Detecting Cacopsylla pyricola (Hemiptera: Psyllidae) in predator guts using COI mitochondrial markers

Cacopsylla pyricola (F?rster) is one of the most important pests of pear in North America, where several native predators have been considered for integrated pest management (IPM) programmes. Two molecular markers of 271 and 188 bp were developed from C. pyricola cytochrome oxidase I (COI) fragments, in order to study the detection of this species in the gut of arthropod predators. Primer sensitivity and the detection period for pear psylla remains in the guts of Anthocoris tomentosus Pericart were determined. The sensitivity threshold was defined at 10-5 dilution of a C. pyricola fifth-instar nymph in all samples. Predator adults were evaluated immediately after ingestion of one to five C. pyricola nymphs (t = 0) and after 2, 4, 6, 8, 16, 24 and 32 h. Detection of the presence of C. pyricola DNA always lasted longer using the shorter fragment and was observed after 32 h of digestion using both markers. The primers amplifying the 188 bp fragment amplified all four psyllid species tested, whereas the primers designed to amplify the 271 bp fragment did so exclusively for C. pyricola and its close relative, Cacopsylla pyri (Linnaeus). Both primers failed to amplify DNA from representative species of the Coccinellidae, Chrysopidae, Hemerobiidae, Anthocoridae, Miridae, Salticidae, Aphididae, Tetranychidae and the Tortricidae, suggesting their suitability for general trophic studies.     

Development of sequence amplified characterized region (SCAR) markers of helicoverpa armigera: a new polymerase chain reaction-based technique for predator gut analysis

A method is described for the development of DNA markers for detection of Helicoverpa armigera (Hubner) (Lepidoptera: Noctuidae) in predator gut analysis, based on sequence characterized amplified regions (SCARs) derived from a randomly amplified polymorphic DNA (RAPD) band. A 1200-bp DNA fragment of H. armigera, absent in the predator band pattern and in other closely related prey species, was identified by RAPD analysis. This fragment was cloned and its extremes sequenced to design extended strand-specific 20-mer oligonucleotide primers. Three pairs of SCAR primers, which amplified three different DNA fragments, were used to study the effect of fragment length on detection of prey in the predator gut. Using the pair of primers that amplified the longest fragment of H. armigera DNA, a single band of 1100 bp was obtained, but its detection was not possible in the predator gut. Detection of the ingested prey was possible with the other two pairs of SCAR primers, obtaining bands of 600 and 254 bp, respectively. Detection of H. armigera DNA in the gut of the predator Dicyphus tamaninii was evaluated immediately after ingestion (t = 0) and after 4 h. Detection of H. armigera DNA after 4 h was only possible using the pair of primers that amplified the shortest fragment (254 bp). The test for specificity, using these last pair of primers, showed that H. armigera was the only species detected. The detection threshold was defined at a 1:8192 dilution of a H. armigera whole egg in all samples.     

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.     

Morphology and Biology of the Preimaginal Stages of Some Species of the Genus Eupithecia Curt. (Lepidoptera,Geometridae)

Entomological Review - The biology, host plants, and statial distribution of 17 species of the genus Eupithecia Curt. (Lepidoptera, Geometridae) from different regions of Asia (Kazakhstan,...     

在韩国危害红松球果的三种蛾类幼虫形态记述（鳞翅目）

详细地记述了韩国红松球果害虫冷杉梢斑螟Dioryctria abietella、赤松梢斑螟D. sylvestrella (螟蛾科)和小花尺蛾Eupithecia abietaria debrunneata (尺蛾科)幼虫的形态特征,并提供了形态特征图。     

致病性大肠埃希菌和沙门菌毒力岛基因的双重PCR检测方法的建立

目的实现对致病性大肠埃希菌（E．coli）、沙门菌（Salmonella）的同时检测,建立快速灵敏的双重PCR检测方法。方法以致病性大肠埃希菌和沙门菌毒力岛基因为研究对象,根据GenBank发表的大肠埃希菌和沙门菌毒力岛基因序列,分别设计合成了大肠埃希菌毒力岛irpl、irl）2和fyuA,沙门菌毒力岛mgtC、sseL和sopB等6对引物,以禽致病性大肠埃希菌（CVCC1565）菌株和沙门菌（ATCC9150）菌株的核酸混合物为模板,经引物特异性试验,引物组合,成功建立了快速鉴别检测致病性大肠埃希菌和沙门菌的双重PCR方法。结果特异性试验结果显示,引物irpl、irp2和fyuA仅能扩增出大肠埃希菌（CVCC1565）的特异性片段,大小分别是799、414和948bp;引物mgtC、sseL和sopB仅能扩增出沙门菌（ATCC9150）的特异性片段,大小分别是500、269和1000bp。敏感性试验结果表明大肠埃希菌和沙门菌的最低检测限分别为2．2×101CFU／mL和2．0×101CFU／mL。结论本研究建立的双重PCR方法具有特异性强、敏感性高、快速简便等特点,可用于致病性大肠埃希菌和沙门菌的联合检测与鉴别诊断。     

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.     

广东地区两种兰花病毒病害的分子鉴定及检测

根据已报道的建兰花叶病毒(CyMV)和齿兰环斑病毒(ORSV)基因组核苷酸序列,在其cp基因上下游设计PCR引物。CyMV预计扩增产物784bp,ORSV预计扩增产物604bp。以采集自广东省顺德的墨兰和文心兰表现病毒病症状的病株叶组织总RNA为模板,进行RT—PCR扩增。对预期大小的5个扩增产物进行克隆和测序,结果表明,来源于不同兰种或同一兰种不同兰场的病样CyMV引物扩增产物核苷酸序列存在少量差异,但均与世界各地的CyMV分离物cp基因高度同源;而来源于不同兰种的病样ORSV引物扩增产物核苷酸序列完全相同,与世界各地的ORSV分离物cp基因高度同源。因此可将侵染广东兰花的两种病毒鉴定为CyMV和ORSV。混合上述两种病毒的PCR引物,采用双重RT—PCR扩增,对采自广东顺德23个兰场共153份样品进行病毒检测,76份(49．7％)检出CyMV,52份(34．0％)检出ORSV,2份(1．3％)同时检出CyMV和ORSV。     

马铃薯瓢虫与茄二十八星瓢虫SCAR标记的建立及应用

为了探索快速鉴定马铃薯瓢虫Henosepilachna vigintioctomaculata(Motschulsky)和茄二十八星瓢虫Henosepilachna vigintioctopunctata(Fabricius)的分子生物学方法,本研究在随机扩增多态性DNA(random amplified polymorphic DNA,RAPD)的基础上,分别设计了可以鉴别两个物种的序列特征扩增区域(sequence characterized amplified regions,SCAR)标记。从随机合成的60条引物中筛选出来2条特异性引物(分别为OPI-6和OPJ-15),引物OPI-6在马铃薯瓢虫中扩增出约750 bp的特异性条带,引物OPJ-15在茄二十八星中扩增出约750 bp的特异性条带,根据测序结果设计了两对SCAR引物对筛选结果进行验证,发现根据OPI-6的测序结果所设计的SCAR引物(OPI-6 test)仅能在马铃薯瓢虫中扩增出645 bp的条带,而根据OPJ-15的测序结果所设计的SCAR引物(OPJ-15 test)仅能在茄二十八星瓢虫中扩增出436 bp的条带。这两对SCAR引物能够准确、稳定且快速地区分马铃薯瓢虫与茄二十八星瓢虫,对这两种害虫的精准防控具有重要意义。     

Molecular analysis of predation by carabid beetles (Carabidae) on the invasive Iberian slug Arion lusitanicus

The invasive Iberian slug, Arion lusitanicus, is spreading through Europe and poses a major threat to horticulture and agriculture. Natural enemies, capable of killing A. lusitanicus, may be important to our understanding of its population dynamics in recently invaded regions. We used polymerase chain reaction (PCR) to study predation on A. lusitanicus by carabid beetles in the field. A first multiplex PCR was developed, incorporating species-specific primers, and optimised in order to amplify parts of the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene of large Arion slugs, including A. lusitanicus from the gut contents of the predators. A second multiplex PCR, targeting 12S rRNA mtDNA, detected predation on smaller Arion species and the field slug Deroceras reticulatum. Feeding trials were conducted to measure the effects of digestion time on amplicon detectability. The median detection times (the time at which 50% of samples tested positive) for A. lusitanicus and D. reticulatum DNA in the foreguts of Carabus nemoralis were 22 h and 20 h, respectively. Beetle activity-densities were monitored using pitfall traps, and slug densities were estimated using quadrats. Predation rates on slugs in the field by C. nemoralis in spring ranged from 16-39% (beetles positive for slug DNA) and were density dependent, with numbers of beetles testing positive being positively correlated with densities of the respective slug species. Carabus nemoralis was shown to be a potentially important predator of the alien A. lusitanicus in spring and may contribute to conservation biological control.     

Can multiple-copy sequences of prey DNA be detected amongst the gut contents of invertebrate predators?

The first experiments to clearly demonstrate that DNA techniques might be used to detect predator-prey interactions between arthropods are reported. The accurate modelling of such interactions has depended until now upon a mixture of laboratory experiments, population monitoring and biochemical tests. The latter involve gut-content analyses, and have most recently depended upon the development of prey-specific monoclonal antibodies. Although these are excellent for detecting predation on a target prey, they are impractical for analysing the prey range of a particular predator. Molecular detection depends upon the ability of DNA to resist digestion in the predator gut and of the polymerase chain reaction (PCR) to amplify prey-specific DNA from semidigested material. As a first step, experiments using carabid beetles, Pterostichus cupreus L., as predators and mosquitoes as prey are reported. The target sequences were fully characterized multiple-copy esterase genes from two laboratory strains of Culex quinquefasciatus Say. Although DNA was extracted from homogenates of whole beetles (minus appendages), a 146 bp product could be amplified from both mosquito strains digested in the beetle gut for 28 h. The larger, 263 bp product was detectable for 28 h in one mosquito strain, but could not be amplified after 5 h from the other. Whether the beetles had eaten one mosquito or six, digested for zero or 28 h, the prey were equally detectable. Having demonstrated that shorter, multiple-copy sequences survive digestion for a considerable period in the gut of a predator, the opportunity exists to develop new detection systems for studying predation in the field.     

Consumption of cereal leaf beetle,Oulema melanopus,by generalist predators in wheat fields detected by molecular analysis

The cereal leaf beetle (CLB), Oulema melanopus L. (Coleoptera: Chrysomelidae), is a major pest of cereal crops that has recently been reported in western Canada. We developed a set of primers to detect CLB DNA in the gut of six common predator taxa in wheat fields: lady beetles (20 positives of 143 individuals), nabid bugs (73 positives of 206 individuals), and wolf spiders (2 positives of 25 individuals). Nabis americoferus Carayon (Hemiptera: Nabidae) and Coccinella septempunctata L. (Coleoptera: Coccinellidae) were the most abundant predators in cereal fields, with 0.35 and 0.05 proportion of samples positive for CLB DNA, respectively. The prey DNA half-lives were used to adjust the estimates for N. americoferus to 0.22, due to its longer DNA detectability relative to C. septempunctata. Overall, Hippodamia parenthesis (Say) (Coleoptera: Coccinellidae) had the highest proportion of positives at 0.43. There was a positive association between CLB abundance and proportion of N. americoferus and C. septempunctata positives for CLB DNA. This study highlights the contribution of generalist predators to CLB mortality and their important role in integrated management for CLB. Furthermore, we provide a molecular tool that can be used to identify predators of CLB and predation frequency in agricultural fields .     

PCR-based gut content analysis of insect predators: using ribosomal ITS-1 fragments from prey to estimate predation frequency

We used polymerase chain reaction to determine whether Ostrinia nubilalis (Hübner) (Lepidoptera: Crambidae) DNA was present in the guts of larvae and adult males and females of the generalist predator Coleomegilla maculata De Geer (Coleoptera: Coccinellidae). The predators were fed Ostrinia nubilalis egg masses and allowed to digest at either 20 degrees C or 27 degrees C for time spans ranging from 0 to 12 h. Four primer pairs, specific for O. nubilalis were developed, using a nuclear ribosomal RNA sequence including part of the 18S gene, the complete internal transcribed spacer (ITS-1) region and part of the 5.8S gene. These primers amplified four sequences that were 492, 369, 256 and 150 base pairs long. We found a significant negative effect of time since feeding on the number of bands that could be detected. The shortest fragment was detected for the longest time after feeding (up to 12 h). We found no effect of predator weight, sex, developmental stage, or meal size on the time course over which bands of varying lengths could be detected.     

Strain-specific SCAR markers for the detection of Trichoderma harzianum AS12-2, a biological control agent against Rhizoctonia solani, the causal agent of rice sheath blight

In order to identify a specific marker for T. harzianum AS12-2, a strain capable of controlling rice sheath blight caused by Rhizoctonia solani, UP-PCR was performed using five universal primers (UP) both separately and in pairwise combinations. The application of two UP primers resulted in the amplification of unique fragments from the genomic DNA of T. harzianum AS12-2, clearly distinguishing it from other Trichoderma strains. The unique fragments had no significant sequence homology with any other known sequence available in databases. Based on the sequences of the unique fragments, 14 oligonucleotide primers were designed. Two primer sets amplified a fragment of expected size from the DNA of strain T. harzianum AS12-2 but not from any other examined strains belonging to T. harzianum, to other Trichoderma species assayed, or to other common fungi present in paddy fields of Mazandaran province, Iran. In conclusion, SCAR (sequence characterized amplified regions) markers were successfully identified and rapid, reliable tools were provided for the detection of an effective biocontrol Trichoderma strain, which can facilitate studies of its population dynamics and establishment after release into the natural environment.     

Rapid screening of invertebrate predators for multiple prey DNA targets

DNA-based techniques are providing valuable new approaches to tracking predator-prey interactions. The gut contents of invertebrate predators can be analysed using species-specific primers to amplify prey DNA to confirm trophic links. The problem is that each predator needs to be analysed with primers for the tens of potential prey available at a field site, even though the mean number of species detected in each gut may be as few as one or two. Conducting all these PCRs (polymerase chain reactions) is a lengthy process, and effectively precludes the analysis of the hundreds of predators that might be required for a meaningful ecological study. We report a rapid, more sensitive and practical approach. Multiplex PCRs, incorporating fluorescent markers, were found to be effective at amplifying degraded DNA from predators' guts and could amplify mitochondrial DNA fragments from 10+ species simultaneously without 'drop outs'. The combined PCR products were then separated by size on polyacrylamide gels on an ABI377 sequencer. New primers to detect the remains of aphids, earthworms, weevils and molluscs in the guts of carabid predators were developed and characterized. The multiplex-sequencer approach was then applied to field-caught beetles, some of which contained DNA from as many as four different prey at once. The main prey detected in the beetles proved to be earthworms and molluscs, although aphids and weevils were also consumed. The potential of this system for use in food-web research is discussed.     

Molecular analysis reveals lowbush blueberry pest predation rates depend on ground beetle (Coleoptera: Carabidae) species and pest density

Molecular gut-content analysis allows determination of pest predation by field-collected predators. Ground beetles (Coleoptera: Carabidae) common in lowbush blueberries may consume blueberry spanworm, Itame argillacearia (Packard) (Lepidoptera: Geometridae), and blueberry flea beetle, Altica sylvia Malloch (Coleoptera: Chrysomelidae), providing pest suppression. Using newly developed pest specific primers, laboratory feeding trials showed that the median detection time (MDT) for blueberry spanworm in the largest beetle, Carabus nemoralis O.F. Müller, was 3.7 h, whereas Poecilus lucublandus (Say) and Pterostichus mutus (Say) had MDTs between 27.1 and 31.6 h for both pests. At a field-site with high pest abundances, the probability of detecting blueberry spanworm and blueberry flea beetle DNA was greater in P. lucublandus, 26 and 39 % respectively, than in P.mutus, 8 and 20 % respectively. Only 0 and 1 % of P. lucublandus and P. mutus, respectively, tested positive for blueberry spanworm DNA at a second site with low abundance. At the first site, the probability of detecting pest DNA in both ground beetle species was positively related to pest density. Higher pest DNA detection rates and captures of ground beetles corresponded to field areas where significant pest reductions occurred from late May to early June. Conservation of predatory carabid beetles could lead to valuable biological control in lowbush blueberries.     

The effects of temperature on detection of prey DNA in two species of carabid beetle

PCR-based techniques to investigate predator-prey trophic interactions are starting to be used more widely, but factors affecting DNA decay in predator guts are still poorly understood. Here, we investigated the effects of time since feeding, temperature and amplicon size on the detectability of prey DNA in the gut content of two closely related predator species. Cereal aphids, Sitobion avenae, were fed to the carabid beetles Pterostichus melanarius and Nebria brevicollis. Beetles were allowed to digest their meal at 12 degrees C, 16 degrees C and 20 degrees C, and batches of beetles were subsequently frozen at time periods from 0-72 h after feeding. Aphid DNA was detected within beetles' gut contents using primers amplifying fragments of 85, 231, 317 and 383 bp. Prey DNA detection rates were significantly higher in N. brevicollis than in P. melanarius, indicating fundamental dissimilarities in prey digestion capacities. High temperatures (20 degrees C) and large amplicons (383 bp) significantly decreased detection rates. The shortest amplicon gave the highest prey DNA detection success, whereas no differences were observed between the 231 bp and the 317 bp fragment. Our results indicate that factors such as ambient temperature, predator taxon and amplicon size should all be considered when interpreting data derived from PCR-based prey detection. Correction for such factors should make calculation of predation rates in the field more accurate and could help us to estimate when predation events occur in the field.     

Optimizing methods for PCR-based analysis of predation

Molecular methods have become an important tool for studying feeding interactions under natural conditions. Despite their growing importance, many methodological aspects have not yet been evaluated but need to be considered to fully exploit the potential of this approach. Using feeding experiments with high alpine carabid beetles and lycosid spiders, we investigated how PCR annealing temperature affects prey DNA detection success and how post-PCR visualization methods differ in their sensitivity. Moreover, the replicability of prey DNA detection among individual PCR assays was tested using beetles and spiders that had digested their prey for extended times postfeeding. By screening all predators for three differently sized prey DNA fragments (range 116-612 bp), we found that only in the longest PCR product, a marked decrease in prey detection success occurred. Lowering maximum annealing temperatures by 4 °C resulted in significantly increased prey DNA detection rates in both predator taxa. Among the three post-PCR visualization methods, an eightfold difference in sensitivity was observed. Repeated screening of predators increased the total number of samples scoring positive, although the proportion of samples testing positive did not vary significantly between different PCRs. The present findings demonstrate that assay sensitivity, in combination with other methodological factors, plays a crucial role to obtain robust trophic interaction data. Future work employing molecular prey detection should thus consider and minimize the methodologically induced variation that would also allow for better cross-study comparisons.     

A new cultivation independent approach to detect and monitor common Trichoderma species in soils

A set of primers was developed for the detection, identification and quantification of common Trichoderma species in soil samples. Based on a broad range master alignment primers were derived to amplify an approximate 540 bp fragment comprising the internal transcribed spacer region 1 (ITS 1), 5.8S rDNA and internal transcribed spacer region 2 (ITS 2) from all taxonomic Clades of the genus Trichoderma. The primer set was applied to test strains as well as community DNA isolated from arable and forest soil. For all tested isolates the corresponding internal transcribed spacer regions of Trichoderma spp. strains were amplified, but none of non-Trichoderma origin. PCR with community DNA from soil yielded products of the expected size. Analysis of a clone library established for an arable site showed that all amplified sequences originated exclusively from Trichoderma species mainly being representatives of the Clades Hamatum, Harzianum and Pachybasioides and comprising most of the species known for biocontrol ability. In a realtime PCR approach the primer set uTf/uTr also proved to be a suitable system to quantify DNA of Trichoderma spp. in soils.     

小麦矮腥黑穗菌差异片段筛选与分子检测体系的建立

采取随机扩增DNA多态性(Random amplified polymorphic DNA,RAPD)引物介导的半特异PCR技术(RAPD primer mediated hemi-specific PCR,RM-PCR),在从不同地域征集的18个小麦矮腥黑穗菌(Tilletia controversa Kühn,TCK)菌株和29个小麦网腥黑穗病菌(Tilletia caries(DC)Tul,TCT)菌株的总基因组DNA中筛选鉴定出TCK独有的大小为1322bp差异基因组片段。根据该片段序列设计筛选出2对特异性引物CQUTCK2/CQUTCK3和CQUTCK4/CQUTCK5,均可以从18个TCK菌株的菌丝体和冬孢子DNA中稳定地扩增出747bp和200bp的单一靶带DNA,而在29个TCT菌株的菌丝体或冬孢子DNA均无任何扩增产物。以腥黑穗菌属通用引物对CQUK6/CQUK7为内置对照,可以确定被检样品是否含PCR抑制物质进而判断检测体系是否正确,同时有效地排除样品检测结果的假阳性和假阴性。采用建立的TCK特异PCR检测技术体系,实现简单而快速地鉴定小麦矮腥黑穗菌冬孢子或罹病小麦组织中侵染菌丝体的目的。