Identification and analysis of JHBP/TO family genes and their roles in the reproductive fitness cost of resistance in Frankliniella occidentalis (Pergande).

The juvenile hormone binding protein (JHBP) and takeout (TO) genes, mediated by the juvenile hormone (JH), play a crucial role in regulating the reproductive physiology of insects. Our previous study revealed that spinosad-resistant Frankliniella occidentalis (NIL-R) exhibited reduced fecundity and significant changes in JHBP/TO family gene expression. We hypothesized that these genes were involved in regulating the fitness costs associated with resistance. In this study, 45 JHBP/TO genes were identified in F. occidentalis, among which FoTO2 and FoTO10 were duplicates. Additionally, eight genes exhibited significant down-regulation in the NIL-R population. Two genes (FoTO6 and FoTO24) that exhibited the most significant differential expression between the spinosad-susceptible (Ivf03) and NIL-R populations were selected to investigate their roles in resistance fitness using RNA interference (RNAi). Following interference with FoTO6, FoTO24, and their combination, the expression levels of vitellogenin (Vg) were downregulated by 3%-30%, 13%-28%, and 14%-32% from the 2nd day to the 5th day, respectively; Krüppel-homolog 1 (Kr-h1) expression was down-regulated by 3%-65%, 11%-34%, and 11%-39% from the 2nd day to the 5th day, respectively; ovariole length was shortened by approximately 18%, 21%, and 24%, respectively; and the average number of eggs decreased from 407 to 260, 148, and 106, respectively. Additionally, a JH supplementation experiment on the NIL-R population revealed that the expression levels of both FoTO6, FoTO24, Vg and Kr-h1 were significantly upregulated compared with those observed in the Ivf03 population, resulting in increased fecundity. These results suggest that FoTO6 and FoTO24 are involved in JH-mediated regulation of the reproductive fitness cost of resistance to spinosad. Further, FoTO6 and FoTO24 can be considered potential target genes for applying RNAi technology in the scientific management of F. occidentalis.

Mutation (G275E) of nAChR subunit Foα6 associated with spinetoram resistance in Australian western flower thrips, Frankliniella occidentalis (Pergande).

Western flower thrips, Frankliniella occidentalis is an economically important agricultural pest. It causes damage by feeding and oviposition or indirectly by plant virus transmission. Australian F. occidentalis are resistant to many insecticides including spinosad and the related chemical spinetoram. Spinetoram resistance to F. occidentalis has been recently reported in three different Australian States, however, mechanisms conferring that resistance have not been investigated. To identify the mechanisms underlying resistance to spinetoram in F. occidentalis, we sequenced the genomic region of nicotinic acetylcholine receptor Foα6 in number of spinosad and spinetoram resistant field-populations. We found that a single nucleotide substitution (G to A) in exon 9 of the α6 subunit was present in resistant strains (G275E) and absent from susceptible. By examining field populations we consider the G275E mutation is the major cause of resistance to spinetoram in Australian F. occidentalis. We developed a real-time PCR diagnostic assay to quickly identify resistant alleles in field-populations. The method was used to test spinetoram resistant F. occidentalis collected from Australian cotton during the 2018-2019. Results show thrips tested carried the G275E mutation and the resistance allele was unusually widely distributed. The wide distribution of G275E mutation was not expected because spinetoram is not extensively used in Australian cotton. We speculate resistance may relate to extensive chemical use in crops nearby such as horticulture where thrips are often targeted for control. Our molecular diagnostic assay can provide timely and precise resistance frequency information that can support sustainable chemical use including spinetoram based IPM.

Constitutive and Inducible Resistance to Thrips Do Not Correlate With Differences in Trichome Density or Enzymatic-Related Defenses in Chrysanthemum.

Western flower thrips (WFT), Frankliniella occidentalis, is a serious insect pest of Chrysanthemum [Chrysanthemum × morifolium Ramat. (Asteraceae)]. Here we have investigated whether genotypic variation in constitutive and inducible resistance to WFT correlates with phenotypic differences in leaf trichome density and the activity of the defense-related enzyme polyphenol oxidase (PPO) in chrysanthemum. Non-glandular and glandular leaf trichome densities significantly varied among ninety-five chrysanthemum cultivars. Additional analyses in a subset of these cultivars, differing in leaf trichome density, revealed significant variation in PPO activities and resistance to WFT as well. Constitutive levels of trichome densities and PPO activity, however, did not correlate with chrysanthemum resistance to WFT. Further tests showed that exogenous application of the phytohormone jasmonic acid (JA) increased non-glandular trichome densities, PPO activity and chrysanthemum resistance to WFT, and that these effects were cultivar dependent. In addition, no tradeoff between constitutive and inducible resistance to WFT was observed. JA-mediated induction of WFT resistance, however, did not correlate with changes in leaf trichome densities nor PPO activity levels. Taken together, our results suggest that chrysanthemum can display both high levels of constitutive and inducible resistance to WFT, and that leaf trichome density and PPO activity may not play a relevant role in chrysanthemum defenses against WFT.

Metabolomics of Thrips Resistance in Pepper (Capsicum spp.) Reveals Monomer and Dimer Acyclic Diterpene Glycosides as Potential Chemical Defenses.

The development of pesticide resistance in insects and recent bans on pesticides call for the identification of natural sources of resistance in crops. Here, we used natural variation in pepper (Capsicum spp.) resistance combined with an untargeted metabolomics approach to detect secondary metabolites related to thrips (Frankliniella occidentalis) resistance. Using leaf disc choice assays, we tested 11 Capsicum accessions of C. annuum and C. chinense in both vegetative and flowering stages for thrips resistance. Metabolites in the leaves of these 11 accessions were analyzed using LC-MS based untargeted metabolomics. The choice assays showed significant differences among the accessions in thrips feeding damage. The level of resistance depended on plant developmental stage. Metabolomics analyses showed differences in metabolomes among the Capsicum species and plant developmental stages. Moreover, metabolomic profiles of resistant and susceptible accessions differed. Monomer and dimer acyclic diterpene glycosides (capsianosides) were pinpointed as metabolites that were related to thrips resistance. Sucrose and malonylated flavone glycosides were related to susceptibility. To our knowledge, this is the first time that dimer capsianosides of pepper have been linked to insect resistance. Our results show the potential of untargeted metabolomics as a tool for discovering metabolites that are important in plant - insect interactions.

Phytochemical Background Mediates Effects of Pyrrolizidine Alkaloids on Western Flower Thrips.

Plants produce an extremely diverse array of metabolites that mediate many aspects of plant-environment interactions. In the context of plant-herbivore interactions, it is as yet poorly understood how natural backgrounds shape the bioactivity of individual metabolites. We tested the effects of a methanol extract of Jacobaea plants and five fractions derived from this extract, on survival of western flower thrips (WFT). When added to an artificial diet, the five fractions all resulted in a higher WFT survival rate than the methanol extract. In addition, their expected combined effect on survival, assuming no interaction between them, was lower than that of the methanol extract. The bioactivity was restored when the fractions were combined again in their original proportion. These results strongly suggest synergistic interactions among the fractions on WFT survival rates. We then tested the effects of two pyrrolizidine alkaloids (PAs), free base retrorsine and retrorsine N-oxide, alone and in combination with the five shoot fractions on WFT survival. The magnitude of the effects of the two PAs depended on the fraction to which they were added. In general, free base retrorsine was more potent than retrorsine N-oxide, but this was contingent on the fraction to which these compounds were added. Our results support the commonly held, though seldom tested, notion that the efficacy of plant metabolites with respect to plant defence is dependent on their phytochemical background. It also shows that the assessment of bioactivity cannot be decoupled from the natural chemical background in which these metabolites occur.

T929I and K1774N mutation pair and M918L single mutation identified in the voltage-gated sodium channel gene of pyrethroid-resistant Thrips tabaci (Thysanoptera: Thripidae) in Japan.

Pyrethroid-resistance in onion thrips, Thrips tabaci, has been reported in many countries including Japan. Identifying factors of the resistance is important to correctly monitoring the resistance in field populations. To identify pyrethroid-resistance related genes in T. tabaci in Japan, we performed RNA-Seq analysis of seven T. tabaci strains including two pyrethroid-resistant and five pyrethroid-susceptible strains. We identified a pair of single point mutations, T929I and K1774N, introducing two amino acid mutations, in the voltage-gated sodium channel gene, a pyrethroid target gene, in the two resistant strains. The K1774N is a newly identified mutation located in the fourth repeat domain of the sodium channel. Genotyping analysis of field-collected populations showed that most of the T. tabaci individuals in resistant populations carried the mutation pair, indicating that the mutation pair is closely associated with pyrethroid-resistance in Japan. Another resistance-related mutation, M918L, was also identified in part of the resistant populations. Most of the individuals with the mutation pair were arrhenotokous while all individuals with the M918L single mutation were thelytokous. The result of differentially expressed gene analysis revealed a small number of up-regulated detoxification genes in each resistant strain which might be involved in resistance to pyrethroid. However, no up-regulated detoxification genes common to the two resistant strains were detected. Our results indicate that the mutation pair in the sodium channel gene is the most important target for monitoring pyrethroid-resistance in T. tabaci, and that pyrethroid-resistant arrhenotokous individuals with the mutation pair are likely to be widely distributed in Japan.

Selection of lethal genes for ingestion RNA interference against western flower thrips, Frankliniella occidentalis, via leaf disc-mediated dsRNA delivery.

The western flower thrips, Frankliniella occidentalis, is a major pest that damages a wide variety of crops and vegetables. Following extensive use of insecticides, it has developed high levels of resistance to almost all groups of insecticides due to its high reproduction rate and short generation time. Therefore, an alternative pest control strategy, such as RNA interference (RNAi)-based control, is essential. To establish an ingestion RNAi-based control, a total of 57 genes involved in various biological processes were selected, and their double-stranded RNAs (dsRNA) were delivered to an insecticide-susceptible strain of F. occidentalis via the leaf disc-feeding method using a bioassay chamber optimized by 3D printing. The mortality of dsRNA-ingested thrips was examined every 24 h until 120 h post-treatment. Of the 57 genes screened, dsRNAs of the Toll-like receptor 6, apolipophorin, coatomer protein subunit epsilon and sorting and assembly machinery component were most lethal when ingested by thrips. The dsRNA-fed thrips showed substantially reduced transcription levels of target genes, demonstrating that the observed mortality was likely due to RNAi. When these genes were tested for ingestion RNAi against an insecticide-resistant strain of F. occidentalis, bioassay results were similar. In conclusion, this study provides the first evidence that ingestion RNAi can be lethal to F. occidentalis, a mesophyll sucking pest, and further suggests that transgenic plants expressing hairpin RNA of these essential genes can be employed to control insecticide-resistant thrips.

α-Ionone, an Apocarotenoid, Induces Plant Resistance to Western Flower Thrips, Frankliniella occidentalis, Independently of Jasmonic Acid.

Apocarotenoids, such as ß-cyclocitral, α-ionone, ß-ionone, and loliolide, are derived from carotenes via chemical or enzymatic processes. Recent studies revealed that ß-cyclocitral and loliolide play an important role in various aspects of plant physiology, such as stress responses, plant growth, and herbivore resistance. However, information on the physiological role of α-ionone is limited. We herein investigated the effects of α-ionone on plant protection against herbivore attacks. The pretreatment of whole tomato (Solanum lycopersicum) plants with α-ionone vapor decreased the survival rate of western flower thrips (Frankliniella occidentalis) without exhibiting insecticidal activity. Exogenous α-ionone enhanced the expression of defense-related genes, such as basic ß-1,3-glucanase and basic chitinase genes, in tomato leaves, but not that of jasmonic acid (JA)- or loliolide-responsive genes. The pretreatment with α-ionone markedly decreased egg deposition by western flower thrips in the JA-insensitive Arabidopsis (Arabidopsis thaliana) mutant coi1-1. We also found that common cutworm (Spodoptera litura) larvae fed on α-ionone-treated tomato plants exhibited a reduction in weight. These results suggest that α-ionone induces plant resistance to western flower thrips through a different mode of action from that of JA and loliolide.

Molecular cloning and characterization of GABA receptor and GluCl subunits in the western flower thrips, Frankliniella occidentalis.

To understand the role of target site insensitivity in abamectin resistance in the western flower thrips (WFT), Frankliniella occidentalis (Pergande), cDNAs encoding gamma-aminobutyric acid receptor subunit (FoRdl) and glutamate-gated chloride channel (FoGluCl) were cloned from WFT, and both single nucleotide polymorphisms (SNPs) and mRNA expression levels of FoRdl and FoGluCl were detected in a susceptible strain (ABA-S) and a laboratory selected strain (ABA-R) displaying 45.5-fold resistance to abamectin. Multiple cDNA sequence alignment revealed three alternative splicing variants of FoRdl and two alternative splicing variants of FoGluCl generated by alternative splicing of exon 3. While sequence comparison of FoRdl and FoGluCl in ABA-S and ABA-R strains identified no resistance-associated mutations, the expression level of FoGluCl in ABA-R strain was 2.63-fold higher than that in ABA-S strain. Thus, our preliminary results provide the evidence that the increased mRNA expression of FoGluCl could be an important factor in FoGluCl-mediated target site insensitivity in WFT.

Male Pheromones Influence the Mating Behavior of Echinothrips americanus.

Two dibasic esters, the dimethyl ester of hexanedioic acid (dimethyl adipate, DBE-6) and the dimethyl ester of pentanedioic acid (dimethyl glutarate, DBE-5) were found in head-thorax extracts of male Echinothrips americanus. DBE-5 induced abdomen wagging and raising in males and females, which is typically exhibited when encountering a male. DBE-6 was avoided by males and was detected on mated, but not on virgin, females. Both substances applied to virgin females lead to females being ignored by males. The role of both substances is discussed with regard to the male mating system.

Protoilludane, Illudane, Illudalane, and Norilludane Sesquiterpenoids from Granulobasidium vellereum.

Two new and seven known sesquiterpene compounds were isolated from an agar plate culture of Granulobasidium vellereum, isolated from a log of Ulmus sp. The two new structures were elucidated with spectroscopic methods as an illudalane derivative, granulolactone (1), and a 15-norilludane, granulodione (9). The acaricidal and insecticidal activities of the isolated compounds were examined in vitro against two major horticultural pests, the two-spotted spider mite Tetranychus urticae and the glasshouse thrips Heliothrips haemorrhoidalis, respectively.

Incidence and characterization of resistance to pyrethroid and organophosphorus insecticides in Thrips tabaci (Thysanoptera: Thripidae) in onion fields in Isfahan, Iran.

Onion thrips, Thrips tabaci Lindeman, is the main pest of onion-growing fields in Isfahan and is mainly controlled by frequently spraying several insecticides. To investigate the resistance status and mechanisms, the susceptibility of ten field populations collected from Isfahan onion-growing regions were tested to several currently used pesticides. Resistance to the tested insecticides was observed in most populations when compared with the susceptible reference population. Enhanced detoxification, implicated by the use of inhibitors of major metabolic detoxification enzymes, was observed in the populations resistant to profenofos and chlorpyrifos. In the deltamethrin resistant populations, the amino acid substitution T929I was detected in the voltage gated sodium channel, which is known to confer pyrethroid resistance. These data are a first step towards more efficient resistance management tactics through early detection of resistant onion thrips in Iran.

Effect of Spinosad Resistance on Transmission of Tomato Spotted Wilt Virus by the Western Flower Thrips (Thysanoptera: Thripidae).

Tomato spotted wilt virus (TSWV) is transmitted by Frankliniella occidentalis (Pergande) in a persistent-propagative manner. We previously observed significant results in terms of feeding behavior of spinosad-susceptible (Ivf03) and -resistant (Spin-R) strains of F. occidentalis using electrical penetration graph. TSWV transmission by the two strains was compared in the present study. The results showed that the titer of TSWV-N RNA (a part of S RNA of TSWV and encoding the nucleocapsid protein) in Ivf03 and Spin-R strains was not significantly different after a 48-h inoculation access period. The TSWV transmission rate did not significantly differ between the two strains and was 51.0% for Ivf03 and 44.4% for Spin-R. The virus transmission rate was significantly higher for males than females of both strains. The virus transmission rate for males and females of Ivf03 was 68.1 and 33.8%, respectively; however, in case of Spin-R, it was 60 and 28.8% for males and females, respectively. Additionally, number of probes and duration of probes were generally greater for viruliferous females of Ivf03 than for viruliferous females of Spin-R but the total number and duration of noningestion probes did not significantly differ between males of the two strains. The latter finding behavior may help explain the similar transmission rates for the susceptible and resistant strains.

Demonstration of an adaptive response to preconditioning Frankliniella occidentalis (Pergande) to sublethal doses of spinosad: a hormetic-dose response.

Sublethal doses of some insecticides have been reported to either stimulate or reduce the survival and fecundity of insects. Many sublethal-effect studies have been conducted after exposure of only one generation to sublethal insecticides, and there is little information about the sublethal effects on insects after long-term exposure to sublethal insecticides. In this study, changes in biological characteristics were investigated in spinosad-susceptible (Spin-S) and sublethal-spinosad-treated (Spin-Sub) strains of Frankliniella occidentalis (Pergande) after exposure to their corresponding sublethal concentrations of spinosad. The results showed that for the Spin-S strain, the LC10 concentration of spinosad slightly affected the biotic fitness both in parents and offspring of F. occidentalis. The LC25 concentration of spinosad prolonged the development time, reduced the fecundity, and significantly reduced the intrinsic rate of increase, the net reproductive rate and the finite rate of increase in the Spin-S strain. However, the negative effects were not as pronounced in the offspring (F1 generation) as in the parent generation. For the Spin-Sub strain, the LC10 and LC25 concentrations of spinosad had little negative effect on the development and fecundity, and no significant difference was found between the effects of the LC10 and LC25 treatments on the Spin-Sub strain. The Spin-Sub strain exhibited a shorter developmental time, and larger intrinsic rates of increase and net reproductive rates, compared with the corresponding treatments of the Spin-S strain. These findings combined with our previous studies suggest that the biotic fitness increased in the Spin-Sub strain and the strain became more adaptable to sublethal doses of spinosad, compared with the Spin-S strain. Physiological and biochemical adaptation may contribute to these changes after long treatment times at sublethal doses.

Sublethal and Transgenerational Effects of Abamectin on the Biological Performance of the Predatory Thrips Scolothrips longicornis (Thysanoptera: Thripidae).

Determination of sublethal and transgenerational effects of pesticides on natural enemies is necessary for successful implementation of biocontrol in integrated pest management programs. In this study, these effects of abamectin on the predatory thrips Scolothrips longicornis Priesner fed on Tetranychus urticae Koch were estimated under laboratory conditions in which adult predators were exposed to pesticide residues on bean leaves. The estimated values of LC50 for female and male predators were 0.091 and 0.067 µg a.i./ml, respectively. Low-lethal concentrations (LC10, LC20 and LC30) of abamectin severely affected fecundity and longevity of treated females of S. longicornis. In addition, transgenerational effects on reproductive and life table parameters of the subsequent generation were observed. The results from this research can be used to develop guidelines for the use of abamectin to minimize the impact on S. longicornis.

Proteomic Analysis Reveals Resistance Mechanism Against Chlorpyrifos in Frankliniella occidentalis (Thysanoptera: Thripidae).

The western flower thrips is an economically important worldwide pest of many crops, and chlorpyrifos has been used to control western flower thrips for many years. To develop a better resistance-management strategy, a chlorpyrifos-resistant strain of western flower thrips (WFT-chl) was selected in the laboratory. More than 39-fold resistance was achieved after selected by chlorpyrifos for 19 generations in comparison with the susceptible strain (WFT-S). Proteome of western flower thrips (WFT-S and WFT-chl) was investigated using a quantitative proteomics approach with isobaric tag for relative and absolute quantification technique and liquid chromatography-tandem mass spectrometry technologies. According to the functional analysis, 773 proteins identified were grouped into 10 categories of molecular functions and 706 proteins were presented in 213 kinds of pathways. Comparing the proteome of WFT-chl with that of WFT-S, a total of eight proteins were found up-regulated and three down-regulated. The results from functional annotation and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses indicated that the differentially expressed protein functions in binding, catalyzing, transporting, and enzyme regulation were most important in resistance development. A list of proteins functioning in biological processes of metabolism, biological regulation, and response to stimulus was found in WFT-chl, suggesting that they are possibly the major components of the resistance mechanism to chlorpyrifos in western flower thrips. Notably, several novel potential resistance-related proteins were identified such as ribosomal protein, Vg (vitellogenin), and MACT (muscle actin), which can be used to improve our understanding of the resistance mechanisms in western flower thrips. This study provided the first comprehensive view of the complicated resistance mechanism employed by WFT-S and WFT-chl through the isobaric tag for relative and absolute quantification coupled with liquid chromatography-tandem mass spectrometry technologies.

Insecticidal quinoline and isoquinoline isoxazolines.

A series of quinoline and isoquinoline isoxazolines have been designed as pesticides for crop protection. Herein we reported the chemical synthesis, biological activity and structure-activity relationships. The isoquinoline derivative, such as 3i, is discovered as potent new class of isoxazoline insecticide which is competitive with commercial insecticide Indoxacarb.

6-Pentyl-2H-pyran-2-one: a potent peach-derived kairomone for New Zealand flower thrips, Thrips obscuratus.

New Zealand flower thrips, Thrips obscuratus (Thysanoptera: Thripidae), are attracted to ripening fruits, especially peaches. Volatiles from unripe and ripe peach fruits were collected and analyzed by gas chromatography/mass spectrometry. Six lactones were found only in ripe peach volatiles: γ-heptalactone, γ-octalactone, γ-nonalactone, 6-pentyl-2H-pyran-2-one, γ-decalactone, and δ-decalactone. When these compounds were tested individually in field-trapping experiments, three of them (γ-octalactone, γ-nonalactone, and 6-pentyl-2H-pyran-2-one) attracted New Zealand flower thrips. In another field-trapping experiment, aimed at testing various combinations of the three active compounds, no synergistic effects were found among all combinations tested; no combination caught more thrips than 6-pentyl-2H-pyran-2-one, alone. A further field-trapping experiment was conducted to determine the dose (10, 100, and 500 mg) of 6-pentyl-2H-pyran-2-one that gave the greatest catch of T. obscuratus, while also comparing it against another attractant, ethyl nicotinate, for T. obscuratus. The greatest catches in traps baited with either attractant were at loadings of 500 mg. At both 10 and 500 mg, traps baited with 6-pentyl-2H-pyran-2-one caught more T. obscuratus than those baited with the same amounts of ethyl nicotinate. 6-Pentyl-2H-pyran-2-one is a potent attractant for New Zealand flower thrips and, therefore, could be used for monitoring and control of New Zealand flower thrips. Work is underway developing monitoring and control options utilizing 6-pentyl-2H-pyran-2-one for this important pest.

The α6 nicotinic acetylcholine receptor subunit of Frankliniella occidentalis is not involved in resistance to spinosad.

Insects evolve resistance which constrains the sustainable use of insecticides. Spinosyns, a class of environmentally-friendly macrolide insecticides, is not an exception. The mode of inheritance and the mechanisms of resistance to spinosad (the most common spinosyn insecticide) in Frankliniella occidentalis (Western flower thrips, WFT) were investigated in this study. Resistance (170,000-fold) was autosomal and completely recessive. Recent studies showed that deletion of the nicotinic acetylcholine receptor α6 subunit gene resulted in strains of Drosophila melanogaster, Plutella xylostella and Bactrocera dorsalis that are resistant to spinosad, indicating that nAChRα6 subunit maybe important for the toxic action of this insecticide. Conversely, a G275E mutation of this subunit in F. occidentalis was recently proposed as the mechanism of resistance to spinosad. We cloned and characterized nAChRα6 from three susceptible and two spinosad resistant strains from China and the USA. The Foα6 cDNA is 1873bp and the open reading frame is 1458bp which encodes 485 amino acid residues with a predicted molecular weight of 53.5-kDa, the 5' and 3' UTRs are 121 and 294bp, respectively. There was no difference in the cDNA sequence between the resistant and susceptible thrips, suggesting the G275E mutation does not confer resistance in these populations. Ten isoforms of Foα6, arising from alternative splicing, were isolated and did not differ between the spinosad-susceptible and resistant strains. Quantitative real time PCR analysis showed Foα6 was highly expressed in the first instar larva, pupa and adult, and the expression levels were 3.67, 2.47, 1.38 times that of the second instar larva. The expression level was not significantly different between the susceptible and resistant strains. These results indicate that Foα6 is not involved in resistance to spinosad in F. occidentalis from China and the USA.

Spinosad resistance, esterase isoenzymes and temporal synergism in Frankliniella occidentalis (Pergande) in Australia.

Spinosad has been widely used in Australia to control western flower thrips Frankliniella occidentalis (Pergande) but spinosad usefulness is now compromised by resistance. Here we studied a highly spinosad resistant strain of F. occidentalis to explore if esterases had a role in spinosad resistance. Enhanced esterase activity in pressured spinosad-resistant F. occidentalis was confirmed via PAGE electrophoresis and estimated to be approximately three times higher than that in a susceptible strain. Spinosad-esterase inhibition data in the resistant strain, showed a concentration effect with significant esterase-spinosad binding occurring at spinosad concentrations from 6.2× 10(-7) to 1.5× 10(-5) M. Similarly, a spinosad-piperonyl butoxide (PBO) inhibition curve showed a concentration effect, with significant esterase-PBO binding occurring in the resistant strain at PBO concentrations between 3.3× 10(-5) M and 8.4× 10(-4) M. No binding of esterase to spinosad or PBO occurred in the susceptible strain. Results of bioassays in which spinosad resistant F. occidentalis were sprayed with a 4h delayed release formulation of cyclodextrin-complexed spinosad with immediately available PBO demonstrated that spinosad resistance was significantly reduced from 577 to 72-fold. With further development the PBO synergism of spinosad using a delayed release formulation, similar to that used here, may provide effective control for spinosad resistant F. occidentalis. Temporal synergism of spinosad may prove to be effective tactic for the control of spinosad resistant F. occidentalis where the main resistance mechanism involved has been confirmed to be esterase based.