Effect of temperature on the development, fecundity, progeny sex ratio and life-table of Campoletis chlorideae, an endolarval parasitoid of the pod borer, Helicoverpa armigera

Development, survival, fecundity, progeny sex ratio (PSR) and age-specific life-table parameters of the parasitoid Campoletis chlorideae Uchida (Hymenoptera: Ichneumonidae) were examined at six different constant temperatures (12, 17, 22, 27, 32 and 37°C) in the laboratory [70 ± 10% RH and 10:14 h (light:dark) photoperiod]. Second instar larvae of Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) were reared on chickpea (Cicer arietinum L.) and used as the host. Development times shortened as the temperature increased from 12 to 37°C. The estimated lower developmental threshold (tL) was 3.4°C. The thermal summation for total immature stages was 379.97 degree-days. A reciprocal relationship between temperature and longevity was observed in the range of 12–17°C. The maximum mortality of pupae (71.8%) occurred at 37°C. At 22°C, the yield of a female parasitoid averaged 137.3 ± 14.7 (mean ± SD) progeny, of which 89.6 ± 7.6 were daughters. The number of daughters produced decreased when the females were kept either above or below 22°C, although the PSR was female biased in the range of 17–27°C. The analyses of life-table parameters, developmental rates, reproduction, mortality and PSR suggest that maximum population growth (r _m ) is near 27°C. There was little variation observed in most of the desired qualities of C. chlorideae in the range of 17–27°C, and it appears that the parasitoid is adapted to a wide range of temperatures. We suggest that for maximum production the parasitoid should be reared at 22 ± 4°C and be released in areas where the temperature ranges between 17° and 27°C, as in the plains of northern India.     

棉铃虫齿唇姬蜂嗅觉受体基因 CchlOrco 的克隆及组织表达谱分析

【目的】昆虫的嗅觉受体（olfactory receptors, ORs）一般以气味分子特异的ORs与共受体（ co-Receptor, Orco）通过形成异质二聚体在嗅觉感受中发挥关键作用,其中Orco由于具有序列的保守性而受到广泛的重视。本研究旨在克隆棉铃虫齿唇姬蜂 Campoletis chlorideae 的Orco基因,并对其组织表达谱进行分析。【方法】利用RT-PCR技术和转录组分析技术克隆棉铃虫齿唇姬蜂的Orco基因,并对其编码的氨基酸序列进行生物信息学分析;利用Real-time PCR技术对该基因在该蜂成虫不同组织中的表达量进行分析。【结果】获得了棉铃虫齿唇姬蜂 Orco 的全长cDNA序列,命名为 CchlOrco（GenBank登录号：KP255444）。序列分析表明, 该基因开放阅读框全长1 437 bp,编码478个氨基酸,预测该氨基酸序列具有7个跨膜区。CchlOrco 主要在成虫触角中表达,且在雄蜂触角中的表达量最高,是雌蜂触角中表达量的8.0倍,而在其他组织中表达量极低。【结论】本研究克隆了棉铃虫齿唇姬蜂 CchlOrco 序列全长,明确了其在成虫不同组织中的表达水平,为进一步研究该基因及其他嗅觉受体基因功能奠定了基础。     

Orientation behaviour of Helicoverpa armigera egg parasitoid,Trichogramma chilonis and the larval parasitoid,Campoletis chlorideae towards different groundnut genotypes

Behavioural responses of Helicoverpa armigera egg parasitoid, Trichogramma chilonis and the larval parasitoid, Campoletis chlorideae towards the leaves of groundnut (Arachis hypogaea) genotypes (ICGV 86699, ICGV 86031, ICG 2271, and ICG 1697-resistant, and the susceptible check-JL 24) were studied by using a Y-tube olfactometer. Orientation was studied in comparison to clean air, to insect resistant genotypes in relation to JL 24 and towards H. armigera damaged and undamaged leaves. Leaves of ICGV 86699, ICGV 86031 and ICG 2271 were more attractive to T. chilonis adults than to the clean air. They were strongly attracted to the leaves of ICGV 86699, ICGV 86031 and ICG 1697 than of JL 24. Insect damaged leaves of ICGV 86699, ICGV 86031 and ICG 1697 were more attractive than the respective uninfested leaves. C. chlorideae showed greater attraction towards leaves of ICGV 86699, ICG 2271 and ICG 1697 than the clean air, and were more attracted towards leaves of ICGV 86699 and ICGV 86031 than those of JL 24. The damaged leaves of ICGV 86699, ICGV 86031 and ICG 2271 were more attractive to C. chlorideae than the respective uninfested leaves. Thus insect resistant genotypes exhibited greater compatibility with the natural enemies in groundnut.     

The effect of parasitization by Trichogramma australicum on Helicoverpa armigera host eggs and embryos

Histological investigations of the pathology of Helicoverpa armigera (Hübner) eggs after attack by the egg parasitoid, Trichogramma australicum (Girault), indicate that the developing embryo is immediately killed by envenomation. Soon afterward the histological staining characteristics of parasitized host embryos change and the embryonic germ band dissociates into a mass of individual rounded cells. Hosts attacked by females sterilized by gamma-irradiation showed the same pathological effects as normally parasitized hosts, indicating that host degeneration is due to female venom rather than factors derived from the parasitoid embryo or larva. Cell death also occurred in older host embryos although tissue breakdown was delayed. These findings have allowed us to determine not just that the host dies but what happens to the cells and tissues, i.e., their physical appearance, the time course of their degeneration, and that the process is retarded in older hosts. These processes can possibly be emulated in artificial diets.     

Eimeria tenella: Effects of diclazuril treatment on microneme genes expression in second-generation merozoites and pathological changes of caeca in parasitized chickens

The effects of diclazuril on mRNA expression levels of invasion-related microneme genes were examined in second-generation merozoites of Eimeria tenella (E. tenella) by quantitative real-time (QRT) PCR. Diclazruil treatment of infected chickens significantly decreased the number of second-generation merozoites by 65.13%, and resulted in downregulation of EtMIC genes: EtMIC1 by 65.63%, EtMIC2 by 64.12%, EtMIC3 by 56.82%, EtMIC4 by 73.48%, and EtMIC5 by 78.17%. SEM images of caecum tissue from uninfected chickens showed regular intestinal villus structure. In infected chickens, a distinct loss of the superficial epithelium, with a flattened mucosa and large-area necrosis and anabrosis, was evident. In diclazruil-treated chickens, a decrease in merozoite number and a visibly improved appearance of the caeca were noted. These improvements appeared to be mediated in part by downregulation of the expression of invasion-related EtMIC genes in response to diclazuril.     

Cloning and characterization of two thermostable xylanases from an alkaliphilic Bacillus firmus

Two genes encoding thermostable xylanases, named xyn10A and xyn11A, from an alkaliphilic Bacillus firmus were cloned and expressed in Escherichia coli. The E. coli harboring either gene showed clear zone with Congo red clearance assay on xylan plate. The Xyn10A and Xyn11A have molecular weights of 45 and 23kDa, respectively, and both show activities on xylan-zymogram. The xyn10A encodes 396 amino acid residues and is very similar to an alkaliphilic xylanase A from alkaliphilic Bacillus halodurans. The Xyn11A contains 210 amino acid residues and only one amino acid different from an endo-beta-1,4-xylanase from B. halodurans. From alignment of the amino acid sequences with other xylanases, Xyn10A and Xyn11A belong to family 10 and 11 glycosyl hydrolases, respectively. Both show activities over the pH range of 4-11 at 37 degrees C and over 80% activities at 70 degrees C. Interestingly both still retain over 70% activities after 16h preincubation at 62 degrees C.     

Influence of the chickpea cropping system onHelicoverpa armigera (Lep.: noctuidae) populations and their rate of parasitism byCampoletis chlorideae (Hym.: ichneumonidae)

The effect of intercropping chickpea with coriander or linseed onHelicoverpa armigera Hübner egg and larval populations and on the rate of larval parasitism was examined. There were significantly moreH. armigera larvae per plot in the sole chickpea treatment than in the intercropped treatments. The reduction was not the result of a negative effect of the companion crops on the numbers of larvae attacking chickpea. Larval density on chickpea was higher in the intercropped plots than the plots of sole chickpea. The presence of the flowering companion crops did not result in either the attraction of otherwise uncommon parasitoids to the crop or an increase in the proportion ofH. armigera larvae parasitized byCampoletis chlorideae Uchida. The results suggest that chickpea is a more suitable host forH. armigera than coriander or linseed, and that the reduction in the density ofH. armigera larvae in the intercroped treatments was due to the lower number of chickpea plants in these treatments.     

Toxicity of Bacillus thuringiensis insecticidal proteins for Helicoverpa armigera and Helicoverpa punctigera (Lepidoptera: Noctuidae), major pests of cotton

The susceptibilities of the major pests of cotton in Australia, Helicoverpa armigera and Helicoverpa punctigera, to some insecticidal proteins from Bacillus thuringiensis were tested by bioassay. A commercial formulation, DiPel, and individual purified insecticidal proteins were tested. H. armigera was consistently more tolerant to B. thuringiensis insecticidal proteins than was H. punctigera, although both were susceptible to only a limited range of these proteins. Only Cry1Ab, Cry1Ac, Cry2Aa, Cry2Ab, and Vip3A killed H. armigera at dosages that could be considered acceptable. There was no significant difference in the toxicities of Cry1Fa and Cry1Ac for H. punctigera but Cry1Fa had little toxicity for H. armigera. The five instars of H. armigera did not differ significantly in their susceptibility to DiPel on the basis of LC(50). However, there were significant differences in the susceptibility to Cry1Ac and Cry2Aa of three strains of H. armigera. Bioassays conducted with Cry1Ac and Cry2Aa showed that there was a small but significant negative interaction between these delta-endotoxins.     

转基因抗虫棉对棉铃虫及其内寄生蜂的双重效应

以含1%转基因（Cry1A+CpTI）抗虫棉“中抗310”棉叶粉的人工饲料为基础,建立一套抗虫棉 棉铃虫Helicoverpa armigera 棉铃虫幼虫内寄生蜂中红侧沟茧蜂Microplitis mediator和棉铃虫齿唇姬蜂Campoletis chlorideae的三级营养关系的研究系统,研究了转基因抗虫棉对棉铃虫及内寄生蜂的双重效应,分析比较了6种状态的棉铃虫生长发育动态,以及寄生蜂的生长状况。结果表明,无论是否被寄生,抗虫棉对棉铃虫生长发育的抑制作用都非常显著;寄生取食抗虫棉饲料的棉铃虫的寄生蜂,其出茧率和茧重都显著下降,对于中红侧沟茧蜂,出茧率和茧重分别下降了26.1%和1.0 mg;对于棉铃虫齿唇姬蜂,分别下降了17.9%和5.1 mg。解剖寄主发现,两种寄生蜂在取食抗虫棉饲料的寄主体内发育缓慢并出现部分畸形幼蜂。棉铃虫幼虫血淋巴总蛋白含量和血淋巴蛋白SDS-PAGE电泳分析表明,取食抗虫棉饲料后,棉铃虫血淋巴总蛋白含量低于相应的对照,推测寄主血淋巴蛋白含量降低是导致寄生蜂生长缓慢、发育不正常的一个重要原因。     

Differential inhibition of Helicoverpa armigera gut proteinases by proteinase inhibitors of pigeonpea (Cajanus cajan) and its wild relatives

The seeds of 36 pigeonpea [Cajanus cajan (L) Millsp.] cultivars, resistant and susceptible to pests and pathogens and 17 of its wild relatives were analysed for inhibitors of trypsin, chymotrypsin, and insect gut proteinases to identify potential inhibitors of insect (Helicoverpa armigera) gut enzymes. Proteinase inhibitors (PIs) of pigeonpea cultivars showed total inhibition of trypsin and chymotrypsin, and moderate inhibition potential towards H. armigera proteinases (HGP). PIs of wild relatives exhibited stronger inhibition of HGP, which was up to 87% by Rhynchosia PIs. Electrophoretic detection of HGPI proteins and inhibition of HGP isoforms by few pigeonpea wild relative PIs supported our enzyme inhibitor assay results. Present results indicate that PIs exhibit wide range of genetic diversity in the wild relatives of pigeonpea whereas pigeonpea cultivars (resistant as well as susceptible to pests and pathogens) are homogeneous. The potent HGPIs identified in this study need further exploration for their use in strengthening pigeonpea defence against H. armigera.     

Comparative pathogenesis of the Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus in noctuid hosts of different susceptibility

Neonate larvae of the noctuid moth Spodoptera exigua were susceptible to an infection by Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus (HaSNPV). Biological activity (LD(50),ST(50)) of the virus was considerably reduced as compared to its activity in the homologous host, H. armigera. Pathogenesis was studied using a recombinant HaSNPV carrying a green fluorescent protein gene, which induces fluorescence in infected cells to mark infection. In larvae of H. armigera, fluorescence was pronounced in the fat body after 2.9 days post infection and could also be detected in several other tissues. In contrast, fluorescence was not observed in tissues of S. exigua until 9 days post infection and was restricted almost exclusively to cells of the ganglia. Examination of serial sections of wildtype HaSNPV-infected S. exigua-larvae revealed a similar pattern of tissue tropism. Apparently, HaSNPV does not undergo the usual steps in host invasion and infection in this insect species, but targets specifically to nervous tissue.     

Inhibition of pheromone biosynthesis in Helicoverpa armigera by pheromonostatic peptides

Male insect accessory glands contain factors that are transferred during mating to the female, some inducing post-mating behavior, including the cessation of pheromone production, non-receptivity and the initiation of oviposition. One such factor is the Drosophila melanogaster sex-peptide (DrmSP). A pheromone suppression peptide, termed HezPSP, was identified in the moth Helicoverpa zea, isolated by HPLC and the active peak sequenced, but the activity of the synthesized peptide has not been reported to date. HezPSP bears no sequence homology to DrmSP. However, both peptides contain a disulfide bridge separated by an equal number, but dissimilar, amino acids. We herein report on the pheromonostatic activity of HezPSP partial peptides in the moth Helicoverpa armigera.     

棉铃虫5型质型多角体病毒RNA聚合酶的确定与功能机制研究

棉铃虫5型质型多角体病毒属于呼肠孤病毒科质型多角体病毒属,以重要农业害虫棉铃虫为其天然宿主,对棉铃虫的生物控制具有重要意义.本文对棉铃虫5型质型多角体病毒第3片段编码的蛋白的功能进行了初步研究.首先通过同源性对比,推测其所编码的蛋白可能行使RNA依赖的RNA聚合酶(RdRP)的功能.通过体外活性研究确定了该蛋白的RdRP活性,并确定了其保守活性位点GDD.随后以病毒基因组RNA和3′-OH封闭的病毒基因组RNA为模板,利用Northern blot方法研究该蛋白起始病毒基因组RNA合成的分子机制.结果表明,该病毒的RdRP主要通过引物非依赖的方式起始病毒基因组RNA的合成,并且该RdRP蛋白并不具有末端转移酶活性.最后,对RdRP行使功能的生化条件进行探索,发现RdRP功能的发挥需要二价金属离子Mg2+的存在.     

八肋游仆虫大核基因组中组蛋白基因 H4A和H4B的克隆及分析

组蛋白作为核小体的基本组分,是染色质的结构和功能必需的。组蛋白的变体和修饰共同参与染色质修饰及基因的表达调控。真核生物细胞中的5种组蛋白在进化中高度保守,然而纤毛虫的组蛋白H4与其他真核生物相比有较大的差异。本实验应用PCR技术从八肋游仆虫(Euplotes octocarinatus)中获得了2种组蛋白H4基因,分别为H4A和H4B,GenBank登录号为:JN715068和JN715069。序列分析表明,H4A基因开放阅读框324 bp,预测编码107个氨基酸,分子量为11.6 ku,等电点为10.99。而H4B基因编码框384 bp,编码127个氨基酸,分子量为14.4 ku,等电点为9.93。Blast结果显示,H4A序列与其他生物中H4的一致性相对较高,达81%～94%,而H4B的一致性为36%～70%。H4A和H4B的一致性仅为44.7%。实时荧光定量PCR表明,H4A的转录本高于H4B。结果提示:在进化过程中八肋游仆虫可能进化出特殊的组蛋白H4基因,不同的组蛋白H4可能发挥不同的功能。     

Bitter gourd proteinase inhibitors: potential growth inhibitors of Helicoverpa armigera and Spodoptera litura

Proteinase inhibitors (PIs) from the seeds of bitter gourd (Momordica charantia L.) were identified as strong inhibitors of Helicoverpa armigera gut proteinases (HGP). Biochemical investigations showed that bitter gourd PIs (BGPIs) inhibited more than 80% HGP activity. Electrophoretic analysis revealed the presence of two major proteins (BGPI-1 and-2) and two minor proteins (BGPI-3 and-4) having inhibitory activity against both trypsin and HGP. The major isoforms BGPI-1 and BGPI-2 have molecular mass of 3.5 and 3.0 kDa, respectively. BGPIs inhibited HGP activity of larvae fed on different host plants, on artificial diet with or without added PIs and proteinases excreted in fecal matter. Degradation of BGPI-1 by HGP showed direct correlation with accumulation of BGPI-2-like peptide, which remained stable and active against high concentrations of HGP up to 3 h. Chemical inhibitors of serine proteinases offered partial protection to BGPI-1 from degradation by HGP, suggesting that trypsin and chymotrypsin like proteinases are involved in degradation of BGPI-1. In larval feeding studies, BGPIs were found to retard growth and development of two lepidopteran pests namely Helicoverpa armigera and Spodoptera litura. This is the first report showing that BGPIs mediated inhibition of insect gut proteinases directly affects fertility and fecundity of both H. armigera and S. litura. The results advocate use of BGPIs to introduce insect resistance in otherwise susceptible plants.