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. suzukiiwere 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,greenhouse snap clamps 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 of bio-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 . LC50 and LC90 values from these analyses are presented in Table 1. Overall, zeta-cypermethrin exhibited the lowest LC50, followed by spinosad and malathion, respectively. However, when comparing LC90s, spinosad exhibited the lowest value of the three materials tested.
These results differ from previous studies which found that, at the adult stage, spinosad lethal concentration values are greater than both malathion and zeta-cypermethrin. Together, these studies suggest that the relative efficacy of insecticides may differ across life stages or as a result of the different exposure routes they experience . This pattern of larval susceptibility does not appear to be caused by the different solvents used to dilute our insecticides, as the number of flies that emerged from ddH2O- and acetone-treated bottles was similar. In all cases, larval LC90 values fell far below the maximum caneberry field application rate of each material. For malathion, the maximum label rate is 139 times greater than the larval LC90 found here; the spinosad label rate is 24 times higher than the larval LC90; and the zeta-cypermethrin label rate is 29 times higher than the larval LC90. However, the concentrations experienced by larvae in the field, within infested fruit, are likely significantly lower than the field application rates. Indeed, a recent study by Andika et al showed that treating D. suzukii infested cherries with the label rate of various insecticides significantly reduced, but did not eliminate, larval survival and adult emergence in most instances. For zeta-cypermethrin, specifically, residue concentrations within the fruit were measured across two years, and high variability was observed. In 2018, an average concentration of 3.7 ppm was detected at the subsurface level, whereas 2017 results detected no zetacypermethrin residue within the fruit . These findings suggest significant overlap in the range of concentrations tested here and those experienced by D. suzukii larvae in the field.The change in adult susceptibility to spinosad due to larval selection was assessed by performing dose-response analysis on Watsonville D. suzukii before and after imposing five generations of selection. Prior to implementing the larval selection protocol, male and female WAT adults exhibited significantly higher LC50 values than their same-sex counterparts from the susceptible Wolfskill population . Specifically, we observed RRs of 4.9 and 6.8 for WAT males and females, respectively .
These values are similar to those reported by Gress and Zalom which tested the same field populations in fall of 2017 and found RRs of 4.3 for males and 5.2 for females. Following larval selection, WAT-S5 males exhibited LC50s 75% higher than pre-selection WAT males , similar to the 86% increase observed using the adult selection method. In contrast, female resistance increased more rapidly with larval selection than with adult selection . As a result, WAT-S5 RRs increased to 12.6 for males and 14.4 for females when compared to the susceptible strain. It is currently unclear what accounts for the sex-specific response patterns observed in these studies, and future work should investigate this question in greater detail. When comparing adult mortality in the Wolfskill population during the first and second assessment periods, we found no significant difference in female susceptibility . Males, however, exhibited a marginally significant decline in LC50 during the second assessment . This finding raises the possibility that laboratory conditions or other sources of error could have increased the potency of spinosad residues during the second assessment period. If true, our results may underestimate the real evolutionary potential for resistance to increase in the near-term. Nevertheless, this study provides the first evidence that larval selection can be used as an effective tool for performing resistance risk assessments in D. suzukii and is capable of increasing both larval and adult tolerance to the target AI. In a previous study, Smirle et al attempted to select for malathion resistance in D. suzukii from British Columbia, Canada using a different larval selection protocol developed for D. melanogaster,snap clamps for greenhouse but the concentration of malathion used had no impact on D. suzukii larval survival or eclosion success. Perhaps unsurprisingly, no malathion resistance was observed despite 30 generations of exposure. By identifying lethal concentrations of commonly used insecticides for susceptible D. suzukii larvae, this study provides a valuable resource for researchers looking to perform resistance monitoring or risk assessments on field populations of interest. We expect that this information will become increasingly more important as the widespread use of these critical insecticides results in the loss of susceptibility throughout North America, South America and Europe. To this point, a recent study by Mishra et al found that D. suzukii collected from commercial blueberry fields in Georgia exhibited a 3-fold increase in tolerance to malathion, spinosad, and zeta-cypermethrin relative to flies from an untreated location, indicating that resistance could soon emerge in other locations. The larval bio-assay, whether used for resistance monitoring or selection studies, also offers several advantages over the adult glass vial approach that makes it appealing across many experimental contexts. First, implementing this protocol requires little time, effort or cost investment beyond what is already required for standard D. suzukii colony maintenance. In contrast, the glass vial approach used for adults requires 1) purchasing large numbers of glass scintillation vials, 2) rolling treated vials throughout the drying process to help ensure residues are evenly distributed, and 3) manually loading adult flies into each vial to perform the bio-assay. The loading process can be performed either using CO2 to anaesthetize the flies or by gently aspirating the adults into the vial, and both methods have the potential to harm or kill the flies in the process. Additionally, 4) adult mortality assessments can be time-consuming and require subjective assessments of how to classify individuals at varying stages of moribundity. With larval bio-assays, survival can easily and objectively be estimated by counting the number of adults to emerge from treated and untreated bottles.Finally, because each adult female is capable of laying multiple eggs per day, a fraction of the number of adult D. suzukii are needed to conduct resistance monitoring and risk assessment studies with the larval protocol. For example, using the adult selection method adopted by Gress and Zalom if a group of 100 female D. suzukii were exposed to their LC90 concentration, on average, 10 survivor females would remain to produce the subsequent generation.
In contrast, if the same 100 females were split among 5 bottles and the larval selection method was implemented using the LC90, our results indicate that, on average, more than 30 survivor adults would emerge. Moreover, because the larval bio-assay does not require killing the parental females, these flies can continue to produce new progeny, thus drastically expanding the number of potential offspring. Although we did not measure the extent to which oviposition rate or larval susceptibility changes with maternal age, previous work has shown that female reproductive output remains high for up to 90 days post-eclosion when maintained on artificial cornmeal diet. Together, these findings indicate that larval bio-assays are a simple, cost-effective approach for measuring the current state of insecticide resistance in field populations as well as the potential for greater resistance to develop. Greater effort to monitor these trends in D. suzukii field populations is needed to ensure that appropriate resistance management actions can be implemented when early susceptibility loss is detected.Chronic metabolic diseases are associated with elevated FFAs and prolonged postprandial hyperlipidemia particularly with TG-rich lipoproteins 10 . High-fat meal-induced postprandial lipidemia is often associated with enhanced concentrations of proinflammatory marker proteins in the plasma . However, the mechanism by which postprandial hyperlipidemia affects inflammatory responses is unclear. Our previous studies revealed that saturated FAs can activate Toll-like receptor 4 and nucleotide-binding oligomerization domain receptor 2-mediated signaling pathways, whereas the n-3 FA DHA and certain plant polyphenols that are abundant in fruits and vegetables can inhibit TLR4 and nucleotide-binding oligomerization domain receptor 2-mediated inflammatory signaling pathways . Animal studies demonstrated that TLR4- or TLR2-deficient mice were protected from high-saturated fat diet-induced inflammation and insulin resistance, suggesting that inflammation and insulin resistance induced by dietary saturated fat are at least in part mediated through the activation of TLR4 and TLR2 . In addition, activation of TLR2 by FFAs released from TGRLs derived from human subjects who consumed a high fat meal leads to inflammasome-mediated secretion of IL-1b in isolated primary blood monocytes or whole blood . FAs derived from dietary saturated fat from people who consume a high-saturated fat meal are transported in TGRLs as TGs in the postprandial state.Our previous mechanistic study revealed that both exogenous palmitic acid and endogenous FFAs released from TGRLs derived from the human subjects who consumed a high-fat meal directly activate TLR2 and induce inflammasome-mediated secretion of IL-1b in isolated primary blood monocytes or whole blood. These results suggest that the plasma concentration of FFAs is an important determinant in modulating TLR-mediated cytokine production in blood. In a microenvironment where secreted LPL can hydrolyze TGs in plasma TGRLs to release FFAs, elevated plasma FFAs can enhance the propensity of monocyte activation.