The involvement of microsomal oxidases in pyrethroid resistance in Helicoverpa armigera from Asia

Five contemporary strains of the bollworm Helicoverpa armigera Hübner from China, Pakistan and India, all with high resistance to pyrethroids, were compared with a standard susceptible strain that originated from the Cote D'Ivoire in the 1970s ('SCD'). Two of the Chinese strains ('YGF' and 'YGFP') were derived by laboratory selection from a third, field collected strain ('YG'). The strain 'YG' exhibited 7-, 14- and 21-fold resistance to fenvalerate, cypermethrin and deltamethrin, respectively. After selection with fenvalerate for 14 generations ('YGF'), this increased to 1690-, 540- and 73-fold. Selection with a mixture of fenvalerate and piperonyl butoxide (PBO) for 14 generations ('YGFP') resulted in resistance ratios of 2510, 2920 and 286. The synergistic ratios to fenvalerate that resulted from pre-treatment of PBO were 5-, 462- and 12-fold in YG, YGF and YGFP strains, respectively. Resistance ratios for a Pakistani strain (PAK) were 2320-, 4100- and 223-fold to fenvalerate, cypermethrin and deltamethrin, respectively. The synergistic ratio of PBO to these pyrethroids was 450-, 950- and 11-fold. The strong synergism of pyrethroids by PBO implied that an oxidative metabolism could be involved in pyrethroid resistance in these resistant strains. The activities of cytochrome P450 monooxygenases from midguts of final instar larvae to p-nitroanisole (PNOD), ethoxycoumarin (ECOD), methoxyresorufin (MROD) significantly increased in all the resistant strains when compared with the susceptible strain. This further implies that cytochrome P450 monooxygenases are involved in pyrethroid resistance in Asian H. armigera. Comparative in vitro studies of the metabolism of 14C-deltamethrin by midgut microsomes of the resistant PAK and susceptible SCD strains showed that the resistant strain had a much greater capacity than the susceptible strain for the metabolic degradation of deltamethrin. This enhanced metabolic degradation occurred in the presence of NADPH which suggested an oxidative detoxification. In the resistant strains, minor increases in glutathione S-transferase activity (to the substrates CDNB and DCNB), and esterase activity (to the substrate alpha-naphthyl acetate) further suggested that, of the putative metabolic mechanisms, oxidases are the most important. This study provides the first evidence that cytochrome P450 monooxygenases are a major metabolic mechanism responsible for pyrethroid resistance in H. armigera from Asia.     

Molecular identification of three novel glutaredoxin genes that play important roles in antioxidant defense in Helicoverpa armigera

Glutaredoxins (Grxs), also known as thioltransferases, play key roles in maintaining intracellular redox balance and protecting cells from oxidative damage in plants and mammals. We tested whether Grxs play important roles in antioxidant defense in insects using the moth, Helicoverpa armigera. We obtained the full-length cDNA sequences of three novel Grx genes, named HaGrx, HaGrx3, and HaGrx5. Sequence analysis indicated that HaGrx shared a high amino acid identity (58%–78%) and a CPYC motif of conserved redox activity with homologues from other selected insect species. In contrast, HaGrx3 and HaGrx5 both shared a CGF(S/G) motif, a conserved catalytic domain, with other orthologous genes. Quantitative real-time PCR results revealed that HaGrx, HaGrx3, and HaGrx5 exhibited temporally- and spatially-dependent patterns of expression. The mRNA expression of HaGrx, HaGrx3, and HaGrx5 was induced by various temperature stresses and H₂O₂ treatments. We further investigated the knockdown of HaGrx, HaGrx3, and HaGrx5 in H. armigera larvae and found that most of the selected antioxidant genes were up regulated. However, Tpx was down regulated, and further interpretation of the complementary functions of these antioxidant genes is still required. We also determined the effect of HaGrx, HaGrx3, and HaGrx5 knockdown on antioxidant enzymatic activity and metabolite content. The enzymatic activities of SOD, CAT, and POD, and the metabolite contents of hydrogen peroxide, ascorbate, protein carbonyl, and total GSH increased after RNAi mediated knockdown of HaGrx, HaGrx3, and HaGrx5. These results supported our hypothesis that HaGrx, HaGrx3, and HaGrx5 play important roles in antioxidant defense of Helicoverpa armigera and provided a theoretical basis for further in-depth study of physiological function in the insect glutaredoxin family genes.     

棉铃虫蛾复眼光反应特性

用视网膜电位图(electroretinogram,ERG)技术研究了棉铃虫Helicoverpa armigera蛾暗适应过程中对单色光和白光刺激的光感受性变化。结果显示：(1)依ERG振幅大小(峰-峰值),在340～605 nm波谱内有3个大小不等的峰-主峰位于绿 黄光区562 nm,次峰在蓝光区483 nm,第3个峰在近紫外区400 nm,显示其至少有3种感受器;(2)性别、日龄及暗适应时间长短对其光谱敏感性有影响,低龄时雄蛾对单色光刺激较雌蛾敏感,高日龄时相反;1～5日龄内, 3日龄蛾的视网膜电位(ERP)值最高;随暗适应时间延长,其复眼对近紫外区400 nm敏感性明显增加;(3)一定光强度范围内,随单色光和白光光强度增强该蛾复眼的ERP值增大,初期增加较缓,中期较快,呈近似S型曲线,显示其复眼具有较强的光强度自调节和适应机制。     

Molecular characterization and functional distribution of N-ethylmaleimide-sensitive factor in Helicoverpa armigera

N-ethylmaleimide-sensitive fusion protein (NSF) is an essential component for the neurotransmitter or neurohormone release apparatus present in all eukaryotic cells. Here, a new NSF orthologue was characterized from the cotton bollworm, Helicoverpa armigera (Har). Northern blot exhibited a high expression in larval brain. Southern analysis indicated that a single copy of the gene is present in a haploid genome. Using antibodies labeled with fluoresceins, we directly proved that NSF is co-localized with two crucial neurohormones, prothoracicotropic hormone and diapause hormone, both of which regulate insect development. These findings suggest that Har-NSF may be involved in regulating insect neurohormone release.     

转双基因抗虫棉对棉铃虫的抗性

将苏云金杆菌 (Ｂａｃｉｌｌｕｓｔｈｕｒｉｎｇｉｅｎｓｉｓ,Ｂｔ)杀虫晶体蛋白 (ｉｎｓｅｃｔｉｃｉｄａｌｃｒｙｓｔａｌｐｒｏｔｅｉｎ ,ＩＣＰ)基因转入棉花植株中获得的抗虫棉花 (下略为Ｂｔ棉 ) ,为棉花害虫综合防治开辟了新的途径。Ｂｔ棉除具有丰产性、纤维品质好和对靶标害虫的良好控制作用等特性外 ,更重要的应用价值在于其高杀虫效果与维护生态环境的协调发展的良性作用上[1] 。但研究结果已表明 ,害虫对ＢｔＩＣＰ的抗性适应 ,将严重威胁Ｂｔ棉的使用寿命[2 ] 。已有的报道证明 ,将两种不同作用机制的杀虫基因转入植物并同时表…     

Molecular cloning and characterization of Hearm caspase-1 from Helicoverpa armigera

Members of the caspase family play a central and evolutionary role in programmed cell death (PCD), which removes unwanted, damaged and dangerous cells during development to maintain homeostasis. In this paper, we describe the cloning and characterization of a caspase from the cotton bollworm, Helicoverpa armigera, named Hearm caspase-1. The 1,350 bp full-length cDNA contains an 885 bp open reading frame (ORF) that encodes a Hearm caspase-1 proenzyme of 294 amino acids. The deduced protein is highly homologous to Spodoptera frugiperda Sf caspase-1 and Drosophila melanogaster ICE and has the highly conserved pentapeptide QACQG, the recognized catalytic site of caspases, suggesting that it is an effector caspase of the cotton bollworm. Northern blot and RT-PCR analyses demonstrate that Hearm caspase-1 is expressed in embryos and the fat body, midgut and haemocytes of feeding and wandering larvae. Expression of Hearm caspase-1 in the haemocytes appears to be correlated with the pulse of ecdysone, and it is up-regulated by ecdysone agonist RH-2485, implying that Hearm caspase-1 activation is regulated by ecdysone.     

Purification and characterization of prophenoloxidase from cotton bollworm,Helicoverpa armigera

Phenoloxidases are oxidative enzymes, which play an important role in both cell mediated and humoral immunity. Purification and biochemical characterization of prophenoloxidase from cotton bollworm, Helicoverpa armigera (Hübner) were carried out to study its biochemical properties. Prophenoloxidase consists of a single polypeptide chain with a relative molecular weight of 85 kDa as determined by SDS–PAGE, MALDI–TOF MS and LC–ESI MS. After the final step, the enzyme showed 71.7 fold of purification with a recovery of 49.2%. Purified prophenoloxidase showed high specific activity and homology with phenoloxidase subunit‐1 of Bombyx mori and the conserved regions of copper binding (B) site of phenoloxidase. Purified prophenoloxidase has pH optima of 6.8 and has high catalytic efficiency towards the dopamine as a substrate in comparison to catechol and L‐Dopa. The PO activity was strongly inhibited by phenylthiourea, thiourea, dithiothreitol and kojic acid.     

Comparison of Bombyx mori and Helicoverpa armigera cytoplasmic actin genes provides clues to the evolution of actin genes in insects

The cytoplasmic actin genes BmA3 and BmA4 of Bombyx mori were found clustered in a single genomic clone in the same orientation. As a similar clustering of the two cytoplasmic actin genes Ha3a and Ha3b also occurs in another lepidopteran, Helicoverpa armigera, we analyzed the sequence of the pair of genes from each species. Due to the high conservation of cytoplasmic actins, the coding sequence of the four genes was easily aligned, allowing the detection of similarities in noncoding exon and intron sequences as well as in flanking sequences. All four genes exhibited a conserved intron inserted in codon 117, an original position not encountered in other species. It can thus be postulated that all of these genes derived from a common ancestral gene carrying this intron after a single event of insertion. The comparison of the four genes revealed that the genes of B. mori and H. armigera are related in two different ways: the coding sequence and the intron that interrupts it are more similar between paralogous genes within each species than between orthologous genes of the two species. In contrast, the other (noncoding) regions exhibited the greatest similarity between a gene of one species and a gene of the other species, defining two pairs of orthologous genes, BmA3 and HaA3a on one hand and BmA4 and HaA3b on the other. However, in each species, the very high similarities of the coding sequence and of the single intron that interrupts it strongly suggest that gene conversion events have homogenized this part of the sequence. As the divergence of the B. mori genes was higher than that of the H. armigera genes, we postulated that the gene conversion occurred earlier in the B. mori lineage. This leads us to hypothesize that gene conversion could also be responsible for the original transfer of the common intron to the second gene copy before the divergence of the B. mori and H. armigera lineages.     

Mechanisms of resistance to Helicoverpa armigera and introgression of resistance genes into F1 hybrids in chickpea

The noctuid pod borer, Helicoverpa armigera is a major pest of chickpea, and host plant resistance is an important component for managing this pest. We evaluated a set of diverse chickpea genotypes with different levels of resistance to H. armigera, and their F₁ hybrids for oviposition non-preference, antibiosis, and tolerance components of resistance under uniform insect infestation under greenhouse/laboratory conditions. The genotypes ICC 12476, ICC 12477, ICC 12478, ICC 12479, and ICC 506EB were non-preferred for oviposition under no-choice, dual-choice, and multi-choice conditions, and also suffered lower leaf damage in no-choice tests as compared to the susceptible check, ICCC 37. Antibiosis expressed in terms of low larval weights was observed in insects reared on ICC 12476, ICC 12478, and ICC 506EB. Weight gain by the third-instars was also low on ICC 12476, ICC 12477, ICC 12478, ICC 12479, and ICC 506EB at the podding stage. Non-preference for oviposition and antibiosis (poor larval growth) were also expressed in hybrids based on ICC 12477, ICC 12476, ICC 12478, ICC 12479, and ICC 506EB as compared to the hybrids based on the susceptible check, ICCC 37, indicating that oviposition non-preference and antibiosis in the F₁ hybrids is influenced by the parent genotype. Loss in grain yield was lower in ICC 12477, ICC 12478, ICC 12479, and ICC 506EB compared to that on ICCC 37. The genotypes ICC 12477, ICC 12478, ICC 12479, and ICC 506EB showing antixenosis, antibiosis, and tolerance mechanism of resistance to H. armigera can be used for developing chickpea cultivars for resistance to this pest.     

Molecular adaptations of Helicoverpa armigera midgut tissue under pyrethroid insecticide stress characterized by differential proteome analysis and enzyme activity assays

Helicoverpa armigera is an insect that causes important economic losses in crops. To reduce this loss, pyrethroids have been commonly used against H. armigera in farming areas. However, excess and continuous usage of pyrethroids cause resistance in H. armigera. Therefore, expressions of midgut proteins of two H. armigera field populations were compared to those of a susceptible strain by 2-D PAGE and MALDI-ToF-MS. Our results indicate that H. armigera reacts to pyrethroid-induced stress mainly by increasing the expression of energy metabolism-related proteins, such as ATP synthase and arginine kinase. NADPH cytochrome P450 reductase, also up-regulated, could play a role in detoxification of toxic pyrethroid metabolites, such as 3-phenoxybenzaldehyde. Interestingly, while GSTs were not found up-regulated in the comparative proteome analysis, biochemical assays showed significant increases of enzyme activities in both field populations as compared to the susceptible strain. Similarly, although esterases were not found differentially expressed, biochemical assays showed significant increases of esterase activities in both field populations. Thus, esterases are also proposed to be involved in metabolic responses towards pyrethroid insecticide-induced stress. In conclusion, we suggest increased energy metabolism in the midgut tissue of H. armigera as a general prerequisite for compensating the costs of energy-consuming detoxification processes.     

棉铃虫雌雄性腺转录组的比较分析

【目的】棉铃虫Helicoverpa armigera(Hübner)是我国重要的农业害虫,生殖力强是其发生为害的内在生物学基础,而已知的与棉铃虫生殖相关的基因信息相对较少,本研究旨在筛选棉铃虫性腺发育的相关基因。【方法】通过高通量测序技术对棉铃虫成虫的卵巢和精巢进行转录组测序。【结果】共获得100 603条unigenes,平均长度为666.05 bp,其中N50为1 114 bp。经同源性比对,52 071条unigenes获得注释信息,其中注释到Nr数据库的序列最多。通过比较转录组分析发现,在棉铃虫精巢和卵巢中存在7 714个差异表达的基因（Differentially expressed genes,DEGs）,其中3 288个DEGs表达水平在卵巢中上调,4 426个DEGs在精巢中上调。差异基因富集结果涉及到生殖系统发育的相关通路有mTOR信号通路、卵母细胞减数分裂、促性腺激素释放激素信号通路和胰岛素信号通路等。同时筛选得到部分与性腺发育相关的基因,包括vitellogenin(Vg)、vitellogeninreceptor(Vg R)、chorion-relatedgene、testis-specific serine/threonine-protein kinase和spermatogenesis-associated protein等。选取其中12个DEGs用实时荧光定量检测其表达量,结果表明RT-qPCR与RNA-Seq的结果一致。【结论】本研究获得了棉铃虫雌雄性腺的转录组数据及主要性腺发育相关基因,将有助于丰富棉铃虫繁殖相关的基因资源,为进一步研究棉铃虫生殖调控机理提供基础数据。     

Wheat methionine sulfoxide reductase genes and their response to abiotic stress

The wheat cultivar Shanrong no. 3 (cv. SR3) tolerates both salinity and drought stress more effectively than does its progenitor cultivar Jinan 177 (cv. JN177). When the cultivars are subjected to stress, a number of genes encoding methionine sulfoxide reductase (MSRs) are known to be upregulated in SR3. Here, a set of 12 full length Triticum aestivum MSR (TaMSR) cDNAs have been isolated from cv. SR3. The genes were transcribed in the wheat root, stem, and leaf in plants sampled at various developmental stages. Those induced by salinity and drought harbored known stress-responsive cis elements in their promoter region. The constitutive expression in Arabidopsis thaliana of four MSRs which were induced by salt and drought in microarray assay showed that the product of one (TaMSRA2) heightened the plant’s tolerance to NaCl, methylviologen (MV), and abscisic acid, that of the second (TaMSRA5) enhanced salinity tolerance, that of the third (TaMSRB1.1) increased tolerance to salinity, MV and H₂O₂, and that of the fourth (TaMSRB5.1) increased tolerance to both salinity and mannitol. The effect of the presence in A. thaliana of TaMSRB1.1 was to suppress the accumulation of reactive oxygen species and to increase the intracellular content of soluble sugars.     

Alterations in hippocampal antioxidant enzyme activities and sympatho-adrenomedullary system of rats in response to different stress models

The study deals with activity of three antioxidant enzymes, copper, zinc-superoxide dismutase (CuZnSOD), manganese superoxide dismutase (MnSOD), catalase (CAT) in hippocampus of rats, following the exposure to single chronic (individual housing or forced swimming) and acute (immobilization or cold) stress, as well as to combined chronic/acute stress. In addition, plasma noradrenaline (NA) and adrenaline (A) concentrations were measured in the same stress conditions, because their autooxidation can add to the oxidative stress. We observed that i) long-term social isolation and repeated forced swimming had minor effects on plasma catecholamines, but in the long-term pretreated groups, acute stressors caused profound elevation NA and A levels, ii) chronic stressors activate antioxidant enzymes, iii) acute stressors decrease catalase activity, their effects on CuZnSOD appear to be stressor-dependent, whereas MnSOD is not affected by acute stressors, and iv) pre-exposure to chronic stress affects the antioxidant-related effects of acute stressors, but this effect depends to a large extent on the type of the chronic stressor. Based on both metabolic and neuroendocrine data, long-term isolation appears to be a robust psychological stressor and to induce a "priming" effect specifically on the CuZnSOD and CAT activity.     

棉铃虫对辛硫磷抗性的风险评估与预报

在室内用辛硫磷对采自江苏东台的棉铃虫Helicoverpa armigera连续筛选了12代,平均成活率为37.6%,抗性上升了4.9倍。据Tabashnik介绍的方法,估计了抗性现实遗传力为0.0865。并预报了棉铃虫对辛硫磷的抗性发展速率。还对抗性风险评估、影响抗性发展速率的因素及抗性治理进行了讨论。     

不同温、湿度下白僵菌对棉铃虫幼虫的致病力

测试了不同温、湿度下白僵菌Beauveria bassiana对棉铃虫Helicoverpa armigera幼虫的致病力。实验设置了10、15、20、25、30℃ 5个温度水平,用高浓度（1×10<.sup8孢子/mL）、中浓度（1×10⁷孢子/mL）、低浓度（1×10⁶孢子/mL）的白僵菌孢子液分别感染1～4龄的棉铃虫。结果表明：三个浓度处理的棉铃虫在温度为25℃时的致死中时（LT₅₀）最短,死亡速度最快,死亡率最高;高于或低于此温度时,棉铃虫的LT₅₀延长,死亡速度减慢。相对湿度发生变化时,感病棉铃虫死亡速度和死亡率明显不同。相对湿度为95%左右时,棉铃虫死亡速度最快,死亡率最高;相对湿度低于70%时,棉铃虫死亡率显著降低。