Cry toxins produced by Bacillus thuringiensis(Bt) are effective biological insecticides against certain insect species.In this study,bioassay results indicated that Cry1B and Cry1C were toxic to Spodoptera exigua.We also identified a cadherin-like gene in S.exigua that could enhance the toxicity of Cry1B and Cry1C.The cadherin-like gene identified from the larvae midgut tissue was cloned by reverse transcription polymerase chain reaction(RT-PCR) and rapid amplification of cDNA ends(RACE).The full-length cDNA of the gene consisted of 5 220 bp encoding 1 740 amino acid with a predicted molecular mass of 196 kD.BLAST search analysis showed that the predicted amino acid sequence had a high sequence identity to the published sequences of cadherin-like proteins from other Lepidoptera insects.Spatial expression of the cadherin-like gene detected by qRT-PCR analysis revealed that the cadherin-like gene was mainly present in the gut of 4th instar larvae and during different life stages.The results suggested that the commercial development of this synergist has the potential to enhance Cry1B and Cry1C toxicity against Lepidoptera insects.
LU QiongZHANG YongojunCAO Guang-chunZHANG Li-liLIANG Ge-meiLU Yan-huiWU Kong-mingGAO Xi-wuGUO Yu-yuan
A chemosensory protein named HarmCSP5 in cotton bollworm Helicoverpa armigera(Hübner) was obtained from antennal cDNA libraries and expressed in Escherichia coli.The real time quantitative PCR(RT-qPCR) results indicated that HarmCSP5 gene was mainly expressed in male and female antennae but also expressed in female legs and wings.Competitive binding assays were performed to test the binding affinity of recombinant HarmCSP5 to 60 odor molecules including some cotton volatiles.The resules showed that HarmCSP5 showed strong binding abilities to 4-ehtylbenzaldehyde and 3,4-dimethlbenz aldehyde,whereas methyl phenylacetate,2-decanone,1-pentanol,carvenol,isoborneol,nerolidol,2nonanone and ethyl heptanoate have relatively weak binding affinity.Moreover,the predicted 3D model of HarmCSP5 consists of six α-helices located among residues 33-38(α1),40-48(α2),62-72(α3),80-96(α4),98-108(α5),and 116-119(α6),two pairs of disulfide bridges Cys49-Cys55,Cys75-Cys78.The two amino acid residues,Ile94 and Trp101,may play crucial roles in HarmCSP5 binding with ligands and need further study for confirmation.
Cry toxins produced by Bacillus thuringiensis (Bt) are effective biological insecticides against certain insect species. However, there are potential risks of the evolved resistance of insects to Cry toxin owing to decreased binding of toxins to target sites in the brush border membranes of the larva midgut. The Cry toxins with different binding sites in the larval midgut have been considered to be a good combination to deploy in delaying resistance evolution. Bioassay results demonstrated that the toxicity of different Cry toxins ranked differently for each species. The toxicity ranking was Cry1Ac>Cry1Ab>Cry2Ab for Helicoverpa armigera, Cry1B>Cry1C>Cry2Ab for Spodoptera exigua, and Cry2Ab>Cry1B> Cry1C for S. litura. Only Cry2Ab was toxic to Agrotis ipsilon. Binding experiments were performed with 125I-Cry1Ab, ^(125)I-Cry1Ac, 125I-Cry1B, 125I-Cry1C, 125I-Cry2Ab and the brush border membranes vesicles (BBMV) from H. armigera, S. exigua, S. litura and A. ipsilon. The binding of Cry1Ab and Cry1Ac was shown to be saturable by incubating with increasing concentrations of H. armigera BBMV (K_d =(45.00±2.01) nmol L-1 and (12.80±0.18) nmol L-1, respectively; B_(max) =(54.95±1.79) ng and (55.44±0.91) ng, separately). The binding of Cry1B was shown to be saturable by incubating with increasing concentrations of S. exigua BBMV (K_d =(23.26±1.66) nmol L-1; B_(max) =(65.37±1.87) ng). The binding of ^(125)I-Cry toxins was shown to be non-saturable by incubating with increasing concentrations of S. litura and A. ipsilon BBMV. In contrast, Cry1B and Cry1C showed some combination with the BBMV of S. litura, and a certain amount of Cry2Ab could bind to the BBMV of A. ipsilon. These observations suggest that a future strategy could be devised for the focused combination of specific cry genes in transgenic crops to control target pests, widen the spectrum of insecticide effectiveness and postpone insect resistance evolution.
LU QiongCAO Guang-chunZHANG Li-liLIANG Ge-meiGAO Xi-wuZHANG Yong-junGUO Yu-yuan
The tea geometrid Ectropis obliqua is one of the most serious leaf-feeding insect pests in tea(Camellia sinensis) in East Asia. Although several volatile chemicals emitted from tea plants have been reported to be attractive to E. obliqua moths, no synthetic attractants for E. obliqua moths have been developed. By measuring the behavioral responses of the moth to a series of chemicals in the lab, we found that a blend containing a ternary mixture containing(Z)-3-hexenal,(Z)-3-hexenyl hexanoate and benzyl alcohol clearly attracted to E. obliqua moths of both sex and that(Z)-3-hexenyl acetate could enhance the attractiveness of the ternary blend. Moreover, we found that the volatiles emitted from the plant-E. obliqua larva complex have the same attractiveness as: 1) the blend of volatiles containing the ternary mixture and 2) the blend containing(Z)-3-hexenyl acetate plus the ternary mixture to both male and female moths. In a field bioassay, more male moths were observed on traps that were baited with the blend containing(Z)-3-hexenyl acetate plus the ternary mixture than on control traps. Our study raises the tantalizing possibility that synthetic blends could be deployed as attractants for pests in the field.
SUN Xiao-lingLI Xi-wangXIN Zhao-junHAN Juan-juanRAN WeiLEI Shu
