Kaolin as a management alternative for insecticide-resistant navel orangeworm (Lepidoptera: Pyralidae).

The production of almonds and pistachios in California's Central Valley employs insecticides for the management of their primary pest, navel orangeworm. The pyrethroid Bifenthrin is commonly used, and now a strain of Amyelois transitella Walker (Lepidoptera: Pyralidae) (R347) obtained from Kern County almond orchards with a history of Bifenthrin use has acquired >110-fold resistance toward pyrethroids. One method to improve control is to use additives and spray adjuvants, which are applied simultaneously with an insecticide to increase coverage and/or duration of control. We tested 2 levels of the naturally occurring clay Kaolin as an additive, alone and in combination with either Bifenthrin or the diamide Chlorantraniliprole, to determine if it could reduce feeding damage and decrease survival of pyrethroid-resistant A. transitella on almonds in the laboratory and improve the efficacy of Chlorantraniliprole in the field. Larval performance was measured for the strains R347 and ALM (34.7-fold resistance compared to susceptible lab strain) reared on treated almonds. Strain R347 had 1.9-fold greater survival and caused 1.3-fold more feeding damage than strain ALM across all treatments, although both strains were susceptible to the combination of Kaolin + insecticide. Kaolin synergized Bifenthrin for R347, decreasing survival by 10.0%. Kaolin did not reduce feeding damage for either strain. When combined with insecticide, feeding damage was similar to insecticide alone, but the addition of Kaolin to the insecticide generally decreased survival more than the insecticide alone. In the field, the addition of Kaolin to Chlorantraniprole during application helped retain activity against this challenging pest.

Characterization of Field-Derived Drosophila suzukii (Diptera: Drosophilidae) Resistance to Pyrethroids in California Berry Production.

The spotted-wing drosophila, Drosophila suzukii (Matsumura), is a global economic pest of berry crops and stone fruit. Since management of this pest primarily relies on calendar insecticide applications, and field-derived resistance to spinosad has already been documented in California caneberry production, there is significant concern for development of resistance to other insecticides. In this study, susceptibility of D. suzukii populations collected from caneberry and strawberry fields to two pyrethroids, zeta-cypermethrin, and bifenthrin, was assessed in 2019 and 2020. Resistance to both pyrethroids were observed in flies from all sampling sites. For flies collected from caneberries in 2019, the LC50 values ranged from 4.5 to 5.2 mg liter-1 with RR50s ranging from 7.5- to 8.7-fold. Our 2020 assays showed that susceptibility of flies to the discriminating dose of zeta-cypermethrin decreased significantly as the season progressed. For flies collected from strawberries in 2020, the LC50s ranged from 19.0 to 36.1 mg liter-1 and from 30.3 to 90.7 mg liter-1 for zeta-cypermethrin and bifenthrin, respectively. The RR50 values varied from 19.0- to 36.1-fold for zeta-cypermethrin and from 15.9- to 47.7-fold for bifenthrin. This study is the first report of field-derived pyrethroid resistance in D. suzukii from two major California berry production areas. Adoption of informed insecticide resistance management practices would be essential to prolong the efficacy of products available to control D. suzukii. Future molecular work is needed to unravel the underlying genetic mechanisms conferring the observed pyrethroid resistance and to develop robust diagnostics that can inform integrated pest management of this pest.

Spatio-temporal Variation of Spinosad Susceptibility in Drosophila suzukii (Diptera: Drosophilidae), a Three-year Study in California's Monterey Bay Region.

Spinosyn insecticides are widely used in conventional berry production, and spinosad is regarded as the most effective insecticide for managing Drosophila suzukii (Matsumura) (Diptera: Drosophilidae), spotted-wing drosophila, in organic berry crops. Following the 2017 identification of spinosad resistance in caneberry fields in the Watsonville area, Santa Cruz Co., California, we conducted a study to examine the seasonal and annual susceptibility of D. suzukii over a three-year period. Adult flies were collected from two conventional and two organic caneberry fields in the Monterey Bay region, California, at 'early', 'middle', and 'late' time points during the 2018-2020 growing seasons, and their susceptibility to spinosad was assessed. Results demonstrated that spinosad susceptibility in the D. suzukii field populations generally decreased during the fruit production season (from June through November), and over consecutive seasons. LC50 values of adults from the conventional sites were determined to be as high as 228.7 mg l-1 in 2018, 665.6 mg l-1 in 2019, and 2700.8 mg l-1 in 2020. For the organically managed fields, LC50s of adults were as great as 300.0 mg l-1 in 2018, 1291.5 mg l-1 in 2019, and 2547.1 mg l-1 in 2020. Resistance ratios based on the LC50 values were as high as 10.7-, 13.2-, and 16.9-fold in 2018, 2019, and 2020, respectively. These results should serve as a caution for growers in other production areas, facilitate informed choice of insecticides used in D. suzukii management, and emphasize the need to develop effective insecticide resistance management strategies for this insect.

Differential regulation of cytochrome P450 genes associated with biosynthesis and detoxification in bifenthrin-resistant populations of navel orangewom (Amyelois transitella).

Pyrethroid resistance was first reported in 2013 for the navel orangeworm, Amyelois transitella, but the genetic underpinnings of pyrethroid resistance are unknown. We investigated the role of cytochrome P450 monooxygenases (P450s) belonging to the CYP3 and CYP4 clans using colonies derived from individuals collected in 2016 from almond orchards in two counties. One colony (ALM) originated from an almond orchard in Madera County with no reported pyrethroid resistance and the second colony (R347) originated from the same Kern County orchard where pyrethroid resistance was first reported. We used high-throughput quantitative real-time PCR (qRT-PCR) analyses of 65 P450s in the CYP3 and CYP4 clans of A. transitella to identify P450s induced by bifenthrin and associated with pyrethroid resistance. Nine P450s were constitutively overexpressed in R347 compared to ALM, including CYP6AE54 (11.7-fold), belonging to a subfamily associated with metabolic pesticide detoxification in Lepidoptera and CYP4G89 (33-fold) belonging to a subfamily associated with cuticular hydrocarbon (CHC) synthesis and resistance via reduced pesticide penetrance. Cuticular hydrocarbons analysis revealed that R347 produced twice as many total CHCs in the egg and adult stages as ALM. Topical toxicity bioassays for R347 determined that egg mortality was reduced at low bifenthrin concentrations and larval mortality was reduced at high concentrations of bifenthrin compared to ALM. Our discovery of both changes in metabolism and production of CHCs for R347 have implications for the possible decreased efficacy of other classes of insecticide used to control this insect. The threat of widespread pyrethroid resistance combined with the potential for cross-resistance to develop through the mechanism of reduced penetrance warrants developing management strategies that facilitate insecticide passage across the cuticle.

Selective Sweeps in a Nutshell: The Genomic Footprint of Rapid Insecticide Resistance Evolution in the Almond Agroecosystem.

Among the most familiar forms of human-driven evolution on ecological time scales is the rapid acquisition of resistance to pesticides by insects. Since the widespread adoption of synthetic organic insecticides in the mid-twentieth century, over 500 arthropod species have evolved resistance to at least one insecticide. Efforts to determine the genetic bases of insecticide resistance have historically focused on individual loci, but the availability of genomic tools has facilitated the screening of genome-wide characteristics. We resequenced three contemporary populations of the navel orangeworm (Amyelois transitella), the principal pest of almond orchards in California, differing in bifenthrin resistance status to examine insecticide-induced changes in the population genomic landscape of this species. We detected an exceptionally large region with virtually no polymorphisms, extending to up to 1.3 Mb in the resistant population. This selective sweep includes genes associated with pyrethroid and DDT resistance, including a cytochrome P450 gene cluster and the gene encoding the voltage-gated sodium channel para. Moreover, the sequence along the sweep is nearly identical in the genome assembled from a population founded in 1966, suggesting that the foundation for insecticide resistance may date back a half-century, when California's Central Valley experienced massive area-wide applications of DDT for pest control.

Impact of Pesticide Resistance on Toxicity and Tolerance of Hostplant Phytochemicals in Amyelois Transitella (Lepidoptera: Pyralidae).

For some polyphagous insects, adaptation to phytochemically novel plants can enhance resistance to certain pesticides, but whether pesticide resistance expands tolerance to phytochemicals has not been examined. Amyelois transitella Walker (navel orangeworm) is an important polyphagous pest of nut and fruit tree crops in California. Bifenthrin resistance, partially attributable to enhanced cytochrome P450 (P450)-mediated detoxification, has been reported in an almond-infesting population exposed to intense pesticide selection. We compared the toxicity of bifenthrin and three phytochemicals-chlorogenic acid, and the furanocoumarins xanthotoxin and bergapten-to three strains of A. transitella: pyrethroid-resistant R347 (maintained in the laboratory for ∼10 generations), fig-derived FIG (in the laboratory for ∼25 generations), and CPQ-a laboratory strain derived from almonds ∼40 years ago). Whereas both Ficus carica (fig) and Prunus dulcis (almond) contain chlorogenic acid, furanocoumarins occur only in figs. Both R347 and FIG exhibited 2-fold greater resistance to the three phytochemicals compared with CPQ; surprisingly, bifenthrin resistance was highest in FIG. Piperonyl butoxide, a P450 synergist, increased toxicity of all three phytochemicals only in CPQ, implicating alternate tolerance mechanisms in R347 and FIG. To test the ability of the strains to utilize novel hostplants directly, we compared survival on diets containing seeds of Wisteria sinensis and Prosopis pallida, two non-host Fabaceae species; survival of FIG was highest and survival of R347 was lowest. Our results suggest that, while P450-mediated pesticide resistance enhances tolerance of certain phytochemicals in this species, it is only one of multiple biochemical adaptations associated with acquiring novel hostplants.

Effect of Piperonyl Butoxide on the Toxicity of Four Classes of Insecticides to Navel Orangeworm (Amyelois transitella) (Lepidoptera: Pyralidae).

Amyelois transitella (Walker) (Lepidoptera: Pyralidae), the navel orangeworm, is a highly polyphagous economic pest of almond, pistachio, and walnut crops in California. Increasing demand for these crops and their rising economic value has resulted in substantial increases of insecticide applications to reduce damage to acceptable levels. The effects of piperonyl butoxide (PBO), a methylenedioxyphenyl compound that can act as a synergist by inhibiting cytochrome P450-mediated detoxification on insecticide metabolism by A. transitella, were examined in a series of feeding bioassays with first-instar A. transitella larvae from a laboratory strain. PBO, however, can have a variety of effects on metabolism, including inhibition of glutathione-S-transferases and esterases and induction of P450s. In our study, PBO synergized the toxicity of acetamiprid, λ-cyhalothrin, and spinosad, suggesting possible involvement of P450s in their detoxification. In contrast, PBO interacted antagonistically with the organophosphate insecticide chlorpyrifos, reducing its toxicity, an effect consistent with inhibition of P450-mediated bioactivation of this pesticide. The toxicity of the anthranilic diamide insecticide chlorantraniliprole was not altered by PBO, suggestive of little or no involvement of P450-mediated metabolism in its detoxification. Because a population of navel orangeworm in Kern County, CA, has already acquired resistance to the pyrethroid insecticide bifenthrin through enhanced P450 activity, determining the effect of adding a synergist such as PBO on detoxification of all insecticide classes registered for use in navel orangeworm management can help to develop rotation practices that may delay resistance acquisition or to implement alternative management practices where resistance is likely to evolve.

Mechanism of Resistance Acquisition and Potential Associated Fitness Costs in Amyelois transitella (Lepidoptera: Pyralidae) Exposed to Pyrethroid Insecticides.

The polyphagous navel orangeworm, Amyelois transitella (Walker) (Lepidoptera: Pyralidae), is the most destructive pest of nut crops, including almonds and pistachios, in California orchards. Management of this insect has typically been a combination of cultural controls and insecticide use, with the latter increasing substantially along with the value of these commodities. Possibly associated with increased insecticide use, resistance has been observed recently in navel orangeworm populations in Kern County, California. In studies characterizing a putatively pyrethroid-resistant strain (R347) of navel orangeworm, susceptibility to bifenthrin and ß-cyfluthrin was compared with that of an established colony of susceptible navel orangeworm. Administration of piperonyl butoxide and S,S,S-tributyl phosphorotrithioate in first-instar feeding bioassays with the pyrethroids bifenthrin and ß-cyfluthrin produced synergistic effects and demonstrated that cytochrome P450 monooxygenases and carboxylesterases contribute to resistance in this population. Resistance is therefore primarily metabolic and likely the result of overexpression of specific cytochrome P450 monooxygenases and carboxylesterase genes. Resistance was assessed by median lethal concentration (LC50) assays and maintained across nine generations in the laboratory. Life history trait comparisons between the resistant strain and susceptible strain revealed significantly lower pupal weights in resistant individuals reared on the same wheat bran-based artificial diet across six generations. Time to second instar was greater in the resistant strain than the susceptible strain, although overall development time was not significantly different between strains. Resistance was heritable and may have an associated fitness cost, which could influence the dispersal and expansion of resistant populations in nut-growing areas in California.