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Agriculture, Ecosystems and Environment 179 (2013) 163– 167

Contents lists available at ScienceDirect

Agriculture, Ecosystems and Environment

jo ur nal ho me page: www.elsev ier .com/ locate /agee

t cotton expressing Cry1Ac/Cry2Ab or Cry1Ac/epsps does not harmhe predator Propylaea japonica through its prey Aphis gossypii

ao Zhaoa, Yan Mab, Lin Niua, Weihua Maa, Amani Mannakkaraa,c,izhen Chena,∗, Chaoliang Leia

Hubei Insect Resources Utilization and Sustainable Pest Management Key Laboratory, Huazhong Agricultural University, Wuhan 430070, Hubei, ChinaInstitute of Cotton Research, Chinese Academy of Agricultural Sciences, Anyang, Henan 455000, ChinaDepartment of Agricultural Biology, Faculty of Agriculture, University of Ruhuna, Kamburupitiya 81100, Sri Lanka

r t i c l e i n f o

rticle history:eceived 2 February 2013eceived in revised form 1 August 2013ccepted 3 August 2013vailable online 13 September 2013

eywords:

a b s t r a c t

To assess the ecological effects of Bt-cotton cultivars (ZMSJ and ZMKCKC, expressing Cry1Ac/Cry2Ab andCry1Ac/EPSPS, respectively), laboratory experiments were performed to evaluate the transmission ofCry1Ac protein in the food chain and the effects of Bt toxin on the ladybeetle predator Propylaea japonicathrough its herbivorous prey Aphis gossypii. Trace amounts of Cry1Ac protein (3.0 ng/g fresh mass [FM]in ZMSJ, 3.8 ng/g FM in ZMKCKC) were detected in A. gossypii feeding on Bt cotton cultivars. Cry1Ac pro-tein was also detected in ladybeetles preying on Bt-fed aphids, and its quantity increased as the adult

t cottonphis gossypiiropylaea japonicary1Ac

period extended (5–20 d). However, there were no distinct differences in the total developmental mor-tality, newly emerged adult weights, or fecundity between predators fed Bt-fed or Bt-free aphids. A delayin development was observed when P. japonica was fed A. gossypii reared on both Bt cottons. The pre-oviposition period was significantly longer in the ZMSJ ladybeetles than in the control ladybeetles. Theseresults suggest that the Cry1Ac protein expressed in these transgenic cotton plants can be transmitted

r her

to predators through thei

. Introduction

Cotton is one of the most important crops worldwide. In 2006, itas grown in more than 75 countries, with a total production of 27illion t, and supplied almost 40% of the global demand for fiber

Naranjo et al., 2008; Naranjo, 2010). Cotton varieties expressingry proteins derived from the soil bacterium Bacillus thuringien-is (Bt) are now grown worldwide to control cotton bollworm andeduce the need for insecticide sprays (Lu et al., 2012). Before these of Bt cotton, cotton crops accounted for an estimated 22.5% ofhe total insecticide used worldwide (Naranjo et al., 2008; Naranjo,010). Since 1996, genetically modified insect-resistant cotton pro-uction has resulted in a 21.8% reduction in the application ofhe active ingredients of insecticides in cotton fields worldwideBrookes and Barfoot, 2011).

Although Bt cotton provides an effective tool for controlling Lep-doptera pests in cotton fields (Liu et al., 2010), it may pose potentialhreats to other non-target herbivorous insects and their natural

nemies (Baur and Boethel, 2003; Liu et al., 2002; Wu and Guo,003; Zhang et al., 2006b).

∗ Corresponding author. Tel.: +86 02787287207; fax: +86 02787287207.E-mail address: lzchen@mail.hzau.edu.cn (L. Chen).

167-8809/$ – see front matter © 2013 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.agee.2013.08.005

bivorous prey, but they do not affect their biology.© 2013 Elsevier B.V. All rights reserved.

The ladybeetle, Propylaea japonica (Coleoptera: Coccinellidae),is native to China and feeds on aphids, thrips, spider mites, eggsand young larval Lepidoptera (Fang and Zhang, 1998). Because P.japonica fills an important ecological role, several studies of theeffects of Bt cotton on P. japonica have been undertaken (Zhanget al., 2006a,b,c; Zhu et al., 2006). For example, the survival, devel-opment, longevity, reproduction, and weight of P. japonica did notdiffer when it was supplied with the aphid Aphis gossypii fed oneither transgenic or non-transgenic cotton (Zhu et al., 2006). Nosignificant differences were found in preimaginal mortality, thepreoviposition period, or fecundity between P. japonica reared onBt-fed or Bt-free A. gossypii, but P. japonica that preyed on Bt-reared aphids developed faster in the preimaginal stages, maturedfaster, and mated more frequently than those fed with Bt-freeaphids (Zhang et al., 2006b). These studies focused predominantlyon Bt cotton expressing Cry1Ac or Cry1Ac/Cry1Ab. No studieshave been conducted on the potential effects of Cry1Ac/Cry2Ab-or Cry1Ac/EPSPS-expressing Bt cotton on the predator P. japon-ica. The Bt toxins (Cry1Ac and Cry2Ab) target Lepidoptera pests(Li et al., 2007), and the EPSPS gene makes the plants toler-ant to the herbicide glyphosate (Liu et al., 2007; Padgette et al.,

1995). The two cotton cultivars (Cry1Ac/Cry2Ab and Cry1Ac/EPSPS)assessed in our study have not been promoted in China, so thisrisk assessment before their commercial release is very impor-tant.

1 s and Environment 179 (2013) 163– 167

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Table 1Mean ± SE Cry1Ac protein content in tender tip of Bt cotton and A. gossypii fed on Btcotton.

Cotton cultivar Cry1Ac protein content (ng/g FM)

Tender tip of cotton plants Aphis gossypii

Emian 24 ND NDZMSJ 99.9 ± 5.7a 3.0 ± 0.5aZMKCKC 110.7 ± 5.3a 3.8 ± 0.5a

64 Y. Zhao et al. / Agriculture, Ecosystem

The work reported here had three objectives: (1) to verify anduantify the presence of Cry1Ac protein in A. gossypii when fed ont cotton; (2) to verify and quantify the presence of Cry1Ac protein

n P. japonica when fed with Bt-cotton-reared aphids; and (3) tovaluate the effects of Bt cotton on P. japonica through its prey A.ossypii.

. Materials and methods

Two transgenic Bt cotton cultivars, ZMSJ (expressingry1Ac/Cry2Ab) and ZMKCKC (expressing Cry1Ac/EPSPS), aift from the Institute of Cotton Research, Chinese Academy ofgricultural Science, together with a local cotton variety, Emian4 (non-Bt cotton), a gift from the National Key Laboratory of Cropenetic Improvement, Huazhong Agricultural University, weresed in this study. Three cotton seeds of each cultivar were sown

n pots (plastic pots, 140 mm diameter and 100 mm tall) filledith humus-rich sterilized soil. Ten pots were set for each cultivar,

nd 15 d later, a new lot of cotton seeds were planted. The Bt andon-Bt cultivars were kept in different greenhouses set at 24 ± 1 ◦C,

n 70 ± 10% relative humidity (RH), and a 16:8 h light:dark cycleL:D).

The cotton aphids used in this study originated from adults andymphs collected in a non-Bt cotton field located at the Huazhonggricultural University in July 2011. The cotton aphids were rearedn the three cotton cultivars in greenhouses under conditions of4 ± 1 ◦C, 70 ± 10% RH, and 16:8 L:D. The ladybeetles used in thistudy originated from adults collected in a non-Bt maize fieldocated in the Huazhong Agricultural University, in May 2011.wo adult pairs were placed in each insect-rearing jar (cylindrical,5 mm diameter, 90 mm tall), fed with Acyrthosiphon pisum, andeared at 25 ± 1 ◦C, 70 ± 10% RH, and 16:8 L:D.

The concentrations of Cry1Ac in the cotton leaves, A. gossypii,nd P. japonica were measured with an enzyme-linked immuno-orbent assay (ELISA) using the Envirologix Qualiplate KitEnviroLogix Inc., Portland, ME, USA). Absorbance was measured at50 nm with a microtiter plate reader (Chromate 4300, Awarenessechnology, Inc., Palm City, FL, USA). The measured absorbance val-es were calibrated to a range of Cry1Ac concentrations preparedith a purified protein solution.

To quantify the Cry1Ac protein in the cotton leaves, five plantsf each cotton cultivar were selected randomly at the five- toeven-leaf stage, and 200 mg pieces from the tender tips (2–3 unde-eloped leaves per tip, each 20–30 mm in diameter) of each plantere homogenized in 1 ml of extract buffer (provided with the

it), resulting in a 1:5 (sample [mg]:buffer [�l]) dilution, whichas centrifuged at 12,000 r.p.m. for 10 min. The supernatant wasiluted 1:100 before it was transferred to the ELISA plates. Touantify the Cry1Ac protein in A. gossypii, five samples (about 500phids per sample) that had been reared on each cotton cultivar for0–50 d were collected. Each sample was checked under a micro-cope to confirm that it was not contaminated with other pestse.g., spider mites or thrips) or leaf pieces. Each sample was theneighed, frozen in liquid nitrogen, and stored at −80 ◦C. Each sam-le (50.11 ± 0.03 [SE] mg) was homogenized in 0.5 ml of extractuffer, resulting in a 1:10 dilution, and centrifuged as above. Thendiluted supernatant (50 �l/well) was transferred to an ELISAlate.

To evaluate the Cry1Ac protein in P. japonica, newly hatched. japonica larvae were fed with aphids reared on each cottonultivar until they developed into adults. Samples were collected

, 10, 15, and 20 d. after emergence, and 10 adults (five femalesnd five males) from each treatment were weighed individually,hen frozen in liquid nitrogen and stored at −80 ◦C. Single adults6.09 ± 0.09 mg) were homogenized in 0.3 ml of extract buffer,

The minimum detectable quantity of Cry1Ac protein of the ELISA kits is 0.1 ng/g FM.ND: not detected.

resulting in a 1:50 dilution, and centrifuged as above. The undilutedsupernatants (50 �l/well) were transferred to ELISA plates.

The newly hatched coccinellid larvae were fed with the aphidsreared on each cotton cultivar for 30–50 d. The young larvae wereindividually reared in glass tubes (cylindrical, 20 mm diameter,80 mm tall, sealed with cotton gauze) at 25 ± 1 ◦C, 70 ± 10% RH, and16:8 L:D. Sixty P. japonica larvae from each cotton cultivar were fed.The P. japonica larvae were examined twice daily (09:00 and 21:00)and their developmental stages and mortality recorded.

When the adults emerged, their sexes were determined, andthey were weighed individually. A male and a female from the samecotton cultivar were paired randomly in an insect-rearing jar andallowed to mate (10 replicates per treatment). They were similarlyfed with the aphids of each cotton cultivar. The length (d) of thepreoviposition period and the number of eggs laid by each femalewere recorded daily. The eggs laid were removed every day.

2.1. Data analysis

Statistical analyses were performed using SPSS 17.0 (SPSS Inc.,Chicago, IL, USA.). Student’s t test was used to analyze the differ-ences in the amounts of Cry1Ac protein in the tender tips of thethree cotton cultivars and in the A. gossypii reared on each cot-ton cultivar. One-way ANOVA and LSD tests were used to analyzethe Cry1Ac protein contents in different day-age adults, the pre-oviposition periods, the weights of newly emerged adult, and thenumbers of eggs laid by individual females in the first 20 d of theegg-laying period. Nonparametric tests (K independent samples:Kruskal–Wallis H test; two independent samples: Mann–WhitneyU test) were used to compare the developmental periods of lady-beetle larvae and pupae. �2 tests were used to compare the totaldevelopmental mortality rates.

3. Results

On average, 99.9 ng Cry1Ac/g plant fresh mass (FM) and 110.7 ng Cry1Ac/g plantFM were detected in the tender tips of cottons ZMSJ and ZMKCKC, respectively(Table 1). ZMSJ A. gossypii and ZMKCKC A. gossypii contained 3.0 ng/g FM and 3.8 ng/gFM of Cry1Ac protein, respectively. No Cry1Ac protein was detected in the tendertips of the control cotton Emian 24 or in A. gossypii reared on this cultivar.

The Cry1Ac protein passed into the predator P. japonica through A. gossypii rearedon Bt cotton. From 5 to 20 d, the amount of Cry1Ac protein increased from 6.3 ng/gpredator FM to 18.7 ng/g predator FM when the adult was fed with ZMSJ aphids,and from 8.9 to 16.8 ng/g predator FM when the adult was fed with ZMKCKC aphids(Fig. 1). No Cry1Ac protein was detected when the adults were fed control aphids.

3.1. Effects of Bt cotton on the development and reproduction of P. japonica

The P. japonica parameters of total developmental mortality (�2 = 1.273,P = 0.529) and newly emerged adult weight (females: F2,85 = 1.743, P = 0.181; males:F2,78 = 1.472, P = 0.236) were not affected significantly compared with the control(Table 2). However, the developmental periods of the ZMSJ and ZMKCKC ladybeetlelarvae were significantly longer than the control period (Mann–Whitney U tests:P < 0.001 and P < 0.001) (Table 2). Similarly, the ZMSJ and ZMKCKC ladybeetle pupae

persisted for significantly more days than the control pupae (Mann–Whitney U tests:P < 0.001 and P < 0.001, respectively; Table 2). The ZMSJ ladybeetles experienced asignificantly longer preoviposition period than the control ladybeetles (F1,18 = 6.486;P = 0.02), but no significant differences were observed between the ZMKCKC lady-beetles and the controls (F1,18 = 2.227; P = 0.153; Table 2). The fecundity of the control

Y. Zhao et al. / Agriculture, Ecosystems and Environment 179 (2013) 163– 167 165

Table 2Effects of non-Bt versus Bt cotton on developmental, weight and reproductive characteristics of P. japonica.

Cotton cultivar Development duration (d) Total developmentalmortality (%)

Weight Preoviposition period (d) Fecundity (no. of eggs/♀)

Larval stage Pupa Females Males

Emian 24 8.1 ± 0.1a 3.6 ± 0.1a 8.3%a 4.8 ± 0.1a 4.2 ± 0.1a 4.8 ± 0.2b 33.1 ± 3.2abZMSJ 8.8 ± 0.1b 4 ± 0.1b 6.7%a 4.5 ± 0.1a 4.0 ± 0.1a 5.6 ± 0.3a 25.7 ± 2.5bZMKCKC 8.7 ± 0.1b 4 ± 0.1b 3.3%a 4.7 ± 0.1a 4.0 ± 0.1a 5.3 ± 0.3ab 38.5 ± 3.1a

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ecundity = numbers of eggs produced by the adult during its egg laying peak perio

adybeetles did not differ from that of the ZMSJ ladybeetles (F1,18 = 3.314, P = 0.085)r the ZMKCKC ladybeetles (F1,18 = 1.5, P = 0.236), but the fecundity of the ZMKCKCadybeetles was significantly higher than that of the ZMSJ ladybeetles (F1,18 = 10.307,

= 0.005; Table 2).

. Discussion

Because A. gossypii mainly feeds on cotton leaves, the levels ofry proteins in the leaves are an important factor. In our study,ry1Ac protein was detected at concentrations of 99.9 ng/g and10.7 ng/g in the tender tips of the ZMSJ and ZMKCKC cotton plants,espectively (Table 1). The protein levels were similar to thosef several other studies (138.2 ng/g in Zhang et al., 2006a, and4.2 ng/g in Zhang et al., 2006b), but lower than those in othertudies (340, 620, and 380 ng/g in Lawo et al., 2009, and 240 ng/gn Torres et al., 2006).

The Cry1Ac protein was taken up by A. gossypii from the trans-enic cottons, and was detected in the aphids by ELISA (3.0 ng/gM in ZMSJ, 3.8 ng/g FM in ZMKCKC; Table 1). Previous studies ofnother Bt (Cry1Ac) cotton revealed similar protein levels (4.0 ng/gM) in A. gossypii (Zhang et al., 2006b). In other studies of Bt maize,uch higher toxin concentrations were found in the herbivores.

he Cry3Bb1 concentration in spider mites Tetranychus urticae fedn Bt maize was 21 �g/g (Li and Romeis, 2010). The concentra-ions of Cry1Ab and Cry3Bb1 in Bt-maize-reared T. urticae were 4.7nd 19.9 �g/g, respectively (Alvarez-Alfageme et al., 2011), and thery1Ab concentrations in three herbivores (Rhopalosiphum padi,podoptera littoralis, and T. urticae) fed on Bt maize were 0.02,.72, and 2.5 �g/g (Dutton et al., 2002). The Cry1Ab concentration

n Bt-maize-reared T. urticae was 1.28 �g/g (García et al., 2010),

ut several studies of other Bt crops detected no Bt protein inerbivores (Lawo et al., 2009; Meissle and Romeis, 2009; Ramirez-omero et al., 2008; Torres et al., 2006). Romeis and Meissle (2011)rgued that low concentrations of Bt Cry proteins in aphids can be

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ig. 1. Mean ± SE Cry1Ac protein content in 5, 10, 15 and 20 d old P. japonica adultsA5, A10, A15, A20) fed with A. gossypii reared on Bt or non-Bt cotton plants through-ut the entire adult life from hatching. Data within same group followed by differentetters are significantly different at P < 0.05 (one-way ANOVA, LSD tests). n.d., notetected. The minimum detectable quantity of Cry1Ac protein of the ELISA kits is.1 ng/g FM.

0.05.e first 20 d.

explained by the contamination of the samples. However, becausethe aphids used in our study were checked under a microscope, itis unlikely that the samples were contaminated with spider mitesor thrips.

In our study, the longer the ladybeetle consumed Bt aphids, themore toxin it accumulated in its body. The amounts of Cry1Acprotein detected in ZMSJ and ZMKCKC P. japonica adults were6.3–18.7 ng/g and 8.9–16.8 ng/g, respectively (Fig. 1). In anotherstudy, similar levels of Cry1Ac toxin were detected in ladybee-tles (9.0–24.0 ng/g) fed Bt-fed aphids, and the quantities increasedas the predatory period increased (5–20 d) (Zhang et al., 2006b).The similar Cry1Ac contents in ZMSJ and ZMKCKC ladybeetles maybe attributable to the similar Cry1Ac contents of the ZMSJ andZMKCKC cotton leaves. In another study, 10-d-old Poecilus cupreuslarvae contained the highest Cry1Ab concentration (79.4 ng/g FM),whereas older larvae (20-d-old larvae and 33–43-d-old larvae) hadconcentrations of 34.8 and 42.2 ng/g FM, respectively, when thelarvae were fed with S. littoralis reared on Bt maize (Meissle et al.,2005). Li and Romeis (2010) measured the Cry3Bb1 concentrationsafter adult Stethorus punctillum beetles had consumed Bt-maize-fedmites (T. urticae) for 5, 10, or 15 d, and much higher concentra-tions were found as the toxin accumulated. In their study, theadults that received Bt-maize-fed spider mites for 5, 10, or 15 d dis-played Cry3Bb1 concentrations ranging from 0.92 to 1.06 �g/g FMin females and from 0.08 to 0.12 �g/g FM in males (Li and Romeis,2010). The Cry1Ab concentration in Atheta coriaria adults fed Bt-maize-raised T. urticae was 0.21 �g/g FM (García et al., 2010).

P. japonica fed on Bt-reared aphids and the control P. japonica didnot differ in their total developmental mortality, newly emergedadult weights, or fecundity. However, the lengths of the develop-ment periods of the Bt cotton ladybeetle larvae and pupae weresignificantly longer than those of the control ladybeetles. Similarly,a developmental delay was observed when the predator Chrysop-erla carnea was fed S. littoralis larvae reared on Bt (Cry1Ab) maize(Dutton et al., 2002). The development of C. sinica was also delayedwhen they were fed Bemisia tabaci reared on Bt (Cry1Ac) cotton(Guo et al., 2004). On the contrary, Zhang et al. (2006b) reported thatthe preimaginal stages of the ladybeetle P. japonica developed fasterwhen fed A. gossypii reared on either the Cry1Ac or Cry1Ac/Cry1AbBt cotton cultivar than those fed control aphids. The nymphal devel-opment of Orius majusculus was shorter when the nymphs were fedon Bt (Cry1Ab)-containing spider mite T. urticae (Lumbierres et al.,2012). In our study, the fecundity of P. japonica was lowest on theZMSJ cultivar, and highest on the ZMKCKC cultivar. Peach-potatoaphids Myzus persicae maintained on transgenic potatoes express-ing Galanthus nivalis agglutinin (a snowdrop lectin that makes themresistant to aphids) caused significantly reduced fecundity whenfed to the two-spot ladybeetle Adalia bipunctata (Birch et al., 1999).Li and Romeis (2010) reported that female S. punctillum beetles that

were fed T. urticae from Bt (Cry3Bb1) maize had increased fecun-dity and increased fertility relative to those of females fed spidermites from non-Bt maize. Our results show that female ZMSJ lady-beetles experienced a longer preoviposition period than the control

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66 Y. Zhao et al. / Agriculture, Ecosystem

adybeetles. In contrast, the female ladybeetle S. punctillum had ahorter preoviposition period when fed the spider mite T. urticaeeared on Bt (Cry3Bb1) maize (Li and Romeis, 2010). The combinednteraction of poor prey quality and an exogenous gene (Cry1Ac,ry2Ab, or EPSPS) may account for the negative effects observed inur experiment. Previous research has indicated that plant allelo-hemicals can affect the nutritional suitability of herbivores, withotential effects on the development, survivorship, body weight,nd fecundity of predators at the third trophic level of the foodhain (Hauge et al., 1998; Francis et al., 2000; Du et al., 2004). Aignificantly shorter larval period and greater adult weight haveeen observed in P. japonica fed aphids reared on the high-gossypolultivar than in those fed aphids reared on the two cultivars withower gossypol contents (Du et al., 2004). The differences observedn our study may also be attributable to cotton cultivars with dif-erent gossypol contents. Furthermore, some unintended changesave been detected in transgenic varieties, including differences inhe concentrations and chirality of some amino acids (Faria et al.,007; Herrero et al., 2007), and changes in the total nitrogen con-ent and in the C–N relationship (Manetti et al., 2006; Griffiths et al.,007). Considering the different plant characteristics of the threeotton cultivars used in our study, all the factors discussed aboveay have exerted negative effects on the ladybeetles.Many other studies of predatory beetles through a tritrophic

hain have found no adverse effects of Bt toxin. For instance, Bt pota-oes caused no adverse effects on the survival, aphid consumption,evelopment, or reproduction of the aphidophagous ladybeetle,ippodamia convergens, feeding on M. persicae reared on Bt potato

Dogan et al., 1996). Larval survival and development, adult sur-ival, and adult dry weight did not differ in S. punctillum fed withhe spider mite T. urticae reared on Bt (Cry3Bb1) or non-Bt maize forver two months (Li and Romeis, 2010). The mortality, weight, andevelopment time of A. bipunctata larvae were not affected whenhe larvae were fed T. urticae reared on Bt (Cry1Ab/Cry3Bb1) maizeAlvarez-Alfageme et al., 2011). Furthermore, the survival, develop-

ent time, adult weight, and fecundity of Coleomegilla maculata didot differ significantly when they were fed with resistant Trichoplu-ia ni larvae reared on either Bt (Cry1Ac/Cry2Ab) or control cottonLi et al., 2011). These results also show that C. maculata is not sensi-ive to Cry1Ac and Cry2Ab at concentrations exceeding the levels int cotton, demonstrating that Bt cotton will pose a negligible risk to. maculata (Li et al., 2011). Nevertheless, it has been reported that

ndirect effects attributable to a reduction in the nutritional qualityf the prey may occur, as in P. cupreus fed with S. littoralis larvaeaised on Bt (Cry1Ab) maize, which experienced higher mortalityhan those fed with larvae raised on conventional maize (Meisslet al., 2005). Stephens et al. (2012) have also reported that Har-onia axyridis can be affected indirectly by Bt (Cry3Bb) maize via

hopalosiphum maidis. They found that the lifespan of H. axyridised on field-collected aphids from Bt (Cry3Bb) maize was reducedy 38% compared with the lifespan of those fed aphids from controlaize (Stephens et al., 2012).Our results indicate that the two transgenic cotton cultivars had

o detrimental effect on the biology of an important natural enemy. japonica, but whether the Bt protein is passed on to the ladybee-le’s progeny or becomes widespread remains unclear. Zhang et al.2006b) reported that small amounts of Bt protein were found inewly hatched, unfed coccinellid larvae when their parents were

ed Bt (Cry1Ac)-cotton-reared aphids (15.0 ng/g FM), but not whenhe parents were fed another Bt (Cry1Ac/1Ab)-cotton-reared prey.

cknowledgements

This research is founded by the National Special Transgenicroject (Project Number: 2013ZX08011-002) from the Chinese

Environment 179 (2013) 163– 167

Ministry of Agriculture. We thank Jie Li for her help during thecourse of these experiments.

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