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\begin{document}

\title{The for gene as one of the drivers of foraging variations in a parasitic
wasp}



\author[1]{aurore gallot}%
\author[2]{Emmanuel Desouhant}%
\author[1]{Vincent Lhuillier}%
\author[1]{david lepetit}%
\author[3]{Adil El-filali}%
\author[2]{Laurence Mouton}%
\author[2]{Cristina Vieira}%
\author[4]{Isabelle Amat}%
\affil[1]{Université Claude Bernard Lyon 1}%
\affil[2]{Université de Lyon}%
\affil[3]{Université Lyon 1, CNRS, UMR 5558, Laboratoire de Biométrie et Biologie Evolutive, Villeurbanne, France}%
\affil[4]{Biometrie et biologie evolutive}%


\vspace{-1em}



  
  \date{May 07, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Foraging behaviours encompass strategies to locate resources and to
exploit them. In many taxa these behaviours are controlled by a major
gene called for, but mechanisms vary between species. In the parasitoid
wasp Venturia canescens, sexual and asexual populations coexist in
sympatry but differ in their foraging behaviours. Here we explored the
molecular bases underpinning this divergence in foraging behaviours by
testing two mutually non-exclusive hypotheses: firstly the divergence in
the for gene results in difference in foraging strategies, and second
this latter is due to a divergence in whole-genome expression. Using
comparative genomics, we showed that the for gene was conserved across
insects considering both sequence as well as gene model complexity.
Polymorphism analysis did not support the occurrence of two allelic
variants diverging across the two populations, yet asexual population
exhibited less polymorphism compared to the sexual one. Sexual and
asexual transcriptomes sharply split up, with 10.9\% of differentially
expressed genes, but these were not enriched in behavioural related
genes. We showed that the for gene was more expressed in asexual female
heads than in sexual ones, and that asexuals were the ones that explored
more the environment and exploited more host patches. Overall, these
results suggested that a fine tuning in the for gene expression between
populations may have led to distinct foraging behaviours. We
hypothesized that reproductive polymorphism and coexistence in sympatry
of sexual and asexual populations specialized to different ecological
niches via divergent optima on phenotypic traits, could imply adaptation
through different expression patterns of the for gene and at many other
loci throughout the genome.%
\end{abstract}%



\sloppy


\textbf{The \emph{for} gene as one of the drivers of foraging variations
in a parasitic wasp}

Genomic bases underlying foraging in parasitoid

Aurore Gallot\textsuperscript{1}*, Emmanuel
Desouhant\textsuperscript{1}, Vincent Lhuillier\textsuperscript{1},
David Lepetit\textsuperscript{1}, Adil El Filali\textsuperscript{1},
Laurence Mouton\textsuperscript{1}, Cristina
Vieira-Heddi\textsuperscript{1}, Isabelle Amat\textsuperscript{1}.

\textsuperscript{1} Universit\selectlanguage{ngerman}é de Lyon, Université Lyon1, LBBE --
Laboratoire de Biométrie et Biologie Evolutive -- UMR 5558, CNRS,
Villeurbanne, France

*Corresponding author:\emph{aurore.gallot@univ-lyon1.fr},
https://orcid.org/ \textbf{0000-0003-4552-090X}

ABSTRACT

Foraging behaviours encompass strategies to locate resources and to
exploit them. In many taxa these behaviours are controlled by a major
gene called \emph{for,} but mechanisms vary between species. In the
parasitoid wasp \emph{Venturia canescens} , sexual and asexual
populations coexist in sympatry but differ in their foraging behaviours.
Here we explored the molecular bases underpinning this divergence in
foraging behaviours by testing two mutually non-exclusive hypotheses:
firstly the divergence in the \emph{for} gene results in difference in
foraging strategies, and second this latter is due to a divergence in
whole-genome expression. Using comparative genomics, we showed that
the\emph{for} gene was conserved across insects considering both
sequence as well as gene model complexity. Polymorphism analysis did not
support the occurrence of two allelic variants diverging across the two
populations, yet asexual population exhibited less polymorphism compared
to the sexual one. Sexual and asexual transcriptomes sharply split up,
with 10.9\% of differentially expressed genes, but these were not
enriched in behavioural related genes. We showed that the \emph{for}gene
was more expressed in asexual female heads than in sexual ones, and that
asexuals were the ones that explored more the environment and exploited
more host patches. Overall, these results suggested that a fine tuning
in the \emph{for} gene expression between populations may have led to
distinct foraging behaviours. We hypothesized that reproductive
polymorphism and coexistence in sympatry of sexual and asexual
populations specialized to different ecological niches\emph{via}
divergent optima on phenotypic traits, could imply adaptation through
different expression patterns of the \emph{for} gene and at many other
loci throughout the genome.

Keywords: behavior, foraging, hymenoptera, transcriptomic, populations.

1 INTRODUCTION

Within a population, individuals tend to exhibit similar traits given
than sexual reproduction, through meiosis and fecundation, homogenizes
genotypes and thus prevent from phenotype divergence. Loss of sexuality
have been frequently recorded in a diverse array of eucaryotic taxa with
three possible origins leading to the emergence of asexual lineages:
mutation, hybridization (Normark, 2003) or endosymbiotic infection
(Stouthamer et al., 1990). When reproductive modes are exclusive;
asexuals start to diverge by accumulating genetic mutations since they
no longer exchange gene flow with the sexual population from which they
originated. Competitive interactions should favor one reproductive mode
over the other (Lively, 2010). Asexuals have a demographic advantage by
producing only females, they avoid the cost of males (Maynard-Smith,
1978). In contrast, sexual populations maintain greater genetic
diversity which may confer a decisive advantage in changing environment
(Otto, 2009). If geographical or ecological heterogeneity allows
ecological specialisation, with each lineage performing better in a
specific habitat, sexual and asexual populations can coexist in
different geographical areas or sympatrically (Bell, 1982; Lynch, 1984).
Such coexistence of sexual and asexual lineages is called reproductive
polymorphism. In this case, adaptations in behavior, morphology or life
history traits, should distinguish sexual from asexual populations.

Such cases of reproductive polymorphisms have been widely reported in
haplodiploid arthropods. Van der Kooi et al. described in an exhaustive
database 765 parthenogenetic species, among which 143 presented evidence
of sexual lineages as well, representing 19\% of the described species
having reproductive polymorphism (van der Kooi et al., 2017). Although
this proportion is likely to be underestimated, this nevertheless
indicates the frequent occurrence of the coexistence between sexual and
asexual populations. Thus, the competitive exclusion of one or the other
reproductive modes is not the norm, the ecological conditions allowing
their coexistence seems to be frequently met. In the hymenopteran
parasitoid \emph{Venturia canescens} (Ichneumonoidea~: Gravenhorst)
asexual populations coexist in sympatry with sexual populations
(Beukeboom et al., 1999; Schneider et al., 2002) each better adapted to
specific ecological niches (Amat et al., 2017). In particular,
divergence in the foraging behaviours have been shown in many population
pairs coming from different localities by a meta-analysis (Amat et al.,
2017). Asexual wasps have a better capacity than sexual wasps to exploit
their environment given their higher capacity to find hosts (Liu et al.,
2009), a larger egg load and their ability to lay eggs faster (Pelosse
et al., 2007). In contrast, sexual wasps explore better their
environment than asexual wasps as they flight longer and faster, have
higher energy content, and live longer (Luká\selectlanguage{polish}š \selectlanguage{english}et al., 2010). \emph{V.
canescens} asexual reproduction involves a central fusion automictic
parthenogenesis, which means that some genetic recombination occurs
during the early stages of oogenesis. Thus, genetic variation still
exists between asexual offspring's even if an irreversible increase of
homozygosity in populations occurs over time (Beukeboom and Pijnacker,
2000; Mateo Leach et al., 2009). The divergence in foraging behaviours
observed between sexual and asexual wasps \emph{V. canescens} is
certainly based on genetic divergence between the two populations, since
no more genetic exchanges through mating occurred in natural populations
(Mateo Leach et al., 2012).

Genetic control of foraging behaviours has been mainly studied
in\emph{Drosophila melanogaster} where two distinct types of strategies
have been characterized (Allen et al., 2017; Anreiter et al., 2017; de
Belle and Sokolowski, 1989; Osborne et al., 1997; Sokolowski, 1980).
While sitters hug the boundaries of a food patch but remain focus within
one food patch, rovers travel greater distances within and between food
patches, thus exploring widely their environment and exploiting more
food resources (Sokolowski, 1980). The two strategies are under the
control of a major gene called \emph{foraging (for)} . Despite the many
genes involved in generating foraging behaviours (Anreiter et al.,
2017), manipulations of the \emph{for} expression are sufficient to
modify them (Osborn et al., 1997). The control of foraging behaviours by
the \emph{for} gene has been demonstrated in drosophila larvae as well
as in adults, and encompass both searching for food resources and
oviposition sites (Edelsparre et al., 2014; McConnell and Fitzpatrick,
2017). The \emph{for} gene has two allelic variants conserved during
evolution: rovers have at least one dominant allele (\emph{for}
\textsuperscript{R}), while sitters have the two recessive alleles
(\emph{for} \textsuperscript{S}). Both alternative behaviours are
maintained by selection; patchy food and high population densities
advantage rovers, while evenly distributed food and low population
densities advantage sitters (Sokolowski et al., 1997). Genotype
differences between rovers and sitters are reflected by differences in
the \emph{for} gene expression, higher in rover heads than in sitters,
and so is the enzymatic activity of the corresponding protein PKG
(Osborn et al., 1997). The role of the \emph{for} gene as a single major
gene influencing foraging behaviours has been characterized in many
animal species. Orthologs of the \emph{D. melanogaster} \emph{for} gene
influence foraging behaviours in taxa as diverse as nematodes (Hao et
al., 2011; Hong et al., 2008) or mammals (Struk et al., 2019). Most of
the studies on genetic control of foraging behaviours has been conducted
on insects species, such as diptera (\emph{D. melanogaster} ,
\emph{Aedes aegypti} (Keating et al., 2013)), hymenoptera \emph{(Apis
mellifera}(Ben-Shahar et al., 2002), \emph{Bombus terrestris} (Tobback
et al., 2011), \emph{Pheidole pallidula} (Lucas and Sokolowski,
2009),\emph{Pogonomyrmex barbatus} (Ingram et al., 2005), \emph{Vespula
vulgaris} (Wenseleers et al., 2008), orthoptera (\emph{Schistocerca
gregaria} (Lucas et al., 2010)), lepidoptera (\emph{Sesamia
nonagrioides} (Chardonnet et al., 2014)). In all those species,
the\emph{for} gene contributes to foraging behaviours. However, the
existence of allelic variants as well as the relationships between
the\emph{for} expression level and foraging behaviours exhibit
variations between species. For example, within Hymenoptera, the
eusocial honey bee (\emph{A. mellifera} ) displays caste division where
young workers take care of the hive, whereas older workers of the colony
are foragers. Albeit only one allele of \emph{for} has been identified
in this species, foragers exhibit higher gene expression associated with
a higher corresponding PKG activity (Ben-Shahar, 2005). A same
overexpression of the \emph{for} gene have been shown in \emph{B.
terrestris} (Tobback et al., 2011), phylogenetically related to honey
bee. In contrast, \emph{for} expression was found to be higher in nurses
than in foragers in three other eusocial hymenopteran species with a
same age-dependent division of labor: the ants (\emph{P. barbatus}
,\emph{P. pallidula} ), and the common wasp (\emph{V. vulgaris} )
(Ingram et al., 2005; Lucas and Sokolowski, 2009; Wenseleers et al.,
2008). Although influence of foraging behaviors by the \emph{for} gene
has been maintained during evolution, the underpinning mechanisms
variate with opposite patterns within eusocial Hymenoptera species.
Studying the genetics of the foraging behaviours on additional
hymenopteran species would make it possible a better understanding of
their evolution in this taxon. \emph{V. canescens} that belongs to the
Ichneumonoidea, a parasitoid superfamily basal within the Apocrita group
which include all other eusocial hymenopteran species (bees, ants and
wasps) (Peters et al., 2017), appears to be a relevant model for
studying the genetic bases underlying the variability of foraging
behaviours and their evolution within Hymenoptera.

Here, we investigated the genetic bases underpinnings the variability of
foraging behaviours observed between sexual and asexual \emph{V.
canescens} populations. We explored two non-mutually exclusive
hypotheses that could explained the divergence observed in foraging
behaviours: firstly, a divergence in the \emph{for} gene, and second a
divergence in whole-genome expression. We first proceed to the
characterization of the \emph{for} gene in the \emph{V. canescens} using
genomic and transcriptomic sequences: 1) we described the
\emph{for}orthologous and reconstructed its evolution in insects; 2) we
annotated the full gene model by analyzing sexual and asexual
transcriptomes; 3) we described allelic variations in sexual and asexual
populations. We explored the second hypothesis by studying the
differential gene expression between sexual and asexual populations,
with a special focus on the behavioral genes. Finally, we tested whether
variations in the\emph{for} gene expression could explain the observed
variations in foraging behaviours between sexual and asexual
populations, by coupling a behavioral experiment with the \emph{for}
gene quantification.

2 MATERIALS AND METHODS

2.1 Field sampling and insect rearing

\emph{V. canescens} is a solitary endoparasitoid of caterpillars of
pyralid moths (Salt, 1976). The females used in the experiments come
from sexual and asexual populations collected annually near Valence
(N44\selectlanguage{ngerman}°58'21'', E4°55'39''). In this unique location, individuals of the
sexual population were usually sampled in an orchard, while individuals
of asexual population were mostly collected close to grain silos.
Caterpillars of \emph{Ephestia kuehniella} (Zeller) were left a week
exposed to parasitoids, then brought back to the laboratory waiting the
emergence of parasitoids. Virgin emerging \emph{V. canescens} females
were isolated and left with hosts in order to sexed their progeny.
In\emph{V. canescens} sex determination is haplodiploid: sexual females
have a parthenogenetic arrhenotokous reproduction, i.e. unfertilized
eggs produce haploid males while diploid females resulted from
fertilized eggs. Thus, virgin arrhenotokous females produce only males.
In contrast, virgin asexual thelytokous females produce only females.
Sexual and asexual wasps were maintained separately on the host \emph{E.
kuehniella} feed on semolina, were they produced kairomones attracting
for parasitoids from mandibular gland secretions (Castelo et al., 2003).
Insects were grown under constant environment (25 ± 1°C, 55 ± 5 \% RH,
12:12 LD).

2.2 Annotation of the \emph{for} gene in \emph{V. canescens} genome

Orthologs, \emph{i.e.} genes descended from the same ancestral sequence
separated by a speciation event, often have the same function, hence we
first searched the orthologs of the \emph{for} gene in the genome
of\emph{V. canescens} . A set of 40 orthologs \emph{for} sequences from
38 insect species, as well as the branchiopoda \emph{Daphnia pulex} and
the mouse (\emph{Mus musculus} ) sequences, were identified using
ortholog annotation in EnsemblMetazoa database and literature (table
S1). To identify \emph{for} ortholog in \emph{V. canescens} genome, we
used the reciprocal best hits with tblastn with a set of 42 \emph{for}
orthologs protein sequences previously described as query, and the
\emph{V. canescens} genome as database
(\emph{http://bipaa.genouest.org/sp/venturia\_canescens/}V.1.0). The
\emph{for} gene that was localized on the scaffold 64, contained the
longest open reading frame (\emph{Vcan27709} ) constituted of 2,445
nucleotides encoding 815 amino acids.

2.3 The \emph{for} gene phylogenetic reconstruction in insects

The putative \emph{V. canescens} \emph{for} sequence was added to the
set of 42 orthologs \emph{for} , then aligned using MUSCLE (Edgar,
2004). The corresponding protein alignment diverged in N-terminal but
was conserved in C-terminal. Alignment was manually curated, most
conserved residues were selected using Gblock, the resulting alignment
consisted of 533 amino acids. To reconstruct the \emph{for} phylogeny
and position the \emph{V. canescens} \emph{for} among other insect
sequences, ProtTest v3.4.2. was used to determine the best-fit model of
protein evolution using AIC (Abascal et al., 2005). JTT model of protein
evolution was used and topology optimization was carried out using best
of NNI and SPR options. The phylogenetic tree was constructed with
maximum likelihood method using PhyML implemented in Seaview (v 4.7)
(Gouy et al., 2010). Default aLRT (SH-like) was used for branch support
(Anisimova and Gascuel, 2006).

2.4 RNA extraction and sequencing

A total of 6 RNA-seq libraries were prepared: sexual and asexual
populations were constituted both by 3 biological replicates. Each
replicate was constituted of a pool of 30 individual heads taken from
emerging females and flash frozen, next used as an input for RNA
extraction. Heads were crushed using steel beads and Qiagen TissueLyser
(45s, 25hz). Total RNAs were extracted using Rneasy Mini Kit (Qiagen)
following the manufacturer protocol and including the DNase step. RNA
integrity was controlled using gel electrophoresis and quantified with
Nanodrop. After integrity control and quantification, polyadenylated
RNAs were enriched from 1 \selectlanguage{greek}μ\selectlanguage{english}g of high-quality total RNAs with oligo-dT
magnetic beads, then fragmented and converted to cDNAs (Illumina TruSeq
Stranded mRNA Library Prep kit). Fragments around 200bp were selected,
adapters ligated, and fragments amplified by PCR to generate DNA
colonies. Each library was labelled, multiplexed and pooled for
sequencing on a HiSeq 2500 Illumina sequencer (Fasteris, Switzerland),
with a paired-end protocol (2x150bp).

2.5 The \emph{for} gene model reconstruction

We identified all the isoforms of \emph{for} transcript and
reconstructed the \emph{for} gene model in \emph{V. canescens} by
screening the 6 RNA-seq libraries from sexual and asexual populations
and focusing on the \emph{for} reads. KisSplice 2.5.4. (Sacomoto et al.,
2012) is a method based on De Bruijn graphs that allows identification
of all variants without using a reference genome, including single
nucleotide polymorphism (SNPs), indels and alternative splicing events.
In parallel, we built a \emph{de novo} transcriptome assembly with
Trinity (Haas et al., 2013).

2.6 Polymorphism analysis at the \emph{for} locus

To evaluate the \emph{for} gene polymorphism, we localized all the SNPs
based on the list of all the SNPs, insertions and deletions across all
the \emph{for} isoforms, previously identified with KisSplice. We then
used KisSplice2RefTranscriptome to position each SNP on
\emph{for}isoforms. Finally, we used the R package KissDE in order to
find SNPs that significantly differed in frequency across sexual and
asexual populations (adjusted \emph{P} -values\textless{}0.05).

2.7 Differential expression analysis

The genome-wide divergence between sexual and asexual populations was
estimated using RNA-seq libraries to identify differentially expressed
genes (DEGs) between the two populations. Reads quality was first
assessed with FastQC, then reads were trimmed and filtered using
trimmomatic with minimum length set to 75pb. After filtering, the
transcriptomic dataset included a total of 91 millions of reads of which
an average of 92\% were successfully aligned on \emph{V.
canescens}transcriptome using HiSat2 (Kim et al., 2019) (table S2).
Genes with differential expression between sexual and asexual
populations were identified using negative binomial GLM implemented in
the program DESeq2 (Love et al., 2014). We tested for differential
expression of all transcripts with an average level of expression
superior to 10 reads per gene (\emph{n} =14,106). A gene was considered
differentially expressed (DE) when the false discovery rate (FDR)
adjusted \emph{p-} value was inferior to 0.05, without applying
supplementary fold change threshold.

2.8 Functional analysis

The \emph{de novo} transcriptome was annotated using BLAST and Gene
Ontology tools to assign biological function to transcripts. Then, we
focused our analysis on the transcripts related to the `behaviour' GO
term or to any of its child terms, thus annotating a functional group of
transcripts related to behaviour. This list of transcripts was crossed
with the previously established lists of DEGs between the two
populations.

2.9 Behavioural experiment

We set up an experimental design to quantify exploitation and
exploration of host patches by individuals from sexual and asexual
populations. Experimental device contained two host patches placed 20 cm
far from each other inside a box (50 x 16 x 8 cm) with 2 side holes
covered with veils to allow ventilation. The two host patches were made
of Petri dish (5.5 cm \selectlanguage{ngerman}Ø) containing six 21-days old larvae of \emph{E.
kuehniella} and semolina to the rim, prepared seven days before the test
and covered with a thin gauze to prevent larvae from escaping. Each host
patch was embedded in clean semolina in the middle of a bigger Petri
dish (13 cm Ø). Every morning, wasps were collected at the emergence and
placed individually in tubes with one drop of water. The day after,
males and females of the sexual strain were gathered in a cage to mate.
Females were observed and gradually picked up in a tube as they have
mated until the behavioural experiment. In the meantime, emerging
asexual females were placed in another cage in the same conditions. At
the beginning of the experiment a single female was inserted in the box
and deposited in the middle of the left patch, called `patch 1', whereas
the right patch was called `patch 2'. A total of 34 females (17
asexuals, 17 sexuals) were tested in random order. Foraging behaviours
were followed during 20 min by recording 4 metrics with Jwatcher
(Blumstein and Daniel, 2007): i) probing, wasps probed the substrate
with ovipositor once presence of hosts detected thanks to kairomones;
ii) cocking, a peculiar movement of the abdomen observed after egg
laying, when the female load of a new egg at the tip of its ovipositor
(Rogers, 1972); iii) moving outside of patches (\emph{i.e.} flying or
walking); and iv) time dedicated to hosts, next called patch residence
time (PRT) \emph{i.e.} sum of the time spent on patch 1 and patch 2. The
female was considered to have left a patch when more than 150 seconds
was spent outside of the patch, hence PRT includes short excursions
outside patch boundaries. Exploitation was considered as the capacity of
females to find hosts within patches. Host patches exploitation has been
measured by the means of 2 parameters: i) total PRT was used as a
synthetic parameter to summarize the exploitation of the two patches
(sum of PRT on patches 1 and 2); ii) the total number of
ovipositions,\emph{i.e.} the number of cockings, used to assess the
success of exploitation. Exploration was considered as the ability to
visit the entire experimental device (i.e. environment) and has been
quantified using two parameters: i) the proportion of females that
manage visiting the two host patches, considered as the aptitude to
locate new resources; ii) the number of switches between the two host
patches, considered as the ability to navigate between different
resources. Immediately after behavioural experiment, all the 34 wasp
heads were individually collected to quantify expression of the
\emph{for} gene, while abdomens were dissected to count the number of
eggs in the ovarioles, next called egg load. Heads were stored on ice in
10 \selectlanguage{greek}µ\selectlanguage{english}l of RNA-later (Sigma-Aldrich), then at -20\selectlanguage{ngerman}°C until RNA extraction.

2.10 Quantification of the \emph{for} gene expression

We quantified the \emph{for} gene in each individual wasp head using
RT-qPCR to be able to correlate foraging behaviours with the
\emph{for}gene expression. Once all samples collected, the 34 RNA
extractions were performed in one batch by series of 12 randomized
samples, using the protocol described above. First-strand cDNA was
synthesized from 70ng of total RNA using SuperScript III first strand
synthesis system (ThermoFisher scientific) with random hexamer primers,
and followed by a RNAse-H step. Quantification was conducted on the
\emph{for} gene, together with two reference genes (\emph{rpl32} and
\emph{gapdh} ) used for normalization between samples to control
variations in extraction yield, reverse transcription yield, efficiency
of amplification. Reactions were performed on a CFX-96 (BioRad) using
1:10 diluted cDNA and SYBR Green master mix (BioRad), according to the
manufacturer instructions. Amplification conditions were a first step of
denaturation (95°C, 1 min) followed by 40 cycles of denaturation (95°C,
10 sec) and elongation (melting temperature, 30 sec). Details on primers
and melting temperatures were listed in table S3. Fluorescence was
quantified at the end of each cycle, and the quantification cycle
(C\textsubscript{q}) corresponding to the start of exponential phase
amplification was measured. Each sample was quantified twice: all
duplicated C\textsubscript{q} values varied less than 0.5 cycle,
indicating an elevated replicability. The expression level of the
\emph{for} gene was determined relatively to the expression level of
both reference genes\emph{rpl32} and \emph{gapdh,} using
\selectlanguage{english}[?]\selectlanguage{english}[?]C\textsubscript{q} method (Livak and Schmittgen, 2001). Results were
consistent whatever the reference gene used, and both \emph{rpl32} and
\emph{gapdh} provided satisfactory quality control (low
C\textsubscript{q} values and low variations across samples) (figure S1
and table S4). Therefore, we finally used the mean C\textsubscript{q}
between \emph{rpl32} and \emph{gapdh} for normalization to increase
precision of the results. The \emph{for}expression values were expressed
using relative values comparing each individual to the median
individual, considered as the value 0. Negative values indicated thus
individuals with \emph{for} expression lower than the median, and
positive values indicated individuals with \emph{for}expression superior
to the median.

2.11 Statistical analysis

The foraging behaviours, decomposed into exploitation and exploration,
each measured by a set of parameters previously defined, were analyzed
using Generalized Linear Models (GLM). The population (sexual or
asexual), the \emph{for} gene expression (fold change), the egg load (a
proxy for parasitoid fitness (West et al., 1996)), and the double
interactions with the variable population were included as predictor
variables. PRT was analyzed with GLM with a Gamma distribution for
errors and inverse link. Number of switches between host patches was
analyzed with GLM with a Poisson distribution and log link. Number of
cockings was also analyzed with GLM with a Poisson distribution for
error and log link. PRT was added to the full model since cocking
probability increase with PRT. The \emph{for} gene expression was
analyzed with linear model using population, egg load and their
interaction as explanatory variables. Least contributive variables in
all models were iteratively removed using backward selection to select
optimal models. All statistical analyses were performed with R (R Core
Team, 2017).

3 RESULTS

3.1 Identification of the \emph{for} gene in \emph{V. canescens} genome
and \emph{for} gene evolution in insects

We identified a sequence candidate to be \emph{for} ortholog in
the\emph{V. canescens} genome, and then aligned it to a set of
\emph{for}orthologs in order to reconstruct the evolutionary history of
the\emph{for} gene. The resulting maximum likelihood tree robustly
related the major represented insect clades: hymenoptera, orthoptera,
coleoptera, diptera, lepidoptera (figure 1A). A majority of one-to-one
orthologous relations was detected, with the exception of rare
duplication events. All ten sequences from hymenopteran species
constituted a monophyletic group highly supported (bootstrap value
\textgreater{} 95\%, figure 1A). Within this group, \emph{V.
canescens}(Ichneumonidae) clustered with \emph{Nasonia vitripennis}
(Chalcidoidea) to constitute the parasitoida group. The phylogenetic
reconstruction confirmed that one unique sequence within the \emph{V.
canescens} genome was ortholog to the \emph{for} gene in \emph{D.
melanogaster} , and was then annotated as the \emph{V. canescens for}
gene (\emph{Vcan\_for)} .

3.2 Characterization of the \emph{for} gene model in \emph{V. canescens}

We produced RNA-seq libraries from sexual and asexual populations with a
triple objective: i) reconstruct the \emph{for} gene model,
\emph{i.e.}the region of the gene that is supposed to be transcribed
into RNA; ii) evaluate polymorphism at the \emph{for} locus within
sexual and asexual populations, and iii) assess genome-wide differences
in gene expression between the two populations. To produce an accurate
model of the\emph{for} gene, we thus screened the RNA-seq libraries from
sexual and asexual female heads searching for all reads mapping on this
locus and reconstructed all the transcripts. We identified four separate
transcription start sites, supporting a gene model that contains four
independent promoters (\emph{pr1} -\emph{pr4} ) corresponding to four
distinct open reading frames (figure 1B). The longest open reading
frame\emph{Vcan27709} started with \emph{pr1} , exhibited 7 isoforms
that mainly differed in their untranslated regions (UTRs).
\emph{Vcan27708}transcript started with \emph{pr2} and showed 4
isoforms, while\emph{Vcan27707} (\emph{pr3} ) possessed 5 isoforms.
Finally, the shortest transcript \emph{Vcan27706} starting with
\emph{pr4} presented one isoform. Overall, a total of 13 exons were
identified whose different combinations constituted 17 different
isoforms. The 9 first exons exhibited alternative splicing, thus
isoforms essentially differed in their 5' UTR and the corresponding
N-terminal coding sequences. In contrast, the last 4 exons were
constitutive of all isoforms (excepted\emph{Vcan27707\_i12,
Vcan27709\_i14, Vcan27708\_i17;} that contained an early stop codon),
and constituted one unique 3' extremity (figure 1B) encoding the
C-terminal part of the protein, containing the two cGMP-binding domains
as well as the kinase domain.

3.3 Allelic variation at the \emph{for} locus between sexual and asexual
populations

We focused on the population polymorphism at the \emph{for} locus, by
screening RNA-seq libraries based on 90 females from sexual and asexual
populations, we identified a total of 15 single nucleotide polymorphim
(SNP) (table 1). Only 3 SNPs were located within the coding region,
including 2 synonymous SNPs and one single non-synonymous mutation. The
12 remaining SNPs were located outside of coding sequences, within UTRs.
Among the 15 SNPs, 14 variants exhibited significant differences in
frequency between sexual and asexual populations (table 1). All those
variants were polymorphic in sexual population, while nine were fixed in
asexual population. Together, these results do not support the existence
of two allelic variants differing between sexual and asexual
populations. We rather described a variety of polymorphic sites
accumulated all along the locus, with an important reduction of
polymorphism detected in the asexual population. Moreover, the protein
sequence was little affected by polymorphism, with only one non
synonymous variant recorded and located outside of the functional sites.
Nonetheless, the numerous polymorphic sites reported all along
the\emph{for} gene could affect the transcription or the alternative
splicing of the gene rather than the sequence of the encoded protein
itself.

3.4 Genome-wide expression divergence across sexual and asexual
populations

Overall, we found that gene expression strongly diverged according to
sexual or asexual population. The principal component analysis based on
the expression of all the genes showed that the first axis separated
sexual from asexual population and explained 82\% of the total variance
(figure 2A). Among the 14,106 transcripts that passed the expression
filter, a total of 1,539 genes were DE (\emph{P} -adj\textless{}0.05)
between sexual and asexual population, representing 10.9\% of the
transcriptome. The \emph{for} transcript, represented in the
transcriptome by its longest isoform (\emph{Vcan27709}transcript\emph{)}
, was not included within this list of DEG (rank 2,507/14,106, \emph{P}
-adj = 0.168) (figure 2B). Although the\emph{P} -adj value being above
the significance level, the analysis of normalized counts of the
\emph{for} transcript across the 6 libraries showed that \emph{for}
expression was about 10\% higher in asexual compared to sexual
population (figure 2C). None of the 3 others\emph{for} transcripts
(\emph{Vcan27706} , \emph{Vcan27708} and\emph{Vcan27709)} exhibited
significant differential expression between sexual and asexual
populations, but all showed the same expression pattern (figure S2).

3.5 Behavioural genes expression divergence between sexual and asexual
populations

The \emph{de novo} transcriptome assembly was constituted of a total of
22,333 transcripts that were annotated using BLAST and Gene Ontology
tools. Among those, 18,316 get a blast hit (82\%) and 12,923 get at
least one GO term annotation (58\%). We selected the `behaviour' Gene
Ontology term, as well as all its child related GO terms. In this way,
we annotated 249 transcripts with putative functions associated with
behavior in \emph{V. canescens} . Among them, we reported 26 transcripts
that were DE between the two populations, which represented potential
candidates in the differences in foraging behaviours observed between
sexuals and asexuals (table 2). The proportion of behavioural genes with
differential expression between the 2 populations was not different
compared to the full transcriptome one (26/249 \emph{vs} 1,539/12,567;
\selectlanguage{greek}χ\selectlanguage{english}\textsuperscript{2}= 0.44, \emph{P} -val=0.50). Among those, we noticed
a majority of transcripts related to sensory behaviour: chemosensory (18
transcripts) or visual (2 transcripts). The other functions detected
were the locomotory behaviour (2 transcripts), learning and memory (2
transcripts), reproductive behaviour (1 transcripts) and rhythmic
behaviour (1 transcript).

3.6 Asexual females exploited more hosts and explored more environment
than sexual females

In the behavioural experiment asexual wasps exploited more hosts than
sexual ones, by allocating more time to hosts (figure 3A;
\selectlanguage{greek}χ\selectlanguage{english}\textsuperscript{2}= 3.81, df=1, \emph{P} \textless{}0.01). On average,
asexual females spent twice more time on host patches compared to
sexuals (655.5 \selectlanguage{ngerman}± 60.8s \emph{vs} 333.3 ± 80.4s). Neither the egg load,
the \emph{for} expression nor their interactions with population were
significantly different. Asexual females laid twice more eggs than
sexual ones (figure 3B; \selectlanguage{greek}χ\selectlanguage{english}\textsuperscript{2}=4.94, df=1,\emph{P}
\textless{}0.05), with on average 1.24 eggs laid by asexuals (\selectlanguage{ngerman}± 0.32)
compared to 0.53 eggs by sexuals (±0.17). Time spent on a patch
determined the number of eggs laid since PRT has positive effect on the
number of cockings (\selectlanguage{greek}χ\selectlanguage{english}\textsuperscript{2}=14.09, df=1,\emph{P}
\textless{}0.001). However, \emph{for} expression has a marginal, though
not statistically significant, effect on the number of cockings
(\selectlanguage{greek}χ\selectlanguage{english}\textsuperscript{2}= 3.01, df=1, \emph{P} =0.08), with a number of
cockings increasing in individuals with higher \emph{for}expression. The
interactions between \emph{for} expression and population, and between
\emph{for} expression and PRT, did not explain the number of cockings.
Asexual females also explored more the environment than sexual ones. We
did not detect differences between sexual and asexual populations in the
proportion of females finding the second hosts patch (11/17 \emph{vs}
14/17 respectively, Fisher exact test, \emph{P} =0.44). However, asexual
females switched more from one patch to another than sexual females
(figure 3C; \selectlanguage{greek}χ\selectlanguage{english}\textsuperscript{2}=4.937, df=1, \emph{P} \textless{}0.05),
with on average 3-fold more changes in asexual females (2.12 \selectlanguage{ngerman}± 0.37 in
asexuals\emph{vs} 0.71 ± 0.14 in sexual females). Switches between host
patches were not influenced by other variables, nor by their
interactions with population. Together these results showed that asexual
females exploited more hosts, with more time spent on host patches and
more eggs laid, and explored more the environment by changing more often
of host patches.

3.7 Expression of the \emph{for} gene and correlations with behaviours
in sexual and asexual females

The \emph{for} gene expression was superior in asexual female heads
(1.06±0.60) compared to sexual ones (-0.98±0.54) (figure 4A; F=7.62,
df=1 and 31, \emph{P} \textless{}0.01). Within each
population,\emph{for} expression decreased with egg load (figure 4B;
F=6.7, df=1 and 31, \emph{P} \textless{}0.05). There was no significant
interaction between egg load and population on the \emph{for} gene
expression. When analyzing both populations separately, the number of
cockings increased with PRT in asexual females
(\selectlanguage{greek}χ\selectlanguage{english}\textsuperscript{2}=6.00, df=1,\emph{P} =0.014) but decreased with
\emph{for} expression (\selectlanguage{greek}χ\selectlanguage{english}\textsuperscript{2}=4.96, df=1, \emph{P} =0.026)
(figure 4C). In sexual females, number of cockings is correlated with
PRT (\selectlanguage{greek}χ\selectlanguage{english}\textsuperscript{2}=8.1061, df=1, \emph{P} =0.004) but not
with\emph{for} expression.

4 DISCUSSION

The \emph{for} gene exhibited a strong sequence conservation across
insects, consistent with the function conservation in influencing the
foraging behaviours described in numerous insects (Reaume and
Sokolowski, 2009). Beyond \emph{for} sequence conservation, we also
showed a conservation of the \emph{for} gene model complexity
between\emph{V. canescens} and \emph{D. melanogaster} with four
alternative promoters encoding four proteins differing in their
N-termini (Allen et al., 2017). The use of alternative promoters
represents a source of diversity and flexibility in the regulation of
gene expression, and ultimately function. This as has been particularly
demonstrated in the\emph{for} gene, whose promoter variations cause
changes in both tissue localization and substrate specificity. Indeed,
\emph{pr1-for} and\emph{pr4-for} transcripts were expressed within
neurons, while\emph{pr2-for} and \emph{pr3-for} transcripts were
localized in glia cells of fruit flies central nervous system (Allen et
al., 2018; Dason et al., 2020). The isoform \emph{pr1-for} was presumed
to be the only transcript necessary to forage since in mutants
\emph{pr1-for}expression in neurons was the only required to rescue
larval foraging behaviours (Allen et al., 2018). Variations in the
N-termini are critical to the specificity of PKG-substrate interactions
(Pearce et al., 2010). PKG phosphorylate serine and threonine residues
on a dozen of proteins known to modulate muscle activity and neuronal
signaling pathways (Edelman et al., 1987; Schlossmann and Desch, 2009).
Such variety of substrates may explain the pleiotropic effects of
the\emph{for} gene. Conservation of gene model complexity between the
diptera \emph{D. melanogaster} and the hymenoptera \emph{V.
canescens}supports the importance of maintaining such complexity to
regulate alternative foraging behaviours. However, our work did not
allow the characterization of qualitative differences in \emph{for}
isoforms between sexual and asexual populations, but rather suggested a
decrease in all \emph{for} isoforms transcription in the sexual
population.

Polymorphism analysis revealed 15 SNPs along the \emph{for} gene, most
of them varying in frequency across populations, and supported a major
reduction of genetic diversity that occurred in asexuals rather than the
presence of two allelic variants diverging between sexual and asexual
populations. Such reduction of polymorphism in asexuals was expected: in
general thelytokous individuals are more homozygous than arrhenotokous
ones (Beukeboom and Pijnacker, 2000) and this has been already shown
in\emph{V. canescens} with a study based on 15 microsatellites, that
were all homozygous (Mateo Leach et al., 2012). In contrast, some
genetic diversity still persisted at the \emph{for} locus in asexuals.
The vast majority of identified SNPs did not affect the protein sequence
itself since occurring outside of coding region or corresponding to
synonymous polymorphism. A single SNP corresponding to a non-synonymous
mutation was located at the N-terminal part of the predicted PKG I,
corresponding to the substrate binding region of the protein, outsides
the kinase and cGMP binding domains. By comparison, \emph{rover} and
\emph{sitter}alleles differed by more than 300 SNPs segregating in
\emph{D. melanogaster} , but also involved regulatory mutations rather
than changes in aminoacid sequence (Allen et al., 2017). In contrast,
the two allelic variants identified in the moth \emph{Sesamia
nonagrioides}differed by only one non-synonymous SNP located within the
kinase domain of the protein, each variant was associated with different
levels of\emph{for} expression, PKG activity, and distinct behaviours
(Chardonnet et al., 2014). However, previous studies in hymenopteran
species have not shown any evidence of the existence of allelic variants
at the\emph{for} locus, neither did the present study in \emph{V.
canescens} .

Differences in foraging behaviours recorded between sexuals and asexuals
should rely on genome divergence since there is no more gene flows
between the 2 populations (Mateo Leach et al., 2012). Previous study
revealed such genome divergence since individuals from sexual and
asexual populations can be distinguished based on microsatellites.
However, how much gene expression diverged at the genome-wide scale
between the two populations has not been studied so far. By comparing
head transcriptomes, we reported that the 2 populations clearly split
up, with a total of 1,539 DEGs. This proportion of 11\% of DEGs between
2 populations from a same species is high and almost as high as that
observed in recently diverged species such as \emph{Drosphila
pseudoobscura pseudoobscura} and \emph{D. pseudoobscura
bogotana}(\textasciitilde{}0.25mya divergence and 14.6\% DEGs) (Gomes
and Civetta, 2015). While behavioural divergences are among the most
remarkable differences between sexual and asexual wasps, the
behaviour-annotated group of genes was not overrepresented within DEGs.
Among the behavioural genes whose expression varies between populations,
transcripts involved in sensory perception (olfactory, sensitive,
visual) were the most numerous. Chemosensory genes evolved rapidly and
played important role in adaptation (Brand et al., 2015). While
the\emph{for} gene has been implicated in foraging behaviours in a
variety of organisms, none of the \emph{for} isoforms were detected as
DE. Analysis of RNA-seq data at this locus showed that all isoforms of
the\emph{for} gene were more expressed in asexual population than in
sexual population, although beyond significance threshold.

Globally the behavioural results were congruent with our predictions,
that is asexuals exploited more host patches since they are faster to
choose and walk between hosts, and have a greater egg load (Amat et al.,
2017). On the opposite, sexual females are better dispersers with both
longer and faster flights, they have higher longevity, and greater
energy content (Amat et al., 2017). They were thus expected to explore
more their environment, yet this prediction was not verified in the
current results. The discrepancy could came from the experimental device
that might be too small for all exploration-related behaviours to be
expressed, in particular dispersal involving long flights with high
energy costs (Amat et al., 2012). Field experiments conducted
with\emph{D. melanogaster} showed that rovers exhibit higher dispersion
with both greater dispersal tendencies and longer distances of flight
than sitter flies, and that artificial increasing of \emph{for}
expression in the brain and nervous system increases dispersal in
sitters (Edelsparre et al., 2014). Our study, conducted in the lab, does
not allow assessing wasps dispersal ability. Nevertheless, by showing
that asexual females switched more frequently between host patches than
sexual ones, the experimental device is relevant to detect differences
in some aspects of the exploration between the two populations.

\emph{V. canescens} asexual females present homology with the rover
phenotype observed in \emph{D. melanogaster} both by exploring and
exploiting more. The \emph{for} gene is more expressed in asexual wasps
than in sexual ones, consistently with the \emph{Drosophila} rover
model. Previous classification of the \emph{for} transcript among
non-DEG might be due to the low number of RNA-seq replicates (3), while
RT-qPCR was conducted on a higher number of individuals (17) thus
increasing statistical power in the detection of DEG. The higher
fecundity is another common characteristic between \emph{D.
melanogaster} rovers (McConnell and Fitzpatrick, 2017) and \emph{V.
canescens} asexuals, here measured by both a higher egg load,
corresponding to their potential fitness, and higher number of eggs
laid, corresponding to their effective fitness but measured during a
short period. Hence in these two species, individuals that exploited and
explored more were also the more fecund, and the ones with the
higher\emph{for} expression. A major contribution of the present study
is the joint analysis of the \emph{for} expression and foraging
behaviours measured at the individual scale, that conferred information
on the inter-individual variations and allowed studying correlations
between these traits, beyond average measures. Two major results emerged
from this approach: the first one is that the egg load decreases in
females with the highest \emph{for} expression; and the second is a
decrease of eggs laid by asexual females with the highest \emph{for}
expression. These two correlations were consistent and suggested that an
increase in the \emph{for} expression may be costly for females and
could result in a decrease of progeny number. In the wasp \emph{V.
canescens} , the cost of reproduction is mostly based on finding hosts
to lay eggs, as the egg itself contains little reserve and is not costly
to produce (Pelosse et al., 2011). In this case, rather than an
energetic cost due to the\emph{for} expression that would directly
induce a decrease in fecundity, the cost might be indirect and related
to the numerous other functions fulfilled by the highly pleiotropic
\emph{for} gene apart from resource searching behaviours, such as
learning, memory, or social interactions (Alwash et al., 2021; Reaume
and Sokolowski, 2009).

Given the extent of transcriptomic divergence, with hundreds of DEGs
between sexual and asexual populations, and in the absence of functional
analysis, we cannot firmly conclude on the functional role played by the
slight differences in the \emph{for} expression recorded in the
differences of foraging behaviours. It is worth mentioning that
comparison between rovers and sitters in \emph{D. melanogaster} showed
that differences in the \emph{for} expression were small but consistent
(Osborne et al., 1997). Drosophila rovers and sitters have shown
differences between their transcriptomes, apart from the single
variation in the \emph{for} expression (Kent et al., 2009). Honey bee
nurses and foragers differed by about 40\% of their brain transcriptome
(Whitfield et al., 2003). However, manipulation of the \emph{for} gene
expression or the corresponding PKG enzyme activity was sufficient to
modify foraging behaviours in the two species (Ben-Shahar et al., 2002;
Osborne et al., 1997). Therefore, the differences in the \emph{for} gene
expression detected in the current study between sexuals and asexuals,
although moderate, might nevertheless have an essential function in the
differences in foraging behaviours reported between \emph{V.
canescens}populations.

The present study that highlights the molecular bases underpinning the
variability in foraging behaviours in the parasitoid wasp \emph{V.
canescens} brings insights on the evolution of the control of foraging
behaviours by the \emph{for} gene in hymenoptera. So far, studies have
focused on social hymenopteran species that acquired eusociality
independently: bees, ants and wasps. All these studies revealed a
caste-specific \emph{for} expression correlated with foraging intensity
but with opposite patterns: honey bee (\emph{A. mellifera} ) and
bumblebee (\emph{B. terrestris} ) foragers exhibit a higher
\emph{for}expression compared to nurses (Ben-Shahar et al., 2002;
Tobback et al., 2011), whereas nurses presented a higher \emph{for}
expression compared to foragers in ants (\emph{P. barbatus, P.
pallidula} ) and common wasp\emph{V. vulgaris} (Ingram et al., 2005;
Lucas and Sokolowski, 2009; Wenseleers et al., 2008). The \emph{for}
gene influences social behavior in a variety of species and has been
postulated to be part of a genetic toolkit involved in the evolution of
eusocial insects (Rittschof and Robinson, 2016): whereas acquisition of
eusociality relies on the emergence of a forager caste specialized on
foraging tasks that appears to be related to differences in the
\emph{for} gene expression. In contrast, selection in bees shows
opposite patterns to the one described in the ancestral groups of ants
and wasps. Parasitoids wasps, which are ancestral to the Apocrita group
that includes all social hymenopteran (Peters et al., 2017), are
solitary species and therefore do not have foragers. This study suggests
that differences in the \emph{for} expression pattern underlying changes
in foraging strategies, could be ancestral to Apocrita and precede the
acquisition of sociality. In this group, variations in the \emph{for}
gene expression would not rely on allelic variants. The present work
illustrates an original case of a divergence in foraging behaviours that
is not based on caste differences but associated with a difference in
the \emph{for} expression between populations that also differ in their
reproductive mode. However, the adaptations observed in numerous life
history traits in the two populations are not limited to the difference
in expression of one gene but could involve differences in the optimum
expression pattern of several hundred genes.

Acknowledgements

We thank Francois Debias for his help during field capture and Elsa Day
for her help in insect rearing and experiment. This work was funded by
the Agence Nationale de la Recherche JCJC AVOIDINBRED
(ANR-17-CE02-0004-01) attributed to A.G. This work was performed using
the computing facilities of the CC LBBE/PRABI.

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Accessibility and Benefit-Sharing section

Data Accessibility statement

Data were deposited in the GEO repositories from NCBI database with the
accession code GSE194171.

Benefit-Sharing Statement

Benefits from this research accrue from the sharing of our data and
results on public databases as described above.

Author Contributions

A.G., E.D., L.M., C.V.H. and I.A. designed research; A.G. and D.L.
performed the research; A.G., V.L., D.L. and A.E.F. analyzed data; A.G.,
E.D., L.M., C.V.H. and I.A. wrote the paper.

Tables and Figures

\textbf{Hosted file}

\verb`image1.emf`  available at \url{https://authorea.com/users/504994/articles/584220-the-for-gene-as-one-of-the-drivers-of-foraging-variations-in-a-parasitic-wasp}

\textbf{Figure 1 -} \textbf{The \emph{for} gene in \emph{V. canescens.}}
A) Maximum likelihood phylogenetic tree of the \emph{for} gene in
insects. Sequences used from 39 species of insects: hemiptera (2),
orthoptera (2), coleoptera (4), lepidoptera (6), diptera (15),
hymenoptera (10). Sequence from \emph{V. canescens} figured in bold
within hymenoptera cluster. One crustacean (\emph{Daphnia pulex} ) and
one mammal (\emph{Mus musculus} ) sequences were used as outgroups.
Phylogeny was constructed from the 533 aminoacids located in the
conserved C-terminal region of the protein. Branch support was estimated
by likelihood-ration test, aLRT values [?]0.90 were indicated with empty
circles, and [?]0.95 with full circles. B) Schematic of the \emph{for}
gene model and 17 associated isoforms identified in \emph{V. canescens.}
The \emph{for} gene located on the scaffold 64 of \emph{V. canescens}
genome was constituted of 13 exons (dark blue boxes). The two conserved
functional protein domains (two cGMP binding domains and protein kinase
domain) were located at the 3' extremity of the gene sequence and were
represented under the genome sequence (in blue and red respectively).
The 5' part of the gene exhibited variations, with 4 distinct promotors
identified (\emph{pr1-4} , dark blue arrows), and the corresponding
transcripts were labelled accordingly (\emph{Vcan27706} to
\emph{Vcan27709} ). A total of 17 isoforms were identified within
RNA-seq libraries issued from sexual and asexual \emph{V. canescens}
heads, and were all represented under the genome sequence with coding
sequences figured in dark grey boxes and non-coding sequences (5' and 3'
UTRs) in pale grey boxes. The amplicon used to quantify \emph{for}
transcript with RT-qPCR was common to almost all isoforms (excepted
\emph{Vcan\_27707i12} ) and figured in green.

\textbf{Table 1 -- Decreased polymorphism at the \emph{for} locus in
asexual population.} Characterisation of \emph{for} SNPs that differed
in frequency across sexual and asexual populations: transcript column
indicated the isoform cluster containing the variant; the region and
position column indicated if the SNP is located within the coding
sequence (CDS) or outside (UTR) and its position considering the longest
transcript sequence figured into bracket; polymorphism indicated the
different bases identified at the SNP position; sexual and asexual
population columns contained the number of reads corresponding to each
variant in brackets. In the last two columns information related to the
type of mutation (synonymous or non-synonymous, when SNPs occurred
within CDS), and aminoacid change (only for the non-synonymous mutation)
are informed.

\textbf{Hosted file}

\verb`image2.emf`  available at \url{https://authorea.com/users/504994/articles/584220-the-for-gene-as-one-of-the-drivers-of-foraging-variations-in-a-parasitic-wasp}

\textbf{Hosted file}

\verb`image3.emf`  available at \url{https://authorea.com/users/504994/articles/584220-the-for-gene-as-one-of-the-drivers-of-foraging-variations-in-a-parasitic-wasp}

\textbf{Figure 2 - Transcriptome comparison between sexual and asexual
population.} A) Two-dimensional principal component analysis based on
head transcriptomes from sexual (black) and asexual (red) populations.
Samples, based on the expression profile of 14,106 genes, clustered
according to population. B) Volcano plot illustrated variations in gene
expression between sexual and asexual populations, the y axis measured
the statistical significance (FDR) while the x axis indicated the
magnitude of change. Grey points represented the 1,539 DEG, while genes
not differentially expressed were represented by blue dots including
the\emph{for} gene pointed in red. C) Comparison of normalized counts
number of the \emph{for} gene between the 3 asexual (red) and 3 sexual
(black) RNA-seq libraries.

\textbf{Table 2 -- Behavioural genes belonging to the DEGs between
sexual and asexual populations.} Among the 1539 DEGs between sexual and
asexual populations, 26 transcripts were annotated has related to
behaviour GO term, or one of its child GO terms. The population in which
the transcript was overexpressed was indicated as well as its fold
change (log2), with positive values when overexpressed in sexual
population, and negative values in asexual population.

\textbf{Hosted file}

\verb`image4.emf`  available at \url{https://authorea.com/users/504994/articles/584220-the-for-gene-as-one-of-the-drivers-of-foraging-variations-in-a-parasitic-wasp}



\textbf{Hosted file}

\verb`image5.emf`  available at \url{https://authorea.com/users/504994/articles/584220-the-for-gene-as-one-of-the-drivers-of-foraging-variations-in-a-parasitic-wasp}

\textbf{Figure 3 - Foraging behaviour differences between sexual and
asexual populations of \emph{V. canescens}.} A) Total time spent on host
patches (PRT) according to the population. B) Number of cockings,
corresponding to the number of eggs deposited into hosts according to
the population. C) Number of switches between the 2 host patches
according to the population. Each population consisted of 17 females
from asexual or sexual population.



\textbf{Hosted file}

\verb`image6.emf`  available at \url{https://authorea.com/users/504994/articles/584220-the-for-gene-as-one-of-the-drivers-of-foraging-variations-in-a-parasitic-wasp}

\textbf{Figure 4 -- Comparison of \emph{for} expression between sexual
and asexual populations, in relation with fertility.} A) Variations in
the \emph{for} gene expression between sexual and asexual populations.
B) Individual \emph{for} expression according to egg load in sexual
(black) or asexual (red) females. Black and red lines corresponded to
the values predicted by the model in sexuals and asexuals, respectively.
C) Individual \emph{for} expression according to the number of eggs laid
in asexual females. Circles represent data measured in females: 17
asexuals (red) and 17 sexuals (black). Expression of the gene
\emph{for}is indicated relatively to the median individual.

\textbf{Appendix - Supplementary data}

Table S1 -- Set of \emph{for} (PKG protein) ortholog sequences used in
the phylogenetic analysis.

Table S2 - RNA-seq librairies information.

Table S3 - Primers sequences used for qPCR.

Table S1- Raw results of qPCR

Figure S1 -- Information about quality of RT-qPCR results.

Figure S2 -- Comparison of the 4 \emph{for} main isoforms across sexual
and asexual population.

\textbf{Table S1- PKG1 sequences used in the phylogenetic analysis.}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image7/image7}

\end{center}
\end{figure}

\textbf{Table S2 - RNA-seq librairies information.}\selectlanguage{english}
\begin{longtable}[]{@{}llll@{}}
\toprule
ID & population & number of paired reads & alignment rate\tabularnewline
\midrule
\endhead
AMXC-1 & sexual & 14,556,391 & 94.12\%\tabularnewline
AMXC-2 & sexual & 14,883,970 & 92.30\%\tabularnewline
AMXC-3 & sexual & 18,465,742 & 93.32\%\tabularnewline
AMXC-4 & asexual & 12,999,549 & 90.76\%\tabularnewline
AMXC-5 & asexual & 15,146,936 & 89.97\%\tabularnewline
AMXC-6 & asexual & 14,967,273 & 91.21\%\tabularnewline
\bottomrule
\end{longtable}

\textbf{Table S3 - Primers sequences used for RT-qPCR.}\selectlanguage{english}
\begin{longtable}[]{@{}llllll@{}}
\toprule
Gene & Description & Forward primer sequence & Reverse primer sequence &
Amplicon size & Tm\tabularnewline
\midrule
\endhead
\emph{Rpl32} & Ribosomal protein L 32 & 5'-GCGTTTCAAGGGTCAGTTCT-3' &
5'-AGCGATCTCTGCACAGAATTT-3' & 159 & 60\tabularnewline
\emph{Gapdh} & Glyceraldehyde 3-phosphate dehydrogenase &
5'-TGTGTCCGGATGTACCTGAGT-3' & 5'-TTAAATACACGCTCGGTCTCG-3' & 149 &
55\tabularnewline
\emph{For} & Foraging gene & 5'-GGGTCGAACTCGTCCAAATA-3' &
5'-CTTGGCCATCGAGCAAAAG-3' & 328 & 60\tabularnewline
\bottomrule
\end{longtable}



\textbf{Table S4 - Raw results of qPCR}\selectlanguage{english}
\begin{longtable}[]{@{}llll@{}}
\toprule
female\_id & \emph{For} (C\textsubscript{q}) & \emph{Rpl32}
(C\textsubscript{q}) & \emph{Gapdh} (C\textsubscript{q})\tabularnewline
\midrule
\endhead
26 & 28 & 27.19 & 28.05\tabularnewline
38 & 27.8 & 25.74 & 26.41\tabularnewline
4 & 30.19 & 27.76 & 28.16\tabularnewline
16 & 31.42 & 27.45 & 28.29\tabularnewline
60 & 30.44 & 27.25 & 27.12\tabularnewline
53 & 31.8 & 27 & 27.65\tabularnewline
5 & 27.13 & 25.43 & 26.52\tabularnewline
14 & 30.94 & 27.76 & 27.42\tabularnewline
52 & 27.77 & 25.84 & 26.74\tabularnewline
40 & 28.18 & 25.66 & 27.64\tabularnewline
32 & 31.26 & 26.78 & 27.62\tabularnewline
50 & 29.27 & 27.52 & 28.31\tabularnewline
2 & 31.9 & 28.21 & 28.55\tabularnewline
19 & 31.13 & 28.34 & 29.21\tabularnewline
55 & 28.13 & 27.18 & 28.23\tabularnewline
59 & 25.97 & 25.96 & 26.89\tabularnewline
7 & 31.21 & 29.12 & 29.95\tabularnewline
48 & 29.95 & 28.34 & 29.29\tabularnewline
\#12 & 34.96 & 33.63 & 33.85\tabularnewline
28 & 31.25 & 27.58 & 28.29\tabularnewline
57 & 26.99 & 27.59 & 29.04\tabularnewline
9 & 29.11 & 26.59 & 27.23\tabularnewline
37 & 28.45 & 26.67 & 27.64\tabularnewline
31 & 29.67 & 27.43 & 27.37\tabularnewline
58 & 30.32 & 26.96 & 27.49\tabularnewline
1 & 29.72 & 26.03 & 26.5\tabularnewline
49 & 28.47 & 26.5 & 27.06\tabularnewline
15 & 29.1 & 26.58 & 27.21\tabularnewline
8 & 28.57 & 28.45 & 29.65\tabularnewline
54 & 27.68 & 25.42 & 27.08\tabularnewline
3 & 31.06 & 27.46 & 27.72\tabularnewline
6 & 27.56 & 27.65 & 28.33\tabularnewline
41 & 30.22 & 27.5 & 27.9\tabularnewline
39 & 29.68 & 27.76 & 28.37\tabularnewline
18 & 29.84 & 28.47 & 28.56\tabularnewline
23 & 30.77 & 27.53 & 27.86\tabularnewline
29 & 29.18 & 27.33 & 27.7\tabularnewline
27 & 31.06 & 28.05 & 28.01\tabularnewline
21 & 26.84 & 26.22 & 27.1\tabularnewline
13 & 26.61 & 26.06 & 27.27\tabularnewline
61 & 26.64 & 26.15 & 27.73\tabularnewline
45 & 26.4 & 26.42 & 27.52\tabularnewline
\bottomrule
\end{longtable}

\textbf{Hosted file}

\verb`image8.emf`  available at \url{https://authorea.com/users/504994/articles/584220-the-for-gene-as-one-of-the-drivers-of-foraging-variations-in-a-parasitic-wasp}

\textbf{Figure S1 -- Information about quality of RT-qPCR
results.}Boxplot of quantification cycle (C\textsubscript{q}) measured
on 34 individual heads for 3 genes: \emph{for} , \emph{rpl32} ,
\emph{gapdh} . The \emph{rpl32} and \emph{gapdh} used as reference gene
to normalised\emph{for} expression exhibited less variation among
samples than the\emph{for} gene (F-test; p-val\textless{}0.05) but do
not differ between them. C\textsubscript{q} values were lower in
reference genes than in\emph{for} gene, indicating that \emph{rpl32} and
\emph{gapdh} have a higher expression compared to \emph{for} gene.

\textbf{Hosted file}

\verb`image9.emf`  available at \url{https://authorea.com/users/504994/articles/584220-the-for-gene-as-one-of-the-drivers-of-foraging-variations-in-a-parasitic-wasp}

\textbf{Figure S2 - Comparison of the 4 \emph{for} main isoforms across
sexual and asexual population.} None of the 4 \emph{for} transcripts
(\emph{Vcan27706, Vcan27707, Vcan27708, Vcan27709} ) exhibited
significant differential expression across asexual and sexual
populations (FDR\textgreater{}0.05).

\selectlanguage{english}
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