize{8}{9.5}\selectfont
\tabcolsep3pt
\begin{tabular}{cccccccccc}
\thead
\xmorerows{2}{Name}
&amp;&amp; \multicolumn{4}{c}{VENOM GLAND's profile} 
&amp; \multicolumn{4}{c}{SPME analysis} \\\cline{3-6}\cline{7-10}
&amp;&amp;&amp;&amp;&amp;&amp; SH &amp; WH &amp; SH &amp; WH \\\cline{7-8}\cline{9-10}
&amp; &amp; Queens &amp; Foundresses &amp; Workers &amp; Prewintering gynes &amp;
\multicolumn{2}{c}{Workers} &amp;
\multicolumn{2}{c}{Prewintering gynes} \\
\endthead
Acetophenone &amp;\textbf{a}                        &amp; $1.7\pm 1   $ &amp; $0.4\pm 0.3 $ &amp; $0.2\pm 0.2 $ &amp; $8\times 10^{-3}\pm 1\times 10^{-3}$ &amp; - &amp; -  &amp;  - 	   &amp; -            \\
8-Nonen-2-one &amp;\textbf{b}                       &amp; $0.4\pm 0.3 $ &amp; $0.8\pm 0.2 $ &amp; $3.7\pm 0.9 $ &amp; $3.2\pm 0.5 $          	  &amp; $1.1\pm 0.9 $ &amp; $1.4\pm 0.4 $ &amp;  $3.1\pm 2.5 $ &amp; $1.1\pm 1.05 $ \\
Nonan-2-one &amp;\textbf{c}                         &amp; $3.4\pm 0.9 $ &amp; $16.4\pm 2.8$ &amp; $18.2\pm 3.2$ &amp; $14.2\pm 3.7$          	  &amp; $7.7\pm 3.8$  &amp; $10.9\pm 3.8$ &amp;  $3.3\pm 2.2$  &amp; $6.3\pm 2.6$   \\
Nonan-2-ol &amp;\textbf{d}                          &amp; $0.3\pm 0.3 $ &amp; $0.4\pm 0.2 $ &amp; $1.6\pm 0.8 $ &amp; $0.8\pm 0.9 $          	  &amp; $1.2\pm 0.7 $ &amp; $1.5\pm 0.5 $ &amp;  $1.8\pm 1.1 $ &amp; $0.6\pm 0.3 $  \\
Isoamyl isovalerate &amp;\textbf{e}                 &amp; $0.1\pm 0.1 $ &amp; $0.8\pm 0.2 $ &amp; $2.0\pm 0.9 $ &amp; $2.5\pm 0.8 $          	  &amp; $1.2\pm 1.3 $ &amp; $0.8\pm 0.5 $ &amp;  $0.4\pm 0.5 $ &amp; $1.2\pm 0.9 $  \\
Unknown 1 (cyclohexanol) &amp;\textbf{f}            &amp; $3\pm 0.4   $ &amp; $0.7\pm 0.1 $ &amp; $1.3\pm 0.9 $ &amp; $0.8\pm 0.7 $          	  &amp; -           &amp; -           &amp;  -           &amp; -            \\
Isoamyl senecioate &amp;\textbf{g}                  &amp; $0.6\pm 0.5 $ &amp; $0.2\pm 0.1 $ &amp; $0\pm 0     $ &amp; $0.1\pm 0.1 $                    &amp; -           &amp; -           &amp;  -           &amp; -            \\
X-C12:1 &amp;\textbf{h}                             &amp; $0.4\pm 0.4 $ &amp; $0.4\pm 0.1 $ &amp; $0.7\pm 0.9 $ &amp; $0.8\pm 1.1 $          	  &amp; -           &amp; -           &amp;  -           &amp; -            \\
Unknown 2 &amp;\textbf{i}                           &amp; $1.4\pm 0.3 $ &amp; $0.2\pm 0.1 $ &amp; $0.8\pm 0.6 $ &amp; $0.5\pm 0.5 $          	  &amp; -           &amp; -           &amp;  -           &amp; -            \\
4,8-dimethyl-7-nonen-2-one &amp;\textbf{j}          &amp; $28.3\pm 5.7$ &amp; $21.8\pm 2.9$ &amp; $23.3\pm 5.3$ &amp; $24.6\pm 3.1$          	  &amp; $34.4\pm 4.8$ &amp; $38.2\pm 3.1$ &amp;  $37.7\pm 7.6$ &amp; $32.5\pm 5.5$  \\
2-Nonanyl acetate &amp;\textbf{k}                   &amp; $1\pm 0.6   $ &amp; $3.7\pm 0.5 $ &amp; $3.4\pm 1.1 $ &amp; $4\pm 0.8   $          	  &amp; $5.6\pm 1.4 $ &amp; $6.7\pm 1.6 $ &amp;  $6.7\pm 1.2 $ &amp; $5.2\pm 1.1 $  \\
$\upbeta$-Citronellol, methyl ether &amp;\textbf{l} &amp; $4.5\pm 2.1 $ &amp; $1.0\pm 0.4 $ &amp; $1.2\pm 0.8 $ &amp; $1.7\pm 0.8 $          	  &amp; $4.1\pm 0.6 $ &amp; $3.5\pm 1.1 $ &amp;  $5.7\pm 2.1 $ &amp; $3.8\pm 0.8 $  \\
2-Phenethyl acetate &amp;\textbf{m}                 &amp; $0\pm 0     $ &amp; $0.4\pm 0.2 $ &amp; $0\pm 0     $ &amp; $0.2\pm 0.3 $          	  &amp; -           &amp; -           &amp;  -           &amp; -            \\
X-Undecen-2-one &amp;\textbf{n}                     &amp; $2.6\pm 0.2 $ &amp; $5.5\pm 0.4 $ &amp; $9.3\pm 2.9 $ &amp; $5.8\pm 1.4 $          	  &amp; $7.1\pm 2.4 $ &amp; $7.5\pm 2.8 $ &amp;  $4.9\pm 1.2 $ &amp; $5.7\pm 2.4 $  \\
X$'$-Undecen-6-one &amp;\textbf{o}                  &amp; $6.7\pm 1.8 $ &amp; $2.3\pm 0.7 $ &amp; $8.5\pm 2   $ &amp; $11.5\pm 2.0$          	  &amp; $1.3\pm 0.8 $ &amp; $0.9\pm 1.3 $ &amp;  $1.5\pm 1.3 $ &amp; $3.5\pm 0.7 $  \\
X$''$-Undecen-2-one &amp;\textbf{p}                 &amp; $0.2\pm 0.3 $ &amp; $0.2\pm 0.1 $ &amp; $1.2\pm 0.9 $ &amp; $1.5\pm 0.5 $          	  &amp; $1.5\pm 0.5 $ &amp; $1.3\pm 0.3 $ &amp;  $0.3\pm 0.2 $ &amp; $1.5\pm 0.4 $  \\
Undecen-2-one &amp;\textbf{q}                       &amp; $2.4\pm 1   $ &amp; $6.1\pm 1   $ &amp; $7.7\pm 1.6 $ &amp; $7.7\pm 2.0 $          	  &amp; $6.9\pm 2.9 $ &amp; $8.1\pm 3.1 $ &amp;  $2.7\pm 0.4 $ &amp; $5\pm 1.4 $    \\
Citronellyl acetate &amp;\textbf{r}                 &amp; $1.6\pm 0.8 $ &amp; $6.9\pm 2   $ &amp; $0.5\pm 0.5 $ &amp; $0.7\pm 0.6 $          	  &amp; $5.8\pm 3.8 $ &amp; $1.8\pm 1.1 $ &amp;  $7.9\pm 3.3 $ &amp; $11.3\pm 4.9$  \\
Unknown 3 &amp;\textbf{s}                           &amp; $23\pm 5    $ &amp; $15.8\pm 2.2$ &amp; $5.2\pm 1.6 $ &amp; $7.2\pm 2.6 $          	  &amp; $21.9\pm 6.1$ &amp; $17.3\pm 5.6$ &amp;  $23.7\pm 10$  &amp; $22.4\pm 5.9$  \\
Geranyl acetate &amp;\textbf{t}                     &amp; $1.2\pm 0.5 $ &amp; $1.7\pm 0.4 $ &amp; $0.1\pm 0.1 $ &amp; $0.1\pm 0.2 $          	  &amp; - 	  &amp; - 	  &amp;  - 	   &amp; -            \\
Decan-2,9-dione &amp;\textbf{u}                   &amp; $0.2\pm 0.2 $ &amp; $0.1\pm 0.2 $ &amp; $0.8\pm 0.6 $ &amp; $0.6\pm 0.4 $          	  &amp; - 	  &amp; - 	  &amp;  - 	   &amp; -            \\
Unknown 4 &amp;\textbf{v}                           &amp; $0.2\pm 0.2 $ &amp; $0.5\pm 0.1 $ &amp; $1.2\pm 0.7 $ &amp; $1\pm 0.3   $          	  &amp; - 	  &amp; - 	  &amp;  - 	   &amp; -            \\
Isopentyl benzoate &amp;\textbf{w}                  &amp; $1.3\pm 0.1 $ &amp; $2.4\pm 1   $ &amp; $0.0\pm 0   $ &amp; $0.3\pm 0.4 $          	  &amp; - 	  &amp; - 	  &amp;  - 	   &amp; -            \\
3-Methyl-3-butenyl benzoate &amp;\textbf{x}         &amp; $1.2\pm 0.3 $ &amp; $2.0\pm 0.8 $ &amp; $0.2\pm 0.2 $ &amp; $0.4\pm 0.6 $          	  &amp; - 	  &amp; - 	  &amp;  - 	   &amp; -            \\
X-Undecen-2,10-dione &amp;\textbf{y}               &amp; $2\pm 0.6   $ &amp; $0.8\pm 0.4 $ &amp; $4.5\pm 2.1 $ &amp; $4.7\pm 1.8 $          	  &amp; - 	  &amp; - 	  &amp;  - 	   &amp; -            \\
Undecen-2,10-dione &amp;\textbf{z}                 &amp; $1.6\pm 0.5 $ &amp; $0.5\pm 0.3 $ &amp; $1.3\pm 0.8 $ &amp; $0.4\pm 0.5 $          	  &amp; - 	  &amp; - 	  &amp;  - 	   &amp; -           
\botline
\end{tabular}
\tabnote{Experimental treatments: SH, stressed hornet; WH, wounded
hornet.{\vspace*{-6pt}}}
\end{table*}

While the 26 compounds were all present in the pre-wintering gynes and
foundresses, 3 were absent from the workers (\textbf{g}, \textbf{m},
and \textbf{w}; Table~\ref{tab2}), and 1 was absent from the queens
(\textbf{m}; Table~\ref{tab2}). In the queens and foundresses, two
compounds, 4,8-dimethyl-7-nonen-2-one (\textbf{j}) and compound
\textbf{s}, accounted for more than 58\% and 42\% of the overall
profile, respectively. In the workers and pre-wintering gynes, the two
most common compounds were 4,8-dimethyl-7-nonen-2-one (\textbf{j}) and
nonan-2-one (\textbf{c}); they accounted for 43\% and 42\% of the
overall profile, respectively. \looseness=-1

\vspace*{3pt}

PCAs were carried out on these 26 compounds. Two rounds of analysis
were performed. The first explored differences among pre-wintering
gynes, foundresses, and queens ($n=7$, 6, and 6 pools, respectively,
Figure~\ref{fig4}). The first and second axes accounted for 40\% and
21\% of the total variance, respectively, and revealed a marked
separation among the three groups. This clustering was confirmed by the
K-means analysis, in which the foundresses and queens were assigned to
distinct groups. Only one pre-wintering gyne (G2) was incorrectly
placed in the queens' group. The queens stood apart from the other two
groups based on the presence of three compounds: acetophenone
(\textbf{a}) and two unknown compounds (\textbf{f} and \textbf{s}). The
foundresses formed a distinct group based on the presence of four
compounds: citronellyl acetate (\textbf{r}), geranyl acetate
(\textbf{t}), isopentyl benzoate (\textbf{w}), and isoprenyl benzoate
(\textbf{x}, {i.e.}, 3-methyl-3-butenyl benzoate). The second round of
analysis added profile information for the workers ($n=15$ pools)
(Figure~\ref{fig5}). No differences were seen between workers and
pre-wintering gynes, a result confirmed by the K-means analysis
resulting in no group separation.\looseness=-1

\begin{figure*}
\includegraphics{fig04}
\caption{\label{fig4}(A) Principal component analysis results and (B)
correlation circle plot for the venom gland profiles of queens,
foundresses, and pre-wintering gynes. Q: queens ($n=6$), F: foundresses
($n=6$), and G: pre-wintering gynes ($n=7$). The ellipses correspond to
the 95\% confidence interval of the data.~Compounds found closer to the
circumference of the circle convey greater amounts of information.}
\end{figure*}

\begin{figure*}
\includegraphics{fig05}
\caption{\label{fig5}(A) Principal component analysis and (B)
correlation circle plot for the venom gland profiles of queens,
foundresses, workers, and pre-wintering gynes. Q: queens ($n=6$), F:
foundresses ($n=6$), W:~workers ($n=15$), and G: gynes ($n=7$). The
ellipses indicate the 95\% confidence interval of the data.}
\end{figure*}

\vspace*{3pt}
The fiber-based extractions revealed that the workers and pre-wintering
gynes released 13 compounds under stressful conditions (SH and WH). All
the compounds were present in the solvent extract obtained from the
venom glands (Table~\ref{tab2}). Two~compounds accounted for more than
50\% of the total profile: 4,8-dimethyl-7-nonen-2-one (\textbf{j}) and
the unknown compound \textbf{s}. A PCA was carried out on the profiles
of the workers ($n=16$ pools) and pre-wintering gynes ($n=8$ pools)
subject to the SH and WH treatments. The first and second axes
accounted for 42\% and 22\% of the total variance, respectively. The
analysis did not show any clear differences based on caste or treatment
(Figure~\ref{fig6}). 

\begin{figure*}
{\vspace*{-2pt}}
\includegraphics{fig06}
{\vspace*{-2pt}}
\caption{\label{fig6}Principal component analysis of the 13 compounds
identified via PDMS-SPME after workers and pre-wintering gynes were
experimentally stressed (SH treatment, G: $n=4$ and W: $n=8$) or
wounded (WH treatment, G: $n=4$ and W:~$n=8$). The ellipses indicate
the 95\% confidence interval of the data.}
{\vspace*{-2pt}}
\end{figure*}

\section{Discussion}

As insects evolved to form organized societies, it became essential for
them to develop communication systems for sharing diverse
messages~\cite{48}. Eusocial insects most commonly employ chemical
communication~\cite{49}, and it can be challenging to study the cues
and signal patterns involved in different behaviors (e.g., recruitment,
dispersal, aggressiveness, or aggregation)~\cite{30,35,50}. Chemical
signals, like pheromones, can rapidly and efficiently disseminate
information and are used by all eusocial insects~\cite{51,52}. Volatile
pheromones are common and may convey information across great
distances~\cite{33,52,53}. Previous studies identified 19 compounds
with chain lengths ranging between 7 and 12 carbons in workers of both
\textit{V. v. auraria} and \textit{V. v.
nigrithorax}~\cite{26,27,42,43}.  The majority were ketones, the most
phylogenetically widespread class of alarm pheromones~\cite{52}. In the
study presented here, 9 new compounds were identified among the 13
compounds found, which displayed the same range of chain lengths as in
previous research (Table~\ref{tab1}). To our knowledge, this is the
first time that three of them ($\upbeta$-citronellol methyl ether
[\textbf{l}], 2-nonanyl acetate [\textbf{k}], and 2,9-decanedione
[\textbf{u}]; Table~\ref{tab2}) could been identified as potential
semiochemicals or as molecules involved in toxic actions. Other
compounds that we observed are known to have communication functions in
Hymenoptera. Geranyl acetate (\textbf{t}) is used by three
\textit{Bombus} species~\cite{54}, and citronellyl acetate
(\textbf{r}), isoamyl isovalerate (\textbf{e}), and isoamyl senecioate
(\textbf{g}) occur in the European hornet \textit{Vespa
crabro}~\cite{55}. Undecan-2-one (\textbf{q}) occurs in
\textit{Dolichovespula maculate}~\cite{56} and \textit{Polistes}
species~\cite{39}. Nonan-2-one (\textbf{c}), 4,8-dimethyl-7-nonen-2-one
(\textbf{j}), and undecan-2-one (\textbf{q}) are known to elicit
different levels of alarm in \textit{Vespa orientalis}~\cite{57}.
Thus, these compounds may be common in Vespidae. Some compounds have
also been found in other insects. For example, phenethyl acetate
(\textbf{m}) is an attractant in \textit{Diachasmimorpha
longicaudata}~\cite{58} and is part of a pheromone released by the
milkweed bug \textit{Neacoryphus bicrucis}~\cite{59}. Others, such as
isopentyl benzoate (\textbf{w}) and isoprenyl benzoate (\textbf{x}),
are volatiles in plants~\cite{60,61}. The presence of undecan-2-ol, in
contrast to previous research in \textit{V.~v.~auraria}~\cite{34} and
\textit{V. v. nigrithorax}~\cite{57}, was not observed. Since a
reference sample of undecan-2-ol was also injected, using the same
method, and present in the profile, we are confident that the absence
of this compound does not reflect a dehydration or thermolysis artifact
in our GC-MS injector.\looseness=-1

This same previous research~\cite{42,43} discovered that four compounds
were common in the venom glands of \textit{V.~velutina} workers:
nonan-2-one (\textbf{c}), 4,8-dimethyl-7-nonen-2-one (\textbf{j}),
X-undecen-2-one (\textbf{n}), and undecan-2-one (\textbf{q}). In our
study, however, the main compound observed in all castes was
4,8-dimethyl-7-nonen-2-one (21--28\%, liquid extraction), not
nonan-2-one. Nonan-2-one was the second most common compound in
workers, pre-wintering gynes, and foundresses but not in queens
(relative representation: 4\%). Instead, the second and third most
common compounds in queens were compound \textbf{s} (23\%, 
Figure~\ref{fig3}) and undecen-6-one (\textbf{o}), respectively. In
Cheng \etal~\cite{42}, \textit{Vespa v. auraria} collected did not
display the peak for compound \textbf{s}. Nevertheless, compound
\textbf{s} might correspond to the unidentified peak placed after the
citronellyl acetate in both \textit{Vespa v. auraria} (seen
by~\cite{62}) and \textit{V. v. nigrithorax} (seen by~\cite{43}).
Additionally, compound quantity differed between our work here and that 
of{~\cite{42}.}
For example, in \textit{V. v. auraria}, Cheng
\etal~\cite{42} found that X-undecen-2-one (\textbf{n}) and undecan-2-one
(\textbf{q}) were the second and third most common compounds,
respectively; in our study, however, they were present at far lower
levels (2.6--9.3\%, liquid extraction). In short, our results differ
quantitatively and qualitatively from those of both~\cite{42}\break
and~\cite{43}.

Considering that undecan-2-ol was absent from our results and that we
observed a different set of common compounds (i.e., like citronellyl
acetate in the French and Spanish hornet populations), it could be that
the hornet's chemical blends are changing at the population scale, as
has been seen in some ants~\cite{63,64,65}. Whether such shifts between
and within subspecies could arise from isolation by distance or
difference in evolutionary lineages is a question that merits further
exploration.

In the venom gland profiles, there were clear differences among the
gynes, queens, and foundresses. This result indicates that the chemical
signature of the female venom gland changes over the course of life
history: from pre-wintering gynes (females without nest) to foundresses
(mated females with small nest and some eggs) to queens (mated females
with workers). However, no differences between the pre-wintering gynes
and workers profiles were observed. This result shows that
pre-wintering gynes start off with profiles similar to those of workers
but that those profiles then change over time. The most obvious change
could be seen in compound \textbf{s}: its mean relative levels were 5\%
in workers versus 7, 15, and 23\% in pre-wintering gynes, foundresses,
and queens, respectively. Unfortunately, no match was found for
compound \textbf{s} in any of the available databases. However, this
compound did have a fragmentation spectrum highly similar to that of
citronellyl acetate (\textbf{r}): fragments 137 and 152 were present,
and fragment 138 was almost completely\break absent.

The differences among the females raise questions about the hornet's
chemical ecology. The blend observed could act as an alarm pheromone
for pre-wintering gynes, which occur in their colony of origin and can
play a defensive role alongside workers. However, the queens displayed
a different compound ratio, which could mean that the blend had a more
queen-specific function. Indeed, this compound ratio could serve as a
specific signal from the queen, potentially informing the colony's
workers that their queen has a problem or is in danger. Alternatively,
it could act as a signal of queen presence and health. Indeed, research
on wasps, bees, and ants has shown that reproductives may chemically
communicate their fertility~\cite{66}. Consequently, it could be that
compound \textbf{s} is a signal generated by mature egg-laying queens.
Further study is needed to explore this hypothesis in \textit{V. v.
nigrithorax}.

Previous studies identified 12 compounds in the crushed venom glands of
workers (\textit{V. v. nigrithorax}; \cite{43}) and in living hornets
(\textit{V. v. auraria}; \cite{42}) using blue PDMS fibers (Supleco).
Here, we used SPME and liquid extracts to characterize the compounds
released by the venom glands of \textit{V.~velutina} hornets as a
result of experimental stress or wounding. 13 out of 26 compounds in
this study were identified in living hornets using red PDMS fibers
(Supleco). We found 4,8-dimethyl-7-nonen-2-one (32--38\%; \textbf{j})
to be the most common compound, followed by compound \textbf{s}. The
results were unaffected by either caste (worker versus
pre-wintering gyne) or treatment. When stressed, both groups of hornets
emitted an alarm signal that was the same as the one emitted when
facing a life-threatening event. Eight of the compounds associated with
this signal had been previously observed in both \textit{V. v. auraria}
and \textit{V. v. nigrithorax}~\cite{42,43}. Recent work by
Rodriguez-Flores \etal~\cite{26} identified 8 compounds in queens ($n=4$)
and workers ($n=3$) using HS-SPME/GC-MS~\cite{29}. Among them were
nonane-2-one, undecan-2-one, and 4,8-dimethyl-7-nonen-2-one, which have
been seen in all studies on the hornet's chemical ecology; in contrast,
nonanal, decanal, geranyl acetone, and 4,8-dimethyl-1,7-nonadiene were
not seen~\cite{26}. The differences between our results and previous
research could be explained by SPME fiber type, collection materials,
and/or chromatography methods and machine systems.

This study represents an important step towards developing
pheromone-based traps. Indeed, the next step is to identify all the
components of \textit{V. v. nigrithorax} pheromones, including compound
\textbf{s}, which might be important in queen signaling. A useful
approach could be to assess the effects of each major compound, first
individually and then in proportionally accurate blends. Indeed,
certain compounds may only elicit a strong response when they occur in
blends, as is the case with alarm pheromones in honey bees~\cite{67} or
sex pheromones in moths~\cite{68}. Moreover, the precise compound ratio
of blends could be of major importance. It is also essential to test
the effects of any target compounds on bees. Given that alarm
pheromones are produced during both defensive and offensive situations,
they could also serve as a kairomone for the hornet's prey. Indeed,
bees may detect certain components of hornet alarm pheromones and
modify their behavior accordingly. \looseness=-1
\vspace*{38pt}

{\vspace*{-1pt}}

\section*{Conflict of interest}

The authors declare no competing interests.

{\vspace*{-1pt}}

\section*{Availability of data and materials}

The datasets generated during the current study are available from the
corresponding author upon reasonable request.

{\vspace*{-1pt}}

\section*{Ethics approval}

The study does not require ethics approval since the yellow-legged
hornet is classified as an invasive species; all research was carried
out in compliance with the relevant national guidelines.

{\vspace*{-1pt}}

\section*{Code availability}

Not applicable.

{\vspace*{-1pt}}

\section*{Authors' contributions}

The experiments and data collection were carried out by LB, JG, MH, and
ED. The chemical assays were carried out by CL, AGB, LB, and AK. Formal
Analysis: LB. The first draft of the manuscript was written by LB. The
manuscript was revised and edited by LB, CL, AGB, and ED. Funding was
obtained by ED. All the authors read the manuscript and gave their
approval for submission. Conceptualization \&amp; Project Administration:
ED. 

{\vspace*{-1pt}}

\section*{Consent to participate}

Not applicable.

{\vspace*{-1pt}}

\section*{Consent for publication}

All the authors have approved the content of this paper and have agreed
to the ``Special Issue:
%{\pagebreak} 
{\mbox{Chemical} Ecology -- Chemical Mediation in the Environment}''
Comptes Rendus de Chimie submission policies. This
manuscript has not been published previously and is not under
consideration for publication elsewhere. 

\section*{Funding}

This study received funding from the Centre-Val de Loire region, in the
form of French regional grants for our FRELON and FRELON 2 projects, as
well as from Veto-pharma and the French administrative department of La
Manche. The TQ-MS was funded by the region Centre-Val de Loire (APR-IA
2012).

\section*{Acknowledgments}

Thanks to R\'{e}mi De La Burgade for the advice and access to the
chemistry platform. This study received funding from the Centre-Val de
Loire region, in the form of French regional grants for our FRELON and
FRELON 2 projects, as well as from Veto-pharma and the French
administrative department of La Manche. We are grateful to everyone who
helped locate the hornets' nests. We would like to thank the chemical
ecology platform of the IRBI (UMR7261 CNRS, University of Tours) for
the services and facilities. Our thanks also go out to Ignacio Ruiz for
the help in the field and Jessica Pearce for the English-language
editing.

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