RNA interference of an antimicrobial peptide, gloverin, of the beet armyworm, Spodoptera exigua, enhances susceptibility to Bacillus thuringiensis

Gloverin is known to be an inducible antimicrobial peptide. This study reports a gloverin gene (Seglv) identified from the beet armyworm, Spodoptera exigua. Seglv encodes 175 amino acids with a signal peptide. Its amino acid sequence is highly homologous (>95%) to other known gloverins. Seglv was expressed from egg to adult stages even without immune challenge. Especially, in larval stage, it was expressed in all tested tissues, such as hemocyte, fat body, gut, and epidermis. However, the constitutive expression level was significantly elevated in response to bacterial challenge. Expression of a Toll gene was required for expression of Seglv. A recombinant Seglv protein was synthesized using a bacterial expression system and purified with an affinity chromatography. The recombinant protein showed a specific antibacterial activity against a Gram-positive bacterium, but no activity against a Gram-negative Escherichia coli. Injection of specific double stranded RNA (dsRNA) against Seglv could suppress its expression. Knockdown of Seglv expression induced a significant developmental retardation and resulted in hypotrophy pupae. The larvae treated with dsRNA were much more susceptible to Bacillus thuringiensis than the control larvae. These results suggest that Seglv acts as an antimicrobial peptide especially against Gram-positive bacteria including B. thuringiensis.     

PGE(2) induces oenocytoid cell lysis via a G protein-coupled receptor in the beet armyworm, Spodoptera exigua

Eicosanoids mediate cellular and humoral immune responses in the beet armyworm, Spodoptera exigua, including activation of prophenoloxidase (PPO). PPO activation begins with release of its inactive zymogen, PPO, from oenocytoids in response to prostaglandins (PGs). Based on the biomedical literature, we hypothesized that PGs exert their actions via specific G protein-coupled receptor(s) in S. exigua. This study reports a G protein-coupled receptor (Se-hcPGGPCR1) gene, which is expressed in the hemocytes of S. exigua. The Se-hcPGGPCR1 consists of 420 amino acids and belongs to rhodopsin-type GPCRs. The high content of hydrophobic amino acid residues within the Se-hcPGGPCR1 protein is explained by prediction of seven-transmembrane domains that are characteristic of these GPCRs. Except for the eggs, Se-hcPGGPCR1 was expressed in all life stages. During the larval stage, it was expressed in hemocytes and gut, but not in fat body nor in epidermis. Real time quantitative RT-PCR showed that bacterial challenge induced more than 20-fold increases in its expression level. Fluorescence in situ hybridization showed that Se-hcPGGPCR1 was expressed in a specific hemocyte type, the oenocytoids. A specific eicosanoid, PGE₂, significantly induced oenocytoid lysis and increased PO activity in the plasma. In contrast, when Se-hcPGGPCR1 expression was suppressed by RNA interference (RNAi), the oenocytoid lysis and the PO activation in response to PGE₂ were not elevated above basal levels. A binding assay using intracellular calcium mobilization showed that the RNAi-treated hemocytes were significantly less responsive to PGE₂ than the control hemocytes. These results support our hypothesis with the specific finding that PGE₂ acts through Se-hcPGGPCR1 to activate PPO by lysing oenocytoids.     

甜菜夜蛾类钙粘蛋白基因全长cDNA的克隆及序列分析

昆虫中肠膜上的类钙粘蛋白(cadherin-like protein)是Bt毒素的一类重要受体,它与Bt毒素对昆虫的杀虫作用机制以及昆虫对Bt毒素的抗性等密切相关。本研究根据已报道的其它昆虫的类钙粘蛋白基因的保守区设计简并引物,应用RT-PCR和RACE技术克隆了迁飞性重要害虫甜菜夜蛾Spodoptera exigua(Hübner)类钙粘蛋白基因全长cDNA序列(命名为SeCAD,GenBank登录号为HQ647122),全长5582bp,编码1739个氨基酸,推导的氨基酸序列包括1个信号肽、1个前蛋白区、11个钙粘蛋白重复、1个近膜区、1个跨膜区和1个胞质区。预测的分子量和等电点分别为196.447ku和4.47。该蛋白与同科的大螟Sesamia inferens、蛀茎夜蛾S.nonagrioides、烟芽夜蛾Heliothi svirescens、烟夜蛾Helicover paassulta亲缘关系更近,氨基酸序列一致性分别为61.28%、60.34%、60.14%、60.08%。这些结果对于揭示转Bt基因作物对非靶标、迁飞性甜菜夜蛾的杀虫作用机制以及评价其潜在的对Bt毒素抗性机制等奠定了基础。     

甜菜夜蛾抗药性研究现状

就有关甜菜夜蛾Spodoptera exigua(Hbner)对常用杀虫剂的抗药性现状作综述。甜菜夜蛾对拟除虫菊酯类、有机磷类、氨基甲酸酯类杀虫剂已产生了较高水平的抗药性,其中山东泰安抗性种群对氯氟氰菊酯的抗性高达2445.5倍;对多杀菌素等生物杀虫剂产生了中低水平的抗性;对昆虫生长调节剂如虫酰肼的敏感性也有所降低,但昆虫生长调节剂依然是比较理想的防治药剂。对交互抗性及抗性治理也作了阐述。     

Functional analysis of two chitinase genes during the pupation and eclosion stages of the beet armyworm Spodoptera exigua by RNA interference

Insect chitinases are a multigene family that is encoded by a rather large and diverse group of genes. The main function of chitinases is to digest the chitin contained in tissues such as the cuticles and gut lining during molting. In this study, we examined the role of a chitinase (SeChi) and a bacterial type chitinase (SeChi-h) during the pupation and eclosion stages of Spodoptera exigua. First, efficient silencing of the SeChi and SeChi-h genes through specific double-stranded RNA (dsRNA) injection led to a significant reduction in the mRNA levels of SeChi and SeChi-h. Additionally, different phenotypic defects were observed at the pupal and adult stages after injection of the SeChi and SeChi-h dsRNAs. After injecting SeChi dsRNA in the pupal stage, the cuticle of the head split open and the pupal cuticle was visible under the old larval cuticle. However, after injecting the SeChi-h dsRNA, animals died without exhibiting any special phenotypes. At the adult death stage, animals injected with dsSeChi could not shed their pupal shell completely, and their old cuticles remained attached to their head or chest. However, the main lethal phenotype was that insects did not emerge after dsSeChi-h injection. Additionally, the average survival rates of S. exigua were 52.02% and 40.38% at the pupal and adult stages, respectively, after injection with SeChi dsRNA. For the insects injected with SeChi-h dsRNA, the survival rates were 72.38% and 48.52%, respectively. These results suggest that SeChi and SeChi-h may have different biologic functions during the pupal-adult molting.     

Nutrient utilization by caterpillars of the generalist beet armyworm, Spodoptera exigua

Abstract.  Beet armyworm, Spodoptera exigua (Hübner) (Lepidoptera: Noctuidae), caterpillars are destructive crop pests responsible for considerable annual economic losses. These generalist herbivores are challenged with a diversity of dietary choices that can impact their survival, development and fecundity. In the present study, dietary choices of S. exigua caterpillars, based on the protein to digestible carbohydrate (P : C) ratio of the food, and the impact of nutritionally unbalanced foods on caterpillar performance are assessed. In choice experiments, individual third-instar caterpillars are offered simultaneously a P-biased and a C-biased food until pupation. Caterpillars feed nonrandomly and select a slightly P-biased diet (22P : 20C). In no-choice experiments, second instar caterpillars are reared until pupation on diets ranging in P : C ratio from extremely P- to extremely C-biased. High mortality and delayed development are observed on the C-rich, P-poor diets, highlighting the potential deleterious effects of excess carbohydrates and the importance of protein for growth and development. Diet-dependent differences in pupal weight or pupal lipid reserves are not observed. This contrasts with closely-related Spodoptera species where pupal mass and lipid stores increase on C-rich, P-poor diets. On the extremely P-biased diet, performance is similar to that of individuals reared on the self-selected diet, suggesting that these caterpillars may efficiently be deaminating excess amino acids to generate carbon skeletons, which are shunted into lipid biosynthesis. Spodoptera exigua caterpillars exhibit flexible and efficient pre-ingestive nutrient intake regulation and post-ingestive utilization, allowing these generalist feeders to cope with the heterogeneous diets they may encounter.     

温度对甜菜夜蛾飞行能力的影响

温度对甜菜夜蛾飞行能力有显著的影响（P<0.05）。在16～32℃内,成虫均能进行正常的飞行活动。24℃下的成虫飞行能力最强,在15 h的吊飞飞行中,成虫飞行距离最远（37.14 km）、飞行速度最快（0.87 m/s）、飞行时间最长（11.73 h）。温度低于２０℃或高于２８℃时,其飞行能力均显著降低。甜菜夜蛾在不同温度下飞行时对主要能源物质（甘油三酯）的利用效率不同。在较适宜的温度下,尽管成虫飞行消耗的甘油三酯较多,但单位飞行距离所消耗的甘油三酯却较少,即利用效率较高,表明成虫飞行能源物质利用效率的不同是导致其在不同温度下飞行能力产生差异的主要原因之一。     

甜菜夜蛾抗寒与越冬能力研究

研究了甜菜夜蛾不同虫态的过冷却点、耐低温能力、可能的越冬虫态及其越冬能力,并结合气象资料的分析,初步确定了我国甜菜夜蛾的越冬北界.甜菜夜蛾3～5龄幼虫的过冷却点较高,分别为-11.17℃、-11.96℃和-10.50℃(平均-11.21℃),预蛹期次之,为-12.47℃,蛹期最低,平均为-17.16℃.在5℃、0℃、-10℃和-5℃条件下,甜菜夜蛾各种虫态的LT50、LT90和LT99.9均随温度的降低而缩短,尽管不同虫态的耐低温能力有很大的差别.其耐低温能力由弱至强的排列顺序依次为卵<成虫<幼虫<蛹.这些结果表明,蛹是甜菜夜蛾的四个虫态中耐低温能力最强的,因而是最可能的越冬虫态.但由于蛹在0℃条件下的LT99.9为38.06d,表明甜菜夜蛾在冬季0℃以下的温度超过38d的地区不能越冬.另外,连续两年冬春的田间试验结果表明,蛹在北京西郊越冬死亡率均为100%,说明甜菜夜蛾在北京地区不能越冬.根据这些结果并结合我国气温的变化规律,初步将我国甜菜夜蛾的越冬北界定于北纬38°左右,即1月份-4℃等温线左右.     

甜菜夜蛾核型多角体病毒在宿主种群中的垂直传播研究

以两种浓度的病毒液感染甜菜夜蛾4龄、5龄幼虫,以研究甜菜夜蛾核型多角体病毒从亲代到子代垂直传递的方式和效率。实验结果表明,该病毒可通过母体介导(卵内、卵表)和父体介导等3种方式传递给子代,造成子代感染。幼虫经口感染后母体介导传递实验表明,病毒垂直传播可引起20%～48%的子代幼虫病死,并能使子代成虫产卵量下降;母体介导传播实验中,以5%福尔马林进行卵表面消毒证实仍有18.8%的幼虫感染病毒死亡,表明了该病毒可经卵内传播,并同时存在着附卵传播;母体介导传递实验还表明病毒对宿主有弱化作用,亲代免于病死的蛹重量减轻,羽化所得雌成虫的产卵量下降,产卵期延长。以交互配对将感染与未感染的雌雄成虫彼此交配,表明雄成虫精子也能垂直传递病毒(父体介导),引起20％～40%子代幼虫感病死亡。成虫经口感染,对所产卵块表面消毒后,仍导致28.6%的子代幼虫死于病毒感染,也表明存在卵内传播。     

An entomopathogenic bacterium, Xenorhabdus nematophila, inhibits hemocyte phagocytosis of Spodoptera exigua by inhibiting phospholipase A(2)

Phagocytosis is a hemocytic behavior against bacterial infection. An entomopathogenic bacterium, Xenorhabdus nematophila, inhibits immune responses of target insects and causes hemolymph septicemia. This study analyzed how X. nematophila could inhibit phagocytosis to increase its pathogenicity. Granular cells and plasmatocytes were the main phagocytic hemocytes of Spodoptera exigua determined by observing fluorescence-labeled bacteria in the cytosol. X. nematophila significantly inhibited phagocytosis of both hemocytes, while heat-killed X. nematophila lost its inhibitory potency. However, co-injection of X. nematophila with arachidonic acid did not show any significant inhibition of hemocyte phagocytosis. In fact, hemocytes of S. exigua infected with X. nematophila showed significant reduction in phospholipase A(2) (PLA(2)) activity. Dexamethasone, a specific PLA(2) inhibitor, significantly inhibited phagocytosis of both cell types. However, the inhibitory effect of dexamethasone was recovered by addition of arachidonic acid. Incubation of hemocytes with benzylideneacetone, a metabolite of X. nematophila, inhibited phagocytosis in a dose-dependent manner. These results suggest that X. nematophila produces and secretes PLA(2) inhibitor(s), which in turn inhibit the phagocytic response of hemocytes.     

Relationship of flight and reproduction in beet armyworm, Spodoptera exigua (Lepidoptera: Noctuidae), a migrant lacking the oogenesis-flight syndrome

The beet armyworm, Spodoptera exigua, undertakes long-distance migration. We used flight mills to investigate the interaction between flight and reproduction in this species given the apparent absence of the oogenesis-flight syndrome. This syndrome, common in many migratory insects, is characterized by a suite of traits including migration during the pre-oviposition period followed by a switch to oogenesis. No negative effects of inter-ovipositional flight on lifetime fecundity were observed. Instead, adult reproductive output suffered when female flight was initiated the first day after eclosion and before oviposition, suggesting that migratory flight overlaps with the oviposition period rather than being confined to the pre-oviposition period. Mating status of both females and males had no negative influence on their flight performance except that flight distance and flight duration of 7-day-old mated females were significantly less than in unmated females. Furthermore, the number of eggs produced and mating frequency of females less than 7 days of age were not significantly correlated with flight performance, suggesting reproductive development paralleled and was independent of migratory behavior. This independent relationship between flight and reproduction of adults is consistent with the very short pre-oviposition period in this species, and suggests that resources are partitioned between these activities during pupal development. Together, our results uncovered neither obvious trade-offs nor mutual suppression between flight and reproduction in S. exigua, which indicates the lack of an oogenesis-flight syndrome for coordination of these two energy-intensive processes. We propose a conceptual model of migration for this species based on the current and previous studies.     

In vivo development of the entomogeneous hyphomycetePaecilomyces farinosus in hostSpodoptera exigua (beet armyworm) larvae

The in vivo development of the entomogenous hyphomycetePaecilomyces farinosus inSpodoptera exigua (beet armyworm) larvae was examined using light and electron microscopic techniques. Blastospores injected into larval hemocoels (500 blastospores/larva) were immediately ingested by phagocytic hemocytes, and no fungal cells were detected in the hemolymph until 36 h post-injection. As indicated by immunocytochemical methods, the in vivo-produced blastosopres, in contrast to in vitro blastospores, lacked a galacto-mannan surface layer required for opsonization by aS. exigua humoral lectin. Therefore, these in vivo cells were not recognized by phagocytic granulocytes and were freely-circulating in the hemolymph. Hyphae differentiating from the blastospores were recognized by the hemocytes and induced formation of multicellular hemocytic nodules. By 72 h post-injection, mycelia were observed emerging from the nodules and by 96 h, larvae had become mummified due to extensive proliferation of the fungus throughout host tissues. Neither phagocytosis of the initially injected in vitro-produced blastospores nor nodule formation around hyphal cells later in the infection process was effective in stopping fungal growth. The in vivo development ofP. farinosus was similar to that of another hyphomycete,Beauveria bassiana except that in the latter case, extensive nodule formation was inhibited by the production of fungal metabolites.     

Identification of a hypertrehalosemic factor in Spodoptera exigua

Trehalose is a major blood sugar in insects with a range of physiological functions, including an energy source and a cryoprotectant. Hemolymph trehalose concentrations are tightly regulated according to physiological conditions. An insulin‐like peptide, SeILP1, downregulates hemolymph trehalose concentrations in Spodoptera exigua. Here, we identified a factor that upregulates hemolymph trehalose concentration in S. exigua. Hemolymph trehalose concentrations were significantly increased after immune challenge or under starvation in a time‐dependent manner. To determine endocrine factors responsible for the upregulation, stress‐associated mediators, such as octopamine, serotonin, or eicosanoids were injected, but they did not upregulate hemolymph trehalose. On the other hand, injection with Schistocerca gregaria adipokinetic hormone (AKH) significantly increased hemolymph trehalose concentration in S. exigua. During upregulation of hemolymph trehalose by AKH injection, trehalose degradation appeared to be inhibited because expression of trehalase and SeILP1 were significantly suppressed while that of trehalose phosphate synthase was not significantly changed. Interrogation of a Spodoptera genome database identified an S. exigua AKH‐like gene and its expression was confirmed. During starvation, its expression concentrations were increased, although RNA interference specific to the AKH‐like hypertrehalosemic factor (SeHTF) gene significantly prevented the upregulation of hemolymph trehalose concentrations during starvation. A synthetic peptide of SeHTF was prepared and injected into S. exigua larvae. At nanomolar concentration, the synthetic SeHTF peptide effectively upregulated hemolymph trehalose concentrations. Here we report a novel hypertrehalosemic factor in S. exigua (SeHTF).     

甜菜夜蛾性信息素的研究与应用

甜菜夜蛾Spodoptera exigua(Hbner)已成为我国的重要农业害虫,其高抗药性和危害具有隐蔽性,致使防治十分困难。文章介绍甜菜夜蛾性信息素的主要成分、活性、配比、诱芯剂型、诱捕器类型和利用性信息素监测、防治甜菜夜蛾技术的研究概况。认为改进现有技术后并应用于实际,将会更有效地监测和防治甜菜夜蛾的发生与危害。     

甜菜夜蛾在四种寄主植物上的生命表参数比较研究

在控制条件下对甜菜夜蛾Spodoptera exigua(Hübner)在白菜、大葱、甘蓝和豇豆上的生命表参数进行了比较研究。结果表明,甜菜夜蛾幼虫在4种寄主植物上均为5个龄期。幼虫和蛹在大葱上的发育历期最长,在白菜上最短。甘蓝叶片饲养的甜菜夜蛾生殖力最高,单雌产卵量为1015.8粒,豇豆叶片上饲养的生殖力最低,为496.1粒。甜菜夜蛾在甘蓝上的内禀增长率和净增殖率最高,分别为0.237和287.82,在大葱上最低,分别为0.172和173.90。在大葱上甜菜夜蛾幼虫存活率较低,在其他3种寄主植物上较高。甜菜夜蛾的特定年龄生殖率在甘蓝叶片上最高,第22天单雌产卵量高达453.6粒。研究结果表明,在选取的4种植物中,甘蓝是甜菜夜蛾的最适寄主。     

氰氟虫腙亚致死剂量对甜菜夜蛾生长发育和繁殖力的影响

本文研究了氰氟虫腙的亚致死剂量LC15(28.423μg/mL)和LC25(41.363μg/mL)对甜菜夜蛾Spodoptera exigua(Hübner)亲代和子1代的一些生物学特性的影响。结果表明:LC15和LC25处理组与对照组相比,甜菜夜蛾亲代的化蛹率,羽化率和单雌产卵量分别降低了49.3%,60.0%;♀(12.65%,25.09%)和♂(13.14%,28.91%);25.6%,65.7%,畸形蛹率分别提高了10.5%,14.4%。子1代的化蛹率,羽化率和单雌产卵量分别降低了11.8%,24.6%;♀(9.48%,21.54%)和♂(11.61%,24.08%);14.4%,54.3%,畸形蛹率分别提高了4.1%,5.8%。另外子1代的孵化率、幼虫的存活率降低,蛹期和成虫期缩短,产卵前期延长,产卵期显著缩短。总体来说,LC15和LC25剂量的氰氟虫腙对甜菜夜蛾亲代与子1代的生物特性都有不同程度的影响,并且LC25剂量对甜菜夜蛾的影响要比LC15剂量大。     

Functional expression and structural characterization of ORF cDNA encoding chitinase of the beet armyworm, Spodoptera exigua (Lepidoptera: Noctuidae)

Chitin is an important component of the exoskeleton and peritrophic matrix in insects. Its bio-degradation is initiated by the endo-splitting chitinase. We cloned an ORF cDNA encoding chitinase from the last instar larva of the beet armyworm, Spodoptera exigua (Lepidoptera: Noctuidae), into E. coli to confirm its functionality. Its amino acid sequence was compared with previously described lepidopteran chitinases. S. exigua chitinase expression enhanced cell growth approx. 1.5 fold in transformed E. coli than in the wild strain in a 1% colloidal chitin-containing medium with insufficient regular nutrients. Compared with the wild strain, the two intracellular chitin degradation derivatives, glucosamine and N-acetylglucosamine, increased approx. 5.8 and 1.5 fold, respectively, and extracellular chitinase activity in the transformed strain was about 1.6 fold higher. The ORF of S. exigua chitinase-encoding cDNA including stop codon was composed of 1674 bp nucleotides and the calculated molecular weight of the deduced 557 amino acid residues was about 62.6 kDa. The ORF consisted of an N-terminal leading signal peptide (AA 1-20), a catalytic domain (AA 21-392), a linker region (AA 393-493), and a C-terminal chitin-binding domain (AA 494-557) showing a typical molting fluid chitinase structure. Phylogenetic analysis determined that all 5 noctuid chitinases were grouped together, while two bombycid enzymes and one tortricid enzyme mapped together in one lineage. In the noctuid group, the sub-lineages reflected their taxonomic relationships at the Genus level.     

甜菜夜蛾防控技术研究与示范——公益性行业(农业)科研专项“甜菜夜蛾防控技术研究与示范”研究进展

针对当前我国蔬菜生产中甜菜夜蛾Spodoptera exigua(Hübner)高抗药性、猖獗为害、防控困难的现状,项目组对甜菜夜蛾的发生规律、越冬与迁飞、抗药性、生物防治与综合防控等进行了系统的研究。明确了甜菜夜蛾在我国各地的发生规律及发生动态,从南到北发生时间呈楔形,而发生量总体东高西低,从南到北呈中部高南北低的马鞍形。阐述了甜菜夜蛾"无卵子发生-飞行拮抗"的迁飞特性,提出了接力棒式季节性南北往返迁飞模式,初步阐明了甜菜夜蛾在我国东部地区的迁飞路线与迁飞时间。提出并认证了甜菜夜蛾在我国的越冬区域。明确了全国各主要发生区甜菜夜蛾对10种杀虫剂的抗药性水平,探明了甜菜夜蛾对茚虫威、甲氨基阿维菌素苯甲酸盐的抗性风险、交互抗性、种群适合度、抗性遗传规律和抗性机理,提出了抗药性治理措施。分别在海南、湖南、湖北、上海、天津等地进行了甜菜夜蛾田间药效试验,筛选出环境友好型高效杀虫剂7种。掌握了马尼拉陡胸茧蜂、夜蛾黑卵蜂和淡足侧沟茧蜂人工大量繁殖技术,改进了甜菜夜蛾核型多角体病毒生产工艺,解决了寄生蜂和病毒规模化生产及田间释放关键技术,达到规模化生产的要求。改进优化了甜菜夜蛾性引诱剂配方、研发出新型诱捕装置,并投入生产应用。集成出4套农业生产轻简化实用技术,华南、华中、华北、华东地区根据当地发生特点制定了4套防控方案。在全国甜菜夜蛾主要发生区建立试验基地16个,核心示范基地29个,年示范面积总计约3800hm2,取得显著的经济、社会和生态效益。