Indoxacarb resistance in Iranian populations of Tuta absoluta (Lepidoptera: Gelechiidae): Cross-resistance, biochemical and molecular mechanisms.

Tuta absoluta (Meyrick) is an invasive tomato pest that occurs worldwide, including Iran. This study investigates the occurrence of resistance to indoxacarb, an oxadiazine insecticide, and the underlying mechanisms in Iranian populations of T. absoluta. Bioassays were performed on second-instar larvae using indoxacarb alone and in combination with three synergists: Piperonyl butoxide (PBO), diethyl maleate (DEM), and triphenyl phosphate (TPP). The activities of the main detoxification enzymes, including glutathione S-transferases (GST), general carboxylesterases (CarEs), and P450 monooxygenases (P450s), were evaluated. In addition, the presence of known amino acid substitutions in the IV segment 6 domain of the T. absoluta sodium channel was investigated. The results showed that resistance rates to indoxacarb in Iranian populations ranged from 2.37- to 14.45-fold. However, pretreatment with synergists did not significantly increase the toxicity of indoxacarb. Enzyme assays showed that Ardabil (Ar) and Kerman (Kr) populations had the highest CarEs activity, while Ar population showed the highest P450 activity. However, the observed increases in enzyme activities were <2-fold. Two indoxacarb resistance mutations, F1845Y and V1848I, were detected. Apart from a significant and positive correlation between LC50 values of indoxacarb and thiocyclam hydrogen oxalate, no cross-resistance between indoxacarb and other insecticides was detected. Overall, these results suggest that populations of T. absoluta in Iran have developed resistance to indoxacarb, primarily through changes at the target site.

Cardinium symbionts are pervasive in Iranian populations of the spider mite Panonychus ulmi despite inducing an infection cost and no demonstrable reproductive phenotypes when Wolbachia is a symbiotic partner.

Maternally transmitted symbionts such as Cardinium and Wolbachia are widespread in arthropods. Both Cardinium and Wolbachia can cause cytoplasmic incompatibility, a reproductive phenotype that interferes with the development of uninfected eggs that are fertilized by infected sperm. In haplodiploid hosts, these symbionts can also distort sex allocation to facilitate their spread through host populations. Without other fitness effects, symbionts that induce strong reproductive phenotypes tend to spread to high and stable infection frequencies, whereas variants that induce weak reproductive phenotypes are typically associated with intermediate and variable frequencies. To study the spread of Cardinium in a haplodiploid host, we sampled Iranian populations of the economically important spider mite Panonychus ulmi in apple orchards. Within several field populations, we also studied the Wolbachia infection frequencies. All P. ulmi field populations carried a Cardinium infection and exhibited high infection frequencies. In contrast, Wolbachia frequency ranged between ca. 10% and ca. 70% and was only found in co-infected mites. To test whether Cardinium induce reproductive phenotypes in P. ulmi, a Cardinium-cured derived line was generated by antibiotic treatment from a co-infected field population. Genetic crosses indicated that Cardinium do not induce demonstrable levels of cytoplasmic incompatibility and sex allocation distortion in co-infected P. ulmi. However, Cardinium infection was associated with a longer developmental time and reduced total fecundity for co-infected females. We hypothesize that Cardinium spread through P. ulmi populations via uncharacterized fitness effects and that co-infection with Wolbachia might impact these drive mechanisms.

Efficiency of plant-based acaricide gels compared to fluvalinate-impregnated strips for control of Varroa destructor in honey bee colonies.

The Varroa mite is one of the most important pests of honey bee colonies. This study compares the efficiency of thymol-based acaricides with the tau-fluvalinate-based commercial treatment to control this mite species. Two experiments were conducted: (1) in the Fall, thymol-based gel was compared with a widely used acaricide, fluvalinate-impregnated strips (Apistan®), and (2) in the Spring, gels containing thymol-only, thymol + menthol + eucalyptus essential oil, and thymol + thyme essential oil were compared with fluvalinate-impregnated strips. In the first experiment, both treatments reduced Varroa mite infestation levels significantly after 70 days of their application compared to the control treatment. The results of the second experiment showed that thymol + thyme essential oil gel, thymol + menthol + eucalyptus essential oil gel, and Apistan strips had always higher efficiency on decreasing mite infestation levels of the selected colonies compared to the control treatment and thymol-only gel during their 7-week application. The results of this study demonstrate that plant-based acaricides for controlling Varroa mites in honey bee colonies have similar efficiency compared to the chemical control methods. Therefore, it is better to use these selected acaricides to reduce mite resistance to chemical control treatments in honey bee colonies.

Rhizoglyphus robini, a pest mite of saffron, is unable to resist extracellular ice formation.

The saffron mite, Rhizoglyphus robini Claparède (Acari, Astigmata: Acaridae), is one of the most important pests of saffron-producing regions in Iran. It causes yellowing and decreases saffron growth, and finally it destroys the bulbs. In this research, the cold tolerance and supercooling point (SCP) of the saffron mite were measured in three populations and two temperature regimes. Our results showed that the mean SCP of the saffron mite was approximately -14.6 °C without significant difference among the populations. On the contrary, acclimation of the mites significantly decreased their SCP to a mean of approximately -16.5 °C. Exposure of the mites for 24 h to 0 and -2.5 °C had no significant effect on the survival of the mites but when the mites were exposed to -5.0 °C for 24 h, survival of the three populations reached the lowest level of roughly 60%. By 24-h exposure to -7.5 °C, survival of the mites was almost negligible. As a large proportion of mortality was observed above the SCP, and LT50 > SCP, it can be inferred that the saffron mite is likely a chill-susceptible species. This suggests that the saffron mite lacks the ability to withstand extracellular ice formation. Overall, the results of the current study suggest no significant physiological differences between populations of the saffron mite.

Hormetic effects of neonicotinoid insecticides on Rhizoglyphus robini (Acari: Acaridae).

The stimulation of biological processes by sublethal doses of insecticides or other stressors is known as hormesis. Here, we have evaluated whether exposure to field-relevant or low concentrations of neonicotinoids induce changes in the reproductive capacity of the bulb mite Rhizoglyphus robini (Acari: Acaridae). Among the tested neonicotinoids imidacloprid, thiamethoxam, and dinotefuran, the highest hormetic effect on the reproduction of R. robini occurred 24 h after the 48 h exposure period to imidacloprid at concentrations of 70 and 140 mg a.i./L. Despite the stimulating effects of imidacloprid on mite reproduction, no significant differences were observed in the offspring (F1) for biological aspects including egg hatch rate, embryonic period and sex ratio, while an increase was found in the duration of development time from egg to adult. Evaluation of the detoxification enzyme activities of treated adults showed that the highest activity of carboxyl/cholinesterases, cytochrome P450s, and glutathione S-transferases was obtained when exposed to 70, 140 and 70 mg a.i./L imidacloprid, immediately after the exposure period, respectively. Also, an increase in the activity of the antioxidant enzyme catalase was observed compared to that of the control. After imidacloprid pretreatment (140 mg a.i./L), the tolerance of adult mites to diazinon was increased about two-fold. This study shows that exposure to imidacloprid can induce hormetic effects on R. robini and could severely complicate its control due to a higher reproduction, enhanced detoxification enzyme activities, and increased tolerance against other pesticides.

CYP6DW3 Metabolizes Imidacloprid to Imidacloprid-urea in Whitefly (Bemisia tabaci).

Bemisia tabaci has developed high resistance to many insecticides and causes substantial agricultural and economic losses annually. The insecticide resistance of whitefly has been widely reported in previous studies; however, the underlying mechanism remains little known. In this study, we cloned two P450 genes: CYP6DW3 and CYP6DW5v1; these genes were markedly overexpressed in imidacloprid-resistant whitefly populations compared with susceptible populations, and knockdown of these genes decreased the imidacloprid resistance of whitefly. Moreover, heterologous expression of whitefly P450 genes in SF9 cells and metabolic studies showed that the CYP6DW3 protein could metabolize 14.11% imidacloprid and produced imidacloprid-urea in vitro. Collectively, the expression levels of CYP6DW3 and CYP6DW5v1 are positively correlated with imidacloprid resistance in B. tabaci. Our study further reveals that cytochrome P450 enzymes affect the physiological activities related to resistance in insects, which helps scholars more deeply understand the resistance mechanism, and contributes to the development of integrated pest management framework.

Biochemical and insecticidal effects of plant essential oils on insecticide resistant and susceptible populations of Musca domestica L. point to a potential cross-resistance risk.

Essential oils (EOs) can provide important alternatives to chemical insecticides in the control of pests. In this study, 12 EOs of native plant species from Iran were evaluated for their adulticidal activity against the house fly. In addition, we examined the insecticidal activity of Zataria multiflora and Rosmarinus officinalis EOs on adult female house flies from pyrethroid and organophosphate resistant and susceptible populations, using both fumigant and topical bioassays. The involvement of detoxification enzymes in susceptibility was investigated with synergism experiments in vivo, while the inhibitory effects of R. officinalis and Zataria multiflora EOs on the activities of cytochrome P450-dependent monooxygenases (P450s), carboxylesterases (CarEs) and glutathione S-transferases (GSTs) were determined by enzymatic inhibition assays in vitro. The EOs of Z. multiflora, Mentha pulegium, R. officinalis and Thymus vulgaris were the most effective against adults in contact topical assays, while oils extracted from Eucalyptus cinerea, Z. multiflora, Citrus sinensis, R. officinalis, Pinus eldarica and Lavandula angustifolia where the most effective in fumigant assays. Rosmarinus officinalis and Z. multiflora EOs were selected for further investigation and showed higher toxicity against a susceptible population, compared to two insecticide-resistant populations. Correlation analysis suggested cross-resistance between these EOs and pyrethroids in the resistant populations. The toxicity of both EOs on the resistant populations was synergized by three detoxification enzyme inhibitors. Further, in vitro inhibition studies showed that R. officinalis and Z. multiflora EOs more effectively inhibited the activities of the detoxification enzymes from flies of the susceptible population compared to those of the pyrethroid resistant populations. Synergistic and enzymatic assays further revealed that increased activities of P450s, GSTs, and CarEs are possibly involved in the cross-resistance between EOs and pyrethroids. Investigating the molecular mechanisms of P450s, GSTs, and CarEs in the resistance to EOs should be subject to further studies.

Fenpyroximate resistance in Iranian populations of the European red mite Panonychus ulmi (Acari: Tetranychidae).

The European red mite, Panonychus ulmi (Koch), is one of the most important apple orchard pests worldwide. Fenpyroximate, a mitochondrial electron transport inhibitor of complex I (METI-I), is a commonly used acaricide to control this pest. In this study, we determined fenpyroximate resistance levels for 11 P. ulmi populations from Iran and a spirodiclofen-resistant strain from Germany (PSR-TK). The LC50 values ranged between 121.8 and 5713.9 mg a.i. L-1 and the highest resistance ratio (RR) was 47-fold for the Padena population. PBO, TPP and DEM synergist ratios (SRs) were the highest for the PSR-TK (SR = 6.7), Shahin Dej (SR = 6.1) and Semirom3 (SR = 3.6) populations, respectively. In vitro enzyme activity measurements also showed that there was a higher glutathione S-transferases (GSTs) activity in the PSR-TK and Shahin Dej population compared to the most susceptible populations, whereas the esterase and P450 monooxygenase activity were not significantly higher in the resistant populations. Last, we screened all populations for the presence of two mutations previously associated with METI-I resistance in spider mites but none of these mutations could be detected. To conclude, moderate to high levels of fenpyroximate resistance were observed in P. ulmi populations from Iran, with increased detoxification most likely underlying fenpyroximate resistance.

A novel, simple, and stable mesoporous silica nanoparticle-based gene transformation approach in Solanum lycopersicum.

In this study, a novel and stable gene transformation system was developed under control of Maize Proteinase Inhibitor (MPI) as an inducible promoter using the Mesoporous Silica Nanoparticles (MSNs). The functionalized MSNs with a proper particle size were synthesized and attached to a recombinant construct (pDNA) containing cryIAb gene under the control of MPI promoter (pPZP122:MPI:cryIAb:MSN [pDNA: MSN]) following transformation of tomato plants through injection of the pDNA: MSN complex into tomato red fruit at early ripening stage and then, putative transgenic seeds were collected. As an initial selection, gentamicin-resistant seedlings of T1 (24.24%) and T2 (61.37%) plants were identified. The transgene integration and expression were confirmed through the PCR, RT-PCR, and western blot approaches in the selected seedlings. PCR analysis showed that transformation frequency was equal to 10.71% in T1 plants. Semi-quantitative RT-PCR analysis confirmed the transcript expression of cryIAb in all the T1 and T2 PCR-positive plants. Western blot analysis confirmed the existence of CryIAb protein in the leaves of T2 putative transgenic plants. Accordingly, the results demonstrated that the transgene has more likely integrated into the tomato genome through homologous recombination. Bioassay was carried out for further assessment of the plant responses to Tuta absoluta resulting in an enhanced tolerance of the plant. In conclusion, the MSN-mediated stable transformation system under the MPI as an inducible promoter can be used as a suitable alternative for conventional genetic transformation methods due to its biodegradability, biocompatibility, cost and time-effectiveness, and positive effect on the plant defense against pathogens and pests.

Molecular and genetic analysis of resistance to METI-I acaricides in Iranian populations of the citrus red mite Panonychus citri.

The citrus red mite, Panonychus citri, is a major pest on citrus all around the world. Mitochondrial Electron Transport Inhibitors of complex I (METI-I) acaricides such as fenpyroximate have been used extensively to control P. citri populations, which resulted in multiple reports of METI-I resistant populations in the field. In this study, biochemical and molecular mechanisms of fenpyroximate resistance were investigated in P. citri. Seven populations were collected from Northern provinces of Iran. Resistance ratios were determined and reached up to 75-fold in comparison to a fenpyroximate susceptible population. Cross-resistance to two additional METI-I acaricides, pyridaben and tebufenpyrad, was detected. PBO synergism experiments, in vivo enzyme assays and gene expression analysis suggest a minor involvement of cytochrome P450 monooxygenases in fenpyroximate resistance, which is in contrast with many reported cases for the closely related Tetranychus urticae. Next, we determined the frequency of a well-known mutation in the target-site of METI-Is, the PSST subunit, associated with METI-I resistance. Indeed, the H92R substitution was detected in a highly fenpyroximate resistant P. citri population. Additionally, a new amino acid substitution at a conserved site in the PSST subunit was detected, A94V, with higher allele frequencies in a moderately resistant population. Marker-assisted back-crossing in a susceptible background confirmed the potential involvement of the newly discovered A94V mutation in fenpyroximate resistance. However, introduction of the A94V mutation in the PSST homologue of D. melanogaster using CRISPR-Cas9 did not result in fenpyroximate resistant flies. In addition, differences in binding curves between METI-Is and complex I measured directly, in isolated transgenic and wildtype mitochondria preparations, could not be found.

Age stage, two-sex life table of Habrobracon hebetor (Braconidae) on Spodoptera exigua (Noctuidae) reared on different sugar beet genotypes.

The present study was carried out to unveil interactive relevance among consecutive and alternate members of a tritrophic system comprised of sugar beet genotypes, beet armyworm, Spodoptera exigua (Hübner), and its parasitoid, Habrobracon hebetor (Say) using demographic parameters. To do so, H. hebetor was reared on S. exigua fed on 10 sugar beet genotypes, including SB26; SB27; SB29; SB33; SB34; (7112*SB36)*Sh-1-HSF-5; FC220; FC301; SBSI006; and HM 1339RZ in a growth chamber at 25 ± 1 °C, 60 ± 5% RH, and 16:8 (L: D) h photoperiod. The data was analyzed based on the age-stage, two-sex life table theory. Our results revealed high variation in duration of different life stages of H. hebetor on S. exigua reared on different sugar beet genotypes examined. The shortest (10.605 days) and longest (13.721 days) pre-adult period of H. hebetor was on S. exigua reared on SB26 and SB34, respectively. The longest (17.2 days) and shortest adult longevity (7.5 days) was on S. exigua reared on SB26 and SB27, respectively. The highest values of the intrinsic rate of increase (r) (0.209 day-1) and finite rate of increase (λ) (1.233 day-1) were observed on S. exigua reared on SB34 and their lowest values (0.159 and 1.172 day-1, respectively) were recorded on SB27. Resistant and susceptible genotypes to S. exigua, FC301 and (7112*SB36)*Sh-1-HSF-5, respectively, were only genotypes on which H. hebetor had greater and approximately equal r compared with S. exigua. This finding indicates high capability of H. hebetor to be successfully employed against S. exigua on sugar beet genotypes which are extremely different in resistance to this pest.

Dichlorvos Resistance in the House Fly Populations, Musca domestica, of Iranian Cattle Farms.

BACKGROUND: Insecticide resistance is one of the most important problems associated with the control of Musca domestica, due to the potential of the rapid development of resistance to different chemical insecticides. The present study was carried out to evaluate dichlorvos resistance in the house fly populations collected from central regions of Iran, Isfahan Province and Chaharmahal and Bakhtiari Province, during 2017 to 2019. METHODS: Bioassays were carried out using a standard topical application method as well as a fumigation method. The Koohrang population (susceptible) with the lowest LD50 values to dichlorvos was chosen to calculate the resistance ratios (RR). Altered sensitivity of acetylcholinesterase (AChE), a target enzyme for dichlorvos, was investigated. RESULTS: According to the results, very high levels of dichlorvos resistance were observed in the Mobarake population (RR= 80.25-fold by topical application and 33-fold by fumigation bioassay), and Isfahan population (RR= 107.30-fold by topical application and 43-fold by fumigation bioassay) compared to the Koohrang population. Acetylcholinesterase of the Koohrang population was the most sensitive to inhibition by dichlorvos based on the determination of median inhibitory concentration (IC50), but AChE of Mobarake and Isfahan populations were 741.93- and 343.94- fold less sensitive to inhibition. CONCLUSION: The insensitivity of AChE was possibly involved in dichlorvos resistance in the house fly populations.

Point mutations in the voltage-gated sodium channel gene associated with pyrethroid resistance in Iranian populations of the European red mite Panonychus ulmi.

The European red mite Panonychus ulmi (Koch) is a major pest of apple trees worldwide and causes significant damage to apple orchards in Iran. Pyrethroid insecticides/acaricides, such as fenpropathrin and fenvalerate, are widely used to control P. ulmi, but their long-term use may lead to low efficacy. Earlier studies investigating pyrethroid resistance in closely related mites such as Tetranychus urticae revealed that pyrethroid resistance was associated with point mutations in the voltage-gated sodium channel gene (vgsc). The aim of this study was to investigate the biochemical and molecular mechanisms of fenpropathrin and fenvalerate resistance in Iranian populations of P. ulmi. Pyrethroid toxicity bioassays were carried out on different P. ulmi field populations. Marand (resistance ratio, RR = 149), Maraqeh (RR = 90) and Mianeh2 (RR = 71) populations exhibited high levels of resistance to fenpropathrin, compared to a susceptible field population (Shahin Dej). Resistance was also observed for fenvalerate with resistance ratio's ranging from 2- to 20-fold. Synergism experiments and enzyme activity assays predicted a minor role for classical detoxification enzymes. In contrast, two amino acid substitutions in the VGSC, L1024V and F1538I, that were previously shown to confer pyrethroid resistance, were detected in all three resistant P. ulmi populations and point towards target-site insensitivity as the most likely resistance mechanism. Furthermore, sequencing after cloning of vgsc fragments from single haploid males revealed the presence of multiple copies of vgsc in a highly resistant strain. The link between resistance mutations and vgsc copy number variation should be the subject of future study, as this might be used to develop molecular markers for monitoring pyrethroid resistance of P. ulmi in the field.

Screening of Potential Sources of Resistance to Spodoptera exigua (Lepidoptera: Noctuidae) in 24 Sugar Beet Genotypes.

Spodoptera exigua (Hübner) is one of the polyphagous pests destroying crops such as sugar beet worldwide. The aim of this experiment was to evaluate the resistance of 24 sugar beet genotypes to S. exigua. In this study, nine sugar beet cultivars (Dorotea, Tous, Persia, Silenta, Jaam, HM1339RZ, Jolgeh, Ekbatan, and SBSI006), three hybrids ((7112*SB36)*Sh-1-HSF-5, (7112*SB36)*Sh-1-HSF-15, and (7112*SB36)*Sh-1-HSF-16), four lines (FC201, FC301, FC220, and FC221), and eight populations (SB26, SB27, SB28, SB29, SB30, SB33, SB34, and SB35) were assessed. The experiments were performed under laboratory conditions at 25 ± 1 °C, 60 ± 5% RH, and a photoperiod of 16:8 (L:D) h using age-stage, two-sex life table procedure. The larval period ranged from 17.09 d on FC301 to 14.47 d on SB30. The total fecundity (F) was highest on (7112*SB36)*Sh-1-HSF-5 (905.13 eggs) and lowest on FC301 (312.46 eggs). (7112*SB36)*Sh-1-HSF-5 and FC220 with 318.02 and 26.76 eggs per individual had the highest and lowest net reproductive rates (R0), respectively. The highest values of the intrinsic rate of increase (r) and finite rate of increase (λ) were on (7112*SB36)*Sh-1-HSF-5 (r: 0.187 d - 1, λ: 1.205 d - 1) and the lowest were on FC301 (r: 0.104 d - 1, λ: 1.11 d - 1). SB26 (r: 0.185 d - 1, λ: 1.203 d - 1) and FC220 (r: 0.114 d - 1, λ: 1.12 d - 1) had no significant difference with (7112*SB36)*Sh-1-HSF-5 and FC301, respectively. The comparison of demographic parameters of S. exigua on 24 sugar beet genotypes suggested that FC220 and FC301 were the most resistant and (7112*SB36)*Sh-1-HSF-5 and SB26 were the most susceptible genotypes to this pest.

Diapause and Cold Hardiness of the Almond Wasp, Eurytoma amygdali (Hymenoptera: Eurytomidae), Two Independent Phenomena.

The almond wasp, Eurytoma amygdali Enderlein (Hymenoptera: Eurytomidae), a key pest of almond, is a univoltine pest diapausing as last instar larvae inside the damaged fruits for almost nine months in a year. In this study, changes in the amount of total simple sugars, lipid, protein, glycogen, trehalose, glucose, supercooling points (SCPs), and cold hardiness of the diapausing larvae were measured from October to March for first year diapause-destined and in August and September for second year diapause-destined larvae. Changes in glycogen content were reversely proportional to changes in total simple sugars and low molecular weight carbohydrates. These changes reflect the interconversion of glycogen to sugar alcohol in order to increase the insect cold tolerance. We found that cold hardiness and diapause of the last instar larvae of the almond wasp have evolved separately. Cold hardiness was highly associated with physiological changes (accumulation of cryoprotectants), but no physiological changes occurred in early diapause of first year diapause-destined and second year diapause-destined larvae. The almond wasp larvae were found to be a freeze-avoidant insect, as no larva survived after SCP determination and crystallization of its body fluids.

The cyclic keto-enol insecticide spirotetramat inhibits insect and spider mite acetyl-CoA carboxylases by interfering with the carboxyltransferase partial reaction.

Acetyl-CoA carboxylase (ACC) catalyzes the committed and rate-limiting step in fatty acid biosynthesis. The two partial reactions, carboxylation of biotin followed by carboxyl transfer to the acceptor acetyl-CoA, are performed by two separate domains in animal ACCs. The cyclic keto-enol insecticides and acaricides have been proposed to inhibit insect ACCs. In this communication, we show that the enol derivative of the cylic keto-enol insecticide spirotetramat inhibited ACCs partially purified from the insect species Myzus persicae and Spodoptera frugiperda, as well as the spider mite (Tetranychus urticae) ACC which was expressed in insect cells using a recombinant baculovirus. Steady-state kinetic analysis revealed competitive inhibition with respect to the carboxyl acceptor, acetyl-CoA, indicating that spirotetramat-enol bound to the carboxyltransferase domain of ACC. Interestingly, inhibition with respect to the biotin carboxylase substrate ATP was uncompetitive. Amino acid residues in the carboxyltransferase domains of plant ACCs are important for binding of established herbicidal inhibitors. Mutating the spider mite ACC at the homologous positions, for example L1736 to either isoleucine or alanine, and A1739 to either valine or serine, did not affect the inhibition of the spider mite ACC by spirotetramat-enol. These results indicated different binding modes of the keto-enols and the herbicidal chemical families.

Molecular analysis of resistance to acaricidal spirocyclic tetronic acids in Tetranychus urticae: CYP392E10 metabolizes spirodiclofen, but not its corresponding enol.

Spirodiclofen is one of the most recently developed acaricides and belongs to the new family of spirocyclic tetronic acids (ketoenols). This new acaricidal family is an important chemical tool in resistance management strategies providing sustainable control of spider mites such as Tetranychus urticae. Spirodiclofen targets lipid biosynthesis mediated by direct inhibition of acetyl coenzyme A carboxylase (ACCase). In this study, we investigated two genetically distant spider mite strains with high resistance to spirodiclofen. Despite the strong resistance levels to spirodiclofen (up to 680-fold), only limited cross-resistance with other members of this group such as spiromesifen and spirotetramat could be detected. Amplification and sequencing of the ACCase gene from resistant and susceptible strains did not reveal common non-synonymous mutations, and expression levels of ACCase were similar in both resistant and susceptible strains, indicating the absence of target-site resistance. Furthermore, we collected genome-wide expression data of susceptible and resistant T. urticae strains using microarray technology. Analysis of differentially expressed genes revealed a broad response, but within the overlap of two resistant strains, several cytochrome P450s were prominent. Quantitative PCR confirmed the constitutive over-expression of CYP392E7 and CYP392E10 in resistant strains, and CYP392E10 expression was highly induced by spirodiclofen. Furthermore, stage specific expression profiling revealed that expression levels were not significantly different between developing stages, but very low in eggs, matching the age-dependent resistance pattern previously observed. Functional expression of CYP392E7 and CYP392E10 confirmed that CYP392E10 (but not CYP392E7) metabolizes spirodiclofen by hydroxylation as identified by LC-MS/MS, and revealed cooperative substrate binding and a Km of 43 µM spirodiclofen. CYP392E10 also metabolizes spiromesifen, but not spirotetramat. Surprisingly, no metabolism of the hydrolyzed spirodiclofen-enol metabolite could be detected. These findings are discussed in the light of a likely resistance mechanism.

Acaricide resistance and resistance mechanisms in Tetranychus urticae populations from rose greenhouses in the Netherlands.

BACKGROUND: Spider mites are important crop pests that rapidly develop resistance to acaricides. To investigate whether acaricide resistance is a threat to greenhouse rose culture in the Netherlands, the susceptibility of 15 strains of Tetranychus urticae was tested to several currently used acaricides, and resistance mechanisms were investigated. RESULTS: Although the observed levels of susceptibility differed between strains and acaricides, resistance was detected in most strains. The activity of detoxifying enzymes was significantly increased in most field-collected strains, and a number of amino acid substitutions known to be involved in resistance were detected. CONCLUSIONS: Resistance levels to traditional acaricides such as bifenthrin and abamectin were prominent, and might result in control failure under field conditions. Resistance to more recently registered compounds was detected in several populations. Resistance levels were generally unstable in the laboratory without selection pressure. The toxicological, biochemical and genetic data in this study will be essential in devising an efficient resistant management for Dutch rose culture.

Acetylcholinesterase point mutations in European strains of Tetranychus urticae (Acari: Tetranychidae) resistant to organophosphates.

BACKGROUND: In Tetranychus urticae Koch, acetylcholinesterase insensitivity is often involved in organophosphate (OP) and carbamate (CARB) resistance. By combining toxicological, biochemical and molecular data from three reference laboratory and three OP selected strains (OP strains), the AChE1 mutations associated with resistance in T. urticae were characterised. RESULTS: The resistance ratios of the OP strains varied from 9 to 43 for pirimiphos-methyl, from 78 to 586 for chlorpyrifos, from 8 to 333 for methomyl and from 137 to 4164 for dimethoate. The insecticide concentration needed to inhibit 50% of the AChE1 activity was, in the OP strains, at least 2.7, 55, 58 and 31 times higher for the OP pirimiphos-methyl, chlorpyrifos oxon, paraoxon and omethoate respectively, and 87 times higher for the CARB carbaryl. By comparing the AChE1 sequence, four amino acid substitutions were detected in the OP strains: (1) F331W (Torpedo numbering) in all the three OP strains; (2) T280A found in the three OP strains but not in all clones; (3) G328A, found in two OP strains; (4) A201S found in only one OP strain. CONCLUSIONS: Four AChE1 mutations were found in resistant strains of T. urticae, and three of them, F331W, G328A and A201S, are possibly involved in resistance to OP and CARB insecticides. Among them, F331W is probably the most important and the most common in T. urticae. It can be easily detected by the diagnostic PCR-RLFP assay developed in this study.

Susceptibility of an organophosphate resistant strain of the two-spotted spider mite (Tetranychus urticae) to mixtures of bifenazate with organophosphate and carbamate insecticides.

Bifenazate, a new and frequently used carbazate, is a pro-acaricide which needs to be activated by carboxylesterases. We evaluated the possible antagonism of organophosphate and carbamate insecticides on bifenazate toxicity in Tetranychus urticae applied in mixtures. Two organophosphate resistant strains were used (WI and MR-VL) and several organophosphate (chlorpyrifos, azinphosmethyl and phosmet) and carbamate (carbaryl and methomyl) insecticides were evaluated. Mixing chlorpyrifos with bifenazate decreased bifenazate toxicity in both tested strains. However, in the strain with a higher esterase activity, antagonism decreased after 2 days. Of all other tested chemicals, only methomyl displayed an antagonistic effect 1 day after treatment. These findings indicate that mixing organophosphate and carbamate insecticides with bifenazate may inhibit bifenazate efficacy under field conditions, especially when resistant strains are present.