Insecticidal activity of the chitinase from the Spodoptera litura nucleopolyhedrovirus

Baculovirus chitinase gene (chiA) is a late gene essential for liquefying the host insect at a late stage of infection for its hydrolyzing chitin function. In a previous report, baculovirus ChiA has been shown to offer many interesting new opportunities for pest control. Recently, a putative chiA gene was identified in the Korean isolate of the Spodoptera litura nucleopolyhedorvirus (SpliMNPV‐K1) genome. The open reading frame (ORF) contains 1692 nucelotides and encodes a protein of 563 amino acids with a predicted molecular weight of about 62.6 kDa. To study the insecticidal activity of ChiA from SpliMNPV‐K1, we constructed a recombinant AcMNPV, Ap‐SlChiA, which is designed to express the ChiA under the control of a polyhedrin promoter. Western blot analysis indicated that ChiA was successfully expressed by this recombinant virus. Chitinase assay revealed that the chitobiosidase and endochitinase activity of the recombinant virus was 2.5‐ and 3.9‐flods higher than those of wild‐type AcMNPV, respectively. In addition, the recombinant virus showed higher evident insecticidal activity against 3rd instar larvae of Spodotera exigua than that of the AcMNPV. These results suggest that the chiA gene from SpliMNPV‐K1 could be successfully applied to improve pathogenicity of baculoviruses.     

Insecticidal activity influence of destruxins on the pathogenicity of Paecilomyces javanicus against Spodoptera litura

Abstract:  The bioactivities of destruxins (dtx), depsipeptides isolated from Metarhizium anisopliae , against Spodoptera litura were tested in laboratory. For contacting toxicities, dtx-E was more effective than dtx-A and dtx-B. The LC₅₀s values of dtx-A, B and E were 197.98, 292.00 and 113.99 mg/l at 48 h after treatment, while the LT₅₀s were 42.65, 59.45 and 23.68 h at 300 mg/l. In the experiment of antifeedant activity, dtx-A, dtx-B and dtx-E at five concentrations (200, 100, 50, 25 and 12.5 mg/l) were bioassayed. Destruxins in a dose-dependent manner gave an apparent antifeedant activity. Generally, dtx-A, over dtx-B and dtx-E had the significant (P < 0.05) larger choice and no-choice antifeedant indexes (CAIs and NCAIs). At the concentration of 200 mg/l, the CAIs or NCAIs of dtx-A, dtx-B and dtx-E were 96.78, 84.93 and 85.90 or 89.75, 62.42 and 72.28 respectively. Furthermore, the synergistic activity of crude destruxin (CD) for pathogenicity of Paecilomyces javanicus strain Pj01 was detected. The LC₅₀s values of single Pj01 and the mixtures of Pj01 plus CD at 100 or 200 mg/l (Pj01-CD100 or Pj01-CD200) were respectively 474.63 × 10⁵, and 197.45 × 10⁵ or 113.11 × 10⁵ spores/ml at the fifth day after treated. Meanwhile, Pj01, Pj01-CD100 and Pj01-CD200 gave the LT₅₀s values of 6.99 day, 5.49 day and 4.21 day at 100 × 10⁵ spores/ml. Clearly, dtx decreased the values of LC₅₀ and LT₅₀ of the strain Pj01.     

In vitro biosynthesis of volicitin in Spodoptera litura

Volicitin [N-(17-hydroxylinolenoyl)-L-glutamine] and N-linolenoyl-L-glutamine, originally identified in the regurgitant of Spodoptera exigua, induce damaged corn leaves to release volatile compounds which enable parasitic wasps to locate host caterpillars. Here we demonstrate the in vitro biosynthesis of volicitin for the first time by using gut tissues of Spodoptera litura larvae, as well as N-linolenoyl-L-glutamine. When crop, midgut tissues, peritrophic membrane and gut contents isolated from S. litura were incubated with sodium linolenate and L-[alpha-15N] glutamine, not only 15N-labeled N-linolenoyl-L-glutamine but 15N-labeled volicitin was detected mainly in the midgut incubation by LCMS and LCMSMS analysis. In contrast, there were negligible amounts of the newly biosynthesized compounds in the gut content incubation. Furthermore, the microsomal fraction obtained from the gut tissues clearly showed specific incorporation of glutamine. This substrate selectivity accounts for the exclusive uptake of glutamine by fatty acid amides (FAAs) in the noctuid caterpillars, even though glutamine was not a major component in the regurgitant. Additionally, intensive chemical analyses revealed that more than 20% of glutamine in hemolymph was present as conjugates in gut contents. These results suggest that FAA compounds are actively synthesized by caterpillar tissues and might play important physiological role(s) in glutamine metabolism.     

杀菌剂对斜纹夜蛾SL细胞系和幼虫的生物活性

以MTT法筛选了19种杀菌剂对斜纹夜蛾Spodoptera litura SL细胞的毒杀活性,并以该方法研究了三唑类杀菌剂对SL细胞的毒力。结果表明: 福美双、烯唑醇、己唑醇、氟硅唑、苯霜灵、苯醚甲环唑、戊唑醇和腈菌唑等杀菌剂对SL细胞具有优异的毒杀活性。三唑类杀菌剂腈菌唑、烯唑醇、己唑醇和戊唑醇处理SL细胞48 h后,LC₅₀值分别为21.94 μg/mL、 23.80 μg/mL、 33.16 μg/mL和47.63 μg/mL。以考马斯亮蓝G₂₅₀法研究了腈菌唑对SL细胞中蛋白质含量和乳酸脱氢酶(LDH)漏出率的影响, 20 μg/mL腈菌唑处理12 h、24 h、48 h和72 h后,SL细胞中蛋白质含量分别降低8.55%、25.95%、42.95%和67.05%;处理24 h和48 h后,SL细胞的LDH漏出率分别为30.66%和32.05%。以浸叶喂食法处理斜纹夜蛾3龄幼虫,三唑类杀菌剂可显著抑制试虫体重增长。以0.5 μg/头、 1.0 μg/头和2.0 μg/头剂量的腈菌唑注射处理72 h后,斜纹夜蛾4龄幼虫血细胞数量分别降低12.31%、 25.96%和25.73%;腈菌唑注射处理48 h和72 h后,对斜纹夜蛾幼虫的LD₅₀值分别为1.59 μg/头和1.53 μg/头。结果显示以离体培养细胞为对象,从现有杀菌剂中寻找新的杀虫剂先导化合物具有良好的研究潜力。     

Antifeedant activity of an anthraquinone aldehyde in Galium aparine L. against Spodoptera litura F

The insect antifeedant anthraquinone aldehyde nordamnacanthal (1,3-dihydroxy-anthraquinone-2-al) was identified in Galium aparine L., and isolated from the root powder of akane (Rubia akane), a member of the Rubiaceae. Structure-activity relationship (SAR) studies using a series of anthraquinone analogues suggested that the aldehyde group on the anthraquinone was more important than the quinone moiety for antifeedant activity against the common cutworm (Spodoptera litura). High levels of nordamnacanthal were found in the seed leaf stage and in callus tissue induced from seedlings of G. aparine, but its concentration decreased with plant development. Since these compounds are natural pigments for dying textiles, we also evaluated the antifeedant activity against the carpet beetle (Attagenus japonicus ), a textile pest was also evaluated. While nordamnacanthal had strong antifeedant activity against the common cutworm, it did not show any antifeedant activity against the carpet beetle. The most effective antifeedant against the carpet beetle was the major constituent in the extract of R. trictorum, lucidin-3-O-primeveroside, a food pigment.     

Decarboxylases for polyamine biosynthesis in Drosophila melanogaster larvae.

Ornithine decarboxylase (L-ornithine carboxy-lase, EC 4.1.1.17) and S-adenosyl-methionine decarboxylase (S-adenosyl-L-methionine carboxy-lase, EC 4.1.1.50) were assayed in Drosophilia melanogaster larvae. The highest enzyme activities were detected in 24 and 48 h larvae, with diminishing activities in subsequent larval stages. Stimulation of S-adenosylmethionine decarboxylase by putrescine was demonstrable in late but not in early stages of larval development.     

Antifeedant and toxic activity of Trichilia americana extract against the larvae of Spodoptera litura

The biological activity of a crude methanolic extract of Trichilia americana (Sesse and Mocino) Pennington (Meliaceae) was assessed using the Asian armyworm, Spodoptera litura (Fabr.) (Lepidoptera: Noctuidae). The extract exhibited strong antifeedant activity in a choice leaf disc bioassay with 0.18 g cm^–2 extract deterring feeding by 50%. In nutritional assays, the crude extract reduced growth, consumption and the utilisation of ingested and digested food in a dose-dependent manner when fed to larvae, suggesting both antifeedant and toxic activities. When relative growth rates were plotted against relative consumption rates, the growth efficiency of the S. litura fed on diet containing T. americana crude extract was significantly less than that of control larvae. This result further indicates that the extract acts as both an antifeedant and chronic toxin. Toxicity is only seen following ingestion and was not observed following topical application or injection into the hemocoel. Larvae reared initially on extract-containing diet then transferred to control diet showed nutritional indices comparable to those of larvae fed continuously on control diet. This suggests that the extract is not permanently damaging the insect's digestive tract. The mode-of-action of the extract as a chronic toxin remains unknown.     

Insecticidal activity of synthetic amides on Spodoptera frugiperda

The phytochemistry of the genus Piper (Piperaceae) has been widely studied due to the biological properties of amides from these plants. In this work, we have synthesized and evaluated the toxic effect of 11 amides against the fall armyworm Spodoptera frugiperda larvae. The naturally occurring piperine was also evaluated. The most active amide was N-[3-(3',4'-methylenedioxyphenyl)-2-(E)-propenoyl]piperidine with a LD50 of 1.07 microg mg(-1) larvae. This amide was also evaluated by ingestion.     

杀菌剂丙环唑对斜纹夜蛾的毒性

在研究比较了11种农药对斜纹夜蛾Spodoptera litura细胞（简称SL细胞）的毒杀活性基础上,选择毒杀活性最高的杀菌剂丙环唑,对其毒理学机理进行进一步研究。结果表明,丙环唑的细胞毒力最高,在100 μg/mL浓度下处理后48 h,SL细胞的死亡率为98.08％。处理后36 h,丙环唑对SL细胞的LC₅₀值为20.31 μg/mL。丙环唑能明显降低SL细胞的蛋白质含量。以0.5 μg/头的丙环唑注射斜纹夜蛾4龄幼虫,处理后72 h,试虫血淋巴总含量及血细胞数分别下降了26.80％和25.26％;在1.0 μg/头的剂量下,则分别下降了37.67%和36.32%。以0.5 μg/头和1.0 μg/头的丙环唑注射处理后,斜纹夜蛾幼虫体重显著降低。此外,丙环唑能降低斜纹夜蛾幼虫血淋巴含糖量及血淋巴蛋白质含量。在注射处理后96 h和120 h,丙环唑对斜纹夜蛾4龄幼虫的LD₅₀值分别为0.59 μg/头和0.45 μg/头。丙环唑对SL细胞和斜纹夜蛾幼虫均具有较好的毒杀活性,显示出丙环唑类似物控制害虫的可能性。     

大豆胰蛋白酶抑制剂和防御信号物质对斜纹夜蛾解毒酶的影响

在昆虫与植物漫长的相互作用中,植物合成多种抗虫物质并采用防御信号转导系统抵御昆虫,昆虫也具有多种解毒酶系统保护其免受植物毒素的毒害.本文研究了人工添加大豆胰蛋白酶抑制剂和植物防御信号物质对斜纹夜蛾幼虫羧酸酯酶和谷胱甘肽-S-转移酶活性的影响.结果表明:持续6代自幼虫2龄或3龄开始喂养含有大豆胰蛋白酶抑制剂的人工饲料,其5龄幼虫中肠和脂肪体内羧酸酯酶、谷胱甘肽-S-转移酶活性显著升高,2、3龄处理的继代幼虫中肠和脂肪体内羧酸酯酶活性均在第二代达到最大值,分别为对照的2.06、2.40倍和1.96、2.70倍;其谷胱甘肽-S-转移酶活性则分别于第4、2代达到最大值,分别为对照的7.03、11.58倍和5.71、3.60倍,并呈现先升高再降低的趋势.预先接触外源信号物质茉莉酸甲酯、水杨酸甲酯48 h和添加大豆胰蛋白酶抑制剂均可使斜纹夜蛾幼虫中肠、脂肪体内羧酸酯酶和谷胱甘肽-S-转移酶的活性显著升高,且预先接触茉莉酸甲酯和水杨酸甲酯48 h可减缓大豆胰蛋白酶抑制剂对幼虫中肠和脂肪体内羧酸酯酶、谷胱甘肽-S-转移酶活性的作用效果.     

斜纹夜蛾天敌作用的评价

通过组建斜纹夜蛾第４代和第８代自然种群生命表,运用排除作用控制指数分析了生物因子对斜纹夜蛾种群的自然控制作用．结果表明,低龄（１～３龄）幼虫的捕食性天敌是影响斜纹夜蛾种群数量动态的重要因子．对第４代和第８代种群的排除作用控制指数分别为１３．９０４和１２．９４６．如果没有捕食性天敌的作用,下代种群数量将分别增长到当代的１５．１２０６和７４．６７８倍．病原微生物是影响第４代斜纹夜蛾种群数量的另一重要因子,其排除作用控制指数为２．４７２６．     

Evaluation of substituted oxime ethers for growth regulatory activity against Spodoptera litura (F.)

Insect growth regulatory activity (IGR) of fifty-two substituted oxime ethers were evaluated against an important polyphagous lepidopteran crop pest, Spodoptera litura (F.). A number of compounds produced symptoms comparable to exogenously applied juvenile hormone. Maximum IGR activity was exhibited by 4'-(2,6,6-trimethyl-2-cyclohexen- -yl)-3'-buten-2'(E)-ketoxime-N-O-alkyl ether with an ED50 (morphological) of 40 microg g(-1) body weight, compared to 20 microg g(-1) of JH III. Two more compounds namely 4'-(2,6,6-trimethyl-2-cyclohexen-1-yl)-3'-buten-2'(Z)-ketoxime-N-O-methyl propyl ether (ED50 192 microg g(-1)) and 4'-(2,6,6-trimethyl-1-cyclohexen-1-yl)-3'-buten-2'(E)-ketoxime-N-O-pentyl ether (ED50 380 microg g(-1)) showed considerable IGR activity, whereas 4'-(2,6,6-trimethyl-2-cyclohexen-1-yl)-3'-buten-2'(E)-ketoxime-N-O-pentyl ether was found to be toxic to the larvae (ED50 268 microg g(-1)). Three compounds used in this study were also synergised by piperonyl butoxide (PBO). The synergistic ratios were found in the range of 1.33 to 4.605. The ovicidal activity of the oxime ethers is not significant.     

Growth inhibitory and antifeedant activity of extracts from Melia dubia to Spodoptera litura and Helicoverpa armigera larvae

The growth inhibitory activity and deterrency of Melia dubia (Meliaceae) extracts to Spodoptera litura and Helicoverpa armigera were investigated. Artificial diet bioassays using neonate larvae of both S. litura and H. armigera indicated that dichloroethane (DCE) and methanol (Me) extracts of M. dubia inhibited growth in a dose dependent manner. DCE and Me-5II fractions also resulted in 50% deterrency at concentrations of 22.5 and 16.8 micrograms/cm2 respectively against S. litura larvae in a leaf disc-choice test. The DCE-5 fraction was found to be more toxic to larvae (LC50 of 0.65%) than the Me-5II (LC50 of 0.8%), 72 hr after topical application. Both fractions lack contact toxicity, but the deterrent effect persisted for at least 60 hr under laboratory conditions. Although salannin was isolated from the DCE fraction to show antifeedant activity, the physico-chemical characteristics of the active fractions DCE-5 and Me-5II were not identical with either salannin or azadirachtin.     

Ni胁迫对斜纹夜蛾幼虫包囊反应的影响

将不同浓度Ni2+添入人工饲料中,研究连续3代取食含不同剂量Ni2+饲料的斜纹夜蛾5、6龄幼虫血细胞对凝胶珠包囊反应的影响。将新蜕皮的5龄幼虫记为0 h,每隔24 h注射凝胶珠处理幼虫,直至6龄末,注射24h后解剖虫体,观察包囊情况。结果表明,5mg/kg及以下处理浓度Ni2+的1代胁迫可提高0-120 h幼虫血细胞对凝胶珠的包囊反应,但2-3代的胁迫只增加发育初期0-72 h幼虫血细胞的包囊反应;10-20 mg/kg处理浓度Ni2+的1代胁迫仅增加0-24 h幼虫血细胞的包囊率,但可降低2-3代幼虫血细胞各发育时间的包囊率。此外,40 mg/kg Ni2+胁迫可连续降低3个世代各个发育阶段幼虫的包囊反应。饲料中Ni2+剂量、胁迫世代数以及两者的交互作用均对幼虫包囊率产生了显著影响。     

番荔枝内酯化合物布拉它辛对斜纹夜蛾的杀虫活性及对SL细胞的致凋亡作用

为了明确番荔枝内酯化合物布拉它辛的杀虫活性和探索杀虫作用机理,本研究采用浸叶法测定该化合物对斜纹夜蛾Spodoptera litura幼虫的生物活性,采用MTT检测法和流式细胞术,研究该化合物对斜纹夜蛾离体培养卵巢细胞（SL细胞）的细胞毒力和致细胞凋亡作用,以及对SL细胞线粒体膜电位的影响。结果表明: 布拉它辛不仅对斜纹夜蛾幼虫具有良好的拒食活性,处理后24 h,对2龄和3龄幼虫的AFC₅₀值分别为60.25 μg/mL和86.73 μg/mL,还对幼虫生长有良好的抑制作用。布拉它辛处理SL细胞后24 h和48 h,IC50值分别为22.32 μg/mL和10.03 μg/mL。流式细胞仪检测结果表明,布拉它辛对SL细胞具有明显的致凋亡作用,可导致SL细胞线粒体膜电位显著下降。本研究表明布拉它辛对斜纹夜蛾幼虫具有良好的拒食活性与生长抑制作用, 并且该化合物能明显抑制SL细胞的增殖,诱导细胞凋亡,降低线粒体膜电位。因此,布拉他辛具有广阔的研究应用前景。     

Alkaline protease in the larvae of the army worm,Spodoptera litura

The alkaline protease activity in the gut of Spodoptera litura was found to increase with the development of larvae and decreased with the onset of pupation. Fasting of the 5th instar larvae caused a slight increase in protease activity at 4 hr, which declined consistently on further starvation. The optimum pH for the gut protease was 11.0, with a shoulder between pH 8.0 and 9.0. The protease was inactivated upon dialysis of the crude enzyme solution at room temperature but not at 4°C. Incubation of the crude enzyme solution at pH 11.0 and 37°C for 22 hr resulted in a three-fold rise in specific activity of the alkaline protease which declined on further incubation. The three-fold purified preparation, obtained by incubation of the crude enzyme solution, was passed through Sephadex G-75 to give a seven-fold purification and 70% yield. The preparation was not completely homogeneous and showed three clearly separable protein bands by polyacrylamide gel electrophoresis. The partially purified protease exhibited no shoulder between pH 8 to 9, like the crude preparation.     

Effect of nucleopolyhedrovirus infection of Spodoptera litura larvae on host discrimination by Microplitis pallidipes

The behaviour and oviposition of solitary endoparasitoid Microplitis pallidipes Szepligeti (Hymenoptera: Braconidae) were monitored to investigate the ability of the parasitoids to distinguish between nucleopolyhedrovirus (NPV)-infected and noninfected Spodoptera litura Fabricius (Lepidoptera: Noctuidae) larvae. The results indicated that the parasitoid searching time and the time until the first parasitoid attack on infected larvae were greater than those recorded on noninfected larvae; the number of infected larvae attacked by parasitoids, the percent of first attacks and parasitism rate in infected larvae were lower than those on noninfected larvae; and these differences were all significant 3 to 5 days postexposure of the larvae to a dose of 1.6 × 10⁸ occlusion bodies (OB)· ml^?1 and significant 4 and 5 days postexposure of the larvae to a dose of 1.6 × 10⁷ OB·ml^?1. The lowest dosage (1.6 × 10⁶ OB·ml^?1) had no significant effect on the above index values. In a field cage experiment, we found that the percentage of infected larvae parasitized by M. pallidipes gradually decreased as the time after NPV inoculation (1.6 × 10⁸ OB·ml^?1) increased, and that M. pallidipes significantly preferred to oviposit in healthy larvae from day 3 to day 5 after virus inoculation. Our research concluded that this parasitoid's ability to discriminate between healthy and infected hosts increased as virus concentration increased and as the time between exposure of hosts to virus and subsequent exposure to parasitoids increased.     

The effect of dietary nickel on the immune responses of Spodoptera litura Fabricius larvae

By exposing Spodoptera litura Fabricius larvae to nickel (Ni) in artificial diets for successive three generations, we investigated the impacts of the dietary Ni on growth and immune response of the fifth and sixth instar larvae at 24 h intervals. The time of newly moulted fifth instar larvae was labelled as 0 h. After exposure to 5 mg/kg Ni for two generations, Ni exposure significantly improved larval phenoloxidase activity and encapsulation grade in fifth instar larvae when compared to controls, except for encapsulation grade at 72-120 h in the second generation. However, higher concentrations of Ni (≥10 mg/kg) only significantly reduced encapsulation grade at 72-120 h. In the third generation, insects given higher dietary levels of Ni (≥10 mg/kg) showed lower immune responses and retarded relative growth rate (RGR) compared to controls, but those exposed to lower Ni levels (≤5 mg/kg) had a significantly improved encapsulation grade at 24-72 h. Larvae at lower Ni level (≤5 mg/kg) treatments had significantly higher RGR in comparison with that in controls. There was no significant difference in food relative consumption rate (RCR) and RGR among any treatment of the fifth instar larvae in three successive generations. These results indicated that the type and extent of effects on growth and immune responses of S. litura varied with the Ni concentrations and exposure periods.     

Control of zoopathogenic fungi in vitro by polyamine biosynthesis inhibitors.

Effect of inhibitors of polyamine (PA) biosynthesis, alpha-difluoromethylornithine (DFMO), methylglyoxal bis (guanylhydrazone)--MGBG and bis (cyclohexylammonium) sulphate (BCHA) on mycelial growth of three clinically important fungi-Trichophyton mentagrophytes, Microsporum gypseum and Aspergillus flavus was examined in vitro. All inhibitors at concentrations 1 to 50 mM produced greater inhibition of mycelial growth in all fungi tested in a dose-dependent manner. MGBG was the most effective inhibitor, and T. mentagrophytes was the most sensitive fungus to all inhibitors followed by M. gypseum and A. flavus. The results suggested that control of fungal diseases in animals and human beings with specific inhibitors of PA biosynthesis is possible.     

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.