ldentification of chorion genes and RNA interference-mediated functional characterization of chorion-1 in Plutella xylostella

DONG Shi-jie ,LlU Bo ,ZOU Ming-min,LlU Li-li,CAO Min-hui,HUANG Meng-qi,LlU Yan,Liette VASSEUR,YOU Min-sheng,PENG Lu

1 State Key Laboratory of Ecological Pest Control for Fujian-Taiwan Crops,Institute of Applied Ecology,Fujian Agriculture and Forestry University,Fuzhou 350002,P.R.China

2 International Joint Research Laboratory of Ecological Pest Control of Ministry of Education,Fujian Agriculture and Forestry University,Fuzhou 350002,P.R.China

3 Ministerial and Provincial Joint Innovation Centre for Safety Production of Cross-Strait Crops,Fujian Agriculture and Forestry University,Fuzhou 350002,P.R.China

4 Key Laboratory of Integrated Pest Management for Fujian-Taiwan Crops,Ministry of Agriculture and Rural Affairs,Fuzhou 350002,P.R.China

5 Fujian Provincial Key Laboratory of Insect Ecology,Fujian Agriculture and Forestry University,Fuzhou 350002,P.R.China

6 Agricultural Genomics Institute at Shenzhen,Chinese Academy of Agricultural Sciences,Shenzhen 518120,P.R.China

7 Department of Biological Sciences,Brock University,St.Catharines,Ontario L2S 3A1,Canada

Abstract Choriogenesis is the last step of insect oogenesis,a process by which the chorion polypeptides are produced by the follicular cells and deposited on the surface of oocytes in order to provide a highly specialized protective barrier to the embryo. The essential features of chorion genes have yet to be clearly understood in the diamondback moth,Plutella xylostella,a worldwide Lepidoptera pest attacking cruciferous crops and wild plants. In this study,complete sequences for 15 putative chorion genes were identified,and grouped into A and B classes. Phylogenetic analysis revealed that both classes were highly conserved and within each,branches are also species-specific. Chorion genes from each class were located in pairs on scaffolds of the P.xylostella genome,some of which shared the common promoter regulatory region. All chorion genes were highly specifically expressed in the P.xylostella adult females,mostly in the ovary with full yolk,which is a crucial period to build the shells of the eggs. RNAi-based knockdown of chorion-1,which is located on the Px_scaffold 6 alone,although had no effect on yolk deposition,resulted in smaller eggs and sharply reduced hatchability. Additionally,inhibition of PxCho-1 expression caused a less dense arrangement of the columnar layers,reduced exochorion roughness and shorter microvilli. Our study provides the foundation for exploring molecular mechanisms of female reproduction in P.xylostella,and for making use of chorion genes as the potential genetic-based molecular target to better control this economically important pest.

Keywords: Plutella xylostella,chorion genes,RNAi,oogenesis,pest control

1.lntroduction

Insect oogenesis undergoes a series of physiological processes,including the accumulation and storage of nutrients (vitellogenin,glycogen,lipid,etc.) for the embryonic development,the transfer of cytoplasmic components between nurse cells and oocytes,the localization of nucleus,protein or mRNA in a specific region,the formation of Anterior/Posterior polarity and Dorsal-Venial polarity of embryo,and the formation of eggshell structure with protective function in the outer layer (Yamauchi and Yoshitake 1984;Sweverset al.2005). After the oocyte is covered by the eggshell,it is ready to be fertilized and laid in the environment (Louet al.2018). The structure and function of eggshell are therefore crucial in insect reproductive biology.

The eggshell plays important functions in embryonic development,such as protecting embryo from mechanical damage and microbial infection,preventing water loss,allowing gas exchange,and participating in embryonic polarity formation (Waring 2000;Woodset al.2005;Zrubek and Woods 2006;Bomfimet al.2017). Although structurally and functionally diverse (Margaritis 1985),insect eggshells consist of common layers with a similar arrangement pattern (Woods 2010;Louet al.2018).From the oocyte to the outer surface,lepidopteran eggshells consist of the vitelline envelope with the wax layer,the crystalline-chorionic and trabecular layers,and the chorion,which is the predominant structural component (Margaritis 1985;Woods 2010).

Choriogenesis is the last stage of the oogenesis where the chorion proteins are produced and secreted by follicular cells on the surface of the oocytes (Huebner and Anderson 1972;Wuet al.2008;Santos and Ramos 2021). Chorion polypeptides typically are low-molecular weight (10-25 kDa) three-part structure that contains a highly conservative central domain and two variable left and right arm flanks (Tsitilouet al.1983). Chorion genes are proposed to be members of a single-gene superfamily,which is divided into mainly two homologous gene families A and B (Lecanidouet al.1986). According to the phylogenetic relationship and the spatiotemporal order of protein synthesis during choriogenesis,A and B families are divided into three subfamilies,ErA/B,A/B and HcA/B (Chenet al.2015). The subfamilies are involved in three steps of choriogenesis. ErA/B proteins are mainly synthesized in early choriogenesis. They form a thin trabecular layer near the oocytes and a fibrous spiral framework to encompasses the eggshell. The second step involves the synthesis of A/B family proteins,which allows for the expansion of the framework and its densification. In the last step,HcA/B proteins infiltrate the chorion to create a dense spiral surface structure(Spoerelet al.1986;Goldsmith 1989;Chenet al.2015).Choriogenesis has been studied in species such asDrosophilamelanogaster,Bombyxmoriand recentlyRhodniusprolixuswith a focus on aspects including the transcriptional activation of chorion genes,biochemical analysis of these proteins,and structure of the chorion(Tootleet al.2011;Velentzaset al.2018;Santos and Ramos 2021).

The highly programmed expression of chorion genes makes it a good model system for studying gene regulation. For example,Mitsialis and Kafatos (1985)examine the differences in the gene regulation of the A and B families and the conservation of thetransandcis-regulatory elements essential for chorion gene expression in both model insects,D.melanogasterandB.mori. Several studies have reported that transcription factors,such as GATA factor and two types of C/EBP,maybe involve in controlling choriogenesis,and act as an activator or repressor or both during choriogenesis (Drevetet al.1994,1995;Sourmeliet al.2003;Papantoniset al.2008). However,these regulatory components have only been identified in model insects. Much remains to study to better understand the complex choriogenesis patterns in other insect species.

Chorion proteins show high variability in number of genes,with 127 inB.mori(Rodakiset al.1982;Chenet al.2015) and 15 inLymantriadispar(Leclerc and Regier 1994). Their functions have also been examined for a few species. They include the chorion geneBrowniethat forms the aeropyle and micropyle structures (Irleset al.2009),and theCitrusgene,which is necessary for the endochorionin formation inBlattellagermanica(Irles and Piulachs 2011). Boutset al.(2007) have identified the specific chorion proteins Rp30 and Rp45,of which Rp45 is related to an antifungal activity. Louet al.(2018)report a novel chorion protein being essential for oocyte maturation and oviposition ofNilaparvatalugens.

Investigating the molecular characteristics and functions of chorion genes in the Lepidoptera pest diamondback moth,Plutellaxylostella(Lepidoptera,Plutellidae) (Liet al.2016),is of great interest to understand its reproduction and find ways to control it.The high fecundity ofP.xylostellaenables it to invade any areas where cruciferous plants grow,making it one of the most widespread lepidopteran pests in the world(Furlonget al.2013). The chorion genes have been predicted from the genome sequences and transcriptome data ofP.xylostella(Youet al.2013;Penget al.2017).However,these genes need to be further identified and characterized to lay the foundation for in-depth analysis of their functions. In the present study,we identified the molecular characteristics,scaffold location,transcription factors ofP.xylostellachorion genes (PxChos) and compared their phylogenetic relationships with other insect chorions. The spatio-temporal expression profiles of thePxChogenes were also analyzed using our RNAseq data and qRT-PCR. Finally,we explored the functions ofPxCho-1,located on the Px_scaffold 6 alone,in the oogenesis and embryonic development ofP.xylostella.This study is conducive to screening and obtaining new targets,and therefore uses CRISPR/Cas9 and gene drive technology to build a new approach for the genetic control ofP.xylostella.

2.Materials and methods

2.1.lnsect rearing

The insecticide susceptibleP.xylostellacolony Geneva 88(thereafter G88) used for this study was provided by Prof.Shelton T from Cornell University,USA in 2016,and has been maintained for over 46 generations in the insectary of the Institute of Applied Ecology at Fujian Agriculture and Forestry University,China,before being used for this experiment. Larvae were reared on artificial diet (#F9772-DBM,Frontier Scientific Serves,USA) at (25±1)°C,(65±5)% relative humidity and L:D=16 h:8 h. Pupae were transferred into a new plastic box (10.4 cm×7.3 cm×8.5 cm)until emergence. After emergence,adults were fed with 10% honey solution for nutrition.

2.2.ldentification of chorion candidate genes in P.xylostella

The chorion amino acid sequences ofB.mori,Manducasexta,Bombyxmandarina,Spodoptera frugiperda,Helicoverpaarmigera,Spodopteralitura,Papiliomachaon,L.dispar,Papillioxuthus,Parargeaegeria,Bicyclusanynana,Danausplexippus,Vanessa tameamea,Galleriamellonella,Antheraeapolyphemus,Ostriniafurnacalis,Pierisrapae,Trichoplusiani,andHyposmocomakahamanoawere downloaded from NCBI GenBank (http://www.ncbi.nlm.nih.gov/),and then used as queries against the DBM database (http://iae.fafu.edu.cn/DBM/) using local BLASTP Program (E-value＜10-5).Further filter candidate sequences were identified through comparison with online LepChorionDB (http://bioinformatics.biol.uoa.gr/LepChorionDB/) database and domain prediction by local Pfam database. Local TBLASTN Program (E-value＜10-5) was used to search omissive sequences,which were identified by the above methods. ExPASy (https://www.expasy.org/) was used to predict protein molecular weight,isoelectric point,hydrophilicity,glycosylation site and amino acid contents.Phosphorylation sites were analyzed using Netphos 3.1 Server (http://www.cbs.dtu.dk/services/NetPhos/),and Conserved regions were predicted using MEME (https://meme-suite.org/).

2.3.Phylogenetic analysis

Each putative amino acid sequence ofP.xylostellachorions was used for online BLASTP (E-value＜10-5) with the amino acid sequences ofA.polyphemus,B.anynana,H.armigera,L.dispar,M.sexta,O.furnacalis,P.machaon,andS.frugiperdaon NCBI,of which sequence with lowest E-value were further classified by LepChorionDB database. Additionally,eachP.xylostellachorion sequence was also applied for local BLASTP(E-value＜10-5) with the chorion sequences ofB.moridownloaded from NCBI database according to Chenet al.(2015). The homologous chorion sequences were aligned with Clustal W2.0. The neighbor-joining (NJ) tree was constructed using MEGA-X with a bootstrap value of 1 000 replicates (Kumaret al.2018).

2.4.Analysis of transcription factor in chorion gene promoters

The bi-and non-bidirectional regulatory regions in the promoter of the chorion gene pairs,and the upstream 2 kb sequence of the unpaired chorion genes were analyzed using the transcription factor database (http://jaspar.genereg.net/) to predict the binding sites of two key transcription factors,GATA and C/EBP (Chen et al.2015).

2.5.RNA extraction and cDNA synthesis

Total RNA was extracted from individuals or tissues using Eastep?Super Total RNA Extraction Kit (Promega,USA) according to the manufacturer’s instructions.RNA concentration was determined using Nano Vue Spectrophotometer (GE-Healthcare,UK),and RNA purity was detected with agarose gel electrophoresis.The cDNA template was synthesized by the FastKing gDNA Dispelling RT SuperMix (TIANGEN,China) with an amount of 1 000 ng RNA.

2.6.Validation of gene expression by qRT-PCR

For the expression profiles,all developmental stages(instar 1 to 4,pupa,and adult) and both sexes (from fourth instar to adult) were sampled. For tissue-specific expression patterns,40 new emerged adults were dissected to extract the head,thorax,midgut+Malpighian tubule,ovariole with full yolk,ovariole with no full yolk,fat body and epidermis in RNA protect reagent (QIAGEN,Germany) (Penget al.2019,2020). Each tissue was then separately placed in 1% PBS and stored at -80°C.RNA isolation and cDNA synthesis were completed as described above. The qRT-PCR was performed with GoTaq?qPCR Master Mix Kit (Promega,USA). Reaction system consisted of 2 μL cDNA template,10 μL 2× realtime PCR Mix,0.2 μmol L-1of each primer,0.2 μL ROX and 7 μL nuclease-free water. PCR was conducted with standard thermal cycle conditions using the twostep qRT-PCR method: 95°C for 30 s,then 95°C for 5 s,and 44 cycles at 60°C for 30 s. The homogeneity of PCR products was detected by melting curve analysis.The ribosomal proteinL32gene (RIBP) was used as endogenous control. There were three biological replicates for each treatment (i.e.,tissue,developmental stage and sex). Specific primers used for the qPCR were listed in Appendix A. The comparative Ct method(2-ΔCt) method was used to analysis the transcription level.

2.7.RNA interference

Specific primers containing the T7 RNA polymerase promoter sequence were designed for dsRNA synthesis(Appendix B). The PCR program was performed as follows: 95°C for 3 min;35 cycles of 95°C for 30 s,55°C for 30 s and 72°C for 30 s;and an additional extension at 72°C for 5 min. The PCR products were further purified by Gel Extraction Kit (Omega,China). Doublestranded RNA (dsRNA) ofPxCho-1andEGFPwas synthesized by the T7 RiboMAXTM Express RNAi System(Promega,Madison,WI,USA) following the manufacturer recommendations. The dsRNAs ofPxCho-1(dsPxCho-1)andEGFP(dsEGFP) were diluted into nuclease-free water at the concentration of 4 000 ng μL-1,and then stored at -80°C. The 150 nL (600 ng) of dsPxCho-1was injected into 3-d pupae with a Nanoliter 2010 Injector(WPI,Sarasota,FL,USA),and the dsEGFPwas injected as a negative control. Five injected individuals were collected at 12,24,36,and 48 h after injection for gene silencing efficiency detection.

2.8.Phenotypic observation and bioassays

Fifteen ovaries were dissected from 48-h old females injected with dsPxCho-1and dsEGFPas previously described in Section 2.6 and rinsed with PBS three times.Then,the number of follicles with complete yolk deposition per ovariole was recorded using digital microscope VHX-2000C (KEYENCE,Japan).

Single pairs of newly emerged adults were placed each in a plastic box (10.4 cm×7.3 cm×8.5 cm) with a parafilm sheet containing the cabbage leaf extract for laying eggs,of which nutrition was provided with 10%honey solution. Fifteen pairs ofP.xylostellaadults of dsPxCho-1(dsPxCho-1♀×WT ♂) and dsEGFP(dsEGFP♀×WT ♂) treatments were used and maintained at(25±1)°C,photoperiod L:D=16 h:8 h,and relative humidity 60-70%,respectively. Each parafilm sheet was removed and replaced with a new one every 24 h. The number of eggs laid in 3 d was recorded as well as the hatching rate of eggs laid by each pair. The length and width of the eggs laid within 1 h after mating by 10 pairs ofP.xylostellaadults of dsPxCho-1and dsEGFPtreatments were measured by digital microscope VHX-2000C (KEYENCE,Japan).

2.9.Microstructure observation of chorion

Eggs laid on the 1st d were collected and pricked with microneedles to remove the contents. The samples were fixed in 2.5% glutaraldehyde in PBS for 24 h. The newly obtained samples were washed with 0.1 mol L-1pH 7.0 1× PBS for three times and fixed with 1% osmium tetroxide in PBS for 1.5 h at room temperature. The fixed samples were washed again with 0.1 mol L-1pH 7.0 1× PBS for three times,and dehydrated in grade series of ethanol up to 100%,and 100% acetone for 30 min.The sample were transferred to Spurr’s resin (Sigma,USA) and acetone (1:1) for 1 h,followed to Spurr’s resin and acetone (3:1) for 3 h. Subsequently,the sample was embedded in Spurr’s resin and polymerized at 70°C for 24 h. Sections were stained with both uranyl acetate and Lead (II) citrate trihydrate to observe the effects ofPxCho-1knockdown on the ultrastructure of chorion layer by transmission electron microscope (TME) (H-7650,HITACHI).

2.10.Statistical analysis

For the relative mRNA expressions of chorion genes among different stages and tissues,comparisons were performed using one-way analysis of variance(ANOVA) with Tukey’s multiple range test (P＜0.05). The comparison between dsPxCho-1and dsEGFPstrains was performed using an independent samplet-test (＊,P＜0.05;＊＊,P＜0.01). Data analyses were completed with SPSS Statistics Software 22.0.

3.Results

3.1.ldentification and characterization of the P.xylostella choiron

Based on the homology alignment method,a total of 15 putative chorion genes were identified in theP.xylostellagenome and further validated by cloning and sequencing(Appendices C-Q). The length of coding sequences and inferred amino acid sequences of chorion genes are provided in Table 1. The predicted molecular mass of chorion proteins ranged from 12.8 to 22.7 kDa and the theoretical isoelectric points varied between 4.0 and 5.1 (Table 1). All of them had signal peptide sequences located in 1-20/21 of the N-terminal except PxCho-8.Chorions were hydrophobic proteins due to the 6-22 O-glycosylation sites but didn’t have N-glycosylation sites(Appendix R). In addition toPxCho-2,the most abundant amino acid in these proteins was glycine with percentages ranging from 16.3 to 25.5% (Appendix S). Chorion proteins also contained relatively high amounts of leucine,serine and asparagine (Appendix S).

Table 1 Description of chorions in the Plutella xylostella genome

3.2.Gene location and structural comparison

The chorion genes ofP.xylostellaincluded two major classes,A and B (Fig.1). Except for thePxCho-1being alone on Px_scaffold 6,the other chorion genes in the different classes were located in pairs on the two scaffolds,Px_scaffold 446 and Px_scaffold 529. The cluster on the Px_scaffold 446 was interrupted by a nonchorion genePx011672betweenPxCho-9andPxCho-10(Fig.1-A). There was a conserved domain of chorion_1 in most chorion genes except forPxCho-8,12,and14(Appendix R). The chorion proteins had conserved motifs.All chorion genes in class B contained three types (a,b,and c) of special motifs except forPxCho-2. All chorion genes in class A had two kinds of special motifs,a and c(Fig.1-B).

3.3.Phylogenetic analysis of chorion genes

The phylogenetic tree was constructed with 74 chorion sequences from 10 species of Lepidoptera. The phylogenetic tree showed that the two classes of chorion genes were clustered well into their relevant phylogenetic branches (Fig.2). In both classes,theP.xylostellachorions were all clustered into a single branch,suggesting the chorion genes were species-specific.

3.4.Cis-elements analysis in the promoters of chorion genes

The gene pairs ofPxCho-3/4,PxCho-8/9andPxCho-10/11shared the same promoter regulatory region,the sizes of which were 350,250 and 305 bp,respectively. The typical C/EBP (5′-HHDVMVMM-3′(M=A/C,H=A/T/C,D=G/A/T,V=G/A/C)) element could be found in all common promoter regions;however,the GATA(5′-TWTTGATAKST-3′ (W=A/T,K=G/T,S=G/C)) element only existed inPxCho-8/9andPxCho-10/11(Table 2).The prediction for the non-bidirectional promoter regions showed that there were one or more C/EBP and GATA transcription factor-binding sites in 2-kb region upstream of other chorion genes (Appendix T).

Table 2 The cis-elements in the bidirectional promoter regions of chorion gene pairs1)

3.5.Expression profiling of the chorion genes

The expression profiles of the chorion genes at different developmental stages ofP.xylostellawere characterized using our unpublished RNA-seq data (Appendix U). The results indicated that all 15PxChoswere significantly highly expressed at the female adult stage,suggesting their potential functions in reproductive process ofP.xylostella(Fig.3). The expression patterns of thePxChosat different developmental stages were also characterized by qRT-PCR. The results showed that all of chorion genes were significantly highly expressed in female adults (P＜0.05) (Fig.4). The expression level ofPxCho-2in male adults was second only to that of female adults (Fig.4). In addition,some chorion genes were slightly expressed in the 4th instar larva and pupal stage,such asPxCho-5,PxCho-6,andPxCho-10(Fig.4).Tissue-specific expression profiles of female adults showed that the relative expression of all chorion genes was significantly higher in the ovary with full yolk than any other tissues (P＜0.05) (Fig.5). Secondly,thePxCho-2,PxCho-4andPxCho-14were slightly expressed in the ovary with no full yolk,whilePxCho-1,PxCho-14andPxCho-15were expressed in small amounts in the fat body (P＜0.05) (Fig.5). The expression levels of chorion genes were negligible in the tissues of head,thorax,midgut+Malpighian tubule and epidermis (P＞0.05) (Fig.5).

3.6.The effects of chorion-1 knockdown on P.xylostella reproduction

After dsPxCho-1injection for 24 h,mRNA expression level ofPxCho-1in females was significantly suppressed by 33.3% compared to the dsEGFPcontrol group (t=5.63,df=4,P=0.005) (Fig.6-A). There was no significant difference in the number of fully developed follicles per ovariole of the newly emerged females from dsPxCho-1and dsEGFPtreatments (8.9 for dsPxCho-1and 8.4 for dsEGFP) (t=-0.95,df=32,P=0.35) (Fig.6-B). Injection of dsPxCho-1had no signification effect on the number of eggs laid per female in 3 d (Fig.6-C). However,the hatching rate was siginificantly reduced by 9.9% for the offspring eggs laid on the 1st d (t=2.82,df=73,P＜0.01)and 16.4% for the offspring eegs laid on the 2nd d (t=3.113,df=60,P＜0.01) (Fig.6-D). The length and width of eggs were significantly reduced from 518.8 and 291.0 μm for dsPxCho-1to 543.9 (t=4.14,df=96,P＜0.01) and 315.5 μm(t=6.14,df=99,P＜0.01) for dsEGFP(Fig.6-E and F).

3.7.The effects of chorion-1 knockdown on the formation of chorion

Chorion-1knockdown did not affect the the formation of vitelline membrane and enochorion,but resulted in a less dense arrangement of the columnar layers that connect the inner and outer enochorion (Fig.7). Meanwhile,inhibition ofPxCho-1expression resulted in reduced exochorion roughness and shorter microvilli covered on the surface of the eggs (Fig.7).

4.Discussion

The chorion polypeptides produced by the follicular cells in ovarioles are important eggshell components as they play the essential function of protecting developing embryo from external agents and providing access of the sperm to the egg (Papantoniset al.2015). In our study,15 chorion genes were identified inP.xylostellagenome. This number of chorion genes is similar to the 15 inL.dispar(Leclerc and Regier 1994). The predicted molecular mass of chorion proteins inP.xylostellaranged from 12.8 to 22.7 kDa with a conserved domain of chorion_1,which is the typical type of chorion polypeptides. Tsitilouet al.(1983) report that classical chorions are of low molecular weight (10-25 kDa) and have a highly conserved central domain. Almost all ofPxChoshad a signal peptide,which is consistent with most chorion proteins (Louet al.2018). These results also confirmed that chorions might be a secretory protein transcribed in follicular cells(Regieret al.1978). O-Glycosylation sites were located in chorions ofP.xylostella,but no N-glycosylation sites were found. Louet al.(2018) also report similar results forN.lugens.

Glycine was the most abundant in chorion proteins ofP.xylostella,followed by leucine,serine,asparagine,alanine and valine. Regieret al.(1978) report that the eggshell proteins ofB.moriare very rich in the nonpolar amino acids,especially in glycine,alanine,valine and leucine,followed by tyrosine and cysteine. Eggshell proteins are usually cross-linked to form chemically and mechanically hard structures (Benakiet al.1998). Crosslinking is common in structural components,such as chorine where resilin is formed between tyrosine residues to produce dityrosine and trityrosine (Petriet al.1976;Epstein and Lamport 1984). Although there was a low percentage of tyrosine residues in the chorion genes ofP.xylostella,only ranging from 1.4 to 8.5%,they were widely distributed in the peptide sequences,suggesting thatPxChosmight be cross-linked by dityrosine and trityrosine.

The chorion genes ofP.xylostellawere categorized as A and B classes,each clustered in its own phylogenetic branch. Papantoniset al.(2015) suggest that chorion genes belong to a single-gene superfamily consisting of homologous gene families from different developmental groups and can be further divided in two branches of Aor B-type proteins. InB.mori,chorion proteins are further subdivided into the classes of early A and B,middle A and B,late high-cysteine A and B (Regieret al.1978;Chenet al.2015). Phylogenetic analysis results showed that the two classes of chorion genes inP.xylostellawere clustered well into the distinct branches. This observation is similar with the results of Leclerc and Regier (1994) who classify the group A/B in chorion genes prior to species differentiation.Both classes of chorion genes inP.xylostella,were clustered into a single sub-branch in phylogenetic analysis different fromB.moriandM.sexta,suggesting that chorion genes may be species-specific.

In our study,chorion genes in the different classes were located in pairs on the scaffolds ofP.xylostellagenome,some of which shared the common promoter regulatory region with fragment length of 250-350 bp.This observation is consistent with the previous reports inB.moriwhere most chorion genes are arranged in pairs ofA-andB-type genes of co-developmental specificity,and undergoing different transcription from a common promoter ((300±30) bp) (Iatrouet al.1984;Spoerelet al.1986;Hibneret al.1988;Regieret al.1994). The conserved C/EBP and GATA elements were found in all common promoter regions in chorion genes ofP.xylostella. Tsukadaet al.(2011) have identified C/EBPs inB.morichorion gene promoter,and BmC/EBP is the important factor to regulate chorion gene expression(Papantoniset al.2015). InD.melanogaster,C/EBP was involved in regulating border cell migration and embryonic development (R?rth 2009). Skeiky and Iatrou (1991)have identified a conservedcis-element for transcription factors of the GATA family in the promoter of the Hc.L12 chorion gene inB.mori. Other studies have shown that there is antagonism between C/EBP and GATA factors in the promoters of early and middle chorion genes,whereas GATA promotes the expression of late-specific chorion genes (Sourmeliet al.2003;Papantoniset al.2008).Overall,these results suggested that similar regulative mechanisms controlled oogenesis in the different insect species.

The expression profile showed that allPxChoswere expressed at extremely high levels inP.xylostellaadult females,and especially in the ovary with full yolk (P＜0.05).Papantoniset al.(2015) suggest that chorion genes are female-specific and may play an significant role in chorionic formation at the late stage of oogenesis. Similar expression patterns have also been observed in other insect species,such asB.moriandD.melanogaster(Parks and Spradling 1987;Chenet al.2015),where chorion genes are found only in specific regions of the eggshell,suggesting the functions in forming micropyle,operculum and dorsal appendages. Additionally,some chorion genes ofP.xylostellawere not only highly expressed in female adults and ovaries,but also in lower expression in other stages (e.g.,adult males) and tissues(e.g.,fat body),implying various potential physiological functions. Carteret al.(2013) report that there are different mechanisms for regulating chorion genes,including splicing variants (Drevetet al.1995),antisense RNA (Skeiky and Iatrou 1990),distinct promoters,and alternatively polyadenylated isoforms (Papantoniset al.2008;Lecanidou and Papantonis 2010a,b). Chenet al.(2015) confirm that the expression of three chorion genes in embryos,follicular cells,and testes ofB.moriare regulated by distinct promoters and alternative splicings.

Chorion proteins,as major structural proteins of eggshell,are essential for oocyte maturation and embryonic development in most invertebrate species(Papantoniset al.2015). Although essential for reproductive development,studies have been confined to model insects. In this study,we used RNAi-based knockdown ofPxCho-1expression,only located on the Px_scaffold 6,to further explore the reproductive functions of chorion genes ofP.xylostella. Here,we found that injection of dsPxCho-1into female pupae ofP.xylostelladid not suppress the yolk deposition of follicles. This result is consistent with the process of egg formation in insects,especially in Lepidoptera (Kafatoset al.1995;Telfer 2009;Papantoniset al.2015). Choriogenesis is involved in the last step of oogenesis,and the transition to choriogenesis occurs after the completion of vitellogenesis (Papantoniset al.2015). At this point,follicular epithelium cells differentiate for the production and secretion of chorion proteins,which eventually form the eggshell (Papantoniset al.2015;Louet al.2018).Louet al.(2018) report,after the knockdown of a chorion gene ofN.lugens,no significant effect on vitellogenin expression,which is the major egg yolk precursor protein.Additionally,after knockdown ofPxCho-1,the length and width of the eggs as well as the hatching rate were lower than the dsEGFP control group. Furthermore,inhibition ofPxCho-1expression caused a less dense arrangement of the columnar layers,reduced exochorion roughness and shorter microvilli. There were also found inN.lugenandB.germanica(Irles and Piulachs 2011;Louet al.2018).

We speculate that these results could be due to knockdown ofPxCho-1causing structurally defective eggshells during oogenesis and leading to rapid water loss. Studies have confirmed that the chorion is the predominant structural constituent of the eggshell,protecting the developing embryo from being abraded and crushed,while allowing gas exchange with minimal water loss (Margaritis 1985;Spradling 1993;Woods 2010). We found that the formation of chorion was not completely inhibited,and that there was no effect on the egg-laying amount after knockdown ofPxCho-1. We speculated that it might be possible that other chorion family members with similar conserved domains might also compensate for the loss ofPxCho-1function.

5.Conclusion

This is a first study characterizing the chorion genes inP.xylostellaand confirming the important role ofPxCho-1on oogenesis and embryonic development. We found that the chorion genes ofP.xylostellawere speciesspecific both in gene number and genetic evolution,andPxCho-1played an important role in oogenesis and embryonic development. These findings may lay the foundation for understanding molecular mechanisms of female reproduction ofP.xylostella,and for making use of chorion genes as the potential genetic-based molecular target to better controlP.xylostellabasing on the endogenous regulatory elements to drive Cas9 and single guide RNA (sgRNA) expression inP.xylostella. Further research needs to subdivide the subclasses of chorion genes inP.xylostella,and identify their transcription patterns to clarify the functions of chorion genes in the female reproductive process such as eggshell formation,as well as other potential physiological processes using CRISPR/Cas9 technology.

Acknowledgements

This work was funded by the National Natural Science Foundation of China (32172404),the Natural Science Foundation of Fujian Province,China (2019J01666),the Fujian Agriculture and Forestry University Fund for Distinguished Young Scholars,China (xjq201903),and the “111” Program -Innovation Center for Ecologically Based Pest Management of Subtropical Crops,Fujian Agriculture and Forestry University,China.

Declaration of competing interest

The authors declare that they have no conflict of interest.

Appendicesassociated with this paper are available on http://www.ChinaAgriSci.com/V2/En/appendix.htm