Transcriptome and population structure of glassy-winged sharpshooters (Homalodisca vitripennis) with varying insecticide resistance in southern California.

BACKGROUND: Homalodisca vitripennis Germar, the glassy-winged sharpshooter, is an invasive insect in California and a critical threat to agriculture through its transmission of the plant pathogen, Xylella fastidiosa. Quarantine, broad-spectrum insecticides, and biological control have been used for population management of H. vitripennis since its invasion and subsequent proliferation throughout California. Recently wide-spread neonicotinoid resistance has been detected in populations of H. vitripennis in the southern portions of California's Central Valley. In order to better understand potential mechanisms of H. vitripennis neonicotinoid resistance, we performed RNA sequencing on wild-caught insecticide-resistant and relatively susceptible sharpshooters to profile their transcriptome and population structure. RESULTS: We identified 81 differentially expressed genes with higher expression in resistant individuals. The significant largest differentially expressed candidate gene linked to resistance status was a cytochrome P450 gene with similarity to CYP6A9. Furthermore, we observed an over-enrichment of GO terms representing functions supportive of roles in resistance mechanisms (cytochrome P450s, M13 peptidases, and cuticle structural proteins). Finally, we saw no evidence of broad-scale population structure, perhaps due to H. vitripennis' relatively recent introduction to California or due to the relatively small geographic scale investigated here. CONCLUSIONS: In this work, we characterized the transcriptome of insecticide-resistant and susceptible H. vitripennis and identified candidate genes that may be involved in resistance mechanisms for this species. Future work should seek to build on the transcriptome profiling performed here to confirm the role of the identified genes, particularly the cytochrome P450, in resistance in H. vitripennis. We hope this work helps aid future population management strategies for this and other species with growing insecticide resistance.

Clothianidin and Imidacloprid Residues in Poa annua (Poales: Poaceae) and Their Effects on Listronotus maculicollis (Coleoptera: Curculionidae).

Competitive enzyme-linked immunosorbent assay was used to quantify the amounts of the neonicotinoids clothianidin and imidacloprid in Poa annua L. clippings from treated golf course fairways. Average clothianidin residues 7 d after application ranged from 674 to 1,550 ng/g tissue in 2012 and 455-2,220 ng/g tissue in 2013. Average clothianidin residues the day of application ranged from 17,100-38,800 ng/g tissue in 2014. Average imidacloprid residues 7 d after treatment ranged from 1,950-3,030 ng/g tissue in 2012 and 7,780-9,230 ng/g tissue in 2013. Average imidacloprid residues the day of application ranged from 31,500-40,400 ng/g tissue in 2014. Neonicotinoid or bifenthrin-neonicotinoid combination products applied in field plots in 2012 did not significantly reduce the numbers of larvae relative to the untreated control. However, in 2013, statistically significant reductions in the numbers of larvae recovered from treated field plots were associated with the presence of bifenthrin alone or when used in combination with neonicotinoid active ingredients. Listronotus maculicollis (Kirby) adults caged on neonicotinoid-, bifenthrin-, and bifenthrin-neonicotinoid-treated P. annua turf plugs fed on P. annua leaves, but mortality was only highly significantly different between treated and untreated foliage when weevils were placed on treated foliage the day after treatment and allowed to feed for 7 d. The modest degree of population suppression with bifenthrin in these experiments may not be adequate to justify the continued use of these products due to the increased risk of insecticide resistance and disruption of biological control.

Geographical survey of the mycobiome and microbiome of Southern California glassy-winged sharpshooters.

The glassy-winged sharpshooter, Homalodisca vitripennis Germar, is an invasive xylem-feeding leafhopper with a devastating economic impact on California agriculture through transmission of the plant pathogen, Xylella fastidiosa. While studies have focused on X. fastidiosa or known symbionts of H. vitripennis, little work has been done at the scale of the microbiome (the bacterial community) or mycobiome (the fungal community). Here, we characterize the mycobiome and the microbiome of H. vitripennis across Southern California and explore correlations with captivity and host insecticide resistance status. Using high-throughput sequencing of the ribosomal internal transcribed spacer 1 region and the 16S rRNA gene to profile the mycobiome and microbiome, respectively, we found that while the H. vitripennis mycobiome significantly varied across Southern California, the microbiome did not. We also observed a significant difference in both the mycobiome and microbiome between captive and wild H. vitripennis. Finally, we found that the mycobiome, but not the microbiome, was correlated with insecticide resistance status in wild H. vitripennis. This study serves as a foundational look at the H. vitripennis mycobiome and microbiome across Southern California. Future work should explore the putative link between microbes and insecticide resistance status and investigate whether microbial communities should be considered in H. vitripennis management practices. IMPORTANCE The glassy-winged sharpshooter is an invasive leafhopper that feeds on the xylem of plants and transmits the devastating pathogen, Xylella fastidiosa, resulting in significant economic damage to California's agricultural system. While studies have focused on this pathogen or obligate symbionts of the glassy-winged sharpshooter, there is limited knowledge of the bacterial and fungal communities that make up its microbiome and mycobiome. To address this knowledge gap, we explored the composition of the mycobiome and the microbiome of the glassy-winged sharpshooter across Southern California and identified differences associated with geography, captivity, and host insecticide resistance status. Understanding sources of variation in the microbial communities associated with the glassy-winged sharpshooter is an important consideration for developing management strategies to control this invasive insect. This study is a first step toward understanding the role microbes may play in the glassy-winged sharpshooter's resistance to insecticides.

Interactions of light intensity, insecticide concentration, and time on the efficacy of systemic insecticides in suppressing populations of the sweetpotato whitefly (Hemiptera: Aleyrodidae) and the citrus mealybug (Hemiptera: Pseudococcidae).

The impact of light intensity on the uptake and persistence of the systemic neonicotinoid insecticides, imidacloprid and dinotefuran, were evaluated in poinsettia (Euphorbia pulcherrima Willd.) and yellow sage (Lantana camara L.). Insecticide residues were measured in leaves sampled from the treated plants at four time intervals after treatment to determine the relationship between insecticide concentration and efficacy against two insect pests: sweetpotato whitefly, Bemisia tabaci Gennadius, and the citrus mealybug, Planococcus citri Risso. The insecticides were evaluated at their respective label rate and at the comparable label rate of the other insecticide under two different light environments: ambient and shade. The uptake of dinotefuran into yellow sage was more rapid at both treatment rates than both rates of imidacloprid, resulting in higher percent mortality of whitefly nymphs (89.8-100) compared with imidacloprid (14.1-89.2) across all 4 wk. Additionally, plants that received both rates of dinotefuran had fewer whitefly pupae (< 1.0) at week 4 compared with imidacloprid-treated plants (23.7-25.3). The uptake of dinotefuran into poinsettia plants was also more rapid and resulted in quicker and higher percent mortality of whitefly nymphs (89.5-99.6) compared with imidacloprid (14.1-89.2) across all 4 wk. However, despite efficient uptake, the efficacy of both systemic insecticides was less for citrus mealybug where percent mortality values were <50% among all the treatments across the 4 wk. The use of the two systemic insecticides evaluated in regards to pest management in horticultural cropping systems is discussed.

Distribution of Bemisia tabaci (Hemiptera: Aleyrodidae) biotypes in North America after the Q invasion.

After the 2004 discovery of the Bemisia tabaci (Gennadius) (Hemiptera Aleyrodidae) Q biotype in the United States, there was a vital need to determine the geographical and host distribution as well as its interaction with the resident B biotype because of its innate ability to rapidly develop high-level insecticide resistance that persists in the absence of exposure. As part of a coordinated country-wide effort, an extensive survey of B. tabaci biotypes was conducted in North America, with the cooperation of growers, industry, local, state, and federal agencies, to monitor the introduction and distribution of the Q biotype. The biotype status of submitted B. tabaci samples was determined either by polymerase chain reaction amplification and sequencing of a mitochondrial cytochrome oxidase I small subunit gene fragment and characterization of two biotype discriminating nuclear microsatellite markers or esterase zymogram analysis. Two hundred and eighty collections were sampled from the United States, Bermuda, Canada, and Mexico during January 2005 through December 2011. Host plants were split between ornamental plant and culinary herb (67%) and vegetable and field crop (33%) commodities. The New World biotype was detected on field-grown tomatoes (Solanum lycopersicum L.) in Mexico (two) and in commercial greenhouses in Texas (three) and represented 100% of these five collections. To our knowledge, the latter identification represents the first report of the New World biotype in the United States since its rapid displacement in the late 1980s after the introduction of biotype B. Seventy-one percent of all collections contained at least one biotype B individual, and 53% of all collections contained only biotype B whiteflies. Biotype Q was detected in 23 states in the United States, Canada (British Columbia and Ontario territories), Bermuda, and Mexico. Forty-five percent of all collections were found to contain biotype Q in samples from ornamentals, herbs and a single collection from tomato transplants located in protected commercial horticultural greenhouses, but there were no Q detections in outdoor agriculture (vegetable or field crops). Ten of the 15 collections (67%) from Canada and a single collection from Bermuda contained biotype Q, representing the first reports of biotype Q for both countries. Three distinct mitochondrial haplotypes of B. tabaci biotype Q whiteflies were detected in North America Our data are consistent with the inference of independent invasions from at least three different locations. Of the 4,641 individuals analyzed from 517 collections that include data from our previous work, only 16 individuals contained genetic or zymogram evidence of possible hybridization of the Q and B biotypes, and there was no evidence that rare hybrid B-Q marker co-occurrences persisted in any populations.

Metagenome-Assembled Genomes of Bacterial Symbionts Associated with Insecticide-Resistant and -Susceptible Individuals of the Glassy-Winged Sharpshooter (Homalodisca vitripennis).

The role of microbes in insecticide resistance is an emerging question. Here, we describe six metagenome-assembled genomes (MAGs) associated with the glassy-winged sharpshooter (Homalodisca vitripennis [Germar, 1821]) (Hemiptera, Cicadellidae). MAGs representing the obligate symbionts Candidatus Sulcia muelleri and Candidatus Baumannia cicadellinicola and the facultative symbiont Wolbachia were obtained from imidacloprid-resistant and imidacloprid-susceptible sharpshooters.

Effects of insecticides on behavior of adult Bactericera cockerelli (Hemiptera: Triozidae) and transmission of Candidatus Liberibacter psyllaurous.

The potato psyllid, Bactericera cockerelli (Sulc) (Hemiptera: Triozidae), is a serious pest of potatoes (Solanum tuberosum L.) that can cause yield loss by direct feeding on crop plants and by vectoring a bacterial pathogen, Candidatus Liberibacer psyllaurous. Current pest management practices rely on the use of insecticides to control the potato psyllid to lower disease incidences and increase yields. Although many studies have focused on the mortality that insecticides can cause on potato psyllid populations, little is known regarding the behavioral responses of the potato psyllid to insecticides or whether insecticides can decrease pathogen transmission. Thus, the objectives of this study were to determine the effects of insecticides on adult potato psyllid behaviors, the residual effects of insecticides on potato psyllid behaviors over time, and effects of these insecticides on Ca. L. psyllaurous transmission. Insecticides tested included imidacloprid, kaolin particle film, horticultural spray oil, abamectin, and pymetrozine. All insecticides significantly reduced probing durations and increased the amount of time adult psyllids spent off the leaflets, suggesting that these chemicals may be deterrents to feeding as well as repellents. Nonfeeding behaviors such as tasting, resting, and cleaning showed variable relationships with the different insecticide treatments over time. The insecticides imidacloprid and abamectin significantly lowered transmission of Ca. L. psyllaurous compared with untreated controls. The implications of our results for the selection of insecticides useful for an integrated pest management program for potato psyllid control are discussed.

Insecticide resistance in California populations of the glassy-winged sharpshooter Homalodisca vitripennis.

BACKGROUND: The glassy-winged sharpshooter, Homalodisca vitripennis (Germar), is a primary vector of Pierce's disease of grapes in California. Systemic imidacloprid treatments have been the mainstay of area-wide treatment programs that were established in the Central Valley (Kern and Tulare Counties) and Southern California (Riverside County) during the 1990s to combat the pest. The programs helped to suppress populations on citrus, a major sharpshooter host, to levels that significantly reduced migration into adjacent vineyards. However, beginning in 2012, there has been a resurgence of glassy-winged sharpshooter populations in Kern and Tulare counties, and hitherto successful treatment strategies have not been as effective. This study investigated the possibility that insecticide resistance was a contributing factor to the population resurgence. RESULTS: Topical application bioassays detected high levels of resistance to imidacloprid in Kern and Tulare populations, and lower levels of resistance (perhaps due to cross-resistance) to the foliar neonicotinoid acetamiprid (20-fold), the pyrethroid fenpropathrin (7.4-fold), and the butenolide flupyradifurone (4-fold). Samples of glassy-winged sharpshooters from citrus groves under organic management also exhibited high levels of imidacloprid resistance. CONCLUSION: The long-term use of imidacloprid has selected for resistance in glassy-winged sharpshooters. The most resistant populations also exhibited resistance to the foliar neonicotinoid acetamiprid, the pyrethroid fenpropathrin, and the butenolide flupyradifurone. High levels of imidacloprid resistance in insects sampled from organic groves indicate that resistant insects are migrating from nearby conventional groves, which could compromise the control of sharpshooters in organic systems with insecticides affected by cross-resistance. © 2021 Society of Chemical Industry.

Improved draft reference genome for the Glassy-winged Sharpshooter (Homalodisca vitripennis), a vector for Pierce's disease.

Homalodisca vitripennis (Hemiptera: Cicadellidae), known as the glassy-winged sharpshooter, is a xylem feeding leafhopper and an important agricultural pest as a vector of Xylella fastidiosa, which causes Pierce's disease in grapes and a variety of other scorch diseases. The current H. vitripennis reference genome from the Baylor College of Medicine's i5k pilot project is a 1.4-Gb assembly with 110,000 scaffolds, which still has significant gaps making identification of genes difficult. To improve on this effort, we used a combination of Oxford Nanopore long-read sequencing technology combined with Illumina sequencing reads to generate a better assembly and first-pass annotation of the whole genome sequence of a wild-caught Californian (Tulare County) individual of H. vitripennis. The improved reference genome assembly for H. vitripennis is 1.93-Gb in length (21,254 scaffolds, N50 = 650 Mb, BUSCO completeness = 94.3%), with 33.06% of the genome masked as repetitive. In total, 108,762 gene models were predicted including 98,296 protein-coding genes and 10,466 tRNA genes. As an additional community resource, we identified 27 orthologous candidate genes of interest for future experimental work including phenotypic marker genes like white. Furthermore, as part of the assembly process, we generated four endosymbiont metagenome-assembled genomes, including a high-quality near complete 1.7-Mb Wolbachia sp. genome (1 scaffold, CheckM completeness = 99.4%). The improved genome assembly and annotation for H. vitripennis, curated set of candidate genes, and endosymbiont MAGs will be invaluable resources for future research of H. vitripennis.

Extraordinary resistance to insecticides reveals exotic Q biotype of Bemisia tabaci in the New World.

A strain of the whitefly Bemisia tabaci (Gennadius) possessing unusually high levels of resistance to a wide range of insecticides was discovered in 2004 in the course of routine resistance monitoring in Arizona. The multiply resistant insects, collected from poinsettia (Euphorbia pulcherrima Willd. ex Klotzsch) plants purchased at a retail store in Tucson, were subjected to biotype analysis in three laboratories. Polyacrylamide gel electrophoresis of naphthyl esterases and sequencing of the mitochondrial cytochrome oxidase I gene (780 bp) confirmed the first detection of the Q biotype of B. tabaci in the New World. This U.S. Q biotype strain, referred to as Poinsettia'04, was highly resistant to two selective insect growth regulators, pyriproxyfen and buprofezin, and to mixtures of fenpropathrin and acephate. It was also unusually low in susceptibility to the neonicotinoid insecticides imidacloprid, acetamiprid, and thiamethoxam, relative to B biotype whiteflies. In 100 collections of whiteflies made in Arizona cotton (Gossypium spp.), vegetable, and melon (Cucumis melo L.) fields from 2001 to 2005, no Q biotypes were detected. Regions of the United States that were severely impacted by the introduction of the B biotype of B. tabaci in the 1980s would be well advised to promote measures that limit movement of the Q biotype from controlled environments into field systems and to formulate alternatives for managing this multiply-resistant biotype, in the event that it becomes more widely distributed.

Distribution of Bemisia tabaci (Hemiptera: Aleyrodidae) biotypes in Florida-investigating the Q invasion.

After the 2004 discovery of the Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) Q biotype in the United States, there was an urgent need to determine its distribution. As part of a coordinated country-wide effort, an extensive survey of B. tabaci biotypes was conducted in Florida, with the cooperation of growers and state and federal agencies, to monitor the introduction and distribution of both the B and Q biotypes. The biotype status of submitted B. tabaci samples was determined by polymerase chain reaction (PCR) amplification and sequencing of a 700-800-bp mitochondrial cytochrome oxidase I small subunit (mtCOI) gene fragment, PCR amplification, and size determination of two unique microsatellite markers and esterase zymogram analysis. One hundred and eighty collections were sampled from 23 counties. Of these samples, 58% were from vegetables, 37% were from ornamentals, and 5% were from peanuts, alfalfa, and weeds. Eighteen percent of all collections were found to be the Q biotype that came from greenhouse grown ornamental and herbs located in six counties. Sequence comparison of the mtCOI gene identified three separate haplotypes within Florida that were defined as Q1, Q2, and Q3. Haplotypes could be used to associate populations known to be related by grower and plant type. For example, collections from five counties were made on hibiscus linked to the same grower and all samples contained only the Q1 haplotype. Other populations contained a mix of the Q2 and Q3 haplotypes, supporting the conclusion that the Q biotype must have entered Florida through at least two separate introductions. Our data also show that two microsatellite markers are a cost-effective diagnostic alternative for biotype identification with 100% concurrence with mtCOI sequence data.

Toxicity of systemic neonicotinoid insecticides to avocado thrips in nursery avocado trees.

The efficacies of four systemic neonicotinoid insecticides applied to potted avocado trees at manufacturer-recommended rates were assessed against the avocado thrips, Scirtothrips perseae Nakahara. At the time of treatment, fully expanded first-flush young leaves were tagged for identification, and a proportion of these leaves was used in bioassays with second-instar thrips. At 7 weeks post-treatment, a second flush of leaves had fully expanded on the trees, and these leaves were included in additional bioassays comparing avocado thrips mortality on both first- and second-flush leaves. In bioassays with first-flush leaves, imidacloprid (273 mg AI pot(-1)) was the most effective insecticide, providing at least 70% mortality of thrips for 14 weeks. Thiamethoxam (137 mg AI pot(-1)), clothianidin (109 mg AI pot(-1)) and dinotefuran (241 mg AI pot(-1)) provided good control in bioassays that were conducted within 4 weeks of treatment, but thereafter their efficacies were inconsistent. In bioassays with second-flush leaves, imidacloprid provided at least 70% mortality up to 9 weeks after the insecticide application. Thereafter, mortality declined to 30% or lower. Bioassays with second-flush leaves collected from trees treated with thiamethoxam, clothianidin and dinotefuran resulted in unacceptably low thrips mortality. Monitoring of imidacloprid and thiamethoxam residues by ELISA showed that the greater persistence of imidacloprid in both first and second leaf flushes was due to a steadier uptake of this material. Although thiamethoxam residues rose quickly within the first leaf flush, levels had already begun to dissipate by the time the second leaf flush had started to develop.

Evaluation of systemic neonicotinoid insecticides for the management of the Asian citrus psyllid Diaphorina citri on containerized citrus.

BACKGROUND: Studies were conducted to evaluate uptake and retention of three systemic neonicotinoid insecticides, dinotefuran, imidacloprid and thiamethoxam, in potted citrus nursery plants treated at standard label rates. Infestation of these plants placed at a field site with moderate levels of Asian citrus psyllid (ACP) was monitored for 14 weeks following treatments, and insecticide residues in leaf tissue were quantified using enzyme-linked immunosorbent assay (ELISA). Bioassays were conducted using leaves harvested on various dates post-treatment to compare the efficacies of residues against adult ACP. RESULTS: Residues of the three neonicotinoids were detected in leaf tissues within 1 week after treatment. Peak concentrations established at 1 week for imidacloprid and dinotefuran and at 2 weeks for thiamethoxam. Imidacloprid and thiamethoxam outperformed the control and dinotefuran treatments at protecting trees from infestations by ACP eggs and nymphs. For a given insecticide concentration in leaf tissue, thiamethoxam induced the highest mortality of the three insecticides, and dinotefuran was the least toxic. CONCLUSION: If the time needed to achieve effective thresholds of a systemic neonicotinoid is known, treatments at production facilities could be scheduled that would minimize unnecessary post-treatment holding periods and ensure maximum retention of effective concentrations after the plants have shipped to retail outlets. The rapid uptake of the insecticides and retention at effective concentrations in containerized citrus suggest that the current 30 day post-treatment shipping restriction from production facilities to retail outlets outside of quarantine could be shortened to 14 days. Thiamethoxam should be added to the list of approved nursery treatments. © 2016 Society of Chemical Industry.

Quantification of imidacloprid toxicity to avocado thrips, Scirtothrips perseae Nakahara (Thysanoptera: Thripidae), using a combined bioassay and ELISA approach.

A competitive ELISA (enzyme-linked immunosorbent assay) technique was evaluated for quantifying titres of imidacloprid in homogenates of leaf discs sampled from avocado plants treated with systemic applications of imidacloprid 240 g litre(-1) SC (Admire). Matrix effects were evident with undiluted leaf tissue homogenates, but these were effectively eliminated by dilution of homogenates in water. In a field trial conducted in a commercial nursery, there was an excellent correlation between imidacloprid residues within leaves and avocado thrips (Scirtothrips perseae Nakahara) mortality. However, with subsequent sampling over a 2-month period, the relationship between mortality and apparent imidacloprid concentration became less robust, suggesting that the material was being degraded within the plant to non-toxic metabolites. Nevertheless, assessments of thrips mortality on leaves that had been recently treated with imidacloprid established a lower threshold of activity for imidacloprid residues of 6 ng cm(-2) leaf. Limitations on the use of ELISA to quantify the impact of systemic insecticides on pest populations are discussed.

Spatial and temporal distribution of imidacloprid and thiamethoxam in citrus and impact on Homalodisca coagulata populations.

Titers of two systemic neonicotinoid insecticides in citrus trees were measured in conjunction with conventional evaluations of their impact on glassy-winged sharpshooter (Homalodisca coagulata (Say); GWSS) populations. Xylem fluid samples were collected at regular intervals and from multiple locations within field-grown citrus trees to determine imidacloprid and thiamethoxam concentrations using commercial ELISA kits. Uptake profiles varied considerably with peak mean titers of imidacloprid occurring 6-8 weeks after application compared with 2 weeks for thiamethoxam. The persistence of each compound also varied as near-peak levels of imidacloprid were sustained for another 6-10 weeks before gradually declining. In contrast, thiamethoxam titers declined more rapidly after the initial peak, possibly reflecting an application rate only one-quarter of that used for imidacloprid. Within-tree distributions were more similar for the two compounds, with no significant effect due to height of the sample (upper or lower half) or to the quadrant location within the tree, with the exception of one quadrant in the thiamethoxam-treated trees. Substantial reductions in GWSS nymphs and adults were observed in imidacloprid-treated trees during the 2001 trial and were sustained for 4-5 months after treatment. Treatment effects on nymphs were not as well pronounced in the 2002 trial, when overall GWSS infestations were much reduced from the previous year. However, consistently lower adult infestations were still observed in 2002 for both treatments compared with untreated trees. Information on the spatial and temporal profiles in citrus trees was obtained for both compounds to complement field impact data and improve understanding of their pest management potential.

Application of competitive enzyme-linked immunosorbent assay for the quantification of imidacloprid titers in xylem fluid extracted from grapevines.

A competitive enzyme-linked immunosorbent assay (ELISA) technique was evaluated for quantifying titers of imidacloprid in xylem fluid extracted from Vitis vinifera L. grapevines that were treated with systemic applications of the neonicotinoid insecticide Admire. Evidence of matrix effects, factors that compromise the precision and accuracy of the ELISA, was present in assays with undiluted xylem fluid. These effects could be eliminated by dilution of extracts in water, resulting in a lower sensitivity of the assay of 4 microg liter(-1). In a field trial conducted in a commercial vineyard, there was an excellent correlation between Admire application rates and xylem fluid concentrations of imidacloprid. At an Admire application rate of 1.17 liter ha(-1) (16 fl oz per acre), uptake of imidacloprid into vines was rapid. Imidacloprid was consistently detected in the xylem for up to 3 mo after application at concentrations known to be effective at managing populations of the sharpshooter Homalodisca coagulata Say, an important vector of Xylella fastidiosa Wells in California vineyards. The ELISA is a sensitive technique that can be used to study the behavior of systemic insecticides within crop systems and their impact on pest populations.

Biochemical study of resistance to imidacloprid in B biotype Bemisia tabaci from Guatemala.

Systemic uptake bioassays using excised cotton leaves confirmed resistance to imidacloprid in a Guatemalan population of the tobacco whitefly Bemisia tabaci Gennadius. Polyacrylamide gel electrophoresis of naphthyl esterases identified the insects as B-types. Upon collection from the field, resistance was determined to be 58-fold relative to a susceptible strain originating in the Imperial Valley of California. Resistance levels increased to 126-fold in this population during its continuous exposure to systemically treated cotton. In biochemical investigations, there was no detectable NADPH-dependent mixed function oxidase metabolism of 14C-imidacloprid at any time during the selection process. In contrast, microsomal preparations from housefly abdomens readily produced significant amounts of the mono-hydroxy and olefin derivatives of the parent compound. Detoxification of imidacloprid by housefly MFOs may account for reports of lower toxicity of the insecticide towards this insect compared with whiteflies, despite similar binding properties between imidacloprid and the nicotinic acetylcholine receptors in both species.

Evaluation of peracid activated organophosphates in studies of insecticide resistance conferred by insensitive acetylcholinesterases.

Peracid activated organophosphorus (OP) insecticides were evaluated against electric eel and a range of insect acetylcholinesterases (AChE). Incubation of chlorpyrifos and sulprofos with meta-chloroperoxybenzoic acid (MCPBA) produced active metabolites capable of distinguishing between AChE enzymes conferring OP susceptibility and resistance in populations of the tobacco whitefly, Bemisia tabaci Gennadius, and the beet armyworm, Spodoptera exigua Hubner. These products were also active against enzymes present in the lygus bug Lygus hesperus Knight and the greenhouse whitefly, Trialeurodes vaporariorum Westwood. Although profenofos was active against electric eel AChE, incubation of the OP with MCPBA did not enhance its anti-AChE properties toward the eel or insect AChEs.

Biochemical monitoring of acetylcholinesterase sensitivity to organophosphorus insecticides in glassy-winged sharpshooter Homalodisca coagulata Say (Homoptera: Cicadellidae) and smoke-tree sharpshooter H. lacerta Fowler.

The glassy-winged sharpshooter Homalodisca coagulata Say (Homoptera: Cicadellidae) is a new pest to California agriculture. It is the principal vector of several plant pathogenic diseases, particularly Pierce's Disease in grapevines, and oleander leaf scorch. A microplate-based assay is described that measures the sensitivity of acetylcholinesterase (AChE) activity to inhibition by organophosphorus (OP) insecticides in this important pest. The technique provides users with an accurate measure of the efficacy of OP binding to this target site, and is a valuable tool for monitoring field populations of the insect to determine whether the use of OP insecticides has selected for resistant individuals. The technique will also measure AChE sensitivity in the smoke-tree sharpshooter, H. lacerta Fowler. This species is native to California and is regarded only as a minor pest. Both inhibition and kinetic measurements for the AChE enzymes in these sharpshooters demonstrate the close phylogenetic relationships between the two species.

Determination of exposure levels of honey bees foraging on flowers of mature citrus trees previously treated with imidacloprid.

BACKGROUND: Field and tunnel cage studies were undertaken to determine the extent to which honey bees foraging on citrus blossoms were exposed to imidacloprid and its metabolites when citrus trees were treated with soil applications of the insecticide. Residues were measured by LC/MS/MS in nectar and pollen samples from trees treated up to 232 days prior to bloom. RESULTS: Imidacloprid, imidacloprid olefin and 5-hydroxy imidacloprid were detected in nectar and pollen sampled from the flowers of citrus trees treated with imidacloprid up to 232 days prior to bloom. In tunnel studies, where foraging was restricted exclusively to citrus, imidacloprid residues in nectar extracted from flowers and from bee crops were similar (<10 ng mL(-1) ) and below the established no-observed-effects limit; however, the residue levels were about threefold higher in nectar sampled from comb. Concentrations of imidacloprid in nectar were higher in trees treated with higher application rates. CONCLUSIONS: Imidacloprid and its metabolites were detected in the nectar and pollen of citrus trees treated up to 232 days prior to the onset of bloom. However, based on published bioassay data, the imidacloprid concentrations in the floral nectar did not surpass levels that would compromise foraging activity under normal use conditions for imidacloprid. Further research is needed to assess the impact of elevated levels of imidacloprid within stored nectar in the comb.