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Evaluation of Bt-toxin uptake by the non-target herbivore, Myzus persicae (Hemiptera: Aphididae), feeding on transgenic oilseed rape

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Evaluation of Bt-toxin uptake by the non-target herbivore, Myzus persicae (Hemiptera: Aphididae), feeding on transgenic oilseed rape
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  SHORT COMMUNICATION Evaluation of  Bt  -toxin uptake by thenon-target herbivore,  Myzus persicae (Hemiptera: Aphididae), feeding ontransgenic oilseed rape G. Burgio   *  , A. Lanzoni, G. Accinelli, G. Dinelli,A. Bonetti, I. Marotti and F. Ramilli Dipartimento di Scienze e Tecnologie Agroambientali, Alma MaterStudiorum-Universita` di Bologna, Viale Fanin 42, 40127 Bologna, Italy Abstract As consequence of the concern about the biosafety of genetically modifiedplants, biological and ecological studies are considered crucial for environmentalrisk assessment. Laboratory experiments were carried out in order to evaluate thetransfer of the Cry1Ac  Bt -toxin from a transgenic  Bt -oilseed rape to a non-targetpest,  Myzus persicae  Sulzer. Cry1Ac protein levels in plants and aphids weredetermined using a double sandwich enzyme-linked immunosorbent assay.Phloem sap from ( Bt + ) and ( Bt x ) oilseed rape plants was collected from leavesusing a standard method of extraction in an EDTA buffer.  Bt -toxin was present inphloem sap, with a mean concentration of 2.7 + 1.46 ppb, corresponding to a24-fold lower level than in oilseed rape leaves. Toxin was also detected in aphidsamples, with a mean concentration in the positive samples of 2.0 + 0.8 ppb. Theevidence that  Bt -toxin remains in herbivores, in this case an aphid, could be usefulto clarify functional aspects linked to possible consequences of   Bt -crops on foodchains involving herbivore–natural enemy trophic systems. Further studies areneeded in order to improve the knowledge on the functional aspects linked to thetransfer of the Cry1Ac  Bt -toxin from GM-oilseed rape to aphids and their possibleconsequence. Keywords:  transgenic oilseed rape, Cry1Ac toxin, phloem sap,  Myzus persicae ,non-target insects Introduction The cultivation of genetically modified (GM) plantsis becoming increasingly important worldwide. In 2003,GM crops covered a surface of approximately 68 millionhectares:theyweregrownmostlyintheUSA(42.8millionha),Argentina (13.9 million ha) and Canada (4.4 million ha)(James, 2003). The most important genetically engineeredcrops that have been introduced contained genes encodinginsect resistance and herbicide tolerance; they were soybean,maize, cotton and canola. In 2003, according to an ISAAAreport (James, 2003), the increase of acreage covered withtransgenic crops was about 15 % , with a 40-fold increase withrespect to 1996. Genetically engineered crops that encode    * Fax: 0039 051 2096281E-mail: gburgio@entom.agrsci.unibo.it Bulletin of Entomological Research  (2007)  97 , 211–215 doi:10.1017/S0007485307004920  toxin genes from bacteria such as  Bacillus thuringiensis Berliner ( Bt ) have shown great promise for pest control(Hilder & Boulter, 1999; Gatehouse & Gatehouse, 2000;Mandaokar  et al ., 2000). However, the cultivation of GMcrops, both for experimental and commercial aims, deter-mined the need for an evaluation of the possible conse-quences for natural- and agro-ecosystems. Directive 2001/18/EC of the European Commission, since the introduction of Directive 90/220/EEC, introduced the basic principles for acorrect evaluation of environmental risk, and it remains the basic methodology to use in risk assessment. In Italy, therule n. 224 of 8 July 2003 considers information of publicopinion as regards deliberate GMO emission in the environ-ment (Sorlini  et al ., 2005). A general methodology for riskassessment, concerning GM organisms, was suggested bythe Italian Ministry of Environment with the aim of reviewing the main risk sources and to propose a monitor-ing programme within a risk evaluation plan (Sorlini  et al .,2005).Similar to other plant protection technology, insectresistant transgenic plants can bear risks and benefits tothe environment. The primary ecological concerns to therelease of transgenic plants include those related to theirpossible invasiveness in ecosystems, out-crossing, horizontalgene transfer, development of pest resistance and effectson non-target organisms (Dutton  et al ., 2003; Knols & Dicke,2003; Scholte & Dicke, 2005). Such evaluations should be carried out with a case-by-case approach, considering thetarget crop, the genetic trait introduced and the environment(Groot & Dicke, 2002; Dutton  et al ., 2003; Scholte & Dicke,2005). Moreover, they need specific and detailed experi-mental activity both in the field and in the laboratory (Lo¨vei& Arpaia, 2005). Negative effects of GM plants on non-target beneficial arthropods have been a major concern becausethese organisms play an important role in controlling pestpopulations and plant pollination (Jervis & Kidd, 1996;Dutton  et al ., 2003). Some reviews were published onecological risk assessment and potential non-target effectsof GM plants on arthropod fauna (Groot & Dicke, 2002;Conner  et al ., 2003; Dutton  et al ., 2003; Lo¨vei & Arpaia, 2005).In particular, Lo¨vei & Arpaia (2005) presented a criticalreview of laboratory studies concerning the impact of GMplants on natural enemies.Since the risk that GM plants pose on non-targetorganism is a function of exposure to the insecticidal proteinand the toxicity of the substance towards the specificorganism, one important component in the risk assessmentis to determine exposure (Raps  et al ., 2001; Dutton  et al ., 2002,2004; Scholte & Dicke, 2005). Exposure will depend on thefeeding behaviour of the organism in question, togetherwith the expression of the transgene in the plant (Dutton et al ., 2004). Another open question is whether the transgeneproduct could be transported in the phloem sap. Aphidsand other pests are obligatory phloem sap feeders, and theyrepresent important prey for beneficial insects such aspredators and parasitoids. For these reasons, the openquestion whether Cry toxins are present in the phloemsap of transgenic plants and whether aphids ingest  Bt -toxin is of great ecological relevance within the task of the evaluation of non-target effects of GM. The presentstudy aimed to evaluate, by laboratory tests, the possibletransfer of   Bt -toxin from a transgenic plant ( Bt -oilseed rape)to a non-target pest,  Myzus persicae  Sulzer (Hemiptera:Aphididae). Materials and methods Plants and insects Transgenic oilseed rape plants ( Brassica napus  L. cv.‘Westar’ lines GT 2-4), expressing a truncated, syntheticversion of the cry1Ac gene from  Bacillus thuringiensis  var. kurstaki  active against Lepidoptera under the constitutive CaMV 35S  promoter (Harper  et al ., 1999), were employed forthis study. Transgenic oilseed rape expressed, as markers,a fluorescence gene ( GFP ) and a kanamycin resistance gene( nptII  ). The corresponding near-isogenic line was employedas control. For the remainder of the manuscript, these twohybrids will be referred to as ( Bt + ) and ( Bt x ). All plantswere planted in plastic pots (15 cm diameter) containing a1:1 (v/v) peat:sand sterile potting mix. Plants were placedin a greenhouse set at 24 + 4  C, 50 + 10 %  relative humidity(RH). Plants were fertilized and irrigated weekly. For allexperiments, plants were used when they had reached aheight of 70 cm (7–10 leaf stage).A stock colony of   M. persicae  was maintained in thelaboratory according to Lanzoni  et al . (2004). Aphid popu-lationswerethentransferredfortheexperimentson( Bt + )and( Bt x ) oilseed rape plants. Two aphid strains were employedin the laboratory tests: the first one (climatic chamberstrain) was reared on ( Bt + ) and ( Bt x ) oilseed rape plantsat ‘Dipartimento di Scienze e Tecnologie Agroambientali’laboratories in separate growth chambers at 20 + 1  C, 50 + 10 %  RH, and L:D 16:8 h photoperiod. The second strain(greenhouse strain) was reared on ( Bt + ) and ( Bt x ) oilseedrape plants at ‘Istituto Nazionale di Apicoltura’, Bolognagreenhouses (mean temperature=25  C, range: 10–35  C;mean RH=60 % , range: 25–100 % ). Bt -toxin analysis Cry1Ac levels in plants and herbivores were determinedusing a double sandwich enzyme-linked immunosorbentassay (ELISA) QuantiPlate kit Cry1Ab/Cry1Ac (EnviroLogixInc. Portland, Maine). Spectrophotometric measurementswere conducted with a microtitre plate reader (LabsystemMultiscan, Dasit, Italy) at 405 nm.Plant expression of Cry1Ac was quantified by analysingleaf material (50 + 3 mg + SD). Leaf pieces from randomlyselected ( Bt + ) and ( Bt x ) oilseed rape plants were weighted,homogenized in a 5 ml extraction buffer, centrifuged and theextract was diluted 1:50 with an extraction buffer.Phloem sap from ( Bt + ) and ( Bt x ) oilseed rape plants wascollected from leaves (two leaves per plant). Leaves were cutand immediately soaked in an EDTA buffer (20 m M ethylendiaminetetraacetate-Na2-salt-dihydrate, pH 7). Aftera 24-h exudation period, samples were frozen at x 80  C untilanalysis. This operation was performed twice.To quantify Cry1Ac toxin in herbivores, protein wasextracted from samples of 200–400 adult aphids reared on( Bt + ) and ( Bt x ) plants, weighted, homogenized in a 200  m lextraction buffer with no dilution and analysed (for sampleweights, see table 1). Bt -toxin concentrations were expressed in ppb ( m gCry1Ac kg x 1 of fresh weight). All samples were centrifugedfor 5 min at 13,000 rpm before they were introduced into theELISA plate. The statistical comparison of the concentrationof Cry1Ac toxin in aphid and phloem sap with the limit of detection of ELISA was carried out by means of a t-test(Berthouex & Brown, 2002). 212  G. Burgio  et al .  Results A mean concentration of 64.3 + 2.9 (mean + SD) ppbof Cry1Ac was found in ( Bt + ) oilseed rape leaves (fig. 1). Bt  toxin was present also in phloem sap, with a meanconcentration of 2.7 + 1.46 ppb (mean + SD), which is 24-foldlower than in leaves. A t-test calculated on the positivesamples above the limit of detection (LOD) (=1.2 ppb)for Cry1Ac reported in the ELISA kit, showed that theconcentration of   Bt -toxin was significantly ( P <0.05) higherthan the LOD.In our analysis, four aphid samples (of a total of fourexamined) fed on  Bt -oilseed rape in the greenhouse, andone aphid sample (of a total of eight examined) fed on  Bt -oilseed rape in the climatic chamber, contained a detectableamount of Cry1Ac (table 1). The concentration of Cry1Acwas 1.9 + 0.8 ppb (mean + SD) for the greenhouse strainand 2.5 + 1.3 ppb (mean + SD) for the climatic chamber strain(fig. 1). The concentration of the toxin in the positive aphidsamples was 2.0 + 0.8 ppb (mean + SD); this value was sig-nificantly higher (t-test,  P <0.05) than the LOD. This value isquite similar to that reported for the concentration of toxin in Bt -oilseed rape phloem sap. Expression of Cry1Ac in theplants on which aphid strains had fed was confirmed. Theseresults seem to corroborate that  Bt -toxin can pass throughfrom  Bt -oilseed rape to aphids. Discussion Phloem sap from  Bt -oilseed rape showed a detect-able amount of Cry1Ac; however, a strong difference in theCry1Ac concentration between leaves and sap was observed.The present results showed that Cry1Ac can be present in Bt -oilseed rape phloem sap, but it is unknown if the toxin isexpressed in phloem tissue or if it is simply translocated inthe sap. Raps  et al . (2001) did not detect Cry1Ab in  Bt -maizesap by microcapillary technique; on the contrary, Cry1Abtoxin was detected by EDTA-method at 1 ppb concentration, but the authors hypothesized that the traces of toxin couldsrcinate from damaged cells (Raps  et al ., 2001). The de-tection of Cry1Ac toxin in phloem from  Bt -oilseed rape inour experiments can not srcinate from damaged cells, as the Table 1. Results of the ELISA test of two  Myzus persicae  strains (G, greenhouse strain; Cc,climatic chamber strain) fed on isogenic ( Bt x ) or transgenic ( Bt + ) oilseed rape.Aphid strain SamplecodeNumber of aphidsAphidweight(mg)ELISAissue m g Cry1Ac kg x 1 fresh weight(ppb)Cc strain fed on ( Bt x ) 109 200 68.8  x  0125 200 60.7  x  0113 200 62.7  x  0117 200 62.9  x  0146 400 116.8  x  0150 445 122.3  x  0164 424 111.5  x  0151 400 104  x  0Cc strain fed on ( Bt + ) 98 200 40  x  0103 200 41.1  x  0106 200 39.6  x  0122 200 44.1  x  0134 400 74.4  x  0129 351 84.8  x  0140 450 130.1  +  2.48139 328 65.5  x  0G strain fed on ( Bt x ) 33 200 76.7  x  041 200 82.9  x  050 200 77.8  x  060 200 65.5  x  0G strain fed on ( Bt + ) 68 200 86.3  +  1.7373 200 95.4  +  3.0584 200 106.8  +  1.1589 200 104.7  +  1.60 110100Leaves Sap Aphids(G)Aphids(Cc)Aphids(pooled)   p  p   b   C  r  y   1   A  c       B      t   -   t  o  x   i  n  +   S   D Fig. 1. Mean ( + SD) Cry1Ac  Bt -toxin concentration ( m g/Kg freshweight) measured in transgenic oilseed rape leaves and sap andin two  Myzus persicae  strains fed on  Bt -oilseed rape(G=Greenhouse strain; Cc=Climatic chamber strain). Bt -toxin uptake by  Myzus persicae  on transgenic oilseed rape  213  exudation from petioles prevents the damage of mesophyllcells. On the other hand, in transgenic tobacco plantsexpressing the snowdrop lectin (GNA) under the control of the  CaMV 35S  promoter, the toxin was detected in phloem,xylem cells, parenchyma cells, mesophyll cells cortex andpith tissue (Shi  et al ., 1994). In the same way, soybean trypsininhibitor expression (SKTI), driven by the  CaMV 35S  pro-moter, enhances the resistance in transgenic potato or riceagainst aphids such as  M. persicae, Aulacorthum solani (Kaltenbach) (Hemiptera: Aphididae) or  Nilaparvata lugens Sta˚l (Hemiptera: Delphacidae), indicating a translocation of toxins from phloem cells to sap (Down  et al ., 1996; Gatehouse et al ., 1996; Lee  et al ., 1999). However, notwithstandingthe control of   CaMV 35S  promoter in the expression of Cry1Ab in maize, no evidence exists of the translocation of Cry1Ab into the phloem sap in maize. One reason could bedue to high Cry1Ab molecular size (65–68 kDa) (Raps  et al .,2001), but this would not explain the transport of the Cry1Actoxin (60 kDa) in oilseed rape phloem sap. Anotherexplanation could be the lack in maize of specific signalsequences for the transport to phloem sap. Concerning thepresent results, specific information about the mechanismundergoing the presence of   Bt -toxin in phloem sap is still to be assessed.Concentrations of Cry1Ac of about 2 ppb in adult  M. persicae , feeding on  Bt -oilseed rape, were easily andreliably detected. The detection of the toxin was appreciablein all greenhouse samples and in 12.5 % of climatic chambersamples. Previous studies about  Bt  effect on non-targetinsects demonstrated that no or little Cry1Ab could bedetected on  Rhopalosiphum padi  (Linnaeus) (Hemiptera:Aphididae) fed on  Bt -maize (Raps  et al ., 2001; Dutton  et al .,2004). However, available studies about the effects of   Bt -crops on aphids are still few. Field tests evidenced no short-term effects of   Bt -maize on aphids, and laboratory testsevidenced the lack of direct toxic effects (Lozzia  et al ., 1998).Raps  et al . (2001) demonstrated that  R. padi , a grainaphid infesting corn, ingested or contained no or very lowconcentrations of Cry1Ab toxin when fed on  Bt -corn; because R. padi  is an important prey for beneficial insects in corn, theauthors concluded that Cry1Ab was unlikely to cause anyharm to the predator guild. Shi  et al . (1994) detected  CaMV 35S  promoter activity in different tissues of transgenictobacco plants, therefore, Cry proteins can also be expectedeven in phloem cells of other  Bt -transformed crops, even if the expression of a protein in a phloem cell does not implythe translocation in phloem sap due to the size and structureof the protein. GNA can be expressed in transgenic tobaccoand rice plants in a phloem specific manner, using a geneconstruct containing the GNA coding sequence driven bythe promoter from the rice sucrose synthase gene  RSs1 (Gatehouse & Gatehouse, 2000). The  RSs1  promoter directsthe expression of a  gus  reporter gene in the phloem tissue of leaves, stems, petioles and roots of transgenic tobaccoplants with no detectable expression in other tissues (Shi et al ., 1994). Transgenic plants containing these constructs areeffective against phloem-feeding homopteran insect pests(Gatehouse & Gatehouse, 2000). Transgenic rice containingan  RSs1 -GNA construct has been shown to accumulate GNAin vascular and epidermal tissue (Sudhakar  et al ., 1998).Expression of GNA from a constitutive promoter (maizeubiquitin) gave similar results (Gatehouse & Gatehouse,2000). However, available information in the literature israther fragmentary, and it has not completely clarified themechanism undergoing the presence of insecticidal toxins inthe phloem sap.The presence of the Cry1Ac toxin in phloem sap from Bt -oilseed rape, expressed under the control of the  CaMV 35S  promoter and in  M. persicae , showed the importance of providing an estimate of the expected environmentalconcentration of Cry1Ac in the diets of non-target organismseating aphids feeding on the transgenic crop. Moreover,our preliminary results improve the knowledge of a trophicsystem not as well-known as  Bt -maize.In the present study, all aphids reared in greenhouseconditions resulted as positive, while aphid colonies rearedin climatic chambers exhibited only 12.5 % positive samples.This difference could be due to environmental conditionsinfluencing either plant physiology or insect behaviour, butthis hypothesis should be investigated in further experi-ments. The present data could improve the knowledge of aspects concerning the transfer of the toxin to organisms belonging to higher trophic levels, including natural enemiesthat are indirectly exposed by feeding on herbivores (Dutton et al ., 2002; Groot & Dicke, 2002; Romeis, 2004; Scholte &Dicke, 2005). Moreover, detailed knowledge of the possiblechronic effects, which involve the uptake of toxin along thefood chain, is considered a basic aspect in the risk-assessment of GM crops (Romeis, 2004; Scholte & Dicke,2005).In conclusion, the evidence that  Bt -toxin is present inherbivores, in this case an aphid, could be useful to clarifyfunctional aspects linked to possible consequences of   Bt -crops on herbivore–natural enemy trophic systems. Besides,transfer of Cry1Ac from  Bt -oilseed rape to aphid increasesexposition of this toxin in the environment, and thispossibility must be taken into account to evaluate thepossible impact of transgenic crops on non-target organisms.Future work should address also bioassay with beneficialspecies (for example predators like coccinellids), includingtests like Western blot to demonstrate if the toxin remainsactive in the aphid. Acknowledgements This work was supported by the Ministero dell’Ambientee della Tutela del Territorio (Direzione generale per lasalvaguardia ambientale). The authors are grateful toFrancesco Cellini and Maria Carola Fiore (MetapontumAgriobios, Matera, Italy) for supplying  Bt  and isogenicoilseed rape seeds, Marcel Dicke (Laboratory of Ento-mology, Wageningen University) for critically reviewing themanuscript and Maria Luisa Dindo (DiSTA-Entomologia,Alma Mater Studiorum-Universita` di Bologna) for usefulsuggestions. 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