Susceptible Laboratory Strain Research Articles

Fall armyworm (Lepidoptera: Noctuidae): practical resistance of 2 Brazilian populations to Cry1A.105 + Cry2Ab and Cry1F Bt maize.

In the Americas, transgenic cropsproducing insecticidal proteins fromBacillus thuringiensis Berliner (Bt,Bacillales: Bacillaceae) have been used widely to manage fall armyworm (FAW, Spodoptera frugiperda [J.E. Smith]). As resistance to Cry1 single-gene Bt maize (Zea mays L.)rapidly evolved in some FAW populations, pyramided Bt maizehybrids producing Cry1, Cry2, or Vip3Aa proteins were introduced in the 2010s. We examined field-evolved resistance to single- and dual-protein Bt maize hybrids in 2 locations in southeastern Brazil, where plant damage by FAW larvae far exceeded the economic threshold in 2017. We collected late-instar larvae in Cry1A.105 + Cry2Ab and Cry1F maize fields and established 2 FAW populations in the laboratory. The F1 offspring reared on the foliage of Bt and non-Bt maize plants (Cry1A.105 + Cry2Ab and Cry1F) showed neonate-to-adult survival rates as high as 70% for both populations. There was no significant difference in the life-table parameters of armyworms reared on non-Bt and Bt maize foliage, indicating complete resistance to Cry1A.105 + Cry2Ab maize. Larval survival rates of reciprocal crosses of a susceptible laboratory strain and the field-collected populations indicated nonrecessive resistance to Cry1F and a recessive resistance to Cry1A.105 + Cry2Ab maize. When relaxing the selection pressure, the armyworm fitness varied on Cry1A.105 + Cry2Ab and non-Bt maize; the resistance was somewhat stable across 12 generations, without strong fitness costs, although one of the lines died confounded by a depleted-quality, artificial rearing diet. To our knowledge, this is the first report documenting the practical resistance of FAW to a pyramided Bt crop. We discuss the implications for resistance management.

Bio-efficacy of Olyset® Plus, PermaNet® 3.0 and Interceptor® G2 on pyrethroid-resistant populations of Anopheles gambiae s.l. prior to the June 2023 net distribution campaign in Benin, West Africa

BackgroundThis study investigates the effectiveness of new-generation mosquito nets, like Olyset® Plus and PermaNet® 3.0, and dual-action nets such as Interceptor® G2, against pyrethroid-resistant Anopheles gambiae mosquitoes following the 2023 mass distribution of long-lasting insecticidal nets in Benin.MethodsWe tested wild mosquito populations from six communes in Benin against various pyrethroid (permethrin 0.75%, alphacypermethrin 0.05%, and deltamethrin 0.05%) using WHO tube tests. Additionally, we exposed mosquitoes to chlorfenapyr 100 µg/ml using the CDC bottle bioassay method. A subset of mosquitoes underwent biochemical and PCR tests to check the overexpression of metabolic enzymes and the Kdr L1014F mutation. We evaluated the effectiveness of Olyset® Plus, PermaNet® 3.0, and Interceptor® G2 nets using cone and tunnel tests on both laboratory and field populations of An. gambiae.ResultsOverall, the highest mortality rate was 60% with pyrethroid and 98 to100% with chlorfenapyr. In cone tests, all three types of nets induced mortality rates above 80% in the susceptible laboratory strain of An. gambiae. Notably, Olyset® Plus showed the highest mortality rates for pyrethroid-resistant mosquitoes in cone tests, ranging from 81.03% (95% CI: 68.59–90.13) in Djougou to 96.08% (95% CI: 86.54–99.52) in Akpro-Missérété. PermaNet® 3.0 had variable rates, from 42.5% (95% CI: 27.04–59.11) in Djougou to 58.54% (95% CI: 42.11–73.68) in Porto-Novo. However, revealed good results for Interceptor® G2, with 94% (95% CI: 87.40–97.77) mortality and 89.09% blood sampling inhibition in local populations of An. gambiae. In comparison, Interceptor® had lower rates of 17% (95% CI: 10.23–25.82) and 60%, respectively.ConclusionThese results suggest that tunnel tests are effective for evaluating dual-active ingredient nets. Additionally, Interceptor® G2 and PBO nets like Olyset® Plus could be considered as alternatives against pyrethroid-resistant mosquitoes.

Physical integrity and bioefficacy of used long-lasting insecticidal nets in Makenene, Centre Region of Cameroon.

Long-lasting insecticide nets (LLINs) are the recommended tools against mosquito-borne diseases. However, their physical integrity and bioefficacy in the field could be affected by several factors. This study evaluated the physical integrity and bioefficacy of nets used in Makenene since 2016. Cross-sectional field surveys were carried out after 6 y. A questionnaire was first administered to the heads of households, and then the physical integrity of the LLINs was determined by calculating the proportional hole index (pHI). WHO cone bioassays were conducted to determine the bioefficacy of LLINs currently being used against wild strains of Anopheles gambiae s.l., Culex pipiens s.l., and laboratory-reared pyrethroid-susceptible strain of Anopheles coluzzii (Ngousso). Of the 167 LLINs examined in households, 39.5% were fairly good, 26.4% were acceptable, and 34.1% were damaged. The most torn faces of the nets were the sides used for entering and exiting. None of the 30 LLINs used for WHO cone bioassays was still effective against An. gambiae s.l. and Cx. pipiens s.l. while up to 85.7% of these LLINs were at least effective against the susceptible strain after 24 h, with a significant difference observed when comparing the mortality rates between wild and laboratory-susceptible strain of Anopheles (P-value < 0.01). Anopheles gambiae s.l. were all (100%) identified as An. gambiae s.s. by PCR. The LLINs distributed in Makenene since the 2016 campaign are only effective on susceptible strain and should be replaced for a better control of residual malaria transmission and the nuisance by Culex mosquitoes in the locality.

CYP4CS5-mediated thiamethoxam and clothianidin resistance is accompanied by fitness cost in the whitefly Bemisia tabaci.

Understanding the trade-offs between insecticide resistance and the associated fitness is of particular importance to sustainable pest control. One of the most devastating pest worldwide, the whitefly Bemisia tabaci, has developed resistance to various insecticides, especially the neonicotinoid group. Although neonicotinoid resistance often is conferred by P450s-mediated metabolic resistance, the relationship between such resistance and the associated fitness phenotype remains largely elusive. By gene cloning, quantitative reverse transcription (qRT)-PCR, RNA interference (RNAi), transgenic Drosophila melanogaster, metabolism capacity in vitro and 'two sex-age stage' life table study, this study aims to explore the molecular role of a P450 gene CYP4CS5 in neonicotinoid resistance and to investigate whether such resistance mechanism carries fitness costs in the whitefly. Our bioassay tests showed that a total of 13 field-collected populations of B. tabaci MED biotype displayed low-to-moderate resistance to thiamethoxam and clothianidin. Compared to the laboratory susceptible strain, we then found that an important P450 CYP4CS5 was remarkably upregulated in the field resistant populations. Such overexpression of CYP4CS5 had a good match with the resistance level among the whitefly samples. Further exposure to the two neonicotinoids resulted in an increase in CYP4CS5 expression. These results implicate that overexpression of CYP4CS5 is closely correlated with thiamethoxam and clothianidin resistance. RNAi knockdown of CYP4CS5 increased mortality of the resistant and susceptible populations after treatment with thiamethoxam and clothianidin in bioassay, but obtained an opposite result when using a transgenic line of D. melanogaster expressing CYP4CS5. Metabolic assays in vitro revealed that CYP4CS5 exhibited certain capacity of metabolizing thiamethoxam and clothianidin. These in vivo and in vitro assays indicate an essential role of CYP4CS5 in conferring thiamethoxam and clothianidin resistance in whitefly. Additionally, our life-table analysis demonstrate that the field resistant whitefly exhibited a prolonged development time, shortened longevity and reduced fecundity compared to the susceptible, suggesting an existing fitness cost as a result of the resistance. Collectively, in addition to the important role of CYP4CS5 in conferring thiamethoxam and clothianidin resistance, this resistance mechanism is associated with fitness costs in the whitefly. These findings not only contribute to the development of neonicotinoids resistance management strategies, but also provide a new target for sustainable whitefly control. © 2023 Society of Chemical Industry.

Characterization of emamectin benzoate resistance in the diamondback moth, Plutella xylostella (Lepidoptera: Plutellidae).

Plutella xylostella (L.) is a destructive pest of cruciferous crops worldwide that has evolved resistance to many insecticides. Here we examined the mode of inheritance, cross-resistance profile, and potential mechanisms of emamectin benzoate resistance in a field-derived strain of P. xylostella from Japan. A field-collected population of P. xylostella, was found to exhibit strong (> 150-fold) resistance to emamectin benzoate in insecticide bioassays when compared with a laboratory susceptible strain. Genetic analysis showed that resistance is inherited as an autosomal, recessive trait, and is conferred by a single or a few closely linked loci. The emamectin benzoate resistant strain also exhibited resistance to abamectin, lepimectin, chlorantraniliprole, lufenuron, spinetoram, indoxacarb, fipronil, dieldrin, endosulfan and lambda-cyhalothrin, demonstrating a remarkable multi-resistance profile. Insecticide bioassays employing inhibitors of detoxification enzymes revealed that piperonyl butoxide (PBO) increased the toxicity of emamectin benzoate in the resistant strain by ten-fold indicating the potential involvement of cytochrome P450 monooxygenases in avermectin resistance. Furthermore, cloning and sequencing of the primary receptor of avermectins, the GluCl channel, revealed the absence of target-site mutations in the resistant strain. Our data on the mode of inheritance and mechanisms of resistance to emamectin benzoate in a P. xylostella strain from Japan provide a foundation for the development of regional resistance management strategies. However, the high levels of phenotypic resistance in this strain to a diverse range of other insecticide classes available for control illustrate the challenges associated with the sustainable control of this important pest. © 2023 Society of Chemical Industry.

The PitA protein contributes to colistin susceptibility in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic pathogen that causes a wide range of problematic infections in individuals with predisposing conditions. Infections can be treated with colistin but some isolates are resistant to this antibiotic. To better understand the genetic basis of resistance, we experimentally evolved 19 independent resistant mutants from the susceptible laboratory strain PAO1. Whole genome sequencing identified mutations in multiple genes including phoQ and pmrB that have previously been associated with resistance, pitA that encodes a phosphate transporter, and carB and eno that encode enzymes of metabolism. Individual mutations were engineered into the genome of strain PAO1. Mutations in pitA, pmrB and phoQ increased the minimum inhibitory concentration (MIC) for colistin 8-fold, making the bacteria resistant. Engineered pitA/phoQ and pitA/pmrB double mutants had higher MICs than single mutants, demonstrating additive effects on colistin susceptibility. Single carB and eno mutations did not increase the MIC suggesting that their effect is dependent on the presence of other mutations. Many of the resistant mutants had increased susceptibility to β-lactams and lower growth rates than the parental strain demonstrating that colistin resistance can impose a fitness cost. Two hundred and fourteen P. aeruginosa isolates from a range of sources were tested and 18 (7.8%) were colistin resistant. Sequence variants in genes identified by experimental evolution were present in the 18 resistant isolates and may contribute to resistance. Overall our results identify pitA mutations as novel contributors to colistin resistance and demonstrate that resistance can reduce fitness of the bacteria.

Assessing the susceptibility and efficacy of traditional neurotoxic (pyrethroid) and new-generation insecticides (chlorfenapyr, clothianidin, and pyriproxyfen), on wild pyrethroid-resistant populations of Anopheles gambiae from southern Benin.

The objective of this study was to determine the susceptibility of wild Anopheles gambiae sensu lato (s.l.) from southern Benin to the new insecticides (chlorfenapyr (CFP), pyriproxyfen (PPF), and clothianidin (CTD)) and assess the efficacy of insecticide-treated bed nets (ITNs) that contain these new products. Wild An. gambiae from the Benin communes of Allada, Ifangni, Akpro-Missérété, and Porto-Novo were tested for their susceptibility to CFP and PPF using the WHO bottle tests, and pyrethroids (alpha-cypermethrin, deltamethrin, and permethrin) and CTD using WHO tube tests. WHO cone tests were used to evaluate the efficacy of Interceptor® (which contains alpha-cypermethrin (ACM) only), Interceptor® G2, (CFP + ACM), and Royal Guard® nets (PPF + ACM). The ovaries of blood-fed An. gambiae from Ifangni exposed to a new PPF net were dissected, and egg development status was examined using Christopher's stages to determine the fertility status of the mosquitoes. Using a standardized protocol, the oviposition rate and oviposition inhibition rate were calculated from live blood-fed An. gambiae placed in oviposition chambers after exposure to PPF. In all four mosquito populations, pyrethroid mortality ranged from 5 to 80%, while chlorfenapyr and clothianidin mortality ranged from 98 to 100%. At Ifangni, all mosquitoes exposed to Royal Guard® nets were infertile (100%) while the majority (74.9%) of mosquitoes exposed to Interceptor® nets had fully developed their eggs to Christopher's stage V. The oviposition inhibition rate after exposure of the mosquitoes to the PPF was 99% for the wild population of An. gambiae s.l. and the susceptible laboratory strain, An. gambiae sensu stricto (Kisumu). The results of this study suggest that pyrethroid-resistant An. gambiae from the selected communes in southern Benin are susceptible to chlorfenapyr, clothianidin, and pyriproxyfen. In addition, based on bioassay results, new and unused Interceptor® G2 and Royal Guard® nets were effective on Ifangni's mosquito populations. Despite the availability of new effective insecticides, continued vigilance is needed in Benin. Therefore, monitoring of resistance to these insecticides will continue to periodically update the Benin national insecticide resistance database and management plan.

Can the use of larviciding with biological compounds contribute in increasing Anopheles gambiae s.l. susceptibility to pyrethroid in a population expressing high resistance intensity?

BackgroundLarviciding using non-insecticide compounds is considered appropriate for controlling outdoor biting mosquitoes and for managing insecticide resistance. However, there is still not enough information on the influence of larviciding in managing pyrethroid resistance. In the present study, we checked whether the introduction of larviciding using the biolarvicide VectoMax G in the city of Yaoundé is contributing in restoring the susceptibility of An. coluzzii populations to pyrethroids. MethodologyThe susceptibility status of field An. coluzzii population was evaluated at different time points before and during larviciding treatments. In addition, An. coluzzii larvae collected in the city of Yaoundé, were split into four groups and exposed to different selection regimes for many generations as follow; (i): deltamethrin 0.05%_only, (ii): Vectomax_only, (iii): Vectomax+deltamethrin 0.05%, (iv): VectoMax+deltamethrin 0.05% + susceptible. Life traits parameters were measured in the progeny and compared between colonies. The control was the susceptible laboratory strain “Ngousso”. Kdr allele frequency and the profile of expression of different detoxification genes and oxidative stress genes was checked using qPCR analysis. Gene's expression was compared between the first and the last generation of each colony and in field populations collected before and during larviciding. ResultsAn increase in mosquito susceptibility to deltamethrin and permethrin was recorded for the field populations after larviciding implementation. Resistance intensity to deltamethrin was found to decrease from high to low in field populations. Only the colony vectomax+deltamethrin+susceptible presented a high susceptibility to deltamethrin after 21 generations. The kdr gene frequency was found to be unchanged in the field population and laboratory colonies. A significant decrease in the overexpression profile of Gste2 was detected in field population after larviciding implementation. Other genes showing a similar pattern though not significant were Cyp6z1, Cyp6p1 and Cyp6g16. Concerning fitness only the colony vectomax+deltamethrin+susceptible was found to display a fitness profile similar to the susceptible colony with high fecundity, high hatching rate, short development time and long adult survival rate. ConclusionThe profile of the field population supported reversal of phenotypic resistance to pyrethroids however no reduction in the frequency of the kdr allele was recorded. Some detoxification genes were detected less overexpressed. The study suggest that reversal may take longer to achieve in a population expressing a very high resistance profile and under continuous insecticide selection pressure.

Insecticide resistance of western flower thrips, Frankliniella occidentalis (Pergande) in China

Frankliniella occidentalis (Pergande) is a polyphagous thrips that feeds on various horticultural crops worldwide. Its unique biological properties make it highly adaptable, and it has developed resistance to various insecticides as a result. Understanding the susceptibility of F. occidentalis field populations to insecticides is necessary to select suitable agents for their control. In the present study, we tested the toxicity of eight insecticides (spinosad, spinetoram, abamectin, emamectin-benzoate, chlorfenapyr, thiamethoxam, β-cypermethrin, and broflanilide) in 21 field populations of F. occidentalis collected from 2019 to 2021. F. occidentalis has shown variable resistance to seven of these insecticides, but it is susceptible to broflanilide. Populations from Changping, Tongzhou and Yanqing in Beijing were found to be the most resistant, while those from Shunyi, Beijing and Hetian in Xinjiang were the least resistant. The population with the highest level of resistance was collected from Yanqing, Beijing; for emamectin-benzoate, the resistance ratio (RR) of this population compared to the susceptible laboratory strain of F. occidentalis was 7455.6. The overall resistance of F. occidentalis has decreased over the past three years, as demonstrated by the reduction in the proportion of highly resistant populations. This may have been due to the implementation of pesticide reduction programs and the increased use of non-chemical control methods.

Toxicity, resistance and metabolic mechanism of resistance to permethrin in larvae of laboratory and field strains of Musca domestica L.

Management of Musca domestica is desirable globally in order to manage public health issues. However, development of insecticide resistance is one of major hindrances in the management of M. domestica. Here, we explored toxicity of permethrin against larvae of laboratory and field strains of M. domestica, resistance comparison, synergistic effect of enzyme inhibitors on permethrin toxicity, and activities of metabolic enzymes in both strains. In case of the Lab-susceptible strain, LC50 values of permethrin were 0.14, 0.52 and 0.94 µg g-1 for first, second and third instars, respectively. In case of the field strain, LC50 values were 28.09, 66.86 and 112.24 µg g-1 for first, second and third instars, respectively. First, second and third instars of the field strain exhibited 200.64, 128.58 and 119.40 fold resistance to permethrin when compared with the first, second and third instars of the Lab-susceptible strain, respectively. Toxicity of permethrin in the field strain synergized in the presence of enzyme inhibitors (PBO or DEF). Enzyme analysis revealed significantly higher activities of MFO, CarE and GST in the field strain than those of the Lab-susceptible strain, suggesting metabolic-based mechanism of permethrin resistance. In conclusion, the field strain exhibited resistance to permethrin, which could be managed by adopting alternate measures for the management of M. domestica.

Persistent variation in insecticide resistance intensity in container breeding Aedes (Diptera: Culicidae) co-collected in Houston, TX.

As observed in many locations worldwide, resistance to pyrethroids is common in Aedes aegypti (L.) in the southern United States and northern Mexico. Strong resistance in Aedes albopictus (Skuse) is less common and is not as well characterized. These 2 species have been undergoing range expansion and are sympatric in many locations including Houston, Texas. They are often collected from the same locations and lay eggs in the same larval habitats. In this study, we colonized both Ae. aegypti and Ae. albopictus from 4 locations in Houston and characterized insecticide resistance using permethrin as a model pyrethroid. We found differences in resistance intensity between the species at all 4 sites. Within the Ae. aegypti, resistance ratios ranged from 3.5- to 30.0-fold when compared to the ORL1952 laboratory susceptible strain. Expression of several P450s was higher than in the ORL1952 strain, but the pattern was similar between the field strains of Ae. aegypti. Higher resistance ratios did correlate with increasing percentages of the dilocus knockdown resistance (kdr) genotype. In contrast, Ae. albopictus from the 4 locations all had very low resistance ratios (<4-fold) when compared to the same laboratory susceptible strain. Five years later, we performed additional collections and characterization from the most resistant location to assess the temporal persistence of this difference in resistance between the species. The same pattern of high resistance in Ae. aegypti and low resistance in sympatric Ae. albopictus remained 5 yr later and this may have implications for operational efficacy.

Assays to Measure Insecticide Toxicity and Insecticide Resistance in Mosquitoes.

Mosquitoes' resistance to commonly used insecticides is now widespread, hampering control efforts and leading to substantial increases in human illness and mortality rates in many areas of the world. Insecticide bioassays are quantitative methodologies used to determine the dose-response relationship of insects to insecticides and to evaluate the susceptibility or resistance of mosquitoes to specific insecticides. They are frequently used to monitor the development of insecticide resistance in mosquitoes for both field resistance diagnoses (surveillance assays), in which the ability of mosquitoes to survive exposure to a standard dose or concentration of an insecticide is measured, and laboratory bioassays, in which responses to insecticides are tested in parallel populations of resistant (field) populations and laboratory susceptible strains using serial doses or concentrations. Metabolic detoxification, in which insecticides are metabolized by enzymes, including cytochrome P450s, hydrolases, and glutathione-S-transferases (GSTs), to become more polar and less toxic, is one resistance mechanism. Piperonyl butoxide (PBO), S,S,S-tributyl phosphorotrithioate (DEF), and diethyl maleate (DEM) are the inhibitors of P450s, hydrolases, and GSTs, respectively, and act as synergists for rapidly testing the involvement of these enzymes in insecticide resistance. Such synergistic assays are used to identify the detoxification enzyme that leads to resistance to a specific insecticide. This introduction and its associated protocols present a detailed discussion of appropriate methodologies and procedures for laboratory larval, adult, and synergistic bioassays and introduces the field surveillance tests used to monitor insecticide resistance as recommended by the latest World Health Organization (WHO) and U.S. Centers for Disease Control (CDC) guidelines.

Mosquito Larval Bioassays.

Insecticide bioassays are frequently used to measure levels of insecticide resistance in mosquito populations, examining the ability of mosquitoes to survive exposure to insecticides. Laboratory bioassays measure insects' responses to insecticides in resistant (field) populations and laboratory susceptible strains using serial doses or concentrations across the range of >0 and <100% mortality. This protocol measures the toxicity of insecticides to mosquito larvae and determines the level of insecticide resistance. Generally, laboratory-reared mosquito larvae of known age or instar are exposed to H2O containing various concentrations of an insecticide, and the response (mortality) is recorded 24 h after the test. Larval bioassay tests can (1) identify the lethal concentrations of larvicide that cause 50% and 90% mortality (LC50 and LC90, respectively); (2) determine the diagnostic concentration needed to monitor susceptibility in mosquito larvae in the field; and (3) investigate the resistance status and the mechanisms governing insecticide resistance to a specific insecticide.

Functional characterization of cytochrome P450s associated with pyrethroid resistance in the olive fruit fly Bactrocera oleae

Resistance to pyrethroid insecticides has evolved in Bactrocera oleae populations in Greece, threatening the efficacy of control interventions based on this insecticide class. Here we report the collection of populations from Crete, with resistance levels reaching up to 132-folds, compared to susceptible laboratory strains and show that pyrethroid resistance is substantially suppressed by the PBO synergist, suggesting the involvement of detoxification enzymes. To identify specific candidate genes implicated in resistance, we performed comparative transcriptomic analysis, between the pyrethroid resistant populations from Crete and the susceptible laboratory strains, using both whole bodies and Malpighian tubules. Several genes were found differentially transcribed between resistant and susceptible flies in each comparison, with P450s being among the most highly over-expressed detoxification genes in pyrethroid resistant populations. Four of the over-expressed P450s (Cyp6A61, Cyp6G6, Cyp4P6 and Cyp6G28) were recombinantly expressed in Escherichia coli and in vitro metabolism assays revealed that CYP6A61 is capable of metabolizing alpha-cypermethrin, while CYP6G6, CYP4P6 and CYP6G28 are capable of metabolizing deltamethrin. No metabolism of neonicotinoid insecticides was recorded. We further silenced CYP6G6 in vivo, via RNAi, which led to a small, but significant increase in deltamethrin toxicity. The study provides valuable information towards the development of molecular diagnostics and evidence-based insecticide resistance management strategies.

The sublethal and&amp;nbsp;transgenerational effects of pyridaben on fitness and induced tolerance in Panonychus citri

Panonychus citri is a dominant citrus pest mite in the world. Pesticide-induced population resurgence is a concern for environmental and resistance risk assessment in mite control. To evaluate the effective mite management strategies, the sublethal and transgenerational effects of pyridaben on P. citri were assessed using life-table parameters. Two segregated pyridaben-resistant and susceptible strains (previously obtained from the crossing between laboratory susceptible strain and pyridaben-resistant population) were used in our study. After exposure to LC30 pyridaben, the fecundity of Pyr_Control and Pyr_Rs strains were both significantly reduced in the parental generation (F0). Life-table results showed that LC30 pyridaben significantly induced the fecundity of both strains in F1 generation (egg stage was prolonged as well). Interestingly, this effect also stimulated the fecundity of F2 generation in Pyr_Control strain. But no significant effects were observed in Pyr_Rs strain. While detoxification enzyme assays indicated that only the activity of P450s was significantly activated by LC30 pyridaben in Pyr_Control and Pyr_Rs strains (1.242- fold and 1.355-fold increase, respectively). The expressions of P450 gene (CYP4CL2) and reproduction-related gene (Pc_Vg) were determined and the results showed that the expression of Pc_Vg of pyridaben treated mites in F0 generation was significantly reduced in both strains. In Pyr_Control and Pyr_Rs strains, the significant upregulation CYP4CL2 (2.029-fold and 2.516-fold increase, respectively) and PcVg (1.771-fold and 2.924-fold increase, respectively) of F1 generation suggested there were delayed hormesis effects on reproduction and developed tolerance to pyridaben. No significant difference was found between LC30 treatments and controls in F2 generation of both strains. These results provided evidence that transgenerational hormesis effects of low concentration of pyridaben might lead to mite population outbreak and resistance risk in the field through stimulating the reproduction and developing tolerance to pyridaben. Furthermore, LC30 pyridaben could affect the offspring populations of susceptible strain in the longer-term as compared to resistant strain.&#x0D;

Selection for resistance to pirimiphos-methyl, permethrin and spinosad in a field strain of Sitophilus oryzae: resistance risk assessment, cross-resistance potential and synergism of insecticides.

Indiscriminate use of insecticides in food storage facilities for controlling insect pests has deleterious effects on the environment and non-targeted organisms in the premises. Continuous use of insecticides may result in resistance development in insect pests, which compel the stakeholders to increase the dosage of insecticides to manage resistant insect pests. The increased dosage of insecticides ultimately may result in contamination of stored food stuff that affects human health. The present study was planned to generate data that will be helpful to delay resistance development and to reduce environmental pollution. A field strain of Sitophilus oryzae, one of the most common insect pests of stored foodstuff, was selected separately with pirimiphos-methyl, permethrin, or spinosad for five consecutive generations. The selected strains were studied for resistance risk assessment, time taken to develop resistance to insecticides after continuous exposure in the selection process, preliminary mechanism of resistance, and whether the development of resistance due to the selection with a particular insecticide could develop cross-resistance to other insecticide or not. In comparison to a laboratory susceptible reference strain, the insecticide-selected strains revealed rapid development of resistance against insecticides as a result of selection process: 31.05-fold resistance to pirimiphos-methyl, 156.49-fold resistance to permethrin, and 65.6-fold resistance to spinosad. The selected strains did not show cross-resistance to insecticides to with these strains were not exposed during selection experiments, i.e., strain selected with pirimiphos-methyl did not show cross-resistance to spinosad and permethrin. In the synergism bioassays, the synergists (S,S,S-tributyl phosphorotrithioate and piperonyl butoxide) significantly reduced resistance of the selected strain against insecticides to with these were selected, revealing the probability of metabolic mechanism of resistance. The present study revealed high risks of resistance development to pirimiphos-methyl, spinosad, and permethrin under consistent selection pressure. Lack of cross-resistance among insecticides provides an opportunity to use insecticides in rotation instead of increasing dosages to manage resistant insects that will ultimately pollute the environment.

Resistance to Multiple Insecticide Classes in the Vinegar Fly Drosophila melanogaster (Diptera: Drosophilidae) in Michigan Vineyards.

Vinegar flies are vectors of pathogens causing fruit rots of grapes, so control of these insects is important for preventing vineyard yield loss. Recent outbreaks of sour rots may be linked to greater challenges controlling vinegar flies, so we investigated the insecticide susceptibility of populations collected from commercial vineyards across Michigan. We first determined the discriminating concentration for phosmet, malathion, methomyl, and zeta-cypermethrin using a laboratory susceptible (Canton-S) strain of D. melanogaster females. The discriminating concentrations were determined as 252.08, 2.58, 0.96, and 1.68 ppm of the four insecticides, respectively. These concentrations were first tested in 2020 against populations from the two major counties for grape production. In 2021, we expanded monitoring to twenty-three populations collected from vineyards across six counties. All populations had significantly lower sensitivity to all four insecticides compared with Canton-S strain, with up to 98.8% lower mortality for phosmet. The LC50, LC90, and LC99 values of the four insecticides for the two populations tested in 2020 were 7-1,157-fold higher than the Canton-S strain. For the twenty-three populations collected in 2021, mortality ranged from 56.3 to 100% when the flies were screened using a 10x concentration of the discriminating concentration of the insecticides, whereas it ranged from 82.4 to 100% when the flies were screened using a 20x concentration. Our results suggest variable levels of resistance to insecticides from multiple chemical classes in D. melanogaster populations in Michigan vineyards, highlighting the need to implement integrated sour rot management approaches that are less dependent on insecticides for control of this species.

Lack of fitness cost and inheritance of resistance to abamectin based on the establishment of a near-isogenic strain of Tetranychus urticae

Many populations of the two-spotted spider mite, Tetranychus urticae Koch, have developed high levels of resistance to the pesticide abamectin in China and other countries. This study developed a near-isogenic line to understand better the inheritance, cross-resistance, and fitness costs associated with abamectin resistance in the field population of T. urticae in China. We introduced the trait that confers extremely high abamectin resistance in a field-collected population of T. urticae into a susceptible laboratory strain (IPP-SS) to generate an abamectin-resistant near-isogenic line (NIL-Aba). This process was carried out through multiple backcrossing to IPP-SS and via parthenogenesis and abamectin screening. Compared with IPP-SS, the NIL-Aba strain had a 25 147-fold resistance to abamectin and a high level of cross-resistance to bifenthrin (288.17-fold), an intermediate level to emamectin benzoate (42.57-fold), and low levels to bifenazate, chlorfenapyr, cyflumetofen, cyenopyrafen, and cyetpyrafen with resistance ranging from 3.18- to 9.31-fold. But it had no cross-resistance to profenofos. The resistance to abamectin in NIL-Aba was autosomal, incompletely dominant, and polygenic. Based on two sex life table parameters, no fitness cost was found in NIL-Aba. Establishing the NIL-Aba strain provides a reliable basis for an in-depth study of abamectin resistance in T. urticae. New information on toxicological characteristics and fitness cost should facilitate the management of abamectin resistance in field populations of T. urticae.

Formulation of abamectin and plant oil-based nanoemulsions with efficacy against the two-spotted spider mite Tetranychus urticae (Acari: Tetranychidae) under laboratory and field conditions

Due to the harmful effects of synthetic chemical acaricides on ecosystems and human health, there is increasing interest in the use of nanotechnology to fabricate eco-friendly nanoemulsions based on plant oils in the field of spider mite control. In this study, nanoemulsions of abamectin, garlic, and neem oils were prepared by a high-energy approach and characterized by transmission electron microscopy. The droplet sizes of all tested nanoemulsions were less than 100 nm. The acaricidal activities of the prepared nanoemulsions compared to abamectin were evaluated against a susceptible laboratory strain of Tetranychus urticae Koch (Acari: Tetranychidae) under laboratory and field conditions. The results showed that abamectin nanoemulsion was the most toxic compound against adult females of T. urticae followed by abamectin emulsion. Neem nanoemulsion had moderate toxicity and garlic nanoemulsion had the lowest toxicity. The effects of tested compound residues on egg deposition and egg hatching in descending order were as follows: abamectin nanoemulsion > abamectin emulsion > neem oil nanoemulsion < garlic oil nanoemulsion. In the field experiment, all tested compounds were effective in reducing the population density of T. urticae in the motile stage, with mean reductions ranging between 66.08% and 95.24% for all compounds. The most effective compound was abamectin nanoemulsion. The results of the present study demonstrate that nanoemulsion enhanced the biological activity of abamectin. Further, neem and garlic oil nanoemulsions have potential utility as environmentally friendly acaricides in integrated pest management programs.

Resistance risk assessment in diamondback moth, Plutella xylostella (L.) to fluxametamide

The diamondback moth Plutella xylostella (L.) (Lepidoptera: Plutellidae) is a devastating pest that infests several cruciferous vegetable crops worldwide and has developed insecticide resistance in most chemical classes. Fluxametamide is a novel neuroactive isoxazoline compound that binds to a unique site on the γ-aminobutyric acid (GABA) receptor and has an exceptional insecticidal activity on a range of lepidopteran caterpillars. The present study aimed to assess the risk of resistance in P. xylostella to fluxametamide under laboratory conditions. After the repeated exposure to fluxametamide for successive 18 generations of P. xylostella, there was 3.095-fold increase in the LC50. When the realized heritability (h2) of P. xylostella to fluxametamide in the open field was assumed to be the laboratory-estimated value (h2 = 0.180) and the mortality was 70–90%, only 22.9–33.1 generations were expected to be required to obtain a 10-fold increase in fluxametamide resistance. Compared with the laboratory susceptible strain (F0), the Flux-SEL (F18) strain exhibited a very low level of cross-resistance to emamectin benzoate (5.17-fold) and no significant cross-resistance to fipronil or flubendiamide. With the F0 and the Flux-SEL test strains (F12, F15 and F18), the specific activities of cytochrome P450 and glutathione S-transferase showed a remarkable increase during selection, while the esterase activity did not differ significantly. The results indicated that a potential risk of resistance development to fluxametamide exists in P. xylostella after continuous exposure. These pieces of information will be crucial in formulating rational fluxametamide application and resistance management guidelines for controlling P. xylostella under field condition.