RNA interference of NADPH-cytochrome P450 reductase increases susceptibilities to multiple acaricides in Tetranychus urticae.

The two-spotted spider mite, Tetranychus urticae, is a polyphagous pest feeding on over 1100 plant species, including numerous highly valued economic crops. The control of T. urticae largely depends on the use of acaricides, which leads to pervasive development of acaricide resistance. Cytochrome P450-mediated metabolic detoxification is one of the major mechanisms of acaricide resistance in T. urticae. NADPH-cytochrome P450 reductase (CPR) plays as a crucial co-factor protein that donates electron(s) to microsomal cytochrome P450s to complete their catalytic cycle. This study seeks to understand the involvement of CPR/P450 in acaricide resistance in T. urticae. The full-length cDNA sequence of T. urticae's CPR (TuCPR) was cloned and characterized. TuCPR was ubiquitously transcribed in different life stages of T. urticae and the highest transcription was observed in the nymph and adult stages. TuCPR was constitutively over-expressed in six acaricide resistant populations compared to a susceptible one. TuCPR transcriptional expression was also induced by multiple acaricides in a time-dependent manner. Down-regulation of TuCPR via RNA interference (RNAi) in T. urticae led to reduced enzymatic activities of TuCPR and cytochrome P450s, as well as a reduction of resistance to multiple acaricides, abamectin, bifenthrin, and fenpyroximate. The outcome of this study highlights CPR as a potential novel target for eco-friendly control of T. urticae and other related plant-feeding pests.

Mating Disruption for Managing Prionus californicus (Coleoptera: Cerambycidae) in Hop and Sweet Cherry.

Larvae of Prionus californicus Motschulsky feed on the roots of many woody perennial plants and are economically important pests of hop Humulus lupulus L. (Urticales: Cannabaceae) and sweet cherry Prunus avium (L.) (Magnoliopsida: Rosaceae) in the United States Pacific Northwest and Intermountain West. Adult males are strongly attracted to a volatile sex pheromone, (3R,5S)-3,5-dimethyldodecanoic acid, produced by females. Here, we summarize the results of field experiments evaluating the synthetic pheromone in a blend of all four possible stereoisomers as a means for managing P. californicus in hop yards and sweet cherry orchards by mating disruption (MD). Mean capture of male beetles was lower, in all 3 yr of the study, from plots in commercial hop yards and sweet cherry orchards treated with synthetic P. californicus pheromone than from similar, untreated plots. Although trap catch was lower in sweet cherry, relative differences between trap catches from MD and nonmating disruption plots were similar to that seen in hop yards. The number of P. californicus larvae recovered from plots in hop yards treated for three consecutive growing seasons with synthetic pheromone was lower than in similar plots that were not treated with the pheromone or treated with the soil fumigant ethoprop. Our research demonstrates that deployment of synthetic P. californicus pheromone effectively reduces mate-finding by males, can effectively reduce larvae populations in pheromone-treated hop yards, and thus, has excellent potential for managing P. californicus in hop, sweet cherry, and perhaps in other crops where it or Prionus species are pests.

Phenotypic and Genotypic Plasticity of Acaricide Resistance in Populations of Tetranychus urticae (Acari: Tetranychidae) on Peppermint and Silage Corn in the Pacific Northwest.

Tetranychus urticae Koch is a generalist pest of economic crops and is notorious for its rapid development of acaricide resistance. This poses a significant threat to the sustainability of integrated pest management (IPM) in cropping systems plagued by T. urticae. It is critical to evaluate the resistance status of T. urticae populations on crops and identify any underlying resistance mechanisms. This study investigated the efficacy of five major acaricides on T. urticae populations on peppermint and silage corn in the Pacific Northwestern United States and identified the underlying resistance mechanisms. Significant variations in acaricide resistance status of T. urticae populations were identified to abamectin, bifenthrin, fenpyroximate, hexythiazox, and spirodiclofen. In most cases, T. urticae populations from silage corn exhibited greater levels of acaricide resistance relative to peppermint populations. We detected known target-site mutations: F1534S and F1538I (conferring resistance to bifenthrin), G126S (linked with resistance to bifenazate), and I1017 (conferring resistance to hexythiazox and etoxazole) in 10, 90, and 90% of the populations, respectively, from peppermint fields. These four mutations were identified in all the populations collected from silage corn fields. Significantly higher transcript levels of metabolic genes associated with resistance to abamectin, fenpyroximate, and spirodiclofen were observed in some T. urticae populations collected from both peppermint and silage corn fields. This study provides evidence of multiple resistance to diverse active ingredients in field populations of T. urticae and the reliability of known molecular markers for active acaricide resistance monitoring. The observed resistance pattern will help in designing a sustainable IPM program for T. urticae.

Pumpkin as an Alternate Host Plant for Laboratory Colonies of Grape Mealybug.

The grape mealybug, Pseudococcus maritimus (Ehrhorn; Hemiptera: Pseudococcidae), is the primary vector of Grapevine Leafroll associated Viruses (GLRaVs) in Washington State vineyards. Rearing laboratory colonies of grape mealybug has proven difficult. Several host plants were tested to determine their suitability for use as an alternate host plants for laboratory colonies of grape mealybug. Of the plants tested, colonies of grape mealybug were successful on pumpkin (Cucurbita pepo cv 'Connecticut field') leaves and vines. Mealybugs were able to develop from the crawler stage, through developmental instars, and adults were able to reproduce viable offspring. To date this is the only successful study to raise grape mealybugs on a cucurbit. Pumpkin appears to be a viable alternate host plant for laboratory colonies of the grape mealybug.

Migratory Bee Hive Transportation Contributes Insignificantly to Transgenic Pollen Movement Between Spatially Isolated Alfalfa Seed Fields.

Contracted commercial beekeeping operations provide an essential pollination service to many agricultural systems worldwide. Increased use of genetically engineered crops in agriculture has raised concerns over pollinator-mediated gene flow between transgenic and conventional agricultural varieties. This study evaluated whether contracted migratory beekeeping practices influence transgenic pollen flow among spatially isolated alfalfa fields. Twelve honey bee (Apis mellifera L.) colonies were permitted to forage on transgenic alfalfa blossoms for 1 wk in Touchet, WA. The hives were then transported 112 km to caged conventional alfalfa plots following one and two nights of isolation (8 and 32 h, respectively) from the transgenic source. Alfalfa seed harvested from the conventional plots was assessed for the presence of the transgene using a new seedling germination assay. We found that 8 h of isolation from a transgenic alfalfa source virtually eliminated the incidence of cross-pollination between the two varieties.

Molecular mechanisms of Tetranychus urticae chemical adaptation in hop fields.

The two-spotted spider mite, Tetranychus urticae Koch is a major pest that feeds on >1,100 plant species. Many perennial crops including hop (Humulus lupulus) are routinely plagued by T. urticae infestations. Hop is a specialty crop in Pacific Northwest states, where 99% of all U.S. hops are produced. To suppress T. urticae, growers often apply various acaricides. Unfortunately T. urticae has been documented to quickly develop resistance to these acaricides which directly cause control failures. Here, we investigated resistance ratios and distribution of multiple resistance-associated mutations in field collected T. urticae samples compared with a susceptible population. Our research revealed that a mutation in the cytochrome b gene (G126S) in 35% tested T. urticae populations and a mutation in the voltage-gated sodium channel gene (F1538I) in 66.7% populations may contribute resistance to bifenazate and bifenthrin, respectively. No mutations were detected in Glutamate-gated chloride channel subunits tested, suggesting target site insensitivity may not be important in our hop T. urticae resistance to abamectin. However, P450-mediated detoxification was observed and is a putative mechanism for abamectin resistance. Molecular mechanisms of T. urticae chemical adaptation in hopyards is imperative new information that will help growers develop effective and sustainable management strategies.

Microsatellite Variation of two Pacific Coast Drosophila suzukii (Diptera: Drosophilidae) Populations.

The vinegar fly, Drosophila suzukii (Diptera: Drosophilidae), is a recent invader in North America that has become a serious threat to small fruit production. It was first detected in California in 2008 and in Washington state in 2009. In this study, D. suzukii populations from the area of the original detection on California's central coast and from eastern Washington, the United States, were sampled over a 3-year period to determine genetic variation in both using microsatellite markers. Six different loci were successfully amplified and included in the analysis. These loci included nanos, elf1, antennapedia, mastermind, z600, and tenA. The population from eastern Washington was highly monomorphic with one locus, mastermind, having multiple alleles. There was greater genetic variation in the coastal California population with all loci having multiple alleles, with the exception of tenA. Owing to the relatively low levels of genetic variation in the eastern Washington population compared with the coastal California population, it appears that the D. suzukii population in the eastern Washington region has undergone a significant bottleneck.

Use of a highly sensitive immunomarking system to characterize face fly (Diptera: Muscidae) dispersal from cow pats.

We tested an immunomarking system that used egg white as marker and enzyme-linked immunosorbent assay as a detection assay to characterize face fly (Musca autumnalis DeGeer) dispersal from cow pats in a pastured beef cattle operation. In microcage assays, adult flies acquired marker after contact with cow pats that were treated with marker and field aged up to 11 d. In arena assays on sprayed full-size cow pats, 77% of eclosed face flies acquired the marker. In a field-marking study, four applications of egg white marker were applied on freshly deposited cow pats over a summer at two peripheral paddocks to a main grazing pasture of ≍50 head of beef cattle. Of the 663 face flies captured, 108 were positive for the egg white marker (16.3%). Of the marked flies, ≍ twofold more male than female flies were captured. Sex-specific dispersal distances were roughly equal up to 450 m, with 11% of female flies dispersing >450 m. Dispersal capability of face flies is discussed in relation to efficacy of rotational grazing and other IPM strategies.

Evaluation of sunlight-exposed pyrethroid-treated netting for the control of face fly and housefly (Diptera: Muscidae).

BACKGROUND: Face flies, Musca autumnalis De Geer (Diptera: Muscidae), and houseflies, Musca domestica L. (Diptera: Muscidae), have a significant impact on livestock and dairy production throughout North America. Pyrethroid insecticide efficacy can be affected by exposure to direct sunlight, and the rate of photodegradation is substrate and formulation dependent. Insecticide-treated netting (ITN) is finding new applications in crop and livestock production systems. A baseline study using long-duration no-choice assays has been carried out to gauge the effectiveness of ITN treated with ß-cyfluthrin, λ-cyhalothrin and bifenthrin on face flies and houseflies. RESULTS: After 12 weeks in direct sunlight, ITN treated with ß-cyfluthrin was still highly insecticidal to face flies and houseflies, producing 100% mortality in petri dish assays. However, sunlight reduced the insecticidal activity of λ-cyhalothrin, with 3% of face flies and 50% of houseflies surviving after exposure to ITN that had been deployed for 10 weeks. Insecticidal activity was greatly reduced on bifenthrin-treated netting, with 20% of face flies and 50% of houseflies surviving in assays with netting deployed for only 3 weeks. CONCLUSION: With careful choice of the pyrethroid applied, treated netting could be an important component of livestock integrated pest management programs focused on sustainable practices.

Identification of an alternative knockdown resistance (kdr)-like mutation, M918L, and a novel mutation, V1010A, in the Thrips tabaci voltage-gated sodium channel gene.

BACKGROUND: Knockdown resistance (kdr) has been identified as a main mechanism against pyrethroid insecticides in many arthropod pests including in the onion thrips, Thrips tabaci. To characterize and identify pyrethroid-resistance in onion thrips in Washington state, we conducted insecticide bioassays and sequenced a region of the voltage gated sodium channel gene from several different T. tabaci populations. RESULTS: Field collected Thrips tabaci were found to have large variations in resistance to the pyrethroid insecticide lambda-cyhalothrin. We identified two single nucleotide substitutions in our analysis of a partial sequence of the T. tabaci voltage-gated sodium channel gene. One mutation resulted in the non-synonymous substitution of methionine with leucine (M918L), which is well known to be responsible for super knockdown resistance in some pest species. Another non-synonymous substitution, a valine (GTT) to alanine (GCT) replacement at amino acid 1010 (V1010A) was identified in our study and was associated with lambda-cyhalothrin resistance. CONCLUSION: We have characterized a known kdr mutation and identified a novel mutation in the voltage-gated sodium channel gene of Thrips tabaci associated with resistance to lambda-cyhalothrin. This gene region and these mutations are expected to be useful in the development of a diagnostic test to detect kdr resistance in many onion thrips populations.

Effect of ensilation of potato on viability of Taenia hydatigena eggs.

A Taenia hydatigena model was used to assess the effect 0, 7, 14, 21, and 28 days of ensilation of minced potato on viability of tapeworm eggs. For infection of lambs, 2,000 T. hydatigena eggs were ensiled for 0, 7, 14, 21 and 28 days in minced potato at 22°C and fed to recently weaned lambs (29.9±0.76 kg). At slaughter, no cysticerci were recovered from lambs infected with eggs ensiled for 28 days while a mean of 5.0±5.0 cysticerci (0.25% of the initial egg dose) were recovered from lambs infected with eggs ensiled for 21 days. For lambs fed eggs ensiled for 0 days (control), 359.3±55.6 cysticerci were recovered (18.0% of the initial egg dose). Regression analysis revealed that a 99.9% reduction in viability was attained after 18.59 days of ensilation.

Effect of heat treatment on viability of Taenia hydatigena eggs.

Effects of heat treatments on activation and infectivity of Taenia hydatigena eggs were assessed. Eggs containing oncospheres were used for in vitro and in vivo studies to determine the response to 5min of heat treatment, ranging from room temperature (22°C) to 60°C. The study demonstrated 99.47% and 100% reduction in oncosphere activation or infectivity after 5min of heat treatment at 60°C and 57.38°C under in vitro and in vivo conditions, respectively. Similar results between the two approaches indicted the appropriateness of the in vitro methods to identify oncosphericidal treatments of practical significance. Similar heat treatments may also be effective against Taenia saginata and help to reduce occurrence of beef cysticercosis.

Identification of Lygus hesperus by DNA barcoding reveals insignificant levels of genetic structure among distant and habitat diverse populations.

BACKGROUND: The western tarnished plant bug Lygus hesperus is an economically important pest that belongs to a complex of morphologically similar species that makes identification problematic. The present study provides evidence for the use of DNA barcodes from populations of L. hesperus from the western United States of America for accurate identification. METHODOLOGY/PRINCIPAL FINDINGS: This study reports DNA barcodes for 134 individuals of the western tarnished plant bug from alfalfa and strawberry agricultural fields in the western United States of America. Sequence divergence estimates of <3% reveal that morphologically variable individuals presumed to be L. hesperus were accurately identified. Paired estimates of F(st) and subsequent estimates of gene flow show that geographically distinct populations of L. hesperus are genetically similar. Therefore, our results support and reinforce the relatively recent (<100 years) migration of the western tarnished plant bug into agricultural habitats across the western United States. CONCLUSIONS/SIGNIFICANCE: This study reveals that despite wide host plant usage and phenotypically plastic morphological traits, the commonly recognized western tarnished plant bug belongs to a single species, Lygus hesperus. In addition, no significant genetic structure was found for the geographically diverse populations of western tarnished plant bug used in this study.

Laboratory survival of Drosophila suzukii under simulated winter conditions of the Pacific Northwest and seasonal field trapping in five primary regions of small and stone fruit production in the United States.

BACKGROUND: Drosophila suzukii was first found in Oregon in August 2009. The threat of this pest to regional small and stone fruit production industries led to investigations on its overwintering capabilities in fruit-growing regions in the Pacific Northwest. Knowledge of its cold tolerance will help in the development of computer models to forecast seasonal population growth and decline. RESULTS: Of 1500 adults or pupae, 22 (1.4%) individuals survived the 84 day experimental chilling period. Most (86%) of the survivors were subjected to 10 °C temperature treatments. Survival decreased significantly at lower temperature treatments. Freezing temporarily increased the mortality rate but did not significantly affect overall mortality over the trial period. Flies that emerged from pupae are estimated to survive for up to 103-105 days at 10 °C and for shorter periods at lower temperatures. Field trapping in five fruit production areas has demonstrated overwintering survival in California and Oregon, but lower survival is predicted in Eastern Washington and Michigan. CONCLUSION: The experiments reported here indicate that long-term survival of D. suzukii is unlikely at temperatures below 10 °C. Field data from five climatic regions indicated extended low initial D. suzukii field presence in 2010 in all regions except California, where field presence was recorded earlier.

Development of a multiplex PCR for identification of vineyard mealybugs.

A simple molecular tool was developed and tested to identify seven mealybug species found in North American vineyards: Pseudococcus maritimus Ehrhorn, Pseudococcus viburni (Signoret), Pseudococcus longispinus (Targioni-Tozzeti), Pseudococcus calceolariae (Maskell), Planococcus ficus (Signoret), Planococcus citri (Risso), and Ferrisia gilli Gullan. The developed multiplex PCR is based on the mitochondrial cytochrome c oxidase subunit one gene. In tests, this single-step multiplex PCR correctly identified 95 of 95 mealybug samples, representing all seven species and collected from diverse geographic regions. To test the sensitivity, single specimen samples with different Pl. ficus developmental stages (egg to adult female and adult male) were processed PCR and the resulting output provided consistent positive identification. To test the utility of this protocol for adult males caught in sex baited pheromone traps, Pl. ficus adult males were placed in pheromone traps, aged at a constant temperature of 26±2°C, and processed with the multiplex each day thereafter for 8 d. Results showed consistent positive identification for up to 6 d (range, 6-8 d). Results are discussed with respect to the usefulness of this molecular tool for the identification of mealybugs in pest management programs and biosecurity of invasive mealybugs.

An endemic population of western poplar clearwing moths (Lepidoptera: Sesiidae) invades a monoculture of hybrid poplar.

Western poplar clearwing, Paranthrene robiniae (Hy. Edwards) (Lepidoptera: Sesiidae), is endemic in Pacific Northwest riparian habitats at low population densities. These moths have colonized commercial hybrid poplar (Populus spp.) plantings. Moth populations increased rapidly and became a major pest. Trap catches of male moths in mid-season surveys increased 190-fold from 95 in 2001 to >18,500 in 2002 across 6597 ha of poplars monitored. The outbreak of western poplar clearwings was widespread in 2002. Pheromone-baited traps placed one trap per 81.75 ha over 13,274 ha of commercial poplars captured >108,000 male moths in 2002. Damage to commercial poplars included girdling of saplings and burrows in limbs and trunks of trees. Repeated applications of chlorpyrifos failed to reduce the abundance of moths in 2002. Two management strategies over two separate plantations of approximately 6500 ha each were contrasted. Future control strategies recommend a halt to the use of contact insecticides that target adult moths. Short-term (3-5 yr) control should involve a pheromone-based mating disruption strategy followed eventually by selection of a clone that is less susceptible to P. robiniae attack.