甜菜夜蛾核多角体病毒泛素基因的克隆及原核表达

甜菜夜蛾核多角体病毒(Spotoptera exigua multi-nucleopolyhedrovirus,SeMNPV)泛素基因ubiquitin被克隆和序列分析,该基因编码区全长243bp,编码80个氨基酸残基,预计蛋白质分子量为9.4kDa.将这一ubiquitin基因克隆到原核表达载体pET-28a上,转化至BL21(DE3)中,用IPTG进行诱导表达,对表达的条件进行了优化.用异源的泛素单克隆抗体检测目的蛋白,Western blot实验证明所表达的蛋白是泛素蛋白.同时,我们制备了特异性的抗体,为以后的研究工作做了基础.通过计算机软件Gendoc对不同来源的泛素进行分析,结果显示,病毒中的泛素与真核细胞中的泛素相比较,泛素的氨基酸序列有较大的变化,杆状病毒的泛素基因在分子进化上可能有比较独特的途径.     

甜菜夜蛾核多角体病毒泛素基因的克隆及原核表达

甜菜夜蛾核多角体病毒（Spotoptera exigua multi-nucleopolyhedrovirus,SeMNPV)泛素基因ubiquitin被克隆和序列分析,该基因编码区全长243bp,编码80个氨基酸残基,预计蛋白质分子量为9.4kDa。将这一ubiquitin基因克隆到原核表达载体pET-28a上,转化至BL21（DE3）中,用IPTG进行诱导表达,对表达的条件进行优化,用异源的泛素单克隆抗体检测目的蛋白,Western blot 实验证明所表达的蛋白是泛素蛋白。同时,我们制备了特异性的抗体,为以后的研究工作做了基础,通过计算机软件Gendoc对不同来源的泛素进行分析,结果显示,病毒中的泛素与真核细胞中的泛素相比较,泛素的氨基酸序列有较大的变化,杆状病毒的深入素基因在分子进化上可能有比较独特的途径。     

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.     

人工饲料成分对甜菜夜蛾核多角体 病毒产量的影响

通过采取单因素和正交组合设计的方法,研究了甜菜夜蛾Spodoptera exigua人工饲料中黄豆粉、酵母粉和麦麸3种主要营养成分对甜菜夜蛾核多角体病毒产量的影响,同时获得甜菜夜蛾核多角体病毒高产优化组合方案：酵母粉7 g、黄豆粉14 g和麦麸6 g。此外,还研究了4种无机盐和1种非蛋白氮对甜菜夜蛾核多角体病毒产量的影响。     

Effect of host plants on the infectivity of nucleopolyhedrovirus to Spodoptera exigua larvae

Previous studies have shown that the infectivity of baculovirus to herbivores is affected by phytochemicals ingested during the acquisition of viral inoculum on the foliage of host plants. Here, we measured the effects of 14 host plant species on the infectivity of Spodoptera exigua nucleopolyhedrovirus (SeNPV) to its larvae. The order of the LD₅₀ values of SeNPV among the host plants was Ipomoea aquatica > Brassica oleracea > Raphanus sativus > Amaranthus tricolor > Spinacia oleracea > Vigna unguiculata > Solanum melongena > Capsicum annuum > Apium graveolens > Allium fistulosum > Lactuca sativa > Brassica chinensis > Zea mays > Glycine max, with 940.1 ± 2.26, 424.0 ± 0.60, 295.2 ± 1.13, 147.3 ± 0.63, 138.6 ± 0.22, 119.9 ± 0.07, 119.8 ± 0.02, 109.2 ± 0.18, 104.8 ± 0.62, 102.1 ± 0.66, 97.9 ± 0.22, 89.9 ± 0.32, 79.0 ± 0.13 and 64.0 ± 0.38 OBs per larva, respectively, and the values of mean time to death of virus‐infected larvae were 6.21 ± 0.11, 7.12 ± 0.10, 7.33 ± 0.21, 6.97 ± 0.02, 7.06 ± 0.01, 7.29 ± 0.03, 7.32 ± 0.05, 7.07 ± 0.08, 7.24 ± 0.11, 7.09 ± 0.13, 7.50 ± 0.06, 7.23 ± 0.01, 7.30 ± 0.02 and 7.19 ± 0.07 days, respectively. The mean time to death of larvae decreased with increasing viral dose, and corrected mortality decreased as the larval mean time to death increased. These findings have significance for understanding the effects of host plants on the infectivity of baculovirus to noctuids.     

An evaluation of some natural enemies of Spodoptera exigua on sugarbeet in northern California

Eggs and small to medium-sizedlarvae of Spodoptera exigua (Hübner)(Lepidoptera: Noctuidae) are exploited by acomplex of natural enemies in spring-plantedsugarbeet fields in northern California. Fieldstudies revealed that predation on sentinel eggmasses ranged from 20 to 100%egg mass;predation rate was lowest in fields previouslytreated with methomyl and highest innon-treated fields. Predators typicallydestroyed all of the eggs in a given egg mass;percentage predation per egg mass was densityindependent (spatial context). Survival of eggs(to neonate larvae) in cages that excludedpredators ranged from 80 to $>$90%. Theegg-predator guild consisted of adults andnymphs of Orius tristicolor(White)(Anthocoridae), Nabis americoferusCarayon (Nabidae), Lygus hesperus Knight(Miridae), and Geocoris punctipes (Say)(Geocoridae); larvae of Chrysoperlacarnea (Stephens) (Chrysopidae); and adults ofCollops vittatus (Say) (Melyridae). Laboratory evaluation revealed that largelarvae of C. carnea and the adults of theother species (except for O. tristicolor)could consume 100 eggs of S. exigua in a48 h period. The parasite guild associatedwith small and medium-sized larvae consisted ofthree species: Hyposoter exiguae(Viereck) and Pristomerus spinator (F.)(Ichneumonidae), both larval endoparasites; andChelonus insularis Cresson (Braconidae),an egg-larval endoparasite. Parasitization in field samples ranged from 30 to 65%. Smalland medium-sized larvae were also infected witha nuclear polyhedrosis virus (NPV); rates ofNPV infection ranged from 0 to 35% in fieldsamples. These results are consistent withanecdotal evidence that natural enemies,primarily generalist predators, are largelyresponsible for maintaining populations ofS. exigua at relatively low levels innontreated sugarbeet fields.     

甜菜夜蛾核型多角体病毒对拟澳洲赤眼蜂的影响：种群参数比较和PCR检测

用生命表方法和PCR检测研究了甜菜夜蛾核型多角体病毒（Spodopteraexiguamulticapsid nucleopolyhedrovirus, SeMNPV）对拟澳洲赤眼蜂Trichogramma confusum Viggiani的影响。结果表明,在25±1℃下,取食含SeMNPV蜂蜜和纯蜂蜜（对照）的两组赤眼蜂雌蜂的寿命、繁殖力、羽化率、产雌率均无显著差异;内禀增长率（r_m）分别为0.3524 和0.3418,周限增长率（λ）为1.42和1.41,非常接近;净繁殖率（RO）分别为 124.34和142.57,平均世代历期为13.6和14.5天, 种群倍增时间分别为1.96和2.03天。对5头取食含SeMNPV蜂蜜的雌蜂的子代的PCR 检测结果也均为阴性。因此,结论认为SeMNPV对拟澳洲赤眼蜂成蜂并无明显影响,体内携带病毒的亲代蜂并不能将病毒传给子代。     

Characterization of the gut microbiome in the beet armyworm Spodoptera exigua in response to the short-term thermal stress

The gut microbiota is critical for energy and nutrient utilization and plays a role in host immunity in response to environmental changes. The beet armyworm Spodoptera exigua is a worldwide polyphagous agricultural pest and has frequently experienced potentially stressful temperature fluctuations under natural environmental conditions. However, little is known about the effects of thermal stress on the gut microbiome of this moth pest. Therefore, we investigated the gut microbiome variations, composition and community structure of S. exigua among low-temperature (10 °C), control (26 °C) and high temperature (35 °C) treatments using 16S amplicon sequencing. Overall, 1,192,707 high-quality reads and 762 operational taxonomic units (OTUs) were detected from 15 samples. A total of 289 genera belonging to 19 bacterial phyla were captured, with Firmicutes and Proteobacteria being the most prominent phyla. Alpha diversity metrics indicated no significant differences in the gut bacterial diversity of S. exigua among the three temperature treatments. Principal coordinates and hierarchical cluster analysis revealed significant differences in the structure of gut microbiota between the low-temperature treatment and the other two temperature treatments. In addition, PICRUSt2 analysis demonstrated that the predicted metagenomes associated with the gut microbiome were amino carbohydrate transport and metabolism, acid transport and metabolism, inorganic ion transport and metabolism and cellular processes. Our study showed that thermal stress induced changes in the gut microbiome of the beet armyworm, which may contribute to better understanding the ecological adaptation of S. exigua under changing temperature trends and to evaluating the use of gut microorganisms as biocontrol agents for this pest.     

Transgenic expression of Dendrolimus kikuchii nucleopolyhedrovirus enhancin in tobacco enhances the mortality and affects the development of Spodoptera exigua larvae

A novel 2551-bp enhancin gene designated En-Dk was isolated from the Dendrolimus kikuchii nucleopolyhedrovirus genome. Sequence analysis revealed that En-Dk encodes a polypeptide chain of 792 amino acid residues containing a conserved enhancin domain and an α-helical transmembrane domain, but no signal peptide. Compared with wild-type tobacco (Nicotiana tabacum L.), transgenic tobacco showed significant phenotypic changes following En-Dk overexpression with slower growth and delayed initial flowering. An interesting finding is that the transgenic tobacco exerted a toxic effect on newly hatched Spodoptera exigua larvae, with a significant inhibitory effect on third-instar larvae in terms of body weight, pupal weight, pupation and the emergence process.     

Competition between wild-type and a marked recombinant baculovirus (Spodoptera exigua nucleopolyhedrovirus) with enhanced speed of action in insect larvae

Competition between virus genotypes in insect hosts is a key element of virus fitness, affecting their long-term persistence in agro-ecosystems. Little information is available on virus competition in insect hosts or during serial passages from one cohort of hosts to the next. Here we report on the competition between two genotypes of Spodoptera exigua nucleopolyhedrovirus (SeMNPV), when serially passaged as mixtures in cohorts of 4th instar S. exigua larvae. One of the genotypes was a SeMNPV wild-type isolate, SeUS1, while the other was a SeMNPV recombinant (SeMNPV-XD1) having a greater speed of kill than SeUS1. SeXD1 lacks a suite of genes, including the ecdysteroid UDP-glucosyl transferase (egt) gene. SeXD1 expresses the green fluorescent protein (GFP) gene, enabling the identification of SeXD1 in cell culture and in insects. The relative proportion of SeUS1 and SeXD1 in successive passages of mixed infections in various ratios was determined by plaque assays of budded virus from infected larvae and by polymerase chain reactions and restriction enzyme analyses. The SeUS1 genotype outcompeted recombinant SeXD1 over successive passages. Depending on the initial virus genotype ratio, the recombinant SeXD1 was no longer detected after 6-12 passages. A mathematical model was developed to characterize the competition dynamics. Overall, the ratio SeUS1/XD1 increased by a factor 1.9 per passage. The findings suggest that under the experimental conditions recombinant SeXD1 is displaced by the wild-type strain SeUS1, but further studies are needed to ascertain that this is also the case when the same baculoviruses would be used in agro-ecosystems.     

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.     

Clastogenic factors in plasma of HIV-1 infected patients

The objective of this study was to investigate the clastogenic activity of plasma ultrafiltrates from HIV-I infected patients. Clastogenic factors are chromosome-damaging agents with low molecular weight (<10,000 daltons) which cause chromosome aberrations, sister chromatid exchanges, DNA strand breakage, and gene mutation. They have first been described in the plasma of irradiated persons, but they are also found in hereditary breakage syndromes and chronic inflammatory diseases with autoimmune reactions. Their formation and their clastogenic effects are modulated by superoxide anion radicals. We analyzed a total of 22 HIV-1 positive patients in comparison to 20 reference plasma samples from healthy HIV negative blood donors of similar age. The plasma ultrafiltrates (filter cutoff 10,000 daltons) from patients induced a statistically significant increase in chromosomal breakage in the cytogenetic test system (20.5 ± 6.8 aberrations per 100 cells), while no increase was observed in test cultures exposed to plasma ultrafiltrates from healthy blood donors (6.3 ± 2.9 aberrations per 100 cells). The breakage values were slightly, but not significantly, lower in the 10 patients with more than 200 T-helper cells/ml (18 ± 4 aberrations per 100 cells), than in the 12 patients with less than 200 T-helper cells/ml (22.3 ± 7.9 aberrations per 100 cells). HIV patients with high clastogenic activity (induction of more than 20 aberrations per 100 cells, range 20 to 39) showed higher plasma levels for malondialdehyde than those with lower clastogenic activity (less than 20 aberrations per 100 cells, range 12 to 18). However, the difference was statistically not significant. Another lipid peroxidation product, 4-hydroxynonenal, was increased equally in both groups. There were no significant differences in water- and lipid-soluble plasma antioxidants between the low- and high-breakage group. In agreement with previous findings, the clastogenic effects of plasma ultrafiltrates in the test cultures were reduced by the antioxidant enzyme superoxide dismutase. The presence of clastogenic factors in the plasma of HIV patients is further evidence for a prooxidant state in these persons. Since clastogenic factor formation appears to occur at an early stage of the disease, it may be significant for virus release or activation, because of the superoxide anion stimulating effects of clastogenic factors. From a practical standpoint, clastogenic factors may be useful for evaluation of promising drugs.     

The impact of co-infection by a nucleopolyhedrovirus and the endoparasitoid Microplitis pallidipes on the total hemocyte count and composition in larval beet armyworm,Spodoptera exigua

The physiological effects of nucleopolyhedrovirus (NPV) infection and parasitism by Microplitis pallidipes (Hymenoptera: Braconidae) on the hemocytes of Spodoptera exigua (Lepidoptera: Noctuidae) larvae were examined. We found that compared to healthy (control) larvae, the total hemocyte count (THC) and granulocyte count in parasitized larvae increased 1 day after parasitization and then decreased, while the plasmatocyte count was not significantly affected for the first 5 days but was significantly enhanced on day 6 after parasitization. In parasitized + infected larvae, both the THC and granulocyte counts began be lower from day 1 compared to parasitized larvae, while the plasmatocyte count was generally lower than in parasitized larvae. Compared to the control, THC, and granulocyte counts of virus-infected larvae were higher 1 day after infection. Compared to that in virus-infected larvae, THC and granulocyte counts in parasitized + infected larvae began to decrease from day 1 while the plasmatocyte count generally decreased. We concluded that the host immune response of cell communities to parasitization by M. pallidipes was elicited during the development of the parasitoid egg, but that immune response was inhibited during larval development of parasitoids in the host body. Meanwhile, we found that NPV infection impeded the regulatory effect of M. pallidipes on host cellular immune responses, and parasitization by M. pallidipes similarly inhibited the host cellular immune response caused by NPV infection.     

Effects of a nucleopolyhedrovirus in mixtures with azadirachtin on Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae) larvae and viral occlusion body production

Abstract

The combined and alone effects of azadirachtin (AZA) and Spodoptera frugiperda multicapsid nucleopolyhedrovirus (SfMNPV) on the mortality of S. frugiperda (J. E. Smith) (Lepidoptera: Noctuidae) were evaluated in the laboratory. For this, diet surface contamination bioassays were performed on S. frugiperda in the third instar. LC₅₀ values for SfMNPV alone were determined to be 430 and 373 viral occlusion bodies (OBs)/mm² at 192 and 216 h after treatment, respectively. LC₅₀ values for AZA alone were estimated for two periods of continuous exposure (4 or 5 days). In this case, LC₅₀ values were 45.5 and 16.8 mg L^?1 at 216 h after treatment (4 or 5 days of larval exposition to insecticide, respectively). We observed that although the interaction of AZA with SfMNPV increased viral pathogenicity, such improvements were of greater magnitude and more consistent at the lower OB concentration used (177 OBs/mm²). Application of SfMNPV (430 OBs/mm²), AZA (26.4 mg L^?1) or SfMNPV–AZA mixtures resulted in a significant reduction in the mean weight of larvae treated in the third instar across the experiment, by 23–41%, 17–95% and 26–97%, respectively, compared to control. The duration of larval development during the third and fourth instars increased significantly in larvae exposed to SfMNPV–AZA mixtures and AZA alone compared to SfMNPV alone and control treatments. The yield of OBs/mg weight of larvae treated with SfMNPV alone was 1.8-fold higher than OB yields from insects inoculated with SfMNPV–AZA mixtures. We conclude that AZA + SfMNPV mixtures are unlikely to be useful for the mass production of this virus and laboratory observations on the value of AZA + SfMNPV mixtures as a potentiator of biological insecticides require validation in field studies under commercial growing conditions.     

植物MYB转录因子与非生物胁迫响应研究

MYB转录因子家族是植物中最大的转录因子家族之一,在植物体内的多种生理生化反应中起着关键性作用,其中一项重要功能就是对非生物逆境的应答。这类转录因子通过调控生长发育,影响代谢产物的合成和影响激素信号等多方面参与非生物逆境的应答。介绍了MYB转录因子的结构特点和分类上的新发现,并综述了近几年MYB转录因子家族在植物响应干旱、高温、低温和高盐等非生物胁迫方面的研究进展。     

The effect of nucleopolyhedrovirus infection and/or parasitism by Microplitis pallidipes on hemolymph proteins, sugars, and lipids in Spodoptera exigua larvae

We determined the physiological effects of joint and separate nucleopolyhedrovirus (NPV) infection and parasitism by the endoparasitoid Microplitis pallidipes Szepligeti on biochemical events in the noctuid Spodoptera exigua (Hübner). The results indicated that in parasitized larvae, compared to healthy larvae, total protein concentration in host hemolymph began to decline and total sugar concentration significantly increased by the first day, while lipid content in the host body significantly increased by the second day after parasitization. Meanwhile, in jointly infected and parasitized hosts, compared to parasitized larvae, total protein concentration was consistently higher, total sugar concentration was consistently lower, and lipid content became higher by the second day after treatment. In virus-infected larvae, compared to healthy larvae, total protein concentration sharply declined during the first two days but increased by the third, while total sugar concentration increased on the second and third days after virus infection but decreased at other observation times, and lipid content began to increase by the second day after virus infection. Finally, in larvae that were both parasitized and virus-infected, compared to just virus-infected larvae, total protein concentration increased during the first two days but decreased by the third, total sugar concentration increased only on the first and fourth days, and lipid content decreased significantly on the first day but began to increase by the second day after treatment. These findings led us to conclude that parasitization inhibited protein mobilization but stimulated sugar mobilization in host hemolymph, and promoted lipid mobilization in the host body, while Spodoptera exigua NPV infection stimulated protein mobilization induced by parasitization but inhibited sugar mobilization induced by parasitization.