小分子热激蛋白基因HaHSP19.8在棉铃虫抗逆反应中的作用

【目的】小分子热激蛋白(small heat shock protein,sHSP)在昆虫抵御外界环境压力中至关重要。本研究旨在探究小分子热激蛋白sHSP19.8基因在棉铃虫Helicoverpa armigera生长发育、抵御高温胁迫和对Cry1Ac杀虫蛋白抗性机制中的作用,为更深入探析该基因作用机理及棉铃虫的防治奠定基础。【方法】通过PCR结合RACE克隆棉铃虫sHSP19.8基因序列,利用生物信息学软件对该基因序列进行分析;通过qRT-PCR测定Cry1Ac敏感棉铃虫5龄幼虫在40℃高温下处理1 h和2 h及饲喂含30μg/mL Cry1Ac的人工饲料1 h和2 h后该基因的表达量,并测定抗感Cry1Ac棉铃虫不同发育阶段(1-5龄幼虫、蛹及成虫)和5龄幼虫不同组织(前肠、中肠、后肠、马氏管及表皮)中该基因的表达模式。【结果】获得了棉铃虫sHSP19.8基因的全长cDNA序列,命名为HaHSP19.8(GenBank登录号:XP_021195228.1),长608 bp,开放阅读框长528 bp,编码175个氨基酸残基,具有小分子热激蛋白的典型α-晶体结构域(α-crystallin domain,ACD)。该基因受40℃高温和30μg/mL Cry1Ac杀虫蛋白诱导时在Cry1Ac敏感棉铃虫5龄幼虫中均过量表达;在Cry1Ac敏感棉铃虫整个发育阶段和5龄幼虫各组织中均表达,其中在成虫和5龄幼虫以及5龄幼虫表皮、马氏管和中肠内表达量较高;但是该基因在Cry1Ac抗性品系各个发育阶段和5龄幼虫各组织中表达量相比敏感品系都显著较低。【结论】结果说明HaHSP19.8参与棉铃虫生长发育和生理生化的过程,帮助昆虫抵御外界环境压力,并可能参与到棉铃虫对Cry1Ac的抗性机制中。

Analysis of phototactic responses in Spodoptera frugiperda using Helicoverpa armigera as control

Light traps are widely utilized to monitor and manage insect pest populations.In late 2018,the fall armyworm(FAW),Spodoptera frugiperda,invaded China through Yunnan Province representing a huge threat to grain production.To estimate the efficiency of light traps on FAW moths,we first identified the opsin genes from FAW by using the transcriptome.Phylogenetic analysis indicated that the four opsins of FAW were clustered with those of other Noctuidae species.The expressed levels of opsins in S.frugiperda were lower than in Helicoverpa armigera,suggesting a different phototactic response between the two species.Then,we determined the phototactic behavior of FAW using H.armigera as a control,which is widely monitored and managed using light traps in China.Our results indicated that the two moths species showed significantly different phototactic behavior and both female and male FAW displayed faster flight-to-light speed than H.armigera.This may be due to a faster flight capacity in FAW compared to H.armigera.However,the capture rate of both female and male of S.frugiperda was significantly lower than that of H.armigera,which was consistent with the expression levels of opsins.These results support the positive phototaxis of S.frugiperda moths and suggest light traps could be used for monitoring and managing the pests,but with a lower efficiency than H.armigera.

Population occurrence of the fall armyworm,Spodoptera frugiperda(Lepidoptera:Noctuidae),in the winter season of China

Fall armyworm(FAW),Spodoptera frugiperda(J.E.Smith),has become an important pest in Chinese agricultural systems since its invasion on 11 December 2018.After its establishment,FAW months in the year-round breeding region have become the main source population migrating to other areas in China.Field investigations were conducted in tropical and subtropical regions to improve understanding of its year-round breeding area in China.The results showed that FAW larval density was significantly correlated with the seasonal temperature of the location surveyed.The FAW larvae maintained a high density in the tropical area and were frequently found in sites of the south subtropical region,but were absent from the north subtropical region and the northern part of the central subtropical region.These results indicated that FAW can reproduce annually in the tropical and south subtropical regions of China,including Hainan,Taiwan and the southern area of Fujian,Guangdong,Guangxi,Guizhou,and Yunnan.Hence,great effort should be made to monitor and control FAW in the year-round breeding region to suppress the population density of this area and to reduce migration of moths into northern parts of China.This study clarifies the occurrence area of the pest in winter in China and provides much valuable information for its population forecasting and management.

A Fragment of Cadherin-Like Protein Enhances Bacillus thuringiensis Cry1B and Cry1C Toxicity to Spodoptera exigua(Lepidoptera:Noctuidae)

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.

Performance of Three Adelphocoris spp.(Hemiptera: Miridae) on Flowering and Non-flowering Cotton and Alfalfa

The genus Adelphocoris（Hemiptera： Miridae） is a group of key insect pests in cotton fields in China that includes three dominant species： A. suturalis（Jakovlev）, A. lineolatus（Goeze） and A. fasciaticollis（Reuter）. Previous field surveys have found that adults of these Adelphocoris species usually move onto specific host plants when the plants enter the flowering stage. To determine the potential trade-offs for this host-plant preference behavior, the performance of these three Adelphocoris spp. on flowering and non-flowering cotton and alfalfa were compared in the laboratory. The results showed that Adelphocoris spp. had significantly higher nymphal developmental and survival rates, along with increased adult longevity and fecundity on flowering cotton and alfalfa than on non-flowering plants of either species. In addition, compared with cotton plants, alfalfa generally promoted better performance of these three Adelphocoris species, especially for A. lineolatus. Simple correlation analysis indicated that female adult longevity was positively correlated to male adult longevity and female fecundity, and female fecundity was positively correlated to nymphal development and survival rates. This study demonstrated a positive correlation between adult preference and offspring/adult performance for all three Adelphocoris species, with no evidence of any trade-offs for this preference for flowering host plants. These findings support the hypothesis that hemimetabolous insects such as mirid bugs have positive adult preference-adult/nymphal performance relationships, which is likely due to the similar feeding habits and nutritional requirements of adults and nymphs.

The Binding Characterization of Cry Insecticidal Proteins to the Brush Border Membrane Vesicles of Helicoverpa armigera, Spodoptera exigua, Spodoptera litura and Agrotis ipsilon

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 CrylAc〉CrylAb〉Cry2Ab for Helicoverpa armigera, CrylB〉CrylC〉Cry2Ab for Spodoptera exigua, and Cry2Ab〉CrylB〉 CrylC for S. litura. Only Cry2Ab was toxic to Agrotis ipsilon. Binding experiments were performed with ^125I-CrylAb, ^125I-CrylAc, ^125I-CrylB, ^125I-CrylC, ^125I-Cry2Ab and the brush border membranes vesicles （BBMV） from H. armigera, S. exigua, S. litura and A. ipsilon. The binding of CrylAb and CrylAc was shown to be saturable by incubating with increasing concentrations of H. armigera BBMV （Kd=（45.00＋2.01） nmol L-1 and （12.80＋0.18） nmol L^-1, respectively; Bmax=（54.95±l.79） ng and （55.44±0.91） ng, separately）. The binding of CrylB was shown to be saturable by incubating with increasing concentrations ofS. exigua BBMV （Kd=（23.26±1.66） nmol L^-1; Bmax=（65.37±1.87） ng）. The binding of ^125I-Cry toxins was shown to be non-saturable by incubating with increasing concentrations of S. litura and A. ipsilon BBMV. In contrast, CrylB and CrylC showed some combination with the BBMV orS. litura, and a certain amount of Cry2Ab could bind to the BBMV ofA. 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.

Characterization and functional analysis of β-1,3-galactosyltransferase involved in Cry1Ac resistance from Helicoverpa armigera(Hübner)

Carbohydrate chains are the principal antigens by which Bacillus thuringiensis（Bt） identify receptor proteins. The interaction between the antigen and Bt causes a pore in the membrane of midgut epithelial cells of insects. Receptor proteins, such as aminopeptidase N and alkaline phosphatase, are glycoproteins. Cadherin is another cell surface receptor protein which has potential glycosylation sites. Glycosyltransferase is very important for the synthesis and modification of receptor proteins. It can indirectly influence the function of Bt. The 1 950 bp full-length c DNA encoding β-1,3-galactosyltransferase was cloned from the the midgut of Helicoverpa armigera by degenerative PCR combined with RACE techniques（GAL-Harm, Gen Bank accession no.： GQ904195.1） with two potential N-glycosylation sites（^157NNTI^160 and ^272NKTL^275）. Protein sequence alignments revealed that H. armigera β-1,3-galactosyltransferase shared high identity with β-1,3-galactosyltransferase in other insect species. The expression level of the β-1,3-galactosyltransferase gene in Cry1Ac-resistant H. armigera larvae was 9.2-fold higher than that in susceptible strain. The function of β-1,3-galactosyltransferase was investigated using RNAi technique. The result showed Cry1 Ac enhanced the toxicity against the si RNA-treated larvae compared with non-si RNA-treated ones, which indicated β-1,3-galactosyltransferase played an important role for the insecticidal toxicity of Cry1 Ac in H. armigera.

Alanine-substituted mutant on Gly^(373) and Asn^(375) of Cry1Ai-h-loop 2 causes reduction in both toxicity and binding against Helicoverpa armigera

Cry1Ai-h-loop 2 is a mutant of Cry1Ai constructed by exchanging loop 2 from Cry1Ah protein and shows insecticidal activity against Helicoverpa armigera. The toxicity of Cry1 Ai-h-loop 2, in contrast to the very low toxicity of Cry1Ai, is closely associated with the eleven residues in the loop 2 region. To characterize the key sites of loop 2 in Cry1Ai-h-loop 2, alaninesubstituted mutants were generated. The toxicity of these mutants against H. armigera indicated that dual-mutant on Gly373 and Asn375 caused a significant decrease in toxic activity. ELISA binding and competition binding assays demonstrated that the reduction of toxicity in the mutant of interest was correlated with decreased binding affinity.

Polycalin is involved in the action mechanism of Cry2Aa toxin in Helicoverpa armigera(Hübner)

Receptor proteins on the brush border membrane of the insect midgut epithelium are involved in the mode of action of insecticidal Cry proteins from Bacillus thuringiensis(Bt). Polycalin has been identified as a binding protein of the Bt Cry1 Ac toxin in several Lepidoptera including Helicoverpa armigera, but its role in the action mechanism of Cry2 Aa is still unclear. In this study, we investigated the binding characteristics of polycalin from the midgut of H. armigera with Cry2 Aa and its role in the toxicity of Cry2 Aa. The results demonstrated that heterologously expressed H. armigera polycalin peptide could bind with Cry2 Aa with high affinity(K_d=32 nmol L^(–1)). The toxicity of Cry2 Aa decreased by 27% after H. armigera larvae ingested polycalin antisera. These results suggested that polycalin could be a potential functional receptor for Cry2 Aa, and it plays an important role in the susceptibility of H. armigera to Cry2 Aa.

Assessment of the potential toxicity of insecticidal compounds to Peristenus spretus,a parasitoid of mirid bugs

With the increased cultivation of Bt crops in China,Apolygus lucorum and other mirid bugs have emerged as important agricultural pests because they are insensitive to the Bt proteins.In addition,the reduction of pesticide applications after planting Bt crops also increases the severity of mirid bug outbreaks.Peristenus spretus is a parasitoid of mirid nymphs,but its sensitivity to Bt proteins is not known.In the current study,we developed a dietary exposure assay to assess the effects of Bt proteins(Cry1Ab,Cry1Ac,Cry1F,Cry2Aa,and Cry2Ab)on P.spretus adults using a diet consisting of a 10% honey solution with or without Bt proteins at 400μg g^(-1) diet.The results showed that the survival and reproduction of P.spretus adults were reduced by the cysteine protease inhibitor E-64(a positive control)but were not affected by any of the five Bt proteins.The activities of digestive,detoxifying,and antioxidant enzymes in P.spretus were also unaffected by diets containing the Cry proteins,but they were significantly affected by the diet containing E-64.We then developed a tritrophic bioassay to determine the effects of the five Bt proteins on P.spretus larvae and pupae.In this assay,A.lucorum nymphs fed an artificial diet containing Cry proteins were used as the hosts for P.spretus.The results of the tri-trophic assay indicated that neither the pupation rate nor the eclosion rate of the P.spretus parasitoids were significantly affected by the presence of high concentrations of Bt proteins in the parasitized A.lucorum nymphs.The overall results indicate that these two assays can be used to evaluate the toxicity of insecticidal compounds to P.spretus and that the tested Cry proteins are not toxic to P.spretus.

Toxicity and binding analyses of Bacillus thuringiensis toxin Vip3A in Cry1Ac-resistant and-susceptible strains of Helicoverpa armigera(Hübner)

The Bacillus thuringiensis vegetative insecticidal protein, Vip3 A, represents a new family of Bt toxin and is currently applied to commercial transgenic cotton. To determine whether the Cry1Ac-resistant Helicoverpa armigera is cross-resistant to Vip3 Aa protein, insecticidal activities, proteolytic activations and binding properties of Vip3 Aa toxin were investigated using Cry1Ac-susceptible（96S） and Cry1Ac-resistant H. armigera strain（Cry1Ac-R）. The toxicity of Vip3 Aa in Cry1Ac-R slightly reduced compared with 96 S, the resistance ratio was only 1.7-fold. The digestion rate of full-length Vip3 Aa by gut juice extracts from 96 S was little faster than that from Cry1Ac-R. Surface plasmon resonance（SPR） showed there was no significant difference between the binding affinity of Vip3 Aa and BBMVs between 96 S and Cry1Ac-R strains, and there was no significant competitive binding between Vip3 Aa and Cry1 Ac in susceptible or resistant strains. So there had little cross-resistance between Vip3 Aa and Cry1 Ac,Vip3A＋Cry proteins maybe the suitable pyramid strategy to control H. armigera in China in the future.

Comparative Analysis of Cry1Ac Toxin Oligomerization and Pore Formation Between Bt-Susceptible and Bt-Resistant Helicoverpa armigera Larvae

With the long-term use of Bacillus thuringiensis （Bt） insecticide and expansion of CrylA-expressing transgenic plants, some insect pests have developed resistance to Bt in open fields, greenhouses, and in the laboratory. Bt resistance is complex and there appear to be different ways for resistance development. Understanding the Bt resistance mechanisms is critical to prolong its usefulness. In this article, Bt receptors, the cadherin and aminopeptidase N （APN）, in brush border membrane vesicles （/3BMV） of Helicoverpa armigera were examined in both CrylAc-susceptible （96S） and CrylAc- resistant （LF120） strains, to compare CrylAc toxin oligomerization and pore formation in these two strains. CrylAc toxin oligomerization and pore formation in these two strains were compared. Results showed that cadherin and aminopeptidase N proteins could express normally in both susceptible and resistant 11. armigera strains. The ability to form CrylAc oligomers and ion channels on BBMVs was also not significantly different between these two strains.

二十一世纪以来棉花害虫治理成就与展望

本文对21世纪以来的20年间我国棉花害虫治理研究进展进行了回顾,从Bt棉花害虫种群地位演替、靶标害虫对Bt棉花抗性演化、棉花-害虫-天敌化学通讯三个方面总结了棉花害虫生物学与生态学研究进展,从预测预报、棉花抗虫性利用、生态调控、理化诱控、生物防治、化学防治等方面梳理棉花害虫防控关键技术研发概况,以棉铃虫、盲蝽两大主要害虫为例介绍了棉花害虫绿色防控技术体系创新应用。最后,从我国棉花产业发展现状出发,对今后一段时间我国棉花害虫防控研究与实践进行了展望。