Male reproductive maturity and diapause termination in the leaf beetle Gastrophysa atrocyanea

Male reproductive potential and growth of testes are studied in the leaf beetle Gastrophysa atrocyanea Motschulsky (Coleoptera: Chrysomelidae) that is exposed to mild temperatures after imaginal ecdysis. Continuous administration of the Juvenile Hormone (JH) in artificial diet is highly effective with respect to stimulating testes growth and mating behaviour. Female reproductive diapause can be induced under standard rearing conditions, although optimum reproductive maturity of males is achieved at 15 °C. Treatment of males with a JH analogue accelerates testes development up to the stage of spermiogenesis (the bundling of spermatozoa) but does not promote the formation of cysts with mature sperm. Treatment with 20‐hydroxyecdysone has no influence on diapause termination and testes function. These results suggest that male reproductive maturation in G. atrocyanea is independent of JH, and moderate temperatures may induce an unknown factor that is responsible for the maturation of sperm cysts.     

Egg-hatching synchrony and larval cannibalism in the dock leaf beetle Gastrophysa viridula (Coleoptera: Chrysomelidae)

Females of leaf beetles and many other herbivorous insects lay eggs in coherent batches. Hatchlings emerge more or less simultaneously and often prey on their late-hatching clutchmates. It is not certain, however, whether this synchrony of hatching is a mere by-product of cannibalism or whether an additional synchronizing factor exists. The following simple experiment was aimed at determining the causal relationship between cannibalism and simultaneous larval emergence. Egg clutches of the dock leaf beetle Gastrophysa viridula were split into two halves. These halves were either kept as coherent groups in two separate dishes or, alternatively, only one half remained whole, whereas the other one was divided into single eggs, each of which was incubated in a separate dish. Halving of a clutch into coherent groups only slightly disrupted the synchrony of emergence. The consequence of individual isolation was more dramatic. Half-clutches consisting of disconnected solitary eggs required almost twice as much time for complete emergence of all larvae, which was significantly more than cannibalism as a sole synchronizing factor might explain. Moreover, survival rates were the same in coherent half-clutches (in the presence of cannibalism) and among isolated individuals. This group effect and the small contribution of cannibalism suggest the existence of an additional synchronizing factor. Possible mechanisms underpinning this phenomenon are discussed.     

Double-stranded RNA in exosomes: Potential systemic RNA interference pathway in the Colorado potato beetle,Leptinotarsa decemlineata

Despite extensive research during the past decade elucidating the mechanism of RNA interference (RNAi) in insects, it is not clear how ingested or injected double-stranded RNA (dsRNA) triggers RNAi response in the whole body or even its progeny, which is referred to as systemic RNAi. In the present study, we aim to understand how the dsRNA delivered into cells causes systemic RNAi using Colorado potato beetle cells (Lepd-SL1). We first tested if dsRNA treatment induces systemic RNAi in Lepd-SL1 cells. Exposure of a new batch of Lepd-SL1 cells to the conditioned medium where Lepd-SL1 cells treated with dsRNA targeting inhibitor of apoptosis were grown for 6 h induced apoptosis in these new batch of cells. We hypothesized the exosomes in the conditioned medium are responsible for RNAi-inducing effect. To test this hypothesis, we isolated exosomes from the conditioned medium from Lepd-SL1 cells that had been treated with dsGFP (dsRNA targeting gene coding for green fluorescent protein) or dsLuc (dsRNA targeting gene coding for the luciferase) were grown. RNA present in the purified exosomes was analyzed to check if long dsRNA or siRNA is accumulated in them. The results from the electrophoretic mobility shift assay clearly showed that the long dsRNAs are present in the exosomes. By knockdown of candidate genes involved in endosome recycling and generation pathways, we found that Rab4 and Rab35 are involved in exosome production and transport.     

沉默类胰岛素多肽(Ilp)基因对褐飞虱翅和卵巢发育及海藻糖代谢的影响

[目的]类胰岛素多肽(insulin-like peptide,Ilp)位于胰岛素信号通路最上游,其在糖类物质调控中发挥关键作用.本研究则旨在探究Ilp在褐飞虱Nilaparvata lugens海藻糖代谢平衡的调控作用.[方法]以褐飞虱5龄若虫为实验材料,采用RNAi技术干扰Ilps的表达,观察RNAi后褐飞虱的表型...     

Inhibition of Colorado potato beetle larvae by a locust proteinase inhibitor peptide expressed in potato

The cDNA for a 73-mer peptide containing two locust serine proteinase inhibitors was cloned, fused to the constitutive CaMV35S promoter and introduced into potato by Agrobacterium-mediated transformation. From 23 independent transgenic lines, three with high mRNA level and proteinase inhibitory activity were propagated in vitro and transferred to pots. The peptide from the leaves was identified by its N-terminal sequence and by K_i values against chymotrypsin and trypsin. Colorado potato beetle larvae reared on transgenic plants grew slightly but significantly more slowly than those on control plants. This supports the notion that expression of multifunctional proteinase inhibitors of insect origin might be a good strategy to improve insect resistance in plants.     

Diapause induction and termination in a commonly univoltine leaf beetle (Phratora vulgatissima)

Abstract The leaf beetle Phratora vulgatissima (Linnaeus 1758) is commonly univoltine in south‐central Sweden but may sometimes initiate a partial second generation. The current study was set out to investigate under what abiotic conditions the beetles initiate a second generation. Using climate chamber experiments, the beetles were shown to have a facultative reproductive diapause induced by declining day‐length. The critical day‐length (CDL) for diapause induction was estimated to be 18 h and 10 min. In the field, first‐generation beetles developing to adulthood before August in 2009 became reproductively active and produced a second generation, but most individuals emerged later and were in reproductive diapause. P. vulgatissima overwinter as adults and diapause was shown to be maintained until mid‐winter in 2008/2009. The cumulative temperature requirement for oviposition after diapause termination was estimated to be 222 day‐degrees with a 5.5°C temperature threshold. Three different day‐degree models that were developed to predict the phenology of female oviposition in the spring were validated by comparing model results with field data on the timing of oviposition in previous years. The study suggests that P. vulgatissima may initiate a second generation in Sweden if development of the first generation is completed before August. Warmer spring and summer temperatures due to ongoing climate change may cause advanced insect phenology and faster completion of insect life‐cycles at northern latitudes, which will affect the proportion of insects that initiate a second generation.     

利用RNA干扰抑制具有多种剪接形式的RNA结合蛋白基因的表达

目的:构建具有多种剪接形式的RNA结合蛋白(RBPMS)基因siRNA的真核表达载体,观察其对RBPMS表达的影响。方法:利用RNA干扰(RNAi)技术,设计并合成了2条针对RBPMS基因的siRNA,将其克隆到siRNA表达载体pSliencer2.1-U6neo上。将重组质粒和带FLAG标签的RBPMS共转染293T人胚肾细胞,通过Western印迹检验RNAi效应。结果:测序证明成功构建了RBPMSsiRNA真核表达载体;Western印迹表明构建的siRNA能有效地抑制RBPMS基因的表达。结论:构建了RBPMSsiRNA的真核表达载体,该siRNA能有效地抑制RBPMS基因的表达。     

Factors affecting body size variation within a population of an herbivorous lady beetle,Henosepilachna niponica (Lewis)

Size variation in newly-emerged adults was examined in two different local populations of an herbivorous lady beetle, Henosepilachna niponica, for 1976–80. Mean adult size of both sexes changed rather synchronously in the two populations over 5 years. Body size of adult beetles apparently decreased with increasing leaf damage of the plants on which they developed. Adult beetles which emerged late in the season, associated with increasing food deterioration, were smaller than those which emerged early. Ecological consequences of adult size variation is discussed in terms of oviposition site selection.     

Age- and diapause-related acid and alkaline phosphatase activities in the intestine and malpighian tubules of the Colorado potato beetle, Leptinotarsa decemlineata (Say)

Specific activities for soluble (s) and membrane (m)-bound acid (ACP) and alkaline phosphatases (ALP) were determined in the midgut, hindgut, and Malpighian tubules for developing, prediapausing, and diapausing adult Colorado potato beetles, Leptinotarsa decemlineata (Say). High ACP activities were found in the hindgut and Malpighian tubules while high ALP activities were found in the Malpighian tubules. Variation in both ACP and ALP activities in each tissue reflects fluctuation in protein synthesis and secretion involved with digestion, excretion, and other unknown functions. Phosphatase activities in the tissues examined show the dynamic nature of diapause in this insect. Diapausing beetles showed increases in phosphatase activity after hormone treatments. JHA treatments increased s-ACP and m-ACP activities in all tissues but 20-HE did not increase activity in any tissue. Allatotropin tended to mimic the effects of JHA treatment. The s-ALP activity was also increased in all tissues whereas m-ALP was increased in the midgut and hindgut by JHA treatment. Malpighian tubule m-ALP activity was only increased by 20-HE treatments. Allatotropin was not as effective in increasing ALP activities as it was with ACP activities.     

Quantitative study of HIV-1 Tat peptide and TAR RNA interaction inhibited by poly(allylamine hydrochloride)

The interaction of poly(allylamine hydrochloride) (PAH) with TAR RNA has been studied by quartz crystal microbalance (QCM) cooperating with capillary electrophoresis (CE). Experimental results showed that PAH had high affinity for TAR RNA. In particular, PAH could disrupt the interaction of Tat peptide with TAR RNA, which is critical for HIV-1 virus replication. The approaches described here indicate that they are powerful for studying the binding processes of Tat peptide-TAR RNA and drug-TAR RNA, having great significance for the design of new drug.     

昆虫RNA沉默抗病毒机制研究进展

RNA沉默是昆虫用来抵御病毒入侵的一种普遍而又进化保守的防御机制, 而昆虫病毒也会相应地编码沉默抑制子来破坏宿主的防御功能。本文主要结合果蝇的相关研究成果对昆虫RNA沉默抗病毒机制、 RNA沉默抑制子的作用特征及宿主与病毒的共进化关系做一综述。研究表明, 由小干扰RNA (small interfering RNAs, siRNA)介导的RNA干扰在果蝇抗病毒防御机制中发挥重要作用。果蝇中Dicer-2（Dcr-2）, argonaute-2（AGO2）和双链RNA结合蛋白R2D2是siRNA干扰途径中的3个关键组分, 这3个基因的缺失或突变会显著提高果蝇对RNA病毒的感受性。此外, 果蝇中还鉴定了其他与RNA干扰密切相关的基因, 如vasa intronic gene, aubergine, armitage, rm62 和piwi, 它们在抗病毒感染中同样发挥重要作用。果蝇病毒中已鉴定出3种RNA沉默病毒抑制子（viral suppressors of RNAi, VSRs）, 分别为果蝇FHV病毒沉默抑制子FHV-B2、 果蝇C病毒沉默抑制子DCV-1A及果蝇CrPV病毒沉默抑制子CrPV-1A。FHV-B2和DCV-1A通过与dsRNA或siRNA结合抑制RNA沉默, 而CrPV-1A通过与AGO2结合阻止RISC的形成抑制RNA沉默。在漫长的进化过程中, 病毒和宿主相互博弈, 协同进化。昆虫抗病毒沉默途径中的关键组分通过保持持续和快速进化来对抗高度变异的VSRs。     

RNA干扰抑制乙型肝炎病毒复制及基因表达研究进展

RNA干扰(RNAinterference,RNAi)是指由21～23个核苷酸组成的双链RNA(dsRNA)所引发的生物细胞内同源基因转录后沉默的现象,是生物体在进化过程中普遍存在的一种基因调控机制。目前对由乙型肝炎病毒(HBV)引起的病毒性肝炎尚无令人满意的治疗效果,而RNA干扰技术的出现为各类慢性HBV感染的治疗开辟了新的途径。本文对RNA干扰抑制HBV复制及基因表达的研究现状、存在问题及应用前景进行了综述。     

RNA interference is ineffective as a routine method for gene silencing in chick embryos as monitored by fgf8 silencing

The in vivo accessibility of the chick embryo makes it a favoured model system for experimental developmental biology. Although the range of available techniques now extends to miss-expression of genes through in ovo electroporation, it remains difficult to knock out individual gene expression. Recently, the possibility of silencing gene expression by RNAi in chick embryos has been reported. However, published studies show only discrete quantitative differences in the expression of the endogenous targeted genes and unclear morphological alterations. To elucidate whether the tools currently available are adequate to silence gene expression sufficiently to produce a clear and specific null-like mutant phenotype, we have performed several experiments with different molecules that trigger RNAi: dsRNA, siRNA, and shRNA produced from a plasmid coexpressing green fluorescent protein as an internal marker. Focussing on fgf8 expression in the developing isthmus, we show that no morphological defects are observed, and that fgf8 expression is neither silenced in embryos microinjected with dsRNA nor in embryos microinjected and electroporated with a pool of siRNAs. Moreover, fgf8 expression was not significantly silenced in most isthmic cells transformed with a plasmid producing engineered shRNAs to fgf8. We also show that siRNA molecules do not spread significantly from cell to cell as reported for invertebrates, suggesting the existence of molecular differences between different model systems that may explain the different responses to RNAi. Although our results are basically in agreement with previously reported studies, we suggest, in contrast to them, that with currently available tools and techniques the number of cells in which fgf8 gene expression is decreased, if any, is not sufficient to generate a detectable mutant phenotype, thus making RNAi useless as a routine method for functional gene analysis in chick embryos.     

Independently recruited oxidases from the glucose-methanol-choline oxidoreductase family enabled chemical defences in leaf beetle larvae (subtribe Chrysomelina) to evolve

Larvae of the leaf beetle subtribe Chrysomelina sensu stricto repel their enemies by displaying glandular secretions that contain defensive compounds. These repellents can be produced either de novo (iridoids) or by using plant-derived precursors (e.g. salicylaldehyde). The autonomous production of iridoids, as in Phaedon cochleariae, is the ancestral chrysomeline chemical defence and predates the evolution of salicylaldehyde-based defence. Both biosynthesis strategies include an oxidative step of an alcohol intermediate. In salicylaldehyde-producing species, this step is catalysed by salicyl alcohol oxidases (SAOs) of the glucose-methanol-choline (GMC) oxidoreductase superfamily, but the enzyme oxidizing the iridoid precursor is unknown. Here, we show by in vitro as well as in vivo experiments that P. cochleariae also uses an oxidase from the GMC superfamily for defensive purposes. However, our phylogenetic analysis of chrysomeline GMC oxidoreductases revealed that the oxidase of the iridoid pathway originated from a GMC clade different from that of the SAOs. Thus, the evolution of a host-independent chemical defence followed by a shift to a host-dependent chemical defence in chrysomeline beetles coincided with the utilization of genes from different GMC subfamilies. These findings illustrate the importance of the GMC multi-gene family for adaptive processes in plant–insect interactions.     

RNA interference: the story of gene silencing in plants and humans

RNA interference is an exciting field of functional genomics that can silence viral genes. This property of interfering RNA can be used to combat viral diseases of plants as well as animals and humans. It is a short sequence of nucleic acid that can bind to the mRNA of the gene and interferes the process of its expression. It is diverse in occurrence as well as in applications. It occurs from nematodes to fungi and can cause gene silencing in plants, animals and human beings. Small interfering RNAs are used to silence plant viral genes and in production of therapeutic drugs against Hepatitis or Immuno-deficiency viruses in human. In this review, we will discuss the history, mechanism and applications of RNA interference in plant, animal and human research.     

First report of the invasive Australian tortoise beetle Trachymela sloanei (Coleoptera: Chrysomelidae: Chrysomelinae) in Asia

The Australian tortoise beetle Trachymela sloanei (Coleoptera: Chrysomelidae: Chrysomelinae), an invasive pest of several species of Eucalyptus has been reported for the first time in Asia. It was first detected in Shenzhen, China where it has become established. Currently, T. sloanei is not a destructive forest pest in Shenzhen. Pupae and adults were frequently found infected by an unknown fungus.