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\DOI{10.5802/crchim.238}
\datereceived{2023-04-03}
\daterevised{2023-06-16}
\dateaccepted{2023-06-20}
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\dateposted{2023-08-09}
\begin{document}
%\copyeditor{RK}

\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{Male antennae of \protect\textit{Armadillidium vulgare}
discriminate \protect\textit{Wolbachia}-infected females}

\alttitle{Les antennes des m\^ales Armadillidium vulgare discriminent
les femelles infect\'ees par Wolbachia}

\author{\firstname{Nicolas} \lastname{Mouret}\IsEqualContrib}
\address{Laboratoire Ecologie et Biologie des Interactions EBI, UMR CNRS 7267, Universit\'{e} de Poitiers, Poitiers, France}
\email[N. Mouret]{nicolas.mouret79@orange.fr}

\author{\firstname{Vincent} \lastname{Tolassy}\IsEqualContrib}
\addressSameAs{1}{Laboratoire Ecologie et Biologie des Interactions EBI, UMR CNRS 7267, Universit\'{e} de Poitiers, Poitiers, France}
\email[V. Tolassy]{tolassy2@hotmail.fr}

\author{\firstname{Freddie-Jeanne} \lastname{Richard}\CDRorcid{0000-0002-2796-1181}\IsCorresp}
\addressSameAs{1}{Laboratoire Ecologie et Biologie des Interactions EBI, UMR CNRS 7267, Universit\'{e} de Poitiers, Poitiers, France}
\email[F.-J. Richard]{freddie.jeanne.richard@univ-poitiers.fr}

\keywords{\kwd{\textit{Wolbachia
pipientis}}\kwd{Electroantennography}\kwd{Scanning electron
microscopy}\kwd{Mate choice}\kwd{Terrestrial isopods}\kwd{Endoparasites}}

\altkeywords{\kwd{\textit{Wolbachia
pipientis}}\kwd{\'Electroantennographie}\kwd{Microscopie \'electronique
\`a balayage}\kwd{Choix du partenaire}\kwd{Isopodes
terrestres}\kwd{Endoparasites}}

\shortrunauthors

\begin{abstract}
In natural populations of \textit{A.~vulgare}, the endosymbiont
\textit{Wolbachia} feminizes genetic males. When confronted with
genetic females and feminized males, males prefer genetic females. We
tested whether the ability of males to discriminate both females
involved the detection of chemical compounds by extracting female
cuticular compounds through various solvent baths and presenting them
to male antennae. We also tested female antennae responses to evaluate
the potential impact of \textit{Wolbachia} on individual detection. Our
results showed that only the ethyl acetate extracts triggered a
significantly different response of male antennae between the two types
of females. No difference between female antennal detection was
recorded. Further experiments are required to identify the involved
molecule explaining the sexual preference of males towards
\textit{Wolbachia}-free females.
{\vspace*{-3pt}}
\end{abstract}

\begin{altabstract}
Dans les populations naturelles d'\textit{A.~vulgare}, l'endosymbiote
\textit{Wolbachia} f\'{e}minise les m\^{a}les g\'{e}n\'{e}tiques.
Lorsqu'ils sont confront\'{e}s \`{a} une femelle g\'{e}n\'{e}tique ou
\`{a} un m\^{a}le f\'{e}minis\'{e}, les m\^{a}les pr\'{e}f\`{e}rent la
femelle g\'{e}n\'{e}tique. Nous avons test\'{e} si la capacit\'{e} des
m\^{a}les \`{a} discriminer les deux femelles implique la d\'{e}tection
de compos\'{e}s chimiques en extrayant les compos\'{e}s cuticulaires
des femelles \`{a} travers diff\'{e}rents bains de solvants et en les
pr\'{e}sentant aux antennes des m\^{a}les. Nous avons \'{e}galement
test\'{e} les antennes des femelles pour \'{e}valuer l'impact potentiel
de \textit{Wolbachia} dans la d\'{e}tection des individus. Nos
r\'{e}sultats montrent que seuls les extraits d'ac\'{e}tate
d'\'{e}thyle ont d\'{e}clench\'{e} une r\'{e}ponse statistiquement
diff\'{e}rente des antennes des m\^{a}les entre les deux types de
femelles. Aucune diff\'{e}rence entre les capacit\'{e}s de
d\'{e}tection antennaire des femelles n'a \'{e}t\'{e} enregistr\'{e}e.
D'autres exp\'{e}riences sont n\'{e}cessaires pour identifier la
mol\'{e}cule en question, expliquant la pr\'{e}f\'{e}rence sexuelle des
m\^{a}les envers les femelles exemptes de \textit{Wolbachia}.
\end{altabstract}

\maketitle

{\vspace*{14pt}}

\twocolumngrid

\end{noXML}

\section{Introduction}\label{sec1}

In the animal and plant kingdoms, intraspecific communication is mainly
mediated by chemical components that are released by a transmitter and
that modify the behaviour of a receiver in an adapted fashion~\cite{1}.
These chemicals---pheromones---can act at various distances (e.g.,
contact chemicals for gustation and long-range chemicals for olfaction)
and mediate numerous behaviours such as sex recognition, copulation and
gregarism~\cite{2}.



%\advance\baselineskip by -.4pt

In invertebrates, pheromones can be present in glands or directly on an
individual's cuticle and thus are easily releasable and accessible at
long distances, at short distances or by contact with potential
receivers~\cite{3}. Cuticular chemical compounds (CCCs) are detected by
peripheral organs called sensilla, housing gustatory or olfactory
receptor neurons and representing the first relay of chemical
detection. Peripheral detection is followed by the central integration
of chemical information, leading to the behavioural response of the
receiver~\cite{4}. In terrestrial isopods, chemoreception is mainly
ensured by the second antenna (the first pair being strongly reduced),
on which is located the apical sensory organ, a tuft of sensilla
gathering both the olfactory and gustatory functions~\cite{5,6,7}.

{\vspace*{5pt}}

Qualitative and/or quantitative modifications of CCC composition can be
observed between sexes~\cite{3}, but other factors (i.e., non-genetic
factors), such as the reproductive status or health of an individual,
can also alter CCC production~\cite{8,9,10,11,12,13,14,15}. Many
studies have shown the negative impact of pathogens or parasites on
host odour~\cite{16}, such as loss of its attractiveness or, to the
extreme, becoming repulsive~\cite{13,17,18,19}, limiting the fitness of
the infected host~\cite{20}. In those cases, the recognition and
avoidance of infected partners by non-infected partners can be
beneficial, directly by decreasing the probability of contracting the
pathogens and indirectly by avoiding transmission to offspring (even
increasing their resistance to it)~\cite{21}. The odour modification of
an individual resulting from its infection by a pathogen can thereby
inform a partner of its health status and thus represent an honest
signal of its quality~\cite{22,23}.

\textit{Wolbachia pipientis} (hereafter \textit{Wolbachia}) is an
intracellular alpha-Proteobacterium maternally transmitted in oocyte
cytoplasm and spread in nearly 75\% of terrestrial arthropods and
nematodes~\cite{24}. In some species, \textit{Wolbachia} infection
leads to deleterious effects on host fitness: in \textit{Tribolium
confusum}, infected females produce fewer offspring than non-infected
females~\cite{25}. In \textit{Drosophila simulans}, \textit{Wolbachia}
strain \textit{w}Ri is responsible for a decrease in infected female
fecundity~\cite{26}. A loss of performance (in terms of sexual
individuals produced) is observed in \textit{Formica truncorum}
colonies exhibiting a high proportion of infected workers~\cite{27}.
\textit{Wolbachia} can also be responsible for breeding behaviour
distortions, as in the spider mite host \textit{Tetranychus urticae},
where uninfected individuals tend to mate together, while infected
individuals promote sib matings in their progeny~\cite{28}.

\textit{Wolbachia} is also known to manipulate the reproduction of its
host, securing its vertical transmission, through four major
strategies: cytoplasmic incompatibility (decreasing the fitness of
uninfected individuals), parthenogenesis, male killing and feminization
(increasing the female proportion in the population)~\cite{29}. This
last strategy describes the conversion of infected genetic males into
intersexed functional females (male genotype, female
phenotype)~\cite{29,30}.

In the terrestrial isopod \textit{Armadillidium vulgare} (commonly
called woodlouse), males discriminate between males and females at
short distances~\cite{31}. Copulation is linked with assortative
mating, and mating probability necessitates a stimulation duration
beyond the threshold~\cite{32}.

The sex ratio of natural \textit{A.~vulgare} populations is, however,
biased towards females by the presence of two \textit{Wolbachia}
strains (\textit{w}VulC and \textit{w}VulM) responsible for the
feminization of infected male embryos~\cite{33}. These infected
individuals (\textit{Wb} females from now on) suffer from decreased
survival and lower fertility and immunity~\cite{34,35,36}.

\textit{Wb females} also show a decrease in their mating
success~\cite{31,37,38}; however, the cause of this decrease is still
unknown. Two main hypotheses can be formulated: (i) In the short range,
the chemical differences in cuticular profiles between \textit{Wb}
females and \textit{Wb-free} females may allow males to assess their
respective health status~\cite{12}, helping them properly orient their
sexual behaviour towards uninfected females. In this scenario, we
hypothesize that \textit{Wb-free} and \textit{Wb} female emit a ``sex
reversal''-specific odour discriminable by males. (ii) After male
courtship starts, \textit{Wb} females display aberrant behaviour
leading to multiple mating interruptions~\cite{35,38}. In this
scenario, we hypothesize that \textit{Wolbachia} presence alters
\textit{Wb} female olfactory detection of male compounds, potentially
leading to aberrant behaviour and the interruption of mating at early
stages.

To that end, we first evaluated the olfactory electrophysiological
responses of \textit{A.~vulgare} male antennae to volatiles derived
from \textit{Wb-free} and \textit{Wb} females using
electroantennography recording~\cite{39,40}. To detect potential
biologically active unpleasant odours, cuticular compounds of
\textit{Wb-free} and \textit{Wb} females were extracted in five
successive solvents (water--methanol--hexane--dichloromethane--ethyl 
acetate) and presented to the male antenna.

We then tested whether \textit{Wolbachia} infection impacted male
detection by females by extracting male cuticular compounds and
presenting them to \textit{Wb} and \textit{Wb-free} female antennae.

\section{Material and methods}\label{sec2}
\subsection{Rearing conditions}

All experiments took place in the Ecology and Biology of Interactions
Laboratory at the University of Poitiers, France. Individuals of
\textit{Armadillidium vulgare} (Crustacea, Isopoda, Oniscidea;
Latreille, 1804) were kept on moistened compost in plastic boxes 
(26~$\times$~13~cm) under laboratory conditions (20\textdegree, photoperiod
of France, 46\textdegree~40$^{\prime}$~N) and supplied \textit{ad
libitum} with fresh carrots and linden tree leaves. Specimens were
derived from individuals initially collected in Helsing\o r (Denmark).
In the current experiment, we used males (ZZ), females (ZW)
later called asymbiotic females and
feminized males (ZZ $+$ \textit{Wo}). To avoid inbreeding, gravid females
were isolated, and their offspring were sexed. Males and females were
then reared in different boxes before sexual maturity, allowing
breeding control. All individuals used in this study were one-year-old\break
and virgin.

\subsection{\textit{Wolbachia} assessment}

\textit{Wolbachia} presence was assessed by DNA extraction and PCR
assays on dissected gonads or legs. Total genomic DNA was obtained by
phenol/chloroform extraction and ethanol precipitation. PCR
amplification was performed using a 650-bp segment of the WOSP gene,
commonly used to amplify a \textit{Wolbachia} surface protein present
in various strains of \textit{Wolbachia} (primers sequences: 81F; 
5$'$-TGG-TCC-AAT-AAG-TGA-TGA-AGA-AAC-3$'$ and 691R;
5$'$-AAA-AAT-TAA-ACG-CTA-CTC-CA-3$'$)~\cite{41}. PCR cycling conditions
were 95~\textdegree C for 2~min; 35 cycles at 95~\textdegree C for
1~min, 55~\textdegree C for 1~min, 72~\textdegree C for 1~min;
72~\textdegree C for 5~min; and then held at 4~\textdegree C. The gene
fragment was finally revealed by ethidium bromide staining on
agarose~gel.\looseness=1

\subsection{Scanning electron microscopy (SEM)}

Scanning electron microscopy was carried out on the antennae of
1-year-old males, females and symbiotic individuals. We selected
individuals just after anterior moulting and kept them individually in
plastic boxes ($\xvarnothing = 8\times 5$ cm) lined with moistened
paper. Two days after full moulting, both antennae of every individual
were collected under a dissection microscope. Antennae were washed for
2~h in a fixing buffer at 4~\textdegree C (3\% glutaraldehyde, 0.1~M
sodium cacodylate, 1\% NaCl, pH adjusted to 7.3) then 2~h in a washing
buffer at 4~\textdegree C (0.26~M sucrose, 0.1~M sodium cacodylate, 1\%
NaCl, pH adjusted to 7.3) followed by 1~h in post-fixing buffer at room
temperature (1.3\% osmium tetroxide, 0.1~M sodium cacodylate, 1.8\%
NaCl). Antennae were then dehydrated in four successive acetone baths
with increasing concentrations (50, 70, 90 and 100\%), dried with a
critical point bypass device (CDP 030 Balzers$^{\text{\textregistered}}$)
gradually replacing the acetone with CO\tsub{2}, and then fixed on a
SEM adapted support with silver lacquer and platinum coated (metallizer
SCD 005 BAL-TEC$^{\text{\textregistered}}$). Observations were finally
carried out on a JEOL 840A at a 10-kV tension.

To support the possible size dimorphism between the two antennae (right
and left) of an individual, the sensilla density per $\upmu$m$^{2}$ for
each antennal segment was determined according to~\cite{42}: the
segment was compared to a cylinder of surface
$\uppi{\cdot}\xvarnothing{\cdot}$h. Sensilla were counted on a portion
corresponding to 1/3 of the visible surface (equivalent to 1/6 of the
total surface of the segment). The density was finally obtained by
dividing the total number of sensilla on the segment by its surface.

\subsection{Silver staining}

The presence of porous sensilla on \textit{A.~vulgare} antennae was
investigated by silver staining~\cite{43}. Two one-year-old males,
females and feminized males (naturally infected females)
in anterior moulting were placed in plastic
boxes ($\xvarnothing = 8\times 5$~cm) on moistened paper. Twenty-four
hours after moulting, both antennae of all individuals were excised and
cleaned with a soft brush soaked in 70\% ethanol. After air-drying,
antennae were immersed in a mixture of 70\% ethanol and 1~M silver
nitrate for 24~h at 4~\textdegree C and protected from light. Antennae
were then cleaned with two successive ethanol baths of 1~h each (90 and
100\%). They were finally placed overnight in xylene
(Merck$^{\text{\textregistered}}$, 95\% purity) and then mounted on
microscope slides with xylene-free mounting medium (Diamount,
DiaPath$^{\text{\textregistered}}$) for optical microscope observations
(Eclipse Ci-S model,~Nikon$^{\text{\textregistered}}$). Observations focused
on the tip of the antenna, displaying the apical sensory
organ~\cite{44}, and the sensilla/aesthetascs distributed over the\break
antenna~\cite{45}.

\subsection{Extraction of cuticular compounds}
\subsubsection{Experiment 1. Assessing male antennal response towards
female cuticular compounds}

We extracted the cuticular profiles of 15 \textit{A.~vulgare}
asymbiotic and 15 naturally infected females. Females were freeze-killed for 
20~min at ${-}$20~\textdegree C prior to extraction. Chemical extractions
were carried out using five solvents of various polarities: water
(polarity $=$\ 1), methanol (polarity $=$\ 0.762), hexane (polarity
$=$\ 0.009, considered apolar), dichloromethane (polarity $=$\ 0.309)
and ethyl acetate (polarity $=$\ 0.228) (Acros
Organics$^{\text{\textregistered}}$)~\cite{46}. Each individual was
successively immersed in 1~ml of solvent under constant agitation for
30~min with a 30-min drying period under a hood between each solvent.
The water extracts were lyophilized at ${-}$80~\textdegree C and
resuspended in 50~$\upmu$l. Methanol, hexane, dichloromethane and ethyl
acetate extracts were dried under high-purity nitrogen flow (N\tsub{2})
up to 50~$\upmu$l.

\subsubsection{Experiment 2. Assessing female antennal response
(asymbiotic and naturally infected) towards male cuticular compounds}

Based on the results of Experiment~1, we modified the protocol for the
second experiment. We extracted the cuticular chemical profiles of 72
\textit{A.~vulgare} males. Males were transferred to filter paper for
cleaning 3~h prior to extraction and then freeze-killed 2~h prior to
it. The cuticular chemical profiles of 3 batches of 24 males were
extracted with solvents of different polarity: dichloromethane, ethyl
acetate, and hexane. Batches were first immersed in 5~ml of Milli-Q
water for 30~min, then dried for 30~min under a hood and finally
immersed in 5~ml of solvent. Regular agitation was performed during
both immersions. Extracts were dried up to 600~${\upmu}$l under
nitrogen flow and stored at ${-}$20~\textdegree C.

\subsection{Electroantennography (EAG)}

EAG allows the recording of voltage variation across an antenna
(differential between the base and the tip) after the detection of a
chemical or a mixture of chemicals~\cite{47}. Here, we measure EAG
recording with an isolated antenna connected on both extremities of the
electrodes on a closed circuit~\cite{39,48}. For each replicate, the
left or right antenna of 1-year-old individuals was randomly sampled by
excision at its base and mounted between the electrodes of an EAG
recording probe (Syntech$^{\text{\textregistered}}$). Electrical contact was
ensured by an electrode conductive gel (Signa
gel$^{\text{\textregistered}}$, Parker), and special attention was given to
the position of the antenna (placed in an arc at the end of the two
electrodes), allowing it to sag over time without getting bogged down.
The recording device was composed of an electronic filter (high-pass
filter to 0.05~Hz) with a stimulus controller (CS-55,
Syntech$^{\text{\textregistered}}$) and an acquisition interface device
\mbox{(IDAC-2,} Syntech$^{\text{\textregistered}}$). The electroantennogram
profiles were visualized using EAD/2014 software
(Syntech$^{\text{\textregistered}}$).\looseness=-1

During the whole experiment, the antenna is exposed to a stable
moistened air flow carried from the stimulus controller, passing
through a humidifier and delivered via a glass tube surrounding the
antenna. Antennae were exposed to a series of chemical extracts
presented in random order deposited on rectangle filter papers (2
$\times$ 0.2 cm, Whatman$\!^{\text{\texttrademark}}$, GE Healthcare).
After solvent evaporation, the filter papers were introduced into a
glass Pasteur pipette (150~mm, WWR$^{\text{\textregistered}}$) equipped
with a rubber pipette bulb. Stimuli presentations were performed by
sending the odours (by pressing) into the glass tube carrying the air
flow, ensuring reception of the volatile compounds by the antenna.
Every stimulus, of 1~s each, was spaced at least 30~s apart to avoid
any adaptation or sensitization of the antenna~\cite{49}.

Four replicates were performed per antenna, with each consisting of 3
controls of volatiles from filter papers exposed to pure solvent
followed by 3 filter papers exposed to solvent plus cuticular extracts.
After the antenna mounting, a control composed of pure methanol was
presented before and after the presentation of 15 stimuli to ensure the
full reactivity of the antenna. In total, we used 15 male antennae to
assess their response towards female-borne volatiles and 14 female
antennae (7 per condition: asymbiotic and naturally infected) to assess
their response towards male-borne volatiles.

\subsection{Statistical analysis}
\subsubsection{Scanning electron microscopy}

For SEM, we calculated the mean of the sensilla density on both
antennae for the same sex and compared it using Wilcoxon signed rank test.

\subsubsection{Comparisons of antennal perception}

Mean responses to a solvent (responses to the water control and water
extracts of asymbiotic and naturally infected females, for example) were
compared using Kruskal--Wallis tests followed by post hoc tests.

The difference in perception between asymbiotic and naturally infected
female antennae towards compounds from the male cuticle was evaluated
with non-parametric independent tests (Wilcoxon rank sum test).

\section{Results}\label{sec3}
\subsection{Scanning electron microscopy of \textit{A.~vulgare}
antennae}

The \textit{A.~vulgare} antenna (4.59 $\pm$ 0.06~mm) is composed of six
segments (Figure~\ref{fig1}) covered with trichodea sensilla and
aesthetacs (Figures~\ref{fig2} and~\ref{fig3}). The apical extremity of
the antenna (segment~I) is capped with the sensorial apical organ,
formed by a tuft of sensilla whose taste function has been investigated
through silver staining (Figure~\ref{fig3}).

\begin{figure}
\includegraphics{fig01}
\caption{\label{fig1}Overview of \textit{A.~vulgare} antennae.}
\end{figure}

\begin{figure*}
\includegraphics{fig02}
\caption{\label{fig2}Visualization of unimodal olfactory sensilla
present along the \textit{A.~vulgare} antenna. (A--C) Scanning electron
microscopy. (D--F) Silver staining; the black colour at the base (E and
F) seems to indicate the entrance of volatile molecules.}
\end{figure*}

\begin{figure*}
\includegraphics{fig03}
\caption{\label{fig3}\textit{A.~vulgare} sensorial apical organ located
at the end of segment~I (see Figure~\ref{fig1})
observed by scanning electron microscopy (A)
composed of apical sensilla (B). Their argyrophilic property (C) allows
us to classify them as taste sensilla.}
\end{figure*}

Counting sensilla allowed us to estimate sensilla density
(sensilla/$\upmu$m$^{2}$) on antennae for males, females free of
\textit{Wolbachia} and naturally infected females (Table~\ref{tab1}). Antenna
fixation and silver lacquering did not allow accurate counting of
sensilla density on the two basal segments, and observations were
mainly carried out on segments I--IV. The mean lengths of the segments
were I (0.59 $\pm$ 0.039 mm), II (0.41 $\pm$ 0.029~mm), III (1.34 $\pm$
0.089 mm) and IV (0.66 $\pm$ 0.035~mm) (Table~\ref{tab1}).

%tab1
\begin{table*}
\caption{\label{tab1}Sensilla density on the right and left antennae of
different \textit{A.~vulgare} sexes{\vspace*{-2pt}}}
\fontsize{9}{10.8}\selectfont
\tabcolsep3.5pt
\begin{tabular}{ccccccccccc}
\thead
&amp; \xmorerows{2}{Segments} &amp; 
\multicolumn{2}{c}{\parbox[t]{3pc}{\centering Counted sensilla}} &amp;
\multicolumn{2}{c}{\parbox[t]{5pc}{\centering Total number of sensilla}} &amp;
\multicolumn{2}{c}{\parbox[t]{6pc}{\centering Surface of segments ($\upmu$m\tsup{2})}} &amp;
\multicolumn{2}{c}{\parbox[t]{5pc}{\centering Density (sensilla/$\upmu$m\tsup{2})}} 
&amp; \xmorerows{2}{Chi\tsup{2}} \\
\cline{3-4}\cline{5-6}\cline{7-8}\cline{9-10}
&amp; &amp; Right &amp; Left &amp; Right &amp; Left &amp; Right &amp; Left &amp; Right &amp; Left &amp; \\
\endthead
\morerows{4}{\parbox[t]{3pc}{\centering Wolbachia-females}} 
&amp; I &amp; 48 &amp; 34 &amp; 288  &amp; 204 &amp; 185,849   &amp; 227,959   &amp; 0.0013\0  &amp; 0.00075 &amp; \\
                  &amp; II    &amp; 40 &amp; 31 &amp; 240  &amp; 186 &amp; 196,705   &amp; 124,149   &amp; 0.0012\0  &amp; 0.0015\0  &amp; \\
                  &amp; III   &amp; 58 &amp; 52 &amp; 348  &amp; 312 &amp; 562,486   &amp; 865,417   &amp; 0.00062 &amp; 0.00036 &amp; \\
                  &amp; IV    &amp; 27 &amp; 40 &amp; 162  &amp; 240 &amp; 442,510   &amp; 509,451   &amp; 0.00037 &amp; 0.00047 &amp; \\
                  &amp; Total &amp;    &amp;    &amp; 1038\0 &amp; 942 &amp; 1,387,552\0\, &amp; 1,726,977\0\, &amp; 0.00075 &amp; 0.00054 &amp; $3.17\times 10^{-5}$; $P&gt;0.05$ \vspace*{5pt}\\
\morerows{4}{\parbox[t]{3pc}{\centering Wolbachia-free females}} 
&amp; I &amp; 34 &amp; 30 &amp; 204 &amp; 180  &amp; 291,482   &amp; 257,368   &amp; 0.0006\0   &amp; 0.00062 &amp; \\
                       &amp; II    &amp; 22 &amp; 29 &amp; 132 &amp; 174  &amp; 235,969   &amp; 207,751   &amp; 0.00056  &amp; 0.00084 &amp; \\
                       &amp; III   &amp; 64 &amp; 60 &amp; 384 &amp; 360  &amp; 1,300,726\0\,  &amp; 1,159,775\0\,  &amp; 0.0003\0   &amp; 0.00031 &amp; \\
                       &amp; IV    &amp; 36 &amp; 49 &amp; 216 &amp; 294  &amp; 661,625   &amp; 687,490   &amp; 0.00033  &amp; 0.00043 &amp; \\
                       &amp; Total &amp;    &amp;    &amp; 936 &amp; 1008 &amp; 2,489,803\0\,  &amp; 2,312,386\0\,  &amp; 0.00038  &amp; 0.00044 &amp; $4.43\times 10^{-6}$; $P&gt;0.05$ \vspace*{5pt}\\
\morerows{4}{Males} 
&amp; I     &amp; 32 &amp; 34 &amp; 192 &amp; 204 &amp; 244,453   &amp; 273,825   &amp; 0.00067 &amp; 0.00062 &amp; \\
      &amp; II    &amp; 26 &amp; 20 &amp; 156 &amp; 120 &amp; 220,134   &amp; 256,111   &amp; 0.00071 &amp; 0.00047 &amp; \\
      &amp; III   &amp; 45 &amp; 39 &amp; 270 &amp; 234 &amp; 1,159,326\0\, &amp; 1,056,725\0\, &amp; 0.00023 &amp; 0.00022 &amp; \\
      &amp; IV    &amp; 36 &amp; 22 &amp; 216 &amp; 132 &amp; 617,350   &amp; 658,544   &amp; 0.00035 &amp; 0.0002\0  &amp; \\
      &amp; Total &amp;    &amp;    &amp; 834 &amp; 690 &amp; 2,241,264\0\, &amp; 2,245,206\0\, &amp; 0.00037 &amp; 0.00031 &amp; $6.18\times 10^{-6}$; $P&gt;0.05$
\botline
\end{tabular}
{\vspace*{-2pt}}
\end{table*}

For all individuals, comparisons between left and right antennae were
not significant (all \textit{Chi}$^{2}$\ $p&gt;0.05$) based on total
density, thus allowing the random selection of left or right antennae
for further EAG experiments.

Antenna electrical responses were recorded for male and female
\textit{A.~vulgare} towards conspecific volatile cues extracted with
different solvents. An example of the amplitude of the antenna response
is provided in Figure~\ref{fig4}.

\begin{figure}
\includegraphics{fig04}
\caption{\label{fig4}Electroantennogram of asymbiotic female antennae
towards control solvent and male volatile extracts. (A) Timestamps of
stimulus delivery. (B) Amplitude of antennal response. C: Control
solvent; T: male extract; Di: dichloromethane; Hex: hexane; EA: ethyl
acetate. Square sides represent an amplitude of 2~mV.}
\end{figure}

\subsection{Assessing male antennal response towards female cuticular
compounds}

We recorded male antennal responses towards cuticular compounds
extracted from asymbiotic and naturally infected females with methanol,
hexane, dichloromethane or ethyl acetate (Figure~\ref{fig5}). Male
antennae showed the same responses to female extracts and pure solvent
for methanol, hexane and dichloromethane ($KW_{(2\mathrm{df})} = 1.53$,
$p=0.46$; $KW_{(2\mathrm{df})} = 1.89$,  $p=0.39$; $KW_{(2\mathrm{df})}
= 0.39$,  $p=0.83$, respectively). However, the antennal response was
significantly different between extracts made with ethyl acetate
($KW_{(2\mathrm{df})} = 63.28$, $p &lt; 0.0001$), with extracts of
naturally infected females eliciting stronger responses than pure
solvent and asymbiotic female extracts (Bonferroni test,  $z =7.95$, $p
&lt; 0.0001$;  $z =-3.79$, $p &lt; 0.0001$,  respectively), and extracts from
asymbiotic females eliciting stronger responses than pure solvent
(Bonferroni test, $z = 4.15$, $p &lt; 0.0001$).

\begin{figure*}
\includegraphics{fig05}
\caption{\label{fig5}Male antennal response (mV) towards pure solvent
and asymbiotic and naturally infected female cuticular extracts.}
\end{figure*}

\subsection{Comparison of antennal response of asymbiotic and naturally
infected females towards male extracts}

We then compared the antennal response of asymbiotic and naturally
infected females towards male cuticular extracts. No significant
differences were found in the antennal response of asymbiotic and
naturally infected females (Figure~\ref{fig6}) towards the ethyl
acetate extract, dichloromethane extract and hexane extract (Wilcoxon
rank sum test, $W=346.5$, $p = 0.6496$; $W=483$,   $p = 0.7212$; 
$W=447.5$, $p = 0.8193$, respectively).

\begin{figure*}
\includegraphics{fig06}
\caption{\label{fig6}Asymbiotic and naturally infected female antennal
response (mV) towards male cuticular extracts.}
\end{figure*}

\subsection{Assessing female antennal response towards male cuticular
compounds}

We recorded female antennal responses towards male volatiles extracted
with three different solvents, with the responses to each pure solvent
used as controls. Male volatiles extracted with ethyl acetate elicited
significantly higher responses than the control (Wilcoxon signed rank
test, $V = 15.5$, $p &lt; 0.0001$). However, female antennae responded
equally towards male dichloromethane and hexane extracts in comparison
with their respective control solvents (Wilcoxon signed rank test, $V =
631.5$, $p = 0.3219$; $V = 511$, $p = 0.4322$, respectively).
Furthermore, the antennal response was significantly different between
extracts of male cuticular compounds obtained with different solvents
($KW_{(2\mathrm{df})} = 99.517$, $p &lt; 0.0001$). The ethyl acetate
extract elicited significantly higher female antennal responses than
the dichloromethane and hexane extracts (Bonferroni test, $z = {-}8.938$,
$p &lt; 0.0001$; $z = 8.304$, $p &lt; 0.0001$, respectively).

\section{Discussion}\label{sec4}

This study is the first attempt to characterize the olfaction of
terrestrial isopods through electroantennography. Few
electroantennographic studies have been carried out on crustaceans with
convincing results on the costal and vent-dwelling shrimps
\textit{Palaemon elegans} and \textit{Mirocaris
fortunate}~\cite{50,51}, on daphniids~\cite{52}, on the coconut crab
\mbox{\textit{Birgus latro}~\cite{53}} and on the hermit crab
\textit{Coenobita clypeatus}~\cite{54}. We recorded a positive EAG
response, which is uncommon in insects but not surprising in
crustaceans. Such a pattern has been reported in shrimp
(\textit{Palaemon elegans}~\cite{51}), in the terrestrial hermit crab
(\textit{Coenobita clypeatus}~\cite{54}) and in a coconut
crab~\cite{53}, even if for the latter, its overall responses are quite
similar to those of insects. In addition, polarity has also been
reported to vary according to the chemical properties of the
odourant~\cite{53,54}. In \textit{C.~clypeatus}, these differences
seemed to be associated with related behaviours and could be the result
of different pathways involved in signal transduction~\cite{54}. Here,
we report electroantennography to be a reliable tool to study olfaction
in terrestrial isopods.

We recorded the antennal response of male, asymbiotic and naturally infected
female \textit{A.~vulgare} towards cuticular compounds from the
opposite sex extracted with different solvents. We found that ethyl
acetate-extracted compounds increased the antennal response in the
tested antennae of both sexes. These compounds may be volatile cues of
interest for intraspecific recognition and communication. In fact, male
antennae responded more strongly towards naturally infected female
cuticular compounds than towards compounds from asymbiotic females.
\textit{A.~vulgare} males were previously found to prefer \mbox{asymbiotic}
females over \textit{Wolbachia}-infected females~\cite{35,38}, and it
was further shown that both females' cuticles had distinct chemical
profiles~\cite{35}. Thus, the female compounds that we extracted with
ethyl acetate could contribute to males assessing female infection
status or changes in attractiveness.

The fact that male antennae react to the ethyl acetate extract of
asymbiotic females may imply that the chemical(s) involved in the
recognition of infected female cuticular compounds is (are) naturally
produced by females but in a quantity dependent on their infectious
state. It is possible that \textit{Wolbachia} alters its host's signal
synthesis pathway~\cite{55} or issuance organ by modifying, for
example, the infected females' cuticle properties and
composition~\cite{56,57,58}, thus releasing more molecules.

It is also possible that \textit{Wolbachia} produces its own signal
directly in the excretory/secretory apparatus of the host~\cite{59,60},
modifying the chemical profile of infected females in a qualitative
fashion.

We also compared antennal responses between asymbiotic and naturally
infected females towards males' cuticle extracts obtained with
different solvents. Similar responses were found between both kinds of
females. Therefore, the presence of \textit{Wolbachia} does not seem to
influence antennal perception of volatiles of the opposite sex,
indicating that the aberrant behaviour displayed by infected females
during mating is not caused by an alteration in the detection of male
odour~\cite{35,38}.

Such a lack of impact of endosymbionts on antennal perception has
already been reported in the pea aphid \textit{Acyrthosiphon pisum},
for which the bacterial symbiont \textit{Hamiltonella defensa} did not
alter the perception of its alarm pheromone~\cite{61}. However, in
\textit{Drosophila simulans}, \textit{Wolbachia} has been shown to
enhance the olfaction of its hosts towards food-related cues~\cite{62}.
It has further been suggested that \textit{Wolbachia} might do so by
mediating the expression of genes encoding odourant
receptors~\cite{63}, thus directly impacting antennal perception.

According to sexual selection theory, variation in female quality
(infected vs. uninfected) in the natural population of
\textit{A.~vulgare} favours male mate choice~\cite{64}, and their
preference for uninfected females is thought to slow the dispersal of
\textit{Wolbachia}~\cite{38}. Our results constitute a new indication
that male selection is mainly mediated through the detection of
chemical variation among sexes. Identification of these chemicals and
behavioural tests to confirm their bioactive role are still needed.

\section*{Conflicts of interest}

Authors have no conflict of interest to declare.

\section*{Acknowledgements}

We wish to thank Alexandra Lafitte for isopod management and Maryline
Raimond for microscopy advice.

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