Interactions between an ectoparasitoid and a nucleopolyhedrovirus when simultaneously attacking Spodoptera exigua (Lepidoptera: Noctuidae)

Insect pathogenic viruses and parasitoids represent distinct biological entities that exploit a shared host resource and have similar effects in suppressing host populations. This study explores the interactions between the ectoparasitoid Euplectrus plathypenae (Hymenoptera: Eulophidae) and the Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV) in larvae of S. exigua (Lepidoptera: Noctuidae). Parasitoid progeny failed to complete development in hosts that had been infected prior to parasitism. However, infection of S. exigua fourth instars at 48 h post‐parasitism had no significant effects on the survival of parasitoid progeny. Larval and pupal development times of E. plathypenae that survived on virus‐infected S. exigua did not differ significantly from that of parasitoids on healthy hosts. Virus‐induced mortality and the production of occlusion bodies were very similar in parasitized and non‐parasitized S. exigua. The virus was genetically stable over three passages in parasitized and unparasitized hosts. These results suggest that applications of SeMNPV‐based insecticides are unlikely to disrupt pest control exerted by the parasitoid E. plathypenae in biological pest control programs as long as virus applications are timed not to coincide with parasitoid releases.     

对甜菜夜蛾高毒苏云金芽孢杆菌菌株的选育*

采用物理诱变——虫体传代模式,选育获得一株对甜菜夜蛾高毒菌株BtCZE 99985。通过摇瓶和40t发酵罐3年10批发酵试验,表明该菌株具有良好的发酵性能。摇瓶试验表明,与出发菌株93005、对照菌株HD-1-580、GC-91相比较,该菌株对甜菜夜蛾的毒效分别提高429％、655％、114％。40t发酵罐发酵试验表明,该菌株对甜菜夜蛾测定的LC50平均值为0．076μL/mL,比GC-91菌株(平均0．213μL/mL)的毒效提高180％。     

不同营养条件下甜菜夜蛾幼虫感染核型多角体病毒的敏感性及取食趋性

为探讨不同营养条件下甜菜夜蛾Spodoptera exigua对甜菜夜蛾核型多角体病毒(Spodoptera exigua multiple nucleopolyhedrovirus,SeMNPV)的敏感性及取食行为响应,本研究通过改变甜菜夜蛾人工饲料配方建立高蛋白低糖(简称高蛋白)、中蛋白中糖(简称中蛋白)、低蛋白高...     

Fast accumulation of few polyhedra mutants during passage of a Spodoptera frugiperda multicapsid nucleopolyhedrovirus (Baculoviridae) in Sf9 cell cultures

Baculoviruses are a group of viruses that infect invertebrates and that have been used worldwide as a biopesticide against several insect pests of the Order Lepidoptera. In Brazil, the baculovirus Spodoptera frugiperda multicapsid nucleopolyhedrovirus (SfMNPV; Baculoviridae) has been used experimentally to control S. frugiperda (Lepidoptera: Noctuidae), an important insect pest of corn (maize) fields and other crops. Baculoviruses can be produced either in insect larvae or in cell culture bioreactors. A major limitation to the in vitro production of baculoviruses is the rapid generation of mutants when the virus undergoes passages in cell culture. In order to evaluate the potential of in vitro methods of producing SfMNPV on a large-scale, we have multiplied a Brazilian isolate of this virus in cell culture. Extensive formation of few polyhedra mutants was observed after only two passages in Sf9 cells.     

A cell strain cloned from Spodoptera exigua cell line (IOZCAS-Spex-II) highly susceptible to S. exigua nucleopolyhedrovirus infection

A cell strain (IOZCAS-Spex-II-A) cloned from IOZCAS-Spex-II, a cell line established from the fat body of Spodoptera exigua (Lepidoptera: Noctuidae) larva, was characterized, and its capability to produce S. exigua nucleopolyhedrovirus was high with infection rate exceeding 90% compared with its parental cell line IOZCAS-Spex-II that scored only 50%. Growth curve of budded virus (BV) in the strain was analyzed and the titer of BV reached the highest of 3.7?×?10⁴ pfu/mL by 96 h after inoculation. Concentration of occlusion bodies (OBs) produced by the cloned cell strain (IOZCAS-Spex-II-A) was 7.1?×?10⁷ OBs/mL, while the parental cell line produced 2.4?×?10⁷ OBs/mL. The average yield of the virus was 176 OBs/cell of IOZCAS-Spex-II-A compared with 211 OBs/cell that of the parental cell line. Significant differences were observed in virus production, growth characters, cell shape, between the parental cell line, and its clone. The cell lines (IOZCAS-Spex-II and IOZCAS-Spex-II-A) were also susceptible to Autographa californica multiple nucleopolyhedrovirus infection. In addition, they were characterized with regard to their growth rates and DNA amplification fingerprinting technique employing polymerase chain reaction.     

Efficacy of Spodoptera exigua multiple nucleopolyhedrovirus as a biological insecticide for beet armyworm control in greenhouses of southern Spain

Chemical control measures targeted at Spodoptera exigua in greenhouse sweet pepper crops in Spain have resulted in pest resistance to virtually all commercially available insecticidal products. A multicapsid nucleopolyhedrovirus (SeMNPV), isolated from diseased S. exigua in Spain, was produced in laboratory reared larvae, tested for insecticidal activity in a laboratory bioassay, and was then applied in eleven commercial greenhouses planted with sweet pepper. Virus occlusion bodies (OBs) were applied on two occasions, at an interval of ～7 days, at a rate of 5×10⁸ OBs/L of spray in a volume of ～600 L/ha, depending on crop phenology and greenhouse area. The percentage of plants showing recent (<48 h old) feeding damage fell dramatically in greenhouses with high infestations of S. exigua; the same pattern was observed, although less dramatically, in greenhouses with low infestations. Average mortality of larvae collected from treated plants at 4 days after each application, and reared in the laboratory until death, was high (70–89%) and was not significantly affected by the degree of crop infestation. In a separate trial, the rate of acquisition of infection was examined in larvae that fed on plants treated with 1×10⁸ or 5×10⁸ OBs/L of spray. Of the 27 and 60% of larvae, respectively, that acquired infection in the 48 h period after spraying, about half became infected in the first 6 h post-application, irrespective of application rate. Acquisition of infection proceeded more slowly during the night-time compared to the daytime period, underlining the advantages of early morning applications of the virus. We conclude that the Spanish SeMNPV isolate merits registration as a biological insecticide for use in greenhouse crops in this region.     

Effect of tinopal LPW on the insecticidal properties and genetic stability of the nucleopolyhedrovirus of Spodoptera exigua (Lepidoptera: Noctuidae)

The influence of an optical brightener, Tinopal LPW, on the activity of a purified genotype of the nucleopolyhedrovirus (SeMNPV) of the beet armyworm, Spodoptera exigua (Hübner), was determined in second to fifth instar (L2-L5) S. exigua. When mixed with viral occlusion bodies (OB) 1% Tinopal LPW significantly reduced the median lethal dose (LD50) of the virus in all instars compared with insects treated with SeMNPV alone. Levels of enhancement, as determined by LD50 values, ranged from 2.6- to 580-fold, depending on the instar. The greatest enhancement occurred on the two later instars, L4 (70-fold) and L5 (580-fold), which show a much higher resistance to SeMNPV infection than earlier instars. The median time to death (MTD) values were not significantly different in any instar among larvae treated with SeMNPV + Tinopal LPW and those treated with SeMNPV alone. Larval development in SeMNPV + Tinopal LPW treated larvae was retarded, in second and fourth instars, compared with controls or larvae treated with SeMNPV alone. The OB yields from SeMNPV treated larvae were almost 1.6-fold greater in second instars (9.3 x 10(6) OBs/larvae), and 1.9-fold greater in fourth instars (1.9 x 10(8) OBs/larvae), than those obtained in larvae treated with SeMNPV + Tinopal LPW. The addition of 1% Tinopal LPW to the virus suspension did not alter the genotypic composition of viral progeny during four successive passages of the virus.     

Insecticidal activities of Ginkgo biloba seed coat extracts against Spodoptera exigua (Lepidoptera: Noctuidae)

The present study relates to a methanol extract of the seed coat of Ginkgo biloba, and tested particularly on the third instar larvae of Spodoptera exigua. The extract was found to have an inhibitory effect on the growth of the larvae besides bringing a change in the nutrient reserves in the body of the insect. Topical application of five different doses of the methanol extract resulted in a mortal effect to third instar larvae of S. exigua that is very much dependent on the dose as well as duration of exposure. Lower doses revealed lower mortality after 24 h of application. At doses of 1.00, 2.00, 4.00, 8.00 and 16.00 ng/larva, mortalities were 9.25, 26.07, 50.32, 56.28 and 92.44%, respectively. The dose for 50% mortality (LD₅₀) of methanol extracts by applied by a topical method with 1 µL of acetone solution was 1.92 ng/larva. Nutrient reserves like protein, glycogen and lipid are known to regulate pupation and adult emergence. These reserves have been found to be lower in treated larvae, indicating the insecticidal role of methanol extracts from G. biloba against third instar larvae of S. exigua.     

Expression of the lef5 gene from Spodoptera exigua multiple nucleopolyhedrovirus contributes to the baculovirus stability in cell culture

Applied Microbiology and Biotechnology - Baculoviruses are a broad group of viruses infecting insects, predominately of the order Lepidoptera. They are used worldwide as biological insecticides and...     

草地贪夜蛾核型多角体病毒对我国草地贪夜蛾不同地理种群毒力的比较分析

草地贪夜蛾Spodoptera frugiperda(J. E. Smith)入侵我国多个地区,逐渐形成地理种群。在草地贪夜蛾核型多角体病毒Spodoptera frugiperda multiple nucleopolyhedrovirus(SfMNPV)生物农药的生产过程中发现,不同地理来源的宿主对SfMNPV的敏感性和产量存在明显差异,显著影响了SfMNPV的生产效率。为解析其敏感性差异及其产生的原因,本研究首先对云南德宏、广东广州、广西钦州、西藏林芝4个地区草地贪夜蛾种群基因型进行了鉴定,采用生物测定法测试SfMNPV对4龄幼虫的口服毒力,然后,通过向幼虫体内注射草地贪夜蛾核多角体病毒出芽型病毒粒子(budded virions, BVs)的方式,越过口服感染中肠的过程,分析敏感性差异发生的阶段。最后比较了高敏感和低敏感种群中肠肠液pH,并基于16S rDNA测序测定了肠道菌群组成。结果表明,广西种群属于纯合玉米型,其余种群为带有水稻型COI标记的杂合玉米型。广西种群对SfMNPV的口服敏感性最低,西藏种群的敏感性最高,但两者注射BV后死亡率差异无统计学意义,暗示病毒敏感性差异...     

Phenotypic characteristics and relative proportions of three genotypic variants isolated from a nucleopolyhedrovirus of Spodoptera exigua

US2A, US2D, and US2F are three in vivocloned genotypic variants from the wild-type strain of a Spodoptera exiguanucleopolyhedrovirus (SeMNPV) isolated in Florida (USA) and is the active component of the commercial bioinsecticide Spod-X^®. These variants were compared in terms of pathogenicity (LD₅₀), speed of kill (expressed as mean time to death) and viral progeny productivity (OBs/larva). LD₅₀values were similar for the three cloned genotypes. The mean time to death value for US2D (113.7 h) was significantly higher than those of US2A (31.7 h) and US2F (27.8 h). Virus yield was determined for L₄larvae infected with the estimated LD₉₉. US2D infected larvae attained higher weight than those infected with US2A and US2F, and produced a higher OB yield than larvae infected with US2A or US2F. An outstanding feature of US2F, in contrast to US2A and US2D, was its inability to disrupt the teguments of NPV-killed larvae. To study the relative proportion of the three genotypic variants throughout successive passages, S. exigualarvae were originally infected with a viral inoculum containing a 1:1:1 mixture of the three genotypes. After three successive passages, US2D was no longer detected in either of the three replicate experiments performed, while US2A was the predominant genotype in all of them, and US2F remained at similar proportions throughout the three passages. The influence of the phenotypic characteristics of the three variants on their relative proportions in mixed infections is discussed.     

A new cell line (NTU-SE) from pupal tissues of the beet armyworm, Spodoptera exigua (Lepidoptera: Noctuidae), is highly susceptible to S. exigua multiple nucleopolyhedrovirus (SeMNPV) and Autographa californica MNPV (AcMNPV)

A new continuous cell line, NTU-SE, was established from the pupal tissues of an economically important pest, the beet armyworm Spodoptera exigua (Lepidoptera: Noctuidae). This cell line contains four major morphologic types: round, polymorphic, spindle-shaped, and comma-shaped cells. The population doubling time of this new line in TNM-FH medium supplemented with 8% fetal bovine serum (FBS) at 28°C is 35.5h. The chromosomal spread from NTU-SE cells is typical to the chromosomal morphology of lepidopteran cell lines. Confidently, NTU-SE cell line is a new cell line that exhibits distinct isozyme patterns of esterase, lactate dehydrogenase (LDH), and malate dehydrogenase (MDH) from those of the other insect cell lines. In addition, the DNA sequence of the nuclear ribosomal internal transcribed spacer (ITS) region of NTU-SE cells is above 96% identical to that sequence of S. exigua larvae, as compared to only 66% identical to that of S. litura larvae. The NTU-SE cell line is highly susceptible to S. exigua multiple nucleopolyhedrovirus (SeMNPV) and Autographa californica MNPV (AcMNPV). Therefore, a highly virulent SeMNPV strain, SeMNPV-1, had been successfully isolated and propagated in NTU-SE cells. We conclude that the NTU-SE cell line will be a useful tool for the selection and mass production of highly virulent SeMNPV strains for the S. exigua biocontrol and the baculovirus based recombinant protein expression systems.     

不同温度下斜纹夜蛾的两性生命表

【目的】斜纹夜蛾Spodoptera litura(Fabricius)是一种鳞翅目夜蛾科食叶害虫,在不同的寄主、饲料等条件下表现出不同的生长发育特点。本实验旨在探究温度对斜纹夜蛾生长发育状况和种群参数的影响。【方法】在室内人工气候箱20±1℃,27±1℃和30±1℃(相对湿度和光周期分别为65%±10%和14L∶10D)条件下饲养斜纹夜蛾,观察和组建斜纹夜蛾在3种温度下的年龄-阶段两性生命表。【结果】斜纹夜蛾各个阶段的发育历期随温度升高变短,成虫产卵前期(adult preoviposition period,APOP)和总产卵前期(total preoviposition period,TPOP)同样表现为随温度升高而变短。此外,20±1℃处理的平均产卵量最高(1 810.36粒/雌),27±1℃处理次之(1 623.51粒/雌),30±1℃处理最低(1 084.60粒/雌)。斜纹夜蛾在不同温度条件下内禀增长率(r)、周限增长率(λ)和净增殖率(R0)有显著差异,20±1℃处理的r和λ显著低于27±1℃处理和30±1℃处理;27±1℃处理的R0显著大于20±1℃和30±1℃处理;20±1℃,27±1℃和30±1℃下平均世代周期(T)分别为52.51,30.63和26.87 d,依次减少且相互间差异显著(P0.05)。【结论】斜纹夜蛾在27±1℃和30±1℃温度条件下均表现出很强的增殖能力,该结果可为田间综合防治斜纹夜蛾提供理论依据。     

不同温度条件下叉角厉蝽对草地贪夜蛾幼虫的捕食作用

为探明叉角厉蝽Eocanthecona furcellata (Wolff)对草地贪夜蛾Spodoptera frugiperda (Smith)的控害潜能,本文研究了不同温度（17℃、22℃、27℃、32℃、37℃）条件下叉角厉蝽3~4龄若虫对草地贪夜蛾2~3龄幼虫的捕食作用,评估了其在不同温度下的生物防治效果。结果表明,在试验温度范围内叉角厉蝽3~4龄若虫对草地贪夜蛾2~3龄幼虫的捕食功能反应模型符合Holling II型方程,捕食效能a/Th和日均捕食量均在32℃时最大。在同一温度下,叉角厉蝽3~4龄若虫的寻找效应与草地贪夜蛾幼虫密度呈负相关,但猎物密度相同时叉角厉蝽3~4龄若虫的寻找效应与温度呈正相关。在17℃~37℃范围内,Hassell-Varley干扰反应模型能较好地反映出不同叉角厉蝽密度条件下对草地贪夜蛾的捕食情况;在相同的猎物密度条件下叉角厉蝽3~4龄若虫个体间存在种内竞争和自我干扰作用,随着自身密度增大,相互干扰作用增强,搜寻系数Q和相互干扰系数m随着温度升高而增大,在37℃时最大,分别为0.4719和0.788。本试验证实了叉角厉蝽若虫在温度为32℃、猎物密度为60（头/皿）条件下对草地贪夜蛾的日捕食量最大,但随着温度升高,若虫的捕食量趋于饱和或出现下降趋势,在37℃温度条件下种内竞争和自我干扰作用最强。     

Chemical and biological stress factors on the activation of nucleopolyhedrovirus infections in covertly infected Spodoptera exigua

Following the consumption of baculovirus occlusion bodies (OBs), insects may succumb to lethal disease, but the survivors can harbour sublethal covert infections and may develop, reproduce and transmit the infection to their offspring. The use of different chemical and biological stressors was examined to determine whether they could be used to activate covert infections in populations of Spodoptera exigua larvae infected by the homologous nucleopolyhedrovirus (SeMNPV). Treatment of covertly infected S. exigua second instars with Tinopal UNPA‐GX, hydroxylamine, paraquat, Bacillus thuringiensis var. kurstaki crystals, spores or mixtures of crystals + spores, or a heterologous nucleopolyhedrovirus (Chrysodeixis chalcites SNPV) did not result in the activation of SeMNPV covert infections. Similarly, virus treatments involving permissive NPVs did not result in greater mortality in covertly infected insects compared with the virus‐free controls. In contrast, 0.1% copper sulphate, 1% iron (II) sulphate and 1 mg/l sodium selenite treatments resulted in 12–41% lethal polyhedrosis disease in covertly infected larvae. A greenhouse trial using copper sulphate and sodium selenite as activation factors applied to covertly infected S. exigua larvae on sweet pepper plants resulted in very low levels of SeMNPV activation (<3%). These results highlight the important roles of copper, iron and selenium in insect immunity and baculovirus‐induced disease. However, these substances seem unlikely to prove useful for the activation of covert SeMNPV infections in S. exigua larvae under greenhouse conditions.     

Pathogenicity of Choristoneura fumiferana nucleopolyhedrovirus propagated in vitro at different incubation temperatures

To optimize the in vitro production of Choristoneura fumiferana nucleopolyhedrovirus (CfMNPV) as a potential microbial pest control agent, the pathogenicity of occlusion bodies (OBs) produced in two cell lines at three incubation temperatures was determined by bioassay. A plaque-purified isolate of CfMNPV was amplified in permissive C. fumiferana cell lines, FPMI-CF-203 and FPMI-CF-2C1, and incubated at 22, 24, and 28 degrees C. Occlusion bodies propagated in FPMI-CF-203 cells at 28 degrees C were significantly larger (17.5 microm(3)) and more pathogenic (LD(50) = 27; LD(95) = 185, where LD(50) and LD(95) are doses required to kill 50 and 95% of the test larvae, respectively) than those produced in either of the cell lines at any of the incubation temperatures tested. Increased temperatures yielded larger OBs from both cell lines. The pathogenicity of OBs propagated in the FPMI-CF-203 cell line increased with incubation temperature, whereas that of OBs produced in FPMI-CF-2C1 cells decreased. Comparison of the pathogenicity of OBs, whether naturally occurring or genetically modified, should be standardized by cell line and incubation temperature used for propagation. Production efficiency decreased with increasing incubation temperature for each cell line. Lower incubation temperatures used for propagation, and standardization of the titer of viral inoculum, should be further investigated to determine the economic feasibility of the in vitro production of CfMNPV as a microbial pest control agent.     

Demographic parameters of Spodoptera exigua (Lepidoptera: Noctuidae) on different soybean cultivars

Life table, reproduction, and population growth parameters of the beet armyworm, Spodoptera exigua (Hübner) (Lepidoptera: Noctuidae), were determined on nine soybean (Glycine max L.) cultivars ('032', '033', 'Hill', 'M4', 'M7', 'M9', 'M11', 'TMS', and 'Zane') at 25°C. The age-specific survival rate of eggs and immature stages was higher on 033 in comparison with other cultivars. The gross fecundity rate ranged from 911 on M9 to 1,296 on Zane, but the differences were not statistically significant. The net fecundity rate was significantly higher on 033 (1,082 eggs per female) than on other cultivars. The gross fertility rate significantly differed on tested cultivars, and was the highest on Zane (1,257 eggs per female) and lowest on Hill (813 eggs per female). The net fertility rate varied from 532 on M11 to 1,082 eggs per female on 033. Both daily number of eggs and daily number of fertile eggs laid per female varied significantly among the different cultivars, and were the highest on TMS (90 for both parameter) and lowest on M7 (47 and 43), respectively. The values of net reproductive rate, intrinsic rate of increase, and finite rate of increase were significantly higher on 033. The shortest generation time (23 d), shortest doubling time (1.66 d), and highest percentage of female offspring (56%) also were obtained on 033. According to population growth parameters, 033 and Hill partially were susceptible and resistant, respectively, to S. exigua. The results of this study provide direction to design a more comprehensive integrated pest management program for this pest.     

Wolbachia在我国甜菜夜蛾中的超感染

Wolbahcia是一类广泛分布于节肢动物体内,可以引起节肢动物生殖行为改变的细胞内共生细菌。甜菜夜蛾Spodoptera exigua(Hübner)是我国常见的鳞翅目害虫,可危害粮、棉、菜等多种作物。已有很多研究表明Wolbachia在鳞翅目昆虫中具有广泛的分布,但是未见有关于甜菜夜蛾中感染Wolbachia的报道,为了研究我国甜菜夜蛾中Wolbachia的分布情况,采集了甜菜夜蛾的野生种群,对其进行DNA提取,通过对的Wolbachia的wsp基因进行PCR扩增及克隆测序,明确了Wolbachia在我国甜菜夜蛾野生种群内的分布情况。在检测的甜菜夜蛾成虫中,都感染了两种类型的Wolbahcia,分别命名为wExiA和wExiB(GenBank注册号分别为EU332343和EU332344)。wExiA属于A组,wExiB属于B组。本研究首次明确了在我国甜菜夜蛾的野生种群内存在着Wolbachia的超感染现象。