Field evaluation of Cucurbit yellow stunting disorder virus transmission by Bemisia tabaci.

Cucurbit yellow stunting disorder virus (CYSDV) is a whitefly-transmitted Crinivirus (Closteroviridae) that impacts melon production in many parts of the world including the USA. It has been responsible for melon crop loss in the southwestern U.S. since 2006 when it was first identified. Control strategies have revolved mainly around chemical control, but research to identify suitable products and approaches to implementing them have lagged. The current study investigated the performance of four systemic insecticides in the field while concurrently tracking CYSDV disease progression after controlled and natural whitefly inoculation of young melon plants. Assessments of virus incidence were made using two different visual observation methods in concert with ELISA analyses of leaf disks samples collected biweekly. Infection rates were consistently lowest in plots treated with the butenolide insecticide flupyradifurone while dinotefuran was second in efficacy measures. Flupyradifurone also held whitefly densities to their lowest numbers relative to the other treatments. Two other insecticides, imidacloprid and cyantraniliprole, exacerbated virus incidence in multiple trials. Further investigation into the anomalous finding of increased virus incidence due to insecticide application is ongoing.

De novo transcriptome assemblies of four xylem sap-feeding insects.

Background: Spittle bugs and sharpshooters are well-known xylem sap-feeding insects and vectors of the phytopathogenic bacterium Xylella fastidiosa (Wells), a causal agent of Pierce's disease of grapevines and other crop diseases. Specialized feeding on nutrient-deficient xylem sap is relatively rare among insect herbivores, and only limited genomic and transcriptomic information has been generated for xylem-sap feeders. To develop a more comprehensive understanding of biochemical adaptations and symbiotic relationships that support survival on a nutritionally austere dietary source, transcriptome assemblies for three sharpshooter species and one spittlebug species were produced. Findings: Trinity-based de novo transcriptome assemblies were generated for all four xylem-sap feeders using raw sequencing data originating from whole-insect preps. Total transcripts for each species ranged from 91 384 for Cuerna arida to 106 998 for Homalodisca liturata with transcript totals for Graphocephala atropunctata and the spittlebug Clastoptera arizonana falling in between. The percentage of transcripts comprising complete open reading frames ranged from 60% for H. liturata to 82% for C. arizonana. Bench-marking universal single-copy orthologs analyses for each dataset indicated quality assemblies and a high degree of completeness for all four species. Conclusions: These four transcriptomes represent a significant expansion of data for insect herbivores that feed exclusively on xylem sap, a nutritionally deficient dietary source relative to other plant tissues and fluids. Comparison of transcriptome data with insect herbivores that utilize other dietary sources may illuminate fundamental differences in the biochemistry of dietary specialization.

Monitoring changes in Bemisia tabaci (Hemiptera: Aleyrodidae) susceptibility to neonicotinoid insecticides in Arizona and California.

Bemisia tabaci (Gennadius) biotype B is a highly prolific and polyphagous whitefly that established in much of North America during the 1980s. Neonicotinoid insecticides have been fundamental in regaining control over outbreak populations of B. tabaci, but resistance threatens their sustainability. Susceptibility of B. tabaci in the southwestern United States to four neonicotinoid insecticides varied considerably across populations within each year over a 3 yr period. Using a variability ratio of highest LC50 to lowest LC50 in field-collected whitefly adults from Arizona and California, the ranges of LC50(s) across all tests within compounds were highest to imidacloprid and lowest to thiamethoxam. Patterns of susceptibility were similar among all four neonicotinoid insecticides, but the greater variability in responses to imidacloprid and significantly higher LC50(s) attained indicated higher resistance levels to imidacloprid in all field populations. Further evidence of differential toxicities of neonicotinoids was observed in multiple tests of dinotefuran against imidacloprid-resistant lab strains that yielded significant differences in the LC50(s) of dinotefuran and imidacloprid in simultaneous bioassays. To test the possibility that resistance expression in field-collected insects was sometimes masked by stressful conditions, field strains cultured in a greenhouse without insecticide exposure produced significantly higher LC50(s) to all neonicotinoids compared with LC50(s) attained directly from the field. In harsh climates such as the American southwest, resistance expression in field-collected test insects may be strongly influenced by environmental stresses such as high temperatures, overcrowding, and declining host plant quality.

Host plant influence on susceptibility of Bemisia tabaci (Hemiptera: Aleyrodidae) to insecticides.

A resistance monitoring program conducted for the polyphagous whitefly, Bemisia tabaci (Gennadius), in Imperial Valley, CA, USA generated a large set of LC50s for adults collected from broccoli, cantaloupe and cotton crops over a four-year period. A vial bioassay and, subsequently, a yellow-sticky card bioassay produced similar temporal profiles of relative susceptibilities to the pyrethroid insecticide bifenthrin. Both bioassays revealed that whiteflies collected from broccoli were significantly less susceptible to bifenthrin compared to the other two crops. A similar finding was observed for endosulfan and the mixture of bifenthrin+endosulfan in the yellow-sticky card bioassay. The possibility that seasonal differences contributed to the observed differences in susceptibility provided the impetus to conduct a reciprocal transfer experiment using broccoli (or kale) and cantaloupe grown simultaneously in the field and greenhouse. Whitefly adults collected from an organic farm over three consecutive weeks had significantly higher LC50s on kale than those collected the same day on cantaloupe. After culturing in the greenhouse on broccoli or cantaloupe and testing again, LC50s remained significantly higher on broccoli after one week and again at the F1 generation. In contrast, whiteflies originating on kale in the field and transferred to cantaloupes in the greenhouse had significantly reduced LC50s at the F1 generation. When tested against the bifenthrin+endosulfan mixture, significantly higher LC50s were generated for whiteflies reared on broccoli in the greenhouse at one week and the F1 compared to the field source from cantaloupes. The consistently higher LC50s for whiteflies on broccoli and other Brassica spp. crops, compared to cantaloupes or cotton, point to statistically significant host-plant influences that are expressed in both field-collected and greenhouse-reared populations of whiteflies.

Baseline susceptibility of Bemisia tabaci B biotype (Hemiptera: Aleyrodidae) populations from California and Arizona to spiromesifen.

Susceptibility to spiromesifen, a tetronic acid derivative, was determined for three imidacloprid-resistant strains and 12 geographically discrete natural populations of Bemisia tabaci (Gennadius) (=Bemisia argentifolii Bellows & Perring) (Hemiptera: Aleyrodidae) from California and Arizona by laboratory bioassays. Newly emerged first instars were sprayed with aqueous serial dilutions of spiromesifen and evaluated for toxicity to establish baseline susceptibility data. Interpopulation variability in susceptibility to spiromesifen was observed among the natural populations of whiteflies up to 29-fold; however, there was only 30-fold difference in susceptibility among natural and resistant populations tested. In general, spiromesifen was quite toxic to first instars across most of their geographic range, with LC50 values ranging from 0.210 to 6.08 microg (AI)/ml. The magnitude of variation was smaller among the three-resistant strains. These results suggest that the observed variability reflect natural variation in spiromesifen susceptibility among all the test populations, possibly due to previous exposure to insecticides at each location. The effectiveness of spiromesifen also was evaluated against all immature stages of whiteflies from three field and two resistant strains. Spiromesifen was significantly more active against early instars of whiteflies based on lower LC50 values recorded compared with the fourth instars. Spiromesifen was effective against the resistant strains including a Q-biotype of B. tabaci from Spain, which is highly resistant to neonicotinoids. Results of this study indicate absence of cross-resistance between spiromesifen and more commonly used neonicotinoids. Our findings suggest that spiromesifen should be considered an ideal candidate for whitefly resistance management programs in rotation with other effective chemistries.

Comparison of sampling methods for determining relative densities of Homalodisca vitripennis (Hemiptera: Cicadellidae) on citrus.

Four sampling methods that included A-Vac, D-Vac, pole-bucket, and beat-net devices were evaluated for estimating relative densities of glassy-winged sharpshooter, Homalodisca vitri-pennis (Germar) (Hemiptera: Cicadellidae) nymphs and adults on citrus (Citrus spp.) trees. All four methods produced similar temporal and spatial distribution profiles, although significant differences in quantities of H. vitripennis adults and nymphs caught by each device were observed. The four sampling methods also showed a consistent male bias in adult populations across a range of densities, suggesting that previously reported male-biased sex ratios in H. vitripennis adult populations are real and not a product of sampling bias. A strong relationship (R2 = 0.95) between the monitoring methods we evaluated and yellow sticky trap catches of female H. vitripennis adults suggest that yellow sticky trap catches may provide a good relative index of infestation levels in citrus trees. Based on quantitative analyses examining precision and cost, the pole bucket was the most efficient method for sampling nymphs, and it was as efficient as the beat-net method for sampling adults and both stages combined. In addition to these analyses, consideration of other sampling characteristics such as added flexibility in sampling and higher sensitivity in detecting infestations within individual trees helped to fortify the conclusion that the pole bucket was the best overall sampling method of those tested.

Amino acid fluctuations in young and old orange trees and their influence on glassy-winged sharpshooter (Homalodisca vitripennis) population densities.

The glassy-winged sharpshooter (GWSS) is an invasive insect pest in California that proliferated in citrus while incorporating many other plant species in its host range. Field studies were conducted from September 2002 to September 2003 in adjacent young and old orange groves to determine the influence of amino acid concentrations in the xylem fluid on relative densities of the xylophagous GWSS. Beginning in early September 2002, higher densities of adult GWSS were observed on young compared to old trees. This difference became increasingly pronounced through late October in conjunction with increasing concentrations of essential amino acids in xylem fluid of the young trees. Greater densities of adult GWSS remained on young trees through early February 2003. Thereafter, the population crashed unexpectedly, leaving only negligible numbers of GWSS on either young or old trees. Mean concentrations of the essential amino acids for insect growth and development were higher in young compared to old trees. Besides these essential amino acids, asparagine, serine, glutamine, and tyrosine were also significantly higher in young trees during the September-February period when GWSS was present. The pattern of elevated amino acid concentrations in young trees continued through the entire year irrespective of the presence or absence of GWSS. Principal component analysis followed by factor analysis revealed three factors for young or old orange trees that accounted for 81.6 and 78.9% of the total variation in the young and old tree analyses, respectively. Various groups of amino acids in different factors displayed peak or elevated levels in young trees corresponding to the increased densities of GWSS. The potential roles of these amino acids in GWSS host selection are discussed.

Toxicity of seven foliar insecticides to four insect parasitoids attacking citrus and cotton pests.

Laboratory studies were carried out to compare the toxicity of seven foliar insecticides to four species of adult beneficial insects representing two families of Hymenoptera: Aphelinidae (Aphytis melinus Debach, Eretmocerus eremicus Rose & Zolnerowich, and Encarsiaformosa Gahan) and Mymaridae (Gonatocerus ashmeadi Girault) that attack California red scale, Aonidiella aurantii (Maskell); sweetpotato whitefly, Bemisia tabaci (Gennadius) (both E. eremicus and E. formosa); and glassy-winged sharpshooter, Homalodisca vitripennis (Germar), respectively. Insecticides from four pesticide classes were evaluated using a petri dish bioassay technique across a range of concentrations to develop dosage-mortality regressions. Insecticides tested included acetamiprid (neonicotinoid); chlorpyrifos (organophosphate); bifenthrin, cyfluthrin, and fenpropathrin (pyrethroids); and buprofezin and pyriproxyfen (insect growth regulators [IGRs]). Chlorpyrifos was consistently the most toxic pesticide to all four species of beneficial insects tested based on LC50 values recorded 24 h posttreatment compared with 48-h LC50 values with the neonicotinoid and pyrethroids or 96 h with the IGRs. Among the three pyrethroids, fenpropathrin was usually less toxic (except similar toxicity to A. melinus) than was cyfluthrin, and it was normally less toxic (except similar toxicity with E. formosa) than was bifenthrin. Acetamiprid was generally less toxic than bifenthrin (except similar toxicity with G. ashmeadi). The IGRs buprofezin and pyriproxyfen were usually less toxic than the contact pesticides, but we did not test for possible impacts on female fecundity. For all seven pesticides tested, A. melinus was the most susceptible parasitoid of the four test species. The data presented here will provide pest managers with specific information on the compatibility of select insecticides with natural enemies attacking citrus and cotton, Gossypium hirsutum L., pests.

Susceptibility of immature stages of Homalodisca coagulata (Hemiptera: Cicadellidae) to selected insecticides.

Susceptibility of immatures of the glassy-winged sharpshooter, Homalodisca coagulata (Say) (Hemiptera: Cicadellidae), to 10 insecticides that included chlorpyrifos, dimethoate, endosulfan, bifenthrin, cyfluthrin, esfenvalerate, fenpropathrin, acetamiprid, imidacloprid, and thiamethoxam was evaluated in the laboratory. All five instars were exposed to different doses of each foliar insecticide by the petri dish technique, whereas a systemic uptake method was used to assess the toxicity to imidacloprid and thiamethoxam. All test insecticides exhibited high toxicity to all immature stages of H. coagulata at concentrations below the field recommended rates of each insecticide. Although all five instars were susceptible to test insecticides, mortality was significantly higher in first instars than in the older immatures based on low LC50 values (ranging from 0.017 to 5.75 ng(AI)/ml) with susceptibility decreasing with each successive stage. Fifth instars were generally the least sensitive (LC50 values ranging from 0.325 to 216.63 ng (AI)/ml). These results show that mortality was directly related to age of the insect and suggest that chemical treatment at early stages is more effective than at late stages. Acetamiprid (neonicotinoid) and bifenthrin (pyrethroid) were the most toxic to all five instars, inducing most mortality within 24 h and showing lower LC50 values ranging from 0.017 to 0.686 ng/ml compared with other insecticides (LC50 values ranging from 0.191 to 216.63 ng(AI)/ml). Our data suggest that a diverse group of very effective insecticides are available to growers for controlling all stages of H. coagulata. Knowledge on toxicity of select insecticides to H. coagulata immatures may contribute to our understanding of resistance management in future for this pest by targeting specific life stages instead of the adult stage alone.

Phenology and demography of Homalodisca coagulata (Hemiptera: Cicadellidae) in southern California citrus and implications for management.

Populations of Homalodisca coagulata (Say) were sampled from citrus orchards in southern California, USA to characterize and quantify seasonal occurrences of nymphs and adults with the goal of identifying management opportunities through well-timed treatments and/or natural enemy releases. Higher densities of H. coagulata in 2001 contributed to a complete seasonal profile that began in early spring with the emergence of first instar nymphs and their progression through five nymphal instars lasting until mid-August. Adult emergence began in mid-June with peak adult densities attained from mid to late August followed by a gradual decline through autumn. A persistent and significant male bias was observed in the adult sex ratio from the time of first emergence through mid-October in oranges; the same trend was present in lemons, but with more variability. Adult densities gradually declined through the winter months into the following spring before rapidly increasing again in June as the 2002 spring generation of nymphs began emerging as adults. The seasonal timing of nymphs and adults in 2002 was nearly identical to that observed the previous year. Phenology data from both years were incorporated into a stochastic, temperature-dependent model that predicts the occurrences of H. coagulata stages through time. Applications of imidacloprid early in the spring generation of nymphs proved very effective at reducing nymphs and sustaining lower densities of adults through summer.

Influence of seasonal nitrogen nutrition fluctuations in orange and lemon trees on population dynamics of the glassy-winged sharpshooter (Homalodisca coagulata).

The glassy-winged sharpshooter (GWSS) is a xylem feeder that develops conspicuous, year-round populations in many citrus-producing regions of California. Field studies were conducted in a combined lemon and orange orchard to determine the influence of changing amino acid concentrations on relative densities of GWSS. Nineteen protein amino acids were detected in xylem fluid of both lemon and orange trees. Although annual profiles of total and essential amino acids were similar for each citrus species, mean concentrations were consistently higher in lemons than in oranges for most of the year, except for one critical period in late winter-early spring when concentrations were higher in oranges. Principal component analysis followed by factor analysis was performed individually on lemon and orange data sets to identify a reduced number of orthogonal factors composed of amino acids having similar seasonal profiles. Four factors were identified for each citrus species that accounted for 85% and 79% of the total variation in the orange and lemon analyses, respectively. These were then examined with respect to shifts in GWSS numbers that occurred asynchronously in lemons and oranges over the annual population cycle. Three distinctive number shifts were identified that included a peak in adult numbers in lemons during August 2001, significantly higher numbers in lemons relative to oranges during midwinter, and finally an increase in oranges of both adults and nymphs during spring 2002. Various groups of amino acids, i.e., factors, displayed peak annual or elevated levels during the intervals when shifts in GWSS numbers were occurring. Soluble protein levels in oranges and lemons did not correspond to shifting GWSS numbers as certain amino acids. However, soluble protein levels were higher in oranges during late winter/early spring when GWSS adults were sexually active. Potential roles of these amino acids and proteins in GWSS host selection are discussed.

Field evaluation of different insecticide use strategies as resistance management and control tactics for Bemisia tabaci (Hemiptera: Aleyrodidae).

Various insecticide use strategies including rotations, sequential use, and mixtures were evaluated experimentally on Bemisia tabaci (Gennadius) in California and Arizona (U.S.A.) cotton fields. Toxicological responses of adult B. tabaci were measured along with preimaginal densities and cotton yields from plots subjected to different insecticide regimens. Weekly monitoring for susceptibility changes over ten consecutive weeks in four different trials failed to detect significant differences between sequential use and rotation regimens, nor in comparison to the control plots. There were, however, significant differences among study-site locations and between study years as well as significant within-season time effects. Relative infestations in insecticide-treated plots expressed as a percentage of preimaginal densities in control plots indicated that better control was obtained by all insecticide treatments in conjunction with higher susceptibility levels observed in the second year. Lower preimaginal densities of B. tabaci were measured in the rotation treatment in comparison to sequential treatments of endosulfan, chlorpyrifos, or amitraz, but all were less effective than sequential treatments of bifenthrin or the mixture of bifenthrin + endosulfan. Cotton lint yields were inversely related to B. tabaci densities, with highest yields in the bifenthrin and mixture plots and lowest yields in the control plots. Suppression of B. tabaci infestations in insecticide-treated plots relative to untreated control plots also improved under conditions of lower B. tabaci pressure. The increases in cotton yield and susceptibility to insecticides seen in the current study support the trend observed in the southwestern USA of improved management of B. tabaci despite continuing intensive use of insecticides.

Inter-regional differences in baseline toxicity of Bemisia argentifolii (Homoptera: Aleyrodidae) to the two insect growth regulators, buprofezin and pyriproxyfen.

A survey of 53 Bemisia argentifolii Bellows & Perring populations from different agricultural regions in California and Arizona was conducted from 1997 to 1999 to establish baseline toxicological responses to buprofezin and pyriproxyfen. Although both compounds proved to be highly toxic even in minute quantities to specific stages, geographical and temporal differences in responses were detected using a leaf spray bioassay technique. Monitoring for three years revealed that six to seven populations had higher LC50 values but not greater survival when exposed to these two insecticides. A significant difference in relative susceptibility to buprofezin was first observed in late season 1997 in San Joaquin Valley populations with LC50s ranging from 16 to 22 microg (AI)/liter(-1) compared with IC50s of 1 to 3 mg (AI)/liter(-1) in Imperial, Palo Verde Valley and Yuma populations. Whiteflies collected in subsequent years from these and other locations showed an increase in susceptibility to buprofezin. Regional differences in susceptibilities to pyriproxyfen were minimal within the same years. Three years of sampling revealed consistently higher LC50s to pyriproxyfen in populations from Palo Verde Valley, CA, compared with whiteflies from Imperial, San Joaquin Valley or Yuma. As was the case with buprofezin, a decline in LC50s to pyriproxyfen was observed in whiteflies from all locations sampled in 1999. However, no correlation was observed between buprofezin and pyriproxyfen toxicity in any of the strains. The variable toxicities observed to both compounds over a period of 3 yr may be due principally to inherent differences among geographical populations or due to past chemical use which may confer positive or negative cross-resistance to buprofezin or pyriproxyfen.

Sensing isothermal changes in the lateral pressure in model membranes using di-pyrenyl phosphatidylcholine.

In this work we present data from a homologous series of di-pyrenyl phosphatidylcholine (dipyPC) probes which can sense lateral pressure variations in the chain region of the amphiphilic membrane (lateral pressures are tangential to the interface). The dipyPC has pyrene moieties attached to the ends of equal length acyl chains on a phosphatidylcholine molecule. Ultraviolet stimulation produces both monomer and excimer fluorescence from pyrene. At low dilutions of dipyPC in model membranes the excimer signal is entirely intra-molecular and since it depends on the frequency with which the pyrene moieties are brought into close proximity, the relative intensity of the excimer to monomer signal, eta, is a measure of the pressure. We synthesised or purchased dipyPC probes with the pyrene moieties attached to acyl chains having 4, 6, 8 and 10 carbon atoms and then measured eta in fully hydrated bilayers composed of dioleoylphosphatidylcholine and dioleoylphosphatidylethanolamine (DOPC and DOPE respectively). Although the resolution of our measurements of lateral pressure as a function of distance into the monolayer was limited, we did observe a dip in the excimer signal in the region of the DOPC/DOPE cis double bond. As we isothermally increased the DOPE composition, and hence the desire for interfacial curvature, we observed, as expected, that the net excimer signal increased. However this net increase was apparently brought about by a transfer of pressure from the region around the glycerol backbone to the region near the chain ends, with the lateral pressure dropping above the cis double bond but increasing at a greater rate beyond the double bond.

Assessment of an in situ rat intestine preparation with perfused vascular bed for studying the absorption and first-pass metabolism of drugs.

The perfused in situ rat jejunum preparation originally described by Hanson and Parsons (1976) was adapted for use in absorption and metabolism studies with drugs. The preparation allows simultaneous perfusion of the gut lumen and associated vasculature and is viable for one hour. The viability of the preparation was assessed, and the application of the method is illustrated by experiments with the opiate analgesic, buprenorphine.