Disruption of plant carotenoid biosynthesis through virus-induced gene silencing affects oviposition...

Disruption of plant carotenoid biosynthesis throughvirus-induced gene silencing affects ovipositionbehaviour of the butterfly Pieris rapae

Si-Jun Zheng1, Tjeerd A. L. Snoeren1, Sander W. Hogewoning2, Joop J. A. van Loon1 and Marcel Dicke1

1Laboratory of Entomology, Wageningen University, PO Box 8031, 6700 EH Wageningen, The Netherlands; 2Horticultural Production Chains Group,

Wageningen University, PO Box 630, 6700 AP Wageningen, The Netherlands

Author for correspondence:Marcel Dicke

Tel: +31 317 484311Email: [email protected]

Received: 8 November 2009

Accepted: 13 January 2010

New Phytologist (2010) 186: 733–745doi: 10.1111/j.1469-8137.2010.03213.x

Key words: Arabidopsis thaliana, Brassica

nigra, Brassica oleracea, carotenoid, insect–plant interactions, Nicotiana benthamiana,phytoene desaturase, virus-induced genesilencing (VIGS).

Summary

• Optical plant characteristics are important cues to plant-feeding insects. In this

article, we demonstrate for the first time that silencing the phytoene desaturase

(PDS) gene, encoding a key enzyme in plant carotenoid biosynthesis, affects insect

oviposition site selection behaviour.

• Virus-induced gene silencing employing tobacco rattle virus was used to knock

down endogenous PDS expression in three plant species (Arabidopsis thaliana,

Brassica nigra and Nicotiana benthamiana) by its heterologous gene sequence

from Brassica oleracea.

• We investigated the consequences of the silencing of PDS on oviposition behav-

iour by Pieris rapae butterflies on Arabidopsis and Brassica plants; first landing of

the butterflies on Arabidopsis plants (to eliminate an effect of contact cues); first

landing on Arabidopsis plants enclosed in containers (to eliminate an effect of vol-

atiles); and caterpillar growth on Arabidopsis plants. Our results show unambigu-

ously that P. rapae has an innate ability to visually discriminate between green and

variegated green-whitish plants. Caterpillar growth was significantly lower on

PDS-silenced than on empty vector control plants. This study presents the first

analysis of PDS function in the interaction with an herbivorous insect.

• We conclude that virus-induced gene silencing is a powerful tool for investigat-

ing insect–plant interactions in model and nonmodel plants.

Introduction

A major challenge for current biology is to integrateresearch approaches that address different levels of biologi-cal organization, that is, from subcellular mechanisms tofunctions in ecological interactions (Zheng & Dicke,2008). To achieve this, the determination of the functionof genes is indispensable. To date, most reverse geneticsapproaches have relied on post-transcriptional gene silenc-ing to study the function of plant genes (Watson et al.,2005). This is especially suitable for nonmodel species,which lack a rich abundance of well-characterized mutants,but is also a useful tool for model species (e.g. Alonso et al.,2003). Virus-induced gene silencing (VIGS) has beendeveloped recently to characterize the function of plantgenes through gene transcript suppression (Burch-Smithet al., 2006; Wang et al., 2006; Pflieger et al., 2008). The

most widely used VIGS vectors are based on the tobaccorattle virus (TRV) (Ratcliff et al., 2001; Liu et al., 2002).TRV-based VIGS vectors have been mainly used to silencegenes in a number of solanaceous plant species, includingNicotiana benthamiana, N. attenuata, Solanum lycopersicumand S. nigrum (Ratcliff et al., 2001; Liu et al., 2002; Bhat-tarai et al., 2007; Kandoth et al., 2007; Senthil-Kumaret al., 2007; Hartl et al., 2008; Wu et al., 2008). The cosi-lencing of LeMPK1 and LeMPK2 has been shown to reducesystemin-mediated resistance to Manduca sexta larvae intomato (Kandoth et al., 2007). The silencing of methyljasmonate (MeJA) esterase expression in N. attenuata hasshown that the resistance elicited by MeJA treatment is notelicited by MeJA, but by its demethylated analogue jas-monic acid (Wu et al., 2008). The application of VIGShas also been used in reverse genetics of floral scent in petu-nia (Spitzer et al., 2007) and in the development of symbi-

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otic root nodules in Pisum sativum (Constantin et al.,2008).

In this article, we investigate insect behavioural responsesto plant pigmentation which is modified through TRV-based VIGS. Adult insects select plants as food for them-selves or as host for their offspring. For example, many but-terfly species select flowers for nectar feeding and greenleaves for oviposition (Prokopy & Owens, 1983). Duringhost plant selection, butterflies are known to utilize visual,tactile, olfactory and gustatory information (Prokopy &Owens, 1983; Schoonhoven et al., 2005). In many animalspecies, foraging and sexual behaviour is guided by visualstimuli (Logothetis & Sheinberg, 1996; Briscoe & Chittka,2001). Investigations of the visual cues that mediate biolog-ical interactions usually employ differently coloured nonliv-ing substrates to circumvent the difficulties in the experi-mental control of the plant or animal pigments involved.Oviposition site selection involves an important behaviouraldecision in the life cycle of an herbivorous insect, becauseneonate larvae have limited dispersal capacity (Chew &Renwick, 1995).

We employed VIGS to knock down endogenous phytoenedesaturase (PDS) gene expression in three plant species(Arabidopsis thaliana, Brassica nigra and Nicotiana benthami-ana) by its heterologous gene sequence from Brassica olera-cea. Although PDS silencing has been used as a marker ofsuccessful gene silencing, studies on the biological functionof PDS have been restricted to its role in carotenoid biosyn-thesis (Giuliano et al., 1993; Fang et al., 2008). In this arti-cle, we address the function of PDS in an ecological context.Defects in PDS activity result in green-whitish variegatedplants that resemble virus-infected or leafminer-infestedplants (Wetzel et al., 1994). The green-whitish variegatedphenotype occurs in many plant species (Evenari, 1989; Lev-Yadun, 2006) and appears to influence the ovipositionbehaviour of insects (Soltau et al., 2009). In this study, wespecifically investigated the effect of PDS expression on theinteraction with an insect, the small cabbage white butterfly,Pieris rapae, as mediated by changes in colour pattern in theplant’s phenotype. This butterfly species is a specialist onplants in the Brassicaceae (syn. Cruciferae) family, andresearch on sensory cues relevant for oviposition behaviourhas focused on plant chemistry, especially the stimulatoryrole of the characteristic secondary metabolites of Brassica-ceae, i.e. glucosinolates (Van Loon et al., 1992; Renwick &Chew, 1994; de Vos et al., 2008). However, chemosensoryinformation is integrated with visual information, as femaleP. rapae butterflies can learn to associate colour with thetaste of glucosinolates (Traynier & Truscott, 1991). The clo-sely related large cabbage white butterfly, Pieris brassicae,learns to associate a difference in green shades with the pres-ence of glucosinolates on artificial substrates (Smallegangeet al., 2006). This study is the first to use living plants inwhich the pigment pattern has been manipulated by disrup-

tion of the function of a single gene, that is, PDS, throughVIGS. We hypothesized that plants with a pigmentation pat-tern deviating from that of wild-type plants would beavoided by the insect in making reproductive decisions. Wedesigned specific experiments to eliminate an effect of poten-tial chemical changes on butterfly behaviour. Furthermore,we tested the performance of the offspring of the butterflieson plants with different pigmentation patterns.

Materials and Methods

Plants and insects

Seeds of Arabidopsis thaliana (L.) Heynh ecotype Col-0were sown and grown at the Laboratory of Entomology(Wageningen University) in a climate chamber at 60–70%relative humidity, 23 ± 1�C, 8 h : 16 h light : dark photo-period under a light intensity of c. 45 lmol m)2 s)1 (lampsused: TLD 58W ⁄ 840HF, type Reflex, Philips, Eindhoven,the Netherlands). Four- to five-leaf seedlings were usedfor VIGS, c. 21–25 d after sowing. Brassica nigra L.CGN06619 and Nicotiana benthamiana Domin were sownand grown at Wageningen University’s Unifarm facility(50–70% relative humidity, 20–30�C, 16 h : 8 h light : -dark photoperiod) for 1 and 4 wk, respectively, before theywere transferred to a climate chamber. Four- to five-leafseedlings of N. benthamiana and seedlings of B. nigra inthe cotyledon stage were used for VIGS in a climate cham-ber at 60–70% relative humidity, 23 ± 1�C, 16 h : 8 hlight : dark photoperiod (c. 45 lmol m)2 s)1; lamps used:Philips, TLD 58W ⁄ 840HF, type Reflex). Arabidopsis andBrassica plants 21 d post-infiltration with TRV were usedin bioassays.

Pieris rapae L. (small cabbage white butterfly) were rou-tinely reared at the Laboratory of Entomology (WageningenUniversity) on cabbage plants (B. oleracea var. gemmifera cvCyrus) in a climatically controlled room at 21 ± 1�C, 50–70% relative humidity, 16 h light : 8 h dark (natural day-light supplemented with fluorescent strip lights, PhilipsTLD 58 ⁄ 840HF).

Isolation of B. oleracea full-length phytoenedesaturase (BoPDS) cDNA

A fragment of the B. oleracea PDS (BoPDS) gene, 643 bplong, was obtained by reverse transcriptase-polymerasechain reaction (RT-PCR) with B. oleracea var. gemmifera cvCyrus cDNA as template and the primer pair 5¢-ATGGT-TGTGTTTGGGAATGTTTC-3¢ and 5¢-TCTCTGGCC-ATGTCAGCATCTCG-3¢. The primer pair was designedon the basis of the A. thaliana PDS gene accessionNM_117498 (At4g14210) and its sequence similarity withthe B. napus expressed sequence tag sequence (CD827969).Total RNA isolation, cDNA synthesis and RT-PCR were

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performed according to the methods described previously(Zheng et al., 2007). The purification, ligation intopGEM-T Easy (Promega Corp., Madison, WI, USA) andbacterial transformation of the PCR products were per-formed according to the manufacturer’s protocol. Ampicil-lin-resistant colonies were identified and plasmid DNA wasisolated using a miniprep kit (Qiagen, Venlo, the Nether-lands) according to the manufacturer’s instructions. Plas-mids were selected which showed inserts of the expectedsize after digestion with EcoRI. Expected inserts based onsize were used for sequencing. Sequencing was performed inan ABI PRISM 310 automated DNA sequencer (PerkinElmer, Foster City, CA, USA).

A full-length BoPDS cDNA was obtained by rapid ampli-fication of cDNA ends (RACE) PCR. Cloning of the 5¢ and3¢ ends of BoPDS cDNA was accomplished by the BDSMART RACE cDNA Amplification Kit (BD BioscienceClontech, Palo Alto, CA, USA) following the manufac-turer’s protocol. 3¢-RACE-Ready cDNA and 5¢-RACE-Ready cDNA synthesis, PCR and touchdown PCR wereused as described previously (Zheng et al., 2007). BoPDSgene-specific primers (forward primer, 5¢-AACAACGA-GATGCTGACATGGCCAGAG-3¢; reverse primer, 5¢-TTCTCTGGCCATGTCAGCATCTCGTTG-3¢) weredesigned on the basis of the above-mentioned partialsequence. The 5¢- and 3¢-RACE products were purified,ligated and sequenced as explained above. The full nucleo-tide sequence of BoPDS has been deposited in GenBank(EU719210).

Construction of TRV plasmids

The pTRV1 (pYL192) and pTRV2 (pYL156) vectorsdescribed previously (Liu et al., 2002) were used in thisstudy. To generate VIGS plasmids, two cDNA fragmentswere amplified by PCR using the above-mentioned B.oleracea var. gemmifera cv Cyrus cDNA and primers F1 (5¢-TCAGAATTCCGATCTCTTCACAAGCGC-3¢) and R1(5¢-TCTCTGGCCATGTCAGCATCTCGTTG-3¢), andF2 (5¢-CAACGAGATGCTGACATGGCCAGAGA-3¢)and R2 (5¢-GCGCCTTCCATGGAAGCTAAGTACTT-CTG-3¢), respectively. The resulting PCR products, 552and 969 bp in length, were cloned into EcoRI-cut pTRV2and generated sense VIGS constructs pTRV2-BoPDS1 andpTRV2-BoPDS2, respectively (Fig. 1). The pYL156 recom-binant constructs obtained were confirmed by PCR usinginsert-specific primers (F1 and R1 or F2 and R2) and vec-tor-specific primers 156F (5¢-GGTCAAGGTACGTAGT-AGAG-3¢) and 156R (5¢-CGAGAATGTCAATCTCG-TAGG-3¢), which span the multiple cloning site in TRV2(Hileman et al., 2005). Both constructs were then purifiedand the identity of the final constructs was verified bysequencing with the 156F and 156R primers. Recombinantconstructs were electroporated into competent cells of

Agrobacterium tumefaciens strain AGL0 as described previ-ously (Zheng et al., 2005) by a microPulser� (Bio-Rad,Hercules, CA, USA). Colonies were screened by PCR usingprimers 156F and 156R for the presence of pTRV2-BoP-DS1 or pTRV2-BoPDS2. Glycerol stocks were made fromsingle positive transformants.

Preparation of Agrobacterium inoculation

Cultures of A. tumefaciens strain AGL0 containing pTRV1,pTRV2 and pTRV2-BoPDS1 or pTRV2-BoPDS2 plasmidwere cultured separately in 20 ml Luria–Bertani mediumcontaining 50 lg ml)1 kanamycin and 50 lg ml)1 rifam-picin, and shaken at 28�C overnight. After centrifugation at2880 g for 15 min, bacterial cell pellets were resuspendedin infiltration buffer containing 1 mM Mes (pH 5.8),10 mM MgCl2 and 100 lM acetosyringone to an opticaldensity at 600 nm (OD600) = 2, and allowed to stand atroom temperature for 2 h before infiltration. Suspensionsof A. tumefaciens carrying pTRV1 and pTRV2-BoPDS1 orpTRV2-BoPDS2 were mixed 1 : 1 and infiltrated into theunderside of the upper leaves of 3–4-wk-old Arabidopsisplants, 4-wk-old N. benthamiana plants or 1-wk-old plantsof B. nigra (the latter only carrying cotyledons) using a2 ml syringe. Suspensions of A. tumefaciens carryingpTRV1 and pTRV2 were also mixed 1 : 1 and infiltratedin the same way as above as an empty vector control(referred to as EV control below).

Generation of PDS-silenced plants in three differentplant species

Agroinfiltration with VIGS constructs pTRV2-BoPDS1and pTRV2-BoPDS2, respectively, resulted in successfulphotobleaching in all three plant species studied. Eitherinsert fragment BoPDS1 or BoPDS2 was sufficient to inducegene silencing in different species to produce photobleach-ing pds phenotypes. However, the timing and extent ofphotobleaching varied among plant species. Nicotianabenthamiana plants developed photobleaching symptoms5 d after infiltration, whereas A. thaliana and B. napusshowed symptoms 7–10 d after infiltration. We furtherexamined whether growing conditions and plant stages forA. thaliana affected the expression of VIGS. We analysedthe number of plants showing a pds phenotype after growthunder long-day (16 h : 8 h light : dark photoperiod) vsshort-day (8 h : 16 h light : dark photoperiod) conditions.Under both long-day and short-day conditions, 80–100%of the plants displayed photobleaching in 10 independentexperiments. After testing differently aged seedlings, wefound that the silencing of AtPDS was equally effective inseedlings inoculated at the two- to three-leaf stage, four- tofive-leaf stage and six- to seven-leaf stage (data not shown).Therefore, four- to five-leaf stage seedlings were used in

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later oviposition experiments under the same conditions asused for the rearing of A. thaliana plants. After agroinfiltra-tion, B. nigra and N. benthamiana plants were also grownunder the same conditions as used for the rearing of thestock plants.

Real-time quantitative PCR

To quantify endogenous PDS transcript levels in PDS-silenced and EV control plants, real-time quantitative PCRwas performed. Leaf samples for real-time quantitativePCRs were harvested from individual PDS-silenced and EVcontrol Arabidopsis plants. Total RNA extraction, purifica-tion and cDNA synthesis and PCRs were the same asdescribed previously (Zheng et al., 2007). Briefly, a 25 llPCR was prepared containing 1 ll of template cDNA, 1 llof forward primer (10 lM), 1 ll of reverse primer(10 lM), 9.5 ll of water and 12.5 ll of 2· iQ�SYBRgreen Supermix (Bio-Rad). The final amount of cDNAtemplate assayed was equivalent to 10 ng of RNA. All sam-ples were amplified in duplicate assays under the followingconditions: 95�C for 3 min for one cycle, followed by 40cycles of 95�C for 15 s and 56�C for 45 s, with data collec-tion at 56�C. The PCR products for each primer set werealso subjected to melt curve analysis. Melt curve analysisensured that the resulting fluorescence originated from a

single PCR product and did not represent primer dimersformed during PCR or nonspecific product. No-templatecontrols, as water and minus RT (10 ng of RNA), were alsoincluded to detect any spurious signals arising from theamplification of any DNA contamination or primer dimerformed during the reaction. The gene-specific primers ofAtPDS (gene accession NM_202816) and AtGAPDH (geneaccession M64116 as reference; Shih et al., 1991) weredesigned with Beacon Designer software (Premier BiosoftInternational, Palo Alto, CA, USA) set to an annealing tem-perature of 56�C. AtPDS primers were F-AtPDS (5¢-CAAT-GACGAT-GGCACGGTTAAGAG-3¢) and R-AtPDS (5¢-CTGGAGC-GGCAAACACATAAGC-3¢). The predictedlength was 83 bp. The AtPDS primers corresponded toregions in the plant mRNAs that were absent from the virusvectors. AtGAPDH primers were F-AtGAPDH (5¢-AAT-GAAGGACTGGAGAGGTGGAAG-3¢) and R-AtGAPDH(5¢-ACGGTTGGGAC-ACGGAAAGAC-3¢). The predictedlength was 138 bp. These primer sequences were blastedagainst the National Center for Biotechnology Informationnucleotide and expressed sequence tag database to ascertainthat they were not homologous to other genes. The quanti-fication of gene expression was performed with a Rotor-gene� 6000 (Corbett, Sydney, Australia) machine. For therelative quantification of gene transcript abundances inPDS-silenced and EV control plants, a standard curve

(a)

(b)

(c)

(d)

(e)

Fig. 1 Tobacco rattle virus (TRV)-basedvirus-induced gene silencing (VIGS) con-structs used in this study. (a) Map of thephytoene desaturase (PDS) gene fromcabbage (Brassica oleracea) BoPDS. The lightblue and dark blue areas are the 552 and969 bp fragments cloned into TRV2 togenerate pTRV2-BoPDS1 andpTRV2-BoPDS2, respectively. The primers(F1 and R1 vs F2 and R2) used foramplification are indicated below. Theasterisk represents the stop codon. 3¢-UTRrepresents the 3¢-untranslated region. (b, c)TRV-based VIGS vector systems. 2 · 35S,duplicated CaMV 35S promoter; NOSt;nopaline synthase terminator; RdRp, RNA-dependent RNA polymerase; 16K, 16 kDacysteine-rich protein; MP, movementprotein; CP, coat protein; LB and RB, left andright borders of T-DNA; Rz, self-cleavingribozyme; MCS, multiple cloning sites. (d)Map of the TRV2-BoPDS1 construct used forVIGS. The light blue area is the 552 bpBoPDS1 fragment. (e) Map of theTRV2-BoPDS2 construct used for VIGS. Thedark blue area is the 969 bp BoPDS2

fragment.

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method was applied according to the manufacturer’s proto-col (Corbett, Australia). AtPDS expression relative to At-GAPDH expression was quantified by comparing thethreshold cycle for each PCR to their respective dilution ser-ies and dividing the resulting quantities.

Butterfly oviposition preference experiments onA. thaliana and B. nigra

Soon after pupation, adult butterflies were transferred intoa small mesh cage (25 · 25 · 35 cm) and supplied with10% sucrose solution in the climate chamber at 60–70%relative humidity, 23 ± 1�C, 16 h : 8 h light : dark photo-period (c. 45 lmol m)2 s)1; lamps used: Philips TLD58W ⁄ 840HF, Reflex) to allow them to mate during 2–4 d.Butterflies that were used in the bioassays had not beenexposed to plants before the experiment and were consid-ered naive. For the experiments, one male and female but-terfly were introduced per oviposition cage (50 · 50 ·50 cm). In these cages, the butterflies were also suppliedwith 10% sucrose solution. In the morning, the differentPDS-silenced and EV control plants of A. thaliana orB. nigra were introduced into the cages, and the butterflieswere allowed to oviposit for 6 or 24 h depending on theexperiment. At the end of each experiment, the numbers ofeggs on the plants were counted. The experiments werecarried out in several cages per day, and this was repeatedover at least 3 d per choice assay. The positions of the plantsin the cages were randomized to avoid positional bias.

Oviposition preference experiment on artificialsubstrate

Green and white paper sheets were sprayed with sinigrin(Bruinsma et al., 2007), a glucosinolate that is a known ovi-position stimulant for P. rapae (Traynier, 1986), to investi-gate whether female P. rapae butterflies discriminatedbetween green and white paper. The green and white papersheets (8 · 12 cm) were pierced and folded onto a T-shaped wire standard. The T-shaped wire (width, 15 cm;height, 19 cm) was positioned on a solid copper socket(Supporting Information Fig. S1). The T-shaped wire wasplaced in a flow cabinet and 1 ml of a 5 mM sinigrin solu-tion was evenly distributed over the upper side of the paperwith a chromatographic sprayer that produces a fine mist(Desaga, Heidelberg, Germany). Sinigrin is not a volatile,but a glucoside that has been amply demonstrated to bedetected by gustatory receptors on the butterfly tarsi afterlanding, rendering paper acceptable for oviposition byP. rapae butterflies (Stadler et al., 1995). The two standardswith green and white paper sheets were placed next to eachother at a distance of 20 cm in the oviposition cage(50 · 50 · 50 cm). In each cage, four female butterflies,each with a colour mark for individual identification on the

underside of their hind wings (e.g. black, blue, green andred), were released. The landings by each marked butterflywere recorded for 2 h. After recording the landing frequen-cies, the butterflies were left for another 4 h, and the eggsdeposited during this time interval were counted on bothsides of the paper in each experiment. The experiments werecarried out in two to three cages per day, and each experi-ment consisted of a total of nine independent replicates.The position of the paper sheets in the cages was random-ized to avoid positional bias.

Butterfly first landing choice on EV green vsPDS-silenced green-whitish plants

The first landing response of P. rapae butterflies was investi-gated in a two-choice test. This experiment was set up toinvestigate the response of the butterflies before they couldcontact the plant, so as to eliminate an effect of contactchemicals. Pairs of 3-wk-old PDS-silenced and EV A. thalianaplants were used to investigate whether P. rapae butterfliesdiscriminated between optically different plants during hostplant selection for oviposition on freely accessible plants.The pot size was such that large parts of the rosette leaveswere presented against the dark background of the pottingsoil. Five female butterflies, marked with different coloursto allow individual recognition, were released in a cage(50 · 50 · 50 cm) with one pair of distinct test plants.The first choice of each butterfly on a green or green-whitish plant was recorded. After a butterfly had depositedan egg, both butterfly and egg were discarded (a videorecording of butterfly choice behaviour is available as Sup-porting Information Movie S1). Another marked butterflywas subsequently introduced into the cage. After five inde-pendent first choices in response to one pair of plants, theplant pair was replaced by a new pair, switching positionsbetween the treated and control plants to prevent potentialpositional bias. On each day, five pairs of plants were tested,and the experiment was repeated on four separate dates toaccumulate data on 100 landing butterflies.

In order to rule out any metabolic changes potentiallyoccurring in PDS-silenced green-whitish plants that mightinfluence butterfly preference behaviour through olfactorycues, additional experiments were carried out with plantsenclosed in air-tight sealed transparent boxes. PDS-silencedand EV plants of A. thaliana were individually placed intransparent cylinders (diameter, 12 cm; height, 6 cm). Eachcylinder (one with a PDS-silenced plant and one with anEV plant) was covered with a Petri dish (145 mm ·20 mm) which was tightly sealed with Parafilm just beforethe two cylinders were placed into the cage. The release ofthe butterfly was performed in the same way as before. Thebutterfly’s first choice was recorded when it landed eitheron the Petri dish cover of the container with the PDS-silenced plant or that of the EV plant. The experiment was






repeated on four separate days to obtain data on the firstlanding of 60 butterflies. The positions of the plants in thecages were randomized to avoid positional bias.

Pieris rapae larval performance

Three-week-old PDS-silenced and EV plants of A. thalianawere offered to P. rapae butterflies to oviposit five eggs perplant. If butterflies laid more than five eggs on the plant,the surplus eggs were removed. The eggs all hatched 4 dafter oviposition on the same day on both plant types. Thefresh body mass of individual larvae from 18 plant pairs wasdetermined 8 d after oviposition using a Sartorius balance(accuracy, 0.1 mg; Gottingen, Germany).

Leaf reflectance measurements

Leaf reflectance was measured using an improved version ofthe system described by Soares et al. (2008). In our system,the USB-2000 spectroradiometers were replaced by USB-4000 spectroradiometers (Ocean Optics, Dunedin, FL,USA) with custom-enlarged slit width, which were cooledto 5�C in order to increase the signal strength and stability.Transmittance measurements are not shown. Two bluelight-emitting diodes (405 and 435 nm peak wavelength)were added to both laboratory-built, stabilized, quartz-halo-gen measuring light sources to increase the signal in the bluepart of the spectrum. Three plants from each type (i.e. EVcontrol, PDS-silenced and wild-type plants) were used forreflectance measurements. Reflectance was measured on atleast five leaf discs per plant, cut from visually healthy,

young mature leaves. For the PDS-silenced plants, separatesamples were taken from the green and from the whitish,chlorophyll-deficient leaf zones (five samples per tissue typeper plant). Reflectance was determined per nanometre inthe range 400–800 nm.

Statistical analysis

Each individual butterfly female was subjected to a dual-choice situation, in which individual butterflies were allowedto oviposit on PDS-silenced and EV plants. The data wereanalysed using the Wilcoxon matched-pair signed-ranks testemploying SPSS 15.0 for Windows. Butterfly first choices inassays offering EV plants and PDS-silenced plants, boththose freely accessible and those enclosed in air-tight trans-parent containers, were analysed by a binomial test. Theperformance of caterpillars of P. rapae on EV plants andPDS-silenced plants was analysed by Student’s t-test. Theresults for the experiments on freely accessible plants andenclosed plants were analysed using a chi-squared test.

Results

Silencing of endogenous PDS gene in A. thaliana,B. nigra and N. benthamiana plants via itsheterologous gene sequence BoPDS from B. oleracea

In the first step, we generated plants in which pigmentexpression was modified by targeting the gene encoding forphytoene desaturase (PDS), a key enzyme in the biosynthe-sis of carotenoids, pigments that prevent photobleaching

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700 800

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Whitish parts ofPDS-silenced plant

Green parts ofPDS-silenced plant

Empty vectorcontrol

Fig. 2 Silencing of the phytoene desaturase(PDS) gene from cabbage (Brassica oleracea)BoPDS using tobacco rattle virus (TRV)-based virus-induced gene silencing (VIGS) inArabidopsis Col-0 plants. (a) Wild-type (left),empty vector negative control (middle) andPDS-silenced plant (right) agroinfiltrated withpTRV1 and pTRV2-BoPDS1. (b) Overview ofPDS-silenced plants. (c) Reflectance spectraof the three different plant types and leafparts of plants. Note that the curves for theempty vector control and the green leaf partsof the PDS-silenced plants are almost indis-tinguishable.

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(Ratcliff et al., 2001; Liu et al., 2002). The silencing ofPDS, and thus the absence of the gene product, resulted in atypical green-whitish variegated pds phenotype. We used theB. oleracea PDS (BoPDS) gene sequence (EU719210) toobtain VIGS constructs via TRV (Fig. 1c–e) and efficientlysilenced plants of three different species, that is A. thaliana(Fig. 2a,b), N. benthamiana (Fig. 3c–e) and B. nigra(Fig. 3h). The pds-silenced leaves in A. thaliana andN. benthamiana plants are completely white along the vas-cular areas of the veins. This pattern differs from thatreported for the im mutant in A. thaliana where most edgeareas of the leaves are white, whereas the vascular areas ofthe veins are still green (Aluru et al., 2006). However, inB. nigra, only a narrow area bordering the veins appearedbleached. EV control plants, which were agroinfiltrated withTRV1 and TRV2 vectors only (Liu et al., 2002), did notshow any photobleaching symptoms and were indistin-guishable from wild-type, noninfiltrated plants (Figs 2a,3b,g), as confirmed by reflectance spectra in the 400–800 nm range (Fig. 2c). However, a dramatic difference inreflectance was observed in the whitish parts of PDS-silenced plants compared with EV control and wild-typeplants (Fig. 2c). All three plant species studied developedphotobleaching symptoms 5–15 d after agroinfiltrationwith TRV1 and TRV2-BoPDS1 or TRV1 and TRV2-BoP-DS2 (Figs 2, 3). These results demonstrate that a heterolo-gous gene sequence from B. oleracea can be used to silenceits orthologues in A. thaliana, B. nigra and N. benthami-ana. We included the Nicotiana plants here because theyshow that the PDS sequence from B. oleracea can evensilence the PDS gene in a nonbrassicaceous plant. However,the timing and extent of photobleaching varied among plant

species. Nicotiana benthamiana plants developed photoble-aching symptoms 5 d after infiltration, whereas A. thalianaand B. napus showed symptoms 10 d after infiltration.

We have shown the expected pds phenotype in all threeplant species (Figs 2, 3). For A. thaliana, we supported thisevidence with quantitative PCR data to demonstrate thatthe PDS transcript level was affected. Silencing the PDSgene in A. thaliana plants reduced significantly the abun-dance of transcripts of AtPDS (6.7-fold compared with thecontrol) (Fig. 4).

We compared the numbers of A. thaliana plants show-ing the pds phenotype when growing under long-day(16 h : 8 h light : dark photoperiod) and short-day (8 h :16 h light : dark photoperiod) conditions. In both long-day and short-day conditions, 80–100% of the plantsdisplayed photobleaching in 10 separate experiments. Fur-thermore, silencing of AtPDS was equally effective in seed-lings inoculated at the two- to three-leaf stage, four- tofive-leaf stage or six- to seven-leaf stage (data not shown).Therefore, four- to five-leaf stage seedlings were used inlater oviposition experiments under short-day conditions(8 h : 16 h light : dark photoperiod) because, under long-day conditions, A. thaliana plants initiate flowering early.Both B. nigra and N. benthamiana plants were grownunder long-day conditions (16 h : 8 h light : dark photo-period) after agroinfiltration.

Effects of PDS silencing on oviposition behaviour byP. rapae butterflies

We investigated the effects of modified leaf pigmentationon the oviposition behaviour of P. rapae butterflies in

(a) (b) (c) (d)

(e) (f) (g) (h)

Fig. 3 Silencing of the phytoene desaturase (PDS) gene using tobacco rattle virus (TRV)-based virus-induced gene silencing (VIGS) inNicotiana benthamiana and Brassica nigra. (a–e) Nicotiana benthamiana: (a) wild-type; (b) empty vector (EV) control plant; (c, d)PDS-silenced plants of N. benthamiana agroinfiltrated with pTRV1 and pTRV2-BoPDS1; (e) PDS-silenced N. benthamiana plantsagroinfiltrated with pTRV1 and pTRV2-BoPDS1 (top two plants), and pTRV1 and pTRV2-BoPDS2 (bottom two plants). (f–h) Brassica nigra:(f) wild-type; (g) EV control; (h) PDS-silenced plant of B. nigra agroinfiltrated with pTRV1 and pTRV2-BoPDS1.






A. thaliana and B. nigra plants. Three-week-old PDS-silenced plants and EV control plants of A. thaliana orB. nigra were offered to P. rapae butterflies in dual-choicebioassays. During bioassays lasting either 6 or 24 h, thenaive butterflies significantly preferred to oviposit on EVplants rather than PDS-silenced plants (A. thaliana inFig. 5a, n = 23, P < 0.001; Fig. 5b, n = 24, P < 0.001;B. nigra in Fig. 5c, n = 14, P < 0.05). These results might

also be explained by possible pleiotropic effects of pigmentalteration on the metabolic pathways of silenced plants,because Pieris butterflies use the presence of secondarymetabolites, such as glucosinolates or their volatile break-down products, as stimuli for oviposition (Van Loon et al.,1992; Loivamaki et al., 2008; de Vos et al., 2008). There-fore, to assess the possible role of contact cues, of either gus-tatory or tactile nature, we recorded whether naivebutterflies landed first on EV plants or on PDS-silencedplants. We observed that as many as 81% of the first land-ings were made on EV Arabidopsis plants (Fig. 6a, n = 100,P < 0.001; see Movie S1 for a video recording of butterflybehaviour in this dual-choice bioassay). Furthermore, to testthe possible effect of differences in plant volatile produc-tion, we offered both EV and PDS-silenced plants in air-tight sealed transparent containers. In this bioassay, 90% ofthe first landings were on the contained EV Arabidopsisplants (Fig. 6b, n = 100, P < 0.001). The results for land-ing behaviour in the experiments on freely accessible plants(Fig. 6a) and enclosed plants (Fig. 6b) were similar(v2 = 2.304, P > 0.05), thus clearly demonstrating that theobserved preference for EV plants was not influenced by apossible difference in plant volatiles. We have not per-formed experiments with B. nigra in containers because the

Fig. 4 Effect of agroinfiltration of Arabidopsis plants with specificconstructs on the abundance of AtPDS transcripts as quantified byquantitative RT-PCR (empty vector (EV) control plants were treatedwith pTRV1 and pTRV2, whereas PDS-silenced plants were treatedwith pTRV1 and pTRV2-BoPDS1; mean ± SE, n = 3).

(a)

(c)

(b)

Fig. 5 Oviposition (number of eggs perplant) by Pieris rapae butterflies in two-choice assays on empty vector (EV) controlplants (closed boxes) and PDS-silenced plants(open boxes) after different exposure dura-tions. (a) Arabidopsis thaliana exposed toP. rapae oviposition during 6 h (total numberof eggs laid: 672 on 23 pairs of plants). (b)Arabidopsis thaliana exposed to P. rapaeoviposition during 24 h (total number of eggslaid: 1642 on 24 pairs of plants). (c) Brassica

nigra exposed to P. rapae oviposition during6 h (total number of eggs laid: 593 on 14pairs of plants). The boxes span the first tothird quartile range; the median is indicatedby a line across the box. Outliers are repre-sented by dots. Asterisks indicate significantdifferences between oviposition responses toEV and PDS-silenced plants (*P < 0.05;***P < 0.001; Wilcoxon matched-pairsigned-ranks test).

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A. thaliana rosette plants were much more amenable forsuch experiments than the B. nigra plants. All previousresults demonstrate that the discrimination by butterflies isbased on visual cues only. These results allow the unambig-uous conclusion that Pieris butterflies possess an innatevisual ability to discriminate green from green–white varie-gated plants. This is the first time that this ability has beendemonstrated in response to living plant material by silenc-ing only one functional gene. Our conclusion is supportedby previous studies employing coloured papers (Traynier,1986) and our own experiments with such papers (Figs S1,S2). Our present experiments with PDS-silenced plantsallow the rigorous conclusion that this ability extends to thevariable colour patterning of living plant leaves.

Performance of Pieris caterpillars on PDS-silencedgreen-whitish plants

We further investigated how the silencing of PDS in A.thaliana influences the performance of Pieris caterpillars thathatch from the eggs. We predicted that caterpillars wouldperform less well on PDS-silenced than on EV A. thalianaplants. To test this hypothesis, we offered 3-wk-old PDS-silenced and EV A. thaliana plants to P. rapae butterflies tooviposit five eggs per plant. On both plant types, the eggsall hatched 4 d after oviposition. After another 4 d of feed-ing, the fresh body mass of individual larvae from 18 plantpairs was determined. Surprisingly, hatched larvae only fedon the green parts of pds-silenced plants and not on thewhite parts of the plants (Fig. 7b). Indeed, caterpillar per-formance was found to be reduced significantly on PDS-silenced plants compared with EV control plants. Pierisrapae larvae grew much more slowly on PDS-silenced com-pared with EV Arabidopsis plants (Fig. 7c; Student’s t-test,F = 11.55, P < 0.001). The reduction in caterpillar growth

by 20% over the first 4 d will probably become larger dur-ing further development, as a caterpillar’s development isimportant for performance later on (Gols et al., 2008).

Discussion

The VIGS vectors that were originally developed in themodel species N. benthamiana (Ratcliff et al., 2001; Burch-Smith et al., 2004 Goodin et al., 2008;) have been usedsuccessfully to decipher the functions of several plant genesin this species, which yields high infection rates, and inother solanaceous species such as tomato (Liu et al.,2002;Bhattarai et al., 2007; Kandoth et al., 2007; Hartlet al., 2008; Shao et al., 2008; Wu et al., 2008). Recently,Senthil-Kumar et al. (2007) have shown that TRV-medi-ated VIGS can be performed in a wide range of solanaceousplant species, and that heterologous gene sequences fromdistantly related plant species can be used to silence theirrespective orthologues in the VIGS-efficient model plantN. benthamiana (Goodin et al., 2008). However, the use ofVIGS in high-throughput functional genomics has thus farbeen limited, mostly as a result of the difficulties of intro-ducing the VIGS vector into plant species other than sola-naceous species. These limitations also exist for the modelplant of molecular genetics, A. thaliana, although opti-mized procedures for the efficiency of agroinoculation ofthis species have been reported recently (Burch-Smith et al.,2006; Wang et al., 2006; Pflieger et al., 2008). However, itwas unknown to date whether heterologous gene sequencesfrom other plant species can be used to silence their respec-tive orthologues in VIGS-refractory plant species, such asB. nigra. In A. thaliana, only seedlings in the two- to three-leaf stage grown under long-day conditions have beenshown previously to display the photobleaching phenotype(Burch-Smith et al., 2006). Here, we report an efficient

Fig. 6 First landing choice by Pieris rapae butterflies in a dual-choice bioassay on empty vector (EV) control and PDS-silenced Arabidopsisplants. (a) Plants exposed to butterfly landing (total number of butterflies observed to land was 100). (b) Plants were offered as optical stimulito butterflies enclosed in air-tight sealed transparent boxes (total number of butterflies observed to land was 60; ***P < 0.001, binomial test).There is no significant difference (ns) between experiment (a) and experiment (b) (v2 = 2.304, P > 0.05), which shows that volatiles played nosignificant role in butterfly landing preference.






protocol for silencing the PDS gene in A. thaliana plantsunder both long-day (16 h light) and short-day (8 h light)conditions for plants in the two- to seven-leaf stages. Fur-thermore, our study shows that heterologous gene sequencesfrom B. oleracea can not only silence its orthologues in therelated brassicaceous plants, such as B. nigra and A. thali-ana, but also in the nonrelated solanaceous model speciesN. benthamiana. In this study, we have exploited theunique opportunities that VIGS provides to investigate therole of a functional PDS in interactions of plants with her-bivorous insects. By employing VIGS, we have unambigu-ously shown that Pieris butterflies discriminate betweengreen plants and green–white variegated conspecifics on thebasis of visual cues only. Although chemical cues are veryimportant for foraging Lepidoptera in general, and forPieris spp. in particular (Chew & Renwick, 1995; Schoon-hoven et al., 2005), a potential role for such cues has beenruled out by our experiments in which the butterflies dis-played the same preference for EV plants over PDS-silencedplants when the plants were enclosed in air-tight containers.To the best of our knowledge, this is the first time that pig-ment-manipulated living plants have been used to study theeffects of variegated plant tissues on insect behaviour. Sofar, studies on the role of vision in host plant selection foroviposition by insects have employed the use of nonlivingsubstrates (paper) and dyes. However, the spectral charac-teristics and patterns of artificial substrates never matchexactly plant pigmentation patterns, as has also been shownexperimentally for coloured papers vs Brassica plants (Smal-legange et al., 2006). In the present study, we have effec-tively silenced one gene, i.e. PDS, and investigated theeffect of this on butterfly behaviour. PDS is a crucial genein carotenoid biosynthesis and thereby in the harvestingof light, the peroxidation of lipids, hormone synthesisand assembly of the photosystem. All of these functions ofPDS have received ample attention in previous studies

(Grotewold, 2006). However, one more phenotypical com-ponent that is influenced by PDS is the plant’s visual char-acteristics to animals, such as butterflies. It is known thatthe inhibition of PDS in the im mutant of A. thalianaresults in white–green variegated plants (Wetzel et al.,1994; Aluru et al., 2009) and white–green variegated formsare also known for many other plant species (Evenari, 1989;Lev-Yadun, 2006). It is interesting to note that a recentstudy has shown that, in the araceous plant Caladium steu-dneriifolium, the degree of variegation, as artificially manip-ulated with white Tipp-Ex� correction fluid, is negativelycorrelated with caterpillar damage (Soltau et al., 2009). Theauthors have suggested that the white–green variegatedplants deter ovipositing moths by mimicry of miner infesta-tion (Soltau et al., 2009). Results supporting this suggestionhave also been reported in a nonmanipulative study withnaturally occurring Hydrophyllum virginianum (Campitelliet al., 2008). Thus, variegation might be beneficial forplants despite the implicated loss of photosyntheticallyactive surface. It remains to be investigated what are the netfitness consequences, and this is likely to depend on localconditions, especially related to herbivore abundance.Indeed, in nature, natural variation in the degree of variega-tion was found in C. steudneriifolium (Soltau et al., 2009),and the role of plant colour variegation in plant anti-herbi-vore defence has been hypothesized previously (Lev-Yadun,2006).

Pieris butterflies probably have an innate preference forgreen because most of their host plants are green (Snell-Rood & Papaj, 2009). Moreover, the green-whitish varie-gated optical appearance of the PDS-silenced plants mimicsthe generally observed yellow-whitish symptoms of virusinfection or the patterns of leafminer infestation. The dataof our larval growth experiment show that the PDS-silencedplants are nutritionally inferior: caterpillars grow morerapidly on EV plants than on PDS-silenced plants, and thus

(a)

(b)

(c)

Fig. 7 Performance of Pieris rapae

caterpillars on empty vector (EV) controlgreen and green-whitish PDS-silenced plants.(a) Arabidopsis plants before butterflyoviposition. (b) Plants 8 d after oviposition(left, control plants; right, silenced pdsplants). (c) Fresh body mass of caterpillars onthe two types of plant (18 plants were usedfor each plant type; mean ± SE;***P < 0.001, Student’s t-test).

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butterfly preference is positively correlated with offspringperformance. It is important to note that, although not dis-playing symptoms that are optically detectable, EV plantsare infected with TRV. One probable cause of the nutri-tional inferiority of PDS-silenced plants is that the abun-dance of transcripts of AtPDS is reduced significantly inPDS-silenced A. thaliana plants (Fig. 4). Low-carotenoidplants are likely to have many chemical differences fromcontrol plants. The absence of carotenoids obviously leadsto oxidation of chlorophyll and probably other compounds(Aluru et al., 2006; Fang et al., 2008). This results in alower carbohydrate content which, in turn, may result inchanges in other nutrients (Aluru et al., 2006). However, inour oviposition experiments on enclosed plants, we explic-itly excluded the possible effects of such chemical changeson butterfly behaviour: here only visual stimuli could beused by the butterflies. Carotenoid deficiency of plants islikely to have an impact on the visual capacity of insectsdeveloping on such plants. Rhodopsins are crucial visualpigment molecules in insect eyes, composed of an opsinmoiety and a retinal-based chromophore (Stavenga & Arik-awa, 2006). The biosynthetic precursors of 11-cis-retinaland 11-cis-3-hydroxyretinal are plant-derived carotene andxanthophylls (Seki & Vogt, 1998).

In this study, we have exploited, for the first time, amolecular genetic approach to investigate the role of visionin insect–plant interactions; this was performed by generat-ing plants that are genetically identical, except for the func-tional expression of one gene, leading to a leaf colourpattern difference. EV plants may differ in more character-istics than only colour from PDS-silenced plants. However,in the current study, we were able to specifically address theoptical aspects and eliminated other aspects through theexperimental set-ups used. Our behavioural data show thatonly the colour change associated with PDS silencing influ-enced P. rapae’s host plant selection behaviour. The PDSgene has been mainly used previously as a marker of success-ful silencing. Mutation of PDS in rice leads to preharvestsprouting and photo-oxidation because of its influence onthe carotenoid precursors of ABA biosynthesis (Fang et al.,2008). PDS is also differentially regulated by photo-oxida-tive stress and developmental mechanisms that controlcarotenoid biosynthesis in leaves, flowers and fruits intomato (Giuliano et al., 1993). However, the effect of mod-ified PDS expression on plant–animal interactions has neverbeen investigated.

It is interesting to note that neonate P. rapae larvae onlyfed on the green parts of pds-silenced plants and avoidedfeeding on the white leaf parts. The white sectors of im phe-notypes of A. thaliana accumulate low levels of sucrose incontrast with the green sectors (Aluru et al., 2006). Tran-scriptome studies have suggested that energy is deprived viaaerobic and anaerobic metabolism of imported sugar forgrowth and development in im white leaf sectors, and that

oxidative stress responses are induced in these sectors (Aluruet al., 2009). These and other effects of defective carotenoidbiosynthesis may explain why the caterpillars avoid feedingon the white parts of the variegated plants through nutri-tional feedbacks. Thus, elimination of the functionalexpression of this particular gene not only affects animalbehaviour through its effect on the production of visualcues, but also affects feeding site selection by the herbivo-rous stages. These data relate to laboratory studies and can-not yet be extrapolated to the field. However, it isinteresting to see that field studies support our conclusionthat variegated plants receive less herbivory than greenplants (Soltau et al., 2009). Our approach allows the studyof putative adaptive shifts in host plant selection and feed-ing behaviour as affected by plant carotenoids.

Although Arabidopsis can be used as a model plant spe-cies to study plant–insect interactions (Kappers et al.,2005), it is important to point out that our protocol can beapplied to other, nonmodel, cruciferous species, such asB. nigra, which is a close relative of other economicallyimportant crops, such as B. oleracea and B. napus. In addi-tion, the variegated phenotype of B. nigra PDS-silencedplants is subtle compared with that of A. thaliana (Figs 2b,3h). It is indeed highly remarkable that the butterflies sig-nificantly avoided pds-silenced B. nigra plants (Fig. 5c).These findings imply that TRV-based VIGS holds promiseas a powerful tool for molecular and behavioural ecology ofplant–insect interactions for which mutant plants are notavailable. Quantitative variation in character expression isan important subject in ecology, and a next step is thedevelopment of VIGS to allow different levels of silencingof the target gene (Zheng & Dicke, 2008). The use ofVIGS can be further extended to provide unique opticalplant phenotypes by the targeted modification of the carot-enoid pathway, allowing controlled modification of carot-enoid types, levels and their patterns of expression withinplant organs. Understanding how herbivorous insectsexploit visual signals during host plant selection may con-tribute to improved insect pest management (Foster &Harris, 1997).

Acknowledgements

We thank L. Koopman, F. van Aggelen and A. Gidding forinsect rearing and Unifarm of Wageningen University forsupplying Brassica nigra and Nicotiana benthamiana plants.We are grateful to S. P. Dinesh-Kumar (Yale University,USA) for kindly providing the VIGS vectors, D. G. Sta-venga and S. S. Liu for comments and discussion, T. Buk-ovinszky for help with data analysis, H. M. Smid fortechnical assistance, and V. C. F. Dupre, C. E. Jongelingand H. P. M. Thoens for help with the experiments. Thecomments on a previous version of the manuscript by fouranonymous reviewers have been very helpful in improving






the manuscript. This work was supported financially by aVICI grant from the Netherlands Organization for Scien-tific Research, NWO (865.03.002).

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Supporting Information

Additional supporting information may be found in theonline version of this article.

Fig. S1 Green and white paper sheets used for mimickingthe visual cues of plant leaves.

Fig. S2 Landing choice and oviposition by Pieris rapae butter-flies on paper.

Movie S1 Host plant selection behaviour displayed by afemale Pieris rapae butterfly, resulting in a first landing onthe empty vector control (EV) plant (right) offered togetherwith a PDS-silenced plant (left) (4378 KB QuickTimeMovie).

Please note: Wiley-Blackwell are not responsible for thecontent or functionality of any supporting information sup-plied by the authors. Any queries (other than missing mate-rial) should be directed to the New Phytologist CentralOffice.






Disruption of plant carotenoid biosynthesis through virus-induced gene silencing affects oviposition...

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