A cell line (NTU-MV) established from Maruca vitrata (Lepidoptera: Pyralidae): Characterization, viral susceptibility, and polyhedra production

Here we describe the establishment of a new cell line, NTU-MV, derived from pupal tissues of an economically important pest, the legume pod borer Maruca vitrata. This cell line contained four major cell types: polymorphic cells, round cells, spindle-shaped cells, and comma cells. The doubling time of MV cells in TNM-FH medium supplemented with 8% FBS at 28 degrees C was 27h. The chromosome numbers of MV cells varied widely from 16 to 268. Compared to other insect cell lines, the MV cell line produced distinct isozyme patterns with esterase, malate dehydrogenase (MDH), and lactate dehydrogenase (LDH). Confirmation that NTU-MV was derived from M. vitrata was demonstrated by showing that the sequence of the internal transcribed spacer regions (ITS) of the MV cells was 98% identical to that of M. vitrata larvae. Two NTU-MV cell strains, NTU-MV1 and NTU-MV56, were selected based on susceptibility to MaviMNPV (M. vitrata multiple nucleopolyhedrovirus). NTU-MV, MV1, and MV56 cells showed a high susceptibility to MaviMNPV and produced high yields of polyhedra (47-50OBs/cell, 4x10(7)-5.96x10(7)OBs/ml) after 2 weeks of MaviMNPV infection. We conclude that the NTU-MV cell line will be a useful tool for studying MaviMNPV as well as for the mass production of MaviMNPV polyhedra for the biocontrol of M. vitrata.     

中国豆野螟性信息素腺体组份的化学结构鉴定

研究中国豆野螟Marucavitrata(Fabricius)性信息素腺体成分。分析证实(反,反)-10,12-十六碳二烯醛(E,E-10,12-16∶ALD)是中国豆野螟性信息素腺体的主要组份;同时利用GC和GC-MS发现(反,反)-10,12-十六碳二烯醇(E,E-10,12-16∶OH)和反-10-十六碳单烯醛(E-10-16∶ALD)也是中国豆野螟性信息素腺体的组份;三者在腺体中分别以51∶4∶45的比例存在。     

Effect of plant lectins on larval development of the legume pod borer, Maruca vitrata

The legume pod-borer Maruca vitrata (Fabricius), [Lepidoptera: Pyralidae] is a major constraint restricting increased cowpea production in tropical Africa and Asia. Since lectins are known to have insecticidal properties against several pests, a survey was undertaken to screen for the effects of 25 lectins from 15 plant families on the development of Maruca pod borer (MPB) larvae. The list included 8 galactose/N-acetylgalactosamine-, 7 mannose-, 5 complex glycan-, 2 sialic acid- and 3, N-acetylglucosamine-specific lectins. Feeding bioassays using artificial diet were carried out at 2% (w/w) topical levels. Although a total of 16 lectins had detrimental effects pertaining either to larval survival, weight, feeding inhibition, pupation, adult emergence and/or fecundity, only the Listera ovata agglutinin (LOA) (Orchidaceae) and Galanthus nivalis (Amaryllidaceae) agglutinin were effective against MPB larvae for all six parameters examined. Larval mortality and feeding inhibition caused by the most active lectin (LOA) was above 60%.     

Toxicity of Bacillus thuringiensis delta-endotoxins against bean shoot borer (Epinotia aporema Wals.) larvae, a major soybean pest in Argentina

The toxicity of seven Bacillus thuringiensis Cry protoxins was tested against neonate larvae of Epinotia aporema, a major soybean pest in Argentina and South America. The most active protoxins were Cry1Ab and Cry1Ac, with LC50 values of 0.55 and 1.39 microg/ml, respectively. Cry1Aa, Cry1Ba, Cry1Ca, and Cry9Ca protoxins were equally toxic with LC50 values about 4 microg/ml, whereas Cry1Da was not toxic. The synergistic activity of different protoxin-mixtures was also analyzed, no synergistic effect between the Cry proteins was observed, with the exception of the poorly toxic Cry1Ba/Cry1Da mixture that was slightly synergistic. The binding capacity of individual Cry1 and Cry9Ca toxins to brush border membranes of E. aporema was also determined. The non-toxic Cry1Da toxin was the only toxin unable to bind to E. aporema membranes. In addition the heterologous competition experiments showed that Cry1Ab and Cry1Ac toxins share a common binding site. Based on these data, we propose that Cry1Ab and Cry1Ac toxins could be used in the biological control of E. aporema.     

Susceptibility of Cry1Ab-resistant and -susceptible sugarcane borer (Lepidoptera: Crambidae) to four Bacillus thuringiensis toxins

Sugarcane borer, Diatraea saccharalis (F.), is a primary corn stalk borer pest targeted by transgenic corn expressing Bacillus thuringiensis (Bt) proteins in many areas of the mid-southern region of the United States. Recently, genes encoding for Cry1A.105 and Cry2Ab2 Bt proteins were transferred into corn plants (event MON 89034) for controlling lepidopteran pests. This new generation of Bt corn with stacked-genes of Cry1A.105 and Cry2Ab2 will become commercially available in 2009. Susceptibility of Cry1Ab-susceptible and -resistant strains of D. saccharalis were evaluated on four selected Bt proteins including Cry1Aa, Cry1Ac, Cry1A.105, and Cry2Ab2. The Cry1Ab-resistant strain is capable of completing its larval development on commercial Cry1Ab-expressing corn plants. Neonates of D. saccharalis were assayed on a meridic diet containing one of the four Cry proteins. Larval mortality, body weight, and number of surviving larvae that did not gain significant weight (<0.1 mg per larva) were recorded after 7 days. Cry1Aa was the most toxic protein against both insect strains, followed in decreasing potency by Cry1A.105, Cry1Ac, and Cry2Ab2. Using practical mortality (larvae either died or no significant weight gain after 7 days), the median lethal concentration (LC₅₀) of the Cry1Ab-resistant strain was estimated to be >80-, 45-, 4.1-, and −0.5-fold greater than that of the susceptible strain to Cry1Aa, Cry1Ac, Cry1A.105 and Cry2Ab2 proteins, respectively. This information should be useful to support the commercialization of the new Bt corn event MON 89034 for managing D. saccharalis in the mid-southern region of the United States.     

豆野螟成虫行为学特征及性信息素产生与释放节律

豆野螟Maruca vitrata (Fabricius)是一种严重的泛热带豆类蔬菜害虫。本文在（29±1）℃、相对湿度75%～80%、光周期14L∶10D条件下研究了豆野螟成虫的羽化、交尾行为以及雌蛾性信息素的释放节律。结果表明：其羽化行为全天可见,在雌蛾中,86%于暗期羽化; 在雄蛾中,73%于暗期羽化。雌雄蛾羽化行为在暗期第4、5和8 h差异达到显著 (t>4; P<0.05)。交尾活动发生在暗期19：00到5：00之间,交尾持续时间最短约为20 min,最长约为90 min,3日龄进入暗期第5 h具有最高的交尾率。1、6和7日龄成虫具有单个交尾高峰,2到5日龄成虫具有两个交尾高峰。同一日龄成虫交尾在暗期前半段平均花费的时间要明显高于在后半段花费的时间。低龄和高龄的成虫用于交尾的时间明显高于中龄的性成熟成虫。成虫的开始交尾时间随着日龄的增加逐渐前移。雄蛾对进入暗期后第5 h和第9 h处女雌蛾的性腺提取物和空气收集性信息素的触角电位反应最强,对 3日龄处女雌蛾的性腺提取物和空气收集性信息素的触角电位反应最强。处女雌蛾田间诱蛾试验表明：23：00-01：00为诱蛾高峰期,3日龄处女雌蛾的诱蛾效果最好。该蛾的羽化、交尾及性信息素产生与释放均存在节律上的一致性。雌蛾的性信息素释放的时间较长,见于整个暗期,然而交尾行为发生时间较短,主要发生于两个交尾高峰之间。     

Geographic distribution of phylogenetically-distinct legume pod borer, Maruca vitrata (Lepidoptera: Pyraloidea: Crambidae)

Maruca vitrata Fabricius is a pantropical lepidopteran pest of legumes. Phylogenetic analysis of a mitochondrial cytochrome c oxidase-I gene (cox1) fragment indicates that three Maruca sp. mitochondrial lineages have unique geographic distributions [lineages 1 and 2: Australia, Taiwan, and West Africa (Niger, Nigeria, and Burkina Faso), and lineage 3: Puerto Rico]. The haplotype (T30, T114) is specific to lineages 1&2 and was assayed by NsiI and SacI polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) within population samples; it was not observed in the Puerto Rican samples, but was nearly fixed among samples from West Africa, Australia and Taiwan (85.5?C100%). Re-sequencing and phylogenetic analyses of PCR-RFLP defined cox1 haplotypes indicate that nucleotide diversity is highest among samples from West Africa. Phylogenetic reconstruction based upon ribosomal DNA (rDNA) internal transcribed spacer-2 (ITS-2) sequences provided additional evidence for three Maruca sp. clades. These data suggest that multiple unique Maruca species or subspecies are present worldwide, which has implications for the management of this pest species-complex.     

豆野螟成虫日龄对交尾的影响及雄蛾对性信息素的触角电位反应

豆野螟Marucavitrata(Fabricius)是一种严重的泛热带豆类蔬菜害虫。在不同温度,光周期14L∶10D,相对湿度75%～80%条件下,研究豆野螟成虫日龄对交尾的影响及雄蛾对性信息素的EAG反应。不同日龄雌蛾与3日龄雄蛾的交尾,交尾率随蛾龄增加呈下降趋势;不同日龄雄蛾与3日龄雌蛾交尾,交尾率随蛾龄增加呈升高趋势;相同日龄成蛾,交尾率先升后降。不同日龄雌蛾交尾百分率的下降在较高温环境比在较低温环境更加显著;但环境温度对不同日龄雄蛾的交尾却没有太大的影响;相同日龄成蛾3日龄以后的交尾在较高温环境下下降的更快。不同日龄雌蛾的交尾研究中,随着雌蛾日龄的增加,交尾持续时间明显延长;不同日龄雄蛾的交尾研究中,较低日龄和较高日龄雄蛾交尾时的持续时间较长。在较低温环境下所有试验各日龄的持续时间均有所延长。不同日龄雌蛾(雄蛾)与3日龄雄蛾(雌蛾)的交尾和相对应的雄蛾对雌蛾性信息素的EAG反应基本一致,说明雄蛾的反应在交尾行为的完成中具有重要的作用。     

Toxicity of Bacillus thuringiensis insecticidal proteins for Helicoverpa armigera and Helicoverpa punctigera (Lepidoptera: Noctuidae), major pests of cotton

The susceptibilities of the major pests of cotton in Australia, Helicoverpa armigera and Helicoverpa punctigera, to some insecticidal proteins from Bacillus thuringiensis were tested by bioassay. A commercial formulation, DiPel, and individual purified insecticidal proteins were tested. H. armigera was consistently more tolerant to B. thuringiensis insecticidal proteins than was H. punctigera, although both were susceptible to only a limited range of these proteins. Only Cry1Ab, Cry1Ac, Cry2Aa, Cry2Ab, and Vip3A killed H. armigera at dosages that could be considered acceptable. There was no significant difference in the toxicities of Cry1Fa and Cry1Ac for H. punctigera but Cry1Fa had little toxicity for H. armigera. The five instars of H. armigera did not differ significantly in their susceptibility to DiPel on the basis of LC(50). However, there were significant differences in the susceptibility to Cry1Ac and Cry2Aa of three strains of H. armigera. Bioassays conducted with Cry1Ac and Cry2Aa showed that there was a small but significant negative interaction between these delta-endotoxins.     

Activity of Bacillus thuringiensis delta-endotoxins against codling moth (Cydia pomonella L.) larvae

Solubilized protoxins of nine Cry1 and one hybrid Cry1 delta-endotoxin from Bacillus thuringiensis were tested for their activity against larvae of the codling moth (Cydia pomonella L). Cry1Da was the most toxic, followed by Cry1Ab, Cry1Ba, and Cry1Ac, while Cry1Aa, Cry1Fa, Cry1Ia, and SN19 were still less active. Cry1Ca and Cry1Cb showed no activity. In vitro trypsin activation increased activity of all eight active delta-endotoxins, and dramatically enhanced toxicity of hybrid SN19, Cry1Aa, Cry1Ac, and Cry1Fa. The differences between toxicity of proteins before and after trypsin digestion suggests that proteolytic activation in the C. pomonella digestive tract plays a critical role for the activity of Cry proteins against this insect.     

Survival and life table parameters of soybean pod borer Maruca vitrata (Geyer) (Lepidoptera: Crambidae) on leguminous crop cultivars

The soybean pod borer, Maruca vitrata is one of the key insect pests of tropical legumes. It damages tender leaf axils, flower buds, flowers and pods by webbing and boring clusters of flowers and pods. In this study, we investigated the survival and life table parameters of M. vitrata on several leguminous crops; soybean (cvs. Daewon, Poongsannamool and Socheongja), azuki bean (cv. Hongeon), mung bean (cv. Sanpo), and cowpea (cv. Jangchae), compared to artificial diet to assess the antibiosis resistance to M. vitrata. The life‐variables of M. vitrata were significantly affected by the tested legume cultivars. None of the larvae fed cowpea cultivar Jangchae survived. The azuki bean cultivar Hongeon and mung bean cultivar Sanpo were found susceptible to M. vitrata, whereas cowpea cultivar Jangchae and soybean cultivar Daewon showed antibiosis resistance to M. vitrata. Further studies should examine the chemicals associated with leguminous crop cultivars and its mechanism to develop a control method against M. vitrata.     

Annual cycle of the legume pod borer Maruca vitrata Fabricius (Lepidoptera: Crambidae) in southwestern Burkina Faso

Maruca vitrata is an economically significant insect pest of cowpea in sub-Saharan Africa. Understanding the seasonal population patterns of M. vitrata is essential for the establishment of effective pest management strategies. M. vitrata larval populations on cultivated cowpea and adult flying activities were monitored in addition to scouting for host plants and parasitoids during 2 consecutive years in 2010 and 2011 in southwestern Burkina Faso. Our data suggest that M. vitrata populations overlapped on cultivated cowpea and alternate host plants during the rainy season. During the cowpea off-season, M. vitrata maintained a permanent population on the wild host plants Mucuna poggei and Daniella oliveri. The parasitoid fauna include three species, Phanerotoma leucobasis Kri., Braunsia kriegeri End. and Bracon sp. Implications of these finding for pest management strategies are discussed.     

Matrix assisted laser desorption ionisation time of flight mass spectrometry (MALDI-TOF MS) for detecting novel Bt toxins

Matrix assisted laser desorption ionisation time of flight mass spectrometry (MALDI-TOF MS) was used to study crystal (Cry) toxins from different Bacillus thuringiensis (Bt) strains. Known Cry toxins such as Cry1Ac and Cry2A, as well as novel toxins for which the protein sequences were predicted by their gene sequences, were used as controls in this study. The peptide masses, obtained after in-gel trypsin digestion for all these proteins, matched correctly to the corresponding proteins. Also, MALDI-TOF MS was able to resolve and identify multiple Cry toxins of very similar molecular weights and highly similar isoelectric points, from a single protein band. Furthermore, in novel Bt strains for which PCR techniques were unable to detect the cognate genes, this method was able to detect novel Cry toxins. Hence, present data clearly suggest that MALDI-TOF MS could be used as a tool for identifying Cry toxins from novel Bt strains.     

豆野螟的生物防治研究进展与展望

豆野螟Maruca vitrata是豇豆等豆类作物的世界性重要害虫,常年在亚热带地区严重发生,防控难度很大。传统的化学防治方法难以对其进行长久且有效的控制,因而造成害虫抗药性增强、超范围使用化学农药等诸多不良现象,严重威胁人民群众身体健康与生态安全。而生物防治作为减少化学农药用量与残留的关键技术之一,在当前我国绿色农业高速发展期更应引起研究人员的高度重视。因此,本文围绕豆野螟生物防治的研究现状与进展,分别从昆虫性信息素、天敌昆虫资源、昆虫病原物、植物源农药等4个方面进行了概述,并深入探讨了我国豆野螟生物防治领域的研究前景与方向,为豆野螟绿色防控体系的全方位建设提供参考,助力我国绿色农业高质量发展。     

Toxicity of several delta-endotoxins of Bacillus thuringiensis against Helicoverpa armigera (Lepidoptera: Noctuidae) from Spain

Toxicity and larval growth inhibition of 11 insecticidal proteins of Bacillus thuringiensis were evaluated against neonate larvae of Helicoverpa armigera, a major pest of important crops in Spain and other countries, by a whole-diet contamination method. The most active toxins were Cry1Ac4 and Cry2Aa1, with LC50 values of 3.5 and 6.3 microg/ml, respectively. At the concentrations tested, Cry1Ac4, Cry2Aa1, Cry9Ca, Cry1Fa1, Cry1Ab3, Cry2Ab2, Cry1Da, and Cry1Ja1, produced a significant growth inhibition, whereas Cry1Aa3, Cry1Ca2, and Cry1Ea had no effect.     

Evaluation of bio-pesticides against legume pod borer,Maruca vitrata Fabricius (Lepidoptera: Pyralidae), in laboratory and field conditions in Thailand

The legume pod borer (Maruca vitrata) is a major destructive insect found on the yard-long bean, causing serious damage from the flowering stage. Because growers mostly rely on synthetic pesticides to control this insect, it has developed resistance to some pesticides. Information on the efficacy of bio-pesticides against this pest in Thailand is scarce. This study was conducted to evaluate the effects of selected bio-pesticides against M. vitrata. Among six commercially available bio-pesticides, only Bacillus thuringiensis subsp. kurstaki and B. thuringiensis subsp. aizawai were found to be more effective against M. vitrata under laboratory conditions, while neem was least effective. In a field trial undertaken between June and September 2011, both B. thuringiensis formulations significantly reduced pod damage compared with untreated plots of the yard-long bean. A similar efficacy pattern was observed during the second field trial between October 2011 and January 2012. These results suggest that B. thuringiensis formulations could become important components in an integrated pest management strategy for controlling M. vitrata on the yard-long bean in Thailand. However, the results of the current study imply the need for additional field trials with combinations of microbial, botanical, and chemical pesticides rather than a single bio-pesticide per treatment in the management of M. vitrata.     

Occurrence and characterization of a nucleopolyhedrovirus from <Emphasis Type="Italic">Maruca vitrata</Emphasis> (Lepidoptera,Pyralidae) isolated in Taiwan

A nucleopolyhedrovirus (MaviMNPV) was isolated from diseased larvae of legume pod borer (LPB), Maruca vitrata, at Tainan in Taiwan. Electron microscopical studies on the ultrastructure of MaviMNPV occlusion bodies (OBs) showed several virions (up to 19) with multiple nucleocapsids (up to 6) packaged within a single viral envelope. The diameter of OBs was 0.9 to 1.3 μm with a mean of 1.152±0.116 μm. The complete sequence of the MaviMNPV polyhedrin (Polh) gene contained 735 nucleotides (GenBank accession number DQ399596). Phylogenetic analyses using the complete sequence of the Polh gene of MaviMNPV indicated that this virus clusters with Group I NPVs. The genome size of MaviMNPV estimated with restriction enzymes viz., HindIII, EcoRI, BglII and PstI was 113.41 ± 1.50 kbp. First instar LPB larvae were the most susceptible stage (LC₅₀ 2.053 × 10² OBs/ml) followed by second, third and fourth instars with the median lethal concentrations (LC₅₀s) 1.410 × 10³, 2.390 × 10³ and 2.636 × 10³ OBs/ml, respectively. This is the first record of this virus from this region. The first and second authors have equal contributions in this paper     

Effects of Bacillus thuringiensis toxin Cry1Ac and Beauveria bassiana on Asiatic corn borer (Lepidoptera: Crambidae)

In this study, interactions between Cry1Ac, a toxic crystal protein produced by Bacillus thuringiensis (Berliner), and Beauveria bassiana on the mortality and survival of Ostrinia furnacalis was evaluated in the laboratory. The results showed that Cry1Ac is toxic to O. furnacalis. Not only were larval growth and development delayed, but pupation, pupal weight and adult emergency also decreased when larvae were fed on artificial diet containing purified Cry1Ac toxin. When third instars O. furnacalis were exposed to combination of B. bassiana (1.8 × 10⁵, 1.8 × 10⁶ or 1.8 × 10⁷ conidia ml⁻¹) and Cry1Ac, (0.2 or 0.8 μg g⁻¹), the effect on mortality was additive, however, the combinations of sublethal concentrations showed antagonism between Cry1Ac (3.2 or 13 μg g⁻¹) and B. bassiana (1.8 × 10⁵ or 1.8 × 10⁶ conidia ml⁻¹). When neonates were reared on sublethal concentrations of Cry1AC until the third instar, and survivors exposed B. bassiana conidial suspension, such treatments showed additive effect on mortality of O. furnacalis except for the combination of Cry1Ac (0.2 μg g⁻¹) and B. bassiana (1.8 × 10⁶ conidia ml⁻¹) that showed antagonism.