A Comprehensive Assessment of the Effects of Bt Cotton on Coleomegilla maculata Demonstrates No Detrimental Effects by Cry1Ac and Cry2Ab

The ladybird beetle, Coleomegilla maculata (DeGeer), is a common and abundant predator in many cropping systems. Its larvae and adults are predaceous, feeding on aphids, thrips, lepidopteran larvae and plant tissues, such as pollen. Therefore, this species is exposed to insecticidal proteins expressed in insect-resistant, genetically engineered cotton expressing Cry proteins derived from Bacillus thuringiensis (Bt). A tritrophic bioassay was conduced to evaluate the potential impact of Cry2Ab- and Cry1Ac-expressing cotton on fitness parameters of C. maculata using Bt-susceptible and -resistant larvae of Trichoplusia ni as prey. Coleomegilla maculata survival, development time, adult weight and fecundity were not different when they were fed with resistant T. ni larvae reared on either Bt or control cotton. To ensure that C. maculata were not sensitive to the tested Cry toxins independent from the plant background and to add certainty to the hazard assessment, C. maculata larvae were fed artificial diet incorporated with Cry2Ab, Cry1Ac or both at >10 times higher concentrations than in cotton tissue. Artificial diet containing E-64 was included as a positive control. No differences were detected in any life-table parameters between Cry protein-containing diet treatments and the control diet. In contrast, larvae of C. maculata fed the E-64 could not develop to the pupal stage and the 7-d larval weight was significantly negatively affected. In both feeding assays, the stability and bioactivity of Cry proteins in the food sources were confirmed by ELISA and sensitive-insect bioassays. Our results show that C. maculata is not affected by Bt cotton and is not sensitive to Cry2Ab and Cry1Ac at concentrations exceeding the levels in Bt cotton, thus demonstrating that Bt cotton will pose a negligible risk to C. maculata. More importantly, this study demonstrates a comprehensive system for assessing the risk of genetically modified plants on non-target organisms.     

Toxicological assessment of pollen from different bt rice lines on Bombyx mori (Lepidoptera: Bombyxidae)

The relative toxicity of Bt rice pollen to domestic silkworm, Bombyx mori Linnaeus (Lepidoptera: Bombycidae), was assessed by a leaf-dip bioassay under laboratory conditions. Silkworm first instars were sensitive to pollen from Bt rice lines, B1 and KMD1, but were not sensitive to pollen from Bt rice line TT9-3. First instars were 1.34-2.12 times more sensitive to B1 pollen than older instars. Bioassays of subacute toxicity under a worst-case scenario suggested that continuous exposure to a sublethal dose of B1 pollen or equivalent doses of non-Bt rice pollen affected silkworm survival and development. Young larvae were more affected by continuous exposure to Bt pollen than older larvae but less affected by non-Bt pollen. Ultrastructural observations showed that Cry proteins associated with Bt pollen were released into the larval lumen and resulted in pathological midgut changes and negative impacts on silkworm survival and development. However, considering that the sublethal dose of Bt pollen (LC15) used in this study is equivalent to the highest detected density of rice pollen on mulberry leaf under field conditions and that the likelihood of such high density of rice pollen occurring in the fields is extremely low, we suggest that the risk of Bt rice pollen on silkworm rearing is negligible.     

Use of maize pollen by adult Chrysoperla carnea (Neuroptera: Chrysopidae) and fate of Cry proteins in Bt-transgenic varieties

We investigated the use of maize pollen as food by adult Chrysoperla carnea under laboratory and field conditions. Exposure of the insects to insecticidal Cry proteins from Bacillus thuringiensis (Bt) contained in pollen of transgenic maize was also assessed. Female C. carnea were most abundant in a maize field when the majority of plants were flowering and fresh pollen was abundant. Field-collected females contained an average of approximately 5000 maize pollen grains in their gut at the peak of pollen shedding. Comparable numbers were found in females fed ad libitum maize pollen in the laboratory. Maize pollen is readily used by C. carnea adults. When provided with a carbohydrate source, it allowed the insects to reach their full reproductive potential. Maize pollen was digested mainly in the insect's mid- and hindgut. When Bt maize pollen passed though the gut of C. carnea, 61% of Cry1Ab (event Bt176) and 79% of Cry3Bb1 (event MON 88017) was digested. The results demonstrate that maize pollen is a suitable food source for C. carnea. Even though the pollen grains are not fully digested, the insects are exposed to transgenic insecticidal proteins that are contained in the pollen.     

A Comprehensive Assessment of the Effects of Transgenic Cry1Ac/Cry1Ab Rice Huahui 1 on Adult Micraspis discolor (Fabricius) (Coleoptera: Coccinellidae)

Micraspis discolor (Fabricius) (Coleoptera: Coccinellidae) is a widely distributed coleoptera predator in southern Asia in rice ecosystem, and adult M. discolor feed on both rice pollen and soft-bodied arthropods. Bitrophic bioassay and tritrophic bioassay were conducted to evaluate the potential impact of Cry1Ac/Cry1Ab-expressing rice Huahui 1 and its non-transgenic counterpart Minghui 63 on fitness parameters of adult M. discolor. The results showed that the survival, and fecundity of this beetle’ adults were not different when they fed on Bt rice or non-Bt rice pollen or Nilaparvata lugens (Stål) reared on Bt rice or non-Bt rice. Toxicity assessment to ensure M. discolor adults were not sensitive to Cry1Ab or Cry1Ac protein independent from the pollen background, M. discolor adults were fed with an artificial diet containing Cry1Ac, Cry1Ab or both protein approximately 10 times higher concentration than in Huahui 1 rice pollen. No difference was detected for any of the life-table parameters tested between Cry protein-containing and pure diet. Artificial diet containing E-64 (N-(trans-Epoxysuccinyl)-L-leucine 4-guanidinobutylamide) was included as a positive control. In contrast, the pre-oviposition and fecundity of M. discolor were significantly adversely affected by feeding on E-64-containing diet. In both bioassays, the uptakes of Cry protein by adult M. discolor were tested by ELISA measurements. These results indicated that adults of M. discolor are not affected by Cry1Ab- or Cry1Ac-expressing rice pollen and are not sensitive to Cry protein at concentrations exceeding the levels in rice pollen in Huahui1. This suggests that M. discolor adults would not be harmed by Cry1Ac/Cry1Ab rice if Bt rice Huahui 1 were commercialized.     

Risk assessment system establishment for evaluating the potential impacts of imported Bacillus thuringiensis maize on a non-target insect,Tenebrio molitor

Large amounts of genetically modified grains producing Bacillus thuringiensis (Bt) toxins have been imported to Korea. Therefore, the establishment of a risk assessment system for evaluating the potential impacts of imported Bt maize on non-target insects is important. Before evaluating the environmental impacts of Bt grains of unknown origin, Cry protein types must first be identified in test Bt grains. Cry toxins of imported Bt maize grains were analyzed by ELISA. Because all tested Bt maize grains contained Cry1A, Tenebrio molitor, a non-lepidopteran species, was selected as the non-target insect species. A domestic maize strain that showed few differences in nutritional composition compared to the Bt maize grain was used as the alternative non-Bt control. Slightly increased survival rate and head capsule width of Bt maize-fed T. molitor were observed, indicating that Bt maize has no sub-chronic adverse effects on T. molitor. An ELISA test revealed that concentrations of Cry1A toxins slowly increased in the body of T. molitor when the insects were fed Bt maize. Such substantial amounts of Cry toxins remaining in the alimentary tract of larvae indicate that Cry toxins can be transferred to the higher trophic level of predatory insects. However, no Cry proteins were detected in the hemolymph of the Bt maize-fed larvae, suggesting that there is little possibility of Cry toxin exposure via T. molitor to the higher endoparasitoids. The risk assessment strategies and protocols established in this study may also be applicable to other imported Bt crops in Korea.     

Laboratory toxicity studies demonstrate no adverse effects of Cry1Ab and Cry3Bb1 to larvae of <Emphasis Type="Italic">Adalia bipunctata</Emphasis> (Coleoptera: Coccinellidae): the importance of study design

Scientific studies are frequently used to support policy decisions related to transgenic crops. Schmidt et al., Arch Environ Contam Toxicol 56:221–228 (2009) recently reported that Cry1Ab and Cry3Bb were toxic to larvae of Adalia bipunctata in direct feeding studies. This study was quoted, among others, to justify the ban of Bt maize (MON 810) in Germany. The study has subsequently been criticized because of methodological shortcomings that make it questionable whether the observed effects were due to direct toxicity of the two Cry proteins. We therefore conducted tritrophic studies assessing whether an effect of the two proteins on A. bipunctata could be detected under more realistic routes of exposure. Spider mites that had fed on Bt maize (events MON810 and MON88017) were used as carriers to expose young A. bipunctata larvae to high doses of biologically active Cry1Ab and Cry3Bb1. Ingestion of the two Cry proteins by A. bipunctata did not affect larval mortality, weight, or development time. These results were confirmed in a subsequent experiment in which A. bipunctata were directly fed with a sucrose solution containing dissolved purified proteins at concentrations approximately 10 times higher than measured in Bt maize-fed spider mites. Hence, our study does not provide any evidence that larvae of A. bipunctata are sensitive to Cry1Ab and Cry3Bb1 or that Bt maize expressing these proteins would adversely affect this predator. The results suggest that the apparent harmful effects of Cry1Ab and Cry3Bb1 reported by Schmidt et al., Arch Environ Contam Toxicol 56:221–228 (2009) were artifacts of poor study design and procedures. It is thus important that decision-makers evaluate the quality of individual scientific studies and do not view all as equally rigorous and relevant.     

Levels of Cry1Ac1 protein in herbivorous and predatory arthropods in fields of Bacillus thuringiensis cabbage

To investigate the extent of exposure and routes of Cry1Ac1 protein through the food chain, we collected Bt cabbage leaves and arthropods that occurred in the field during two trials. Protein levels in the transgenic leaves were significantly higher during the early stages of plant growth, ranging from 209.1 to 553.6?ng?g^?1 in spring and from 208.2 to 402.8?ng?g^?1 in autumn. Enzyme-linked immunosorbent assays were used to measure protein levels in the arthropods. Among the 16 taxa collected in the field, Cry1Ac1 was detected in the bodies of 10. Concentrations were higher in lepidopteran larvae than in the other taxa. In particular, we found a significant correlation between Cry1Ac1 protein levels in cabbage leaves and in Pieris rapae and Mamestra brassicae. Furthermore, this protein was detected in five out of nine taxa of predators (spiders and coleopterans) and parasitoids. These results will be useful as we identify the arthropods that are directly or indirectly exposed to Bt toxin within the food web and the degree to which they are exposed during the cultivation of Bt cabbage.     

A proteomic approach to study the mechanism of tolerance to Bt toxins in Ostrinia furnacalis larvae selected for resistance to Cry1Ab

A Cry1Ab-resistant population of Asian corn borer (ACB-AbR) exhibiting approximately 100 times greater resistance to activated Cry1Ab than a susceptible population (Ostrinia furnacalis; ACB-BtS), was previously shown to exhibit high levels of cross-resistance to Cry1Ah (131-fold), but no cross-resistance to Cry1Ie. It was suggested that the proposed mechanism of resistance was due to the alteration of specific receptors for Cry toxins in the midgut brush border membrane. In the present study a proteomic-based approach was used to identify proteins from brush border membrane vesicles (isolated from both resistant and susceptible Ostrinia furnacalis larvae) interacting with biotinylated Cry1Ab, Cry1Ah, and Cry1Ie. 2D-Electrophoresis in combination with ligand blots were employed and putative protein identities obtained using MALDI-ToF/ToF mass spectrometry. The V-type proton ATPase catalytic subunit A and heat shock 70 kDa proteins were identified as interacting with the Cry toxins tested in the ACB-AbR and ACB-BtS larvae. The biotinylated Cry toxins showed markedly stronger interactions with proteins in the resistant compared to the susceptible larvae, suggesting an up-regulation of the V-type proton ATPase catalytic subunit A and heat shock 70 kDa proteins in the resistant (ACB-AbR) larvae. Interestingly, Cry1Ie interactions with the V-type proton ATPase catalytic subunit A in the ACB-BtS larvae appeared to be absent.     

Transgenic Cry1Ab rice does not impact ecological fitness and predation of a generalist spider

Background

The commercial release of rice genetically engineered to express a Cry1Ab protein from Bacillus thuringiensis (Bt) for control of Lepidoptera in China is a subject of debate. One major point of the debate has focused on the ecological safety of Bt rice on nontarget organisms, especially predators and parasitoids that help control populations of insect pests.

Methodology/Principal Findings

A tritrophic bioassay was conducted to evaluate the potential impact of Cry1Ab-expressing rice on fitness parameters of a predaceous ground spider (Pardosa pseudoannulata (Bösenberg et Strand)) that had fed on Bt rice-fed brown planthopper (Nilaparvata lugens (Stål)) nymphs. Survival, development time and fecundity of this spider were not different when they were fed with Bt rice-fed or non-Bt rice-fed prey. Furthermore, ELISA and PCR gut assays, as well as a functional response trial, indicated that predation by P. pseudoannulata was not significantly different in Bt rice or non-Bt rice fields.

Conclusions/Significance

The transgenic Cry1Ab rice lines tested in this study had no adverse effects on the survival, developmental time and fecundity of P. pseudoannulata in the laboratory or on predation under field conditions. This suggests that this important predator would not be harmed if transgenic Cry1Ab rice were commercialized.     

Bt水稻"克螟稻”花粉对家蚕生长发育的影响

本试验以家蚕为供试对象研究Bt水稻"克螟稻”花粉对家蚕生长发育的影响.结果发现,与正常无花粉处理相比,无论是非Bt水稻花粉处理,还是Bt水稻花粉处理,对初孵家蚕幼虫的致死率无多大影响,而对家蚕的体重有较大影响,其中三龄期家蚕体重存在极显著差异.还发现Bt水稻花粉处理组家蚕在3龄时期大小很不一致,最轻体重为18.1mg,而最重体重为183.8mg.这是由于采用人工抖粉桑叶上花粉浓度不均匀造成的.鉴于实际生态条件下桑叶上的花粉浓度可能远远低于试验条件,因此,在实际稻桑共作环境下,Bt水稻"克螟稻”花粉对家蚕生长发育可能不会造成太大的影响。 Abstract:The effect on the development of silkworm larvae of Bt transgenic rice pollen containing cry1 Ab gene from Bacillus thuringiensis were investigated.Compared with normal treatment,mortality of newly hatched silkworm lar vae in either Bt rice pollen or susceptible rice pollen treatment were not significantly different,while the variances of silkworm larvae weight at third instar were significant at 0.01level.In addition,the weight of each silkworm larva at third instar in Bt rice pollen treatment showed a big difference,the biggest and smallest silkworm larvae were 183.8rug and 18.lmg respectively,which was probably caused by the difference of Bt rice pollen concentration on the mulberry leaves.As pollen concentration on the mulberry leaves in actual field was lower than in lab,the influence on the development of newly hatched silkworm larvae of Bt rice pollen is not likely significant in actual ecological cnvi ronment.     

Effect of Bt maize on the reproduction and development of saprophagous Diptera over multiple generations

A laboratory experiment was used to quantify the effects of Bt maize on Drosophila melanogaster and Megaselia scalaris, representatives of two saprophagous dipteran families (Drosophilidae, Phoridae). Freshly hatched larvae were reared on a diet containing decaying maize leaves. Two transgenic maize varieties, expressing Cry3Bb1 or Cry1Ab, and their corresponding isolines were tested. In an additional treatment, a solution of pure Cry1Ab was added to the maize diet. According to quantitative ELISA analyses, all Bt diets and all larvae feeding on Bt maize contained low concentrations of Cry proteins but Cry proteins were not detected in adults, thus, predators of the larvae are exposed to Cry proteins whereas predators of adult flies are not. Highest concentrations were in larvae feeding on a maize diet supplemented with a Cry1Ab protein solution. The developmental time and fertility (offspring/female) were measured over four generations for D. melanogaster and over three generations for M. scalaris. Only a few significant differences were found between transgenic and non-transgenic treatments but the differences were not consistent and did not indicate any negative effects of Bt proteins. We conclude that D. melanogaster and M. scalaris larvae are not affected in the long term when feeding and developing on decaying Cry1Ab and Cry3Bb1 maize leaves.     

Using field-evolved resistance to Cry1F maize in a lepidopteran pest to demonstrate no adverse effects of Cry1F on one of its major predators

Spodoptera frugiperda (JE Smith) represents the first documented case of field-evolved resistance to a genetically engineered crop expressing an insecticidal protein from Bacillus thuringiensis (Bt). In this case it was Cry1F-expressing maize (Mycogen 2A517). The ladybird beetle, Coleomegilla maculata, is a common and abundant predator that suppresses pest populations in maize and many other cropping systems. Its larvae and adults are polyphagous, feeding on aphids, thrips, lepidopteran eggs and larvae, as well as plant tissues. Thus, C. maculata may be exposed to Bt proteins expressed in genetically engineered crops by several pathways. Using Cry1F-resistant S. frugiperda larvae as prey, we evaluated the potential impact of Cry1F-expressing maize on several fitness parameters of C. maculata over two generations. Using Cry1F resistant prey removed any potential prey-mediated effects. Duration of larval and pupal stages, adult weight and female fecundity of C. maculata were not different when they were fed resistant S. frugiperda larvae reared on either Bt or control maize leaves during both generations. ELISA and insect-sensitive bioassays showed C. maculata were exposed to bioactive Cry1F protein. The insecticidal protein had no effect on C. maculata larvae, even though larvae contained 20?C32?ng of Cry1F/g by fresh weight. Over all, our results demonstrated that the Cry1F protein did not affect important fitness parameters of one of S. frugiperda??s major predators and that Cry1F protein did not accumulate but was strongly diluted when transferred during trophic interactions.     

Discovery of a novel Bacillus thuringiensis Cry8D protein and the unique toxicity of the Cry8D-class proteins against scarab beetles

A novel cry gene, cry8Db, highly toxic to scarab beetles such as the Japanese beetle, Popillia japonica Newman, was cloned from an isolate of Bacillus thuringiensis(Bt), BBT2-5. The cry8Db gene has 3525 bp nucleotides and codes for a protein of 1174 amino acid residues. The protein, Cry8Db, has typical Bt characteristics such as the 8-block, conserved sequences and the three-domain 3 D toxin structure as defined with Cry3Aa. When the amino acid sequence of Cry8Db was compared with that of Cry8Da whose gene was cloned and characterized in our laboratory earlier, substantial sequence diversities were found in their Domain III. The cry8Db gene was expressed in an acrystalliferous B. thuringiensis strain, BT51. BT51 expressing cry8Db formed a spherical crystal like the natural crystal of BBT2-5. The Cry8Db protein was assayed along with the other scarab active Cry8Da and Cry8Ca against the Japanese beetle. While Cry8Da and Cry8Db had toxicity against both adults and larvae of the Japanese beetle, Cry8Ca was toxic to only larvae. Cry8Ca showed no toxicity against the adult beetle up to 30 μg per 1 cm² of leaf discs on which the protein was applied. The activation process of Cry8Db by adult and larval gut juice was compared in vitro with the processes of Cry8Da and Cry8Ca. All three proteins, Cry8Db, Cry8Da and Cry8Ca, produced a toxic core of approximately 70 kDa equally indicating that the activation process does not inactivate the adult activity of Cry8Ca. We concluded that the adult activity of Cry8D proteins is encoded in Domain II. Further tests against other beetle species showed a significant difference between Cry8D’s and Cry8Ca but no difference between Cry8Da and Cry8Db. Comparison of 3D structural models of Cry8Ca, Cry8Da and Cry8Db, which were constructed by using Cry3Bb as the structural template, indicated significant structural differences, especially between Cry8Ca and Cry8D proteins, in three major surface-exposed loops of Domain II that may be involved in determining the adult beetle activity.     

Response of Danaus plexippus to pollen of two new Bt corn events via laboratory bioassay

Laboratory bioassays were conducted to evaluate the response of first instar larvae of the monarch butterfly, Danaus plexippus L. (Lepidoptera: Danaidae), a non‐target species, to pollen from corn, Zea mays L. (Commelinales: Poaceae), from two new corn hybrids genetically modified to express different types of insecticidal proteins derived from the bacterium Bacillus thuringiensis Berliner (Bacillales: Bacillaceae) (Bt). One hybrid expresses both Cry1Ab and Cry2Ab2 proteins (MON 810 × MON 84006), active against lepidopteran pests, and the other expresses Cry3Bb1 protein (MON 863), targeted against coleopteran pests. First instar larvae were placed on milkweed leaves (Asclepias syriaca L.) (Gentianales: Asclepiadaceae) dusted with doses of either Bt pollen or its nonexpressing (isoline) pollen counterpart ranging from 50 to 3200 grains cm^?2 of milkweed leaves, or no pollen at all. Larvae were exposed to pollen for 4 days, then moved to pollen‐free leaves and observed for another 6 days. Survival was observed after 2, 4, and 10 days. Weight gain was estimated after 4 and 10 days, leaf consumption after 2 and 4 days, and larval development after 10 days. Exposure to pollen of the Cry1Ab/Cry2Ab2‐Bt expressing hybrid reduced larval survival approximately 7.5–23.5% at the dose ranges tested relative to a no pollen control. Larval weight gain and consumption were reduced for larvae exposed to pollen of this hybrid and a small minority of larvae (3.1%) never developed past the third instar after 10 days of observation. Exposure to pollen of the Cry3Bb1‐Bt expressing hybrid had no negative effects on larval mortality, weight gain, consumption, or development relative to the consumption of Bt‐free corn pollen. The relevance of these findings to the risk that these Bt corn hybrids pose to monarch populations is discussed.     

Molecular cloning and characterization of the α-glucosidase II from Bombyx mori and Spodoptera frugiperda

The α-glucosidase II (GII) is a heterodimer of α- and β-subunits and important for N-glycosylation processing and quality control of nascent glycoproteins. Although high concentration of α-glucosidase inhibitors from mulberry leaves accumulate in silkworms (Bombyx mori) by feeding, silkworm does not show any toxic symptom against these inhibitors and N-glycosylation of recombinant proteins is not affected. We, therefore, hypothesized that silkworm GII is not sensitive to the α-glucosidase inhibitors from mulberry leaves. However, the genes for B. mori GII subunits have not yet been identified, and the protein has not been characterized. Therefore, we isolated the B. mori GII α- and β-subunit genes and the GII α-subunit gene of Spodoptera frugiperda, which does not feed on mulberry leaves. We used a baculovirus expression system to produce the recombinant GII subunits and identified their enzyme characteristics. The recombinant GII α-subunits of B. mori and S. frugiperda hydrolyzed p-nitrophenyl α-d-glucopyranoside (pNP-αGlc) but were inactive toward N-glycan. Although the B. mori GII β-subunit was not required for the hydrolysis of pNP-αGlc, a B. mori GII complex of the α- and β-subunits was required for N-glycan cleavage. As hypothesized, the B. mori GII α-subunit protein was less sensitive to α-glucosidase inhibitors than was the S. frugiperda GII α-subunit protein. Our observations suggest that the low sensitivity of GII contributes to the ability of B. mori to evade the toxic effect of α-glucosidase inhibitors from mulberry leaves.     

Single amino acid insertions in extracellular loop 2 of Bombyx mori ABCC2 disrupt its receptor function for Bacillus thuringiensis Cry1Ab and Cry1Ac but not Cry1Aa toxins

In a previous report, seven Cry1Ab-resistant strains were identified in the silkworm, Bombyx mori; these strains were shown to have a tyrosine insertion at position 234 in extracellular loop 2 of the ABC transporter C2 (BmABCC2). This insertion was confirmed to destroy the receptor function of BmABCC2 and confer the strains resistance against Cry1Ab and Cry1Ac. However, these strains were susceptible to Cry1Aa. In this report, we examined the mechanisms of the loss of receptor function of the transporter by expressing mutations in Sf9 cells. After replacement of one or two of the five amino acid residues in loop 2 of the susceptible BmABCC2 gene [BmABCC2_S] with alanine, cells still showed susceptibility, retaining the receptor function. Five mutants with single amino acid insertions at position 234 in BmABCC2 were also generated, resulting in loop 2 having six amino acids, which corresponds to replacing the tyrosine insertion in the resistant BmABCC2 gene [BmABCC2_R(+²³⁴Y)] with another amino acid. All five mutants exhibited loss of function against Cry1Ab and Cry1Ac. These results suggest that the amino acid sequence in loop 2 is less important than the loop size (five vs. six amino acids) or loop structure for Cry1Ab and Cry1Ac activity. Several domain-swapped mutant toxins were then generated among Cry1Aa, Cry1Ab, and Cry1Ac, which are composed of three domains. Swapped mutants containing domain II of Cry1Ab or Cry1Ac did not kill Sf9 cells expressing BmABCC2_R(+²³⁴Y), suggesting that domain II of the Cry toxin is related to the interaction with the receptor function of BmABCC2. This also suggests that different reactions against Bt-toxins in some B. mori strains, that is, Cry1Ab resistance or Cry1Aa susceptibility, are attributable to structural differences in domain II of Cry1A toxins.     

Binding Site Concentration Explains the Differential Susceptibility of Chilo suppressalis and Sesamia inferens to Cry1A-Producing Rice

Chilo suppressalis and Sesamia inferens are two important lepidopteran rice pests that occur concurrently during outbreaks in paddy fields in the main rice-growing areas of China. Previous and current field tests demonstrate that the transgenic rice line Huahui 1 (HH1) producing a Cry1Ab-Cry1Ac hybrid toxin from the bacterium Bacillus thuringiensis reduces egg and larval densities of C. suppressalis but not of S. inferens. This differential susceptibility to HH1 rice correlates with the reduced susceptibility to Cry1Ab and Cry1Ac toxins in S. inferens larvae compared to C. suppressalis larvae. The goal of this study was to identify the mechanism responsible for this differential susceptibility. In saturation binding assays, both Cry1Ab and Cry1Ac toxins bound with high affinity and in a saturable manner to midgut brush border membrane vesicles (BBMV) from C. suppressalis and S. inferens larvae. While binding affinities were similar, a dramatically lower concentration of Cry1A toxin binding sites was detected for S. inferens BBMV than for C. suppressalis BBMV. In contrast, no significant differences between species were detected for Cry1Ca toxin binding to BBMV. Ligand blotting detected BBMV proteins binding Cry1Ac or Cry1Ca toxins, some of them unique to C. suppressalis or S. inferens. These data support that reduced Cry1A binding site concentration is associated with a lower susceptibility to Cry1A toxins and HH1 rice in S. inferens larvae than in C. suppressalis larvae. Moreover, our data support Cry1Ca as a candidate for pyramiding efforts with Cry1A-producing rice to extend the activity range and durability of this technology against rice stem borers.     

Effects of Bacillus thuringiensis δ-endotoxins Cry1Ab and Cry1Ac on the coccinellid beetle,Cheilomenes sexmaculatus (Coleoptera,Coccinellidae) under direct and indirect exposure conditions

Genes encoding cry1Ab and cry1Ac δ-endotoxins from the bacterium, Bacillus thuringiensis (Bt) that have been incorporated in several crops to enhance their resistance to insect pests may possibly influence the activity and abundance of natural enemies of insect pests. The ladybird beetle, Cheilomenes sexmaculatus (L.) might ingest Bt toxins expressed by genetically modified plants by feeding on aphids, early instar larvae of lepidopterans, and other soft bodied insects feeding on transgenic plants. Therefore, we studied the effects of Cry1Ab and Cry1Ac Bt toxins on C. sexmaculatus under direct and indirect exposure conditions. For direct exposure, the neonate C. sexmaculatus larvae were fed either pure 2M sucrose (control) or sucrose solution containing Cry1Ab or Cry1Ac (0.1%), and on alternate days with aphids till pupation. Direct exposure of C. sexmaculatus larvae to Bt toxins resulted in reduced larval survival and adult emergence as compared to the controls, which might be due to long-term direct exposure. However, there were no adverse effects of the Bt toxins on C. sexmaculatus when the larvae were reared on Aphis craccivora Koch fed on different concentrations of Cry1Ab or Cry1Ac in the artificial diet. A significant and positive correlation was observed between the presence of Bt toxins in aphids, and coccinellid larvae and adults (r=0.53** to 0.86**). The results suggested that a direct exposure to Bt toxins expressed in transgenic plants or predation on H. armigera on Bt-transgenic plants will have little effect on the activity and abundance of the ladybird, C. sexmaculatus.     

Suppression of phototaxis in silkworm larvae

A study of the olfactory and visual organs of the larvae of the silkworm (Bombyx mori), using electrophysiological and surgical techniques, indicates that olfactory stimuli from mulberry leaves, conducted through the antennae or the maxillary palps, cause continuous suppression of the phototactic response, and that the central nervous system plays an important role in this ‘control’ of phototaxis. Such phototactic suppression lasts for 30 h in fifth instar larvae, even after mulberry leaves have been removed.     

Preventive effects of N‐acetyl‐l‐cysteine against imidacloprid intoxication on Bombyx mori larvae

Imidacloprid, a widely used neonicotinoid insecticide, is toxic to silkworm (Bombyx mori). To explore whether N‐acetyl‐l ‐cysteine (NAC) has an effect on preventing silkworm (B. mori) from toxification caused by imidacloprid, we fed the fifth‐instar larvae with mulberry leaves dipped in 200 mg/L NAC solution before exposing in imidacloprid, and investigated the silkworm growth, survival rate, feed efficiency, cocoon quality, and the activities of antioxidant enzymes in midgut. The results showed that addition of NAC could significantly increase body weight, survival rate, and feed efficiency of imidacloprid poisoned silkworm larvae (P < 0.05), as well as cocoon mass, cocoon shell mass, and the ratio of cocoon shell (P < 0.05). Furthermore, it could significantly promote the activities of the antioxidant enzymes including superoxide dismutase, catalase, and glutathione peroxide in the midgut of fifth‐instar larvae under imidacloprid exposure at the late stage of treatment. In addition, it also could downregulate the malondialdehyde content. The results of our findings proved that the added NAC may have some beneficial effects on protection or restoration of antioxidant balance in imidacloprid exposed larvae.