The resulting Coulomb blockade phenomenon gives nanoSQUID sensors a very slight electric field sensitivity. Gating exfoliated heterostructures tends to produce large electric fields, and these are detectable as variations in the current through the nanoSQUID as a result of Coulomb blockade in parallel with the SQUID on the tip. Droplets functioning as quantum dots and producing a parasitic Coulomb blockage are so common that we observe them on nearly every nanoSQUID sensor, and we almost always have finite electric field sensitivity . This can be useful for finding the edges of devices in the absence of magnetism, but it is important to remember that not all nanoSQUID signals can be understood as local magnetic fields. Other parasitic contrast mechanisms do exist, but they are rarely dominant over magnetic or electric field sensitivity. At high tuning fork amplitudes, interactions between the nanoSQUID tip and the surface can produce local variations in oscillation amplitude and appear as parasitic signals at the tuning fork frequency. Of course, the nanoSQUID is highly sensitive to local temperature, so systems with thermal gradients will generally have backgrounds associated with that. But by far the most important parasitic contrast mechanism in the nanoSQUID campaigns discussed here is electric field contrast through parasitic Coulomb blockade. Drosophila suzukii , commonly known as the spotted-wing drosophila, is an invasive pest of small fruit and berry production native to Southeast Asia. Starting in 2008, growing strawberries vertically this species underwent a rapid geographic range expansion, spreading across North America, South America, and Europe.
Following its arrival to the USA, growers in California, Oregon, and Washington reported yield losses of up to 50% in raspberries and blackberries, 40% inblueberries, and 33% in cherries. Unlike other Drosophila species that typically breed on overripe and rotting fruit, D. suzukii females readily lay eggs into firm, still-ripening fruit using specialized serrated ovipositors. After hatching, larvae continue to feed and develop within the fruit, causing rapid decay and increased susceptibility to subsequent infestation by other drosophilids. Efforts to control D. suzukii and prevent infestation have led to a drastic increase in the use of broad-spectrum insecticide applications in susceptible host crops. Specifically, three main classes of insecticide–organophosphates, pyrethroids, and spinosyns–have shown high efficacy and now form the basis of most conventional D. suzukii management programs. Carbamates and some diamides have also shown moderate efficacy against D. suzukii, but the use of these materials is more limited throughout North America and Europe. The continued reliance on chemical sprays to control this pest, combined with its high fecundity and rapid development time, have elicited concerns that insecticide resistance could quickly develop. For organic growers, the risk of resistance is even greater. Currently, spinosad is the only insecticide approved by the Organic Materials Review Institute with high efficacy against D. suzukii, meaning that growers must rotate spinosad sprays with low-efficacy materials such as pyrethrins and azadirachtin in order to comply with label requirements. Consequently, D. suzukii in organic fields are primarily challenged by only one class of insecticide further increasing the risk of resistance. Indeed, recent work by Gress and Zalom reported low to moderate levels of spinosad resistance in D. suzukii collected from commercial caneberry fields near Watsonville, CA. Specifically, this study utilized dose-response analysis of adult D. suzukii to quantify the degree of resistance and found that Watsonville females exhibited LC50s 5–8 times higher than females from a second, untreated location in California.
This finding demonstrates an immediate need to expand our understanding of resistance evolution in D. suzukii and to develop novel tools that aid in the early detection and assessment of resistant populations so that appropriate management actions can be implemented. When decreased susceptibility to an insecticide is first detected in a pest field population, laboratory selection studies, often referred to as resistance risk assessments, represent a critical first step towards understanding the potential for resistance to develop. These studies are performed by exposing large numbers of individuals from the suspect field population to moderately lethal concentrations of insecticide for multiple generations such that the most susceptible individuals are eliminated and the most tolerant survive. Dose-response analyses are performed both before and after implementing the selection protocol, and the change in LC50 is used to quantify the response to selection and better understand the potential for resistance to develop. This approach, however, is not without its limitations. For example, because laboratory colonies contain limited genetic variation relative to large field populations, these studies can understate the evolutionary potential for resistance to develop in a field setting. Conversely, selection imposed in laboratory settings may be more intense and consistent than in the field, leading to possible overestimation of resistance development. Nevertheless, an increase in resistance due to laboratory selection provides unambiguous evidence that further loss of susceptibility in the field is possible. Additionally, if laboratory selection is successful and resistant colonies are generated, these strains can assist researchers in the development of novel tools for detecting, monitoring, and managing resistance in the field. For example, resistant colonies can be used to identify the molecular mechanism that produces the resistant phenotype, and these markers can, in some instances, be used to track spatiotemporal patterns of resistance development in the field.
Laboratory-generated resistant colonies can also be used to assess whether insecticide resistance in a particular population is associated with fitness costs , and this information can be used to develop programs to manageresistance in the field. The presence of such costs under benign conditions would suggest that efforts to temporarily eliminate the target insecticide from spray programs could successfully maintain susceptibility in the field, at least in the short-term. After decreased spinosad susceptibility in the Watsonville, CA population was identified, Gress and Zalom performed a resistance risk assessment by continuing to expose adults from this population to a discriminating dose of spinosad using a standard glass vial bio-assay protocol developed for D. suzukii. After implementing this technique for 5 generations, Gress and Zalom found that LC50 values increased by 86% for males and 49% for females. This selection protocol, however, is highly labor intensive and costly, making it impractical for long-term selection programs which are needed to generate and maintain resistant lines. Additionally, this method requires that researchers kill off much of their adult population each generation, increasing the risk that selection lines will be lost in the process. Here, we describe a simple larval bio-assay that overcomes these challenges and requires little effort or cost beyond what is already needed for basic laboratory stock maintenance. We then perform a series of larval dose-response bio-assays to calibrate the selection protocol and identify baseline susceptibility for three commonly used insecticides. Finally, we implement the larval bio-assay protocol to perform resistance risk assessments using the same Watsonville, CA field strain previously tested by Gress and Zalom. Changes in the susceptibility at both the larval and adult life stages were quantified, drainage planter pot and results from this study are compared to those obtained with the original adult selection method.Here we explain the general larval bio-assay protocol used throughout the paper. First, groups of n = 20 mated females were transferred into plastic 6 oz Drosophila stock bottles containing fresh Bloomington standard Drosophila cornmeal diet. Once females were removed, 400 μL of insecticide solution was pipetted onto the surface of the diet, and each bottle was shaken side to-side to distribute the solution. This ensured that all larvae were uniformly exposed to the insecticide, both through external contact and internally while feeding on the treated diet. Control bottles were treated with either ddH2O or acetone, depending on the insecticide treatment , but otherwise were handled using the same protocol as experimental bottles. All bottles were then re-plugged with a cotton stopper and stored in a climate-controlled walk in chamber at 22–23˚C and 14–10 light-dark cycle. Because the first larvae typically crawl out of the diet to pupate after 6 days under these conditions , treatment timing is critical to ensure that all larvae are exposed. Bottles were monitored for up to 18 days following treatment for the emergence of adult D. suzukii, and all newly enclosed adults were transferred to fresh bottles and counted.To quantify baseline susceptibility of D. suzukii larvae, we used the larval bio-assay protocol to perform dose-response analyses for three insecticide AIs, each representing a different insecticide class, commonly used against this pest. This work was conducted using a susceptible laboratory strain of D. suzukii originally collected from USDA Wolfskill Germplasm Repository in Winters, CA in fall of 2017.
Wolfskill is an experimental, mixed-fruit orchard, and crops at this location receive no insecticide treatment. This strain of D. suzukii was previously used as a susceptible control by Gress and Zalom when quantifying resistance in the Watsonville population. The three formulated insecticides used in this study were malathion , spinosad , and zeta-cypermethrin . Serial dilutions were performed for each product using either ddH2O or acetone to obtain a range of concentrations , malathion: 0–40 ppm, and zeta-cypermethrin: 0–25 ppm. In total, 5–8 concentrations were used per insecticide, and each concentration was replicated across 2–5 bottles.Approximately n = 250 D. suzukii were live-trapped from four commercial caneberry fields near Watsonville, CA on October 23, 2018 using plastic McPhail traps baited with a mixture of approximately 7 g yeast, 113 g sugar, and 355 ml water. Traps were modified by fitting a mesh barrier such that D. suzukii adults could enter the trap unobstructed but were prevented from reaching the liquid lure. Traps were transported back to a laboratory facility on the University of California, Davis campus where all flies were transferred into 6 oz Drosophila bottles containing standard cornmeal diet, with approximately 30 flies per bottle. Every fourth day, Watsonville D. suzukii were turned over into new bottles with fresh diet, and all progeny were reared to adulthood under standard laboratory conditions . Starting in the F3 and F4 generations, we generated two selection lines: a spinosad selection line and a malathion selection line . Each generation, larvae were treated with a discriminating dose of insecticide following the larval selection protocol outlined above. All adult flies that emerged from treated bottles were collected and transferred to fresh diet to produce the next generation. The number of adults to emerge from each insecticide-treated bottle was counted during the first generation of selection and again after five generations to measure change in larval susceptibility. Survival in the malathion selection line was again assessed after approximately 20 generations of selection . At each time point, bottles treated with either ddH2O or acetone only were included to allow estimation of baseline eclosion in the absence of insecticide. Additionally, WOLF bottles were treated with insecticide to serve as a susceptible point of comparison and ensure that the insecticide treatment was effective.To assess the impact of larval selection on adult susceptibility, we used pre-selection F1 and F2 WAT and post-selection WAT-S5 adults to perform spinosad dose-response analysis, with concentrations ranging from 0–1000 ppm. Prior to testing WAT-S5 adults, insecticide treatment was removed for one generation to expand the size of the colony. Adults from the WOLF population were also tested concurrently with WAT and WAT-S5 and served as a susceptible point of comparison. During the pre-selection phase ofbio-assays, n = 275 WOLF adults of each sex were bio-assayed, and during the post-selection phase n = 295 per sex were tested. Adult bio-assays were performed using the protocol developed by Van Timmeren et al and used by Gress & Zalom in which 3–8-day old laboratory-reared flies were exposed to insecticide residues within glass scintillation vials. In brief, 1 mL of insecticide solution was added to each 20-mL glass scintillation vial, and vials were then capped and shaken for five seconds before pouring out the excess liquid. To prevent the remaining insecticide solution from settling at the bottom, vials were inverted and rolled every 60 minutes for four hours during the drying process. The next morning, n = 5 male and n = 5 female D. suzukii were gently aspirated into each vial and left for 8 hrs. Mortality assessments were performed by individually classifying each fly as living or dead .Data from the larval dose-response analysis, which used susceptible D. suzukii from the Wolfskill population, show that mortality increased in a log-dose manner, enabling the use of probit models to identify lethal concentrations for each insecticide .