Exaptation of I4760M mutation in ryanodine receptor of Spodoptera exigua (Lepidoptera: Noctuidae): Lessons from museum and field samples.

Since 2007, diamide insecticides have been widely used in Korea to control various types of lepidopteran pests including Spodoptera exigua. For nearly a decade, diamide resistance in field populations of S. exigua across 18 localities has been monitored using bioassays. Despite their short history of use, resistance to diamide insecticides has emerged. Based on the LC50 values, some field populations showed a higher level of resistance to chlorantraniliprole, a diamide insecticide, compared to that of the susceptible strain, although regional and temporal variations were observed. To investigate resistance at a molecular level, we examined three mutations (Y4701C, I4790M, and G4946E) in the ryanodine receptor (RyR), which is the primary mechanism underlying diamide insecticide resistance. DNA sequencing showed that only the I4790M mutation was found in most field populations. As resistance levels varied significantly despite the uniform presence of the I4790M mutation, we considered the presence of another resistance factor. Further, the I4790M mutation was also found in S. exigua specimens collected prior to the commercialization of diamide insecticides in Korea as well as in other countries, such as the USA. This finding led us to hypothesize that the I4790M mutation were predisposed in field populations owing to selection factors other than diamide use. For further clarification, we conducted whole-genome sequencing of S. exigua (449.83 Mb) and re-sequencing of 18 individual whole genomes. However, no additional non-synonymous mutations were detected in the RyR-coding region. Therefore, we concluded that the high level of diamide insecticide resistance in Korean S. exigua is not caused by mutations at the target site, RyR, but is attributed to other factors that need to be investigated in future studies.

Ultra-deep sequencing of 45S rDNA to discern intragenomic diversity in three Chrysodeixis species for molecular identification.

Species identification is necessary to prevent introductions of exotic plant pests through global trade. Many of these pests are understudied and lack publicly available DNA sequence data on which rapid molecular identification methods can be based. One such lineage is the genus Chrysodeixis, which includes three species of potential concern for United States trade initiatives: C. includens, C. chalcites, and C. eriosoma. Here we describe a method to generate robust 45S rDNA profiles using long read sequencing in order to clarify evolutionary relationships and develop a real-time PCR identification technique. Such an identification tool will be useful in rapidly differentiating between Chrysodeixis species of quarantine concern where traditional morphological identification methods are insufficient. Molecular methods such as this greatly reduce the time spent identifying each specimen, allow for detection of eDNA, vastly increase throughput, and increase the probability of detection. The methods presented here will be generally adaptable to any understudied lepidopteran taxa that necessitates a molecular diagnostic assay and, with adjustment or testing of the primers, could be applied to any group for which development of rDNA profiles in a benchtop setting would prove useful.

Gibbalaria : A new genus of Olethreutini from the Afrotropical Region (Lepidoptera: Tortricidae: Olethreutinae), and a new combination in Cosmopoda Diakonoff.

Gibbalaria Brown and Aarvik, new genus, is described to accommodate the following species: G. divergana Brown and Aarvik, new species (TL: South Africa); G. occidentana Brown and Aarvik, new species (TL: South Africa); G. chiloanei Brown and Aarvik, new species (TL: South Africa); G. scabellana Zeller, 1852, new combination (TL: South Africa); G. mabalingwae Razowski, 2008, status revised, new combination (TL: South Africa); G. longiphallus Brown and Aarvik, new species (TL: South Africa); G. bagamoyo (Razowski & Wojtusiak, 2014), new combination (TL: Tanzania); and G. analcis (Razowski, 2015), new combination (TL: Republic of Cameroon). Gibbalaria sistrata (Meyrick, 1911), new combination (TL: South Africa), is synonymized with G. scabellana, new synonymy. Although the male genitalia of Gibbalaria form two moderately divergent groups, the two groups share extremely similar female genitalia, which feature a broad, somewhat cylindrical antrum, followed immediately anterad by a conspicuously elbowed region of the ductus bursae forming a weak diverticulum on the left side, and a signum in the form of a patch of small arrowhead-shaped sclerites, usually with a shallow pocket. Male genitalia morphology shows many similarities with the Neopotamia group (Olethreutini), Afroploce Aarvik in particular. The monophyly of Gibbalaria is supported by the raised, curled, opalescent scales on the forewing in both sexes, which occurs in a few other genera scattered through the subfamily (e.g., Thylacandra Diakonoff in Grapholitini; Astronauta Diakonoff and Cosmopoda Diakonoff in Olethreutini); the angled diverticulum in the ductus bursae in the female genitalia; the subrectangular flange from the lower margin of the valva immediately basal to the cucullus; and the setose, protruding ridge immediate above the basal cavity, which appears to be homologous with the subbasal process of the valva in many members of the Neopotamia group of genera. In addition, Diakonoffiana graziani Razowski, 2015 (TL: Republic of Cameroon) is transferred to Cosmopoda Diakonoff, resulting in C. graziani (Razowski), new combination.

CO1 barcodes resolve an asymmetric biphyletic clade for Diabrotica undecimpunctata subspecies and provide nucleotide variants for differentiation from related lineages using real-time PCR.

Diabrotica undecimpunctata is a multivoltine polyphagous beetle species that has long been documented as a significant agricultural pest throughout its native range in North America. This beetle can vector bacterial and viral plant pathogens that result in major losses to crops such as cucumber and soybean. Many countries outside the Americas treat D. undecimpunctata as a species of quarantine importance, while in the USA only the subspecies D. u. duodecimnotata is subject to quarantine, to prevent introduction from Mexico. Identification of D. undecimpunctata on the basis of morphology alone can be complicated given the use of conflicting characters in the description of some subspecific taxa. To better understand relationships among D. undecimpunctata subspecies and other related species, we sequenced mitochondrial cytochrome oxidase 1 (CO1) and nuclear internal transcribed spacer 2 (ITS2) DNA from individuals in different subspecific taxa and across different parts of the species range using museum samples and interceptions. When our data were combined with publicly available Diabrotica data, no pattern of divergence consistent with the currently recognized subspecific designations was found. In addition, we compared phylogenetic patterns in CO1 data from the congener D. virgifera to demonstrate the utility of mitochondrial data in resolving subspecies. From the CO1 data, a diagnostic real-time PCR assay was developed that could successfully identify all haplotypes within the large D. undecimpunctata clade for use in surveys and identification at ports of entry. These findings underscore the need to resolve molecular and morphological datasets into cogent, lineage-based groupings. Such efforts will provide an evolutionary context for the study of agriculturally important attributes of Diabrotica such as host preferences, xenobiotic metabolism, and natural and anthropogenic patterns of dispersal.

A Droplet Digital PCR (ddPCR) Assay to Detect Phthorimaea absoluta (Lepidoptera: Gelechiidae) in Bulk Trap Samples.

The moth species Phthorimaea absoluta (Meyrick) (formerly Tuta absoluta) is serious threat to tomato and other Solanaceous crops worldwide and is invasive throughout Europe, Asia, and Africa. While P. absoluta has not yet been found in the U.S. recent detections in the Caribbean have raised concerns that the species could be introduced to mainland North America. To improve detection capacity, a droplet digital PCR (ddPCR) assay was developed that employs a nondestructive bulk DNA extraction method able to detect one P. absoluta sample among 200 nontargets. Such high-throughput and sensitive molecular assays are essential to preventing introductions through early detection and response. This assay can also be used in areas where P. absoluta is established to monitor outbreaks and track migratory patterns.

Assay Optimization Can Equalize the Sensitivity of Real-Time PCR with ddPCR for Detection of Helicoverpa armigera (Lepidoptera: Noctuidae) in Bulk Samples.

Helicoverpa armigera (Hübner) is one of the most important agricultural pests in the world. This historically Old World species was first reported in Brazil in 2013 and has since spread throughout much of South America and into the Caribbean. Throughout North America, H. armigera surveys are ongoing to detect any incursions. Each trap is capable of capturing hundreds of native Helicoverpa zea (Boddie). The two species cannot be separated without genitalic dissection or molecular methods. A ddPCR assay is currently used to screen large trap samples, but this equipment is relatively uncommon and expensive. Here, we optimized a newly designed assay for accurate and repeatable detection of H. armigera in bulk samples across both ddPCR and less costly, and more common, real-time PCR methods. Improvements over previously designed assays were sought through multiple means. Our results suggest bulk real-time PCR assays can be improved through changes in DNA extraction and purification, so that real-time PCR can be substituted for ddPCR in screening projects. While ddPCR remains a more sensitive method for detection of H. armigera in bulk samples, the improvements in assay design, DNA extraction, and purification presented here also enhance assay performance over previous protocols.

Comparative transcriptomics of Diuraphis noxia and Schizaphis graminum fed wheat plants containing different aphid-resistance genes.

The molecular bases of aphid virulence to aphid crop plant resistance genes are poorly understood. The Russian wheat aphid, Diuraphis noxia, (Kurdjumov), and the greenbug, Schizaphis graminum (Rondani), are global pest of cereal crops. Each species damages barley, oat, rye and wheat, but S. graminum includes fescue, maize, rice and sorghum in its host range. This study was conducted to compare and contrast the transcriptomes of S. graminum biotype I and D. noxia biotype 1 when each ingested phloem from leaves of varieties of bread wheat, Triticum aestivum L., containing no aphid resistance (Dn0), resistance to D. noxia biotype 1 (Dn4), or resistance to both D. noxia biotype 1 and S. graminum biotype I (Dn7, wheat genotype 94M370). Gene ontology enrichments, k-means analysis and KEGG pathway analysis indicated that 94M370 plants containing the Dn7 D. noxia resistance gene from rye had stronger effects on the global transcriptional profiles of S. graminum and D. noxia relative to those fed Dn4 plants. S. graminum responds to ingestion of phloem sap from 94M370 plants by expression of unigenes coding for proteins involved in DNA and RNA repair, and delayed tissue and structural development. In contrast, D. noxia displays a completely different transcriptome after ingesting phloem sap from Dn4 or 94M370 plants, consisting of unigenes involved primarily in detoxification, nutrient acquisition and structural development. These variations in transcriptional responses of D. noxia and S. graminum suggest that the underlying evolutionary mechanism(s) of virulence in these aphids are likely species specific, even in cases of cross resistance.

Modeling Aceria tosichella biotype distribution over geographic space and time.

The wheat curl mite, Aceria tosichella Keifer, one of the most destructive arthropod pests of bread wheat worldwide, inflicts significant annual reductions in grain yields. Moreover, A. tosichella is the only vector for several economically important wheat viruses in the Americas, Australia and Europe. To date, mite-resistant wheat genotypes have proven to be one of the most effective methods of controlling the A. tosichella-virus complex. Thus, it is important to elucidate A. tosichella population genetic structure, in order to better predict improved mite and virus management. Two genetically distinct A. tosichella lineages occur as pests of wheat in Australia, Europe, North America, South America and the Middle East. These lineages are known as type 1 and type 2 in Australia and North America and in Europe and South America as MT-8 and MT-1, respectively. Type 1 and type 2 mites in Australia and North America are delineated by internal transcribed spacer 1 region (ITS1) and cytochrome oxidase I region (COI) sequence differences. In North America, two A. tosichella genotypes known as biotypes are recognized by their response to the Cmc3 mite resistance gene in wheat. Aceria tosichella biotype 1 is susceptible to Cmc3 and biotype 2 is virulent to Cmc3. In this study, ITS1 and COI sequence differences in 25 different populations of A. tosichella of known biotype 1 or biotype 2 composition were characterized for ITS1 and COI sequence differences and used to model spatio-temporal dynamics based on biotype prevalence. Results showed that the proportion of biotype 1 and 2 varies both spatially and temporally. Greater ranges of cropland and grassland within 5000m of the sample site, as well as higher mean monthly precipitation during the month prior to sampling appear to reduce the probability of occurrence of biotype 1 and increase the probability of occurrence of biotype 2. The results suggest that spatio-temporal modeling can effectively improve A. tosichella management. Continual integration of additional current and future precipitation and ground cover data into the existing model will further improve the accuracy of predicting the occurrence of A. tosichella in annual wheat crops, allowing producers to make informed decisions about the selection of varieties with different A. tosichella resistance genes.

A Real-Time PCR Assay for Rapid Identification of Tuta absoluta (Lepidoptera: Gelechiidae).

The tomato leafminer, Tuta absoluta (Meyrick), is a highly destructive pest of tomatoes, causing damage to leaves, stalks, buds, and fruits. Native to South America, T. absoluta is now found throughout Europe, South Asia, Africa, parts of Central America, and the Caribbean. Adults are small, with a wingspan of approximately one cm and lack distinctive markings, making morphological identification difficult. Larvae are also difficult to identify and resemble those of many other gelechiids. Due to the extensive time spent and expertise required for morphological identification, and the imminent threat to the North American tomato crop, we have developed a rapid molecular test for discriminating individual specimens of T. absoluta using a probe-based real-time polymerase chain reaction (PCR) assay. The assay is able to quickly distinguish T. absoluta from similar-sized moth specimens that are attracted to T. absoluta pheromone lures in the United States and is also able to identify larvae of T. absoluta. Decreased identification time for this critical pest will lead to more rapid identification at ports of entry and allow for more efficient trap screening for domestic monitoring programs.

Identification of Heliothine (Lepidoptera: Noctuidae) Larvae Intercepted at U.S. Ports of Entry From the New World.

Heliothine larvae, especially early instars, are difficult to identify, and determinations sometimes rely on indirect information such as origin and host data. The introduction of Helicoverpa armigera (Hübner) into the New World has undermined the reliability of host and origin data to identify intercepted Helicoverpa larvae, and suspect Heliothinae/Helicoverpa larvae intercepted at U.S. ports of entry are now screened for H. armigera and Helicoverpa zea (Boddie) using molecular methods. Here, we analyze heliothine larvae intercepted during 2014-2106 to identify nontargets and evaluate morphological characters traditionally used to separate taxa. In total, nine species were identified, with Chloridea virescens (Fabricius) making up the bulk of interception records. The majority of heliothine suspects originate from Mexico and Peru on pigeon pea, chickpea, tomatillo, pea, and corn. Helicoverpa armigera is commonly intercepted from Peru on pea. Chloridea virescens is recorded from every country where interceptions were identified for this study except Guatemala and is found on multiple hosts. Identification issues and specific host/origin associations are discussed in detail.

A ddPCR Assay for Identification of Autographa gamma (Noctuidae: Plusiinae) in Bulk Trap Samples.

The silver Y moth [Autographa gamma (Linneaus) (Noctuidae: Plusiinae)] is a pervasive crop pest in its native range but has not been found in moth surveys in the United States. Specimens of A. gamma are often intercepted at U.S. ports of entry, so the risk of introduction of this invasive species is high. Currently, identification of Plusiinae adults captured in domestic surveys is done by morphlogical comparison; however, this method is time consuming and misidentifications have occurred in the past. A recent study outlined a real-time PCR assay capable of rapidly identifying individual A. gamma specimens using CO1. This same study provided preliminary data for a droplet digital PCR (ddPCR) assay capable of processing bulk trap samples. Here, we develop and test a ddPCR assay for detecting a single A. gamma in a trap sample of 200 individual moths. This assay will drastically reduce the time and cost needed to screen domestic trap samples for A. gamma.

A droplet digital PCR (ddPCR) assay to detect Helicoverpa armigera (Lepidoptera: Noctuidae) in bulk trap samples.

Moths in the genus Helicoverpa are some of the most important agricultural pests in the world. Two species, H. armigera (Hübner) and H. zea (Boddie), cause the majority of damage to crops and millions of dollars are spent annually on control of these pests. The recent introduction of H. armigera into the New World has prompted extensive survey efforts for this species in the United States. Surveys are conducted using bucket traps baited with H. armigera pheromone, and, because the same pheromone compounds attract both species, these traps often capture large numbers of the native H. zea. Adult H. armigera and H. zea are very similar and can only be separated morphologically by minor differences in the genitalia. Thus, a time consuming genitalic dissection by a trained specialist is necessary to reliably identify either species, and every specimen must be dissected. Several molecular methods are available for differentiating and identifying H. armigera and H. zea, including two recently developed rapid protocols using real-time PCR. However, none of the published methods are capable of screening specimens in large batches. Here we detail a droplet digital PCR (ddPCR) assay that is capable of detecting a single H. armigera in a background of up to 999 H. zea. The assay has been tested using bulk extractions of 1,000 legs from actual trap samples and is effective even when using poor quality samples. This study provides an efficient, rapid, reproducible, and scalable method for processing H. armigera survey trap samples in the U.S. and demonstrates the potential for applying ddPCR technology to screen and diagnose invasive species.

Validating sonication as a DNA extraction method for use with carrion flies.

Entomological evidence can be critical in establishing a postmortem interval estimate. DNA-based species identification can be an extremely valuable tool for forensic entomology. The problem of processing samples in a consistent, cost-effective manner that retains the morphological attributes of the specimen for vouchering has led us to investigate sonication as a primary means of non-destructive DNA extraction from carrion flies. We analyze the efficacy of this technique and compare it to an established DNA extraction technique - the Qiagen DNeasy tissue kit. Our results indicate that sonication produces a significant reduction in the sequence length and lower PHRED quality scores when compared to sequences using DNA obtained using the DNeasy kit, but species identification and phylogenetic inferences between sonication and DNeasy extractions are equivalent.

Virulent Diuraphis noxia Aphids Over-Express Calcium Signaling Proteins to Overcome Defenses of Aphid-Resistant Wheat Plants.

The Russian wheat aphid, Diuraphis noxia, an invasive phytotoxic pest of wheat, Triticum aestivum, and barley, Hordeum vulgare, causes huge economic losses in Africa, South America, and North America. Most acceptable and ecologically beneficial aphid management strategies include selection and breeding of D. noxia-resistant varieties, and numerous D. noxia resistance genes have been identified in T. aestivum and H. vulgare. North American D. noxia biotype 1 is avirulent to T. aestivum varieties possessing Dn4 or Dn7 genes, while biotype 2 is virulent to Dn4 and avirulent to Dn7. The current investigation utilized next-generation RNAseq technology to reveal that biotype 2 over expresses proteins involved in calcium signaling, which activates phosphoinositide (PI) metabolism. Calcium signaling proteins comprised 36% of all transcripts identified in the two D. noxia biotypes. Depending on plant resistance gene-aphid biotype interaction, additional transcript groups included those involved in tissue growth; defense and stress response; zinc ion and related cofactor binding; and apoptosis. Activation of enzymes involved in PI metabolism by D. noxia biotype 2 aphids allows depletion of plant calcium that normally blocks aphid feeding sites in phloem sieve elements and enables successful, continuous feeding on plants resistant to avirulent biotype 1. Inhibition of the key enzyme phospholipase C significantly reduced biotype 2 salivation into phloem and phloem sap ingestion.

Reference gene selection for quantitative real-time PCR normalization in larvae of three species of Grapholitini (Lepidoptera: Tortricidae).

Despite the agricultural importance of species in the Grapholitini (Lepidoptera: Tortricidae), and the value of gene expression analysis for improved population management, few gene expression studies based on quantitative real-time PCR (qPCR) have been conducted for this tribe. Part of the reason for this lack of information is that suitable reference genes, which are fundamental for accurate normalization of qPCR studies, have not been identified for the tribe. Thus, the expression stability of six potential reference genes (ACT, AK, COI, EF1, ENO and TUB) was assessed in three different tissues (whole body, midgut and cuticle) of Cryptophlebia peltastica (Meyrick), Cydia pomonella (L.) and Thaumatotibia leucotreta (Meyrick). Additionally, these reference genes were tested using T. leucotreta at different temperatures (15°C, 25°C and 35°C) with and without baculovirus infection. Suitable reference genes were identified for the whole body and midgut tissue of all three species, and for cuticle tissue of Cy. pomonella and T. leucotreta. When T. leucotreta was infected with the virus at all temperature conditions ACT, AK and EF1 were found to be the most suitable reference genes for experimental normalization. In general, for all tissue types, species and stress conditions, AK and EF1 were the best-performing reference genes. However, even though the three species analysed were closely related and within the same tribe, each species required varying gene combinations for suitable normalization. This study provides the first reference gene evaluation for the Tortricidae, and paves the way for future qPCR analysis in Tortricidae.

A new genus of Grapholitini from Africa related to Thaumatotibia (Lepidoptera, Tortricidae).

Thaumatovalva gen. n. is described and illustrated from the Afrotropical region. As currently defined the genus includes four species: T. deprinsorum sp. n. from the Democratic Republic of Congo; T. albolineana sp. n. (type species) from the Democratic Republic of Congo; T. spinai (Razowski & Trematerra), comb. n., from Ethiopia and Nigeria; and T. limbata (Diakonoff), comb. n., from the Seychelles and Kenya. Thaumatovalva limbata has been reared from the fruit of Cordia somaliensis Baker and C. monoica Roxb. (Boraginaceae) in Kenya. Although structures of the male and female genitalia are extremely similar among three of the four species, male secondary scales on the under surface of the hindwing easily distinguish them.

DNA-based identification of forensically important Lucilia (Diptera: Calliphoridae) in the continental United States.

Correct species identification is critical when dipteran larvae are used for inference of the postmortem interval. To facilitate DNA-based identification of forensically important flies of the genus Lucilia in the continental United States, we develop a vouchered reference collection and DNA sequence database. A total of 122 specimens were collected for nine of the 10 species of Lucilia reported to occur in the continental United States. Using the polymerase chain reaction and DNA sequencing, data were obtained for an 1100-bp region of the mitochondrial gene encoding cytochrome oxidase I (COI). We consider a species suitable for DNA-based identification if it is exclusively monophyletic in >95% of bootstrap pseudoreplicate phylogenetic analyses. Seven of the nine species meet that criterion. Two species (Lucilia coeruleiviridis and Lucilia mexicana) share COI sequence and cannot be distinguished using our reference database. We conclude that DNA-based identification is likely to be successful for the other seven species.

Genetic analysis of Cydia pomonella (Lepidoptera: Tortricidae) populations with different levels of sensitivity towards the Cydia pomonella granulovirus (CpGV).

Microsatellite (simple sequence repeats, SSR) and mitochondrial DNA markers were used to assess the structure of European codling moth populations showing different levels of susceptibility towards one of the most important biocontrol agents used in apple production, the Cydia pomonella granulovirus CpGV-M. In 638 C. pomonella individuals from 33 different populations a total of 92 different alleles were scored using six SSR loci. The global estimate of genetic differentiation for all 33 populations was not significantly different from zero, thus indicating a lack of genetic differentiation. AMOVA analysis revealed a very weak but significant variance among C. pomonella populations from different geographic regions, however, no significant variation was evident between CpGV-M resistant or susceptible C. pomonella populations. Sequence analysis of a fragment of the cytochrome oxidase subunit 1 in eight C. pomonella populations resulted in 27 haplotypes, which were grouped in two distinct clusters. Again, no genetic differentiation between CpGV-M resistant and susceptible codling moth populations was detectable. In addition, Structure analysis using microsatellites and association tests with mtDNA haplotypes found neither population-level nor individual correlations associated with CpGV-M resistance. Accordingly, this lack of population structure does not allow discriminating between one or several, separate origins of CpGV-M resistance.

mtDNA-based identification of Lucilia cuprina (Wiedemann) and Lucilia sericata (Meigen) (Diptera: Calliphoridae) in the continental United States.

Existing data suggest that the forensically important dipteran species Lucilia cuprina (Wiedemann) and Lucilia sericata (Meigen) may be particularly difficult to discriminate using DNA sequence data. L. cuprina is paraphyletic with respect to L. sericata in mtDNA phylogenies, with some L. cuprina having mtDNA haplotypes that are very similar to those of L. sericata. We examine this problem by providing the first DNA data for L. cuprina from North America, including portions of both the mitochondrial COI gene and the nuclear 28S rRNA gene. With the new data, L. cuprina remains monophyletic for 28S but paraphyletic with respect to L. sericata for COI. However, we find that all flies that are identified as L. cuprina by morphology and have L. sericata-like mtDNA form a distinctly monophyletic mtDNA clade. This clade may possibly have originated by hybridization between L. cuprina and L. sericata, but its wide geographic distribution strongly suggests a singular origin as opposed to repeated incidents of hybridization. The phylogenetic results strongly support the hypothesis that L. cuprina and L. sericata can be discriminated using mtDNA sequence data. We find that a fragment of COI spanning approximately 1200 base pairs is sufficient to discriminate between the two species with greater than 95% bootstrap support.