Genetic Adaptations Spodoptera Global Dispersal Invasion Xiao 2020

Accepted Article
DR. HUAMEI XIAO (Orcid ID : 0000-0003-0165-7410)
DR. XINHAI YE (Orcid ID : 0000-0002-0203-0663)
PROF. FEI LI (Orcid ID : 0000-0002-8410-5250)
Article type : Resource Article
The genetic adaptations of fall armyworm Spodoptera frugiperda
facilitated its rapid global dispersal and invasion
Huamei Xiao 1,2, #,, Xinhai Ye 2, #, Hongxing Xu3, #, Yang Mei2, #, Yi Yang2, Xi Chen2, Yajun
Yang3, Tao Liu4,, Yongyi Yu4, Weifei Yang4, Zhongxian Lu3, Fei Li2,
1College of Life Sciences and Resource Environment, Key Laboratory of Crop Growth and
Development Regulation of Jiangxi Province, Yichun University, Yichun 336000, China.

2State Key Laboratory of Rice Biology & Ministry of Agricultural and Rural Affairs Key Laborato
ry of Molecular Biology of Crop Pathogens and Insects, Institute of Insect Sciences, Zhejiang Uni
versity, Hangzhou, 310058, China.

3Institute of Plant Protection and Microbiology, Zhejiang Academy of Agricultural Sciences,
Hangzhou, 310021, China.

4Annoroad Gene Technology (Beijing) Co., Ltd, Beijing, 100176, China
#These authors contributed equally to this work.
Corresponding author: Dr. Fei Li, Email: lifei18@zju.edu.cn, Dr. Huamei Xiao, Email:
xiaohuamei625@163.com; Dr. Tao Liu, Email: taoliu@genome.cn;
This article has been accepted for publication and undergone full peer review but has not been
through the copyediting, typesetting, pagination and proofreading process, which may lead to
differences between this version and the Version of Record. Please cite this article as doi:
10.1111/1755-0998.13182
This article is protected by copyright. All rights reserved
Abstract
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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 understood. 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 heterozygosity. 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 chromosomes
were identified by genome re-sequencing 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 armyworm. Transcriptomic and phylogenetic analyses focused on these
gene families revealed the potential roles of the genes in polyphagia and invasion of fall
armyworm. 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.
Key words: Fall armyworm, Chromosome-level genome, Comparative genomics, Polyphagia,
Insecticide resistance

1 Introduction
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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 to 20.6 million 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 (Prasanna, Huesing, Eddy, & Peschke, 2018). In addition, 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 reportedly dispersed to a record distance of 1,600 km in 30
hours (Martinelli et al., 2006).
The fall armyworm survives year-round in the tropical and subtropical areas of the Americas
and was also wide spread in the northern areas of the United States and as far north as southern
Canada (Todd & Poole, 1980) before 2016. In January 2016, an outbreak of this pest occurred in
the rainforest zone of South-Western Nigeria of Africa (Goergen, Kumar, Sankung, Togola, &
Tamo, 2016). Forty-four 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 armyworm 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 armyworm 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 August 17, 2019, the fall

armyworm had spread to 1,366 counties (cities and districts) across 24 provinces of China
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(NATESC, 2019b). Such a rapid spread of fall armyworm 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 chemical insecticides and transgenic
Bacillus thuringiensis (Bt) crops, and reproductive behaviors (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 (Tpi) (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 indiscriminate use of
insecticides and transgenic Bt corn has led to the development 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
chlorpyriphos, 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).
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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 less than 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
et al., 2019; Nam et al., 2019; Zhang et al., 2019). In addition, all of these versions 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 chromosome-level genome of a female pupa collected from
a corn field 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 genome available to date. Furthermore, we assembled a portion of the W
chromosome. To the best of our knowledge, this is the first report of W chromosome sequence
from the fall armyworm. We also analyzed 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 genomic analysis provide new insights into the mechanism of fall armyworm
invasion, polyphagia and insecticide resistance.
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℃, 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, California, USA) to sequence

the genome of the fall armyworm. High-quality genomic DNA was extracted from a female pupa
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(Supporting Information Table S1) using a DNeasy Blood & Tissue Kit (QIAGEN, Hilden,
Germany). The integrity of the DNA was determined with the Agilent 4200 Bioanalyzer (Agilent
Technologies, Palo Alto, California, USA). Genomic DNA was sheared using g-Tubes (Covaris,
Woburn, USA), and concentrated with AMPure PB magnetic beads (Pacific Biosciences,
California, USA). The SMRT bell library was constructed using the PacBio SMRTbell Express
Template Prep Kit 2.0 (Pacific Biosciences, California, USA). Finally, one SMRT cell was run for
genome sequencing. PacBio sub-reads were initially cleaned using Canu v1.8 (Koren et al., 2017)
(https://github.com/marbl/canu) for sequence error correction. Then, the PacBio corrected reads
were assembled by SMARTdenovo (https://github.com/ruanjue/smartdenovo) as described by
Istace et al. (2017). The redundans pipeline (https://github.com/lpryszcz/redundans) (Pryszcz &
Gabaldón, 2016) was used to remove the redundant contigs from the initial de novo assembly
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 (Supporting Information 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 minutes. The reaction
was quenched by 5 minute incubation with 2.5M glycine solution.
For extracting the chromatin, the supernatant was removed and the tissues were grounded in
liquid nitrogen. The tissues were resuspended 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, Missouri, USA) and then was filtered
through miracloth (Calbiochem, Darmstadt, Germany). The filtrate was centrifuged at 4000 rpm at
4℃ 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, Missouri, USA),
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followed by centrifuging at 14,000 rpm at 4℃ 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, Missouri, USA). The solution was centrifuged at 14,000 rpm at 4℃ for
10 min.
The chromatin was washed with the following steps. First, removing the supernatant and
resuspending the pellet in 500 μl precooling 1x CutSmart buffer, washing it for twice, and then
centrifuged 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℃ for 10 min. After quenching the SDS by Triton X-100, the chromatin was
digested with 400 units MboI at 37℃ on a rocking platform for overnight.
Next, the DNA was labelled with biotin-14-dCTP (Invitrogen, California, USA) and
blunt-end ligated with crosslinked fragments. Then, the proximal chromatin DNA was re-ligated
by ligation enzyme at 16℃ for overnight. The nuclear complexes were reversed crosslinked by
incubating with proteinase K (Invitrogen, California, USA) at 65℃. DNA was purified with
phenol chloroform extraction method. Biotin-C was removed from non-ligated fragment ends
using T4 DNA polymerase (NEB, Massachusetts, USA). Fragments was sheared to a size of
100-500 base pairs by sonication. The fragment ends were repaired by the mixture of T4 DNA
polymerase (NEB, Massachusetts, USA), T4 polynucleotide kinase (NEB, Massachusetts, USA)
and Klenow DNA polymerase (NEB, Massachusetts, USA). Biotin labeled 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 (Beijing, China).
2.4 Scaffolding with Hi-C

Hi-C scaffolding was performed according to the pipeline reported in Servant et al. (2015)
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and Burton et al. (2013). The HiC-Pro v2.7.8 (Servant et al., 2015) pipeline
(https://github.com/nservant/HiC-Pro) was used to identify valid read pairs. In this pipeline, each
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 Bowtie2 v2.2.3 (Langmead & Salzberg, 2012) with the parameters
“--very-sensitive -L 20 --score-min L,-0.6,-0.2 --end-to-end --reorder --rg-id BMG
--phred33-quals -p 5”. The processed 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
assembly contigs into chromosome-scale scaffolds (hereafter pseudo-chromosomes) with
LACHESIS (https://github.com/shendurelab/LACHESIS) (Burton et al., 2013). To access the
accuracy of the scaled-up genome assembly, we cut the pseudo-chromosomes 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
produced by HiC-Pro and then visualized as a heatmap to show the diagonal patches of strong
linkages.
2.5 Transcriptome sequencing and analysis
The transcriptomes of the larva (from first instar to six instar), female pupa, female adult and
male adult of fall armyworm (Supporting Information 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). Then, the clean reads were mapped to
the genome assembly using Hisat2 v2.1.0 (Kim, Landmead, & Salzberg, 2015) and StringTie 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 Bowtie2 v2.3.5 (Langmead &
Salzberg, 2012), and transcript abundances were estimated by RSEM v1.3.1 (Li & Dewey, 2011).

2.6 Assessment of genome assembly
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We used the BUSCO v3.0 (Waterhouse et al., 2018) (Benchmarking Universal Single-Copy
Orthologs) software to scan 1,658 universal 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 predictions,
RepeatMasker v4.0.5 (Tarailo-Graovac & Chen, 2009) was used with Repbase library (Bao,
Kojima, & Kurtz, 2015).
We annotated the protein coding genes by integrating the evidence of de novo,
homology-based and RNA-Seq-based annotations. First, Augustus v2.5.5 (Stanke, Diekhans,
Baertsch, & Haussler, 2008) and SNAP v2013-11-29 (Korf, 2004) were used to generate the de
novo annotation with internal gene models. Then, Exonerate v2.2.0 (Slater & Birney, 2005) and
GenomeThreader 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/refseq/) to the
genome assembly with default parameters. The transcripts of the fall armyworm were obtained by
Hisat2 v2.1.0 (Kim et al., 2015) and StringTie v2.0 (Pertea et al., 2015) pipeline with default
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 Blast2GO 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 BlastKOALA 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
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study), Spodoptera litura (GCA_002706865.2, from NCBI), Trichoplusia ni (GCF_003590095.1,
from NCBI), Helicoverpa armigera (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/dataset/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/dataset/100538),
Drosophila melanogaster (GCF_000001215.4, from NCBI), Anopheles gambiae (from
VectorBase, https://www.vectorbase.org/organisms/anopheles-gambiae), Tribolium castaneum
(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.ensemblgenomes.org/pub/metazoa/release-38/fasta/nasonia_vitripennis/dna/),
Melanaphis sacchari (GCF_002803265.2, from NCBI) and Rhodnius prolixus (from VectorBase,
https://www.vectorbase.org/organisms/rhodnius-prolixus).
In total, 328 single-copy genes were obtained from OrthoMCL results and were used for
phylogeny reconstruction. First, the protein 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 bootstrap replicates. To estimate the
divergence time of the fall armyworm, we applied three calibration points based on fossil records
in Paleobiology Database (www.paleobiology.org): 1) stem Trichoptera (Phryganea solitaria) at
311.45–314.6 mya; 2) stem Lepidoptera (fossil unnamed) at 201.3–208.5 mya; and 3) stem

Noctuoidea (Noctuites incertissima) at 28.1–33.9 mya. The divergence time was estimated by
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using MCMCtree in PAML v4.9e (Yang, 2007) with the topology of these insects we built above.
The tree was visualized using FigTree v1.4.4 (http://tree.bio.ed.ac.uk/software/figtree/).
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 package of SPINES with default parameters
(https://www.broadinstitute.org/genome-sequencing-and-analysis/spines). Synteny blocks were
plotted across chromosomes using Circos 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 protein sequences from twenty-two insects were aligned
to the TreeFam v9 (Schreiber, Patricio, Muffato, Pignatelli, & Bateman, 2014) database to obtain
the TreeFam ID for each protein. The TreeFam v9 results 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 (blast 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 Exonerate v2.2.0 (Slater & Birney, 2005) were used to define the gene structure.
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
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regarded as expressed in this development 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) (blast 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 annotation, 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 transporters (ABC transporters), olfactory receptors (ORs),
ionotropic receptors (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 manually confirmed. Then, we used
BLASTP to determine candidate sequences 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 MAFFT 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
(1000 ultrafast bootstrap approximation replicates) (Kalyaanamoorthy et al., 2017). The tree was
visualized using FigTree v1.4.4 (http://tree.bio.ed.ac.uk/software/figtree/). 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 armyworm that has invaded

the Zhejiang province of China. We identified the Tpi gene and determined the strain from the
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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 armyworm, one female
pupa and one male pupa were re-sequenced using Illumina HiSeq platforms to obtain an
approximate 40X coverage. 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/schatzlab/genomescope; Vurture, et al., 2017) for assessment of genomic
heterozygosity 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 between
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 fqtools v0.1.8 (Droop, 2016), genome re-sequencing
reads were aligned to the fall armyworm genome assembly using Bowtie2 v2.3.5 (Langmead &
Salzberg, 2012) with default 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-project.org/package=changepoint).
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 selection analysis. Protein sequences of each single-copy
gene family were aligned using MAFFT v7 (Katoh & Standley, 2013), and then the protein
alignments were converted to their corresponding nucleotide alignments by the Perl script
PAL2NAL v14 (Suyama, Torrents & Bork, 2006). The dN/dS ratio was estimated for each
homologous 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
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The GO and KEGG enrichment analyses were conducted using Omicshare CloudTools under
this tool’s default instructions (http://www.omicshare.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 sequencing by PacBio long-read
technology, yielding ~126 Gb PacBio sub-reads. The PacBio reads were self-corrected using Canu
v1.8 (Koren et al., 2017) and finally assembled into 361 contigs using SMARTdenovo
(Supporting Information Table 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 assembled 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% (Supporting Information Figure S1). An inflated
assembly size relative to k-mer based estimates has also been observed in other highly
heterozygous species assemblies such as Ancherythroculter nigrocauda, Pyrocoelia pectoralis,
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 genome size variation might be due to the following reasons: (1) different sequencing
technologies, sequencing depth, and assembly approaches and (2) fall armyworm samples are
from different areas/habitats. It has been reported that variable genome size of different strains
within the same species may be a result of the amplification, 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.
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According to our Hi-C interaction information, we cut the primary assembly into 618 contigs,
then anchored 556 (97.4% in length) contigs to 31 chromosomes (30 autosomes and Z
chromosome) (Fig. 1A, Supporting Information Table S4-S7, and Supporting Information Figure
S2). Hi-C correction and scaffolding 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. armigera (Pearce
et al., 2017) and S. litura (Cheng et al., 2017) (Supporting Information Table 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 indicated that 93.1% complete genes (1,658 universal single-copy orthologous 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 et al. (2019) (2.8% in male and 3.1% in female) (Supporting
Information Table S5). The complete and duplicated BUSCO component of the ZJ-version
genome was 20.3%, which was higher than that in Liu 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 armyworm genome assembly will provide a solid foundation for future
analyses.
Using the genome re-sequencing data of a single male pupa and a single female pupa, we
calculated the sequencing coverage of each scaffold and identified the Z chromosome and a
portion of the W chromosome (Fig. 1B, Supporting Information 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 (Fig. 1B, Supporting Information Table S9).

Because the Lepidopteran W chromosome is enriched in repeat sequences, it is difficult to
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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. pomonella with a
length of about 5 Mb (Wan et al., 2019), as well as from 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 genome conservation at the chromosome level in Noctuidae insects
(Fig. 2A and 2B, Supplementary Information Table S10). The syntenic relationship between the
silkworm 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 (Fig. 2C, Supporting Information 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) (Supporting Information
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 (Supporting
Information 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
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in five previously published versions of the genome, but more than that in the genome of Sf21 cell
line. Furthermore, we manually annotated several 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 (Supporting Information Table 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 include the sites E4-165,
E4-168 and E4-183 (Nagoshi, 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 (Supporting Information Figure S3) (Liu et al., 2019; Zhang et al.,
2019).
3.4 Gene orthologs and comparative genomic analysis
Comparative genomics analysis was carried out using 22 insect genomes covering six insect
orders (Lepidoptera, Trichoptera, Diptera, Coleoptera, Hymenoptera, and Hemiptera). A
phylogenetic tree was constructed using 238 single-copy genes (Fig. 3). In addition, 3,076 N: N: N
genes, 6,160 Lepidoptera-specific genes, and 2,608 Noctuidae-specific genes were identified (Fig.
3, Supporting Information 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
(Supporting Information Table S12) and 494 were found to be tandemly duplicated on the
chromosomes (Supporting Information 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 ancestor approximately 9.8 Mya ago (Fig. 3).
The gene family evolution analysis indicated that the fall armyworm genome displayed 774
expanded and 1,048 contracted gene families compared with gene families of the common
ancestor of fall armyworm and S. litura (Fig. 3). The common ancestor of Noctuidae species

showed 449 expanded and 288 contracted gene families compared to that of the common ancestor
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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 x 10-19, Hypergeometric test, FDR-adjusted), glycerolipid metabolism (ko00561, p =
2.63951 x 10-18), and fructose and mannose metabolism (ko00051, p = 3.279737 x 10-9)
(Supporting Information Table S13-S14). In addition, the fall armyworm expanded gene families
were enriched not only in nutrition metabolism but also in transport system, such as ABC
transporters (ko02010, p = 3.012781 x 10-3) (Supporting Information Table S15-S16). Noctuidae
diverged from the Bombycoidae superfamily 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 metabolism
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.ni and H. armigera), 5,410 single-copy genes were used for positive
selection analyses. As a result, we identified 835 positive selected genes in the fall armyworm
using the Branch-site model in PAML, including the GRs (p < 0.05, FDR-adjusted, Supporting
Information Table S17). The GO and KEGG enrichment analyses indicated that the significant
terms and pathways were involved in metamorphosis (GO: 0007552, p = 1.813556 x 10-4), instar
larval or pupae development (GO: 0002165, p = 1.824223 x 10-3), glycerophospholipid
metabolism (ko00564, p = 0.01394475) and sphingolipid metabolism (ko00600, p = 0.01753068)
(Supporting Information Table S18-S19).
3.5 The expansion and wide-spread expression of cytochrome P450 gene family in fall
armyworm
Insect pests, especially the polyphagous insects, can adapt to tolerate the plant toxic defense

chemicals induced after the insect feeds on the host (Gatehouse, 2002). These insects have
Accepted Article
evolved a strong detoxification system in response to the plant defense system, such as
overproducing detoxification enzymes to metabolize the toxins (Despres, David, & Gallet, 2007)
and enhancing the excretion activity (Dermauw & Van Leeuwen, 2014). Using comparative
genomics and a BAC library, Giraudo et al. (2015) identified 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, Ye, Xiao, & Li,
2019). Here, with the ZJ-version chromosome-level genome, we predicted 169 cytochrome P450
genes by TBLASTN and Genewise (Supporting Information 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 armyworm
comparing with that in the model insect of Lepidoptera, the silkworm (Fig. 4A, Supporting
Information Table S20). However, P450 clans Mito and 2 were strongly conserved between the
fall armyworm and silkworm (Fig. 4A, Supporting Information 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 armyworm genome (Fig. 4B). The largest P450 cluster was
located on Chr14 and consisted of 39 CYP340 genes (Fig. 4A and 4B).
We used the RNA-Seq data, which covered major developmental 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 repetitions). Moreover, the
CYP321A (7-9) gene family tended to express in the 5th and 6th instar larva (Fig. 5) and
CYP321A1 reportedly is induced to metabolize xanthotoxin in Helicoverpa zea (Rupasinghe, Wen,
Chiu, & Schuler, 2007). We found that P450 genes tended to be widely expressed in all
developmental stages (Supporting Information 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
Accepted Article
in all of the nine stages (Supporting Information Table S21 and Table S22). Meanwhile, 52 P450
genes (including CYP4, CYP340 and CYP341 gene family) in clan 4 were expressed in all nine
stages (Supporting Information Table S21 and Table S22). Most of the universally expressed P450
genes are important in metabolizing plant secondary metabolites in insects. CYP6B1 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 detoxification 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 likely
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 et al., 2019). Here, with the ZJ-version genome
assembly, we identified 221 gustatory receptors genes which includes 189 bitter receptors, 24
sugar receptors and 8 CO2 receptors using a manual annotation pipeline (Supporting Information
Table S11 and S23). These numbers were much more than what we identified from our previous
studies. Phylogenetic analysis and gene distribution analysis indicated that bitter receptors were
significantly expanded in the fall armyworm than in the silkworm. Two large GR clusters were
found on Chr9 and 24 (Fig. 6A and 6B). The sugar receptors were also tandemly duplicated on
chromosome 4 of the fall armyworm (Fig. 6A and 6B). Transcriptome analysis 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 (Fig. 7), which is similar to GR gene expression
Accepted Article
patterns in H. armigera (Xu, Papanicolaou, Zhang, & Anderson, 2016). Fast host-recognition is
important to maintain the energy requirements for fall armyworm in long distance migration, the
expansion of GR genes likely facilitates host-recognition.
3.7 The expansion of β-fructofuranosidase genes
As a sucrase, β-FFase, is responsible for cleaving sucrose to maintain cell metabolism and
growth in bacteria and plants and was assumed 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 transfer from bacteria and function in insect avoidance of
plant secondary metabolites and glycometabolism modulation (Daimon et al., 2008; Gan et al.,
2018; Zhao, Doucet, & Mittapalli, 2014). In the fall armyworm, five β-FFase genes (SfruSuc1,
SfruSuc2, SfruSuc3, SfruSuc4 and SfruSuc5) were identified (Supporting Information 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-imino-D-arabinitol
(D-AB1) and 1-deoxynojirimycin (DNJ) (Daimon et al., 2008). The β-FFase gene expansion
might be an efficient solution 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 (Supporting Information Figure S5). SfruSuc1 (the ortholog gene of
BmSuc1) was highly expressed in the six instar larvae and pupa, because the six instar
larvae feeding on the maximum amount of food. This result is consistent with our result from
SfruSuc5 (Supporting Information Figure S5) and that from Pedezzi et al.’s (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 (Supporting
Information Figure S5).

Conclusion
Accepted Article
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, gustatory
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 world-wide.
Acknowledgements
This work was supported by Key Project of Zhejiang Provincial Natural Science Foundation
(LZ18C060001) and the National Science Foundation of China (31972354 and 31760514). We
greatly thank the handling editor and anonymous reviewers for their critical comments in
improving 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. assembled 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. performed the sex
chromosome analysis. H.X.X., Z.X.L and Y.J.Y. collected 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 Accessibility Statement
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 genome and transcriptome data are publicly available at NCBI with the BioProject accession
Accepted Article
number PRJNA590312. All data mentioned in this paper can also be accessed at
http://www.insect-genome.com/Sfru/.
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Figures and Tables
Accepted Article
Figure legend
Fig. 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 non-overlapping windows; IV, GC
contents (green) across the genome, drawn in 0.1 Mb non-overlapping 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.
Fig. 2 Whole-genome synteny between S. frugiperda and S. litura, T. ni and B. mori. The fall
armyworm genome shares high synteny with S. litura (A), T. ni (B). 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 (C). Synteny analysis was carried out using Satsuma and viewed with
Circos v 0.69.
Fig. 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 1000 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 TreeFam and Café 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.

Fig. 4 Cytochrome P450s in fall armyworm. (A) Maximum-likelihood phylogenetic analysis of
Accepted Article
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.
Fig. 5 Expression profiles of fall armyworm P450 genes from individuals at different
developmental stages. F: female; M: male.
Fig. 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 colors.
Fig. 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.

Accepted Article
Table 1. Comparison of fall armyworm genome assemblies of this and previous studies.
Nam et al., Nandakuma Kakumani
ZJ-version Liu et al., 2019 Gouin et al., 2017
2019 Zhang et al., 2019 r et al., 2017 et al., 2014
(This study)
C strain SFynMstLFR SFynFMstLFR C strain R strain Sf9 Sf21
Sequencing info
Single female Fourth instar Single female Fourth instar Single Sf21 cell
DNA source Single male moth Single male adult Sf9 cell line
pupa male larvae adult male larvae male larva line
PacBio + PacBio + Illumina
Assembly approach PacBio + Hi-C Illumina + + Hi-C MGISEQ + Hi-C MGISEQ + Hi-C Illumina Illumina PacBio Illumina
Hi-C
Genome assembly
Chromosomes Chromosomes Chromosomes Chromosomes Chromosomes Scaffolds Scaffolds Scaffolds Scaffolds
Assembly level
(30A+Z+W) (30A+Z) (30A+Z) (30A+Z) (30A+Z)
Genome size (Mb) 486.3 384.46 393.25 542.4 530.8 437.9 371.0 514.2 358.0
Number of contigs 618 - 777 - - - - 2,844 97,607
Number of scaffolds 93 125 311 - - 41,562 29,127 2,396 37,235
Gaps number 525 - - - - 13,694 3,818 2,635 95,454
Gap length (kb) 53 346 - 37,947 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 14,883.7 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 - 29.2 29.1 28.1 20.3
SINEs (%) 0.7 - 1.0 - - 12.5 12.9 0.7 -

Accepted Article 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
7,818 - - 16,072 - - - 9,213 4,220
annotations

chr24 chr25
A chr2
3 chr26
chr2
7
10
10
0
chr
ch
10
0
10
r28
22
10
0
ch
10
0
ch
r2 r2
10
9
0
1
0
10
10
ch
ch
r3
0
0
r2
0
10
0
ch
0
r3
0
1
ch
ctg
r 19
10
37
II
0
(W)
0
III
chr1
0
10
chr1(Z
IV
8
0 10
)
chr17
10 20
0
0
chr2
10
chr16
10
0
0
From outer to inner circles:
I. 32 chromosomes at the Mb scale
chr3
10 10
chr1
II. repeat density across the genome

5
10
10
(in 0.1 Mb non-overlapping windows)
chr
ch
r4
0
14
0 III. gene density across the genome

(in 0.1 Mb non-overlapping windows)
10
10
ch
ch
0
r1
r5 IV. GC content across the genome

3
0
10
ch (in 0.1 Mb non-overlapping windows)

10
r1
0
ch
2 r6
0
10
chr
10
0
11
chr
10
0
0
7
10
10
chr10
chr9 chr8
B
2
1
Log2(Mean M:F read counts)
−1 Autosome
Z
−2
6.6 6.8 7.0 7.2
Log10(Scaffold Length)
A B C
S. frugiperda S. frugiperda S. frugiperda
S. litura T. ni B. mori
Spodoptera frugiperda +774 / -1,048
Spodoptera litura +769 / -603
Noctuidae 1:1:1
+449 / -288 Helicoverpa armigera +716 / -1,332
N:N:N
Heliothis virescens +1,042 / -974
Lep.
Trichoplusia ni +2,092 / -1,682
Operophtera brumata +914 / -1,522 Noc.
Antheraea yamamai +2,415 / -2,035 Patchy
Bombyx mori +978 / -1,503 S.D.
Manduca sexta +1,898 / -2,443 N.D.
Danaus plexippus +616 / -1,327
Gene families Lepidoptera Papilio xuthus +755 / -1,417
Expansion / Contraction Cydia pomonella +1,695 / -3,280
Plutella xylostella +1,697 / -3,078
Trichoptera
Stenopsyche tienmushanensis +669 / -3,594
Diptera Anopheles gambiae +1,161 / -1,517
Drosophila melanogaster +1,228 / -928
Coleoptera Leptinotarsa decemlineata +978 / -784
Tribolium castaneum +1,689 / -1,017
Hymenoptera Apis mellifera +1,062 / -1,169
Nasonia vitripennis +1,114 / -1,438
MRCA
(9,266) Melanaphis sacchari +675 / -617
Hemiptera Rhodnius prolixus +701 / -632
500 400 300 200 100 0 Million years age
0
0
0
,0
,0
,0
10
20
30
Gene number
Chr
A 14 c
luste
r
S. frugiperda
B. mori
Sfru_P450_077_CYP340AB1
Sfru_P450_061_CYP340AA1
Sfru_P450_048_CYP340AA1
Sfru_P450_087_CYP340AB1
Sfru_P450_012_CYP3
340AA1
_CYP340AA1
Sfru_P450_011_
Sfru_P450_0
B1
Sfru_P450
340AB1
Sfru_P45
B1
30_CYP340A
0J1
YP340A
0L1
340L1
1
Bmor_XP
Sfru_P450_101_CYP
_CYP34
1
340L
Bmor_
_CYP34
_147_CYP
340L
Bmor_
Bmor_
L1
0_120_C
4C1
Bmor_
_028_CYP
L1
5_CYP
Bmor_
50_017_C
Sfru
_CYP
P340
L1
76_CYP340A
Bmor
Sfru_P450_128
_CYP
Sfru
P340
XP_021
1
Bmor
_CYP
P340
XP_02
Sfru
50_135
340L
_P45
0L1
XP_00
Sfru_P450_1
CYP340AA1
50_117
_02120
XP_0
Sfru
3_CY
_P45
0L1
_103
NP_0
_XP_
450_12
8_CY
_P45
_104
Sfru_P450
YP340AA
063_ YP34
_XP_
0L1
3_CY
YP
_086
0_14
_P
Sfru
P34
1
340AA1
120429
40AA1
204297
Sfru_P4
0_06
2120
0_02
493409
0L
Sfru_P4
P450
0_C
Sfr
P34
450_
0125
0127
Sfru_P4
1
0_14
P450
0_13
4301.1
8_C
0L
0212
CY
_P
P34
P450
0_13
1_CY
Sfru_P
u_
1
A1
Bm
0_16
CY
9_CY 340G
0L
P34
4179
450_
_P45
P45
Sfru_
062_
1
5257
0_06
2_C
4763
CY
_P45
Bm
Sfru_
4.1
34
0L
or_X P_01 2036 7.1
0417
.1
110_
_P45
4.3
Sfru_
1
CY
P340 1
Bm r_XP
1_
Sfr _P4 0_09 _CYP
_C P34
0L
_P45
0_
or_ P_02 203 4C1
102_ YP4C
.1
P340 1
YP
450_
Sfru
CY
L1
_P45
Bm
3.1
.1
8_
34
05
Sfru
P_0 254
or_
09
450_
Sfr
4.1
X
CY
0
Sfru
1
P34
10
YP
G1
0_
34
o
1_
0L
Sfru
4
CY
u_
Sfr
_P
G
12
4_
08
0_
Sfru
1
P45
YP
P2
34
_P
P4 0_0
u_
0L
Sfr
0K
P4C
Sfru
54
0_
P45
YP
1
Sfr u_P 50_ 82_C
35
50
P
34
Sfru
_0
u_
0L
u_
Sfr
57
45
1
45
Sfr _P4 0_0
AB
_1
YP
3
P4
21
u_
34
Sfr
76
Sfr
59
5
u_
Sfr u_P 50_ 81_

Sfr _P4
3
_0
24 CYP C1
40
u_
_0 9_C
YP
50
Sfr
u_ or_X _09 010
19
5
P4
1
.1
0L
50
_C YP3
_C
0
39 CYP 4C1
Sfr
_
P4
21
_C
CY
.2
50
Bm 45
u
06
34
L1
Sfr
Sfr
YP
1
2
50
.1
89
_1
_C
5_
YP
40
u_ or_
4
_0 004 YP 78.1
Sfr
23
Sfr _P
u
_C YP3
63
13
P
4
3
u_
Bm
50
_
4C
6A
P_ 8_C 77
_1
u
6_ 923 18A
CY 18B
32
P
C
4
0
4
1
YP
Sfr u_P 450
1
CY 21
.3
Bm 450
_0 46_
_1
7B
P
Sfr 61
NP
Sfr u_P 450

P
43 P36
Sfr _P
_0
B1
1
u_ o
P4 50_
74
P4 r_N 01 93
_0
67
u
Y
P_ _C 04 _C YP3
4
Bm 50
6.1
50
Sfr _0
or_ _05 001 YP3 52 .2 6
u_
XP 0_0 _C 93 67A
0
P4 XP 7_ 10 06 66
u_
or_ 45 21
Bm 50_ _0 CY 622 A1 _1 55 P3 A1
Sfr or_ 13 04 P3 2 Bm u_P 450 012 _CY 67
u_ NP 4_C 932
03 .1 P3 A1
Sfr u_P _ 68 CY 67
P YP 78
A1 XP _1 P3
Bm 450 _0 Sfr or_ 450 9_
Sfr or_ _14 011 30 9.1 1 5 CY 3.1
u_ 0_ 0 5B Bm u_P 0_ 4_ 34
Sfr P4 NP
CY 622 1 fr P 45 _16 0 8 L4
u_ 5 0_ _ 00 0 S u_ 0 1 P4
P4 07 11 P15 .1 45 01 CY 4L4
50 1_ 4 C1 Sfr _P P_0 8_
Bm _ CY 019 fr u 0 P 0
or_ 021_ 7 S N
or_ 50_
0 CY L1
P3
07 .1 5_ P4
Bm N C Bm _P4 0_11 _CY 0
or_ P_00 YP3 A u P4
L1
NP 11 07 1 Sfr _P4
5 07
0 A _0 CY 9
_0
011 4833 1
u 50 3_ P4L
Sfr _P4 0 8
08 .1 u 0_ CY 9
34
1.1 Sfr _P45 10
7_
YP
4L
u 0_ C
Sfr P45 2_ .1
CYP
16
Sfr
u_
45
0_ 29 6528
u_
P 01 03.1
Sfr P_0 9276 4S8
or _N 04
Bm _XP_0 2_C
YP
or 1
Bm 45 0_02 600.
_P 1204 6.1
Sfru P_02
Sfru or_X 25 4492
_P45
0_09
Bm P_01 5233
8.1
4
Sfru_ 5_CY or_X 0125 G1
Bm
P450 P6AB _XP_ YP4C
Sfru_ _047 31 Bmor 18_C
P450 _CYP 50_0 4CG1
_045 6AB3
Sfru_
P4 _CYP
Sfru_ _CYP 1 _053 .1
P450 6AB1 P450 3134
_038 2 Sfru_ 0107
Sfru_P _CYP
6AB1 NP_0 4M14
450_12
9_C 2 Bmor_ _15 5_CYP
Sfru_P4 YP6A 450 17
50_014 B14 Sfru_P _CYP4M
_CYP6A 50_005 18
Bmor_X B60 Sfru_P4 _CYP4M
P_0049 50_058
Bmor_XP
34109.1 Sfru_P4 833 .1
_0049341 _001103
66.1 Bmor_NP
Bmor_XP
_0125452 _004 934250.2
38.1 Bmor_XP 1
Bmor_NP_0 001106223.
01073135.1 Bmor_NP_
Sfru_P450_11 0_CYP4G75
1_CYP6AB61 Sfru_P450_15
Bmor_NP_0011040 YP4G75
07.1 Sfru_P450_156_C
Sfru_P450_167_CYP6AN4 Bmor_XP_021204692.1
Sfru_P450_070_CYP6AN4 Bmor_NP_001106221.1
Bmor_NP_001266427.1 Sfru_P450_113_CYP4G74
Bmor_NP_001296467.1
Bmor_XP_021206485.1
2A1 Sfru_P450_059_CYP4G74
Sfru_P450_032_CYP33
Sfru_P450_137_C
YP332A1 YP4AU1
Sfru_P450_006_C Sfru_P450_16
1108340.1 9_CYP4AU1
Bmor_NP_00 Bmor_XP_
24A1 021207354.
_033_CYP3 1
Sfru_P450 Sfru_P450
P324A6 _153_CY
_016_CY Sfru_P4 P4AU14
Sfru_P450 4906.1 50_165
_01254 Sfru_P4 _CYP4A
Bmor_XP 1A7 50_002 U14
_CYP32 Bmor_ _CYP34
50_151 321A7 XP_00 1A11
Sfru_P4 4_CYP Bmor_ 492652
450_06 A8 XP_00 4.1
Sfru_P YP321 Bmor_ 492653
450 _049_C 1A8 XP_0 5.1
Sfru_P YP32 Bmor_ 0493
31_C 9 4325
P4 50_0 YP 321A Bmor_
XP_0
2120
.2
Sfru_ _C 9 3900
_122 321A XP_0 .1
P450 _CYP Bmor 2120
Sfru_ _019 A7 _XP_ 3884
P450 P321 Bm 0212 .1
Sfru_ 8_CY A7 or_X 0388
0_ 11 P321 Sfru P_00 7.1
_P45 9_CY B1 _P45 4933
Sfru 0_09 P321 1 Sfru 0_04 974.
45 CY _P45 4_CY 3
Sfru
_P 100_ YP321B 1 Bm
450_ C or_N 0_078_ P341
_P 7_ 321B Sfru P_0 C B2
Sfru 0_09 YP 15 _P 0126 YP341B
_P45 16 1_C 321A Sfr 450_ 6437 1
ru P 1 u_ 013_
Sf 450_ 3_CY 1B Sfr P45 .1
_P P32 4 u_ 0_ CY
Sfru 0_ 00 CY 1B Sfr P45 04
3_ P34
45 0_ P32 5 u 0_ CY 1B
06
Sfru
_P 0_ CY 337B Bm _P45 13
6_ P34 1
P45 02
9_
YP 37B
5 or_ 0_
15 CY 1B
2
u_ 0_ _C Sfr P34
Sfr 3 XP 8_
P45 _144 CYP 7.1 u
Sfr _P4 _021 CY 1B
u_ 0 _ 82 .1 P 1
Sfr 5 09 04 u 5 2 34
P4 _1 11 6314 7 Sfr _P4 0_00 0240 1B
B
u_ 50 00 u 5 4_ 8.1 1
Sfr 4 _ 26 E4 Sfr _P4 0_0 CY
u_
P NP 01 P6A 50 u 50 P3
Sfr or_ P_0 AE Sfr _P 50_ _C 41
Bm r_N CY 4 0 YP
o 6 _ P6 04.1 Sfr u_P 50_ 30_ 34
B2
6
11 _CY 0 8 45 0 CY
Bm 50_ 4 04 E6 Bm u_P 0_ 41_ P3
1B
26
P4 11 11 6A 4.1 45 07 CY 41
u_ 0_ 00 P 9 Sfr or_ 0_ 2_ P3 D1
Sfr P 45 N P_ _CY 523 7.1 Sfr u_P NP_ 079 CY 41
u_ or_ 12
1 5 78 6.1 Bm u_P 450 001 _CY
P3 D1
Sfr 12 22 0 41
Bm 50_ P_0 49 040 63.2 or_ 45 _0 2 P3 B2
P 4 X 00 1 0 N 0 25 691 41 7
P_ _14
Bm or_ NP_ 14
u_ or_ XP_ 001 552 _C 51 B2
54 39.2
Sfr YP .1 7
Bm or_ 450
or_ NP
Bm or_ 00 3_
.1
_ 2
0
NP _01 C
14
3
01 34
XP _00 112
11
Bm u_P
06 YP3 38B
YP 708 .1
Bm or_
_0 49
14
_0
P
Sfr or_
YP AE .1
X 4
13
21
0
3
Bm or_
38
21 036
9.1 8A1 2
_0
Bm or_
YP AE 3
20
10
4
XP
Bm
Bm
43
75
01
1
07
00 5_C
or_
NP
Sfr or_X 0_0
30
_0
NP 02
01
E9
_
Bm
or_
Bm _P4 0_13
.1
9
NP
u_
.1
6
_0
53
_0 120
A
_C
11 P33
Sfr _P4 _001
5
P_
P4
Bm
P45 _127 CYP B58

or_
6
NP
.1
_C 400
01
92
57
Sfr r_NP
_C
CY
58
50
Bm
27 861
or_
.1
Y
P6
Bm
_1
P6B 48
66
_C
u
6B
_0
P_
47
11
Sfr
Bm
42
50
Y
_0
56
or_ 040
02
59
00
Bm
8
4826 .1
or_N 026_ YP6
9A
P4
u_
50
_C 69.1
Sfru
.1
12
.1
P4 NP_
26
_
52
1
u_
or_X
P4
P45
Bm _XP_00 9_CYP
50
50
0_
YP
10
06
_C
.1
62
CY
_1
Bm
Sfr
0_02 129652 1
u_
_P
P4
2
P3
P6B
_C
3
Sfru
W
30
or_X
0.1
10
0_
50
_0
1_
P_0
Sfr
Sfru
1
or
u_
450_
P_00 YP6A
0110
1A
YP 9A1
Bm
or_N _001
Sfru
06
P6CT
50
CY
26
CY
Sfr
Bmor
_P45
P6B1
P6CV
1
P_0
04
49
0
Sfru_
7_
P4
6B1
u_
_P45
64
0_
P
Sfru_
45
.1
092_
P_0
085_
_P45
Bmor_
A1
93
P
9A21
4
CY
Sfr
5_C
u_
19
Bmor_
9A21
2909
P
7_CY
0492 105.1
Sfru_P4
58
0_14
_CYP
_NP_
Sfru_P4
21
60
9_CY
u_
Sfr
0_CY
Bmor_XP
YP9A59
.1
P42
P450
Sfru_P450
0_04
9A21
Sfru_P450_0
05_CYP9A21
CY
Sfru_P450_073
4928
_CYP9A
CYP9A39
450_
0_05
P450
Sfru_P450_075_CYP
A9
Bmor_XP_012546019.1
Bmor_NP_001095934.1
u_
Bmor_XP_012546023.1
26
Bmor_XP_012546027.1
0_14
Sfr
_CYP9A
4_CYP
Bm
2120
7968
9_CYP
XP_0
8A
Sfr
.1
P33
Sfru_P450_037_CYP9
NP_00
0012
Bm
0_13
_034
2_CY
0_08
or_N
_088
_106_CYP
_P
_P45
1
_126
8_CY
50_096
ito
50_112
3A
.1
XP_0
0_046_C
Sfru
_P45
2120
450_15
6629
_0125462
_P45
P450
50_093
Bm
6
_P45
450_00
_CYP
333B
P333
50_090
Sfru
_024_CYP
Sfru_P450_015_
110845
_CYP
P333
Bmor_
Sfru
2508
54_CYP9A40
_CYP9G
5.1
Sfru_P450_1
_CYP6A
Sfru
Sfru_
Sfru
4
B3
m
354A
Sfru_P450
Sfru_P
_CYP9A40
B3
35
Sfru_P4
Sfru_P45
Sfru_P
Sfru_P4
6.1
.1
50.1
4A3
14
9AJ3
5
9A40
13
CYP2
CYP4
CYP3
mito
1(Z) 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 ctg37(W)
va
rva
va
ins arva
va
va
lar
lar
lar
lar
4 th ar la
l
tar
tar
tar
tar
tar
ult
a
lt
CYP2
t
ins
up
du
ins
ins
ins
ins
ad
Fp
Fa
2 nd
3 rd
5 th
th
1 st
M
6
Sfru P450 001 CYP306A1

High
Sfru P450 036 CYP18B3

P450 gene expression
Sfru P450 140 CYP15C1

Sfru P450 027 CYP6CT1
Sfru P450 025 CYP338B42
Sfru P450 003 CYP321A15
Low

Sfru P450 055 CYP6AW1
Sfru P450 088 CYP354A14
Sfru P450 096 CYP9G5
Sfru P450 024 CYP9AJ3
CYP3

Sfru P450 080 CYP6CV6
Sfru P450 114 CYP6AE50
Sfru P450 111 CYP6AB61
Sfru P450 070 CYP6AN4
Sfru P450 167 CYP6AN4
Mito.

Sfru P450 002 CYP341A11
Sfru P450 004 CYP341B26
Sfru P450 050 CYP341B26
Sfru P450 030 CYP341D1
Sfru P450 041 CYP341D1
Sfru P450 072 CYP341B27
Sfru P450 079 CYP341B27
Sfru P450 137 CYP4AU1
Sfru P450 022 CYP4S8
Sfru P450 018 CYP4CG1
Sfru P450 053 CYP4CG1
Sfru P450 058 CYP4M18
Sfru P450 008 CYP4L4
CYP4

Sfru P450 062 CYP340K4
Sfru P450 135 CYP340J1
Sfru P450 101 CYP340AA1
CYP340 cluster
r
A cluste
Chr4 Ch
r9
clu
ste
r
ar
NP_001124346.1
XP_021209
Sfru_GR_007
Sfru_GR_141
7.1
Sfru_GR_095
Sfru_GR_120
BAW33744.1
Sfru_GR_034
ACD85125.1
Sfru_GR_056
Sfru_GR_
ACD85124.1
117
_166
Sfru_GR
Sfru_GR
_176
g
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123321
Sfru_G
375.1
Sfru_G _072
XP_0 _GR_
Sfru_GR_
69
Sfru_
2800.1
_GR_ 66
Sfru_
Su
Sfru_GR
Sfru
183
Sfru_GR
GR_0
060
GR_0
042.1
_GR_ 201
Sfru
DAA06
2120
NP_00
_048
Sfru
GR
_GR 114
R_
_121
_GR_ .1
XP
205
R_100
GR_0 8
_004
_158
Sfru
_GR_
BAK5
_GR_
Sfru
Sfru_
Sfr 0639 1
_021 A0637 68
Sfru_
_GR_ 70
8998
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1.1
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u_ R_013
Sfru
13
D
_G
42
53
Sfru
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A
R_0
Sfru
2086 4.1
046
Sfr _GR 092

Sfr _GR 22.1
Sfru
_0
R_1 .1
099
u_
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Sfr D85 44.1
Sfr _GR _118

NP
A
u
GR
u_G
4
GR 012
A
DA
XP _001 551 75.1
AC 243 86.1
74
u
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77
_ 0 120 345
Sfr u_G _16 4

NP
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011 79
5
XP _01 D85 991 8
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u
2
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S. frugiperda
R
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u
_
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Sfr _G _0
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0
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12
12 8 8 1
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R
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25
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21 G
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1
06 75 09
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2
7
N
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38 7.1
2
B. mori
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0
0.1
B
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CO
2
Sfr AS 7.1
1
u_ 18 37 .1
9
Sfr
G 8 06 379 .1
Sfr u_G R_ 17.1
9
AA 6 78
Sfr u_G R_ 106 D A0 63 1.1
u 1 DA AA0 638 202
XP Sfr _G R_0 26 D A0 R_ 6.1
_0 u_G R_ 87
12 1 DA u_G 512 .1
Sfr 55 R_0 44 Sfr D8 382 .1
1 5
Sfr u_G 334 7 AC A06 396
R .1
Sfr u_G _19 .1 DA A06 376 0
u_ R_ 1
3 DA A06 R_2 5
XP Sfru GR_ 052 DA u_G _14
_ _ 1
XP 0049 GR_ 78 Sfr _G _20
R 6
_0 0 u
04 3226 68 R
Sfr u_G _221
93
14 3.1 Sfr _GR 6.1
09 u 8
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A _0
DA GR
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Sfr
u_G R_062
Sfr
u_G _075
Sfr R
_G _018
Sfru R
_G
Sfru _059
_GR 184
Sfru
Sfru _GR_ 127
_GR_ Sfru
_GR_
Sfru
_G
134 Sfru 194
Sfru_ R_080 _GR_ 82
Sfru
GR_1 GR_1
Sfru_ 55 Sfru_ 11
GR_1 u_ GR_2
Sfru_ 08 Sfr 2
GR GR_11
Sfru_G _011 Sfru_
R_214 R_035
Sfru_G
Sfru_G
R_219 _G R_132
Sfru
Sfru_GR 45.1
_162 BAW337
Sfru_GR 83.1
_084 DAA063
Sfru_GR_ 217
203 Sfru_GR_
Sfru_GR_1 20
91 Sfru_GR_2
Sfru_GR_082 Sfru_GR_215
Sfru_GR_163 BAW33746.1
BAW33756.1
Sfru_GR_076
DAA06384.1
Sfru_GR_016
BAW33748.1
Sfru_GR_047
BAW33750.1
Sfru_GR_179 DAA06385.
14 1
Sfru_GR_0 BAW3375
_192 3.1
Sfru_GR BAW337
Bitter
_094 49.1
Sfru_GR BAW337
_098 51.1
Sfru_GR BAW337
R_148 55.1
Sfru_G BAW3
3752.1
R_180 NP_0
Sfru_G _137 01
Sfru_
GR BAK5 233216.1
39 2799
GR_0 XP_0 .1
Sfru_ 88 12
GR_0 BAK5 546109
Sfru_ R_032 27 .1
_G DAA0 98.1
Sfru 079 Sfru 6395.1
_GR_ 172 _GR_
Sfru Sfru
ru _GR_ 01 _GR 038
Sf _1 Sfru
_GR 74 _G
_164
Sfru R_0
B
Sfru R_0
_G _124 02
Sfru R Sfru _GR_2
fru _G _143 Sfr _GR_1 13
S R u_G
u_G _136 Sfr 50
Sfr R u_ R_200
u_G R_154 0 DA GR
Sfr G A _1
u_ _11 DA 063 23
Sfr _GR 129 A 8
u _ DA 063 8.1
Sfr _GR 207 Sfr A06 87.1
u _ 3
Sfr _GR _033 Sfr u_G 89.1
u 4 R
Sfr _GR _02 DA u_G _15
u 2
Sfr _GR _12 9 Sfr A06 R_1 3
9
u R
Sfr u_G _1 1
0 Sfr u_G 394 9
.1
R
Sfr u_G R_1 03
7 Sfr u_G R_0
Sfr u_G R_ 36
Sfr u_G R_1 86 Sfr u_G R_ 97
0
1
Sfr u_G R_ 47 Sfr u_ R_ 156
1
Sfr u_G R_ 083 Sfr u_ GR 015
Sfr u_G R_ 01 7
0 Sfr u_ GR _04
Sfr u_G R_ 19 u_ GR _01 0
G _ G _1 7
Sfr u_ GR
Sfr u_ GR 00
0
R
_0 75
Sfr u_G R_ 19
u_ GR _06 8
Sfr u_
Sfr u_
Sfr u_G R_ 149
_
22
G
R
Sfr u_G R_ 025

Sfr u_G R_ 104
Sfr
R 20 7
G
Sfr u_G R_ 59
_0 8
Sfr u_
Sfr u_G R_1 93

G
85
Sfr _G _
Sfr
_
Sfr _G _2 3
Sfr u_
Sfr u_G R_1 27

Sfr u_G _07 2
1
R_ 049
Sfr u_G R_0 7
Sfr _G _02 7
u
Sfr u_G _18 8
6
R
Sfr u_G _02

5
R
Sfr _GR _10
Sfr u_G _09

Sfr _GR _152
R
R
Sfr _GR _160

Sfr _GR 065
u
1
R
Sfr
Sfr _GR 055
Sfr _GR
Sfr _GR_ 089
_
0
u
R
Sfr
7
u
2
_
u_
10
Sfr 0639
R
Sfru GR_1 61
_
u
u
DA
19
GR
u
u_ R_0
NP 85127.
u_
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AC
u
A
33
GR 3.1
Sfru
_GR_ 37
_001
u
Sfru
G
_0
_GR_ 116
0
R_1
D
Sfru
u
_GR_ 113
_0
_1
u_
Sfru
146
44
_G
_G
Sfru
Sfru_ R_031
1243
_GR
_GR_ 115
95
Sfru_
61
Sfru_
Sfru_G _054
_GR_
Sfr
Sfru_
R_1 9
R_170
_GR_
Sfru_G
R_030
GR_1
Sfru_GR
Ch
_003
Sfru
_GR_ 58
Sfru_GR
_167
_140
DAA0639
Sfru
Sfru_GR_
47.1
89
1
Sfru_GR_1
Sfru_GR_050
Sfru_GR_125
Sfru_GR_063
Sfru_GR_157
Sfru
Sfru_GR_023
GR_0
Sfru_GR_188
Sfru_GR_029
Sfru_GR_151
Sfru_GR_009
30
Sfru_GR_198
13
_G
GR
GR_0
Sfru
GR_0
Sfru_GR_1
Sfru
196
Sfru_GR
Sfru_GR
Sfru
001
Sfru_G
Sfru_GR
r24
Sfru_
R_006
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19
_091
51
2.1
073
69
clu
ste
r
Sugar
Bitter
CO2
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 ctg37
(Z) (W)
va
rva
va
rva
va
va
lar
lar
lar
lar
4 th ar la
6 th ar la
tar
tar
tar
tar
ult
a
lt
t
t
ins
up
du
ins
ins
ins
ins
ins
ad
Fp
Fa
2 nd
3 rd
5 th
1 st
Sfru GR 001
Sfru GR 058
Sfru GR 019
Sfru GR 051
Sfru GR 006
Sfru GR 078
Sfru GR 091
Sfru GR 115
High
Sfru GR 196
Sfru GR 130
Sfru GR 139
Sfru GR 167
Sfru GR 140
Sfru GR 050
GR gene expression
Sfru GR 189
Sfru GR 157
Sfru GR 125
Sfru GR 188
Sfru GR 198
Sfru GR 029
Sfru GR 009
Sfru GR 151
Sfru GR 023
Sfru GR 063
Sfru GR 169
Sfru GR 003
Sfru GR 030
Sfru GR 073
Sfru GR 031
Sfru GR 146
Sfru GR 170
Sfru GR 054
Sfru GR 161
Sfru GR 113
Sfru GR 116
Sfru GR 037
Sfru GR 133
Sfru GR 142
Sfru GR 119
Sfru GR 061
Sfru GR 107
Sfru GR 105
Sfru GR 026
Sfru GR 077
Sfru GR 212
Low
Sfru GR 173
Sfru GR 093
Sfru GR 104
Sfru GR 149
Sfru GR 190
Sfru GR 197
Sfru GR 036
Sfru GR 097
Sfru GR 089
Sfru GR 055
Sfru GR 065
Sfru GR 152
Sfru GR 010
Sfru GR 083
Sfru GR 147
Sfru GR 186
Sfru GR 103
Sfru GR 171
Sfru GR 109
Sfru GR 122
Sfru GR 024
Sfru GR 033
Sfru GR 207
Sfru GR 129
Sfru GR 110
Sfru GR 154
Sfru GR 014
Sfru GR 179
Sfru GR 148
Sfru GR 098
Sfru GR 094
Sfru GR 192
Sfru GR 039
Sfru GR 137
Sfru GR 180
Sfru GR 136
Sfru GR 143
Sfru GR 079
Sfru GR 032
Sfru GR 088
Sfru GR 047
Sfru GR 016
Sfru GR 076
Sfru GR 071
Sfru GR 081
Sfru GR 204
Sfru GR 043
Sfru GR 163
Sfru GR 084
Sfru GR 162
Sfru GR 219
Sfru GR 082
Sfru GR 191
Sfru GR 203
Sfru GR 214
Sfru GR 011
Sfru GR 108
Sfru GR 155
Sfru GR 080
Sfru GR 134
Sfru GR 124
Sfru GR 074
Sfru GR 101
Sfru GR 172
Sfru GR 208
Sfru GR 067
Sfru GR 008
Sfru GR 049
Sfru GR 025
Sfru GR 159
Sfru GR 102
Sfru GR 027
Sfru GR 187
Sfru GR 022
Sfru GR 085
Sfru GR 028
Sfru GR 015
Sfru GR 040
Sfru GR 017
Sfru GR 175
Sfru GR 160
Sfru GR 195
Sfru GR 044
Sfru GR 090
Sfru GR 156
Sfru GR 007
Sfru GR 069
Sfru GR 066
Sfru GR 183
Sfru GR 106
Sfru GR 128
Sfru GR 111
Sfru GR 131
CO2
Sfru GR 068
Sfru GR 178
Sfru GR 193
Sfru GR 052
Sfru GR 087
Sfru GR 144
Sfru GR 057
Sfru GR 126
Sfru GR 046
Sfru GR 138
Sfru GR 042
Sfru GR 072
Sfru GR 177
Sfru GR 070
Sfru GR 099
Sfru GR 096
Sfru GR 012
Suger
Sfru GR 168
Sfru GR 048
Sfru GR 205
Sfru GR 100
Sfru GR 121
Sfru GR 004
Sfru GR 041
Sfru GR 120
Sfru GR 141
Sfru GR 034
Sfru GR 095
Sfru GR 153
Sfru GR 199
Sfru GR 117
Sfru GR 056
Sfru GR 166
Sfru GR 176
Sfru GR 038
Sfru GR 164
Sfru GR 150
Sfru GR 200
Sfru GR 002
Sfru GR 213
Sfru GR 165
Sfru GR 060
Sfru GR 201
Sfru GR 114
Sfru GR 158
Sfru GR 013
Sfru GR 053
Sfru GR 092
Sfru GR 174
Sfru GR 064
Sfru GR 118
Sfru GR 005
Sfru GR 181
Sfru GR 185
Sfru GR 209
Sfru GR 045
Sfru GR 086
Sfru GR 135
Bitter
Sfru GR 123
Sfru GR 221
Sfru GR 210
Sfru GR 145
Sfru GR 206
Sfru GR 202
Sfru GR 216
Sfru GR 218
Sfru GR 215
Sfru GR 217
Sfru GR 220
Sfru GR 020
Sfru GR 021
Sfru GR 062
Sfru GR 075
Sfru GR 018
Sfru GR 059
Sfru GR 184
Sfru GR 127
Sfru GR 194
Sfru GR 182
Sfru GR 211
Sfru GR 112
Sfru GR 035
Sfru GR 132

Genetic Adaptations Spodoptera Global Dispersal Invasion Xiao 2020

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Genetic Adaptations Spodoptera Global Dispersal Invasion Xiao 2020

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DR. HUAMEI XIAO (Orcid ID : 0000-0003-0165-7410)

DR. XINHAI YE (Orcid ID : 0000-0002-0203-0663)

PROF. FEI LI (Orcid ID : 0000-0002-8410-5250)

Article type : Resource Article

The genetic adaptations of fall armyworm Spodoptera frugiperda

facilitated its rapid global dispersal and invasion

Development Regulation of Jiangxi Province, Yichun University, Yichun 336000, China.

versity, Hangzhou, 310058, China.

Hangzhou, 310021, China.

xiaohuamei625@163.com; Dr. Tao Liu, Email: taoliu@genome.cn;

identified. Comparative genomics revealed the expansion of the detoxification-associated gene

families, chemoreception-associated gene families, nutrition metabolism and transport system

well as for pest management of fall armyworm.

Key words: Fall armyworm, Chromosome-level genome, Comparative genomics, Polyphagia,

This article is protected by copyright. All rights reserved

hours (Martinelli et al., 2006).

other Asian countries (Ganiger et al., 2018; Li et al., 2019).

This article is protected by copyright. All rights reserved

wheat production in China.

differ in composition of sex pheromones, susceptibility to chemical insecticides and transgenic

strain-specific sites in the fourth exon of the Z-chromosome-linked gene Triosephosphate

2019; Nagoshi et al., 2017).

chlorpyriphos, permethrin, flubendiamide, chlorantraniliprole, methomyl, and thiodicarb

This article is protected by copyright. All rights reserved

chromosome-level genomes lack the information of the W chromosome.

invasion, polyphagia and insecticide resistance.

2 Materials and Methods

Fall armyworms were collected in Dongyang (29.27°N, 120.23°E, Zhejiang province,

light/dark photoperiod and relative humidity of 70–80%.

2.2 Genome sequencing and de novo assembly

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were assembled by SMARTdenovo (https://github.com/ruanjue/smartdenovo) as described by

Istace et al. (2017). The redundans pipeline (https://github.com/lpryszcz/redundans) (Pryszcz &

of the fall armyworm that we named the ZJ-version.

2.3 Hi-C library preparation

by incubation at room temperature in a 2% formaldehyde solution for 10 minutes. The reaction

was quenched by 5 minute incubation with 2.5M glycine solution.

mM Tris HCl, pH 8.0, 10 mM MgCl2, 5 mM β-mercaptoethanol, 0.1 mM phenylmethylsulfonyl

This article is protected by copyright. All rights reserved

8.0, 0.15% Triton X-100, 2 mM MgCl2, 5 mM β-mercaptoethanol, 0.1 mM PMSF, and 1 μl

polymerase (NEB, Massachusetts, USA), T4 polynucleotide kinase (NEB, Massachusetts, USA)

performed by Annoraod Gene Technology Co. Ltd (Beijing, China).

2.4 Scaffolding with Hi-C

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“--very-sensitive -L 20 --score-min L,-0.6,-0.2 --end-to-end --reorder --rg-id BMG

assembly contigs into chromosome-scale scaffolds (hereafter pseudo-chromosomes) with

LACHESIS (https://github.com/shendurelab/LACHESIS) (Burton et al., 2013). To access the

accuracy of the scaled-up genome assembly, we cut the pseudo-chromosomes predicted by

2.5 Transcriptome sequencing and analysis

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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

Kojima, & Kurtz, 2015).

We annotated the protein coding genes by integrating the evidence of de novo,

homology-based and RNA-Seq-based annotations. First, Augustus v2.5.5 (Stanke, Diekhans,

proteins obtained from NCBI invertebrate RefSeq (https://www.ncbi.nlm.nih.gov/refseq/) to the

Morishima, 2016) online service.

2.8 Phylogenetic reconstruction

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from NCBI), Helicoverpa armigera (GCF_002156985.1, from NCBI), Heliothis virescens

(GCA_002382865.1, from NCBI), Bombyx mori (GCF_000151625.1, from NCBI), Antheraea