Insecticide susceptibility of Cnaphalocrocis medinalis (Lepidoptera: Pyralidae) in China

Insecticide control is the major measure for suppression of Cnaphalocrocis medinalis (Guenée) (Lepidoptera: Pyralidae) damage, and a few insecticides used for long time have proved to fail to control this pest in China. Several new chemicals have been introduced for control of C. medinalis. However, there was no baseline susceptibility data of C. medinalis to insecticides used or will be in use. In this study, a seedling dipping method was developed for bioassay of insecticide susceptibility of C. medinalis. Dose responses of C. medinalis to 11 insecticides were tested. Interpopulation sensitivity to insecticides was compared. Based on LC50 values, C. medinalis was most susceptible to antibiotic insecticides (abamectin, emamectin benzoate, and spinosad) and least sensitive to monosultap and a Bacillus thuringiensis (Bt) product. Chlorantraniliprole and insect growth regulator (IGR) insecticides (tebufenozide and hexaflumuron) exhibited great efficacy against C. medinalis. No susceptibility difference was observed for antibiotic insecticide and IGR insecticides among three populations. Narrow variation in tolerant level was detected for organophosphates insecticides, chlorantraniliprole, monosultap, and Bt. The results in this study provided baseline susceptibility data of C. medinalis to 11 insecticides and also offered useful information for choice of alternative insecticide and for integrated resistance management of C. medinalis.     

纵卷叶螟绒茧蜂对稻纵卷叶螟幼虫的功能反应

在室内测定了纵卷叶螟绒茧蜂Apanteles cypris对稻纵卷叶螟Cnaphalocrocis medinalis幼虫的功能反应和寻找效应.结果表明,纵卷叶螟绒茧蜂对稻纵卷叶螟幼虫的功能反应可用Michaelis-Menten-Ⅱ型功能反应模型Na=AN0/（F+N0）拟合,一头纵卷叶螟绒茧蜂雌蜂在24h内对稻纵卷...     

Baseline susceptibility of tobacco budworm (Lepidoptera: Noctuidae) to Cry1F toxin from Bacillus thuringiensis

Transgenic cotton, Gossypium hirsutum L., lines expressing both Cry1F and Cry1Ac insecticidal proteins from Bacillus thuringiensis (Bt) have been commercially available in the United States since 2005. Both Bt proteins are highly effective against tobacco budworm, Heliothis virescens (F.), and other lepidopteran pests of cotton. Although CrylAc has been available in Bt cotton since 1996, the Cry1F component is relatively new. As part of the proactive resistance management program for Cry1F/Cry1Ac cotton, a susceptibility-monitoring program is being implemented. Baseline variation in the susceptibility to Cry1F in field populations of tobacco budworm was measured. There was a three-fold variation in the amount of Cry1F needed to kill 50% of the neonates from 15 different field populations from the southern and central United States. Future variation in susceptibility of tobacco budworm populations to Cry1F or even resistance evolution could be documented based on this baseline data. A candidate diagnostic concentration was determined that may be efficiently used to identify individuals that potentially carry major alleles conferring field-relevant resistance to Cry1F before such alleles spread through field populations.     

Characterization of the complete mitochondrial genomes of Cnaphalocrocis medinalis and Chilo suppressalis (Lepidoptera: Pyralidae)

The complete mitochondrial genomes (mitogenomes) of Cnaphalocrocis medinalis and Chilo suppressalis (Lepidoptera: Pyralidae) were determined and analyzed. The circular genomes were 15,388 bp long for C. medinalis and 15,395 bp long for C. suppressalis. Both mitogenomes contained 37 genes, with gene order similar to that of other lepidopterans. Notably, 12 protein-coding genes (PCGs) utilized the standard ATN, but the cox1 gene used CGA as the initiation codon; the cox1, cox2, and nad4 genes in the two mitogenomes had the truncated termination codons T, T, and TA, respectively, but the nad5 gene was found to use T as the termination codon only in the C. medinalis mitogenome. Additionally, the codon distribution and Relative Synonymous Codon Usage of the 13 PCGs in the C. medinalis mitogenome were very different from those in other pyralid moth mitogenomes. Most of the tRNA genes had typical cloverleaf secondary structures. However, the dihydrouridine (DHU) arm of the trnS1(AGN) gene did not form a stable stem-loop structure. Forty-nine helices in six domains, and 33 helices in three domains were present in the secondary structures of the rrnL and rrnS genes of the two mitogenomes, respectively. There were four major intergenic spacers, except for the A+T-rich region, spanning at least 12 bp in the two mitogenomes. The A+T-rich region contained an 'ATAGT(A)'-like motif followed by a poly-T stretch in the two mitogenomes. In addition, there were a potential stem-loop structure, a duplicated 25-bp repeat element, and a microsatellite '(TA)(13)' observed in the A+T-rich region of the C. medinalis mitogenome. A poly-T motif, a duplicated 31-bp repeat element, and a 19-bp triplication were found in the C. suppressalis mitogenome. However, there are many differences in the A+T-rich regions between the C. suppressalis mitogenome sequence in the present study and previous reports. Finally, the phylogenetic relationships of these insects were reconstructed based on amino acid sequences of mitochondrial 13 PCGs using Bayesian inference and maximum likelihood methods. These molecular-based phylogenies support the traditional morphologically based view of relationships within the Pyralidae.     

稻纵卷叶螟肠道细菌群落结构与多样性分析

【目的】为明确水稻主要害虫稻纵卷叶螟Cnaphalocrocis medinalis(Guenée)幼虫肠道细菌的群落结构和多样性。【方法】利用Illumina Mi Seq技术对稻纵卷叶螟4龄幼虫肠道细菌的16S r DNA V3-V4变异区序列进行测序,应用USEARCH和QIIME软件整理和统计样品序列数和操作分类单元(operational taxonomic unit,OTU)数量,分析4龄幼虫肠道细菌的物种组成、丰度和多样性。【结果】稻纵卷叶螟4个数量不同的4龄幼虫样本(1,2,3和5头)共得165 386条reads,在97%相似度下可将其聚类为604个OTUs。总共注释到22个门,43个纲,82个目,142个科,204个属,244个种。其中在门水平上,主要优势菌为变形菌门(Proteobacteria)(相对丰度26%~34%)和放线菌门(Actinobacteria)(23%~32%);在纲水平上,主要优势菌为放线菌纲(Actinobacteria)(相对丰度23%~32%)、酸杆菌纲(Acidobacteria)(9%~11%)、α-变形菌纲(Alphaproteobacteria)(10%~13%)、β-变形菌纲(Betaproteobacteria)(6%~8%)和γ-变形菌纲(Gammaproteobacteria)(6%~12%);在科水平上,共有优势菌为类诺卡氏菌科(Nocardioidaceae)、丛毛单胞菌科(Comamonadaceae)、黄单胞菌科(Xanthomonadaceae)、鞘脂单胞菌科(Sphingomonadaceae)和芽单胞菌科(Gemmatimonadaceae)等。在属水平上,4个样本前5位优势属中,类诺卡氏属Nocardioides和鞘脂单胞菌Sphingomonas为共有优势属。稻纵卷叶螟肠道细菌Simpson指数、Shannon指数、Ace指数和Chao指数分别为0.16~0.65,0.94~3.22,212~488和210~490。【结论】稻纵卷叶螟幼虫肠道细菌多样性比较丰富,个体间微生物群落结构和多样性有差异。不同数量样本数据与总体数据有助于综合反映稻纵卷叶螟种群肠道微生物状况。本研究结果为进一步研究稻纵卷叶螟肠道微生物的功能及其在防治中的应用奠定了基础。     

Baseline susceptibility of the corn earworm (Lepidoptera: Noctuidae) to the Cry1Ab toxin from Bacillus thuringiensis

Susceptibility to Cry1Ab toxin from Bacillus thuringiensis (Bt) was determined for 12 field populations of neonate corn earworm, Helicoverpa zea (Boddie), from the United States. Earworm larvae were exposed to artificial diet treated with increasing Bt concentrations, and mortality and growth inhibition were evaluated after 7 d. The range of variation in Bt susceptibility indicated by growth inhibition was very similar to that indicated by mortality. Although interpopulation variation in susceptibility to both proteins was observed, the magnitude of the differences was small (less than or equal to fivefold). These results suggest that the observed susceptibility differences reflect natural variation in Bt susceptibility among corn earworm populations rather than variation caused by prior exposure to selection pressures. Therefore, corn earworms apparently are susceptible to Bt toxins across most of their geographic range.     

Transgenic Bt Rice Does Not Challenge Host Preference of the Target Pest of Rice Leaffolder,Cnaphalocrocis medinalis (Lepidoptera: Pyralidae)

Background

Transgenic Bt rice line T2A-1 expresses a synthesized cry2A gene that shows high resistance to Lepidoptera pests, including Cnaphalocrocis medinalis (Guenée) (Lepidoptera: Pyralidae). Plant volatile orientation cues and the physical characteristics of the leaf surface play key roles in host location or host-plant acceptance of phytophagous insects. These volatile compounds and physical traits may become altered in Bt rice and it is not known whether this influences the behavior of C. medinalis when searching for oviposition sites.

Results

The results of electronic nose analysis showed that the Radar map of Bt rice cultivars was analogous to the non- Bt rice cultivars at each growing stage. PCA analysis was able to partly discriminate between some of the Bt vs. non-Bt rice sensors, but could not to separate Bt cultivars from non-Bt cultivars. The total ion chromatogram between Bt and non-Bt rice cultivars at the seedling, booting and tillering stages were similar and 25 main compounds were identified by GC-MS. For most compounds, there was no significant difference in compound quantities between Bt and non-Bt rice cultivars at equivalent growth stages. The densities of the tubercle papicles and the trichomes on the upper and lower surfaces were statistically equal in Bt and non-Bt rice. The target pest, C. medinalis, was attracted to host rice plants, but it could not distinguish between the transgenic and the isogenic rice lines.

Conclusions

There were no significant differences between the Bt rice line, T2A-1 and the non-Bt rice for volatiles produced or in its physical characteristics and there were no negative impacts on C. medinalis oviposition behavior. These results add to the mounting evidence that Bt rice has no negative impact on the target insect oviposition behavior.     

稻纵卷叶螟海藻糖酶基因的克隆、分子特性和表达分析

【目的】海藻糖酶(trehalase,Tre)是昆虫体内海藻糖代谢的一个关键酶,包括可溶型(Tre1)和膜结合型(Tre2)两种类型,在昆虫发育和能量调节中具有重要作用。本研究旨在克隆稻纵卷叶螟Cnaphalocrocis medinalis海藻糖酶基因(Cm Tre),解析其在稻纵卷叶螟不同发育阶段和不同组织中的表达模式,分析该基因及酶蛋白的分子特征。【方法】通过稻纵卷叶螟转录组数据结合RACE技术,克隆稻纵卷叶螟Cm Tre的全长c DNA序列,并对该基因进行生物信息学分析;采用实时荧光定量PCR(RT-q PCR)解析Cm Tre在稻纵卷叶螟不同发育阶段及成虫不同组织部位的m RNA表达模式。【结果】获得两种类型的稻纵卷叶螟Cm Tre基因,即可溶型海藻糖酶基因Cm Tre1和膜结合型海藻糖酶基因Cm Tre2。Cm Tre1的全长c DNA长度为2 364 bp,开放阅读框长1 704 bp,编码567个氨基酸;Cm Tre2的全长c DNA长度为2 079 bp,开放阅读框长1 923 bp,编码640个氨基酸。生物信息学分析表明,Cm Tre前端有一个信号肽,Cm Tre1无跨膜结构,Cm Tre2有一个跨膜结构。同源性和聚类分析表明,Cm Tre1和Cm Tre2的氨基酸序列与竹蠹螟Omphisa fuscidentalis海藻糖酶Tre1和Tre2氨基酸序列的一致性最高,分别为74%和79%。同源建模预测结果显示,Cm Tre1的三维分子结构包含19个α螺旋和2个β折叠片;Cm Tre2的三维分子结构含有23个α螺旋,没有β折叠片。RT-q PCR结果显示,Cm Tre在稻纵卷叶螟整个发育历期都有表达,在成虫期表达水平最高,在整个幼虫期都有相对稳定的表达;Cm Tre1在蛹期表达水平最低,Cm Tre2在5龄幼虫时期表达水平最低。Cm Tre在所检测的成虫组织(中肠、体壁、马氏管、头、卵巢、脂肪体、肌肉和精巢)中均有表达;Cm Tre1在中肠和体壁中的表达水平较高,Cm Tre2在肌肉和中肠中的表达水平较高。【结论】本研究克隆了稻纵卷叶螟两种类型的海藻糖酶基因,分析了其基因特征和表达模式。研究结果为阐明海藻糖酶基因的功能进而以海藻糖酶基因为靶标防治害虫奠定了基础。     

The effect of leaffolder Cnaphalocrocis medinalis (Guenee) [Lepidoptera: Pyralidae] injury on the plant physiology and yield loss in rice

Influence of leaffolder feeding on chlorophyll, PS II activity and plant–water relations, effect of larval density on leaf damage and time course studies on larval feeding behaviour on altered physiological changes in TN1 rice culture were studied. Quantification of yield losses in the field caused by leaffolder was also assessed. Leaffolder damage resulted in 57% reduction in chlorophyll content, 23% reduction in PS II activity and 23% reduction in relative water content in comparison with control. Rice leaffolder larva folds the leaf and scrapes the green tissue from within the fold resulting in scorching and drying of the leaves. Larval density had differentially influenced effective leaf area of rice crop. Larval densities of more than 3 larvae per hill at maximum tillering stage resulted up to 20% unfilled grains, 28–57% reduction in PS II activity and 23% reduction in relative water content in comparison with the control. At flowering stage, flag leaf area damage of above 25% resulted in more than 50% unfilled grains over control, indicating direct effect of yield reduction in rice. Thus, a cumulative effect of loss in chlorophyll, reduced photosynthate availability and altered water relations caused by the leaffolder injury to flag leaf lead to greater yield loss in rice.     

Resistance performances of transgenic bt rice lines T(2A)-1 and T1c-19 against Cnaphalocrocis medinalis (Lepidoptera: Pyralidae)

The transgenic Bacillus thuringiensis (Bt) rice, Oryza sativa L., lines T(2A)-1 and T1c-19 expressing Cry2A* and Cry1C* from 'Minhui 63' (MH63) were evaluated for resistance to newly hatched and third-instar larvae of Cnaphalocrocis medinalis (Lepidoptera: Pyralidae), by using detached leaf laboratory bioassays. Both T(2A)-1 and T1c-19 rice showed high C. medinalis resistance; however, the lethal time (LT)50 of larvae fed with T(2A)-1 rice was significantly longer than that of larvae fed with T1c-19 rice, implying T1c-19 rice was more toxic to C. medinalis larvae. Larval mortality after 4 d on nitrogen-free MH63 was 25.5% compared with 2.4% mortality on high nitrogen fertilizer (250 kg N/ha) plants. Larval mortality on high nitrogen T(2A)-1 plants declined by 20% compared with nitrogen-free plants. However, resistance in T1c-19 plants was unaffected by nitrogen fertilizer. C. medinalis moths preferred MH63 at both the seedling and grain milk stages for oviposition but not the T1c-19 and T(2A)-1 Bt rice lines.     

Infectivity and ecology of Pseudomonas spp. from natural epizootics in the rice leaf folder, Cnaphalocrocis medinalis (Lepidoptera: Pyralidae) in India

In 2001, populations of the leaf folder (LF) (Cnaphalocrocis medinalis Guenee) reached 19-30 larvae/hill in Rabi rice (variety Lalat) fields at Choudwar, Baranga, Alani and Tangi, Orissa, India infesting 61-98% of the plants. Three bacteria viz. Pseudomonas aeruginosa (Pa1 and Pa2) and P. fluorescens (Pf) infected 62-98% of the larvae in different fields. No correlation was found among populations of the Pseudomonas spp. on the rice phyllosphere and larvae harboring the bacteria at different fields. The median lethal concentrations (LC₅₀s; ×10³ bacteria/mL) of Pa1, Pa2 and Pf were 4-4.2, 4.8-4.9 and 5.7-5.9, respectively, for the leaf piece assay and 4.8-4.9, 5-5.1 and 6.3-6.7, respectively, for the potted plant assay against second to fifth instar larvae. The median lethal times (LT₅₀s) were 2.1-2.4, 3.2-3.5 and 5.3-6 days for the leaf piece assay, and 4-4.7, 4.7-4.9 and 6.1-6.8 days for the potted plant assay with Pa1, Pa2 and Pf, respectively. The Pa1 and Pa2 were compatible, exhibited synergistic effect and co-inoculation reduced the LC₅₀ and LT₅₀ as compared to inoculation with individual organisms. The Pf had no synergism with Pa1 or Pa2, and therefore, the lethal values for Pf alone or with the other bacteria were similar. Fulfillment of Koch's postulates confirmed that Pa1, Pa2 and Pf were pathogens of C. medinalis.     

Effects of transgenic Bacillus thuringiensis maize grain on B. thuringiensis-susceptible Plodia interpunctella (Lepidoptera: Pyralidae)

Percentage survivorship, developmental time, adult body length, and sex ratio of Plodia interpunctella (Hübner) reared on field-produced grain from sixteen cultivars of maize, Zea mays L., including several transgenic Bacillus thuringiensis (Bt) Berliner hybrids and selected non-Bt isolines, were evaluated under laboratory conditions. Compared with isolines, development was delayed and survivorship reduced for P. interpunctella reared on grain from transgenic hybrids with the CaMV/35s promoter that express Cry1Ab protein. Similarly, compared with non-Bt hybrids, a transgenic hybrid with the CaMV/35s promoter that expresses Cry9C protein delayed development, decreased survivorship, and caused reductions in adult body length of P. interpunctella. In contrast, no significant differences in P. interpunctella developmental times or survivorship were observed between transgenic hybrids with the PEPC promoter expressing Cry1Ab and their isolines. Additionally, developmental time, survivorship, and adult body length were similar between P. interpunctella reared on a transgenic hybrid with the CaMV/35s promoter expressing Cry1Ac and non-Bt hybrids. Our data demonstrate that transgenic Bt maize grain, especially grain from hybrids with the CaMV/35s promoter expressing Cry1Ab or Cry9C, can significantly affect B. thuringiensis-susceptible P. interpunctella populations up to 4 or 5 mo after harvest.     

Genetic variation for resistance to Bacillus thuringiensis toxins in Helicoverpa zea (Lepidoptera: Noctuidae) in eastern North Carolina

To evaluate resistance to Bacillus thuringiensis Berliner (Bt) toxins, adult female bollworms, Helicoverpa zea (Boddie) (Lepidoptera: Noctuidae), were collected from four light trap locations in two eastern North Carolina counties from August to October during 2001 and 2002. Females were allowed to oviposit, and upon hatching, 24 neonates from each female (F1 lines) were screened for survival and growth rate on each of three diets: non-Bt diet, diet containing 5.0 microg/ml Cry1Ac toxin, or diet containing 5.0 microg/ml Cry2Ab toxin. These screens were designed to identify nonrecessive Bt resistance alleles present in field populations of bollworm. Of 561 and 691 families screened with both Cry1Ac- and Cry2Ab-containing diets in 2001 and 2002, respectively, no F1 lines were identified that seemed to carry a gene conferring substantial resistance to either Cry1Ac or Cry2Ab. Adults from F1 lines with growth scores in the highest (R) and lowest (S) quartiles were mated in four combinations, RxR, SxR, RxS, and SxS. Differences in growth rates of larvae from these crosses demonstrated that there is substantial quantitative genetic variation in eastern North Carolina populations for resistance to both Cry1Ac and Cry2Ab toxins. These findings, in addition to results suggesting partially dominant inheritance of resistance to Cry1Ac and Cry2Ab, are critically important for determining appropriate resistance management strategies that impact the sustainability of transgenic cotton, Gossypium hirsutum (L.).     

Genetic variability of Spodoptera frugiperda Smith (Lepidoptera: Noctuidae) populations from Latin America is associated with variations in susceptibility to Bacillus thuringiensis cry toxins

Bacillus thuringiensis strains isolated from Latin American soil samples that showed toxicity against three Spodoptera frugiperda populations from different geographical areas (Mexico, Colombia, and Brazil) were characterized on the basis of their insecticidal activity, crystal morphology, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of parasporal crystals, plasmid profiles, and cry gene content. We found that the different S. frugiperda populations display different susceptibilities to the selected B. thuringiensis strains and also to pure preparations of Cry1B, Cry1C, and Cry1D toxins. Binding assays performed with pure toxin demonstrated that the differences in the toxin binding capacities of these insect populations correlated with the observed differences in susceptibility to the three Cry toxins analyzed. Finally, the genetic variability of the three insect populations was analyzed by random amplification of polymorphic DNA-PCR, which showed significant genetic diversity among the three S. frugiperda populations analyzed. The data presented here show that the genetic variability of S. frugiperda populations should be carefully considered in the development of insect pest control strategies, including the deployment of genetically modified maize in different geographical regions.     

Susceptibility of Cry1Ab-resistant and -susceptible sugarcane borer (Lepidoptera: Crambidae) to four Bacillus thuringiensis toxins

Sugarcane borer, Diatraea saccharalis (F.), is a primary corn stalk borer pest targeted by transgenic corn expressing Bacillus thuringiensis (Bt) proteins in many areas of the mid-southern region of the United States. Recently, genes encoding for Cry1A.105 and Cry2Ab2 Bt proteins were transferred into corn plants (event MON 89034) for controlling lepidopteran pests. This new generation of Bt corn with stacked-genes of Cry1A.105 and Cry2Ab2 will become commercially available in 2009. Susceptibility of Cry1Ab-susceptible and -resistant strains of D. saccharalis were evaluated on four selected Bt proteins including Cry1Aa, Cry1Ac, Cry1A.105, and Cry2Ab2. The Cry1Ab-resistant strain is capable of completing its larval development on commercial Cry1Ab-expressing corn plants. Neonates of D. saccharalis were assayed on a meridic diet containing one of the four Cry proteins. Larval mortality, body weight, and number of surviving larvae that did not gain significant weight (<0.1 mg per larva) were recorded after 7 days. Cry1Aa was the most toxic protein against both insect strains, followed in decreasing potency by Cry1A.105, Cry1Ac, and Cry2Ab2. Using practical mortality (larvae either died or no significant weight gain after 7 days), the median lethal concentration (LC₅₀) of the Cry1Ab-resistant strain was estimated to be >80-, 45-, 4.1-, and −0.5-fold greater than that of the susceptible strain to Cry1Aa, Cry1Ac, Cry1A.105 and Cry2Ab2 proteins, respectively. This information should be useful to support the commercialization of the new Bt corn event MON 89034 for managing D. saccharalis in the mid-southern region of the United States.     

Frequency of alleles conferring resistance to a Bacillus thuringiensis toxin in a Philippine population of Scirpophaga incertulas (Lepidoptera: Pyralidae)

Using the F2 screen methodology, we estimated the frequency of alleles conferring resistance to the Cry1Ab toxin of Bacillus thuringiensis Berliner in a Philippine population of the stem borer Scirpophaga incertulas (Walker). Evaluation of >450 isofemale lines for survival of F2 larvae on cry1Ab plants did not detect the presence of an allele conferring a high level of resistance. The frequency of such an allele in the sampled population was conservatively estimated to be <3.6 x 10(-3) with 95% confidence and a detection probability of 94%. However, there was evidence of the presence of alleles conferring partial resistance to Cry1Ab. The frequency of alleles for partial resistance was estimated as 4.8 x 10(-3) with a 95% CI between 1.3 x 10(-3) and 1.04 x 10(-2) and a detection probability of 94%. Our results suggest that the frequency of alleles conferring resistance to Cry1Ab in the population of S. incertulas sampled is not too high to preclude successful implementation of the high dose/refuge resistance management strategy.     

Interaction of Bacillus thuringiensis toxins with larval midgut binding sites of Helicoverpa armigera (Lepidoptera: Noctuidae)

In 1996, Bt-cotton (cotton expressing a Bacillus thuringiensis toxin gene) expressing the Cry1Ac protein was commercially introduced to control cotton pests. A threat to this first generation of transgenic cotton is the evolution of resistance by the insects. Second-generation Bt-cotton has been developed with either new B. thuringiensis genes or with a combination of cry genes. However, one requirement for the "stacked" gene strategy to work is that the stacked toxins bind to different binding sites. In the present study, the binding of (125)I-labeled Cry1Ab protein ((125)I-Cry1Ab) and (125)I-Cry1Ac to brush border membrane vesicles (BBMV) of Helicoverpa armigera was analyzed in competition experiments with 11 nonlabeled Cry proteins. The results indicate that Cry1Aa, Cry1Ab, and Cry1Ac competed for common binding sites. No other Cry proteins tested competed for either (125)I-Cry1Ab or (125)I-Cry1Ac binding, except Cry1Ja, which competed only at the highest concentrations used. Furthermore, BBMV from four H. armigera populations were also tested with (125)I-Cry1Ac and Cry1Ab to check the influence of the insect population on the binding results. Finally, the inhibitory effect of selected sugars and lectins was also determined. (125)I-Cry1Ac binding was strongly inhibited by N-acetylgalactosamine, sialic acid, and concanavalin A and moderately inhibited by soybean agglutinin. In contrast, (125)I-Cry1Ab binding was only significantly inhibited by concanavalin A. These results show that Cry1Ac and Cry1Ab use different epitopes for binding to BBMV.     

Susceptibility of Eldana saccharina (Lepidoptera: Pyralidae), Busseola fusca and Sesamia calamistis (Lepidoptera: Noctuidae) to Bacillus thuringiensis Cry toxins and potential side effects on the larval parasitoid Cotesia sesamiae (Hymenoptera: Braconidae)

Laboratory trials were conducted to determine the efficacy of four Bacillus thuringiensis (Bt) Cry toxins at five different concentrations (0.016, 0.08, 0.4, 2, 10 μg/mL) for controlling three lepidopteran stem-borer species (i.e., the pyralid Eldana saccharina and the noctuids Busseola fusca and Sesamia calamistis) as well as to evaluate their indirect effect on the braconid larval parasitoid Cotesia sesamiae. In addition, larvae from the treatments above, after having been parasitized, were either fed a contaminated (group 1) or a toxin-free (group 2) diet and compared with a control (i.e., parasitized larvae which have never fed on Bt-toxin). All Bt Cry toxins induced larval feeding inhibition. Compared with the control, significant mortality resulted at all concentrations and for all species of Lepidoptera. Cry1Ab was the most toxic with 10 days post treatment mortalities ranging from 81% in B. fusca and S. calamistis to 100% in E. saccharina. In contrast, Cry1Ac had comparatively low toxicity particularly for B. fusca and S. calamistis (e.g., respectively, 54 and 74% mortality at 10 days at the highest concentration). In the toxin-treated group 1, percentages of C. sesamiae cocoon-producing moth larvae were higher compared to group 2 and the control, whereas clutch size was higher than in group 2 but similar to the control. In both groups, 0.016, 0.08 μg/mL CrylAc yielded lower female-biased sex ratios than the control. It was suggested that paralyses of the moth larvae caused by the toxin may have facilitated parasitization leading to higher parasitism and clutch size.     

Common features of Bacillus thuringiensis toxins specific for Diptera and Lepidoptera

The complete nucleotide sequence of a cloned gene encoding a 130-kDa crystal protein of Bacillus thuringiensis (B.t.) subspecies israelensis has been determined. The recombinant protein (Bt8) was purified and shown to be a mosquito-specific toxin with a LC50 value of 43 ng/ml to third-instar larvae of Aedes aegypti. Bt8 is processed by proteases or midgut extracts of mosquito larvae into toxic fragments of 68-78 kDa. Deletion mapping indicated that the active fragment of Bt8 is localized in the N-terminal half of the protoxin molecule. The deduced amino acid sequence of Bt8 has been compared with that of Bt2, a Lepidoptera-specific toxin, previously cloned from Bacillus thuringiensis berliner. Highly homologous amino acid stretches are present in the C-terminal half of the proteins. The N-terminal parts show much less sequence homology but they display a strikingly similar distribution of hydrophilic and hydrophobic amino acids. In addition, Bt8 and Bt2 show a significant immunological cross-reaction. The data indicate that although these B.t. delta endotoxins exhibit a different insect-host specificity, they are structurally related and might use a similar mechanism to interact with insect cell membranes.