Gene expression profiling of ovary identified eggshell proteins regulated by 20-hydroxyecdysone in Bactrocera dorsalis

Comparative Biochemistry and Physiology - Part D 30 (2019) 206–216

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Gene expression profiling of ovary identified eggshell proteins regulated by 20-hydroxyecdysone in Bactrocera dorsalis

T

Dong Weia,b,c, Ying-Xin Zhanga,b, Yu-Wei Liua,b, Wei-Jun Lia,b, Zhi-Xian Chena,b, ⁎ Guy Smagghea,b,c, Jin-Jun Wanga,b, a

Key Laboratory of Entomology and Pest Control Engineering of Chongqing, College of Plant Protection, Southwest University, Chongqing 400715, China International Joint Laboratory of China-Belgium on Sustainable Crop Pest Control, Academy of Agricultural Sciences, Southwest University, Chongqing 400715, China c Department of Plants and Crops, Faculty of Bioscience Engineering, Ghent University, Ghent 9000, Belgium b

ARTICLE INFO

ABSTRACT

Keywords: Oriental fruit fly Ovary Vitelline membrane protein Chorion 20-Hydroxyecdysone Vitellogenesis Choriogenesis

The oriental fruit fly, Bactrocera dorsalis, is one of the most destructive pests worldwide. The frequent use of chemical insecticides has led B. dorsalis to develop resistance to many insecticides in recent decades. New highthroughput-sequenced transcriptomes, as well as genomes, have revealed a large number of reference genes for functional target identification. Here, we performed digital gene expression profiling of ovary and testis of B. dorsalis adults. Various genes were identified to be highly expressed in B. dorsalis ovary. The genes encoding components of eggshell, vitelline membrane proteins (Vmps) and chorion-related proteins, were identified to be tissue-specifically expressed in ovary. Five cytochrome P450 genes were also identified to be highly expressed in ovary. Three of them were ecdysone synthesis pathway genes indicating the ovary as a potential synthesis site of female. The up-regulated expression of Vmps by exogenous 20-hydroxyecdysone implied the hormonal regulation of eggshell formation during ovarian development. Many other genes with potential functions in ovarian development were also identified, including vitellogenin receptor, insulin receptor, NASP protein, and odorant binding protein. These findings should promote our understanding of the regulation of vitellogenesis and eggshell formation and enable exploration of potentially novel pest control targets.

1. Introduction As one of the most destructive agricultural pests worldwide, the oriental fruit fly, Bactrocera dorsalis, can infect hundreds of crops including fruit and vegetables because of its high reproductive capacity and invasiveness (Clarke et al., 2005; Wei et al., 2015a). In association with the frequent use of chemical pesticides, B. dorsalis has developed resistance to many chemicals, such as formothion (Kuo et al., 2015), malathion (Wang et al., 2013), cyantraniliprole (Zhang et al., 2014), βcypermethrin, and avermectin (Jin et al., 2011). The development of resistance poses a serious threat to current efforts to manage this species. As such, there is substantial demand for novel, environmentally safe insecticides and/or methods for the control of B. dorsalis. It is also important to identify new targets for developing novel pest control strategies. Not only novel chemicals against new targets but also genespecific double-stranded RNA insecticides are anticipated to produce the next generation of insecticides. Knowledge of the biology and genetics of B. dorsalis would expectedly facilitate the development of new

targets for novel techniques. The tissues for insect reproduction, the ovary and testis, play important roles in generation continuation. Tissue-specific processes take place in such tissues, such as ovary/testis development and sex maturation. Information on the potential functions of the key genes involved in these processes should help to find new targets for pest control by manipulating their reproduction. Many functional genes have been demonstrated to be involved in insect reproduction, especially in ovarian development and oogenesis. For example, the nucleoporin gene nup154 is required in both male and female germlines for successful gametogenesis and its mutant flies lack differentiated sperm and lay abnormal eggs (Riparbelli et al., 2007). The extracellular matrix is a pivotal component of adult tissues, including the ovaryspecific stem cell niches (Pearson et al., 2016). A deep understanding of gene expression in the gonads should help us identify the tissue-specific genes that play critical roles in reproductive behavior, which can be used as new pesticide targets. Transcriptomic and proteomic data have increasingly been used to identify novel target genes related to insect

⁎ Corresponding author at: Key Laboratory of Entomology and Pest Control Engineering of Chongqing, College of Plant Protection, Southwest University, Chongqing 400715, China. E-mail address: [email protected] (J.-J. Wang).

https://doi.org/10.1016/j.cbd.2019.03.006 Received 20 January 2019; Received in revised form 13 March 2019; Accepted 16 March 2019 Available online 19 March 2019 1744-117X/ © 2019 Elsevier Inc. All rights reserved.

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reproduction. In the brown planthopper, Nilaparvata lugens, 115 genes were selected from the transcriptomic and proteomic data of high- and low-fecundity populations to investigate their functions in reproduction by regulating vitellogenin expression (Qiu et al., 2016). In addition, the molecular chaperone Hsp90 was also identified to be involved in oogenesis in Drosophila by a functional proteomic approach (Pisa et al., 2009). High-throughput transcriptomic analysis was also performed to identify the ovary-specific genes related to egg maturation in mosquitoes (Telang et al., 2013). In present study, we performed digital gene expression profiling as a powerful tool to investigate the mechanism behind the complex processes that occur in the gonads of B. dorsalis. The previously acquired reassembled transcriptomes of different tissues and developmental stages were used as references for gene annotation (Wei et al., 2018). Many genes were identified to be differentially expressed in the tested tissues, such as ovary and testis of adults. Genes highly expressed in ovary were selected for subsequent analysis, and some genes specifically expressed in ovary were identified and validated by quantitative real-time quantitative polymerase chain reaction (qRT-PCR), for example, those encoding eggshell proteins, vitelline membrane proteins (Vmps), choriogenesis-related genes, as well as Halloween genes. Insect eggshell proteins provide the conserved protection from physical and biological insults during the egg development. It has been reported that the expression of Vmps and outer chorion, are sequentially expressed during vitellogenesis and choriogenesis in ovary, and the transition occurs via a conserved cascade that requires a decline ecdysone signal (Papantonis et al., 2015). After the identification, three Vmps were cloned from B. dorsalis. The extremely high expression of Halloween spook and phantom indicate that B. dorsalis ovary is a potential synthesis site of 20-hydroxyecdysone (20E) which regulates Vmp synthesis in the ovarioles.

synthesized by reverse transcription using random hexamer primers, buffer, dNTPs, RNase H, and DNA polymerase I. The double-stranded cDNA was purified with magnetic beads, end-repaired, and ligated to adaptors at the ends of the fragments. The ligated products were sizeselected and purified on Tris–acetate–agarose gels. Finally, the fragments were enriched by PCR amplification, then purified using magnetic beads and dissolved in the appropriate amount of elution buffer. After the quantification and integrity validation using an Agilent 2100 Bioanalyzer, the sample library was used for sequencing via the Ion Proton platform by Beijing Genomics Institute (BGI, Shenzhen, China). 2.4. Gene annotation mapping and quantitative analysis The original image data produced by the sequencer were first cleaned by base calling. Then, data filtering was performed to obtain “clean reads” for further analysis. The reassembled transcriptomes from four developmental stages and tissues of fat body, middle gut, testis, male accessory gland, male antenna, and female antenna were used as the reference gene dataset (Wei et al., 2018). Clean reads from the DEG profiling in this study were mapped to the reference sequences using the SOAPaligner tool of SOAP2 with mismatch of no more than two bases allowed in the alignment (Li et al., 2009). Gene expression levels were calculated using the reads per kilobase per million reads (RPKM) method (Mortazavi et al., 2008). If there was more than one transcript for a given gene, the longest transcript was used to calculate the expression level and coverage. To identify genes differentially expressed between two samples, the false discovery rate (FDR) was used to determine the threshold of the P-value in multiple tests (Audic and Claverie, 1997). We used FDR ≤0.001 and the absolute value of log2 ratio ≥1 as the thresholds to judge the significance of differences in gene expression. Using Enzyme Commission number (EC) terms, biochemical pathway information was analyzed for differentially expressed genes (Kanehisa and Goto, 2000). This database contains systematic data on intracellular metabolic pathways and functions of individual gene products. The transcriptomes of fat body from female and male adults in previous study were also compared with those in ovaries and testes in the present study (Wei et al., 2018).

2. Materials and methods 2.1. Insect culture B. dorsalis flies were collected as pupae from Haikou, Hainan Province, China, in 2008, and maintained in our laboratory with an artificial diet as described previously (Wang et al., 2013). Adults were kept in 40 × 30 × 30 cm stock cages covered with fine synthetic mesh. All cages were kept under constant condition of 27.5 ± 0.5 °C, 75 ± 5% relative humidity, and 14:10 h light:dark. Artificial diet was supplied with cotton ad libitum and was replaced daily (Wei et al., 2015a).

2.5. Validation of gene expression by qRT-PCR To validate the ovary-specific gene expression, fat body, middle gut, Malpighian tubule, as well as ovary and testis were dissected from 5day-old male and female adults (20 flies/sample), respectively. After isolating total RNA as described above, DNA was digested with RQ1 DNase (Promega). Then, RNA (1 μg/sample) was reverse-transcribed into first-strand cDNA using PrimeScript RT Reagent Kit (TaKaRa, Dalian, China). The qRT-PCR reactions were performed in a 10-μL reaction volume including 5 μL of GoTaq qPCR Master Mix (Promega), 3.5 μL of nuclease-free water, 0.5 μL of template cDNA, and 0.5 μL of each primer (10 μM). Reactions were performed on a StepOne Plus Real-Time PCR System (Life Technologies, Woodlands, Singapore) under the following conditions: 95 °C for 2 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 30 s. Sequences of the primers used in these reactions are listed in Table S1. For reference purposes, a fragment of the B. dorsalis ribosomal protein subunit 3 open reading frame (ORF) was also amplified, as described previously (Wei et al., 2015b). Three biological replicates were performed for each tissue. The relative gene expression levels were calculated using the 2−ΔΔCt method (Livak and Schmittgen, 2001).

2.2. Total RNA isolation Newly emerged adults were separated by sex and reared in new cages. Ovaries of females and testes of males were dissected from 5-dayold virgin adults (n = 50 flies/sex) and immediately immersed in RNA storage reagent (Tiangen, Beijing, China). Then, the samples were powdered in liquid nitrogen and total RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer's instructions. The concentration and purity of extracted RNA were measured using a NanoVue UV–Vis spectrophotometer (GE Healthcare Bio-Science, Uppsala, Sweden), and the integrity of RNA was confirmed by separation on a 1% agarose gel by electrophoresis. 2.3. Construction of the cDNA library and sequencing Similar to a previous study (Wei et al., 2018), the total RNA of each sample was treated with DNase I (Promega, Madison, WI, USA) to eliminate DNA contamination, followed by mRNA purification using magnetic beads. The mRNA was enriched using oligo(dT) magnetic beads (for eukaryotes) and mixed with fragmentation buffer to fragment the mRNA into ~200 bp fragments. Then, first-strand cDNA was

2.6. Reverse transcription PCR In this study, only the genes that were highly expressed in the ovary of B. dorsalis female were focused on and screened for further analysis. Three Vmp genes were identified to be extremely highly expressed in 5day-old ovary. Their ORF sequences including Vmp26Ab, Vmp26Aa, and 207

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were identified to be differentially expressed among the tissues (Fig. 2B, C). Among these DEGs, 13,337 Unigenes were found between ovary and testis. In total, 13,375 and 12,699 Unigenes were found to be differentially expressed in the comparisons of ovary vs. female fat body and testis vs. male fat body, respectively (Fig. 2B). Most of the above DEGs were differentially expressed in gonads (i.e., ovary and testis). There were far fewer DEGs in the fat body of both sexes than in the gonads (Wei et al., 2018). All the DEGs were mapped to KEGG pathways. The genes were involved in reproduction, such as the categories of “oocyte meiosis”, “JAK-STAT signal pathway”, and “insulin signal pathway” (Fig. S1). Upon performing combined analysis, it was shown that 8448 DEGs in ovary were also significantly differently expressed in female fat body and testis (Fig. 2C). In this study, we were only interested in DEGs that were highly expressed in ovary compared with the levels in testis and female fat body. Finally, 6469 Unigenes were found to be highly expressed in ovary (Fig. 2D, Table S2). Screening identified 114 DEGs with an RPKM value ≥100 of an expression change fold ≥16 (log2 ratio ≥ 4) relative to that in testis and female fat body. These Unigenes were considered to be ovaryspecific genes. All of these genes were manually annotated against B. dorsalis genome in the NCBI nr database. Finally, 69 Unigenes were functionally annotated (Table 2), 29 were uncharacterized, and the remaining 16 were not annotated against B. dorsalis in the NCBI nr database with a credible hit. Only the manually annotated Unigenes were thereafter analyzed in this study. Many potential functional genes, such as Vmps, chorion, ATP-dependent RNA helicase me31b, vitellogenin receptor, odorant binding protein (Obp), Thioredoxin 2, nuclear autoantigenic sperm protein (NASP), nanos, cytochrome P450, juvenile hormone epoxide hydrolase 3, and insulin receptor, were identified to be extremely highly expressed in B. dorsalis ovary.

Vmp26Aa-like were determined by ORF Finder (https://www.ncbi.nlm. nih.gov/orffinder) and also confirmed by reverse-transcription PCR (RT-PCR) from the female ovary using gene-specific primers (Table S1). The PCR conditions and procedures were as follows: initial denaturation at 95 °C for 3 min; followed by 35 cycles of 95 °C for 30 s, 56 °C for 30 s, and 72 °C for 40 s; and then final extension at 72 °C for 10 min. Following purification by agarose gel electrophoresis and a gel extraction kit (Takara), the purified products were cloned into the pGEMT Easy Vector (Promega, Madison, WI) and transformed into Escherichia coli DH5α cells (Vazyme, Nanjing, China). The transformants were screened on Luria–Bertani (LB) agar plates containing 100 μg/mL ampicillin. Plasmids extracted from the positive clones were sequenced by Invitrogen (Shanghai, China). 2.7. Vmp gene expression induced by 20E After the identification of Vmps, we observed their expressions induced by 20E. A droplet (~0.50 μL/fly) of 20E in different dosages of ethyl alcohol (200, 500, and 1000 ng) was applied onto the thoracic tergum of 5-day-old virgin female insects with a hand micro-applicator (Hamilton, Reno, NV, USA) as described previously (Wang et al., 2013). The adults treated with the ethyl alcohol only were used as control. Then, the treated insects were thereafter reared separately, and fed with the artificial diet and maintained in the same condition as stock flies. Ovarian tissue was dissected 24 and 48 h later for total RNA isolation and reverse transcription. The gene expression of Vmps was evaluated by qRT-PCR after 24 and 48 h of induction, as described above. Alphatubulin was used as a reference gene, as in previous study (Table S1) (Shen et al., 2010). 3. Results and discussion

3.3. Genes encoding eggshell component proteins

3.1. Gene mapping to reference dataset

Insect eggshells protect the embryo from physical and biological insults and ensure its survival. For example, the eggshell protects the developing embryo from being abraded, crushed, attacked by parasites, and dehydrated, but allows gas exchange. The eggshell is usually divided into two major layers: the inner vitelline membrane and the outer chorion (Pascucci et al., 1996). The Vmps and chorions are the most important components of the eggshell in insects (Pascucci et al., 1996; Amenya et al., 2010; Marinotti et al., 2014). In this study, four Vmp Unigenes were annotated to three genes named Vmp26Ab (Unigene21308), Vmp26Aa (CL1424.Contig1), and Vmp26Aa-like (CL1424.Contig4). In addition to the Vmps, four chorion-related Unigenes annotated to three genes, namely, chorion peroxidase (Unigene20857), defective chorion-1 protein FC125 (Unigene20394), and defective-chorion protein FC106 (Unigene20691), were also identified to be highly expressed in ovary (Table 1). Most current knowledge on insect eggshell morphology and composition is derived from studies of the important research model D. melanogaster (Cavaliere et al., 2008). Recently, high-throughput technologies have been used to identify the Vmps and chorions in other insects. For instance, integrated transcriptomic and proteomic data identified three new Vmps in Aedes aegypti (Marinotti et al., 2014). Two Vmps and seven chorion proteins were also identified in Anopheles gambiae eggshell by proteomic analysis (Cavaliere et al., 2008; Amenya et al., 2010; Papantonis et al., 2015).

In total, 14,924,152 and 16,434,613 reads were generated from the ovary and testis, respectively (Table 1). Among them, we mapped 98.66% and 98.57% of clean reads to the reference sequences, respectively. These reads were assembled into 23,993 and 25,546 Unigenes in ovary and testis, respectively. Ultimately, a total of 28,526 Unigenes were identified including genes from previous male and female fat body samples. In total, 37%, 37%, 21%, and 25% of Unigenes showed gene coverage of > 90% in ovary, testis, female fat body, and male fat body, respectively (Fig. 1). This indicated that the sequence coverage of genes in reproductive tissue was higher than that in fat body. Almost 50% of sequences showed > 80% sequence coverage in ovary and testis. 3.2. Quantitative expression analysis Upon quantification using the RPKM value, 1399 Unigenes were specifically expressed in ovary, and 2523, 123, and 172 Unigenes were in testis, female and male fat body, respectively (Fig. 2A). A total of 16,371 Unigenes were expressed in all four tissues. Compared with the gene expression in fat body tissues (Wei et al., 2018), 19,603 Unigenes Table 1 Summary of the sequencing and annotation of transcripts. Sample

Total reads

Total basepairs

Mapped reads %

Unigenes

f-OV m-TE f-FB m-FB

14,924,152 16,434,613 13,740,836 15,702,303

1,974,964,164 2,224,160,593 1,873,927,611 2,139,262,814

98.66 98.57 98.59 98.05

23,993 25,546 19,527 20,651

3.3.1. Vitelline membrane protein-coding genes Four Vmps and six chorions have been identified in Drosophila eggshell (Cavaliere et al., 2008), while five Vmps have been identified in A. aegypti (Edwards et al., 1998; Marinotti et al., 2014). In contrast, only three Vmps were identified in this study, including Vmp26Aa-like, which was also identified previously by proteomic approach (Uniprot Accession Number: A0A034WHP3) (Wei et al., 2017). The expression of these three Vmps was subsequently validated by qRT-PCR among various tissues at the transcriptional level (Fig. 3). All genes were highly

Note: f-OV, m-TE, f-FB, and m-FB represent the tissues of ovary (OV), testis (TE), and fat body (FB) from 5-day-old female (f) and male (m) adults. Data of fFB and m-FB are from Wei et al., 2018. 208

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Fig. 1. Distribution of gene coverage in four tissues. A, B, C, and D represent ovary, testis, and fat body from 5-day-old female and male adults, respectively.

Fig. 2. Quantitative analysis of gene expression from ovary, testis, and fat body of female and male Bactrocera dorsalis adults. A, Venn diagrams of Unigenes quantified in four tissues. B, Statistical analysis of genes differentially expressed between tissues. C, Venn diagram of differentially expressed Unigenes in four comparisons. D, Venn diagram of Unigenes highly expressed in ovary compared with the levels in testis and female fat body. The Unigenes of fat body were retrieved from a previous study (Wei et al., 2018).

expressed in ovary, which was consistent with the RPKM values, exhibiting an extremely ovary-specific expression profile. Eggshell formation is a complex process that requires time-coordinated synthesis, cleavage, and transport of various proteins and finally cross-linking

mediated by particular functional domains in the cells of follicular epithelium (Cavaliere et al., 2008). In Drosophila and Bombyx, Vmps are synthesized in the middle stage of oogenesis and form functional proteins just prior to choriogenesis (Kendirgi et al., 2002; Cavaliere et al., 209

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Table 2 Unigenes highly expressed in Bactrocera dorsalis ovary. Gene ID

Gene length

TE-RPKM

OV-RPKM

f-FB-RPKM

log2 ratio (OV/ TE)

log2 ratio (OV/fFB)

Accession number

Unigene21308 CL1424.Contig1 Unigene20658 Unigene10468 Unigene17590 Unigene20681 Unigene20691 Unigene18717 Unigene1320

728 790 638 1755 5565 754 1311 772 950

0.23 0.00 0.13 45.02 0.25 0.00 0.13 0.64 39.02

3407.56 2383.13 1131.10 993.35 765.89 712.24 704.70 701.39 638.87

0.12 0.22 0.00 16.91 0.03 0.00 0.00 0.00 8.45

13.88 21.18 13.10 4.46 11.57 19.44 12.45 10.10 4.03

14.79 13.40 20.11 5.88 14.58 19.44 19.43 19.42 6.24

XP_011204938 JAC53666 AEW66914 XP_011197588 XP_011208480 AKI29000 XP_011203878 XP_011199127 XP_011207167

CL677.Contig2 Unigene20692 Unigene7067 Unigene17826 Unigene6057 Unigene7007 Unigene18713 CL1424.Contig4 Unigene8302 Unigene20943 Unigene20394 Unigene7093 Unigene9136 Unigene17648 CL833.Contig2 Unigene5858 Unigene21139 Unigene8007

2675 328 2222 3901 1209 1416 464 512 1617 1746 2389 1767 2953 3402 4804 2470 1558 1388

2.53 0.00 27.25 0.82 4.97 12.68 1.07 0.00 23.33 0.00 0.10 0.37 23.13 0.29 13.43 15.94 0.05 9.91

637.08 601.34 465.81 442.15 424.19 421.27 417.04 402.95 386.50 384.08 378.60 376.59 372.39 346.22 319.28 319.06 316.15 302.34

20.30 0.00 21.96 0.09 0.00 1.60 6.96 0.00 12.96 0.25 0.00 1.93 3.70 0.05 6.16 0.21 0.06 11.26

7.98 19.20 4.10 9.07 6.41 5.05 8.61 18.62 4.05 18.55 11.84 9.98 4.01 10.22 4.57 4.32 12.55 4.93

4.97 19.20 4.41 12.27 18.69 8.04 5.90 18.62 4.90 10.59 18.53 7.61 6.65 12.72 5.70 10.56 12.46 4.75

JAC38786 JAC55210 XP_011210962 XP_011212199 XP_011200282 XP_011197560 XP_011213414 JAC53665 XP_011198135 AFH54181 XP_011203877 AEN03029 XP_011209691 XP_011202029 XP_019848334 XP_011202013 XP_011208931 JAC57016

CL2292.Contig1 Unigene18025 CL2822.Contig2 Unigene1310 CL2822.Contig1 Unigene20857 Unigene2440

1029 846 1245 1854 1073 2861 2618

0.16 0.19 0.00 2.36 0.00 0.00 14.35

302.04 280.97 257.35 254.45 238.60 237.50 233.02

0.08 0.41 0.07 2.07 0.08 0.06 9.80

10.88 10.49 17.97 6.76 17.86 17.86 4.02

11.80 9.41 11.84 6.94 11.52 11.93 4.57

AGE83235 JAC57553 XP_011197310 XP_011214408 XP_011197310 JAC48831 JAC51635

Unigene2275 Unigene20978 Unigene7466 Unigene5991 Unigene19750 Unigene20479 Unigene4990 Unigene2209 Unigene8048 Unigene21155 Unigene20490 Unigene7098 Unigene17819 Unigene1388 Unigene5637 CL274.Contig2 CL746.Contig3 Unigene20940 Unigene10102

2458 937 2149 1878 476 1052 1499 4297 1226 1545 722 1634 2345 1601 2530 1468 835 692 4107

13.17 0.44 8.05 0.26 0.35 0.08 3.85 9.43 8.80 0.75 0.23 2.52 0.42 2.26 4.07 6.68 1.48 0.36 2.01

231.76 225.88 215.80 205.41 201.39 182.33 181.18 178.85 176.55 175.44 171.33 150.70 145.32 145.08 144.39 140.90 130.67 129.41 127.73

7.25 0.09 9.02 0.09 0.73 0.08 0.00 7.62 6.05 0.00 0.00 0.00 0.07 3.93 4.52 7.67 0.63 0.00 4.40

4.14 9.01 4.74 9.61 9.18 11.19 5.56 4.24 4.33 7.88 9.55 5.90 8.43 6.00 5.15 4.40 6.46 8.50 5.99

5.00 11.24 4.58 11.11 8.10 11.10 17.47 4.55 4.87 17.42 17.39 17.20 10.93 5.21 5.00 4.20 7.70 16.98 4.86

XP_011207472 JAC52106 XP_011212012 XP_011202004 JAC54858 XP_011198855 XP_011213567 JAC45464 XP_011207001 JAC49796 AGT79088 XP_011212670 XP_011197435 XP_011212563 XP_011199409 XP_014102203 JAC40357 JAC51763 JAC41948

CL2862.Contig1 Unigene21221 Unigene7657 Unigene20897 Unigene3484 CL1424.Contig2 Unigene21124 Unigene17578 Unigene2336 Unigene17883 Unigene17723

2270 2542 1090 905 2479 699 2121 1259 1901 1919 1400

6.13 0.06 0.30 0.91 5.62 0.00 0.08 0.72 4.46 0.26 0.35

126.93 126.74 124.42 124.10 122.54 116.92 115.26 114.21 113.30 108.71 108.62

4.54 0.00 0.56 0.00 2.57 0.00 0.00 0.14 1.42 0.00 0.00

4.37 10.93 8.68 7.09 4.45 16.84 10.53 7.31 4.67 8.72 8.27

4.81 16.95 7.79 16.92 5.57 16.84 16.81 9.69 6.31 16.73 16.73

XP_011201673 JAC46426 JAC49414 AFI99080 JAC36282 JAC53666 JAC46935 JAC36701 JAC49261 JAC36701 XP_011212552

Manual annotation Vitelline membrane protein Vm26Ab-like Vitelline membrane protein Vm26Aa Protein disulfide isomerase Putative ATP-dependent RNA helicase me31b Putative vitellogenin receptor Odorant binding protein 19c Defective-chorion protein FC106 Thioredoxin 2 Deoxyuridine 5′-triphosphate nucleotidohydrolase Maltase 2 Defective chorion-1 protein, FC125 Ribosomal L1 domain-containing protein Protein bicaudal c Protein matrimony Protein amalgam Acidic proline-rich protein PRP-like Vitelline membrane protein Vm26Aa Protein nasp homolog Cytochrome p450, partial Defective chorion fc125 Juvenile hormone epoxide hydrolase 3 Protein aubergine Insulin-like peptide receptor Protein LSM14 homolog B isoform X1 Importin subunit alpha Peptidyl-prolyl cis-trans isomerase D Ubiquitin-like domain-containing CTD phosphatase 1 Vitellogenin receptor Protein lethal(2)k10201 Skin secretory protein xp2-like isoform x1 Fatty-acid amide hydrolase 2-b Skin secretory protein xp2-like isoform x1 Chorion peroxidase Ribonucleoside-diphosphate reductase large subunit DNA replication licensing factor Mcm7 Protein tailless Protein maelstrom 1 Cytochrome P450 307a1-like Phormicin, partial Gametocyte-specific factor 1 homolog Protein nanos Myotubularin-related protein 14 Trehalose-phosphate phosphatase B Major royal jelly protein 1 Cytochrome P540 Cyp306a1, partial Tubulin alpha-4 chain Insulin receptor Diphthine methyl ester synthase Inositol-trisphosphate 3-kinase homolog High mobility group protein Z Heat shock protein 23 DNA replication inhibitor plutonium, partial Cerebellar degeneration-related protein 2-like protein Putative adenosylhomocysteinase 2 Protein swallow Retinol dehydrogenase 14 Glutathione-s-transferase epsilon class 1 Serine/threonine-protein kinase Warts, partial Vitelline membrane protein Vm26Aa Serine protease gd, partial Mitosis initiation protein fs(1)Ya Serine/threonine-protein kinase Warts, partial Mitosis initiation protein fs(1)Ya Protein draper isoform X1

(continued on next page)

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Table 2 (continued) Gene ID Unigene17816 Unigene17790 Unigene6235 Unigene8794 Unigene17604

Gene length 1091 5095 872 489 2313

TE-RPKM

OV-RPKM

f-FB-RPKM

log2 ratio (OV/ TE)

log2 ratio (OV/fFB)

Accession number

0.23 0.63 2.65 0.67 0.00

108.57 106.92 106.06 105.11 103.88

0.00 0.00 0.70 3.03 0.00

8.90 7.41 5.33 7.29 16.66

16.73 16.71 7.24 5.11 16.66

AFH54204 JAC37158 XP_011198107 JAC54320 JAC47938

Manual annotation Cytochrome P450, partial Serine protease nudel, partial Heat shock protein 67B3 Sarcocystatin-A UDP-glucuronosyltransferase 1-1, partial

Note: Only Unigenes highly expressed in ovary with a RPKM ≥100, an expression change of log2 ratio ≥4 and functional annotated in NCBI nr database were listed in this table.

2008; Xu et al., 2012). While not all Vmps were predominantly expressed at this stage before choriogenesis. In B. mori, there is a Vmp named Vmp25 that highly expressed in the early stage during choriogenesis (Xu et al., 2012). In terms of the abundance of Vmp26Aa protein during ovarian development, it showed high abundance at the middle of vitellogenic stage in B. dorsalis ovary, as revealed by shotgun proteomic analysis (Wei et al., 2017). After the identification, we cloned the full length of three Vmps in B. dorsalis by RT-PCR and deposited them in the NCBI GenBank database with the accession numbers MG386248 (Vm26Ab), MG386249 (Vm26Aa), and MG386250 (Vm26Aa-like). The cDNA of Vmp26Ab is 728 bp, including an ORF of 438 bp that encodes 145 amino acid residues (Fig. 4A). The cDNAs of Vmp26Aa and Vmp26Aa-like are 790 and 513 bp, including ORFs of 366 and 345 bp that encode 121 and 114 amino acid residues, respectively (Fig. 4B and C). The first 16, 19, and 19 amino acid residues of the precursor were predicted as a signal peptide for secretion, upon analysis by SignalP 4.1 (Fig. 4). The highly conserved 38-amino-acid domain, which is flanked by unrelated regions, was also predicted by Blast in NCBI's conserved domain database (Marchler-Bauer et al., 2016). The domain contains three highly conserved cysteines.

extremely highly expressed in ovary of B. dorsalis at the middle stage of sex maturation (Table 2), including a chorion peroxidase (Unigene20857) and two defective chorion proteins (Unigene20394 and Unigene20691). The validation of expression by qRT-PCR also showed the ovary-specific expression pattern (Fig. 5). In addition, four chorion protein-encoding genes were also identified in ovary, namely, s15 (Unigene20150), s16 (Unigene20763), s18 (Unigene17914), and s19 (Unigene20874), with a low but ovary-specific expression level at the vitellogenesis stage (Table S2). All four genes are conserved and homologous to the genes associated with the middle (s15 and s19) and late (s16 and s18) stages of choriogenesis in Drosophila (Papantonis et al., 2015). The expression of these genes may increase at the late choriogenesis stage, although they also showed high expression in ovary of 5-day-old B. dorsalis (Fig. 5). In a previous proteomic analysis of B. dorsalis, chorion protein s36 was also identified to be highly expressed and abundant in sexually mature ovary at both transcriptional and protein levels (Wei et al., 2017). Therefore, a total of five chorion proteins have thus far been identified in B. dorsalis. In Drosophila, s36 protein plays a crucial role in regulating the morphogenetic integrity of dorsal appendages in follicles, inducing severe structural irregularities on the chorion's surface and completely abolishing fly fertility (Velentzas et al., 2016). A similar function in regulating female fertility should be well addressed to evaluate its target in B. dorsalis. Meanwhile, a chorion peroxidase (CP) gene was identified to be highly expressed in ovary (Table 2, Fig. 5). This chorion peroxidase was also found to be abundant in mature ovary of B. dorsalis (Wei et al., 2017), possibly functioning in the chorion-hardening process through protein crosslinking. The ovarian-specific expression in the follicle cell layer during the late stages of oogenesis was also observed in in situ hybridization for B. oleae (Konstandi et al., 2006). Chorion peroxidase is primarily responsible for the irreversible insolubilization of the three major endochorion proteins after oviposition (Li and Li, 2006).

3.3.2. Chorion related protein coding genes The chorion is the outer layer of the eggshell, which consists of the endochorion and exochorion. In Drosophila, six chorion protein-coding genes have been identified to be organized in two clusters: one at the X chromosome (s36 and s38) and the other at the third chromosome (s15, s16, s18, and s19) (Cavaliere et al., 2008). In the present study, we sequenced the total RNA from 5-day-old female ovary, which is the vitellogenic stage of oogenesis during the development of the laboratory strain, as described previously (Wei et al., 2015a; Wei et al., 2017). Two defective chorions were specifically expressed in 5-day-old ovary, as shown in Table 2. In Drosophila, six chorion proteins were identified to be transcriptionally expressed in the choriogenesis stage, named s15, s16, s18, s19, s36, and s38 (Cavaliere et al., 2008). These genes are prominently expressed in the late stages of oogenesis, such as stages 11–14 (Papantonis et al., 2015). Meanwhile, in B. dorsalis ovary, we identified three chorion-related protein-coding genes that were

3.4. Cytochrome P450 genes Ecdysteroid plays an important role in male and female reproduction (Brown et al., 2009). It has been demonstrated that ovarian development is triggered by the steroid hormone 20E in fruit fly (Soller

Fig. 3. Relative expression of vitelline membrane protein (Vmp)-coding genes among various tissues of Bactrocera dorsalis. MG, FB, MT, OV, and TE stand for midgut, fat body, Malpighian tubule, ovary, and testis, respectively. “♀” and “♂” indicate female and male adults. The expression of each gene was calculated relative to that in female midgut. 211

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Fig. 4. The deduced amino acid sequences of three vitelline membrane proteins (Vmps) transcripts identified in B. dorsalis ovary. A, B and C represent Vmp26Ab, Vmp26Aa and Vmp26Aa-like, respectively. The black underlined amino acids indicate the signal peptides predicted by the online tool SignalP 4.1. The red wavy lines represent the conserved vitelline membrane region predicted by Blast in NCBI's conserved domain database.

et al., 1999; Swevers and Iatrou, 2003; Ameku et al., 2017). Ecdysteroids are multifunctional hormones in female arthropods that are stored in oocytes for use during embryogenesis (Brown et al., 2009). The genes involved in the biosynthesis of ecdysone are called Halloween genes, which encode Cytochrome P450 enzymes. In this study, nine Unigenes annotated as P450 were found to be highly expressed in female ovary. These Unigenes were further manually annotated into five P450 genes, namely, P450 6a13 (Unigene20943), P450 306a1 (Unigene20490), P450 307a1 (Unigene5991), P450 314a1 (Unigene1348), and P450 18a1 (CL2015.contig2), two of which showed ovary-specific expression (Table 2). Three out of the five P450s are Halloween genes, namely,

P450 306a1 (phantom), P450 307a1 (spook), and P450 314a1 (shade). Two of them showed high expression in ovary, as confirmed by qRTPCR, while the P450 314a1 was highly expressed in Malpighian tubule (Fig. 6). Of the other two P450 genes, P450 6a13 and P450 18a1, were both highly expressed in ovary. A high expression of P450 18a1 was also determined in fat body of female, showing a difference between sexes. In insect adults, the gonads (i.e., ovary and testis) are the potential site of ecdysteroid synthesis. In mosquito, Aedes aegypti, the ecdysteroid level is high in ovary after a blood meal, indicating that this is the site of ecdysteroid synthesis (Sieglaff et al., 2005). Similarly, a high titer of

Fig. 5. Relative expression of genes involved in eggshell formation that were highly expressed in Bactrocera dorsalis ovary among various tissues. MG, FB, MT, OV, and TE stand for midgut, fat body, Malpighian tubule, ovary, and testis. “♀” and “♂” indicate female and male adults. The expression of each gene was calculated relative to that in female midgut. 212

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Fig. 6. Relative expression of cytochrome P450 genes that were highly expressed in Bactrocera dorsalis ovary among various tissues. MG, FB, MT, OV, and TE stand for midgut, fat body, Malpighian tubule, ovary, and testis. “♀” and “♂” indicate female and male adults. The expression of each gene was calculated relative to that in female midgut. Genes not highly expressed in ovary were analyzed by independent Student's t-test using SPSS software 19.0 (IBM Inc., Chicago, IL). “ns” and “*” indicate no significant difference and a significant difference between tissues. A value of P < 0.05 was considered to be statistically significant.

their roles are not yet clear.

ecdysteroid in testis was also observed in Spodoptera littoralis and B. mori (Fugo et al., 1996; Polanska et al., 2009; Iga et al., 2013). Three Halloween genes were confirmed to be highly expressed in B. dorsalis ovary, which was consistent with the profiling data, suggesting that the ovary is one of 20E producing sites in this species. Similarly, the male accessory gland was also shown to potentially be the tissue in which ecdysone is synthesized in B. dorsalis male (Wei et al., 2016). Recently, the increased expression of P450 306a1 during the ovarian maturation of B. dorsalis was also investigated (Wei et al., 2017), as well as the juvenile hormone epoxide hydrolase (Unigene7093) highly expressed in ovary. Cytochrome P450 is a large family with multiple roles in detoxification and ecdysone synthesis, among other physiological functions. In this study, two P450 genes, P450 6a13 (Unigene20943) and P450 18a1 (CL2025.Contig2), were highly transcriptionally expressed in female ovary, as presented above. Most studies of P450 performed to date focused on their functions in detoxification and ecdysone synthesis in insects. Few studies have investigated their expression and roles in ovary in terms of an involvement in female reproduction. In Heteropneustes fossilis, a P450 gene, P450 19a1a, was identified to be highly expressed in ovary and also in brain, with a sex-biased and tissue-specific expression profile (Chaube et al., 2015). In situ localization analysis showed that this gene was expressed in ovarian follicular cells. Cyp19a1a was also found to play a crucial role in sex differentiation and sex change in fish (Guiguen et al., 2010). However, the functions of the two ovary-specific P450 genes in ovary remained unknown. Specific CYP6 genes have been identified to be involved in phytotoxin resistance in white fly and aphid (Halon et al., 2015; Peng et al., 2016). One CYP6 gene named P450 6a13 was also identified in resistant and susceptible strains of the aphid Aphis gossypii, but with no significant relationship with resistance to imidacloprid (Kim et al., 2015). After a change of host to a more toxic plant, an increased expression of both P450 18a1 and P450 6a13 were observed in the milkweed-specialist aphid, Aphis nerii (Birnbaum et al., 2017). Meanwhile, in Locusta migratoria, CYP 6a13 was also found to be differentially expressed in non-diapause eggs compared with that in diapause eggs (Hao et al., 2017). Although these two genes were identified in some insect species,

3.5. Vmp gene expression induced by 20-hydroxyecdysone We observed the changes of gene expression of three Vmp genes identified in this study. Specifically, these three genes were significantly up- or down-regulated by different dosages of 20E (Fig. 7). In detail, Vmp26Ab and Vmp26Aa-like were up-regulated at 24 h after 20E treatment, but no differences were observed in Vmp26Aa after this time point. Interestingly, the expression of the three Vmps decreased at 48 h compared with that at 24 h. In a previous study, it was investigated that the ovary of B. dorsalis develops into the later choriogenic stage of maturation at 6th day (Wei et al., 2017). The self-regulation may play an important role for this decrease during the transition from vitellogenesis to choriogenesis. This self-regulation of the systematic gene expression and 20E titer in reproductive organs was reported in insects (Papantonis et al., 2015). This decrease might be critical for the transition to choriogenesis. It has been demonstrated that Vm26Ab (also known as sV23) is the most abundant Vmp in Drosophila, which is essential for female fertility (Manogaran and Waring, 2004; Wu et al., 2010). The absolute expression as reflected in the RPKM value of Vmp26Ab was also higher than those of the other two Vmps, suggesting the need for its abundance at this stage to meet requirement of female fertility. The different expression among three Vmps indicated a similar function of Vmp26Ab in B. dorsalis. 3.6. Other proteins Many protein-encoding genes were also shown to exhibit differential expression during ovarian development, indicating their critical roles in female reproduction (Wei et al., 2017), such as importin-α (Unigene5858), ATP-dependent RNA helicase Me13B (Unigene10468), vitellogenin receptor (VgR) (CL2292.cintig1), heat shock protein 23 (CL746.contig1), and myotubularin-related protein 14 (Unigene2209). Some functional genes, such as vitellogenin receptor (Unigene17590), NASP (Unigene8302), and protein matrimony (Unigene6057), also showed higher expression in ovary than in other tested tissues, as reflected by the KPRM value and also qRT-PCR validation (Fig. 8). Some 213

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Fig. 7. Gene expression of three Vmps in ovary after induction by 20-hydroxyecdysone for 24 and 48 h. Relative expression was calculated compared with that upon treatment with an equal volume of ethyl alcohol. An asterisk above a bar indicates a significant difference compared with the control, as analyzed by Student's t-test using SPSS 19.

genes were previously discussed to potentially have a role in and be a target for manipulating female fertility (Wei et al., 2017). The high expression of VgR in ovary involved in yolk protein uptake has been well studied in female insects, including in the oriental fruit fly (Cong et al., 2015; Klinbunga et al., 2015; Lu et al., 2015; Qian et al., 2015). The high expression of NASP in the late stage of ovarian development was also observed in shrimp (Karoonuthaisiri et al., 2009). Together with the insulin signaling pathway, the disulfide isomerase protein (Unigene20658)-coding gene also showed high expression in ovary. Bicaudal C may function in the absorption of nutrients and in regulating female fecundity via the insulin and TOR signaling pathways (Gamberi et al., 2017). The function of matrimony proteins in meiosis has also been investigated in embryogenesis (Whitfield et al., 2013). Only a small number of B. dorsalis eggshell components were identified in this study. Using spectrometry-based proteomic analysis, 44 proteins were identified as putative components of An. gambiae and A. aegypti (Amenya et al., 2010; Marinotti et al., 2014). The most prominent group was the Obps. A total of 7 and 26 Obps were identified in the eggshell of An. gambiae and A. aegypti, respectively. The abundance of Obp in ovary was also investigated in A. aegypti (Costa-da-Silva et al., 2013), showing tissue-specific expression. In our previous study, we identified some Obps exhibiting dynamic expression during ovarian development (Wei et al., 2017), but their differences in expression among tissues are unclear. In this study, one odorant binding protein (Unigene20681) was found to be extremely highly and ovary-

specifically expressed in female. Similar to the case in mosquito, some other Obps should exist in eggshell, although their roles in female fertility remain unknown. Greater knowledge of the proteins that comprise eggshell is required to understand their roles and differences, and how they contribute to a successful reproductive strategy. Integrated identification of eggshell proteins should be conducted in the future, as well as determination of their roles and regulatory mechanisms. 4. Conclusions In conclusion, the genes expressed in important tissues related to reproduction of B. dorsalis were sequenced and identified. Quantitative analysis of the expression of the genes showed that a large number of genes were highly expressed in B. dorsalis ovary. Most of the potential ovary-specific genes had already been identified and functionally analyzed in other insects. Specifically, genes encoding eggshell proteins were identified to exhibit ovary-specific expression, including three Vmps and four chorions. Moreover, five P450 genes including three Halloween genes were found to be highly expressed in ovary, indicating that this is the site of 20E synthesis. The change in expression due to exogenous 20E indicated that 20E is involved in sensitive regulation of the transition from vitellogenesis to choriogenesis during ovary development. The identification of all of these tissue-specific genes should contribute to our understanding of ovarian development, and also aid in the identification and evaluation of novel target genes.

Fig. 8. Examples of transcriptional expression of genes that were highly expressed in Bactrocera dorsalis ovary among various tissues. MG, FB, MT, OV, and TE stand for midgut, fat body, Malpighian tubule, ovary, and testis. “♀” and “♂” indicate female and male adults. The expression of each gene was calculated relative to that in female midgut. 214

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Supplementary data to this article can be found online at https:// doi.org/10.1016/j.cbd.2019.03.006.

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Acknowledgments We thank Zheng Zhang for his help with the sample preparation for qPCR. We also thank Liwen Bianji, Edanz Group China (www. liwenbianji.cn/ac), for editing the English text of a draft of this manuscript. This work was supported in part by the Chongqing Research Program of Basic Research and Frontier Technology (CSTC2016jcyjA0019), the National Key Research and Development Program (2016YFD0200501-9), the PhD Research Funding of Southwest University (SWU118110), and the 111 Project (B18044). Conflicts of interest The authors declare no conflict of interest. The founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results. References Ameku, T., Yoshinari, Y., Fukuda, R., Niwa, R., 2017. Ovarian ecdysteroid biosynthesis and female germline stem cells. Fly 11, 185–193. Amenya, D.A., Chou, W., Li, J., Yan, G., Gershon, P.D., James, A.A., Marinotti, O., 2010. Proteomics reveals novel components of the Anopheles gambiae eggshell. J. Insect Physiol. 56, 1414–1419. Audic, S., Claverie, J.M., 1997. The significance of digital gene expression profiles. Genome Res. 7, 986–995. Birnbaum, S., Rinker, D., Gerardo, N., Abbot, P., 2017. Transcriptional profile and differential fitness in a specialist milkweed insect across host plants varying in toxicity. Mol. Ecol. 26, 6742–6761. Brown, M.R., Sieglaff, D.H., Rees, H.H., 2009. Gonadal ecdysteroidogenesis in arthropoda: occurrence and regulation. Annu. Rev. Entomol. 54, 105–125. Cavaliere, V., Bernardi, F., Romani, P., Duchi, S., Gargiulo, G., 2008. Building up the Drosophila eggshell: first of all the eggshell genes must be transcribed. Dev. Dyn. 237, 2061–2072. Chaube, R., Rawat, A., Joy, K.P., 2015. Molecular cloning and characterization of brain and ovarian cytochrome P450 aromatase genes in the catfish Heteropneustes fossilis: sex, tissue and seasonal variation in, and effects of gonadotropin on gene expression. Gen. Comp. Endocrinol. 221, 120–133. Clarke, A.R., Armstrong, K.F., Carmichael, A.E., Milne, J.R., Raghu, S., Roderick, G.K., Yeates, D.K., 2005. Invasive phytophagous pests arising through a recent tropical evolutionary radiation: the Bactrocera dorsalis complex of fruit flies. Annu. Rev. Entomol. 50, 293–319. Cong, L., Yang, W.J., Jiang, X.Z., Niu, J.Z., Shen, G.M., Ran, C., Wang, J.J., 2015. The essential role of vitellogenin receptor in ovary development and vitellogenin uptake in Bactrocera dorsalis (Hendel). Int. J. Mol. Sci. 16, 18368–18383. Costa-da-Silva, A.L., Kojin, B.B., Marinotti, O., James, A.A., Capurro, M.L., 2013. Expression and accumulation of the two-domain odorant-binding protein AaegOBP45 in the ovaries of blood-fed Aedes aegypti. Parasit. Vectors 6, 364. Edwards, M.J., Severson, D.W., Hagedorn, H.H., 1998. Vitelline envelope genes of the yellow fever mosquito, Aedes aegypti. Insect Biochem. Mol. Biol. 28, 915–925. Fugo, H., Yamauchi, M., Dedos, S.G., 1996. Testicular ecdysteroids in the silkmoth, Bombyx mori. Proc. Jpn. Acad. Ser. B 72, 34–37. Gamberi, C., Hipfner, D.R., Trudel, M., Lubell, W.D., 2017. Bicaudal C mutation causes myc and TOR pathway up-regulation and polycystic kidney disease-like phenotypes in Drosophila. PLoS Genet. 13, e1006694. Guiguen, Y., Fostier, A., Piferrer, F., Chang, C.-F., 2010. Ovarian aromatase and estrogens: a pivotal role for gonadal sex differentiation and sex change in fish. Gen. Comp. Endocrinol. 165, 352–366. Halon, E., Eakteiman, G., Moshitzky, P., Elbaz, M., Alon, M., Pavlidi, N., Vontas, J., Morin, S., 2015. Only a minority of broad-range detoxification genes respond to a variety of phytotoxins in generalist Bemisia tabaci species. Sci. Rep. 5. Hao, K., Wang, J., Tu, X.B., W., Douglas W., Zhang, Z.H., 2017. Transcriptomic and proteomic analysis of Locusta migratoria eggs at different embryonic stages: comparison for diapause and non-diapause regimes. J. Integr. Agric. 16, 1777–1788. Iga, M., Blais, C., Smagghe, G., 2013. Study on ecdysteroid levels and gene expression of enzymes related to ecdysteroid biosynthesis in the larval testis of Spodoptera littoralis. Arch. Insect Biochem. Physiol. 82, 14–28. Jin, T., Zeng, L., Lin, Y.Y., Lu, Y.Y., Liang, G.W., 2011. Insecticide resistance of the oriental fruit fly, Bactrocera dorsalis (Hendel) (Diptera: Tephritidae), in mainland China. Pest Manag. Sci. 67, 370–376. Kanehisa, M., Goto, S., 2000. KEGG: Kyoto encyclopedia of genes and genomes. Nucleic Acids Res. 28, 27–30. Karoonuthaisiri, N., Sittikankeaw, K., Preechaphol, R., Kalachikov, S., Wongsurawat, T., Uawisetwathana, U., Russo, J.J., Ju, J., Klinbunga, S., Kirtikara, K., 2009. ReproArray GTS: a cDNA microarray for identification of reproduction-related genes in the giant

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