Susceptible Laboratory Strain Research Articles

Three years of insecticide resistance evolution and associated mechanisms in Aedes aegypti populations of Ouagadougou, Burkina Faso

Background Resistance to insecticides is spreading among populations of Aedes aegypti, the primary vector of important human arboviruses. The escalating insecticide resistance poses a significant threat to dengue vector control, with an expanding number of countries affected by the disease. To gain a deeper insight into the evolution of insecticide resistance, it is essential to have longitudinal surveillance results, which are currently lacking, particularly from African Ae. aegypti populations. Here we report on three-years of surveillance of Ae. aegypti susceptibility to insecticide resistance phenotypes and associated kdr mutations in Burkina Faso, a country with regular dengue outbreaks. Methods Ae. aegypti susceptibility to insecticides and the V410L, V1016I, and F1534C kdr target site mutations linked to pyrethroid insecticide resistance were monitored in Ouagadougou from 2016 to 2018. Larvae were collected from artificial containers at two sites and reared to adulthood in an insectary. Bioassays were conducted on female adults, along with a laboratory-susceptible strain, following standard WHO protocols. Allele-specific PCR genotyping assays were utilized to identify the V410L, V1016I, and F1534C kdr pyrethroid target site mutations. Results Bioassays revealed a high level of resistance to permethrin and deltamethrin that progressively increased over the three-year period in both localities. The 1534C mutation was nearly fixed throughout the three years at each locality, and while the closely-related 410L and 1016I mutations did not vary between localities, their frequency notably increased from 2016 to 2018. Interestingly, Ae. aegypti populations in both areas remained susceptible to bendiocarb, fenitrothion, and malathion. Modelling the mortality data further confirmed the escalating resistance trend over the years and emphasized the significant role played by the three kdr mutations in conferring resistance to pyrethroids. Conclusion Mortality rates indicate that Ae. aegypti populations from Ouagadougou are becoming increasingly resistant to pyrethroid insecticides, likely due to an increase in the frequencies of the 410L and 1016I kdr mutations. Organophosphate insecticides are likely to be better alternative options for control.

Knockdown resistance in Stomoxys calcitrans stable fly populations on German dairy farms: kdr alleles explain susceptibility of individual flies to deltamethrin

AbstractThe stable fly, Stomoxys calcitrans, is a globally important pest causing stress, economic losses and transmission of pathogens in livestock. Control on commercial farms relies predominantly on the use of insecticides, with pyrethroids being the most frequently used class of insecticides in industrialised countries. Here, laboratory isolates were obtained from four dairy farms in Brandenburg (Germany) and tested for phenotypic resistance to deltamethrin in comparison to a susceptible reference isolate using topical application. Individual flies were subsequently genotyped using allele-specific real-time PCRs. Phenotypic resistance was observed in all four field isolates with resistance ratios between 46 and 119 compared to the susceptible laboratory strain. At position 1014 of the voltage-sensitive sodium channel, allele-specific PCRs detected the wild-type, kdr-his and kdr genotypes encoding leucine, histidine and phenylalanine, respectively. In the susceptible laboratory isolate, only the wild-type was identified. On the farms with very high LD50 values, the kdr variant was most prevalent and logistic regression analysis revealed that the kdr variant increased the odds to survive exposure to deltamethrin more than the kdr-his genotype. Flies carrying two resistance alleles were less susceptible than flies that also carried one wild-type allele. In three out of four field isolates, the allele frequencies were significantly different from the expectations of the Hardy–Weinberg equilibrium suggesting ongoing selection. The data show that the phenotype can be largely explained by the kdr genotype and represent high frequencies of the L1014F kdr and L1014H kdr-his variants conferring high levels of resistance in northern Germany.

Exposure to copper metal enhances the tolerance of An. gambiae s.s. over multiple generations while reducing both fertility and fecundity in this primary malaria vector.

Background Anopheles s.l. displays the potential to develop tolerance to heavy metals, particularly copper, this may occur at a significant biological cost, which can adversely affect its ecological fitness. This study investigated the larval metal exposure on larval development and reproduction of An. gambiae s.s., a laboratory susceptible strain, kisumu. Methods Stage 2 larvae of Anopheles gambiae, Kisumu were exposed to C1 = 484 μg L-1, C2 = 300 μg L-1 and 0 μg L-1 (control) of copper chloride. Larval mortality, pupation time, pupation rate, gonotrophic cycle length, fecundity and fertility of larvae/adults were assessed over six generations. Results Results revealed that larval mortality rate was significantly higher in the C1 groups of each group (p = 0.000), but this mortality rate decreased over generations. Pupation time was extended to 13 and 14 days respectively for C2 and C1 groups (p = 0.000) compared to the control group. Similar results were observed for the gonotrophic cycle, which increased from 4 days at G0 to more than 6 days at generation 5 in adults of C1. The pupation rate in generation 4 (C1) and generation 5 of the same group (p = 0.000) as well as the emergence rate in generation 4 (C2, p = 0.000) and generation 5 (C1 and C2, p = 0.000) decreased significantly compared to the control group. The average number of eggs laid was lower in the test groups from generation 4 to generation 5 (C1 and C2, p = 0.00) and egg fertility was also negatively affected by exposure of the larval stage of An. gambiae s.s. to copper. Conclusion Although studies have already shown that copper induces resistance in An. gambiae s.l. to insecticides, this study has shown that this adaptation requires a non-negligible biological cost in the life of the insect.

A rapid tick exposure test for monitoring acaricide resistance in Rhipicephalus sanguineus sensu lato ticks on dogs

BackgroundBrown dog ticks (Rhipicephalus sanguineus sensu lato) are vectors of pathogens adversely affecting the health of dogs in many regions of the world. The three-host life cycle of R. sanguineus s.l., with all stages feeding on dogs, can lead to an uncontrolled build-up of large tick populations if not controlled by acaricides. However, frequent tick control on dogs using acaricides has led to the emergence of resistance to permethrin and fipronil. Currently, the larval packet test (LPT) is the standard tick resistance test, which is laborious, requires laboratory facilities, and takes at least 6 weeks before larvae derived from engorged female ticks can be tested. Our novel approach is to expose semi-engorged adult ticks to acaricides immediately after removing them from dogs, obtaining results within 24 h.MethodsAdult ticks from three laboratory colonies of R. sanguineus s.l. were tested in RaTexT®, a rapid tick exposure test in which ticks were confined to small compartments and exposed to an acaricide-impregnated, specially designed matrix. Resistance was confirmed by testing larvae derived from the same laboratory colonies using the LPT. RaTexT® was also used to determine the susceptibility of R. sanguineus acaricides in dog shelters.ResultsRaTexT® detected resistance to permethrin in adult R. sanguineus s.l. ticks from two Brazilian laboratory colonies compared to a susceptible laboratory strain originating in Greece. Resistance was confirmed by LPT testing of larvae from the same colonies with resistance factors between 2.2 and 3.1. All laboratory strains were susceptible to fipronil. A suspected case of fipronil resistance at a dog shelter in Caxias do Sul, Brazil, was resolved within 24 h by testing adult ticks in RaTexT® and could be attributed to improper treatment.ConclusionsRaTexT® is a valuable tool for monitoring the development of resistance to synthetic pyrethroids or phenylpyrazoles in tick-infested dogs.Graphical

Identification and functional validation of P450 genes associated with pyrethroid resistance in the malaria vector Anopheles sinensis (Diptera Culicidae)

Cytochrome P450 monooxygenases (P450s), a multifunctional protein superfamily, are one of three major classes of detoxification enzymes. However, the diversity and functions of P450 genes from pyrethroid-resistant populations of Anopheles sinensis have not been fully explored. In this study, P450 genes associated with pyrethroid resistance were systematically screened using RNA-seq in three field pyrethroid-resistant populations (AH-FR, CQ-FR, YN-FR) and one laboratory resistant strain (WX-LR) at developmental stages, tissues, and post blood-meal in comparison to the laboratory susceptible strain (WX-LS) in An. sinensis. Importantly, the expression of significantly upregulated P450s was verified using RT-qPCR, and the function of selected P450s in pyrethroid detoxification was determined with RNA interference using four laboratory pyrethroid-resistant strains (WX-LR, AH-LR, CQ-LR, YN-LR). Sixteen P450 genes were significantly upregulated in at least one field-resistant population, and 44 were significantly upregulated in different developmental stages, tissues or post blood-meal. A total of 19 P450s were selected to verify their association with pyrethroid resistance, and four of them (AsCYP6P3v1, AsCYP6P3v2, AsCYP9J10, and AsCYP9K1) demonstrated significant upregulation in laboratory pyrethroid-resistant strains using RT-qPCR. Knockdown of these four genes all significantly reduced pyrethroid resistance and increased the mortality by 57.19% (AsCYP6P3v1 and AsCYP6P3v2 knockdown group), 38.39% (AsCYP9K1 knockdown group) and 48.87% (AsCYP9J10 knockdown group) in An. sinensis by RNAi, which determined the pyrethroid detoxification function of these four genes. This study revealed the diversity of P450 genes and provided functional evidence for four P450s in pyrethroid detoxification in An. sinensis for the first time, which increases our understanding of the pyrethroid resistance mechanism, and is of potential value for pyrethroid resistance detection and surveillance.

Development of resistance monitoring for Helicoverpa armigera (Lepidoptera: Noctuidae) resistance to pyramided Bt cotton in China.

The cotton bollworm, Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae), is a significant cotton pest worldwide. Bacillus thuringiensis (Bt) cotton producing Cry1Ac has been used since 1997 for the control of this pest in China and a significant increase in H. armigera resistance to Cry1Ac has occurred in northern China. To mitigate resistance evolution, it is necessary to develop and plant pyramided 2- and 3-toxin Bt cotton to replace Cry1Ac cotton. For sustainable use of pyramided Bt cotton, we used diet overlay bioassays to measure the baseline susceptibility of H. armigera to Cry2Ab in 33 populations collected in 2017, 2018, and 2021 in 12 locations from major cotton-producing areas of China. The lethal concentration killing 50% (LC50) or 99% (LC99) of individuals from the populations ranged from 0.030 to 0.138 µg/cm2 and 0.365 to 2.964 µg/cm2, respectively. The ratio of the LC50 for the most resistant and susceptible population was 4.6, indicating moderate among-population variability in resistance. The susceptibility of H. armigera to Cry2Ab did not vary significantly over years. A diagnostic concentration of 2 µg/cm2 was calculated as twice the LC99 from an analysis of pooled data for the field-collected populations. This concentration discriminated well between susceptible and resistant individuals, as it killed all larvae from a susceptible laboratory strain and 0%, 0%, and 23% of larvae from 3 laboratory strains with > 100-fold resistance to Cry2Ab. These baseline susceptibility data and diagnostic concentration for Cry2Ab will be useful for monitoring the evolution of H. armigera resistance to pyramided Bt cotton in China.

Extreme resistance to S-methoprene in field-collected Culex pipiens (Diptera: Culicidae) across the Chicago, IL region

Insect growth regulators, like S-methoprene, are heavily relied upon worldwide for larval mosquito chemical control due to their target specificity and long-lasting effects. In this study, susceptibility to S-methoprene was evaluated in Culex pipiens, a globally important vector species. Populations from 14 sites throughout the Chicago area with a long history of S-methoprene use and two sites with minimal use in Wisconsin were examined. Using a bioassay methodology and probit analyses, LC50 and LC90 values were calculated and compared to a susceptible laboratory strain to develop resistance ratios, then categorized for resistance intensity. The resistance ratios observed required the addition of another category, termed ‘extreme’ resistance, indicating resistance ratios greater than 100. ‘Low’ to ‘extreme’ levels of resistance to S-methoprene were detected throughout Illinois populations, with resistance ratios ranging from 2.33 to 1010.52. Resistance was not detected in populations where S-methoprene pressure has been very limited. These 'extreme’ resistance ratios observed have never been documented in a wild vector species mosquito population. The relationships between historical S-methoprene use, resistance detected with laboratory bioassays, and the potential for field product failure remain unclear. However, the profound resistance detected here demonstrates a potential critical threat to protecting public health from mosquito-borne diseases.

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.