A viral chitinase enhances oral activity of TMOF

A viral chitinase enhances oral activity of TMOF
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  This article appeared in a journal published by Elsevier. The attachedcopy is furnished to the author for internal non-commercial researchand education use, including for instruction at the authors institutionand sharing with colleagues.Other uses, including reproduction and distribution, or selling orlicensing copies, or posting to personal, institutional or third partywebsites are prohibited.In most cases authors are permitted to post their version of thearticle (e.g. in Word or Tex form) to their personal website orinstitutional repository. Authors requiring further informationregarding Elsevier’s archiving and manuscript policies areencouraged to visit:  Author's personal copy A viral chitinase enhances oral activity of TMOF Luisa Fiandra a , Irma Terracciano b , Paolo Fanti c , Antonio Garonna d , Lia Ferracane e ,Vincenzo Fogliano e , Morena Casartelli a , Barbara Giordana a , Rosa Rao b , Francesco Pennacchio d , * a Dipartimento di Biologia, Università di Milano, Milano, Italy b Dipartimento di Scienze del Suolo, della Pianta, dell ’   Ambiente e delle Produzioni Animali, Università di Napoli  “  Federico II  ”  , Portici, Italy c Dipartimento di Biologia, Difesa e Biotecnologie Agro-Forestali, Università della Basilicata, Potenza, Italy d Dipartimento di Entomologia e, Zoologia Agraria  “  Filippo Silvestri ”  , Università di Napoli  “  Federico II  ”  , Via Università, 100, 80055 Portici (NA), Italy e Dipartimento di Scienza degli Alimenti, Università di Napoli  “  Federico II  ”  , Portici, Italy a r t i c l e i n f o  Article history: Received 14 March 2010Received in revised form30 April 2010Accepted 3 May 2010 Keywords: BioinsecticideGut absorptionPeptidePeritrophic membraneTransgenic plants a b s t r a c t In this study we investigate the combined effect on  Heliothis virescens  (Lepidoptera, Noctuidae) larvae of Aedes aegypti-Trypsin Modulating Oostatic Factor (  Aea -TMOF), a peptide that inhibits trypsin synthesisby the gut, impairing insect digestive function, and  Autographa californica  nucleopolyhedrovirus Chiti-nase A (AcMNPV ChiA), an enzyme that is able to alter the permeability of the peritrophic membrane(PM).  Aea -TMOF and AcMNPV ChiA were provided to the larvae by administering transgenic tobaccoplants, co-expressing both molecules. Experimental larvae feeding on these plants, compared to thosealimented on plants expressing only one of the two molecules considered, showed signi fi cantly strongernegative effects on growth rate, developmental time and mortality. The impact of AcMNPV ChiA on thePM of   H. virescens  larvae, measured as increased permeability to molecules, was evident after fi ve days of feeding on transgenic plants expressing ChiA. This result was con fi rmed by  in vitro  treatment of PM withrecombinant ChiA, extracted from the transgenic plants used for the feeding experiments. Collectively,these data indicate the occurrence of a positive interaction between the two transgenes concurrentlyexpressed in the same plant. The hydrolytic activity of ChiA on the PM of tobacco budworm larvaeenhances the permeation of TMOF molecules tothe ectoperitrophic space, and its subsequent absorption.The permeation through the paracellular route of   Aea -TMOF resulted in a spotted accumulation on thebasolateral domain of enterocytes, which suggests the occurrence of a receptor on the gut side facing thehaemocoel. The binding of the peptide, permeating at increased rates due to the ChiA activity, isconsidered responsible for the enhanced insecticide activity of the transgenic plants expressing bothmolecules. These data corroborate the idea that ChiA can be effectively used as gut permeation enhancerin oral delivery strategies of bioinsecticides targeting haemocoelic receptors.   2010 Elsevier Ltd. All rights reserved. 1. Introduction The reduction of chemical insecticide use is one of the majorissues for safe food production. The importance of this objective inmodern agriculture has fostered signi fi cant research effortstowards the development of innovative technologies based on theuse of biological control agents (Bale et al., 2008), natural insecti-cides, which include small organic molecules (Dayan et al., 2009)and peptide or protein toxins, deriving from plants and insectnatural antagonists (Whetstone and Hammock, 2007).Thesuccessandsafetyof pestmanagementtechnologieslargelydepends on the ef  fi cacy of the delivery methods used to distributethe insecticide molecules in the environment. When dealing withpeptide/protein toxins, the choice of the most appropriate deliveryvector is directed by the localization of the receptor to be targeted,which can be in the gut or behind the gut wall. The delivery of biopesticides through oral ingestion, for example by transgenicplant expression, is considered more appropriate for moleculesexertingtheiractivityinthegut,whilethosetargetinghaemocoelicreceptors are more ef  fi ciently delivered via insect-speci fi c symbi-onts and pathogens (Inceoglu et al., 2006; Whetstone andHammock, 2007). This conceptual dichotomy is largely motivatedby the assumption that most macromolecules are unable to passacross the gut barrier in signi fi cant amounts, but can easily cross itif expressed in recombinant baculoviruses (Liu et al., 2006).However, a growing number of exceptions to this assumption canbe found in the literature, with cases of parasitoid (Maiti et al.,2003) and predator derived toxins (Liu et al., 2006), which have *  Corresponding author. Tel.:  þ 39 081 253 9195; fax:  þ 39 081 775 5145. E-mail address: (F. Pennacchio). Contents lists available at ScienceDirect Insect Biochemistry and Molecular Biology journal homepage: 0965-1748/$  e  see front matter    2010 Elsevier Ltd. All rights reserved.doi:10.1016/j.ibmb.2010.05.001 Insect Biochemistry and Molecular Biology 40 (2010) 533 e 540  Author's personal copy conferred a signi fi cant protection level when expressed in trans-genic plants. However, none of these studies provided directevidence that the toxins passed from the gut lumen to thehaemocoel of the target insects (reviewed in Liu et al., 2006), eventhough they indirectly indicated that gut absorption of macro-molecules in insects is likely possible and of practical value for pestcontrol. The possibility of delivering intrahaemocoelic toxins withfood opens very interesting new perspectives in the  fi eld of biotechnology for insect control, and is certainly worthy of furtherresearch efforts.In spite of these promising perspectives, the study of thephysiological mechanisms mediating the absorption of macro-molecules in the insect gut has received limited attention ( Jefferesand Roe,2008). Overthe last fewyears, wehavecontributedtothisresearch area, by focusing our interest on the absorption pathwaysof peptides and proteins in the midgut of lepidopteran larvae,demonstrating that the paracellular route (Fiandra et al., 2006) ismostly exploited by small peptides (Fiandra et al., 2009), whiletranscytosis is the main route of entrance for proteins (Casartelliet al., 2005, 2007). The absorption pathway of peptides can bemodulated by manipulating the intracellular concentration of cAMP and Ca þþ (Fiandra et al., 2006); the ligand speci fi city of thereceptorinvolvedintheinternalizationofalbumincanbeexploitedfor promoting the uptake of fusion proteins, bearing toxic domainsalong with domains which are involved in the receptor-mediatedendocytosis (Casartelli et al., 2008). This information provides thebackground on which new strategies for enhancing the rate of gutabsorption can be developed.However, the gut epithelium is only one of the two majorintestinal barriers to be crossed by ingested macromolecules, andthe peritrophic membrane (PM) represents the  fi rst physical layerwith pores that discriminates the passage of large molecules(Lehane, 1997; Barbehenn, 2001). In  Bombyx mori  larvae, forinstance, the PM was largely permeable to methylene blue,a molecule with a molecular mass of 320 Da, and almost imper-meable to PEG 4000, while the Trypsin Modulating Oostatic Factorfrom  Aedes eagypti  (  Aea -TMOF) had an intermediate permeabilitycoef  fi cient, in linewith its molecular mass (1005 Da) (Fiandra et al.,2009). Therefore, the structural disruption of the PM can facilitatethe passage of molecules, as naturally occurs in the case of infec-tion by baculoviruses, which use speci fi c metalloproteases fordisrupting the peritrophic membrane, to allow the contact of viralparticles with midgut epithelial cells (Slavicek and Popham, 2005;Liu et al., 2006).In the framework of a coordinated effort towards the devel-opment of new delivery strategies and combinations of bion-secticides, we discovered that the Chitinase A (ChiA) of   Autographa californica  nucleopolyhedrovirus (  Ac  MNPV), whichhas a key-role in the post-mortem liquefaction of the infectedlarval host cadaver (Bonning, 2005), determined structuralalterations on lepidopteran larvae PM (Rao et al., 2004), and hada signi fi cant negative effect on insect biological performance andsurvival when the recombinant protein was delivered either witharti fi cial diet or with transgenic plants (Rao et al., 2004; Corradoet al., 2008). The same studies also clearly showed a strongincrease of the permeability to molecules of the PMs treated  invitro  with ChiA. These results stimulated the idea of using ChiAin combination with  Aea -TMOF, which targets receptorsexpressed in the basolateral membrane of epithelial midgut cellsand causes the inhibition of trypsin synthesis, thus impairing theinsect digestive processes (Borovsky et al., 1994; Nauen et al.,2001; Borovsky and Meola, 2004).  Aea -TMOF exerts mild insec-ticide activity on  Heliothis virescens  larvae when expressed intransgenic tobacco plants (Tortiglione et al., 2002, 2003), andnegatively interferes with larval growth of the tobacco budworm( H. virescens ), when fused to Tobacco Mosaic Virus coat protein(Borovsky et al., 2006).In this study we demonstrate that tobacco plants co-expressingboth  Aea -TMOF and AcMNPV ChiA show a signi fi cantly strongerimpactthanparentallines,expressingonlyoneofthetwogenes,onthe development and survival of the tobacco budworm larvae,which is associated with a higher permeability to  Aea -TMOF of theperitrophic membrane of larvae fed on transgenic plants. Thiscorroborates the hypothesis that the use in tandem of gut perme-ating agents and insecticide molecules targeting haemocoelicreceptors can result in a more ef  fi cient insect control activity, asa consequence of functional complementation of the moleculesused and reduced risk of resistance in the target population. 2. Material and methods  2.1. Production of hybrid tobacco plants co-expressing  AcMNPV ChiA and polyTMOF  Transgenic tobacco plants constitutively expressing the poly-TMOF synthetic gene (line R1-2) and ChiA gene (ChiA HDEL line 9)were obtained as reported in Tortiglione et al. (2002) and Corrado et al. (2008), respectively. Both transgenic lines were screened forresistance to kanamycin on Murashige Skooge medium, supple-mented with 100 mg/l kanamycin, and then transferred to soil andgrown under containment glass house conditions. Crosses of thetwo transgenic lines srcinated tobacco genotypes co-expressingboth genes, ChiA and polyTMOF, which, for simplicity reasons, arehereafter denoted as hybrids.  2.2. Molecular characterization of tobacco hybrid genotypes The presence of both polyTMOF and ChiA mRNA in the tobaccohybrids was detected by Northern blot, with the appropriate cDNAprobes, as previously described (Tortiglione et al., 2002; Corradoet al., 2008).Furthermore, the expression of the ChiA proteinwas monitoredby Western blot. Total proteins were isolated from leaves, quanti- fi ed and resolved by SDS-PAGE (Sambrook et al., 1989). Westernanalysiswascarriedouton40  m gofwatersolubleproteins,usingasprimary antibody the anti-myc (Santa Cruz Biotechnology, CA),diluted 1:500, and anti-rabbit IgG conjugated with horseradishperoxidase, diluited 1:2000, as secondary antibody, according tothe procedures already published (Corrado et al., 2008).  2.3. ChiA puri  fi cation from tobacco transgenic plants The recombinant ChiA protein was puri fi ed from transgenictobacco leaves as described in Di Maro et al. (2010). The enzymaticactivity of the isolated protein was assayed using the substrate 4-methyl-umbelliferyl- b - D - N  - N  0 - N  00 -acetyl-chitotriose [4MU-(GlcNAe) 3 , Sigma e Aldrich, Italy] for the detection of endo-chiti-nolytic activity, as reported elsewhere (McCreath and Gooday,1992; Rao et al., 2004). Brie fl y, for the ChiA protein puri fi cationprocedure, leaves were homogenized in 1   PBS, in presence of EDTA 5 mM, PMSF 1 mM and PVP-40 1.5%, by 20-s bursts at fullpower using a Waring Blender (Waring Products, CT, USA). Theproteins were subjected to ammonium sulfate precipitation,followed by ion exchange and gel  fi ltration chromatography. Thepuri fi cation was monitored by analyzing the chromatographyfractions by SDS-PAGE and Western blot.ChiA, separated by SDS-PAGE, was transferred onto PVDFmembrane and directly subjected to Edman degradation on a Pro-cise Model 491 sequencer (Applied Biosystems), for N-terminalsequencing, as previously described (Di Maro et al., 2001). L. Fiandra et al. / Insect Biochemistry and Molecular Biology 40 (2010) 533 e 540 534  Author's personal copy  2.4. Feeding bioassay on transgenic plants The insecticidal activity of transformed tobacco plants wasassayed  in vivo  on larvae of the tobacco budworm  H. virescens .Selected transformants expressing either the TMOF peptide (linepolyTMOF R1-2) or ChiA (line ChiA HDEL 9) or both of them andcontrol plants (NN) were daily supplied as leaf disks to newlyhatched larvae. Experimental larvae were singly maintained at29  1   C, in multiwell plastic trays, bottom lined with a thinlayer of a 2% agar solution and closed with transparent plasticcovers provided by the commercial supplier (CD International).Two different well sizes were used: 4    4    2 cm (for instars1st e 4th) (CD International BIO-RT-32) and 8    8    2 (for 5thinstars) (CD International BIOSMRT- 8). Larvae were weighedevery other day, starting on day 4 from the beginning of thebioassay. Mortality was daily checked during the whole larvalfeeding period. In each of the 4 replicate, 16 larvae were assayedfor each treatment.The larval development was compared by combining the larvalgrowth and survival into a single parameter, the total larvalbiomass,calculatedeveryotherday, asthesumof theweightof thesurviving larvae in each treatment. The growth curves of the larvalbiomass of individuals fed on control or transformed plants werecompared by Repeated Measures Analysis of Variance (Sokal andRohlf, 1995). The interaction of diet and the within-factor timewas tested using linear, quadratic and cubic order polynomialcontrasts, in order to assess differences in the slope of the growthcurves. Compound symmetry was checked by Huynh-Feldt statis-tics (Systat 12, Systat Software Inc.).Developmental times and survival rates were analyzed by One-Way Analysis of Variance, and mean comparison (Tukey ’ s test) wasperformed when statistical signi fi cance ( a ¼ 0.05) occurred.Percentages were arcsine transformed before analysis (Zar, 2009).Meanpercentages presented in fi gures were transformed back intoproportions after analysis. Because the con fi dence limits are notsymmetrical about the means when expressed again in propor-tions,in the result sectionwereportthe meanvalues and the meanvalues plus and minus the SE.All data analyses were performed with the statistical packageSystat 12 (Systat Software Inc.).  2.5. Evaluation of the peritrophic membrane permeability To assess the impact of feeding on transgenic plant lines,experimental larvae of   H. virescens , fed with arti fi cial diet until theend of the third instar, were divided into four groups of 16 larvaeeach and then reared from the  fi rst day of the fourth instar on thefollowing tobacco genotypes: NN, polyTMOF R1-2, ChiA HDEL 9and hybrids. Their survival and body weight were monitoredduringthe fi fthinstarat120,132and144 hourssincethebeginningof treatment. After 132 h, randomly selected larvae from eachexperimental group were used to study  in vitro  the permeability of their peritrophic membrane. The PM was isolated as described indetail in Rao et al. (2004). Brie fl y, the PM was carefully extractedfrom the dissected midgut, and cut longitudinally on a thin cottongauze, which maintained the PM extended, avoiding its  fl utteringonce mounted in the experimental apparatus. A portion of the PMwas mounted as a  fl at sheet in Ussing chambers (World PrecisionInstruments, Berlin, Germany), with an exposed surface area of 12.6 mm 2 . Thus, the PM separated the endoperitrophic and ecto-peritrophic compartments, both  fi lled with 500  m l of PBS (137 mMNaCl, 2.7 mM KCl, 4.3 mM Na 2 HPO 4  and 1.4 mM KH 2 PO 4 , pH 7).The PMs explanted from experimental larvae fed on differentplant lines were incubated for 90 min, in the presence of 1 mg/mlmethylene blue in the endoperitrophic compartment. The totalamount of dye diffused to the ectoperitrophic compartment wascollected and determined spectrophotometrically (Ultrospec 3000Pharmacia Biotech, Cambridge, UK), at the wavelength of 661 nm.A calibration curve was carried out with known amounts of themolecule dissolved in the incubation buffer.To demonstrate that the increased permeability was due to  Ac  MNPV ChiA produced by transgenic plants, this enzyme,extracted and puri fi ed from ChiA HDEL 9 genotypes, was used inPM permeability assays to TMOF. PMs were explanted from larvaecontinuouslyrearedonarti fi cialdiet.The fl uxofTMOF(synthesizedby GenScript Coorporation, USA) was measured by adding thepeptide (1 mg/ml) to the endoperitrophic compartment in theabsence (control) or in the presence of 40  m g/ml ChiA and byrecovering the solution in the ectoperitrophic compartment after90 min of incubation. The amount of permeated TMOF, detected byZonal Capillary Electrophoresis (Beckman Coulter P/ACE MDQ Capillary System), was determined using a suitable calibrationcurve.The calculated methylene blue and TMOF  fl ux values wereexpressed as nmol/cm 2 /h. Mean values were compared byStudent ’ s  t   test.  2.6. Detection of TMOF in the haemolymph of experimental larvae Experimentallarvaefed for 132 h onpolyTMOFR1-2 and hybridtobacco genotypes, as described in the previous section, were usedfor haemolymph collection. Fifty  m l of haemolymph, collected fromthe cut proleg of 5 larvae using capillary glass tubes, were diluted1:10 in methanol and stored at   20   C. Samples to be analyzedwere centrifuged at 4000 rpm for 10 min and cleaned up ona reversed phase Strata C18-E 500 mg cartridge (Phenomenex,Torrance, CA, USA). Avolumeof 500  m l was loaded on the cartridge,previouslyconditioned with methanol (3 ml) and water (3 ml). Thecolumnwasthenwashedwithwater(3 ml),andelutedwith3 mlof pure methanol. The eluate was dried under a gentle nitrogenstream, dissolved in 50  m l of methanol, centrifuged at 12,000 rpmfor3 minandusedforliquidchromatographycoupledwithtandemmass spectrometry (LC/MS/MS) analyses. Chromatographic sepa-ration was obtained using an HPLC apparatus, equipped with twoMicropumps Series 200 (Perkin Elmer, Shellton, CT, USA), a UV/VISdetector series 200 set at 220 nm and an Aquapore RP300 C8, 7  m m220  2.1 mm column (Brownlee, CT). The eluents were: A: H 2 O,0.1% formic acid; B: CH 3 CN, 0.1% formic acid. The gradient programwas as follows: 0 e 50% B (13 min), 50 e 100% B (3 min), 100% B(4 min), 100 e 0% B (5 min) at a constant  fl ow of 0.2 ml/min. Injec-tion volume was 20  m l and all samples were centrifuged, before theanalysis, at 12,000 rpm for 3 0 using a centrifuge 5415 R (Eppendorf,Germany).MS and MS/MS analyses were performed on an API 3000 triplequadrupole mass spectrometer (Applied Biosystems, Canada),equipped with a TurboIonSpray. Acquisition was in positive ionmode, in MRM (Multiple Reaction Monitoring). The analyses wereperformedusingthefollowingsettings: dryinggas(air)washeatedto 350   C, capillary voltage (IS) was set at 5500 V. The declusteringpotential (DP), focus potential (FP) and the collision energy (CE)wereoptimizedinfusingdirectlyintothemassspectrometeraTMOFpeptide standard solution (10  m g/ml) at a constant fl ow rate of 6  m l/min using a model 11 syringe pump (Harvard Apparatus, Holliston,MA, USA).The detection limit (LOD with a signal tonoise ratio of 3)was 2 ng/ml. TMOF peptide showed an [M þ H] þ ion at  m/z   1047.6and a [M þ 2H] 2 þ ion at  m/z   524.6. The LC/MS/MS characteristics of TMOFare reported in Table 1.The recovery of TMOF was about 100% and was assessed byspikingasampleofhaemolymphwithasolutionofstandardTMOF,at a  fi nal TMOF concentration of 22 ng/ml. L. Fiandra et al. / Insect Biochemistry and Molecular Biology 40 (2010) 533 e 540  535  Author's personal copy  2.7. Incubation of larval midguts in Ussing chambers and  fl uorescence analysis of FITC-TMOF in whole mount tissues Larvae reared on the arti fi cial diet weresacri fi ced on the secondday of the last instar, and the midgut, deprived of the peritrophicmembrane, was mounted as a sheet in the Ussing chambers, aspreviously described (Fiandra et al., 2006). Tissues were perfusedwith2.5 mlofthefollowingphysiologicalsolution(inmM):5CaCl 2 ,24MgSO 4 ,20Kgluconate,190sucroseand5TrisadjustedtopH7inthe haemolymph compartment, or 5 CAPS adjusted to pH 10 in theluminal one. The solutions, connected  via  Ag e AgCl voltage elec-trodes in series with agar bridges (3 M KCl, 5.5% Agar) to a volt-meter for the measurementof the transepithelial voltage( V  t ), werecirculatedbygasin fl ux(100%O 2 )andmaintainedat25   Cinwater- jacketed reservoirs.One hundred thirty  m M of FITC-TMOF (GenScript Coorporation,USA) was added to the luminal solution, which contained a cock-tail with the following peptidase inhibitors: 1 mM 1 e 10 phenan-throline, 10  m M bestatine and 10  m M amastatine (Sigma e Aldrich,Italy). After 2 h of incubation, the midgut was removed from theUssing chambers, washed  fi ve times with the physiological solu-tion and  fi xed for 30 min in 4% paraformaldehyde. After further fi ve rinsing in PBS, the samples were mounted in DABCO (Sigma)-Mowiol (Calbiochem). The tissues covered with a coverslip wereexamined with a confocal laser scanning microscope imagingsystem (CLSM TCS SP2 AOBS- Leica Microsystems, Heidelberg,GmbH, Germany), equipped with an argon-krypton laser and anUV laser. 3. Results  3.1. Production and characterization of tobacco plantsco-expressing ChiA protein and polyTMOF peptide Tobaccoplantsco-expressingChiAandTMOFpeptides,obtainedby crossing the two parental transformants, were subjected toNorthern blot analysis, to monitor the expression of the twotransgenes. The hybridization of the total RNA extracted from thehybrids showed the presence of both transcripts of the expectedsize, in 5 out of the 10 hybrids analyzed (Fig. 1A). The presence of the two bands, one of 0.4 kb (  polyTMOF   transcript) and the other of 2 kb ( ChiA  transcript), separately present in the parental lines andabsent in the control plants, con fi rmed the success of the hybrid-ization between transgenic lines. The presence of the recombinantChiAproteinin thehybridswas showedbyWesternblot(Fig.1B).Asingle band, with an estimated molecular mass of 60 kDa, wasdetected in all the lines where the ChiA gene was actively tran-scribed. The immunodetection of TMOF was not performed, for thetechnical reasons already discussed elsewhere (Tortiglione et al.,2002).  3.2. Biological performance and survival of H. virescenslarvae fed on transformed tobacco plants We compared the larval development bycombining the growthand survival into a single parameter, the total experimentalbiomass, which, after the maximum larval weight was attained,included also the weight of the pupae. The effect of the experi-mental conditions considered on this parameter is shown in Fig. 2.The mean total experimental biomass obtained on the transformedand control plants was different in a highly signi fi cant way(analyzed with Repeated Measures ANOVA until day 10:  F  ¼ 8.824;df 3,12;  p ¼ 0.002). The interaction between diet and time was alsohighlysigni fi cant( F  ¼ 8.802;df9,36;  p < 0.001),indicatingthatthe  Table 1 MS-MS Instrumental parameters optimized for the detection of TMOF (DP Declus-tering potential, CE Collision Energy).Product ions  (m/z)  DP (V) CE (V)Precursor ion [M þ H] þ ( m /  z  )1047,6 601,6 68 46504,4 59407,7 53Precursor ion [M þ 2H] þ 2 ( m /  z  )524,6 385,4 47 29213,0 20279,4 25 Fig. 1.  (A) Northern blot hybridization of ChiA and polyTMOF transcripts. 1-10: ChiA-polyTMOF hybrids; R1-2: tobacco parental line expressing the  polyTMOF   syntheticgene; ChiA: tobacco parental line expressing  ChiA  gene; NN: untransformed tobaccoplant. (B) Western blot analysis of ChiA protein. NN: untransformed tobacco plant, R1-2: transgenic tobacco parental line expressing  polyTMOF   synthetic gene; ChiA: tobaccoparental line expressing  ChiA  gene; 1, 2, 6, 8 and 10: ChiA polyTMOF hybrids. The blotwas probed with the rabbit anti-c-myc as primary antibody and anti-rabbit IgGconjugated with horseradish peroxidase as a secondary antibody. Fig. 2.  Growth curves of the total biomass (Mean  SEM) of   Heliothis virescens  larvae asaffected by feeding with leaf disks obtained from transgenic or control plants(Repeated Measures ANOVA:  F  ¼ 8.82; df 3,12;  p ¼ 0.002). Total larval biomasscombines larval growth and survival and is calculated as the sum of the weight of thesurviving larvae in each treatment. For each treatment,  n ¼ 16 per replicate, for a totalof 4 replicates. L. Fiandra et al. / Insect Biochemistry and Molecular Biology 40 (2010) 533 e 540 536
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