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\DOI{10.5802/crchim.233}
\datereceived{2023-03-28}
\daterevised{2023-05-10}
\dateaccepted{2023-05-12}
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\dateposted{2023-06-13}
\begin{document}

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

\title{Plant volatiles play differential roles in pre and post
alighting phases in a specialist phytophagous insect}

\alttitle{R\^ole diff\'erents des compos\'es volatiles de plantes
impliqu\'es dans les phases pr\'e- et post-atterrissage d'un insecte
phytophage sp\'ecialiste}

\author{\firstname{Kathleen} \lastname{Menacer}\CDRorcid{0000-0002-8212-2196}\IsCorresp}
\address{IGEPP-UMR 1349, INRAE, Institut Agro, Universit\'e Rennes, 35000 Rennes, France}
\email[K. Menacer]{kathleen.menacer@gmail.com}

\author{\firstname{Maxime} \lastname{Herv\'e}\CDRorcid{0000-0002-9257-3687}}
\addressSameAs{1}{IGEPP-UMR 1349, INRAE, Institut Agro, Universit\'e Rennes, 35000 Rennes, France}
\email[M. Herv\'e]{maxime.herve@univ-rennes.fr}

\author{\firstname{Benoit} \lastname{Lapeyre}}
\address{CEFE, Univ Montpellier, CNRS, EPHE, IRD, Univ Paul Val\'{e}ry Montpellier 3, Montpellier, France}
\email[B. Lapeyre]{benoit.lapeyre@cefe.cnrs.fr}

\author{\firstname{Manon} \lastname{Vedrenne}}
\addressSameAs{1}{IGEPP-UMR 1349, INRAE, Institut Agro, Universit\'e Rennes, 35000 Rennes, France}
\email[M. Vedrenne]{manon.vedrenne.17@gmail.com}

\author{\firstname{Anne Marie} \lastname{Cortesero}}
\addressSameAs{1}{IGEPP-UMR 1349, INRAE, Institut Agro, Universit\'e Rennes, 35000 Rennes, France}
\email[A. M. Cortesero]{anne-marie.cortesero@univ-rennes.fr}

\shortrunauthors

\keywords{\kwd{Volatile organic compounds
(VOCs)}\kwd{Electro-antennogram detection (EAD)}\kwd{Exploration
behavior stimulation}\kwd{Distance effect}\kwd{\textit{Delia radicum}}}

\altkeywords{\kwd{Compos\'es organiques volatils (COV)}\kwd{D\'etection
par \'electro-antennographie (EAD)}\kwd{Stimulation du comportement
d'exploration}\kwd{Effet \`a distance}\kwd{\textit{Delia radicum}}}

\begin{abstract} 
Plant volatile organic compounds (VOCs) can influence the behavior of
insects but distance and contact effects are rarely distinguished. We
aimed to identify VOCs that mediate attraction and oviposition
behaviors of the cabbage root fly, and to distinguish whether their
effect applies at distance or at contact. Headspace collections of
host-emitted VOCs were performed and analyzed in combination with
electro-antennogram detection. The behavioral effect of the
electrophysiologically-active VOCs was assessed in behavioral
experiments. We show that VOCs act mainly when in mixtures, and have
effects either at distance or at contact, but not both.
\end{abstract}

\begin{altabstract} 
Les compos\'es organiques volatils (COV) des plantes peuvent influencer
le comportement des insectes, mais les effets de ces compos\'es \`a 
distance et au contact des plantes sont rarement distingu\'es. Nous
avons cherch\'e \`a identifier  les COV qui influencent les
comportements d'attraction et d'oviposition de la mouche du chou
\textit{Delia radicum}, et \`a d\'eterminer si leur effet s'applique \`a
distance ou au contact.  Les COV \'emis par diff\'erents h\^otes ont
\'et\'e \'echantillonn\'es dans l'espace de t\^ete  des plantes et
analys\'es en combinaison avec des analyses d'\'electro-antennographie
(EAD). L'effet des COV actifs sur le plan \'electrophysiologique a
\'et\'e \'evalu\'e dans des exp\'eriences comportementales. Nous
montrons que les COV agissent principalement en m\'elange et qu'ils ont
des effets soit \`a distance, soit au contact, mais pas les deux \`a la
fois.
\end{altabstract}

\maketitle
\vspace*{15pt}
\twocolumngrid

\end{noXML}

\section{Introduction}

{Plants emit complex mixtures of volatile compounds that may contain up
to a hundred different molecules}~\cite{1}{. These volatile organic
compounds (VOCs) strongly influence ecological interactions between
plants and insects, as the latter highly rely on olfaction to perceive
their external environment}~\cite{2,3}{. VOCs can be used by plants to
attract pollinators and seed dispersers, and can also participate to
the defense against herbivores, for \mbox{example} by recruiting natural
enemies}~\cite{1}{. Each plant species, and at a finer scale each
population or even genotype, emits VOCs in particular amounts and
ratios, such that not two ever exhibit strictly identical VOC
blends}~\cite{4,5}{. These differences are used by phytophagous insects
to identify and select their host plants. From an evolutionary
perspective, there thus is a strong selection pressure on insects to
develop efficient ways to detect host-specific blends, as their
survival and reproduction depend on it}~\cite{6}{. Although attraction
can be induced by one or a few taxonomically characteristic compounds,
such as observed with specific VOCs of Brassicaceae} \cite{7,8,9},
Asteraceae~\cite{10}, Rosaceae~\cite{11} or Alliaceae~\cite{12},
insects of several orders have been shown to possess olfactory
receptors able to detect ubiquitous VOCs such as fatty acid
derivatives, phenylpropanoids and isoprenoids \cite{13,14}{. Studies
have shown that host recognition is mostly based on the detection of a
host-specific combination of either specific or ubiquitous VOCs, rather
than on the detection of a single host-specific compound. Indeed, many
examples in Diptera} \cite{15,16}{, Lepidoptera}~\cite{17,18,19} {or
Hymenoptera}~\cite{20} {show that combinations of VOCs can be
significantly more attractive than individual compounds. Furthermore,
individually repulsive VOCs can become attractive once in mixtures}
\cite{21,22,23} {and attractive mixtures can become repulsive if VOC
relative proportions change}~\cite{20}.

{Although VOCs are known to play a key role in distance host
location}~\cite{14}{, they can also act synergistically with contact
compounds and influence insect behavior once in contact with the
plant}~\cite{24,25}{. \mbox{Furthermore,} it has been shown in many species
such as} {\textit{Bombyx mori}}~\cite{26}, {\textit{Drosophila
melanogaster}}~\cite{27}, {\textit{Bactrocera dorsalis}}~\cite{28,29},
{\textit{Anopheles arabiensis}} {and} {\textit{A.
coluzzii}}~\cite{30}{, that certain VOCs alone or in mixtures can
stimulate female oviposition on their own and thus be important in the
contact phase of the host selection process. As an example, M\"{u}ller
and Hilker}~\cite{31} {tested the effect of VOCs on the behavior of the
monophagous chrysomelid} {\textit{Cassida stigmatica}}{, by comparing}
{\textit{Tanacetum vulgare}} {petioles wrapped with perforated filter
paper tubes to dummy petioles. They observed that walking duration was
longer on} {\textit{T. vulgare}} {petioles than on dummy ones, showing
that VOCs alone can influence host assessment and thus trigger contact
exploration behaviors. However, in most examples where the effect of
VOCs on host selection was investigated, the distance effect (i.e.
attraction of the insect to the oviposition site) and contact effect
(i.e. stimulation of exploration or oviposition once in contact) are
poorly distinguished, which does not allow to understand precise cues
involved at each step of the host selection behavioral sequence.} 

{In the cabbage root fly,} {\textit{Delia radicum}}{, receptive females
exhibit a stereotypical host exploration behavior before accepting a
host plant as a suitable oviposition site}~\cite{32}{, which involves
several types of cues, both physical and chemical. Physical cues such
as color, shape or texture}~\cite{33,34,35}{, as well as contact
chemical cues}~\cite{36,37,38,39} {have been extensively studied. Other
studies have shown the involvement of plant VOCs in mediating the
female behavior}~\cite{40,41}{. For example, allyl isothiocyanate has
been shown to be involved in long-range attraction}~\cite{41,42} {while
other VOCs such as dimethyl disulfide (DMDS)}~\cite{43} {or}
{\textit{cis}}{-3-hexenyl acetate}~\cite{44,45} {influence
oviposition.} {Both simultaneously perceived volatile and contact cues
synergistically mediate host plant acceptance}~\cite{46}{. In parallel,
studies have shown contrasts in attractiveness between different host
species}~\cite{47,48} {and others have characterized VOCs emitted by
these species}~\cite{47,49,50,51}{. However, to our knowledge, no
studies have both characterized the compounds emitted by plants in the
host range of} {\textit{D. radicum}} {and identified those involved in
either attracting females or stimulating their host exploration
behavior prior to oviposition. In this study, we aimed to (i)~identify
VOCs involved in host recognition by females of the cabbage root fly}
{\textit{Delia radicum}}{, and (ii)~determine their involvement in
either female attraction at distance or stimulation of host
pre-ovipositional exploration behavior. Experiments were conducted on
three host species of} {\textit{D. radicum}}{, namely}
{\textit{Brassica oleracea}}, {\textit{B. rapa}} {and} {\textit{Sinapis
alba}}{, which are known to be contrasted both in terms of
attractiveness}~\cite{47,48} {and oviposition}~\cite{52}{. At a finer
scale, two cultivars of} {\textit{B. rapa}} {and} {\textit{S. alba}}{,
also contrasted for oviposition}~\cite{52}{, were also included. These
contrasts suggest fine-tuned detection and recognition abilities in}
{\textit{D. radicum}} {females, but the influence of volatiles on these
discrimination abilities remains unknown. First, headspace collection
of VOCs emitted by the different host plants was performed and the
compounds present in the blends were identified by} {gas chromatography
coupled to mass spectrometry} {(GC-MS). In parallel,} {gas
chromatography coupled to} {electro-antennography detection (GC-EAD)
was performed to identify, among all VOCs present in the blends, those
that are detected by the females' antennae. Finally, the behavioral
effect of the different compounds detected, both taken individually and
in combination, was tested using an artificial leaf with synthetic VOCs
emissions.} 

\section{Materials and methods} 
\subsection{Plants}

{Experiments were carried out with five different cultivars, all of
which being commercial: two cultivars of} {\textit{Sinapis alba}}
{(``Verte'' and ``Sarah''), two cultivars of} {\textit{Brassica rapa}}
{ssp.} {\textit{pekinensis}} {(``Richi'' and ``Tabaluga'') and one
cultivar of} {\textit{B. oleracea}} {(``Parthenon''). Single seeds were
sown in individual $3.5 \times 3.5 \times 5$~cm clumps filled with
potting soil (Premier Tech Horticulture) and plants were grown in a
climatic chamber at optimum conditions ($20 \pm 1$~\textdegree C,
16L:8D and $70 \pm 10\%$ RH) where they were watered twice a week with
a nutritive solution}~\cite{53}{. One week before the experiments
started, plants were repotted individually in $7 \times 7 \times
6.5$~cm plastic pots containing the same substrate. All plants were at
the 3--4 true leaves stage when used for experiments.} 

\subsection{Insects} 

{Females of} {\textit{D. radicum}} {originated from a laboratory
rearing initially constituted from a field population collected in the
field in 2019 (Pleumeur-Gautier, Brittany, France). Flies were reared
on rutabagas as described in}~\cite{54} {and fed with a milk powder:
yeast: sugar (1:1:1) mixture. One week after emergence, females were
considered fertilized and used for experiments until they were 12 days
old. Females were identified based on the sexual dimorphism in the size
of the eye-spacing, which is much more pronounced in females than in
males}~\cite{55}.

\subsection{Volatile collection} 

Volatiles from the five cultivars tested were collected using dynamic
headspace. The aboveground part of an individual plant was enclosed in
a PET bag ($35 \times 43$~cm, Alfapac), in which an air flow was
maintained by two pumps (KNF, Neuberger) connected with PTFE tubing.
The airflow was purified by an activated charcoal filter placed between
the upstream pump and the bag. The entrance and exit flow rates were
regulated by flowmeters at respectively 300 and
200~ml${\cdot}$min$^{-1}$. Volatiles (from plants or an empty bag as
control) were collected during 24 h on a cartridge filled with 30~mg of
Porapak Q (80:100 mesh, Sigma-Aldrich) placed downstream from the
sampling bag. After sampling, all cartridges were kept in the dark at
$-$20~\textdegree C until further use. Six to eight replicates were
performed per cultivar.

\subsection{Volatile identification and quantification}

{Samples were analysed at the ``Platform for Chemical Analyses in
Ecology'' (PACE), with the \mbox{support} of the LabEx CeMEB (}{\textit{Centre
M\'{e}diterran\'{e}en pour l'Environnement et la Biodiversit\'{e}}}{,
Montpellier, France).} {Sampling cartridges were solvent desorbed using
200}~${\rmmu}${l of hexane} {(${\geq}$98.5\%; Carlo Erba reagents)}
{and stored at ${-}$20~\textdegree C until analysis. Separation and
semi-quantitative analyses of volatiles peaks were achieved using a}
{Shimadzu QP2010Plus gas chromatograph coupled to a quadrupole mass
spectrometer (GC-MS, Shimadzu Scientific Instruments). The GC was
equipped with an Optima 5-MS fused silica capillary column (length: 
30~m; diameter: 0.25~mm; film thickness: 0.25~${\rmmu}$m,
Macherey-Nagel), with \mbox{helium} as carrier gas
(1~ml${\cdot}$min}$^{-1}${). The temperature ramp started at
40~\textdegree C hold for 1~min, then increased to 220~\textdegree C at
12~\textdegree C${\cdot}$min}$^{-1}${, and finally held at 
220~\textdegree C for 2~min.} {One microliter of sample was injected
into a splitless injector set at 200~\textdegree C. Mass spectra were
recorded in electronic impact mode (EI) at 70~eV over a $m/z$ mass
ranging from  38 to 350. The temperature of the transfer line and the
ion source were set to 250~\textdegree C. Data were processed according
to Kidyoo} {\etal}~\cite{56} {using the MZmine{\texttrademark}
software version 2.53}~\cite{57}{. The control samples analyzed were
used to subtract} {potential contaminant compounds from the plant
samples. Compound identification was based on computer matching of mass
spectra with a database (NIST 2005 MS library, Wiley 9}{th} {edition),
on retention times and mass spectra of external synthetic standards.
The retention times of a series of} {\textit{n}}{-alkanes (}{nC8 to
nC20 alkane solution}{, 04070, Sigma
Aldrich$^{\text{\textregistered}}$) were used to calculated the
retention index of compounds and compare with those reported in the
literature}~\cite{58}{. Peak areas were used for statistical analyses.}

\subsection{Electrophysiological recordings}

{Elutions from all samples of the same cultivar were pooled together
and then concentrated under nitrogen flow for the recordings.}
{Electroantennography assays were performed using} {\textit{D.
radicum}} {females only}{. Each female was immobilized with forceps,
its head was cut and separated from the thorax, then put into an
adapted glass capillary. It was obtained from glass capillary (length:
76~mm, diameter: 1.12~mm; World Precision Instrument) pulled and cut
using a vertical micropipette-puller (P-30 model, World Precision
Instruments). The capillary was previously filled with an electrolytic
solution of Ringer (6.0~g${\cdot}$l}$^{-1}$ {NaCl,
0.4~g${\cdot}$l}$^{-1}$ {KCl,  0.3~g${\cdot}$l}$^{-1}$ {CaCl}$_{2}${,
3~g${\cdot}$l}$^{-1}$ {NaHCO}$_{3}${) and connected to a reference
electrode. The tip of the antenna was in contact with the recording
electrode, also filled with the electrolytic solution, at the opposite
side of the reference electrode. A stimulus controller (CS-55 Syntech
Ockenfels) was set to provide a continuous flow of purified and
humidified air, passing through a glass tube to send the VOCs one by
one to the antenna at a flow rate of 245~ml${\cdot}$min}$^{-1}${.
Electroantennography assays were performed using coupled gas
chromatography-electroantennographic \mbox{detection} (GC-EAD). A \mbox{volume} of
2~${{\rmmu}}$l of the solution was injected in a 8890 GC system gas
chromatograph (Agilent) equipped with a flame ionization detector
(FID), a splitless injector kept at  200~\textdegree C and an Optima
5-MS capillary column (length: 30~m; diameter: 0.25~mm; film thickness:
0.25~${\rmmu}$m). Helium was used as carrier gas
(1~ml${\cdot}$min}$^{-1}${). The temperature ramp was set as above. The
effluent was split into two deactivated fused silica capillary columns
(100~cm $\times$ 0.25~mm), one leading 10\% of the outlet flow to a FID
set \`{a} 300~\textdegree C and a second one leading 90\% of the outlet
flow into a heated EAD transfer line kept at 200~\textdegree C (Syntech
Ockenfels). Antennal electrical responses to volatile compounds were
tested and digitized using an IDAC board (acquisition controller
IDAC-2; Syntech Ockenfels). Data were processed with a PC-based
interface and software package (GcEad 1.2.5, Syntech Ockenfels)}{.
Electrophysiological measurements were conducted on 8--12 females per
cultivar. A compound was considered olfactively active in} {\textit{D.
radicum}} {females when it elicited a clear depolarization response in
at least half of the females tested.}\looseness=-1

\subsection{Behavioral experiments}

{The effect of the VOCs detected by the females' antennae on their
behavior was assessed using a dual-choice setup with artificial leaves
(Supplementary Figure~A). Leaves were made of a 10~cm green-paper straw
(Utopia$^{\text{\textregistered}}$) on which a green-paper limb
(Cultura$^{\text{\textregistered}}$) was stapled at 6 cm from the straw bottom.
To reproduce a plant waxy cuticle, which is mandatory for} {\textit{D.
radicum}} {to lay eggs}~\cite{34} {and thus to induce an exploration
behavior, each leaf was briefly immersed in a 1~l glass beaker
containing 800~ml of water and 40~g of paraffin wax heated at 
85~\textdegree C in a water bath. Leaves were then placed individually
in a Petri dish containing a 7~mm layer of river sand. Two dishes each
containing one leave were placed in a 30~cm cubic nylon cage
(BugDorm$^{\text{\textregistered}}$, Taichung, Taiwan). A $2 \times 2$~cm filter
paper was placed on a wooden rod inserted in the paper straw of each
leaf, so that the filter paper overlaid the artificial leaf without
touching it. On one filter paper, 10~${{\rmmu}}$l of mineral oil
containing a single VOC or a VOC mixture determined by the authors in
the course of the present work and some earlier ones was deposited
(each VOC at $1~{{\rmmu}}\mathrm{g}/{\rmmu}\mathrm{l}$), whereas the
second filter paper with only 10~${{\rmmu}}$l of mineral oil served as
\mbox{control.} After \mbox{impregnating} the filter paper with volatiles for 2~min,
it was placed in the cage. Immediately thereafter, one female was
released and the experiment started. Every 5~min during 90~min, it was
recorded  (i)~whether the female was on a leaf and (ii)~whether it was
showing an exploration behavior. As preliminary experiments showed that
VOCs alone were not sufficient to induce oviposition in these
experimental conditions, the number of eggs laid was not recorded.
Seven experiments were conducted, one with each single VOC detected by
the females antennae, and three with VOC mixtures characterizing the
three plant species studied (hereafter called ``}{\textit{B. oleracea}}
{like'', ``}{\textit{S. alba}} {like'' and ``}{\textit{B. rapa}}
{like''). All experiments took place at $20 \pm 2$~\textdegree C,
16L:8D, and $60 \pm 10\%$ RH, and 10--15 replicates were performed per
experiment.} 

\begin{figure*}
\includegraphics{fig01}
\caption{\label{fig1}Volatile Organic Compound (VOC) emission profiles
of five cultivars of three Brassicaceae species. (a)~Heatmap of VOCs
emitted by each cultivar (mean value per cultivar). The emission level
of each compound is compared between cultivars and represented by a
color gradient from light yellow (low) to dark red (high). (b)~Score
plot of the redundancy analysis performed on the VOCs emitted by
individual plants (orange: \textit{Brassica rapa} (cv. Tabaluga and
Richi); blue: \textit{Sinapis alba} (cv. Verte and Sarah); green:
\textit{B. oleracea} (cv. Parthenon)). All species differed
significantly from each other (respectively \textit{B. rapa} vs.
\textit{S. alba}: $p = 0.003$; \textit{B. rapa} vs. \textit{B.
oleracea}: $p = 0.005$; \textit{S. alba} vs. \textit{B. oleracea}: $p =
0.008$).}
\end{figure*}

\subsection{Statistical analyses}

{All analyses were performed using the R software version
4.1.3}~\cite{59}{. Volatile profiles were compared multivariately using
a redundancy analysis (RDA) on quadratic-root transformed and
autoscaled data}~\cite{60}{, with species and cultivar as independent
factors. The effect of these factors was tested using a permutation F
test with 9999 permutations (R package ``}{vegan''}~\cite{61}{).
Pairwise comparisons between species and cultivars were performed also
using permutation $F$-tests and $p$-values corrected with the False
Discovery Rate method}~\cite{62}{. Analyses were based on the mean area
of the extracted VOCs per plant. In each behavioral experiment, the
probability of being present on a leaf or showing an exploration
behavior was compared between treatments (control} {{vs.}} {VOC
or control} {{vs.}} {VOC mixture) using Wald tests applied on
Generalized Linear Mixed Models (GLMM, distribution: binomial, link:
logit), in which \mbox{treatment,} time and their interaction were considered
as fixed factors and the cage as a random factor. Estimated Marginal
Means (EMMeans) were computed for interpretation (R package
``emmeans''}~\cite{63}{). Three analyses were performed per experiment:
one on the probability for the fly of being attracted to the artificial
leaf (test of the attractiveness of the treatments at distance, i.e.
pre-contact effect), one on the probability of triggering an
exploratory behavior (test of the ability of the treatments to
stimulate an exploration behavior at contact, i.e. post-contact effect)
and one on the global probability of exploring the leaf \mbox{(integration} of
the two previous effects, i.e. test of the global ability of the
treatments to lead to an exploration behavior) (Supplementary
Table~I).} 

\section{Results}

\subsection{VOC emission profiles are characteristic of species but not
of cultivars}

{GC-MS analyses allowed the detection and identification of 12
different compounds, 10 being \mbox{terpenes} (eight monoterpenes and two
sesquiterpenes) and the two others being ethylacetophenone and hexenyl
acetate (Figure~\ref{fig1}a). Significant differences in volatile
profiles were observed between species ($F = 12.66$, $p &lt; 0.001$). An
almost significant difference in volatile profiles was observed between
cultivars of} {\textit{S. alba}} {but not} {\textit{B. rapa}}.
({\textit{S. alba}}{: $p = 0.065$,} {\textit{B. rapa}}{: $p = 0.363$) 
(Figure~\ref{fig1}b). Each species was characterized by a specific
profile. The specific signature of} {\textit{B. oleracea}} {consisted
of ${\rmalpha}$-farnesene and all monoterpenes. The specific signature
of} {\textit{S. alba}} {mostly consisted of ${\rmalpha}$-farnesene and
hexenyl acetate (with all VOCs but hexenyl acetate emitted in greater
amounts in cv. Sarah compared to cv. Verte). The specific signature of}
{\textit{B. rapa}} {mostly consisted of} ${\rmbeta}${-myrcene,}
${\rmbeta}${-ocimene,} ${\rmbeta}${-caryophyllene and ethylacetophenone
(with all VOCs except} ${\rmbeta}${-caryophyllene emitted in greater
amounts in cv. Richi compared to cv. Tabaluga).} \looseness=-1

\begin{figure*}
{\vspace*{-4pt}}
\includegraphics{fig02}
{\vspace*{-6pt}}
\caption{\fontsize{9}{10}\selectfont\label{fig2}Examples of antennal
responses of {\textit{Delia radicum}} females to Volatile Organic
Compounds (VOCs) from headspaces of three Brassicaceae species.
(a)~\textit{Brassica oleracea}~(cv. Parthenon), (b)~\textit{Sinapis
alba}~(cv. Sarah) and (c)~\textit{B. rapa}~(cv. Richi). Grey circles
show true antennal responses, with the respective VOCs identified on
the chromatogram.}
\end{figure*}

\begin{figure*}
\includegraphics{fig03}
{\vspace*{-3pt}}
\caption{\label{fig3}Volatile Organic Compounds (VOCs) emitted by three
Brassicaceae species and {detected by antennae of} {\textit{Delia
radicum}} {females.} (a)~Heatmap of VOCs emitted by each cultivars and
detected by females (mean value per cultivar). The emission level of
each compound is compared between cultivars and represented by a color
gradient from light yellow (low) to dark red (high). (b)~Score plot
1--2 of the redundancy analysis performed on the VOCs emitted by
individual plants (orange: \textit{Brassica rapa} (cv.~Tabaluga and
Richi); blue: \textit{Sinapis alba} (cv. Verte and Sarah); green:
\textit{B. oleracea} (cv.~Parthenon)). All species differed
significantly from each other (respectively \textit{B. rapa} vs.
\textit{S. alba}: $p = 0.003$; \textit{B. rapa} vs. 
\textit{B.~oleracea}: $p = 0.004$; \textit{S. alba} vs.
\textit{B.~oleracea}: $p = 0.004$).} 
\vspace*{-5pt}
\end{figure*}

\subsection{Four compounds among those present are detected by D.
radicum females}

{GC-EAD analyses revealed that females responded to four compounds
present in the VOC blends of the different cultivars: two monoterpenes
(${\rmbeta}$-ocimene and sabinene) and two sesquiterpenes
(${\rmalpha}$-farnesene and ${\rmbeta}$-caryophyllene)
(Figures~\ref{fig2},~\ref{fig3}a). The response to these compounds was
confirmed with synthetic compounds}~\cite{64}{. Four females also
responded to ${\rmbeta}$-myrcene but only with VOC blends from cv.
Tabaluga. This compound was more abundant in the blend from cv.
Parthenon than from cv. Tabaluga (Figure~\ref{fig1}a), and since it is
detected more intensely by the female antennae of} {\textit{D.
radicum}} {with increasing dose}~\cite{64}{, it should also have been
detected by GC-EAD in the VOC mixture from cv. Parthenon. This compound
was therefore removed from the following analyses, as its detection by
female antennae was considered inconsistent and unreliable in the
present experiment. A statistical analysis based on the detected
compounds only, still highlighted interspecific differences in volatile
profiles ($F = 14.77$, $p &lt; 0.001$), but none at the intraspecific
scale (}{\textit{S.~alba}}{: $p = 0.103$,} {\textit{B. rapa}}{: $p =
0.338$) (Figure~\ref{fig3}b). A species-specific signature was shown
again (Figure~\ref{fig3}a), with a blend of ${\rmbeta}$-ocimene,
sabinene and ${\rmalpha}$-farnesene for} {\textit{B. oleraceae}}
{(Figure~\ref{fig2}a), sabinene and ${\rmalpha}$-farnesene for}
{\textit{S. alba}} {(Figure~\ref{fig2}b)} {and ${\rmbeta}$-ocimene,
sabinene and ${\rmbeta}$-caryophyllene for} {\textit{B. rapa}}
{(Figure~\ref{fig2}c)}.

\subsection{VOCs have biological effects mostly when combined, with
effects being either at distance or at contact} 

\begin{figure*}
\includegraphics{fig04}
\caption{\label{fig4}Behavioral response of \textit{Delia radicum}
{females to the VOCs emitted by three Brassicaceae species and
eliciting an antennal response, when tested alone (left of the dotted
line) or in mixtures (right of the dotted line).} (a)~Mean probability
(${\pm}$ SE) of being attracted to the leaf (pre-contact effect). 
(b)~Mean probability (${\pm}$ SE) of triggering to an exploratory
behavior (post-contact effect). (c)~Global probability (${\pm}$~SE) of
exploration (global effect). Black dots indicate the presence of the
VOC in the treatment tested. NS: ${p} &gt; 0.1$, .: ${p} &lt; 0.1$, *: ${p}&lt;
0.05$, **: ${p} &lt; 0.01$, ***: ${p} &lt; 0.001$. $N = 10$--15 per
experiment.}
{\vspace*{2pt}}
\end{figure*}

Following results of the GC-EAD recordings, four VOCs were retained for
behavioral experiments (${\rmbeta}$-ocimene, sabinene,
${\rmalpha}$-farnesene and ${\rmbeta}$-caryophyllene) as well as the
mixtures ${\rmbeta}$-ocimene $+$ sabinene $+$\ ${\rmalpha}$-farnesene
(``\textit{B. oleracea} like''), sabinene $+$\ ${\rmalpha}$-farnesene
(``\textit{S. alba} like'') and ${\rmbeta}$-ocimene $+$ sabinene $+$\ 
${\rmbeta}$-caryophyllene (``\textit{B. rapa} like''). When tested
alone, one of the four VOCs (sabinene) did induce neither pre- or
post-contact effects and two (${\rmbeta}$-ocimene,
${\rmalpha}$-farnesene) seemed to induce \mbox{repulsion} as their associated
probability for the fly of being attracted to the leaf was lower than
that of the control (Figure~\ref{fig4}a). None of these three compounds
induced a higher global probability of exploration when considered
alone (Figure~\ref{fig4}c). Only ${\rmbeta}$-caryophyllene induced
attraction (Figure~\ref{fig4}a), but not stimulation of exploratory
behavior at contact (Figure~\ref{fig4}b). Overall,
${\rmbeta}$-caryophyllene led to increase the global probability of
exploration, but this probability was very low (Figure~\ref{fig4}c).
Contrary to most single VOCs, all VOC mixtures increased the
\mbox{probability} of triggering to an exploratory behavior and the global
probability of exploration, significantly or nearly
(Figure~\ref{fig4}b,c). However, VOC mixture had contrasted effects:
the ``\textit{B. rapa} like'' mixture induced significant pre-contact
effect but not post-contact one, the ``\textit{B. oleracea} like'' had
opposite effect and the ``\textit{S.~alba} like'' induced significant
repulsion but a high stimulation of a post-contact exploratory behavior
(Figure~\ref{fig4}). 

\section{Discussion}

{Plant-emitted VOCs are well known to mediate distance attraction of
phytophagous insects}~\cite{14}{. However, much less studied is the
fact that VOCs can also act on insect behavior once in contact with the
plant} (but see~\cite{28,29}){. Moreover, when the effect of VOCs on
the host selection process is investigated, the distinction between
distance and contact effects is only rarely made. The present study
highlights the importance of making this distinction by showing that
pre- and post-contact effects of VOCs can be independent.} 
\looseness=-1

{Olfactometer experiments had already shown that} {\textit{D. radicum}}
{females are attracted differently to the three plant species tested
here}~\cite{47,48}{. Using GC-MS identification and subsequent
multivariate analyses, we show that these species could be
distinguished based on their VOC blends, and each characterized by a
specific emission profile. These results are in agreement with the
literature since species-specific blends are usually observed
(}{{e.g.}}~\cite{65,66,67}{). The specificity of the blends
characterized in our study has already been reported}~\cite{47}{, and
although species-specific mixtures identified here are not exactly
similar to those described previously, all VOCs identified here, but
the aromatic ethylacetophenone, have already been shown to be emitted
by these species}~\cite{47,68}{. Among the compounds identified in this
study, ${\rmalpha}$-farnesene and ${\rmbeta}$-caryophyllene may be
involved in distance attraction of females~\cite{47}. \mbox{Indeed,} our
behavioural observations showed that ${\rmbeta}$-caryophyllene (alone
or in combination) induced a significant pre-contact effect,} {{i.e.}}
{attraction. Eucalyptol and} {\textit{cis}}{-3-hexenyl acetate, also
present in the different volatile profiles but not detected by female
antennae in our experiments (but see}~\cite{64}{), reduce and stimulate
oviposition, respectively, in field situations}~\cite{43}{. Other
volatile sulphur compounds also produced and emitted by Brassicaceae
but not present in our blends are also known to affect the female
behavior. For example, allyl isothiocyanate is attractive to gravid
females and stimulates their oviposition}~\cite{41,69}{, whereas DMDS
has a deterrent effect on oviposition}~\cite{45}{. In any case, the
specificity of the mixtures emitted by each plant species appears
logically as a starting point for phytophagous insects to evolve
interspecific discrimination abilities.} 

{Electrophysiological recordings revealed that not the whole blends are
detected by females' antennae, but only four ubiquitous terpenes which
combinations are species-specific. While a blend of plant VOCs may
contain more than a hundred compounds, electrophysiological antennal
recordings have shown that only a subset of these VOCs is usually
detected by insects}~\cite{14}{, which is confirmed by our results.
Among the olfactively active compounds, behavioral experiments showed
that ${\rmbeta}$-caryophyllene had an attractive pre-contact effect on}
{\textit{D. radicum}} {females, which supports an important role of
this compound in plant-insect interactions as it has been shown
attractive to a diversity of generalist and specialist phytophagous
insects (}{{e.g.}}~\cite{70,71,72}{). However, it is the only VOC which
induced pre- or post-contact effect when applied alone, while all three
VOC mixtures had such effect. Indeed, ${\rmbeta}$-ocimene,
${\rmalpha}$-farnesene and sabinene appeared neutral or even repellent
alone, whereas their combinations increased the probability to observe
an exploration behavior. Consistently, although the behavioral pattern
was quite similar between ${\rmbeta}$-caryophyllene alone and the only
mixture that included it, the global probability of exploration
appeared higher when ${\rmbeta}$-caryophyllene was combined with
${\rmbeta}$-ocimene and sabinene. These results are in line with the
many experiments showing that stronger behavioral responses are
obtained with specific blends or combinations of VOCs rather than with
individual compounds}~\cite{6}{. Furthermore, the striking example of
Webster}  {\etal}~\cite{21,22,23} {clearly demonstrates that the
signaling value of a mixture of VOCs is determined by the whole mixture
rather than by the sum of the signaling values of its individual
components. Although not all possible combinations were tested in our
study, our results clearly support this idea. Nonetheless, further
behavioral studies with more realistic concentrations and ratios of
VOCs would be necessary to conclude about natural host selection
processes in} {\textit{D.~radicum}}{, as these two factors are known to
be of\break importance}~\cite{6,20,21,70}.

{VOCs have long been recognized as key cues mediating distance
attraction in phytophagous insects (e.g.}~\cite{73,74,75}{). Their
involvement in oviposition stimulation once in contact with the plant
has also been shown in several species, although this aspect was much
less studied}~\cite{28,29,76,77}{. Our behavioral \mbox{experiments} confirm
that in} {\textit{D. radicum}}{, VOCs induce pre-ovipositional
exploration behaviors at contact. Moreover, although the global
probability of} {\textit{D. radicum}} {to explore artificial leaves,
which results from both the distance attractiveness and the ability to
stimulate exploration behaviors once in contact, was (nearly)
significantly increased by all three VOC mixtures, the effect of these
mixtures was strikingly different: for the} ``{\textit{B. rapa}}
{like'' mixture this effect is due to an increased attractiveness,
while for the ``}{\textit{S. alba}} {like'' and ``}{\textit{B.
oleracea}} {like'' mixtures it is due to a higher stimulation of
exploration behaviors once in contact with the leaf. This demonstrates
the relevance of distinguishing between pre- and post-contact phases
when studying the effect of VOCs on host selection processes. Indeed,
it shows that a given behavior can be initiated by different and
independent mechanisms, although these are mediated by the same kind of
plant cues.}

In conclusion, this study provides a better understanding of the action
of plant-emitted VOCs on the behavior of a specialist phytophagous
insect, and confirms the importance of VOC combinations in mediating
plant-insect interactions. More importantly, it demonstrates that the
effects of these chemical cues may be independent when considered at
distance and once the insect makes contact with the plant, and that a
given type of cues (here, VOCs) can mediate several aspects of host
recognition processes of phytophagous insects. 

\section*{Conflicts of interest}
Authors have no conflict of interest to declare.
%\vspace*{30pt}

\section*{Funding}
{This work was supported by a doctoral fellowship from Universit\'{e}
Rennes 1 and a} {Research grant from the GDR MediatEC, France.}

\section*{Acknowledgements} 

We are grateful to Magali Proffit and Florence Nicol\`{e} for their
precious help in the development of the volatile capture device, and to
Nicolas Barth\`{e}s for his help in GC-MS data analysis. We also
acknowledge the GDR MediatEC, which grant partly funded this work.

\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-233-suppl.pdf}}

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