Journal Pre-proof

Identification and phylogenetics of Spodoptera frugiperda

chemosensory proteins based on antennal transcriptome data

Lin Qiu, Li He, Xiaoping Tan, Zhengbing Zhang, Yong Wang,

Xinwen Li, Hualiang He, Wenbing Ding, Youzhi Li

PII: S1744-117X(20)30027-7
DOI: https://doi.org/10.1016/j.cbd.2020.100680
Reference: CBD 100680

Comparative Biochemistry and Physiology - Part D: Genomics and

To appear in:
Proteomics

Received
9 November 2019
date:
Revised
28 February 2020
date:
Accepted
1 March 2020
date:

Please cite this article as: L. Qiu, L. He, X. Tan, et al., Identification and phylogenetics
of Spodoptera frugiperda chemosensory proteins based on antennal transcriptome data,
Comparative Biochemistry and Physiology - Part D: Genomics and Proteomics(2020),
https://doi.org/10.1016/j.cbd.2020.100680

This is a PDF file of an article that has undergone enhancements after acceptance, such
as the addition of a cover page and metadata, and formatting for readability, but it is
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© 2020 Published by Elsevier.

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Identification and phylogenetics of Spodoptera frugiperda

chemosensory proteins based on antennal transcriptome data

Lin Qiua , Li He a , Xiaoping Tanc, Zhe ngbing Zhangc, Yong Wangc, Xinwen Lic，

Hualiang He a , Wenbing Dinga, b and Youzhi Lia, b, *

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a
Hunan Provincial Key Laboratory for Biology and Control of Plant Diseases and

Insect Pests, College of Plant Protection, Hunan Agricultural University, Changsha

410128, China.
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b
Hunan Provincial Engineering & Technology Research Center for Biopesticide and
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Formulation Processing, Changsha 410128, China.

c
Plant Protection and Inspection Station, Agriculture and Rural Department of Hunan
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Province, Changsha 410005, China.

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* Correspondence to: Youzhi Li, Hunan Agricultural University, Changsha 410128,

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China. E-mail: liyouzhi@hunau.edu.cn

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Abstract
Understanding the interaction between the insect olfactory system and the

environment is crucial for fully explaining the molecular mechanisms underlying

insect behavior, and providing new strategies for integrated pest management.

Although there is good evidence that olfactory proteins play a vital role in mediating

insect behaviors, the olfactory mechanism of insects remains poorly understood. We

identified a total of 71 chemosensory genes; 25 odorant-binding proteins (OBPs), 27

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odorant receptors (ORs), 8 ionotropic receptors (IRs), 8 chemosensory proteins (CSPs)

and 3 sensory neuron membrane proteins (SNMPs), in the antennae of male and
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female fall armyworms, Spodoptera frugiperda, an invasive global pest that causes
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significant economic damage worldwide. We used differential gene expression (DGE)
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and fragments per kilobase per million fragments (FPKM) values to compare the

transcript levels of candidate chemosensory genes, and qRT-PCR to compare the

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expression levels of the OR gene, in male and female antennae. The expression of
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candidate OR genes in male and female antennae was consistent with the DGE data,

and the expression of the SfruCL4419.Contig1-All and SfruUnigene1070-All genes

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was sex-biased. These results not only provide new information on the olfactory

mechanism of S. frugiperda, and insects in general, but also suggests new gene targets

for pest control.

Keywords : Spodoptera frugiperda; Transcriptome; Chemosensory genes; Odorant

receptors; Transcript pattern

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Introduction
Olfactory and gustatory systems are essential for foraging, orientation, mate

recognition, oviposition and host-identification in insects (Leal, 2013; Vet and Dicke,

1992). Understanding insect olfaction is an important first step for research in the

chemical ecology and chemosensory biology of insects (Jaquiery et al., 2012; Lei and

Vickers, 2008). Diverse olfactory proteins, including odorant-binding proteins (OBPs),

odorant receptors (ORs), chemosensory proteins (CSPs), ionotropic receptors (IRs),

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sensory neuron membrane proteins (SNMPs) and odorant-degrading enzymes (ODEs),

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are responsible for capturing volatiles from the environment (Leal, 2013; Olivier et al.,

The mechanisms underlying the

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2011; Rutzler and Zwiebel, 2005; Touhara and Vosshall, 2009).

identification of chemical cues by

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Lepidopteran larvae have been well established (Tanaka et al., 2009; Zhang et al.,
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2015a). It is commonly accepted that hydrophobic odorant molecules first interact

with OBPs/CSPs which then cross the aqueous sensillum lymph to the sensory neurons.
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The odorants then activate the ORs/IRs expressed in olfactory sensory neurons where
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the chemical signals are converted into electrical signals (Leal, 2013; Touhara and
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Vosshall, 2009). In addition, SNMPs are also involved in the signal transduction
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pathway and ODEs in subsequent volatile clearance from the lymph (Leal, 2013;

Pelosi et al., 2006).

Insect olfactory proteins were first identified in Drosophila melanogaster and

later in other insects, including Bombyx mori, Aphis gossypii, Manduca sexta, Chilo

suppressalis, Helicoverpa armigera, Spodoptera littoralis and Sesamia inferens

(Andersson et al., 2014; Cao et al., 2014a; Cao et al., 2014b; Liu et al., 2012; Mitsuno et

al., 2008; Sakurai et al., 2011; Van et al., 2009; Zhang et al., 2013). OBPs are small,

water-soluble carrier proteins that are thought to reversibly bind odorant molecules

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such as host volatiles, and transport hydrophobic odorants to receptors through the

hemolymph (Leal, 2013). In the Lepidoptera, OBPs are usually classified into general

odorant-binding proteins (GOBPs), pheromone-binding proteins (PBPs) and antennal

binding proteins (ABP) (Krieger et al., 1996; Vogt and Riddiford, 1981; Vogt et al.,

1991a; Vogt et al., 1991b). Previous studies have found that most OBPs are mainly

expressed in the antennae (Glaser et al., 2013; Zhang et al., 2015b), indeed the

transcript profiles of ABPs are antenna-specific and have characteristics typical of

OBPs. However, this may not be universals as some species may have different

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physiological processes (Graham et al., 2003; Pelosi et al., 2006). By comparison,

CSPs are smaller than OBPs and may play a role in insect development in addition to
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general odorant and sex pheromone recognition (Maleszka et al., 2007).
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ORs and IRs, which are involved in the recognition of different volatiles, were
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first identified in D. melanogaster (Benton et al., 2009; Clyne et al., 1999, 2000).

Furthermore, ORs tetramerize with a conserved subunit and a universal co-receptor,

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referred to as Orco (Butterwick et al., 2018) which was first identified in D.

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melanogaster (DmelOR83b), and which together with OR form a receptor complex

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that functions as an odorant-gated ion channel within insects (Larsson et al., 2004).
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IRs are a new insect chemosensory family that belong to the ionotropic glutamate

receptor superfamily (iGluRs). They are further classified into two subfamilies;

antennal IRs and divergent IRs, which are tuned to food odor, acid and other odorants

(Benton et al., 2009; Croset et al., 2010; Liu et al., 2014). Finally, SNMPs are a smaller

family comprised of SNMP1 and SNMP2, which are thought to be involved in sex

pheromone and general odorant recognition (Pregitzer et al., 2014; Rogers et al., 2001;

Vogt et al., 2009).

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The fall armyworm, Spodoptera frugiperda, is a major agricultural pest native

to the Americas (Goergen et al., 2016; Otim et al., 2018) that has recently spread to

Africa and Asia, including India, Myanmar, Thailand, Sri Lanka and Bangladesh

(CABI, 2019; Mallapur et al., 2018). It has also spread rapidly in China (Zhang et al.,

2019). S. frugiperda attacks a large number of cultivated plant species causing major

economic losses throughout its range (Sena Jr et al., 2003). For example, it causes

losses of 400 million US dollars a year to the Brazilian corn industry (Figueiredo et al.,

2005; Lima et al., 2010; Sena Jr et al., 2003) and the cost of controlling it is more than

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600 million dollars per year (Sisay et al., 2018). S. frugiperda has two morphological

host-plant strains, the “corn strain” (C strain), which feeds mostly on maize and other
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large grasses, and the “rice strain” (R strain) that preferentially feeds on rice and
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various pasture grasses (Meagher et al., 2012; Nagoshi et al., 2004; Pashley et al.,
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1986).

Synthetic insecticides have been the main method of controlling S. frugiperda

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but environmental pollution from pesticide residues and the rapid evolution of
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resistance have now limited their use. Genetically modified (GM) maize that produces
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Bacillus thuringiensis (Bt) proteins, first commercialized in 1996, was initially

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resistant to S. frugiperda (James, 2017) but continuous planting has led to the rapid

evolution of resistance to Bt proteins. It has been reported that S. frugiperda is now

resistant to Cry proteins in the United States, Puerto Rico and Brazil (Farias et al.,

2014; Huang et al., 2014; Omoto et al., 2016; Storer et al., 2010). Therefore, it is now

necessary to find new, environmentally friendly and sustainable pest management

strategies to control this pest. Research on olfaction could help identify such

strategies.

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We used the transcriptome approach to identify candidate chemosensory genes in

the S. frugiperda antenna. 25 OBPs, 27 ORs, 8 IRs, 8 CSPs and 3 SNMPs were

identified and phylogenetic trees of the relationships between these and homologous

genes in other insects constructed which allowed us infer their function. Furthermore,

quantitative real-time RT-PCR (qPCR) was used to compare the expression profiles

of OR genes in male and female antennae. The results provide useful, new

information on the molecular mechanism of chemosensory genes, and suggest

potential avenues of research for developing sustainable pest management tools.

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Materials and methods

Insect rearing and sample preparation pr

S. frugiperda larvae were collected from Changsha City, Hunan province, China,
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in 2019 and transferred to a laboratory where they were kept at 25 ± 1°C, 70 ± 10%
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relative humidity, under a long photoperiod (12 light: 12 dark h). Male and female

antennae were obtained from 2-day-old adults, 90 pairs (30 pairs per sample) of adult
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antennae from each sex were used with three replicates for each group. All samples
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were immediately frozen in liquid nitrogen and stored at - 80 °C until used.

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cDNA library preparation and sequencing

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Dissected antennal tissue was homogenized with a micro-pestle and total RNA

from each sample individually extracted using TRIzol reagent (Invitrogen, Carlsbad,

CA, USA) according to the manufacturer’s instructions. The quantity of RNA

obtained was verified using a Qubit® RNA Assay Kit in Qubit® 2.0 Flurometer (Life

Technologies, CA, USA). 1.5 μg of male and female antennal RNA was used to

construct a cDNA (Complementary DNA) library for each sample (3 for each sex)

which were sequenced using Illumina HiSeq 2000 (Illumina, San Diego, CA, USA).

Paired-end reads were also obtained (BGI, Shenzhen, China).

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Assembly and functional annotation

Raw reads were pre-processed to remove adaptor sequences, ploy-N and

low-quality reads. Clean read assembly was then carried out with the assembling

program Trinity (Grabherr et al., 2011). The raw data have been uploaded to the

National Center for Biotechnology Information (NCBI), under the accession numbers,

SRR10271166 (S. frugiperda male-1), SRR10273598 (S. frugiperda male-2),

SRR10276370 (S. frugiperda male-3), SRR10276647 (S. frugiperda female-1),

SRR10276682 (S. frugiperda female-2) and SRR10276898 (S. frugiperda female-3).

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Unigenes were annotated by aligning them against non-redundant nucleotide (Nt) and

non-redundant protein (Nr) sequences in the NCBI database, and sequences in the
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KOG/COG (Clusters of Orthologous Groups of proteins), KO (KEGG Ortholog
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database) and GO (Gene Ontology), databases.
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Chemosensory gene identification and phylogenetic analysis

Candidate olfactory genes were verified by using BLASTX to manually check

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their sequences against those in a local non-redundant database with e-values <1e-5.
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The putative ORF (Open reading frame) of candidate olfactory genes was identified
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using the ORF finder tool (https://www.ncbi.nlm.nih.gov/orffinder/). The ClustalW

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method was used to align the amino acid sequences of olfactory genes (Larkin et al.,

2007). A phylogenetic tree was constructed in IQ-TREE using the best-fitting

substitution-model and maximum- likelihood method (Trifinopoulos et al., 2016).

Branch support was assessed with 1000 bootstrap replicates. The program FigTree

(Version 1.4.2) was used to color and arrange the phylogenetic tree. CSP sequences

were obtained from Spodoptera exigua, S. litura and H. armigera. The OR, IR, and

OBP sequences data sets contained sequences from S. exigua, S. litura and D.

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melanogaster. The SNMP data set contained SNMP sequences from Plutella

xylostella, S. exigua, C. suppessalis and D. melanogaster.

Differential gene expression

The expression levels of olfactory genes in male and female antennal tissues were

measured using the Fragments Per Kilobase of transcripts per Million mapped reads

(FPKM) method (Mortazavi et al., 2008). Differential expression of genes between

male and female antennae was measured using DESeq (version 1.18.0) based on

P-values (< 0.05) and fold-changes (|fold change| > 2) (Anders and Huber, 2010).

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Quantitative real-time PCR

cDNA was synthesized from 1 μg of total RNA using a PrimeScript RT reagent

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kit with gDNA eraser (perfect real time) (TaKaRa, Dalian, China) according to the
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manufacturer’s instructions. qRT-PCR primers were designed using the National
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Center for Biotechnology Information’s profile server (https://www.ncbi.nlm.

nih.gov/tools/primer-blast/) (Table S2). The S. frugiperda β-Actin and Rpl32 genes

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were used as housekeeping reference genes (Rodriguez-de et al., 2019; Visconti et al.,
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2019). qRT-PCR was performed with SYBR® Premix Ex Taq™ (TaKaRa, Dalian,
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China) according to the manufacturer’s protocols, and a CFX-96™ PCR Detection

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System (Bio-Rad, Hercules, California, USA) was used for PCR reactions. The qPCR

cycling parameters were as follows: 95°C for 30 s, 40 cycles at 95°C for 10 s, then

59°C for 30 s. Melting curve analysis was performed from 55 °C to 95 °C to determine

the specificity of qPCR primers. To determine the efficiency of the qPCR primers, a

5-fold dilution series of antennal tissues cDNA corresponding to one microgram total

RNA was produced a standard curve (cDNA concentration vs. Ct). Primer efficiencies

were then calculated according to the equation: E =(10 [−1/slope] −1)*100 (Pfaffl, 2001;

Radonic et al., 2004). The expression profiles of OR genes were calculated using the

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2−ΔΔCt method (Pfaffl et al., 2001). The statistical significance of differences between

group means was assessed using a one-way ANOVA implemented in the SPSS

program (SPSS, Chicago, IL, USA).

Results

De novo transcriptome assembly

An Illumina HiSeq 2000 platform combined with a Trinity assembly were used

to sequence S. frugiperda antennal transcriptomes. We obtained 49.08 million

(Male-1), 45.57 million (Male-2) and 45.57 million (Male-3) raw-reads from male

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antennae, and 45.57 million (female-1), 47.33 million (female-2) and 47.33 million

(female-3) from female antennae (Additional file 1: Table S1). After filtering, 45.88
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million (male-1), 42.43 million (male-2) and 42.59 million (male-3) clean-reads were
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obtained from male antenna, and 42.18 million (female-1), 44.07 million (female-2)
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and 44.36 million (female-3), from female antennae (Additional file 1: Table S1).

Pooled female and male data were assembled into 74,269 unigenes with an average
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length of 1,412 and an N50 length of 2,691.

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Homology and gene ontology (GO) annotation

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A total of 38,238 (51.49%) S. frugiperda unigenes were found to be similar to

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known proteins in the NCBI non-redundant (nr) protein database when using a cut-off

E-value value of 1e-5 . The highest homology was with S. litura (67.25%) followed by

H. armigera (6.92%), Heliothis virescens (3.51%) and Trichoplusia ni (3.44%) (Fig.

S1a).

Gene ontology (GO) annotations were used to categorize genes into functional

groups. Most genes were classified into the “Biological Process”, “cellular component”

and “molecular function” groups (Fig. S1b).

Analysis of odorant receptor genes

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We identified 27 OR genes coding candidate OR proteins from S. frugiperda

male and female antennae. The phylogenetic tree of these OR genes and those from S.

exigua, S. litura and D. melanogaster shows that most S. frugiperda OR proteins

cluster with those from S. litura. BLASTx indicated that SfruCL3673.Contig1-All and

SfruCL2815.Contig1-All were best matches to S. litura OR15 and OR8, respectively

(Fig 1). Similarly, SfruUnigene13107-All, SfruUnigene5236-All,

SfruUnigene16479-All and SfruCL1456.Contig4-All clustered with S. litura OR29,

and BLASTx indicated that SfruUnigene11529-All and SfruUnigene23117-All best

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matched S. litura OR27. Some S. frugiperda OR proteins clustered with S. exigua

ORs, and SfruUnigene16373-All and SfruCL28.Contig2-All best matched SexiOR4

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and SexiOR10, respectively. The S. frugiperda co-receptor, Orco best matched
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DmelOrco (Dmel83b) from D. melanogaster and SlitOR2 from S. litura. Despite the
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fact that SfruCL4419.Contig1-All best matched DmelOR56a, few S. frugiperda OR

proteins clustered with D. melanogaster OR proteins (Fig. 1). Some genes were
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relatively dissimilar to those from other species; for example SfruCL4266.Contig2-All

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and SfruCL1747.Contig1-All clustered on separate branches of the phylogenetic tree,

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(Fig. 1).
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Identification of odorant binding proteins

We obtained 25 OBP transcripts from S. frugiperda male and female antennae. A

phylogenetic tree of S. frugiperda, S. exigua, S. litura and D. melanogaster OBP

amino acid sequences indicates that most S. frugiperda OBPs are most closely related

to those of S. exigua; for example SfruCL835.Contig4-All, SfruCL4266.Contig15-All

and S. exigua OBP16 are all relatively highly conserved (Fig. 2).

SfruUnigene20826-All and SfruCL6779.Contig2-All best matched S. exigua OBP8

(Fig. 2). SfruUnigene8932-All, SfruUnigene16653-All and SfruCL6337.Contig3-All

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clustered with SlitOBP19, and SfruCL3156.Contig2-All best matched SlitOBP8 (Fig.

2). With the exception of classic OBPs, including 2 GOBPs (GOBP1-2), PBPs and

ABPs in the OBP superfamily, as expected, GOBPs and PBPs from S. exigua and S.

litura clustered on the same branch (Fig. 2). SfruCL5477.Contig2-All best matched

SlitGOBP1 and these two genes cluster on the same a branch (Fig. 2).

SfruCL1778.Contig5-All SlitPBP2 and SexiPBP2 appear highly conserved (Fig. 2).

Analysis of ionotropic receptor genes

We identified 8 IRs from S. frugiperda male and female antennae. A

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phylogenetic tree of the relationship between these and orthologous sequences from S.

exigua, S. litura and D. melanogaster shows that, except for SfruCL1795.Contig5-All

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and SfruUnigene7068-All, all S. frugiperda IRs are closely related to S. litura IR
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proteins (Fig. 3). For example, SfruCL809.Contig3-All and SfruCL2942.Contig3-All
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cluster with SlitIR41a and SlitIR21a, respectively (Fig. 3).

Identification of chemosensory proteins

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We obtained 8 CSP proteins from S. frugiperda male and female antennae from
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bioinformatic analysis. A phylogenetic tree of the relationship between these and CSP
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proteins from S. exigua, S. litura, H. armigera shows that four SfruCSPs closely
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match S. litura CSPs and the remainder cluster with S. exigua CSPs (Fig. 4).

Specifically, SfruCL7252.Contig3-All, SfruCL2908.Contig2-All and SlitCSP1 appear

conserved, which suggests that these three genes may play a similar role in olfaction.

Analysis of sensory neuron membrane proteins

We identified 3 SNMPs in S. frugiperda antennae; SfruCL1720.Contig10-All

and SfruUnigene24219-All are similar to SexiSNMP2 and SexiSNMP3, respectively

(Fig. 5), whereas SfruUnigene7573-All was placed on a separate branch of the

phylogenetic tree (Fig. 5).

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Differentially expressed genes (DEGs)

Fragments per kilobase per million fragments (FPKM) values were used to

compare the expression levels of chemosensory genes in male and female antennae.

With respect to OBP genes, SfruCL6520.Contig11-All, SfruCL5477.Contig2-All and

SfruCL49.Contig2-All were more highly expressed in female than male antennae.

There was no significant difference in the expression of the remaining OBP genes

between male and female antennae (Fig 6a). With respect to OR genes,

SfruUnigene1070-All was more highly expressed in male antennae, whereas

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SfruCL4419.Contig1-All was more abundant in female antennae, in addition, the

SfruOrco co-receptor gene had slightly lower expression in female antennae (Fig. 6b).
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As was the case for OBP genes, most OR genes had similar expression profiles in
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males and females.
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With respect to CSP genes, 4 genes (SfruCL2778.Contig.1-All,

SfruCL7252.Contig.3-All, SfruCL2908.Contig.2-All and SfruUnigene587-All) were

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more highly expressed in female antennae (Fig. 6c) and two,

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SfruCL5794.Contig.2-All and SfruCL5479.Contig.2-All, were more highly expressed

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in male antennae (Fig. 6c). The IR genes SfruUnigene7068-All and

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SfruCL1795.Contig4-All were more highly expressed in male than female antennae

(Fig. 6d). In addition, SfruUnigene7573-All and SfruUnigene24219-All were also

more highly expressed in female antennae (Fig. 6e).

Expression of odorant receptor genes in male and female antennae

qRT-PCR was used to analyze the expression of candidate odorant receptor

genes in S. frugiperda male and female antennae. Expression of

SfruCL4419.Contig1-All was significantly higher in female antennae, whereas that of

SfruUnigene1070-All was markedly lower (Fig. 7). With the exception of

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SfruCL5893.Contig3-All which was more highly expressed in female antennae, these

results are consistent with our DGE results on OR genes based on FPKM values. With

the exception of the above OR genes, there were no significant differences in OR gene

expression in male and female antennae. Consistent with the results of our DGE

analysis the SfruOrco co-receptor gene showed slightly low abundance in female

antennae (Fig. 7).

Discussion

S. frugiperda, is a polyphagous insect pest native to the Americas that has

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recently become an invasive species in Africa and Asia (Mallapur et al., 2018; Otim et

al., 2018; Zhang et al., 2019). Although information on the sex pheromones of S.
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frugiperda has been published, little is known about its olfactory mechanism
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(Andradea et al., 2000; Groot et al., 2008). In the Lepidoptera, many olfactory genes
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play a central role in the modulation of behavior. Therefore, to fully characterize the

chemosensory repertoire of S. frugiperda, we first identified chemosensory proteins

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from antennal transcriptomes and constructed phylogenetic trees of the relationships

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between these candidate chemosensory proteins and orthologous genes from other
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insect species. We then used qRT-PCR to measure the expression of OR genes in

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male and female antennae. We identified a total of 71 chemosensory genes in S.

frugiperda antennae, including 25 OBPs, 27 ORs, 8 IRs, 8 CSPs and 3 SNMPs; fewer

than those found in B. mori (Tanaka et al., 2009; Wanner and Robertson, 2008). The

relative paucity of chemosensory genes in S. frugiperda was discovered in previous

study which identified 50 OBPs, 22 CSPs, 69 ORs and 42 IRs in the entire S.

frugiperda genome (Gouin et al., 2017). However, we found similar numbers of

candidate chemosensory genes in S. frugiperda as have been found in S. litura (Feng

et al., 2015), H. armigera (Zhang et al., 2015a) and O. furnacalis (Yang et al., 2015;

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Zhang et al., 2015b). Differences in the numbers of genes found in different studies

may be due to different methods of analysis.

Insect OBPs are known to act as the first gate in odorant recognition, carrying

odorants, including plant volatiles and pheromones, through the hemolymph to

olfactory receptor neurons (ORNs) (Leal, 2013). We identified 25 OBPs in the

antennae of male and female S. frugiperda, phylogenetic analysis showed that most S.

frugiperda OBPs cluster with S. exigua OBP proteins, whereas partial OBPs match

those of S. litura, although the degree of conservation is not as high as that with D.

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melanogaster. This suggests physiological and evolutionary differences among the

OBPs of different species. The SfruCL6520.Contig11_All gene was more highly

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expressed in female antennae but additional research is required to determine its
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function. CSPs are regarded as a subfamily of OBPs and it has been demonstrated that
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both OBPs and CSPs are specifically expressed in the antennae of Cerapachys biroi

(McKenzie et al., 2014). Compared with OBPs, we only identified 8 CSPs in S.

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frugiperda, fewer than those identified in other insects such as H. armigera and
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Heliothis assulta (Wanner et al, 2004; Zhang et al., 2015a). In addition, there is no
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evidence that CSP genes are differentially expressed in olfactory organs, which
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suggests that their function is not restricted to olfaction (Gong et al., 2012;

Kitabayashi et al., 1998; Zhang et al., 2013).

In contrast to OBPs, ORs are the best characterized group of chemoreceptors in

insects. ORs are localized in the dendritic membranes of ORNs where they transduce

olfactory signals. A conserved odorant receptor co-receptor (Orco) is necessary for

ORs to localize and function correctly. We detected 27 ORs in male and female

antennae of S. frugiperda. SfruCL6333.Contig4-All clustered with the Orco protein

from D. melanogaster, and there is evidence to show that downregulation of Orco

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can reduce the EAG response and alter preferences for host volatiles and

pheromones (DeGennaro et al., 2013; Fan et al., 2015; Lin et al., 2015; Zhang et al.,

2016; Zheng et al., 2012; Zhou et al., 2014). In our DGE analysis, the

SfruUnigene1070-All was highly expressed in male antennae whereas

SfruCL4419-Contig1-All was more highly expressed in female antennae. These

qRT-PCR results were consist with our DGE analysis of OR transcripts based on

FPKM values. These two proteins are likely to be sex- biased and involved in mating

or oviposition (Liu et al., 2012; Zhang et al., 2014; Zhang et al., 2015b). The other

f
oo
ORs were equally expressed in both sexes, which suggests that they don’t have a

sex- biased function. Further functional characterization of these OR proteins would

pr
improve our understanding of the S. frugiperda olfactory mechanism.
e-
IRs are thought to play a key role in the synaptic ligand gated ion channels
Pr

involved in the detection of host odors. We identified 8 contigs encoding putative S.

frugiperda IRs and phylogenetic analysis indicates that SfruCL1705-Contig2-All

al

matched SlitIR25a, which has broad expression in S. littoralis (Olivier et al., 2011),
rn

similar to that of IR25a, the transcripts of which are highly abundant in the antennae
u

and/or maxillary palps of mosquitoes (Bohbot et al., 2014; Matthews et al., 2016;
Jo

Pitts et al., 2011). IRs that are mainly expressed in antennae have been investigated

in other insect species (Benton et al., 2009; Liu et al., 2014; Rytz et al., 2013). Some

D. melanogaster and S. littoralis IRs are, however, expressed in the proboscis and

mouthparts, which suggests that they play a role in gustatory recognition in these

species (Croset et al., 2010; Olivier et al., 2011). Finally, we identified 3 contigs

coding SNMPs in male and female antennae. in the Lepidoptera, SNMPs are

comprised of two conserved groups; SNMP1 and SNMP2, which are involved in

pheromone detection (Rogers et al., 2001; Vogt et al., 2009). We found a new class

15
 Journal Pre-proof

of candidate SNMP, and a phylogenetic tree indicates that S. frugiperda SNMPs and

SexiSNMPs are conserved. Additional research is required to reveal the olfactory

function of SfruSNMPs.

In conclusion, we identified a total of 71 chemosensory genes including, 25

OBPs, 27 ORs, 8 IRs, 8 CSPs and 3 SNMPs from transcriptomes of male and

female S. frugiperda antennae, many of which were differentially expressed in

males and females. These findings not only provide new insights into the molecular

basis of olfactory genes in S. frugiperda and other insects, but also facilitate the

f
oo
development of new tools for controlling S. frugiperda and other insect pests.

Acknowledgments pr
This work was supported by Natural Science Foundation of Hunan Province of China
e-
(2018JJ2171) and Double first-class construction project of Hunan Agricultural
University (SYL2019029).
Pr

Competing interests
al

The authors have declared that no competing interest exists.

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Figure legends
Figure 1: Maximum-likelihood phylogenetic tree of relationships between

putative Spodoptera frugiperda ORs and those of Spodoptera exigua, Spodoptera

litura and Drosophila melanogaster. Tree was constructed using IQ-TREE, branch

support was assessed with 1000 bootstrap replicates. Sfru = Spodoptera frugiperda

(highlighted in pink), Sexi = Spodoptera exigua (highlighted in blue), Slit =

Spodoptera litura (highlighted in dark) and Dmel = Drosophila melanogaster

f
(highlighted in green).

oo
pr
Figure 2: Maximum-likelihood phylogenetic tree of relationships between

putative Spodoptera frugiperda OBPs and those of Spodoptera exigua, Spodoptera

e-
litura and Drosophila melanogaster. Tree was constructed using IQ-TREE, branch
Pr

support was assessed with 1000 bootstrap replicates. Sfru = Spodoptera frugiperda

(highlighted in pink), Sexi = Spodoptera exigua (highlighted in blue), Slit =

al

Spodoptera litura (highlighted in dark) and Dmel = Drosophila melanogaster

rn

(highlighted in green).
u
Jo

Figure 3: Maximum-likelihood phylogenetic tree of relationships between

putative Spodoptera frugiperda IRs and those of Spodoptera exigua, Spodoptera

litura and Drosophila melanogaster. Tree was constructed using IQ-TREE, branch

support was assessed with 1000 bootstrap replicates. Sfru = Spodoptera frugiperda

(highlighted in pink), Sexi = Spodoptera exigua (highlighted in blue), Slit =

Spodoptera litura (highlighted in dark) and Dmel = Drosophila melanogaster

(highlighted in green).

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Figure 4: Maximum-likelihood phylogenetic tree of relationships between

putative Spodoptera frugiperda CSPs and those of Spodoptera exigua, Spodoptera

litura and Drosophila melanogaster. Tree was constructed using IQ-TREE, branch

support was assessed with 1000 bootstrap replicates. Sfru = Spodoptera frugiperda

(highlighted in pink), Sexi = Spodoptera exigua (highlighted in red), Slit =

Spodoptera litura (highlighted in blue) and Harm = Helicoverpa armigera

(highlighted in green).

f
oo
Figure 5: Maximum-likelihood phylogenetic tree of relationships between

putative Spodoptera frugiperda SNMPs and those of Spodoptera exigua,

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Spodoptera litura and Drosophila melanogaster. Tree was constructed using
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IQ-TREE, branch support was assessed with 1000 bootstrap replicates. Sfru =
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Spodoptera frugiperda (highlighted in red), Sexi = Spodoptera exigua (highlighted in

blue), Csup = Chilo suppressalis (highlighted in dark), Pxyl = Plutella xylostella

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(highlighted in green) and Dmel = Drosophila melanogaster (highlighted in pink).

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Figure 6: Differential expression of Spodoptera frugiperda chemosensory genes

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(OBPs, ORs, CSPs, IRs and SNMPs) in male and female antennae. Red bands

indicate high gene expression levels, and blue bands low gene expression levels.

Figure 7: Transcript profiles of Spodoptera frugiperda chemosensory genes from

male and fe male antennae as confirme d by Quantitative Real-time PCR

(qRT-PCR). Relative amounts of chemosensory genes were normalized to the

expression of Rpl32 and β -Actin. Bars with asterisks indicate P- values < 0.05

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(ANOVA).

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Graphical abstract

Highlights
1: The total of seventy-one Spodoptera frugiperda chemosensory genes were
identified from male and female antennal tissue.

2: The first analysis of expression levels of candidate S. frugiperda chemosensory

genes in male and female antenna based on FPKM values.

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3: The Contig gene SfruCL4419.Contig1-All (OR) was highly expressed in female
antennae whereas the SfruUnigene1070-All (OR) was highly expressed in male
antenna.
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