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Many countries operate continuous trapping programs to detect invasive fruit flies (Diptera: Tephritidae) that threaten fruit and vegetable production. Food-based attractants are an important component of these programs, as they are generalized baits that attract both sexes of all target species. However, food baits are considered weak attractants, and the development of improved food attractants would benefit surveillance efforts. Prior work on protein bait sprays demonstrated that the addition of ammonium acetate increased their attractiveness to tephritid fruit flies, and the present study was conducted to determine whether adding ammonium acetate to torula yeast borax solution (a commonly used food bait) similarly increased its attractiveness. Four field experiments were performed in Hawaii comparing captures of Ceratitis capitata (Wiedemann), Bactrocera dorsalis (Hendel), and Zeugodacus cucurbitae (Coquillett) in Multilure traps baited with standard torula yeast borax solution or with the standard solution plus 1% ammonium acetate. Captures of C. capitata and B. dorsalis were significantly higher for the ammonium acetate-supplemented solution, while results for Z. cucurbitae were inconsistent. In one experiment involving only C. capitata, an additional bait having 5% ammonium acetate was included, and captures for both 1% and 5% ammonium acetate exceeded the standard solution but did not differ from one another. Sex ratios of captured flies were similar between food baits for all three species. These results are compared with similar studies on Anastrepha spp., and implications for surveillance programs are discussed., (Published by Oxford University Press on behalf of Entomological Society of America 2024.)

Certain species of true fruit flies (Diptera: Tephritidae) cause tremendous damage to commercially important fruits and vegetables, and many countries operate continuous trapping programs which rely on male-specific lures such as trimedlure (TML), methyl eugenol (ME), and cue-lure (CL). Traditionally, these attractants have been applied as liquids to cotton wicks inside traps, although this results in high evaporative loss of the lure. Slow-release, polymeric plugs have been widely adopted for TML, but such devices are not widely used for ME or CL. Recent data, however, suggest that ME and CL plugs may be attractive for as long as 12 wk in the field. The present study investigates whether ME and CL plugs weathered for 18 or 24 wk are effective in capturing males of Bactrocera dorsalis (Hendel) and Zeugodacus cucurbitae (Coquillett), respectively. For B. dorsalis, 6 g ME plugs were as effective as the control treatment (fresh liquid on a wick) after 12 wk of weathering but not after 18 or 24 wk. For Z. cucurbitae, 3 g CL plugs were as effective as the control treatment (fresh CL plugs) after 12 and 18 wk of weathering but not after 24 wk. The residual content and release rate of the 2 lures were also measured over time, but, with the exception of the residual content of ME, we did not find a direct correlation between these parameters and numbers of flies captured., (Published by Oxford University Press on behalf of Entomological Society of America 2024.)

Anastatus orientalis Yang & Choi (Hymenoptera: Eupelmidae), an egg parasitoid of spotted lanternfly, Lycorma delicatula (White) (Hemiptera: Fulgoridae), has been documented emerging from host eggs in both autumn and spring, at the beginning and end of the period that spotted lanternfly eggs are present in the field, suggesting parasitoid-host specificity and synchrony. This study was designed to test whether, under conditions that simulate native and introduced ranges of spotted lanternfly, (a) A. orientalis has 2 and only 2 generations per year, (b) A. orientalis adults sometimes emerge when only nontarget species would be available for parasitization, and (c) emerging parasitoid adults can parasitize unhatched host eggs in the spring. Parasitized spotted lanternfly eggs were collected in 2019 and 2020 from Beijing, China and in 2020 and 2021 from Yantai, China. They were shipped for laboratory study in growth chambers programmed to simulate temperature and daylength for collection locations in China and in the invaded range in Pennsylvania, United States. Anastatus orientalis had a flexible lifecycle depending on environmental conditions and possibly genetic makeup, with 1-3 generations per year, and parasitoid emergence was not always synchronous with host egg availability. Additionally, given the cooler temperatures in Pennsylvania, autumn parasitoid emergence was often delayed until late October or November, no progeny were produced, and parasitoid populations died out. Anastatus orientalis does not exhibit host synchrony characteristics that would make it a good candidate for a classical biological control program., (Published by Oxford University Press on behalf of Entomological Society of America 2024.)

Drosophila suzukii is a pest native to Southeast Asia that causes significant economic losses to soft fruit crops. Phytosanitary irradiation is a promising treatment for D. suzukii hosts; yet an internationally recognized irradiation protocol is lacking. To fulfil specific requirements for proposing an irradiation treatment for D. suzukii, naturally infested blueberries and cherries containing a total of 37,489 late pupae were irradiated with a maximum absorbed dose of 80 Gy. Infested hosts containing a total of 9578 late pupae were considered unirradiated controls. Prevention of egg laying by females that emerged from treated pupae was considered the treatment endpoint. The fecundity and egg viability of females that emerged from treated pupae mated with their siblings were evaluated using blueberries. While females from unirradiated pupae laid a total of 43,142 eggs, no egg was laid by females that emerged from irradiated pupae. In addition, 1-day-old adults were irradiated with nominal doses of 20 and 72 Gy to evaluate whether egg laying could be prevented in flies emerging before the irradiation treatment. Females irradiated with 72 Gy laid eggs that did not hatch. Our findings suggest the minimum absorbed dose of 80 Gy as a phytosanitary irradiation treatment against D. suzukii and may support its inclusion as a treatment option in the annex of the International Standard for Phytosanitary Measures 28 (ISPM 28)., (© 2024. The Author(s).)

The citrus long-horned beetle, Anoplophora chinensis (Coleoptera: Cerambycidae), is a highly polyphagous species native to eastern and southeastern Asia. Detection of these beetles is dependent on visual surveys, which are inefficient and labor-intensive. The identification and development of pheromone-based lures would help improve survey efforts for A. chinensis should it become established within the US. We identified three antennally active male-produced chemical components (nonanal, 4-(n-heptyloxy)butan-1-ol, and 4-(n-heptyloxy)butanal), which were then tested for behavioral activity at our USDA Quarantine laboratory. When tested together in Y-tube behavioral assays, a three-component blend of the identified compounds was shown to be attractive to adult female A. chinensis . Pheromone production and behavior of A. chinensis and other closely related Anoplophora cerambycid species are discussed in relation to mate finding and potential monitoring.

Heteroplasmy, the presence of multiple mitochondrial genotypes (mitotypes) within an individual, has long been thought to be a rare aberrance that is quickly removed by selection or drift. However, heteroplasmy is being reported in natural populations of eukaryotes with increasing frequency, in part due to improved diagnostic methods. Here, we report a seemingly stable heteroplasmic state in California populations of the polyphagous shothole borer (PSHB), Euwallacea fornicatus; an invasive ambrosia beetle that is causing significant tree dieback. We develop and validate a qPCR assay utilizing locked nucleic acid probes to detect different mitotypes, and qualitatively assess heteroplasmy in individual PSHB. We prove the utility of this assay by: (1) mitotyping field-collected PSHB, documenting the prevalence of heteroplasmy across its range in California; and, (2) measuring relative titers of each mitotype across multiple generations of heteroplasmic laboratory colonies to assess the stability of transmission through the maternal germline. We show that our findings are unlikely to be explained by the existence of NUMTs by next generation sequencing of contiguous sections of mitochondrial DNA, where each of the observed heteroplasmic sites are found within fully functional coding regions of mtDNA. Subsequently, we find heteroplasmic individuals are common in Californian field populations, and that heteroplasmy persists for at least 10 generations in experimental colonies. We also looked for evidence of the common occurrence of paternal leakage, but found none. In light of our results, we discuss competing hypotheses as to how heteroplasmy may have arisen, and continues to perpetuate, in Californian PSHB populations., (© 2024. The Author(s).)

Virginia creeper ( Parthenocissus quinquefolia [L.] Planch.) is a deciduous flowering vine in the Vitaceae family. Native to eastern North America, it is often used ornamentally as a climbing vine or as ground cover due to its rapid growth and foliage color in the fall. In July of 2022, along exterior walls of a private property in Lanham, MD, two Virginia creeper (VC) vines were observed displaying symptoms of yellow mottling and premature reddening of leaves. To investigate the cause of these symptoms, two symptomatic leaf samples and one asymptomatic leaf samples from a third vine in the same vicinity were collected for further analysis. A Qiagen DNeasy Plant Mini Kit was used to extract total DNA from leaf tissues according to the manufacturer's instructions. A phytoplasma-specific real-time PCR (Hodgetts et al. 2009) was used to test the DNA extracts, which detected the presence of phytoplasmas in the two DNA samples derived from symptomatic vines. The near full-length of the 16S ribosomal RNA gene was then amplified by seminested PCR from these samples with primers P1/16S-SR followed by P1A/16S-SR (Deng, and Hiruki 1991; Lee et al. 2004) and Sanger sequenced using primers P1A and 16S-SR. Analysis of the obtained 16S rDNA sequences revealed no variation between the two plant samples, and one sequence was deposited in GenBank representing the phytoplasma strain named VC-MD1 (GenBank PP746981). A BLASTn search of the 16S rRNA gene sequence in the NCBI nucleotide database, showed 99.93% sequence identity with the phytoplasma strain AldY-WA1 (GenBank MZ557341) from red alder in Washington, a phytoplasma associated with VC plants in southern Florida (GenBank AF305198) (Harrison et al. 2001), and other strains detected in grapevines in Europe described as "flavescence dorée" phytoplasma (GenBank AF176319) (Davis, and Dally 2001). The virtual restriction fragment length polymorphism pattern derived from i PhyClassifier (Zhao et al. 2009), indicated that VC-MD1 is indeed a member of the 16SrV-C phytoplasma subgroup. To confirm the identification, the partial spc operon and the partial tuf gene were amplified as previously described (Lee et al. 2010; Makarova et al. 2012). Specifically, the spc operon region was amplified using a nested PCR approach with primer set L15F1A-a/MapR1 followed by L15F1A-b/MapR1A-b. Sequence data obtained from the two loci were deposited to GenBank with accession numbers PP746982 ( spc ) and PP746983 ( tuf ). BLAST searches querying the nucleotide sequences of the spc operon and tuf gene showed 95.39% and 99.05% identity, respectively, to the corresponding loci of ' Candidatus Phytoplasma rubi' strain RS (GenBank CP114006) and hemp dogbane yellows phytoplasma strain HD1 (GenBank FR686506). Phylogenetic analysis based on secY and tuf gene sequences suggest that the VC-MD1 strain is evolutionary closest to 16SrV-C phytoplasma strains detected in various hosts in the United States, including HD1 and AldY-WA1. These North American strains cluster together on a distinct branch within the elm yellows group phytoplasmas. For the State of Maryland, this detection represents the first report of a phytoplasma strain member of the16SrV-C subgroup infecting VC plants. A phytoplasma of the same subgroup was previously detected in Florida in asymptomatic VC vines (Harrison et al. 2001). The 16S rRNA gene sequences of the two VC phytoplasma strains are nearly identical, differing by just a single nucleotide. The disease transmission vectors of the VC-MD1 strain and the prevalence of the disease in the region remains undetermined.

Background: Honey bees are the principal commercial pollinators. Along with other arthropods, they are increasingly under threat from anthropogenic factors such as the incursion of invasive honey bee subspecies, pathogens and parasites. Better tools are needed to identify bee subspecies. Genomic data for economic and ecologically important organisms is increasing, but in its basic form its practical application to address ecological problems is limited., Results: We introduce HBeeID a means to identify honey bees. The tool utilizes a knowledge-based network and diagnostic SNPs identified by discriminant analysis of principle components and hierarchical agglomerative clustering. Tests of HBeeID showed that it identifies African, Americas-Africanized, Asian, and European honey bees with a high degree of certainty even when samples lack the full 272 SNPs of HBeeID. Its prediction capacity decreases with highly admixed samples., Conclusion: HBeeID is a high-resolution genomic, SNP based tool, that can be used to identify honey bees and screen species that are invasive. Its flexible design allows for future improvements via sample data additions from other localities., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

The remarkably diverse plant communities of the Neotropics are the result of diversification driven by multiple biotic (for example, speciation, extinction and dispersal) and abiotic (for example, climatic and tectonic) processes. However, in the absence of a well-preserved, thoroughly sampled and critically assessed fossil record, the associated processes of dispersal and extinction are poorly understood. We report an exceptional case study documenting patterns of extinction in the grape family (Vitaceae Juss.) on the basis of fossil seeds discovered in four Neotropical palaeofloras dated between 60 and 19 Ma. These include a new species that provides the earliest evidence of Vitaceae in the Western Hemisphere. Eight additional species reveal the former presence of major clades of the family that are currently absent from the Neotropics and elucidate previously unknown dispersal events. Our results indicate that regional extinction and dispersal have substantially impacted the evolutionary history of Vitaceae in the Neotropics. They also suggest that while the Neotropics have been dynamic centres of diversification through the Cenozoic, extant Neotropical botanical diversity has also been shaped by extensive extinction over the past 66 million years., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Using semiochemicals collected from spotted lanternflies Lycorma delicatula (Hemiptera: Fulgoridae) (SLF) and deployed in the field with circle traps, we demonstrated that SLF responded to SLF pheromones: in particular, this was the case for males while seeking mates and for females while ovipositing. The attractants consisted of SLF body extract emitted from diffuser lures and SLF honeydew on burlap ribbons, collected from heavily infested locations. Traps with attractants were deployed in field sites with very light SLF infestations to avoid competing signals of pre-existing aggregations. The number of SLF equivalents emitted by each diffuser per trapping period was used in a dose-response analysis. Three trees per block received either (1) a control hexane lure and a clean ribbon, (2) a lure containing SLF extract and a clean ribbon, or (3) a lure containing SLF extract and a honeydew-laden ribbon. Ten blocks were sampled three times per week for twelve weeks. We found a significant positive dose-response by males to SLF body extract only in the presence of SLF honeydew, indicating a synergistic effect between honeydew volatiles and body volatiles. This dose-response occurred for five weeks after mating started, after which males no longer responded. Subsequently, females had a significant positive dose-response to SLF extract only in the presence of honeydew when oviposition was their primary activity, continuing for two weeks, suggesting that females may use pheromones to aggregate for oviposition. The extract in the absence of honeydew did not result in a positive dose-response, nor did the hexane control. These findings suggest that SLF respond synergistically to the combination of pheromones present in both SLF honeydew and SLF bodies. Thus, combining key components from both sources may aid the development of semiochemical lures for SLF.

Microbes and enzymes play essential roles in soil and plant rhizosphere ecosystem functioning. However, fungicides and plant root secretions may impact the diversity and abundance of microbiota structure and enzymatic activities in the plant rhizosphere. In this study, we analyzed soil samples from the rhizosphere of four cannabinoid-rich hemp ( Cannabis sativa ) cultivars (Otto II, BaOx, Cherry Citrus, and Wife) subjected to three different treatments (natural infection, fungal inoculation, and fungicide treatment). DNA was extracted from the soil samples, 16S rDNA was sequenced, and data were analyzed for diversity and abundance among different fungicide treatments and hemp cultivars. Fungicide treatment significantly impacted the diversity and abundance of the hemp rhizosphere microbiota structure, and it substantially increased the abundance of the phyla Archaea and Rokubacteria. However, the abundances of the phyla Pseudomonadota and Gemmatimonadetes were substantially decreased in treatments with fungicides compared to those without fungicides in the four hemp cultivars. In addition, the diversity and abundance of the rhizosphere microbiota structure were influenced by hemp cultivars. The influence of Cherry Citrus on the diversity and abundance of the hemp rhizosphere microbiota structure was less compared to the other three hemp cultivars (Otto II, BaOx, and Wife). Moreover, fungicide treatment affected enzymatic activities in the hemp rhizosphere. The application of fungicides significantly decreased enzyme abundance in the rhizosphere of all four hemp cultivars. Enzymes such as dehydrogenase, dioxygenase, hydrolase, transferase, oxidase, carboxylase, and peptidase significantly decreased in all the four hemp rhizosphere treated with fungicides compared to those not treated. These enzymes may be involved in the function of metabolizing organic matter and degrading xenobiotics. The ecological significance of these findings lies in the recognition that fungicides impact enzymes, microbiota structure, and the overall ecosystem within the hemp rhizosphere.

Background: Dinotefuran, a systemic neonicotinoid insecticide, is approved for control of hemlock woolly adelgid (HWA) (Adelges tsugae Annand), an invasive sap-feeding insect that can kill eastern hemlocks (Tsuga canadensis). Dinotefuran is highly water soluble, facilitating more rapid translocation and HWA control than other neonicotinoids, but its persistence is not well-known. Samples of needles and twigs were collected in spring 2021 from 50 hemlocks treated with a dinotefuran basal trunk spray in 2018 or 2019 (131-145 weeks and 85-93 weeks before sampling, respectively). Processed samples were analyzed with ultra-performance liquid chromatography-tandem mass spectrometer (UPLC-MS/MS) and enzyme-linked immunosorbent assays (ELISA)., Results: Dinotefuran residues were 4.6-6.1 times higher in needles than in twigs collected from the same trees. Average (±SE) residues in foliage samples collected from trees treated in 2019 ranged from 0.663 ± 0.243 to 0.564 ± 0.119 mg kg -1 , compared with 0.213 ± 0.033 and 0.225 ± 0.132 mg kg -1 in foliage from trees treated in 2018. Foliage residues from UPLC-MS/MS were consistently lower but strongly related to those from ELISA. Matrix effects appeared to disrupt ELISA analysis of twigs. None of the 25 trees treated in 2019 had live HWA when samples were collected in 2021 while low densities of HWA were observed on 52% of trees treated in 2018., Conclusions: Dinotefuran was recovered from hemlock foliage, and to a lesser extent twigs, >2 years post-treatment. This, along with its relatively rapid translocation, suggests dinotefuran is a viable option for protecting declining or heavily infested hemlocks from HWA. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry., (© 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.)

Aconitum napellus L. is a popular medicinal plant extensively used in homeopathy. This article provides detailed morphology and microscopy, including the anatomical and histochemical features of the herb, to aid authentication and quality control. In cross-section, the root in secondary growth shows the phloem surrounded by pericyclic fibers and a well-developed xylem. The stem is irregular in outline, displaying unicellular trichomes and many free collateral vascular bundles encircling the pith. The leaf is dorsiventral, hypostomatic with anomocytic and anisocytic stomata, and shows non-glandular trichomes. The floral parts are characterized by uniseriate epidermises, homogeneous mesophyll, anomocytic stomata on the abaxial surface, trichomes, and oval pollen grains. The tissue fragments in powdered herbs show these characteristics and have numerous starch grains with thimble-shaped, linear or star-shaped hilum. The detailed macroscopic and microscopic analysis provided in this study can help in the authentication and quality control of A. napellus raw materials. RESEARCH HIGHLIGHTS: Key anatomical, micromorphological, and microchemical features of Aconitum napellus are described. The results of the study can support the taxonomy of the genus Aconitum. Morphological standardization of the species reported here is helpful in the quality control of this herb., (© 2023 Wiley Periodicals LLC.)

In herbivorous insects, antennae play a crucial role in chemical communication and orientation when locating host plants and mates. To evaluate antennal sensitivity in response to odor stimuli, electroantennography (EAG) has been a practical technique. In the current study of the invasive spotted lanternfly (SLF), Lycorma delicatula (Hemiptera: Fulgoridae), we evaluated and compared their antennal sensitivity to a series of volatile chemicals collected from their bodies, honeydew, and host plants. To do this, we exposed the antennae of SLF fourth-instar and adult males and females to individual chemicals at a fixed dose of 50 ng. Further, a series of dose-response tests were carried out within a range of 0.5 to 100 ng. Although the amplitude of antennal responses varied among stages and sexes, adult males generated the strongest antennal responses in both experiments. In dose-response experiments, increased doses of chemicals up to 50 ng revealed the saturation points except in adult females which required a higher dose (100 ng) to reveal the saturation point. Although EAG does not provide any information on behavioral responses, our results are consistent with the olfactory bioassays in previous publications in which adult males, not females, were attracted to natural volatiles of their conspecifics. EAG indicated a higher sensitivity of adult male antennae to odor stimuli, particularly conspecific volatiles, than female antennae and highlighted sexual differences in the perception of chemical cues in SLF.

Helicoverpa armigera , the cotton bollworm moth, is one of the world's most important crop pests, and is spreading throughout the New World from its original range in the Old World. In Brazil, invasive H. armigera has been reported to hybridize with local populations of Helicoverpa zea . The correct identification of H. armigera-H. zea hybrids is important in understanding the origin, spread and future outlook for New World regions that are affected by outbreaks, given that hybridization can potentially facilitate H. zea pesticide resistance and host plant range via introgression of H. armigera genes. Here, we present a genome admixture analysis of high quality genome sequences generated from two H. armigera-H. zea F1 hybrids generated in two different labs. Our admixture pipeline predicts 48.8% and 48.9% H. armigera for the two F1 hybrids, confirming its accuracy. Genome sequences from five H. zea and one H. armigera that were generated as part of the study show no evidence of hybridization. Interestingly, we show that four H. zea genomes generated from a previous study are predicted to possess a proportion of H. armigera genetic material. Using unsupervised clustering to identify non-hybridized H. armigera and H. zea genomes, 8511 ancestry informative markers (AIMs) were identified. Their relative frequencies are consistent with a minor H. armigera component in the four genomes, however its origin remains to be established. We show that the size and quality of genomic reference datasets are critical for accurate hybridization prediction. Consequently, we discuss potential pitfalls in genome admixture analysis of H. armigera-H. zea hybrids, and suggest measures that will improve such analyses., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer MT declared a shared affiliation with the author JV to the handling editor at the time of review., (Copyright © 2024 Trujillo, Mastrangelo, Estevez de Jensen, Verle Rodrigues, Lawrie and Massey.)

Two new species, Tetralicia sawyeri n. sp. and Aleuroplatus martini n. sp. found on avocado (Persea americana) trees in Peru are described and illustrated. The synonymy of Aleuropleurocelus Drews & Sampson, 1956 n. syn. with Tetralicia Harrison, 1917 is proposed and a review and an illustrated key to the 39 species of Tetralicia of the world based on characteristics of the puparia is provided. Thirty-three species are transferred from Aleuropleurocelus to Tetralicia. A lectotype is designated for Aleurodes abnormis Quaintance, 1900. Aleurotrachelus gratiosus Bondar, 1923 is transferred to the genus Aleuroplatus. Paracarniella mexicana (Distant) and Adparaproba cf. yungensis Carvalho (Hemiptera: Miridae) are recorded for the first time in Peru and are important predators of both new whitefly species. Other natural enemies of these whiteflies found in the study include predatory species: Nephaspis isabelae Gonzlez (Coccinellidae), Chrysoperla sp., Ceraeochrysa sp. (Chrysopidae) and Gasteracantha cancriformis L. (Araneidae) as well as Encarsia brasiliensis Hempel, a common whitefly parasitoid in the Neotropics. A key to the New World genera of whiteflies similar to Aleuroplatus and Tetralicia is provided.

This chapter discusses methods for incorporating DNA barcode information into formal taxonomic descriptions. We first review what a formal description entails and then discuss previous attempts to incorporate barcode information into taxonomic descriptions. Several computer programs are listed that extract diagnostics from DNA barcode data. Finally, we examine a test case (Astraptes taxonomy)., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Rapid identification of organisms is essential for many biological and medical disciplines, from understanding basic ecosystem processes, disease diagnosis, to the detection of invasive pests. CRISPR-based diagnostics offers a novel and rapid alternative to other identification methods and can revolutionize our ability to detect organisms with high accuracy. Here we describe a CRISPR-based diagnostic developed with the universal cytochrome-oxidase 1 gene (CO1). The CO1 gene is the most sequenced gene among Animalia, and therefore our approach can be adopted to detect nearly any animal. We tested the approach on three difficult-to-identify moth species (Keiferia lycopersicella, Phthorimaea absoluta and Scrobipalpa atriplicella) that are major invasive pests globally. We designed an assay that combines recombinase polymerase amplification (RPA) with CRISPR for signal generation. Our approach has a much higher sensitivity than real-time PCR assays and achieved 100% accuracy for identification of all three species, with a detection limit of up to 120 fM for P. absoluta and 400 fM for the other two species. Our approach does not require a sophisticated laboratory, reduces the risk of cross-contamination, and can be completed in less than 1 h. This work serves as a proof of concept that has the potential to revolutionize animal detection and monitoring., (© 2023 John Wiley & Sons Ltd.)

Lycorma delicatula (White) (Hemiptera: Fulgoridae), spotted lanternfly (SLF), is an invasive pest that feeds and oviposits on numerous woody and herbaceous plants important to agricultural, forest, ornamental, and nursery industries. Describing and understanding SLF movements is key to implementing surveillance and control strategies for this pest and projecting population spread. We used radio telemetry (RT) and harmonic radar (HR) to track the movements of individual SLF at field sites in eastern Pennsylvania and northwestern New Jersey. SLF equipped with HR or RT tags were tracked in 2019 and 2020 from adult emergence until oviposition time, and their movements are described. Although the bulkier RT tags disproportionately affected the distance traveled by males, which are smaller than females, both males and females were more likely to be lost due to signal attenuation when affixed with the lighter-weight HR tags. Females were tracked moving longer distances than males, with maximum distances of 434 m by a single female and 57 m by a single male. A significant positive relationship was found between their height in trees and the distance of subsequent movement. Adult SLF were found in trees predominantly at heights between 6-9 m high. For the fraction of SLF found at eye level, males, but not females, significantly moved above eye level in the weeks prior to mating, likely resulting in the observed sex ratio shift that defines the Early-2 stage. During mating time, tracked SLF were significantly higher than 8 m and oriented to trees where tight aggregations of SLF were present. This orientation towards tight aggregations started when mating began and peaked in the following 2.5 weeks for males in Late-1 and the beginning of Late-2 (after oviposition began), whereas females started this orientation behavior a half-week after males, and this activity peaked for two weeks. Male and female SLF adults exhibited slight differences in host preference, and strong preferences for wild grape, black walnut, sweet birch, and tree-of-heaven were observed. The HR-tagged nymphs moved up to 27.6 m over a five-day period in a cornfield. Nitinol wire HR tags performed better than Wollaston process or tungsten wire tags. SLF movement parameters in the field are described.

Candidatus Liberibacter asiaticus (CLas) is associated with Citrus Huanglongbing (HLB), a devastating disease in the US. Previously, we conducted a two-year-long monthly HLB survey by quantitative real-time PCR using root DNA fractions prepared from 112 field grapefruit trees grafted on sour orange rootstock. Approximately 10% of the trees remained CLas-free during the entire survey period. This study conducted 16S metagenomics using the time-series root DNA fractions, monthly prepared during twenty-four consecutive months, followed by microbial co-occurrence network analysis to investigate the microbial factors contributing to the CLas-free phenotype of the aforementioned trees. Based on the HLB status and the time when the trees were first diagnosed as CLas-positive during the survey, the samples were divided into four groups, Stage H (healthy), Stage I (early), II (mid), and III (late) samples. The 16S metagenomics data using Silva 16S database v132 revealed that HLB compromised the diversity of rhizosphere microbiota. At the phylum level, Actinobacteria and Proteobacteria were the predominant bacterial phyla, comprising >93% of total bacterial phyla, irrespective of HLB status. In addition, a temporal change in the rhizosphere microbe population was observed during a two-year-long survey, from which we confirmed that some bacterial families differently responded to HLB disease status. The clustering of the bacterial co-occurrence network data revealed the presence of a subnetwork composed of Streptomycetaceae and bacterial families with plant growth-promoting activity in Stage H and III samples. These data implicated that the Streptomycetaceae subnetwork may act as a functional unit against HLB.

Background: Detection of exotic plant pathogens and preventing their entry and establishment are critical for the protection of agricultural systems while securing the global trading of agricultural commodities. High-throughput sequencing (HTS) has been applied successfully for plant pathogen discovery, leading to its current application in routine pathogen detection. However, the analysis of massive amounts of HTS data has become one of the major challenges for the use of HTS more broadly as a rapid diagnostics tool. Several bioinformatics pipelines have been developed to handle HTS data with a focus on plant virus and viroid detection. However, there is a need for an integrative tool that can simultaneously detect a wider range of other plant pathogens in HTS data, such as bacteria (including phytoplasmas), fungi, and oomycetes, and this tool should also be capable of generating a comprehensive report on the phytosanitary status of the diagnosed specimen., Results: We have developed an open-source bioinformatics pipeline called PhytoPipe (Phytosanitary Pipeline) to provide the plant pathology diagnostician community with a user-friendly tool that integrates analysis and visualization of HTS RNA-seq data. PhytoPipe includes quality control of reads, read classification, assembly-based annotation, and reference-based mapping. The final product of the analysis is a comprehensive report for easy interpretation of not only viruses and viroids but also bacteria (including phytoplasma), fungi, and oomycetes. PhytoPipe is implemented in Snakemake workflow with Python 3 and bash scripts in a Linux environment. The source code for PhytoPipe is freely available and distributed under a BSD-3 license., Conclusions: PhytoPipe provides an integrative bioinformatics pipeline that can be used for the analysis of HTS RNA-seq data. PhytoPipe is easily installed on a Linux or Mac system and can be conveniently used with a Docker image, which includes all dependent packages and software related to analyses. It is publicly available on GitHub at https://github.com/healthyPlant/PhytoPipe and on Docker Hub at https://hub.docker.com/r/healthyplant/phytopipe ., (© 2023. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Tephritid fruit fly pests pose an increasing threat to the agricultural industry due to their global dispersion and a highly invasive nature. Here we showcase the feasibility of an early-detection SEPARATOR sex sorting approach through using the non-model Tephritid pest, Ceratitis capitata. This system relies on female-only fluorescent marker expression, accomplished through the use of a sex-specific intron of the highly-conserved transformer gene from C. capitata and Anastrepha ludens. The herein characterized strains have 100% desired phenotype outcomes, allowing accurate male-female separation during early development. Overall, we describe an antibiotic and temperature-independent sex-sorting system in C. capitata, which, moving forward, may be implemented in other non-model Tephritid pest species. This strategy can facilitate the establishment of genetic sexing systems with endogenous elements exclusively, which, on a wider scale, can improve pest population control strategies like sterile insect technique., (© 2023. Crown.)

Many members of the Oomycota genus Phytophthora cause economic and environmental impact diseases in nurseries, horticulture, forest, and natural ecosystems and many are of regulatory concern around the world. At present, there are 223 described species, including eight unculturable and three lost species. Twenty-eight species need to be redescribed or validated. A lectotype, epitype or neotype was selected for 20 species, and a redescription based on the morphological/molecular characters and phylogenetic placement is provided. In addition, the names of five species are validated: P. cajani , P. honggalleglyana (Synonym: P. hydropathica ), P. megakarya , P. pisi and P. pseudopolonica for which morphology and phylogeny are given. Two species, P. ×multiformis and P. uniformis are presented as new combinations. Phytophthora palmivora is treated with a representative strain as both lecto- and epitypification are pending. This manuscript provides the updated multigene phylogeny and molecular toolbox with seven genes (ITS rDNA, β-tub , COI , EF1 α , HSP90 , L10 , and YPT1 ) generated from the type specimens of 212 validly published, and culturable species (including nine hybrid taxa). The genome information of 23 types published to date is also included. Several aspects of the taxonomic revision and phylogenetic re-evaluation of the genus including species concepts, concept and position of the phylogenetic clades recognized within Phytophthora are discussed. Some of the contents of this manuscript, including factsheets for the 212 species, are associated with the " IDphy : molecular and morphological identification of Phytophthora based on the types" online resource (https://idtools.org/tools/1056/index.cfm). The first version of the IDphy online resource released to the public in September 2019 contained 161 species. In conjunction with this publication, we are updating the IDphy online resource to version 2 to include the 51 species recently described. The current status of the 223 described species is provided along with information on type specimens with details of the host (substrate), location, year of collection and publications. Additional information is provided regarding the ex-type culture(s) for the 212 valid culturable species and the diagnostic molecular toolbox with seven genes that includes the two metabarcoding genes (ITS and COI ) that are important for Sanger sequencing and also very valuable Molecular Operational Taxonomic Units (MOTU) for second and third generation metabarcoding High-throughput sequencing (HTS) technologies. The IDphy online resource will continue to be updated annually to include new descriptions. This manuscript in conjunction with IDphy represents a monographic study and the most updated revision of the taxonomy and phylogeny of Phytophthora, widely considered one of the most important genera of plant pathogens. Taxonomic novelties: New species: Phytophthora cajani K.S. Amin, Baldev & F.J. Williams ex Abad, Phytophthora honggalleglyana Abad, Phytophthora megakarya Brasier & M.J. Griffin ex Abad, Phytophthora pisi Heyman ex Abad, Phytophthora pseudopolonica W.W. Li, W.X. Huai & W.X. Zhao ex Abad & Kasiborski; New combinations: Phytophthora ×multiformis (Brasier & S.A. Kirk) Abad, Phytophthora uniformis (Brasier & S.A. Kirk) Abad; Epitypifications (basionyms): Peronospora cactorum Lebert & Cohn, Pythiacystis citrophthora R.E. Sm. & E.H. Sm., Phytophthora colocasiae Racib., Phytophthora drechsleri Tucker, Phytophthora erythroseptica Pethybr., Phytophthora fragariae Hickman, Phytophthora hibernalis Carne , Phytophthora ilicis Buddenh. & Roy A. Young , Phytophthora inundata Brasier et al. , Phytophthora megasperma Drechsler , Phytophthora mexicana Hotson & Hartge, Phytophthora nicotianae Breda de Haan, Phytophthora phaseoli Thaxt., Phytophthora porri Foister, Phytophthora primulae J.A. Toml., Phytophthora sojae Kaufm. & Gerd., Phytophthora vignae Purss, Pythiomorpha gonapodyides H.E. Petersen; Lectotypifications (basionym): Peronospora cactorum Lebert & Cohn, Pythiacystis citrophthora R.E. Sm. & E.H. Sm., Phytophthora colocasiae Racib., Phytophthora drechsleri Tucker, Phytophthora erythroseptica Pethybr., Phytophthora fragariae Hickman, Phytophthora hibernalis Carne , Phytophthora ilicis Buddenh. & Roy A. Young , Phytophthora megasperma Drechsler , Phytophthora mexicana Hotson & Hartge, Phytophthora nicotianae Breda de Haan, Phytophthora phaseoli Thaxt., Phytophthora porri Foister, Phytophthora primulae J.A. Toml., Phytophthora sojae Kaufm. & Gerd., Phytophthora vignae Purss, Pythiomorpha gonapodyides H.E. Petersen; Neotypifications (basionym): Phloeophthora syringae Kleb., Phytophthora meadii McRae Citation: Abad ZG, Burgess TI, Bourret T, Bensch K, Cacciola S, Scanu B, Mathew R, Kasiborski B, Srivastava S, Kageyama K, Bienapfl JC, Verkleij G, Broders K, Schena L, Redford AJ (2023). Phytophthora : taxonomic and phylogenetic revision of the genus. Studies in Mycology 106 : 259-348. doi: 10.3114/sim.2023.106.05., Competing Interests: The authors declare that there is no conflict of interest., (© 2023 Westerdijk Fungal Biodiversity Institute.)

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Production and hosting by Elsevier B.V.)

Rapid and accurate identification of Anthonomus grandis subspecies is crucial for effective management and eradication. Current diagnostic methods have limitations in terms of time to diagnosis (up to seven days) and can yield ambiguous results. Here, we present the validation of a custom TaqMan SNP Genotyping Assay for the rapid and accurate identification of A. grandis grandis (boll weevil) and A. g. thurberiae (thurberia weevil) subspecies. To validate the assay, we conducted three main experiments: (1) a sensitivity test to determine the DNA concentration range at which the assay performs, (2) a non-target specificity test to ensure no amplification in non-target weevils (false positives), and (3) an accuracy test comparing the results of the new assay to previously established methods. These experiments were carried out in parallel at three independent facilities to confirm the robustness of the assay to variations in equipment and personnel. We used DNA samples from various sources, including field-collected specimens, museum specimens, and previously isolated DNA. The assay demonstrated high sensitivity (PCR success with ≥0.05 ng/µL DNA template), specificity (0.02 false positive rate), and accuracy (97.7%) in diagnosing boll weevil and thurberia weevil subspecies. The entire workflow, including DNA extraction, assay preparation, PCR run time, and data analysis, can be completed within a single workday (7-9 h) by a single technician. The deployment of this assay as a diagnostic tool could benefit boll weevil management and eradication programs by enabling same-day diagnosis of trap-captured or intercepted weevil specimens. Furthermore, it offers a more reliable method for identifying unknown specimens, contributing to the overall effectiveness of boll weevil research and control efforts.

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.

Global agricultural trade has accelerated the emergence and re-emergence of new plant pathogens. In the United States, the fungal pathogen Colletotrichum liriopes is still considered a foreign quarantine pathogen that affects ornamental plants (i.e., Liriope spp.). Even though this species has been reported in East Asia on various asparagaceous hosts, its first and only report in the United States was in 2018. However, that study used only ITS nrDNA for identification, and no available culture or voucher specimen was maintained. The main objective of the present study was to determine the geographic and host distribution of specimens identified as C. liriopes . To accomplish this, new and existing isolates, sequences, and genomes obtained from various hosts and geographic locations (i.e., China, Colombia, Mexico, and the United States) were compared with the ex-type of C. liriopes . Multilocus phylogenetic (ITS, Tub2, GAPDH, CHS-1, and HIS3), phylogenomic, and splits tree analyses revealed that all the studied isolates/sequences form a well-supported clade with little intraspecific variation. Morphological characterizations support these findings. The minimum spanning network, low nucleotide diversity, and negative Tajima's D from both multilocus and genomic data suggest that there was a recent movement/invasion of a few East Asian genotypes to other countries where the ornamental plants are produced (e.g., South America) and subsequently to the importing countries, such as the United States. The study reveals that the geographic and host distribution of C. liriopes sensu stricto is expanded to the United States (i.e., at least Maryland, Mississippi, and Tennessee) and on various hosts in addition to Asparagaceae and Orchidaceae. The present study produces fundamental knowledge that can be used in efforts to reduce costs or losses from agricultural trade and to expand our understanding of pathogen movement., Competing Interests: The author(s) declare no conflict of interest.

Cotton leafroll dwarf virus (CLRDV) is emerging across the major cotton-producing states of the southern United States. Because it was detected in nearly all cotton-producing states within a few years of its initial detection in the United States, the spread of the virus has apparently occurred rapidly. In this study spanning three growing seasons in South Carolina, we collected CLRDV isolates from symptomatic and asymptomatic cotton plants in 10 counties. The genomic region encoding P0, the viral suppressor of RNA silencing, was sequenced and compared among CLRDV isolates. Low variability among CLRDV P0 sequences from South Carolina isolates with similarities to other United States isolates was revealed by amino acid sequence alignment and phylogenetic analysis. Low variability among South Carolina isolates was also confirmed by sequencing a subset of eight near-complete genomes of CLRDV isolates. Although sequence variability was low among South Carolina isolates, this data should be taken in the context of all United States isolates, for which diversity may be higher than initially expected. Sequences gathered in this study add to the body of knowledge on CLRDV diversity in the United States., Competing Interests: The author(s) declare no conflict of interest.

We studied the taxonomy of Pluteus podospileus and similar species using morphological and molecular (nrITS, TEF1-α ) data, including a detailed study of the type collections of P. inflatus var. alneus , Pluteus minutissimus f. major, and P. granulatus var. tenellus . Within the P. podospileus complex, we phylogenetically confirmed six species in Europe, five in Asia, and eight in North America. Based on our results, we recognize P. seticeps as a separate species occurring in North America, while P. podospileus is limited to Eurasia. We describe six new species and a new variety: P. absconditus , P. fuscodiscus , P. gausapatus , P. inexpectatus , P. millsii, and P. notabilis and its variety, P. notabilis var. insignis . We elevate Pluteus seticeps var. cystidiosus to species rank as Pluteus cystidiosus . Based on the holotype of P. inflatus var. alneus , collections of P. inflatus identified by Velenovský, and several modern collections, we resurrect the name P. inflatus . Based on molecular analyses of syntypes of Pluteus minutissimus f. major and a holotype of Pluteus granulatus var. tenellus , we synonymize them under P. inflatus . We also increase our knowledge about the morphology and distribution of P. cutefractus ., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study, in the collection, analysis, or interpretation of data, in the writing of the manuscript, or in the decision to publish the results.

Analysis of ecological and evolutionary aspects leading to durability of resistance in soybean cultivars against species Soybean vein necrosis orthotospovirus (SVNV) (Bunyavirales: Tospoviridae) is important for the establishment of integrated pest management (IPM) across the United States, which is a leading exporter of soybeans in the world. SVNV is a seed- and thrips- (vector)-borne plant virus known from the USA and Canada to Egypt. We monitored the resistance of soybean cultivars against SVNV, surveyed thrips species on various crops including soybeans in Pennsylvania, and studied thrips overwintering hibernation behavior under field conditions. Field and lab experiments determined disease incidence and vector abundance in soybean genotypes. The impact of the virus, vector, and their combination on soybean physiology was also evaluated. Seed protein, fiber, oil, and carbohydrate content were analyzed using near infra-red spectroscopy. We found that the variety Channel3917R2x had higher numbers of thrips; hence, it was categorized as preferred, while results showed that no variety was immune to SVNV. We found that thrips infestation alone or in combination with SVNV infection negatively impacted soybean growth and physiological processes.

Multiple Diaporthe spp. cause root and fruit rots or stem lesions on Cucumis spp.: D. cucurbitae, D. melonis, D. longicolla (syn. D. eres ), D. pterocarpi, D. sclerotioides, D. sojae , and D. ueckerae (Broge et al. 2020; Fukada et al. 2018; Udayanga et al. 2012, 2015). From May-August 2021, cucumbers ( Cucumis sativus ) 'Katrina' and 'Alcazar' were grown in a 24-plant, commercial Bato bucket system with rockwool blocks on a perlite substrate in a research greenhouse in Wooster, Ohio. At maturity, plants collapsed rapidly from stem lesions without foliar chlorosis (25% of 'Katrina' and 17% of 'Alcazar'). Lesions were 7.5 to 15 cm in length, tan to golden-brown with black pycnidia and located 5 to 15 cm above the crown. Stems shredded easily with vascular discoloration around the lesion. Two identical fungal strains were isolated on ½ acidified potato dextrose agar (APDA) following surface disinfestation with 0.6% sodium hypochlorite for 30 s and sterile water rinse. Fungal cultures were floccose, white to tan mycelia with pycnidia. Oblong, elliptical, biguttulate, aseptate alpha conidia were observed with mean dimensions: 8.0 µm (5.2-9.8 µm) by 3.1 µm (2.5-3.8 µm) on ½ APDA and 9.8 µm (6.6-12.4 µm) by 3.0 µm (1.9-5.3 µm) on petioles. On prune extract agar, beta conidia mean dimensions were: 19.7 µm (12.0-27.7 µm) by 1.2 µm (0.8-1.8 µm). Fungal DNA was amplified and sequenced bidirectionally with ITS (ITS4/ITS5), CAL (CAL228F/737R), HIS (CYLH3F/H3-1B), TEF1 (EF1-728f/EF1-986R), and TUB2 (Bt1a/Bt1b) primers (Carbone and Kohn 1999; Glass and Donaldson 1995) (GenBank: OP265712-13, OP288460-65, OQ418506-07). Based on a maximum likelihood phylogenetic tree of concatenated genes, this novel Diaporthe sp., most closely related to D. stewartii, has not been reported on Cucumis spp. Strains were deposited in the USDA-ARS Culture Collection (NRRL# 64461-62). Koch's postulates were conducted in a greenhouse with mean day temperature of 25°C and 12 hr supplemental lighting. One-month old cucumbers 'Katrina,' grown in rockwool cubes (5 plants per isolate) and potting mix (6 plants per isolate), were inoculated with a one-week-old culture of either strain. The second true leaf was cut and a pipette tip containing an inoculated plug of ½ APDA was placed on the remaining petiole (Mathew et al. 2018). Non-inoculated ½ APDA was used for controls. Plants were tarped for 24 hours to increase humidity and pipette tips removed after one week. After two weeks, petioles were shrunken, tan to golden brown with pycnidia. After 3-4 weeks, stem lesions matching those above were observed on inoculated plants, and plants collapsed. For fruit rot, three Beit Alpha cucumbers were rinsed with tap water, dried, a 5 mm plug was removed from the fruit and replaced with a 5 mm plug of one-week-old fungus on ½ APDA. After 3 days, fruits were water soaked and soft. For root rot, two plates of one-week-old cultures were macerated in 500 mL of sterile water and mixed with 1500 mL of vermiculite. Two seeds of cucumber 'Katrina' were planted into three reps of each isolate and control. All control seeds germinated, but all inoculated seeds experienced pre- or post-emergence damping off. No symptoms were ever observed on any controls. Fungi were isolated from all inoculated tissues as described above. Based on morphology, Diaporthe sp. was isolated from all inoculated plants but never from controls. This Diaporthe sp. may be a new constraint to hydroponic cucumber production, but incidence needs to be determined globally.

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., (© 2023. Springer Nature Limited.)

Mass-reared sterile Mexican fruit flies (Anastrepha ludens [Loew]) are released along the US-Mexico border to control fruit fly damage and spread. It is more cost effective for the mass rearing program if males mature at a younger age because they can be held for a shorter time at the facility before release. In this study, adult male Mexican fruit fly diets were tested to determine if the current diet used at the mass rearing facility promotes younger mating and optimal sperm transfer compared to other diet formulations. Hydrolyzed yeast presentation methods were examined: an agar-free dry mixture of yeast and sugar (Y+S), yeast embedded in the diet during the agar boiling process (current method used) and sifted dry on the ager gel surface. Methoprene, a juvenile hormone analog was also tested as a supplement to agar gel diets with and without yeast. A greater proportion of males fed Y+S mated 1 day earlier than males fed other diets. Male mating age and diet did not significantly affect the quantity of sperm transferred; however, a non-significantly higher percentage of males fed diets with embedded yeast transferred enough sperm to fill all 3 spermathecae. The results suggest that the current diet used is optimal for mass-rearing the flies and that yeast presentation method has a significant effect on A. ludens male mating age but not on the quantity of sperm transferred to females., (Published by Oxford University Press on behalf of Entomological Society of America 2023.)

The Mormon cricket, Anabrus simplex, is a flightless katydid, one of the major devastating rangeland pests in several states of the western United States. During the past few years, their sudden and periodic outbreaks into massive migratory bands caused significant economic losses to the rangeland forage and agricultural crops, particularly grain crops. Current population management methods rely heavily on broad-spectrum chemical insecticides, which could be toxic to nontargets, and even the targeted species might develop resistance in the long run. Therefore, we assessed the potential of RNA interference (RNAi)-based alternative management strategies that could supplement the current methods. In insects, RNAi efficiency varies with the method of double-stranded RNA (dsRNA) delivery. We tested 2 different methods of dsRNA delivery: injection and oral feeding of dsRNA. The results showed that Mormon crickets are sensitive to injection of dsRNA in a dose-dependent manner, but refractory to the oral feeding of dsRNA. Further, we confirmed the high nuclease activity in the insect midgut. In order to protect the dsRNA from the dsRNase activity and facilitate its uptake in the midgut, we encapsulated dsRNA inside poly lactic-co-glycolic acid (PLGA) nanoparticles and studied its release kinetics and RNAi efficiency by oral feeding. The release kinetics clearly suggested that the PLGA nanoparticle permeates from the insect digestive system to the hemolymph; however, it failed to induce an efficient RNAi response of the targeted genes. In conclusion, our findings suggest the different responses to dsRNA delivery methods in Mormon crickets, and further investigations involving dsRNA stability and its uptake mechanism are required to use RNAi as an alternative Mormon cricket population management strategy., (© The Author(s) 2023. Published by Oxford University Press on behalf of Entomological Society of America.)

Food-based baits are an important component of trapping networks designed to detect invasive tephritid fruit flies (Diptera: Tephritidae). An aqueous solution of torula yeast plus borax (TYB) is widely used, but synthetic food lures have been developed to facilitate field procedures, ensure standard composition, and lengthen the interval of bait attractiveness. Cone-shaped dispensers, containing ammonium acetate, putrescine, and trimethylamine (so-called 3C food cones), are currently being used in some large-scale trapping systems (e.g., Florida). Prior work in Hawaii showed that traps baited with 3C food cones capture similar numbers of Mediterranean fruit flies (medflies), Ceratitis capitata (Wiedemann), as TYB-baited traps after 1-2 wk of weathering but capture fewer medflies thereafter. In addition, 3C food cones attract fewer oriental fruit flies, Bactrocera dorsalis (Hendel), and melon flies, Zeugodacuscucurbitae (Coquillett) than TYB even when the food cones are freshly deployed. The current study describes an additional trapping experiment that expands upon earlier work by (i) presenting 3C food cones either unbagged (as done previously) or in nonporous or breathable bags to possibly reduce volatilization and lengthen bait effectiveness and (ii) measuring the content of the 3 components over time to potentially associate fruit fly captures with the loss of these food cone constituents. Implications of these findings for fruit fly surveillance programs are discussed., (Published by Oxford University Press on behalf of Entomological Society of America 2023.)

Since its discovery in North America in 2014, the spotted lanternfly (SLF), Lycorma delicatula , has become an economic, ecological, and nuisance pest there. Developing early detection and monitoring tools is critical to their mitigation and control. Previous research found evidence that SLF may use pheromones to help locate each other for aggregation or mating. Pheromone production necessitates specific conditions by the insects, and these must be investigated and described. A chemical process called photo-degradation has been described as a final step in the production of pheromones in several diurnal insect species, in which cuticular hydrocarbons were broken down by sunlight into volatile pheromone components. In this study, photo-degradation was investigated as a possible pheromone production pathway for SLF. Extracts from SLF mixed-sex third and fourth nymphs and male or female adults were either exposed to simulated sunlight to produce a photo-degradative reaction (photo-degraded), or not exposed to light (crude), while volatiles were collected. Behavioral bioassays tested for attraction to volatiles from photo-degraded and crude samples and their residues. In third instars, only the volatile samples from photo-degraded mixed-sex extracts were attractive. Fourth instar males were attracted to both crude and photo-degraded residues, and volatiles of photo-degraded mixed-sex extracts. Fourth instar females were attracted to volatiles of crude and photo-degraded mixed-sex extracts, but not to residues. In adults, only males were attracted to body volatiles from crude and photo-degraded extracts of either sex. Examination of all volatile samples using gas chromatography coupled with mass spectrometry (GC-MS) revealed that most of the identified compounds in photo-degraded extracts were also present in crude extracts. However, the abundance of these compounds in photo-degraded samples were 10 to 250 times more than their abundance in the crude counterparts. Results from behavioral bioassays indicate that photo-degradation probably does not generate a long-range pheromone, but it may be involved in the production of a short-range sex-recognition pheromone in SLF. This study provides additional evidence of pheromonal activity in SLF.

Helicoverpa armigera (Hübner) is a major crop pest native to Europe, Asia, Australia, and Africa which has recently invaded South America and has caused billions of dollars in agricultural losses. Because of challenges in differentiating between H. armigera and Helicoverpa zea (Boddie), a closely related species native to North and South America, genetic tests have previously been developed to detect H. armigera DNA in pooled samples of moth legs. In this study, a field-based recombinase polymerase amplification (RPA) assay using a lateral flow strip and a qPCR melt curve assay were developed for specific detection of H. armigera DNA in pooled moth samples. In addition, a crude DNA extraction protocol for whole moths was developed to allow rapid preparation of DNA samples. The RPA field test was able to detect ≥ 10 pg of purified H. armigera DNA and the crude DNA of one H. armigera sample in a background of 999 H. zea equivalents. The qPCR assay was able to detect ≥ 100 fg of purified H. armigera DNA and the crude DNA of one H. armigera sample in a background of up to 99,999 H. zea equivalents. Both RPA and qPCR assays detected H. armigera in the crude DNA extracted in the field from a pool of one H. armigera moth and 999 H. zea moths. These newly developed molecular assays to detect H. armigera will contribute to large-scale surveillance programs of H. armigera., (© The Author(s) 2023. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Tembrock, Wilson, Zink, Timm, Gilligan, Konstantinov and Tishechkin.)

Here, we report the detection and characterization of the genome of a novel poacevirus isolated from Zoysia matrella (Merrill) imported into the United States from Japan. The novel virus, tentatively named "zoysia mosaic virus" (ZoMV), is a single-stranded RNA virus with a genome of 9,728 nucleotides (nt) in length, encoding a large putative polyprotein of 3,119 amino acids (aa). The ZoMV genome is closely related to the triticum mosaic virus (TriMV; FJ263671) genome, with 57.18% nt and 51.74% aa sequence identity in the polyprotein region. Moreover, phylogenetic analysis showed that ZoMV is closely related to all other members of the genus Poacevirus. A survey of imported grasses showed that ZoMV was detected only in zoysiagrass. This is the first report of the complete genome sequence of a novel viral pathogen of zoysiagrass of the genus Poacevirus, for which we propose the binomial species name "Poacevirus zoisiae"., (© 2023. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Phytophthora , with 203 species, is a genus of high importance in agriculture worldwide. Here, we present the online resource " IDphy ", developed to facilitate the correct identification of species of Phytophthora using the type specimens from the original descriptions wherever possible. IDphy emphasizes species of high economic impact and regulatory concern for the United States. IDphy presents an interactive Lucid key and a tabular key for 161 culturable species described as of May 2018, including 141 ex-types and 20 well-authenticated specimens. IDphy contains standard operating procedures for morphological and molecular characterization, as well as a glossary, image gallery, and numerous links. Each of the 161 factsheets includes access to nomenclature and morphological and molecular features, including sequences of the internal transcribed spacer ribosomal DNA, cytochrome C oxidase subunit I (barcoding genes), YPT1, β-tubulin, elongation factor 1a, L10, heat shock protein 90, and other genes. IDphy contains an innovative in silico BLAST and phylogenetic sequence analysis using NCBI. The IDphy mobile app, released in August 2021 (free for Android or iOS), allows users to take the Lucid key into the laboratory. IDphy is the first online identification tool based on the ex-types implemented for plant pathogens. In this article, we also include information for 21 new species and one hybrid described after the publication of IDphy , the status of the specimens of the types and ex-types at international herbaria and culture collections, and the status of genomes at the GenBank (currently 153 genome assemblies which correspond to 42 described species, including 16 ex-types). The effectiveness of the IDphy online resource and the content of this article could inspire other researchers to develop additional identification tools for other important groups of plant pathogens.

Virome analysis via high-throughput sequencing (HTS) allows rapid and massive virus identification and diagnoses, expanding our focus from individual samples to the ecological distribution of viruses in agroecological landscapes. Decreases in sequencing costs combined with technological advances, such as automation and robotics, allow for efficient processing and analysis of numerous samples in plant disease clinics, tissue culture laboratories, and breeding programs. There are many opportunities for translating virome analysis to support plant health. For example, virome analysis can be employed in the development of biosecurity strategies and policies, including the implementation of virome risk assessments to support regulation and reduce the movement of infected plant material. A challenge is to identify which new viruses discovered through HTS require regulation and which can be allowed to move in germplasm and trade. On-farm management strategies can incorporate information from high-throughput surveillance, monitoring for new and known viruses across scales, to rapidly identify important agricultural viruses and understand their abundance and spread. Virome indexing programs can be used to generate clean germplasm and seed, crucial for the maintenance of seed system production and health, particularly in vegetatively propagated crops such as roots, tubers, and bananas. Virome analysis in breeding programs can provide insight into virus expression levels by generating relative abundance data, aiding in breeding cultivars resistant, or at least tolerant, to viruses. The integration of network analysis and machine learning techniques can facilitate designing and implementing management strategies, using novel forms of information to provide a scalable, replicable, and practical approach to developing management strategies for viromes. In the long run, these management strategies will be designed by generating sequence databases and building on the foundation of pre-existing knowledge about virus taxonomy, distribution, and host range. In conclusion, virome analysis will support the early adoption and implementation of integrated control strategies, impacting global markets, reducing the risk of introducing novel viruses, and limiting virus spread. The effective translation of virome analysis depends on capacity building to make benefits available globally., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Alcalá Briseño, Batuman, Brawner, Cuellar, Delaquis, Etherton, French-Monar, Kreuze, Navarrete, Ogero, Plex Sulá, Yilmaz and Garrett.)

A mark-release-recapture experiment was conducted to evaluate the orientation of spotted lanternfly (SLF) Lycorma delicatula White (Hemiptera: Fulgoridae) nymphs when released equidistant between two trees. The experiment was repeated weekly for eight weeks in a heavily infested area with mature tree-of-heaven Ailanthus altissima (Mill.) Swingle (Sapindales: Simaroubaceae) planted in rows as ornamental street trees in Beijing, China. One tree in each pair received a methyl salicylate lure, and the lure was rotated between trees every week as it aged. Two additional independent variables for each tree were also analyzed: size and SLF population density. Marked-released SLF significantly chose trees with higher SLF population density over trees with lower density populations, and they also chose larger trees significantly more than smaller trees. Population density and tree size were better predictors of attraction than lures, but when those factors were controlled, SLF significantly chose trees with methyl salicylate lures over control trees for the first 4 weeks of lure life. Wild SLF distribution was assessed weekly, revealing strong aggregation in first and second instars that diminished with development to the third and fourth instars. Thus, nymphal SLF aggregate, and orientation is strongly guided by the presence of other SLF and tree size.

Nanoparticles have been proposed as tunable delivery vehicles for targeted treatments and, in some cases, the active therapeutic agents themselves. Despite the promise of such customizable impacts, little evidence exists to support these claims in the realm of antibiotics. Exploration of the silver and copper nanoparticle antibacterial impacts have been reported with inconsistent results. Here, we investigate the physical, chemical, and bacterial properties of silver and copper core particles stabilized with commonly used surface coatings, namely, polyvinylpyrrolidone (PVP, to confer a neutrally charged surface), cetrimonium bromide (CTAB, positively charged surface), citrate (Cit, negatively charged surface for silver nanoparticles), and ascorbic acid (AA, negatively charged surface for copper nanoparticles. The impacts of these potential antibacterial nanoparticles are measured against three bacterial species spanning deep divisions in the bacterial tree of life and include Escherichia coli , Staphylococcus aureus , and Sphingobacterium multivorum . Varying dose, core composition, surface coating, and bacterial species revealed that nanoparticle surfaces accounted for most of the variation in antibacterial activity. In all experiments, dose produced a linear inhibitory effect. Surprisingly, bacterial species reacted similarly regardless of evolutionary relatedness. There is a high degree of consistency, effectiveness, and efficacy among PVP silver and copper nanoparticle. These findings have implications for the intentional use of nanotechnology in environmental systems., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Ameh, Zarzosa, Braswell and Sayes.)