Population genomics of the invasive Northern Giant Hornet Vespa mandarinia in North America and across its native range.

The northern giant hornet Vespa mandarinia (NGH) is a voracious predator of other insect species, including honey bees. NGH's native range spans subtropical and temperate regions across much of east and southeast Asia and, in 2019, exotic populations of the species were discovered in North America. Despite this broad range and invasive potential, investigation of the population genomic structure of NGH across its native and introduced ranges has thus far been limited to a small number of mitochondrial samples. Here, we present analyses of genomic data from NGH individuals collected across the species' native range and from exotic individuals collected in North America. We provide the first survey of whole-genome population variation for any hornet species, covering this species' native and invasive ranges, and in doing so confirm likely origins in Japan and South Korea for the two introductions. We additionally show that, while this introduced population exhibited strongly elevated levels of inbreeding, these signatures of inbreeding are also present in some long-standing native populations, which may indicate that inbreeding depression alone is insufficient to prevent the persistence of NGH populations. As well as highlighting the importance of ongoing monitoring and eradication efforts to limit the spread of this species outside of its natural range, our data will serve as a foundational database for future genomic studies into introduced hornet populations.

Viral Prevalence and Genomic Xenology in the Coevolution of HzNV-2 (Nudiviridae) with Host Helicoverpa zea (Lepidoptera: Noctuidae).

Insect viruses have been described from numerous lineages, yet patterns of genetic exchange and viral prevalence, which are essential to understanding host-virus coevolution, are rarely studied. In Helicoverpa zea, the virus HzNV-2 can cause deformity of male and female genitalia, resulting in sterility. Using ddPCR, we found that male H. zea with malformed genitalia (agonadal) contained high levels of HzNV-2 DNA, confirming previous work. HzNV-2 was found to be prevalent throughout the United States, at more than twice the rate of the baculovirus HaSNPV, and that it contained several host-acquired DNA sequences. HzNV-2 possesses four recently endogenized lepidopteran genes and several more distantly related genes, including one gene with a bacteria-like sequence found in both host and virus. Among the recently acquired genes is cytosolic serine hydroxymethyltransferase (cSHMT). In nearly all tested H. zea, cSHMT contained a 200 bp transposable element (TE) that was not found in cSHMT of the sister species H. armigera. No other virus has been found with host cSHMT, and the study of this shared copy, including possible interactions, may yield new insights into the function of this gene with possible applications to insect biological control, and gene editing.

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.

Mediating a host cell signaling pathway linked to overwinter mortality offers a promising therapeutic approach for improving bee health.

INTRODUCTION: Honey bees provides valuable pollination services for world food crops and wild flowering plants which are habitats of many animal species and remove carbon dioxide from the atmosphere, a powerful tool in the fight against climate change. Nevertheless, the honey bee population has been declining and the majority of colony losses occur during the winter. OBJECTIVES: The goal of this study was to understand the mechanisms underlying overwinter colony losses and develop novel therapeutic strategies for improving bee health. METHODS: First, pathogen prevalence in overwintering bees were screened between 2015 and 2018. Second, RNA sequencing (RNA-Seq) for transcriptional profiling of overwintering honey bees was conducted and qRT-PCR was performed to confirm the results of the differential expression of selected genes. Lastly, laboratory bioassays were conducted to measure the effects of cold challenges on bee survivorship and stress responses and to assess the effect of a novel medication for alleviating cold stress in honey bees. RESULTS: We identified that sirtuin signaling pathway is the most significantly enriched pathway among the down-regulated differentially expressed genes (DEGs) in overwintering diseased bees. Moreover, we showed that the expression of SIRT1 gene, a major sirtuin that regulates energy and immune metabolism, was significantly downregulated in bees merely exposed to cold challenges, linking cold stress with altered gene expression of SIRT1. Furthermore, we demonstrated that activation of SIRT1 gene expression by SRT1720, an activator of SIRT1 expression, could improve the physiology and extend the lifespan of cold-stressed bees. CONCLUSION: Our study suggests that increased energy consumption of overwintering bees for maintaining hive temperature reduces the allocation of energy toward immune functions, thus making the overwintering bees more susceptible to disease infections and leading to high winter colony losses. The novel information gained from this study provides a promising avenue for the development of therapeutic strategies for mitigating colony losses, both overwinter and annually.

A Chromosome-Scale Genome Assembly of a Helicoverpa zea Strain Resistant to Bacillus thuringiensis Cry1Ac Insecticidal Protein.

Helicoverpa zea (Lepidoptera: Noctuidae) is an insect pest of major cultivated crops in North and South America. The species has adapted to different host plants and developed resistance to several insecticidal agents, including Bacillus thuringiensis (Bt) insecticidal proteins in transgenic cotton and maize. Helicoverpa zea populations persist year-round in tropical and subtropical regions, but seasonal migrations into temperate zones increase the geographic range of associated crop damage. To better understand the genetic basis of these physiological and ecological characteristics, we generated a high-quality chromosome-level assembly for a single H. zea male from Bt-resistant strain, HzStark_Cry1AcR. Hi-C data were used to scaffold an initial 375.2 Mb contig assembly into 30 autosomes and the Z sex chromosome (scaffold N50 = 12.8 Mb and L50 = 14). The scaffolded assembly was error-corrected with a novel pipeline, polishCLR. The mitochondrial genome was assembled through an improved pipeline and annotated. Assessment of this genome assembly indicated 98.8% of the Lepidopteran Benchmark Universal Single-Copy Ortholog set were complete (98.5% as complete single copy). Repetitive elements comprised approximately 29.5% of the assembly with the plurality (11.2%) classified as retroelements. This chromosome-scale reference assembly for H. zea, ilHelZeax1.1, will facilitate future research to evaluate and enhance sustainable crop production practices.

Insights into the prey of Vespa mandarinia (Hymenoptera: Vespidae) in Washington state, obtained from metabarcoding of larval feces.

The northern giant hornet, Vespa mandarinia (Hymenoptera: Vespidae), was detected for the first time in North America in 2019. Four nests have since been located and removed in northwestern Washington State as part of an extensive survey and eradication program. This recent introduction into North America has prompted new research on the biology and ecology of V. mandarinia to help inform management strategies. In its native range, V. mandarinia is known to prey on a variety of insects including the economically important honey bee species Apis cerana and Apis mellifera. Although A. cerana has developed defense mechanisms against attack by V. mandarinia, A. mellifera have no such defenses and an entire hive can be quickly destroyed by only a few hornets. In North America the hornet has been observed foraging on paper wasps (Polistes dominula) and honey bees, but little else is known about prey use in its novel range. To address this knowledge gap, we employed a DNA metabarcoding approach to characterize species detected in larval feces collected from 3 of the 4 Washington V. mandarinia nests found to date. Sequences were recovered for 56 species across fourteen orders, of which 36 species were likely prey items and 20 were suspected inquilines. The most frequently detected species were other social Hymenoptera, with Dolichovespula maculata, P. dominula, and A. mellifera present in most samples. All of the species detected, except for A. mellifera, represent new prey records for V. mandarinia, with eight families of insects newly associated with giant hornets. These results suggest that V. mandarinia in Washington preys on an assortment of insects similar to those documented in its native range, and that this new invader has readily incorporated novel species into its foraging and diet.

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.

Biogeographical Patterns of Herbivore Arthropods Associated with Chenopodium quinoa Grown along the Latitudinal Gradient of Chile.

Identifying the particular guilds of herbivore arthropods that affect the production of crops is key to developing sustainable pest-management strategies; however, there is incomplete information about the identity of herbivore arthropods that could potentially damage the production of both highland and lowland quinoa landraces grown in Chile. By both reviewing the literature and conducting field collections across a large latitudinal gradient, we generated an updated list of 43 herbivore arthropods associated with quinoa production in Chile. In general, most species are polyphagous feeders, and only seven are specialists. The number and identity of species varied in relation with the latitude, such that four distinctive assemblages of herbivores were identified, each containing 32, 27, 34, and 22 species between latitudes 18-26, 26-32, 32-40, and 40-44° S, respectively. The most northern production area (18-26° S) is affected by nine unique species, including the major quinoa pest Eurysacca quinoae Povolný (Lepidoptera: Gelechiidae). Similarly, the central area (32-40° S) contains four unique species, including Eurysacca media Povolný (Lepidoptera: Gelechiidae) and Orthotylus flavosparsus (Sahlberg) (Hemiptera: Miridae). The particular species assemblages described here will help further development of local pest-management practices.

Novel real-time PCR based assays for differentiating fall armyworm strains using four single nucleotide polymorphisms.

The fall armyworm, Spodoptera frugiperda, is a polyphagous global pest with a preference for gramineous crops such as corn, sorghum and pasture grasses. This species is comprised of two morphologically identical but genetically distinct host strains known as the corn and rice strains, which can complicate pest management approaches. Two molecular markers are commonly used to differentiate between strains, however, discordance between these markers can lead to inconclusive strain identification. Here, we used double digest restriction site associated DNA sequencing to identify diagnostic single nucleotide polymorphisms (SNPs) with alleles unique to each strain. We then used these strain-specific SNPs to develop four real-time PCR based TaqMan assays to rapidly and reliably differentiate between strains and interstrain hybrids. These assays provide a new tool for differentiating between strains in field-collected samples, facilitating future studies on strain population dynamics and interstrain hybridization rates. Understanding the basic ecology of S. frugiperda strains is necessary to inform future management strategies.

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.

A novel method for the detection and diagnosis of virus infections in honey bees.

In terms of infectious diseases caused by a variety of microorganisms, the ability to promptly and accurately identify the causative agents is the first step on the path to all types of effective management of such infections. Among the various factors that are affecting global bee health, viruses have often been linked to honey bee colony losses and they pose a serious threat to the fraction of agriculture that depends on the service of pollinators. Over the past few decades, PCR-based molecular methods have provided powerful tools for rapid, specific, and sensitive detection and the quantification of difficult-to-grow pathogenic microorganisms such as viruses in honey bees. However, PCR-based methods require nucleic acid extraction and purification, which can be quite laborious and time-consuming and they involve the use of organic solvents and chaotropic agents like phenol and chloroform which are volatile and highly toxic. In response, we developed a novel and non-sacrificial method for detecting viral infections in honey bees. As little as 1 µl of hemolymph was collected from adult workers, larvae, and queens of bee colonies by puncturing the soft inter-tergal integument between the second and third dorsal tergum with a fine glass capillary. The hemolymph was then diluted and subjected to RT-PCR analysis directly. The puncture wound caused by the glass capillary was found to heal automatically and rapidly without any trouble and the lifespan of the experimental workers remained unaffected. Using this method, we detected multiple viruses including Deformed wing virus (DWV), Black queen cell virus (BQCV), and Sacbrood virus (SBV) in infected bees. Furthermore, expressed transcripts that indicate the induction of innate immune response to the virus infections were also detected in the hemolymph of infected bees. The simplicity and cost-effectiveness of this innovative approach will allow it to be a valuable, time-saving, safer, and more environmentally friendly contribution to bee disease management programs.

Immigrant Tortricidae: Holarctic versus Introduced Species in North America.

In support of a comprehensive update to the checklist of the moths of North America, we attempt to determine the status of 151 species of Tortricidae present in North America that may be Holarctic, introduced, or sibling species of their European counterparts. Discovering the natural distributions of these taxa is often difficult, if not impossible, but several criteria can be applied to determine if a species that is present in both Europe and North America is natively Holarctic, introduced, or represented by different but closely related species on each continent. We use DNA barcodes (when available), morphology, host plants, and historical records (literature and museum specimens) to make these assessments and propose several taxonomic changes, as well as future areas of research. The following taxa are raised from synonymy to species status: Acleris ferrumixtana (Benander, 1934), stat. rev.; Acleris viburnana (Clemens, 1860), stat. rev.; Acleris pulverosana (Walker, 1863), stat. rev.; Acleris placidana (Robinson, 1869), stat. rev.; Lobesia spiraeae (McDunnough, 1938), stat. rev.; and Epiblema arctica Miller, 1985, stat. rev. Cydia saltitans (Westwood, 1858), stat. rev., is determined to be the valid name for the "jumping bean moth," and Phiaris glaciana (Möschler, 1860), comb. n., is placed in a new genus. We determine that the number of Holarctic species has been overestimated by at least 20% in the past, and that the overall number of introduced species in North America is unexpectedly high, with Tortricidae accounting for approximately 23-30% of the total number of Lepidoptera species introduced to North America.

Comparative Toxicity of Helicoverpa armigera and Helicoverpa zea (Lepidoptera: Noctuidae) to Selected Insecticides.

Until recently, the Old World bollworm (OWB) Helicoverpa armigera (Hübner) and the corn earworm Helicoverpa zea (Boddie) (Lepidoptera: Noctuidae) were geographically isolated. Both species are major pests of agricultural commodities that are known to develop insecticide resistance, and they now coexist in areas where H. armigera invaded the Americas. This is the first study to compare the susceptibility of the two species to conventional insecticides. The susceptibility of third instar H. armigera and H. zea larvae to indoxacarb, methomyl, spinetoram, and spinosad was determined using a diet-overlay bioassay in a quarantine laboratory in Puerto Rico. Mortality was assessed at 48 h after exposure for up to eight concentrations per insecticide. Spinetoram exhibited the highest acute toxicity against H. armigera, with a median lethal concentration (LC50) of 0.11 µg a.i./cm2, followed by indoxacarb and spinosad (0.17 µg a.i./cm2 for both) and methomyl (0.32 µg a.i./cm2). Spinetoram was also the most toxic to H. zea (LC50 of 0.08 µg a.i./cm2), followed by spinosad (0.17 µg a.i./cm2) and methomyl (0.18 µg a.i./cm2). Indoxacarb was the least toxic to H. zea, with an LC50 of 0.21 µg a.i./cm2. These findings could serve as a comparative reference for monitoring the susceptibility of H. armigera and H. zea to indoxacarb, methomyl, spinetoram, and spinosad in Puerto Rico, and may facilitate the detection of field-selected resistance for these two species and their potential hybrids in areas recently invaded by H. armigera.

Taxonomic issues related to biological control prospects for the ragweed borer, Epiblema strenuana (Lepidoptera: Tortricidae).

The ragweed borer, Epiblema strenuana (Walker, 1863), has a long history of use as a biological control agent against important weed pests in the family Asteraceae. Recently, E. strenuana has been reported feeding on the invasive perennials Ambrosia confertiflora and A. tenuifolia in Israel. The geographic location of Israel has raised concern over the possibility that the moth may spread to areas such as Ethiopia where the oil-seed crop Guizotia abyssinica is cultivated, as this is a potential host for E. strenuana. However, the taxonomic status of E. strenuana and a current synonym, E. minutana (Kearfott, 1905) is unclear. These taxa have been treated as separate species in the past, and they potentially have different feeding habits and damage different parts of the plant. We analyzed DNA data and adult morphology and determined that E. minutana, stat. rev., is a valid species which we raise from synonymy with E. strenuana. Wing coloration, the shape of the female sterigma, and COI DNA barcodes are consistently different between the two species. We also determined that the species previously identified as E. strenuana in Israel is actually E. minutana. While detailed host range tests have been conducted on the E. strenuana populations released in Australia and China, the host range of E. minutana remains to be clarified. We discuss the history of biological control using E. strenuana and the implications for finding E. minutana in Israel. We also provide species redescriptions for E. strenuana and E. minutana and illustrate diagnostic characters.

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.

The myth of monophagy in Paralobesia (Lepidoptera: Tortricidae)? A new species feeding on Cypripedium reginae (Orchidaceae).

The genus Paralobesia Obraztsov, 1953 is found primarily in eastern North America and consists of 18 described and several undescribed species. Prior to 1900, all North American Paralobesia were assumed to be P. viteana (Clemens). However, rearing experiments by William Kearfott in the early 1900s suggested that species of Paralobesia were monophagous and could be separated by host. Recently, a species of Paralobesia was reared from showy lady's slipper, Cypripedium reginae Walter (Orchidaceae), during a study of two populations of this orchid in eastern Ontario and southwestern Québec. Although originally assumed to be P. cypripediana (Forbes), which was described from specimens reared from Cypripedium in Manitoba, DNA barcode data and genital morphology confirmed that this was a new species similar to P. cypripediana and P. monotropana (Heinrich). Herein, we describe P. marilynae, sp. n., and provide specifics of its discovery and life history. Rearing records indicate that Paralobesia can span the range from strictly monophagous to polyphagous, even for very similar species with similar feeding habits, and that host records should be combined with morphological and molecular data when circumscribing species in this genus. This work is part of a complete systematic revision of Paralobesia currently in progress.

Two new species of Eucosmini from the Bahamas (Lepidoptera: Tortricidae).

Recent surveys to document the poorly known moth fauna of the Bahamas have resulted in the collection of more than 60 morphospecies of Tortricidae. Among these are several Eucosmini, including undescribed representatives of Eucosma and Pelochrista that are similar to several species present in the southeastern United States. Eucosma bahamae, sp.n., a member of the E. refusana group, and Pelochrista wrighti, sp.n., a member of the P. canana group, are described and illustrated.

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.