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Sex pheromone receptors (PRs) are key players in chemical communication between mating partners in insects. In the highly diversified insect order Lepidoptera, male PRs tuned to female-emitted type I pheromones (which make up the vast majority of pheromones identified) form a dedicated subfamily of odorant receptors (ORs). Here, using a combination of heterologous expression and in vivo genome editing methods, we bring functional evidence that at least one moth PR does not belong to this subfamily but to a distantly related OR lineage. This PR, identified in the cotton leafworm Spodoptera littoralis, is highly expressed in male antennae and is specifically tuned to the major sex pheromone component emitted by females. Together with a comprehensive phylogenetic analysis of moth ORs, our functional data suggest two independent apparitions of PRs tuned to type I pheromones in Lepidoptera, opening up a new path for studying the evolution of moth pheromone communication.
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*For correspondence:; (EJ-J);
Present address:
d’Ethologie Expe´ rimentale et
Compare´ e (LEEC), Universite´
Paris 13, Sorbonne Paris Cite´ ,
Villetaneuse, France
Competing interest: See
page 13
Funding: See page 14
Received: 01 July 2019
Accepted: 01 November 2019
Published: 10 December 2019
Reviewing editor: Kristin Scott,
University of California, Berkeley,
United States
Copyright Bastin-He´ line et al.
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A novel lineage of candidate pheromone
receptors for sex communication in moths
Lucie Bastin-He
, Arthur de Fouchier
, Song Cao
, Fotini Koutroumpa
Gabriela Caballero-Vidal
, Stefania Robakiewicz
, Christelle Monsempes
Marie-Christine Franc¸ois
, Tatiana Ribeyre
, Annick Maria
, Thomas Chertemps
Anne de Cian
, William B Walker III
, Guirong Wang
Emmanuelle Jacquin-Joly
*, Nicolas Montagne
Sorbonne Universite´ , Inra, CNRS, IRD, UPEC, Universite´ Paris Diderot, Institute of
Ecology and Environmental Sciences of Paris, Paris and Versailles, France;
Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant
Protection, Chinese Academy of Agricultural Sciences, Beijing, China;
7196, INSERM U1154, Museum National d’Histoire Naturelle, Paris, France;
Department of Plant Protection Biology, Swedish University of Agricultural
Sciences, Alnarp, Sweden
Abstract Sex pheromone receptors (PRs) are key players in chemical communication between
mating partners in insects. In the highly diversified insect order Lepidoptera, male PRs tuned to
female-emitted type I pheromones (which make up the vast majority of pheromones identified)
form a dedicated subfamily of odorant receptors (ORs). Here, using a combination of heterologous
expression and in vivo genome editing methods, we bring functional evidence that at least one
moth PR does not belong to this subfamily but to a distantly related OR lineage. This PR, identified
in the cotton leafworm Spodoptera littoralis, is highly expressed in male antennae and is specifically
tuned to the major sex pheromone component emitted by females. Together with a comprehensive
phylogenetic analysis of moth ORs, our functional data suggest two independent apparitions of
PRs tuned to type I pheromones in Lepidoptera, opening up a new path for studying the evolution
of moth pheromone communication.
The use of pheromone signals for mate recognition is widespread in animals, and changes in sex
pheromone communication are expected to play a major role in the rise of reproductive barriers and
the emergence of new species (Smadja and Butlin, 2009). Since the first chemical identification of
such a pheromone in Bombyx mori (Butenandt et al., 1959), moths (Insecta, Lepidoptera) have
been a preferred taxon for pheromone research (Carde
´and Haynes, 2004;Kaissling, 2014). The
diversification of pheromone signals has likely played a prominent role in the extensive radiation
observed in Lepidoptera, which represents almost 10% of the total described species of living organ-
isms (Stork, 2018).
Female moths generally release a species-specific blend of volatile molecules, which attract males
over a long distance (Carde
´and Haynes, 2004). Four types of sex pheromones have been described
in moths (types 0, I, II and III), with distinct chemical structures and biosynthetic pathways
¨fstedt et al., 2016). 75% of all known moth sex pheromone compounds belong to type I and are
straight-chain acetates, alcohols or aldehydes with 10 to 18 carbon atoms (Ando et al., 2004). Type
I pheromones have been found in most moth families investigated, whereas the other types are
restricted to only a few families (Lo
¨fstedt et al., 2016).
Bastin-He´ line et al. eLife 2019;8:e49826. DOI: 1 of 17
In moth male antennae, pheromone compounds are detected by dedicated populations of olfac-
tory sensory neurons (OSNs). Each type of OSN usually expresses one pheromone receptor (PR)
responsible for signal transduction. PRs are 7-transmembrane domain proteins belonging to the
odorant receptor (OR) family and, as ORs, are co-expressed in OSNs together with the conserved
co-receptor Orco (Chertemps, 2017;Fleischer and Krieger, 2018).
Since the first discovery of moth PRs (Krieger et al., 2004;Sakurai et al., 2004), numerous pher-
omone receptors tuned to type I pheromone compounds have been characterized through different
hererologous expression systems, and most appeared to be specific to only one compound
(Zhang and Lo
¨fstedt, 2015). More recently, a few receptors tuned to type 0 and type II pheromones
have also been characterized (Zhang et al., 2016;Yuvaraj et al., 2017). Type I PRs belong to a ded-
icated monophyletic subfamily of ORs, the so-called ‘PR clade’, suggesting a unique emergence
early in the evolution of Lepidoptera (Yuvaraj et al., 2018). Another hallmark of type I PRs is their
male-biased expression (Koenig et al., 2015). The phylogenetic position and the expression pattern
have thus been the main criteria used to select candidate PRs for functional studies.
Likewise, we selected PRs from the male transcriptome of the cotton leafworm Spodoptera littor-
alis (Legeai et al., 2011), a polyphagous crop pest that uses type I sex pheromone compounds
˜oz et al., 2008) and that has been established as a model in insect chemical ecology
(Ljungberg et al., 1993;Binyameen et al., 2012;Saveer et al., 2012;Poivet et al., 2012;
de Fouchier et al., 2017). Through heterologous expression, we characterized two PRs tuned to
minor components of the S. littoralis pheromone blend (Montagne
´et al., 2012;de Fouchier et al.,
2015), but none of the tested candidate PRs detected the major pheromone component (Z,E)-9,11-
eLife digest Many animals make use of chemical signals to communicate with other members of
their species. Such chemical signals, called pheromones, often allow males and females of the same
species to recognize each other before mating. Since the discovery of the very first pheromone in
the silkworm moth Bombyx mori at the end of the 1950s, moths have been a model for pheromone
research. The sex pheromone communication system in these insects has thus been well described:
females emit a mixture of volatile chemicals, which can be detected by the antennae of males up to
several hundred meters away. This detection is achieved through neurons with specialized proteins
known as pheromone receptors that bind to the chemical signals produced by the females.
Recognizing mates by detecting a very specific pheromone signature prevents moths from
interbreeding with other species. The evolution of pheromone signals and their corresponding
receptors can therefore lead to the rise of new reproductive barriers between populations, and
eventually to the emergence of new species. The rate at which sex pheromones have diversified is
likely one reason for the existence of over 160,000 species of moths. But how did moths’ sex
pheromone receptors evolve in the first place?
Previous studies suggested that moth pheromone receptors had appeared just once during
evolution. Specifically, they revealed that these receptors belong to the same branch or lineage in
the ‘family tree’ of all receptors that detect chemical compounds in moths. This meant that when
researchers looked for pheromone receptors in a new species of moth, they always focused on this
lineage. But Bastin-He´ line et al. have now found that one pheromone receptor from a pest moth
called Spodoptera littoralis does not belong to this established group.
First, Bastin-He´ line et al. inserted this receptor into animal cells grown in the laboratory to
confirm that it responds to a specific pheromone produced by S. littoralis. Next, they genetically
modified moths of this species and showed that males need this receptor in order to mate. An
evolutionary analysis showed that the receptor belongs to a different lineage than all the other
known pheromone receptors. Together these results indicate the receptors for sex pheromones
must have evolved multiple times independently in moths.
These results will open new avenues for deciphering pheromone communication in moths, and
lead to further research into this newly discovered lineage of candidate pheromone receptors. Such
studies may foster the development of new strategies to control agricultural pests, given that some
species of moths can have devastating effects on the yields of certain crops.
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Research article Evolutionary Biology Neuroscience
tetradecadienyl acetate (hereafter referred as (Z,E)-9,11-14:OAc), which is necessary and sufficient to
elicit all steps of the male mate-seeking behavioral sequence (Quero et al., 1996).
In order to identify new type I PR candidates, we focused on male-biased ORs, whether they
belong to the PR clade or not. Notably, a preliminary analysis of S. littoralis OR expression patterns
led to the identification of such a receptor, SlitOR5, which was highly expressed in male antennae
but did not belong to the PR clade (Legeai et al., 2011). Furthermore, a recent RNAseq analysis
showed that SlitOR5 was the most abundant OR in S. littoralis male antennae (Walker et al., 2019).
Here, we demonstrate that SlitOR5 is the receptor for (Z,E)-9,11-14:OAc using a combination of het-
erologous expression and in vivo genome editing methods. Based on a comprehensive phylogenetic
analysis of lepidopteran ORs, we show that SlitOR5 belongs to an OR subfamily that is distantly
related to the PR clade but harbors numerous sex-biased ORs from distinct moth families. Alto-
gether, these results suggest that PRs detecting type I pheromones evolved at least twice in Lepi-
doptera, which offers a more detailed and complex panorama on moth PR evolution.
SlitOr5 is highly expressed in males but does not belong to the type I
pheromone receptor clade
We first used quantitative real-time PCR to compare the relative expression levels of the SlitOr5
gene in S. littoralis male and female adult antennae. We found SlitOr5 expressed with a more than
SlitOr5 expression level
(relative to SlitRpl13)
type I pheromone
receptor clade
type II pheromone
type 0 pheromone
Figure 1. SlitOr5 is highly expressed in males but does not belong to the pheromone receptor clade. (A) Expression levels of SlitOr5 in adult male and
female antennae of S. littoralis, as measured by real-time qPCR. Expression levels have been normalized to the expression of SlitRpl13. Plotted values
represent the mean normalized expression values ±SEM (n= 3). Raw results are available in Figure 1—source data 1. (B) Unrooted maximum
likelihood phylogeny of lepidopteran ORs, based on 506 amino acid sequences from nine species, each belonging to a different superfamily. The
position of SlitOR5 and of receptors for type 0, type I and type II pheromone compounds is highlighted. Circles on the nodes indicate the distinct
paralogous OR lineages, supported by a transfer bootstrap expectation (TBE) >0.9. All the PR-containing lineages grouped within a large clade
(highlighted in grey) also supported by the bootstrap analysis. The sequence alignment file is available in Figure 1—source data 2.
The online version of this article includes the following source data for figure 1:
Source data 1. Mean normalized expression values of SlitOr5 measured in the three biological replicates.
Source data 2. Alignment of amino acid sequences used to build the phylogeny (FASTA format).
Bastin-He´ line et al. eLife 2019;8:e49826. DOI: 3 of 17
Research article Evolutionary Biology Neuroscience
50-fold enrichment in the male antennae (Figure 1A), thus confirming previous observations
(Legeai et al., 2011;Walker et al., 2019).
We reconstructed a maximum likelihood phylogeny of lepidopteran ORs, based on entire OR rep-
ertoires from nine different species. Among the 20 paralogous lineages identified (each having
evolved in principle from an ancestral OR present in the last common ancestor of Lepidoptera), Sli-
tOR5 belonged to a lineage distantly related to the type I PR clade, as well as to the lineages con-
taining type 0 and type II PRs (Figure 1B). These four paralogous lineages grouped within a larger
clade highly supported by the bootstrap analysis (highlighted in grey in Figure 1B). This clade has
been previously shown to contain ORs tuned to terpenes and aliphatic molecules – including sex
pheromones – and exhibits higher evolutionary rates compared to more ancient clades that contain
many receptors for aromatics (de Fouchier et al., 2017).
SlitOR5 binds (Z,E)-9,11-14:OAc with high specificity and sensitivity
We next used two complementary heterologous systems to characterize the function of SlitOR5 and
assess whether it is the receptor to (Z,E)-9,11-14:OAc, the major component of the S. littoralis sex
pheromone blend. First, we expressed SlitOR5 in Drosophila melanogaster OSNs housed in at1 tri-
choid sensilla, in place of the endogenous PR DmelOR67d (Kurtovic et al., 2007). Single-sensillum
recordings were performed to measure the response of at1 OSNs to 26 type I pheromone com-
pounds (Supplementary file 1), including all the components identified in the pheromones of Spo-
doptera species (El-Sayed, 2018). SlitOR5-expressing OSNs strongly responded to (Z,E)-9,11-14:
OAc (65 ±15 spikes.s
), whereas there was no significant response to any other compound
(Figure 2A).
Then, we co-expressed SlitOR5 with its co-receptor SlitOrco in Xenopus oocytes and recorded
the responses to the same panel of pheromone compounds using two-electrode voltage-clamp. A
strong current was induced in SlitOR5-expressing oocytes when stimulated with (Z,E)-9,11-14:OAc
(3.9 ±0.3 mA), whereas only minor currents were recorded in response to (Z,E)-9,12-14:OAc and (Z)
9-12:OAc (Figure 2B). SlitOR5 sensitivity was assessed with a dose-response experiment that
showed a low detection threshold (10
M) and an EC
of 1.707 10
M (Figure 2D–E).
We compared the response spectra of heterologously expressed SlitOR5 with that of S. littoralis
male OSNs housed in type one long trichoid sensilla (LT1A OSNs, Figure 2C), known to detect (Z,E)-
9,11-14:OAc (Ljungberg et al., 1993;Quero et al., 1996). When stimulated with the 26 pheromone
compounds, LT1A OSNs significantly responded to (Z,E)-9,11-14:OAc (55 ±4 spikes.s
) and to a
lesser extent to its stereoisomer (Z,Z)-9,11-14:OAc, which is absent from any Spodoptera phero-
mone. This mirrored the response spectra of heterologously expressed SlitOR5, especially the one
observed in Drosophila OSNs (Figure 2A).
In vivo response to (Z,E)-9,11-14:OAc is abolished in SlitOr5 mutant
To confirm in vivo that SlitOR5 is the receptor for the major sex pheromone component of S. littora-
lis, we carried out a loss-of-function study by generating mutant insects for the gene SlitOr5. The
CRISPR/Cas9 genome editing system was used to create a mutation in the first exon of SlitOr5 with
the aim of disrupting the open-reading frame. Guide RNAs were injected along with the Cas9 pro-
tein in more than one thousand eggs. We genotyped 66 G0 hatched larvae and found seven individ-
uals carrying at least one mutation in SlitOr5. These 7 G0 individuals were back-crossed with wild-
type individuals to create 7 G1 heterozygous mutant lines. We selected a line carrying a single muta-
tion that consisted of a 10 bp deletion at the expected location, introducing a premature STOP
codon within the SlitOr5 transcript after 247 codons (Figure 3A).
We next generated G2 homozygous mutant males (SlitOr5
) and compared their ability to detect
(Z,E)-9,11-14:OAc to that of wild-type and heterozygous (SlitOr5
) males, using electroantenno-
gram (EAG) recordings (Figure 3B). When stimulated with (Z,E)-9,11-14:OAc, wild-type and
antennae exhibited similar EAG amplitudes (0.89 mV and 1.16 mV, respectively), whereas
the response was completely abolished in SlitOr5
antennae (0.02 mV). Control experiments using a
S. littoralis minor pheromone component and plant volatiles known to induce EAG responses in S.
littoralis (Saveer et al., 2012;Lo
´pez et al., 2017) showed that antennal responses were not
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Research article Evolutionary Biology Neuroscience
SlitOR5 in Drosophila OSNs
0 25 50 75 100
SlitOR5 in Xenopus oocytes
Current (µA)
0 1 2 3 4 5
Response (spikes.s )
0 25 50 75 100
S. littoralis LT1A OSNs
Response (spikes.s )
Current (µA)
10 10 10 10 10
Concentration of
-9 -8 -7 -6 -5
500 nA
2 min
10 3 10 10 10 10
-9 -8 -7 -6 -5
Figure 2. SlitOR5 is the receptor for the major component of the S. littoralis pheromone blend. (A) Action potential frequency of Drosophila at1 OSNs
expressing SlitOR5 (n= 8) after stimulation with 26 type I pheromone compounds (10 mg loaded in the stimulus cartridge). ***p<0.001, significantly
different from the response to solvent (one-way ANOVA followed by a Tukey’s post hoc test). (B) Inward current measured in Xenopus oocytes co-
expressing SlitOR5 and SlitOrco (n= 13–16) after stimulation with the same panel of pheromone compounds (10
M solution). ***p<0.001, **p<0.01,
significantly different from 0 (Wilcoxon signed rank test). (C) Action potential frequency of LT1A OSNs from S. littoralis male antennae (n= 8–16) after
stimulation with pheromone compounds (1 mg loaded in the stimulus cartridge). ***p<0.001, *p<0.1, significantly different from the response to solvent
(one-way ANOVA followed by a Tukey’s post hoc test). (D) Representative trace showing the response of a Xenopus oocyte co-expressing SlitOR5 and
SlitOrco after stimulation with a range of (Z,E)-9,11-14:OAc doses from 10
M to 10
M. (E) Dose-response curve of SlitOR5/Orco Xenopus oocytes
Figure 2 continued on next page
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Research article Evolutionary Biology Neuroscience
impaired in SlitOr5
mutants, as these odorants elicited similar responses in wild-type, heterozygous
and homozygous moths (Figure 3B).
Then, we analyzed the courtship behavior of SlitOr5
and wild-type males in the presence of (Z,
E)-9,11-14:OAc, and found a strong behavioral defect in mutants. Whereas more than 80% of wild-
type males initiated a movement toward the pheromone source in the first 8 min, only a minority of
SlitOr5 mutants initiated such a movement (even after 30 min of test), a response similar to that of
control wild-type males not stimulated with the pheromone (Figure 3C). All the steps of the court-
ship behavior were similarly affected (Figure 3—figure supplement 1). We also verified whether Sli-
tOr5 knock-out would result in mating inability. When paired with a wild-type virgin female, only 1
out of the 13 SlitOr5
males tested was able to mate, compared to ~75% for wild-type males
(Figure 3D). This behavioral defect was further confirmed by analyzing the number of eggs laid by
the females and the number of offspring (Figure 3E). Overall, these results confirm that SlitOR5 is
the receptor responsible for the detection of the major component of the S. littoralis female phero-
mone blend.
A novel lineage of candidate moth pheromone receptors
In view of these results and the unexpected phylogenetic position of SlitOR5, we rebuilt the phylog-
eny of the lepidopteran OR clade containing SlitOR5 and the known receptors for type 0, type I and
type II pheromones (highlighted in grey in Figure 1B), adding all ORs showing a strong sex-biased
expression (at least 10-fold in one sex compared to the other) and ORs whose ligands were known
as of September 2018 (Supplementary file 2). ORs grouped within eight different paralogous line-
ages, four of which including PRs (Figure 4). One was the so-called PR clade that, as previously
observed, contained all type I PRs characterized so far (except SlitOR5) as well as two type II PRs
(Zhang et al., 2016). The other three lineages harboring PRs consisted of one containing SlitOR5,
one containing EgriOR31 (a type II PR from the geometrid Ectropis grisescens;Li et al., 2017) and
one containing EsemOR3 and 5 (type 0 PRs from the non-dytrisian moth Eriocrania semipurpurella;
Yuvaraj et al., 2017). Interestingly, most sex-biased lepidopteran ORs identified to date clustered
within the PR clade and the lineage containing SlitOR5. While sex-biased ORs within the PR clade
were mainly male-biased, the SlitOR5 lineage contained an equal proportion of male and female-
biased receptors, identified from species belonging to different families of Lepidoptera. Deep nodes
within the phylogeny were highly supported by the bootstrap analysis, enabling us to state that
these two PR-containing clades were more closely related to clades harboring receptors for plant
volatiles than to each other. This suggests that receptors tuned to type I pheromone compounds
emerged twice independently during the evolution of Lepidoptera, and that the clade containing Sli-
tOR5 may constitute a novel lineage of candidate PRs.
While moth sex pheromone receptors have been the most investigated ORs in Lepidoptera, with
more than 60 being functionally characterized (Zhang and Lo
¨fstedt, 2015), it remains unclear how
and when these specialized receptors arose. Type I PRs have been proposed to form a monophy-
letic, specialized clade of ORs, the so-called ‘PR clade’, which emerged early in the evolution of Lep-
idoptera (Yuvaraj et al., 2017;Yuvaraj et al., 2018). Here, we bring functional and phylogenetic
evidence that type I PRs are not restricted to this clade and likely appeared twice independently in
Lepidoptera. We focused on an atypical OR, SlitOR5, which exhibited a strong male-biased expres-
sion in antennae of the noctuid moth S. littoralis but did not group with the PR clade. We demon-
strated, using a combination of heterologous expression and loss-of-function studies, that this OR is
responsible for the detection of (Z,E)-9,11-14:OAc, the major component of the S. littoralis sex pher-
omone blend. Due to the unexpected phylogenetic position of SlitOR5 outside of the previously
Figure 2 continued
(n= 9) stimulated with (Z,E)-9,11-14:OAc (EC
= 1.707 10
M). Plotted values in (A–C and E) are mean responses ±SEM. Raw results for all
experiments are available in Figure 2—source data 1.
The online version of this article includes the following source data for figure 2:
Source data 1. Raw results of electrophysiology experiments.
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Research article Evolutionary Biology Neuroscience
SlitOr5 gene
target sequence for gRNAPAM
0 0.2 0.4 0.6 0.8 1 1.2 1.4
EAG amplitude (mV)
benzyl alcohol
wild-type mRNA
mutant mRNA
SlitOr5 ORF
SlitOr5 ORF
1000 50
25 75
mating rate (%)
0 100 200 300
number of offspring
0 100 200 300
number of eggs
wild-type + solvent
movement toward the source
0 500 1000 1500 2000
time (s)
cumulative occurence (%)
+ (Z,E)-9,11-14:OAc
*** ***
Figure 3. Response to the major pheromone component is abolished in SlitOr5 mutants. (A) Location of the 10 bp deletion induced in the first exon of
the SlitOr5 gene by the CRISPR/Cas9 system. The sequence complementary to the RNA guide is indicated in blue, and the protospacer adjacent motif
(PAM) in red. The frameshift created in the SlitOr5 open-reading frame (ORF) induced a premature stop codon. (B) Electroantennogram (EAG)
amplitude measured in S. littoralis male antennae isolated from wild-type animals (light grey, n= 14), heterozygous SlitOr5 mutants (dark grey, n= 18)
and homozygous SlitOr5 mutants (purple, n= 8) after stimulation with pheromone compounds (1 mg in the stimulus cartridge) and plant volatiles (10 mg
in the stimulus cartridge). Plotted values represent the normalized mean response ±SEM (response to the solvent was subtracted). ***p<0.001,
significantly different from the response of the other genotypes; n.s.: not significantly different (one-way ANOVA, followed by a Tukey’s post hoc test).
Raw results for the EAG experiment are available in Figure 3—source data 1. (C) Cumulative proportion of S. littoralis males initiating a movement
toward the odor source in homozygous SlitOr5 mutants (purple, n= 14) stimulated with the major pheromone component (100 ng in the stimulus
Figure 3 continued on next page
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Research article Evolutionary Biology Neuroscience
defined PR clade, the question arose whether SlitOR5 is an exception or belongs to a previously
unknown clade of moth PRs. This latter hypothesis is supported by the observation that the paralo-
gous lineage containing SlitOR5 harbored many other sex-biased ORs, identified in species from six
distinct lepidopteran families. Notably, male-biased ORs have been found in Lasiocampidae, Sphin-
gidae, Noctuidae and Tortricidae. Among these, two ORs from Ctenopseustis obliquana and C. her-
ana (Tortricidae) have been functionally studied by heterologous expression in cell cultures, but no
ligand could be identified (Steinwender et al., 2015). In the Lasiocampidae species Dendrolimus
punctatus, these male-biased ORs have been referred to as ‘Dendrolimus-specific odorant recep-
tors’, with the suspicion that they would represent good PR candidates since in Dendrolimus species,
there is no OR clustering in the PR clade (Zhang et al., 2014;Zhang et al., 2017). No functional
data yet confirmed this suspicion.
Conversely, within the SlitOR5 lineage, almost half of the sex-biased ORs were female-biased (13
out of 28, compared to 4 out of 63 in the classical PR clade). Female-biased ORs have been gener-
ally proposed to be involved in the detection of plant-emitted oviposition cues, as demonstrated in
Bombyx mori (Anderson et al., 2009). However, another interesting hypothesis is that they could be
tuned to male sex pheromones. In moths, little attention has been put on male pheromones, which
are known to be involved in various mating behaviors such as female attraction, female acceptance,
aggregation of males to form leks, mate assessment or inhibition of other males (reviewed in
Conner and Iyengar, 2016). The use of male pheromone systems has been selected multiple times
in distinct moth families, as reflected by the chemical diversity of male pheromone compounds and
of the disseminating structures (Birch et al., 1990;Phelan, 1997;Conner and Iyengar, 2016). It is
thus expected that this polyphyletic nature of male pheromones would result in a large diversity of
female PR types. Accordingly, female-biased ORs were found in different clades within the phylog-
eny. However, most remain orphan ORs, including BmorOR30 that does belong to the SlitOR5 line-
age but for which no ligand could be identified (Anderson et al., 2009). Although the most
common male-emitted volatiles are plant-derived pheromones (Conner and Iyengar, 2016), some
male courtship pheromones are long-chained hydrocarbons related to type I female pheromone
compounds (Hillier and Vickers, 2004) that could be detected by female-biased type I PRs such as
those identified within the SlitOR5 lineage.
The ancestral protein from which the so-called ‘PR clade’ would have arisen is thought to be an
OR tuned to plant-emitted volatiles (Yuvaraj et al., 2017;Yuvaraj et al., 2018). Here, we evidence
that SlitOR5 is a new type I PR that belongs to a distinct early diverging lineage for which a role in
pheromone detection had never been demonstrated. Together with the findings that PRs for Type 0
(Yuvaraj et al., 2017) and one PR for type II pheromones (Li et al., 2017) group in distinct paralo-
gous lineages also unrelated to the PR clade, our data suggest that lepidopteran PRs have evolved
four times in four paralogous lineages. Whether the SlitOR5 lineage has evolved from ORs that
detected structurally related plant volatiles - as has been proposed for type 0 (Yuvaraj et al., 2017)
and classical type I PRs - remains elusive. Yet, no OR tuned to plant volatiles has been identified in
closely related lineages. More functional data on SlitOR5 paralogs and orthologs in different moth
species, possibly revealing more type I PRs, will help in understanding evolutionary history of this
lineage, as to when and how these receptors have evolved, and confirm that we are facing a novel
type I PR clade.
Figure 3 continued
cartridge) and in wild-type animals stimulated with the pheromone (blue, n= 36) or with solvent alone (light grey, n= 44). ***p<0.001, significantly
different from the other distributions; n.s.: not significantly different (log-rank test). Results obtained for other behavioral items are presented in
Figure 3—figure supplement 1. (D) Proportion of wild-type S. littoralis males (light grey, n= 49) and homozygous SlitOr5 mutants (purple, n= 13) that
mated with a wild-type female during a period of 6 hr in the scotophase. *p<0.05, significant difference between the two genotypes (Fisher’s exact
test). (E) Number of eggs laid (left panel) and of offspring (right panel) obtained per female after the mating experiment. Plotted values represent the
mean ±SEM. ***p<0.001, **p<0.005, significant difference between the two genotypes (Mann-Whitney Utest). Raw results for all the behavioral
experiments are available in Figure 3—source data 2.
The online version of this article includes the following source data and figure supplement(s) for figure 3:
Source data 1. Raw results of the EAG experiment.
Source data 2. Raw results of behavioral experiments.
Figure supplement 1. Behavioral response of wild-type and homozygous SlitOr5 mutants to the major pheromone component.
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Research article Evolutionary Biology Neuroscience
type I pheromone receptors
type II pheromone receptors
type 0 pheromone receptors
plant volatile receptors
female-biased receptor
male-biased receptor
pheromone receptor clade
novel lineage of candidate
pheromone receptors
Figure 4. SlitOR5 may define a novel lineage of candidate pheromone receptors in Lepidoptera. Maximum likelihood phylogeny of the lepidopteran
OR clade that includes all the paralogous lineages containing pheromone receptors. 360 sequences from 34 lepidopteran species were included.
Functional and expression data shown on the figure have been compiled from the literature (Supplementary file 2). Branch colors indicate OR
function, when characterized: PRs for type I pheromones are depicted in red, those for type II pheromones in blue and those for type 0 pheromones in
Figure 4 continued on next page
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Materials and methods
Key resources table
Reagent type
(species) or resource Designation Source or reference Identifiers
(Spodoptera littoralis)
SlitOr5 GenBank GB:MK614705
(Spodoptera littoralis)
SlitOrco Malpel et al. (2008);
Kurtovic et al. (2007);
PMID: 17392786
FLYB:FBal0210948 kindly provided
by B. Dickson
2A w*; M{3xP3-
Bischof et al. (2007);
PMID: 17360644;
Stock Center
UAS-SlitOr5 This study See Materials
and methods
DNA reagent
Bischof et al. (2007);
PMID: 17360644;
GB:EF362409 kindly provided
by J. Bischof
DNA reagent
SlitOr5 (plasmid)
This study See Materials
and methods
DNA reagent
pCS2+ (plasmid) Turner and Weintraub (1994);
PMID: 7926743
kindly provided
by C. He
DNA reagent
This study See Materials
and methods
DNA reagent
This study See Materials
and methods
based reagent
based reagent
based reagent
Rpl13up This study PCR primers GTACCTGCCGCTCTCCGTGT
based reagent
Rpl13do This study PCR primers CTGCGGTGAATGGTGCTGTC
based reagent
based reagent
Or5_forward This study PCR primers CCAAAAGGACTTGGACTTTGAA
based reagent
Or5_reverse This study PCR primers CCCGAATCTTTTCAGGATTAGAA
Figure 4 continued
orange. ORs tuned to plant volatiles are depicted in green. Symbols at the edge indicate expression data: male-biased ORs are highlighted with black
squares and female-biased ORs with black dots. Circles on the nodes indicate the distinct paralogous OR lineages. Support values on basal nodes are
transfer bootstrap expectation (TBE) values. The tree has been rooted using an outgroup as identified in the lepidopteran OR phylogeny shown in
Figure 1. The scale bar indicates the expected number of amino acid substitutions per site. The sequence alignment file is available in Figure 4—
source data 1.
The online version of this article includes the following source data for figure 4:
Source data 1. Alignment of amino acid sequences used to build the phylogeny (FASTA format).
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Research article Evolutionary Biology Neuroscience
Animal rearing and chemicals
S. littoralis were reared in the laboratory on a semi-artificial diet (Poitout and Bue
`s, 1974) at 22˚C,
60% relative humidity and under a 16 hr light:8 hr dark cycle. Males and females were sexed as
pupae and further reared separately. D. melanogaster lines were reared on standard cornmeal-
yeast-agar medium and kept in a climate- and light-controlled environment (25˚C, 12 hr light:12 hr
dark cycle). The 26 pheromone compounds used for electrophysiology experiments
(Supplementary file 1) were either synthesized in the lab or purchased from Sigma-Aldrich (St Louis,
MO) and Pherobank (Wijk bij Duurstede, The Netherlands). Paraffin oil was purchased from Sigma-
Aldrich and hexane from Carlo Erba Reagents (Val de Reuil, France).
Quantitative real-time PCR
Total RNA from three biological replicates of 15 pairs of antennae of two-day-old virgin male and
female S. littoralis was extracted using RNeasy Micro Kit (Qiagen, Hilden, Germany), which included
a DNase treatment. cDNA was synthesized from total RNA (1 mg) using Invitrogen SuperScript II
reverse transcriptase (Thermo Fisher Scientific, Waltham, MA). Gene-specific primers were designed
TTATCACTAT-3’) and for the reference gene SlitRpl13 (Rpl13up: 5’-GTACCTGCCGCTCTCCGTGT-
3’, Rpl13do: 5’-CTGCGGTGAATGGTGCTGTC-3’). qPCR mix was prepared in a total volume of 10
mL with 5 mL of LightCycler 480 SYBR Green I Master (Roche, Basel, Switzerland), 4 mL of diluted
cDNA (or water for the negative control) and 0.5 mM of each primer. qPCR assays were performed
using the LightCycler 480 Real-Time PCR system (Roche). All reactions were performed in triplicate
for the three biological replicates. The PCR program began with a cycle at 95˚C for 13.5 min, fol-
lowed by 50 cycles of 10 s at 95˚C, 15 s at 60˚C and 15 s at 72˚C. Dissociation curves of the amplified
products were performed by gradual heating from 55˚C to 95˚C at 0.5 ˚C.s
. A negative control
(mix without cDNA) and a fivefold dilution series protocol of pooled cDNAs (from all conditions)
were included. The fivefold dilution series were used to construct relative standard curves to deter-
mine the PCR efficiencies used for further quantification analyses. Data were analyzed with the Light-
Cycler 480 software (Roche). Normalized expression of the SlitOr5 gene was calculated with the
Q-Gene software (Joehanes and Nelson, 2008) using SlitRpl13 as a reference, considering it dis-
plays consistent expression as previously described in Durand et al. (2010).
Heterologous expression of SlitOR5 in Drosophila
The SlitOr5 full-length open-reading frame (1191 bp, GenBank acc. num. MK614705) was subcloned
into the pUAST.attB vector. Transformant UAS-SlitOr5 balanced fly lines were generated by Best-
Gene Inc (Chino Hills, CA), by injecting the pUAST.attB-SlitOr5 plasmid (Endofree prepared, Qiagen)
into fly embryos with the genotype y
M{vas-int.Dm}ZH-2A w*; M{3xP3-RFP.attP}ZH-51C
(Bischof et al., 2007), leading to a non-random insertion of the UAS-SlitOr5 construct into the locus
51C of the second chromosome. The UAS-SlitOr5 balanced line was then crossed to the Or67d
line (Kurtovic et al., 2007) to obtain double homozygous flies (genotype w; UAS-SlitOr5,w
) expressing the SlitOr5 transgene in at1 OSNs instead of the endogenous Drosophila receptor
gene Or67d. The correct expression of SlitOr5 was confirmed by RT-PCR on total RNA extracted
from 100 pairs of antennae.
Single-sensillum recordings
Single-sensillum extracellular recordings on Drosophila at1 OSNs were performed as previously
described (de Fouchier et al., 2015). OSNs were stimulated during 500 ms, using stimulus car-
tridges containing 10 mg of pheromone (1 mg/ml in hexane) dropped onto a filter paper. Single-sen-
sillum recordings on S. littoralis LT1A OSNs were performed using the tip-recording technique, as
previously described (Pe
´zier et al., 2007). Briefly, the tips of a few LT1 sensilla were cut off using
sharpened forceps and a recording glass electrode filled with a saline solution (170 mM KCl, 25 mM
NaCl, 3 mM MgCl
, 6 mM CaCl
, 10 mM HEPES and 7.5 mM glucose, pH 6.5) was slipped over the
end of a cut LT1 sensillum. OSNs were stimulated with an air pulse of 200 ms (10 L.h
), odorized
using a stimulus cartridge containing 1 mg of pheromone (diluted at 1 mg/ml in hexane). Odorants
were considered as active if the response they elicited was statistically different from the response
elicited by the solvent alone (one-way ANOVA followed by a Tukey’s post hoc test).
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Heterologous expression of SlitOR5 in Xenopus oocytes and two-
electrode voltage-clamp recordings
Open-reading frames of SlitOr5 and SlitOrco (GenBank acc. num. EF395366, Malpel et al., 2008)
were subcloned into the pCS2+ vector (Turner and Weintraub, 1994). Template plasmids were fully
linearized with PteI for pCS2+-SlitOr5 and NotI for pCS2+-SlitOrco and capped cRNAs were tran-
scribed using SP6 RNA polymerase. Purified cRNAs were re-suspended in nuclease-free water at a
concentration of 2 mg/mL and stored at 80˚C. Mature healthy oocytes were treated with 2 mg/ml
collagenase type I in washing buffer (96 mM NaCl, 2 mM KCl, 5 mM MgCl
and 5 mM HEPES, pH
7.6) for 1–2 hr at room temperature. Oocytes were later microinjected with 27.6 ng of SlitOr5 cRNA
and 27.6 ng of SlitOrco cRNA. After 4 days of incubation at 18˚C in 1 Ringer’s solution (96 mM
NaCl, 2 mM KCl, 5 mM MgCl2, 0.8 mM CaCl2, and 5 mM HEPES, pH 7.6) supplemented with 5%
dialyzed horse serum, 50 mg/ml tetracycline, 100 mg/ml streptomycin and 550 mg/ml sodium pyru-
vate, the whole-cell currents were recorded from the injected oocytes with a two-electrode voltage
clamp. Oocytes were exposed to the 26 pheromone compounds diluted at 10
M in Ringer’s solu-
tion, with an interval between exposures which allowed the current to return to baseline. Data acqui-
sition and analysis were carried out with Digidata 1440A and pCLAMP10 software (Molecular
Devices, San Jose, CA). Odorants were considered as active if the mean response they elicited was
statistically different from 0 (Wilcoxon signed rank test). Dose–response experiments were per-
formed using pheromone concentrations ranging from 10
up to 10
M and data were analyzed
using GraphPad Prism 5.
SlitOr5 knock-out via CRISPR/Cas9
A guide RNA (gRNA sequence: AGCATAAATACTGGACCCAG TGG) was designed against the first
exon of the SlitOr5 gene using the CRISPOR gRNA design tool (; Haeussler et al.,
2016) and transcribed after subcloning into the DR274 vector using the HiScribe T7 High Yield RNA
Synthesis Kit (New England Biolabs, Ipswich, MA). The Cas9 protein was produced in Escherichia coli
as previously described (Me
´noret et al., 2015). A mix of Cas9 protein and gRNA was injected in
freshly laid eggs using an Eppendorf - Transjector 5246, as previously described
(Koutroumpa et al., 2016). Individual genotyping at every generation was performed via PCR on
genomic DNA extracted from larvae pseudopods (Wizard Genomic DNA Purification Kit, Promega,
Madison, WI) using gene-specific primers (Or5_forward: 5’-CCAAAAGGACTTGGACTTTGAA-3’;
Or5_reverse: 5’-CCCGAATCTTTTCAGGATTAGAA-3’) amplifying a fragment of 728 bp encompass-
ing the target sequence. Mutagenic events were detected by sequencing the amplification products
(Biofidal, Vaulx-en-Velin, France). G0 larvae carrying a single mutagenic event were reared until
adults and crossed with wild-type individuals. Homozygous G2 individuals were obtained by crossing
G1 heterozygous males and females.
Phenotyping of CRISPR/Cas9 mutants by electroantennogram
Electroantennogram recordings were performed as previously described (Koutroumpa et al., 2016)
on isolated male antennae from wild-type animals and from heterozygous and homozygous SlitOr5
mutants. Antennae were stimulated using stimulus cartridges loaded with 10 mg of linalool or benzyl
alcohol diluted in paraffin oil, and 1 mg of (Z,E)-9,11-14:OAc or (Z,E)-9,12-14:OAc diluted in hexane.
Stimulations lasted for 500 ms (30 L/h). Negative controls consisted of paraffin oil and hexane alone.
The maximum depolarization amplitude was measured using the pCLAMP10 software. Normalized
mean responses were calculated (response to the solvent was subtracted) and data were analyzed
using a one-way ANOVA followed by a Tukey’s post hoc test.
Behavioral experiments
For courtship monitoring, 2-day-old wild-type or homozygous SlitOr5 mutant males were placed
individually in a plastic squared petri dish (size = 12 cm) before the onset of the scotophase, and
experiments started at the middle of the scotophase (22˚C, 70% relative humidity). Odorant stimula-
tion was performed using Pasteur pipettes containing a filter paper loaded with 100 ng of (Z,E)-9,11-
14:OAc (10 ng/ml in hexane), or hexane alone as control. The narrow end of the pipette was inserted
into the petri dish, and a constant stream of charcoal-filtered, humidified air (0.2 L.min
) passed
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Research article Evolutionary Biology Neuroscience
through the pipette during all the experiment. Male courtship behavior was recorded under dim red
light during 30 min using a webcam (Logitech QuickCam Pro 9000). The latency of each of the fol-
lowing stereotyped behavioral items was individually screened: antennal flicking, movement toward
the odor source, wing fanning, abdomen curving and extrusion of genitalia. Results were presented
as cumulative occurrence of each item along the time of the experiment. Statistical analysis of the
distributions obtained for each treatment were compared using a log-rank (Mantel-Cox) test. For
mating experiments, 2-day-old wild-type or homozygous SlitOr5 mutant males were paired with a 2-
day-old wild-type virgin female in a cylindrical plastic box (diameter 8 cm, height 5 cm), the walls of
which were covered up with filter paper. Experiments started at the onset of the scotophase and
lasted over 6 hr. Copulation events were visually inspected every 20 min under dim red light. After
the end of the experiment, females were kept in the boxes for 12 hr. Then, females were discarded
and filter papers were examined for the presence of egg clutches. When present, eggs were trans-
ferred to a rearing plastic box, hatch was monitored every day during 7 days and second-instar lar-
vae were counted, if any. Statistical analysis of mating rates was done using a Fisher’s exact test, and
analyses of the number of eggs and offspring were done using a Mann-Whitney Utest.
Phylogenetic analyses
The dataset of lepidopteran amino acid OR sequences used to build the phylogeny shown in Fig-
ure 1 included entire OR repertoires from the following nine species, each belonging to a different
lepidopteran superfamily: Bombyx mori (Bombycoidea; Tanaka et al., 2009), Dendrolimus punctatus
(Lasiocampoidea; Zhang et al., 2017), Ectropis grisescens (Geometroidea; Li et al., 2017), Epiphyas
postvittana (Tortricoidea; Corcoran et al., 2015), Eriocrania semipurpurella (Eriocranioidea;
Yuvaraj et al., 2017), Heliconius melpomene (Papilionoidea; Heliconius Genome Consortium,
2012), Ostrinia furnacalis (Pyraloidea; Yang et al., 2015), Plutella xylostella (Yponomeutoidea;
Engsontia et al., 2014) and Spodoptera littoralis (Noctuoidea; Walker et al., 2019). The dataset
used to build the phylogeny shown in Figure 4 contained amino acid sequences from the same nine
species falling into that clade, plus all the sequences of ORs within that clade showing a marked sex-
biased expression (threshold of a 10-fold difference in expression rate between male and female
antennae, based on RNAseq or qPCR experiments) and/or for which ligands have been identified as
of September 2018 (Supplementary file 2). Alignments were performed using Muscle (Edgar, 2004)
as implemented in Jalview v2.10.5 (Waterhouse et al., 2009). Phylogenetic reconstruction was per-
formed using maximum likelihood. The best-fit model of protein evolution was selected by SMS
(Lefort et al., 2017) and tree reconstruction was performed using PhyML 3.0 (Guindon et al.,
2010). Node support was first assessed using 100 bootstrap iterations, then the file containing boot-
strap trees was uploaded on the Booster website (Lemoine et al., 2018) to obtain the TBE (Transfer
Bootstrap Expectation) node support estimation. Figures were created using the iTOL web server
(Letunic and Bork, 2016).
We are grateful to Lixiao Du and Franc¸ oise Bozzolan for technical assistance, Bin Yang for his help
and advice, and Pascal Roskam and Philippe Touton for insect rearing. We also thank Christophe
He´ ligon (CRB Xe´ nope, Rennes) for providing the pCS2+ plasmid. This work has been funded by the
French National Research Agency (ANR-16-CE02-0003-01 and ANR-16-CE21-0002-01 grants), the
National Natural Science Foundation of China (31725023, 31621064) and a PRC NSFC-CNRS 2019
grant. Research was conducted as part of the CAAS-INRA Associated International Laboratory in
Plant Protection.
Additional information
Competing interests
Arthur de Fouchier, Emmanuelle Jacquin-Joly, Nicolas Montagne´ : The intellectual property rights of
SlitOR5 have been licensed by Inra, Sorbonne Universite´ and CNRS for the purpose of developing
novel insect control agents. The other authors declare that no competing interests exist.
Bastin-He´ line et al. eLife 2019;8:e49826. DOI: 13 of 17
Research article Evolutionary Biology Neuroscience
Funder Grant reference number Author
Agence Nationale de la Re-
ANR-16-CE02-0003-01 Nicolas Montagne
Agence Nationale de la Re-
ANR-16-CE21-0002-01 Emmanuelle Jacquin-Joly
National Natural Science
Foundation of China
31725023 Guirong Wang
National Natural Science
Foundation of China
31621064 Guirong Wang
PRC NSFC-CNRS Guirong Wang
The funders had no role in study design, data collection and interpretation, or the
decision to submit the work for publication.
Author contributions
Lucie Bastin-He´ line, Data curation, Investigation, Writing—original draft; Arthur de Fouchier, William
B Walker III, Investigation, Writing—review and editing; Song Cao, Data curation, Investigation, Writ-
ing—review and editing; Fotini Koutroumpa, Gabriela Caballero-Vidal, Stefania Robakiewicz, Marie-
Christine Franc¸ois, Tatiana Ribeyre, Annick Maria, Thomas Chertemps, Investigation; Christelle Mon-
sempes, Data curation, Investigation; Anne de Cian, Resources; Guirong Wang, Conceptualization,
Funding acquisition, Writing—review and editing; Emmanuelle Jacquin-Joly, Conceptualization,
Funding acquisition, Writing—original draft; Nicolas Montagne´ , Conceptualization, Data curation,
Funding acquisition, Writing—original draft
Author ORCIDs
William B Walker III
Emmanuelle Jacquin-Joly
Nicolas Montagne´
Decision letter and Author response
Decision letter
Author response
Additional files
Supplementary files
.Supplementary file 1. List of synthetic compounds used for electrophysiology experiments.
.Supplementary file 2. Functional and sex-biased expression data available for lepidopteran phero-
mone receptors (as of September 2018).
.Transparent reporting form
Data availability
All data generated or analysed during this study are included in the manuscript and supporting files.
Source data files have been provided for Figures 1, 2, 3 and 4.
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... Accordingly, numerous moth PRs tuned to type-I sex pheromones have been functionally characterized using different heterologous expression systems, and most PRs are specific to only one pheromone com-pound (Zhang & Löfstedt, 2015;Cao et al., 2020). However, an OR that is highly expressed in male antennae of Spodoptera littoralis (SlitOR5), specifically detecting the major sex pheromone component of this species, was identified in a clade that is distantly related to the type-I PR clade, and was named the novel PR clade (Bastin-Héline et al., 2019). This result was further confirmed by the identification, in the same clade, of PRs from other moth species (Shen et al., 2020;Yuvaraj et al., 2022), suggesting that PRs detecting type-I sex pheromones have emerged at least twice independently during evolution. ...
... To better understand the PR relationships in this extended novel PR clade, a phylogenetic tree was built based on amino acid sequences of 97 ORs in 12 moth species from 8 different lepidopteran families (Fig. 5). In the novel PR clade, 2 ORs (EposOR30 and EposOR34) in E. postvittana (Yuvaraj et al., 2022), 2 ORs (DpunOR45 and DpunOR4) in D. punctatus (Shen et al., 2020), and 1 OR in S. frugiperda (SfruOR5) and in S. littoralis (SlitOR5) have been identified to respond to type-I sex pheromones (Bastin-Héline et al., 2019;Li et al., 2023). Among the 12 moth species, 9 ORs from 6 moth species clustered in the same clade to form the SfruOR23 lineage. ...
... Previous studies have suggested that type-I PRs have multiple independent origins in the Lepidoptera, with 2 separate clades (the classical PR clade and the so-called novel PR clade) identified as type-I PR clades (Zhang & Löfstedt, 2015;Bastin-Héline et al., 2019;Cao et al., 2020;Shen et al., 2020;Yuvaraj et al., 2022). In this study, we investigated 7 SfruOR5 paralogous ORs in the novel PR clade and in closely related clades, and we found that a male-biased OR (SfruOR23) belonging to one of these clades serves as a moth PR. ...
In moths, pheromone receptors (PRs) are crucial for intraspecific sexual communication between males and females. Moth PRs are considered as an ideal model for studying the evolution of insect PRs, and a large number of PRs have been identified and functionally characterized in different moth species. Moth PRs were initially thought to fall into a single monophyletic clade in the odorant receptor (OR) family, but recent studies have shown that ORs in another lineage also bind type‐I sex pheromones, which indicates that type‐I PRs have multiple independent origins in the Lepidoptera. In this study, we investigated whether ORs of the pest moth Spodoptera frugiperda belonging to clades closely related to this novel PR lineage may also have the capacity to bind type‐I pheromones and serve as male PRs. Among the 7 ORs tested, only 1 ( SfruOR23 ) exhibited a male‐biased expression pattern. Importantly, in vitro functional characterization showed that SfruOR23 could bind several type‐I sex pheromone compounds with Z ‐9‐tetradecenal ( Z 9‐14:Ald), a minor component found in female sex pheromone glands, as the optimal ligand. In addition, SfruOR23 also showed weak responses to plant volatile organic compounds. Altogether, we characterized an S. frugiperda PR positioned in a lineage closely related to the novel PR clade, indicating that the type‐I PR lineage can be extended in moths.
... Additionally, the pheromone perception systems of different moths also exhibit high diversity corresponding to their species-specific sex pheromones. In the peripheral olfactory system of moths, pheromone signals are detected and processed by several proteins, including pheromone binding proteins (PBPs) [17,18], sensory neuron membrane proteins (SNMPs) [19], pheromone degrading enzymes (PDEs) [20,21], and pheromone receptors (PRs) [22][23][24]. Of these, PRs determine the selectivity and specificity of the sex pheromone reception. ...
... The functional differentiation of essential PRs results in behavioral preference divergence in closelyrelated moth species [25,26]. So far, numerous studies have been conducted on the identification [27][28][29][30] and functional deorphanization [24,[31][32][33][34] of PRs in moths, which together open an avenue for the discovery of genetic architectures and evolution in pheromone preferences on the molecular level. However, lacking information on PR function in closely-related moth species greatly limits our understanding of the relationships between PR evolution and speciation. ...
... The two-electrode voltage-clamp (TEVC) recording in combination with the Xenopus laevis oocytes heterologous expression system has been widely used to deorphanize the ligands of insect odorant receptors (ORs) [24][25][26]. The caped RNA (cRNA), encoding the candidate OR and Orco were co-injected into Xenopus oocytes. ...
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Male moths utilize their pheromone communication systems to distinguish potential mates from other sympatric species, which contributes to maintaining reproductive isolation and even drives speciation. The molecular mechanisms underlying the evolution of pheromone communication systems are usually studied between closely-related moth species for their similar but divergent traits associated with pheromone production, detection, and/or processing. In this study, we first identified the functional differentiation in two orthologous pheromone receptors, OR14b, and OR16, in four Helicoverpa species, Helicoverpa armigera, H. assulta, H. zea, and H. gelotopoeon. To understand the substrate response specificity of these two PRs, we performed all-atom molecular dynamics simulations of OR14b and OR16 based on AlphaFold2 structural prediction, and molecular docking, allowing us to predict a few key amino acids involved in substrate binding. These candidate residues were further tested and validated by site-directed mutagenesis and functional analysis. These results together identified two hydrophobic amino acids at positions 164 and 232 are the determinants of the response specificity of HarmOR14b and HzeaOR14b to Z9-14:Ald and Z9-16:Ald by directly interacting with the substrates. Interestingly, in OR16 orthologs, we found that position 66 alone determines the specific binding of Z11-16:OH, likely via allosteric interactions. Overall, we have developed an effective integrated method to identify the critical residues for substrate selectivity of ORs and elucidated the molecular mechanism of the diversification of pheromone recognition systems.
... However, in the two sibling species S. littoralis and S. litura, the major component of the sex pheromone is (Z,E)-9,11-tetradecadienyl acetate (hereafter referred to as (Z,E)-9,11-14:OAc), which is lacking or present only in minute amounts in other species (SI Appendix, Fig. S1). Detection of this compound is necessary and sufficient to trigger male attraction (17,18). This means that a major change in the pheromone communication system occurred in a common ancestor of S. littoralis and S. litura. ...
... This means that a major change in the pheromone communication system occurred in a common ancestor of S. littoralis and S. litura. We recently discovered in S. littoralis a receptor tuned to (Z,E)-9,11-14:OAc, named OR5 (17). Interestingly, this receptor is distantly related to other moth PRs, suggesting independent origins. ...
... As moth sex PRs are generally overexpressed in male antennae (17), we then sought to compare expression levels of Or5 and Or75 in adult antennae of the four Spodoptera species. In S. exigua and S. frugiperda, SexiOr5 and SfruOr5 were expressed in both male and female antennae with a twofold enrichment in males (Fig. 1E). ...
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Pheromone communication is an essential component of reproductive isolation in animals. As such, evolution of pheromone signaling can be linked to speciation. For example, the evolution of sex pheromones is thought to have played a major role in the diversification of moths. In the crop pests Spodoptera littoralis and S. litura, the major component of the sex pheromone blend is (Z,E)-9,11-tetradecadienyl acetate, which is lacking in other Spodoptera species. It indicates that a major shift occurred in their common ancestor. It has been shown recently in S. littoralis that this compound is detected with high specificity by an atypical pheromone receptor, named SlitOR5. Here, we studied its evolutionary history through functional characterization of receptors from different Spodoptera species. SlitOR5 orthologs in S. exigua and S. frugiperda exhibited a broad tuning to several pheromone compounds. We evidenced a duplication of OR5 in a common ancestor of S. littoralis and S. litura and found that in these two species, one duplicate is also broadly tuned while the other is specific to (Z,E)-9,11-tetradecadienyl acetate. By using ancestral gene resurrection, we confirmed that this narrow tuning evolved only in one of the two copies issued from the OR5 duplication. Finally, we identified eight amino acid positions in the binding pocket of these receptors whose evolution has been responsible for narrowing the response spectrum to a single ligand. The evolution of OR5 is a clear case of subfunctionalization that could have had a determinant impact in the speciation process in Spodoptera species.
... There have been extensive studies on the ORs detecting female-released sex pheromones, the so-called pheromone receptors (PRs), in moths that are well-known for their selective and sensitive sex pheromone communication systems (Zhang & Löfstedt, 2015). The majority of the PRs characterized so far are responsive to Type I pheromones (fatty alcohols, aldehydes or fatty alcohol acetates) ; mostly clustered in the classical PR clade (Zhang & Löfstedt, 2015), with some fall into the so-called novel PR clade (Bastin-Héline et al., 2019;Yuvaraj et al., 2022). It seems that the PRs for the Type II epoxide pheromones and the ancestral Type 0 pheromones (short-chain secondary alcohols or ketones) also cluster in respective clades (Li et al., 2017;Yuvaraj et al., 2017), while the receptors for Type II hydrocarbon pheromones reside in classical PR clade (Zhang et al., 2016). ...
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Insects rely on their highly efficient and precise olfactory systems to find suitable mates, host plants and oviposition sites, and adapt to the changing environment. The odorant receptors (ORs) including pheromone receptors (PRs) play a vital role in this process. While extensive studies have been focusing on deorphanization of lepidopteran PR genes, the information on the ligand profiles of general ORs is still sparse. In the present study, we identified a repertoire of 61 ORs including the co-receptor Orco from antennal and ovipositor transcriptomes of the turnip moth Agrotis segetum , which clustered in all the major lepidopteran OR clades. We characterized the function of eight female-biased expressed ORs in Xenopus oocytes and found three ORs differentially tuned to plant volatile compounds that might be repulsive or attractive to the moths. AsegOR13 was broadly tuned to a number of herbivore-induced plant volatiles (HIPVs) while AsegOR20 was specific to citral; AsegOR17 was narrowly tuned to the alcohols, isoamyl alcohol, pentanol and benzyl alcohol, that are potentially attractive to moths. The orthologues of the three ORs in other moth species seem to share the conserved function. Our results support the hypothesis that insects recognize their host plants mostly by detecting the mixture of ubiquitous compounds, instead of taxonomically characteristic host compounds. The combination of narrowly and broadly tuned ORs will ensure both the accuracy of the most important odor signals and the plasticity of the olfactory system to the changes in the environment.
... In addition, no butterfly PR has been identified and functionally deorphanized although abundant PRs have been characterized in different moth species (Krieger et al., 2005;Mitsuno et al., 2008). It is therefore likely that the butterfly PRs are not phylogenetically closely related to the moths' PRs, but might rather be an independent lineage that detect compounds with a different chemical structure (Bastin-Heline et al., 2019). ...
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Sensory processes have often been argued to play a central role in the selection of ecological niches and in the formation of new species. Butterflies are among the best studied animal groups with regards to their evolutionary and behavioral ecology and thereby offer an attractive system to investigate the role of chemosensory genes in sympatric speciation. We focus on two Pieris butterflies with overlapping host-plant ranges: P. brassicae and P. rapae . Host-plant choice in lepidopterans is largely based on their olfactory and gustatory senses. Although the chemosensory responses of the two species have been well characterized at the behavioral and physiological levels, little is known about their chemoreceptor genes. Here, we compared the chemosensory genes of P. brassicae and P. rapae to investigate whether differences in these genes might have contributed to their evolutionary separation. We identified a total of 130 and 122 chemoreceptor genes in the P. brassicae genome and antennal transcriptome, respectively. Similarly, 133 and 124 chemoreceptors were identified in the P. rapae genome and antennal transcriptome. We found some chemoreceptors being differentially expressed in the antennal transcriptomes of the two species. The motifs and gene structures of chemoreceptors were compared between the two species. We show that paralogs share conserved motifs and orthologs have similar gene structures. Our study therefore found surprisingly few differences in the numbers, sequence identities and gene structures between the two species, indicating that the ecological differences between these two butterflies might be more related to a quantitative shift in the expression of orthologous genes than to the evolution of novel receptors as has been found in other insects. Our molecular data supplement the wealth of behavioral and ecological studies on these two species and will thereby help to better understand the role of chemoreceptor genes in the evolution of lepidopterans.
Pheromone receptors (PRs) are key proteins in the molecular mechanism of pheromone recognition, and exploring the functional differentiation of PRs between closely related species helps to understand the evolution of moth mating systems. Pheromone components of the agricultural pest Mythimna loreyi have turned into (Z)-9-tetradecen-1-yl acetate (Z9-14:OAc), (Z)-7-dodecen-1-yl acetate (Z7-12:OAc), and (Z)-11-hexadecen-1-yl acetate, while the composition differs from that of M. separata in the genus Mythimna. To understand the molecular mechanism of pheromone recognition, we sequenced and analyzed antennal transcriptomes to identify 62 odorant receptor (OR) genes. The expression levels of all putative ORs were analyzed using differentially expressed gene analysis. Six candidate PRs were quantified and functionally characterized in the Xenopus oocytes system. MlorPR6 and MlorPR3 were determined to be the receptors of major and minor components Z9-14:OAc and Z7-12:OAc. MlorPR1 and female antennae (FA)-biased MlorPR5 both possessed the ability to detect pheromones of sympatric species, including (Z,E)-9,12-tetradecadien-1-ol, (Z)-9-tetradecen-1-ol, and (Z)-9-tetradecenal. Based on the comparison of PR functions between M. loreyi and M. separata, we analyzed the differentiation of pheromone recognition mechanisms during the evolution of the mating systems of 2 Mythimna species.
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Les effets observés et prédits du réchauffement climatique placent les études sur la biologie thermique au cœur des travaux sur le vivant. La température est en effet connue pour influencer quasiment tous les paramètres des ectothermes tels que les insectes. Cette thèse sur la biologie thermique du ravageur de culture Spodoptera littoralis est basée sur l’étude expérimentale d’aspects clefs touchant aux effets d’insecticides, aux paramètres de valeur sélective et à la sensibilité olfactive. Les apports de ce travail ont été méthodologiques et conceptuels. Une première contribution méthodologique a été de montrer que l’effet négatif de températures élevées sur la valeur sélective est souvent sous-estimé par l’étude de températures constantes. Une seconde contribution a été de montrer l’intérêt de tester des normes de réaction inter-familiales en écotoxicologie. Une troisième contribution a été d’illustrer l’importance de prendre en compte la masse corporelle pour l’étude des réponses à la température et aux insecticides. La thèse a également eu trois apports conceptuels d’importance. Une influence de la température sur la toxicité d’insecticides a été montrée, mais elle a interagi avec des effets transgénérationnels. L’ampleur de la fluctuation journalière de température a également eu un impact sur la toxicité d’insecticides. Enfin, la température de développement a affecté la sensibilité olfactive de mâles adultes, effet qui a été dépendant de leur masse corporelle. En conclusion, tous les paramètres étudiés de S. littoralis ont été sensibles à la température mais de façon plus complexe que généralement attendu dans la littérature.
Under nutrient-poor conditions, animals must save energy by adjusting their behavior and physiology in order to survive. Although the impact of feeding state on olfactory sensory neuron activity has been well studied, the regulatory mechanisms underlying the transcriptional changes in odorant receptors (Ors) induced by feeding signals are seldom mentioned. Here, we showed that starvation could attenuate antennal responses of Bactrocera dorsalis toward multiple odorants, which could be reverted by sugar re-feeding, but not by a protein-rich diet. Using methyl eugenol (ME) as a paradigm, our study provided molecular evidence that Forkhead Box protein O (FoxO) can be expressed in antennal tissues to govern starvation-induced olfactory modifications by binding to the upstream regulatory regions of ME-responsive Ors and regulating their expressions. Since the consensus FoxO binding motif was also identified in other 17 Ors whose expression levels were also significantly altered upon FoxO knockdown and starvation, our data suggest that FoxO-dependent binding is likely a universal regulatory mechanism for Or genes during starvation and re-feeding cycles. Taken together, the FoxO-Ors axis elucidated in this study provides an improved understanding of how the insulin signaling pathway senses the feeding state and certain macronutrient composition to shape olfactory plasticity, allowing flies to dynamically alter chemosensory sensitivities toward different odors. Our study also highlights sugar as a satiety signal, which could increase ME-mediated trap efficiency in the field.
The Asian corn borer Ostrinia furnacalis (Lepidoptera: Crambidae) is a serious corn pest with widespread distribution in East Asia. Its olfactory mechanism is a focus of scientific study, aiming to find good ways to control this pest. Molecular events are considered to be important in olfactory mechanism. Current understandings of olfactory molecular events in O. furnacalis, mainly involving sex pheromones and olfactory proteins, were summarized to provide a reference for further studies. O. furnacalis sex pheromone contains two components E-12-tetradecenyl acetate and Z-12-tetradecenyl acetate, which may be recognized and bound by the pheromone binding proteins OfurPBP3 and OfurPBP2, and then transported to the odorant receptors (ORs) OfurOR4 and OfurOR6 to activate them. The ORs OfurOR8, OfurOR7 and OfurOR5b mainly respond to the sex pheromone components of other Ostrinia species, E-11-tetradecenyl acetate, Z-11-tetradecenyl acetate and Z-9-tetradecenyl acetate. The OR OfurOR27 responds strongly to plant odorants nonanal, octanal and 1-octanol. Much work remains to be done to fully understand odorants with olfactory activity to O. furnacalis and the functions of its olfactory proteins. These studies will help to reveal olfactory mechanism in O. furnacalis, with the aim of regulating its behaviors to control this pest.
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Background: Deciphering the molecular mechanisms mediating the chemical senses, taste, and smell has been of vital importance for understanding the nature of how insects interact with their chemical environment. Several gene families are implicated in the uptake, recognition, and termination of chemical signaling, including binding proteins, chemosensory receptors and degrading enzymes. The cotton leafworm, Spodoptera littoralis, is a phytophagous pest and current focal species for insect chemical ecology and neuroethology. Results: We produced male and female Illumina-based transcriptomes from chemosensory and non-chemosensory tissues of S. littoralis, including the antennae, proboscis, brain and body carcass. We have annotated 306 gene transcripts from eight gene families with known chemosensory function, including 114 novel candidate genes. Odorant receptors responsive to floral compounds are expressed in the proboscis and may play a role in guiding proboscis probing behavior. In both males and females, expression of gene transcripts with known chemosensory function, including odorant receptors and pheromone-binding proteins, has been observed in brain tissue, suggesting internal, non-sensory function for these genes. Conclusions: A well-curated set of annotated gene transcripts with putative chemosensory function is provided. This will serve as a resource for future chemosensory and transcriptomic studies in S. littoralis and closely related species. Collectively, our results expand current understanding of the expression patterns of genes with putative chemosensory function in insect sensory and non-sensory tissues. When coupled with functional data, such as the deorphanization of odorant receptors, the gene expression data can facilitate hypothesis generation, serving as a substrate for future studies.
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Pheromones are chemicals that serve intraspecific communication. In animals, the ability to detect and discriminate pheromones in a complex chemical environment substantially contributes to the survival of the species. Insects widely use pheromones to attract mating partners, to alarm conspecifics or to mark paths to rich food sources. The various functional roles of pheromones for insects are reflected by the chemical diversity of pheromonal compounds. The precise detection of the relevant intraspecific signals is accomplished by specialized chemosensory neurons housed in hair-like sensilla located on the surface of body appendages. Current data indicate that the extraordinary sensitivity and selectivity of the pheromone-responsive neurons (PRNs) is largely based on specific pheromone receptors (PRs) residing in their ciliary membrane. Besides these key elements, proper ligand-induced responses of PR-expressing neurons appear to generally require a putative co-receptor, the so-called “sensory neuron membrane protein 1” (SNMP1). Regarding the PR-mediated chemo-electrical signal transduction processes in insect PRNs, ionotropic as well as metabotropic mechanisms may be involved. In this review, we summarize and discuss current knowledge on the peripheral detection of pheromones in the olfactory system of insects with a focus on PRs and their specific role in the recognition and transduction of volatile intraspecific chemical signals.
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The odorant receptors (ORs) of insects are crucial for host and mate recognition. In moths (Lepidoptera), specialized ORs are involved in male detection of the sex pheromone produced by females. Most moth sex pheromones are C10-C18 acetates, alcohols, and aldehydes (Type I pheromones), and most pheromone receptors (PRs) characterized to date are from higher Lepidoptera (Ditrysia), responding to these types of compounds. With few exceptions, functionally characterized PRs fall into what has been called the "PR-clade", which also contains receptors that have yet to be characterized. While it has been suggested that moth PRs have evolved from plant odor-detecting ORs, it is not known when receptors for Type I pheromones arose. This is largely due to a lack of functionally characterized PRs from non-ditrysian Lepidoptera. The currant shoot borer moth, Lampronia capitella (Prodoxidae), belongs to a non-ditrysian lineage, and uses Type I pheromone compounds. We identified 53 ORs from antennal transcriptomes of this species, and analyzed their phylogenetic relationships with known lepidopteran ORs. Using a HEK293 cell-based assay, we showed that three of the LcapORs with male-biased expression (based on FPKM values) respond to Type I pheromone compounds. Two of them responded to pheromone components of L. capitella and one to a structurally related compound. These PRs are the first from a non-ditrysian moth species reported to respond to Type I compounds. They belong to two of the more early-diverging subfamilies of the PR-clade for which a role in pheromone detection had not previously been demonstrated. Hence, our definition of the monophyletic lepidopteran PR-clade includes these receptors from a non-ditrysian species, based on functional support.
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Felsenstein's application of the bootstrap method to evolutionary trees is one of the most cited scientific papers of all time. The bootstrap method, which is based on resampling and replications, is used extensively to assess the robustness of phylogenetic inferences. However, increasing numbers of sequences are now available for a wide variety of species, and phylogenies based on hundreds or thousands of taxa are becoming routine. With phylogenies of this size Felsenstein's bootstrap tends to yield very low supports, especially on deep branches. Here we propose a new version of the phylogenetic bootstrap in which the presence of inferred branches in replications is measured using a gradual 'transfer' distance rather than the binary presence or absence index used in Felsenstein's original version. The resulting supports are higher and do not induce falsely supported branches. The application of our method to large mammal, HIV and simulated datasets reveals their phylogenetic signals, whereas Felsenstein's bootstrap fails to do so.
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The insect chemosensory system is pivotal for interactions with their environments, and moths have especially sensitive olfaction. Exploration of the connection between the plasticity of olfactory-guided and molecular level pathways in insects is important for understanding the olfactory recognition mechanisms of insects. The pine caterpillar moth, Dendrolimus punctatus Walker, is a dominant conifer defoliator in China, and mating is the priority for adults of this species, during which sex pheromone recognition and oviposition site location are the main activities; these activities are all closely related to chemosensory genes. Thus, we aimed to identify chemosensory related genes and monitor the spectrum of their dynamic expression during the entire mating process in D. punctatus. In this study, we generated transcriptome data from male and female adult D. punctatus specimens at four mating stages: eclosion, calling, copulation, and post-coitum. These data were analyzed using bioinformatics tools to identify the major olfactory-related gene families and determine their expression patterns during mating. Levels of odorant binding proteins (OBPs), chemosensory proteins (CSPs), and odorant receptors (ORs) were closely correlated with mating behavior. Comparison with ORs from other Dendrolimus and Lepidoptera species led to the discovery of a group of ORs specific to Dendrolimus. Furthermore, we identified several genes encoding OBPs and ORs that were upregulated after mating in females; these genes may mediate the location of host plants for oviposition via plant-emitted volatiles. This work will facilitate functional research into D. punctatus chemosensory genes, provide information about the relationship between chemosensory genes and important physiological activities, and promote research into the mechanisms underlying insect olfactory recognition.
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Tea grey geometrid (Ectropis grisescens), a devastating chewing pest in tea plantations throughout China, produces Type-II pheromone components. Little is known about the genes encoding proteins involved in the perception of Type-II sex pheromone components. To investigate the olfaction genes involved in E. grisescens sex pheromones and plant volatiles perception, we sequenced female and male antennae transcriptomes of E. grisescens. After assembly and annotation, we identified 153 candidate chemoreception genes in E. grisescens, including 40 odorant-binding proteins (OBPs), 30 chemosensory proteins (CSPs), 59 odorant receptors (ORs), and 24 ionotropic receptors (IRs). The results of phylogenetic, qPCR, and mRNA abundance analyses suggested that three candidate pheromone-binding proteins (EgriOBP2, 3, and 25), two candidate general odorant-binding proteins (EgriOBP1 and 29), six pheromone receptors (EgriOR24, 25, 28, 31, 37, and 44), and EgriCSP8 may be involved in the detection of Type-II sex pheromone components. Functional investigation by heterologous expression in Xenopus oocytes revealed that EgriOR31 was robustly tuned to the E. grisescens sex pheromone component (Z,Z,Z)-3,6,9-octadecatriene and weakly to the other sex pheromone component (Z,Z)-3,9-6,7-epoxyoctadecadiene. Our results represent a systematic functional analysis of the molecular mechanism of olfaction perception in E. grisescens with an emphasis on gene encoding proteins involved in perception of Type-II sex pheromones, and provide information that will be relevant to other Lepidoptera species.
Insects and arachnids display the most impressive diversity of mating and social behaviour among all animals. This book investigates sexual competition in these groups, and the variety of ways in which males and females pursue, persuade, manipulate, control and help one another, enabling us to gain a better understanding of how conflicts and confluences of interest evolve together. Each chapter provides a comprehensive review of mating systems in particular insect and arachnid groups, discusses intrinsic and extrinsic factors responsible for observed mating strategies, and suggests fruitful avenues for further research. The book culminates in a synthesis, reviewing the date in terms of the theory of sexual conflict. This broad-based book will be of immense value to students and researchers interested in reproductive strategies, behavioural ecology, entomology and arachnology.
In the last decade, new methods of estimating global species richness have been developed and existing ones improved through the use of more appropriate statistical tools and new data. Taking the mean of most of these new estimates indicates that globally there are approximately 1.5 million, 5.5 million, and 7 million species of beetles, insects, and terrestrial arthropods, respectively. Previous estimates of 30 million species or more based on the host specificity of insects to plants now seem extremely unlikely. With 1 million insect species named, this suggests that 80% remain to be discovered and that a greater focus should be placed on less-studied taxa such as many families of Coleoptera, Diptera, and Hymenoptera and on poorly sampled parts of the world. DNA tools have revealed many new species in taxonomically intractable groups, but unbiased studies of previously wellresearched insect faunas indicate that 1-2% of species may be truly cryptic. Expected final online publication date for the Annual Review of Entomology Volume 63 is January 7, 2018. Please see for revised estimates.