Sequence characterized amplified region markers for identifying biotypes of Bemisia tabaci (Hem., Aleyrodidae)

Abstract:  Bemisia tabaci (Gennadius) is an important pest of agriculture and horticulture crops that includes many morphologically indistinguishable biotypes. Molecular markers can provide a scientific method to rapidly identify biotypes. In this study, we attempted to develop specific primer sets for rapidly and stably identifying various biotypes of B. tabaci . We developed sequence characterized amplified region (SCAR) markers for each of six B. tabaci (Gennadius) biotypes (A, B, Q, Nauru, An and S). Two primer sets (BaAF/BaAR and BaQF/BaQR) were designed from random amplified polymorphic DNA-specific fragments for the A and Q biotypes. Four forward primers, BaBF, BaNaF, BaANF and BaSF, with a common reverse primer, L2-N-3014, for the other four biotypes of B, Nauru, An and S, respectively, were used based on their individual mitochondrial cytochrome oxidase I sequences. Six of these SCARs were useful in distinguishing each biotype from the others. Application of these SCARs will be helpful in rapidly identifying biotypes for quarantine pest control to prevent the invasion of the various biotypes.     

Taenia solium and Taenia saginata: identification of sequence characterized amplified region (SCAR) markers

Cysticercosis is one of the most important zoonosis, not only because of the effects on animal health and its economic consequences, but also due to the serious danger it poses to humans. The two main parasites involved in the taeniasis-cysticercosis complex in Brazil are Taenia saginata and Taenia solium. Differentiating between these two parasites is important both for disease control and for epidemiological studies. The purpose of this work was to identify genetic markers that could be used to differentiate these parasites. Out of 120 oligonucleotide decamers tested in random amplified polymorphic DNA (RAPD) assays, 107 were shown to discriminate between the two species of Taenia. Twenty-one DNA fragments that were specific for each species of Taenia were chosen for DNA cloning and sequencing. Seven RAPD markers were converted into sequence characterized amplified region (SCAR) markers with two specific for T. saginata and five specific for T. solium as shown by agarose gel electrophoresis. These markers were developed as potential tools to differentiate T. solium from T. saginata in epidemiological studies.     

Development of molecular diagnostic markers for sharpshooters Homalodisca coagulata and Homalodisca liturata for use in predator gut content examinations

To aid in identifying key predators of Proconiini sharpshooter species present in California, we developed and tested molecular diagnostic markers for the glassy‐winged sharpshooter, Homalodisca coagulata (Say), and smoke‐tree sharpshooter, Homalodisca liturata (Ball) (Homoptera: Cicadellidae). Two different types of markers were compared, those targeting single‐copy sequence characterized amplified regions (SCAR) and mitochondrial markers targeting the multicopy cytochrome oxidase subunit genes I (COI) and II (COII). A total of six markers were developed, two SCAR and four mitochondrial COI or COII markers. Specificity assays demonstrated that SCAR marker HcF5/HcR7 was H. coagulata specific and HcF6/HcR9 was H. coagulata/H. liturata specific. COI (HcCOI‐F/R) and COII (HcCOII‐F4/R4) markers were H. coagulata specific, COII (G/S‐COII‐F/R) marker was H. coagulata/H. liturata specific, and lastly, COII marker (Hl‐COII‐F/R) was H. liturata specific. Sensitivity assays using genomic DNA showed the COI marker to be the most sensitive marker with a detection limit of 6 pg of DNA. This marker was 66‐fold more sensitive than marker Hl‐COII‐F/R that showed a detection limit of 400 pg of DNA. In addition, the COI marker was 4.2‐fold more sensitive than the COII marker. In predator gut assays, the COI and COII markers demonstrated significantly higher detection efficiency than the SCAR markers. Furthermore, the COI marker demonstrated slightly higher detection efficiency over the COII marker. Lastly, we describe the inclusion of an internal control (28S amplification) for predation studies performing predator gut analyses utilizing the polymerase chain reaction (PCR). This control was critical in order to monitor reactions for PCR failures, PCR inhibitors, and for the presence of DNA.     

Detection of Bemisia tabaci remains in predator guts using a sequence-characterized amplified region marker

Bemisia tabaci (Gennadius) (Homoptera: Aleyrodidae) B biotype is an invasive species (biotype) in China. In order to understand the role that native natural enemies might play in its control, techniques were developed for detecting B. tabaci DNA within the gut of predators. A species-specific DNA fragment, ca. 350 bp, was identified by random amplified polymorphic DNA analysis. This fragment was absent in other closely related or co-occurring prey species, cotton, and other select predator species. After cloning and sequencing the fragment, one pair of sequence-characterized amplified region (SCAR) primers was developed, which amplified a single band of 240 bp. Specificity tests performed with the primers showed the presence of the 240-bp band for B. tabaci in all developmental stages and both sexes, in adult Propylaea japonica (Thunberg) (Coleoptera: Coccinellidae) fed on B. tabaci nymphs in the laboratory, and in predators collected in cotton fields. Following consumption of a single red-eyed B. tabaci nymph, prey DNA was detectable in 100% of P. japonica at t = 0, decreasing to 20% after 12 h of digestion, and no B. tabaci DNA detected at t = 24 h. In total, we analyzed the gut contents of 185 field-collected predators, representing four different orders. All nine field-collected predator species (namely, P. japonica, Harmonia axyridis, Scymnus hoffmanni, Coccinella septempunctata, Orius sauteri, Chrysopa pallens, Chrysopa formosa, Erigonnidium graminicolum, and Neoscona doenitzi) contained DNA from B. tabaci and are assumed predators of this pest insect. Overall, the B. tabaci was eaten by more than 50% of field-collected predator individuals, including larvae of the coccinellids (P. japonica and H. axyridis) and lacewings (C. pallens and C. formosa) and adults of O. sauteri and the spiders (E. graminicolum and N. doenitzi). There was a trend of a higher percentage of larval than adult coccinellids and lacewings that preyed on B. tabaci in the field. This study provides a framework for the future use of molecular gut content analysis in arthropod conservation ecology and food web research, with considerable potential for quantifying threats to invasive or endemic pest species in China and elsewhere.     

棉铃虫细胞色素P450 cDNA片段的克隆与序列分析

以 5龄实验室敏感品系棉铃虫Helicoverpaarmigera的总RNA为模板 ,采用简并性引物 ,利用反转录 -多聚酶链式反应 (RT PCR)扩增出了 2个新的长度分别为 2 3 7bp和 2 40bp的cDNA片段。序列分析表明 ,2 3 7bp的cDNA片段与棉铃虫P45 0CYP4家族有较高的相似性 ,最高达 73 %;而 2 40bp的cDNA片段与CYP6家族有较高的相似性 ,最高达 49%。     

Genetic differentiation of Helicoverpa armigera (Hübner) and H. assulta (Guenée) (Lepidoptera: Noctuidae) based on AFLP markers

Abstract Here we use amplified fragment length polymorphism (AFLP) to assess genetic differentiation of Helicoverpa armigera and H. assulta . The results indicated that both species-specific fingerprints and cluster analysis showed the ability of AFLP technique to discriminate the two sibling species; among a total 1963 AFLP markers amplified from nine primer combinations: 777 (39.6%) were H. armigera -specific, 602 (30.7%) were H. assulta -specific, and 584 (29.7%) were common bands. The mean number of H. armigera -specific bands was significantly more than that of H. assulta -specific bands for nine primer combinations ( P < 0.05); the intraspecific distance of H. armigera and H. assulta was 0.123 0 and 0.110 7 respectively, and the interspecific distance was 0.178 3. In addition, the percentage of polymorphic loci and estimated average heterozygosity were used to estimate genetic diversity of the two species. This study therefore demonstrates that AFLP analysis is a sensitive and reliable technique to study genetic differentiation and genetic relationships between species and provides sufficient molecular markers for future linkage map construction, location and eventual cloning of genes involved in traits differentiation.     

Group-specific primers for DNA-based detection of springtails (Hexapoda: Collembola) within predator gut contents

Group‐specific, degenerate polymerase chain reaction primers for DNA‐based detection of springtails (Hexapoda: Collembola) within predator gut contents have been developed for the first time. Primers were designed from 18S rDNA and amplified fragments of 272 bp and 177 bp from 17 springtail species collected in agricultural habitats. Specificity tests against 41 nontarget species revealed no cross‐reactivity. Group‐specific polymerase chain reaction is advantageous when working in species‐rich habitats and these primers could facilitate studies of trophic links between springtails and generalist arthropod predators worldwide.     

Novel polymorphic microsatellite markers developed in the cotton bollworm Helicoverpa armigera （Lepidoptera：Noctuidae）

A novel set of five polymorphic di- or trinucleofide microsatellite loci suitable for population genetic study were developed from an enriched genomic library for the pest insect cotton bollworm, Helicoverpa armigera, and cross-amplifiability of these and other published loci was tested in a closely related species, the tobacco budworm, H.assulta. The expected heterozygosity at these loci ranges from 0.62 to 0.91 in the cotton bollworm. The observed allele numbers varies from 4 to 12 in the limited number of individuals tested. Although a large proportion of cloned microsatellite sequences are present in multi-copy in the cotton bollworm genome, the overwhelming majority of the finalized polymorphic diallelic loci are tri-nucleotide microsatellites - an unexpected outcome, which should facilitate subsequent genotyping analysis.     

Development of a SCAR (sequence characterised amplified region) marker for molecular tagging of the dwarf BREIZH (Bzh) gene in Brassica napus L.

 A SCAR (sequence characterised amplified region) has been developed for optimal tagging of the dwarf Bzh gene in Brassica napus. A RAPD marker named OPMO7-730 was previously found closely linked to the dwarf locus at 0.8±0.7 cM. The DNA band corresponding to this marker was cloned and sequenced, and specific PCR primers were designed. The PCR test allowed us to amplify the locus corresponding to OPM07-730. With a restriction endonuclease digest and optimal electrophoresis conditions, three allelic forms of this marker have been recovered on the 40 B. napus accessions tested. The usefullness of this marker in breeding dwarf rapeseed lines is discussed. Received: 20 April 1998 / Accepted: 29 April 1998     

七氟菊酯和溴氰菊酯对棉铃虫肠道菌群的影响

【目的】本研究旨在探究拟除虫菊酯类杀虫剂对棉铃虫Helicoverpa armigera幼虫肠道菌群结构及代谢的影响,丰富对杀虫剂作用机理的认识。【方法】分别对棉铃虫2和3龄幼虫饲喂普通人工饲料(对照组, SS)、含2%七氟菊酯(Ⅰ型拟除虫菊酯)粉剂饲料(七氟菊酯处理组,Te)和含2.5%溴氰菊酯(Ⅱ型拟除虫菊酯)乳油饲料(溴氰菊酯处理组, DM),然后提取3龄幼虫肠道菌群基因组DNA;利用Illumina MiSeq二代高通量测序技术对肠道细菌的16S rDNA的V3-V4变异区进行测序,分析其肠道细菌的多样性和丰富度;利用qPCR验证16S rDNA测序分析结果。取2和3龄幼虫肠道,匀浆后进行Biolog-Eco实验,分析肠道细菌对Eco板上31种碳源的代谢情况。【结果】16S rDNA测序结果表明,棉铃虫3龄幼虫肠道细菌主要是厚壁菌门(Firmicutes)、变形菌门(Proteobacteria)、拟杆菌门(Bacteroidetes)和蓝藻菌门(Cyanobacteria)。与对照组相比,溴氰菊酯处理组和七氟菊酯处理组的棉铃虫幼虫肠道细菌的α多样性指数没有显著性改变,但是菌群结构发生了变化：在门水平,拟杆菌门的相对丰度减少,厚壁菌门和蓝藻菌门的相对丰度增加,qPCR验证结果亦支持16S rDNA测序分析的这个结果;在属水平,拟杆菌属Bacteroides、普氏菌属Prevotella和假单胞菌属Pseudomonas等的相对丰度降低,狭义梭菌属Clostridium sensu stricto 1、埃希菌属-志贺氏菌属Escherichia-Shigella和盐单胞菌属Halomonas等的相对丰度增加,其中盐单胞菌属Halomonas的相对丰度显著增加。Biolog-Eco结果表明,与对照组相比,溴氰菊酯处理组中2龄幼虫对羧酸类碳源的代谢能力下降;溴氰菊酯处理组和七氟菊酯处理组中3龄幼虫对DL-α-磷酸甘油、肝糖和L-苯丙氨酸等碳源的利用能力下降。【结论】结果显示,拟除虫菊酯类杀虫剂对棉铃虫肠道菌群的结构和代谢能力有明显影响,拟除虫菊酯类杀虫剂使棉铃虫肠道有益菌的相对丰度下降,而使致病菌的相对丰度增加。短时间拟除虫菊酯处理未造成抗药性菌群的丰度增加。qPCR检测结果与16S rDNA测序分析结果相似。Ⅰ型和Ⅱ型拟除虫菊酯类杀虫剂对棉铃虫肠道菌群结构和代谢功能的影响不同。     

Authentication of snakes used in Chinese medicine by sequence characterized amplified region (SCAR)

Random amplified polymorphic DNA fingerprints characteristic of thethree snakes Zaocys dhumnades, Agkistrodonacutus and Bungarus multicinctus multicinctuswere generated using primer OPF-14. Z. dhumnades is anendangered species included in the Convention on International Trade inEndangered Species of Wild Fauna and Flora, and A. acutus,B. multicinctus multicinctus and Z. dhumnades are listed inthe Chinese Pharmacopoeia. The species-specific polymorphic bands Aa specific toA. acutus, Bmm specific to B. multicinctusmulticinctus and Zd specific to Z. dhumnadeswere identified and the sequences of these bands were used to design polymerasechain reaction primers for sequence characterized amplified region (SCAR)analysis of the three snakes. A multiplex SCAR analysis was established toauthenticate the snakes used in Chinese medicine reliably and efficiently.     

棉铃虫酚氧化酶原基因的克隆、序列分析和组织表达

利用RT-PCR和RACE方法,获得了棉铃虫Helicoverpa armigera酚氧化酶原(prophenoloxidase,PPO)基因一个亚型cDNA的完整序列。该序列全长2 405 bp,含有一个2 097 bp的开放阅读框,编码一个由698个氨基酸残基组成的蛋白质。推导的氨基酸序列与其他鳞翅目昆虫PPO2基因相应氨基酸序列有较高的同源性(76％～80％),同时该序列具有铜离子结合位点等PPO基因所具有的典型特征。组织特异性表达分析表明,该基因在棉铃虫血细胞、体壁和中肠中均有表达。     

棉铃虫P450基因HarmCYP9A33的克隆、 序列分析及原核表达

夜间活动昆虫如夜蛾类主要通过嗅觉来寻找配偶、 寄主植物和产卵场所, 是研究昆虫嗅觉分子机制的理想材料。P450为多功能单加氧酶, 在昆虫对各种内源与外源物质的代谢中起重要作用。为研究P450在昆虫嗅觉中的作用, 本研究采用RT-PCR和RACE技术, 从夜蛾科昆虫棉铃虫Helicoverpa armigera (Hübner)雄蛾触角中扩增得到一条全长1 772 bp的P450基因, 命名为HarmCYP9A33 (GenBank登录号为JX486677)。序列分析表明, HarmCYP9A33开放阅读框全长1 590 bp, 编码529个氨基酸残基, 预测蛋白质分子量和等电点分别为61. 62 kD和7. 97; HarmCYP9A33与甘蓝夜蛾Mamestra brassicae触角毛形感器中高表达的MbraCYP9A13蛋白的氨基酸序列一致性最高, 达75%, 蛋白二级结构相似, 6个底物识别位点(substrate recognition sites, SRSs)序列一致性达61%, 其中底物与酶结合通道开关Ⅰ螺旋中SRS4序列完全相同, 与棉铃虫CYP9A亚家族蛋白有一定的结构相似性。Real-time PCR检测表明, HarmCYP9A33在雌、 雄蛾各组织中均有表达, 以腹部表达量最高, 其次为头部; 在卵至成虫各个时期也均表达, 以蛹中表达量最高; 在触角中的表达量随羽化时间而变化, 且多高于卵和幼虫中的表达量。SDS-PAGE检测和Western blot鉴定表明HarmCYP9A33体外融合表达成功。本研究为深入探讨该基因在棉铃虫触角感器细胞中的定位及其生物学功能奠定了基础。     

Postglacial recolonization routes for Picea abies K. in Italy as suggested by the analysis of sequence-characterized amplified region (SCAR) markers

The routes through which Norway spruce recolonized the Alps after the last ice age were investigated at the genetic level. Seven populations along the Alpine range plus one Apennine population were characterized for seven sequence-characterized amplified region (SCAR) loci, detecting an overall FST = 0.118. This rather high value for forest species reflects an uneven distribution of genetic variability, and was analysed through different statistical methods. Alternative hypotheses were tested under the isolation-by-distance model and using the analysis of molecular variance (AMOVA) frame. We conclude that the hypothesis of the existence of a glacial refugium in the Apennines should be rejected, while a putative relict population is identified in the Maritime Alps. The Alpine range of Norway spruce appears to be split in two parts across a north-south line. The results are discussed in comparison with data based on morphological markers, isozymes, chloroplast microsatellites and mitochondrial markers.     

Impact of Cheiracanthium pelasgicum (Araneae: Miturgidae) and Chrysoperla carnea (Neuroptera: Chrysopidae) intraguild predation on the potential control of cotton pest Helicoverpa armigera (Lepidoptera: Noctuidae)

Antagonist interactions such as intraguild predation (IGP) or cannibalism among predatory arthropods can reduce the impact of these invertebrates on pest limitation in agroecosystems. Here, the effects of IGP between two major natural enemies of cotton pests, the cursorial spider Cheiracanthium pelasgicum (C.L. Koch) and the common green lacewing Chrysoperla carnea (Stephens), were studied under laboratory conditions. First, a feeding preference test was carried out to determine the degree of C. pelasgicum preference for lacewing larvae, using second-instar Helicoverpa armigera larvae as alternative prey. In a second bioassay, the effects of predator interactions on potential predation of H. armigera larvae were analysed using three treatment combinations (plus a control with no predator): (1) spider alone, (2) lacewing larvae alone, (3) spider + lacewing larvae. Potential predation by C. pelasgicum on lacewing eggs was also studied. C. pelasgicum showed no significant preference for either of the two species, indicating that this spider may impact negatively on the green lacewing population. Findings revealed no additive effects and an antagonist interaction between C. pelasgicum and green lacewing larvae, which adversely affected H. armigera suppression; both predators displayed lower predation rates when kept together than either predator alone. However, presence of lacewing larvae and subsequent unidirectional IGP did not affect the predation capacity of C. pelasgicum. Finally, predation rates of C. pelasgicum on lacewing eggs were very low (mean 2.35 ± 0.71 eggs, 24 h after offering) indicating that the impact of C. pelasgicum on lacewing populations may be limited.     

An in vitro method for increasing UV-tolerance in a strain of Helicoverpa armigera (Lepidoptera: Noctuidae) nucleopolyhedrovirus

A method for increasing tolerance to ultraviolet (UV) radiation in a strain of nucleopolyhedrovirus of cotton bollworm, Helicoverpa armigera (Hübner) (HearNPV) using a solar simulator is described. The Coimbatore isolate (CBE I) of HearNPV was subjected to a five-step sequence of selection to increase its UV tolerance. Each step consisted of irradiation of wet deposits of the virus to near UV (at energy level of 300W/m²), bioassay against second instar H. armigera larvae and propagation in early fifth instar larvae. Selection steps carried out at 15, 30, 60 and 90 minutes of exposure revealed that the continuous exposure of HearNPV-CBE I at low doses of UV irradiation (270–540 KJ/m²) did not significantly affect the virus activity as measured by its biological activity against second instar larvae. Selection at higher doses (1620 KJ/m²) led to loss of viral activity in the first two exposure cycles; however, there was retention of virulence coupled with increased tolerance to UV doses from third cycle onwards. Further, studies on the persistence of UV tolerant strain of HearNPV-CBE I in comparison with original strain showed that the tolerant strain had more persistence even after 7 days of weathering both under exposed (18% original activity remaining) and shaded (26% original activity remaining) condition on potted cotton plant.     

Functional response of Chrysoperla carnea (Neuroptera: Chrysopidae) to Helicoverpa armigera (Lepidoptera: Noctuidae): Effect of prey and predator stages

Abstract Understanding predator–prey interactions has a pivotal role in biological control programs. This study evaluated the functional response of three larval instars of the green lacewing, Chrysoperla carnea (Stephens), preying upon eggs and first instar larvae of the cotton bollworm, Helicoverpa armigera Hübner. The first and second instar larvae of C. carnea exhibited type II functional responses against both prey stages. However, the third instar larvae of C. carnea showed a type II functional response to the first instar larvae of H. armigera, but a type III functional response to the eggs. For the first instar larvae of C. carnea, the attack rate on H. armigera eggs was significantly higher than that on the larvae, whereas the attack rate of the second instar C. carnea on H. armigera larvae was significantly higher than that on the eggs. For the third instar larvae of C. carnea, the attack rate on the larvae was 1.015 ± 0.278/h, and the attack coefficient on the eggs was 0.036 ± 0.005. The handling times of the third instar larvae on larvae and eggs were 0.087 ± 0.009 and 0.071 ± 0.001 h, respectively. The highest predation rate was found for the third instar larvae of C. carnea on H. armigera eggs. Results of this study revealed that the larvae of C. carnea, especially the third instar, had a good predation potential in controlling H. armigera eggs and larvae. However, for a comprehensive estimation of the bio‐control abilities of C. carnea toward H. armigera, further field‐based studies are needed.     

棉铃虫幼虫唾液腺cDNA文库的构建及EST分析

棉铃虫Helicoverpa armigera (Hübner)幼虫唾液中的各种酶类及各种生化组分在棉铃虫与植物相互作用及协同进化中起到重要作用; 唾液腺是棉铃虫唾液成分的合成器官。本研究通过构建棉铃虫幼虫唾液腺全长cDNA文库, 测序得到1 502条EST序列, 聚类分析后获得821个unigenes, 为筛选棉铃虫与寄主互作信号因子提供基因信息资源。使用Blast2 GO软件对821个unigenes进行了比对和功能注释, 初步获得棉铃虫幼虫唾液腺中mRNA的构成特征。结果显示, 在棉铃虫唾液腺ESTs文库中, 鉴定得到脂类相关消化酶基因17个, 糖类相关消化酶基因5个, 半胱氨酸蛋白酶基因1个, 丝氨酸蛋白酶基因20个（其中16个为新发现）, 提示唾液腺的主要功能是分泌消化酶进行预消化; 还发现在棉铃虫幼虫唾液腺中存在表皮蛋白、 气味结合蛋白和化学感受蛋白基因。结果为研究棉铃虫预消化系统打下基础。