缺失宿主Vta1蛋白的MIT结构域对杆状病毒AcMNPV复制的影响

【目的】克隆草地贪夜蛾(Spodoptera frugiperda)Vta1基因,检测Vta1在苜蓿银纹夜蛾核多角体病毒(Autographa californica multiple nucleopolyhedrovirus,AcMNPV)复制中的作用。【方法】利用反转录-PCR与PCR方法筛选草地贪夜蛾Vta1基因及缺失Vta1N-端MIT结构域的突变体并构建其瞬时表达质粒,通过转染Sf9细胞检测表达;构建Vta1及其突变体的双分子荧光互补表达质粒,并通过瞬时转染检测其与Vps4及ESCRT-III亚基Vps46与Vps60的相互作用;共转染gp64与Vta1及其突变体瞬时表达质粒,检测瞬时表达Vta1突变体对AcMNPV出芽型病毒产量及病毒基因启动子指导报告基因表达的影响。【结果】获得了草地贪夜蛾Vta1基因。氨基酸序列相似性分析表明,昆虫、酵母与人类Vta1同源蛋白的相似性分别约为20%与50%。Western blotting分析表明GFP标签的Vta1及其突变体均能在瞬时转染的Sf9细胞中表达。双分子荧光互补分析发现,缺失第1个或第2个MIT结构域显著降低Vta1突变体与Vps4、Vps46或Vps60的相互作用。此外,瞬时表达Vta1突变体显著降低了AcMNPV感染性出芽型病毒的产量,但并未影响AcMNPVie1基因早期启动子和p6.9基因晚期启动子指导的LacZ和GUS报告基因的表达。【结论】Vta1可能参与杆状病毒AcMNPV子代病毒粒子的组装和/或出芽释放过程。     

四种鳞翅目害虫精氨酸激酶基因的表达谱及基于RNAi的功能分析

【目的】精氨酸激酶(arginine kinase, AK)(EC 2.7.3.3)是昆虫体内重要的磷酸原激酶(能量代谢调节因子),也是唯一能够形成有效ATP的磷酰基供体,起着与脊椎动物中肌酸激酶相同的作用。本研究旨在了解鳞翅目害虫AK基因的表达和功能。【方法】利用qRT-PCR方法测定AK基因在大螟Sesamia inferens、二化螟Chilo suppressalis、甜菜夜蛾Spodoptera exigua和斜纹夜蛾Spodoptera litura 这4种鳞翅目害虫不同发育阶段和3龄幼虫不同组织中的表达谱;通过终点法检测了这4种害虫不同发育阶段和幼虫不同组织中的AK酶活性;采用RNAi技术抑制该基因的表达并分析其功能。【结果】AK基因在大螟、二化螟、甜菜夜蛾和斜纹夜蛾这4种鳞翅目昆虫的不同发育阶段和3龄幼虫不同组织中均有表达,说明该基因的表达不具有发育时期和组织特异性。不同发育时期和3龄幼虫不同组织中AK酶活性与基因表达量变化趋势大体一致。注射以AK基因为靶标的dsRNA 6 d后,4种害虫体内AK基因的mRNA表达下降30%~50%,AK酶活性降低30%左右;14 d后幼虫的死亡率达50%左右,显著高于对照组幼虫的死亡率。【结论】AK基因在上述4种鳞翅目害虫中为组成型表达,RNAi抑制AK基因的表达可导致4种害虫的幼虫死亡,研究结果为开发以AK基因为靶标的鳞翅目害虫防治新技术提供了理论依据。     

基于转录组测序分析甜菜夜蛾卵巢细胞离体培养成系进程

【目的】本研究旨在分析甜菜夜蛾Spodoptera exigua蛹卵巢细胞建立细胞系的整个过程,探究细胞由体内到体外培养过程中其基因表达在转录水平的变化,为昆虫体外培养模型的建立提供理论基础。【方法】利用Illumina Hiseq测序平台对甜菜夜蛾蛹卵巢细胞离体培养过程中各阶段的细胞分别进行转录组测序,对获得注释的差异表达基因及其相关信号通路进行分析;通过荧光定量PCR对部分细胞周期相关基因(cycd和cdk4)、调控基因(cdc20,apc1,skp2和mad1)、增殖相关分子标志物(mcm4和pcna)在甜菜夜蛾卵巢细胞离体培养过程中的转录进行验证。【结果】甜菜夜蛾蛹卵巢细胞离体培养过程包括5个阶段:解剖获得离体的卵巢组织,卵巢组织贴壁培养后游离出原代细胞,细胞转化重新具备增殖能力,成功首次传代,以及能够连续传代15代以上建立细胞系。上述5个阶段的细胞经转录组测序、数据组装后共获得46 796条unigenes序列,组装得到序列长度完整性好;转录本unigenes序列拼接长度分布合理,样本碱基Q30均在94%以上。通过KEGG数据库获得注释的unigenes有1 473条,参与细胞过程紧密相关的20条信号通路,其中有92条unigenes在细胞周期信号通路中获得注释。聚类分析表明,在体内处于快速发育状态的卵巢细胞与同样处于增殖状态的细胞系基因表达模式非常接近。原代细胞由短暂停滞生长至成簇细胞的转化关键期,筛选到差异表达基因619个,cdk4在离体培养期表达量显著降低,cycd在细胞转化关键期之后表达量显著升高,cdc20,apc1,skp2和mad1在卵巢组织和细胞系的表达量显著高于原代细胞、转化关键期细胞和首次传代细胞的。从原代细胞至传代后,cycd的表达显著升高8.7倍,显著高于mcm4和pcna的变化水平。【结论】甜菜夜蛾卵巢细胞离体培养过程中5个阶段的细胞转录组测序获得的序列质量符合数据分析的基本要求。筛选获得了甜菜夜蛾蛹卵巢细胞经离体培养过程中的差异表达基因。原代细胞逆转增殖可能与cdk4,cycd,skp2和mad1等细胞周期调控基因表达有关。另外,cycd可作为原代细胞具备传代能力的标志物。     

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.     

The angiotensin converting enzyme inhibitor captopril reduces oviposition and ecdysteroid levels in Lepidoptera

The role of angiotensin converting enzyme (ACE, peptidyl dipeptidase A) in metamorphic- and reproductive-related events in the Egyptian cotton leafworm, Spodoptera littoralis (Lepidoptera, Noctuidae) was studied by using the selective ACE inhibitor captopril. Although oral administration of captopril had no effect on larval growth, topical administration to new pupae resulted in a large decrease of successful adult formation. Oviposition and overall appearance of adults emerging from treated larvae did not differ significantly from those emerging from non-treated larvae. In contrast, topical or oral administration of captopril to newly emerged adults caused a reduction in oviposition. By evaluating the effect of captopril on ecdysteroid titers and trypsin activity, we revealed an additional physiological role for ACE. Captopril exerted an inhibitory effect on ecdysteroid levels in female but not in male adults. Larvae fed a diet containing captopril exhibited increased trypsin activity. A similar captopril-induced increase in trypsin activity was observed in female adults. In male adults, however, captopril elicited reduced levels of trypsin activity. Our results suggest that captopril downregulates oviposition by two independent pathways, one through ecdysteroid biosynthesis regulation, and the other through regulation of trypsin activity. Apparently, fecundity is influenced by a complex interaction of ACE, trypsin activity, and ecdysteroid levels.     

Distribution and diversity of cry genes in native strains of Bacillus thuringiensis obtained from different ecosystems from Colombia

Colombia is a tropical country located at the north of South America. It is considered to be one of the most important countries in terms of its biodiversity worldwide. One hundred and eight soil samples obtained from agricultural crops and wild ecosystems were evaluated in terms of the presence of Bacillus thuringiensis (Bt) native strains. One hundred and eight different Bt strains were isolated and characterized by the presence of crystal proteins by SDS-PAGE and a multiplex PCR with general and specific primers for cry1 and cry3, cry7, and cry8 gene detection. Most of the Bt strains (73%) reacted with the cry1 general primers; 27.8% of the Bt strains reacted with cry3, cry7, and cry8 general primers and 17.8% of strains did not react with any of these two sets of primers. Thirty different PCR profiles were found in the strains with cry1 genes when they were analyzed with specific primers (cry1A to cry1F). A high frequency of joint occurrence was observed for cry1Aa/cry1Ab, cry1Aa/cry1Ac, cry1Ab/cry1Ac, and cry1C/cry1D genes with a Pearson coefficient of 0.88, 0.74, 0.76, and 0.87, respectively. Other distinctive characteristics were found in the Colombian collection as the presence of 22.2% of native strains which presented, at the same time, lepidopteran and coleopteran active genes. Interesting relations were found as well between the cry gene distribution and the geographical areas sampled. Finally, some strains with moderate to high biopesticide activity against Spodoptera frugiperda (Lepidoptera) and Premnotrypes vorax (Coleoptera) insects were identified, this being important to explore future microbial strategies for the control of these crop pests in the region.     

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.     

Effects of individual Bacillus thuringiensis insecticidal crystal proteins on adult Heliothis virescens (F.) and Spodoptera exigua (Hubner) (Lepidoptera: Noctuidae)

Bacillus thuringiensis (Bt) is a grampositive, spore forming bacterium, which is principally distinguishedfrom other bacilli by the production of large, insecticidal,protein crystals (Insecticidal Crystal Proteins, or ICPs). Theseproteins are usually thought to act only on the actively feedinglarvae of susceptible species by a mechanism which involvesconsumption and proteolytic processing of the protein followed bybinding to, and lysis of, midgut epithelial cells. However, few authorshave reported Bt toxicity to adult insects. In the followingpaper, we expand on previous reports of toxicity to adult insects andpresent data which demonstrate that: (1) proteolytically activated ICPssignificantly reduce the lifespans of adult Heliothis virescensand Spodoptera exigua at concentrations of 500 g/ml, butnot 167 or 25 g/ml, (2) individual activated ICPs are differentiallytoxic to adult H. virescens and S. exigua, and (3)adult S. exigua are sensitive to Cry1C protoxin at aconcentration of 1 mg/ml.Deceased     

Movement of spermatozoa in the reproductive tract of adult male Spodoptera litura: daily rhythm of sperm descent and the effect of light regime on male reproduction

Sperm production and movement from the fused testes into the male reproductive tract of the common cutworm Spodoptera litura were studied in insects maintained in a 12 h:12 h light dark (LD) regime. Two types of sperm bundles, eupyrene (nucleated) and apyrene (anucleate) were present in the adult testes. Eupyrene bundles constituted about 25% of the total. Descent of spermatozoa from the testes into the upper vas deferens (UVD) first occurred about 24-30 h before adult eclosion. On entering the reproductive tract, eupyrene spermatozoa remained in bundles while apyrene bundles became dissociated before they reached the UVD. Downward movement of both eupyrene and apyrene spermatozoa within the male tract occurred in a daily rhythm. Sperm descent from the testes into the UVD occurred during the early scotophase, followed by their further descent into the seminal vesicle (SV) during the photophase. Spermatozoa remained in the SV for only a short duration, whence sperm quickly passed through the lower vas deferens into the duplex, which acted as the main sperm storage organ until mating was initiated. During mating 80% of sperm left the duplex, but mating did not influence the number of sperm bundles that subsequently descended into the duplex or the rate of their descent. There was no evidence of sperm reflux. Rearing in constant light (LL) and in constant dark (DD) reduced the number of eupyrene sperm present in the testes of adults that emerged in LL and DD compared to controls (LD), although there was no significant effect on the number of apyrene sperm in the testes. The rhythmic pattern of sperm descent was suppressed in both LL and DD regimes, and the number of sperm in the duplex was adversely affected, with a marked impact in LL reared insects. Male longevity, mating behaviour, oviposition and fertility were found to be more severely affected in LL than in DD.