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Contents lists available at SciVerse ScienceDirect

Journal of Insect Physiology

journal homepage: www.elsevier .com/ locate/ j insphys

RNAi-mediated knockdown of Shade negatively affects ecdysone-20-hydroxylationin the desert locust, Schistocerca gregaria

Elisabeth Marchal 1, Heleen Verlinden, Liesbeth Badisco, Pieter Van Wielendaele, Jozef Vanden Broeck ⇑Molecular Developmental Physiology and Signal Transduction, Department of Animal Physiology and Neurobiology, Zoological Institute, K.U. Leuven, Naamsestraat 59,B-3000 Leuven, Belgium

a r t i c l e i n f o a b s t r a c t

242526272829303132333435

Article history:Received 8 February 2012Received in revised form 21 March 2012Accepted 22 March 2012Available online xxxx

Keywords:Cytochrome P450 enzymeEcdysteroidHormoneCYP6H1RNA interference

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0022-1910/$ - see front matter � 2012 Published byhttp://dx.doi.org/10.1016/j.jinsphys.2012.03.013

⇑ Corresponding author. Tel.: +32 16 323978; fax: +E-mail addresses: elisabeth.marchal@bio.kuleuven

onto.ca (E. Marchal), heleen.verlinden@bio.kuleuvenbadisco@bio.kuleuven.be (L. Badisco), pieter.van(P. Van Wielendaele), jozef.vandenbroeck@bio.kuleuv

1 Current address: Department of Cell and Systems B25 Harbord Street, Toronto, Ontario, Canada M5S3G5

Please cite this article in press as: Marchal, E., etSchistocerca gregaria. Journal of Insect Physiolog

A major breakthrough in elucidating the ecdysteroid biosynthetic pathway in insects was realized withthe molecular identification and further functional characterization of the ‘Halloween’ genes. These geneswere found to encode cytochrome P450 enzymes catalysing the final steps of ecdysteroid biosynthesis inthe dipteran, Drosophila melanogaster, and in the Lepidoptera, Manduca sexta and Bombyx mori. A recentreport focused on the identification of Halloween orthologs in the desert locust, Schistocerca gregaria, amember of the hemimetabolous insect order of the Orthoptera. In the present study, an additionalHalloween gene Shade, is identified in the desert locust. In Diptera and Lepidoptera, this gene encodesa 20-hydroxylase, catalysing the conversion of ecdysone (E) to 20-hydroxyecdysone (20E). However, thisenzymatic function has previously been suggested for CYP6H1 in another locust species, the migratorylocust, Locusta migratoria. Using q-RT-PCR, the spatial and temporal transcript profiles of S. gregariaorthologs for Shade as well as CYP6H1 were analysed in last larval stage desert locusts. An RNA interfer-ence (RNAi)-based approach was employed to study whether these genes could possibly encode a func-tional 20-hydroxylase in the desert locust.

� 2012 Published by Elsevier Ltd.

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1. Introduction

Post-embryonic development of insects is highly dependent ontwo types of lipophilic hormones, ecdysteroids and juvenilehormones (JH). The balance between these two insect hormonesdefines the nature of each developmental transition. In immatureinsects, surges in the circulating ecdysteroid titer precede eachmoult. The steroid moulting hormone ecdysone (E) is mainlyproduced by the prothoracic glands (PG, paired endocrine organslocated in the insect’s prothorax) or its homologs (such as dipteranring glands) in larval insects. Following secretion into the haemo-lymph, E is converted in peripheral tissues to the physiologicallymore active form, 20-hydroxyecdysone (20E). This enzymatic20-hydroxylase activity has been associated with mitochondrialor microsomal fractions of the fat body, Malphighian tubules andthe midgut (Johnson and Rees, 1977). Recent discoveries havenow unravelled large parts of the 20E biosynthetic pathway indipteran and lepidopteran insects. A major breakthrough was theidentification and molecular characterization of the ‘Halloween’

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.be (H. Verlinden), liesbeth.wielendaele@bio.kuleuven.been.be (J. Vanden Broeck).iology, University of Toronto,

al. RNAi-mediated knockdowny (2012), http://dx.doi.org/10.1

genes. These genes encode cytochrome P450 enzymes catalysingthe final steps of ecdysteroid biosynthesis in the dipteran,Drosophila melanogaster, and the Lepidoptera, Manduca sexta andBombyx mori. High transcript levels of spook, spookier, phantom, dis-embodied and shadow are found in the PG or prothoracic cells of thering gland in immature insects (Chavez et al., 2000; Niwa et al.,2004; Rewitz et al., 2007; Warren et al., 2002, 2004). The finalhydroxylation from E to the active hormone, 20-hydroxyecdysone(20E), occurs through the action of a 20-hydroxylase encoded byshade (shd, CYP314A1) (Petryk et al., 2003) (SupplementaryFig. A). Unlike the other Halloween genes, transcription of this genemainly takes place in peripheral tissues, such as midgut, epidermis,fat body and Malpighian tubules of immature insect stages (Petryket al., 2003; Rewitz et al., 2006a,b). The description/prediction ofthe Halloween genes in other insect species is mostly based onsequence similarity (Christiaens et al., 2010; Iga and Smagghe,2010; Yamazaki et al., 2011). However, the functional characteriza-tion of this class of genes within the hemimetabolous insects wasonly very recently described; two Halloween genes, spook (spo) andphantom (phm), were discovered in a hemimetabolous insect, thedesert locust Schistocerca gregaria, and an RNAi-based silencingapproach confirmed the role of these genes in the locust’s ecdyster-oid biosynthesis (Marchal et al., 2011a). A similar approach wasused in this study with the aim to identify the functional20-hydroxylase gene in the desert locust, S. gregaria. This orthop-teran swarming pest insect belongs to the family of Acrididae or

of Shade negatively affects ecdysone-20-hydroxylation in the desert locust,016/j.jinsphys.2012.03.013

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grasshoppers but is distinguished by a potentially devastating abil-ity to migrate over large distances and form marching (immaturestages) or flying (adults) insect armies eating everything on theirpath. Because the use of the current control strategies (mainly byapplying huge amounts of non-selective insecticides) causes detri-mental effects on non-target organisms and their habitats, explor-ing new potential targets for selective locust control will be crucial.The search for efficient inhibitors of the Halloween enzymes, cor-rupting normal insect ecdysteroidogenesis may prove to be onepossible way to tackle this issue.

Although no shade sequence information was available inS. gregaria, there were already some reports on a gene encodingan E 20-hydroxylase in another locust species, Locusta migratoria.In a very original study, Winter et al. characterised a cytochromeP450 enzyme, CYP6H1, using two-step affinity chromatographywith Type II (binding to the heme iron) and I (binding in the sub-strate binding site and replacing water as sixth ligand to the hemeiron) ligands (Winter et al., 1999, 2001). The gene encoding thisCYP6H1 was then cloned from the migratory locust’s Malpighiantubules and showed the typical motifs associated with a micro-somal cytochrome P450 enzyme. The expression of Cyp6h1 wasmainly localised in larval Malpighian tubules. It is however unfor-tunate that there exists no biochemical or physiological evidenceunquestionably proving that this CYP6H1 truly is an E 20-hydroxy-lase. Since the S. gregaria ortholog of this CYP-gene was recentlyshown to be differentially expressed in a microarray study compar-ing gregarious and solitarious locusts (Badisco et al., 2011c) andsince recently two other Halloween genes were functionally char-acterized in the desert locust (Marchal et al., 2011a), it was ofinterest to examine CYP6H1 further in this insect species.

The aims of the present study were to study the expression pro-file and to verify the possible ecdysteroidogenic role of CYP6H1 andthe Halloween gene shade in S. gregaria. Therefore, both transcriptswere partially cloned and their tissue distribution was studiedusing real time q-RT-PCR. In addition, RNA interference (RNAi)was employed to downregulate SgCYP6H1 and SgSHD mRNA levelsand the possible effects of this knock down on the ecdysteroid ti-tres were evaluated.

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2. Materials and methods

2.1. Animals

Gregarious desert locusts were reared under crowded condi-tions in large cages (38 � 38 � 38 cm), whereby temperature(32 ± 1 �C), ambient relative humidity (40–60%) and light (photo-period L:D = 13:11) were kept as constant as possible. The animalswere fed daily with dried oat flakes and fresh cabbage ad libitum.After breeding, mature females were able to deposit their eggs inpots filled with slightly humidified sand. Each week, these potswere collected and set in empty cages, where eggs were allowedto hatch into first instar larvae. In the described experiments, fifthlarval locusts were collected at the time of ecdysis to obtain poolsof synchronised animals.

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2.2. Tissue collection and sample extraction

Tissues were micro-dissected in locust ringer solution (1L:150 mM NaCl; 1.7 mM CaCl2; 10 mM KCl; 4.3 mM MgCl2; 4 mMNaHCO3; 90 mM sucrose; 5 mM trehalose; pH 7.2) under a binoc-ular microscope and immediately transferred to RNAlater� reagent(Ambion, Austin, TX, USA) or liquid nitrogen to prevent RNA degra-dation. Tissues were stored at �80 �C until further processing.

The dissected tissues were transferred to reaction tubes con-taining ‘‘green beads’’ (Roche, Indianapolis, IN, USA) and homoge-

Please cite this article in press as: Marchal, E., et al. RNAi-mediated knockdownSchistocerca gregaria. Journal of Insect Physiology (2012), http://dx.doi.org/10.1

nised using a MagNA Lyser instrument (Roche, Indianapolis, IN,USA). Total RNA was subsequently extracted from the tissuehomogenate with the RNeasy Lipid Tissue Kit (Qiagen, Valencia,CA, USA) according to the manufacturer’s instructions. To preventgenomic DNA contamination, an additional DNase treatment(RNase-free DNase set, Qiagen, Valencia, CA, USA) was includedin the protocol. Because of the relatively small size of the PG,RNA from these tissues was extracted using the RNAqueous-MicroKit (Ambion, Austin, TX, USA), followed by the recommendedDNase step. Concentration and quality of the resulting RNA sam-ples were measured with a Nanodrop spectrophotometer (ThermoFisher Scientific Inc., Waltham, MA, USA). cDNA was synthesizedusing Superscript III and random hexamers following the manufac-turer’s protocol (Invitrogen Life Technologies, Carlsbad, CA, USA).This 20 ll reaction was subsequently diluted ten-fold.

2.3. PCR amplification of SgShd and SgCyp6h1

Upon searching the S. gregaria EST database (Badisco et al.,2011a) using LmCYP6H1 as a query, a S. gregaria ortholog wasfound.

Unfortunately, no hits were found for a S. gregaria Shade ortho-log in the S. gregaria EST database. Therefore, all known DNA dat-abases including the available arthropod genomic and ESTdatabases were analysed. Degenerate primers corresponding tohighly conserved short amino acid sequences were designed basedon protein alignments of the already known SHD sequences. Fol-lowing degenerate primers were used: forward 50-GCN GGN ATHGAR ACN YT-30 based on AGIETL and reverse 50-YTT NCC NGGRCA CAT NCK-30 based on RMCPGK. Partial sequences were pickedup using these primers in a temperature-gradient polymerasechain reaction (PCR), using REDTaq� DNA polymerase (Sigma–Al-drich Co., St. Louis, MO, USA) and S. gregaria cDNA as a template.Following thermocycling profile was used: 3 min at 95 �C followedby 25–35 cycles of 30 s at 94 �C (denaturation), 2 min at 55 �C (pri-mer annealing, with an 18 �C gradient) and 3 min at 72 �C (elonga-tion). PCR products were then loaded on a 1.2% agarose gel,separated during a 1 h gel electrophoresis and finally visualisedusing UV. Bands of the expected size were cut out and extractedwith a GenElute™ Gel extraction Kit (Sigma–Aldrich Co., St. Louis,MO, USA). Resulting DNA fragments were subcloned into a pCR4-TOPO vector using the TOPO� TA Cloning Kit (Invitrogen Life Tech-nologies, Carlsbad, CA, USA). DNA sequences were determined bymeans of the ABI PRISM 3130 Genetic Analyzer (Applied Biosys-tems, Foster City, CA, USA) following the protocol outlined in theABI PRISM BigDye Terminator Ready Reaction Cycle SequencingKit (Applied Biosystems, Foster City, CA, USA).

2.4. q-RT-PCR

Making use of the Primer Express software (Applied Biosystems,Foster City, CA, USA), suitable q-RT-PCR primers for referencegenes and target genes were designed. Validation of these primersets was performed by designing relative standard curves for genetranscripts with serial (5�) dilutions of a cDNA sample, while theefficiency of q-RT-PCR and correlation coefficient (R2) were re-corded for each primer pair. The stability of several previously de-fined housekeeping genes (Van Hiel et al., 2009) was tested prior toq-RT-PCR transcript profiling (Table 1a). Optimal housekeepinggenes were selected using the geNorm tool (Vandesompele et al.,2002). All q-RT-PCR reactions were performed using 10 ll fastSYBR� Green, 1 ll Forward and Reverse primer (10 lM), 3 llMilli-Q water and 5 ll cDNA. Every reaction was performed induplicate in 96-well plates on a StepOne Plus System (ABI Prism,Applied Biosystems, Foster City, CA, USA) using following thermo-cycler conditions: 50 �C for 2 min, 95 �C for 10 min, followed by 40

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Table 1Oligonucleotide sequences for primers used in (a) q-RT-PCR (EF1a: elongation factor 1 alpha; GAPDH: glyceraldehyde 3-phosphate dehydrogenase; Rp49: ribosomal factor 49; Sg:Schistocerca gregaria) and (b) dsRNA construct design.

Reference genes F-primer R-primer

(a) q-RT-PCR primer sequencesb-actin 50-AATTACCATTGGTAACGAGCGATT-30 50-TGCTTCCATACCCAGGAATGA-30

CG13220 50-TGTTCAGTTTTGGCTCTGTTCTGA-30 50-ACTGTTCTCCGGCAGAATGC-30

EF1a 50-GATGCTCCAGGCCACAGAGA-30 50-TGCACAGTCGGCCTGTGAT-30

GAPDH 50-GTCTGATGACAACAGTGCAT-30 50-GTCCATCACGCCACAACTTTC-30

Rp49 50-CGCTACAAGAAGCTTAAGAGGTCAT-30 50-CCTACGGCGCACTCTGTTG-30

Tubulin 50-TGACAATGAGGCCATCTATG-30 50-CGCAAAGATGCTGTGATTGA-30

Ubiquitin 50-GACTTTGAGGTGTGGCGTAG-30 50-GGATCACAAACACAGAACGA-30

Target genesSgShd 50-CCGCCGTCATTGACTTCATA-30 50-GTGAGCTCCCAAGCGTGG-30

SgCYP6H1 50-GCGCTCTGTGCTGGTGAAG-30 50-TGCTCATCGCTGGTGTTGTAG-30

(b) Primer sequences for dsRNA construct designRNAi constructsSgShd 50-TAATACGACTCACTATAGGGAGACTAGTGCCTCATGGCGCTC-30 50-TAATACGACTCACTATAGGGAGATGAGGAGTTCAGGACTGTGGTTT-30

SgCYP6H1 50-TAATACGACTCACTATAGGGAGACAACCTGCTGCTGCTGCG-30 50-TAATACGACTCACTATAGGGAGATCACCTCCTTGATGTCCAGCT-30

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cycles of 95 �C for 15 s and 60 �C for 60 s. Finally a melt curve anal-ysis was performed to check for primer dimers. For all transcriptsonly a single melting peak was found during the dissociation pro-tocol. Additionally, PCR products were run on a 1.2% agarose gelcontaining GelRedTM (Biotium, Hayward, CA, USA). After electro-phoresis only a single band could be se6en which was furthercloned and sequenced (TOPO� TA cloning kit for sequencing, Invit-rogen Life Technologies, Carlsbad, CA, USA) to confirm target spec-ificity. For each tested cDNA sample the normalisation factor forthe housekeeping genes relative to a calibrator sample was calcu-lated and used to determine the normalised expression levels ofthe target genes relative to the calibrator as was previously de-scribed (Vandesompele et al., 2002). The data for the spatial anddevelopmental expression profile were measured from six individ-ual locusts (n = 6). The data shown in Figs. 3 and 4 represent themean mRNA quantities from seven to eight individual locusts(n = 7–8). Since a different calibrator sample was used in q-RT-PCR measurement of the RNAi experiment, the relative expressionlevels of SgShd and SgCYP6H1 in Fig. 3 cannot be compared to thelevels given in Figs. 1 and 2. Furthermore, even though locustbreeding conditions are controlled, seasonal differences in larvaldevelopmental rates may exist between batches of synchronisedlocusts used in the different experiments.

2.5. RNAi

dsRNA was prepared through the use of Ambion’s MEGAscript�

RNAi Kit, which is designed for the construction of dsRNA largerthan 200 bp. The procedure is based on the high-yield transcriptionreaction of a user-provided linear transcript with a T7 promoter

Fig. 1. Graphic representation of the relative SgShd (A) and SgCYP6H1 (B) transcript levdissected from 6-day old fifth larval female locusts, except for the male gonads (G M). OMal: Malpighian tubules; PG: prothoracic glands and MG: midgut. The data represent meand EF1a transcript levels (n = 6). The vertical bars indicate S.E.M.

Please cite this article in press as: Marchal, E., et al. RNAi-mediated knockdownSchistocerca gregaria. Journal of Insect Physiology (2012), http://dx.doi.org/10.1

sequence. T7 promoter sites were added to the coding sequenceof interest in a PCR reaction using primers to which a T7 site (TAA-TACGACTCACTATAGGGAGA) is attached (Table 1b). This PCR prod-uct can then be directly used in a single RNA polymerasetranscription reaction. Two complementary RNA transcripts aremade, which are hybridized together to form the dsRNA. Anyremaining DNA and ssRNA are removed in a nuclease digestionand the construct is further purified to remove contaminating pro-tein and mono- and oligonucleotides. In designing dsRNA con-structs, possible off-target effects of the dsRNAs were minimizedby blasting their sequences to all sequences available in the locustEST database. No long stretches of nucleic acid identity were ob-served (except with their specific target gene sequences). ThisRNAi study (data shown in Figs. 3–5) was performed during a veryhot period in the summer time, when conditions were optimal forlocust growth and development. Practically, this meant that the fi-nal instar stage only lasted about 6 days (instead of 8 days, asshown in Fig. 2), after which they moulted into the adult form.Freshly moulted fifth larval female locusts were injected on day0 with 2.5 lg of SgShd or SgCyp6h1 dsRNA dissolved in 6 ll elutionbuffer. Control animals were simply injected with 6 ll of this elu-tion solution. A first group of 8 animals was sacrificed on day 4 ofthe final instar. On the same day injection with another 2.5 lgdsRNA was performed. A second group of 8 animals was sacrificedon day 6. Malpighian tubules were dissected and haemolymphsamples were taken as will be described below. A final group ofanimals was injected according to the same scheme, but was leftintentionally to evaluate possible disruptions of the moulting pro-cess. The control and treated groups and their time frame to adultmoult were carefully observed throughout 5th larval development.

els measured in different fifth larval (L5) tissues using q-RT-PCR. All tissues werether abbreviations on the X-axis: Epi: epidermis; Fb: fat body; G F: Female gonads;ans of six individual 6 day-old L5 locusts, run in duplicate and normalised to b-actin

of Shade negatively affects ecdysone-20-hydroxylation in the desert locust,016/j.jinsphys.2012.03.013

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Fig. 2. Graphic representation of the relative SgShd (A) and SgCYP6H1 (B) transcript levels measured in the Malpighian tubules of S. gregaria during fifth larval development.Expression of both genes was measured in six individual animals in duplicate using q-RT-PCR and normalised to b-actin and EF1a transcript levels (n = 6). Measurementswere taken every other day during final larval development. The columns represent averages with vertical bars indicating S.E.M.

Fig. 3. Efficiency of RNAi-mediated knockdown in fifth larval female S. gregaria. Relative quantity of SgShd (a) or SgCYP6H (b) in Malpighian tubules dissected from controlanimals compared to Malpighian tubules from larvae injected with SgShd of SgCYP6H1 dsRNA. Both control and treated animals were sacrificed on days 4 and 6 of the finallarval stage. The data represent averages of eight individual animals run in duplicate using q-RT-PCR and normalised to b-actin and EF1a transcript levels (n = 8). Vertical barsindicate S.E.M. Significant differences (p < 0.01; p < 0.001) are indicated by asterisks (⁄⁄ and ⁄⁄⁄, resp.).

Fig. 4. The effect of downregulating SgShd on the expression of SgSpo (A) and SgPhm (B), possibly involved in the same pathway. The data represent averages of 7–8 individualfemale animals of 6 days old, run in duplicate using q-RT-PCR and normalised to b-actin and EF1a transcript levels (n = 7–8). Vertical bars indicate S.E.M. Significantdifferences (p < 0.05) are indicated by an asterisk (⁄).

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2.6. Ecdysteroid measurements – Enzyme ImmunoAssay (EIA)

For this study, it was essential to measure the content of E and20E present in a haemolymph sample separately. Haemolymphsamples were collected from S. gregaria larval stages by piercingthe insect’s cuticle behind its hind leg and holding a capillary tothe wound. 10 ll were collected from each animal and immedi-ately transferred to 90 ll of methanol (100%). These haemolymphsamples were stored at �20 �C until they were extracted as fol-lows: the samples were vortexed and centrifuged for 10 min atmaximum speed, after which the supernatant was transferred toa fresh eppendorf tube. The remaining pellet was extracted twomore times with 100 ll of methanol (100%). The combined extractswere then dried in a SpeedVac concentrator. These methanolic ex-tracts of 10 ll S. gregaria haemolymph from last instar larvae were

Please cite this article in press as: Marchal, E., et al. RNAi-mediated knockdownSchistocerca gregaria. Journal of Insect Physiology (2012), http://dx.doi.org/10.1

dried and redissolved in 20% ACN (acetonitrile, dissolved in MilliQwater), 0.1% TFA (Trifluoroacetic acid) prior to separation by highpressure liquid chromatography (HPLC). All runs were carried outusing a Beckman HPLC System Gold (Beckman Coulter Inc., Brea,CA, USA) and a symmetry C18 (4.6 � 250 mm) column (Waters,Milford, MA, USA) and monitored with the UV detector at240 nm. A pilot run was carried out with E and 20E (Sigma–AldrichCo., St-Louis, MO, USA) as standards and with following HPLC pro-file: (1) isocratic with 20% ACN for 5 min, (2) a linear gradient of20–35% ACN for another 20 min and finally (3) a gradient of35–80% ACN. The flow rate was 1 mL/min. In this pilot run, 20Eand E were found to elute after 15 and 22 min, respectively. Allsubsequent runs were carried out identically, fractions were col-lected (one per minute, during 30 min) and individually analysedwith the enzyme immunoassay (EIA), as described below. Fractions

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Fig. 5. E/20E ratio in extracted haemolymph samples from (a) control and SgShd dsRNA-treated or (b) control and SgCYP6H1 dsRNA-treated day 4 and day 6 5th larval locusts.The data represent averages of 7–8 individual female animals. Vertical bars indicate S.E.M. Significant differences (p < 0.05; p < 0.01) are indicated by (an) asterisk(s) (⁄ and ⁄⁄,resp.).

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were dried and redissolved in EIA sample buffer. Between columncalibration by standard compounds and the analysis of the biolog-ical samples, blank injections with complete runs were performed,to prevent contamination of the HPLC-column with referenceecdysteroids.

Ecdysteroid levels in the fractions corresponding to elutiontimes for E and 20E standards were measured using an EIA, modi-fied from Porcheron et al. (1989) and discussed by Pascual et al.(1995) and Lafont et al. (2005). In this EIA protocol a peroxidaseconjugate of 20E was used as tracer together with rabbit L2 poly-clonal antibodies. This L2 antiserum has a strong affinity for E, 3-deoxyecdysone and 2-deoxyecdysone and a 6- to 8-fold loweraffinity for 20E. For a detailed description of the EIA protocol, thereader is referred to Marchal et al. (2011a).

Since the experiments with SgShd and SgCYP6H1 were not per-formed at the same time, controls were also different and data rep-resented in Fig. 5a and b should not be compared.

2.7. Statistical analysis

All statistical analysis was performed using GraphPad Prism 4.When comparing relative transcript levels in controls anddsRNA-treated animals by means of q-RT-PCR (RNAi experiments),a two-tailed unpaired t test was used to check for significantdownregulation. The same test was performed when comparingE/20E ratios between control and treated animals.

3. Results

3.1. PCR cloning and sequence analysis of SgShd and SgCyp6h1

Partial sequences were obtained for the CYP6H1 and Shade ortho-logs in S. gregaria. Domain searches in the NCBI database revealedthat they contain conserved motifs found in cytochrome P450enzymes. The sequences have the typical characteristics of themicrosomal P450 enzymes, i.e. a hydrophobic N-terminal sequencepreceding a proline–glycine rich region. Both partial sequenceswere uploaded on NCBI’s (National Center for Biotechnology) Gen-Bank and received accession numbers JN982228 and JN982229,respectively. Protein alignments of these S. gregaria sequences withother known/predicted proteins are given in Supplementary Figs. B& C. We have tried to complete the partial sequences obtained fromthe S. gregaria EST database (CYP6H1) using degenerate primes (Shd)by means of Rapid Amplification of cDNA Ends (RACE), but theseRACE-reactions were only partly successful (as can be seen fromSupplementary Figs. B & C). Upon blastx NCBI database searches,the respective orthologs manifestly showed highest similarity tothe previously described Shd and LmCYP6H1 sequences.

Please cite this article in press as: Marchal, E., et al. RNAi-mediated knockdownSchistocerca gregaria. Journal of Insect Physiology (2012), http://dx.doi.org/10.1

3.2. q-RT-PCR profiling

3.2.1. Tissue distributionThe highest relative transcript levels for SgShd and SgCYP6H1

were found in the Malpighian tubules, while lower levels weremeasured in the larval fat body and in several other tissues (Fig. 1).

3.2.2. Temporal profiling 5th larval stageThe results from the q-RT-PCR profiling can be compared to cir-

culating ecdysteroid levels in the 5th larval stage (SupplementaryFig. D). For both female and male insects, the relative transcriptlevels of SgShd rise and peak on day 6 of the final larval instar(Fig. 2). From the SgCYP6H1 transcript levels and fifth larval ecdy-steroid titer profiles, no clear peak can be derived. A next step inthe study will be trying to silence these genes and evaluating theknockdown effect on the E/20E ratio.

3.3. RNA interference

3.3.1. q-RT-PCR resultsThe q-RT-PCR results of the SgShd and SgCYP6H1 RNAi experi-

ment are given in Fig. 3. The graph shows clearly that – on bothtime points at which samples were taken (day 4 and 6) – the RNAimediated knockdown was successful, reaching a 96% (day 4) – 94%(day 6) for SgShd and a 94% (day 4) – 83% (day 6) for SgCYP6H1downregulation compared to control animals.

Since SgSHD is expected to act in the same pathway as the otherpreviously identified Halloween enzymes SgPHM and SgSPO(Marchal et al., 2011a), the possible effect of the SgShd knockdownwas examined on the transcript levels of the other Halloween genesSgSpo and SgPhm. Relative mRNA quantities of these genes weremeasured in the PG of day 6-control and treated animals to deter-mine whether the downregulation of the probable final Cyp genecould have a (positive) feedback effect on other transcripts in-volved in the same pathway. These q-RT-PCR results are given inFig. 4. SgSpo was significantly upregulated in the PG of SgShddsRNA-treated animals compared to control locusts. For SgPhmno significant differences were found.

3.3.2. Effect on circulating ecdysteroid levelsThe E/20E ratio measured by the EIA in control and SgShd

dsRNA-treated females is given in Fig. 5a. At both time points mea-sured (day 4 and 6) the E/20E ratio was found to be significantlyhigher in the SgShd dsRNA-treated animals compared to controls.This is in sharp contrast with SgCYP6H1 RNAi. The E/20E ratiosdid not differ significantly when comparing control and SgCYP6H1dsRNA-injected locusts as can be observed in Fig. 5b.

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An extra group of locusts (controls and SgShd or SgCYP6H1dsRNA-treated) was injected according to the same injectionscheme as described above and was not sacrificed but left inten-tionally to assess any moulting difficulties. In neither case wasthe moulting process negatively affected.

4. Discussion

Partial sequences were obtained for Shade and CYP6H1 ortho-logs in S. gregaria. SgCYP6H1 closely resembles a B. germanicaCyp6k1, which was previously described to have a high identitypercentage to LmCYP6H1 (Wen and Scott, 2001). During the cock-roach’s life cycle, BgCYP6K1 expression was shown to start in theembryonic stage, to peak at fifth and sixth larval stages and wasfound in both female and male adults. Like LmCYP6H1, BgCYP6K1also encodes a typical microsomal cytochrome P450 enzyme, butthe substrate is currently unknown. In the SgSHD sequence, thecharged residues at the beginning of Helix-L appear to beconserved, which is a signature for mitochondrial targeting(Werck-Reichhart and Feyereisen, 2000). Correspondingly, E20-hydroxylase activity in the Malpighian tubules of S. gregariawas found to be mainly mitochondrial in an earlier study (Green-wood and Rees, 1984). However, prior cell fractionation work inL. migratoria showed E 20-hydroxylase activity in the microsomalfraction (Feyereisen and Durst, 1978). Moreover, since all otherSHD sequences in the alignment are characterised by an N-termi-nal hydrophobic microsomal targeting sequence, followed by aP/G rich region, the SgSHD may possibly also be thought of as beinga hybrid cytochrome P450 enzyme.

The presence of Halloween orthologs in the crustacean Daphniasuggests that these genes became functionally specialised beforethe crustacean and insect split and have been under selective pres-sure since (Rewitz and Gilbert, 2008; Rewitz et al., 2007). Thiswould imply that orthologs probably exist in the hemimetabolousdesert locust as well. We were indeed successful in finding partialorthologous sequences, which again underlines the conservation ofthis family of genes within the Arthropoda.

The spatial and temporal expression profiles of SgShd were inline with earlier studies of Halloween gene transcripts in dipteranand lepidopteran insects. The expression of lepidopteran and dip-teran Shd genes was mainly localised in peripheral tissues, suchas immature midgut, Malpighian tubules and fat body and notin the PG (in contrast to the other Halloween genes (Gilbert,2004; Huang et al., 2008; Petryk et al., 2003; Rewitz et al.,2006c). In final larval S. littoralis, the highest expression wasfound in the Malpighian tubules (Iga and Smagghe, 2010). More-over, in L. migratoria, the Malpighian tubules were indeed shownto have higher 20-hydroxylase activity than any other tissue (fatbody or midgut). Northern analysis of LmCYP6H1 showed a highexpression in the fifth larval Malpighian tubules (Winter et al.,1999). This seemed consistent with E being produced in the larvalPG, being released and being converted to 20E in tissues periph-eral to the PG (Huang et al., 2008). A similar spatial expressionprofile for SgShd and SgCYP6H1 was found in larval S. gregaria.The temporal expression profile of SgShd appeared to correlatewell with the circulating ecdysteroid titer in the haemolymph,while this was not the case with the transcript level data ofSgCYP6H1.

Injection of SgShd dsRNA and SgCYP6H1 dsRNA resulted in a sig-nificant downregulation of the transcript levels in the Malpighiantubules compared to control injected animals. In previous reports,successful RNAi studies were performed in S. gregaria targeting dif-ferent genes in a wide variety of tissues and developmental stages(Badisco et al., 2011b; Marchal et al., 2011b; Ott et al., 2011; Tob-back et al., 2011; Van Wielendaele et al., 2012). Injection of SgShddsRNA and SgCYP6H1 dsRNA resulted in a significant downregula-

Please cite this article in press as: Marchal, E., et al. RNAi-mediated knockdownSchistocerca gregaria. Journal of Insect Physiology (2012), http://dx.doi.org/10.1

tion of the transcript levels in the Malpighian tubules compared tocontrol injected animals. Since different calibrator samples for q-RT-PCR were used in the RNAi experiment compared to the spatialand developmental profiling, it is incorrect to compare absolutemeasures of the ‘‘relative quantities’’ depicted in Fig. 3 comparedto Figs. 1 and 2. For practical and logistic reasons, these distinctexperiments were not performed at the same time, and samplingoccurred in different periods of the year from different batches oflocusts. It is well known that rearing conditions (nutrition,winter–summer, health condition, etc. cause some differences inthe timing of larval development between different batches oflocusts. This is another reason why it is not correct to fully com-pare data obtained from different experiments. The relative mRNAquantity of SgSpo, the Halloween gene involved in the rate-limiting‘black box’ of the ecdysteroid biosynthetic pathway, was shown tobe significantly upregulated in the PG of the SgShd dsRNA-treatedanimals. It seems reasonable to assume that the downregulationof one gene in a particular pathway could result in the upregula-tion of another in an attempt to maintain comparable amountsof the end product.

Our results indicate that the knockdown of SgShd negativelyaffected the conversion of E to 20E, resulting in a higher E/20Eratio. However, the decline in circulating 20E appeared not suffi-cient to effectively disturb the moulting process, since the dsRNA-treated animals still moulted within a similar time frame as thecontrol locusts. As measured with the EIA, there was still 20E cir-culating in the haemolymph, indicating that the RNAi-basedsilencing process did not completely eliminate SgShd transcriptionor the activity of the 20-hydroxylase. It is possible that a suffi-cient threshold concentration of 20E was reached in the targettissues, still allowing for a successful moult. Moreover, accumu-lating levels of E may have contributed to reaching a thresholdactivity, since it can also act as a moulting hormone, albeit farless than 20E itself (Lafont et al., 2005). Another possibility isthat a portion of the ecdysteroids was hidden from detectionusing the EIA since conjugated forms of ecdysteroids are notdetected by the antiserum. The E/20E ratio in the SgCYP6H1dsRNA-treated animals, did not differ significantly from the ratiomeasured in the control animals. From our observations, it can beconcluded that in the desert locust no definite clues werefound for a direct involvement of SgCYP6H1 in ecdysteroidbiosynthesis.

The dsRNA-mediated knockdown of both cytochrome P450encoding genes, SgShd and SgCYP6H1, appeared very robust at thetranscript level. The injections with dsRNA constructs resulted ina significant downregulation of the respective mRNA levels. Basedon the ecdysteroid measurements, it can be concluded that in thedesert locust, the Halloween gene SgShd plays a role in the biosyn-thesis of 20E. Moreover, the conserved SgShd sequence and its spa-tial and temporal transcript level profiles also indicate that thisgene is orthologous to the previously characterised lepidopteranand dipteran shd genes. This report offers a first functional charac-terisation of a shd ortholog in a more primitive hemimetabolous in-sect. While the knockdown was successful at the transcript leveland significant reductions in the 20E titer were measured, no cleareffects of the dsRNA treatments were observed on the actualmoulting process itself. This could be due to compensatory effectswhich may have reduced the requirement for high 20E titers tocontrol moulting (as previously discussed by Marchal et al.,2011a). RNAi may be used in follow-up research to silence moregenes in the ecdysteroid pathway in one injection and may offernew insights in this process. A thorough analysis of the food-dependency of moulting and ecdysteroidogenesis as well as ofother components of the ecdysteroid signalling pathway (forexample RxR and EcR) may constitute interesting future researchtopics.

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5. Uncited references

(Maeda et al., 2008; Sieglaff et al., 2005).

Acknowledgements

The authors would like to thank Roger Jonckers, Sofie Van Soestand Joost Van Duppen for technical assistance and gratefullyacknowledge the Research Foundation–Flanders (FWO), theAgency for Innovation by Science and Technology (IWT), theResearch Foundation of K.U. Leuven (GOA/11/02) and the Interuni-versity Attraction Poles program (Belgian Science Policy Grant, IAPP6/14) for funding. They also acknowledge Prof. Jean-PaulDelbecque (Université de Bordeaux, France) for his kind gifts ofantiserum and tracer. The authors declare no conflict of interest,financial or otherwise, that might potentially bias this work.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.jinsphys.2012.03.013.

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