Differentiation between Aphis pomi and Aphis spiraecola using multiplex real‐time PCR based on DNA barcode sequences

The green apple aphid (Aphis pomi) and the spirea aphid (Aphis spiraecola) are pests of apples in North America. Although management regimes exist to effectively control these pests, they differ significantly because of varying susceptibility of each species to common pesticides and differences in their life cycles. Therefore, accurate identification of the species present is essential for pest control. However, the identification process is complicated because of the morphological similarity between these two species. As a result, confusion between A. pomi and A. spiraecola often occurs. DNA barcoding has been proven to accurately identify species of Aphididae. A further study demonstrated that DNA barcodes could be used to accurately differentiate A. pomi and A. spiraecola. DNA barcoding represents an important step towards rapid identification of these pests as distinctions can be easily made between morphologically similar species as well as from eggs and immature individuals in addition to adults. However, samples must still be sent to specially equipped facilities for sequence analysis, which can take between several hours and days. Real‐time PCR is emerging as a useful tool for more rapid pest identification. The purpose of this study was to develop a real‐time PCR assay for differentiation of A.pomi from A. spiraecola based on DNA barcode sequences from the Barcode of Life Data System. This assay was designed on the portable SmartCycler II platform and can be used in field settings to differentiate these species quickly and accurately. It has the potential to be a valuable tool to improve pest management of A. pomi and A. spiraecola.     

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).     

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.     

Comparative studies on species identification of Noctuoidea moths in two nature reserve conservation zones (Beijing,China) using DNA barcodes and thin‐film biosensor chips

Rapid and accurate identification of species is required for the biological control of pest Noctuoidea moths. DNA barcodes and thin‐film biosensor chips are two molecular approaches that have gained wide attention. Here, we compare these two methods for the identification of a limited number of Noctuoidea moth species. Based on the commonly used mitochondrial gene cytochrome c oxidase I (the standard DNA barcode for animal species), 14 probes were designed and synthesized for 14 species shared by two national nature reserves in Beijing and Hebei, China. Probes ranged in length from 18 to 27 bp and were designed as mismatch probes to guarantee that there were at least three base differences between the probe and nontarget sequences. The results on the chip could be detected by the naked eye without needing special equipment. No cross‐hybridizations were detected although we tested all probes on the 14 target and 24 nontarget Noctuoidea species. The neighbour‐joining tree of the 38 species based on COI sequences gave 38 highly supported independent groups. Both DNA barcoding and thin‐film biosensor chips, based on the COI gene, are able to accurately identify and discriminate the 14 targeted moth species in this study. Because of its speed, high accuracy and low cost, the thin‐film biosensor chip is a very practical means of species identification. Now, a more comprehensive chip will be developed for the identification of additional Noctuoidea moths for pest control and ecological protection.     

Real‐time PCR assays for rapid detection of Zeugodacus cucumis and Bactrocera jarvisi (Diptera: Tephritidae) for quarantine application

Zeugodacus cucumis and Bactrocera jarvisi are pests of fruit and vegetable crops and cause damage to horticulture industries. Immature stages of these two fruit fly species have been intercepted in New Zealand a number of times. Identification to species was not possible using morphological characters; thus, it is important to develop an assay for their species‐level identification. Here, the real‐time PCR assays for rapid identification of Z. cucumis and B. jarvisi were developed and validated. The PCR protocols demonstrated their specificity by amplifying the two target species successfully, with no cross‐reactions observed in the tested tephritid species. The in silico test of the primer and probe binding sites of the two assays also demonstrated the assays’ specificity by no mismatches present in the binding regions of the target species, but 1–4 mismatches in the binding regions of the non‐target fruit fly species. The thresholds of detection for the two assays are as low as 10 copies/µl of the target DNA, indicating that the assays have a very high sensitivity. The application of the real‐time PCR assays has greatly assisted in routine pest identifications at the New Zealand border and surveillance programme. Therefore, the assays have the potential to be used by diagnostic agencies and research organizations worldwide.     

Introduced ants reduce interaction diversity in a multi‐species,ant–aphid mutualism

Mutualisms contribute in fundamental ways to the origin, maintenance and organization of biological diversity. Introduced species commonly participate in mutualisms, but how this phenomenon affects patterns of interactions among native mutualists remains incompletely understood. Here we examine how networks of interactions among aphid‐tending ants, ant‐tended aphids, and aphid‐attacking parasitoid wasps differ between 12 spatially paired riparian study sites with and without the introduced Argentine ant Linepithema humile in southern California. To resolve challenges in species identification, we used DNA barcoding to identify aphids and screen for parasitoid wasps (developing inside their aphid hosts) from 170 aphid aggregations sampled on arroyo willow Salix lasiolepis. Compared to uninvaded sites, invaded sites supported significantly fewer species of aphid‐tending ants and ant‐tended aphids. At invaded sites, for example, we found only two species of ant‐tended aphids, which were exclusively tended by L. humile, whereas at uninvaded sites we found 20 unique ant–aphid interactions involving eight species of ant‐tended aphids and nine species of aphid‐tending ants. Ant–aphid linkage density was thus significantly lower at invaded sites compared to uninvaded sites. We detected aphid parasitoids in 14% (28/198) of all aphid aggregations. Although the level of parasitism did not differ between invaded and uninvaded sites, more species of wasps were detected within uninvaded sites compared to invaded sites. These results provide a striking example of how the assimilation of introduced species into multi‐species mutualisms can reduce interaction diversity with potential consequences for species persistence.     

Simultaneous identification of Grapholita molesta Busck and Grapholita dimorpha Komai by PCR‐RFLP

Although several molecular diagnostic techniques are available for the identification of the apple‐feeding pests Grapholita molesta Busck and Grapholita dimorpha Komai, these pests are severely affecting apple orchards in Korea. These two pests may be misidentified or the available molecular diagnostic techniques may not facilitate the simultaneous identification of the morphological features of both species. In this study, we developed a multiplex assay for these two species using the polymerase chain reaction – restriction fragment length polymorphism (PCR‐RFLP) method. Sixty‐two specimens were collected from apples presumed infested with moth larvae and from pheromone traps from 2013 to 2014. Both species were identified morphologically, and a partial region of the cytochrome b gene was sequenced to design primers for PCR‐RFLP. Digestion profiles of G. molesta and G. dimorpha, using the Sau3A1 restriction enzyme, were characterized using three DNA fragments each for G. molesta (363 bp, 91 bp and 31 bp) and G. dimorpha (220 bp, 234 bp and 31 bp). The RFLP assay developed for both species in this study was more efficient and accurate than other currently used diagnostic assays and would be helpful to identify field‐collected specimens for pest control research.     

Ant attendance of the cotton aphid is beneficial for okra plants: deciphering multitrophic interactions

相似文献   

Identification and expression profiles analysis of odorant‐binding proteins in soybean aphid,Aphis glycines (Hemiptera: Aphididae)

The soybean aphid, Aphis glycines, is an extreme specialist and an important invasive pest that relies on olfaction for behaviors such as feeding, mating, and foraging. Odorant‐binding proteins (OBPs) play a vital role in olfaction by binding to volatile compounds and by regulating insect sensing of the environment. In this work we used rapid amplification of complementary DNA ends technology to identify and characterize 10 genes encoding A. glycines OBPs (AglyOBPs) belonging to 3 subfamilies, including 4 classic OBPs, 5 Plus‐C OBPs, and one Minus‐C OBP. Quantitative real‐time polymerase chain reaction demonstrated variable specific expression patterns for the 10 genes based on developmental stage and aphid tissue sampled. Expression levels of 7 AglyOBPs (2, 3, 4, 5, 7, 9, and 10) were highest in the 4th instar, indicating that the 4th nymphal instar is an important developmental period during which soybean aphids regulate feeding and search for host plants. Tissue‐specific expression results demonstrated that AglyOBP2, 7, and 9 exhibited significantly higher expression levels in antennae. Meanwhile, ligand‐binding analysis of 5 OBPs demonstrated binding of AglyOBP2 and AglyOBP3 to a broad spectrum of volatiles released by green leaf plants, with bias toward 6‐ to 8‐carbon chain volatiles and strong binding of AglyOBP7 to trans‐β‐farnesene. Taken together, our findings build a foundation of knowledge for use in the study of molecular olfaction mechanisms and provide insights to guide future soybean aphid research.     

A Real‐time PCR Assay to Identify Meloidogyne minor

Meloidogyne minor is a small root‐knot nematode that causes yellow patch disease in golf courses and severe quality damage in potatoes. It was described in 2004 and has been detected in The Netherlands, England, Wales, Northern Ireland, Ireland and Belgium. The nematode often appears together with M. naasi on grasses. It causes similar symptoms on potato tubers as M. chitwoodi and M. fallax, which are both quarantine organisms in Europe. An accurate identification method therefore is required. This study describes a real‐time PCR assay that enables the identification of M. minor after extraction of nematodes from soil or plant samples. Alignments of sequences of rDNA‐ITS fragments of M. minor and five other Meloidogyne species were used to design a forward primer Mminor_f299, a specific primer Mminor_r362 and the specific MGB TaqMan probe P_Mm_MGB321. PCR with this primers and probe results in an amplicon of 64 bp. The analytical specificity of the real‐time PCR assay was assessed by assaying it on six populations of M. minor and on 10 populations of six other Meloidogyne species. Only DNA from M. minor gave positive results in this assay. The assay was able to identify M. minor using DNA from a single juvenile independent from the DNA extraction method used.     

Effects of Rag1 aphid‐resistant soybean on mortality,development, and preference of brown marmorated stink bug

The brown marmorated stink bug, Halyomorpha halys (Stål) (Hemiptera: Pentatomidae), poses a new threat to soybean, Glycine max (L.) Merrill (Fabaceae), production in the north central USA. As H. halys continues to spread and increase in abundance in the region, the interaction between H. halys and management tactics deployed for other pests must be determined. Currently, the soybean aphid, Aphis glycines Matsumura (Hemiptera: Aphididae), is the most abundant and damaging insect pest of soybean in the region. Aphid‐resistant soybean, mainly with the Rag1 gene, is commercially available for management of A. glycines. Here, experiments were performed to evaluate the effects of Rag1 aphid‐resistant soybean on the mortality, development, and preference of H. halys. In a no‐choice test, mortality of H. halys reared on Rag1 aphid‐resistant soybean pods was significantly lower than when reared on aphid‐susceptible soybean pods (28 vs. 53%). Development time, adult weight, and proportion females of surviving adults did not differ when reared on Rag1 aphid‐resistant or aphid‐susceptible soybean pods. In choice tests, H. halys exhibited a preference for Rag1 aphid‐resistant over aphid‐susceptible soybean pods after 4 h, but not after 24 h. Halyomorpha halys exhibited no preference when tested with vegetative‐stage or reproductive‐stage soybean plants. The preference by H. halys for Rag1 aphid‐resistant soybean pods and the decreased mortality when reared on these pods suggests that the use of Rag1 aphid‐resistant soybean may favor this emerging pest in the north central USA.     

DNA‐based discrimination between the sibling species Aphis gossypii Glover and Aphis frangulae Kaltenbach

Abstract Morphologically similar species occur in various groups of insects, including aphid pests. In Europe, Aphis frangulae Kaltenbach and Aphis gossypii Glover (sometimes considered as subspecies) are differentiated usually on the basis of life cycle and host plant. We used a sexual population of A. frangulae collected on the primary host and samples of A. gossypii collected on cucurbits or cotton for the development of molecular markers. DNA sequence data for the gene encoding cytochrome b and for the barcode region of cytochrome oxidase I, as well as a length polymorphism for an intron in the sodium channel para‐type gene discriminated unambiguously between the two taxa. These markers were also used as identification keys for aphids collected on crops belonging to the Solanaceae. The cytochrome b marker differentiates host‐related Aphis gossypii haplotypes, and the para‐type gene intron might be suitable for the resolution of taxonomic problems in other aphid species complexes.     

Life history characteristics of Orius insidiosus (Say) fed Aphis glycines Matsumura

The soybean aphid, Aphis glycines Matsumura, is a new invasive pest of soybeans throughout most of the soybean production areas of North America. Field studies have demonstrated that the indigenous predator, Orius insidiosus (Say), is an important natural enemy of the soybean aphid early the soybean crop season. Because soybean aphid is newly introduced into North America, the life history characteristics of predators fed this aphid are not known. In laboratory assays, we measured the survival, development, longevity and reproduction of O. insidiosus fed 1, 3, 6 or 12 seconds to third instars of soybean aphid. O. insidiosus nymphal development decreased from 34.0 to 21.4 days as the number of soybean aphid nymphs provided increased from 1 to 6 aphid nymphs daily. Stage-specific mortality was highest at 68% for first instar O. insidiosus nymphs fed 1 soybean aphid nymph per day. Adult longevity (43.9 days) and fecundity (49.7 eggs per female) was highest for O. insidiosus fed 6 soybean aphid nymphs daily, but longevity (23.5 days) and fecundity (10.1 eggs per female) declined for adults fed 1 soybean aphid nymph daily. The intrinsic rate of increase of O. insidiosus ranged from 0.048 to 0.133. Compared to other prey species, soybean aphid is an adequate prey item for O. insidiosus. Our results suggest that O. insidiosus will be most effective in suppressing soybean aphid population growth in the initial phase of the aphid’s colonization of soybeans.     

Invasion genetics from eDNA and thousands of larvae: A targeted metabarcoding assay that distinguishes species and population variation of zebra and quagga mussels

Identifying species and population genetic compositions of biological invasions at early life stages and/or from environmental (e)DNA using targeted high‐throughput sequencing (HTS) metabarcode assays offers powerful and cost‐effective means for early detection, analysis of spread patterns, and evaluating population changes. The present study develops, tests, and applies this method with a targeted sequence assay designed to simultaneously identify and distinguish between the closely related invasive Eurasian zebra and quagga mussels (Dreissena polymorpha and D. rostriformis) and their relatives and discern their respective population genetic patterns. Invasions of these dreissenid mussel species have markedly changed freshwater ecosystems throughout North America and Europe, exerting severe ecological and economic damage. Their planktonic early life stages (eggs and larvae) are morphologically indistinguishable, yet each species exerts differential ecological effects, with the quagga often outcompeting the zebra mussel as adults. Our targeted assay analyzes genetic variation from a diagnostic sequence region of the mitochondrial (mt)DNA cytochrome oxidase I (COI) gene, to assess temporal and spatial inter‐ and intra‐specific genetic variability. The assay facilitates analysis of environmental (e)DNA from water, early life stages from thousands of individuals, and simultaneous analysis of 50–100 tagged field‐collected samples. Experiments evaluated its accuracy and performance using: (a) mock laboratory communities containing known DNA quantities per taxon, (b) aquaria with mixed‐species/haplotype compositions of adults, and (c) field‐collected water and plankton versus traditional sampling of adult communities. Results delineated species compositions, relative abundances, and population‐level diversity differences among ecosystems, habitats, time series, and life stages from two allopatric concurrent invasions in the Great Lakes (Lake Erie) and the Hudson River, which had separate founding histories. Findings demonstrate application of this targeted assay and our approach to accurately and simultaneously discern species‐ and population‐level differences across spatial and temporal scales, facilitating early detection and ecological understanding of biological invasions.     

The gnd gene of Buchnera as a new,effective DNA barcode for aphid identification

DNA barcoding uses a standard DNA sequence to facilitate species identification. Although the COI gene has been adopted as the standard, COI alone is imperfect due to several shortcomings. The primary endosymbiont of aphids, Buchnera, has higher evolutionary rates and interspecies divergence than its co‐diverging aphid hosts, making it a potential tool for resolving the ambiguities in aphid taxonomy. We compared the effectiveness of employing two different DNA regions, gnd and COI, for the discrimination of over 100 species of aphids. The mean interspecific divergence of the gnd region was significantly higher than the mean intraspecific variation; there were nearly nonoverlapping distributions between the intra‐ and interspecific samples. In contrast, COI showed a lower interspecific divergence, which led to difficulties in identifying closely related species. Our results show that gnd can identify species in the Aphididae, which suggests that the gnd region of Buchnera is a potentially effective barcode for aphid species identification. We also recommend the 2‐locus combination of gnd + COI as the aphid barcode. This will provide a universal framework for the routine use of DNA sequence data to identify specimens and contribute toward the discovery of overlooked species of aphids.     

Molecular phylogeny reveals the existence of two sibling species in the aphid pest Brachycaudus helichrysi (Hemiptera: Aphididae)

Piffaretti, J., Vanlerberghe‐Masutti, F., Tayeh, A., Clamens, A.‐L., C?ur d’Acier, A. & Jousselin E. (2012). Molecular phylogeny reveals the existence of two sibling species in the aphid pest Brachycaudus helichrysi (Hemiptera: Aphididae). —Zoologica Scripta, 41, 266–280. Brachycaudus helichrysi is a worldwide polyphagous aphid pest that seriously damages its primary hosts (Prunus spp.) and the various cultivated plants among its secondary hosts (e.g. sunflower). A recent study of the Brachycaudus genus suggested that this species might encompass two differentiated lineages. We tested this hypothesis, by carrying out a phylogenetic study of this aphid pest based on worldwide sampling and the evaluation of mitochondrial, nuclear and Buchnera aphidicola DNA markers. We show that this species is actually an amalgamation of two sibling taxa, B. helichrysi H1 and B. helichrysi H2, that seem to have overlapping geographic ranges and herbaceous host plant preferences. These two taxa displayed levels of genetic divergence as great as those generally found between sister species in the Brachycaudus genus, suggesting that they actually correspond to two distinct species. Our phylogenetic reconstructions revealed a degree of incongruence between the topologies obtained with the aphid gene data set and with data for a DNA marker from its primary endosymbiont. We identified possible reasons for this observation and discuss the ecological and genotypic data suggesting that B. helichrysi H1 and B. helichrysi H2 have different life cycles.     

Rapid diagnosis of the invasive species,Frankliniella occidentalis (Pergande): a species‐specific COI marker

The western flower thrips, Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae), is an invasive species and currently occurs in only a few areas in China. An easy, accurate and developmental‐stage independent method to identify F. occidentalis would be a valuable tool to facilitate pest management decision making and, more importantly, to provide an early warning so actions can be taken to prevent its introduction into non‐infested areas. Morphological identification of thrips adults and, to a lesser extent, of second‐stage larvae is the main method currently available to identify F. occidentalis. Molecular identification, however, can be easily carried out by a non‐thrips‐specialist with a little training. In this study, DNA sequence data [within the mitochondrial cytochrome oxidase I gene (COI)] and polymerase chain reaction (PCR) were utilized to develop a molecular diagnostic marker for F. occidentalis. A primer set and PCR cycling parameters were designed for the amplification of a single marker fragment (340 bp) of F. occidentalis COI mtDNA. Specificity tests performed on 28 thrips species, efficacy tests performed on five immature developmental stages as well as on male and female adults and tests on primer sensitivity all demonstrated the diagnostic utility of this marker. Furthermore, the primer set was tested on seventeen F. occidentalis populations from different countries and invaded areas in China and proved to be applicable for all geographic populations. It was used successfully to clarify the distribution of F. occidentalis in the Beijing metro area. These results suggested that this diagnostic PCR assay provides a quick, simple and reliable molecular technique for the identification of F. occidentalis.