Dietary RNAi toxicity assay suggests α and γ subunits of HvCOPI as novel molecular targets for Henosepilachna vigintioctopunctata,an emerging coccinellid pest

Henosepilachna vigintioctopunctata is a highly polyphagous pest of solanaceous plants in Asia and has been predominantly controlled using synthetic insecticides. The intensive use of insecticides has led to the development of resistance, creating an urgent need for new control strategies, for this insect pest. RNA interference (RNAi) has been explored as a pest management strategy in multiple insect pests. In this study, we explored RNAi as a potential alternative for controlling H. vigintioctopunctata. Based on preliminary research, we hypothesized that HvCOPI, a coat protein complex that facilitates retrograde transport, is a promising novel molecular target for H. vigintioctopunctata control. To test this overarching hypothesis, we examined the toxicity of double-stranded RNAs (dsRNAs) towards H. vigintioctopunctata using dietary RNAi toxicity assays targeting eight HvCOPI subunits, including α-, β-, β′-, γ-, δ-, ε-, ζ-, and Arf1-COPI using in vitro synthesized and bacterially expressed dsRNAs. The results demonstrated that ingestion of dsHvCOPIs induced acute feeding cessation in both larvae and adults, which led to significant mortality. In this study, H. vigintioctopunctata showed similar sensitivity to both types of dsHvCOPIs production. Ingestion of bacterially expressed dsHvαCOPI and dsHvγCOPI led to the highest mortality in both larvae and adults. Interestingly, there was a significant positive relationship between the knockdown efficiency of different HvCOPI subunits and the corresponding mortality on day 2 and 4, respectively. These combined results suggest that α and γ subunits of HvCOPI are promising targets for the development of RNAi-based biopesticides for the management of H. vigintioctopunctata.

     

The attractiveness of phagostimulant formulations of a nucleopolyhedrovirus-based insecticide depends on prior insect diet

The multiply-enveloped nucleopolyhedrovirus (SeMNPV) of Spodoptera exigua (Hübner) is being employed on an increasing scale as the basis for bioinsecticidal products for control of this pest in greenhouse crops in Europe. The mortality of diet-reared S. exigua larvae was determined after feeding on lettuce leaf discs contaminated by mixtures of SeMNPV occlusion bodies and 1 of 13 substances reported to have phagostimulant properties. Of the substances tested, wheatgerm and soya flour resulted in significantly increased mortality compared to the virus alone. However, these preferences disappeared when larvae were reared on lettuce. We conclude that (1) the activity of potential phagostimulant substances depends on the prior feeding experience of S. exigua and, (2) laboratory tests using diet-reared insects require validation using plant-reared larvae before being developed for field testing.     

Targeting a coatomer protein complex-I gene via RNA interference results in effective lethality in the pollen beetle Brassicogethes aeneus

The pollen beetle Brassicogethes aeneus is a serious pest of oilseed rape (Brassica napus) in Europe. Management of this pest has grown difficult due to B. aeneus’s development of resistance to pyrethroid insecticides, as well as the pressure to establish control strategies that minimise the impact on nontarget organisms. RNA interference represents a nucleotide sequence-based, and thus potentially species-specific, approach to agricultural pest control. The present study examined the efficacy of targeting the coatomer gene coatomer subunit alpha (αCOP), via both microinjection and dietary exposure to exogenous complementary dsRNA, on αCOP-silencing and subsequent mortality in B. aeneus. Beetles injected with dsRNA targeting αCOP (at 0.14 µg/mg) showed 88% and 100% mortality at 6 and 10 days post-injection, respectively; where by the same time after dietary exposure, 43%–89% mortality was observed in the 3 µg dsRNA/µL treatment, though the effect was concentration-dependent. Thus, the effect was significant for both delivery routes. In working towards RNA-based management of B. aeneus, future studies should include αCOP as a target of interest.

     

Flight patterns of the peach twig borer,Anarsia lineatella zell. (Lep., Gelechiidae) in central europe as observed using pheromone traps

Pheromone traps were used for monitoring phenology of the peach twig borer,Anarsia lineatella, in Central Bohemia. The pest has two generations a year, the first one (owerwintering) is usually more numerous than the second one. The peak of the flight of the first generation begins by day-degrees (DD) 400–450 °C and the peak of the flight of the second generation at DD 900–960 °C. DD data can be used for supervised control both in the case of timing the chemical treatment and for deciding on using pheromones for male confusion. The suitable term for chemical treatment in the given locality in Central Bohemia is DD 360°C (above the developmental threshold 10 °C). The injury to fruits and shoots of peach trees similar to that caused byA. lineatella does also the oriental fruit moth,Cydia molesta. According to very low catches in pheromone trapsC. molesta does not seem to be economically important in Central Bohemia at present.With 2 tables and 3 figures     

A trial to detect optimal pin-pricking timing in evaluating the ability to form traumatic resin canals of <Emphasis Type="Italic">Cryptomeria japonica</Emphasis> for selecting resistant trees to <Emphasis Type="Italic">Semanotus japonicus</Emphasis> (Coleoptera: Cerambycidae)

A trial to detect optimal pin-pricking timing in evaluating the ability to form traumatic resin canals (TRCs) of Cryptomeria japonica was examined to select resistant trees to Semanotus japonicus using 14 clones in 2001 and 2002. Resistance to S. japonicus and the ability to form TRCs in the phloem was evaluated by inoculating newly hatched larvae in the bark and by a pin prick, which was conducted every 10 days (four times) on the trunk around the larval phloem-feeding period, respectively. The larval survivorship varied greatly among clones for both years. The mean appearance of newly formed TRCs was generally higher in late treatment for both years, and the tangential width of them was also higher in 2001, whereas those of pre-formed TRCs were not higher for either year. The larval survivorship did not show significant correlations with the appearance and the width of pre-formed TRCs on all treatments. However, it showed significant negative correlations with the appearance of newly formed TRCs on the second and third treatments on the 2-year-old layer, although this was not significant with the width of them. This suggests that resistant clones might have the mechanism of rapidly forming TRCs when just at the stage of newly hatched larval entering the phloem. Thus, although the relationship between the appearance and the width of newly formed TRCs is not clear, pin-pricking treatment when the newly hatched larvae just enter the bark may be one of optimal times for the evaluation of the resistance of C. japonica to S. japonicus. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Behavioural responses of Frankliniella occidentalis Pergande larvae to methyl jasmonate and cis-jasmone

The larval stages of Frankliniella occidentalis Pergande (Thysanoptera: Thripidae) cause more direct feeding damage to plants than the adults. We, therefore, investigated the behaviour-modifying effects on second instar larvae of two jasmonic acid derivatives. The artificial application of methyl jasmonate and cis-jasmone, both at 1 % concentration, deterred the larvae from settling in a dual choice bean leaf disc assay. We observed a dose-dependent feeding deterrence of both jasmonates and calculated the concentration required to reduce the feeding damage by 50 % relative to the control treatment (FDC₅₀) for each jasmonate. The feeding damage was reduced by the application of cis-jasmone at 1 % concentration, but not by the jasmonates at the respective FDC₅₀ in no-choice leaf disc bioassays. However, significantly more larvae left jasmonate-treated whole potted bean plants by migrating to the soil compared with control plants. Our results may be exploited extending behavioural manipulation by using plant compounds in thrips control programmes to the full lifecycle of the pest. Plant compounds could be used in integrated and biological pest management strategies against F. occidentalis in combination with the application of various above and below ground control measures.     

Damage characteristics of three boring pests in Artemisia ordosica

The population density and distribution of the three major boring pests, Holcocerus artemisiae, Sphenoptera sp. and Adosopius sp. were studied in their host plant Artemisia ordosica. Results show that the larvae of H. artemisiae mainly bore the rhizome of A. ordosica, but also the larvae of Sphenoptera sp. and Adosopius sp. bore the rhizome of A. tordosica. The adults of Sphenoptera sp. and Adosopius sp. feed on the leaves of A. ordosica as a nutritional supplement. The distribution of the larvae of three pests in their host plant is completely different. H. artemisiae and Sphenoptera sp. are distributed in the whole A. ordosica plant. The newly hatched larvae first feed on the phloem and xylem of stem and then burrow to the roots before the winter of the same year. However, Adosopius sp. is distributed mainly in the roots. The newly hatched larvae move from the phloem to the xylem, and gradually damage the lower part of the trunk. The larvae of the three pests sometimes coexist in a single A. ordosica plant. However, the probability of the coexistence of the three pests is very small. The highest probability of coexistence of two pests was observed in Sphenoptera sp. and H. artemisiae, while the probability of coexistence of Sphenoptera sp. and Adosopius sp. as well as H. artemisiae and Adosopius sp. was smaller. The reasons for coexistence or its avoidance are not only related to the amount of food provided nor the volume of larval living space in a single A. ordosica plant, but also related to the development characteristics of different kinds of larvae and interspecific competition.     

白蜡虫ws基因RNAi载体构建及原核表达dsRNA

[目的]构建白蜡虫(Ericerus pela)蜡酯合酶(wax synthase,WS)基因干扰载体并建立其体外dsRNA(doublestranded RNA,dsRNA)原核表达体系,低成本大量制备白蜡虫ws基因的dsRNA。[方法]克隆白蜡虫蜡酯合酶基因ws片段,连入L4440载体,将重组质粒转入大肠杆菌HT115感受态细胞,经IPTG诱导获得与目的片段相对应的dsRNA。[结果]白蜡虫ws基因RNA干扰(RNA interference,RNAi)载体成功构建,重组质粒转入HT115感受态细胞经IPTG诱导后菌体成功表达dsRNA,dsRNA的平均获得量1 705 ng·m L~(-1)。[结论]该研究通过原核表达白蜡虫ws基因的dsRNA,为后续利用RNAi实验研究白蜡虫ws基因功能及作用机理奠定基础。     

The effect of grape vine cultivars on <Emphasis Type="Italic">Lobesia botrana</Emphasis> (Lepidoptera: Tortricidae) population levels

The European vine moth Lobesia botrana Denis & Schiff. (Lepidoptera: Tortricidae) is a key pest in the vineyards of Israel and Europe. Traps baited with the female sex pheromone are commonly used to monitor the pest population. However, the role of the vine cultivars on monitoring the moth population using pheromone-baited traps was not yet studied. The present study aimed to identify the effect of grape cultivars on L. botrana adult distribution patterns in the field, in order to achieve a better understanding of the monitored data in respect to male and female’s host preference. The 3-year study in commercial vineyards used four cultivars: Carignan, Emerald Riesling, French Colombard, and Cabernet Sauvignon. The moth population of each generation was estimated from male counts in pheromone traps; three generations were observed in all experimental orchards, each year. Female host choice was studied by monitoring freshly deposited eggs and newly hatched larvae on vines. The study showed a significant effect of cultivar on numbers of trapped males in most years, and a cultivar effect on female host choice. For both males and females and for all phenological stages of the grapes, Carignan and French Colombard attracted the most and Cabernet Sauvignon the fewest specimens. The results show that cultivar blend within the vineyard can affect the distribution pattern of the pest. Knowledge of the expected choice of the female moth, and of the timing of its decision could lead to an improved monitoring system, with the preferred cultivar as an indicator.     

油松毛虫产卵、取食偏嗜行为及油松针叶内含物测定

[目的]为明确油松毛虫在油松上的产卵和取食部位以及可能机理,为未来害虫发生预测模拟提供基础信息数据。[方法]通过室内模拟试验,统计油松毛虫在盆栽油松幼苗不同部位上的产卵量和取食不同油松针叶的偏嗜情况及死亡率;测定油松针叶内含物含量并与油松毛虫偏嗜行为进行了相关性分析。[结果]在一年生油松针叶上平均产卵块数最多(3.10±0.28),显著高于二年生针叶和三年生针叶;取食一年生油松针叶的油松毛虫数量最多(13.40±0.91)、死亡率最低(13.60%±4.87%),显著优于取食二年生油松针叶和三年生油松针叶的幼虫。油松毛虫幼虫取食选择、成虫产卵选择和幼虫体长与油松针叶内含水量成显著正相关(P0.01);与寄主总酚含量(P0.01)和碳氮比(P0.05)成显著负相关。[结论]油松毛虫对一年生油松针叶具有明显的产卵和取食偏嗜行为。一年生油松针叶内含水量最高、总酚含量最低、有机碳含量最低可能是造成寄主偏嗜行为的原因。     

补充营养对白蜡吉丁肿腿蜂寄生效率和发育进程的影响

[目的]为了探明补充营养是否对白蜡吉丁肿腿蜂雌蜂的寄生效率、生殖能力和寿命有显著影响。[方法]本文通过在白蜡吉丁肿腿蜂搜寻到寄主前期为其提供寄主血淋巴和蜂蜜补充营养,调查有前期补充营养经历的肿腿蜂对寄主麻天牛的寄生率、母蜂生殖能力和子代蜂发育进程的变化。同时,给予刚羽化的肿腿蜂雌蜂短期的补充营养的过程,调查其寿命长短的变化情况。[结果]研究结果表明,寄主血淋巴和糖类物质均可以作为供肿腿蜂补充营养的潜在食物源。寄生前期取食寄主血淋巴和蜂蜜的肿腿蜂寄生率显著高于前期仅补充蒸馏水的个体。补充营养后可促使寄生蜂卵的成熟,可以使肿腿蜂更快的开始产卵进程。前期取食寄主血淋巴的肿腿蜂生殖能力显著高于寄生前期仅补充蒸馏水的个体,取食蜂蜜的肿腿蜂产卵量较前2组均无显著差异。寄生蜂母蜂是否补充营养对其子代的发育过程和性比无显著影响。此外,生存分析结果表明,刚羽化的肿腿蜂补充营养后,其寿命显著延长。取食寄主血淋巴或蜂蜜的寄生蜂寿命均约为羽化后无营养补充经历雌蜂个体的2倍。[结论]以上结果表明,白蜡吉丁肿腿蜂在寄生过程开始前期的补充营养过程可以显著的增强其对寄主害虫的控制作用和延长其寿命。取食蜂蜜或寄主血淋巴对肿腿蜂生殖和寿命的增强作用相近。建议在害虫的生物防治中,可以通过在天敌释放区周边配植一定的蜜源植物以此实现对天敌昆虫资源更有效的利用和保护。     

Continuous nondestructive monitoring of larval feeding activity and development of the bamboo powderpost beetle <Emphasis Type="Italic">Dinoderus minutus</Emphasis> using acoustic emission

The life history and feeding biology of the bamboo powderpost beetle Dinoderus minutus remain poorly understood because the beetles’ oviposition, development, and feeding take place inside bamboo culms. In this study, acoustic emission (AE) monitoring was applied for continuous nondestructive analysis of larval feeding activity and development from the first instar to adult eclosion. Newly hatched larvae were inoculated individually into pieces of madake (Phyllostachys bambusoides) culms. AE hits were recorded using single AE sensors fixed onto the bamboo pieces. Generation of AE hits indicated that larval feeding activity had begun after inoculation. Based on the time course of the hourly AE hit rate, the larvae were feeding constantly during each instar, and feeding activity only ceased during periods of ecdysis and pupation. Half of the individuals examined underwent seven instars and the other half underwent eight instars. The time course of AE hit rate per 5 min exhibited periodic cycles, where continuous meals were separated by inactive phases of ca. 5 min, with an average dominant period of each instar ranging between 0.76 and 2.19 h. After correcting the AE data based on distance attenuation of AE waves, the tendency that AE amplitude increased as the larvae developed through ecdysis events became more apparent. AE monitoring continued after adult eclosion, and the feeding activity of the newly emerged adults continued almost ceaselessly during the Reifungsfrass period.     

Antifeedant activity and toxicity of leaf extracts from <Emphasis Type="Italic">Porteresia coarctata</Emphasis> Takeoka and their effects on the physiology of <Emphasis Type="Italic">Spodoptera litura</Emphasis> (F.)

Antifeeding and toxic effects of chemical extracts from Porteresia coarctata Takeoka on the insect pest Spodoptera litura (F.) were investigated in the laboratory. Hexane extracts of P. coarctata leaves were re-dissolved in dimethyl sulfoxide and applied in water to leaves of Ricinus communis L. (castor). A concentration of 1,000 and 2,000 ppm extract exhibited significant antifeeding and toxic effects to third instar larvae when compared to the same concentration of dimethyl sulfoxide in water as control. One day after feeding on extract-treated castor leaves, the mortality of S. litura larvae was 50%. Mortality increased to 95% after 11 days at a dosage of 2,000 ppm. Compared to the control, third instar larvae fed on castor leaves treated with P. coarctata leaf extract at different concentrations showed significant reduction in protein and DNA content in the fat body and midgut tissues. Results demonstrate the insecticidal and antifeedant properties of P. coarctata leaf extract against the generalist pest S. litura leading to biochemical changes in the insect body. Thus, P. coarctata contains phytochemicals, and the extract, in crude and pure form, has potential as a botanical insecticide in alternative control strategies against Lepidoptera pests.     

Insect-resistant mechanism of transgenic triploid of Chinese white poplar

The activities of antidotal enzymes and digestive enzymes of Clostera anachoreta (Fabricius) instar larvae, feeding on leaves of three kinds of insect-resistant clones of transgenic triploid of Chinese white poplar, after 4, 12, 24, 48, 72 and 96 h, were investigated. The results showed that, feeding on clone 7, the activity of esterase, carboxylesterase, and mixed-function oxidases in the midgut of the larvae was very much decreased. Feeding on clone 10, those results were less than those of clone 7 and there were few changes on the larvae, which fed on clone 26. The changes of the amylase in the midgut of larvae were the same as those described above. However, the activities of glutathione S-transferase and proteinase were complex, increased markedly after 24 h feeding on clone 7, and then declined rapidly. The same changes were taking place on the larvae feeding on clone 10. There were many slight changes in glutathione S-transferase of the larvae, feeding on clone 26; no changes occurred in the proteinases of the midgut. Thus, the antidotal enzymes and digestive enzymes in the midgut of the larvae were inhibited. This may be the main mechanism of the transgenic triploid of Chinese white poplar. __________ Translated from Journal of Agricultural University of Hebei, 2005, 28(5) [译自: 河北农业大学学报, 2005, 28(5)]     

松材线虫Bxpel2基因的克隆及RNA干扰载体构建

[目的]为了构建松材线虫(Bursaphelenchus xylophilus)果胶酶Bxpel2基因干扰载体.[方法]通过Trizol 法提取松材线虫总RNA,反转录合成cDNA,设计带T₇启动子的果胶酶Bxpel2基因引物,以cDNA为模板扩增出果胶酶Bxpel2基因片段,连接到RNA干扰载体,再以干扰载体为模板,PCR扩增出目的片段后进行测序鉴定,合成果胶酶Bxpel2基因双链RNA(dsRNA),采用RT-PCR检测松材线虫Bxpel2基因干扰后的表达情况.[结果]表明:1)提取的松材线虫总RNA完整性好,无降解;2)成功克隆出松材线虫果胶酶Bxpel2基因片段(790 bp)并将其连接至pMD19-T载体;3)以RNA干扰载体为模板合成dsRNA,浓度分别为1.313 mg·mL^-1和1.152 mg·mL^-1;4)RT-PCR结果显示,松材线虫经过dsRNA干扰后,Bxpel2基因表达基本受到抑制.[结论]成功构建松材线虫果胶酶Bxpel2基因干扰载体,为进一步研究Bxpel2基因在松材线虫致病过程中的作用和功能奠定了良好的基础.     

Control of a new turf pest, <Emphasis Type="Italic">Dorcadion pseudopreissi</Emphasis> (Coleoptera: Cerambycidae), with the entomopathogenic nematode <Emphasis Type="Italic">Heterorhabditis bacteriophora</Emphasis>

The use of biological control in turf has increased to avoid possible negative effects on humans. Entomopathogenic nematodes (EPNs) belonging to the families Steinernematidae and Heterorhabditidae have control potential against many economically important insect pests. In the present study, the efficacy of Heterorhabditis bacteriophora against a new pest on turf, Dorcadion pseudopreissi, was examined in the field. Cages (1 × 1 × 1 m) with female and male D. pseudopreissi were placed on two kinds of turf: Lolium perenne and Festuca arundinacea. After beetles had deposited eggs into the moist soil surface, the cages were removed and H. bacteriophora was applied at 0.5 million infective juveniles/m² to half the plots. Application of H. bacteriophora caused a statistically significant reduction in numbers of beetle larvae in L. perenne plots and a non-significant reduction in F. arundinacea plots. The area damaged by D. pseudopreissi was significantly reduced by nematode application in both turf species. The number of nematodes declined after application, but small numbers could still be detected after 6 months.