Use of a thin film biosensor for rapid visual detection of PCR products in a multiplex format

Rapid, sensitive assays for nucleic acid amplification products have utility for the identification of bacterial or viral infections. We have developed a nucleic acid hybridization assay utilizing thin film technology that permits visual detection of hybrids. The silicon-based biosensor detects the presence of target sequences by enzymatically transducing the formation of nucleic acid hybrids into molecular thin films. These films alter the interference pattern of light on the biosensor surface, producing a perceived color change. We have applied this technology to the development of a chip containing capture probes specific for human respiratory virus sequences including respiratory syncytial virus, influenza virus A and B, parainfluenza virus types 1 and 3, and rhinovirus. In a ten-minute assay, the biosensor permits unambiguous identification of viral-specific RT/PCR products from infected cell lysates.     

西花蓟马检测鉴定技术研究进展

西花蓟马是一种世界性的入侵害虫,2003年首次在北京发现,目前仅在我国局部地区发生。其危害严重,潜在适生区广,且具有进一步扩散蔓延的趋势。快速准确的检测鉴定技术是防止其进一步扩散蔓延,保障我国农业生产健康发展的必要前提。综合相关报道,用于西花蓟马的检测鉴定技术包括传统的形态学特征鉴定法、由此衍生的计算机软件识别法,以及分子检测技术,如RAPD-PCR法、DNA序列分析法、PCR-RFLP法、SCAR分子标记技术、实时荧光定量PCR检测技术以及蛋白质分析技术等。其中,基因芯片和DNA条形编码技术作为新兴的物种鉴定手段,在西花蓟马等入侵物种的检测鉴定中具有广阔的应用前景。     

Potential for controlling codling moth Cydia pomonella (Linnaeus) (Lepidoptera: Tortricidae) in Argentina using the sterile insect technique and egg parasitoids

Codling moth is the main pest affecting apples and pears worldwide. Most pest control strategies used against this insect have relied on the use of broad‐spectrum insecticides which have led to non‐desirable effects like pesticide resistance, residues in the environment, human health concerns and the reduction of access to international markets. Therefore, alternative pest control strategies that would result in sustainable fruit production systems while taking care of the environment are strongly promoted. The use of the sterile insect technique has proven to be a valuable pest control tactic within area‐wide integrated pest management strategies, and its synergistic effect for Lepidoptera pests when combined with other biological control tactics such as parasitoids has been documented. The purposes of this research were to evaluate the response of an Argentinean codling moth strain to a sub‐sterilizing radiation dose of 100 Gy and to assess the acceptability and suitability of sterile codling moth eggs by the egg parasitoids, Trichogramma cacoeciae (Marchal) and Trichogramma nerudai (Pintureau and Gerding). Irradiated female moths survived better than irradiated male moths and non‐irradiated male and female moths. Also, the fecundity of irradiated female moths was reduced by more than 30% as compared to non‐irradiated ones whereas their fertility was close to zero. The F₁ generation was male biased with a lower fertility (inherited sterility) than the parental generation. Trichogramma cacoeciae and T. nerudai parasitized both fertile and sterile eggs. However, there was a significant reduction in acceptability for sterile eggs. Trichogramma nerudai parasitized more eggs than T. cacoeciae, but egg acceptability for this species was proportionally lower than for T. cacoeciae especially on eggs oviposited by irradiated females. Development to adult of both parasitoids species was not substantially affected by the origin of the eggs and the wasps had acceptable levels of adult emergence, survival and fecundity. These results provided useful information on the potential for controlling the codling moth using egg parasitoids and the sterile insect technique in Argentina.     

DNA Barcoding as useful tool to identify crop pest flea beetles of Turkey

Flea beetles (Chrysomelidae: Galerucinae: Alticini), with ~8,000 species worldwide, include pest species causing substantial economic damage to crops. The genera Phyllotreta and Chaetocnema include both pest and non‐pest species. An accurate and fast taxonomic identification approach is required for discriminating among taxa for non‐expert taxonomists; moreover, the utility of this approach spans from biodiversity conservation to the monitoring of pest species. DNA barcoding represents a reliable and easy identification tool based on the use of short DNA sequences. In this study, 45 new COI sequences of 13 Phyllotreta and five Chaetocnema species, representing ~30% and ~20% of the Turkish species belonging to these genera, were provided. These sequences increased by ~18% and ~25% the number of species of these genera whose sequences are available in BOLD. In order to test DNA barcoding efficiency in Phyllotreta and Chaetocnema species identification, we created a data set consisting of sequences belonging to species present in the Middle East and available in BOLD plus the sequences developed in this study (36 species). The efficiency of species identification, estimated using best close match analysis (with the ad hoc calculated optimal distance threshold of 1.5%), was 99%. The overall intraspecific and interspecific mean nucleotide divergences were 1.4% and 20%, respectively. Interestingly, COI sequences of Phyllotreta nigripes clustered into two well‐separated groups with a high value of the between‐group nucleotide distance (11.4%), which suggests the presence of cryptic species. In addition, information was provided on the crops exploited by the collected organisms and the observed damage.     

A simple and reliable assay for detecting specific nucleotide sequences in plants using optical thin-film biosensor chips

Here we report the adaptation and optimization of an efficient, accurate and inexpensive assay that employs custom-designed silicon-based optical thin-film biosensor chips to detect unique transgenes in genetically modified (GM) crops and SNP markers in model plant genomes. Briefly, aldehyde-attached sequence-specific single-stranded oligonucleotide probes are arrayed and covalently attached to a hydrazine-derivatized biosensor chip surface. Unique DNA sequences (or genes) are detected by hybridizing biotinylated PCR amplicons of the DNA sequences to probes on the chip surface. In the SNP assay, target sequences (PCR amplicons) are hybridized in the presence of a mixture of biotinylated detector probes and a thermostable DNA ligase. Only perfect matches between the probe and target sequences, but not those with even a single nucleotide mismatch, can be covalently fixed on the chip surface. In both cases, the presence of specific target sequences is signified by a color change on the chip surface (gold to blue/purple) after brief incubation with an anti-biotin IgG horseradish peroxidase (HRP) to generate a precipitable product from an HRP substrate. Highly sensitive and accurate identification of PCR targets can be completed within 30 min. This assay is extremely robust, exhibits high sensitivity and specificity, and is flexible from low to high throughput and very economical. This technology can be customized for any nucleotide sequence-based identification assay and widely applied in crop breeding, trait mapping, and other work requiring positive detection of specific nucleotide sequences.     

DNA条形码技术在北京百花山地区夜蛾科物种鉴定中的应用

为了探讨DNA条形码技术在夜蛾物种鉴定中的可行性, 本研究利用条形码通用引物扩增了北京百花山地区43种夜蛾75个样本的线粒体细胞色素C氧化酶亚基I （mitochondrial cytochrome c oxidase subunit I, COI）基因序列, 以Kimura双参数模型进行种内种间遗传距离分析、 使用邻接法（neighbor-joining, NJ）和最大简约法（maximum parsimony, MP）分别构建系统发育树, 并利用分子序列差异阈值对样本进行分子可操作分类单元（molecular defined operational taxonomic units, MOTU）划分。结果表明： 所有夜蛾种类通过系统发育树可以成功区分; 种内平均遗传距离(0.03%)远远小于种间平均遗传距离(11.29%); 采用较为保守的1%的序列差异阈值将75个夜蛾样本分为42个MOTU, 正确率为95%, 除了MOTU04包含2个物种外, 剩余41个MOTU与形态种呈现一一对应的关系。结果显示, 基于COI基因的DNA条形码对于本研究中所涉及的夜蛾具有较好的区分, 可以作为一种有效的工具在夜蛾科昆虫物种鉴定中进行应用。     

Enhanced detection of staphylococcal genomes in positive blood cultures using a polymeric enzyme complex

This article describes a simple and inexpensive signal amplification method, termed polymeric enzyme detection (PED), which permits rapid and sensitive detection of conserved sequences in the tuf gene that identify Staphylococcus genus, conserved sequences in the femB gene that specifically detect Staphylococcus aureus species, and the methicillin resistance gene mecA directly from positive blood culture bottles. Microbe-specific capture probes were immobilized onto microtiter plates or silicon chips. Target sequences and biotin-labeled, target-specific probes were hybridized to complementary capture probes to create a biotin-labeled, surface-immobilized tripartite complex. In a two-step process, signal was amplified by incubating the surface-immobilized biotin with streptavidin followed by the addition of a 500-kDa dextran polymer conjugated with approximately 80 biotins. Signal was then developed by binding of a streptavidin-horseradish peroxidase conjugate followed by incubation with the substrate tetramethylbenzidine. Use of the PED method improved the lower limit of detection 10- to 100-fold in model DNA hybridization assays with limits of detection as low as 1 fmol/L target DNA. This level of sensitivity permits detection of genomic DNA from methicillin-resistant S. aureus positive blood cultures within 25 to 35 min using either a thin film biosensor chip or a microtiter plate-based assay.     

Nutrition,sex and season contribute to variation in fat and glycerol levels in the long‐lived moth Caloptilia fraxinella

The ash leaf cone roller Caloptilia fraxinella Ely (Lepidoptera: Gracillariidae) is an invasive leaf‐mining moth pest of horticultural ash Fraxinus spp. in the Canadian Prairie Provinces. Caloptilia fraxinella overwinter as adults in reproductive diapause and mating occurs after overwintering in the spring. The effect of a carbohydrate food source on fat and glycerol reserves throughout the long adult life stage of this moth is investigated. Insects collected as pupae are given access to either water or sugar water upon adult eclosion. Moths held under the different feeding regimes are sampled before (summer and autumn) and after overwintering in the spring. Analysis of either glycerol or lipid content is conducted for male and female moths from each collection period. Both moth weight and glycerol concentration are affected by moth sex, food regime and season of collection. Although female moths are heavier than males, a higher glycerol concentration occurs in males. Moths fed sugar are heavier and have a higher glycerol concentration than water‐fed moths late in reproductive diapause and after overwintering. Moths collected in the spring after overwintering are lighter and have a lower glycerol content than moths collected before winter. There is a significant influence of feeding regime and season on moth body lipid content, with sugar‐fed moths having more fat than water‐fed moths; however, this difference is smaller in the summer than the autumn or spring. An initial understanding of the overwintering biology and diapause of this pest is provided in the present study.     

Effect of rearing strategy and gamma radiation on fecundity and fertility of codling moth Cydia pomonella (Linnaeus) (Lepidoptera: Tortricidae)

The codling moth Cydia pomonella (Linnaeus) (Lepidoptera: Tortricidae) is a serious pest of pome fruit worldwide. In an effort to reduce the use of pesticides to control this pest, the sterile insect technique (SIT) is used or considered for use as a component of area‐wide integrated pest management programmes. Rearing codling moths through diapause has been shown to improve the competitiveness of sterile moths released in orchards, and provides management alternatives that would allow mass‐rearing facilities to increase their yearly production of sterile moths. Because radiosensitivity in insects can be influenced by numerous biological factors, laboratory tests were conducted to examine whether the response to increasing doses of radiation, as expressed in the fecundity and fertility of cohorts of moths, is similar for adult moths mass‐reared through diapause or through standard (non‐diapause) production protocols. Our data revealed that the effect of increasing doses of radiation on fecundity and fertility of codling moths reared through both rearing strategies was similar. In the case of fertility, this is a particularly important finding for the expanded application of codling moth SIT. If mass‐rearing facilities use year‐round diapause rearing, the dose required to treat the insects prior to release will be similar to that used when codling moths are reared through standard production protocols.     

Construction,validation, and application of nocturnal pollen transport networks in an agro‐ecosystem: a comparison using light microscopy and DNA metabarcoding

1. Moths are globally relevant as pollinators but nocturnal pollination remains poorly understood. Plant–pollinator interaction networks are traditionally constructed using either flower‐visitor observations or pollen‐transport detection using microscopy. Recent studies have shown the potential of DNA metabarcoding for detecting and identifying pollen‐transport interactions. However, no study has directly compared the realised observations of pollen‐transport networks between DNA metabarcoding and conventional light microscopy. 2. Using matched samples of nocturnal moths, we constructed pollen‐transport networks using two methods: light microscopy and DNA metabarcoding. Focussing on the feeding mouthparts of moths, we developed and provide reproducible methods for merging DNA metabarcoding and ecological network analysis to better understand species interactions. 3. DNA metabarcoding detected pollen on more individual moths, and detected multiple pollen types on more individuals than microscopy, although the average number of pollen types per individual was unchanged. However, after aggregating individuals of each species, metabarcoding detected more interactions per moth species. Pollen‐transport network metrics differed between methods because of variation in the ability of each to detect multiple pollen types per moth and to separate morphologically similar or related pollen. We detected unexpected but plausible moth–plant interactions with metabarcoding, revealing new detail about nocturnal pollination systems. 4. The nocturnal pollination networks observed using metabarcoding and microscopy were similar yet distinct, with implications for network ecologists. Comparisons between networks constructed using metabarcoding and traditional methods should therefore be treated with caution. Nevertheless, the potential applications of metabarcoding for studying plant–pollinator interaction networks are encouraging, especially when investigating understudied pollinators such as moths.     

Application of DNA barcoding to the identification of Hymenoptera parasitoids from the soybean aphid (Aphis glycines) in China

Aphis glycines Matsumura is an important pest of soybean in Asia and North America. Hymenoptera parasitoids play a key role in the control of the soybean aphid. The correct identification of parasitoids is a critical step that precedes the assessment of their potential biological control agents. Accurate identification of the majority of the species attacking the soybean aphid often requires elaborate specimen preparation and expert taxonomic knowledge. In this study, we facilitated the identification of soybean aphid parasitoids by applying a DNA barcoding approach following a preliminary morphological identification. We generated DNA sequence data from the mitochondrial COI gene and the D2 region of 28S rDNA to assess the genetic variation within and between parasitoid species emerging from the soybean aphid in China. Fifteen Hymenoptera parasitoid species belonging to 10 genera of five families were identified with little intra‐specific variation (0.09% ± 0.06% for 28S and 0.36% ± 0.18% for COI) and large inter‐specific divergence (30.46% ± 3.42% for 28S and 20.4% ± 1.20% for COI).     

Pheromone‐trapping the nun moth,Lymantria monacha (Lepidoptera: Lymantriidae) in Inner Mongolia,China

The nun moth, Lymantria monacha L., is one of the most important defoliators of Eurasian coniferous forests. Outbreaks during 2011–2015 in the natural/planted larch, and larch‐birch mixed forests of the Greater Khingan Range in Inner Mongolia, China, caused tremendous timber losses from severe defoliation and tree mortality. A series of trapping experiments were conducted in these outbreak areas to evaluate the efficacy of a synthetic species‐specific pheromone lure based on the female pheromone blend of European nun moth populations. Our results clearly show that the nun moth in Inner Mongolia is highly and specifically attracted to this synthetic pheromone, with few gypsy moths (Lymantria dispar) captured. Flight activity monitoring of L. monacha male moths using pheromone‐baited Unitraps at 2 locations during the summer of 2015 indicated that the flight period started in mid‐July, peaking in early August at both locations. Based on male moth captures, there was a strong diurnal rhythm of flight activity throughout the entire scotophase, peaking between 22:00 and 24:00. Unitraps and wing traps had significantly and surprisingly higher catches than the gypsy moth traps. Unitraps fastened to tree trunks 2 m above ground caught significantly more male moths than those at the ground level or at 5 m height. Male L. monacha moths can be attracted to pheromone‐baited traps in open areas 150–200 m distant from the infested forest edge. Our data should allow improvement on the performance of pheromone‐baited traps for monitoring or mass‐trapping to combat outbreaks of this pest in northeastern China.     

Large moths captures by a pest monitoring system depend on farmland heterogeneity

As intensive farmlands cover increasing areas of the world, associated biotic richness is crucial for the biodiversity of entire regions. Using data on non‐target Macrolepidopteran moths captured by a crop pest monitoring system, we compared local (100 m perimeter)‐ and landscape‐scale (1000 m perimeter) predictors of the numbers of moth individuals and moth species richness. During a single year (2009), eighteen light traps captured 91 726 individuals of 564 moths species. Typically for biotically impoverished habitats, the catches were dominated by a few superabundant species. Even in these impoverished assemblages, numbers of species increased with increasing herb and woody plants diversity (100 m around the traps), crop diversity (1000 m perimeter), landscape composition (1000 m) and configuration (100 and 1000 m). Abundance of the catches increased only with woody plants diversity in 100 m perimeters. In separate analyses of two species‐rich families, the presumably less mobile Geometridae increased with landscape configuration (i.e. density of edges) within 100 m perimeters around the traps, whereas the species richness of more mobile Noctuidae also reflected the landscape composition (i.e. proportional representation of land covers) at 100 m perimeters. Proportional representation of pest species decreased with increasing richness of herb and woody plants. Taken together, farmland heterogeneity increases moth species richness, whereas abundance of the catches mainly depends on local factors in the vicinity of light traps, and the local factors are more important for presumably less mobile Geometridae than for more mobile Noctuidae.     

DNA芯片制作原理及其杂交信号检测方法

文章讨论了DNA芯片的制作原理和杂交信号的检测方法。依其结构,DNA芯片可分为两种形式,DNA阵列和寡核苷酸微芯片。DNA芯片的制作方法主要有光导原位合成法和自动化点样法。DNA芯片与标记的探针或DNA样品杂交,并通过探测杂交信号谱型来实现DNA序列或基因表达的分析。适应于DNA芯片的发展,同时出现了许多新型的杂交信号检测方法。主要有激光荧光扫描显微镜、激光扫描共焦显微镜、结合使用CCD相机的荧光显微镜、光纤生物传感器、化学发生法、光激发磷光物质存储屏法、光散射法等。     

Compatibility of codling moths Cydia pomonella (Linnaeus) (Lepidoptera: Tortricidae) from South Africa with codling moths shipped from Canada

The sterile insect technique (SIT) has been successfully applied against codling moth Cydia pomonella (Linnaeus) (Lepidoptera; Tortricidae) in British Columbia since 1992 where the mass‐rearing facility produces between 15 and 16 million moths per week. Due to the seasonality of this pest, the facility is only fully utilized for part of the year. The time and expense of implementing SIT against codling moth in South Africa may be substantially reduced if moths from Canada were sexually compatible with those from South Africa. In addition, because the pome fruit‐growing season in both countries is opposite, the programme in Canada might benefit by maintaining moth production year‐round and selling moths to South Africa. Semi‐field studies in small cages and release‐recapture studies were conducted in an unsprayed apple orchard in South Africa to assess mating compatibility of laboratory‐reared codling moth from Canada and wild codling moths from South Africa. The results suggest that Canadian codling moth males were equally attracted to calling Canadian and South African females despite the fact that Canadian moths had been transported (from Canada to South Africa) for 48 h as both pupae and adults. The data also suggest that at lower field temperatures Canadian moths were more active than South African moths. Results from the release‐recapture field trials indicated that Canadian and South African males were equally attracted to Canadian and South African females. These results suggest that codling moths from Canada and South Africa are fully compatible and indicate that Canadian moths can be used for SIT studies in South Africa. As these studies were conducted with moths from two very different climatic and time zones, it is proposed that populations of codling moth in other pome fruit production areas may also be compatible with the Canadian moths.     

Improved quality management to enhance the efficacy of the sterile insect technique for lepidopteran pests

Lepidoptera are among the most severe pests of food and fibre crops in the world and are mainly controlled using broad spectrum insecticides. This does not lead to environmentally sustainable control and farmers are demanding alternative control tools which are both effective and friendly to the environment. The sterile insect technique (SIT), within an area‐wide integrated pest management (AW‐IPM) approach, has proven to be a powerful control tactic for the creation of pest‐free areas or areas of low pest prevalence. Improving the quality of laboratory‐reared moths would increase the efficacy of released sterile moths applied in AW‐IPM programmes that integrate the (SIT). Factors that might affect the quality and field performance of released sterile moths are identified and characterized in this study. Some tools and methods to measure, predict and enhance moth quality are described such as tests for moth quality, female moth trapping systems, ‘smart’ traps, machine vision for recording behaviour, marking techniques, and release technologies. Methods of enhancing rearing systems are discussed with a view to selecting and preserving useful genetic traits that improve field performance.     

山西翅果油树上三种重要鳞翅目害虫的形态和分子鉴定及生活史观察

[目的]明确山西翅果油树Elaeagnus mollis上发生危害的3种鳞翅目害虫形态鉴定特征及生活史特性,并基于mtDNA COI基因DNA条形码对这3个种进行快速物种识别鉴定.[方法]通过观察山西翅果油树上3种鳞翅目害虫成虫外部形态和解剖拍照雌、雄性外生殖器特征,利用PCR扩增对待测样本COI基因DNA条形码序列进...     

Pollination of Platanthera chlorantha (Orchidaceae): new video registration of a hawkmoth (Sphingidae)

Documenting species interactions is a time consuming enterprise, in particular for rare interaction events and interactions taking place at night. Pollinators foraging on orchids have traditionally been monitored by discovering pollen vectors on collected insects, recording traces left by moths on the orchid, direct observations and recently by continuous video monitoring. Direct observations in the wild of orchids with low visitation rates is time demanding. In the present study I monitored greater butterfly‐orchids (Platanthera chlorantha), by using a event triggered video monitoring system. A total of 23 nights of monitoring were conducted, whereas only 6 nights had visits by one moth species, namely the pine hawk‐moth (Hyloicus pinastri). The total numbers of pine hawk‐moths registered were 18. In addition to species identification, the video recordings also enabled detection of pollinaria on the pine hawk‐moths. Most of the pine hawk‐moth visits took place around midnight. The visit lasted on average for 38.0 sec and the average number of flowers visited was 9.6. In future studies, this video system could give more details on interactions between orchids and insects and even link it to environmental factors (e.g. varying weather conditions).     

Expansion of the winter moth outbreak range: no restrictive effects of competition with the resident autumnal moth

1. Both direct and indirect competition can have profound effects on species abundance and expansion rates, especially for a species trying to strengthen a foothold in new areas, such as the winter moth (Operophtera brumata) currently in northernmost Finland. There, winter moths have overlapping outbreak ranges with autumnal moths (Epirrita autumnata), who also share the same host, the mountain birch (Betula pubescens ssp. czerepanovii). Competitive interactions are also possible, but so far unstudied, are explanations for the observed 1–3 years phase lag between the population cycles of the two moth species. 2. In two field experiments, we studied host plant‐mediated indirect inter‐specific competition and direct interference/exploitation competition between autumnal and winter moths. The experimental larvae were grown either with the competing species or with the same number of conspecifics until pupation. Inter‐specific competition was judged from differences in pupal mass (reflecting lifespan fecundity), larval development time and larval survival. 3. Larval performance measurements suggested that neither direct nor indirect inter‐specific competition with the autumnal moth reduce the growth rate of winter moth populations. Winter moths even had a higher probability of survival when reared together with autumnal moths. 4. Thus, we conclude that neither direct nor indirect inter‐specific competition is capable of suppressing the spread of the winter moth outbreak range and that both are also an unlikely cause for the phase lag between the phase‐locked population cycles of the two moth species.     

Identification of insect pests as food foreign materials using DNA barcodes

Insect foreign materials in food are of great economic and hygienic significance. However, identifying these species with any certainty requires an expert taxonomist and can be a time consuming process. Furthermore, insects are found as body parts or they are immature specimens that cannot be identified by conventional means. For these reasons, a reference database and efficient means of identification by non‐specialists are necessary to control insect pests. In this study, we chose 15 important insect pest species, because they had a higher probability of being included in human foods. We tested the utility of the cytochrome c oxidase I (COI) DNA barcodes for the identification. A 658‐bp fragment of the COI gene was sequenced, aligned, and a sequence data bank was constructed. As a result, the COI barcode sequence was suitable for identifying insect pests as food foreign materials. Photographs of morphological key characters by stereoscopic microscope and a pictorial key of the species are provided.