Transcriptome profiling of the Plutella xylostella (Lepidoptera: Plutellidae) ovary reveals genes involved in oogenesis

Accepted Manuscript Transcriptome profiling of the Plutella xylostella (Lepidoptera: Plutellidae) ovary reveals genes involved in oogenesis

Lu Peng, Lei Wang, Yi-Fan Yang, Ming-Min Zou, Wei-Yi He, Yue Wang, Qing Wang, Liette Vasseur, Min-Sheng You PII: DOI: Reference:

S0378-1119(17)30727-8 doi: 10.1016/j.gene.2017.09.020 GENE 42169

To appear in:

Gene

Received date: Revised date: Accepted date:

14 December 2016 21 July 2017 8 September 2017

Please cite this article as: Lu Peng, Lei Wang, Yi-Fan Yang, Ming-Min Zou, Wei-Yi He, Yue Wang, Qing Wang, Liette Vasseur, Min-Sheng You , Transcriptome profiling of the Plutella xylostella (Lepidoptera: Plutellidae) ovary reveals genes involved in oogenesis, Gene (2017), doi: 10.1016/j.gene.2017.09.020

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ACCEPTED MANUSCRIPT Transcriptome profiling of the Plutella xylostella (Lepidoptera: Plutellidae) ovary reveals genes involved in oogenesis Lu Peng1,2,3,4,5†, Lei Wang1,2,3,4,5†, Yi-Fan Yang1,2,3,4,5†, Ming-Min Zou1,2,3,4,5, Wei-Yi He1,2,3,4,5, Yue Wang1,2,3,4,5, Qing Wang1,2,3,4,5, Liette Vasseur1,2,3,4,5,6, Min-Sheng You1,2,3,4,5*

State Key Laboratory of Ecological Pest Control for Fujian-Taiwan Crops and

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College of Life Science, Fujian Agriculture and Forestry University, Fuzhou

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350002, China

Institute of Applied Ecology, Fujian Agriculture and Forestry University, Fuzhou

Fujian-Taiwan Joint Innovation Centre for Ecological Control of Crop Pests, Fujian

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350002, China

Agriculture and Forestry University, Fuzhou 350002, China Key Laboratory of Integrated Pest Management for Fujian-Taiwan Crops, Ministry

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of Agriculture, Fuzhou 350002, China Fujian Provincial Key Laboratory of Insect Ecology, Fujian Agriculture and Forestry

Department of Biological Sciences, Brock University, St. Catharines, Ontario L2S

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University, Fuzhou 350002, China

3A1, Canada †

These authors contributed equally to this work

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Corresponding author: [email protected]

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ACCEPTED MANUSCRIPT Abstract Background: As a specialized organ, the insect ovary performs valuable functions by ensuring fecundity and population survival. Oogenesis is the complex physiological process resulting in the production of mature eggs, which are involved in epigenetic

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programming, germ cell behavior, cell cycle regulation, etc. Identification of the genes involved in ovary development and oogenesis is critical to better understand the

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reproductive biology and screening the potential molecular targets in Plutella

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xylostella, a worldwide destructive pest of economically major crops.

Results: Based on transcriptome sequencing, a total of 7.88 Gb clean nucleotides was

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obtained, with 19,934 genes and 1,861 new transcripts being identified. Expression profiling indicated that 61.7% of the genes were expressed (FPKM≥1) in the P.

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xylostella ovary. GO annotation showed that the pathways of multicellular organism reproduction and multicellular organism reproduction process, as well as gamete

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generation and chorion were significantly enriched. Processes that were most likely relevant to reproduction included the spliceosome, ubiquitin mediated proteolysis,

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endocytosis, PI3K-Akt signaling pathway, insulin signaling pathway, cAMP signaling pathway, and focal adhesion were identified in the top 20 „highly represented‟ KEGG pathways. Functional genes involved in oogenesis were further analyzed and validated by qRT-PCR to show their potential predominant roles in P. xylostella reproduction.

Conclusions: Our newly developed P. xylostella ovary transcriptome provides an 2

ACCEPTED MANUSCRIPT overview of the gene expression profiling in this specialized tissue and the functional gene network closely related to the ovary development and oogenesis. This is the first genome-wide transcriptome dataset of P. xylostella ovary that includes a subset of functionally activated genes. This global approach will be the basis for further studies

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on molecular mechanisms of P. xylostella reproduction aimed at screening potential

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molecular targets for integrated pest management.

Keywords: Ovary development, Oogenesis, Transcriptome analysis, Reproductive

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regulation, Plutella xylostella

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ACCEPTED MANUSCRIPT Background Oogenesis is the process by which female gametes are developed and includes ovary and egg development, as well as reproductive regulation (Schuetz, 1985). This process involves a large number of genes and signal transduction pathways related to

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epigenetic programming (Skora and Spradling, 2010), germ cell behavior (Dansereau and Lasko, 2008), cell cycle regulation (Bastock and St Johnston, 2008) and

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developmental patterning mechanisms (Lynch et al., 2010; Wilson et al., 2011). In

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insects, female reproductive genes, such as chorion, vitelline membrane proteins, Vg

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and VgR (Sdralia et al., 2012; Chen et al., 2015; Upadhyay et al., 2016), and signal transduction pathways including insulin and hormone pathways (Fox et al., 2011;

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Jindra et al., 2013) have been studied in Anopheles gambiae, Drosophila melanogaster, Nilaparvata lugens, and Apis mellifera (Mack et al., 2006; Rogers et al.,

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2008; Zhai et al., 2013; Niu et al., 2014). Current results indicated that these genes and pathways play major roles in insect reproduction but greatly vary among different

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species (Attardo et al., 2005).

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The advent of high throughput sequencing technologies has provided an opportunity to comprehensively examine all genes or a subset of functionally active genes from the genome of a specific species or tissue (Wang et al., 2009). These techniques, especially transcriptomic analyses, have become important to better understand the mechanisms behind insect fecundity. For example, transcriptome sequencing of reproductive tissues has been performed in insects, such as accessory glands and testis of Bactrocera dorsalis (Wei et al., 2015; Wei et al., 2016) and A. gambiae (Dottorini 4

ACCEPTED MANUSCRIPT et al., 2013), and the ovary of N. lugens, A. mellifera and Venturia canescens (Leach et al., 2009; Zhai et al., 2013; Niu et al., 2014). These studies have been useful to identify genes related to sexual gland development, spermatogenesis, and oogenesis.

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The diamondback moth (DBM), Plutella xylostella (L.) (Lepidoptera, Plutellidae), is a

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Lepidoptera pest that mainly attacks cruciferous plants (Thorsteinson, 1953). It has a

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very high reproductive capacity allowed it to invade all regions where cruciferous plants, mainly crops, grow (Zalucki et al., 2012; Furlong et al., 2013). The

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fundamental reproductive biology of DBM has been widely investigated (Peng et al.,

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2015), but the molecular mechanisms of reproduction remain unclear. It is therefore important to explore genes and major signal pathways involved in reproduction,

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including ovary development and oogenesis, which is of significance for screening

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potential molecular targets to better control P. xylostella.

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In this study, we used deep sequencing to examine P. xylostella genes involved in

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ovary development and oogenesis. More specifically, we aimed to get a comprehensive view of the transcriptional profiles and putative roles of genes referred to as several crucial reproductive processes of embryogenesis, vitellogenesis, and choriogenesis. This global approach provided valuable insights into the molecular mechanisms related to female reproduction of this species.

Materials and methods

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ACCEPTED MANUSCRIPT P. xylostella rearing and sample preparations A susceptible P. xylostella strain (i.e. not resistant to pesticides) collected from a Fuzhou (province of Fujian, China) cabbage (Brassica oleracea var. capitata) field (26.08°N, 119.28°E) in 2004 has been maintained since in laboratory at the Fujian

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Agriculture and Forestry University. The colony has been on 3-5 weeks old radish

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seedlings (Raphanus sativus) at 25 ± 1 °C, 65 ± 5% RH and L:D = 16:8 h without

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exposure to insecticides.

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Based on a preliminary experiment, we found that the P. xylostella ovary began to

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develop at the pupa stage. The organ was fully formed on the second day of pupation and could be dissect as an independent organ. Therefore, for this study, we collected

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ovaries from 2-day old pupae to the 5-day old virgin female adults at every 24 h

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interval (n= 30 per age) as a replication. There were three biological replications. Dissections were performed in sterile PBS-DEPC. The dissected materials were

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immediately soaked in the 1.5 ml centrifuge tube (Axygen) with RNA-later (Qiagen)

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solution on ice, and subsequently stored at −80 °C for RNA extraction.

RNA extraction

Total RNA extraction of individuals at each age was conducted with RNeasy mini kit (Qiagen, Valencia, CA, USA) as described in the manufacturer‟s manual. RNA contamination and degradation were analyzed using 1% agarose gel electrophoresis. RNA samples were assessed for purity at absorbance ratios of OD260/280 and

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ACCEPTED MANUSCRIPT OD260/230 using the NanoDrop2000® spectrophotometer (Thermo, USA). To prepare the library, we pooled an equal quantity of total RNA from samples of the different ages.

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cDNA library construction and Illumina sequencing

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Each sample with an amount of 3 μg RNA was used for the sample preparation. The

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library was developed using the NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (NEB, USA) according to manufacturer‟s instructions. Its quality was then

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assessed on the Agilent Bioanalyzer 2100 system. The sequences of each replicate

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were marked by the index codes, and the index-coded samples were clustered on a cBot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS (Illumina)

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following the manufacturer‟s instructions. After clustering, the libraries were

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reads (Figure S1).

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sequenced on an Illumina Hiseq platform to obtain the 125 bp/150 bp paired-end

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Transcriptome data analysis Quality control

Raw data (raw reads) in FASTQ format were first modified into clean data (clean reads) through Perl scripts. This was done by (1) filtering out adapter-only reads, (2) removing reads containing more than 10% poly-N, and (3) removing low quality reads to ensure PHRED quality scores<=20. The methods used in our experiment were also consistent with those reported elsewhere, such as in Shi et al. (2016) and

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ACCEPTED MANUSCRIPT Huang et al. (2015). Meanwhile, Q20, Q30, and GC contents were calculated. All of the analyses were carried out using the high-quality clean data.

Mapping and assembly of clean reads

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Sequence files used for reference and annotation were downloaded from the

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diamondback moth Genome Database (DBM-DB;

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http://iae.fafu.edu.cn/DBM/index.php). Bowtie v2.2.3 software was used to build the reference genome index (Langmead and Salzberg, 2012), and the clean paired-end

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reads obtained from RNA-seq were aligned to the reference genome with TopHat

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v2.0.12 (Trapnell et al., 2009). After TopHat alignment, known and novel transcripts were constructed and identified based on Reference Annotation Based Transcript

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(RABT) assembly method of Cufflinks v2.1.1 (Trapnell et al., 2010; Trapnell et al.,

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2012).

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Transcripts expression profiling

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The calculations of read numbers mapped for each transcript were performed using HTSeq v0.6.1 and the transcript quantification was calculated with FPKM (expected number of Fragments Per Kilobase of transcript sequence per Million base pairs sequenced) (Trapnell et al., 2010). FPKM value „1‟ was defined as the threshold for the transcript expression. We took the average values of the three biological replicates as the actual expression value for each of the transcripts. Additionally, based on our RNA-seq data, stage-specific expression of the oogenesis genes were profiled.

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GO and COG annotation The GOseq R package was applied for Gene ontology (GO) annotation of all expressed genes (Young et al., 2010) and the GO terms were categorized by WEGO.

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For Clusters of Orthologous Groups (COG) annotation, all expressed genes were

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compared to COG database with BLASTX (E-value ≤1e-3).

GO and KEGG enrichment analyses

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GO enrichment analyses were also completed by GOseq R package (Young et al.,

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2010), and the Fisher‟s exact test and Benjamini-Hochberg FDR correction (false discovery rate (FDR) < 0.05) were used to compare the enrichment differences among

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GO terms. We used the KOBAS 2.0 with a hypergeometric test and

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Benjamini-Hochberg FDR correction to identify the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, which were significantly enriched (Xie et al., 2011).

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The P-value of enriched pathway was calculated as follows:

where N representing the total gene number with pathway annotations; n, the gene number expressed in N; M, the total gene number in a specific annotation pathway, and; m, the gene number expressed in M. A corrected P-value < 0.05 was defined as significantly enriched.

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ACCEPTED MANUSCRIPT Quantitative real-time PCR (qRT-PCR) validation The expression profiling of oogenesis-related genes was completed using qRT-PCR technique. Total RNAs of different developmental stages and different tissues (head, thorax, midgut+malpighian tubule, ovary, testis and residues) were isolated from the

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P. xylostella with RNeasy Mini Kit (Qiagen, USA). GoScript Reverse Transcription

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System (Promega, Madison, WI, USA) was used to synthesize the First-stand cDNA.

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The reactions of qRT-PCR were performed with the GoTaq qPCR Master Mix Kit (Promega, Madison, WI, USA). The reaction system of 20 μL total volume consisted

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of 10 μL GoTaq qPCR Master Mix, 2 μL of template cDNA, 0.4 μL of each primer

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(Table S1), 7 μL of ddH2O, and 0.2 μL CXR. The reaction was executed by QuantStudio™ 6 Flex Real-Time PCR System (ABI, USA) with the procedure as

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follows: preheating at 95 °C for 10 min, then denaturing at 95 °C for 15 s and

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annealing at 58 °C for 30 s with 40 cycles. The specificity of PCR products was evaluated through a melting curve analysis going from 60 to 95 °C. The expression

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level of the transcripts was quantified according to the 2−Δt cycle threshold value

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method (Pfaffl, 2001). One-way analysis of variance (ANOVA) with post hoc multiple range test was used to test the differences (P < 0.05) using SPSS 17.0 (SPSS Inc., Chicago, IL, USA).

Phylogenetic analysis Chorion protein sequences were aligned with ClustalX 2.0 (Larkin et al., 2007), and manually modified to remove gaps and missing data. These protein sequences were

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ACCEPTED MANUSCRIPT further subjected to phylogenetic analysis with MEGA 6.06 (Tamura et al., 2013) using the neighbor-joining method with a bootstrap value of 1000 replicates. Poisson correction model of amino acids and gaps pairwise deletion were applied to

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reconstruct the phylogenetic tree.

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Results and discussion

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RNA-sequencing and identification of novel transcripts

Here, the cDNA sample pooled with different female developmental stages of P.

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xylostella was sequenced. After quality control, a total of 56,754,304, 48,122,060, and

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52,668,652 clean reads containing 8.51, 7.22, and 7.9 giga base (Gb) pairs of clean nucleotides were obtained in the three replicates. The quality of the transcriptome

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sequences was high, with Q20 percentages of 96.78 %, 96.90%, and 97.06% for the

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three replicates, while all of the GC contents were about 50% (Table 1). In addition, RNA-seq data among the three replicated ovary libraries were highly correlated (r≥

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0.90, Pearson correlation) (Figure 1).

There were altogether 1,861 novel transcripts detected by Cuffcompare, among that 301 transcripts had no homology with the known proteins deposited in the protein non-redundant (nr) database (http://www.ncbi.nlm.nih.gov/) through BLASTX search (Table S2). The locations and number of exons of each novel gene were defined (see Table S3).

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ACCEPTED MANUSCRIPT Gene expression profiles A total of 19,934 genes was expressed in P. xylostella ovary transcriptome (Table S4). To better assess the ovary transcription information, FPKM values were used to classify the numerical distribution of genes according to five levels of gene expression.

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We found that in the ovary of P. xylostella, 61.7% of the genes were expressed in the

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class FPKM>1, while 2,533 genes (12.7%) were extremely highly expressed (in class

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FPKM > 60) (Figure 2).

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GO and COG analysis

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A total of 8,489 annotated genes were obtained by GO annotation, and they were assigned to three primary GO terms: biological process, cellular component, and

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molecular function (Figure 3). These genes were further assigned to 45 functional

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groups, and this was slightly fewer than for the whole body transcriptome of P. xylostella (n=50; (He et al., 2012)). In biological process, the most abundant groups

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were cellular process (GO: 0009987) with 4,683 genes and metabolic process (GO:

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0008152) with 4,287 genes. Cell (GO: 0005623) and cell part (GO: 0044464) with both 2,527 genes were the most abundant groups in cellular component. Binding (GO: 0005488) with 5,336 genes was most dominant in molecular function, followed by catalytic activity (GO: 0003824) with 3,967 genes. These data offer a precious resource to explore the molecular mechanisms of P. xylostella reproduction.

The GO enrichment analysis indicated that 74 GO terms in biological process, 31 in

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ACCEPTED MANUSCRIPT cellular component, and 38 in molecular function were significantly enriched (corrected P value ≤ 0.05) (Table S5). Among these, the multicellular organism reproduction and multicellular organismal reproductive process on the level 2, as well as gamete generation and chorion on the level 3 were significantly enriched, which

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might be closely related to the reproduction of P. xylostella.

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Furthermore, we classified the potential functions of all expressed genes based on COG annotation and found that 1,180 genes were allocated in at least one COG class

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(Figure 4). Among the 23 COG classes, the largest two groups were posttranslational

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modification, protein turnover, and chaperones (166, 14.1%), and translation, ribosomal structure and biogenesis (164, 13.9%). The third largest groups was general

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function prediction only (119, 10.1%). Amino acid transport and metabolism (89,

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8.3%), replication, recombination and repair (78, 6.6%), energy production and conversion (78, 6.6%), and transcription (77, 6.5%) were also common. By contrast,

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genes involved in defense mechanisms (1, 0.08%) and nuclear structure (3, 0.25%)

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represented the smallest groups. In addition, 44 (3.7%) sequences were assigned to the unknown functional class.

Metabolic pathways based on the KEGG analysis When the metabolic pathways of the P. xylostella ovary genes were analyzed, 2,966 genes were matched to 293 predicted KEGG pathways (Table S6). Based on gene numbers, the top 20 „highly represented‟ pathways- are presented in Table 2. The

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ACCEPTED MANUSCRIPT primary KEGG pathways including spliceosome (n=116), ubiquitin mediated proteolysis (n=101), endocytosis (n=101), PI3K-Akt signaling pathway (n=95), insulin signaling pathway (n=91), cAMP signaling pathway (n=87), and focal adhesion (n=96) were felt to be relevant to oogenesis and reproduction. These

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predicted pathways will be of significance for future study on reproductive functions

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of P. xylostella.

Genes involved in reproduction

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Based on the functional annotation, we identified 162 transcripts presumably related

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to reproduction in P. xylostella ovary (Table S7). According to the function category in Telang et al. (2013), these transcripts were associated with cytoskeletal proteins,

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signal transduction, nuclear regulation, transcriptional machinery, and protein

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synthesis and modification. Most of them may play crucial functions in the formation and differentiation of germline stem cell and oocyte, ovarian development,

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vitellogenesis, egg maturation (Siaussat et al., 2007; Parthasarathy et al., 2010; Belles and Piulachs, 2015). All 162 transcripts could be expressed at each developmental

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stage. They also, showed gene-differential and stage-specific patterns based on our RNA-seq data (Figure S2). Here, we described some potential roles of these transcripts in the oogenesis process of P. xylostella.

Chorion-related proteins. The follicular cells in ovarioles produce chorion polypeptides that are important eggshell components during eggshell development

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ACCEPTED MANUSCRIPT (Papantonis et al., 2015). Based on our transcriptome data, we identified 22 expressed transcripts encoding putative chorion proteins (Table 3). The chorion genes of P. xylostella included two major classes, A and B, which are also found in other insects (Lecanidou et al., 1986; Papantonis et al., 2015), as well as two other subfamilies, CA

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and CB (Table 3). Two distinct branches were found in the phylogenetic tree, which

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effectively separated the chorion genes, chorion peroxidases Pxds (Px016921,

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Px009205, Px008866 and Novel00541) and chorion transcription factor Cf2 (Px005057). Two subclasses of chorion genes were clustered well into the relevant

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phylogenetic branches (Figure 5). In addition, the four Pxds diverged from the Cf2,

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and they may have different functions. The latter is involved in regulation of chorion genes expression and transcription by binding with the cis-regulatory sites of

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promoter (Shea et al., 1990).

When gene expressions were analyzed for different developmental stages of P.

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xylostella based on our RNA-seq data, fifteen chorions were expressed only in female

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adults (Figure 6, Table S8), and most tended to be expressed at extremely high levels in ovary (FPKM > 400) (Table 3). These genes are female-specific and may play important roles in insect oogenesis (Papantonis et al., 2015). Furthermore, the qRT-PCR results also validated their specificity, which showed that the two chorion genes (Px011665 and Px011666) were expressed at extremely high levels in P. xylostella adult females, and especially in the ovary (P < 0.05) (Figure 7). This observation is consistent with reports from other insect species, such as D.

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ACCEPTED MANUSCRIPT melanogaster (Parks and Spradling, 1987) and B. mori (Chen et al., 2015), where chorion genes are only located in specific regions of the eggshell, suggesting the roles in forming special appendages, such as micropyle, operculum, and dorsal appendages. Interestingly, a chorion gene, Px011664, was not only highly expressed in female

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adults, but also with moderate expression in male adults based on our RNA-seq data

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(Figure 6, Table S8). Chorion genes expression is regulated by different mechanisms

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(Carter et al., 2013), including antisense RNA (Skeiky and Iatrou, 1990), splicing variants (Drevet et al., 1995), alternatively polyadenylated isoforms, and distinct

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promoters (Papantonis et al., 2008; Lecanidou and Papantonis, 2010). In B. mori, the

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expression of three chorion genes in embryos, testes and follicular cells are regulated by alternative splicing and distinct promoter (Chen et al., 2015). In addition, there

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were significant differences between the expression levels of four Pxds, varying from

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101.9 (Novel00541) to 1.2 (Px008866) (Table 3). This suggests that Pxds in the ovary of P. xylostella may have different functions in the chorion assembly (Konstandi et al.,

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2004).

Nups. Nuclear pore complexes (NPCs) are large nuclear envelope (NE)-embedded protein assemblies contain multiple copies of nucleoporins (Nups). They represent a selective transport channel between the nucleus and the cytoplasm (D‟Angelo and Hetzer, 2008). At least two categories of Nups can be identified: scaffold Nups, forming a stable core ring-like structure in the NPC (D‟Angelo and Hetzer, 2008; Toyama et al., 2013) and peripheral Nups creating a permeability barrier to mediate

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ACCEPTED MANUSCRIPT the translocation of cargo through the NPC (Wente and Rout, 2010).

Here, we identified 33 Nup genes expressed in the ovary of P. xylostella (Table S9). There were significant differences between the expression levels of Nups, varying

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from 1.3 for Nup62 (Px012577) to 176.7 for Nup50 (Px007082), suggesting

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diversified functions of Nups in ovary development of P. xylostella. It has been

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confirmed that Nups play a vital function on reproductive development of some model insects, mammals, even humans. For example, RNAi of Nup107 in gonadal cells and

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follicle cells can lead to Drosophila female sterility attribute to the defects in

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oogenesis (Sassone-Corsi et al., 2011). A structural nucleoporin Seh1, which is the NUP107-160 complex, is serving an essential function in germ cells during

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Drosophila oogenesis (Senger et al., 2011). In D. melanogaster, Nup154 has a

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cell-type-specific role during the development of egg-chamber (Grimaldi et al., 2007). In mouse, Nup50 is involved in sustaining primordial germ cells in embryos, and

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deficiency of Nup50 can cause embryonic death (Park et al., 2016; Smitherman et al.,

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2000). However, the reproductive functions of Nups remain elusive in non-model insects. Further experimentation should explore the molecular mechanisms of the Nups during oogenesis in P. xylostella.

VMPs. The vitelline membrane is the inner layer of eggshell and is formed halfway during vitellogenesis (Tootle et al., 2011). Vitelline membrane proteins (VMPs) are the predominant constituents of vitelline membrane. The follicular epithelium secrete

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ACCEPTED MANUSCRIPT VMPs, which form a continuous layer around the oocyte. Here, we identified three VMPs (VMP25, VMP30 and EP80), that were similar to those in B. mori (Kendirgi et al., 2002; Sdralia et al., 2012; Chen et al., 2013). They had low degrees of similarity with other Lepidoptera species (Table S10), suggesting diversification of VMPs

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among insects. Carter et al. (2013) indicate there is no orthologs for D. melanogaster

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VMPs outside the genus Drosophila.

These three P. xylostella VMPs were expressed only in female adults based on our

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RNA-seq data, and with extremely high levels in the ovary (134~265 FPKM). This

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was validated by qRT-PCR with the VMPs (Px000306 and Px016343) being predominantly expressed in the ovary of P. xylostella (P < 0.05) (Figure 7),

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suggesting that these genes were female-specific and might play important roles in

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oogenesis of P. xylostella. Sdralia et al. (2012) and Kendirgi et al. (2002) indicate that knockdown of BmVMP30 and BmVMP90 compromise the integrity of follicular

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epithelium, thus preventing chorion deposition. Similarly, RNAi of BmEP80 in pupae

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leads to production of fragile eggs that collapse under the conditions of low humidity (Xu et al., 2011). BmEP80 mutant lay eggs that can easily dehydrate, rapidly leading to death (Chen et al., 2009). BmEP80 can indirectly influence oogenesis and eliminating BmEP80 can reduce expression levels of many genes in follicular cells, such as ecdysone oxidase and vitellogenin receptor (VgR). However, the specific functions of the three VMP genes in P. xylostella oogenesis remain unknown. Further studies are required to investigate the functions of these VMPs during oogenesis in P.

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HSPs. Heat shock proteins (HSPs) have been confirmed as the critical factors in cellular defense mechanisms to maintain cell survival under adverse environmental

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conditions (Wei et al., 2015). Generally, the transcription of HSP genes, and their

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molecular chaperones, cannot only be influenced by environmental factors, but also

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plays an important role during some developmental processes, including ovary

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development and oogenesis (Sarkar and Lakhotia, 2008).

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In our transcriptome, we identified 26 HSP genes expressed in ovary of P. xylostella, including small HSPs, HSP60, HSP70, and HSP90 families (Table S11). Most of the

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HSPs were highly expressed in the ovary of P. xylostella, particularly those small

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HSP (Px003390) and HSP83 (Px010238), with the values of FPKM reaching 1,199 and 3,952, respectively (Table S11). Furthermore, qRT-PCR results also show

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Px010238 significantly highly expressed in female adult and ovary of P. xylostella

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(Figure 7). Carter et al. (2013) also report a large number of HSPs and their orthologs transcribed in the Pararge aegeria ovarioles. The functions of HSPs are diverse in reproductive development. In Caenorhabditis elegans, HSP90 is associated with cell cycle control of oogenesis (Inoue et al., 2006), while in mice HSP90 is positively correlated with ovarian follicles development (Choudhury and Khole, 2015). In D. melanogaster, HSP60C is necessary for organizing and maintaining cytoskeleton and cell adhesion of follicle and germline cells during oogenesis. HSP70 regulates the

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ACCEPTED MANUSCRIPT border cell migration in ovaries (Cobreros et al., 2008; Sarkar and Lakhotia, 2008). Okada et al. (2014) report that HSP genes may also have positive effects on early reproduction. In addition, the knock-down of HSP83 negatively affects the oocytes maturity in T. castaneum (Xu et al., 2010). Nevertheless, further research is

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needed to explore the specific functions of HSPs genes in ovary development and

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oogenesis of P. xylostella.

Cyclin-related proteins. The progression of the meiotic cell cycle in oogenesis can be

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influenced during two stages: at prophase I, permitting oocyte differentiation, and at

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metaphase I and metaphase II, coordinating the completion of meiosis and fertilization. Both of these stages are controlled by several cell cycle regulators, such

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as cyclins, cyclin-dependent kinases (Cdks), and cyclin-dependent kinase inhibitors

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(Cdk inhibitors) (Alekseev et al., 2009).

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Here, we identified 53 cyclin-related genes, with 31 cyclin genes, 20 Cdks genes, and

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2 Cdk inhibitor genes expressed in the P. xylostella ovary (Table S12). The expression levels varied among the different types of cyclins, with FPKM ranging from 1.6 to 1,046.7. Two G2/mitotic-specific cyclin-B genes (Px001947 and Px015145) had the highest expression in the P. xylostella ovary, followed by two G2/mitotic-specific cyclin-B3 genes (Px003066 and Px015088) (Table S12). Px015088 exhibited significantly high expressions in egg and female stages, as well as in the ovary of P. xylostella (P < 0.05) (Figure 7). These results suggest that the

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ACCEPTED MANUSCRIPT cyclin B genes may be essential for oogenesis and the ovary development of P. xylostella, as reported in Drosophila (Jacobs et al., 1998). The expression levels of different types of Cdk also significantly varied, with the values of FPKM ranging from 1.4 (Px001420) to 301.2 (Px003866) (Table S12). In addition, the expression

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level of Cdk inhibitor 1 (Px007724, 118.6) was obviously higher than that of Cdk

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oogenesis of P. xylostella than inhibitor 4 (Table S12).

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inhibitor 4 (Px007723, 9.6), suggesting the Cdk inhibitor 1 may play a greater role in

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The specific functions of the Cyclin-related genes on oogenesis and ovary

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development have focused on vertebrate and model species (Fox et al., 2011; Adhikari et al., 2012; Atikukke et al., 2014). Jacobs et al. (1998) indicate that in D.

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melanogaster, cyclin B is essential for female reproduction, and Cyclin B/CDK1 may

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take part in oocyte maturation (Vardy et al., 2009). Cyclin A can control mitotic divisions early in oogenesis of Drosophila, which is related to the formation of

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oocytes and nurse cells (Lilly et al., 2000). CycG controls an early step of meiotic

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recombination repair, thus regulating dorso-ventral axis formation of Drosophila oocyte during oogenesis (Nagel et al., 2012). CycJ is required to regulate egg chamber production or maturation. CycE/Cdk2 kinase activity influences the type of cell produced during oogenesis in Drosophila and C. elegans (Morris et al., 2004; Fox et al., 2011). However, the reproductive functions of Cyclin-related genes remain unknown in non-model insects. The roles of cyclin-related genes during oogenesis in P. xylostella would require further analyses.

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The present study has added insights on how P. xylostella could be controlled using omics. Target genes, as those identified here, would need to further examine to define their exact functions. Ovary development is an important factor of insect reproduction,

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thus obtaining the target genes associated with ovary development and oogenesis is

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one of the effective ways to control pest population reproduction. The target genes

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related to the insect growth, metamorphosis and reproduction are already being screened by omics for pest management (Qiu et al., 2013; Gunaratna and Jiang, 2013).

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Xu (2016) identified the female-specific gene follicle cell protein 3C by N. lugens

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transcriptome, which provides the reproduction-related target gene for RNAi-based

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control of this rice pest.

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Conclusion

In this study, approximately 7.88 Gb of clean nucleotides were obtained from the

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ovary of P. xylostella, and a great number of ovary-expressed genes and major

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signaling pathways closely related to the process of ovary development and oogenesis were identified. Our newly developed P. xylostella ovary transcriptome represents the first genome-wide transcriptome dataset of the ovary of P. xylostella and provides a comprehensive resource for the studies on reproductive biology. More significantly, it gives an overview for the gene expression profiling in specific tissues and major signaling pathways closely related to the ovary development and oogenesis. Their potential functions are discussed considering the current knowledge of other insect

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ACCEPTED MANUSCRIPT species and demonstrate the importance of these genes in ovary and oogenesis.

In this study, we examine the possible molecular mechanisms involved in female reproduction of this species. It also allowed us to further our understanding of the

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complicated molecular regulatory networks that promote the biological processes of

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insect oogenesis, as well as the evolutionary mechanisms behind these processes.

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However, the spatio-temporal expression profiles and functions of the key genes associated with ovary development and oogenesis of P. xylostella will need to be

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analyzed to confirm some of the results presented in this study for potential

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integration into tools such as RNAi or CRISPER-based pest control.

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Ethics approval and consent to participate

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Since P. xylostella is considered a pest and in not under any conservation legislation

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in China, no permit for specimen collection and animal care clearance were required.

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Consent for publication Not applicable.

Competing interests The authors declare no conflict of interest.

Authors’ contributions

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ACCEPTED MANUSCRIPT The study was designed by LP and MY in collaboration with LV and WL. Experiments and data collection were performed by LP, WL, and YY. Data analysis was completed by LP with the help of ZM, HW and WY. The first draft of the manuscript was written by LP after discussion of the results with MY. All the authors

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were actively involved in writing and revising the manuscript.

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Acknowledgments

We are grateful to Qian Zhao for their technical assistance on computer graphics. This

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work was supported by the National Natural Science Foundation of China

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(31320103922, 31230061, 31401744), and the National Key Project of Fundamental Scientific Research in China (“973” Program, 2011CB100404), National Natural

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Science Foundation of Fujian Province (2015J01088). LV is supported by the

State Key Laboratory of Ecological Pest Control of Fujian-Taiwan Crops and

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1

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Author details

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Minjiang Scholars program of Fujian Province (PRC).

College of Life Science, Fujian Agriculture and Forestry University, Fuzhou 350002, China. 2Institute of Applied Ecology, Fujian Agriculture and Forestry University, Fuzhou 350002, China. 3Fujian-Taiwan Joint Innovation Centre for Ecological Control of Crop Pests, Fujian Agriculture and Forestry University, Fuzhou 350002, China. 4Key Laboratory of Integrated Pest Management for Fujian-Taiwan Crops, Ministry of Agriculture, Fuzhou 350002, China. 5Fujian Provincial Key Laboratory of

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ACCEPTED MANUSCRIPT Insect Ecology, Fujian Agriculture and Forestry University, Fuzhou 350002. 6

Department of Biological Sciences, Brock University, St. Catharines, Ontario L2S

3A1, Canada.

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Figure 1 Correlations of FPKM values between the replicated ovary RNA-seq

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Figure 2 Numerical distribution based on different gene expression levels (or FPKM values) in the ovary of P. xylostella. The gene expression levels were defined as no expression (FPKM = 0~1), low expression (1~3), moderate expression (3~15), high expression (15~60), and extremely high expression (>60).

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Figure 3 Functional classification of the P. xylostella ovary genes based on the

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Figure 4 Functional classification of the P. xylostella ovary genes based the Clusters of Orthologous Groups (COG) analysis

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Figure 5 Phylogenetic tree of the chorion genes from P. xylostella constructed

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with the deduced amino acids based on the Neighbor-joining (NJ) method with MEGA 6.06.

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Figure 6 Expression profiling of the chorion genes at different developmental stages of P. xylostella

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MA

NU

SC

RI

PT

ACCEPTED MANUSCRIPT

CE

Figure7 qRT-PCR-based expression profilings of the oogenesis-related genes in different stages and tissues of P. xylostella.

AC

The bars among the developmental stages or tissues labeled with different letters are significantly different (P < 0.05). OV: ovary, TE: testis, HD: head, TX: thorax, MG: midgut+malpighian tubule, RE: residues

40

ACCEPTED MANUSCRIPT Table 1 Summary of the sequence assemblies according to the RNA-seq data Sample

Raw reads

Clean

Error rate

Q20

Q30

GC

reads

bases

(%)

(%)

(%)

content (%)

58937558

56754304

8.51G

0.02

96.78

92.19

50.25

Pxovary2

49860028

48122060

7.22G

0.02

96.9

92.45

50.45

Pxovary3

54576352

52668652

7.9G

0.02

97.06

92.74

49.97

AC

CE

PT E

D

MA

NU

SC

RI

Pxovary1

PT

name

Clean

41

ACCEPTED MANUSCRIPT Table 2 Top 20 pathway-based gene numbers as determined by KEGG analysis of the transcriptome of the P. xylostella ovary Pathways

No. of genes

ID

1

Huntington's disease

126 (4.2%)

ko05016

2

Protein processing in endoplasmic reticulum

125 (4.2%)

ko04141

3

Pathways in cancer

120 (4.0%)

ko05200

4

Spliceosome

116 (3.9%)

ko03040

5

Ribosome

106 (3.6%)

ko03010

6

Purine metabolism

105 (3.5%)

ko00230

7

HTLV-I infection

104 (3.5%)

ko05166

8

Alzheimer's disease

9

Ubiquitin mediated proteolysis

10

Endocytosis

11

RNA transport

12

Epstein-Barr virus infection

13

PI3K-Akt signaling pathway

14

Insulin signaling pathway

15

Lysosome

16

Viral carcinogenesis

17

SC

RI

PT

No.

ko05010

101 (3.4%)

ko04120

101 (3.4%)

ko04144

95 (3.2%)

ko03013

95 (3.2%)

ko05169

95 (3.2%)

ko04151

91 (3.1%)

ko04910

88 (3.0%)

ko04142

87 (2.9%)

ko05203

Proteoglycans in cancer

87 (2.9%)

ko05205

18

cAMP signaling pathway

87 (2.9%)

ko04024

19

Focal adhesion

86 (2.9%)

ko04510

20

Carbon metabolism

86 (2.9%)

ko01200

MA

D

PT E

AC

CE

NU

104 (3.5%)

42

ACCEPTED MANUSCRIPT

Table 3 List of the chorion genes that were transcribed in the ovary of P. xylostella Sequence ID Px011665 Px011667 Px011669 Px011671 Px013131 Novel01507 Px011668 Px013130 Px011664 Px011666 Px011670 Px011673 Px013132 Novel00861 Px011674 Px013129 Px013965 Novel00541 Px008866 Px009205 Px016921 Px006057

Protein Chorion class A protein L11 Chorion class A protein L11 Chorion class A protein L11 Chorion class A protein L11 Chorion class A protein L11-like Chorion class A protein L11-like Chorion class B protein L12 Chorion class B protein Ld10 Chorion class B protein Ld34 Chorion class B protein Ld34 Chorion class B protein Ld34 Chorion class B protein Ld34 Chorion class B protein Ld34 Chorion class B protein Ld34 Chorion class CA protein ERA.1 Chorion class CA protein ERA.3 Chorion class CB protein M5H4-like Chorion peroxidase Chorion peroxidase Chorion peroxidase Chorion peroxidase Chorion transcription factor Cf2

D E

T P E

A

C C

FPKM 457.0 693.1 97.2 2239.4 945.8 649.3 782.9 829.3 515.4 679.4 418.7 27.4 469.5 153.7 425.8 490.9 1023.6 101.9 1.2 3.8 32.0 31.9

Species Plutella xylostella Plutella xylostella Plutella xylostella Plutella xylostella Plutella xylostella Plutella xylostella Plutella xylostella Danaus plexippus Plutella xylostella Danaus plexippus Danaus plexippus Danaus plexippus Danaus plexippus Danaus plexippus Bombyx mori Bombyx mori Bombyx mori Papilio xuthus Papilio xuthus Papilio xuthus Amyelois transitella Papilio machaon

M

Nt. ID 92.45 100 100 72.73 67.27 100 60.16 41.67 100 30.99 45 31.94 31.88 53.33 48.21 51.92 40.91 65 80 87 75 38

T P

I R

C S U

N A

E value 6.00E-25 2.00E-46 4.00E-36 3.00E-38 3.00E-18 7.00E-37 1.00E-35 7.00E-07 9.00E-39 1.00E-07 5.00E-06 4.00E-06 1.00E-06 2.00E-13 2.00E-06 1.00E-07 8.00E-07 0 0 5.00E-172 0 3.00E-70

Accession no. XP_011562125.1 XP_011563672.1 XP_011563686.1 XP_011563686.1 XP_011563686.1 XP_011562125.1 XP_011564732.1 EHJ74666.1 XP_011563675.1 EHJ71795.1 EHJ71400.1 EHJ71795.1 EHJ71793.1 EHJ71400.1 XP_004933234.1 XP_004933652.1 XP_012551830.1 KPI99509.1 KPJ02570.1 KPJ02570.1 XP_013192839.1 XP_014366977.1 43

ACCEPTED MANUSCRIPT

Highlights 

The first genome-wide transcriptome dataset of the ovary of P. xylostella was reported



The pathways of multicellular organism reproduction and multicellular organism reproduction process, as well as gamete generation and

T P

I R

chorion were significantly enriched

C S U

 Processes related to reproduction including the spliceosome, ubiquitin mediated proteolysis, endocytosis, PI3K-Akt signaling pathway, insulin signaling pathway, cAMP signaling pathway, and focal adhesion were mostly represented

 Functional genes involved in oogenesis were further analyzed

D E

N A

M

T P E

C C

A

44

ACCEPTED MANUSCRIPT

List of abbreviation: bp: base pair(s) BmCho: Bombyx mori chorion

T P

cAMP: Cyclic adenosine 3‟,5‟-monophosphate

I R

Cdks: Cyclin-dependent kinases cDNA: DNA complementary to RNA

C S U

COG: Clusters of Orthologous Groups FDR: false discovery rate

N A

FPKM: Fragments Per Kilobase of transcript sequence per Million base pairs sequenced Gb: giga base GO: Gene Ontology

D E

HD: head HSPs: Heat shock proteins

T P E

KEGG: Kyoto Encyclopedia of Genes and Genomes MG: midgut NE: Nuclear envelope

C C

NPCs: Nuclear pore complexes Nups: Nucleoporins OV: ovary

M

A

piRNA: Piwi-interacting RNA Pxds: Peroxidases Q20 percentages: percentage of sequences with sequencing error rate lower than 1%

45

ACCEPTED MANUSCRIPT

qRT-PCR: Quantitative Real-Time PCR RABT: Reference Annotation Based Transcript RNAi: RNA interference

T P

RNA-seq: RNA Sequencing

I R

TE: testis TX: thorax

C S U

Vg: Vitellogenin VgR: Vitellogenin receptor VMPs: Vitelline membrane proteins

N A

WEGO: Web Gene Ontology Annotation Plot Δ: deletion

D E

M

T P E

C C

A

46