Characterization of carboxylesterase PxαE8 and its role in multiinsecticide resistance in Plutella xylostella (L.)

LI Ran,SUN Xi,LIANG Pei,GAO Xi-wu

Department of Entomology,China Agricultural University,Beijing 100193,P.R.China

Abstract Carboxylesterase (CarE) was considered as important phase-I detoxifying enzymes which participated in detoxification of different types of insecticides. Up-regulation of CarE genes has been proved playing a major role in insecticide resistance in many pest insects,but its involvement in resistance to insecticides in Plutella xylostella has been rarely reported. In this study,a CarE cDNA named PxαE8 was identified in P.xylostella,which has an open reading frame of 1 599 nucleotides and putatively encodes 532 amino acids. The investigation of spatial expression profiles of PxαE8 revealed that it was expressed in all developmental stages,especially in larvae and adults. The body part/tissuespecific expression profiles showed that the PxαE8 mainly expressed in fat body,malpighian tubule and hemolymph of larvae. Further,the relative expression of PxαE8 in two multi-resistant field populations,Hainan (HN) and Guangdong(GD) populations,was found 24.4-and 15.5-fold higher than that in susceptible population,respectively. Knockdown of PxαE8 by RNA interference dramatically increased the mortalities of larvae of HN population treated with LC50 of betacypermethrin and phoxim by 25.3 and 18.3%,respectively. These results suggested that up-regulation of PxαE8 was involved in resistance to both beta-cypermethrin and phoxim in P.xylostella,which shed light on further understanding of molecular mechanisms of multi-insecticide-resistance in P.xylostella and other pest insects.

Keywords:Plutella xylostella,carboxylesterase,insecticide resistance,beta-cypermethrin,phoxim

1.Introduction

Plutellaxylostella(L.) is a notorious insect pest and causes serious damages to most of cruciferous plants worldwide (Furlonget al.2013). Currently,chemical insecticides play the most important role inP.xylostellacontrol. However,extensive and unreasonable applications of insecticides have fastened the development of resistance to vast majority of insecticides inP.xylostella(Liet al.2016) and led to the emergence of multi-insecticides resistant field populations (Zhanget al.2016). Our previous work also revealed that a field population collected from Guangdong Province,China had developed high resistance to nine insecticides (abamectin,beta-cypermethrin,chlorantraniliprole,chlorfenapyr,indoxacarb,metaflumizone,phoxim,tebufenozide and spinosyns) which belong to nine different classes with different mechanisms of action (Liet al.2018),while the mechanisms of multi-resistance were rarely reported.Target insensitivity and enhanced detoxification are two most important mechanisms of insecticide resistance,and the former usually causes cross-resistance among insecticides sharing the same target protein,while the latter has the potential to lead to multi-resistance because of the rich diversity of detoxifying enzymes and broadspectrum of their substrates (Yu 2015).

Carboxylesterase (CarE),which belongs to a superfamily of α/β hydrolase,is one of major detoxifying enzymes and can catalyze the hydrolysis of carboxyl esters into alcohols and carboxylic acids (Wheelocket al.2005). CarE has been reported to be involved in detoxification of many insecitcides (Yanget al.2018;Zhanget al.2018;Liet al.2020) in different insect pests.For instance,overproduction of CarEs was responsible for resistant to organophosphates,carbamates and pyrethroids inMyzuspersicaethrough enhanced degradation and sequestration (Basset al.2014).Overexpression of CarE genes was also associated with organophosphates resistance inNilaparvatalugens(Small and Hemingway 2000),Aphisgossypii(Caoet al.2008),andP.xylostella(Xieet al.2017),and was involved in detoxification of pyrethroids in resistantHelicoverpa armigera(Wuet al.2011) andMuscadomestica(Feng and Liu 2018,2020). It was also found involved in spirodiclofen resistance inTetranychusurticae(Weiet al.2020). Upregulation of CarE mRNAPxCCE016bmay contribute to resistance to chlorantraniliprole inP.xylostella(Huet al.2016) and nitenpyram inN.lugens(Maoet al.2020). All these proved that overexpression of CarE played important roles in insecticide resistance.Therefore,we proposed that the overexpression of CarE possibly contribute to multi-insecticide resistance inP.xylostella.

2.Materials and methods

2.1.Insects

A total of three populations ofP.xylostellawere used in this study. Two resistant populations,Hainan (HN) and Guangdong (GD) populations,were sampled respectively from vegetable fields of Hainan Province and Guangdong Province of China in 2019,and a laboratory susceptible population (SS) was collected from Beijing suburb and reared indoor without any insecticides for more than 10 years. All populations were reared on radish seedlings at (27±1)°C,relative humidity (RH) of 40-60% and a photoperiod of 16 h L:8 h D. The adults were supplied with 10% (w/v) honey solution as supplementary food,and allowed to oviposit on radish seedlings.

2.2.Bioassays

The leaf dipping method (Guoet al.2014) was used to determine the toxicities of beta-cypermethrin (technical grade 95.3%;Jiangsu Changlong Chemical Industry Ltd.,Jiangsu,China),chlorantraniliprole (technical grade 95%;DuPont Agriculture Chemical Ltd.,Shanghai,China),metaflumizone (technical grade 95%;Shandong Aodeli Chemical Ltd.,Shandong,China),phoxim(technical grade 95%;Shandong Lunan Pesticide Ltd.,Shandong,China) and tebufenozide (technical grade 96.8%;Shandong Lunan Pesticide Ltd.,Shandong,China) to different populations ofP.xylostella. The stock solutions of the insecticides were prepared with acetone and then diluted to five serials concentrations with distilled water containing 0.1% Triton X-100. The clean Chinese cabbage leaves were cut into 6 cm×6 cm discs,and immersed in suitable concentrations of insecticides for 10 s,then allowed to air dry indoor for 30 min.Then the leaf disc was placed into a Petri dish (6 cm in dimeter) with filter paper and a total of 15-20 neonate third-instar larvae were gently added into the Petri dish with a brush. Three replications were prepared for each concentration. Leaves treated with distilled water containing 0.1% Triton X-100 were used as the control.Mortality was recorded 48 h after treatment and the larva showed no response to stimulus of a needle was considered as dead.

2.3.RNA extraction and cDNA synthesis

The total RNA from 10 third-instar larvae was extracted using TRIzol Reagent Kit (Invitrogen,CA,USA) according to the manufacturer’s instructions. The integrity of the total RNA was examined on 1% agarose gels,and the concentration was determined with NanoDrop 2000(Thermo Scientific,Wilmington,USA). The cDNA for PCR was synthesized from 1 mg total RNA using Prime Script RT Reagent Kit with gDNA eraser (TaKaRa Biotechnology,Dalian,China) following the manufacturer’s instructions.

2.4.Gene cloning and sequence analysis

The gene specific primers were designed according to the sequence ofPxαE8(GenBank number:XM_011555505.1) and the complete open reading frame(ORF) was amplified using following procedure:95°C of initial incubation for 5 min,followed by 35 cycles of 95°C for 30 s,56°C for 30 s,then 72°C for 1 min 50 s and 72°C of final extension for 10 min. The purified amplicons were cloned into the PMd19-T cloning vector(TaKaRa) and transformed intoEscherichiacoliDH5α.The transformants were selected with Luria-Bertani (LB)agar plates containing 0.1% ampicillin,and positive clones were sequenced (Sangon Biotech,Shanghai,China).Complete sequence was analyzed using the BLAST web tools of NCBI (http://www.ncbi.nlm.nih.gov/blast/Blast.cgi).

The isoelectric point (pI) and molecular mass ofPxαE8were predicted using ExPASy tools (http://web.expasy.org),and the glycosylation of the protein was predicted by NetNGlyc 1.0 server (Carageaet al.2007). The catalytic triad and substrate binding pocket ofPxαE8were identified with BlastP analysis in NCBI. The signal peptide was also predicted using the SignalP 4.1 Program(http://www.cbs.dtu.dk/services/signalp/).

2.5.Quantification of PxαE8 by RT-qPCR

The relative expression ofPxαE8in HN and GD populations were compared with that of the SS population by qRT-PCR using a Platinum?SYBR?Green qPCR SuperMix-UDG Kit (Invitrogen) following the manufacturer’s instructions. A total of 10 third-instar larvae were randomly sampled for the total RNA isolation and cDNA synthesis as described in Section 2.3.

For determination of expression profiles in different developmental stages,the eggs,first-to fourth-instar larvae,pupae and adults ofP.xylostellawere randomly collected and stored at -80°C for the total RNA extraction.For body part/tissue-specific expression profile analysis,six different body parts/tissues (head,midgut,malpighian tubule,fat body,haemolymph and integument) were dissected from the fourth-instar larvae of the SS and washed in cold Ringer’s solution (96 mmol L-1NaCl,2 mmol L-1KCl,5 mmol L-1MgCl2,0.8 mmol L-1CaCl2and 5 mmol L-1Hepes;pH 7.6) for three times,then frozen in liquid nitrogen and stored at -80°C for the total RNA isolation.

The total RNA of the above cold stored samples was isolated and the cDNA was synthesized as described in Section 2.3. Then the qPCR was performed on an ABI 7500 Real Time PCR System (Applied Biosystems,Foster,CA). The cyclic thermal procedure contained an initial denaturation step at 94°C for 30 s,following 40 cycles at 94°C for 30 s and 60°C for 34 s,then a dissociation step was performed,the specificity of the PCR products was assessed by melting curve analysis for all samples. Primers used for RT-qPCR were listed in Table 1,andribosomalproteinL32was used as an internal control according to Liet al.(2017). Three biological replicates were set up for all treatments.

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2.6.Functional analysis of PxαE8 gene by RNAi

The cDNA sequence ofPxαE8was amplified using the gene-specific primers conjugated with the T7 RNA polymerase promoter (Table 1),the product was used as templates for double-stranded RNA (dsRNA)synthesis using Transcript Aid T7 High Yield Transcription Kit (Thermo Fisher Scientific,Massachusetts,USA)according to the protocol. The dsRNA of enhanced green fluorescent protein (EGFP) was also synthesized similarly.The synthesized products were dissolved in nucleasefree water,quantified with NanoDrop2000 and stored at-20°C until use. Approximately 350 ng of dsRNA was injected into each third-instar larvae from HN population using a microinjector (Nanoliter 2000 Injector;WPI Inc.,FL,USA),and then reared on clean cabbage leaves under laboratory conditions. Ten larvae were randomly collected at 48 h after dsRNA injection and used for qRTPCR analysis,and the third larvae injected with dsEGFPwere used as the control. To evaluate the function ofPxαE8in insecticide resistance,both dsPxαE8and dsEGFPinjected larvae were treated with LC50of each insecticide 12 h post injection,50 individuals were treated in each replicate and three replications were set up for each insecticide. Mortalities were recorded 48 h post treatment.

Table 1 Primers used for RT-PCR,qPCR and dsRNA in this study1)

2.7.Statistical analysis

Concentration-mortality data were assessed by probit analysis using POLO Plus 2.0 (LeOra Software,Petaluma,CA,USA). The resistance ratio (RR) was given as the ratio of the resistant population LC50/SS population LC50.Relative expression ofPxαE8was calculated according to the 2-ΔΔCTmethod (Livak and Schmittgen 2001). The relative expression ofPxαE8among different populations,developmental stages,and body part/tissues as well as the mortalities among different treatments were compared by one-way ANOVA followed by Tukey’s multiple comparison test at 0.05 level using GraphPad InStat 3.05(GraphPad Software,San Diego,California,USA).

3.Results

3.1.Resistance of field populations of P.xylostella to five insecticides

The bioassays results showed that both the HN and GD populations showed resistance to all the five insecticides tested,and the HN population showed much higher resistance than GD,especially to chlorantraniliprole,metaflumizone and phoxim (Table 2).

Table 2 Toxicity of five insecticides to different populations of Plutella xylostella

3.2.Characterization of PxαE8

The full-length ofPxαE8cDNA has an ORF of 1 599 nucleotides encoding 532 amino acid residues with a molecular mass of 59.4 kDa and a pI of 5.94. SignalP 4.1 analysis showed thatPxαE8 had no signal peptide.The amino acid sequence of PxαE8contains several conserved motifs including a CarE-superfamily-specific catalytic triad and substrate binding pocket. The analogous triad was ser187-Glu322-His439 inPxαE8(Fig.1),in which Ser187 was part of Gly185-Glu186-Ser187-Ala188-Gly189 motif,which was the conserved Gly-Xaa-Ser-Xaa-Gly (Xaa can be any amino acid) motif in all esterases/lipases. The non-contiguous residues such as Gly106,Gly108,Glu186,Ala188,Ala191,Leu349,Val353,Thr354,Val387,Leu440 and Leu443 inPxαE8may form the substrate binding pockets according to Cygleretal.(1993).

Fig.1 Nucleotide and deduced amino acid sequences of PxαE8. The conserved Gly-X-Ser-X-Gly motif was boxed. Underlined letters denote the catalytic triad,which is necessary for carboxylesterase activity;letters in grey shadow represent the substrate binding pocket;the boxed single letters represents the glycosylation site.

3.3.Expression of PxαE8 in different populations,developmental stages and body parts/tissues

The relative expression ofPxαE8in GD and HN populations were 15.5-and 24.4-fold higher than that in SS population,respectively (Fig.2-A). The stage-specific and body part/tissue-specific expression patterns ofPxαE8were also analyzed using RT-qPCR (Fig.2-B and C). The results showed thatPxαE8was expressed in all stages ofP.xylostellawith the larvae and adults exhibited significantly higher levels compared with that of eggs and pupae. The expression ofPxαE8was also detected in all six body parts/tissues tested,and abundantly distributed in fat body,malpighian tubule and hemolymph,whichwas 9.04-,5.04-and 4.99-fold higher than that in head(Fig.2-C).

Fig.2 Relative expression of PxαE8 in different populations (A),different developmental stages (B) and different body parts/tissues(C) of Plutella xylostella. SS,susceptible population;GD,Guangdong population;HN,Hainan population. L1-L4 represent first to fourth instars,respectively. The data were presented as mean±SD (n=3). Different letters above the error bars indicated significant difference (one-way ANOVA followed by Tukey’s test,P＜0.05).

3.4.Effect of gene silencing on susceptibility of P.xylostella to five insecticides

The results of qRT-PCR showed that injection of dsPxαE8significantly decreased the expression ofPxαE8in HN population by 75.53% at 48 h after injection (Fig.3). Then the susceptibility of HN population to the five insecticides was assessed after injection of dsRNA. Knockdown ofPxαE8dramatically increased the mortalities of larvae treated with LC50of beta-cypermethrin and phoxim by 25.3 and 18.3%,respectively,but showed no effect on susceptibilities to chlorantraniliprole,metaflumizone and tebufenozide (Fig.4).

Fig.3 Relative expression of PxαE8 in third-instar larvae injected with dsEGFP and dsPxαE8. The data were presented as mean±SD (n=3). ＊ indicates significant difference between the dsPxαE8-and dsEGFP-injected groups (Student’s t-test,P＜0.05).

Fig.4 Mortality of dsRNA-injected third-instar larvae 48 h after treatment with LC50 of each of five insecticides. dsEGFP and dsPxαE8 represent the larvae injected with dsEGFP and dsPxαE8,respectively;CYP,beta-cypermethrin. CHL,chlorantraniliprole;MET,metaflumizone;PHO,phoxim;TEB,tebufenozide. The data were presented as mean±SD (n=3). Bars marked with different lowercase letters are significantly different (one-way ANOVA followed by Tukey’s multiple comparison test,P＜0.05).

4.Discussion

In this study,a carboxylesterase cDNAPxαE8was found overexpressed in two multi-insecticide resistant field populations,and the results of RNAi combined with that of bioassays provided evidence that the up-regulation ofPxαE8was involved in beta-cypermethrin and phoxim resistance inP.xylostella,which lay a foundation for further exploration of the function ofPxαE8in multiresistance inP.xylostella.

As one of the most resistant insect pests,P.xylostellahas evolved varying resistance to as many as 96 insecticides (Sparkset al.2020),and because of the complexity of insecticides application as well as the short generation time and high fecundity of this pest,the field populations ofP.xylostellaoften developed resistance to multiple insecticides with different mechanisms of action(Cao and Han 2006;Puet al.2010;Zhanget al.2016;Shenet al.2017). Similarly,here we found the two field populations ofP.xylostella(GD and HN) also possessed different levels of resistance to all five commonly used insecticides. And it is much difficult for local farmers in Guangdong and Hainan Province to find an effective insecticide to use. Therefore,it is very important to study the mechanisms of multi-insecticide resistance in diamondback moth.

As a superfamily of detoxifying enzyme,CarE has been associated to resistance to many insecticides,and a single CarE gene could cause high resistance to insecticides (Smallet al.2000;Basset al.2014;Feng and Liu 2018;Zhanget al.2018;Weiet al.2020).When examine our transcriptome data of several field populations ofP.xylostella,a CarE gene namedPxαE8was found highly expressed in all resistant field populations and all these populations showed resistance to multiple insecticides. Therefore,we proposed that thisPxαE8may play a role in multi-resistance. So,the complete cDNA sequence ofPxαE8was cloned and the deduced amino acid sequence contained the catalytic triad (Ser-Glu-His) and the pentapeptide (Gly-X-Ser-X-Gly) motif,which were the typical molecular characteristics for carboxylesterase performing detoxification function (Oakeshottet al.2005). Then the developmental stage-and body part/tissue-specific expression profiles ofPxαE8were investigated. The results showed that this gene expressed in all stages ofP.xylostella,especially highly expressed in larval stage when theP.xylostellahas a great chance to be exposed to insecticides. Except the larval stage,high expression ofPxαE8was also found in adults.Considering the adults have less chance of exposing to insecticides,it implied thatPxαE8may have other functions in adult stage. Among the six body parts/tissues tested,PxαE8was found highly expressed in fat body,malpighian tubule and hemolymph (Fig.2-C).Fat body and malpighian tubule have been reported as the primary detoxification tissues and responsible for insecticide resistance (Birner-Gruenbergeret al.2012;Shenet al.2013). The hemolymph is important for transportation of insecticide molecular to target sites,thus high expression ofPxαE8in hemolymph indicated that it may play a role in hydrolysis of insecticide during the transportation of insecticide by hemolymph,but this need further investigation. Put the results of developmental stage-and body part/tissue-specific expression profiles ofPxαE8together,it implied that this gene is possibly involved in response to insecticides inP.xylostella.

To further investigate the function ofPxαE8in multiinsecticide resistance,RNAi was used in this study and has been proved an effective tool for exploring the functions of individual genes (Zhuet al.2019;Fuet al.2020) especially the gene functions in insecticide resistance (Gonget al.2014;Gellatlyet al.2015;Liuet al.2020;Menget al.2020). In this study,we found that among the five insecticides tested,knockdown the expression ofPxαE8by RNA interference significantly increased the toxicity of phoxim and beta-cypermethrin to the larvae,which indicatedPxαE8was involved in resistance to these two different classes of insecticides inP.xylostella.

However,though the increase of mortality were statistically significant,they were relatively low (only 25.3 and 18.3% for phoxim and beta-cypmethrin,respectively),indicating that the overexpression ofPxαE8is involved in resistance to these two insecticides but may not be so important. Therefore,the roles of other CarE genes as well as other detoxifying enzymes such as cytochrome P450s,glutathioneS-transferase(GSTs) and UDP-glycosyltransferase (UGTs) should be further examined.

5.Conclusion

The results of this study provided fundamental evidence thatPxαE8was involved in resistance to phoxim and beta-cypmethrin,which lay a foundation for further study of multi-resistance mechanisms inP.xylostellaand other insect pests.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (31371956,31572023 and 31772186).

Declaration of competing interest

The authors declare that they have no conflict of interest.