Real‐time PCR for identification of the soybean aphid,Aphis glycines Matsumura

The soybean aphid (Aphis glycines Matsumura) is an economically significant pest in North America, causing extensive damage to soybean crops through direct feeding damage and disease transmission. If unchecked, this pest could cause billions of dollars of damage to soybean crops. Identification of the soybean aphid can be difficult due to its small size, complex life cycle and morphological plasticity. Generally, an expert is required to identify a specimen. Additionally, identification of some life stages, such as eggs, is impossible. DNA barcoding has been successfully used to differentiate aphid species, including A. glycines, based on sequencing of a standardized gene region. Although this method represents an important step towards accurate identification, samples must still be sent to specialized facilities for analysis. Using existing DNA barcode sequences in the publically accessible Barcode of Life Data System (BOLD; www.boldsystems.org ), species‐specific differences were identified and used to develop a real‐time PCR assay to identify soybean aphids. This assay can be run on portable systems for rapid, accurate and simple identification in the field. The use of a non‐destructive DNA extraction protocol allows the original insect to be vouchered and therefore available for further study if necessary. This work represents an important step in soybean aphid management.     

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‐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.     

Effects of prey aphid species on the abundance of a parasitoid and two predator species in aphid colonies attended by ants on citrus

This study focused on three species of enemies, the parasitoid wasp Lysiphlebus japonicus Ashmead (Hymenoptera: Aphidiidae), the ladybird Scymnus posticalis Sicard (Coleoptera: Coccinellidae) and the predatory gall midge Aphidoletes aphidimyza (Rondani) (Diptera: Cecidomyiidae), all of which are able to exploit aphids attended by ants. I experimentally evaluated the effects of prey aphid species on the abundance of each of the three enemy species in ant‐attended aphid colonies on citrus. The aphids compared were Aphis gossypii Glover versus Aphis spiraecola Patch in late spring, and Toxoptera citricidus (Kirkaldy) versus A. spiraecola in late summer (all, Hemiptera: Aphididae). Colonies of the three aphid species were attended by the ant Pristomyrmex punctatus Smith (Hymenoptera: Formicidae). The initial number of attending ants per individual aphid did not differ significantly between the colonies of the two aphid species compared in each season. Between A. gossypii and A. spiraecola, there was no significant difference in the number of mummies formed by the parasitoid or foraging larvae of each of the two predators per aphid colony. A significant difference was detected between T. citricidus and A. spiraecola for each of the three enemy species, with a far greater number of L. japonicus mummies in T. citricidus colonies and distinctly more larvae of each of the two predators in A. spiraecola colonies. Thus, the abundance of each of the three enemy species in ant‐attended aphid colonies was significantly influenced by the species of the prey aphids, with the three enemies showing different responses to the three aphid species.     

Why the aphid Aphis spiraecola is more abundant on clementine tree than Aphis gossypii?

Aphis spiraecola and Aphis gossypii cause harmful damages on clementine tree orchards. Weekly surveys measured the abundance of aphids (larvae, winged and wingless adults) as well as of auxiliary insects and parameters of energy metabolism. Correlatively, soluble carbohydrates, total free amino acids, free proline and condensed tannins were quantified in control and infested leaves. Both aphid species showed parallel temporal variations, but A. spiraecola was consistently more abundant regardless of the stage. Amino acids had a positive effect on both aphid species abundance, but neither condensed tannins nor auxiliary insects seemed to modulate aphid populations. Interestingly, the leaf carbohydrate content was positively correlated with the abundance of A. spiraecola, but not with that of A. gossypii. Moreover, A. gossypii's abundance was significantly down-regulated by high proline concentrations. Thus, the higher abundance of A. spiraecola could be explained by a better tolerance to high proline contents and a better conversion of foliar energy metabolites.     

Cucumber mosaic virus epidemiology in pepper: Aphid dispersal,transmission efficiency and vector pressure

Cucumber mosaic virus (CMV) causes significant damage and yield losses in peppers. The objective of this study is to determine the efficiency of prevalent aphid species occurring in pepper fields to transmit this virus within pepper plants and to identify their vector pressure in order to target the critical species implicated in CMV epidemics spread. Alatae and apterae were monitored in an experimental pepper field in northern Tunisia for 3 years. Sixty-eight species were captured in winged form in yellow water traps. The most abundant species were Myzus persicae, Aphis gossypii, Aphis fabae, Aphis spiraecola, Acyrthosiphon pisum, Metopolophium dirhodum, Rhopalosiphum maidis, Aphis craccivora, Aphis nerii, Hyalopterus pruni, Sitobion avenae and Rhopalosiphum padi constituting 90% of aphid populations in the field. Their temporal dynamic showed a high period of flight activity from April to June and a second peak in September was registered. Two of these species, M. persicae and A. gossypii were also found in their wingless form on pepper leaves with a prevalence of 99.5% and 0.5%, respectively. The 12 most abundant aphid species were tested for their transmission efficiency of CMV (CMV-pepp2 isolate) with A. gossypii as a reference vector. All aphids tested, including colonizing and non-colonizing species on pepper, were verified to be vectors of this isolate. However, significant differences in the transmission efficiency were found between the aphid species (p < .001, SE = 7.29). M. persicae (60%) scored the highest transmission efficiency rate. Additionally, A. fabae solanella (50%) had higher transmission efficiency than the reference vector, A. gossypii (40%). H. pruni (16.67%) was documented as a new CMV vector to pepper. The single-aphid transmission probabilities ranged from 0.7% to 16.7%. The calculated mean Vector Pressure Index (VPI) for these 12 species showed a stronger relationship with the specific aphid population variance (R = 0.89, p < .01) than the variation of the specific single-aphid transmission probabilities (R = 0.62, p < .05). Indeed, for alate non-colonizer vectors, A. spiraecola has recorded the highest mean VPI (27.5), despite its moderate transmission efficiency (23.33%). Nevertheless, for colonizer vectors in both winged and wingless forms, M. persicae had the highest mean VPI (49.26) of all vector species and was mostly present in its apterous form. The 12 vector species contributed to a total mean VPI of 133.48 during the surveyed periods. This research determined key features of CMV epidemiology in pepper crops that might be helpful for CMV disease management at an early stage.     

X-linked heterochromatin distribution in the holocentric chromosomes of the green apple aphid <Emphasis Type="Italic">Aphis pomi</Emphasis>

Chromatin organization in the holocentric chromosomes of the green apple aphid Aphis pomi has been investigated at a cytological level after C-banding, NOR, Giemsa, fluorochrome staining and fluorescent in situ hybridization (FISH). C-banding technique showed that heterochromatic bands are exclusively located on X chromosomes. This data represents a peculiar feature that clearly contradicts the equilocal distribution of heterochromatin typical of monocentric chromosomes. Moreover, silver staining and FISH carried out with a 28S rDNA probe localized rDNA genes on one telomere of each X chromosome; CMA₃ staining reveals that these silver positive telomeres are the only GC-rich regions among A. pomi heterochromatin, whereas all other C-positive bands are DAPI positive thus containing AT-rich DNA.     

Evaluation of biorational insecticides and DNA barcoding as tools to improve insect pest management in lablab bean (Lablab purpureus) in Bangladesh

Lablab bean (Lablab purpureus) is a popular vegetable crop in Bangladesh, but farmers growing this crop experience significant losses to insect pests despite heavy reliance on conventional insecticides. We conducted field studies to improve pest management in lablab bean by testing biorational insecticides as alternatives to conventional insecticides for the control of pod borers (Maruca vitrata) and aphids (Aphis craccivora), and characterizing flower-inhabiting thrips as an emerging pest in this crop. In field experiments, spinosad was the most promising biorational we tested, suppressing pod-boring caterpillars more effectively than thiamethoxam or quinalphos. In contrast, azadirachtin (neem) did not significantly suppress any of the insect pests we measured, although target aphid populations were generally low at our research site. Using DNA barcoding at the coxI locus combined with morphological identification, we found eight thrips taxa inhabiting lablab bean flowers, dominated by Megalurothrips usitatus and M. distalis/peculiaris. A preliminary regression analysis indicated that these flower-inhabiting thrips reduced lablab bean yield. Our results suggest that spinosad may be useful within lablab bean IPM programs, and that these programs will likely need to incorporate tactics against thrips to effectively protect yield. Finally, we found that DNA barcoding was a valuable tool for pest identification in an understudied crop and region, but that to reach its full potential will require an investment in more comprehensive reference libraries.     

Coccinellid immigration to infested host patches influences suppression of Aphis glycines in soybean

Generalist natural enemies may be well adapted to annual crop systems in which pests and natural enemies re-colonize fields each year. In addition, for patchily-distributed pests, a natural enemy must disperse within a crop field to arrive at infested host patches. As they typically have longer generation times than their prey, theory suggests that generalist natural enemies need high immigration rates to and within fields to effectively suppress pest populations. The soybean aphid, Aphis glycines Matsumura, is a pest of an annual crop and is predominantly controlled by coccinellids. To test if rates of coccinellid arrival at aphid-infested patches are crucial for soybean aphid control, we experimentally varied coccinellid immigration to 1 m² soybean patches using selective barriers and measured effects on A. glycines populations. In a year with low ambient aphid pressure, naturally-occurring levels of coccinellid immigration to host patches were sufficient to suppress aphid populations, while decreasing coccinellid immigration rates resulted in large increases in soybean aphid populations within infested patches. Activity of other predators was low in this year, suggesting that most of the differences in aphid population growth were due to changes in coccinellid immigration. Alternatively, in a year in which alate aphids continually colonized plots, aphid suppression was incomplete and increased activity of other predatory taxa contributed to adult coccinellid predation of A. glycines. Our results suggest that in a system in which natural enemy populations cannot track pest populations through reproduction, immigration of natural enemies to infested patches can compensate and result in pest control.     

Importance of early arrival of adult <Emphasis Type="Italic">Harmonia axyridis</Emphasis> for control of <Emphasis Type="Italic">Aphis spiraecola</Emphasis> on apple

The biological control of aphid populations may only be possible when natural enemies arrive soon after aphid colonization. This study was done to identify how quickly adult Harmonia axyridis (Pallas) (Coleoptera: Coccinellidae) need to arrive at newly established spirea aphid [Aphis spiraecola Patch (Homoptera: Aphididae)] colonies on apple (Malus domestica Borkh.) to provide population control. A total of 100 newly established spirea aphid colonies were caged in an experimental apple orchard in West Virginia, USA. A single adult H. axyridis was added to each of ten caged colonies at day 0, 5, 10, 15 and 20 days after caging. An additional ten caged colonies were opened for exposure to natural levels of predation at each of the treatment intervals as a control. The single H. axyridis eliminated the aphid colonies significantly more quickly than natural predation for up to ten days after colony establishment. The probability of an aphid colony producing alates was significantly lower in the presence of a single H. axyridis adult than when exposed to natural predation for the first ten days. Adult H. axyridis beetles are capable of completely controlling individual spirea aphid colonies on apple only if they are abundant enough to find colonies within one week of colony establishment.     

Developmental rates of the aphid Aphis pomi (Aphidoidea: Aphididae) and its parasitoid Aphidius ervi (Hymenoptera: Aphidiidae)

Developmental rates of the aphid Aphis pomi and its parasitoid Aphidius ervi were compared at four constant temperatures (15, 20, 25, 30°C). Aphis pomi required 159.07 degree-days (DD) above lover developmental threshold (LDT) of 0.3°C. Aphidius ervi required 229.73 DD above LDT of 5.98°C. Developmental rate of A. pomi is higher than the developmental rate of A. ervi at the same conditions     

Seasonal Phenology and Species Composition of the Aphid Fauna in a Northern Crop Production Area

Background

The species diversity of aphids and seasonal timing of their flight activity can have significant impacts on crop production, as aphid species differ in their ability to transmit plant viruses and flight timing affects virus epidemiology. The aim of the study was to characterise the species composition and phenology of aphid fauna in Finland in one of the northernmost intensive crop production areas of the world (latitude 64°).

Methodology/Principal Findings

Flight activity was monitored in four growing seasons (2007–010) using yellow pan traps (YPTs) placed in 4–8 seed potato fields and a Rothamsted suction trap. A total of 58,528 winged aphids were obtained, identified to 83 taxa based on morphology, and 34 species were additionally characterised by DNA barcoding. Seasonal flight activity patterns analysed based on YPT catch fell into three main phenology clusters. Monoecious taxa showed early or middle-season flight activity and belonged to species living on shrubs/trees or herbaceous plants, respectively. Heteroecious taxa occurred over the entire potato growing season (ca. 90 days). Abundance of aphids followed a clear 3-year cycle based on suction trap data covering a decade. Rhopalosiphum padi occurring at the end of the potato growing season was the most abundant species. The flight activity of Aphis fabae, the main vector of Potato virus Y in the region, and Aphis gossypii peaked in the beginning of potato growing season.

Conclusions/Significance

Detailed information was obtained on phenology of a large number aphid species, of which many are agriculturally important pests acting as vectors of plant viruses. Aphis gossypii is known as a pest in greenhouses, but our study shows that it occurs also in the field, even far in the north. The novel information on aphid phenology and ecology has wide implications for prospective pest management, particularly in light of climate change.     

Spatial and temporal spread of Citrus tristeza virus and its aphid vectors in the North western area of Morocco

First report of Citrus tristeza virus (CTV,Closterovirus) in Morocco datesback to 1961 in collections of citrus varieties. An exhaustive survey of citrus in the north of the country in 2009 revealed that CTV was spread all over the citrus production area. We attempted to evaluate the relative contribution of different aphid species in the spread of CTV disease in a Citrus reticulata orchard at the Loukkous region during 2 years (2012 and 2013). The overall CTV incidence estimated in the experimental site increased from 17.8% in 2012 to 31.15% in 2013. The most abundant aphid species colonising clementine trees was Aphis spiraecola and A. gossypii. Both aphid species reached their maximum peaks during the spring season. The rate of viruliferous aphids, estimated by real‐time RT‐PCR of single aphid, revealed that 35.4% of winged A. gossypii and 28.8% of winged A. spiraecola were viruliferous, confirming a high inoculum pressure in the area surrounding the experimental site. The aphid species Toxoptera citricida, which is able to transmit the aggressive isolates of CTV, was not found in the Loukkous region. The study of the spatial distribution of the CTV showed that in general, the disease was randomly distributed in the field. Overall, the results seem to indicate that A. spiraecola may be considered as the major aphid species contributing to CTV spread in our experimental conditions. The prevalence of mild strains in the region and the high level of aphid flight activity could explain the rapid evolution of CTV incidence in the experimental area.     

Efficacy of using DNA barcoding to identify parasitoid wasps of the melon-cotton aphid (<Emphasis Type="Italic">Aphis gossypii</Emphasis>) in watermelon cropping system

Parasitoid wasps have received a great deal of attention in the biological control of melon-cotton aphid (Aphis gossypii Glover). The species of parasitoids are often difficult to identify because of their small body size and profound diversity. DNA barcoding offers scientists who are not expert taxonomists a powerful tool to render their field studies more accurate. Using DNA barcodes to identify aphid parasitoid wasps in specific cropping systems may provide valuable information for biological control. Here, we report the use of DNA barcoding to confirm the morphological identification of 14 species (belonging to 13 genera of 7 families) of parasitoid wasps from two-year field samples in a watermelon cropping system. We generated DNA sequences from the mitochondrial COI gene and the nuclear D2 region of 28S rDNA to assess the genetic variation within and between parasitoid species. Automatic Barcode Gap Discovery (ABGD) supported the presence of 14 genetically distinct groups in the dataset. Among the COI sequences, we found no overlap between the maximum K2P distance within species (0.49%) and minimum distance between species (6.85%). The 28S sequences also showed greater interspecific distance than intraspecific distance. DNA barcoding confirmed the morphological identification. However, inconsistency and ambiguity of taxonomic information available in the online databases has limited the successful use of DNA barcoding. Only five species matched those in the BOLD and GenBank. Four species did not match the entries in GenBank and five species showed ambiguous results in BOLD due to confusing nomenclature. We suggested that species identification based on DNA barcodes should be performed using both COI and other genes. Nonetheless, we demonstrate the potential of the DNA barcoding approach to confirm field identifications and to provide a foundation for studies aimed at improving the understanding of the biocontrol services provided by parasitoids in the melon ecosystem.     

Behavioural response of alate Aphis craccivora Koch (Homoptera: Aphididae) to volatiles from different cowpea cultivars

The cowpea aphid, Aphis craccivora, is a major insect pest of cowpea in Africa. Volatile organic compounds (VOCs) mediate plant–arthropod interactions that could be used in the management of insect pests. In this study, we established the VOC profile involved in the interaction between A. craccivora and four cowpea cultivars, namely Ex‐Luanda, Katumani 80, Machakos 66 and Ken Kunde 1. Behavioural assays were conducted to study host preference and gas chromatography‐mass spectrometry (GC/MS) for chemical analysis of volatiles. In preference assays, alate A. craccivora had no significant preference for any of the four cowpea cultivars tested. However, in the olfactometer assays, the aphids showed a significant preference for odours from cultivar Ex‐Luanda compared to Katumani 80. Machakos 66 and Ken Kunde 1 elicited neutral responses. In pairwise comparisons, alate A. craccivora did not distinguish between odours of respective cowpea cultivars. GC/MS analysis identified 23 compounds in the volatiles of the four cowpea cultivars. Not all compounds were detected in all cowpea cultivars, and the detected compounds amounts varied in each cultivar. Of these, only four compounds (hexanal, (E)‐2‐hexenal, 1‐octen‐3‐ol and p‐xylene) were emitted in significantly different quantities in the four cultivars. A blend of hexanal and (E)‐2‐hexenal added to cowpea cultivar Ex‐Luanda decreased its attractiveness to A. craccivora compared to the control. Our findings showed differential attractiveness of VOCs of cowpea cultivars to A. craccivora, suggesting that VOCs could be used in the management of A. craccivora.     

Genetic variability of green citrus aphid populations from Tunisia,assessed by RAPD markers and mitochondrial DNA sequences

The green citrus aphid Aphis spiraecola (Patch) is one of the major pests of several plant species including economically important crops such as citrus. In this study, we used random amplified polymorphic DNA (RAPD) markers and mitochondrial cytochrome oxidase subunit I sequences to assess the level and distribution of genetic diversity of A. spiraecola populations reared from Rutaceae and Rosaceae in different regions in Tunisia. RAPD analysis conducted on 141 individuals with 5 primers revealed only 50 polymorphic RAPD markers, indicating a low genetic diversity that might result from the lack of sexual phase for this species in Tunisia. Analysis of molecular variance (amova ) showed that the genetic structure was not associated with geographic location or year of collection (P = 0.70 and 0.34, respectively); however, the host‐plant had a significant effect on the partitioning of the total genetic diversity (P < 0.01). Multidimensional scaling analysis indicated that the distribution of genetic variability was significantly influenced by the host‐plant with no evidence of spatial differentiation. Based on 20 barcode sequences of the mitochondrial cytochrome‐c oxidase subunit I (COI) gene, we revealed the occurrence of two haplotypes in association with the host‐plant. Results reported here suggest the occurrence of a limited gene flow between A. spiraecola populations from Rosaceae and Rutaceae and, therefore, a possible host‐race status that could be considered in the development of an integrated controlling strategy.     

QTL analysis for aphid resistance and growth traits in apple

The rosy apple aphid (Dysaphis plantaginea), the leaf-curling aphid (Dysaphis cf. devecta) and the green apple aphid (Aphis pomi) are widespread pest insects that reduce growth of leaves, fruits and shoots in apple (Malus × domestica). Aphid control in apple orchards is generally achieved by insecticides, but alternative management options like growing resistant cultivars are needed for a more sustainable integrated pest management (IPM). A linkage map available for a segregating F₁-cross of the apple cultivars ‘Fiesta’ and ‘Discovery’ was used to investigate the genetic basis of resistance to aphids. Aphid infestation and plant growth characteristics were repeatedly assessed for the same 160 apple genotypes in three different environments and 2 consecutive years. We identified amplified fragment length polymorphism (AFLP) markers linked to quantitative trait loci (QTLs) for resistance to D. plantaginea (‘Fiesta’ linkage group 17, locus 57.7, marker E33M35–0269; heritability: 28.3%), and to D. cf. devecta (‘Fiesta’ linkage group 7, locus 4.5, marker E32M39–0195; heritability: 50.2%). Interactions between aphid species, differences in climatic conditions and the spatial distribution of aphid infestation were identified as possible factors impeding the detection of QTLs. A pedigree analysis of simple sequence repeat (SSR) marker alleles closely associated with the QTL markers revealed the presence of the alleles in other apple cultivars with reported aphid resistance (‘Wagener’, ‘Cox’s Orange Pippin’), highlighting the genetic basis and also the potential for gene pyramiding of aphid resistance in apple. Finally, significant QTLs for shoot length and stem diameter were identified, while there was no relationship between aphid resistance and plant trait QTLs. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Influence of vermicompost and cucumber cultivar on population growth of Aphis gossypii Glover

The melon aphid, Aphis gossypii Glover (Hem., Aphididae), is one of the most important pests of cucumber throughout the world. This aphid has a short generation time and high fecundity that result in an enormous reproductive potential, especially in cucumber‐growing greenhouses. Vermicomposts, which are produced by exploiting interactions between earthworms and microorganisms, may enhance plant growth and plant resistance against some pests and disease. In this study, the effects of vermicompost and cucumber cultivar (Cucumis sativus L.) on infestation levels with A. gossypii were evaluated. We conducted a factorial experiment with two cucumber cultivars (Royal and Storm) and five concentrations of vermicompost in the soil, including 0% (control), 10%, 20%, 30% and 50%, employing a randomized complete block design with four replicates. The experiment was conducted in a growth chamber at 25 ± 2°C, 65 ± 10% RH and a photoperiod of 14 L: 10 D h. The number of aphids was counted 3, 5, 7, 9, 12, 15, 18 and 21 days after infestation of cucumber seedlings by aphids. We found that in all vermicompost‐amended treatments, aphid numbers were lower than when plants were grown in soil without any vermicompost. The highest and lowest aphid counts occurred in the control treatment on cucumbers of the Royal cultivar and in the 30% and 50% vermicompost treatments on the storm cultivar, respectively. Overall, our study showed that the application of vermicompost has a high potential for reducing A. gossypii populations in cucumber cultures.     

DNA barcoding of Bradysia (Diptera: Sciaridae) for detection of the immature stages on agricultural crops

We investigated the usefulness of mitochondrial cytochrome c oxidase (COI) DNA barcoding of the genus Bradysia for the detection of immature stages and cryptic species complex. Although the larvae of some species in this genus are agricultural pests, immature stages are rarely identified due to the lack of key morphological characteristics. We constructed partial sequences of the COI gene for 25 species of Bradysia as a first step towards a DNA barcode. Using these data, Bradysia impatiens, B. procera and B. peraffinis were identified from larval specimens collected, respectively, from paprika, ginseng and oak sawdust beds used for cultivating shiitake. Our findings reveal a complex of three species within the B. tilicola group. These species were all identified as important pest B. ocellaris based on the morphology of male genital structures; however, the interspecific genetic divergence of the COI region was significantly greater (16.1–19.4%) than the intraspecific variation in each species. Therefore, B. ocellaris may consist of at least three species. The results demonstrate that COI DNA barcodes are useful for Bradysia species identification.