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\begin{document}

\title{Chromosome-level genome of the peach fruit moth Carposina sasakii
(Lepidoptera: Carposinidae) provides a resource for evolutionary studies
on moths}



\author[1]{Li-Jun Cao}%
\author[1]{Wei Song}%
\author[1,1]{Lei Yue}%
\author[1]{Shao-Kun Guo}%
\author[1,1]{Jin-Cui Chen}%
\author[1]{Ya-Jun Gong}%
\author[2]{Ary Hoffmann}%
\author[1]{Shu-Jun Wei}%
\affil[1]{Beijing Academy of Agriculture and Forestry Sciences}%
\affil[2]{The University of Melbourne Bio21 Molecular Science and Biotechnology Institute}%


\vspace{-1em}



  
  \date{May 24, 2024}


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\selectlanguage{english}
\begin{abstract}
The peach fruit moth (PFM), Carposina sasakii Matsumura, is a major
phytophagous orchard pest widely distributed across Northeast Asia.
Here, we report the chromosome-level genome for the PFM, representing
the first genome for the family Carposinidae, from the lepidopteran
superfamily Copromorphoidea. The genome was assembled into 404.83 Mb
sequences using PacBio long-read and Illumina short-read sequences,
including 275 contigs, with a contig N50 length of 2.62 Mb. All contigs
were assembled into 32 linkage groups assisted by the Hi-C technique,
including 30 autosomes, a female-specific W chromosome and a Z
chromosome. BUSCO analysis showed that 98.2\% genes were complete and
0.4\% of genes were fragmented, while 1.4\% of genes were missing in the
assembled genome. In total, 23,218 protein-coding genes were predicted,
of which 82.72\% were functionally annotated. Because of the importance
of diapause triggered by photoperiod in PFM, five circadian genes in the
PFM as well as in the other related species were annotated, and
potential genes related to diapause and photoperiodic reaction were also
identified from transcriptome sequencing. In addition, manual annotation
of detoxification gene families was undertaken and showed a higher
number of ABC and GST genes in PFM than in most other lepidopterans, in
contrast to a lower number of UGT genes, suggesting different
detoxication pathways in this moth. The high-quality genome provides a
resource for comparative evolutionary studies of this moth and its
relatives within the context of radiations across Lepidoptera.%
\end{abstract}%



\sloppy




\textbf{Introduction}

The peach fruit moth (PFM), \emph{Carposina sasakii} Matsumura
(Lepidoptera: Carposinidae, superfamily Copromorphoidea), is a major
phytophagous orchard pest of fruit such as apple, pear, peach, apricot
and jujube from the families of Rosaceae and Rhamnaceae (\textbf{Fig. 1}
). The hatched larvae directly bore into fruit to feed, causing losses
in fruit production. PFM is one of the most severe borers on deciduous
fruit in northeast Asia. It is also considered a potential risk to fruit
production in most parts of the world, although PFM is currently
restricted to northeast Asia and far east Russia (D. Kwon, Kwon, Kim, \&
Yang, 2018; Y. Z. Wang et al., 2017).

One possible of reason for the currently restricted distribution of PFM
is its sensitivity to environmental factors. PFM has evolved diapause to
cope with cold winter conditions and to synchronize its phenology with
host plants (Toshima, Honma, Masaki, \& Zoology, 1961). Both long-day
and short-day photoperiods induce diapause in the last instar of PFM
larvae, resulting in a diapausing cocoon (B.-Z. Hua, Zeng, \& Zhang,
1998; Huang, Wang, Ye, Zhang, \& Zhang, 1976). The life cycle of PFM can
be univoltine or bivoltine, depending on photoperiods encountered and
environmental factors like humidity (Chiba \& Kobayashi, 1985; D.-S.
Kim, Lee, \& Yiem, 2000; Sato \& Ishitani, 1976). Temperature also
affects the occurrence of PFM through effects on development rate and
the emergence of the overwintering generation from diapause (D. S. Kim,
Lee, \& Yiem, 2001; B. Zhang et al., 2016).

The effects of environmental factors as well as photoperiod on the life
history of PFM provides an opportunity to investigate the genomic basis
of adaptation to temperate environments in the Copromorphoidea
superfamily and across Lepidoptera more generally. Candidate genes
involved in climatic adaptation could then also be investigated at the
geographic level, given that a combination of mtDNA and microsatellite
variation indicates strong genetic differentiation among populations of
the PFM among geographical populations across its native range in China
(Y. Z. Wang et al., 2017). The highly variable life history of PFM on
different host plants may reflect different host-associated biotypes as
supported by an analysis of esterase isozyme patterns (L. Hua \& Hua,
1995) and random amplified polymorphic DNA (RAPD) (Xu \& Hua, 2004),
although this is not yet been confirmed by direct studies on population
differentiation in PFM (D. H. Kwon, Kim, Kim, Lee, \& Yang, 2017; J.
Wang et al., 2015).

Well-assembled genomes are increasingly becoming available as resources
for tracing evolutionary adaptation across the Lepidoptera. Already
there are substantial genomic resources for many moths (W. Chen et al.,
2019; Cheng et al., 2017; Kanost et al., 2016; Lange et al., 2018; Ma et
al., 2020; Pearce et al., 2017; Wan et al., 2019; Xia et al., 2004;
Xiang et al., 2018; Xiao et al., 2020; You et al., 2013; S. Zhang et
al., 2020) and butterflies (Ahola et al., 2014; Cong, Borek, Otwinowski,
\& Grishin, 2015; Dasmahapatra et al., 2012; Lu et al., 2019; Nishikawa
et al., 2015; Zhan, Merlin, Boore, \& Reppert, 2011) which are being
used in comparative analyses to link genomic changes to phenotypes like
the detoxification of compounds encountered in hosts (Rane et al.,
2019). Available genomes provide abundant reference points for
investigating evolution across the Lepidoptera, although most species
sequences so far are from the Papilionoidea, Noctuoidea, Bombycoidea and
Pyraloidea, with less genomic information available for the Carposinidae
(Copromorphoidea) despite the importance of this group as agricultural
pests.

In the present study, we report on a chromosome-level genome of PFM
which was \emph{de novo} assembled based on sequences obtained from the
PacBio and Illumina platforms and assembled at the chromosome level with
the Hi-C technique. We compare features of the PFM genome with those of
eleven other moths, focusing particularly on detoxification gene
families important in host adaptation and pesticide resistance,
contributing to ecological niches occupied by species (Rane et al 2019).
As an initial study using the newly assembled genome, we investigate
transcriptomic changes induced by long-day and short-day photoperiods
that induce diapause in PFM larvae, and we identify genes involved in
these responses which are critical to climatic adaptation by PFM.

\textbf{Materials and methods}

Sample collection and rearing

We established a laboratory strain of PFM from 30 larvae collected from
an apple orchard in the Beijing area of China in July 2018. This strain
was maintained for five generations on apple (\emph{Malus}
\emph{pumila}Mill) in the laboratory under 25 \selectlanguage{ngerman}± 1 °C, a relative
humidity of 75 ± 5\%, and a photoperiod of 15L : 9D (ND, normal-day
condition). Eggs were laid on filter paper and moved to ripe apples
before hatching. Larvae developed in the apples and the last (fifth)
instar larvae left the fruit to pupate on prepared sawdust. Samples used
in genome sequencing and RNA-seq were from this strain.

In order to induce diapause in larvae, we moved batches of newly hatched
larvae to long-day and short-day photoperiodic conditions before they
bored into apple for feeding. The long-day condition (LD) was set to a
photoperiod of 22 L : 2 D, while the short-day condition (SD) was set to
a photoperiod of 8 L : 16 D. Both treatments were conducted under 25 ± 1
°C, and a relative humidity of 75 ± 5\%. The last instar larvae leaving
the fruit were collected and stored in RNAlater at \selectlanguage{english}-80 degC
(Sigma-Aldrich, St. Louis, USA) for subsequent RNA-seq library
construction.

Genome sequencing

We extracted genomic DNA from 12 pupae using MagAttract HMW DNA kit
(Qiagen, Hilden, Germany) for Illumina library and PacBio library. The
paired-end Illumina library with insert sizes of about 500 bp, was
constructed using VAHTS\textsuperscript{TM} Universal DNA Library Prep
Kit for Illumina(r) V2 (Vazyme, Nanning, China) and sequenced on an
Illumina Novaseq platform to obtain 150-bp paired-end reads. The raw
reads generated were filtered by the software Trimmomatic v0.38 (Bolger,
Lohse, \& Usadel, 2014). After filtering, we obtained 31.02 Gb of short
clean reads (coverage: 77.24X). The sequencing data was used to survey
genome feature and polish \emph{de novo} assemblies.

For long-read sequencing, SMRTbell libraries were constructed with
Sequel(r) Sequencing Kit 3.0 (Pacific Biosciences, Menlo Park, CA, USA).
Long DNA fragments of approximately 20 kb were sequenced on a PacBio
Sequel sequencer (Pacific Biosciences, Menlo Park, CA, USA). Four SMRT
cells were processed and 55.52 Gb subreads (mean subread length: 18.13
kb, subread N50 length: 32.84 kb, coverage: 138.2X) were obtained for
contig-level genome assembly.

To assist the chromosome-level assembly, we used the Hi-C
(High-throughput chromosome conformation capture) technique to capture
genome-wide chromatin interactions (Belaghzal, Dekker, \& Gibcus, 2017).
Twenty 5\textsuperscript{th} instar larvae were ground in 2\%
formaldehyde for cross-linking of cellular protein. Chromatin was
digested with restriction enzyme \emph{MboI} overnight. Then, the DNA
ends were flatted, marked with biotin-14-dCTP and ligated with bridge
linker. The samples were digested with proteinase K and purified by
phenol-chloroform extraction. Biotins on unligated DNA fragments ends
were removed with T4 DNA polymerase. Fragments were sheared into 200-600
base pairs using an S220 Focused-ultrasonicator (Covaris, U.S.). Biotin
marked DNA fragments were enriched using streptavidin C1 magnetic beads.
Illumina library was constructed from the enriched fragments using
VAHTS\textsuperscript{TM} Universal DNA Library Prep Kit for Illumina(r)
V2 (Vazyme, Nanning, China) and sequenced on an Illumina Novaseq
platform to obtain 150-bp paired-end reads. After removing the
low-quality reads, 1,509 million clean reads were retained (coverage:
559.3X).

\textbf{Genome survey}

We used the k-mer method to survey the genome features of the PFM. The
k-mer count histogram was obtained from Illumina paired-end sequencing
data using Jellyfish v2.99 (Marcais \& Kingsford, 2011) with 17, 21, 25
and 35 mers. Genome size, heterozygosity and rate of duplication were
estimated by GenomeScope v1.0 (Vurture et al., 2017).

\textbf{Genome assembly and evaluation}

Long reads generated from PacBio sequencing were corrected and assembled
using CANU version 1.8 (Koren et al., 2017) with default parameters. The
initial assembly was polished using Pilon v1.22 (Walker et al., 2014)
with short reads from Illumina paired-end sequencing for three times.
Two haplotypes in part of the genome might be assembled as separate
primary contigs due to the high degree of heterozygosity (Roach,
Schmidt, \& Borneman, 2018). To corrected these possible allelic
contigs, we reassigned the polished assembly using the pipeline Purge
Haplotigs to identify pairs of contigs that are syntenic and removed one
of them (Roach et al., 2018), resulting in a contig-level genome.

Clean reads sequenced from the Hi-C library were aligned to the
contig-level genome with an end-to-end algorithm implemented in Bowtie
v2.3.5 (Langmead \& Salzberg, 2012) according to the HiC-Pro strategy
(Langmead \& Salzberg, 2012; Servant et al., 2015). The Juicer v1.5 and
3D \emph{de novo} assembly (3D-DNA) pipelines were used to assemble the
contigs into a chromosome-level genome (Dudchenko et al., 2017; Durand
et al., 2016). The completeness of the genome was evaluated through the
analysis of single-copy orthologs (Simao, Waterhouse, Ioannidis,
Kriventseva, \& Zdobnov, 2015), implemented in Benchmarking Universal
Single-Copy Orthologs (BUSCO) v3.0.2 (Simao et al., 2015), based on the
insecta\_odb9 database (1,658 genes). Synteny between PFM and
\emph{Cydia pomonella} (Lepidoptera: Tortricidae) (Assembly accession:
GCA\_003425675.2) (Wan et al., 2019) and \emph{Spodoptera litura}
(Assembly accession: GCF\_002706865.1) (Cheng et al., 2017) genomes were
analyzed using TBtools v0.58 (C. Chen et al., 2020).

\textbf{Transcriptome sequencing and assembly}

To provide evidence of transcripts for genome structure annotation, we
conducted RNA-seq for four developmental stages of egg, larva, pupae,
and adults (male and female) reared under normal conditions as described
above. To identify the differentially expressed genes between normal
(ND) and diapausing larvae, we constructed another two RNA-seq libraries
for the long-day (LD) and short-day (SD) induced
5\textsuperscript{th}instar larvae. In total, 7 RNA-seq libraries were
constructed, including one for eggs, three for larvae, one for pupae,
one for male adults and one for female adults of PFM. All libraries were
prepared using VAHTSTM mRNA-seq V2 Library Prep Kit for Illumina
according to the manufacturer's instructions (Vazyme, NanJing, China)
and sequenced on an Illumina Novaseq platform to obtain 150-bp
paired-end reads. After removing the low quality reads with Trimmomatic
v0.38 (Bolger et al., 2014), the reads were mapped to the
chromosome-level genome using Hisat v2.2.0 (D. Kim, Paggi, Park,
Bennett, \& Salzberg, 2019) and assembled with StringTie v2.1.2 (Pertea
et al., 2015). FPKM (Fragments Per Kilobase per Million) values of each
annotated gene in each RNA-seq were estimated with cufflinks v2.2.1 (D.
Kim et al., 2013).

Differential gene expression among larvae reared at different
photoperiod (SD, ND and LD) was assessed using cufflinks v2.2.1 (D. Kim
et al., 2013). Genes with a fold-change \selectlanguage{english}[?] 2 and q-value \selectlanguage{english}[?] 0.05 were
considered significant differentially expression genes (DEGs) between
samples. For significantly expressed genes, up-regulated or
down-regulated genes in both comparisons (ND vs. LD and ND vs. SD) were
considered as genes related to diapause, while the FPKM values
specifically high in the SD or LD condition were considered as
light-induced genes. Gene expression visualization of DEGs were
conducted with the \emph{Pheatmap} R package.

\textbf{Repeat element and non-coding RNA annotation}

Repeats and transposable element families in the PFM genome were first
detected by RepeatMasker v4.0.7 pipeline (Tarailo\selectlanguage{english}-Graovac \& Chen, 2009)
against the Insecta repeats within RepBase Update
(http://www.girinst.org) and Dfam database (20170127), with RMBlast
v2.10.0 as a search engine. The noncoding RNAs (ncRNA) were annotated by
aligning the genomic sequence against RFAM v14.2 (http://rfam.xfam.org/)
with BLASTN. The tRNAs and rRNAs were predicted by tRNAscan-SE and
RNAmmer (Lagesen et al., 2007; Lowe \& Eddy, 1997).

\textbf{Protein-coding gene annotation and filtering}

We annotated protein-coding genes using \emph{ab initio} ,
RNA-seq-based, and homolog-based methods in the MAKER v2.31.10 genome
annotation pipeline (Cantarel et al., 2008). Augustus v3.2.3 (Stanke \&
Waack, 2003) and SNAP v2013-02-16 (Korf, 2004) were used for the
\emph{ab initio} gene prediction. For Augustus, we used the retrained
parameters obtained in the above BUSCO analysis of genome assembly by
invoking the Augustus retraining option. In the first round of
annotation, we ran MAKER by providing transcriptome assemblies of PFM,
protein sequences from eight lepidopteran species (\emph{Bombyx mori} ,
\emph{Trichoplusia ni} , \emph{Ostrinia furnacalis} , \emph{Bombyx
mandarina} , \emph{Galleria mellonella} , \emph{Spodoptera litura} ,
\emph{Helicoverpa armigera} ,\emph{Plutella xylostella} ) and the
Augustus model as evidence. The GFF3 file of first round annotation was
used to train parameters of SNAP. In the next three rounds of
annotation, GFF3 from the last round, Augustus and SNAP models were used
as evidence.

The annotation results from the MAKER pipeline were filtered by using
gene expression evidence, functional annotation results and Annotation
Edit Distance (AED) value. Genes that had a FPKM value great than 0 in
any RNA-seq were considered as real genes and retained in further
analysis. Functional domains for proteins were identified using
InterproScan 5.34-74.0 (Jones et al., 2014) against Pfam database v32.0
(S. El-Gebali et al., 2019). The gene models were filtered based on
domain content and evidence support following Campbell, Holt, Moore, and
Yandell (2014). Finally, the annotations with AED \textless{} 0.75 were
removed (Campbell et al., 2014).

Functions of the protein-coding genes were annotated using the software
eggNOG-Mapper v1.0.3 (Jaime Huerta-Cepas et al., 2017), a tool for fast
functional annotation of novel sequences using precomputed eggNOG-based
orthology assignments, against the database EggNOG v5.0 (J. Huerta-Cepas
et al., 2019).

\textbf{Orthology identification and phylogenetic inference}

Protein-coding genes from another 11 species of Lepidoptera as well as
two species of Coleoptera and two species of Diptera were obtained from
the NCBI genomes database for comparative analysis (\textbf{Table 1} ).
Orthologs were identified using OrthoFinder version 2.2.7 (Emms \&
Kelly, 2015) under default parameters. The phylogenetic tree was
inferred in the OrthoFinder pipeline with an
approximately-maximum-likelihood method implemented in FastTree v2.1.10
(Price, Dehal, \& Arkin, 2009) based on a concatenated multiple sequence
alignment (MSA) of single-copy genes. The most likely category for each
site was set using a Bayesian approach with a gamma prior. Amino acid
sequences were aligned in MAFFT v7.450 (Katoh \& Standley, 2013) with
the G-INS-I algorithm.

\textbf{Manual annotation of circadian genes}

We further manually annotated well-studied circadian genes:\emph{period}
(PER), \emph{timeless} (TIM), \emph{Clock} (CLK),\emph{cycle} (CYC) and
cryptochrome (CRY), using BLAST v2.2.31 (Altschul, Gish, Miller, Myers,
\& Lipman, 1990). Reference protein sequences of insect circadian genes
were obtained from the Uniprot database. Conserved domains within
proteins were annotated against the conserved domain database (Lu et
al., 2020). Circadian genes of the other 15 insect species were
annotated in the same way. For a common domain of three genes (CLK, PER
and CYC), a neighbor-joining tree was constructed using MEGA7 (Kumar,
Stecher, \& Tamura, 2016) with 500 bootstrap replicates.

\textbf{Manual annotation of detoxification gene families}

We manually annotated five detoxification gene families of cytochrome
P450 monooxygenase (P450s), glutathione S-transferase (GSTs),
carboxyl/cholinesterases (CCEs), UDP-glycosyltransferases (UGTs) and
ATP-binding cassette (ABC) transporters. We used the bioinformatic
pipeline BITACORA (Vizueta, Sanchez-Gracia, \& Rozas, 2019) to conduct
HMMER v3.3 (Finn, Clements, \& Eddy, 2011)and BLAST v2.2.31 (Altschul et
al., 1990) analyses under a full mode. Hits were filtered with a default
cut-off E-value of 10e-5. The HMMs of P450 were downloaded from Pfam
v32.0 (Sara El-Gebali et al., 2018), while other HMMs of detoxification
gene families were created by HMMER v3.3 (Finn et al., 2011). Orthologs
from \emph{Bombyx mori} and \emph{D. melanogaster} were used as
evidence. The annotated genes were further filtered manually based on
gene length and the presence of conserved domains. Genes with a length
shorter than 80 amino acids were removed. Orthologs were aligned with
the G-INS-I algorithm implemented in MAFFT v7.450 (Katoh \& Standley,
2013). A neighbor-joining tree was constructed for each gene family
using MEGA7 (Kumar et al., 2016) with 500 bootstrap replicates.

\textbf{Results and discussion}

\textbf{Features of the assembled genome}

The genome size of PFM is estimated to be 338.52-352.59 Mb through k-mer
analysis depend on the k-mers used (k = 17, 21, 25, 35). The k-mer
distributions showed double peaks, indicating that this genome has a
high rate of duplication and heterozygosity. The estimated
heterozygosity ranges from 1.06\% to 1.15\% and rate of duplication
ranges from 1.95\% to 2.06\% (Fig. 2a).

At the contig level, we assembled the PFM genome into 404.83 Mb
sequences, including 275 contigs, with a contig N50 length of 2.62 Mb.
Based on contig interaction frequency calculated from the pairs aligned
to the contigs, the 275 contigs were clustered into 32 linkage groups
(Fig. 2b). The longest contig group was 19.1 Mb while the shortest one
was 2.63 Mb, with an N50 of 14.39 Mb. BUSCO analysis showed that 98.2\%
(single-copied gene: 97.2\%, duplicated gene: 1.0\%) of 1,658 genes were
identified as complete, 0.40\% of genes were fragmented, while 1.4\% of
genes were missing in the assembled genome. The genome comprised 36.96\%
GC base pairs.

Synteny analysis showed that the PFM, \emph{S. litura} and \emph{C.
pomonella} genome have a highly conserved gene order (Fig. 2c). PFM has
similar chromosomes as \emph{S. litura} , including 30 autosomes, a Z
chromosome (Chr01) and a female specific W chromosome, while \emph{C.
pomonella} has undergone three fusion events, resulting in 27 autosomes,
a W chromosome, and a neo-Z chromosome arising from a Z-autosomal fusion
(Wan et al., 2019). The chromosome-level assembly of the PFM genome
provides resources for understanding chromosome evolution in the
Lepidoptera (Ahola et al., 2014).

\textbf{Genome annotation}

We identified 29,228 protein-coding genes in the 1st round of MAKER
annotation. BUSCO analysis revealed 91.9\% of the evaluated single-copy
genes were identified as complete. After three rounds of MAKER
annotation, the number of genes increased to 52,667, while the
proportion of complete single-copy genes was up to 95.2\%. After
filtering based on gene expression analysis, functional domains and AED
values, 23,218 genes remained. BUSCO analysis showed that 95.0\%
(single-copied gene: 94.1\%, duplicated gene: 1.1\%) of the evaluated
single-copy genes were identified as complete, 1.6\% of the genes were
fragmented, and 3.2\% of the genes were missing in the annotated gene
set. In total, 19,206 genes (82.72\%) were functionally annotated, of
which 5,970 (25.71\%) and 3,134 (13.50\%) genes annotated to GO terms
and KEGG KOs respectively. We predicted 53 rRNAs, 11,076 tRNAs, 20 small
nuclear RNAs, and 48 micro RNAs in the PFM genome based on Rfam
databases.

In total, 45.5 Mb (11.33\%) of the genome was identified to be repeat
DNA. Overall, 259,729 transposable elements (TEs) including 125,601
retroelements (17,962 short interspersed nuclear elements (SINEs),
95,657 long interspersed nuclear elements (LINEs) and 11982 long
terminal repeats (LTR)) and 34,478 DNA transposons were identified.

Orthology and phylogenetic relationships of lepidopterans

OrthoFinder assigned 320,821 genes (93.41\% of total) to 15,076
orthogroups for the 16 species compared. Fifty percent of the assigned
genes were in orthogroups with 28 or more genes (G50 was 28) and were
contained in the largest 3,174 orthogroups (O50 was 3,174).

There were 947 single-copy genes with 364,262 reliable sites retained
for phylogenetic inference. The topology is congruent with previously
inferred phylogenetic relationships of Lepidoptera, in which no
representative of the Copromorphoidea was included (Wan et al., 2019).
Current molecular phylogenetic studies have not resolved the
phylogenetic relationship between Copromorphoidea and Papilionoidea
(Mitter, Davis, \& Cummings, 2017). Our result supports the notion that
PFM from the Copromorphoidea forms a sister-group relationship to the
butterfly \emph{D. plexippus} (Papilionoidea), rather than a sister
group between Copromorphoidea/Papilionoidea and Pyraloidea + (Noctuoidea
+ Bombycoidea) (Fig. 3a).

We investigated orthogroups shared by PFM and four species of
Lepidoptera representing different clades of the phylogenetic tree of
Lepidoptera (Fig 3b). There were 7,827 orthogroups (60.5\% of 12,938
orthogroups) shared by all five lepidopteran species and 1,549
orthogroups shared by four species except for \emph{C. pomonella} . We
identified 357 orthogroups specific to PFM, fewer than that of \emph{B.
mori} (406), but higher than other three lepidopteran species (Fig. 3b).

\textbf{Evolution of circadian genes}

Five circadian genes were annotated in the PFM genome and the other
reference insect species. The PER gene was not found in currently
assembled genomes of \emph{Cydia pomonella} and \emph{Anoplophora
glabripennis} . Two types of CRY gene were annotated in 16 species,
mammalian-type cryptochrome (CRY-m) and Drosophila type cryptochrome
(CRY-d). For most of the 16 insects, two types of CRY gene were found,
while only CRY-m was found in two Coleoptera species and only CRY-d was
found in \emph{Drosophila melanogaster} . In the PFM, FPKM values of
CRY-m were higher than CRY-d in each stage, indicating that CRY-m may be
a major element in the circadian clock of PFM. Domains of circadian
genes were conserved among the 16 species (Fig. 4). PAS domains were
common in CLK, CYC and PER genes. The phylogenetic tree of PAS domains
revealed six clades, corresponding to two domains of three genes (Fig.
5).

Gene expression in diapause and non-diapause PFM

Compared with larvae that developed under a normal day photoperiod, 11
genes were significantly up-regulated and 9 genes were down-regulated in
larvae that developed under long-day or short-day photoperiods
(\textbf{Table S1, Fig. S1} ). Genes highly expressed in pre-diapause
larvae (SD and LD photoperiod) included genes encoding CUSOD2
(CS\_07203), an enzyme that destroys radicals, and that plays an
important role in diapause and cold tolerance of insect (Bi, Yang, Yu,
Shu, \& Zhang, 2014; He, Meng, Yang, \& Hua, 2013; Isobe et al., 2006;
Y. I. Kim et al., 2010; Sim \& Denlinger, 2011; Zhao \& Shi, 2009). We
identified a cytochrome P450 gene (CS\_20496) showing weak expression in
pre-diapause larvae and high expression in the other stages, which was
also found in diapausing larvae of the wild silk moth, Antheraea yamamai
(Yang, Tanaka, Kuwano, \& Suzuki, 2008).

We identified 44 genes specifically up-regulated under a long-day
photoperiod, and 14 genes specifically up-regulated under a short-day
photoperiod (\textbf{Table S1, Fig. S1} ). Four genes (CS\_04235,
CS\_05017, CS\_15183, CS\_01854) related to digestion of proteins were
up-regulated in larvae developing under a long-day photoperiod. This is
congruent with previous reports suggesting that photoperiod had
significant effects on digestive enzyme activity (Espinosa-Chaurand,
Vega-Villasante, Carrillo-Farnes, \& Nolasco-Soria, 2017; Ramzanzadeh,
Yeganeh, JaniKhalili, \& Babaei, 2016; Shan, Xiao, Huang, \& Dou, 2008;
Subala \& Shivakumar, 2017). The functional link of many of these genes
to diapause is not really clear. The circadian genes, which are
important in diapause in another moth (Kozak et al., 2019), did not show
significant changes for larvae under different photoperiods.

\textbf{Evolution of detoxification genes}

We manually identified 96 P450s, 77 GSTs, 63 CCEs, 28 UGTs, and 104 ABCs
in the PFM genome (Table 2; Fig. S2). PFM had the highest number of GST
genes and the second highest number of ABCs following the \emph{O.
furnacalis} when compared to the other lepidopterans. The number of P450
and CCE genes in PFM are at an intermediate level. We found that PFM had
the lowest number of UGT genes, along with two other moths located at
basal lineages of the Lepidoptera. These results suggest that PFM may
have a unique way of detoxication with a reduced importance of UGT when
compared to the other moths. This may have implications for pesticide
responses in PFM give that these detoxification genes can respond in
different ways to various pesticides in moths (Hu et al., 2019).

\textbf{Conclusions}

We assembled the chromosome-level genome for the PFM using PacBio
long-read and Hi-C technology. This is the first assembled genome for
the superfamily Copromorphoidea. This novel genomic resource allowed us
to explore possible genes in PFM associated with adaptation to
environmental factors. We identified five core genes relating to
circadian rhythm in PFM and annotated models for each gene. Using the
genome as a reference, we identified DEGs related to diapause of OFM
which may point to candidate genes. Given the expression of long-day and
short-day diapause by PFM, this moth species will be a useful model to
further investigate adaptive shifts involving diapause, particularly by
combining genomic information with intraspecific comparisons across
geographic gradients (Ragland, Armbruster, \& Meuti, 2019). The
assembled genome provides a resource for further comparative studies of
moths and butterflies particularly with respect to life cycle evolution
and parallel evolution in detoxification functions.

\textbf{Data Availability Statement}

The Whole Genome assembly has been deposited in the Genome repository of
NCBI (accession numbers: CP053148-CP053179) under BioProject PRJNA627116
(reviewer
link:\emph{https://dataview.ncbi.nlm.nih.gov/object/PRJNA627116?reviewer=rvjh98ap96u9pqguc8k17dl0cb}).
Raw reads obtained for genome assembly were deposited in the Sequence
Read Archive (SRA) repository (accession numbers: SRR12328811 and
SRR12336732). Gene sequences of manually annotated families were
deposited in the Dryad repository
(\emph{https://doi.org/10.5061/dryad.m0cfxpp1j}).

\textbf{Author contributions}

Shu-Jun Wei conceived and designed the study; Jin-Cui Chen and Ya-Jun
Gong conducted the collection and rearing of the insect; Li-Jun Cao
conducted the molecular works; Li-Jun Cao, Wei Song, Lei-Yue, Shao-Kun
Guo and Shu-Jun Wei analyzed the data; Li-Jun Cao, Shu-Jun Wei and Ary
Hoffmann discussed the results and wrote the manuscript.

\textbf{Acknowledgments}

We thank Qiang Gao for his help on the assembly of Hi-C data and Qiang
Gong for the assistance of insect rearing. This research was supported
by National Natural Science Foundation of China (31901884), the Natural
Science Foundation of Beijing Municipality (6184037), Beijing Academy of
Agriculture and Forestry Sciences (BAAFS) - The University of Melbourne
(UOM) Joint Laboratory on Pest Control Research, and the Beijing Key
Laboratory of Environmentally Friendly Pest Management on Northern
Fruits (BZ0432).

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\textbf{Tables}

Table 1 Features of chromosome-level genomes in the Lepidoptera\selectlanguage{english}
\begin{longtable}[]{@{}llllllll@{}}
\toprule
\textbf{Features} & \textbf{Csas} & \textbf{Cpom} & \textbf{Tni} &
\textbf{Bmor} & \textbf{Slit} & \textbf{Mcin} &
\textbf{Hmel}\tabularnewline
\midrule
\endhead
Genome size (Mb) & 401.67 & 772.89 & 368.2 & 431.7 & 438.32 & 393 &
269\tabularnewline
Karyotype & 2n=64 & 2\emph{n}=56 & 2\emph{n}=54 & 2\emph{n}=56 &
2\emph{n}=62 & 2\emph{n}=62 & 2\emph{n}=42\tabularnewline
No. contigs & 275 & 2221 & 26,605 & 15,018 & 13,636 & 49,851 &
NA\tabularnewline
No. scaffolds & NA & 1717 & 6181 & 7397 & 3597 & 8262 &
3807\tabularnewline
No. CHR* & 31A+Z+W & 27A+Z+W & 26A+Z+W & 27A+Z & 30A+Z & 30A+Z &
20A+Z\tabularnewline
Contig N50 (kb) & 2620 & 862.49 & 621.9 & 15.5 & 68.35 & 13 &
51\tabularnewline
Scaffold N50 (Mb) & NA & 8.92 & 14.2 & 3.7 & 0.915 & 0.119 &
0.277\tabularnewline
BUSCO genes (\%) & 98.20\% & 98.5 & 97.8 & 97.7 & 98.3 & 91.5 &
97.4\tabularnewline
Repeat (\%) & 11.33 & 42.87 & 20.5 & 43.6 & 31.83 & 28 &
24.94\tabularnewline
G+C (\%) & 36.96 & 37.43 & 35.6 & 37.3 & 36.5 & 33.0 & NA\tabularnewline
No. genes & 23,227 & 17,184 & 14,043 & 14,623 & 15,317 & 16,667 &
12,669\tabularnewline
\bottomrule
\end{longtable}

Csas, \emph{Carposina} \emph{sasakii} ; Cpom, \emph{Cydiapomonella} ;
Tni, \emph{Trichoplusia} \emph{ni} ; Bmor,\emph{Bombyx} \emph{mori} ;
Slit, \emph{Spodoptera} \emph{litura} ; Mcin,\emph{Melitaea}
\emph{cinxia} ; Hmel, \emph{Heliconius} \emph{melpomene} ; * A
represents auto chromosome; Z and W represent sex chromosomes; NA, not
available. Data for all species except for Csas were summarized by Wan
et al. (2019).

Table 2 Number of genes in five detoxification families across species
of Lepidoptera\selectlanguage{english}
\begin{longtable}[]{@{}lllllll@{}}
\toprule
Species & P450 & GST & CCE & ABC & UGT & Reference for
genome\tabularnewline
\midrule
\endhead
\emph{Carposina sasakii} & 96 & 77 & 63 & 104 & 28 & This
study\tabularnewline
\emph{Plutella xylostella} & 74* & 11* & 55* & 97* & 38 & You et al.
(2013)\tabularnewline
\emph{Cydia pomonella} & 137** & 30** & 73** & 47** & 30** & Wan et al.
(2019)\tabularnewline
\emph{Danaus plexippus} & 107 & 35 & 73 & 76 & 47 & Zhan et al.
(2011)\tabularnewline
\emph{Trichoplusia ni} & 143 & 51 & 122 & 71 & 68 & W. Chen et al.
(2019)\tabularnewline
\emph{Spodoptera litura} & 138* & 47* & 110* & 54* & 64 & Cheng et al.
(2017)\tabularnewline
\emph{Helicoverpa armigera} & 122 & 57 & 105 & 76 & 54 & Song, Downes,
Parker, Oakeshott, and Robin (2015)\tabularnewline
\emph{Bombyx mandarina} & 94 & 37 & 94 & 64 & 48 & Xiang et al.
(2018)\tabularnewline
\emph{Bombyx mori} & 83* & 26* & 76* & 51* & 50 & Xia et al.
(2004)\tabularnewline
\emph{Manduca sexta} & 97* & 17* & 86* & 47* & 51 & Kanost et al.
(2016)\tabularnewline
\emph{Galleria mellonella} & 137 & 44 & 75 & 72 & 58 & Lange et al.
(2018)\tabularnewline
\emph{Ostrinia furnacalis} & 126 & 48 & 115 & 112 & 46 & Ma et al.
(2020)\tabularnewline
\bottomrule
\end{longtable}

*, data from S. Zhang et al. (2020); **, data from Wan et al. (2019).
The other data were manually identified in our study.

\textbf{Figure legends}

\textbf{Fig. 1} Eggs (A), larva (B), cocoons (C) and adult (D) of the
peach fruit moth \emph{Carposina} \emph{sasakii} (A-D) and the damage
symptoms to apple (E-G). The hatched larva bores into apple usually near
the calyx with white secreta near the boring hole (E); the damaged apple
showing shrinkage (F); damage from larvae boring and developing in the
apple (G).

\textbf{Fig. 2} Genome features of \emph{Carposina sasakii.} (a)
GenomeScope analysis of genome size, heterozygosity and duplicate rate
using k-mers (K = 17) count histogram, indicating a genome size of
338.52 Mb, a heterozygosity of 1.06\%, and a duplication rate of 2.06\%;
(b) Genome-wide all-by-all Hi-C interaction identified 32 linkage
groups; (c) Synteny between \emph{Carposina sasakii} (Csas)
and\emph{Cydia pomonella} (Cpom) and \emph{Spodoptera} \emph{litura}
(Slit) genomes reveal highly conserved gene order and chromosomal fusion
or split events in the three moths.

\textbf{Fig. 3} Comparative genomics of \emph{Carposina sasakii.} (a)
Phylogenetic tree of PFM with 15 insect genomes including 11 other
Lepidoptera. The phylogeny was inferred from 947 single-copy genes with
364,262 reliable sites by an approximately-maximum-likelihood method.
All nodes received bootstrap support of 100. (b) Orthogroups shared by
five Lepidoptera species of \emph{Carposina} \emph{sasakii}
,\emph{Cydia} \emph{pomonella} , \emph{Bombyx} \emph{mori} ,
\emph{Ostrinia furnacalis} and \emph{Helicoverpa armigera.}

\textbf{Fig. 4} Schematic arrangement of the domains of five circadian
genes including \emph{period} (PER), \emph{timeless} (TIM),
\emph{Clock}(CLK), \emph{cycle} (CYC) and cryptochrome (CRY-m, CRY-d)
in\emph{Carposina sasakii} and other 15 insects. Boxes in different
color show different domains, while numbers under boxes show the postion
of domains on protein sequences. Species and their taxonomic status are
shown on the left: Tcas, \emph{Tribolium castaneum} ;
Agla,\emph{Anoplophora glabripennis} ; Agam, \emph{Anopheles gambiae} ;
Dmel,\emph{Drosophila melanogaster} ; Pxy, \emph{Plutella xylostella} ;
Cpom,\emph{Cydia pomonella} ; Csas, \emph{Carposina sasakii} ;
Dple,\emph{Danaus plexippus} ; Tni, \emph{Trichoplusia ni} ;
Slit,\emph{Spodoptera litura} ; Harm, \emph{Helicoverpa armigera} ;
Bmor,\emph{Bombyx mori} ; Bman, \emph{Bombyx mandarina} ; Msex,
\emph{Manduca sexta} ; Gmel, \emph{Galleria mellonella} ; Ofur,
\emph{Ostrinia furnacalis} .

\textbf{Fig. 5} Phylogenetic relationships of two PAS domains in three
circadian genes: \emph{period} (per), \emph{Clock} (clk) and\emph{cycle}
(cyc). Each tip is labeled by the name of domain, gene and species.
Abbreviations of species are same as in Fig. 4. Six clades shaded in
different color reveal two domains of three genes, while one domain of
\emph{clk} gene has two different type among species. Tips in red show
the position of \emph{Carposina sasakii} .\selectlanguage{english}
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