Insecticide-susceptible Strain Research Articles

The effect of larval exposure to acids and detergents on the life history of the major malaria vector Anopheles arabiensis Patton (Diptera: Culicidae).

Anopheles arabiensis, a highly adaptable member of the Anopheles gambiae complex, poses a challenge for control efforts due to its outdoor biting and resting behaviour. Consequently, indoor insecticide-based control methodsare ineffective against An. arabiensis. Furthermore, An. arabiensis are adapting to breeding in polluted waters, and may be contributing to residual malaria and malaria in urban areas. There have been some advances in understanding the effect of rural pollutants on Anopheles mosquitoes, but the effect of urban pollutants is poorly understood. Thus, in this study, the effect of acidic pollutants [nitric acid (HNO3) and hydrochloric acid (HCl)] and alkaline pollutants (phosphate-free and phosphate-containing detergent) on two laboratory-reared An. arabiensis strains - an insecticide susceptible strain (SENN) and an insecticide-resistant strain selected from SENN (SENN-DDT) - were determined. The median lethal concentration (LC50) and larval exposure on larval development, adult longevity and insecticide tolerance were evaluated. Nitric acid and phosphate-containing detergent were found to be more toxic than HCl and phosphate-free detergent in terms of LC50 values. Detergent exposure (both phosphate-containing and phosphate-free) increased adult longevity of both strains. Nitric acid reduced larval development time in both SENN and SENN-DDT, whereas HCl reduced larval development time in SENN only. By contrast, both phosphate-containing and phosphate-free detergents increased larval development time of both strains. Furthermore, HNO3 and phosphate-containing detergent increased insecticide tolerance the most. The two An. arabiensis strains responded to urban pollutants differently. Thus, this study provides insight into the adaptation of An. arabiensis to acidic and alkaline urban pollutants. © 2024 The Author(s). Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Mechanisms of transcriptional regulation in Anopheles gambiae revealed by allele-specific expression.

Malaria control relies on insecticides targeting the mosquito vector, but this is increasingly compromised by insecticide resistance, which can be achieved by elevated expression of detoxifying enzymes that metabolize the insecticide. In diploid organisms, gene expression is regulated both in cis, by regulatory sequences on the same chromosome, and by trans acting factors, affecting both alleles equally. Differing levels of transcription can be caused by mutations in cis-regulatory modules (CRM), but few of these have been identified in mosquitoes. We crossed bendiocarb-resistant and susceptible Anopheles gambiae strains to identify cis-regulated genes that might be responsible for the resistant phenotype using RNAseq, and CRM sequences controlling gene expression in insecticide resistance relevant tissues were predicted using machine learning. We found 115 genes showing allele-specific expression (ASE) in hybrids of insecticide susceptible and resistant strains, suggesting cis-regulation is an important mechanism of gene expression regulation in A. gambiae. The genes showing ASE included a higher proportion of Anopheles-specific genes on average younger than genes with balanced allelic expression.

Piperonyl butoxide elicits a robust transcriptional response in adult Drosophila melanogaster

While much attention has been devoted to understanding the transcriptional changes underlying resistance to insecticides, comparatively little is known about the transcriptional response of naive insects to agrochemicals. In this study, we analyze the transcriptomic response of an insecticide susceptible strain of Drosophila melanogaster to nine agrochemicals using a robust method that goes beyond classical replication standards. Our findings demonstrate that exposure to piperonyl butoxide (PBO), but not to eight other compounds, elicits a robust transcriptional response in a wild-type strain of Drosophila melanogaster. PBO exposure leads to the upregulation of a subset of Cyps, GSTs, UGTs and EcKls. This response is both time and concentration-dependent, suggesting that the degree of inhibition of P450 activity correlates with the magnitude of the transcriptional response. Furthermore, the upregulation of these enzymes is excluded from reproductive organs. Additionally, different sets of genes are regulated in the digestive/secretory tract and the carcass. Our results suggest that P450s play a role in metabolizing yet unidentified endogenous compounds and are involved in an as-yet-unknown physiological regulatory feedback loop.

Transcriptome mining and expression analysis of ABC transporter genes in a monophagous herbivore, Leucinodes orbonalis (Crambidae: Lepidoptera)

Insecticide resistance is a global concern and requires immediate attention to manage dreadful insect pests. One of the resistance mechanisms adopted by insects is through ATP-binding cassette (ABC) transporter proteins. These proteins rapidly transport and eliminate the insecticidal molecules across the lipid membranes (Phase III detoxification mechanism). In the present study, we investigated the potential role of ABC transporter genes in imparting insecticide resistance in field-collected insecticide resistant larvae of eggplant shoot and fruit borer (Leucinodes orbonalis; Crambidae: Lepidoptera). Dose-mortality bioassays against five insecticidal molecules revealed moderate to high levels of insecticide resistance (32.2. to 134.1-fold). Thirty-one genes encoding ABC transporter proteins were mined from the transcriptome resources of L. orbonalis. They were classified under eight sub-families (ABCA to ABCH). Phylogenetic analysis indicated ABCG is the most divergent, composed of nine genes as compared to many other insects. Transcriptome analysis of the insecticide resistant and susceptible strains of L. orbonalis revealed differential expression of 13 ABC transporter genes. The altered expression of these genes was further validated using qRT-PCR. The knockdown studies indicated the involvement of ABCD1 and ABCG2 genes in chlorantraniliprole resistance in the insecticide-resistant strain of L. orbonalis. This study unveils the additional mechanisms employed by L. orbonalis in resisting insecticide toxicity through accelerated excretion mode. These ABCD and ABCG family genes could be candidate targets in developing genome-assisted pest management strategies in the future.

Enhancing toxicity of pyrethroids by oxidase and esterase inhibitors in Spodoptera littoralis (Boisd.) larvae

Although pyrethroids are increasingly being used to control a number of agricultural insect pests, especially the cotton leafworm, Spodoptera littoralis (Boisd.), pyrethroid resistance is a major obstacle limiting effective control. With the aim of maintaining the effectiveness of pyrethroids in managing pests, a study was undertaken to evaluated the effectiveness of oxidase and esterase inhibitors for synergizing pyrethroids in S. littoralis larvae. Compared with the insecticide-susceptible strain (L-SS) of S. littoralis, the resistance ratio (RR) in the field population (F-RS) was 271.43–fold to cypermethrin. The use of profenofos as an esterase inhibitor significantly increased larval susceptibility to cypermethrin in the F-RS strain, with a synergy ratio (SR) of up to 192.57–fold. Significant inhibition of esterase by profenofos in the F-RS strain was found in vivo. Also, piperonyl butoxide (PB) as an oxidase inhibitor had slight effect of cypermethrin toxicity, so its addition is not a solution for pyrethroid resistance. Thus, modifying the toxicity of cypermethrin by mixing it with organophosphorus compounds (OPs) increased its toxicity and decreased the population of S. littoralis, which is a successful strategy for managing pyrethroid resistance.

Characterization of the Tissue and Strain-Specific Microbiota of Anopheles funestus Giles (Diptera: Culicidae).

The mosquito microbiota is a critical determinant of mosquito life history. It is therefore a target for novel vector control strategies like paratransgenesis. However, the microbiota in Anopheles funestus, a major African malaria vector, is poorly characterized. Thus, the study aimed to investigate the overall bacterial landscape in the salivary glands, ovaries and midguts of three laboratory strains of An. funestus differing in insecticide-resistant phenotype by sequencing the V3-V4 hypervariable region of bacterial 16S rRNA genes. When examining alpha diversity, the salivary glands harbored significantly more bacteria in terms of species richness and evenness compared to ovaries and midguts. On the strain level, the insecticide-susceptible FANG strain had significantly lower bacterial diversity than the insecticide-resistant FUMOZ and FUMOZ-R strains. When looking at beta diversity, the compositions of microbiota between the three tissues as well as between the strains were statistically different. While there were common bacteria across all three tissues and strains of interest, each tissue and strain did exhibit differentially abundant bacterial genera. However, overall, the top five most abundant genera across all tissues and strains were Elizabethkingia, Acinetobacter, Aeromonas, Cedecea and Yersinia. The presence of shared microbiota suggests a core microbiota that could be exploited for paratransgenesis efforts.

Monitoring of Thiamethoxam Resistance in Turkish House Fly Strains, Musca domestica (Diptera: Muscidae).

The house fly, Musca domestica, is vector for pathogens and parasites and causes economic damage to livestock by reducing forage conversion efficiency, negatively impacting weight gain and milk production. It has shown resistance to multiple insecticide classes. The aim of this research was to determine the susceptibility levels of seventeen field M. domestica strains to thiamethoxam, a neonicotinoid insecticide, in Türkiye. Insecticide susceptibility of the house flies to thiamethoxam was determined using the WHO glass jar method. A probit analysis program was used to determine LD50 values, and then the resistance ratios were compared with insecticide-susceptible strain. All strains were ≥18.5-fold resistant to thiamethoxam. The data showed that 10 out of 17 strains had either high or very high resistance levels. Our findings revealed that house flies from solid waste landfills in Samsun, Ankara, and Kocaeli exhibited higher resistance ratios compared to those found in animal shelters. Conversely, in Gaziantep, Antalya, İzmir and Erzurum, the exact opposite trend was observed. Regarding the LD50 values among solid waste storage areas, the lowest rate was obtained from Gaziantep (0.72 gr ai/m2), and the highest rate was obtained from Ankara (9.35 gr ai/m2). Furthermore, regarding the LD50 values among animal shelters, the lowest was obtained from Samsun (0.37 gr ai/m2), and the highest was obtained from Denizli (21800 gr ai/m2). The use of integrated control systems is recommended for controlling house fly populations, including insecticide class rotations for preventing, or at least, delaying the onset of resistance.

Differences in excito-repellent behavior between susceptible and permethrin-resistant strains of Aedes aegypti upon exposure to insecticide-treated fabric.

An important component of the biological activity of pyrethroids, when used in disease vector control, is excito-repellency. In this study, behavioral differences between insecticide susceptible (Orlando) and pyrethroid resistant (Puerto Rican) strains of Aedes aegypti were explored in a round glass arena using fabrics treated with permethrin, etofenprox, deltamethrin, or DDT. Repellency was evaluated across several variables, including the time to first flight (TFF), number of landings (NOL), total flight time (TFT), and maximum surface contact (MSC), all by video analysis. Results from the Orlando strain indicated they were less likely than the Puerto Rican strain to tolerate tarsal contact with the treated fabrics. All four response variables indicated that the mosquito flight and landing behavior was most affected by pyrethroid resistance [knockdown resistance (kdr)] status. In other experiments, mosquitoes were surgically altered, with antennae ablated bilaterally, and these mosquitoes were more likely to stay on the treated surfaces for longer periods of time, irrespective of any chemical exposure. There were also differences in the responses to antennal ablation between the two strains of mosquitoes, indicating that resistance factors, probably kdr, influence the reactivity of mosquitoes to pyrethroid and DDT treatments, and that it was not completely negated by antennal ablation. These findings confirm the role of antennal olfactory components in the expression of excito-repellent behaviors, and also support the hypothesis that excito-repellency from pyrethroid/DDT exposure is probably due to a combination of sublethal neurotoxic excitation and interactions with the olfactory system. © 2023 Society of Chemical Industry.

Effects of next-generation, dual-active-ingredient, long-lasting insecticidal net deployment on insecticide resistance in malaria vectors in Tanzania: an analysis of a 3-year, cluster-randomised controlled trial

Insecticide resistance among malaria-vector species is a pervasive problem that might jeopardise global disease-control efforts. Novel vector-control tools with different modes of action, including long-lasting insecticidal nets (LLINs) incorporating new active ingredients, are urgently needed to delay the evolution and spread of insecticide resistance. We aimed to measure phenotypic and genotypic insecticide-resistance profiles among wild Anopheles collected over 3 years to assess the longitudinal effects of dual-active-ingredient LLINs on insecticide resistance. For this analysis, data nested in a 3-year, four parallel-arm, superiority cluster-randomised controlled trial (cRCT) in Tanzania, collected from 84 clusters (39 307 households) formed of 72 villages in the Misungwi district, were used to measure insecticide-resistance profiles among female Anopheles mosquitoes via insecticide-resistance bioassays and quantitative RT-PCR of metabolic-resistance genes. Wild, blood-fed, indoor-resting mosquitoes were collected annually during the rainy seasons from house walls in clusters from all four trial groups. Mosquitoes were morphologically identified as An gambiae sensu lato (SL) or An funestus SL before separate bioassay testing. The primary outcomes were lethal-dose values for α-cypermethrin, permethrin, and piperonyl butoxide pre-exposure plus permethrin-resistance intensity bioassays, mortality 72 h after insecticidal exposure for chlorfenapyr bioassays, fertility reduction 72 h after insecticidal exposure for pyriproxyfen bioassays, and fold change in metabolic-enzyme expression relative to an insecticide-susceptible laboratory strain. All primary outcomes were measured in An funestus SL 1 year, 2 years, and 3 years after LLIN distribution. Primary outcomes were also assessed in An gambiae SL if enough mosquitoes were collected. The cRCT is registered with ClinicalTrials.gov (NCT03554616). Between May 24, 2019, and Oct 25, 2021, 47 224 female Anopheles were collected for resistance monitoring. In the pyrethroid (PY)-LLIN group, there were significant increases in α-cypermethrin-resistance intensity (year 1 LD50=9·52 vs year 2 76·20, p<0·0001) and permethrin-resistance intensity (year 1 13·27 vs year 2 35·83, p=0·0019) in An funestus SL. In the pyriproxyfen PY-LLIN group, there was similar increase in α-cypermethrin-resistance intensity (year 1 0·71 vs year 2 81·56, p<0·0001) and permethrin-resistance intensity (year 1 5·68 vs year 2 50·14, p<0·0001). In the piperonyl butoxide PY-LLIN group, α-cypermethrin-resistance intensity (year 1 33·26 vs year 3 70·22, p=0·0071) and permethrin-resistance intensity (year 1 47·09 vs year 3 2635·29, p<0·0001) also increased over time. In the chlorfenapyr PY-LLIN group, there were no effects on α-cypermethrin-resistance intensity (year 1 0·42 vs year 3 0·99, p=0·54) or permethrin-resistance intensity (data were not estimable due to nearly 100% mortality). There were also minimal reductions in chlorfenapyr susceptibility. However, in the chlorfenapyr PY-LLIN group, a significant decline in piperonyl-butoxide synergy was seen by year 3 (year 1 0·02 vs year 3 0·26, p=0·020). Highly over-expressed detoxification enzymes showed dynamic patterns of selection throughout the trial. Our phenotypic data supports trial epidemiological findings; chlorfenapyr PY-LLINs provided superior protection from malaria across multiple transmission seasons, with few effects on insecticide-resistance selection. Rapid pyrethroid-resistance intensification in the piperonyl butoxide PY-LLIN group and pre-existing tolerance of pyriproxyfen in vector populations might explain the poorer performance of these two interventions regarding malaria outcomes. Further work is required to elucidate the potential mechanisms driving cross-resistance between pyrethroids and novel active ingredients to better inform the design of pre-emptive resistance-management strategies. UK Department for International Development; UK Medical Research Council; Wellcome Trust; UK Department of Health and Social Care; UK Foreign, Commonwealth and Development Office; and The Bill and Melinda Gates Foundation via the Innovative Vector Control Consortium.

Mode of inheritance of field-evolved resistance to pirimiphos-methyl in Sitophilus oryzae (Linnaeus) (Coleoptera: Curculionidae)

Sitophilus oryzae is an economic pest of stored grain worldwide with the ability to transmit mycotoxigenic fungi in the infested commodities. Recently this pest has shown field-evolved resistance to pirimiphos-methyl in Pakistan. Understanding mode of inheritance of insecticide resistance is of prime importance in order to implement a successful resistance management program. Here, we studied the mode of inheritance of resistance to pirimiphos-methyl in two field-evolved resistant strains (LR-SO and HR-SO) of S. oryzae. LR-SO and HR-SO strains exhibited 31.05 and 219.18 fold resistance to pirimiphos-methyl compared with an insecticide susceptible reference strain (Lab-SO). Reciprocal progenies revealed an indistinguishable difference in their response (LC50s) to pirimiphos-methyl pointing out the absence of maternal effects or sex-linkage and autosomal inheritance pattern. The degree of dominance values for all the reciprocal and pooled progenies ranged from −0.47 to −0.78 suggesting that resistance to pirimiphos-methyl was expressed as an incompletely recessive trait. Chi-square and log-dose probit line analyses of self-bred and backcross progenies exhibited significant variation between the expected and observed mortality, which suggest that resistance was governed by more than one gene. In conclusion, field-evolved resistance to pirimiphos-methyl in S. oryzae was autosomal, incompletely recessive, and multigenic. Our results provide basic information about the nature of resistance to pirimiphos-methyl that will be helpful to formulate successful management strategies for controlling S. oryzae and to alleviate the allied threats to environment and human health.

Reduced synergistic efficacy of piperonyl butoxide in combination with alpha-cypermethrin in vitro in an insecticide-resistant strain of the sheep blowfly, Lucilia cuprina

Control of flystrike on sheep relies on the use of insecticides. The present study used in vitro assays to examine the potential for increasing the efficacy of synthetic pyrethroids against sheep blowfly larvae using the synergist piperonyl butoxide (PBO). We examined the potency of alpha-cypermethrin (ACP) / PBO combinations against a reference insecticide-susceptible strain (LS) and a field-derived strain showing resistance to dicyclanil and imidacloprid. Co-treatment of the insecticide-susceptible strain with ACP/PBO resulted in increasing levels of synergism as the PBO concentration was increased, with synergism ratios (SRs) of up to 114-fold. Treatment with PBO/ACP combinations at ratios of 20:1 and 5:1 resulted in significant levels of synergism: SRs of 13.5- and 7.6-fold, respectively. However, the levels of synergism were significantly less for the insecticide-resistant strain: SRs of 4.6- and 2.6-fold for the 20:1 and 5:1 ratios, respectively. The resistant strain showed no resistance to ACP when administered alone, however, was 2-fold less sensitive than the LS strain to the toxic effects of PBO alone. This insensitivity to PBO was removed by co-treatment with the P450 inhibitor aminobenzotriazole, suggesting an increased level of P450-mediated metabolism of the PBO in this strain compared to the LS strain, and hence providing a likely explanation for the reduced synergistic efficacy of PBO on ACP toxicity in the resistant strain. While PBO was able to synergise ACP with both of the blowfly strains examined here, the reduced synergistic efficacy observed with the field-derived insecticide-resistant strain lessens the potential usefulness of such a combination for blowfly control in the field.

Potential of ivermectin as an active ingredient of the attractive toxic sugar baits against the Indian malaria vectors Anopheles culicifacies and Anopheles stephensi.

Attractive toxic sugar bait (ATSB) is a novel vector control tool that exploits the sugar feeding behavior of mosquitoes. The current study aims to evaluate the efficacy of ivermectin-based ATSB against insecticide susceptible and resistant strains of major Indian malaria vectors - Anopheles culicifacies and Anopheles stephensi. ATSB with different concentrations of ivermectin were tested against mosquito vectors under standard laboratory conditions. Dose-response analysis of ivermectin-ATSB showed 7.8 and 19.8ppm as 50% and 90% lethal concentration (LC50 and LC90 ) values for insecticide susceptible An. culicifacies. In the case of insecticide susceptible An. stephensi, the LC90 value was 35 ppm which was significantly higher in comparison to the LC90 for An. culicifacies. The LC50 of insecticide-resistant An. culicifacies and An. stephensi were 10.6 and 15.9ppm respectively whereas LC90 values were 36.9 and 61.0. Ivermectin-ATSB resulted in 99 ± 0.8% mortality of An. culicifacies and 93 ± 3.8% mortality of An. stephensi at an ivermectin concentration of 25 ppm. In another set of experiments, the ATSB solution containing standardized dose of ivermectin was sprayed on Allysum plant and mortality of both Anopheline vectors was recorded. Here, we observed > 90% mortality in both An. stephensi and An. culicifacies. Our study demonstrates the potential of ivermectin-based ATSB in killing Indian malaria vectors irrespective of the method of application. Further field trials with ivermectin containing ATSB may pave the way for its usage in the national vector control program. © 2022 Society of Chemical Industry.

Brazilian Populations of Aedes aegypti Resistant to Pyriproxyfen Exhibit Lower Susceptibility to Infection with Zika Virus.

Zika virus (ZIKV) infection has caused devastating consequences in Brazil as infections were associated with neurological complications in neonates. Aedes aegypti is the primary vector of ZIKV, and the evolution of insecticide resistance (IR) in this species can compromise control efforts. Although relative levels of phenotypic IR in mosquitoes can change considerably over time, its influence on vector competence for arboviruses is unclear. Pyriproxyfen (PPF)-resistant populations of Ae. aegypti were collected from five municipalities located in Northeast of Brazil, which demonstrated different resistance levels; low (Serrinha, Brumado), moderate (Juazeiro do Norte, Itabuna), and high (Quixadá). Experimental per os infection using ZIKV were performed with individuals from these populations and with an insecticide susceptible strain (Rockefeller) to determine their relative vector competence for ZIKV. Although all populations were competent to transmit ZIKV, mosquitoes derived from populations with moderate to high levels of IR exhibited similar or lower susceptibility to ZIKV infection than those from populations with low IR or the susceptible strain. These observations suggest an association between IR and arbovirus infection, which may be attributable to genetic hitchhiking. The use of PPF to control Brazilian Ae. aegypti may be associated with an indirect benefit of reduced susceptibility to infection, but no changes in disseminated infection and transmission of ZIKV among PPF-resistant phenotypes.

Resistance to grain protectants and synergism in Pakistani strains of Sitophilus oryzae (Coleoptera: Curculionidae)

The widespread use of insecticides for the management of insect pests in storage facilities and food industries have caused insecticide resistance a frequent issue worldwide. Nonetheless, this issue has been little explored in Pakistan that resulted in control failures and increased dosage of insecticides. In the present study, insecticide resistance to chlorpyrifos-methyl, pirimiphos-methyl, permethrin and spinosad was surveyed in five field strains of Sitophilus oryzae: FSD-SO, GJR-SO, DGK-SO, MTN-SO and BWP-SO, collected from five different localities of Punjab, Pakistan, and contrasted with an insecticide susceptible reference strain (Lab-SO). Dose-mortality bioassays were performed in glass vials containing insecticide-treated rice grains, and lethal doses (LD50 and LD95) were calculated and compared using the ratio tests. In comparison to the Lab-SO strain at LD50 and LD95 levels, field strains exhibited: 24.51 to 52.80 and 36.55 to 69.31 resistance ratios (RRs), respectively, for chlorpyrifos-methyl; 15.89 to 45.97 and 55.12 to 194.93 RRs, respectively, for pirimiphos-methyl; 39.76 to 108.61 and 61.33 to 130.12 RRs, respectively, for permethrin; 4.23 to 27.50 and 6.28 to 41.00 RRs, respectively, for spinosad. In the synergism experiments using the Lab-SO and the most resistant strains against each insecticide, the enzyme inhibitors (PBO and DEF) failed to synergize toxicity of insecticides in the Lab-SO strain; however, toxicity of chlorpyrifos-methyl, pirimiphos-methyl and permethrin significantly enhanced in the resistant strains of S. oryzae, suggesting possibility of metabolic mechanism of resistance. In addition, activities of detoxification enzymes (CarE, MFO and GST) were significantly higher in resistant strains compared to the Lab-SO strain. The results revealed presence of insecticide resistance in field strains of S. oryzae that necessitate the need to develop a resistance management strategy.

Новые стратегии борьбы с резистентными к инсектицидам рыжими тараканами

In a review of foreign literature, the issues of biological methods for controlling synanthropic cockroaches, including the use of entomopathogenic fungi such as Metarhizium and Beauveria, are considered, due to high biodegradability and species-specificity, is a potential alternative to the chemical method. It has been shown that the combination of chemical and biological agents in cockroach control can slow down the emergence of insect resistance and enhance the insecticidal effect. The densonucleosis viruses BgDNV and PfDNV may be required for the biological control of cockroaches due to such features as rapid replication, high specificity, and pathogenicity for the host. In view of the close relationship between symbiotic microorganisms and host physiology, insect control strategies using transgenic symbionts have become the subject of research. Significant differences in the composition of the intestinal microbiota between the pyrethroid-resistant and insecticide-susceptible strains of the German cockroach were found. A new potential direction for insect resistance management has been identified. Keywords: entomopathogenic fungi, German cockroach, microbiota, symbiotic microorganisms.

Mode of action and toxicological effects of the sesquiterpenoid, nootkatone, in insects

Nootkatone, a sesquiterpenoid isolated from Alaskan yellow cedar (Cupressus nootkatensis), is known to possess insect repellent and acaricidal properties and has recently been registered for commercial use by the Environmental Protection Agency. Previous studies failed to elucidate the mechanism of action of nootkatone, but we found a molecular overlay of picrotoxinin and nootkatone indicated a high degree of structural and electrostatic similarity. We therefore tested the hypothesis that nootkatone was a GABA-gated chloride channel antagonist, similar to picrotoxinin. The KD50 and LD50 of nootkatone on the insecticide-susceptible strain of Drosophila melanogaster (CSOR) showed resistance ratios of 8 and 11, respectively, compared to the cyclodiene-resistant strain of RDL1675, indicating significant cross-resistance. Nootkatone reversed GABA-mediated block of the larval CSOR central nervous system; nerve firing of 78 ± 17% of baseline in the CSOR strain was significantly different from 24 ± 11% of baseline firing in the RDL1675 strain (p = 0.035). This finding indicated that the resistance was expressed within the nervous system. Patch clamp recordings on D. melanogaster central neurons mirrored extracellular recordings where nootkatone inhibited GABA-stimulated currents by 44 ± 9% at 100 μM, whereas chloride current was inhibited 4.5-fold less at 100 μM in RDL1675. Taken together, these data suggest nootkatone toxicity in D. melanogaster is mediated through GABA receptor antagonism.

Insecticidal effect of ethnobotanical plant extracts against Anopheles arabiensis under laboratory conditions

BackgroundThe emergence and spread of resistant strains of malaria vectors to chemical insecticides are becoming major problems for malaria vector management. Natural plant products have a vital role to play in the current challenge of malaria control. The current study was conducted to evaluate insecticidal effect of ethnobotanical plant extracts against the primary malaria vector, Anopheles arabiensis in northwestern Ethiopia.MethodsPrimarily, ethnobotanical plants used for Anopheles mosquito control were surveyed in Dangur district, northwestern Ethiopia. Insecticide-susceptible strains of Anopheles arabiensis mosquito were reared in the insectary of the Tropical and Infectious Diseases Research Centre, Assosa University. After surveying plants used for mosquito control in local people, four frequently used plants were identified for extraction. The larvicidal and adulticidal potential of frequently used plant extracts against susceptible strains of the laboratory colony were evaluated.ResultsA total of 15 plants were identified as ethnobotanical plants that help local people with mosquito control. Azadirachta indica, Ocimum lamiifolium, Ocimum americanum, Moringa olifeira leaf, and Moringa olifeira seed species of local plants were found to be frequently used to kill and/or repel mosquitoes in the study district. All the plant extracts were found to have potential larvicidal activity against fourth instar larvae of An. arabiensis and only ethanol and methanol extract of Azadirachta indica and Ocimum lamiifolium were found to have potential adulticidal effect against adult of An. arabiensis. The highest larvicidal activity was observed in ethanol extract of Azadirachta indica with 95% larval mortality and lowest Lethal Concentration 50 (LC 50) of 40.73parts per million (ppm) and LC90 of 186.66 ppm. The highest adulticidal activity was observed in methanol extract of Azadirachta indica with 75% adult mortality at 300 ppm and lowest LC50 of 106.65 ppm and LC90 of 1,293 ppm. The lowest larvicidal and adulticidal activity was observed in methanol extracts of Ocimum lamiifolium with 63.35% larval mortality and leaf extract of Moringa olifeira with 50% adult mortality at 300 ppm, respectively.ConclusionEthanol extract of Azadirachta indica exerted a remarkable larvicidal effect against An. arabiensis and thus it can be used for botanical mosquito insecticide development. Since the current finding is based on susceptible strain of An. arabiensis, further work on wild mosquitoes is recommended.

Characterisation of the epigenetic architecture of the major malaria vector Anopheles arabiensis (Diptera: Culicidae) after treatment with epigenetic modulators and heavy metals

Anopheles arabiensis (a member of the An. gambiae species complex) is a major vector of malaria in sub-Saharan Africa. Despite its disease vector status, there is currently a paucity of epigenetic information for this species. The aim this study was therefore to analyse global epigenetic markers and their response to metal exposure in insecticide susceptible and resistant laboratory strains of An. arabiensis. This was done using commercially available epigenetic marker quantification kits. In order to validate the efficacy of the kits, several kits were assessed to determine whether changes induced by known epigenetic modulators were detectable using these platforms. The efficacy of the dosages used were determined by examining the effect of the dosages used on insecticide resistant phenotypes. Upon confirmation that the dosages used were sufficient to induce a phenotypic change, the effect on epigenetic markers was assessed. Commercial kits were used to quantify 5-methylcysteine (5-mC) and 5-hydroxymethylcysteine (5-hmC) methylation in DNA, m6A methylation in mRNA as well as Histone Acetyl Transferase (HAT) activity. There was a marked difference in the phenotypic response in adult mosquitoes of the insecticide susceptible strain compared to that of its’ resistant counterpart. For males and females of the resistant strain, exposure to nucleic acid modifying drugs typically increased their tolerance to insecticides. The patterns of changes in 5-mC methylation by epigenetic modulators was congruent with previous studies which quantified by mass spectrometry. The two strains differed in methylation patterns under control conditions and responded differentially to larval metal exposure. In the resistant strain, which previously was demonstrated to show increased detoxification enzyme activity and insecticide tolerance after the same treatment, the potential increase in transcriptional activity appeared to be modulated by reduced methylation and increased HAT activity. This study suggests that the commercial epigenetic quantification kits can be used to characterise phenotypic changes in An. arabiensis, and also shows that epigenetic regulation of the response to metal exposure is regulated at the DNA as opposed to the RNA level.

Negative cross-resistance of a pyrethroid-resistant Drosophila mutant to Phryma leptostachya-derived haedoxan A.

Voltage-gated sodium channels are the primary target of pyrethroid insecticides. Mutations in sodium channel confer knockdown resistance (kdr) to pyrethroids in various arthropod pests. Haedoxan A (HA) is the major insecticidal component from Phryma leptostachya. It has been shown that HA alters electrical responses at the Drosophila neuromuscular junction and modifies the gating properties of cockroach sodium channels expressed in Xenopus oocytes. However, whether sodium channel mutations that confer pyrethroid resistance also affect the action of HA is unknown. In this study, we conducted bioassays using HA and permethrin in two Drosophila melanogaster strains: w1118 , an insecticide-susceptible strain, and parats1 , a pyrethroid-resistant strain due to a I265N mutation in the sodium channel, and identified a new case of negative cross-resistance (NCR) between permethrin and HA. Both parats1 larvae and adults were more resistant to permethrin, as expected. However, both parats1 larvae and adults were more sensitive to HA compared to w1118 . We confirmed that the I265N mutation reduced the sensitivity to permethrin of a Drosophila sodium channel variant, DmNav 22, expressed in Xenopus oocytes. Interestingly, the I265N mutation also abolished the effect of HA on sodium channels. Further characterization showed that I265 on the sodium channels is critical for the action of both pyrethroids and HA on sodium channels, pointing to an overlapping mode of action between pyrethroids and HA on the sodium channel. Overall, our results suggest an I265N-independnt mechanism(s) in parats1 flies that is responsible for the NCR between permethrin and HA at the whole insect level.

A whole transcriptomic approach provides novel insights into the molecular basis of organophosphate and pyrethroid resistance in Anopheles arabiensis from Ethiopia

The development of insecticide resistance in malaria vectors is of increasing concern in Ethiopia because of its potential implications for vector control failure. To better elucidate the specificity of resistance mechanisms and to facilitate the design of control strategies that minimize the likelihood of selecting for cross-resistance, a whole transcriptomic approach was used to explore gene expression patterns in a multi-insecticide resistant population of Anopheles arabiensis from Oromia Region, Ethiopia. This field population was resistant to the diagnostic doses of malathion (average mortality of 71.9%) and permethrin (77.4%), with pools of survivors and unexposed individuals analyzed using Illumina RNA-sequencing, alongside insecticide susceptible reference strains. This population also demonstrated deltamethrin resistance but complete susceptibility to alpha-cypermethrin, bendiocarb and propoxur, providing a phenotypic basis for detecting insecticide-specific resistance mechanisms. Transcriptomic data revealed overexpression of genes including cytochrome P450s, glutathione-s-transferases and carboxylesterases (including CYP4C36, CYP6AA1, CYP6M2, CYP6M3, CYP6P4, CYP9K1, CYP9L1, GSTD3, GSTE2, GSTE3, GSTE4, GSTE5, GSTE7 and two carboxylesterases) that were shared between malathion and permethrin survivors. We also identified nineteen highly overexpressed cuticular-associated proteins (including CYP4G16, CYP4G17 and chitinase) and eighteen salivary gland proteins (including D7r4 short form salivary protein), which may be contributing to a non-specific resistance phenotype by either enhancing the cuticular barrier or promoting binding and sequestration of insecticides, respectively. These findings provide novel insights into the molecular basis of insecticide resistance in this lesser well-characterized major malaria vector species.