Mol Ecol Resour. 2020;00:1–19. wileyonlinelibrary.com/journal/men
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1© 2020 John Wiley & Sons Ltd
Received: 12 December 2019
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Revised: 24 April 2020
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Accepted: 27 April 2020
DOI: 10.1111/1755-0998.13182
RESOURCE ARTICLE
The genetic adaptations of fall armyworm Spodoptera
frugiperda facilitated its rapid global dispersal and invasion
Huamei Xiao1,2 | Xinhai Ye2 | Hongxing Xu3 | Yang Mei2 | Yi Yang2 | Xi Chen2 |
Yaj u n Ya n g3 | Tao Liu4 | Yongyi Yu4 | Weifei Yang4 | Zhongxian Lu3 | Fei Li2
Huamei X iao, Xinhai Ye, Hongxing Xu and Yang Mei contrib uted equally to t his work.
1Key Laboratory of Crop Growth and
Development Regulation of Jiangxi Province,
College of Life Sciences and Resource
Environment, Yichun University, Yichun,
China
2State Key Laboratory of Rice Biology &
Ministry of Agricultural and Rural Affairs
Key Laboratory of Molecular Biology of
Crop Pathogens and Insects, Institute
of Insect Sciences, Zhejiang Universit y,
Hangzhou, China
3Institute of Plant Protection and
Microbiology, Zhejiang Academy of
Agricultural Sciences, Hangzhou, China
4Annoroad Gene Technology (Beijing) Co
Ltd, Beijing, China
Correspondence
Huamei Xiao, Key Laboratory of Crop
Growth and Development Regulation of
Jiangxi Province, College of Life Sciences
and Resource Environment , Yichun
University, Yichun, China.
Email: xiaohuamei625@163.com
Tao Liu, Annoroad Gene Technology
(Beijing) Co., Ltd, Beijing, China.
Email: taoliu@genome.cn
Fei Li, State Key Laborator y of Rice Biology
& Ministry of Agricultural and Rural Affairs
Key Laboratory of Molecular Biology of
Crop Pathogens and Insects, Institute
of Insect Sciences, Zhejiang Universit y,
Hangzhou, China.
Email: lifei18@zju.edu.cn
Funding information
Key Project of Zhejiang Provincial Natural
Science Foundation, Grant/Award Number:
LZ18C060001; the National Science
Foundation of Chinathe National Science
Foundation of China, Grant/Award Number:
31972354 and 31760514
Abstract
The fall armyworm (Spodoptera frugiperda) is a lepidopteran insect pest that causes
huge economic losses. This notorious insect pest has rapidly spread over the world in
the past few years. However, the mechanisms of rapid dispersal are not well under-
stood. Here, we report a chromosome-level assembled genome of the fall armyworm,
named the ZJ-version, using PacBio and Hi-C technology. The sequenced individual
was a female collected from the Zhejiang province of China and had high heterozy-
gosity. The assembled genome size of ZJ-version was 486 Mb, containing 361 contigs
with an N50 of 1.13 Mb. Hi-C scaffolding further assembled the genome into 31
chromosomes and a portion of W chromosome, representing 97.4% of all contigs and
resulted in a chromosome-level genome with scaffold N50 of 16.3 Mb. The sex chro-
mosomes were identified by genome resequencing of a single male pupa and a single
female pupa. About 28% of the genome was annotated as repeat sequences, and
22,623 protein-coding genes were identified. Comparative genomics revealed the
expansion of the detoxification-associated gene families, chemoreception-associated
gene families, nutrition metabolism and transport system gene families in the fall ar-
myworm. Transcriptomic and phylogenetic analyses focused on these gene families
revealed the potential roles of the genes in polyphagia and invasion of fall army-
worm. The high-quality of the fall armyworm genome provides an important genomic
resource for further explorations of the mechanisms of polyphagia and insecticide
resistance, as well as for pest management of fall armyworm.
KEYWORDS
chromosome-level genome, comparative genomics, fall armyworm, insecticide resistance,
polyphagia
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1 | INTRODUCTION
The fall armyworm, Spodoptera frugiperda (Lepidoptera, Noctuidae),
is a highly invasive noctuid insect pest that causes huge agricultural
production and economic losses (Martinelli, Barata, Zucchi, Silva-
Filho Mde, & Omoto, 2006). According to the report of Centre for
Agriculture and Biosciences International, the fall armyworm has
the potential to cause maize losses of approximately 8.3–20.6 mil-
lion tons per year in Africa (Abrahams et al., 2017). Several features
of fall armyworm biology contribute to the outbreak of population
growth in newly established regions. These include high fecundity,
long adult life span and high spawning rate, with an adult female
estimated to produce an average of 1,500 eggs over her typically
10 days lifespan (P ras ann a, Hu esing, Edd y, & Pes chke, 2018). In addi-
tion, due to a strong ability to fly, the fall armyworm has the capacity
to migrate long distances with air currents, e.g., at least 500 km per
generation across Africa (Westbrook, Nagoshi, Meagher, Fleischer,
& Jairam, 2016). With a suitable air current, moths have report-
edly dispersed to a record distance of 1,600 km in 30 hr (Martinelli
et al., 2006).
The fall armyworm survives year-round in the tropical and
subtropical areas of the Americas and was also widespread in the
northern areas of the United States and as far north as southern
Canada (Todd & Poole, 1980) before 2016. In January 2016, an out-
break of this pest occurred in the rainforest zone of South-Western
Nigeria of Africa (Goergen, Kumar, Sankung, Togola, & Tamo, 2016).
A total of 44 countries had officially reported the invasion of this
pest by 2018 (Feldmann, Rieckmann, & Winter, 2019). In May
2018, Sharanabasappa et al. (2018) reported invasions of fall ar-
myworm in various districts of Karnataka state in India for the first
time. Subsequent invasions were reported in Yemen, Myanmar,
Thailand, Bangladesh, Sri Lanka, and other Asian countries (Ganiger
et al., 2018; Li et al., 2019).
By January 2019, the presence of fall army worm was confirmed
in southwest Yunnan province of China (China National Agricultural
Technology Extension and Service Center,NATESC, 2019a; Wu,
Jiang, & Wu, 2019). Due to fast dispersal rates and available suitable
habitats (central, southwest and northern regions in China), by 17
August 2019, the fall armyworm had spread to 1,366 counties (cities
and districts) across 24 provinces of China (NATESC, 2019b). Such a
rapid spread of fall army worm poses a serious threat to maize and
wheat production in China.
The polyphagous fall armyworm feeds on more than 350 plants
in such families as Poaceae, Asteraceae and Fabaceae (Montezano
et al., 2018). For many years, the fall armyworm has been known to
have two haplotypes, the “rice strain” (R strain) preferring to feed
on rice and grasses and “corn strain” (C strain) preferring to feed on
corn and sorghum (Pashley, 1988; Pashley, Johnson, & Sparks, 1985).
The two strains are morphologically identical, but they significantly
differ in composition of sex pheromones, susceptibility to chem-
ical insecticides and transgenic Bacillus thuringiensis (Bt) crops,
and reproductive behaviours (Adamczyk, Holloway, Leonard, &
Graves, 2013; Cruz-Esteban, Rojas, Sanchez-Guillen, Cruz-Lopez, &
Malo, 2018; Lima & McNeil, 2009; Schofl, Heckel, & Groot, 2009). A
molecular method used to distinguish the two strains is based on the
sequence of mitochondrial Cytochrome Oxidase Subunit I (COI) and
strain-specific sites in the fourth exon of the Z-chromosome-linked
gene Triosephosphate isomerase (Tp i ) (Juarez et al., 2014; Nagoshi,
Goergen, Du Plessis, van den Berg, & Meagher, 2019; Nagoshi
et al., 2017).
Application of chemical insecticides and planting transgenic
Bt corn have been the main strategies used to control this pest
(Carvalho, Omoto, Field, Williamson, & Bass, 2013; Yu, Nguyen, &
Abo-Elghar, 2003). Unfortunately, the widespread and indiscrimi-
nate use of insecticides and transgenic Bt corn has led to the de-
velopment of high levels of resistance to these control methods.
In the Americas, the fall armyworm has developed resistance to at
least 29 insecticidal active ingredients in six mode-of-action groups
(Mota-Sanchez & Wise, 2017). In Puerto Rico and Mexico, the fall
armyworm has developed field-evolved resistance to chlorpyri-
phos, permethrin, flubendiamide, chlorantraniliprole, methomyl,
and thiodicarb (Gutierrez-Moreno et al., 2019). Furthermore, the fall
armyworm has developed resistance to different Bt proteins, such
as Cry1F, Cry1Ac and Cry1Ab in Puerto Rico (Storer et al., 2010),
Cry1A.105 and Cry1F in the United States (Jakka et al., 2016), Cry1F
and Cry1Ab in Brazil (Omoto et al., 2016) and Cry1F Argentina
(Chandrasena et al., 2018).
In developing new pest control strategies, it is necessary to
understand the genetic information of the fall armyworm. Though
several versions of the fall armyworm genome have been reported
before (Gouin et al., 2017; Kakumani, Malhotra, Mukherjee, &
Bhatnagar, 2014; Nandakumar, Ma, & Khan, 2017), these versions
of genome assemblies are of low quality with a scaffold N50 of
<700 kb. Recently, three new versions of chromosome-level genome
assemblies have been reported in the pre-print journal bioRxiv, but
the sequences are not fully released to date (Liu, Lan, et al., 2019;
Nam et al., 2019; Zhang et al., 2019). In addition, all of these ver-
sions of chromosome-level genomes lack the information of the W
chromosome.
To uncover the genetic background of the fall armyworm found
in Zhejiang Province of China, we sequenced and assembled a chro-
mo s ome-l evel ge nome of a fe male pu pa co llecte d fro m a corn fie ld in
Zhejiang Province. Henceforth, we refer to this genome assembly as
the ZJ-version. The scaffold N50 of this genome is ~16 Mb, making
it a high quality and potentially the best quality fall armyworm ge-
nome available to date. Furthermore, we assembled a portion of the
W chro mos ome . To the best of our kn owledge, this is the first repo r t
of W chromosome sequence from the fall army worm. We also anal-
ysed the haplotype of the fall armyworm invading Zhejiang Province
and the expanded gene families associated with invasiveness, such
as cytochrome P450, gustatory receptor and β-fructofuranosidase.
This high-quality fall armyworm genome and the comparative ge-
nomic analysis provide new insights into the mechanism of fall ar-
myworm invasion, polyphagia and insecticide resistance.
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2 | MATERIALS AND METHODS
2.1 | Insects
Fall armyworms were collected in Dongyang (29.27°N, 120.23°E,
Zhejiang province, China), in June 2019 and reared on artificial diets
under laboratory conditions of 25°C, 16:8 light/dark photoperiod
and relative humidity of 70%–80%.
2.2 | Genome sequencing and de novo assembly
We applied the PacBio SMRT platform (Pacific Biosciences) to se-
quence the genome of the fall armyworm. High-quality genomic DNA
was extracted from a female pupa (Table S1) using a DNeasy Blood &
Tissue Kit (QIAGEN). The integrity of the DNA was determined with
the Agilent 4,200 Bioanalyser (Agilent Technologies). Genomic DNA
was sheared using g-Tubes (Covaris), and concentrated with AMPure
PB magnetic beads (Pacific Biosciences). The SMRT bell library was
constructed using the PacBio SMRTbell Express Template Prep Kit
2.0 (Pacific Biosciences). Finally, one SMRT cell was run for genome
sequencing. PacBio subreads were initially cleaned using canu v1.8
(Koren et al., 2017) (https://github.com/marbl /canu) for sequence
error correction. The PacBio corrected reads were then assembled
by SMARTdenovo (https://github.com/ruanj ue/smart denovo) as
described by Istace et al. (2017). The redundans pipeline (https://
github.com/lprys zcz/redun dans) (Pryszcz & Gabaldón, 2016) was
used to remove the redundant contigs from the initial de novo as-
sembly genome with the parameters “--identity 0.5, --overlap 0.75”,
yielding the final assembled genome of the fall armyworm that we
named the ZJ-version.
2.3 | Hi-C library preparation
A sixth instar larva was used for Hi-C library preparation (Table S1).
The Hi-C library preparation was performed following the protocol
published by Shi et al. (2019). After washing diced larval tissue in
cooled phosphate buffered saline, crosslinking was performed by
incubation at room temperature in a 2% formaldehyde solution for
10 min. The reaction was quenched by 5 min incubation with 2.5 M
glycine solution.
For extracting the chromatin, the supernatant was removed and
the tissues were grounded in liquid nitrogen. The tissues were re-
suspended in 25 ml of extraction buffer I (0.4 M sucrose, 10 mM
Tris HCl, pH 8.0, 10 mM MgCl2, 5 mM β-mercaptoethanol, 0.1 mM
phenylmethylsulfonyl fluoride [PMSF], and 1 μl protease inhibitor,
Sigma) and then was filtered through miracloth (Calbiochem). The
filtrate was centrifuged at 4,000 rpm at 4°C for 20 min. Next, the
supernatant was removed and the pellet was resuspended in 1 ml
extraction II buffer (0.25 M sucrose, 10 mM Tris HCl, pH 8.0, 10 mM
MgCl2, 1% Triton X-100, 5 mM β-mercaptoethanol, 0.1 mM PMSF,
and 1 μl protease inhibitor, Sigma), followed by centrifuging at
18,400 g at 4°C for 10 min. Again, the supernatant was removed
and the pellet was resuspended in 300 μl extraction buffer III (1.7 M
sucrose, 10 mM Tris HCl, pH 8.0, 0.15% Triton X-100, 2 mM MgCl2,
5 mM β-mercaptoethanol, 0.1 mM PMSF, and 1 μl protease inhibitor,
Sigma). The solution was centrifuged at 18,400 g at 4°C for 10 min.
The chromatin was washed with the following steps. First, re-
moving the supernatant and resuspending the pellet in 500 μl
precooling 1× CutSmart buffer, washing it for twice, and then cen-
trifuged at 2,500 g for 5 min. Second, the nuclei were washed with
0.5 ml restriction enzyme buffer and transferred to a safe lock tube.
Next, the chromatin was solubilized with dilute SDS by incubating
at 65°C for 10 min. After quenching the SDS by Triton X-100, the
chromatin was digested with 400 units MboI at 37°C on a rocking
platform overnight.
Next, the DNA was labelled with biotin-14-dCTP (Invitrogen)
and blunt-end ligated with crosslinked fragments. Then, the prox-
imal chromatin DNA was religated by ligation enzyme at 16°C for
overnight. The nuclear complexes were reversed crosslinked by in-
cubating with proteinase K (Invitrogen) at 65°C. DNA was purified
with phenol chloroform extraction method. Biotin-C was removed
from nonligated fragment ends using T4 DNA polymerase (NEB).
Fragments was sheared to a size of 100–500 bp by sonication. The
fragment ends were repaired by the mixture of T4 DNA polymerase
(NEB), T4 polynucleotide kinase (NEB) and Klenow DNA polymerase
(NEB). Biotin labelled Hi-C sample were specifically enriched using
streptavidin magnetic beads. The fragment ends were subjected to
A-tailing by exo-Klenow and Illumina paired-end sequencing adapter
were added by ligation. At last, the Hi-C libraries were amplified by
10–12 cycles of PCR amplification and sequenced using Illumina
HiSeq platform with 2 × 150 bp reads. Hi-C library preparation and
sequencing was performed by Annoraod Gene Technology Co. Ltd.
2.4 | Scaffolding with Hi-C
Hi-C scaffolding was performed according to the pipeline reported
in Servant et al. (2015) and Burton et al. (2013). The hi c-pro v2 .7. 8
(Servant et al., 2015) pipeline (https://github.com/nserv ant/HiC-
Pr o) wa s used to id enti f y valid re ad pai r s. In thi s pipel ine, ea ch read
in the pair is mapped independently and, where ligation sites are
detected by exact matching, the 3′ sequence is trimmed from the
read and the 5′ portion remapped. The sequence alignments were
made using bow tie 2 v2.2.3 (Langmead & Salzberg, 2012) with the
parameters “--very-sensitive -L 20 --score-min L,-0.6,-0.2 --end-
to - end --re ord er --r g-id BMG --ph red 33- q u als -p 5”. The proce sse d
mappings were then merged into a single alignment file with valid
interaction pairs expected to involve two different restriction
fragments. Then the valid interaction pairs were used to build the
interaction matrices and we scaled up the primary genome assem-
bly contigs into chromosome-scale scaffolds (hereafter pseudo-
chromosomes) with LACHESIS (https://github.com/shend urela
b/LACHESIS) (Burton et al., 2013). To access the accuracy of the
scaled-up genome assembly, we cut the pseudo-chromosomes
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XIAO et Al.
predicted by LACHESIS into bins with 100 kb lengths. Then we
constructed a heatmap based on the interaction signals that were
revealed by valid mapped read pairs between bins. The matrix was
produ ced by HiC-Pro and th en visua lized as a he atm ap to sh ow the
diagonal patches of strong linkages.
2.5 | Transcriptome sequencing and analysis
The transcriptomes of the larva (from first instar to six instar), fe-
male pupa, female adult and male adult of fall armyworm (Table S1)
were sequenced using the Illumina HiSeq 2000 platform with
paired-end libraries. Three biological replicates were obtained for
each RNA-Seq sample type. Low-quality bases in the RNA-Seq raw
reads were first filtered using trimmomatic v0.38 (Bolger, Lohse, &
Usadel, 2014). The clean reads were then mapped to the genome
assembly using hisat2 v2.1.0 (Kim, Landmead, & Salzberg, 2015) and
string tie v2.0 (Pertea et al., 2015) to obtain putative transcripts. To
determine gene expression levels, the RNA-Seq clean reads were
mapped to the genome assembly using bow tie2 v2.3.5 (Langmead &
Salzberg, 2012), and transcript abundances were estimated by r sem
v1.3.1 (Li & Dewey, 2011).
2.6 | Assessment of genome assembly
We used the busco v3.0 (Waterhouse et al., 2018) (Benchmarking
Universal Single-Copy Orthologues) software to scan 1,658 univer-
sal single-copy orthologous genes selected from insecta_db 9 data
sets in genome assembly with default parameters.
2.7 | Genome annotation
We identified repeat sequences and transposable elements (TEs) by
both homology-based and de novo prediction methods. For de novo
predictions, repeatmodeler v1.0.7 was used to construct a de novo
repeat library with default parameters. For homology-based predic-
tions, repe atmasker v4.0.5 (Tarailo-Graovac & Chen, 2009) was used
with Repbase library (Bao, Kojima, & Kohany, 2015).
We annotated the protein coding genes by integrating the evi-
dence of de novo, homology-based and RNA-Seq-based annotations.
First, augustus v2. 5.5 (Stan ke, Di ekhans, Baer tsc h, & Haussl er, 2008)
and snap v2013-11-29 (Korf, 2004) were used to generate the de
novo annotation with internal gene models. Then, exoner ate v2.2.0
(Slater & Birney, 20 05) and genomethr eader v1.7.1 (Gremme, Brendel,
Sparks, & Kurtz, 2005) were used to align the proteins obtained
from NCBI invertebrate RefSeq (https://www.ncbi.nlm.nih.gov/
refse q/) to the genome assembly with default parameters. The tran-
scripts of the fall armyworm were obtained by h isat2 v2.1.0 (Kim
et al., 2015) and strin gtie v2.0 (Pertea et al., 2015) pipeline with de-
fault parameters. We next integrated these three types of evidences
with different weights (the weight for de novo annotation is “1”, for
homology-based annotation is “5”, for RNA-Seq-based annotation
is “10”) for each by EVidenceModeler (EVM) (Haas et al., 2008) to
obtain the official gene set (OGS). Gene Ontology (GO) analysis was
carried out using the software bla st2go v5.2 (Conesa et al., 2005).
We further mapped these genes to data from the Kyoto Encyclopedia
of Genes and Genomes (KEGG) database using the bla stkoal a v2.2
(Kanehisa, Sato, & Morishima, 2016) online service.
2.8 | Phylogenetic reconstruction
Proteins sequences of 22 insect species were clustered using
the orthomcl v2.0.9 pipeline with default parameters (Li,
Stoeckert, & Roos, 2003). These accessions were: S. frugiperda
(this study), Spodoptera litura (GCA_002706865.2, from NCBI),
Trichoplusia ni (GCF_003590095.1, from NCBI), Helicoverpa ar-
migera (GCF_002156985.1, from NCBI), Heliothis virescens
(GCA_002382865.1, from NCBI), Bombyx mori (GCF_000151625.1,
from NCBI), Antheraea yamamai (from GigaDB, http://gigadb.org/
datas et/100382), Manduca sexta (GCF_000262585.1, from NCBI),
Operophtera brumata (GCA_001266575.1, from NCBI), Cydia
pomonella (from InsectBase; Yin et al., 2016), Danaus plexippus
(GCA_000235995.2, from NCBI), Papilio xuthus (GCF_000836235.1,
from NCBI), Plutella xylostella (GCF_000330985.1, from NCBI),
Stenopsyche tienmushanensis (from GigaDB, http://gigadb.org/
datas et/100538), Drosophila melanogaster (GCF_000001215.4,
from NCBI), Anopheles gambiae (from VectorBase, https://www.
vecto rbase.org/organ isms/anoph eles-gambiae), Tribolium cas-
taneum (GCF_000002335.3, from NCBI), Leptinotarsa decemlineata
(GCF_000500325.1, from NCBI), Apis mellifera (GCF_003254395.2,
from NCBI), Nasonia vitripennis (OGS2, from Ensemble Database,
ftp://ftp.ensem blgen omes.org/pub/metaz oa/relea se-38/fasta /
n a s o n i a _ v i t r i p e n n i s / d n a / ) , Melanaphis sacchari (GCF_002803265.2,
from NCBI) and Rhodnius prolixus (from VectorBase, https://www.
vecto rbase.org/organ isms/rhodn ius-prolixus).
In total, 328 single-copy genes were obtained from OrthoMCL
results and were used for phylogeny reconstruction. First, the pro-
tein sequences of each gene family were independently aligned
by mafft v7 (Katoh & Standley, 2013). Then, trimal v1.2 (Capella-
Gutierrez, Silla-Martinez, & Gabaldon, 2009) was used to clean each
alignment and extract the conserved block. Next, we concatenated
all single-copy genes to create one super gene for each species. We
used ModelFinder (Kalyaanamoorthy, Minh, Wong, von Haeseler,
& Jermiin, 2017) to select the best model. iq-tree v1.5.5 (Nguyen,
Schmidt, von Haeseler, & Minh, 2015) was used to construct the
phylogenetic tree using the LG + F + I + G4 model and 1,000 boot-
strap replicates. To estimate the divergence time of the fall army-
worm, we applied three calibration points based on fossil records in
Paleobiology Database (www.paleo biolo gy.org): (a) stem Trichoptera
(Phryganea solitaria) at 311.45–314.6 mya; (b) stem Lepidoptera (fos-
sil unnamed) at 201.3–208.5 mya; and (c) stem Noctuoidea (Noctuites
incertissima) at 28.1–33.9 mya. The divergence time was estimated
by using MCMCtree in paml v4.9e (Yang, 2007) with the topology of
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these insects we built above. The tree was visualized using figtre e
v1.4.4 (http://tree.bio.ed.ac.uk/softw are/figtr ee/).
2.9 | Whole-genome synteny
Whole-genome synteny between S. frugiperda, S. litura, and B. mori
were estimated using satsuma v3.1.0 (Grabherr et al., 2010), a pack-
age of SPINES with default parameters (https://www.broad insti
tute.org/genom e-seque ncing -and-analy sis/spines). Synteny blocks
were plotted across chromosomes using ci rcos v0.69-9 (Krzywinski
et al., 2009).
2.10 | Gene family expansion and contraction
We used café v4.2.1 (De Bie, Cristianini, Demuth, & Hahn, 2006) to
perform a gene family expansion and contraction analysis. The pro-
tein sequences from twenty-two insects were aligned to the tr eefam
v9 (Schreiber, Patricio, Muffato, Pignatelli, & Bateman, 2014) data-
base to obtain the TreeFam ID for each protein. The tr eefam v9 re-
sults and a tree with estimated divergence time were used as inputs
of CAFÉ. We used a criterion of p < 0.05 for significantly changed
gene families.
2.11 | Gene family analysis
For the P450 gene family, we first downloaded reference protein
sequences of Lepidoptera P450s from NCBI GenBank and manually
confirmed these sequences to obtain a clean reference sequences
for Lepidoptera P450s. TBLASTN (blas t v2.9.0) was used to search
P450 candidate sequences in the fall armyworm genome assembly
(E-value < 1E−5). genewise v2.4.1 (Madeira et al., 2019) and e xonerate
v2.2.0 (Slater & Birney, 2005) were used to define the gene struc-
ture. And we also confirmed the P450 candidate sequences using
hmmer v3.2.1 (Potter et al., 2018) against sequences from the Pfam
database (Pfam domain PF00067, E-value < 1E−5) (Finn et al., 2014).
The fall armyworm P450 sequences were compared to P450 genes
of S. litura and B. mori by phylogenetic studies for name assignment.
rsem v1.3.0 (Li & Dewey, 2011) was used for gene expression level
(FPKM) calculation. In this study, if the expression level of a given
gene with FPKM > 0.4 in all three RNA-Seq repetitions of a given
development stage, this gene was regarded as expressed in this de-
velopment stage.
For the gustatory receptor (GR) gene family, we searched GR
candidate sequences in the fall armyworm genome assembly using
TBLASTN (E-value < 1E−5) (bl ast v2.9.0) with a set of GR reference
sequences obtained from NCBI GenBank. Then, genewise v2.4.1
(Madeira et al., 2019) and exonerate v2.2.0 (Slater & Birney, 2005)
were used to define the gene structure. For GR subfamily anno-
tation, we compared the fall armyworm GR sequences with GRs
from S. litura and B. mori by phylogenetic studies. rsem v1.3.0 (Li
& Dewey, 2011) was used for GR gene expression level (FPKM)
calculation.
For other gene families, including glutathione-S-transferases
(GSTs), carboxylesterases (COEs), ATP-binding cassette transport-
ers (ABC transporters), olfactory receptors (ORs), ionotropic recep-
tors (IRs), odorant-binding proteins (OBPs), chemosensory proteins
(CSPs), and β-fructofuranosidase (β-FFase), we identified each gene
family's genes using a two-step method in OGS. First, we collected
the reference protein sequences of each gene family from NCBI
GenBank. And the reference protein sequences were further man-
ually confirmed. Then, we used BLASTP to determine candidate se-
quences from OGS of each insect (E-value < 1E−5). Next, HMMER
was used to align the candidate sequences to the Pfam database (E-
value < 1E−5) (Finn et al., 2014).
For the phylogenetic analysis of gene families, we aligned
protein sequences of each gene family using maff t v7 (Katoh &
Standley, 2013) and filtered sequences with trimal v1.2 (Capella-
Gutierrez et al., 2009) to obtain the conserved blocks. iq-tree v1.5.5
(Nguyen et al., 2015) was used to construct the phylogenetic tree
with the best model estimated by ModelFinder (1,000 ultrafast
bootstrap approximation replicates) (Kalyaanamoorthy et al., 2017).
The tree was visualized using figtree v1.4.4 (http://tree.bio.ed.ac.
uk/softw are/figtr ee/). An R package RIdeogram v.0.1.1 was used to
map and visualize genes in chromosomes (Hao et al., 2019).
2.12 | Determination of the fall armyworm strain in
Zhejiang Province
The Tpi gene was used as a marker to identify the strain of fall ar-
myworm that had invaded the Zhejiang province of China. We
identified the Tpi gene and determined the strain from the Zhejiang
population using sites in the fourth exon of Tpi (TpiE4-165, TpiE4-
168 and TpiE4-183).
2.13 | Sex chromosomes
To identify the sex chromosomes (Z and W chromosomes) in fall ar-
myworm, one female pupa and one male pupa were resequenced
using Illumina HiSeq platforms to obtain an approximate 40× cover-
age. The paired-end sequencing data of the female pupa was used
as an input to jellyfish v2.2.0 (Marcais & Kingsford, 2011) with k-mer
length = 17 and genomescope (https://github.com/schat zlab/genom
escope; Vurture et al., 2017) for assessment of genomic heterozygo-
sity and genomic size. Normalized coverage levels of sequence reads
from the Z chromosome in males should be twice that of females.
In contrast, males do not have any DNA contribution from the W
chromosome, while the autosomes should have equal coverage be-
tween males and females. Thus, a difference in sequencing coverage
ratio is expected for both Z and W chromosomes between sexes, but
not autosomes and this difference can be used to identify sex-linked
scaffolds. After filtering with fqto ols v0.1.8 (Droop, 2016), genome
6
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XIAO et Al.
TABLE 1 Comparison of fall armyworm genome assemblies of this and previous studies
ZJ-version
(this study)
Nam et al. (2 019)
Zhang et al. (2019)
Liu, Lan, et al. (2019) Gouin et al. (2017)
Nandakumar
et al. (2017)
Kakumani
et al. (2014)
C strain SFynMstLFR SFynFMstLFR C strain R strain Sf9Sf21
Sequencing info
DNA source Single female
pupa
Fourth instar
male larvae
Single male moth Single male adult Single female
adult
Fourth instar
male larvae
Single male
larva
Sf9 cell line Sf21 cell line
Assembly approach PacBio + Hi-C PacBio + Illumina
+Hi-C
PacBio + Illumina
+Hi-C
MGISEQ + Hi-C MGISEQ + Hi-C Illumina Illumina PacBio Illumina
Genome assembly
Assembly level Chromosomes Chromosomes Chromosomes Chromosomes Chromosomes Scaffolds Scaffolds Scaffolds Scaffolds
(30A + Z + W) (30A + Z) (30A + Z) (30A + Z) (30A + Z)
Genome size (Mb) 486.3 384.46 393.25 542.4 530.8 4 37.9 371.0 514 .2 358.0
Number of contigs 618 -777 – – – – 2,844 97,607
Number of scaffolds 93 125 311 – – 41,562 2 9,127 2,396 37,235
Gaps number 525 – – – – 13,694 3,818 2,635 95,454
Gap length (kb) 53 346 –37,94 7 35,693 11, 378 131 891 27,685
Quality assessment
Contig N50 (kb) 1,130.0 –5,606.9 92.0 125.0 21.6 25.4 516.1 7. 8
Scaffold N50 (kb) 16,346.9 13,151. 2 13, 317.1 14,162.8 14,88 3.7 52.7 28.5 601 .1 53.7
BUSCO genes (%) 93.1 96. 6 98.2 95.0 94.5 88.6 89. 3 90.8 –
Genomic features
Protein-coding genes 22,623 –23,281 22,201 –21,700 26,329 25,699 11,595
Repeat (%) 28.0 –27.2 28.2 –2 9. 2 29.1 28.1 20.3
SINEs (%) 0.7 –1.0 – – 12.5 12.9 0.7 –
LINEs (%) 9.1 –8.7 – – 1.9 1.7 10.7 0 .1
LTR elements (%) 0.3 –1.4 – – 0.08 0.07 1.1 0.09
DNA elements (%) 1.7 –2.7 – – 0.3 0.3 1.7 0.03
G + C (%) 36.4 –36.4 36.5 36.6 35.1 36.1 36.5 24.3
Annotation
Genes with GO terms 13,044 – – – – 13,369 9, 261 15,623 5,713
Genes with KEGG
annotations
7,818 – – 16,072 – – – 9, 213 4,220
|
7
XIAO et Al .
resequencing reads were aligned to the fall armyworm genome as-
sembly using bow tie2 v2.3.5 (Langmead & Salzberg, 2012) with de-
fault parameters. Analysis and visualization of the log2 of the male:
female (M:F) coverage ratio were performed using the r package
changepoint v2.2.2 (https://CRAN.R-proje ct.org/packa ge=chang
epoint).
2.14 | Positive selection analysis
All 5,410 single-copy genes shared by four Noctuidae insects, S.
frugiperda, S. litura, T. ni and H. armigera were used for positive se-
lection analysis. Protein sequences of each single-copy gene fam-
ily were aligned using mafft v7 (Katoh & Standley, 2013), and then
the protein alignments were converted to their corresponding nu-
cleotide alignments by the Perl script pal2na l v14 (Suyama, Torrents,
& Bork, 2006). The dN/dS ratio was estimated for each homolo-
gous cluster using the CodeML program in the paml v4.9e package
(branch-site model) (Yang, 2007). We calculated the significances of
obtained positive-selected genes using the Chi-square test with a
false discovery rate (FDR) cutoff of 0.05.
2.15 | Enrichment analysis
The GO and KEGG enrichment analyses were conducted using omic-
share cloudto ols under this tool's default instructions (http://www.
omics hare.com/).
3 | RESULTS AND DISCUSSION
3.1 | Chromosome-level genome assembly of fall
armyworm
A female pupa of the fall armyworm was used for genome se-
quencing by PacBio long-read technology, yielding ~126 Gb PacBio
subreads. The PacBio reads were self-corrected using canu v1.8
(Koren et al., 2017) and finally assembled into 361 contigs using
SMARTdenovo (Tables S1–S3, see Methods) (Istace et al., 2017).
There were 194 contigs (126 Mb) identified as representing allelic
variants of sequence already present in the assembly and these were
removed. We named this genome assembly the ZJ-version. The as-
sembled genome size was 486 Mb with a contig N50 of 1.13 Mb.
Surprisingly, the assembled genome size was 155 Mb larger than
the genome size estimated by 17-mer analysis. The k-mer analysis
showed that the fall armyworm has high heterozygosity of 3.45%
(Figure S1). An inflated assembly size relative to k-mer based esti-
mates has also been observed in other highly heterozygous species
assemblies such as Ancherythroculter nigrocauda, Pyrocoelia pecto-
ralis, and Oncopeltus fasciatus (Fu et al., 2017; Panfilio et al., 2019;
Zhang et al., 2020). Previously released genomes indicated the
genome size of the fall armyworm ranges from 358 Mb to 542 Mb
(Table 1). There is also a big difference between the genome sizes
of two cell lines derived from pupal ovaries of the fall armyworm
(Table 1) (Kakumani et al., 2014; Nandakumar et al., 2017). The ge-
nome size variation might be due to the following reasons: (a) dif-
ferent sequencing technologies, sequencing depth, and assembly
approaches; and (b) fall armyworm samples are from different areas/
habitats. It has been reported that variable genome size of differ-
ent strains within the same species may be a result of the amplifica-
tion, deletion and divergence of repetitive sequences; colonization
of new environments; variation of environmentally-dependent life
history traits (Ellis et al., 2014; Nardon et al., 2005). Further study is
needed to determine the reason of the genome size variation in the
different strains of fall armyworm.
According to our Hi-C interaction information, we cut the pri-
mary assembly into 618 contigs, then anchored 556 (97.4% in length)
contigs to 31 chromosomes (30 autosomes and Z chromosome)
(Figure 1a, Tables S4–S7, and Figure S2). Hi-C correction and scaf-
folding did not change the genome size or contig N50, but increased
the scaffold N50 to 16.3 Mb, which was much higher than any other
published genome of the fall armyworm (Table 1). The length of N50
for both contigs and scaffolds were much longer than those of other
published Noctuidae insects including T. ni (Chen et al., 2019), H. ar-
migera (Pear ce et al ., 2017 ) and S. litura (Chen g et al. , 2017) (Tab le S8).
The ZJ-version genome assembly had only 525 gaps and the gap
lengths were estimated to be 53 kb, suggesting that the ZJ-version
genome assembly was highly complete (Table 1). BUSCO analysis in-
dicated that 93.1% complete genes (1,658 universal single-copy or-
thologous genes of insects) exist in the ZJ-version genome assembly
(Table 1), and the fragmented BUSCOs (1.0%) was apparently less
than that in Liu, Lan, et al. (2019) (2.8% in male and 3.1% in female)
(Table S5). The complete and duplicated BUSCO component of the
ZJ-version genome was 20.3%, which was higher than that in Liu,
Lan, et al. (2019) (9.8% in male and 7.8% in female), and also higher
than that in Nam et al. (2019) (1.7%), suggesting that the potential
allelic duplication might be present in the ZJ-version genome. Taken
together, these results suggest that we obtained a robust fall army-
worm genome assembly will provide a solid foundation for future
analyses.
Using the genome resequencing data of a single male pupa and a
single female pupa, we calculated the sequencing coverage of each
scaffold and identif ied the Z chromoso me and a por tion of the W chro-
mosome (Figure 1b, Table S9, see Methods). The largest super-scaffold
(Chr1) yielded two-fold greater male coverage, as expected for the Z
chromosome. Although we failed to obtain an intact W chromosome
using Hi-C scaffolding, we have identified 4.7 Mb W-linked sequences
in the unanchored contigs, including a long W-linked contig (ctg37,
contig 37) of a length of 3.5 Mb (Figure 1b, Table S9). Because the
Lepidopteran W chromosome is enriched in repeat sequences, it is dif-
ficult to assemble a complete W chromosome with present sequencing
and assembly methods (Sahara, Yoshido, & Traut, 2012). Although a
number of chromosome-level genomes of Lepidoptera insects have
been released, the W chromosome has only been reported from C. po-
monella with a length of about 5 Mb (Wan et al., 2019), as well as from
8
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XIAO et Al.
chr1(Z)
0
10
20
chr2
0
10
chr3
0
10
chr4
0
10
chr5
0
10
chr6
0
10
chr7
0
10
chr8
0
10
chr9
0
10
chr10
0
10
chr11
0
10
chr12
0
10
chr13
0
10
chr14
0
10
chr15
0
10
chr16
0
10
chr17
0
10
chr18
0
10
chr19
0
10
chr20
0
10
chr21
0
10
chr22
0
10
chr23
0
10
chr24
0
10
chr25
0
10
chr26
0
10
chr27
0
10
chr28
0
10
chr29
0
10
chr30
0
chr31
0
ctg37(W)
0
−2
−1
0
1
2
6.6 6.8 7.07.2
Log
10
(scaffold length)
Log2(Mean M:F read counts)
Autosome
W
Z
(a)
(b)
From outer to inner circles:
I. 32 chromosomes at the Mb scale
II. repeat density across the genome
(in 0.1 Mb non-overlapping windows)
III. gene density across the genome
(in 0.1 Mb non-overlapping windows)
IV. GC content across the genome
(in 0.1 Mb non-overlapping windows)
I
II
III
IV
|
9
XIAO et Al .
the T. ni Hi5 germ cell line (Fu et al., 2018). Here, we report the first
partial W chromosome for Noctuidae insects.
The fall armyworm genome shares high synteny with other
Lepidopteran insect genomes showing a strong evidence for ge-
nome conservation at the chromosome level in Noctuidae insects
(Figure 2a,b, Table S10). The syntenic relationship between the silk-
worm and fall armyworm revealed three fusion events in autosomes:
Chr17 and Chr31, Chr22 and Chr29, and Chr23 and Chr26 in the fall
armyworm were fused to Chr11, Chr23, and Chr24 in the silkworm,
respectively (Figure 2c, Table S10).
3.2 | Genome annotation
In total, 28% of the ZJ-version fall armyworm genome was annotated
as repeat sequences (Table 1). Although the total genome size varies
between different S. frugiperda genome assemblies, the proportions
of repeat sequences were similar (Table 1). The content of repeat
sequences in the fall armyworm were larger than that in T. ni (Fu
et al., 2018) and in H. armigera (Pearce et al., 2017), but less than that
in S. litura (Cheng et al., 2017) (Table S8). After masking these repeat
sequences, the EVidenceModeler (EVM) pipeline (Haas et al., 2008)
was used to predict protein-coding genes by integrating the evidence
of protein homology, de novo predictions, and RNA-Seq transcripts
(first instar to six instar larvae, female pupae, male and female adult)
(see Methods). In total, 22,623 protein-coding genes were annotated
in the ZJ-version fall armyworm genome (Table 1), 14,123 (62.4%) of
which were detected in at least one sample of RNA-Seq data. The
number of protein-coding genes in the fall armyworm is the largest
set of genes in the published Noctuidae insect genomes (Table S8).
Of these annotated genes, 13,044 (57.7%) genes have GO terms and
7,818 (34.6%) genes have homology in the KEGG database (Table 1).
The number of annotated protein-coding genes in the ZJ-version fall
armyworm genome was similar to the genes identified in five previ-
ously published versions of the genome, but more than that in the
genome of Sf21 cell line. Furthermore, we manually annotated sev-
eral gene families associated with insect adaptation, including 169
P450s, 59 GSTs, 98 COEs, 79 ABC transporters, 70 ORs, 221 GRs,
41 IRs, 35 OBPs, and 29 CSPs (able S11).
3.3 | The Zhejiang Province fall armyworm is the
C strain
The Z chromosome-linked gene Tpi is commonly used to identify the
strain of fall armyworm (Nagoshi, 2010). The strain-specific sites in the
fourth exon of Tpi in clu de th e sites E4-16 5, E4-168 and E4-18 3 (N a goshi
et al., 2019). Based on these sites, we determined that the strain of fall
armyworm indicated by the ZJ-version genome is the C strain, which
is the same strain found in the Yunnan population (Figure S3) (Liu, Lan,
et al., 2019; Liu, Xiao, et al., 2019; Zhang et al., 2019).
3.4 | Gene orthologues and comparative
genomic analysis
Comparative genomics analysis was carried out using 22 insect ge-
nomes covering six insect orders (Lepidoptera, Trichoptera, Diptera,
FIGURE 1 Chromosome-level genome assembly of the ZJ-version fall armyworm. (a) Circos plot showing the genomic landscape of
the 32 fall armyworm chromosomes. From outer to inner circles: I, 32 chromosomes at the Mb scale; II and III, repeat density (blue) and
gene density (yellow) across the genome, respectively, drawn in 0.1 Mb nonoverlapping windows; IV, GC contents (green) across the
genome, drawn in 0.1 Mb nonoverlapping windows. (b) Male: female coverage ratios for each chromosome. Each point represents a single
chromosome. The dotted red line shows the expectation for the Z chromosome
FIGURE 2 Whole-genome synteny between S. frugiperda and S. litura, T. n i and B. mori. (a) The fall armyworm genome shares high
synteny with S. litura, (b) T. ni. (c) Three fusion events were founded in autosomes between the fall armyworm and silkworm, Chr17 and
Chr31, Chr22 and Chr29, and Chr23 and Chr26 in the fall armyworm were fused to Chr11, Chr23, and Chr24 in the silkworm, respectively.
Synteny analysis was carried out using Satsuma and viewed with Circos v 0.69
S. frugiperda S. frugiperd
aS
. frugiperda
T. ni B. moriS. litura
(a) (b) (c)
10
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XIAO et Al.
Coleoptera, Hymenoptera, and Hemiptera). A phylogenetic tree
was constructed using 238 single-copy genes (Figure 3). In addition,
3,076 N:N:N genes, 6,160 Lepidoptera-specific genes, and 2,608
Noctuidae-specific genes were identified (Figure 3, Table S12).
Compared to that of four other moth species in Noctuidae, the fall
armyworm has the largest number of Noctuidae orthologous genes,
species-specific genes and species-specific duplicated genes, 1,646
species-specific duplicated genes were identified (Table S12) and
494 were found to be tandemly duplicated on the chromosomes
(Figure S4), suggesting that gene expansion might have occurred in
the fall armyworm genome. Based on our phylogenetic tree, the fall
armyworm and S. litura may have diverged from their common an-
cestor approximately 9.8 Mya ago (Figure 3).
The gene family evolution analysis indicated that the fall army-
worm genome displayed 774 expanded and 1,048 contracted gene
families compared with gene families of the common ancestor of fall
armyworm and S. litura (Figure 3). The common ancestor of Noctuidae
species showed 449 expanded and 288 contracted gene families com-
pared to that of the common ancestor of Noctuidae species and O.
brumata. Notably, Noctuidae expanded gene families were enriched
in nutrition metabolism pathways, including protein digestion and
absorption (ko04974, p = 1.269365 × 10–1 9, Hypergeometric test, FDR-
adjusted), glycerolipid metabolism (ko00561, p = 2.63951 × 10–18 ), and
fructose and mannose metabolism (ko00051, p = 3.279737 × 10
–9)
(Tables S13–S14). In addition, the fall armyworm expanded gene fam-
ilies were enriched not only in nutrition metabolism but also in trans-
port system, such as ABC transporters (ko02010, p = 3.012781 × 10–3)
(Tables S15–S16). Noctuidae diverged from the Bombycoidae super-
family ca. 94 million years ago (Wahlberg, Wheat, & Pena, 2013), and
most of the pests in Noctuidae are polyphagous, while the silkworm
in Bombycoidae is a monophagous species. For the polyphagous fall
armyworm, the number of host plants is as much as 353 species among
76 families (Montezano et al., 2018). The expansion of nutrition me-
tabolism and transport system genes might facilitate the absorption of
nutrients from different plant hosts and the detoxification of natural
xenobiotics from plants. We suspect that the expansion of these genes
may have facilitated the high invasion of fall armyworm.
Based on orthologous gene annotation by OrthoMCL across
four Noctuidae insects (S. frugiperda, S. litura, T. n i and H. armigera),
5,410 single-copy genes were used for positive selection analy-
ses. As a result, we identified 835 positive selected genes in the
fall armyworm using the Branch-site model in PAML, including
FIGURE 3 Genome evolution of fall armyworm. The phylogeny tree of 22 insects covering six insect orders was calculated by maximum-
likelihood analyses using 238 single-copy proteins. Bootstrap values based on 1,000 replicates are equal to 100 for each node. Divergence
times calculated by MCMCtree are indicated by yellow bars at the internodes, and the bars indicate the 95% confidence intervals of the
divergence time. The number of expanded gene families (green) and contracted gene families (red) obtained from tr eefam and caf é software
are shown on the branches. Bars are subdivided to represent different types of orthology across 22 insects: “1:1:1” indicates universal
single-copy genes present in all species, absence and/or duplication in, at most, one genome is included; “N:N:N” indicates other universal
genes; “Lep.” indicates common unique genes in Lepidoptera; “Noc.” indicates common unique genes in the family Noctuidae; “S.D.”
indicates species-specific duplication; “N.D.” indicates species-specific genes; “Patchy” includes all remaining genes
Drosophila melanogaster
Stenopsyche tienmushanensis
Plutella xylostella
Danaus plexippus
Papilio xuthus
Helicoverpa armigera
Heliothis virescens
Spodoptera frugiperda
Spodoptera litura
Trichoplusia ni
Operophtera brumata
Bombyx mori
Antheraea yamamai
Manduca sexta
Cydia pomonella
Anopheles gambiae
Leptinotarsa decemlineata
Tribolium castaneum
Apis mellifera
Nasonia vitripennis
Melanaphis sacchari
Rhodnius prolixus
0100200300400 Million years age500
Hemiptera
Hymenoptera
Coleoptera
Diptera
Trichoptera
Lepidoptera
Noctuidae
+1,062/–1,169
+1,114/–1,438
+978/–784
+1,689/–1,017
+2,415/–2,035
+978/–1,503
+1,898/–2,443
+716/–1,332
+1,042/–974
+774/–1,048
+769/–603
+2,092/–1,682
+914/–1,522
+616/–1,327
+755/–1,417
+1,695/–3,280
+1,697/–3,078
+669/–3,594
+1,161/–1,517
+1,228/–928
+675/–617
+701/–632
+449/–288
Gene families
Expansion/Contraction
MRCA
(9,266)
1:1:1
N:N:N
Lep.
Noc.
Patchy
S.D.
N.D.
0
10,000
20,000
30,000
Gene number
|
11
XIAO et Al .
FIGURE 4 Cytochrome P450s in fall armyworm. (a) Maximum-likelihood phylogenetic analysis of P450 genes in fall armyworm and
silkworm. The largest CYP340 cluster on Chr14 is shown. (b) Distribution of 166 P450 genes in the fall armyworm chromosomes
(a)
1(Z) 2345678910 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 ctg37(W)
(b)
CYP2
CYP4
CYP3
mito
Bmor_NP_001077080.1
01077080.1
Sfru_P450_049_CYP321A8
Sfru_P450_167_CYP6AN4
Sfru_P450_160_CYP340L1
Sfru_P450_065_CYP4C1
Sfru_P450_071_CYP307A1
Sfru_P450_003_CYP321A15
S
5
Sfru_P450_001_CYP306A1
Sfru_P450_106_CYP9A21
Sfru_P450_156_CYP4G75
Sfru_P450_079_CYP341B27
27
Sfru_P450_099_CYP321A7
Sfru_P450_104_CYP340L1
Sfru_P450_084_CYP340L1
Sfru_P450_128_CYP340AA1
u_P450_128_CYP340AA1
u_
Bmor_NP_001073134.1
Sfru_P450_107_CYP4L9
Sfru_P450_021_CYP307A1
Bmor_NP_00
1
140197.1
Sfru_P450_087_CYP340AB1
Sfru_P450_087_CYP340AB1
Sfru_P450_041_CYP341D1
Bmor_XP_004933974.3
B
B
Sfru_P450_130_CYP340AB1
1
Sfru_P450_141_CYP340G1
Sfru_P450_120_CYP340AA1
S
Bmor_XP_012545238.1
Bmor_XP_012544926.1
Sfru_P450_126_CYP354A3
Bmor_NP_00
1
104833.1
Sfru_P450_140_CYP15C1
Sfru_P450_122_CYP321A9
Bmor_XP_021203627.1
Sfru_P450_009_CYP9A21
Sfru_P450_125_CYP340L1
Bmor_NP_001266295.1
Sfru_P450_095_CYP6AB31
Sfru_P450_035_CYP340L1
Sfru_P450_145_CYP339A1
Sfru_P450_163_CYP340AB1
B1
Sfru_P450_024_CYP9AJ3
Sfru_P450_080_CYP6CV6
Bmor_NP_001266314.1
Bmor_XP_021203900.1
Bmor_NP_00
1
12
1
192.1
Bmor_NP_001036953.1
Sfru_P450_053_CYP4CG1
Sfru_P450_029_CYP321B4
1B4
1B4
Bmor_XP_021204174.1
Sfru_P450_054_CYP9A40
Bmor_NP_00
1
104005.1
Bmor_XP_004934361.3
Bmor_XP_012547619.2
Sfru_P450_168_CYP367A6
Bmor_NP_001274763.1
Sfru_P450_031_CYP321A8
Sfru_P450_147_CYP340AB1
1
Bmor_NP_001266437.1
Sfru_P450_014_CYP6AB60
Sfru_P450_133_CYP340L1
Sfru_P450_0
S
1
1_CYP340AA1
Bmor_XP_012552573.1
Bmor_XP_021202909.1
9.1
9.1
Bmor_XP_004934094.3
Sfru_P450_124_CYP4C1
Sfru_P450_151_CYP321A7
Bmor_XP_004934039.2
Sfru_P450_150_CYP4G75
Sfru_P450_015_CYP9A39
Bmor_NP_001296467.1
Sfru_P450_108_CYP340L1
S
S
Bmor_NP_00
1
108343.1
Sfru_P450_063_CYP340L1
Sfru_P450_
1
12_CYP6A13
Sfru_P450_039_CYP4C1
Bmor_XP_012552394.1
Bmor_XP_004923216.1
Bmor_NP_00
1
103814.1
Sfru_P450_082_CYP340L1
Sfru_P450_123_CYP4C1
Sfru_P450_016_CYP324A6
324A
Sfru_P450_152_CYP6B58
58
Sfru_P450_042_CYP333B3
Bmor_XP_021208861.1
Bmor_NP_00
1
103833.1
Bmor_XP_004934166.1
Sfru_P450_017_CYP340AB1
ru_P450_017_CYP340AB1
Sfru_P450_096_CYP9G5
Sfru_P450_037_CYP9A9
Sfru_P450_036_CYP18B3
Sfru_P450_144_CYP337B5
YP337B5
Bmor_XP_004927603.1
Sfru_P450_002_CYP341A
1
1
Sfru_P450_044_CYP341B2
Sf
Bmor_NP_00
1
104826.1
Sfru_P450_018_CYP4CG1
Bmor_NP_001266419.1
Bmor_XP_021203887.1
Bmor_XP_012545759.1
Sfru_P450_010_CYP49A1
Sfru_P450_131_CYP49A1
1
1
B
A
0
4
3
P
Y
C
_
7
7
0
_
0
5
4
P
_
u
r
f
S
Sfru_P450_068_CYP340L1
Sfru_P450_132_CYP340L1
Sfru_P450_146_CYP340L1
Sfru_P450_093_CYP9A58
Sfru_P450_025_CYP338B42
42
Sfru_P450_169_CYP4AU1
Sfru_P450_134_CYP305B1
1
.
7
2
0
6
4
5
2
1
0
_
P
X
_
r
o
m
B
Sfru_P450_142_CYP340G1
Sfru_P450_153_CYP4AU14
Bmor_XP_004926535.1
Sfru_P450_
Sfru
1
15_CYP4L4
Sfru_P450_066_CYP6AE43
Sfru_P450_101_CYP340AA1
1
Sfru_P450_057_CYP303A1
Sfru_P450_088_CYP354A14
4
Sfru_P450_033_CYP324A1
Bmor_XP_012546250.1
Sfru_P450_040_CYP6AE97
Sfru_P450_032_CYP332A1
Sfru_P450_159_CYP367A1
Bmor_XP_021203621.1
Sfru_P450_026_CYP6B42
Bmor_NP_00
1
106226.1
Bmor_XP_004922787.1
Sfru_P450_056_CYP301A1
S
Bmor_NP_001073135.1
Sfru_P450_023_CYP340L1
Sfru_
Sfru_
Sfru_P450_092_CYP333A6
Sfru_P450_020_CYP6B58
58
Sfru_P450_038_CYP6AB12
Sfru_P450_055_CYP6
A
W1
A
A
Sfru_P450_060_CYP321B1
1B
S
fru
_
P450
_
1
13
_
CYP4G7
4
Sfru_P450_013_CYP341B1
S
Sfru_P450_105_CYP9A21
Bmor_XP_004934250.2
1
A
A
A
0
4
3
P
Y
C
_
1
6
0
_
0
5
4
P
_
u
r
f
S
S
Sfru_P450_085_CYP6B50
Sfru_P450_154_CYP9A21
_CYP9A21
Sfru_P450_072_CYP341B27
Sfru_P450_072_CYP341B27
Bmor_NP_001274759.1
Bmor_NP_001266427.1
Sfru_P450_162_CYP4L9
Sfru_P450_
fru_
1
17_CYP340L1
Bmor_NP_00
1
104007.1
Bmor_XP_021204179.1
Sfru_P450_064_CYP321A7
Bmor_XP_021206485.1
Bmor_XP_004934325.2
Sfru_P450_006_CYP332A1
Bmor_NP_00
1
108341.1
Sfru_P450_097_CYP321B1
Sfru_P450_129_CYP6AB14
Sfru_P450_166_CYP367B1
Sfru_P450_16
Sfru_P450_050_CYP341B26
26
Bmor_NP_00
1
106222.1
Sfru_P450_158_CYP341B1
Sfru_P450_076_CYP340AA1
S
Sfru_P450_
11
1_CYP6AB61
Sfru_P450_062_CYP340K4
Sfru_P450_
1
14_CYP6AE50
Sfru_P450_008_CYP4L4
Bmor_XP_004927968.1
Sfru_P450_127_CYP6B48
P6B48
P6B48
Sfru_P450_047_CYP6AB31
Sfru_P450_149_CYP333B4
Sfru_P450_089_CYP340L1
Sfru_P450_109_CYP337B5
Bmor_NP_00
1
104827.1
Sfru_P450_058_CYP4M18
Sfru_P450_069_CYP340G1
G
G
Bmor_XP_004926524.1
Sfru_P450_027_CYP6CT1
1
.
3
2
0
6
4
5
2
1
0
_
P
X
_
r
o
m
B
Sfru_P450_007_CYP4L10
S
S
Sfru_P450_139_CYP6B1
Bmor_XP_012552338.1
Bmor_NP_001077078.1
Sfru_P450_
1
18_CYP321A7
Sfru_P450_052_CYP367B1
Sfru_P450_091_CYP4C3
Sfru_P450_045_CYP6AB12
Sfru_P450_048_CYP340AA1
S
Bmor_XP_012552063.2
Bmor_XP_004932789.1
Sfru_P450_005_CYP4M17
Bmor_NP_001296528.1
Bmor_XP_004928105.1
Sfru_P450_102_CYP4C3
Sfru_P450_070_CYP6AN4
Bmor_XP_021203884.1
Bmor_XP_004932126.1
Sfru_P450_135_CYP340J1
Bmor_XP_004934109.1
Sfru_P450_028_CYP340AA1
S
Sfru_P450_081_CYP340L1
Bmor_XP_021207530.1
Bmor_XP_021207354.1
Sfru_P450_012_CYP340AA1
S
Sfru_P450_022_CYP4S8
Sfr
Bmor_NP_001269151.1
Sfru_P450_165_CYP4AU14
Bmor_NP_00
1
108456.1
Sfru_P450_075_CYP9A40
Bmor_NP_00
1
104006.1
Sfru_P450_143_CYP338A1
Sfru_P450_161_CYP321B1
B
Sfru_P450_019_CYP321A9
Bmor_XP_021206369.1
Sfru_P450_073_CYP9A40
Sfru_P450_157_CYP6AE43
3
Sfru_P450_083_CYP4L10
Bmor_NP_00
1
135414.1
Bmor_XP_021202508.1
Sfru_P450_138_CYP340L1
Sfru_P450_164_CYP367A1
S
Sfru_P450_043_CYP341B2
Bmor_XP_021202408.1
Sfru_P450_155_CYP4M14
Sfru_
Sfru_P450_
Sf
1
10_CYP340L1
Sfru_P450_067_CYP428A1
Sfru_
Bmor_NP_00
1
106219.1
Sfru_P450_103_CYP340L1
fru_
Sfru_P450_094_CYP340L1
Sfru_P450_
Bmor_NP_001296520.1
Sfru_P450_030_CYP341D1
Sfru_P450_004_CYP341B26
26
Sfru_P450_
1
19_CYP340L1
Sfru_P450_121_CYP6AE68
Sfru_P450_046_CYP9A59
Sfru_P450_078_CYP341B1
Bmor_NP_00
1
104004.1
Bmor_XP_012552193.2
B
B
Bmor_XP_021204600.1
Sfru_P450_051_CYP340L1
Sfru_P450_074_CYP6A13
Bmor_XP_021204692.1
Sfru_P450_059_CYP4G74
Bmor_XP_012544906.1
4906
4906
Sfru_P450_086_CYP4C1
Bmor_XP_021204297.1
Sfru_P450_137_CYP4AU1
_P4
Bmor_XP_021204301.1
Bmor_XP_012546019.1
Bmor_NP_00
1
106221.1
Sfru_P450_100_CYP321B1
1B
Sfru_P450_136_CYP341B1
Sfru_P450_098_CYP18A1
Bmor_NP_00
1
106223.1
Bmor_NP_00
1
108340.1
1
.
4
3
9
5
9
0
1
0
0
_
P
N
_
r
o
m
B
Sfru_P450_090_CYP9A60
Sfru_P450_034_CYP6B1
Bmor_XP_021204294.1
Bmor_NP_00
1
106220.1
Sfru_P450_
1
16_CYP6AE47
Sfru_P450_148_CYP333B3
S. frugiperda
B. mor
i
CYP2
Chr 14 cluste
r
CYP4
mito
CYP3
12
|
XIAO et Al.
the GRs (p < .05, FDR-adjusted, Table S17). The GO and KEGG
enrichment analyses indicated that the significant terms and
pathways were involved in metamorphosis (GO: 0,007,552,
p = 1.813556 × 10
–4), instar larval or pupae development (GO:
0,002,165, p = 1.824223 × 10–3), glycerophospholipid metabolism
(ko00564, p = .01394475) and sphingolipid metabolism (ko00600,
p = .01753068) (Tables S18–S19).
3.5 | The expansion and widespread expression of
cytochrome P450 gene family in fall armyworm
Insect pests, especially the polyphagous insects, can adapt to tol-
erate the plant toxic defense chemicals induced after the insect
feeds on the host (Gatehouse, 2002). These insects have evolved
a strong detoxification system in response to the plant defense
system, such as overproducing detoxification enzymes to metabo-
lize the toxins (Despres, David, & Gallet, 2007) and enhancing the
excretion activity (Dermauw & Van Leeuwen, 2014). Using com-
parative genomics and a BAC library, Giraudo et al. (2015) identi-
fied 42 P450 genes in the fall armyworm. Their allelochemicals
and xenobiotics inducing experiment indicated 29 P450 genes
were induced by plant secondary metabolites, insecticides and
model inducers. We also have reported the expansion of the P450
gene family using the genome of the Sf9 cell line (Mei, Yang, Ye,
Xiao, & Li, 2019). Here, with the ZJ-version chromosome-level
genome, we predicted 169 cytochrome P450 genes by TBLASTN
and Genewise (Table S11). This number is more than previously
reported numbers, suggesting the ZJ-version genome contains a
more complete gene set of higher quality. The number of P450
genes is almost twice that of the silkworm. Phylogenetic analysis
indicated P450 clans 3 and 4 show a large expansion in the fall ar-
myworm comparing with that in the model insect of Lepidoptera,
the silkworm (Figure 4a, Table S20). However, P450 clans Mito
and 2 were strongly conserved between the fall armyworm and
silkworm (Figure 4a, Table S20). A total of 163 P450 genes were
mapped to the 23 chromosomes of fall armyworm. Distribution
analysis showed at least 19 P450s clusters exist in the fall army-
worm genom e (Figure 4b). The lar ge st P450 cluster was lo cated on
Chr14 and consisted of 39 CYP340 genes (Figure 4a,b).
We us ed the RNA-Se q dat a, whic h cove red major developm en-
tal stages of the fall armyworm, to study the expression profile
of all identified P450 genes. In total, 166 out of 169 P450 genes
were detected as expressed genes (FPKM > 0.4 in all three repe-
titions). Moreover, the CYP321A (7-9) gene family tended to ex-
press in the fifth and sixth instar larva (Figure 5) and CYP321A1
reportedly is induced to metabolize xanthotoxin in Helicoverpa zea
(Rupasinghe, Wen, Chiu, & Schuler, 2007). We found that P450
FIGURE 5 Expression profiles of fall armyworm P450 genes from individuals at different developmental stages. F, female; M, male
CYP340 cluster
P450 gene expression
Low High
Sfru P450 146 CYP340L1
Sfru P450 042 CYP333B3
Sfru P450 134 CYP305B1
Sfru P450 094 CYP340L1
Sfru P450 155 CYP4M14
Sfru P450 077 CYP340AB1
Sfru P450 004 CYP341B26
Sfru P450 062 CYP340K4
Sfru P450 169 CYP4AU1
Sfru P450 121 CYP6AE68
Sfru P450 109 CYP337B5
Sfru P450 101 CYP340AA1
Sfru P450 039 CYP4C1
Sfru P450 163 CYP340AB1
Sfru P450 068 CYP340L1
Sfru P450 002 CYP341A11
Sfru P450 040 CYP6AE97
Sfru P450 013 CYP341B1
Sfru P450 044 CYP341B2
Sfru P450 038 CYP6AB12
Sfru P450 016 CYP324A6
Sfru P450 100 CYP321B1
Sfru P450 018 CYP4CG1
Sfru P450 158 CYP341B1
Sfru P450 110 CYP340L1
Sfru P450 050 CYP341B26
Sfru P450 102 CYP4C3
Sfru P450 090 CYP9A60
Sfru P450 154 CYP9A21
Sfru P450 165 CYP4AU14
Sfru P450 131 CYP49A1
Sfru P450 019 CYP321A9
Sfru P450 116 CYP6AE47
Sfru P450 125 CYP340L1
Sfru P450 029 CYP321B4
Sfru P450 067 CYP428A1
Sfru P450 076 CYP340AA1
Sfru P450 119 CYP340L1
Sfru P450 105 CYP9A21
Sfru P450 008 CYP4L4
Sfru P450 043 CYP341B2
Sfru P450 137 CYP4AU1
Sfru P450 079 CYP341B27
Sfru P450 024 CYP9AJ3
Sfru P450 054 CYP9A40
Sfru P450 120 CYP340AA1
Sfru P450 144 CYP337B5
Sfru P450 020 CYP6B58
Sfru P450 007 CYP4L10
Sfru P450 049 CYP321A8
Sfru P450 135 CYP340J1
Sfru P450 093 CYP9A58
Sfru P450 064 CYP321A7
Sfru P450 153 CYP4AU14
Sfru P450 117 CYP340L1
Sfru P450 078 CYP341B1
Sfru P450 138 CYP340L1
Sfru P450 139 CYP6B1
Sfru P450 022 CYP4S8
Sfru P450 148 CYP333B3
Sfru P450 111 CYP6AB61
Sfru P450 087 CYP340AB1
Sfru P450 006 CYP332A1
Sfru P450 140 CYP15C1
Sfru P450 141 CYP340G1
Sfru P450 114 CYP6AE50
Sfru P450 103 CYP340L1
Sfru P450 005 CYP4M17
Sfru P450 081 CYP340L1
Sfru P450 017 CYP340AB1
Sfru P450 118 CYP321A7
Sfru P450 045 CYP6AB12
Sfru P450 080 CYP6CV6
Sfru P450 157 CYP6AE43
Sfru P450 031 CYP321A8
Sfru P450 034 CYP6B1
Sfru P450 156 CYP4G75
Sfru P450 084 CYP340L1
Sfru P450 166 CYP367B1
Sfru P450 107 CYP4L9
Sfru P450 047 CYP6AB31
Sfru P450 108 CYP340L1
Sfru P450 124 CYP4C1
Sfru P450 075 CYP9A40
Sfru P450 055 CYP6AW1
Sfru P450 159 CYP367A1
Sfru P450 132 CYP340L1
Sfru P450 162 CYP4L9
Sfru P450 106 CYP9A21
Sfru P450 104 CYP340L1
Sfru P450 003 CYP321A15
Sfru P450 048 CYP340AA1
Sfru P450 133 CYP340L1
Sfru P450 030 CYP341D1
Sfru P450 098 CYP18A1
Sfru P450 015 CYP9A39
Sfru P450 150 CYP4G75
Sfru P450 089 CYP340L1
Sfru P450 073 CYP9A40
Sfru P450 028 CYP340AA1
Sfru P450 082 CYP340L1
Sfru P450 001 CYP306A1
Sfru P450 168 CYP367A6
Sfru P450 112 CYP6A13
Sfru P450 128 CYP340AA1
Sfru P450 086 CYP4C1
Sfru P450 074 CYP6A13
Sfru P450 085 CYP6B50
Sfru P450 009 CYP9A21
Sfru P450 113 CYP4G74
Sfru P450 092 CYP333A6
Sfru P450 115 CYP4L4
Sfru P450 052 CYP367B1
Sfru P450 070 CYP6AN4
Sfru P450 060 CYP321B1
Sfru P450 071 CYP307A1
Sfru P450 149 CYP333B4
Sfru P450 127 CYP6B48
Sfru P450 027 CYP6CT1
Sfru P450 151 CYP321A7
Sfru P450 014 CYP6AB60
Sfru P450 023 CYP340L1
Sfru P450 063 CYP340L1
Sfru P450 041 CYP341D1
Sfru P450 129 CYP6AB14
Sfru P450 096 CYP9G5
Sfru P450 056 CYP301A1
Sfru P450 147 CYP340AB1
Sfru P450 145 CYP339A1
Sfru P450 010 CYP49A1
Sfru P450 069 CYP340G1
Sfru P450 035 CYP340L1
Sfru P450 051 CYP340L1
Sfru P450 097 CYP321B1
Sfru P450 065 CYP4C1
Sfru P450 152 CYP6B58
Sfru P450 088 CYP354A14
Sfru P450 130 CYP340AB1
Sfru P450 053 CYP4CG1
Sfru P450 032 CYP332A1
Sfru P450 057 CYP303A1
Sfru P450 058 CYP4M18
Sfru P450 046 CYP9A59
Sfru P450 025 CYP338B42
Sfru P450 160 CYP340L1
Sfru P450 061 CYP340AA1
Sfru P450 012 CYP340AA1
Sfru P450 167 CYP6AN4
Sfru P450 164 CYP367A1
Sfru P450 033 CYP324A1
Sfru P450 099 CYP321A7
Sfru P450 091 CYP4C3
Sfru P450 136 CYP341B1
Sfru P450 126 CYP354A3
Sfru P450 122 CYP321A9
Sfru P450 037 CYP9A9
Sfru P450 026 CYP6B42
Sfru P450 036 CYP18B3
Sfru P450 142 CYP340G1
Sfru P450 123 CYP4C1
Sfru P450 011 CYP340AA1
Sfru P450 095 CYP6AB31
Sfru P450 143 CYP338A1
Sfru P450 072 CYP341B27
Sfru P450 021 CYP307A1
Sfru P450 066 CYP6AE43
Sfru P450 059 CYP4G74
Sfru P450 161 CYP321B1
Sfru P450 083 CYP4L10
1st instar larva
2nd instar larva
3rd instar larva
4th instar larva
5th instar larva
6th instar larva
F pupa
F adult
M adult
CYP4
Mito.
CYP3
CYP2
FIGURE 6 Gustatory receptor gene family expansion in fall armyworm. (a) Maximum-likelihood phylogenetic analysis of GR genes in fall
armyworm and silkworm. Two large bitter GR clusters on Chr9 and Chr24 and one sugar GR cluster are shown. (b) Distribution of 221 GR
genes in the fall armyworm chromosomes. The three main types of GR genes (sugar = green, bitter = purple, and CO2 = yellow) are indicated
in different colours
|
13
XIAO et Al .
S. frugiperda
B. mor
i
Sfru_GR_099
Sfru_GR_099
Sfru_GR_177
Sfru_GR_177
Sfru_GR_068
Sfru_GR_068
Sfru_GR_038
Sfru_GR_038
Sfru_GR_130
Sfru_GR_13
B
A
W33756.1
A
A
Sfru_GR_135
Sfru_GR_
B
A
W33754.1
A
A
Sfru_GR_161
u_GR_161
Sfru_GR_208
Sfru_GR_208
Sfru_GR_047
Sfru_GR_047
B
A
W33752.1
A
A
ACD85124.1
Sfru_GR_052
Sfru_GR_052
Sfru_GR_036
Sf
Sfru_GR_036
Sfru_GR_018
Sfru_GR_
Sfru_G
Sfru_GR_074
Sfru_GR_074
XP_021208998.1
XP_021208998.
021208998.
81
Sfru_GR_097
Sfru_GR_097
DAA06388.1
Sfru_GR_186
Sfru_GR_186
Sfru_GR_199
Sfru_GR_19
Sfru_GR_146
Sfru_GR_146
ACD85123.1
8
9
1
_
R
R
G
G
_
_
u
r
f
f
S
S
Sfru_GR_032
Sfru_GR_032
Sfru_GR_143
R_143
Sfru_GR_143
Sfru_GR_168
Sfru_GR_168
Sfru_GR_168
Sfru_GR_056
fru_GR_056
ACD85127.1
Sfru_GR_063
u_GR_063
Sfru_GR_121
Sfru_GR_12
Sfru_GR_144
Sfru_GR_144
XP_021207991.1
XP
XP_021207991.1
Sfru_GR_101
Sfru_GR_10
Sfru_GR_10
Sfru_GR_
fru_GR_
1
17
XP_012551334.1
XP_012551334.1
XP_012551334.1
DAA06377.1
D
Sfru_GR_057
u_GR_057
R_057
Sfru_GR_054
fru_GR_054
DAA06379.1
BAK52799.1
BAK5279
Sfru_GR_073
Sfru_GR_0
B
A
W33747.1
A
A
Sfru_GR_172
Sfru_GR_172
Sfru_GR_189
u_G
u_GR_189
Sfru_GR_025
Sfru_GR_
Sfru_GR_02
Sfru_GR_046
GR_046
Sfru_GR_046
1
1
4
4
1
_
_
R
G
_
_
u
r
f
S
XP_012546109.1
XP_012546109.1
Sfru_GR_217
Sfru_GR_2
Sfru_GR_215
Sfru_GR_2
Sfru_GR_154
fru_GR_154
fru_GR_154
Sfru_GR_043
_GR_043
Sfru_GR_209
Sfru_GR_209
B
A
W33757.1
A
A
Sfru_GR_148
u GR_148
Sfru_GR_072
Sfru_GR_072
Sfru_GR_041
u_GR_041
u_GR_04
Sfru_GR_145
Sfru_GR_145
Sfru_GR_145
XP_021208674.1
XP_021208674.
Sfru_GR_100
Sfru_GR_100
Sfru_GR_10
Sfru_GR_134
u_GR_134
XP_012551875.1
XP_0125
51
012551875.1
Sfru_GR_023
_ _023
023
ACD85125.1
ACD8
ACD8
Sfru_GR_127
S
Sfru_GR_12
Sfru_GR_051
Sfru_GR_05
Sfru_GR_126
fru_GR_126
126
Sfru_GR_164
S
Sfru_GR_164
S
ACD85122.1
122.1
122.1
Sfru_GR_005
Sfru_GR_005
Sfru_GR_180
Sfru_GR_18
Sfru_GR_18
Sfru_GR_149
9
Sfru_GR_149
Sfru_GR_214
fru_GR_214
fru_GR_214
214
Sfru_GR_150
Sfru_GR_150
XP_021209042.1
X
20904
0421
Sfru_GR_064
Sfru_GR_064
Sfru_GR_213
Sfru_GR_213
Sfru_GR_213
Sfru_GR_019
Sfru_GR_01
Sfru_GR_01
S
9
2
0
_
R
G
_
u
r
f
S
Sfru_GR_082
_GR_082
_GR_082
DAA06374.1
Sfru_GR_091
Sfru_GR_
DAA06375.1
Sfru_GR_219
ru_GR_219
Sfru_GR_178
Sfru_GR_178
Sfru_GR_178
DAA06396.1
DAA06387.1
Sfru_GR_194
Sfru_GR_19
Sfru_GR_055
u_GR_055
Sfru_GR_137
u_GR_137
u_GR_13
Sfru_GR_106
Sfru_GR_106
Sfru_GR_001
Sfru_GR_
Sfru G
Sfru_GR_059
Sfru_GR_059
Sfru_GR_166
Sfru_GR_166
Sf
fru_GR_166
Sfru_GR_128
Sfru_GR_128
Sfru_GR_162
fru_GR_162
162
Sfru_GR_026
Sfru_GR_026
DAA06385.1
Sfru_GR_081
ru_GR_081
Sfru_GR_
ru_GR_
ru_GR
1
1
16
16
Sfru_GR_167
fru_GR_167
Sfru_GR_105
ru_GR_105
Sfru_GR_105
Sfru_GR_155
155
u_GR_155
Sfru_GR_002
Sfru_GR_002
ACD85126.1
A
A
XP_004931409.2
XP_004931409.2
1
.
4
4
7
3
3
W
A
W
W
B
Sfru_GR_079
9
Sfru_GR_079
9
Sfru_GR_195
Sfru_GR_1
DAA06380.1
DAA06394.1
DA
DA
Sfru_GR_207
Sfru_GR_207
Sfru_GR_207
07
Sfru_GR_087
87
Sfru_GR_087
Sfru_GR_109
u_GR_109
109
Sfru_GR_109
Sfru_GR_
Sfru_GR_
Sfru_GR_
1
10
Sfru_GR_173
Sfru_GR_173
3
Sfru_GR_173
Sfru_GR_080
Sfru_GR_080
B
A
W33746.1
A
A
DAA06392.1
DAA0
Sfru_GR_220
Sfru_GR_2
Sfru_GR_2
DAA06395.1
B
A
W33755.1
A
A
Sfru_GR_184
Sfru_GR_184
S
Sfru_GR_151
_ _151
_ _151
Sfru_GR_169
Sfru_GR_
Sfu
Sfru_GR_
Sfru_GR_098
R_09
GR 098
R_09
Sfru_GR_175
Sfru
Sfru_GR_17
Sfru_GR_131
u_GR_131
R 131
Sfru_GR_010
u_GR_010
Sfru_GR_010
Sfru_GR_040
Sfru_GR_040
DAA06393.1
DAA0
DAA0
Sfru_GR_218
Sfru_GR_2
Sf G
NP_00
_00
_00
1
124346.1
Sfru_GR_132
Sfru_GR_13
Sfru_GR_061
Sfru_GR_
Sfru_GR_061
Sfru_GR_179
Sfru_GR_17
Sfru_GR_205
Sfru_GR_20
Sfru_GR_200
_200
Sfru_GR_200
Sfru_GR_153
Sfru_GR_153
Sfru_GR_035
Sfru_GR_03
Sfru_GR_187
Sfru_GR_187
Sfru_GR_075
Sfru_GR_075
S
B
B
A
A
W33749.1
W
A
A
A
A
Sfru_GR_156
Sfru_GR_
Sfru_GR_
Sfru_G
Sfru_GR_159
Sfru_
Sfru_GR_159
Sfru_GR_096
fru_GR_096
096
Sfru_GR_058
Sfru_GR_0
Sfru_GR_192
Sfru_GR_19
Sfru_GR_066
Sfru_GR_066
Sfru_GR_066
Sfru_GR_027
Sfru_GR_027
Sfru_GR_027
Sf
B
A
W33748.1
A
A
Sfru_GR_181
Sfru_GR_181
Sfru_GR_206
Sfru_GR
Sfru_GR_
Sfru_GR
Sfru_GR_
1
15
Sfru_GR_182
Sfru_GR_1
XP_012551881.1
XP_012551881.1
Sfru_GR_086
Sfru_GR_086
Sfru_GR_048
Sfru_GR_04
Sfru_GR_124
Sfru_GR_124
Sfru_GR_185
u_GR_1
Sfru_GR_1
Sfru
BAK52800.1
B
Sfru_GR_183
Sfru_GR_183
Sfru_GR_136
Sfru_GR_136
Sfru_GR_171
Sfru_GR_171
Sfru_GR_
Sfru_GR_
1
14
Sfru_GR_003
fru_GR_003
Sfru_GR_020
Sfru_GR_020
Sfru_GR_102
Sfru_GR_102
Sfru_GR_012
Sfru_GR_012
Sfru_GR_138
Sfru_GR_138
Sfru_GR_129
Sfru_GR_129
Sfru_GR_201
Sfru_GR_201
Sfru_GR_201
Sfru_GR_083
ru_GR_083
Sfru_GR_050
u_GR_050
Sfru_GR_062
Sfru_GR_
Sfru_GR_196
Sfru_GR_19
Sfru_GR_015
Sfru_GR_01
9
9
0
0
0
0
_
_
R
R
G
G
_
u
r
f
S
Sfru_GR_147
fru_GR_147
fru_GR_147
147
DAA06386.1
Sfru_GR_094
Sfru_GR_094
Sfru_GR_176
Sfru_GR_
Sfru_GR_060
Sfru_GR_060
NP_00
N
0
1
124345.1
51
Sfru_GR_030
R 030
_GR_030
Sfru_GR_008
Sfru_GR_008
Sfru_GR_152
Sfru_GR_152
Sfru_GR_039
Sfru_GR_039
DAA06389.1
NP_00
NP_00
NP_00
1
124347.1
1
Sfru_GR_024
Sfru_GR_024
24
Sfru_GR_022
Sfru_GR_02
Sfru_GR_108
8
fru_GR_108
DAA06381.1
DAA
Sfru_GR_125
Sfru_GR_125
Sfru_GR_142
Sfru_GR_142
Sfru_GR_174
Sfru_GR
Sfru_GR_071
1
_ _
Sfru_GR_037
_GR_037
Sfru_GR_090
Sfru_GR_
Sfru_GR_204
_GR_204
_GR_204
B
A
W33753.1
A
A
DAA06378.1
D
DAA
NP_001091791.1
NP_001091791.1
Sfru_GR_212
u_GR_212
Sfru_GR_
fru_GR_
Sfru_GR_
Sfru G
1
18
NP_001233217.1
1
P_001233217.1
Sfru_GR_092
Sfru_GR_09
Sfru_GR_0
Sfru_GR_0
1
1
XP_004932263.1
XP_004932263.1
Sfru_GR_016
Sfru_GR_016
Sfru_GR_
Sfru_GR_
Sfru_GR_
1
1
1
Sfru_GR_197
Sfru_GR_197
Sfru_GR_033
Sfru_GR_033
Sfru_GR_053
Sfru_GR_053
Sfru_GR_053
Sfru_GR_076
_GR_076
_
Sfru_GR_095
Sfru_GR_
B
A
W33750.1
A
A
Sfru_GR_045
Sfru_GR_045
Sfru_GR_203
Sfru_GR_203
DAA06384.1
Sfru_GR_123
Sfru_GR_123
Sfru_GR_034
Sfru_GR_034
Sfru_GR_170
R_170
u_GR 170
_170
Sfru_GR_004
Sfru_GR_00
Sfru_GR_00
Sfru_GR_190
90
Sfru_GR_190
90
NP_00
NP_00
1
124344.1
1
Sfru_GR_107
_GR_107
Sfru_GR_085
S
Sfru_GR_085
Sfru_GR_031
1
Sfru_GR_031
1
Sfru_GR_089
ru_GR_089
ru_GR_089
089
Sfru_GR_088
88
Sfru_GR_088
Sfru_GR_139
Sfru_G
Sfru_GR_139
Sf
Sfru_GR_157
Sfru_GR_15
Sfru_G _
Sf
Sfru_GR_093
fru_GR_093
093
Sfru_GR_103
fru_GR_103
Sfru_GR_103
103
BAK52798.1
Sfru_GR_210
Sfru_GR_210
Sfru_GR_133
Sfru_GR_133
Sfru_GR_140
40
ru_GR_140
DAA06391.1
DA
DA
Sfru_GR_070
Sf
Sfru_GR_070
DAA06390.1
Sfru_GR_216
Sfru_GR_
Sf GR
Sfru_GR_065
Sfru_GR_065
Sfru_GR_065
5
Sfru_GR_165
Sfru_GR_165
Sfru_GR_104
_GR_104
Sfru_GR_193
193
Sfru_GR_193
Sfru_GR_188
G _
Sfru_G _
Sfru_GR_017
Sfru_GR_017
DAA06376.1
D
D
Sfru_GR_122
Sfru_GR_122
Sfru_GR_078
Sfru_GR_0
S
Sfru_GR_0
BAS18817.1
1
Sfru_GR_202
Sfru_GR_2
Sfru_GR_158
Sfru_GR_15
Sfru
Sfru_GR_069
Sfru_GR_069
Sfru_GR_069
Sfru_GR_221
Sfru_G
Sfru_GR
Sfru_GR_013
Sfru_GR_013
Sfru_GR_2
Sfru_GR_2
1
1
Sfru_GR_
Sfru_GR_
Sfru GR_
Sf
1
1
1
12
Sfru_GR_120
R_
Sfru_GR_
Sfru_GR_
_GR_
Sfru_GR_
1
13
B
A
W33745.1
A
A
Sfru_GR_044
Sfru_GR_0
Sfru_GR_0
Sfru
XP_021207986.1
XP_021207986.1
XP_021207986.1
NP_001233216.1
NP_001233216.1
NP_001233216
Sfru_GR_021
GR 0
Sfru_G
Sfru_GR_0
Sfru_GR_191
u_G
u_GR_191
DAA06383.1
DAA
Sfru_GR_014
Sfru_GR_01
Sfru_GR_077
Sfru_GR_077
77
Sfru_GR_077
7
0
0
0
0
_
R
R
G
G
_
u
r
f
f
S
S
B
A
W33751.1
A
A
Sfru_GR_028
Sfru_GR_028
Sfru_GR_028
DAA06382.1
Sfru_GR_
Sfru_GR_
1
19
Sfru_GR_049
Sfru_GR_049
Sfru_GR_049
Sf
Sfru_GR_163
_ _ 63
_ _ 63
Sfru_GR_042
2
2
Sfru_GR_042
Sfru_GR_006
Sfru_GR_00
Sfr
Sfru_GR_160
Sfru_GR_160
Sfr
Sfru_GR_084
ru_G
ru_GR_084
Sfru_GR_067
Sfru
Sfru_GR_0
Sfru
CO
2
Sugar
Chr4 cluster
Chr9 cluster
Bitter
Chr24 cluster
1
(Z)
2345678910 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 ctg37
(W)
(a)
(b)
Suga
r
Bitte
r
CO2
14
|
XIAO et Al.
genes tended to be widely expressed in all developmental stages
(Table S21), suggesting the importance of P450 genes in all life
stages of the fall armyworm. Particularly in the P450 clan 3 and
clan 4, 51 P450 genes in clan 3 (including the family of CYP6AE,
CYP6B, CYP6AB, and CYP9) were expressed in all of the nine
stages (Tables S21 and S22). Meanwhile, 52 P450 genes (including
CYP4, CYP340 and CYP341 gene family) in clan 4 were expressed
in all nine stages (Tables S21 and S22). Most of the universally
expressed P450 genes are important in metabolizing plant second-
ary metabolites in insects. CY P6B1 can be induced to metabolize
xanthotoxin in Papilio polyxenes (Petersen, Niamsup, Berenbaum,
& Schuler, 2003). In Cnaphalocrocis medinalis, CYP6AE enzymes
are implicated in the detoxification of rice phytochemicals (Liu,
Chen, & Yang, 2010), and CYP6AE14 is involved in gossypol detox-
ification in H. armigera (Krempl et al., 2016). Similarly, plant hosts
of fall armyworm contain secondary metabolites that are toxic to
the insects, such as cyclic hydroxamic acids (CHx) in the maize,
wheat and rye; DIMBOA in maize (Kojima, Fujii, Suwa, Miyazawa,
& Ishikawa, 2010); hemiterpene aldehyde in cotton (Stipanovic,
Lopez, Dowd, Puckhaber, & Duke, 2006); and nicotine in tobacco
(Booker et al., 2010). The expansion of P450 in clan 3 and clan 4,
and the wide-spread expression of these P450 genes in almost all
developmental stages are probably important for fall armyworm
to detoxify the plant xenobiotics.
3.6 | Transcriptome and phylogenetic analysis of
gustatory receptors in fall armyworm
Chemoreception is vital for insects to quickly find plant hosts, and
is thus important for the spread and invasion of pest populations.
We have reported chemoreception genes from a previous genome
assembly from the Sf9 cell line (Liu, Xiao, et al., 2019). Here, with
the ZJ-version genome assembly, we identified 221 gustatory re-
cept ors ge nes wh ich in clu des 189 bit ter re cep tor s, 24 sugar recep-
tors and eight CO2 receptors using a manual annotation pipeline
(Tables S11 and S23). These numbers were much more than what
we identif ied from our previous studies. Phylogenetic analysis and
gene distribution analysis indicated that bitter receptors were sig-
nificantly expanded in the fall armyworm than in the silkworm.
Two large GR clusters were found on Chr9 and 24 (Figure 6a,b).
The sugar receptors were also tandemly duplicated on chromo-
some 4 of the fall armyworm (Figure 6a,b). Transcriptome analy-
sis indicated that 152 out of 189 bitter GR genes were detected
as expressed genes (FPKM > 0.4 in all three repetitions), the GR
genes tended to express in the adult (Figure 7), which is similar
to GR gene expression patterns in H. armigera (Xu, Papanicolaou,
Zhang, & Anderson, 2016). Fast host-recognition is important to
maintain the energy requirements for fall armyworm in long dis-
tance migration, the expansion of GR genes probably facilitates
host-recognition.
3.7 | The expansion of β-fructofuranosidase genes
As a sucrase, β-FFase, is responsible for cleaving sucrose to main-
tain cell met abolism and grow th in bac teria and plants an d was as-
sumed to have not existed in animals for many years (Koch, 2004;
Liebl, Brem, & Gotschlich, 1998). Recent studies show that in
insects, β-FFase genes were acquired via horizontal gene trans-
fer from bacteria and function in insect avoidance of plant sec-
ondary metabolites and glycometabolism modulation (Daimon
FIGURE 7 Expression profiles of fall armyworm GR genes from individuals at different developmental stages. The GR genes tended to
express in the adult. F, female; M, male
Sfru GR 170
Sfru GR 139
Sfru GR 146
Sfru GR 152
Sfru GR 174
Sfru GR 009
Sfru GR 049
Sfru GR 128
Sfru GR 194
Sfru GR 083
Sfru GR 206
Sfru GR 088
Sfru GR 027
Sfru GR 021
Sfru GR 078
Sfru GR 105
Sfru GR 053
Sfru GR 217
Sfru GR 076
Sfru GR 104
Sfru GR 172
Sfru GR 164
Sfru GR 039
Sfru GR 106
Sfru GR 050
Sfru GR 161
Sfru GR 186
Sfru GR 188
Sfru GR 073
Sfru GR 007
Sfru GR 201
Sfru GR 182
Sfru GR 218
Sfru GR 180
Sfru GR 160
Sfru GR 112
Sfru GR 149
Sfru GR 220
Sfru GR 070
Sfru GR 158
Sfru GR 126
Sfru GR 185
Sfru GR 195
Sfru GR 145
Sfru GR 091
Sfru GR 008
Sfru GR 216
Sfru GR 211
Sfru GR 012
Sfru GR 143
Sfru GR 177
Sfru GR 024
Sfru GR 032
Sfru GR 026
Sfru GR 081
Sfru GR 103
Sfru GR 018
Sfru GR 056
Sfru GR 166
Sfru GR 189
Sfru GR 165
Sfru GR 171
Sfru GR 151
Sfru GR 107
Sfru GR 131
Sfru GR 067
Sfru GR 079
Sfru GR 048
Sfru GR 037
Sfru GR 129
Sfru GR 200
Sfru GR 003
Sfru GR 055
Sfru GR 120
Sfru GR 045
Sfru GR 179
Sfru GR 154
Sfru GR 033
Sfru GR 084
Sfru GR 061
Sfru GR 144
Sfru GR 191
Sfru GR 136
Sfru GR 041
Sfru GR 114
Sfru GR 011
Sfru GR 036
Sfru GR 074
Sfru GR 156
Sfru GR 062
Sfru GR 207
Sfru GR 212
Sfru GR 058
Sfru GR 043
Sfru GR 015
Sfru GR 031
Sfru GR 028
Sfru GR 208
Sfru GR 140
Sfru GR 117
Sfru GR 181
Sfru GR 137
Sfru GR 077
Sfru GR 157
Sfru GR 004
Sfru GR 085
Sfru GR 016
Sfru GR 119
Sfru GR 199
Sfru GR 060
Sfru GR 215
Sfru GR 001
Sfru GR 196
Sfru GR 121
Sfru GR 019
Sfru GR 072
Sfru GR 093
Sfru GR 068
Sfru GR 183
Sfru GR 042
Sfru GR 109
Sfru GR 173
Sfru GR 040
Sfru GR 086
Sfru GR 094
Sfru GR 203
Sfru GR 046
Sfru GR 153
Sfru GR 095
Sfru GR 155
Sfru GR 163
Sfru GR 118
Sfru GR 071
Sfru GR 069
Sfru GR 080
Sfru GR 122
Sfru GR 187
Sfru GR 125
Sfru GR 135
Sfru GR 190
Sfru GR 141
Sfru GR 023
Sfru GR 123
Sfru GR 176
Sfru GR 159
Sfru GR 059
Sfru GR 192
Sfru GR 198
Sfru GR 116
Sfru GR 168
Sfru GR 014
Sfru GR 017
Sfru GR 202
Sfru GR 102
Sfru GR 022
Sfru GR 110
Sfru GR 047
Sfru GR 221
Sfru GR 169
Sfru GR 219
Sfru GR 052
Sfru GR 029
Sfru GR 087
Sfru GR 097
Sfru GR 044
Sfru GR 178
Sfru GR 113
Sfru GR 013
Sfru GR 124
Sfru GR 064
Sfru GR 132
Sfru GR 167
Sfru GR 065
Sfru GR 209
Sfru GR 210
Sfru GR 063
Sfru GR 101
Sfru GR 054
Sfru GR 148
Sfru GR 096
Sfru GR 020
Sfru GR 005
Sfru GR 066
Sfru GR 034
Sfru GR 108
Sfru GR 130
Sfru GR 075
Sfru GR 184
Sfru GR 090
Sfru GR 100
Sfru GR 134
Sfru GR 147
Sfru GR 051
Sfru GR 030
Sfru GR 205
Sfru GR 175
Sfru GR 213
Sfru GR 082
Sfru GR 138
Sfru GR 025
Sfru GR 111
Sfru GR 193
Sfru GR 127
Sfru GR 057
Sfru GR 133
Sfru GR 038
Sfru GR 204
Sfru GR 002
Sfru GR 089
Sfru GR 006
Sfru GR 098
Sfru GR 115
Sfru GR 150
Sfru GR 099
Sfru GR 035
Sfru GR 197
Sfru GR 142
Sfru GR 162
Sfru GR 010
Sfru GR 092
Sfru GR 214
Bitter Suger
CO2
1st instar larva
2nd instar larva
3rd instar larva
4th instar larva
5th instar larva
6th instar larva
F pupa
F adult
M adult
GR gene expression
Low High
|
15
XIAO et Al .
et al., 2008; Gan et al., 2018; Zhao, Doucet, & Mittapalli, 2014).
In the fall armyworm, five β-FFase genes (Sf ruSuc1, SfruSuc2,
SfruSuc3, SfruSuc4 and SfruSuc5) were identified (Figure S5) and
they exhibited significant gene expansion compared with those
of the silkworm. Only one β-FFase gene, BmSuc1, was identified
in the silkworm to facilitate the avoidance of the toxic effects of
alkaloidal sugar mimic glycosidases, such as 1, 4-dideoxy-1, 4-im-
ino-D-arabinitol (D-AB1) and 1-deoxynojirimycin (DNJ) (Daimon
et al., 2008). The β-FFase gene exp ansion might be an effi cient so-
lution for the fall armyworm to adapt and tolerate a high number
of plant secondary metabolites, as well as maintain the balance in
glucose metabolism.
Transcriptome analysis showed that all five β-FFase genes were
expressed in different developmental stages (Figure S5). SfruSu c1
(the orthologue gene of BmSuc1) was highly expressed in the six instar
larvae and pupa, because the six instar larvae feeding on the maxi-
mum amount of food. This result is consistent with our result from
SfruSuc5 (Figure S5) and that from the study of Pedezzi et al. (2014)
study of Sl-β-fruct in Sphenophorus levis. In contrast, SfruSuc2 highly
expressed from the first instar to the fifth instar larva, and SfruSuc3
and SfruSuc4 highly expressed in the adult (Figure S5).
In conclusion, we present data from a chromosome-level genome
assembly of fall armyworm and validated the genome, ZJ-version,
as a C strain. Furthermore, cytochrome P450 gene family, gusta-
tory receptors, and β-fructofuranosidase genes were significantly
expanded in the fall armyworm, revealing the genetic adaptations
which may have facilitated the recent rapid invasion of this notorious
insect pest worldwide.
ACKNOWLEDGEMENTS
This work was supported by Key Project of Zhejiang Provincial
Natural Science Foundation (s) and the National Science Foundation
of China (31972354 and 31760514). We greatly thank the handling
editor and anonymous reviewers for their critical comments in im-
proving our manuscript.
CONFLICT OF INTEREST
The authors have no competing interests to declare.
AUTHOR CONTRIBUTIONS
F.L. conceived and designed the whole project. X.H.Y., and Y.M. as-
sembled and annotated the genome. X.H.Y., and Y.Y. performed the
comparative genomics analysis. Y.M., and H.M.X. performed the
gene family expression analysis and improved the figures. X.C. per-
formed the sex chromosome analysis. H.X.X., Z.X.L., and Y.J.Y. col-
lected and provided all insect samples. H.M.X., and X.H.Y. prepared
the DNA and RNA samples for sequencing. T.L., Y.Y.Y., and W.F.Y.
contributed to the genome sequencing. X.H.Y. drafted a part of the
manuscript. Z.X.L contributed his efforts (discussion and editing the
manuscript) in the first-round of manuscript revision. H.M.X., and
F. L. improved the whole manuscript. All authors approved the final
manuscript.
DATA AVAILAB ILITY STATE MEN T
The Whole Genome Shotgun project has been deposited at DDBJ/
ENA/GenBank under the accession WMCG00000000. The version
described in this paper is version WMCG01000000. The raw ge-
nome and transcriptome data are publicly available at NCBI with the
BioProject accession number PRJNA590312. All data mentioned in
this paper can also be accessed at http://www.insec t-genome.com/
Sfr u /.
ORCID
Huamei Xiao https://orcid.org/0000-0003-0165-7410
Xinhai Ye https://orcid.org/0000-0002-0203-0663
Fei Li https://orcid.org/0000-0002-8410-5250
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SUPPORTING INFORMATION
Additional supporting information may be found online in the
Supporting Information section.
How to cite this article: XiaoH, Ye X, Xu H, et al. The genetic
adaptations of fall armyworm Spodoptera frugiperda
facilitated its rapid global dispersal and invasion. Mol Ecol
Resour. 2020;00:1–19. https://doi.org/10.1111/1755-
0998 .13182
