Using targeted sequencing and TaqMan approaches to detect acaricide (bifenthrin, bifenazate, and etoxazole) resistance associated SNPs in Tetranychus urticae collected from peppermint fields and hop yards.

Tetranychus urticae (Koch) is an economically important pest of many agricultural commodities world-wide. Multiple acaricides, including bifenazate, bifenthrin, and extoxazole, are currently registered to control T. urticae. However, populations of T. urticae in many different growing regions have developed acaricide resistance through multiple mechanisms. Within T. urticae, single nucleotide polymorphisms (SNPs) have been documented in different genes which are associated with acaricide resistance phenotypes. The detection of these mutations through TaqMan qPCR has been suggested as a practical, quick, and reliable tool to inform agricultural producers of acaricide resistance phenotypes present within their fields and have potential utility for making appropriate acaricide application and integrated pest management decisions. Within this investigation we examined the use of a TaqMan qPCR-based approach to determine genotypes which have been previously associated with acaricide resistance in field-collected populations of T. urticae from peppermint fields and hop yards in the Pacific Northwest of the United States and confirmed the results with a multiplex targeted sequencing. The results suggest that a TaqMan qPCR approach accurately genotypes T. urticae populations for SNPs that have been linked to Bifenazate, Bifenthrin, and Etoxazole resistance. The results also demonstrated that different populations of mites in Washington and Idaho displayed varying frequencies of the examined SNPs. While we were able to detect the SNPs associated with the examined acaricides, the mutation G126S was not an appropriate or accurate indicator for bifenazate resistance.

Multiplex Polymerase Chain Reaction Reveals Unique Trends in Pathogen and Parasitoid Infestations of Alfalfa Leafcutting Brood Cells.

The alfalfa leafcutting bee Megachile rotundata Fabricius (HYMENOPTERA: Megachilidae) is an important pollinator for multiple agricultural seed commodities in the United States. M. rotundata is a solitary cavity nesting bee that forms brood nests where its larvae can develop. During the developmental stages of growth, brood can be preyed upon by multiple different fungal pathogens and insect predators and parasitoids, resulting in the loss of the developing larvae. Larval loss is a major concern for alfalfa (Medicago sativa L.) seed producers because they rely on pollination services provided by M. rotundata. Reduced pollination rates result in lower yields and increased production costs. In the present study, we examined the taxonomic composition of organisms found within M. rotundata brood cells using a multiplex PCR assay which was developed for the detection of bacterial, fungal, and invertebrate pests and pathogens of M. rotundata larvae. Known pests of M. rotundata were detected, including members of the fungal genus Ascosphaera, the causative agent of chalkbrood. The presence of multiple Ascosphaera species in a single brood cell was observed, with potential implications for chalkbrood disease management. The multiplex assay also identified DNA from more than 2,400 total species, including multiple predators and pathogenetic species not previously documented in association with M. rotundata brood cells.

Low-Cost Live Insect Scouting Drone: iDrone Bee.

Unmanned aerial vehicles (UAVs, e.g., drones) are a common tool for many civil applications, including precision agriculture, transportation, delivery services, rescue missions, law enforcement, and more. Remote sensing technologies used in conjunction with drones are a dominant application in precision agriculture. Multispectral instrumentation attached to UAVs allows the user to observe multiple parameters, including the normalized difference vegetation index which can represent crop stresses induced by various factors (e.g., drought, insect outbreak, nutrient loss, and other diseases). However, little research has been done to apply drones to accomplish a mission-oriented actionable task in agriculture, such as insect sampling. We propose a low-cost, open source-based live insect scouting drone named 'iDrone Bee' to benefit the integrated pest management (IPM) community by minimizing time and efforts of human interventions while collecting live insects in agricultural fields. Herein we present instruction and operation procedures to build and operate an iDrone Bee for insect scouting in an agricultural ecosystem and validate the system in an alfalfa seed field. The findings of this investigation demonstrate that a drone-based insect scouting method may be a valuable tool to benefit the IPM community.

Examining Historical Rates of Leafcutting Bee Brood Cell Pathogens, Parasitoids, and Predators to Establish Baseline Infectivity Rates for Alfalfa Seed Growers.

The alfalfa leafcutting bee Megachile rotundata (Fabricius) (Hymenoptera: Megachilidae) is the primary pollinator for the alfalfa seed industry. It is a solitary cavity nesting bee that utilizes leaf lined brood cells provisioned with pollen for larval development and pupation into the adult stage. During development, multiple pathogens, parasitoids, and predators can prey upon or use the larvae as a host, resulting in the loss of the future adult bee. As such, the presence of invertebrate pests and fungal pathogens presents a major concern for commercial alfalfa seed growers. In the present study, we used historic data from the Parma Cocoon Diagnostic Laboratory to determine baseline rates of pathogens, parasitoids, and predators of Megachile rotundata brood cells and used this analysis to determine cutoffs for management practices to inform growers when the purchase of new bee stocks should be considered. Additionally, we compared the presence of chalkbrood, predators, and parasitoids in samples collected from both grower-produced stocks and newly purchased Canadian bees. The results of the investigation provide historic averages of the presence of chalkbrood, predators, and parasitoids, show a significant increase in chalkbrood and predators in 2007-2011, and find a significant difference in rates of chalkbrood and predators between samples from Canadian suppliers and grower stocks. We speculate that these differences may have resulted from economic conditions that increased the cost of Canadian Megachile rotundata cells and likely resulted in increased reliance on 2nd-year U.S. grower stocks and subsequently increased infection rates during this time period.

Targeted RNA sequencing reveals differential patterns of transcript expression in geographically discrete, insecticide resistant populations of Leptinotarsa decemlineata.

BACKGROUND: The Colorado potato beetle (Leptinotarsa decemlineata Say) is a major agricultural pest of commercial potatoes, partially due to its ability to rapidly develop resistance to multiple insecticide modes of action. Patterns of L. decemlineata insecticide resistance in the contiguous United States have been linked to geographic location and regional management practices. Several previous studies have classified enzymes that are overexpressed following L. decemlineata exposure to commercial pesticides, many of which have been linked to xenobiotic metabolism. Studies have further associated geographic disparities in resistance patterns to cross-resistance driven by fungicide exposure in the East Coast and Midwest. RESULTS: In this study, our objective was to investigate transcript expression of 38 previously classified detoxification enzymes induced by imidacloprid (an insecticide) and chlorothalonil (a fungicide) within five discrete populations of L. decemlineata obtained from areas in the USA representing eastern, midwestern and western production regions. We found unique patterns of transcript expression in different geographic locations, including overexpression of transcripts related to insecticide metabolism within insecticide-resistant populations. CONCLUSION: The results suggest the genetic response of these populations may be partially linked to geographic location and corresponding management practices. © 2021 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Selection for high levels of resistance to double-stranded RNA (dsRNA) in Colorado potato beetle (Leptinotarsa decemlineata Say) using non-transgenic foliar delivery.

Insecticidal double-stranded RNAs (dsRNAs) silence expression of vital genes by activating the RNA interference (RNAi) mechanism in insect cells. Despite high commercial interest in insecticidal dsRNA, information on resistance to dsRNA is scarce, particularly for dsRNA products with non-transgenic delivery (ex. foliar/topical application) nearing regulatory review. We report the development of the CEAS 300 population of Colorado potato beetle (Leptinotarsa decemlineata Say) (Coleoptera: Chrysomelidae) with > 11,100-fold resistance to a dsRNA targeting the V-ATPase subunit A gene after nine episodes of selection using non-transgenic delivery by foliar coating. Resistance was associated with lack of target gene down-regulation in CEAS 300 larvae and cross-resistance to another dsRNA target (COPI ß; Coatomer subunit beta). In contrast, CEAS 300 larvae showed very low (~ 4-fold) reduced susceptibility to the Cry3Aa insecticidal protein from Bacillus thuringiensis. Resistance to dsRNA in CEAS 300 is transmitted as an autosomal recessive trait and is polygenic. These data represent the first documented case of resistance in an insect pest with high pesticide resistance potential using dsRNA delivered through non-transgenic techniques. Information on the genetics of resistance and availability of dsRNA-resistant L. decemlineata guide the design of resistance management tools and allow research to identify resistance alleles and estimate resistance risks.

'Candidatus Phytoplasma asteris' subgroups display distinct disease progression dynamics during the carrot growing season.

Aster Yellows phytoplasma (AYp; 'Candidatus Phytoplasma asteris') is an obligate bacterial pathogen that is the causative agent of multiple diseases in herbaceous plants. While this phytoplasma has been examined in depth for its disease characteristics, knowledge about the spatial and temporal dynamics of pathogen spread is lacking. The phytoplasma is found in plant's phloem and is vectored by leafhoppers (Cicadellidae: Hemiptera), including the aster leafhopper, Macrosteles quadrilineatus Forbes. The aster leafhopper is a migratory insect pest that overwinters in the southern United States, and historical data suggest these insects migrate from southern overwintering locations to northern latitudes annually, transmitting and driving phytoplasma infection rates as they migrate. A more in-depth understanding of the spatial, temporal and genetic determinants of Aster Yellows disease progress will lead to better integrated pest management strategies for Aster Yellows disease control. Carrot, Daucus carota L., plots were established at two planting densities in central Wisconsin and monitored during the 2018 growing season for Aster Yellows disease progression. Symptomatic carrots were sampled and assayed for the presence of the Aster Yellows phytoplasma. Aster Yellows disease progression was determined to be significantly associated with calendar date, crop density, location within the field, and phytoplasma subgroup.

Genetic Variation Among Geographically Disparate Isolates of Aster Yellows Phytoplasma in the Contiguous United States.

Aster Yellows phytoplasma (AYp; Candidatus Phytoplasma asteris) is associated with diseases of herbaceous plants, including ornamentals and important commercial vegetable and grain crops. The aster leafhopper (ALH; Macrosteles quadrilineatus Forbes) is the predominant vector of these bacteria, though other leafhopper species can acquire and transmit AYp. Potentially inoculative leafhoppers are reported to overwinter in the southern United States and migrate to northern latitudes in the spring. Examining the genetic similarities and differences in AYp associated with southern and northern populations of ALH may provide insight into the role that migrating ALH play in AYp disease development. To investigate similarities among geographically distinct populations of ALH and characterize the variation in AYp associated within these populations, we identified genetic variations in subgroup designation and the relative proportions of secreted AY-WB proteins from field-collected populations of AYp isolated from ALH from select locations in the southern (Arkansas, Kansas, Oklahoma, and Texas) and the northern United States (Wisconsin) in 2016, 2017, and 2018. Isolated phytoplasma were tested for variation of AYp genotypes, numbers of potentially inoculative (AYp-positive) ALH, and presence of specific AYp virulence (effector) genes. Geographically distinct populations of ALH collected in northern and southern regions were similar in CO1 genotype but carried different proportions of AYp genotypes. While similar AYp strains were detected in geographically distinct locations, the proportion of each genotype varied over time.

Changes in emergence phenology, fatty acid composition, and xenobiotic-metabolizing enzyme expression is associated with increased insecticide resistance in the Colorado potato beetle.

The Colorado potato beetle (Leptinotarsa decemlineata) is a major agricultural pest of solanaceous crops. An effective management strategy employed by agricultural producers to control this pest species is the use of systemic insecticides. Recent emphasis has been placed on the use of neonicotinoid insecticides. Despite efforts to curb resistance development through integrated pest management approaches, resistance to neonicotinoids in L. decemlineata populations continues to increase. One contributing factor may be alterations in insect fatty acids, which have multiple metabolic functions and are associated with the synthesis of xenobiotic-metabolizing enzymes to mitigate the effects of insecticide exposure. In this study, we analyzed the fatty acid composition of L. decemlineata populations collected from an organic production field and from a commercially managed field to determine if fatty acid composition varied between the two populations. We demonstrate that a population of L. decemlineata that has a history of systemic neonicotinoid exposure (commercially managed) has a different lipid composition and differential expression of known metabolic detoxification mechanisms relative to a population that has not been exposed to neonicotinoids (organically managed). The fatty acid data indicated an upregulation of Δ6 desaturase in the commercially managed L. decemlineata population and suggest a role for eicosanoids and associated metabolic enzymes as potential modulators of insecticide resistance. We further observed a pattern of delayed emergence within the commercially managed population compared with the organically managed population. Variations in emergence timing together with specific fatty acid regulation may significantly influence the capacity of L. decemlineata to develop insecticide resistance.

Conjugated linoleic acid as a novel insecticide targeting the agricultural pest Leptinotarsa decemlineata.

The Colorado Potato Beetle, Leptinotarsa decemlineata, is a major agricultural pest of solanaceous crops in the United States. Historically, a multitude of insecticides have been used to control problematic populations. Due to increasing resistance to insecticides, novel compounds and methodologies are warranted for the control of beetle populations. Mixed-isomer conjugated linoleic acid has been studied in-depth for its beneficial properties to mammalian systems. At the same time, studies have demonstrated that conjugated linoleic acid can manipulate fatty acid composition in non-mammalian systems, resulting in embryo mortality. Consequently, experiments were conducted to assess the effects of foliar-applied conjugated linoleic acid on larval growth, embryogenesis, and feeding preference in Colorado potato beetle. Both maternal and deterrent effects of dietary conjugated linoleic acid were assessed. Conjugated linoleic acid demonstrated desirable insecticidal properties, including increased larval mortality, slowed larval development, antifeedant effects, and decreased egg viability after maternal ingestion.

Transcriptomic analysis reveals similarities in genetic activation of detoxification mechanisms resulting from imidacloprid and chlorothalonil exposure.

The Colorado potato beetle, Leptinotarsa decemlineata (Say), is an agricultural pest of commercial potatoes in parts of North America, Europe, and Asia. Plant protection strategies within this geographic range employ a variety of pesticides to combat not only the insect, but also plant pathogens. Previous research has shown that field populations of Leptinotarsa decemlineata have a chronological history of resistance development to a suite of insecticides, including the Group 4A neonicotinoids. The aim of this study is to contextualize the transcriptomic response of Leptinotarsa decemlineata when exposed to the neonicotinoid insecticide imidacloprid, or the fungicides boscalid or chlorothalonil, in order to determine whether these compounds induce similar detoxification mechanisms. We found that chlorothalonil and imidacloprid induced similar patterns of transcript expression, including the up-regulation of a cytochrome p450 and a UDP-glucuronosyltransferase transcript, which belong to protein families associated with xenobiotic metabolism. Further, transcriptomic responses varied among individuals within the same treatment group, suggesting individual insects' responses vary within a population and may cope with chemical stressors in a variety of manners.

Agricultural fungicides inadvertently influence the fitness of Colorado potato beetles, Leptinotarsa decemlineata, and their susceptibility to insecticides.

The Colorado potato beetle (CPB), Leptinotarsa decemlineata (Say), is an agricultural pest of solanaceous crops which has developed insecticide resistance at an alarming rate. Up to this point, little consideration has been given to unintended, or inadvertent effects that non-insecticide xenobiotics may have on insecticide susceptibility in L. decemlineata. Fungicides, such as chlorothalonil and boscalid, are often used to control fungal pathogens in potato fields and are applied at regular intervals when L. decemlineata populations are present in the crop. In order to determine whether fungicide use may be associated with elevated levels of insecticide resistance in L. decemlineata, we examined phenotypic responses in L. decemlineata to the fungicides chlorothalonil and boscalid. Using enzymatic and transcript abundance investigations, we also examined modes of molecular detoxification in response to both insecticide (imidacloprid) and fungicide (boscalid and chlorothalonil) application to more specifically determine if fungicides and insecticides induce similar metabolic detoxification mechanisms. Both chlorothalonil and boscalid exposure induced a phenotypic, enzymatic and transcript response in L. decemlineata which correlates with known mechanisms of insecticide resistance.

A model species for agricultural pest genomics: the genome of the Colorado potato beetle, Leptinotarsa decemlineata (Coleoptera: Chrysomelidae).

The Colorado potato beetle is one of the most challenging agricultural pests to manage. It has shown a spectacular ability to adapt to a variety of solanaceaeous plants and variable climates during its global invasion, and, notably, to rapidly evolve insecticide resistance. To examine evidence of rapid evolutionary change, and to understand the genetic basis of herbivory and insecticide resistance, we tested for structural and functional genomic changes relative to other arthropod species using genome sequencing, transcriptomics, and community annotation. Two factors that might facilitate rapid evolutionary change include transposable elements, which comprise at least 17% of the genome and are rapidly evolving compared to other Coleoptera, and high levels of nucleotide diversity in rapidly growing pest populations. Adaptations to plant feeding are evident in gene expansions and differential expression of digestive enzymes in gut tissues, as well as expansions of gustatory receptors for bitter tasting. Surprisingly, the suite of genes involved in insecticide resistance is similar to other beetles. Finally, duplications in the RNAi pathway might explain why Leptinotarsa decemlineata has high sensitivity to dsRNA. The L. decemlineata genome provides opportunities to investigate a broad range of phenotypes and to develop sustainable methods to control this widely successful pest.

RNA interference of three up-regulated transcripts associated with insecticide resistance in an imidacloprid resistant population of Leptinotarsa decemlineata.

The Colorado potato beetle, Leptinotarsa decemlineata (Say), is a major agricultural pest of potatoes in the Central Sands production region of Wisconsin. Previous studies have shown that populations of L. decemlineata have become resistant to many classes of insecticides, including the neonicotinoid insecticide, imidacloprid. Furthermore, L. decemlineata has multiple mechanisms of resistance to deal with a pesticide insult, including enhanced metabolic detoxification by cytochrome p450s and glutathione S-transferases. With recent advances in the transcriptomic analysis of imidacloprid susceptible and resistant L. decemlineata populations, it is possible to investigate the role of candidate genes involved in imidacloprid resistance. A recently annotated transcriptome analysis of L. decemlineata was obtained from select populations of L. decemlineata collected in the Central Sands potato production region, which revealed a subset of mRNA transcripts constitutively up-regulated in resistant populations. We hypothesize that a portion of the up-regulated transcripts encoding for genes within the resistant populations also encode for pesticide resistance and can be suppressed to re-establish a susceptible phenotype. In this study, a discrete set of three up-regulated targets were selected for RNA interference experiments using a resistant L. decemlineata population. Following the successful suppression of transcripts encoding for a cytochrome p450, a cuticular protein, and a glutathione synthetase protein in a select L. decemlineata population, we observed reductions in measured resistance to imidacloprid that strongly suggest these genes control essential steps in imidacloprid metabolism in these field populations.

Temporal patterns of imidacloprid resistance throughout a growing season in Leptinotarsa decemlineata populations.

BACKGROUND: The Colorado potato beetle, Leptinotarsa decemlineata (Say), is a major agricultural pest of commercial potatoes. Pest managers use a combination of control tactics to limit populations, including multiple insecticides. Finding a window of insecticide susceptibility and understanding genetic responses to insecticide exposure during a growing season may provide novel management recommendations for L. decemlineata. RESULTS: We examined temporal changes (during one growing season) in phenotypic response between a susceptible population and an imidacloprid-resistant population. Beetles remained more susceptible to imidacloprid in the susceptible population throughout the growing season. Estimated mean LC50 values varied throughout the growing season in the resistant population, with increased susceptibility among overwintered and recently emerged adult beetles compared with a heightened level of resistance in the second generation. RNA transcript abundance was compared among multiple time points through the growing season, showing that cuticular proteins and cytochrome p450s were highly upregulated during peaks of measured resistance. CONCLUSION: Temporal variation in imidacloprid susceptibility of L. decemlineata was observed, which included early time points of susceptibility and later peaks in resistance. Heightened resistance occurred during the second generation and correlated to increased transcript abundance of multiple mechanisms of resistance, including multiple cuticular protein and cytochrome p450 transcripts. © 2016 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Characterizing Molecular Mechanisms of Imidacloprid Resistance in Select Populations of Leptinotarsa decemlineata in the Central Sands Region of Wisconsin.

The Colorado potato beetle, Leptinotarsa decemlineata (Say), is a major agricultural pest in the Central Sands region of Wisconsin. Imidacloprid, a neonicotinoid insecticide, has commonly been used for control of L. decemlineata since its registration in 1995. In the last 10 years, many field populations of L. decemlineata have begun to show increasing imidacloprid resistance. We studied resistance phenotype as a phenomenon that reduces neonicotinoid efficacy and has practical consequences for potato pest management. Although we have not observed complete field failure following the use of these products, multiple studies have demonstrated that the lethal concentration to kill 50% of the test organisms (LC50) in different field populations of L. decemlineata varies greatly which may suggest that resistance of L. decemlineata is heritable and involves genetic changes. An important challenge in understanding resistance is assessing the genetic mechanisms associated with resistance and classifying up-regulated genes that may be involved in combating an insecticide insult. In this study we uncovered trends in imidacloprid phenotypic response that have developed in the region by estimating the LC50 values among different field populations against a range of imidacloprid doses. The LC50 values collected in 2008-2011, and more recently in 2013 and 2014, show that some field locations remain susceptible to imidacloprid, while nearby fields (<100km) have developed high levels of resistance. We also sought to uncover potential mechanisms of resistance at each field location. We compiled a transcriptome for populations, characterized as phenotypically 'susceptible' and 'resistant', by isolating mRNA from adult beetles and analyzing gene expression level differences. Strong differences were observed in constituently up and down-regulated genes among different field populations. Most significantly, the up-regulation of 3 cytochrome p450s and a glutathione synthetase related protein in multiple resistant populations provide a mechanistic explanation of resistance evolution in L. decemlineata.