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Aedes aegypti (Linnaeus) plays an important role as a vector of different deadly diseases particularly dengue fever. Insecticides are used as a primary tool to control Ae. aegypti. However, due to the excessive use of insecticides on agricultural, public health, and industrial levels, mosquitoes have developed resistance. In this study, the current susceptibility status of Ae. aegypti mosquitoes against different insecticides (Temephos, DDT, dieldrin, Malathion, Bendiocarb, Permethrin, Cypermethrin, and Lambda-cyhalothrin) was evaluated in district Lahore and district Muzaffargarh of Punjab, Pakistan. For this purpose, WHO bioassays and biochemical assays were performed on Ae. aegypti population from Lahore (APLa) and Aedes population from Muzaffargarh (APMg). Results of APLa and APMg showed high levels of resistance against the larvicide Temephos. Resistance against all adulticides was also observed in APLa and APMg (% mortality < 98%). The biochemical assays indicated statistically significant elevated levels of detoxification enzymes in APLa and APMg. APLa showed slightly higher levels as compared to APMg. Mosquitoes were also screened for the presence of kdr mutations. The results revealed no mutation in domain II while the presence of mutation F1534C in domain III was found in both field populations. The results showed the presence of moderate to high grade resistance against all insecticides in Ae. aegypti in district Lahore and district Muzaffargarh of Punjab, Pakistan.

Aedes aegypti , the primary vector of dengue, has developed widespread resistance to insecticides, posing a threat to the efficacy of vector control programs. This study assessed the susceptibility status of Ae. aegypti across Malaysian dengue hotspots and characterized knockdown resistance ( kdr ) mutations, including the first detection of T1520I. Adult female mosquitoes were collected from seven localities across five Malaysian states. Susceptibility to deltamethrin (0.03%), permethrin (0.4%), pirimiphos-methyl (60 mg/m 2 ), and malathion (5%) was evaluated using bioassay protocols. Knockdown times (KT 50 , KT 95 ) and resistance ratios (RR) were estimated using probit analysis, and the voltage-gated sodium channel gene was sequenced to detect kdr mutations. All field populations remained susceptible to malathion, with mortality ≥ 95%, though one Johor strain (ABS) showed suspected resistance (95%). Pirimiphos-methyl resistance was widespread, with mortality as low as 6% (ABS). Whereas resistance to pyrethroids was pronounced, with deltamethrin mortality ranging from 22% (ABS) to 95% (AA), and permethrin from 0% (TMB, ABS) to 95% (AA). Knockdown assays revealed high resistance ratios, particularly for permethrin (TMB: RR 50 =9.98, RR 95 =14.98; ABS: RR 95 =41.46). Sequencing identified multiple kdr mutations, including S989P, V1016G, F1534C, A1007G, and the novel detection of T1520I in Malaysian populations. F1534C was strongly associated with permethrin resistance, while V1016G and S989P predominated under deltamethrin exposure. Aedes aegypti populations in Malaysia exhibit widespread pyrethroid resistance and emerging organophosphate resistance, underpinned by multiple kdr mutations. The first detection of T1520I underscores the evolving genetic basis of resistance. These findings highlight the urgent need for integrated resistance management, including molecular surveillance and insecticide rotation, to sustain effective dengue control.

Introduction The Plasmodium transmission-blocking endosymbiont Microsporidia MB was previously identified in Anopheles gambiae s.l., but its association with the carriage of the genotypes of the L1014F kdr mutation, as well as the ecological factors driving its geographical distribution remain understudied. Methods Adult mosquitoes were field-collected using human landing catches (HLCs) across 60 villages in the Covè, Ouinhi, and Zangnanado communes of southern Benin. After morphological identification, a sub-sample of An. gambiae s.l. were molecularly speciated, and genotypied for the L1014F kdr mutation by Polymerase Chain Reaction (PCR). Enzyme-Linked Immunosorbent Assay (ELISA) and qPCR were also used to assess infection with Plasmodium falciparum sporozoites and Microsporidia MB, respectively. The environmental variables that drive the habitat suitability for Microsporidia MB were also assessed using Maximum Entropy (MaxEnt) modelling. Results The An. gambiae complex ( N  = 1040) was composed of 93.7% An. coluzzii , 4.4% An. gambiae s.s., 0.2% An. gambiae s.s./ coluzzii hybrids, while the rest failed to amplify. Infection prevalence with Microsporidia MB was 1.6% (95% CI: 0.7–3.3) in An. coluzzii and 2.2% (95% CI: 0.1–13.2) in An. gambiae s.s. The P. falciparum sporozoite rate was 2% (95% CI: 1.2–3.1, N  = 974) in An. coluzzii , and null in An. gambiae s.s. ( N  = 46). None of the mosquitoes infected with Microsporidia MB were infected with P. falciparum . The frequency of the L1014F kdr mutation was 75.1% (95% CI: 73.1–76.9) in An. coluzzii and 91.3% (95% CI: 83.1–95.9) in An. gambiae s.s. Microsporidia MB was absent in kdr -SS mosquitoes but was present in low proportions in both kdr -RS and kdr -RR mosquitoes (1.9%, 95% CI: 0.6–5.1). The mean load of Microsporidia MB DNA was higher in kdr -RR (23.23 ng/µl, 95% CI: 18.77–28.48) compared to kdr -RS (10.13 ng/µl, 95% CI: 7.36–13.91) mosquitoes. The elevation and soil contributed to explain, at 78% and 20% respectively, the habitat suitability for Microsporidia MB. Conclusion In this study, we demonstrated that An. gambiae s.l. mosquitoes bearing the L1014F kdr resistant allele was associated with a higher prevalence and load of Microsporidia MB than their susceptible counterparts. Moreover, the geographical distribution of Microsporidia MB was found to be influenced by certain environmental conditions, which warrant further large-scale investigations.

The Ae. albopictus mosquito has gained global attention due to its ability to transmit viruses, including the dengue and zika. Mosquito control is the only effective way to manage dengue fever, as no effective treatments or vaccines are available. Insecticides are highly effective in controlling mosquito densities, which reduces the chances of virus transmission. However, Ae. albopictus has developed resistance to pyrethroids in several provinces in China. Pyrethroids target the voltage-gated sodium channel gene ( VGSC ), and mutations in this gene may result in knockdown resistance ( kdr ). Correlation studies between resistance and mutations can assist viruses in managing Ae. albopictus , which has not been studied in Guizhou province. Nine field populations of Ae. albopictus at the larval stage were collected from Guizhou Province in 2022 and reared to F1 to F2 generations. Resistance bioassays were conducted against permethrin, beta-cypermethrin, and deltamethrin for both larvae and adults of Ae. albopictus . Kdr mutations were characterized by PCR and sequencing. Additionally, the correlation between the kdr allele and pyrethroid resistance was analyzed. All nine populations of Ae. albopictus larvae and adults were found to be resistant to three pyrethroid insecticides. One kdr mutant allele at codon 1016, one at 1532 and three at 1534 were identified with frequencies of 13.86% (V1016G), 0.53% (I1532T), 58.02% (F1534S), 11.69% (F1534C), 0.06% (F1534L) and 0.99% (F1534P), respectively. Both V1016G and F1534S mutation mosquitoes were found in all populations. The kdr mutation F1534S was positively correlated with three pyrethroid resistance phenotypes (OR > 1, P  < 0.05), V1016G with deltamethrin and beta-cypermethrin resistance (OR > 1, P  < 0.05) and F1534C only with beta-cypermethrin resistance (OR > 1, P  < 0.05). Current susceptibility status of wild populations of Ae. albopictus to insecticides and a higher frequency of kdr mutations from dengue-monitored areas in Guizhou Province are reported in this paper. Outcomes of this study can serve as data support for further research and development of effective insecticidal interventions against Ae. albopictus populations in Guizhou Province.

The peach–potato aphid, Myzus persicae (sulzer), is an important arable pest species throughout the world. Extensive use of insecticides has led to the selection of resistance to most chemical classes including organochlorines, organophosphates, carbamates and pyrethroids. Resistance to pyrethroids is often the result of mutations in the para-type sodium channel protein (knockdown resistance or kdr). In M. persicae, knockdown resistance is associated with two amino-acid substitutions, L1014F (kdr) and M918T (super-kdr). In this study, the temporal and spatial distributions of these mutations, diagnosed using an allelic discriminating polymerase chain reaction assay, were investigated alongside other resistance mechanisms (modified acetylcholinesterase (MACE) and elevated carboxylesterases). Samples were collected from the UK, mainland Europe, Zimbabwe and south-eastern Australia. The kdr mutation and elevated carboxylesterases were widely distributed and recorded from nearly every country. MACE and super-kdr were widespread in Europe but absent from Australian samples. The detection of a strongly significant heterozygote excess for both kdr and super-kdr throughout implies strong selection against individuals homozygous for these resistance mutations. The pattern of distribution found in the UK seemed to indicate strong selection against the super-kdr (but not the kdr) mutation in any genotype, in the absence of insecticide pressure. There was a significant association (linkage disequilibrium) between different resistance mechanisms, which was probably promoted by a lack of recombination due to parthenogenesis.

Background The emergence and spread of insecticide resistance in malaria vectors threatens vector control efforts. The use of spatial repellent products (SR) containing volatile insecticides such as transfluthrin offer a promising complementary strategy to current vector control tools. Here, we employed whole transcriptome analysis to investigate the molecular mechanisms underlying reduced behavioral sensitivity to transfluthrin in two pyrethroid-resistant populations of Anopheles gambiae s.s. Using a high-throughput screening system (HITSS), we evaluated 600 mosquitoes across three populations (Bungoma field population, the insecticide-resistant Pimperena lab strain, and the susceptible Kisumu lab strain), categorizing them as responders or non-responders based on their SR avoidance behavior. Non-responders exhibited significantly reduced repellency (spatial activity index < 0.1) at standard transfluthrin concentrations (0.0025 μg/ml). Results RNA sequencing of pooled samples ( n  = 10 mosquitoes per pool, three replicates per condition) revealed distinct transcriptional profiles between responders and non-responders. The cytochrome P450 gene CYP12F12 showed significant overexpression (FC = 36.6389, p  < 0.001) in Bungoma non-responders, suggesting its potential role in transfluthrin metabolism. Additionally, we observed population-specific distributions of voltage-gated sodium channel mutations, with fixation of kdr L995F in Pimperena non-responders and elevated frequency (80–100%) of kdr L995S in Bungoma non-responders. Conclusions These findings provide the first molecular evidence linking both metabolic and target-site mechanisms to reduced behavioral sensitivity to transfluthrin in malaria vectors. The co-occurrence of CYP12F12 overexpression and kdr mutations suggests multiple resistance mechanisms may affect spatial repellent efficacy, highlighting the need for resistance monitoring in spatial repellent deployment strategies. Graphical Abstract

We searched for evidence of knockdown resistance (kdr) mutations in the voltage-gated sodium channel gene of Aedes aegypti (Linnaeus) (Diptera: Culicidae) and Aedes albopictus (Skuse) (Diptera: Culicidae) mosquitoes from Panama. Conventional PCR was performed on 469 Ae. aegypti and 349 Ae. albopictus. We did not discover kdr mutations in Ae. albopictus, but 2 nonsynonymous kdr mutations, V1016I (found in 101 mosquitoes) and F1534C (found in 29 of the mosquitoes with the V1016I), were detected in Ae. aegypti.These kdr mutations were present in all specimens that were successfully sequenced for both IIS5-S6 and IIIS6 regions, which included samples collected from 8 of the 10 provinces of Panama. No other kdr mutations were found in Ae. aegypti, including V1016G, which has already been reported in Panama. Findings suggest that the V1016I-F1534C variant is prevalent in Panama, which might be related to the introduction and passive movement of mosquitoes as part of the used-tire trade. However, we cannot rule out the possibility that selection on de novo replacement of kdr mutations also partially explains the widespread distribution pattern of these mutations.These 2 ecological and evolutionary processes are not mutually exclusive, though, as they can occur in tandem. Research in Panama needs to calculate the genotypic and allelic frequencies of kdr alleles in local Ae. aegypti populations and to test whether some combinations confer phenotypic resistance or not. Finally, future studies will have to track the introduction and spreading of new kdr mutations in both Aedes species. Graphical Abstract

Background Malaria is still endemic in South Korea. However, limited information is available on the current Anopheles breeding sites and the occurrence of insecticide resistance-associated genetic mutations and their distribution needed to control the malaria vector efficiently. Methods This study explored breeding sites of Anopheline adults in Gimpo-si, near the demilitarized zone (DMZ) in Gyeonggi-do province, South Korea, from 2022 to 2023. Genetic diversity was investigated based on the internal transcribed spacer (ITS2), cytochrome c oxidase subunit I (COI), and knockdown resistance ( kdr ) genes of Anopheles mosquitoes. A natural environment associated with the seasonal abundance of Anopheles larvae was characterized. Results Two breeding sites of Anopheles larvae and adults were found at a stream margin or shallow freshwater near the forest in Wolgot-myeon in Gimpo-si without cattle shed within 1 km and in Naega-myeon in Ganghwa-gun with cow shed within 100 m in 2022 and 2023, respectively. Both sites were located between the newly cultivated lands and the forest. Besides, both breeding sites were in the valley at a slight elevation of 60–70 m from ground lands and maintained the shadow all day. Overall, the Wolgot-myeon breeding site showed various Anopheles spp. larvae, including Anopheles sinensis . Naega-myeon, an additional breeding site found in 2023, had Anopheles sineroides larvae, and approximately 59.7% (89/149) of An. sinensis adults inhabited within a 100-m distance. The total collection, including larvae and adults, revealed that An. sinensis , Anopheles pullus , Anopheles kleini , An. sineroides , Anopheles belenrae , and Anopheles lindesayi accounted for 44.2% (118/267), 0.7% (2/267), 0.7% (2/267), 22.1% (59/267), 1.9% (5/267), and 30.3% (81/267), respectively. Furthermore, various kdr mutant genotypes (F/F, C/C, L/F, L/C and F/C) in An. sinensis , and the first kdr allele mutant (L/F1014) in An. belenrae were identified in South Korea. Conclusions Two breeding sites of Anopheles larvae were studied in Wolgot-myeon and Naega-myeon. Various Anopheles spp. larvae were detected in both habitats, but overall, An. sinensis was the most prevalent adults in both study sites. The occurrence of kdr allele mutant of An. belenrae in South Korea was reported. Rigorous larvae monitoring of Anopheles spp., continuously updating information on Anopheles breeding sites, and understanding the environmental conditions of Anopheles habitats are required to develop an effective malaria control programme in South Korea.

Background Dengue is a serious public health problem worldwide, including in Selangor, Malaysia. Being an important vector of dengue virus, Aedes aegypti are subjected to control measures which rely heavily on the usage of insecticides. Evidently, insecticide resistance in Ae. aegypti , which arise from several different point mutations within the voltage-gated sodium channel genes, has been documented in many countries. Thus, this robust study was conducted in all nine districts of Selangor to understand the mechanisms of resistance to various insecticides in Ae. aegypti. Mosquitoes were collected from dengue epidemic and non-dengue outbreak areas in Selangor. Methods Using the Center for Disease Control and Prevention (CDC) bottle assays, the insecticide resistance status of nine different Ae. aegypti strains from Selangor was accessed. Synergism tests and biochemical assays were conducted to further understand the metabolic mechanisms of insecticide resistance. Polymerase chain reaction (PCR) amplification and sequencing of the IIP-IIS6 as well as IIIS4-IIIS6 regions of the sodium channel gene were performed to enable comparisons between susceptible and resistant mosquito strains. Additionally, genomic DNA was used for allele-specific PCR (AS-PCR) genotyping of the gene to detect the presence of F1534C, V1016G and S989P mutations. Results Adult female Ae. aegypti from various locations were susceptible to malathion and propoxur. However, they exhibited different levels of resistance against dichlorodiphenyltrichloroethane (DDT) and pyrethroids. The results of synergism tests and biochemical assays indicated that the mixed functions of oxidases and glutathione S-transferases contributed to the DDT and pyrethroid resistance observed in the present study. Besides detecting three single kdr mutations, namely F1534C, V1016G and S989P, co-occurrence of homozygous V1016G/S989P (double allele) and F1534C/V1016G/S989P (triple allele) mutations were also found in Ae. aegypti . As per the results, the three kdr mutations had positive correlations with the expressions of resistance to DDT and pyrethroids. Conclusions In view of the above outcomes, it is important to seek new tools for vector management instead of merely relying on insecticides. If the latter must be used, regular monitoring of insecticide resistance should also be carried out at all dengue epidemic areas. Since the eggs of Ae. aegypti can be easily transferred from one location to another, it is probable that insecticide-resistant Ae. aegypti can be found at non-dengue outbreak sites as well.

From 2008 to 2017, the city of Abidjan, Côte d'Ivoire experienced several Aedes-borne disease epidemics which required control of the vector mosquito population based on the reduction of larval habitats and insecticidal sprays for adult mosquitoes.This study was undertaken to assess the insecticide susceptibility status of Aedes aegypti (Linnaeus) in the city of Abidjan. Immature Ae. aegypti were sampled from several larval habitats within seven communes of Abidjan and reared to adults.Three to five days old F1 emerged adults were tested for susceptibility using insecticide-impregnated papers and the synergist piperonyl butoxide (PBO) following WHO bioassay guidelines. The results showed that Ae. aegypti populations from Abidjan were resistant to 0.1% propoxur, and 1% fenitrothion, with mortality rates ranging from 0% to 54.2%. Reduced susceptibility (93.4–97.5% mortality) was observed to 0.05% deltamethrin, 0.75% permethrin, 0.05% lambda-cyhalothrin, 5% malathion, and 0.8% chlorpyrifos-methyl. This reduced susceptibility varied depending on the insecticide and the collection site. The restoration of mortality when the mosquitoes were pre-exposed to the synergist PBO suggests that increased activity of oxidases could be contributing to resistance. Three kdr mutations (V410L, V1016I, and F1534C) were present in populations tested, with low frequencies for the Leu410 (0.28) and Ile1016 (0.32) alleles and high frequencies for the Cys1534 allele (0.96). These findings will be used to inform future arbovirus vector control activities in Abidjan.